Journal of Muscle Research and Ceil Motility 13, 475-490 (1992)

Abstracts of the I99I Annual Meeting on Muscle and Cell Motility Physiology TEIKYO UNIVERSITY SCHOOL

OF M E D I C I N E ,

TOKYO,

29-30

NOVEMBER,

1991

MECHANICS, ENERGETICS, BIOCHEMISTRY A N D STRUCTURE OF SKELETAL MUSCLE

Effect of antibody to m y o s i n subfragment-2 on the contraction characteristics and the ATPase activity of glycerinated skeletal muscle fibres T. Kobayashi 1, T. Gross 2, K. Noguchi 1 and H. Sugi ~

Theoretical studies on the f o r c e - v e l o c i t y relation of the myosin-coated beads on actin cables and on the tension responses to sudden length change in muscle T. Mitsui I and H. Ohshima z

*Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173, Japan and 2Physiologisches Institute, Bereich Medizin, Humboldt-Universitdt zu Berlin, 1040 Berlin, Germany

Department of Physics, School of Science and Technology, Meiji University, Higashimita, Tama-ku, Kawasaki 214, Japan and 2Faculty of Pharmaceutical Sciences, Science University of Tokyo, khigaya, Shinjuku-ku 162, Tokyo, Japan

To explore the possibility suggested by Harrington and colleagues that the subfragment-2 region of myosin is involved in muscle contraction, we studied the effect of polyclonal antibody directed against the subfragment-2 (S-2) region of myosin, which were supplied to us from Harrington's laboratory, on the contraction characteristics and the ATPase activity of glycerinated muscle fibres prepared from rabbit psoas. Single muscle fibres were mounted horizontally between a force transducer (Akers, AMES01) and a servo-motor (General Scanning, PDI00) at sarcomere lengths around 2.2 I,tm. Muscle fibre stiffness was measured by applying sinusoidal vibrations (1 kHz, peak-to-peak amplitude 0.1% of fibre length) with the servo-motor. After a steady isometric force was reached in response to contracting solutions containing 10-SM Ca2+, the force-velocity relation was obtained by applying a ramp decrease in load from the steady force to zero and recording the resulting fibre shortening with a differential transformer incorporated in the servo-motor. The Mg-ATPase activity of small fibre bundles (about 2 mm diameter) was also measured during the Ca2+-activated force development by measuring the decrease of NADH as an ADP production. All experiments were made at room temperature (23-25~ When the fibres were kept in relaxing solutions containing the anti-S-2 antibody (1.5-3 mg ml i) for some time and then made to contract by contracting solutions, the magnitude of the CaZ+-activated steady force decreased by 20-80% depending on the time of previous treatment with the antibody. The stiffness of the fibres decreased in parallel with the Ca2+-activated force so that the linear force versus stiffness relation remained unchanged before and after the antibody treatment. Despite the marked decrease in Ca2+-activated force, the maximum shortening velocity of the fibres remained the same before and after the antibody treatment, and the force-velocity curves were superimposed when they were normalized with respect to the maximum steady force. These results indicate that when the anti-S-2 antibody combines with the S-2 region of a crossbridge it can no longer contribute to force generation and fibre stiffness; in other words, it no longer attaches to the thin filament. Measurement of the Mg-ATPase activity of the fibres showed, however, that the ATPase activity did not change appreciably despite ~he marked decrease of the Ca2+-activated force development. The above findings suggest that it is necessary to reconsider the mechanism of 'actin activation of myosin ATPase' derived from the ATPase kinetics of myosin and actomyosin.

0142-4319 9

1992 Chapman & Hall

We have extended and refined the model of myosin head motion along the actin filament proposed by us in 1988 [J. Muscle Res. Cell Motility 9, 248-60] and discussed on the experimental results reported by Chaen and colleagues (Abstracts in this journal) and by Ford and colleagues [J. Physiol. 269 (1977) 441-515]. We assumed that the height of the induced potential discussed in our above-mentioned paper depends on tension with a maximum around the isometric tension so that efficiency of transfer of ATP hydrolysis energy to mechanical energy is increased. We obtained a force-velocity relation similar to the observation by Chaen and colleagues in some speicmens that the velocity of the myosin head along the actin cables decreases first with increasing centrifugal force applied in the direction of bead movement ('negative load') and then tends to increase approaching asymptotically to a certain value. As to the centrifugal force applied opposite to the bead movement ('positive load'), our theory indicated that the bead velocity could change with an upward convex and then decrease to zero if the maximum of the induced potential would shift to lower tension, in accordance with the observations for some specimens. We assumed that binding of an ATP-activated myosin head to an actin filament causes a local structural change extending roughly 20 nm long on the actin filament, which provides a potential well over about 16 nm for the myosin head, and the rest of the modified structure (of roughly 20 nm minus 16 nm) is responsible for producing the induced potential. The potential well of more than 16 nm long covers three actin molecules on the strand of actin fibre and thus three binding sites for the myosin head. Therefore, we postulated further that within the potential well there are three additional potential wells with progressively lower potential energies, similar to the model discussed by Huxley and Simmons [Nature 233 (1971) 533-8]. We studied the kinetics of the myosin head in the potential well about 16 nm tong to explain the early tension recovery .after the sudden change of muscle length as observed b y Ford and colleagues. Calculation was done without further approximations, by solving differential equations for the kinetics and by numerical integration to obtain tension as a function of time and the amplitude of the length change step. It was difficult to explain the experimental results when we assumed that depths of the three potential wells are insensitive to tension. Good agreement with the experimental data was obtained by the additional assumption that depths of the three potential wells

476 increase linearly as functions of amplitude of the length change step.

ABSTRACTS

Fluctuation of sarcomere length during shortening in frog skeletal muscle examined b y laser diffraction method M. Matsumura

Shortening of single sarcomeres in a scallop striated muscle myofibril activated b y rapid solution exchange

Department of Physiology, Kawasaki Medical School, Kurashiki 701-0I, Japan

T. Tameyasu

The changes in sarcomere length, SL, during isotonic contraction were measured using a laser diffraction method. A single muscle fibre was prepared from the tibialis anterior muscle or semitendinosus muscle of the bullfrog. The principles of the method for continuous measurement of SL were the same as originally devised by Iwazumi and Pollack (1979), and the change in SL was recorded simultaneously with the whole length change and tension development. The length of the fibre illuminated by the laser beam was minimized to < 1 ram. As the illuminated portion moved during muscle shortening, only the fibres that showed consistent diffraction patterns along the longitudinal axis were selected. The muscle was stimulated by short tetanic pulses. In isometric contraction at SL of 2.5 ~tm, rapid and expansive SL fluctuations were observed at the onset of contraction, and they were followed by slow and small ones during the tension plateau. The initial rapid SL fluctuation was considered to result from asynchronous activation of the myofilaments along the radial direction of the muscle fibre. In isotonic contraction, the rapid SL fluctuation remained as it was observed in the isometric one. A slow SL fluctuation lasted throughout shortening phase when the muscle shortened under a load of 0.2 P0 at 20-22~ The extent of peak-to-peak SL fluctuation attained + 0.05 ~tm with 2-5 ms periodicity. Increasing afterload to 0.6 P0 or total load of 0.8 P0 did not alter the extent of the SL fluctuation. Lowering the temperature of the bathing solution to 3~ the maximum SL fluctuation during steady-state shortening decreased to -}-0.02 ~tm (peak-to-peak) or less. The sarcomere shortening velocity was 1.15 ~tm s -~ under 0.2 P0 and 0.37 ~tm s -1 under 0.6 P0, and the extent of the SL fluctuation was independent of the amplitude of afterload or shortening velocity. The present study showed that the changes in the average SL within the portion illuminated by the laser beam were variable during shortening, although the sarcomeres at the resting state were arranged uniformly. It is considered that some sarcomeres shorten faster by 2% (3~ or approximately 5% (20~ than other adjacent sarcomeres in one moment and the latter shorten faster than the former in the next intervals. The independent SL fluctuations of mass of load or shortening velocity suggest that the fluctuations resulted from a different degree of activation among the sarcomeres rather than asynchronized movements of crossbridges.

Department of Physiology, School of Medicine, St. Marianna University, Kawasaki 216, Japan The time course of unloaded shortening of single sarcomeres was studied with myofibrils (1 ~tm thick, 4-5 ~m wide and (10 mM EGTA) was induced by photorelease of approximately 130 gM ATP in skinned fibres from rat psoas at 15-16~ pH 7.0 and F/2 = 200 raM. The magnitude of the contraction did not depend on [EGTA] (1-30 raM). In each experiment, two force transients were recorded: one (s) stretching the muscle fibre (approximately 0.4%) before photolysis, and the other (i) under the same conditions but without stretch. The difference between s and i was assumed to represent the time course of detachment (d) of the rigor crossbridges. The time course of force development (f) by reattached crossbridges could be estimated by subtracting an appropriately scaled d from s. Neither EG (ethylene glycol, 20%) nor Ca 2+ (approximately 50,M) significantly affected d. On the other hand, EG made f half the control, slowing down its time course, while Ca 2+ (approximately 50 ~tM)made f twice as large as the control without changing its time course. The effect of Ca2+ was the same in both the presence and absence of EG. The independence of the effects of Ca 2+ and EG suggests that although EG suppresses contractile activity it has little effect on the mechanism of its regulation.

Ligand-dependent rotation of myosin head crosslinked to actin with EDC in skinned rabbit skeletal muscle fibres H. Iwamoto and R. J. Podolsky

Laboratory of Physical Biology, NIAMS, NIH, Bethesda, MD 20892, USA Actomyosin complex is largely classified into two categories: weakbinding and strong-binding (rigor-like) species. Rotatability of the strong-binding myosin head has been tested by straining rigor muscles with negative results [Naylor & Podolsky (1981) Proc. Natl. Acad. Sci. USA 78, 5559-63]. The test of the rotatability of weak-binding myosin head is hampered by the tendency of the head to detach from actin easily. Crosslinking of myosin heads to actin with EDC provides a practical means to overcome this difficulty. The crosslinking site on actin is known to be the acidic N-terminal segment [Sutoh (1982) Biochemistry 21, 3654-61]. Studies using the antibody raised against this segment and the actin-binding fragment of caldesmon [Adams et al. (1990) ]. Biol. Chem. 265, 2231-7; Brenner et al. (1991) Proc. Natl. Acad. Sci. USA 88, 5739-43] suggest that the site for EDC crosslinking is identical to that for the weak interaction, and therefore crosslinking can produce weak-binding myosin heads which do not dissociate from actin in the presence of nucleotides like ATP.

ABSTRACTS

477

When uncrosslinked fibres were stretched by 1% of their length, little changes were observed in the equatorial reflection intensities I~,0 and I~,I . After EDC-crosslinking, however, a decrease of up to 10% was observed in Ii,x. in the presence of nucleotides (ATP, ATP-7-S, pyrophosphate (PPi), and AMPPNP), while Ii.0 stayed relatively constant. In the presence of ADP or in rigor, the decrease of I],~ was either very slow or absent. This behaviour of equatorial reflection is consistent with a three-dimensional form of myosin head rotation. The rate of I~,~ decrease paralleled the dissociation rate constant of actomyosin or the mechanical relaxation rate in the presence of each ligand. This result is best explained if one assumes that the ligands do not dissociate the crosslinked actomyosin complex but trap it in a weak-binding state, in which the head is more elastic than a strong-binding head and rotatable when stressed. To test the possibility that the present observations are the result of the mixed population of crosslinked and uncrosslinked myosins, the KC1 or K-acetate concentration was raised from 120 mM to 500 mM in the presence of PPi to dissociate all the myosins which remained uncrosslinked [Ishiwata et al. (1986) Biophys. J. 49 821-8; Brenner et al. (1986) Biophys. J. 50, 1101-8]. With our standard crosslinking procedure (8 mM EDC, 20~ 20 rain reaction), the increased potassium concentration did not appreciably affect the equatorial pattern or the mechanical response to stretch. This indicates that most of the myosin molecules were crosslinked and the observed changes in I~,~ can be ascribed to the crosslinked myosins. Independent evidence for almost complete crosslinking has been obtained through mechanical experiments [Iwamoto & Podolsky (1992) (in press)].

Direct experimental method to determine the protein friction exerted by protein motors through a weak-binding interaction in the in vitro motility. K. Tawada

Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Fukuoka 812, Japan In our previous work [Biophys. J. (1991) $9, 343-56; J. Theor. Biol. (1991) 150, 193-200], we showed that a weak-binding interaction of protein motors with a corresponding cytoskeletal polymer results in a viscous-like protein friction. The protein friction exerted on a sliding polymer is proportional to the sliding velocity and more than ten times larger than the hydrodynamic viscous friction. The coefficient of the protein friction is a function of the polymer length, the lifetime of the single weak-binding interaction, the elastic stiffness constant of a motor protein and the density of protein motors on the solid surface. With the protein friction hypothesis we could explain many characteristics of the in vitro motility of myosin motors and microtubule-associated motors. With a diffusion constant (Dp) related to the coefficient of the protein friction (~p) by the Einstein equation (Dp = kBT/~p), we could explain the one-dimensional Brownian movement of a microtubule placed on dynein-coated solid surface in the presence of vanadate and ATP as well. Hence, the sliding velocity (Vs) in the in vitro motility can be given by

,

v~=r;G/GT)

(1)

where Fp is the sliding force generated by protein motors. Note that once Dp is experimentally determined as shown below, Fp can also be estimated by using Equation 1. Although both Fp and Dp are caused by the protein motors unlike the usual case, Equation 1 suggests that we can regard in vitro motility as a diffusion process with drift. If we do so and consider the sliding movement of a cytoskeletal polymer driven by protein motors in position space rather than in velocity space, we can derive the following. The average of the sliding distance in a period of time (t)

is given by (X(t)) = [FpDp/(kBT)]t, which corresponds to Equation 1, and the standard deviation of the sliding distance is given by x/ =

x/~pt.

(2)

As X(t)/t is a drift velocity (Va) and its average is the same as V~ in Equation 1, the standard deviation of the drift velocity is given by - < vd >)2 > = , / ~ p / t (3)

,/ (contraction-gelsolin). The passive tension versus extension relation in (contraction-gelsolin) was similar to that in intact myofibrils under a relaxing condition, indicating that the resting tension is mainly ascribed to the extension of connectin (titin); this result confirms the previous one obtained by using muscle fibres [Funatsu et al. (1990)]. The passive tension versus extension relation in (rigor-gelsolin) suggested that nebulin is attached side-by-side to thin filaments in an intact form but shows elasticity after the partial removal of thin filaments.

Measurement of force of actomyosin in in vitro motility assay using bead-tailed F-actin and optical tweezers N. Suzuki I, S. Ishiwata 2 and K. Kinosita, JrJ

Department of Physics, Faculty of Scienceand Technology, Keio University, Kohoku-ku, Yokohama 223, Japan and ZDepartment of Physics, School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo I69, Japan We have developed a new system for measuring the forces produced by HMM interacting with a single actin filament in in vitro motility assay system. We succeeded in covalently crosslinking gelsolin to latex beads (1 I,tm in diameter). The gelsolin-coated bead selectively bound to the barbed end of F-actin. We call this bead-bound F-actin 'bead-tailed F-actin' as the F-actin slid over HMM pulling the bead at the tail when the movement was observed in an in vitro motility assay system described by Kron and colleagues [Methods Enzymol. (1991) 196, 399-416]. The bead-tailed F-actin was manipulated by trapping the bead in a light spot formed by tightly focusing the laser beam from a Nd/YAG laser ('optical tweezers'). When the power of the laser beam was high, we were able to stop sliding of F-actin by trapping

ABSTRACTS the bead bound to F-actin. Under this high power state, it was also possible to pull up sliding F-actin from an HMM-coated surface. But we failed to pick up F-actin bound to HMM in rigor condition. We reduced the power of the laser beam to a state in which the trapping force and the forces produced by HMM were comparable. Under this low power state, there existed a critical length of F-actin depending on the power of laser beam. The bead pulled by F-actin longer than the critical length overcame the trapping force and escaped. But the bead pulled by F-actin shorter than the critical length was trapped. In the latter case, we observed the position of bead fluctuating in the direction parallel to the filament axis of F-actin. We were able to trace the position of the bead with a time resolution of 33 ms and spatial resolution of a few nm using a digital image processor (DIPS, Hamamatsu Photonics). The size of the bead fluctuation parallel to the filament axis was about 50-100 nm and the perpendicular fluctuation about 2-5 nm. This latter value was of the same extent as the position fluctuation of a bead, without F-actin, undergoing Brownian motion under the same laser power. We derived a potential energy profile of the trapping force from an analysis of Brownian motion of a bead. We estimated the force produced by HMM interacting with a single actin filament in length about 4 btm was 3-4 pN.

Elementary distance of the ATP-induced actin-myosin sliding under very small load studied using an in vitro force-movement assay system K. Oiwa, T. Kawakami, S. Chaen and H. Sugi

Department of Physiology, School of Medicine, Teikyo University, Itabashiku, Tokyo I73, Japan Using an in vitro force-movement assay system which consists of a myosin-coated glass needle and actin cables of a green alga, Nitellopsis obtusa, we studied the distance of 'elementary' actin-myosin sliding under very small load in response to iontophoretically applied ATP. To avoid complications arising from large numbers of myosin heads involved in the ATP-induced sliding along actin cables, we coated the needle with copolymers of myosin and myosin rod (molar ratio, 1 : 10) in contrast with our previous experiments, in which the needle was simply coated with filaments of rabbit skeletal muscle myosin [Chaen et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1510-14; Oiwa et al. (1991) ]. Physiol. 437, 751-63]. The maximum isometric force exerted by these copolymers on the needle was reduced from 500-1000 pN to < 100 pN with decreasing content of myosin in the copolymers. The elastic coefficient of the needle used was 56 pN ~tm i. The experimental solution contained 50 units per ml hexokinase and 2 mM glucose to remove ATP quickly around the needle. When a tip of the ATP electrode was placed 20-30 ~tm distant from that of the needle and ATP was applied as a negative pulse lasting I s (total charge, 2-80 nC), the needle typically responded with brief ( < 3 s) and short ( < 0.5 ~m) sliding movement. When the amount of ATP released from the ATP-electrode was reduced by decreasing amount of charge passed through the electrode from 80 nC to 2 nC, the distance of the ATP-induced actin-myosin sliding decreased almost linearly from about 100 nm to about 10 nm. No needle movement was detectable when the amount of charge passed through the ATP electrode was further reduced. With decreasing amount of charge through the ATP-electrode, both the amount of ATP delivered to myosin heads and the period in which the delivered ATP stays around myosin heads are expected to decrease. As the force exerted by the myosin-coated needle is only 0.56 pN when its tip is displaced by 10 nm by the actin-myosin sliding, the minimum actin-myosin sliding observed (about 10 nm) takes place under very small load (about 0.6% of the maximum isometric force of 100 pN). It is therefore suggested that when a limited number ( ~ 100) of myosin heads are synchronously activated to interact with actin cables, their elementary distance of sliding along actin cables under very small load is of the order of 10 nm.

479

ABSTRACTS

Motility of fluorescent F-actin on heavy meromyosin in vitro in the absence of phalloidin

of Applied Chemistry, Muroran Institute of Technology, Hokkaido 050, Japan

N. Oishi

To study the structural characteristics of myosin head, we investigated structural changes of myosin head induced by tryptic digestion by use of IH-NMR technique. Myosin subfragment-1 was prepared by chymotryptic digestion of myosin isolated from back and leg muscles of rabbit. Limited tryptic digestions of myosin subffagment-1 were made to produce myosin heads with nicks at specific loci in its heavy chain, one with a nick between 75 kDa and 20 kDa domain and another with nicks between 23 kDa and 50 kDa domain and between 50 kDa and 20 kDa domain. NMR measurements were made with an NMR spectrometer (JEOL GX-400) at 400 MHz for protons. The temperature of myosin subfragment-1 solutions was changed by purging temperature-controlled nitrogen gas over the NMR sample tube during measurement. The changes of IH-NMR spectra associated with a mild trypsin treatment of myosin subfragment-1 indicated that an N-terminal peptide fragment of light chain was split off while the SDS-PAGE indicated that a nick was formed between 75 kDa and 20 kDa domain of its heavy chain. Further extensive trypsin treatment of myosin subfragment-1 produced another nick between 23 kDa and 50 kDa domain of its heavy chain which was confirmed by SDS-PAGE and split off a loop between 23 kDa and 50 kDa domain which was confirmed by the changes of ~H-NMR spectra. The i H-NMR spectra of myosin subfragment-1 with nicks was almost the same with that of intact myosin subfragment-1. When temperature was changed between 4 and 60~ NMR spectra of myosin subfragment-1 with nicks changed in almost the same fashion as those of intact myosin subfragment-1, suggesting that the stability of overall structure of myosin head is not affected by nick formations in the heavy chain of myosin head. Small changes of mobile amino acid residues including the exposure of hydrophobic residues, were found associated with the trypsin treatments which possibly related with the changes of ATPase and actin binding activities of myosin heads owing to nick formation in its heavy chain.

Radioisotope Research Centre, School of Medicine, Teikyo University, Tokyo I73, Japan Although it is known that depletion or substitution of the nucleotide bound to actin causes some conformational change in F-actin, no differences in the sliding movement have been observed among the nucleotide-modified F-actins (NM-actins) by an in vitro motility assay using rhodamine-phalloidin [Oishi et al. (1991) J. Muscle Res. Cell MotiI. 12, 310]. Because the nucleotide-binding site is close to phalloidinbinding site in an actin monomer, phalloidin might possibly change the structural properties of F-actin and cancel the effect of the modification of the bound nucleotide. So I examined the sliding movement of NM-actins in vitro in the absence of phalloidin. Rabbit skeletal muscle F-actins were labelled with tetramethylrhodamine iodoacetamide. Three kinds of NM-actins were prepared: F-actins polymerized from G-actin containing ADP, F-actins containing a small fraction of nucleotide (ADP K 1%, AMP K 8% determined from HPLC analysis), F-actins containing 8-bromoADP. In order to obtain long filaments, the F-actins were concentrated to 100 ~M and preincubated with 2~tM heavy meromyosin overnight. Soon after dilution of the F-actins with a solution containing 0.1 ~M cytochalasin D, sliding movement on heavy meromyosin was examined in vitro. Without phalloidin, F-actins often moved interruptedly or arrested and rapidly depolymerized under the diluted sliding condition. However, all kinds of NM-actins were able to slide. The results suggest that the F-actinbound nucleotide might have little effect on the sliding movement.

Direct effects of lactic acid and pH in glycerinated muscle fibres H. Aoki ~, Y. Nakayama I and M. Yamaguchi 2

Department of PhysiolOgy & Anatomy, Kyoritsu Collegeof Pharmaceutical Science, Tokyo 105, Japan and 2Department of Biochemistry and Nutrition, School of Health and Physical Education, Juntendo University, Chiba 270-I6, Japan It is well known that muscle fatigue after heavy exercise is accompanied by an increased concentration of lactic acid (LA), and a decreased pH, in skeletal muscle tissues. We examined the direct effects of LA and pH, using a MES buffer solution, on the contractile protein system in glycerinated muscle fibre. The tension development of fibre declined with treatment in LA for 30 min at 35~ concentration of 10, 20, 30 mM, and pH 6.5, 6.0, 5.5, respectively. However, only decreasing pH from 6.8 to 5.5 with no LA, under the same condition of LA-pH-[reatments, tension also declined, particularly remarkable at pH 5.5, although it was milder than the effects of LA and pH together. When the tension height decreased with LA and pH, the rate of tension rise tended to be slower considerably than normal. However, the reduced tension height and rate of rise recovered to normal after soaking in a MES buffer solution. On the other hand, keeping pH at 6.8 in the presence of LA (containing 30 mM), hardly affected contractility in muscle fibre. Furthermore, sucrose solution, 0.1-0.4 M added to buffer solution, could fairly protect against the declining effects of LA and pH, preserving hydrogen bonds in the contractile protein. From these results, it was assumed that there is a chemical obstruction from LA and pH together which is produced reversibly at the molecular level of actin-myosin interaction in the myofilaments of glycerinated muscle fibre.

~H-NMR studies of structural changes of myosin subfragment-1 of rabbit skeletal muscle induced by limited trypsin digestion T. Yamada ~, Y. O k a m o t o 2 and H. Sugi 1

I Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173, Japan and 2Division of Bioengineering Department

Water structure is stabilized well in the relaxed muscle M. Ogata

Institute of Health Science, Kyushu University, Kasuga ,516, Japan Dynamic ~H-NMR study on the water structure in the muscle suggested that the water structure in muscle is more stable in relaxation than in contraction. At a magnetic field of 500 MHz (FT NMR) the bandshapes of I H-NMR for a skeletal muscle bundle (m. semitensinosus, bullfrog), packed in a TEFRON tube without any external solutions, are asymmetrical and suggest that in muscle there are two different levels of water structures corresponding to the different resonance frequencies. One (Wf) is a major signal related to a fleer water group, and the other (Wa) is a minor signal related to a more highly structured water group. From the theoretical point of view the bandshape of the muscle water fits unequally populated and uncoupled two-site exchange case. Vast varieties of bandshapes can be simulated by employing the general equation. Parameters needed to calculate are: (1) population (Pwf, Pwa), (2) frequency difference (c~v), (3) transverse relaxation time (Twf, Twa), and (4) rate constant (Kwf, Kwa). As the equation expressed by Rogers and Woodbrey is so strict plausible values of all parameters can be obtained. As a result of a precise simulation, it is concluded that in the relaxed muscle Twf is 8.4 ms and it increases up to 159 ms during the contraction. This marked change of transverse relaxation time indicates that even a majority group of water (Wf) is well structured in relaxation and it becomes almost as free as pure water in contraction as if the water structure has collapsed. The muscle cell contains a considerable amount of protein. The skinned fibre shows a jelly-like structure. A dense F-actin solution is just a jelly indicating that water structure around actin are stabilized. A transverse relaxation time of water in the F-actin pellet is K 10 ms. Myoplasmic resistance is high in relaxation and

480 suddenly decreases by 46% during sustained contraction. These facts seem to coincide with behaviour of water structure in the muscle.

Vanadate-dependent photochemical cleavage of myosin heavy chain from rabbit skeletal muscle Y. O k a m o t o and C. Cremo

Division of Bioengineering, Department of Applied Chemistry, Muroran Institute of Technology, Muroran 050 Japan and Biochemistry and Biophysics DepartmenL Washington State University, Pullman WA 99164-4660 USA Our knowledge of the myosin head region, where the principal energy transducing process takes place, is far more atomic resolution. We need to know more about the structural and functional organization of the myosin head. Previously, we have worked mainly on two subjects. Photoaffinity labelling techniques have been used to map key sites for nucleotide binding on the primary sequence. Second, we have tried to isolate a smaller piece of the head fragment from myosin after making a specific proteolytic nick within the head. The smaller fragment is then dissociated from the remaining molecule by a heat treatment. It has proved difficult to isolate a pure form of this smaller active fragment because of incomplete inactivation of the protease activity during the heat treatment. This is why we have to use a similar protocol to prepare a cleaved myosin but without the use of protease. As a first step to prepare such a myosin, myosin from rabbit skeletal muscle has been photochemically cleaved in the presence of vanadate ion and ATP. Two cleavage sites termed V1 and V2 within the $1 head region were studied. In the presence of magnesium ion both sites were cleaved but in the absence of divalent cation cleavage only occurred at the V2 site. From the previous work on nucleotide trapping in the active site of myosin, it is known that in the presence of EDTA, nucleotide cannot be stably trapped in the active site. However, the added ATP appears to protect the V1 site in the active site against vanadate interaction while the V2 site can be cleaved exclusively, cleaved myosin had higher K+-EDTA-ATPase and actin activated Mg>-ATPase activity than V1, V2 cleaved mysoin. From these observations the V1 site is confirmed to be the important site for nucleotide binding in the catalytic cycle of the ATPase reaction. Immunochemical characterization shows that the photochemical cleavage is more specific than that of proteolytic cleavage as breakdown of light chains was not observed for the photochemical method. What is the relevance of the vanadate binding and cleavage at the V2 site? Thus, the V2 site may be a nucleotide-independent phosphate binding site. It would now be useful to test the V2-cleaved myosin using a more biological method such as an in vitro motility assay. Vanadatedependent photochemical cleavage reaction could be a powerful way to prepare a single site alteration in myosin which may be superior in specificity to that of pfoteolytic enzyme.

Complex between non-polymerizable actin and myosin T. Arata

Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan Polymerization of G-actin in the presence of salt was blocked by treatment of G-actin with m-maleimidebenzoic acid N-hydroxysuccinimide ester (MBS) (designated as m-actin). The actin dimer produced by chemical crosslinking of F-actin with N,N'-p-phenylenedimaleimide did not polymerize and was still dimeric or tetrameric after further treatment with MBS (designated as d-actin). However, m-actin polymerized during long incubation with phalloidin and stimulated myosin $1 ATPase-like native F-actin (considerably higher Vm and lower Km), suggesting no serious damage. These actins retained the ability to bind DNase I; a 1:1 actin monomer-DNase I complex for m-actin whereas a 2: I actin monomer-DNase I complex for d-actin. The m- and d-actins also retained the ability to bind to myosin heads with apparent dissociation constants of 3-8 x 10 0 M and 3-5 x 10 7 M, respectively. d-Actin formed a 1:1 actin monomer-myosin head complex. How-

ABSTRACTS ever, m-actin formed a 2:1 m-actin head complex, suggesting at least two latent actin-binding sites on a myosin head. ATP weakens only two to six-fold the binding of these complexes. One of two m-actins on a myosin head was replaced by d-actin. Native F-actin blocked the binding of both m- and d-actins on myosin heads in the presence and absence of ATP, although the affinities of myosin head for MBStreated actins and F-actin are similar in the presence of ATP. These results suggest that there are at least three actin-binding sites on a myosin head: one is responsible for F-, m- and d-actins, the second for F- and m-actins, and the third for F-actin at least in the presence of ATP. F-actin binding to the third site may be transmitted in some way to block the first and second binding sites [Arata, J. Biochem. 109, 335-40]. m-Actin was covalently crosslinked to $I or HMM by MBS. The resulting complex did not bind to F-actin and showed no F-actinactivated ATPase, suggesting that m-actin- and F-actin-binding sites overlap or interact with each other on myosin head. Similar complex was produced to the less extent in the presence of ATP (on SDS gels). The electron microscopic examination of these monomeric acto-HMM complex is now in progress to explore the spatial location of actin binding site on a myosin head. The preliminary experiments showed that the location is near the distal end of the head and 14.3 nm away from the head-rod junction.

Synthesis of a highly bright terbium-chelate and its application to actin T. Anflo I, N. Kobayashi 2 and E. Munekata 2

~Department of Physics, Faculty of Science, Kanazawa University, Kanazawa 920, Japan and 2Department of Applied Biochemistry, Tsukuba University, Tsukuba 305, Japan Terbium (Tb) is a fluorescent lanthanide ion. Owing to its unusually long excited lifetime (in ms), resonance fluorescence energy transfer can occur in the rapid-diffusion limit. Diffusion-enhanced energy transfer has been utilized for obtaining the distance of closest approach of the donor and acceptor. The energy transfer is susceptible to electrostatic interaction between the donor and charged acceptor which allows us to study electrostatic circumstances around a Tbbound site on protein surface [J. Muscle Res. Cell Motil. 12, 310]. The small absorbance coefficient of Tb (in the order of 0.1 M-~ cm-~), and thereby the very dim fluorescence emission, however, limit its applicability. Previously, we synthesized Tb-DTPA-phalloidin to introduce Tb to actin filaments within muscle fibres []. Muscle Res. Ceil Motil. 12, 310]. We needed a bundle consisting of about 10-20 single muscle fibres for obtaining sufficient photon counts from the Tbstained sample. Neither DTPA nor phalloidin could sensitize the emission of Tb much. SO, we decided to try to combine an aromatic ring with the Tb-chelate. Energy absorbed by an aromatic ring of a such chelate can be efficiently transferred to the adjacent lanthanide ion. DTPA dianhydride can react specifically with amine, and has capacity for reacting with two amines, retaining its ability to chelate Tb 3+ (the resulting DTPA chelator possesses three carboxyl groups). We screened 20 kinds of compounds for their ability to sensitize the Tb emission. All of the compounds tested had both a six-membered ring and an amino group. Among them, cytosine was the best sensitizer. With peak-to-peak ratio, the fluorescence intensity of a solution of Tb-DTPA-cytosine-phalloidin-labelled actin was about one-third of an equimolar solution of Rhodamine B. The excitation peak was around 305 nm. A single muscle fibre stained with this new Tb-chelate emitted so intense fluorescence that we could accumulate sufficient photon counts in a short time. The new Tb-chelate has additional expediencies. It is electrically neutral, which is quite advantageous when it is used for studying electrostatic circumstances around Tb-site on protein. Tb 3§ itself is optically isotropic. However, cytosine of the Tb-chelate has optical anisotropy. Therefore, in conjunction with the long excited lifetime, the newly synthesized Tb-chelate also serves to probe slow rotational relaxation of macromolecules in solution.

ABSTRACTS

Antibodies against head A and B of the myosin molecule S. Mural, T. Arata and A. Inoue

Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan We studied the function of two heads of myosin using antibodies which bind specifically to each of the heads of the myosin molecule. We showed previously that two heads of myosin have a different function, one head (head B) forms the myosin-P-ADP complex and the other head (head A) forms the myosin-ATP complex as a stable intermediate. We also showed that the amino acid sequence around specific Lys (Lys 86 in N-terminal 25 kDa fragment) are different between head A and B. Then, we synthesized chemically these peptides and prepared antibodies against them. These antibodies bound to S-1. The binding occurs only on A-B specific structure: the binding of anti-A antibody to S-I was blocked by A-peptide but was unaffected by B-peptide, while the binding of anti-B antibody to S-1 was blocked by B-peptide and was unaffected by A-peptide. By Western blotting on tryptic S-1 it was shown that both anti-A and anti-B antibodies bound to the 25 kDa fragment. Anti-A antibody also bound to the 27 kDa fragment. When S-1 was applied on the column of immobilized anti-A or anti-B antibodies, only half of S-1 was trapped by the column. However, the EDTA-ATPase activity is trapped only by the column of anti-B antibody. We studied the amount of antibody bound to myosin filament by the centrifugation method. Thus, 0.5 mole of anti-A or anti-B IgG bound to I mole of myosin head. However, when the mixture of anti-A and anti-B antibodies is added to the myosin filament, 1 mole of IgG bound to I mole of myosin head. The present result showed that there are two kinds of myosin head with different structure and function.

Shape change of the myosin head induced by MgATP, studied by small-angle synchrotron X-ray scattering i n solution K. Wakabayashi I, M. Tokunaga 2, I. Kohno 1, T. Hamanaka ~, Y. Sugimoto 1, Y. Takezawa ~, T. Wakabayashi 2 and Y. Amemiya 3

1Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560, Japan, 2Department of Physics, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 153, Japan and 3Photon Factory, National Laboratory for High Energy Physics, Tsukaba, Ibaraki 305, Japan We have investigated conformational change of the myosin head (subfragment 1 ($1)) in the presence of MgATP by small-angle X-ray scattering in solution using synchrotron radiation. Myosin $1 was isolated from chicken pectoralis muscle and cleaved with papain. This $1 preparation has both classes of light chains and a heavy chain for a total molecular weight of 130 kDa. Nucleotide-free and various nucleotides-bound Sls were prepared and purified by an HPLC just before X-ray experiments. Small-angle synchrotron X-ray scattering was performed by using the diffractometer at BL-15A1 of the Photon Factory [Amemiya et al. (1983) Nucl. Instrum. Methods 208, 471]. Scattering data were collected in a static mode using a 1D-PSD and/or an Imaging Plate at protein concentrations of 3-7 mg ml ~ at 18.5~ The specimen-to-detector distance was 254 cm to cover the smallest angle region as soon as possible. The exposure time was set at 180 s to avoid the radiation damage of the sample. The scattering profile from SI solutions had a feature with a gentle bump centred around S = 0.012 ~ ~ and a distinct shoulder around S = 0.032 .i.-~, where S = 2 sin 0/d, 20, the scattering angle and 5~,the wavelength of X-rays. The Guinier plots of the net scattering data from all $1 samples gave straight lines in the range of 0.00279 ,/~-~ ~< S ~< 0.00472 ~-1 (Smax)"The plots deviated upwardly from the straight lines beyond Sm,~. The deviation was smaller in $1 inthe presence of MgATP (5 mg ml-~) than in free $1, corresponding to the decrease in height of the bump. That of ADP + Vi (vanadate)bound $1 (S1.ADP.Vi) was similar to that of $1 in the MgATP

481 solution. In contrast, those of ATPTS-bound (S1.ATPTS) or ADPtrapped $1 by a pPDM crosslinking (S1.ADP-pPDM) and ADP-bound $1 (S1.ADP) were similar to that of free $1 with slight differences. When extrapolated to zero protein concentration, the radius of gyration (Rg) of free $1 was 47.8 ___0.4 A [see Garrigos and Vachette (1989) Biophys. J. 55, 80a; Mendelson et al. (1991) Adv. Biophys. 27, 143]. The Ra of $1 in the MgATP solution decreased by about 3 ]~, being slightly smaller than but closer to that of S1.ADP.Vi. On the other hand, the Rg of S1.ADP-pPDM was almost the same as that of free $1 and that of S1.ADP was slightly smaller (by about 1 ~) than but closer to that of free $1. The scattering curve of $1 after brought to completion of ATP hydrolysis resembled that of S1.ADP, giving the Rg value close to that of S1.ADP. The pair distance distribution function (P(r)) calculated from the free $1 data had a highly asymmetric profile with a peak at 37 A and a distinct shoulder around 80 ~, giving the maximum particle dimension (Dmax) of about 170 .~.. The P(r) of $1 in the MgATP solution showed a peak at the same position, with a less distinct shoulder and intersecting the free $1 curve at 80 ~, then decaying with smaller values than in free $1. The resulting Dmax became shorter by about 10 A. The P(r) of S1.ADP.Vi was very similar to that of $1 in the MgATP solution. Those of SI.ADP-pPDM (or S1.ATPTS) and S1.ADP were very similar to that of free $1. These results strongly indicate that the shape of $1 becomes compact or rounding in the presence of MgATP, agreeing with the recent studies by electron microscopy [Tokunaga et al. (1991) Adv. Biophys. 27, 157]. Assuming that S1.ATPTS and/or S1.ADP-pPDM are an analogue of the Sl-bound ATP state and S1.ADP.Vi is an analogue of the Sl-bound ADP.Pi (phosphate) state, this change of $1 in the MgATP solution probably occurs in the S1.ADP.Pi intermediate state of the ATP hydrolysis cycle.

Configuration of myosin heads during acto-myosin superprecipitation; an in vitro model of muscle contraction E. Katayama

Department of Fine Morphology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, 108 Japan With quick-freeze deep-etch electron microscopy coupled with micaflake technique, I showed previously that myosin subfragment-1 ($1) attached to F-actin in the presence of ATP is short and rounded, in contrast to its elongated and tilted appearance under the rigor condition. Actin-associated $1 with bound ADP.Vi (inorganic vanadate) was also in a rounded shape [J. Biochem. (1989) 106, 751-70]. Later, I indicated that each head of heavy meromyosin (HMM) changes its configuration in a likely manner as above by the addition of various nucleotides; i.e. heads were conventionally pear-shaped in the absence of nucleotide, but strongly kinked to the particular direction (clockwise, ff followed from the neck to the tip of the head) in the presence of ATP or ADP.Vi [J. Muscle Res. Cell Motil. (1991) 12, 313]. Such morphological data corroborate the independent biochemical and biophysical evidence suggesting possible gross conformational changes of myosin head on binding ATP or ADP .Vi; e.g. the estimated distance by energy transfer between two fluorophores, each labelled to actin and alkali light chain of $1 respectively, was less than half when $1 took active conformation as compared with the value under rigor condition [Bhandari et al. (1985); Trayer & Trayer (1988)]; Sl's radius of gyration became significantly smaller when ADP.Vi was bound instead of ADP [Aguirre et al. (1989); Highsmith and Eden (1990)]. Negatively-stained image of chemically cross-linked acto-S1 also included many heads sharply kinked to a uniform direction, confirming the above observation. Further attempts were made to examine if such conformational change of myosin head occurs during actomyosin superprecipitation, a good in vitro model of muscle contraction. Samples were quick-frozen at the time-point during rapid turbidity-increasing phase where actin filaments actively slid past

482 myosin heads along thick filaments. The image obtained showed conspicuous actin filaments occasionally associated with myosin heads protruding out from thick filament backbones. Among myosin molecules which were in close contact with F-actin, some of the heads showed a definable configuration. Such heads appeared all in a kinked configuration with the same polarity as that of HMM in the presence of ATP or ADP-Vi. These observations suggest the possibility that myosin heads could be in a kinked configuration while supporting the active sliding movement of actin filaments. As superprecipitation is an in vitro model of no-load shortening of muscle contraction, population among the conformers of the heads could be different under forcedeveloping conditions.

Effect of chemical modification of myosin heads on the ultrastructure of glycerinated rabbit psoas muscle fibres R. Shibayama, S. Suzuki, Y. Oshimi and H. Sugi

Department of Physiology, School of Medicine, Teikyo University, Itabashiku, Tokyo 173, Japan It has been known that a bifunctional thiol reagent, p-phenylenedimaleimide (PDM), reacts with reactive sulphydryls on the myosin head which results in the loss of its ability to combine with F-actin and to hydrolyze ATP. When one of the two myosin heads is modified with PDM in glycerinated rabbit psoas muscle fibres, the other native head still hydrolyzes ATP but no longer participates in force generation [Chaen et al. (1986) J. Biol. Chem. 261, 13632-6]. Recently, the possibility that the PDM modification induces orientation changes of myosin heads in glycerinated rabbit psoas muscle fibres was suggested by time-resolved X-ray diffraction studies [Tanaka et al. (1991) J. Muscle Res. Cell Motil. 12, 307-8]. To make clear whether PDM modification of myosin heads induces the ultrastructural change in myofilaments, the PDM-treated glycerinated rabbit psoas muscle fibres were examined with electron microscope.

ABSTRACTS Glycerinated rabbit psoas muscle fbres (approximately 10 fibres) were treated with the relaxing solution including 200 I,tM PDM for 15 min at room temperature. In these PDM-treated fibres, the Caactivated isometric force could not be generated. The PDM-modified and unmodified fibres were fixed with 2.5% glutaraldehyde and 2% OsO 4. On the other hand, these fibres were also frozen rapidly by pressing on a polished block of pure copper cooled to 4 K, and freeze-substitution was applied in absolute acetone containing 2% OsO4 (-80~ Longitudinal ultrathin sections were made and observed with a JEOL IOOCX electron microscope. To examine the structural changes of myofilaments, the electron micrographs were analysed with a Toshiba Tospix II digital image processor. In all prepared fibres, the sarcomeres were about 2.3 btm in length, and well arrayed along the fibre axis. The Z-bands of each sarcomere were opposed in parallel. Electron-dense crosslink structures extending radially from the thick filament shaft were frequently observed in the overlap region of A-band, where the thick and thin filaments were running in parallel alternately. When the fibres were modified with PDM, the number of such crosslink structures seemed to increase. They appeared sometimes with regular interval of about 43 nm along the fibre axis, indicating that they might be myosin heads. Furthermore, electron-dense particles, which also seemed to be myosin heads, were occasionally observed on the surface of thick filament shaft, and were well ordered by PDM-modification. These structural changes caused by the PDM-modification were more conspicuous in the fibres prepared by the freeze-substitution than those fixed with glutaraldehyde-OsO 4. By digital image processing of electron micrographs, Fourier transforms of the overlap regions between thick and thin filaments demonstrated that the intensity of 14.3 nm meridional reflection was enhanced remarkably by the PDM-modification. These results are consistent with those found by the X-ray diffraction studies, and suggest that the orientation of myosin heads along the thick filament axis is more ordered by PDM-modification.

CELL MOTILITY A N D CYTOSKELETON

Mechanoreception and cellular contraction in Vorticella K. Katoh and Y. Naitoh

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305, Japan The peritrich ciliate Vorticella exhibits a quick contraction of all-ornothing type (shrinkage of the cell body and coiling of the stalk) in response to a mechanical agitation of the cell. The primary objectives of the research were to examine localized difference in the mechanosensitivity for evoking a cellular contraction on the cell surface and to find out factors of a mechanical stimulus effective in evoking a cellular contraction. A stroke of the tip of a microneedle against the cell of Vorticella evoked a contraction. The mechanical threshold for evoking a contraction was lower in the cell body than the stalk. In the stalk it was lower in a portion closer to the cell body. A mechanical stroke against the stalk did not evoke a contraction if the stalk was mechanically clamped in a region between the site of stroke and the cell body. A quick drawing of a small portion of the cell body into a micropipette by suction evoked a contraction, whereas drawing of a portion of the stalk did not. It is therefore concluded that a mechanical stroke against the stalk evokes a contraction because it exerts mechanical agitation on the mechanosensitive cell body. Tilting of the stalk against the cell body evoked a contraction. The threshold degree of tilting, 0 t was inversely proportional to the angular velocity of the tilting, cot. That is, a contraction occurred when the product, 0~. co was over a threshold value. A localized depression of the cell body caused by a stroke of a microneedle against it evoked a contraction. The threshold extent of depression, x~ was inversely proportional to the rate of depression, dx/dt. That is, a contraction occurred when xt.dx/dt was over a threshold value. A tilting of the stalk is assumed to cause a localized depression of the surface

membrane of the cell body around the stalk. The threshold extent of the depression, dx(0) t and the rate of depression, dx(O)/dt were estimated from 0~ and r A contraction occurred when dx(O)t.dx(O)/dt was over a threshold value. A depression of the cell body is assumed to cause an expansion of the surface membrane of the cell body. xt.dx/dt has dimension of [LZT i], which corresponds to rate of change in the area. The rate of expansion of a membrane in the cell body is responsible for activation of a hypothetical mechanoreceptor mechanism for evoking a contraction in Vorticella.

Role of calcium in catch connective tissue, the connective tissue with mutable mechanical properties T. Motokawa

Biological Laboratory, Faculty of Science, Tokyo Institute of Technology, Meguro-ku, Tokyo, 152, Japan Catch connective tissue is a collagenous connective tissue that can alter its mechanical properties by several orders of magnitude within a minute through neuronal control. Its main function is in posture control of echinoderms. The body wall dermis of sea cucumbers is a typical catch connective tissue. The dermis contains no muscle cells nor any other cellular elements that possibly exert force. There are neural elements in the dermis. Mechanical tests were performed on the isolated dermis. Stress-relaxation test revealed that the change in the mechanical properties appeared mainly in viscosity and not in elasticity. Creep test showed that the viscosity was quite sensitive to the concentration of cations in the bathing media. The greatest effect was found when calcium concentration was altered. Tenfold increase in Ca concentration increased the viscosity ten times and Ca removal

ABSTRACTS with addition of 5 mM EGTA decreased the viscosity to 1/100 when artificial sea water was used as a standard medium. ASW containing 100 mM K (high K) increased the viscosity ten times. A dermis extracted with Triton X-100 did not respond to high K whereas it responded to high Ca. The response to high Ca was similar in extent and in time course to that of the intact dermis. This suggests that Ca directly affected the mechanical properties of the extracellular materials whereas K exerted its effect through stimulating cellular elements that control the mechanical properties of the extracellular materials. I found in the dermis of the sea cuctunber a vacuole cell which possibly controls the calcium concentration in the extracellular matrix. The cell has long processes which are packed with vacuoles. When fixed with pyroantimonate, precipitates appeared in the vacuoles in relaxed samples. No precipitates were found in samples at catch (stiff) state. Analysis with an energy-dispersive X-ray microanalyser suggested that the precipitate contained calcium. With these results, I propose that the mechanical properties of catch connective tissue is regulated by calcium ions. Calcium released from the vacuole cells possibly make crossbridges between the negative charges of glycosaminoglycans and thus increases viscosity of the dermis.

Cytoplasmic streaming in characean internodes under centrifugal acceleration E. Kamitsubo ~ and M. Kikuyama z

1BiologicaI Laboratory, Hitotsubashi University, Kunitachi and 2BiologicaI Laboratory, The University of the Air, Wakaba, Chiba Cytoplasmic streaming in intemodal cells of Characeae under centrifugal acceleration was studied using a centrifuge microscope of the stroboscopic type [Kamitsubo et al. (1989) Protoplasma 152, 148-55]. The 40X (NA 0.55) objective lens was used with a LWD condenser lens (NA 0.65). The data recorded with video-enhanced contrast optics and VTR system were analysed with an improved streak camera technique [Kaneda et al. (1990) Develop. Growth Differ. 32, 15-22]. The speed of endoplasm flowing contiguous to the actin cables ('peripheral flow') in Nitella axilliformis under terrestrial gravity was about 50 ],tm s -~ at 21~ Centrifugal force did not accelerate peripheral flow in the centrifugal direction nor retard centripetal flow in the range 4-1500 g. The thickness of the endoplasm was estimated to be about 2 lam. To increase the effect of centrifugal force on the peripheral flow of endoplasm, we introduced an artificial cell sap of high density into the cell by means of vacuolar perfusion [Tazawa Plant Cell Physiol. 5, 33-43 (1964)]. For this purpose, internodes of Chara australis were used. The density difference between the endoplasm and the cell sap was made to be 0.044 g cm 3 approximately ten times of the original one (0.004 g cm 3). In this case, too, peripheral flow was not affected at all by centrifugal force up to 1400g. The thickness of the endoplasm was again estimated to be around 2 ~tm. This result indicated that the rate of the peripheral flow of endoplasm in an intact internodal cell having the natural cell sap should be unchanged even under centrifugal acceleration at 14,000 g. Then, an intact internodal cell of N. orientalis was set tangentially to the edge of centrifuge rotor and was subjected to lateral centrifugal force. The rate of peripheral flow decreased nearly proportionally to the increase of centrifugal force. At 1100g, the rate was about 26 p_m s -I, 43% of the control. The interaction between the actin cables and putative myosin in the endoplasm is considerably weak to the lateral force in contrast to the parallel one.

Force--velocity relation in the sliding movement of Chara myosin-coated beads on actin cables studied with a centrifuge microscope S. Chaen, J. Inoue and H. Sugi

Department of Physiology, School of Medicine, Teikyo University, Tokyo 1;'3, Japan To examine the mechanical properly of Chara myosin interacting with actin, the force-velocity relation in the sliding movement of Chara

483 myosin along actin cables was studied with a centrifuge microscope. Chara is a kind of a green alga, and its myosin has been thought to cause cytoplasmic streaming in an alga. The velocity of cytoplasmic streaming is ten times faster than the sliding velocity between skeletal myosin and actin. After perfusing the vacuole of the Chara internodal cell with MgATP medium (30 mM PIPES pH 7.0, 5 mM EGTA, 6 mM MgC12, 2 mM ATP and 200 mM Sorbitol), tosyl-activated polystyrene beads (Dyna beads, 2.8 I.tm in diameter, specific gravity 1.3) were introduced to attach the endoplasm containing myosin. This internodal cell preparation was then mounted in the chamber of a rotor in the centrifuge microscope. Under constant centrifugal forces opposite to the bead movement, the beads moved at constant velocities, which decreased with increasing centrifugal forces. When the centrifugal force was increased to balance the maximum force generated by Chara myosin molecules, the bead stopped moving for 5-10 s and then suddenly detached from the actin cable to flow away in the direction of centrifugal force. Force-velocity relation of Chara myosin was found to be less curved than that of skeletal muscle myosin, a/Po were ranging from 1 to 20, and Po from 1 to 11 pN (n =6). Less curved feature of force-velocity relation from Chara myosin suggests that association rate constant in crossbridge cycle is large compared to the dissociation rate constant, as a high value of association rate constant keeping the number of crossbridges high at intermediate speed makes the curve flat. We applied the centrifugal force in the same direction as that of the bead sliding (negative load). As the negative load increased, the velocity decreased, then increased slightly over Vm~x, and finally detached from the actin cable. This is qualitatively similar to that from skeletal muscle myosin.

Intracellular localization and functions of Tetrahymena 14-nm filament-forming protein O. Numata, I. Takagi and Y. Watanabe

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan The Tetrahymena 14-nm filament-forming protein (49 KDa protein) is a structural protein involved in pronuclear behaviour during conjugation. We previously reported cloning and sequencing of the 49 KDa protein gene. Its primary structure not only exhibits low homologies with vertebrate vimentin and desmin but exhibits a high sequence identity (51.5%) with porcine heart citrate synthase. The 49KDa protein actually possesses citrate synthase activity, and is detected in mitochondria. These results suggest that the 49 KDa protein has dual functions as both a TCA cycle enzyme and a structural protein in the cytoskeleton. Having now purified Tetrahymena citrate synthase from isolated mitochondria, we have found that the citrate synthase and the 49 KDa protein are identical by five criteria: molecular weight, antigenicity, properties of enzyme activity, amino-terminal amino acid sequence and filament formation. Furthermore, we investigated immunoelectron microscopical localization of the 49 KDa protein in mitochondria with anti-49 KDa protein antiserum. We obtained the results that the 49 KDa protein formed filament bundles in mitochondrial matrix and that size of each filament was approximately 14 nm. The results support our notion that polymerized 49 KDa protein (14 nm filament) functions as citrate synthase in mitochondria.

Expression of Tetrahymena actin gene and chimeric actin genes in Dictyostelium cells M. Hirono I, K. Kurifa I, J. Kurashima I, T. O h n o ~, K. Sutoh 2 and Y. Watanabe 3

1Cell Bank, Institute of Physiology and Chemical Research (RIKEN), Tsukuba, 2Department of Pure and Applied Sciences, University of Tokyo, Tokyo and 3Institute of Biological Science, University of Tsukuba, Tsukuba, Ibaraki 305, Japan We previously reported that Tetrahymena actin has a divergent primary structure and some unusual properties. Transient expression

484 of the Tetrahymena actin gene in COS-I cells slightly affected to division of the cells. To know more about the influence of Tetrahymena actin on cell division, we transfected Tetrahymena actin gene on expression vector to Dictyostelium cells which have the usual type of actin. We obtained some clones that constitutively express large amounts of recombinant Tetrahymena actin. Expressed Tetrahymena actin could be extracted with a low ionic strength buffer from acetone powder of transformed cells, suggesting that the recombinant actin is produced as native form. Celt shapes of transformants with Tetrahymena actin gene were less stretched than those of transformants with Dictyostelium actin gene. The average growth rate of transformants with Tetrahymena actin gene was three times slower than that of transformants with Dictyostelium actin gene. These results suggested that expression of Tetrahymena actin gene affects the shape and division of Dictyosteliumcells, probably because Tetrahymenaactin has some unusual properties. Using this expression system, we tried to make two kinds of chimeric actin molecules composed of Tetrahymena actin and Dictyosielium actin. A DNA fragment coding for amino acid residues 1-83 of Tetrahymena actin was ligated to a fragment coding for residues 84-374 of Dictyostelium actin (T83D). The other chimeric actin gene was 1-83 for Dictyostelium actin and 84-374 for Tetrahymena actin (D83T). These chimeric actin genes were inserted to expression vector and transfected to Dictyostelium cells. Transformants expressing the chimeric actin T83D formed tight colonies although wildtype Cells formed relatively loose colonies. This result suggests that

ABSTRACTS locomotion of T83D transformant was partially inhibited. Average growth rate of T83D transformants was approximately the same with that of transformants with Tetrahymena actin, but that of D83T transformants was four times slower than that of T83D transformants. We are presently analysing the products of these chimeric actin genes.

Site-directed mutagenesis of

Dictyostelium actin

K. Sutoh

Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Komaba, Tokyo 153, Japan Mutations were introduced into subdomain 1 of Dictyostelium actin gene 15. These mutant actins were expressed in Dictyosteliumcells by using expression vectors B10 and pnDeI. Some of the expressed actins were purified by a novel single-step HPLC procedure to homogeneity. All mutant actins prepared so far formed normal filaments. Biochemical properties of these actin filaments were examined by measuring actin-activated ATPase activity of myosin and by myosin-driven sliding movement of the filaments on a cover slide. Disruption of negative charges at the N-terminus of actin (DIH, D4H, DIH/D4H, DIH/E3H/D4H) resulted in loss of actin-activation of myosin ATPase activity. Sliding movement of actin filaments was inhibited by these mutations. Similar results were obtained by other mutations such as D24H/D25H, E99H/E100H. However, mutations E360/E361 and E363/D364 did not affect the sliding motion.

MECHANICS, ENERGETICS BIOCHEMISTRY A N D STRUCTURE OF C A R D I A C A N D S M O O T H MUSCLES

Effects of MgATP on myocardial crossbridge kinetics Y. Saeki I, M. Kawai z and Y. Zhao z

1Department of Physiology, Tsurumi UniversitySchool of Dental Medicine, Yokohama 230, Japan and ZDepartmentof Anatomy, Collegeof Medicine, The University of Iowa, Iowa City, IA 52242, USA The role of the substrate (MgATP) in myocardial crossbridge kinetics was investigated by small sinusoidal length oscillations (peak to peak amplitude: 0.25% of muscle length) and by following amplitude change and phase shift in tension time courses. The range of the discrete frequencies used for this investigation was 0.13 to 135 Hz, which corresponds to 1.2-1200 ms in time domain. The experiments were performed in maximally activated (pCa 4.82) Triton X-100treated chemically-skinned ferret papillary or trabecular muscles at 20~ and at the ionic strength 200 raM. We identified three exponential processes in the sinusoidal tension response to the imposed length oscillation, and these were labelled process (B), (C) and (D) in the order of increasing speed. A slow process (A), which is normally present in fast twitch skeletal muscles, was either very small or absent. Process (B) is an exponential delay, and the muscle produces oscillatory work on the forcing apparatus; Processes (C) and (D) are exponential advances in which the muscle absorbs work. The rate constants 2~b, 2nc and 2~d deduced from these processes increased and saturated hyperbolically with an increase in MgATP concentration (0.I-10 mM). The isometric tension decreased with an increase in the MgATP concentration. These results were analysed in terms of a crossbridge scheme with five states, and the following kinetic parameters were deduced: Ks = 2.5 mM i, k1a = 1000 s -I, k_~a= 350 s -z, kz = 20 s -z, k z = 30 s -z, k3= 14 s -z, k_3 = 8 s -~. In this scheme, it was assumed that )(i was in rapid equilibrium with MgATP to form X~a, the transition between Xia and X2 was sensed by process (D), the transition between Xz and X3 by process (C), the transition between X3 and X4 by process (B), and that the transition from X4 to X1 was the slowest of all reactions (the rate limiting step). By correlating the scheme with the biochemical cycle of hydrolysis of ATP by actomyosin in solution, it can be considered that X~=AM (A, actin: M, myosin), X~,=AMS

(S = MgATP), X2 = AM*S (isomerized), X3 = A + MS or A + MDP (detached; D = MgADP, P = phosphate) or AMDP (weakly attached and non-force generating), and X4 = AM*DP or AMD (attached force generating) states.

Effect of ADP treatment on the contraction of skinned aortic smooth muscle fibres S. M o r i m o t o and Y. O g a w a

Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113, Japan To explain the latch state, i.e. high stress maintenance with low level of myosin phosphorylation, in intact vascular smooth muscle, Murphy and colleagues proposed a model postulating that latch bridges are produced by the dephosphorylation of crossbridges attached to the thin filaments. To test this hypothesis we examined the effect of substitution of ADP for ATP (ADP-solution) following the maximal isometric contraction of Triton X-I00-treated rabbit aortic skinned smooth muscle fibres in an activating solution (pCa 4.9). Under acidic conditions (pH 6.0) in the ADP solution, 30-60% of tension generated in the activating solution was maintained even 20-30 min after the ADP substitution, and myosin phosphorylation was reduced to the resting level, suggesting the dephosphorylation of attached crossbridges, i.e. the formation of latch bridges. Subsequent immersion into a relaxing solution of pCa 7.5 (pH 7.0) which is below the threshold for tension development induced rapid development of large tension, and the tension was then sustained irrespective of Ca2+ concentrations. Two-dimensional gel electrophoresis, however, showed that the CaZ+-insensitive sustained contraction Was accompanied by an extremely high level of myosin light chain phosphorylation in contrast to the latch state. After treatment with neutral or alkaline ADP solutions, however, sustained tension in the relaxing solution cannot be observed. Trifluoperazine at 0.1 raM, an indirect myosin light chain kinase inhibitor, reduced the rate of tension development in the relaxing solution after ADP treatment, but did not change the maximal

ABSTRACTS level of contraction. On the other hand, okadaic acid (1 laM),a myosin light chain phosphatase inhibitor, made the tension development after ADP treatment transient. Staurosporine, a protein kinase C inhibitor, also made the tension development after ADP treatment transient at 1 I.tMand completely suppressed at I0 I.tM. These results suggest that the myosin light chain phosphorylation by protein kinase C as well as by myosin light chain kinase is involved in this Ca2+-insensitive sustained contraction.

Can calcium wasting effect of ryanodine be assessed by cardiac energetics in dog heart? T. Takasago I, Y. Goto 2, K. Hata ~, A. Saeki ~, T. Nishioka ~, and H. Suga 3

1Department of Cardiovascular Dynamics, National Cardiovascular Centre, Suita, Osaka 565, Japan, 2Department of Internal Medicine, National Cardiovascular Centre, Suita, Osaka 565, Japan and 3The 2nd Department of Physiology, Okayama University Medical School, Okayama 700, Japan Ryanodine (RYA) at a low concentration (several tens of nM) is known to bind to calcium (Caz+) release channels in sarcoplasmic reticulum (SR) and fix it open. These channels are assumed to have an important role for the regulation of cardiac contractility. However, the relationship between changes in their channel activity and cardiac contractility of the whole heart is still obscure. Therefore, we studied the mechanism of negative inotropism of RYA at a low concentration in terms of cardiac energetics using isovolumic contractions of the isolated, blood-perfused, cross-circulated dog left ventricle (LV). First, we enhanced Emax (an index of LV contractility) in steps with CaCI2 given into coronary circulation and obtained the LV oxygen consumption (Vo;)-total mechanical energy (TME) relation at a constant LV volume in 11 hearts. Next, we depressed Emax with RYA (up to 30--q-13 nM) and obtained the VozTME relation at the same LV volume. Both VQ and TME decreased with the gradually decreased Er,~. RYA significantly depressed Em~xby 42.1 + 14.8% (p < 0.001) and TME by 44.4 + 15.3% (p < 0.00I). However, RYA depressed Vo~ only by 7.4 4- 12.1%; this change was not statistically significant. We calculated TME-independent Vo~ values at varied Em~. levels in both CaC12 and RYA runs and obtained the relation between TME-independent VQ and Er~,. We have already found that TME-independent Vo~ is primarily related to the total sequestrated Ca2+ by SR, and that TME-independent Vo~ and Er~• are proportionally altered by ordinary inotropic drugs such as CaC12, dobutamine and propranolol [Suga (1990) Physiol. Rev. 70, 247]. In contrast, we found that TME-independent Vo~ remained disproportionately high despite the gradually d e c r e a s e d Ema• in RYA run. Similar results were obtained using a r-blocker propranolol to eliminate the fl-stimulatory effect of catecholamines released from the support dog. The VQ-TME relations were also obtained in steady-state contractions at five to seven different LV volume in the presence and absence of RYA (Volume-Loading run). RYA did not change the contractile efficiency calculated from the inverse value of the slope of the Vo~-TME relation in Volume-Loading run. These results indicate that RYA suppresses the coupling from the total sequestrated Ca2+ by SR to the force generation of cardiac muscle and does not affect the coupling from ATP to mechanical force in the myofilament. We speculate about the underlying mechanism that RYA makes SR leaky for Ca 2+ and thereby decreases the efficiency of Ca2+ sequestration by SR.

Velocity of skeletal and cardiac actin filament on rat cardiac myosin in vitro S. Sugiura, H. Yamashita, T. Serizawa and T. Sugimoto

2nd Department of Internal Medicine, School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan In adult rat myocardium, cardiac-type 0r is exclusively expressed. With the introduction of pressure-overload hypertrophy, however, skeletal type ~-actin is expressed transiently. Furthermore, this actin

485 isoform redistribution coincides with the shift of myosin isozyme distribution. Although the time course and the extent of this change has been extensively studied, its functional implication is still not clear. We investigated the effect of isoform change on the sliding velocity of actin filament on myosin in vitro. Both cardiac and skeletal actin was obtained from adult Wistar rat myocardium and leg muscle, respectively. Considering the concomitant change in myosin isozyme, two different myosin isozymes were used. V I isozyme with high ATPase activity was obtained from normal young Wistar rat hearts, and V3 isozyme with low ATPase activity from the hearts of hypothyroid rats treated with methimazole. Myosin was fixed on a glass coverslip coated with nitrocellulose. Actin filament was labelled with rhodamine-phalloidin and made to slide on myosin layer in the presence of Mg-ATP (30~ pH 7.8). The sliding movement was observed by fluorescence microscope and the velocity was measured. On V3 myosin skeletal-type actin filaments moved faster (average 3.1 ~m s-~) than cardiac-type ones (2,8 I.tms-1). There was a tendency for skeletal-type actin to move faster also on V1 myosin. These results suggest that actin isozyme redistribution may also be an adaptation response to the different loading condition imposed on the heart.

Effect of mechanical vibration on myocardial mechanics and energetics in isolated, blood-perfused dog heart T. Nishioka I, Y. Goto I, K. Hata 1, T. Takasago I, A. Saeki ~ and H. Suga 2

~Department of Cardiovascular Dynamics, National Cardiovascular Centre, 5 Fujishiro-dai, Suita, Osaka, 565, Japan and 2The 2nd Department of Physiology, Okayarna University Medical School, 2 Shikatacho, Okayarna, 700, Japan It has been reported that mechanical vibration inhibits contractility of various types of isolated muscle, e.g. skeletal muscle, vascular smooth muscle and cardiac muscle. Moreover, some investigators reported the inhibitory effect of mechanical vibration on the whole heart. However, the mechanism of this inhibitory effect is unknown. To elucidate this inhibitory mechanism, we studied the relation between myocardial mechanics and energetics during mechanical vibration in the isolated, cross-circulated dog left ventricle (LV) using the frameworks of E~ax (a contractility index) and systolic pressure-volume area (PVA). PVA is a measure of LV total mechanical energy expenditure and has been shown to correlate linearly with myocardial oxygen consumption (VQ). The Vo2-intercept (PVA-independent Vo2) of the VovPVA relation reflects VQ for excitation--contraction coupling and basal metabolism, whereas VQ above the intercept (PVA-dependent VQ) indicates VQ for crossbridge cycling. In this study we used 12 isolated, cross-circulated dog heart preparations and applied mechanical vibration of 2 mm in amplitude and 70 Hz in frequency to the anterolateral part of LV wall. We performed two experimental protocols. First, measurements for LV pressure, volume, and Vo2 were made steady-state, isovolmic contractions to determine the Vo2-PVA relations in both control and vibration-induced depressed contractile states (volume loading run, n = 12). Next, we altered contractility with mechanical vibration, calcium, or propranolol at a fixed LV volume to compare oxygen cost of contractility (the slope of the relation between PVA-independent VQ and Em,x) (inotropic run, n = 6). The response of LV end-systolic pressure (LVESP) to mechanical vibration was quite similar as previously reported, that is, LVESP was depressed instantaneously on mechanical vibration and recovered to the initial level without delay by cessation of the mechanical vibration. In a volume loading run, despite a 19% (p < 0.01) decrease in Emax with mechanical vibration, the Vo2-PVA relation during mechanical vibration was superimposable on that during the control period. The oxygen cost of contractility during mechanical vibration was considerably lower than that during calcium or propranolol (p < 0.01), and moreover, did not significantly differ from zero. This indicates that the mechanism of the inhibitory effect of mechanical vibration differs from that of the negative inotropic effect of reduced calcium or propranolol,

486 which alters intracellular calcium handling in proportion to changes in Em~x. From these results, we conclude that mechanical vibration decreases PVA-dependent Vo2 without decreasing PVA-independent 17o2. Thus, suppressed crossbridge cycling appears to be the main mechanism of the inhibitory effect of mechanical vibration on LV contractility.

Chemical crosslinking studies on acto-smooth muscle heavy meromyosin and smooth muscle subfragment 1 H. Onishi I, T. Maita g, G. Matsuda 2, and K. Fujiwara 1

IDepartment of Structural Analysis, National Cardiovascular Centre Research Institute, Suita, Osaka 565, Japan and 2Department of Biochemistry, Nagasaki University School of Medicine, Nagasaki ,~52, Japan When chicken gizzard heavy meromyosin (HMM) in its rigor complex with F-actin was reacted with a zero-length crosslinker, 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC), the two heads of the HMM molecule were crosslinked [Onishi et aI., (1989) Biochemistry 28, 1898-1904; 1905-1912]. The sequence analysis of the two isolated crosslinked peptides, one consisting of the HMM heavy chain residues from 53 to 66 and the other from 164 to 182, indicated that crosslinking occurred between Lys-65 and Glu-168 [Onishi et al. (1990) J. Biol. Chem. 265, 19,362-8]. As EDC catalyses the formation of a covalent bond between a carboxyl and an amino groups, our result suggested that Lys-65 in one HMM head and Glu-168 of the other were in contact with each other in the acto-gizzard HMM rigor complex. Electron microscopic studies of head-to-head crosslinked HMM molecules indicated that crosslinking occurred between the two heads of a single HMM molecule and that both crosslinking sites were located within a distal, more globular half of each head [Onishi, H. and Fujiwara, K. (1990) Biochemistry 29, 3013-23]. Results described here made it possible for us to predict the geometry of the myosin heads when they are attached to the F-actin filament. Although different myosin fragments are involved, our model agrees well with the three-dimensional structure of acto-rabbit skeletal myosin subfragment 1 (S-1) reconstituted from the fluorescence resonance energy transfer data [Botts eta/. (1989) Proc. Natl. Acad. Sci. USA 86, 2204-8]. The interaction between the heavy and the regulatory light chains within chicken gizzard myosin heads was also investigated by using the same crosslinker. Gizzard S-1 used was treated with papain so that the heavy chain was partly cleaved into the NH 2-terminal 72 KDa and the COOH-terminal 24 KDa fragments and the regulatory light chain into the 16 KDa fragment. When S-1 was reacted with EDC either alone or in "the presence of ATP or F-actin, the 16 KDa fragment of

ABSTRACTS the regulatory light chain formed a covalent crosslink with the 24 KDa heavy chain fragment but not with the 72 KDa fragment. The location of the crosslinking sites on the amino acid sequence of the heavy and the light chains was investigated by proteolytic fragmentation of the crosslinked peptides. Our data indicate that the regulatory light chain is located near the COOH-terminal end of the S-1 heavy chain.

Structure and contractile properties of paramyosin-containing muscle in sea urchin,

Asthenosoma ijimai T. Tsuchiya 1, T. Mori 2, E. Suzuki 3 and S. Amemiya 4

~Department of Physiology, School of Medicine, Teikyo University, Tokyo 173, 2Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, 3Department of Fine Morphology, Institute of Medical Science, University of Tokyo, Tokyo 108 and 4Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Kanagawa 238-02, Japan In a typical molluscan catch smooth muscle, e.g. ABRM (the anterior byssus retractor muscle of Mytilus), the thick filaments which are much thicker and longer than those in skeletal muscle, are known to contain paramyosin in their cores and it has long been discussed whether this protein is respor/sible for catch mechanism. The radial muscle of sea urchin Asthenosoma ijimai has been shown to contain paramyosin by immunoblot analysis [Tsuchiya et al., J. Exp. Biol. (in press)] and therefore it is of interest to know the structure and the contractile properties of this muscle from the viewpoint of catch. Each smooth muscle cell contained thick and thin filaments. The thick filaments, 20--60 nm in diameter and 2-5 ~tm in length, were surrounded by many thin filaments, 5--6 nm in diameter. The thin filaments appeared to run into and join dense bodies which were often intimately associated with cell membrane. A small bundle of the radial muscle (0.3-1.0 mm in diameter and 6-8 mm in length) was dissected out together with a piece of test plate at the distal end and with connective tissue at the proximal end. One end of the muscle preparation was connected to a force transducer and the other end to a length step generator and stimulated by the application of acetylcholine or by repetitive or direct electrical current stimulation. After any stimulation, the developed force was not maintained unlike the ABRM and when the muscle was quickly released by 0.8-1.2% lo (rest length) during force development, force redeveloped clearly after a sudden drop, suggesting this muscle does not have the nature of catch. Paramyosin does not seem to be involved in catch as far as the radial muscle of Asthenosoma is concerned.

EC COUPLING OF SKELETAL, CARDIAC A N D S M O O T H MUSCLES

Time course of recovery of twitch and tetanus tension after a sudden interruption of potassium contracture at low temperatures K. Noguchi, A. Suzuki and H. Sugi

Department of Physiology, School of Medicine, Teikyo University, Itabashiku, Tokyo 173, Japan Caputo [(1972) J. Physiol. 223, 483-505] found that the repriming process after a potassium (K) contracture was markedly slowed down at low temperatures; when a K contracture in single frog muscle fibres was interrupted by a sudden reduction of [K]0 to normal value and another contracture was produced by re-exposure to high [K]0, the sum of the time integral of tension in the two successive contractures was equal to the time integral of tension in a non-interrupted contracture. To obtain information about the mechanism of the repriming process, we examined the time course of recovery of twitch and tetanus tension after the sudden interruption of a K contracture. Single fibres isolated from the anterior tibialis muscle of the frog (Rana japonica) were first made to contract by increasing [K]0 from 2.5

to 190 mM, and at 20-30 s after the maximum contracture tension was reached, the contracture was interrupted by reducing [K]0 from 190 to 2.5 mM in a few seconds. The temperature of experimental solutions was kept at 1-3~ After the completion of relaxation of the interrupted contracture, the fibres were stimulated with transversely applied single or repetitive 3 ms current pulses through a pair of Pt plate electrodes. The amplitude of isometric twitch tension recovered to the normal value in 2 min after the contracture interruption, while the amplitude of isometric tetanus produced by repetitive current pulses at 20 Hz recovered much more slowly, being complete in about 10 rain after the contracture interruption. The delayed recovery of tetanic tension compared with the rapid recovery of twitch tension was associated with the incomplete fusion of individual twitches in response to 20 Hz repetitive current pulses, indicating a prolonged refractory period of action potentials. Recordings of action potentials of the fibres with intracellular microelectrodes showed that this was actually the case; although both resting membrane potentials and action potential amplitudes recovered to their normal values in approximately 2 rain after the contracture interruption, the falling

ABSTRACTS phase of action potentials was markedly prolonged to result in a markedly prolonged refractory period (more than 100 ms). The falling phase of action potential is known to be markedly prolonged when Ca ions are injected into squid giant axons (Yamagishi, personal communication). This analogy may be taken to suggest that the repriming process includes binding of activator Ca to the inner surface of the transverse tubules, in which the delayed rectifier conductance responsible for the falling phase of action potential is believed to exist.

Precise mechanism to initiate contraction in single skeletal muscle fibres of frog, Rana japonica T. Arima, M. Fujino, C. Hasegawa, K. Harano, M. Takahashi, S. Sano and T. Ito

Department of Physiology, Defense Medical College, Tokorozawa 359, Japan Our morphophysiological studies using concanavalin A-ferritin (Con A-F) have indicated that: (1) an out- and upward movement of a movable structure at the luminal surface portion of the T-tubular membrane opposite the feet initiates contraction, (2) the grade of the movement depends on that of depolarization, and (3) the movable structure is essentially a 'moving arm', which is fixed in wall of T-tubules at its fixed end and is able to be bound to the Con A-moiety of Con A-F particle about at its free end. Calculation based on molecular morphology and behaviour of Con A-F particle revealed following points. If (a) the origin of coordinate be the intersection of longitudinal centre line and the surface of T-tubular membrane in the transverse section of the tubules, (b) the fixed point of the arm is exactly on the surface of T-tubular membrane, and (c) the movement takes place in the transverse direction to the longitudinal axis of T-tubules, then (1) the location of the centre point of the movement of the moving arm is at 5.4 nm in the outside direction from the origin, and (2) the arm is about 4 nm in length and moves by about 2.4 nm up- and outward at its free end on about complete depolarization.

Inactivation and complexity of input apparatus responsible for excitation-contraction (EC) coupling in single fast muscle fibres of frog, Rana japonica M. Fujino, C. Hasegawa, T. Arima, K. Harano, M. Takahashi, S. Sano and T. Ito

Department of Physiology, Defense Medical College, Tokorozawa 359, Japan Our morphophysiological studies using concanavalin A-ferritin (Con A-F) in fast fibres have revealed that: (1) apparatus responsible for surface control over contraction exist at the luminal surface of the T-membrane opposite the feet, (2) the apparatus are divided to two kinds, which are located at respective sites, relatively inward and outward with respect to foot; (3) each apparatus consists of movable structures which behave rather oppositely to each other, (4) the inward apparatus responds fast and moves outward on depolarization, has higher affinity for Con A, and is bound with phenylglyoxaL (5) the outward apparatus responds slowly and inward on depolarization, has higher affinity for wheat germ agglutinin, and is bound to nitrendipine; and (6) the grade of movement of outward apparatus depends on that of depolarization.

Effect of ryanodine on contraction of crayfish skeletal muscle fibre H. Ushio x, S. Watabe I, N. Fusetani I and M. Iino z

1Laboratory of Marine Biochemistry, Faculty of Agriculture and 2Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan It has been shown that external Ca2+ influx is necessary for contraction of crustacean skeletal muscle. However, it is still unclear whether influx of external Caz+ is sufficient to elicit contraction or Ca2+ release from intracellular Ca2+ stores is required for contraction. To answer these

487 questions we studied the effects of pharmacological agents such as ryanodine, caffeine, diltiazem and nicardipine on the electrically stimulated contractions of crayfish single muscle fibres. Single skeletal muscle fibres were obtained from the flexor muscle of crayfish (Cambarus clarki) carpodite and mounted in a chamber at 10~ filled with a Ringer solution specially prepared for crayfish. Isometric contraction was elicited by electrical stimulation with external platinum electrodes. The membrane potential was measured by using a microelectrode containing 3 M KC1 (about 10 mY2) impaled near one end of the fibres. In another set of experiments, second microelectrode was impaled to pass currents to stimulate the fibres. A straingauge transducer was used to measure isometric tension. The tension elicited by external electrodes was reduced by the removal of Caz+ or by the addition of 25 ].I,Mnicardipine in the crayfish Ringer solution. However, 100 ~M diltiazem had no effect on the contraction or high K+ (70 mM) contracture. An application of 30 ~I.M ryanodine with I mM caffeine induced contracture, the tension of which was I0-20% of that of high K§ contracture. The ryanodinecaffeine-induced contraction was transient, and subsided to pretreatment level in about 30 rain. After the treatment, 30 mM caffeine failed to induce contraction. This suggests that intracellular Ca2§ stores are exhausted by the ryanodine-caffeine treatment, probably owing to fixation by ryanodine of the Ca2+ release channels in the sarcoplasmic reticulum at an open state. Although the resting potential ( - 7 0 to - 8 5 mV) was not changed by the ryanodine-caffeine treatment, contraction elicited by external electrodes was abolished. Action potentials were not observed in the crayfish Ringer solution, but in the solution where a half of Na + was replaced by tetraethyl ammonium ion (TEA) single fibres produced prolonged action potentials, the duration of which was about 300-500 ms. In these actionpotential-induced contractions the peak tension was attained at about 300 ms after the stimulation. The TEA-induced action potential was completely inhibited by the removal of Ca 2+ or by the addition of nicardipine, while 20 [LI,M tetrodotoxin had no effect. Some fourfold increase in the duration of action potential was observed after ryanodine-caffeine treatment. Although this prolonged action potential elicited contraction, the rate of rise of tension was much slower than that before the ryanodine-caffeine treatment, and the tension reached a peak in about 10 s. These results suggest the following. In contractions induced by brief depolarizations, Ca2+ release from the sarcoplasmic reticulum seems important in crayfish carpodite muscle, and for the Ca z+ release Ca 2+ influx via nicardipine sensitive Ca 2+ channels is required. In prolonged depolarizations, the Ca 2+ influx itself seems capable of inducing contraction.

Histochemical studies on the mode of innervation of the swimbladder muscle of a sound-producing teleost fish T. Kobayashi ~, T. Daimon 2, A. Goto ~, D. Kondo ~ and H. Sugi I

1Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo I73, and 2Departmentof Anatomy, Schoolof Medicine, Teikyo University, Itabashi-ku, Tokyo I73, Japan The mode of motor nerve innervation in the swimbladder muscle (SBM) of a teleost fish (Sebasticus marmoratus) was studied to obtain information about the neuromuscular mechanism for the rapid contractions to produce sounds. The swimbladder muscle consists of about 600 muscle fibres of nearly the same size, while the nerve innervating the muscle contained about I00 axons of nearly the same diameter, suggesting the absence of sensory axons. In accordance with the result that endplate potentials can be recorded along the entire length of the SBM fibres, a rapid Golgi silver impregnation of the SBM fibres showed that nerve branches run along the entire fibre length to form endplates at many points. Each endplate region clearly exhibited acetylcholine esterase activity, indicating cholinergic nature of neuro-

488 muscular transmission. All muscle fibres showed a weak succinic dehydrogenase activity and a high ATPase activity at pH 10.4 but not at pH 4.35, indicating that the SBM is composed only of muscle fibres histochemically similar to mammalian first-type muscle fibres. As the ratio between the number of muscle fibres and the number of axons innervating them is only about six, it seems likely that each muscle fibre is innervated by more than one axon, although more experimental work is needed to actually prove the polyneuronal innervation.

Effects of tonicity of external solutions on contractile tension, muscle fibre volume and membrane potential in rat cardiac muscle T. Kobayashi, D. Kondo, K. Kikuchi and H. Sugi

Department of Physiology,Schoolof Medicine, Teikyo University,Itabashiku, Tokyo 173, Japan Although the effect of tonicity of external solution on the contractile tension of cardiac muscle has been studied from time to time, little information is at present available about how the contractile tension changes over a wide range of tonicities including both hyper- and hypotonic solutions. We studied the effects of hyper- and hypotonic solutions on the contractile force, muscle fibre volume and the resting and action potentials in the papillary muscle of rat heart. The papillary muscle preparations were stimulated with I ms current pulses at 0.5 Hz given through a pair of Pt wire electrodes, and the isometric contractile force in response to each stimulus was recorded under varying tonicities. The resting and action potentials were recorded with conventional intracellular microelectrodes. Hypertonic solutions were prepared by adding sucrose to the standard solution, while hypotonie solutions were made by partial removal of Na ions from the standard solution. Tonicities were measured with an osmometer (type 3MO, Advanced Instruments). When the tonicity was increased to 300-4000 mOsm I ~ from the standard value (280 mOsm i-*), the contractile tension showed a transient increase, reaching a maximum (120-130% of the initial value). The force then decreased to reach a steady value smaller than the initial value. With hypertonic solutions above 500 mOsm l -~ the contractile force simply decreased to a low steady value ( < 20% of the initial value). Estimation of muscle fibre volume from the blotted weight of the preparation indicated that the fibre volume always decreased monotonically to a steady value irrespective of the degree of hypertonicity, and both the fibre volume and the contractile force appeared to reach their steady values with the same time course. No appreciable changes were observed in both the resting and action potentials in hypertonic solutions, expect for the tendency of the falling phase of action potential to shorten with increasing tonicities. These results indicate that the decrease in the contractile force in hypertonic solutions results from the increased intracellular ionic strength when the fibre volume decreases; the transient increase in the contractile force in the mild hypertonic solutions may be from an increased rate of Caz+ release from the sarcoplasmic reticulum, known to take place in skeletal muscle fibres. On the other hand, a decrease of tonicity to 250-150mOsml -I caused a steep increase of the contractile force to 130-220% of the initial value. As the marked potentiation of the contractile force was associated with a distinct increase of the baseline tension, the above contractile force potentiation may result largely from an increased intracellular Ca2+ concentration owing to the Na-Ca exchange mechanism.

ABSTRACTS Although it has been shown that cytochalasin D relaxes smooth muscle contractions, the site of action has not been determined yet and this is the purpose of the present experiments. Cytochalasin D inhibited the contraction in rat aorta induced by high K + in concentration-dependent manner. In the fura-2 loaded muscle, high K+ and norepinephrine caused sustained increase in both [CaZ+]i and muscle tensions. Addition of cytochalasin D inhibited the high K +- and norepinephrine-induced contraction without changing [Ca2+]i . In the absence of external Caz+, I2-deoxyphorbol 13-isobutylate (DPB) (1 I.tM) induced sustained contraction. Cytochalasin D also inhibited this contraction. The concentrations of cytochalasin D to inhibit these contractions were almost identical. In contrast, cytochalasin D did not change the muscle tension induced by passive stretch. These results suggest that cytochalasin D inhibits contractile elements without changing [Ca2+]i . In the liquid nitrogen-treated skinned fibre of chicken gizzard, cytochalasin D inhibited CaZ+-induced contraction. The inhibitory effect was not modifed by changing the Ca z§ concentration (1-10 p,M). Cytochalasin D did not inhibit myosin light chain phosphorylation owing to increase in Ca z+ in the native actomyosin preparation. In the myosin light chain that was thiophosphorylated by a treatment with Ca2+ and ATPyS in the absence of ATP, application of ATP caused sustained contraction in the absence of Ca2+. This Ca2+-independent contraction was regarded as the results of an interaction between thiophosphorylated myosin and actin filaments. Cytochalasin D (I0 ~M) inhibited this contraction to a similar degree as to inhibit the Ca>-induced contraction. These results suggest that cytochalasin D directly inhibits actin-myosin interaction without affecting Ca2+-calmodulin-myosin light chain kinase system. In the native actomyosin preparation, cytochalasin D decreased velocity and maximum level of superprecipitation. It has been shown that sonication cleaves actin filaments although it does not increase actin monomers. Sonication of the preparation for 2-3 s decreased velocity but not the maximum level of superprecipitation. In contrast, cytochalasin D changed neither the maximum level nor the time course of the Mg2+-ATPase activity measured by a release of inorganic phosphate in native actomyosin preparation. Thus, the inhibitory effect of cytochalasin D to inhibit superprecipitation is not attributable to the inhibition of actin-myosin interaction. The decrease in velocity of superprecipitation may be attributable to the shortening of actin filaments, whereas the decrease in the maximum level of superprecipitation may be from the effect of cytochalasin D to increase the number of actin monomers. In summary, cytochalasin D inhibited smooth muscle contractions without changing [Ca2+]i. Cytochalasin D also inhibited muscle contraction in skinned fibre and the superprecipitation of native actomyosin without changing Mg2+-ATPase activity, suggesting that actin-myosin interaction was not affected. These results suggest that cytochalasin D acts on actin filaments to shorten the filament length and thus inhibiting the force transduction to extracellular matrix. In addition, inhibition by cytochalasin D of the polymerization--depolymerization homeostasis may also contribute to inhibit the contractile tension because this homeostasis may play a role in force maintenance in smooth muscle.

Membrane currents in smooth muscle cells isolated from the guinea pig gastric antrum N. Gotoh, H. Tokuno and T. Tomita

Cytochalasin D relaxes smooth muscle contraction by direct inhibition of contractile element

Department of Physiology,School of Medicine, Nagoya University,Nagoya 466, Japan

S. Saito, M. Hori, H. Ozaki and H. Karaki

Membrane currents in single smooth muscle cells freshly dispersed from guinea pig gastric antrum were analysed using the whole-cell clamp method. The bathing medium contained 2.4 mM Ca z+ and patch electrodes contained 130 mM KC1 (or 100 mM Cs-aspartate + 30 mM CsCl), 5 mM EGTA, and 10 mM HEPES buffer, and experiments were carried out at 35~ Holding potential was - 6 0 mV and command pulses of 400 ms were applied. With KCl-filled electrodes, depolarizing

Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo, 113, Japan Cytochalasins induce various biological actions in many cells such as phagocytosis, exocytosis, secretion and cell division. These actions are attributable to the effect of cytochalasins to shorten actin fibre by capping barbed end and thus inducing depolymerization of F-actin.

ABSTRACTS pulses elicited a small transient inward current followed by a noisy outward current which decayed very slowly. The voltage-current relationship showed normal rectification and the membrane conductance was 0.6 -I- 0.2 nS (the mean -}- SD, n = 5) near holding potential. TEA (30raM) decreased the conductance and markedly reduced rectification. When CaCl-filled electrodes were used, a clear inward current was observed in most cells. The threshold for inward current was about - 3 0 mV and inward current decayed rapidly, being fully inactivated in 200 ms. The noise in outward current was not observed with Cs-electrodes, but outward currents were small in some (type A) but large in other cells (type B). The outward current observed with Cs-electrodes did not decay in 400 ms and weakly inhibited by TEA. In excess K+ solution (20-60 raM) a transient inward current was followed by a sustained inward current which was increased by increasing K+ concentration in type A cells. The transient inward current was reduced when K + concentration was increased over 40 raM. On the other hand, in typeB cells, effects of excess K § on outward current was relatively small, although a slow large inward current appeared on cessation of depolarizing command pulse. This off-response was blocked by TEA. The difference in response may be because the type A cell has a C1- conductance much lower than the type B cell.

Osmotic effects on mechanical activity in the circular muscle of the guinea pig gastric muscle T. Tomita and M. Md. Syed

Department of Physiology, School of Medicine, Nagoya University, Nagoya 466, Japan Circular muscle strips taken from the fundic region have sustained muscle tone which is produced by endogenous prostaglandins, whereas those from the antral region produce rhythmic phasic contractions on top of a weak basal tone. When the NaC1 concentration (127 raM) in the medium was reduced to 95-70% (121-89 mM), keeping other composition constant, a tonic contraction developed and the rhythmic activity was inhibited in the presence of atropine, tetrodotoxin and meclofenamate, a prostaglandin biosynthesis inhibitor. This contraction was not caused by a decrease in Na concentration by itself but to hyposmolarity, because the tonic contraction was prevented when the osmolarity was compensated by N-methylglucamine, choline or sucrose. The contraction was larger with a greater decrease in osmolarity and it was also larger in the fundus than antrum. Hyperosmolarity of the bathing solution had opposite effects. The hyposmotic contraction was easily blocked by removing the external Ca 2+, but it was resistant to verapamil (5 p,M). Using fura-2, an increase in intracellular Ca 2+ concentration could be demonstrated during the hyposmotic contraction. These results suggest that cell swelling increases Ca z+ influx through a pathway (probably stretch-activated) which is insensitive to organic Caz+ channel blockers and that the density of this channel is higher in the fundic region than the antral region. Hyposmotic contractions were not significantly affected by excess K + (60 raM) in the absence of verapamil, but it was markedly inhibited in the presence of verapamil. It appears that membrane depolarization increases the sensitivity of the Caz+ influx pathway to Ca2+ channel blockers.

Release of stored Ca and contraction in vascular smooth muscle of spontaneously hypertensive rats K. Shimamura, K. Moriyama and S. Sunano

Research Institute of Hypertension, Kinki University, Osaka-sayama, 589 Japan It has been shown in smooth muscles that Ca released from intracellular store site as well as Ca influxed from extracellular space contributes to the contraction. In the contraction of vascular smooth muscle from hypertensive animals, changes in the reactivity to Ca or Ca handling have been reported. However, evidence concerning changes in Ca

489 store or contraction induced by released Ca is still controversial. Previously we have reported that contraction induced by caffeine or noradrenaline in the absence of extracellular Ca is greater in vasculature from hypertensive rats than normotensive rats. In the present study, we compared the role of released Ca from store in noradrenaline-induced contraction in normotensive and hypertensive rats. Noradrenaline is known to produce IP3 and C-kinase as second messengers; the former induces Ca release from store site and the latter promotes contraction through increases of sensitivity to intracellular Ca. In the present study we examined contractions and free Ca signal induced by noradrenaline or C-kinase activator in the absence of extracellular Ca. The mesenteric arteries were isolated from 4-month-old male normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Spirally-cut strips of 300 ].tM width and 10 mm length were made from secondary branches of the artery and their endothelium was removed by rubbing the intimal surface. They were suspended in a modified Tyrode's solution and isometric tension was recorded. Intracellular free calcium was measured by Fura-2 method simultaneously with mechanical activity. After 80raM K-induced contraction, the preparation was incubated in Ca-flee medium for 10 min, and noradrenaline was then applied to the tissue in Ca free condition. The changes in fluorescence intensity induced by the drug were expressed as percentages of those induced by 80 mM K. Maximal contraction induced by noradrenaline was greater in the preparation from SHRSP than that from WKY. Noradrenaline-induced increases of fluorescence in Fura-2 loaded preparation in Ca-flee solution were also greater in SHRSP than those of WKY when expressed as a percentage of 80 mM K-induced increase. It is known that a part of the noradrenaline-induced contraction is mediated by C-kinase-induced change in Ca sensitivity of contractile proteins. Therefore, 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of C-kinase, for example, induces contraction witho'ut changing intracellular Ca concentration. It was also demonstrated in the present experiment that TPA induced contractions in mesenteric arteries from WKY and SHRSP without change of intracellular free Ca signal change, The contractions induced by C-kinase were not different between WKY and SHRSP. Thus, it can be concluded that the increased noradrenaline-induced contraction is not caused by the difference in protein kinase-C-induced change in Ca sensitivity of contractile proteins but to the increased release of stored Ca from IP3-sensitive sites.

Release of intracellular Ca 2+ by mechanical stimulation in vascular tissues Y, Tanaka, S. Hata, H. Ishiro, K. Ishii and K. Nakayama

Department of Pharmacology, School of PharmaceuticalSciences, University of Shizuoka, Yada, Shizuoka-City, Shizuoka 422, Japan Our previous study has demonstrated that myogenic contraction of cerebral and coronary arteries of various animal species, including dog and pig, produced by quick stretch (a rate of 10 cm s i, an amount of 140% of the slack length = 100%, and a stimulus period of 30 s) is always preceded by the increase in intracellular Ca2- concentrations ([CaZ+]~), which is attributable to not only the transmembrane influx of Ca 2+ through 1,4-dihydropyridine-sensitive Ca 2- channels but also the release of Ca2+ from intracellular storage sites. The present study was undertaken to elucidate the mechanism for release of Ca 2involved in the stretch-induced contraction. The contractile component on quick stretch that was resistant to nicardipine was reduced to half after treatment with ryanodine. The nicardipine-resistant contractile component produced by quick stretch was mostly suppressed by a putative phospholipase C inhibitor, NCDC (2-nitro-4carboxyphenyl-N, N,-diphenylcarbamate) or by lowering the temperature from 35~ to 20~ Moreover, intracellular formation of 1,4,5triphosphate (IP3) in pig coronary artery was augmented by quick stretch up to approximately three-fold of the control. The results

490 suggest that Ca2~ released from both ryanodine-sensitive and -insensitive storage sites participates in the myogenic contraction of vascular tissues produced by quick stretch, and that IP3 is a common mediator of the release of Ca2; from both storage sites.

Effects of phosphatase inhibitors on calcium channel current of guinea pig taenia coli smooth muscle cells K. Obara and H. Yabu

ABSTRACTS [Ishihara et at. (1989) BBRC, 159, 87i-7]. In the absence of ATP in pipette solution, OA and CL-A did not affect the inward current. These results suggest that the increasing effects of higher concentrations of OA and CL-A on the inward current may involve phosphatase type-1 activity and the decreasing effects of lower concentrations of OA and CL-A may involve phosphatase type2A activity, and that the effects of OA and CL-A may be dependent on the balance between phosphatase type-I and type-ZA activities.

Department of Physiology, Sapporo Medical College, Sapporo 060, Japan It has been reported that the activities of dihydropyridine-sensitive L-type Ca2~ channel are modulated by the phosphorylation responding to the physiological stimuli. The steady-state level of phosphorylation of any cellular protein is dependent on the activity balance of the protein kinase and protein phosphatase. The phosphorylation reaction is well studied but the reverse reaction is not well documented. Okadaic acid (OA) and calyculin A (CL-A) are cytotoxic compounds isolated from marine sponges and they are potent and specific inhibitors of protein phosphatase type-1 and type-2A. In this study, we examined the effects of OA and CL-A on the voltagedependent Ca2§ channel current of smooth muscle cells isolated from guinea pig taenia coli. Single smooth muscle cells were enzymatically isolated from guinea pig taenia coli. The inward currents carried by Ba2+ were recorded by using a whole-cell parch clamp technique. The pipette filled with 140mM CsCI, 4raM EGTA, 5 mM HEPES and 4raM Na2ATP (pH 7.4). The bath solution contained 135 mM NaC1, 5.4 mM KC1, 1.8 mM BaCI2, 1 mM MgCI2 and 5 mM HEPES (pH 7.4). Drugs were applied by addition them to the perfusing bath solution. OA and CL-A had a dual effect on the inward current that is dependent on the concentrations of them; at higher concentrations they increased the inward current and at lower concentrations they decreased the inward current. OA (5 x I0 ~-5 x 10 -6 M) and CL-A (I0-9-10 -7 M) dose-dependently increased the inward current and the maximum responses of OA and CL-A were observed at approximately 10 6M OA and 10-8M CL-A, respectively. The EDs0 values for OA and CL-A were approximately 2 x 10-TM and 3 x 10-9M, respectively. CL-A appeared to be about 100-fold more potent in increasing the inward current than OA. This result is consisted with report of Ishihara and colleagues [BBRC, 159, 871-7 (1989)] that OA and CL-A are potent inhibitors of phosphatase type-1 and type-2A, but CL-A is about 100 times more effective inhibitor of phosphatase type-1 than OA. OA (10-~~ and CL-A (10-~-10-9M) decreased the inward current. The maximum inhibitory effects of OA and CL-A were observed at I0 -~ M OA and 5 x 10 10M CL-A, respectively. In these concentration ranges, OA and CL-A inhibit phosphatase type-2A activity but they have no or little effect on phosphatase type-1 activity

Specific potentiation by endothelin-1 and neuropeptide Y of contractile activity in vascular tissues K. Nakayama, S. Hata, H. Ishiro and Y. Tanaka

Department of Pharmacology,Schoolof PharmaceuticalSciences, University of Shizuoka, Yada, Shizuoka-City, Shizuoka 422, Japan Endothelin-1 (ET-1) and neuropeptide Y (NPY) are well-known vasoconstrictor peptides of endogenous origins. The present study was undertaken to provide some new insights into the pharmacological synergy between potential mediators of vascular spasms in coronary and cerebral circulations. Isometric tension and cytosolic Ca2+ concentration ([CaZ+]i) of porcine coronary and canine cerebral arteries loaded with fura-2 were simultaneously measured. Of various coronary vasoconstrictors, such as acetylcholine, histamine, and 5-hydroxytryptamine (5-HT), a small amount of ET-1 (30-100 pM) specifically potentiated the 5-HT-induced contraction of porcine coronary artery without causing a significant increase in [CaZ+]v which seems to be qualitatively similar to DPB (12-deoxyphorbol-13-isobutyrate), a tumour-promoting phorbol ester. On the other hand, NPY (10-30 nM) potentiated the contractions of canine cerebral artery produced by practically all of vasoconstrictors used in the present study, including histamine, norepinephrine, 5-HT, and prostaglandin F2~. Bay K 8644, a Ca2+ agonist of 1,4-dihydropyridine type also showed a non-specific potentiation of agonist-induced contractions. The contractile potentiation produced by ET-1 was not affected by 20 mM KC1, and partially inhibited by Ca2+ antagonist such as nisoldipine, while that by NPY became more prominent under a partially-depolarized state. Furthermore, NPY-induced contractile potentiation was accompanied with the increase in [Ca2+]~, and very susceptible to nisoldipine and cromakalim. The results suggest that ET-1 in combination with 5-HT augments the Ca2+ sensitivity of the contractile elements through a possible mechanism for activation of protein kinase C, while the NPY-induced contractile potentiation when combined with other agonists is attributable to enhanced transmembrane influx of Caz+. It seems possible that the transducer pathways coupled to contractile activity are differently overlapped for different receptor systems, depending on vascular tissues and agonistic stimuli.