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Annu. Rev.

FLASH PHOTOLYSIS STUDIES OF EXCITATION-CONTRACTION COUPLING, REGULATION, AND CONTRACTION IN SMOOTH MUSCLE

Andrew P. Somlyo and Avril V. Somlyo Department of Physiology, University of Virginia Health Sciences Center, Box 449, Charlottesville, Virginia 22908 KEY WORDS:

caged compounds, muscle physiology. contraction delays, muscle agonist­

receptors, pharmacomechanical coupling

INTRODUCTION The binding of an excitatory transmitter to its receptor on smooth muscle leads to contraction through the combined mechanisms of excitation­ contraction coupling, contractile regulation, and crossbridge cycling (re­ viewed in 6, 23, 63, 68). Typical smooth muscle preparations consist of numerous cells with large surface/volume ratios and with geometries com­ plicated by tortuous extracellular spaces lined with proteins. In such pre­ parations, but even in single cells, the interpretation of any phenomenon evoked by the addition of a diffusible transmitter, messenger, or substrate is greatly complicated by diffusional delays. Photolysis of caged compounds overcomes such diffusional delays and, although still in its infancy, the application of this method to the study of excitation-contraction coupling, 857

0066-4278/90/0315-0857$02.00

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SOMLYO & SOMLYO

contractile regulation, and contraction in smooth muscle, has already pro­ duced valuable new information. The term "caged compound" was introduced to denote caged ATP (40), an ATP molecule that was rendered inaccessible as a substrate to the Na, K-ATPase by esterifying its tenninal phosphate with a photolabile nitrobenzyl group. This general strategy of caging with a photolabile group has since been successfully applied to a number of adenine and guanine nucleotides, inositol 1, 4, 5 trisphosphate, and other compounds, particularly since a single step method of caging the substrate became available (77, 78). Photolysis of such a caged compound yields (within ms) an active compound instantaneously accessible to its ligand and a nitroso-ketone "leaving" group. The structure and photochemistry of these and other types of caged compounds have been reviewed elsewhere (20, 41, 47). The general concept underlying their design is to produce an inactive compound that can be diffused to and equilibrated at its site of action "at leisure", and then activated, within micro/milliseconds, by a light flash. Alternatively, an active photolabile compound, such as a dihydropyridine calcium-entry blocker, can be inactivated by flash photoly­ sis. A recent novel approach called fluorescence photoactivation and dissipa­ tion allows one to produce foci of fluorescence within a cell and track the movement of the fluorescent molecules of interest. This technique is based on the removal, through photolysis, of the quenching effect of the photosensitive groups that were covalently attached to fluorescent compounds (81). A recent and significant improvement in the application of caged com­ pounds has been their introduction into cells penneabilized with staphylococ­ cal alpha-toxin or with the saponin ester beta-escin. Smooth muscles (10, 42) and other cells (1) exposed to toxin become permeable to low molecular weight (Mr ::; ] ,000 dalton) solutes while retaining high molecular weight compounds, including enzymes and calmodulin. Pe�eabilization with beta­ escin creates larger holes that are penneable to calmodulin (Mr 17,000 dalton). Since the majority of caged compounds are charged molecules, they generally do not penetrate the plasma membranes, and in earlier studies they had to be introduced into mechanically (skeletal) or saponin (smooth) skinned muscle fibers (18, 25,71, 79). These methods led to the loss of a number of regulatory substances, soluble enzymes and others and, in the case of smooth muscle, uncoupled excitatory (e.g. alpha-adrenergic, muscarinic) receptors. PenneabiIization with either alpha-toxin or beta-escin does not uncouple these receptors (42, 43) and permits the exploration of the effects of caged com­ pounds under more nearly physiologic conditions. The caged compounds that we have thus far introduced into alpha-toxin and/or beta-escin permeabilized smooth muscles include caged ATP, GTP'YS, and inositol 1, 4, 5 trisphos­ phate (InsP3). =

SMOOTH MUSCLE

859

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EXCITATION-CONTRACTION COUPLING AND THE LATENCY OF ACTIVATION: THE USE OF CAGED InsP3 AND CAGED PHENYLEPHRINE Electromechanical coupling and phannacomechanical coupling are the major modalities of excitation-contraction coupling in smooth muscle (69, 73 re­ viewed in 70). Electromechanical coupling is mediated by changes in surface membrane potential: the influx of Ca2+ through voltage gated channels, depolarization induced Ca2+ release, or the inhibition of these Ca2+ move­ ments by hyperpolarization. Pharmacomechanical coupling is also mediated primarily by agonist-induced Ca2+ release or Ca2+ influx through ligand gated or second messenger gated channels (reviewed in 70, 76). According to its original definition (73), however, phannacomechanical coupling may also include a component that modulates the sensitivity of the regulatory­ contractile apparatus to Ca2+. The main applications of caged compounds to the study of excitation­ contraction coupling in smooth muscle have been directed to phanna­ comechanical coupling: Ca2+ release and, most recently, modulation of Ca2+ sensitivity. The major source of intracellular Ca2+ released by pharma­ comechanical coupling is the sarcoplasmic reticulum (SR), although there may be another small nonmitochondrial, InsPrinsensitive and GTP")'S­ sensitive compartment in some smooth muscles (44). Caged compounds have been particularly useful in exploring the kinetics and thus the mechanism of phannacomechanical Ca2+ release from the SR. The rapid force development by smooth muscle responding to release of InsP3 by photolysis from caged InsP3 provides strong evidence of the role of InsP3 as a physiologic messenger of phannacomechanical Ca2+ release (70, 79). The time elapsed (delay or latency) between excitation of the surface membrane and contraction is related to the mechanism of excitation­ contraction coupling. Long, temperature-sensitive latencies are considered characteristic of chemical transmission, while short latencies, such as observed in fast striated muscles (48), are thought to be suggestive, though not diagnostic, of mechanical or electrical coupling mechanisms. Early evi­ dence (5) revealed a very long (1 s at 39°C) delay between stimulation of excitatory (adrenergic) nerves and contraction of (rabbit pulmonary artery) smooth muscle, but did not resolve the question whether or not this was due to diffusional delays preceding the binding of the agonist to its receptor or to post-receptor events. With current technology, the latency can be separated into two components: the first, between binding of an agonist to its receptor and the rise in cytoplasmic Ca2+, and a second, between the rise of Ca2+ and contraction. In the case of modulation of Ca2+ -sensitivity by an agonist, the

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SOMLYO & SOMLYO

relevant delay is the one between agonist-receptor interaction and the change in force. The latency between agonist-receptor interaction and force-development has been explored with caged phenylephrine (70). The long (1 .5±0.26 s at 20°C) interval between photolysis of this compound (Figure 1 ) and its high QIO (2.7) are consistent with a chemical mechanism, presumably the hydroly­ sis of phosphatidylinositol biphosphate (PIP2) to yield InsP3 as the Ca2+­ releasing messenger (4). The relative contributions of Ca2+ influx or Ca2+ release to such agonist-induced contractions remain to be quantitated, but the results clearly show that neither of these processes is initiated by phenyleph­ rine on a very fast (Le.: ms) time scale. Two technical aspects of these experiments should be noted. First, it was necessary to use relatively high (50 pM) concentrations of caged phenylephrine in order to reach threshold con-

100

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Laser Pulse

o

! Time

Figure 1

Two force transients recorded after photolysis of caged phenylephrine (50 ILM) in

an

intact muscle strip and caged InsO ,4,5)P3 in a permeabilized muscle strip of guinea-pig portal vein at 20°C. A 50 ns laser pulse at 347 nm is indicated by the arrow. The peak force and t1/2 to peak force for Ins(l,4,5)P3 and phenylephrine were 177 /LN, 1.4 s and 205 /LN, 1.4 s, respectively. The lag phase preceding force development was 0.4 s for Ins (l ,4,5)P3 and 1.8 s for phenylephrine. The intact strip used for the caged phcnylephrine experiment had been treated with 6-hydroxy-dopamine for 20 min to produce adrenergic denervation. This experiment was done in the presence of 143 roM potassium to depolarize the cell membrane. The Ins( l,4,5)P, response was obtained in a muscle strip permeabilized with 50 /Lg ml-I saponin for 15 min and calcium loaded for 5 min at pCa6.6, 1 roM EGTA, followed by a 2 min wash in 0 calcium containing 1 mM EGTA solution, and subsequent incubation with 10 /LM caged Ins(l,4,5)P, (esterified on the pS position) in a solution containing 0.1 roM EGTA and 90 ILM calmodulin for 3 min before the laser flash. Approximately 10% Ins(I,4,5)P3 and phenylephrine were released from the caged precursors (from reference 70).

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SMOOTH MUSCLE

861

centration sufficiently fast, given the rather slow dark reactions of this caged compound. Control experiments with (uncaged) phenylephrine jumps showed similar results, however, indicating that the long delay was not due to slow dark reactions or to an alpha-adrenergic antagonist action of the caged com­ pound itself. Second, the experiments were done in high K+ depolarizing solution to prevent activation of the surface membrane by the laser pulse itself. Under these conditions the responses were completely blocked by an alpha-adrenergic antagonist. Considering the high QlO of the delay of con­ tractions initiated by photolysis of caged phenylephrine, the increase in InsP3 found after 500 ms in trachealis smooth muscle neurally stimulated at 37°C (49) is consistent with the time course of contraction induced by photorelease of caged agonist. Excitatory receptors in smooth muscle (44), as in other systems (for review, 9), are coupled by G proteins to phospholipase C. Pharmacomcchani­ cal Ca2+ release can be directly evoked with nonhydrolyzable analogues of GTP, such as GTPyS (44, 45). The delay between the application of GTPyS and Ca2+ release and the physiologic response is rate-limited by the off-rate of GOP from the G proteins (e.g. 8, 9), however, and can be longer than the physiologic delay that is shortened through acceleration of the GOP off-rate by agonists. Therefore, the delays observed following the addition of GTPyS to permeabilized smooth muscle are longer than normally seen following the application of an agonist to intact muscle. Subthreshold concentrations of an agonist to accelerate the exchange of GTPyS for GDP on G protein would be expected to shorten the delay following photolysis of GTPyS. Ca2+ is thought to be released through InsPTsensitive SR channels that are specifically blocked by heparin (14, 44, 45). The opening of channels, as contrasted to the operation of active transport systems and carriers, is general­ ly thought to be associated with faster kinetics and less likely to require gating times of tens of msec. The upper limit of the delay between photorelease of high concentrations (up to about 50 /LM) of released InsP3 and the rise in Ca2+ measured with FIuo 3 (39, 50) in guinea pig portal vein smooth muscle (in the presence of 2 mM AMPPCP and the absence of ATP), is approximate­ ly 30 ms (K. Horiuti, A. V. Somlyo, O. R. Trentham, et aI, in preparation). Longer delays were found at low InsP3 concentrations. The delays were shortened by ATP and also by its nonhydrolyzable analogue AMPPCP, although both the delay and rate of release were very much depressed in the absence of nucleotides. InsPrinduced Ca2+ release is reported to be absolute­ ly dependent on ATP or one of its nonhydrolyzable analogues in cultured smooth muscle cells (64), but not in taenia caeci (33) or in basophilic leukemic cells (47a). These results suggest that the adenine nucleotides play a role other than as phosphate donors in modulating the InsPTgated channel. An InsPrgated channel could open with delay if, for example, the InsP3

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862

SOMLYO & SOMLYO

receptor at rest was not free, but occupied by an inactive compound, perhaps ATP or an InsP3 metabolite. In such a case, the off-rate of this compound could rate-limit the on-rate of InsP3 to its receptor and cause a delay in Ca2+ release. Alternatively, the delay in basophilic leukemia cells has been attrib­ uted (47a) to the kinetics of binding of multiple InsP3 molecules thought to be required for opening Ca2+ channels. Approximately 0.3-0.5 s (at 22°C) of the delay between activation, whether electromechanical or pharmacomechanical, and force is due to steps following the increase in [Ca2+]j. In contrast to Ca2+ release, the delay in force development following photolysis of caged InsP3 (Figure 2) is relatively long (about 0.5±0.12 s at 22°C) and has a low QIO (70). There is a similar (about 0.2-0.3 s at 22°C) delay between the rise in [Ca2+]j and contraction in spontaneously active or electrically stimulated intact smooth muscle (29,83). Similar latencies were found between photolysis of caged ATP and force initiated from the rigor state in the presence of Ca2+ (31). This delay was shorter, by nearly an order of magnitude, when ATP was released by pho­ tolysis into the myofilament lattice under conditions where the myosin light chains were prephosphorylated by treatment with ATPyS or the phos­ phatase inhibitor okadaic acid (31). In intact smooth muscle, there is also a close temporal relationship between myosin light chain phosphorylation .

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Ca2+ transients upon photolysis of caged InsP3 in guinea pig ileum. The muscle strip

was mounted in a 0.3 mm i.d. quartz capillary. One end of the muscle was attached to a force transducer via a tungsten wire and the other end attached to a tungsten wire anchored to the inner wall of the capillary tube. The muscle was permeabilized with staphylococcal alpha-toxin, the sarcoplasmic reticulum loaded with calcium by incubation at pCa6.3, 10 mM EGTA for 10 min, followed by rapid removal ofCa and EGTA and subsequent exposure to 30 fLM caged InsP3 (with the nitrobenzyl group on the P5 position) and 10 fLM Fluo 3 at 25°C for 3 min. before photolysis with the 2 ms UV light from the flash lamp. indicated by the arrowhead. Approximately 15% of the caged InsP3 was photolyzed. The force and Ca2+ (Fluo) responses are shown on a slow and fast time base. Note the delay of 350 and 30 ms for the force and Ca z + transients. respectively (from K. Horiuti, et al. in preparation).

LL_--

863

SMOOTH MUSCLE

and stiffness (37). In view of the short delay between light chain phosphoryla­ tion and the mechanical events, the longer latencies between the rise in Ca2+ and contraction have been ascribed variously to prephosphorylation reactions between Ca2+, calmodulin, and myosin light chain kinase (71, 83), a me­ chanical effect of series elastic elements, and to the kinetics of the (minimal­

ly) two-step reaction of myosin light chain phosphorylation and attachment of

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crossbridges into force-generating states

(31).

CONTRACTILE REGULATION: CONTRACTION KINETICS OF PHASIC AND TONIC SMOOTH MUSCLES AND THE MODULATION OF Ca2+ SENSITIVITY The

primary

regulatory

mechanism

of

smooth

muscle

contraction is

phosphorylation of myosin light chain (LC2o) by myosin light chain kinase

that is activated by its regulatory subunit,Ca2T -calmodulin, as the result of an increase in cytoplasmic Ca2+ (reviewed in 23,58). Phosphorylation of LC 0, 2 usually in response to an increase in [Ca2+]j above its resting value (80-140 nM; reviewed in 66), is sufficient for the activation of myosin ATPase by actin (35). Relaxation, according to this mechanism, results from de­ phosphorylation of LC20 by myosin light chain phosphatases (21, 30, 59), usually, though not always, as a result of a decline in [Ca2+]i' The possibility of secondary, perhaps thin filament-associated, mechanisms of regulation (reviewed in 38) is outside the scope of this presentation,as decisive evidence of the physiologic role of such a mechanism has yet to be obtained, and its operation has not been explored with caged compounds. The fact that myosin light chain phosphorylation is the major mechanism initiating smooth muscle contraction raises the question whether this process, rather than the kinetics of the crossbridge cycle, limits the rate of contraction under physiologic conditions. Related questions concern the manner in which myosin light chain phosphorylation may also be regulated by factors other than [Ca2+]i and how variations in the regulatory mechanism contribute to the phasic or tonic properties (27, 28, 69) of smooth muscle. Activation of permeabilized smooth muscles through photolysis of caged ATP was particu­

larly useful in answering some of these questions, as it was necessary to eliminate the effects of diffusion of ATP, required both as a phosphate donor for myosin phosphorylation and as a substrate of actomyosin ATPase The initiation of contraction from rigor, in the presence of maximally activating concentrations of Ca2+ -calmodulin, represented the condition in which phosphorylation of myosin LC 0 was a prerequisite for contraction: 2 force development under these conditions occurred at a rate similar to that observed in intact smooth muscles (Figure 3; 31, 71). In contrast, photolysis of caged ATP could tum on the already phosphorylated crossbridges without .

864

SOMLYO & SOMLYO

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photolysis Figure 3

Kinetics of contraction initiated by flash photolysis of caged ATP in trachealis smooth

In the upper trace the myosin light chains were pre-thiophosphorylated with ATPyS. In the lower trace the muscle had not b een pre-phosphorylated before photolysis. Each force record was normalized to its amplitude of response. An HPLC analysis revealed that the amount of liberated ATP was (mM) 1.4 in the pre-phosphorylated and 1.5 in the non-phosphorylated muscle. The asterisk indicates the 2 ms pulse ofUV light from the flash lamp (data from reference 31).

muscle.

the intervening steps of phosphorylation in smooth muscles that, prior to photolysis, were thiophosphorylated with ATPyS or maintained with the phosphatase inhibitor okadaic acid (75), in a normally phosphorylated (with ATP) state during rigor. Under the latter conditions, force development was several times faster than when contraction was initiated in non­ phosphorylated muscles, which indicates that myosin light chain phosphorylation is usually slower than crossbridge turnover and limits the rate of the force development. Force development, initiated by photolysis of caged ATP, is faster in phasic than in tonic smooth muscles, regardless of whether the muscles are unphosphorylated or prephosphorylated before releasing ATP (31). These findings suggest that both the regulatory and the contractile apparatus are slower in tonic than in phasic smooth muscles. Differences in the maximum shortening velocity have also been found in intact smooth muscles (55). Whether such differences in mechanical properties are expressions of differ­ ent myosin isozymes (13, 1 6), as suggested by recent observations (24), is yet to be established (74). Mechanisms other than a change in cytoplasmic Ca2+ can also regulate smooth muscle contraction. This conclusion is supported by several lines of

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SMOOTH MUSCLE

865

evidence. The force/Ca2+ ratio is higher during agonist-induced than high K+-induced contractions (7,26,52,57,61),and the relationship between the [Ca2+]j transient and force can diverge during a K+ contraction (27, 28); in tonic smooth muscle, following its initial increase, [Ca2+;] declines, while force continues to rise over a 30 min period. In contrast, in phasic smooth muscle the phasic decline in force is greater than would be predicted on the basis of the fall in [Ca2+]j (27, 28). That such phasic fall in force need not be due to changes in [Ca2+]j is also shown by the similarly phasic response of (ileum) smooth muscle (permeabilized with alpha toxin) to "clamped", sub­ maximally activating concentrations of Ca2+ (42, 67). Inhibition of myosin light chain phosphatase converts such phasic contractions to more tonic ones (67). These findings suggest that, at least in some smooth muscles, phasic contractile properties and modulation of Ca2+ sensitivity may be the conse­ quence of regulated of myosin light chain phosphatase(s) activity. According to this model, desensitization (27) of the regulatory/contractile apparatus of phasic smooth muscles to Ca2+ reflects regulated changes in the myosin light chain kinase/phosphatase activity ratio, most probably because of regulation of phosphatase. While this hypothesis will require considerable testing, it is supported by the very rapid and transient phosphorylation of phasic guinea pig ileum smooth muscle during K+ contractions (28). Sensitization to CaH, observed in permeabilized smooth muscle, provides further evidence of the existence of regulatory mechanisms other than, or in addition to, changes in [Ca2+]i. Muscarinic (42, 43) and alpha-adrenergic (42, 43, 54) agonists in the presence of GTP or GTP"S and GTP"S itself (Figure 4 and 17, 42, 43) can markedly increase the contractile response to a given submaximal level of Ca2+. In view of the sensitizing action of GTP"S and of the inhibition of the sensitizing effects of agonists and GTP"S by GDPf3S, it is probable that sensitization is mediated by G protein (s). The question currently investigated is whether this G protein(s) is identical with or dissimilar to the Gp thought to couple agonists to phospholipase C. The possible role of kinase C in sensitizing smooth muscle to [Ca2+]i (e.g. 36, 60; briefly reviewed in 66) remains to be clarified. Evidence supporting such a mechanism is based on the increased Ca2+ sensitivity evoked by stimulators of kinase C, phorbol ester, and phosphatydil serine and by its inhibition by H7, a moderately specific kinase C inhibitor. The effects of these compounds on intact smooth muscle are complex and may or may not be associated with changes in cytoplasmic Ca2+ (reviewed in 66), due to a variety of effects, including some on receptor-effector coupling (3a, 36). Even in permeabilized smooth muscles, the ca2+ -sensitizing action of phor­ bol esters has been variably reported to be associated with (56) or uncorrelated with (12) myosin light chain phosphorylation. Interestingly, in the sole study in which the effect of (brain) kinase C on permeabilized muscle

SOMLYO & SOMLYO

866

.

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GTPyS at constant Ca2+. Force development and Ca2+ were per­ meabilized with alpha-toxin, incubated with 77 /LM caged GTPyS, 50 /LM Fluo-3, pCa6.3, 10 mM EGTA, for 3 min prior to photolysis with a 2 msec pulse (arrowhead) of 360 nm light from a

Figure 4

Modulation of force by

monitored in a small strip of portal vein, mounted in a 0.3 mm i.d. quartz capillary,

flash lamp. Note the approximately 10 sec delay before force rises and that there is no detectable change in Ca2+. (From Ref. 72).

was studied, this effect was relaxation (34), rather than contraction. This is consistent with the inhibitory effect of phosphorylation of the kinase C sites on smooth muscle myosin (53). In view of this, the possibility arises that some of the effects of phorbol esters may be umelated to protein phosphoryla­ tion, or that the effects observed are due to phosphorylation of proteins other than myosin. Finally, in view of the very slow time course of contractions induced by phorbol esters, there does not appear to be an urgent need for the use of caged compounds to explore their effects in smooth muscle.

MECHANISM OF CONTRACTION: CROSSBRIDGE CYCLE, COOPERATIVITY, AND FORCE DEVELOPMENT BY DETACHMENT OF NEGATIVELY STRAINED CROSSBRIDGES Caged compounds have been particularly valuable in relating the biochemical kinetics of myosin ATPase to the mechanical transients of the crossbridge

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SMOOTH MUSCLE

867

cycle in striated muscle (e.g. 18, 19, reviewed in 25). The rapid and syn­ chronous manner in which photolysis of caged ATP provides the substrate to crossbridges has proven similarly useful in studies of smooth muscle. In addition to revealing the kinetics of crossbridge detachment and attachment into force-developing states, the use of caged ATP also provides unexpected evidence of cooperativity among crossbridges and of force development due to the detachment of negatively strained crossbridges. Rigor is a state mechanically characterized by increased stiffness, in which myosin crossbridges are bound to actin in the absence of ATP. The existence of rigor in smooth muscle, inferred some 20 years ago (69), was clearly established by demonstrating that crossbridges present on myosin filaments in smooth muscle (65) assume, in the absence of ATP, an "arrowhead" con­ figuration typical of rigor (71). The structural change in rigor, also detected in the X-ray pattern (3), is associated with the appearance of rigor stiffness and force (2, 71). According to the crossbridge cycle mechanism of contraction, the initial step of the cycle following the binding of ATP to the rigor bridge is the detachment of myosin from actin. This is followed by ATP hydrolysis, attachment, transition to force-generating state(s), product release, and de­ tachment. Photolysis of caged ATP permitted the precise measurement of the rapid phase of detachment and the detection of the somewhat complicated kinetics of relaxation from rigor (71). One of the major conclusions to emerge from this study was that the detachment rate, approximately 105M-1s-1, was too fast to be rate-limiting the ATPase cycle. Thus in the presence of physiologic, millimolar concentrations of ATP, detachment would occur at a rate of approximately 100 s -I, compared to the total hydrolysis rate of approximately 1-2 S-I (62). This detachment rate, however, is one order of magnitude slower than in solution (106M-1s-1) (46), as it is in striated muscle (18), which suggests a mechanochemical effect (strain dependence) of organized (filamentous) actin and myosin on the chemical cycle. In the presence of Ca2+, the rapid phase of detachment is followed by force de­ velopment (Figure 5) at a rate determined by the existing state of phosphoryla­ tion (see above). Photolysis of caged ATP in the absence of Ca2+ revealed a second, slower phase of detachment (Figure 5). Based on analogy with striated muscle (18), it is likely that this slow phase of detachment or bump represents the reattachment of initially detached crossbridges. If so, the biochemical scheme of the crossbridge cycle (25) suggests that the addition of inorganic phosphate (Pi) will drive crossbridges into the detached or more weakly bound (15) states, thus reducing the probability of cooperative reattachment. The addition of Pi to muscles prior to photolysis markedly accelerates relaxation (2) by reducing the slow phase of detachment or bump (71). This and the effect of mechanical manipulations [releasing or restretch-

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868

SOMLYO & SOMLYO

o Figure 5

2

4

sec

Two superimposed tension transients after liberation of ATP by photolysis of caged

ATP in the presence (+Ca2+) and absence (-Ca2+) ofCa2+ . The muscle was stretched by 1 % of its length before liberation of ATP. Both transients show a similar initial rapid fall in force caused by the detachment of rigor crossbridges by the released ATP. In the absence of Ca2+, after the rapid detachment, the transient has a complex shape with a plateau of -400 ms, followed by a slow fall to the relaxed baseline (from reference 70.

ing the muscles prior to photolysis; (71 )] support the notion that the slow phase of detachment and the bump, accentuated in prereleased muscles (figure 8 in 71), are due to reattachment of p reviously detached crossbridges. The fact that crossbridges can reattach in the absence of Ca2+ and, con­ sequently, in the absence of myosin light chain phosphorylation, implies the reattach ment of nonphosphorylated crossbridge s Cooperativity (82) can sup­ port such reattachment and cause contraction in the absence of Ca2+ , at ATP con centration lower than the concentration of myosin heads in rigor. Under these conditions, the cooperative effect of rigor bridges facilitates the reattachment of bridges detached by micromolar ATP into force-generating states. Photolysis of micromolar quantities of ATP in smooth muscles in rigor revealed the existence of such cooperative contractions, which could be up to 40% of maximal Ca2+ -activated force (71). The use of CTP confirmed that this was not because of myosin being phosphorylated by a Ca2+ -insensitive kinase produced as the result of partial proteolysis (35, 80). CTP is a substrate for the myosin ATPase, but is not a phosphate donor in the myosin light chain kinase reaction (1 1 ). Nevertheless, in the absence of Ca2+, photolysis of caged CTP, like that of caged ATP, caused bumps during detachment and .

SMOOTH MUSCLE

869

contractions at micromolar concentrations. Therefore, the evidence for coop­ erative attachment of crossbridges in smooth muscle appears reasonably strong. Based on these findings and on the similar effects of Pi on the bump and relaxation, we have suggested

(71) that the latch state (22) of high

force/low phosphorylated myosin LCzo ratio may possibly reflect cooperative attachment of dephosphorylated crossbridges; in intact smooth muscle the cooperative action is thought to be mediated not by rigor, but by attached, phosphorylated crossbridges. This hypothesis is also compatible with the

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length-dependence of force maintenance at low levels of phosphorylation

(51). The absence of latch at long (1.4Lo) lengths (Lo

=

optimal length for

force development), and presumably reduced filament overlap, could be due to an insufficient number of attached and phosphorylated crossbridges avail­ able for cooperativity and/or to negative strain-dependence of cooperative reattachment. In his

1957 model of contraction, Huxley (32) proposed that crossbridges

can bear negative strain: the detachment of negatively strained crossbridges could result in force development

(19, 32). If smooth muscles in rigor are

prereleased to place negative strain on crossbridges, photolysis of caged ATP causes a very rapid early phase of force development (figure

11 in 71),

coinciding with a fall in stiffness. The early, rapid contraction is significantly faster than the attachment rates, and it is thought to reflect force development caused by the detachment of negatively strained crossbridges.

SUMMARY AND CONCLUSION 1. Flash photolysis of caged compounds of phenylephrine, inositol 1, 4, 5 trisphosphate (InsP3), GTPyS, ATP, and CTP has been successfully used to study excitation-contraction coupling, contractile regulation, and con­ traction in smooth muscle. Major processes explored with this method were

(a) the delay between agonist-receptor interaction and contraction (b) the effect

and between the rise in InsP3, Caz+ release and contraction;

of myosin light chain phosphorylation on the rate of force development and the respective contributions of phosphorylation and crossbridge kinet­ ics to differences between phasic and tonic smooth muscles;

(c) the

kinetics of the crossbridge cycle. We have also reviewed recent results obtained by other methods and bearing on the mechanisms of pharma­ comechanical Ca2+ release and modulation of the Ca2+ sensitivity of the regulatory/contractile apparatus.

2. The long delay (1.5 s at 22°C) following activation of alpha I-adrenergic receptors through photolysis of caged phenylephrine and the high

QIO of

this process are consistent with the hypothesis that activation of phospholi­ pase C is the major mechanism of alpha-adrenergic pharmacomechanical Ca2+ release.

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3. The delay between photolysis of caged InsP3 and Ca2+ release is short: 30

ms or less, while the latency of contraction is significant (0.3--0.5 s at 22°C) and similar to the lag between the rise in [Ca2+]i and force develop­ ment in intact smooth muscles. The latency of contraction following photolysis of caged ATP in permeabilized muscles in rigor, in the presence of Ca2+ and calmodulin, is similar, about 0.2--0.5 s at 22°C. 4. In muscles in which the myosin light chains are maintained in a phos­ phorylated state during rigor, photolysis of caged ATP initiates con­ tractions with a short delay (10 ms or less). This result and those summa­ rized above (2 and 3) suggest that the major portion of the delay between agonist-receptor interaction and contraction is due to activation of phos­ pholipase C and InsP3 production, and about 0. 2--0.5 s of the delay (22°C) can be ascribed to prephosphorylation reactions between Ca2+, calmodu­ lin, and myosin light chain kinase, and/or to mechanical processes, or to the chemical kinetics of two-step reactions. 5. Force development from rigor, initiated by photolysis of caged ATP in the presence of Ca2+ -calmodulin, is rate-limited by myosin light chain phos­ phorylation; it is significantly accelerated if the myosin light chains are already phosphorylated prior to photolysis. 6. The rate of force development is faster in phasic than in tonic smooth muscles, whether contraction is initiated by photolysis of caged ATP in muscles that are unphosphorylated or prephosphorylated. This result sug­ gests that the rate-limiting processes of both the regulatory and of the contractile mechanisms maybe quantitatively different in the two types of smooth muscle. , 7. Evidence obtained with Ca2+ indicators in intact smooth muscles and in smooth muscles permeabilized by staphylococcal alpha-toxin or bcta-escin indicates the existence of mechanisms mediated by G protein(s) that can modulate the Ca2+ sensitivity of the regulatory/contractile apparatus. Permeabilization with these methods has also been useful for the introduc­ tion of caged compounds into cells in which receptors remain coupled to their effectors. Sensitization of permeabilized smooth muscle to Ca2+ by photolysis of caged GTPyS has a very long (10---1 5 s) delay. 8. The detachment rate of crossbridges from the rigor state, measured follow­ ing photOlysis of caged ATP, is approximately 105M-I S -I; this rate is too fast to be rate limiting the crossbridge cycle. 9. A slow component of relaxation following crossbridge detachment, observed following photolysis of either caged ATP or caged CTP, is attributed to reattachment of unphosphorylated crossbridges through cooperativity of the remaining, attached rigor bridges. It is suggested that the state of high force/low phosphorylation observed during tonic force maintenance in intact smooth muscles (latch) may be due to similar reattachment of dephosphorylated cross bridges through the cooperative

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action of remaining phosphorylated bridges. The existence of cooperativ­ ity in smooth muscles in rigor is revealed through the contractions evoked by photolytic release of micromolar concentrations of caged ATP or caged CTP into smooth muscles in rigor. 10. A very rapid phase of contraction, observed in the absence of Ca2+ in

smooth muscles that were mechanica lly released during rigor in order to negatively strain crossbridges, is attributed to force development caused

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by the detachment of negatively strained bridges. ACKNOWLEDGMENTS

The authors' research was supported by National Institutes of Health Grant H215835 to the Pennsylvania Muscle Institute. We thank Ms. Ruby Raines and Mrs. Betty Ferguson for preparation of the manuscript and Ms. Mary Alice Spina for illustrations.

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