186
Biochimica et Biophysica Acta, 1034(1990) 186-194 Elsevier
BBAGEN 23299
Rapid activation by photolysis of nitr-5 in skinned fibres of the striated adductor muscle from the scallop Trevor J. Lea 1, Mark J. Fenton 1, James D. Potter 2 and Christopher C. Ashley 1 JUniversity Laboratory of Physiology, Oxford (U.K.) and 2Departmentof Pharmacology, MiamL FL (U.S.A.) (Received 9 October, 1989) (Revised manuscript received 18 January 1990) Key words: Skinned muscle fibre; Caged calcium; Laser photolysis; Muscle contraction; Calcium ion; (Scallop muscle)
Photolysis of nitr-5, a caged calcium molecule, has been used for rapid activation of skinned fibre bundles of a myosin-regulated muscle, the striated adductor of the scallop, Pecten maximus. Chemically skinned fibre bundles (diameter 7 0 - 2 0 0 /tin) were equilibrated in solutions containing 1 - 3 m M nitr-5 (pCa 6.1) and then activated by ultraviolet laser pulse (25 ns). Pulse energies of 6 0 - 9 5 mJ gave contractions of over 90% maximum tension and a mean half-time for tension rise of 43 ms ( n - - 4 ) at 12°C. Electrically stimulated bundles of intact fibres develop a tetanus with a rise half-time of 60.2 ms at 1 0 ° C ( n = 5 ) (Rail, J.A. (1981) J. Physiol. 321, 287-295, and personal communication). At lower pulse energies the skinned fibres gave smaller amplitude contractions with slower rates of rise (up to 260 ms half-time). In addition, a slower component of tension development (mean rise half-time 13.3 s) was often observed. In ATP-free solutions containing hexokinase and glucose, rigour tension developed with a delayed onset. Rapid release of A T P (0.47-0.59 mM) from photolysis of caged ATP (2 raM) at pCa 4.5 then caused a rapid contraction with a mean half-time for tension development of 17 ms (n = 4). The fast activation rates obtained by the photorelease of Ca 2+ from nitr-5 are similar to those obtained with skinned skeletal fibres of actin-regulated muscle. The results imply that the rate-limiting step in excitation-contraction coupling of the scallop muscle is not the increase in sarcoplasmic Ca 2+, but rather the activation of the muscle in response to this increase. The half-times of ATP-induced contractions at pCa 4.5 suggest that in a contraction activated by a rapid Ca 2+ jump the process comprising ATP hydrolysis and cross-bridge cycling occurs at a somewhat faster rate than the Ca2+-dependent activation process which precedes it. Introduction In vertebrate skeletal muscle, calcium regulation of contraction is primarily mediated via the thin filament [1] as a result of Ca 2÷ binding to troponin C [2]. By contrast, the regulation of some invertebrate muscles is via the thick filaments and one of the best examples of this is molluscan fast, striated muscle [3]. The Ca 2+ regulation appears to be via two light chains on the myosin molecule; in isolation these regulatory light chains do not bind Ca 2÷, but in association with myosin they do so: one Ca 2÷ per light chain [4]. If the light chains are removed from a skinned muscle fibre bundle of scallop muscle by using EDTA, the tension of the bundle becomes Ca-insensitive [5]. Ca 2÷ control is then restored by addition of the regulatory light chains to the solution bathing the skinned fibres. We have used laser-flash photolysis of the caged Ca 2÷ molecule nitr-5 [6,7] to produce a very fast rise in
Correspondence: C.C. Ashley, University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, U.K.
free C a 2+ (half-time 1 ms) within skinned fibre bundles of the scallop striated adductor muscle, so that rapid activation kinetics of a myosin-regulated muscle can be studied without the delays due to Ca 2÷ diffusion which have been present in previous studies. This technique has been previously used with permeabilised (demembranated) fibres of vertebrate skeletal muscle [8-10].
Materials and Methods Preparation and skinning of fibre bundles Adult specimens of the great scallop (Pecten maximus) were obtained from either Plymouth Marine Laboratory or Millport Marine Laboratory, U.K. They were kept in circulating seawater at 8°C. Skinned muscle fibre bundles were prepared from the striated adductor muscle using a method similar to that described by Simmons and Szent-GyiSrgyi [5]. Muscle strips about 3 mm wide and 2 cm long were removed from the muscle at room temperature and immersed in relaxing solution. Thinner bundles of 7 0 - 2 0 0 / z m diameter and 5-10 mm were then prepared. T-shaped
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
187 aluminium foil clips made by a photo-etching process [11] were folded around each end of a bundle, so that the length of exposed bundle between the clips was about 3 mm. The bundle was then mounted horizontally in an experimental set-up, the design of which has been described previously [12,13]. One end of the myofibrillar bundle was attached to a stainless steel hook fixed to a tension transducer (silicone strain gauge model AE 801, AME, Norway) and the other end was attached to a fixed hook. The positions of both ends of the bundle could be adjusted by micro positioners. A set of six stainless steel troughs, cooled by circulating coolant to 12°C, contained a series of bathing solutions (25 #1). Solution changes were made by automatically lowering the trough beneath the preparation, moving a selected trough into position and then raising the trough. The front trough had a quartz glass window which allowed direct focusing of the laser pulse onto the preparation immersed in bathing solution. The fibre bundle was skinned by immersion in relaxing solution containing 1% Triton X-100 at 12°C for 15-60 rain. In early experiments bundles were first prepared with T clips while immersed in paraffin oil instead of relaxing solution. However, this procedure was found to prevent subsequent skinning by Triton. In some experiments the average sarcomere length of the mounted fibre bundle was measured from the diffraction pattern obtained from a He-Ne laser beam focused on the bundle. The bundle was initially stretched to give a mean sarcomere length of 2.8-3.2 #m.
Laser assembly Laser pulses of 25 ns nominal duration and 347 nm wavelength were produced from a frequency-doubled Ruby laser (Lumonics, Warwickshire, U.K.). The laser beam, 0.8 cm in diameter, was focused with a cylindrical lens (focal length 12 cm) to give a narrow horizontal beam, 0.07 cm in the vertical direction and typically 2-3 mm wide. The width was adjusted with a metal slit device so that the whole length of the muscle preparation was illuminated, but illumination of the T-clips was avoided. The alignment was checked by examining the burn pattern on a piece of exposed and
developed photographic paper placed behind the hooks. The energy of the laser pulse was varied from 20 to 100 mJ by varying the capacitor voltage which drives the laser flashlamps. A diode energy monitor (Lumonics) placed on line with the main beam, recorded the energy of each pulse. The firing of the laser, lowering of the trough assembly and data acquisition were controlled by a CED 1401 Interface (CED, Cambridge, U.K.) and an IBM PC AT computer. The output of the tension transducer was recorded on a pen recorder and as a filtered signal (time constant 1 ms) on a Nicolet 'Explorer' digital oscilloscope (206).
Solutions Table I shows the composition of the bathing solutions. Nitr-5 solutions. Nitr-5 (the tetrasodium salt) was bought from Calbiochem, La Jolla, CA, or was a gift from Dr. R.Y. Tsien. It was made up as a stock solution of 150 mM in 100 mM KTes (pH 7.0). It was added to solution LR from which the E G T A was omitted. Required pCa values were obtained by the addition of CaC12 (pH 7.0), and were confirmed using a calciumsensitive Microelectrode (MI-600, Microelectrodes, Londonderry, NH, U.S.A.) connected to an Ion83 Ion Meter (Radiometer, Copenhagen, Denmark). Caged A TP solution. This solution also contained 2.0 mM caged ATP (disodium salt from Calbiochem) and 10.0 mM reduced glutathione. The latter was included to protect the fibre bundles from the effects of a byproduct of the photolysis of caged ATP, 2-nitrosoacetophenone [15]. Free Mg 2÷* = 1.0 mM assuming photolytic release of 1.0 mM free ATP. In experiments with the caged ATP solution a rigour solution (pCa 8) was used to deplete the muscle preparation of ATP; it contained (mM): 5.0 MgC1 z, 5.0 EGTA, 20 Tes, 100 KC1, 1.0 glucose, 20 units/ml hexokinase (pH 7.0). Intermediate pCa activating solutions. These were made by mixing H R and HA in the appropriate ratio according to a computer program [14] for the binding of metal ions to ligands. This used the following apparent affinity constants (M -1) (pH 7.0) and 1 2 ° C :
TABLE I
Composition of muscle solutions (raM) Abbreviations: LR = low relaxing; H R = high relaxing; HA = high activating; prop = propionate; Mg(t) = total Mg; Mg2+(f) = free Mg; CP = creatine phosphate. In addition each solution contained: 60 mM Tes (pH 7.0) at 1 2 ° C . Creatine kinase (20 u n i t s / m l ) was added in some experiments to act with creatine phosphate as an ATP-regenerating system. The ionic strength of the solutions was 0.20-0.24 M. Solution:
K+
Na ÷
prop -
CI -
Mg(t)
Mg 2 + (f)
EG TA
ATP
CP
LR (pCa 6.8) H R (pCa 9) HA (pCa 4) Caged ATP (pCa 4.5)
150.0 131.0 131.0 137.0
30.0 30.0 30.0 0
130.0 50.0 50.0 60.0
11.0 12.0 69.6 63.5
5.5 6.0 4.8 1.8
1.0 1.0 1.0 1.0 *
0.1 30.0 30.0(Ca) 30.0(Ca)
5.0 5.0 5.0 0
10.0 10.0 10.0 0
188 K ( C a E G T A ) = 2.96- 106, K ( M g E G T A ) = 39, K(CaATP) = 2800, K ( M g A T P ) = 4200, K(Ca,MgCP) = 20, K(Ca,MgTES) = 1. Skinning solutions. H R + 0.5 - 1.0% Triton X - 100.
(b)
(a)
tl/2 34 m I
:
~ 0.10 s
A TP assay The amount of ATP released from photolysis of caged ATP was assessed using the luciferin-luciferase assay [15,16]. The muscle solution containing 2 m M caged ATP (pCa 4) was added to the fibre trough with the front window and exposed to either one or two laser pulses. The A T P concentration of this solution was then measured by adding 2.5 #1 of it to 400 #1 KTes (0.1 M, p H 7.0)+ 25 #1 40 m g / m l luciferin-luciferase reagent (Chrono-lume no. 395, Chrono-log, Havertown, PA, U.S.A.). Light output after mixing was measured conventionally with a photomultiplier tube, which was enclosed in a light-tight case and operated with the photocathode at - 6 0 0 V. The A T P concentration of the solution was estimated from a calibration curve of peak light versus ATP concentration. This value was then multiplied by 10 (the ratio between the total volume of solution and the volume which was illuminated by the laser pulse) to give the estimated ATP concentration which would have existed within the fibre bundle following photolysis of the caged ATP.
Results
Contractions induced by photolysis of caged calcium In order to obtain contractions of scallop skinned fibres by nitr-5 photolysis, a skinned fibre bundle was first pre-equilibrated in a solution containing 3 m M nitr-5, to which a total of 2.15 m M Ca 2+ had been added, so that the solution pCa was 6.1 before photolysis of the nitr-5. In some bundles a small amount of tension developed during this equilibration period; the pCa of the solution was adjusted so that this initial tension was less than 10% maximal tension. In the typical experiment shown in Fig. la, c, a 95 mJ laser pulse focused onto a 140 /xm diameter fibre bundle which was suspended in air produced a contraction of 90% maximal amplitude. It had an initial fast component which was approx, exponential with a halfrise time of 34 ms (Fig. la) and an amplitude of 45.5 k N / m 2. Following the fast rise in tension, a slight fall was observed and this was followed by a slow component in tension development with a half-time of several seconds. The bundle was then transferred to an activating solution (5.0 m M EGTA, pCa 4.5) to obtain maxim u m tension (50.0 k N / m 2) and it was then relaxed in the relaxing solution. In the foregoing experiments, the fibre bundle was kept suspended in air for up to 20 s. In separate experiments this time was reduced to 2 s by automatically lowering the solution trough 150 ms before the
(c)
HA (pCa4.5)
0.7
~
HR
~
mN 0
.
.
.
.
. i
95
mJ
.
lOs
Idl 1.0
0.5
0 1 55
md
10 s
60 s
Fig. 1. Contractions of two skinned fibre bundles from the scallop striated adductor muscle resulting from the laser-induced photolysis of nitr-5. In each experiment the bundle was first equilibrated in a solution containing 3.0 m M nitr-5 (initial pCa 6.1) at 1 2 ° C with no resulting development of tension. The solution trough was then lowered beneath the bundle and a laser pulse was fired at the bundle. The resulting contraction was recorded simultaneously on the Nicolet oscilloscope (a, b) and at a slower speed on the pen recorder (c, d). M a x i m u m tension was then obtained in a pCa 4.5 activating solution (HA) and then relaxed in a relaxing solution (HR). Fibre bundle diameter: 140 # m (a, c), 115 btm (b, d). In (a) the bundle remained suspended in air for about 15 s; in (b) the solution trough was lowered beneath the bundle 150 ms before the laser pulse (first arrow) and then raised 2 s later (second arrow in (d)).
laser pulse and then raising it again (Fig. lb, d). This protocol gave essentially the same results as the first method. In a series of similar experiments using laser pulses of 60-95 mJ energies, the average half-time for the rate of tension development in the fast phase was 49.7 ms (S.E. = 5.6 ms for n = 9 bundles). The average tension developed in the fast phase was 63.7% m a x i m u m tension. For a group of seven fibre bundles of estimated cross-section areas of 0.010-0.023 m m 2, maximum tension in the E G T A activating solution of pCa 4.5 was in the range of 48.8-120.8 k N / m 2 (mean = 77.3, S.E. = 9.9, n = 7). The purpose of suspending the fibre bundle in air at the time of the laser pulse was to avoid absorption of the pulse of light by nitr-5 in the bulk solution. It was,
189 however, possible to obtain contractions from bundles illuminated by the laser pulse while immersed in solution if the nitr-5 concentration was reduced to 1.0 mM, keeping the initial pCa approximately the same. The laser pulse was focused onto the preparation through the front quartz glass window of the muscle trough. The contraction which resulted from the laser pulse had a time course similar to those obtained from bundles in air (Fig. 2). A fast phase (mean half-time 75 ms, n = 3) was again followed by a slower component. The average tension which developed in the fast component was 70.7 k N / m 2 (S.E. = 12.4, n = 4). In addition, the experiment in Fig. 2 shows two properties which were found in some of the scallop skinned muscle preparations and which were first observed by Simmons and SzentGy~Srgyi [5]. After the maximal contraction, relaxing solutions were unable to reduce the tension to the starting value, and a subsequent transfer to a pCa 4.5 solution failed to elicit the same maximal tension as first recorded with nitr-5 photolysis. The slow component in tension development was observed in bundles activated either in air or in the nitr-5 solution by the laser pulse. It occurred over a range of laser pulse energies (12-95 rnJ) and bundle diameters, although in some preparations it was absent. The time for the slow component to reach half its peak value varied from 5.7 to 23.1 s for six bundles (mean 13.3 s). The temperature of the fibre bundle when it was suspended in air was a potential problem. Ferenczi [13], using the Ca2÷-dependent tension of rabbit skinned
muscle fibres as an indicator of fibre temperature in air, showed that when the fibre bundle is removed from solution and suspended in air, it rapidly attains the dew-point temperature. In our laboratory (air temperature = 18 ° C, relative humidity of 40-50%) this temperature would have been about 12°C. Therefore it is assumed in our experiments that the bundle temperature remained relatively constant when the bundle was lifted into air from a bathing solution which was being maintained at 12 ° C.
Varying beam energies The flash photolysis technique can produce, in frog skinned muscle fibres, fast intermediate levels of activation by variation of the b e a m energy of the laser pulse. This is because increasing the laser pulse energy linearly increases the percentage photolysis of nitr-5 (Ferenczi, M.A. and Barsotti, R.J., personal communication) and the resulting free Ca 2÷ concentration increases (non-linearly) with percentage photolysis of nitr-5 (see below). Fig. 3 shows that 12 mJ and 35 mJ pulses produced in a scallop fibre bundle, first equilibrated in 3.0 m M nitr-5 (pCa 6.1), contractions which were 18% and 43% maximum tension respectively with corresponding rise half-times for tension development of 98 and 66 ms, respectively. The relationship between the half-time for the rise in tension and beam energy for a series of fibre bundles is given in Fig. 4a, showing a fall in the half-time with increasing pulse energy. In Fig. 4b the half-times are plotted against relative tension (pCa 4.5 activating solutions are assumed to give a relative ten-
,Sms/
halftime
, ./ LR ~ 60 mJ
L
1.0
pC" 4.5
mN
l
laser pulse 60 mJ
30 s
1 rain
Fig. 2. A contraction of a scallop skinned fibre bundle (diameter 170 pm) resulting from a 60 mJ laser pulse focused onto the preparation while it was immersed in a bathing solution containing 1.0 mM nitr-5 at pCa 6.1 at 12° C. The oscilloscope record of the fast component of the contraction is shown in the inset. Following development of a slow component of tension transfer of the bundle to a low relaxing solution did not return the tension to the original relaxed level. A pCa 4.5 solution (20 mM EGTA) then gave a contraction which was smaller than the response to nitr-5 photolysis (note the slower recording speed).
190 (a)
relative tension of 0.5 was given by a pCa of approx. 5.8. In the experiments using photolysis of 3.0 mM nitr-5, a relative tension of 0.5 was given by laser pulses of 50-70 mJ energy, and the relative tension increased approximately linearly with pulse energy for energies up to 100 mJ (Fig. 5b). In separate experiments, myofibrillar bundles were prepared from split single fibres of an arthropod striated muscle, that of the giant barnacle Balanus nubulus (Lea, T.J. and Ashley, C.C., unpublished data). The amplitude of contraction of a bundle following nitr-5 photolysis was taken as an indicator of the post-photolysis pCa within the bundle. It was found that postphotolysis pCa decreased non-linearly with laser pulse energy. A computer program for calculating the binding of metal ions to ligands [14] estimated the pCa of a solution containing known concentrations of photolysed and unphotolysed nitr-5 ( K d = 12 /~M and 0.27 #M, respectively). This allowed the theoretical relation between post-photolysis pCa and percentage photolysis of nitr-5 to be derived. The observed relation between post-photolysis pCa and pulse energy was consistent with a laser pulse of 100 mJ (with the focusing employed in the present experiments) producing 50% photolysis of Ca nitr-5 (and Mg nitr-5). Since the quantum efficiency for the unbound nitr-5 has been found to be half that for Ca nitr-5 (Ferenczi, M.A. and Barsotti, R.J., unpublished data), a 100 mJ pulse would photolyse 25% of the free nitr-5. For a 3.0 mM nitr-5 solution at pCa 6.1 containing a total of 2.3 mM nitr-5 bound to the divalent cations, this means that a 100 mJ pulse would photolyse 44% of the total nitr-5. In Fig. 5a the data of Fig. 5b have been replotted by calculating from the computer program the expected post-photolysis pCa for each pulse energy, assuming the
35mJ
0.25mN[ 12mJ
lO00ms
A laser pulse
HR
1
! 0.5
HR
t
t
12 m J
35 mJ
los
Fig. 3. (a) Two superimposed contractions of a skinned fibre bundle (diameter ]40/~m) resulting from partial photolysis of nitr-5 (3.0 raM,
pCa 6.1) at two laser pulse energies (12 and 35 mJ). The bundle was suspended in air for the laser pulses. (b) The same contractions shown in (a), recorded on the pen recorder at a slower time scale. In each case the bundle was first equilibrated in the solution containing nitr-5 without any development of tension and then lifted into air prior to the laser pulse. Relaxation was by transfer of the bundle to the relaxing solution HR. M a x i m u m tension was elicited at the end of the experiment by a pCa 4.5 activating solution (HA).
sion of 1.0). It shows that the fastest contractions from nitr-5 photolysis are obtained as the amplitude of contraction approaches maximum tension. A conventional pCa-tension curve for the scallop skinned fibre preparation was obtained using activating solutions containing 30 mM total EGTA (Fig. 5a). A
scallop adductor: 3.0 mM nitr-5, pCa 6.1 300
300
(a)
(b)
o
E
.E_ 200
200
•
I
(9
J~ I
O
100
•c. 100
•
•
•
• tt •
0
o
0
20
i
i
°o i
40 60 80 pulse energy mJ
I
1O0
L
0
0.5
I
1.0
relative tension
Fig. 4. The half-time for the rise in tension following nitr-5 photolysis as a function of laser pulse energy in (a) and as a function of the relative tension generated ( m a x i m u m tension = 1.0) in (b). Data from 10 fibre bundles, which were first equilibrated in 3.0 m M nitr-5, pCa 6.1 at 1 2 ° C and then suspended in air for the laser pulse. Each point is a single measurement. The triangle is from the experiment in which the bundle was immersed in a solution containing 1.0 m M nitr-5 (pCa 6.1) and the laser pulse was focused on it via the front window (see Fig. 2).
191 1.0
(a) o
0.5 n=4 ._...~//e
~
o
/.~'q-'--=• n 6
i
6.0
7.0
5.0
4.0
pCa 3.0 mM nitr-5, pCa 6.1
(b)
1.0
e
e
rate constant for the release of ATP is 51 s -1 at 12°C and an ionic strength of 0.2 M. This value has been calculated by correcting the rate at 20 ° C (118 s-1) for both temperature, using an activation energy of 55 k J / m o l , and for ionic strength [19]. Fibre bundles of the scallop striated adductor were pre-equilibrated in a rigour solution which contained no ATP but included hexokinase and glucose to utilize any residual ATP in the skinned fibres. In this solution rigour tension began to develop after 2-3 min and reached a peak within 4 - 6 min (Fig. 6a). When the rigor tension had reached a constant value, the fibre bundle was transferred to a solution containing 2 m M caged ATP at pCa 4.5, with no significant change in the tension. After equilibration in this solution, a laser pulse was fired at the bundle while it remained immersed in this solution. The photolysis of caged ATP by a laser pulse of 50-65 mJ produced a rapid rise in
e
U~ r-
e •
0.5
:
(a)
e
0.5
caged ATP
¢
e
mN rigor solution
0
0
t
I
20
40
laser pulse
I
I
I
60
80
100
energy (rnJ)
Fig. 5. (a) Tension-pCa relationships for the scallop skinned fibre preparation obtained by two methods: (1) conventionally with activating solutions containing EGTA (O) (combined data from three bundies); (2) by photolysis of nitr-5 (
Biochimica et Biophysica Acta, 1034(1990) 186-194 Elsevier
BBAGEN 23299
Rapid activation by photolysis of nitr-5 in skinned fibres of the striated adductor muscle from the scallop Trevor J. Lea 1, Mark J. Fenton 1, James D. Potter 2 and Christopher C. Ashley 1 JUniversity Laboratory of Physiology, Oxford (U.K.) and 2Departmentof Pharmacology, MiamL FL (U.S.A.) (Received 9 October, 1989) (Revised manuscript received 18 January 1990) Key words: Skinned muscle fibre; Caged calcium; Laser photolysis; Muscle contraction; Calcium ion; (Scallop muscle)
Photolysis of nitr-5, a caged calcium molecule, has been used for rapid activation of skinned fibre bundles of a myosin-regulated muscle, the striated adductor of the scallop, Pecten maximus. Chemically skinned fibre bundles (diameter 7 0 - 2 0 0 /tin) were equilibrated in solutions containing 1 - 3 m M nitr-5 (pCa 6.1) and then activated by ultraviolet laser pulse (25 ns). Pulse energies of 6 0 - 9 5 mJ gave contractions of over 90% maximum tension and a mean half-time for tension rise of 43 ms ( n - - 4 ) at 12°C. Electrically stimulated bundles of intact fibres develop a tetanus with a rise half-time of 60.2 ms at 1 0 ° C ( n = 5 ) (Rail, J.A. (1981) J. Physiol. 321, 287-295, and personal communication). At lower pulse energies the skinned fibres gave smaller amplitude contractions with slower rates of rise (up to 260 ms half-time). In addition, a slower component of tension development (mean rise half-time 13.3 s) was often observed. In ATP-free solutions containing hexokinase and glucose, rigour tension developed with a delayed onset. Rapid release of A T P (0.47-0.59 mM) from photolysis of caged ATP (2 raM) at pCa 4.5 then caused a rapid contraction with a mean half-time for tension development of 17 ms (n = 4). The fast activation rates obtained by the photorelease of Ca 2+ from nitr-5 are similar to those obtained with skinned skeletal fibres of actin-regulated muscle. The results imply that the rate-limiting step in excitation-contraction coupling of the scallop muscle is not the increase in sarcoplasmic Ca 2+, but rather the activation of the muscle in response to this increase. The half-times of ATP-induced contractions at pCa 4.5 suggest that in a contraction activated by a rapid Ca 2+ jump the process comprising ATP hydrolysis and cross-bridge cycling occurs at a somewhat faster rate than the Ca2+-dependent activation process which precedes it. Introduction In vertebrate skeletal muscle, calcium regulation of contraction is primarily mediated via the thin filament [1] as a result of Ca 2÷ binding to troponin C [2]. By contrast, the regulation of some invertebrate muscles is via the thick filaments and one of the best examples of this is molluscan fast, striated muscle [3]. The Ca 2+ regulation appears to be via two light chains on the myosin molecule; in isolation these regulatory light chains do not bind Ca 2÷, but in association with myosin they do so: one Ca 2÷ per light chain [4]. If the light chains are removed from a skinned muscle fibre bundle of scallop muscle by using EDTA, the tension of the bundle becomes Ca-insensitive [5]. Ca 2÷ control is then restored by addition of the regulatory light chains to the solution bathing the skinned fibres. We have used laser-flash photolysis of the caged Ca 2÷ molecule nitr-5 [6,7] to produce a very fast rise in
Correspondence: C.C. Ashley, University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, U.K.
free C a 2+ (half-time 1 ms) within skinned fibre bundles of the scallop striated adductor muscle, so that rapid activation kinetics of a myosin-regulated muscle can be studied without the delays due to Ca 2÷ diffusion which have been present in previous studies. This technique has been previously used with permeabilised (demembranated) fibres of vertebrate skeletal muscle [8-10].
Materials and Methods Preparation and skinning of fibre bundles Adult specimens of the great scallop (Pecten maximus) were obtained from either Plymouth Marine Laboratory or Millport Marine Laboratory, U.K. They were kept in circulating seawater at 8°C. Skinned muscle fibre bundles were prepared from the striated adductor muscle using a method similar to that described by Simmons and Szent-GyiSrgyi [5]. Muscle strips about 3 mm wide and 2 cm long were removed from the muscle at room temperature and immersed in relaxing solution. Thinner bundles of 7 0 - 2 0 0 / z m diameter and 5-10 mm were then prepared. T-shaped
0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
187 aluminium foil clips made by a photo-etching process [11] were folded around each end of a bundle, so that the length of exposed bundle between the clips was about 3 mm. The bundle was then mounted horizontally in an experimental set-up, the design of which has been described previously [12,13]. One end of the myofibrillar bundle was attached to a stainless steel hook fixed to a tension transducer (silicone strain gauge model AE 801, AME, Norway) and the other end was attached to a fixed hook. The positions of both ends of the bundle could be adjusted by micro positioners. A set of six stainless steel troughs, cooled by circulating coolant to 12°C, contained a series of bathing solutions (25 #1). Solution changes were made by automatically lowering the trough beneath the preparation, moving a selected trough into position and then raising the trough. The front trough had a quartz glass window which allowed direct focusing of the laser pulse onto the preparation immersed in bathing solution. The fibre bundle was skinned by immersion in relaxing solution containing 1% Triton X-100 at 12°C for 15-60 rain. In early experiments bundles were first prepared with T clips while immersed in paraffin oil instead of relaxing solution. However, this procedure was found to prevent subsequent skinning by Triton. In some experiments the average sarcomere length of the mounted fibre bundle was measured from the diffraction pattern obtained from a He-Ne laser beam focused on the bundle. The bundle was initially stretched to give a mean sarcomere length of 2.8-3.2 #m.
Laser assembly Laser pulses of 25 ns nominal duration and 347 nm wavelength were produced from a frequency-doubled Ruby laser (Lumonics, Warwickshire, U.K.). The laser beam, 0.8 cm in diameter, was focused with a cylindrical lens (focal length 12 cm) to give a narrow horizontal beam, 0.07 cm in the vertical direction and typically 2-3 mm wide. The width was adjusted with a metal slit device so that the whole length of the muscle preparation was illuminated, but illumination of the T-clips was avoided. The alignment was checked by examining the burn pattern on a piece of exposed and
developed photographic paper placed behind the hooks. The energy of the laser pulse was varied from 20 to 100 mJ by varying the capacitor voltage which drives the laser flashlamps. A diode energy monitor (Lumonics) placed on line with the main beam, recorded the energy of each pulse. The firing of the laser, lowering of the trough assembly and data acquisition were controlled by a CED 1401 Interface (CED, Cambridge, U.K.) and an IBM PC AT computer. The output of the tension transducer was recorded on a pen recorder and as a filtered signal (time constant 1 ms) on a Nicolet 'Explorer' digital oscilloscope (206).
Solutions Table I shows the composition of the bathing solutions. Nitr-5 solutions. Nitr-5 (the tetrasodium salt) was bought from Calbiochem, La Jolla, CA, or was a gift from Dr. R.Y. Tsien. It was made up as a stock solution of 150 mM in 100 mM KTes (pH 7.0). It was added to solution LR from which the E G T A was omitted. Required pCa values were obtained by the addition of CaC12 (pH 7.0), and were confirmed using a calciumsensitive Microelectrode (MI-600, Microelectrodes, Londonderry, NH, U.S.A.) connected to an Ion83 Ion Meter (Radiometer, Copenhagen, Denmark). Caged A TP solution. This solution also contained 2.0 mM caged ATP (disodium salt from Calbiochem) and 10.0 mM reduced glutathione. The latter was included to protect the fibre bundles from the effects of a byproduct of the photolysis of caged ATP, 2-nitrosoacetophenone [15]. Free Mg 2÷* = 1.0 mM assuming photolytic release of 1.0 mM free ATP. In experiments with the caged ATP solution a rigour solution (pCa 8) was used to deplete the muscle preparation of ATP; it contained (mM): 5.0 MgC1 z, 5.0 EGTA, 20 Tes, 100 KC1, 1.0 glucose, 20 units/ml hexokinase (pH 7.0). Intermediate pCa activating solutions. These were made by mixing H R and HA in the appropriate ratio according to a computer program [14] for the binding of metal ions to ligands. This used the following apparent affinity constants (M -1) (pH 7.0) and 1 2 ° C :
TABLE I
Composition of muscle solutions (raM) Abbreviations: LR = low relaxing; H R = high relaxing; HA = high activating; prop = propionate; Mg(t) = total Mg; Mg2+(f) = free Mg; CP = creatine phosphate. In addition each solution contained: 60 mM Tes (pH 7.0) at 1 2 ° C . Creatine kinase (20 u n i t s / m l ) was added in some experiments to act with creatine phosphate as an ATP-regenerating system. The ionic strength of the solutions was 0.20-0.24 M. Solution:
K+
Na ÷
prop -
CI -
Mg(t)
Mg 2 + (f)
EG TA
ATP
CP
LR (pCa 6.8) H R (pCa 9) HA (pCa 4) Caged ATP (pCa 4.5)
150.0 131.0 131.0 137.0
30.0 30.0 30.0 0
130.0 50.0 50.0 60.0
11.0 12.0 69.6 63.5
5.5 6.0 4.8 1.8
1.0 1.0 1.0 1.0 *
0.1 30.0 30.0(Ca) 30.0(Ca)
5.0 5.0 5.0 0
10.0 10.0 10.0 0
188 K ( C a E G T A ) = 2.96- 106, K ( M g E G T A ) = 39, K(CaATP) = 2800, K ( M g A T P ) = 4200, K(Ca,MgCP) = 20, K(Ca,MgTES) = 1. Skinning solutions. H R + 0.5 - 1.0% Triton X - 100.
(b)
(a)
tl/2 34 m I
:
~ 0.10 s
A TP assay The amount of ATP released from photolysis of caged ATP was assessed using the luciferin-luciferase assay [15,16]. The muscle solution containing 2 m M caged ATP (pCa 4) was added to the fibre trough with the front window and exposed to either one or two laser pulses. The A T P concentration of this solution was then measured by adding 2.5 #1 of it to 400 #1 KTes (0.1 M, p H 7.0)+ 25 #1 40 m g / m l luciferin-luciferase reagent (Chrono-lume no. 395, Chrono-log, Havertown, PA, U.S.A.). Light output after mixing was measured conventionally with a photomultiplier tube, which was enclosed in a light-tight case and operated with the photocathode at - 6 0 0 V. The A T P concentration of the solution was estimated from a calibration curve of peak light versus ATP concentration. This value was then multiplied by 10 (the ratio between the total volume of solution and the volume which was illuminated by the laser pulse) to give the estimated ATP concentration which would have existed within the fibre bundle following photolysis of the caged ATP.
Results
Contractions induced by photolysis of caged calcium In order to obtain contractions of scallop skinned fibres by nitr-5 photolysis, a skinned fibre bundle was first pre-equilibrated in a solution containing 3 m M nitr-5, to which a total of 2.15 m M Ca 2+ had been added, so that the solution pCa was 6.1 before photolysis of the nitr-5. In some bundles a small amount of tension developed during this equilibration period; the pCa of the solution was adjusted so that this initial tension was less than 10% maximal tension. In the typical experiment shown in Fig. la, c, a 95 mJ laser pulse focused onto a 140 /xm diameter fibre bundle which was suspended in air produced a contraction of 90% maximal amplitude. It had an initial fast component which was approx, exponential with a halfrise time of 34 ms (Fig. la) and an amplitude of 45.5 k N / m 2. Following the fast rise in tension, a slight fall was observed and this was followed by a slow component in tension development with a half-time of several seconds. The bundle was then transferred to an activating solution (5.0 m M EGTA, pCa 4.5) to obtain maxim u m tension (50.0 k N / m 2) and it was then relaxed in the relaxing solution. In the foregoing experiments, the fibre bundle was kept suspended in air for up to 20 s. In separate experiments this time was reduced to 2 s by automatically lowering the solution trough 150 ms before the
(c)
HA (pCa4.5)
0.7
~
HR
~
mN 0
.
.
.
.
. i
95
mJ
.
lOs
Idl 1.0
0.5
0 1 55
md
10 s
60 s
Fig. 1. Contractions of two skinned fibre bundles from the scallop striated adductor muscle resulting from the laser-induced photolysis of nitr-5. In each experiment the bundle was first equilibrated in a solution containing 3.0 m M nitr-5 (initial pCa 6.1) at 1 2 ° C with no resulting development of tension. The solution trough was then lowered beneath the bundle and a laser pulse was fired at the bundle. The resulting contraction was recorded simultaneously on the Nicolet oscilloscope (a, b) and at a slower speed on the pen recorder (c, d). M a x i m u m tension was then obtained in a pCa 4.5 activating solution (HA) and then relaxed in a relaxing solution (HR). Fibre bundle diameter: 140 # m (a, c), 115 btm (b, d). In (a) the bundle remained suspended in air for about 15 s; in (b) the solution trough was lowered beneath the bundle 150 ms before the laser pulse (first arrow) and then raised 2 s later (second arrow in (d)).
laser pulse and then raising it again (Fig. lb, d). This protocol gave essentially the same results as the first method. In a series of similar experiments using laser pulses of 60-95 mJ energies, the average half-time for the rate of tension development in the fast phase was 49.7 ms (S.E. = 5.6 ms for n = 9 bundles). The average tension developed in the fast phase was 63.7% m a x i m u m tension. For a group of seven fibre bundles of estimated cross-section areas of 0.010-0.023 m m 2, maximum tension in the E G T A activating solution of pCa 4.5 was in the range of 48.8-120.8 k N / m 2 (mean = 77.3, S.E. = 9.9, n = 7). The purpose of suspending the fibre bundle in air at the time of the laser pulse was to avoid absorption of the pulse of light by nitr-5 in the bulk solution. It was,
189 however, possible to obtain contractions from bundles illuminated by the laser pulse while immersed in solution if the nitr-5 concentration was reduced to 1.0 mM, keeping the initial pCa approximately the same. The laser pulse was focused onto the preparation through the front quartz glass window of the muscle trough. The contraction which resulted from the laser pulse had a time course similar to those obtained from bundles in air (Fig. 2). A fast phase (mean half-time 75 ms, n = 3) was again followed by a slower component. The average tension which developed in the fast component was 70.7 k N / m 2 (S.E. = 12.4, n = 4). In addition, the experiment in Fig. 2 shows two properties which were found in some of the scallop skinned muscle preparations and which were first observed by Simmons and SzentGy~Srgyi [5]. After the maximal contraction, relaxing solutions were unable to reduce the tension to the starting value, and a subsequent transfer to a pCa 4.5 solution failed to elicit the same maximal tension as first recorded with nitr-5 photolysis. The slow component in tension development was observed in bundles activated either in air or in the nitr-5 solution by the laser pulse. It occurred over a range of laser pulse energies (12-95 rnJ) and bundle diameters, although in some preparations it was absent. The time for the slow component to reach half its peak value varied from 5.7 to 23.1 s for six bundles (mean 13.3 s). The temperature of the fibre bundle when it was suspended in air was a potential problem. Ferenczi [13], using the Ca2÷-dependent tension of rabbit skinned
muscle fibres as an indicator of fibre temperature in air, showed that when the fibre bundle is removed from solution and suspended in air, it rapidly attains the dew-point temperature. In our laboratory (air temperature = 18 ° C, relative humidity of 40-50%) this temperature would have been about 12°C. Therefore it is assumed in our experiments that the bundle temperature remained relatively constant when the bundle was lifted into air from a bathing solution which was being maintained at 12 ° C.
Varying beam energies The flash photolysis technique can produce, in frog skinned muscle fibres, fast intermediate levels of activation by variation of the b e a m energy of the laser pulse. This is because increasing the laser pulse energy linearly increases the percentage photolysis of nitr-5 (Ferenczi, M.A. and Barsotti, R.J., personal communication) and the resulting free Ca 2÷ concentration increases (non-linearly) with percentage photolysis of nitr-5 (see below). Fig. 3 shows that 12 mJ and 35 mJ pulses produced in a scallop fibre bundle, first equilibrated in 3.0 m M nitr-5 (pCa 6.1), contractions which were 18% and 43% maximum tension respectively with corresponding rise half-times for tension development of 98 and 66 ms, respectively. The relationship between the half-time for the rise in tension and beam energy for a series of fibre bundles is given in Fig. 4a, showing a fall in the half-time with increasing pulse energy. In Fig. 4b the half-times are plotted against relative tension (pCa 4.5 activating solutions are assumed to give a relative ten-
,Sms/
halftime
, ./ LR ~ 60 mJ
L
1.0
pC" 4.5
mN
l
laser pulse 60 mJ
30 s
1 rain
Fig. 2. A contraction of a scallop skinned fibre bundle (diameter 170 pm) resulting from a 60 mJ laser pulse focused onto the preparation while it was immersed in a bathing solution containing 1.0 mM nitr-5 at pCa 6.1 at 12° C. The oscilloscope record of the fast component of the contraction is shown in the inset. Following development of a slow component of tension transfer of the bundle to a low relaxing solution did not return the tension to the original relaxed level. A pCa 4.5 solution (20 mM EGTA) then gave a contraction which was smaller than the response to nitr-5 photolysis (note the slower recording speed).
190 (a)
relative tension of 0.5 was given by a pCa of approx. 5.8. In the experiments using photolysis of 3.0 mM nitr-5, a relative tension of 0.5 was given by laser pulses of 50-70 mJ energy, and the relative tension increased approximately linearly with pulse energy for energies up to 100 mJ (Fig. 5b). In separate experiments, myofibrillar bundles were prepared from split single fibres of an arthropod striated muscle, that of the giant barnacle Balanus nubulus (Lea, T.J. and Ashley, C.C., unpublished data). The amplitude of contraction of a bundle following nitr-5 photolysis was taken as an indicator of the post-photolysis pCa within the bundle. It was found that postphotolysis pCa decreased non-linearly with laser pulse energy. A computer program for calculating the binding of metal ions to ligands [14] estimated the pCa of a solution containing known concentrations of photolysed and unphotolysed nitr-5 ( K d = 12 /~M and 0.27 #M, respectively). This allowed the theoretical relation between post-photolysis pCa and percentage photolysis of nitr-5 to be derived. The observed relation between post-photolysis pCa and pulse energy was consistent with a laser pulse of 100 mJ (with the focusing employed in the present experiments) producing 50% photolysis of Ca nitr-5 (and Mg nitr-5). Since the quantum efficiency for the unbound nitr-5 has been found to be half that for Ca nitr-5 (Ferenczi, M.A. and Barsotti, R.J., unpublished data), a 100 mJ pulse would photolyse 25% of the free nitr-5. For a 3.0 mM nitr-5 solution at pCa 6.1 containing a total of 2.3 mM nitr-5 bound to the divalent cations, this means that a 100 mJ pulse would photolyse 44% of the total nitr-5. In Fig. 5a the data of Fig. 5b have been replotted by calculating from the computer program the expected post-photolysis pCa for each pulse energy, assuming the
35mJ
0.25mN[ 12mJ
lO00ms
A laser pulse
HR
1
! 0.5
HR
t
t
12 m J
35 mJ
los
Fig. 3. (a) Two superimposed contractions of a skinned fibre bundle (diameter ]40/~m) resulting from partial photolysis of nitr-5 (3.0 raM,
pCa 6.1) at two laser pulse energies (12 and 35 mJ). The bundle was suspended in air for the laser pulses. (b) The same contractions shown in (a), recorded on the pen recorder at a slower time scale. In each case the bundle was first equilibrated in the solution containing nitr-5 without any development of tension and then lifted into air prior to the laser pulse. Relaxation was by transfer of the bundle to the relaxing solution HR. M a x i m u m tension was elicited at the end of the experiment by a pCa 4.5 activating solution (HA).
sion of 1.0). It shows that the fastest contractions from nitr-5 photolysis are obtained as the amplitude of contraction approaches maximum tension. A conventional pCa-tension curve for the scallop skinned fibre preparation was obtained using activating solutions containing 30 mM total EGTA (Fig. 5a). A
scallop adductor: 3.0 mM nitr-5, pCa 6.1 300
300
(a)
(b)
o
E
.E_ 200
200
•
I
(9
J~ I
O
100
•c. 100
•
•
•
• tt •
0
o
0
20
i
i
°o i
40 60 80 pulse energy mJ
I
1O0
L
0
0.5
I
1.0
relative tension
Fig. 4. The half-time for the rise in tension following nitr-5 photolysis as a function of laser pulse energy in (a) and as a function of the relative tension generated ( m a x i m u m tension = 1.0) in (b). Data from 10 fibre bundles, which were first equilibrated in 3.0 m M nitr-5, pCa 6.1 at 1 2 ° C and then suspended in air for the laser pulse. Each point is a single measurement. The triangle is from the experiment in which the bundle was immersed in a solution containing 1.0 m M nitr-5 (pCa 6.1) and the laser pulse was focused on it via the front window (see Fig. 2).
191 1.0
(a) o
0.5 n=4 ._...~//e
~
o
/.~'q-'--=• n 6
i
6.0
7.0
5.0
4.0
pCa 3.0 mM nitr-5, pCa 6.1
(b)
1.0
e
e
rate constant for the release of ATP is 51 s -1 at 12°C and an ionic strength of 0.2 M. This value has been calculated by correcting the rate at 20 ° C (118 s-1) for both temperature, using an activation energy of 55 k J / m o l , and for ionic strength [19]. Fibre bundles of the scallop striated adductor were pre-equilibrated in a rigour solution which contained no ATP but included hexokinase and glucose to utilize any residual ATP in the skinned fibres. In this solution rigour tension began to develop after 2-3 min and reached a peak within 4 - 6 min (Fig. 6a). When the rigor tension had reached a constant value, the fibre bundle was transferred to a solution containing 2 m M caged ATP at pCa 4.5, with no significant change in the tension. After equilibration in this solution, a laser pulse was fired at the bundle while it remained immersed in this solution. The photolysis of caged ATP by a laser pulse of 50-65 mJ produced a rapid rise in
e
U~ r-
e •
0.5
:
(a)
e
0.5
caged ATP
¢
e
mN rigor solution
0
0
t
I
20
40
laser pulse
I
I
I
60
80
100
energy (rnJ)
Fig. 5. (a) Tension-pCa relationships for the scallop skinned fibre preparation obtained by two methods: (1) conventionally with activating solutions containing EGTA (O) (combined data from three bundies); (2) by photolysis of nitr-5 (
