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1. Ca2(+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum was measured by isometric tension recording from barnacle myofibrillar bundles. Laser-induced photolysis of the caged calcium molecule, nitr-5, was used to generate a rapid jump in free Ca2+ (within 1 ms) at the site of the sarcoplasmic reticulum, thus overcoming delays due to Ca2+ diffusion from the bathing solution. 2. The method consisted of equilibrating a myofibrillar bundle (100 micrograms diameter) in a solution containing 0.1 mM-nitr-5 (initial pCa 6.8-6.6) and then exposing it to a UV laser pulse. The resulting phasic contraction had an amplitude of up to 100% maximum tension (P0) in some preparations and a mean half-time for the rise of tension of 2.3 s at 12 degrees C. Longer half-times were obtained at low pulse energies. 3. Pre-treatment of the myofibrillar bundles with ryanodine (10(-4) M) or the detergent Triton X-100 abolished a large part of the phasic contraction, confirming its dependence on SR Ca2+ release. The small tonic response which remained had a shorter rise half-time than the Ca2(+)-induced Ca2+ release response and was attributed to direct activation of the myofibrils by Ca2+ released from the nitr-5. 4. The size of the photolytic Ca2+ jump was estimated from the amplitude of the fast tension component. By increasing the laser pulse energy or the initial Ca2+ loading of the nitr-5, the post-photolysis pCa was varied from 6.7 to 6.0; the CICR response increased in size over this pCa range. 5. Direct activation of Triton-treated myofibrils by photolysis of 2.0 mM-nitr-5 (initial pCa 6.4) gave contractions of up to 100% P0 and a mean rise half-time of 164 ms at 12 degrees C (n = 9 for contractions greater than 40% P0). Both the amplitude and the rate of these contractions were dependent on the laser pulse energy. 6. The Ca2(+)-induced Ca2+ release responses obtained with nitr-5 photolysis were significantly slower than the fastest rate of tetanus development which has been recorded from intact fibres of barnacle muscle (mean half-time = 177 ms at 12 degrees C). This could mean that either Ca2(+)-induced Ca2+ release is less efficient in isolated myofibrillar bundles than in intact fibres or that Ca2(+)-induced Ca2+ release is not the primary Ca2+ releasing mechanism in excitation-contraction coupling in barnacle muscle.

1. An intracellular glass scintillator (Caldwell & Lea, 1973) has been used to obtain a continuous record of the influx of 45Ca into single muscle fibres of the barnacle, Balanus nubilus. 2. In the presence of intracellular EGTA (final concentration greater than 3 mM/kg), the scintillator detected an initial fast phase to the influx (half-time = 18.3 min, compartment size = 4.1% fibre volume) followed by a slow, linear phase which gave a value for the Ca influx of 1.2 p-mole . cm-2 . sec-1. The efflux of 45Ca was also measured with the scintillator by transferring a 45Ca-loaded fibre into 45Ca-free saline. Two exponential phases of efflux were detected with half-times of 16.2 and 500 min. 3. The characterisitics of the fast phase of the influx and efflux are similar to those of the influx of the impermeant sucrose and inulin, suggesting that the fast phase represents exchange with the extracellular 'cleft space'. This phase was insensitive to external La3+ (2 mM). 4. The slow phase is considered to represent the flux of Ca across the surface membrane. It was inhibited by external La3+ (2 mM) and stimulated by replacing external Na+ with Li+. 5. When EGTA-injected fibres were depolarized using an axial, intracellular electrode the Ca influx, measured from the slow phase, was increased. At higher concentrations of intracellular EGTA (6--22 mM/kg), the extra Ca influx due to a rectangular, depolarizing current pulse was proportional to the number of Ca spikes it produced. A single Ca spike gave an extra Ca influx of 19--48 p-mole . cm-2. External D600 (5 x 10(-4)M) inhibited both Ca spike and the extra Ca influx. 6. At lower intracellular EGTA concentrations (3.6--11 mM/kg), a 50 mV depolarization of 250 msec duration gave a mean extra Ca influx of 80 p-mole . cm-2. The upper value was 145 p-mole . cm-2 and this would increase the total internal Ca by 4.1 micrometer/kg. It is calculated that if all this extra Ca was bound to the myofibrillar sites for tension, it would only produce 6.2% of the force expected for a similar depolarization in a fibre with no intracellular EGTA.

1. Internal microinjection of the Ca-sensitive photoprotein aequorin or the isotope (45)Ca have been used to assess Ca movements in single muscle fibres from the barnacle Balanus nubilus and the crab Maia squinado.2. Progressive isosomotic replacement of external Na by Li, choline, sucrose or Tris was associated with a rapid increase in the level of light emission from internally injected aequorin. This response was dependent upon the presence of external Ca. The light output was maximal for Na concentrations < 50 mM in the replaced salines, while Na concentrations > 350 mM produced no apparent increase in the resting light emission.3. If the Ca concentration in the external saline was altered, no effect was observed in Na replaced salines when the Ca concentration was < 1 mM, but maximal effects were observed at concentrations of Ca of ca. 100 mM.4. The increased light emission from aequorin in Na-replaced salines was substantially inhibited by 1 mM-La(3+) applied externally, but not by N-ethyl maleimide (NEM), propanolol or D-600.5. Following microinjection of (45)Ca and 2 hr equilibration, the residual efflux of Ca was shown to be sensitive to the removal of external Ca and Na, the extent of each component being variable.6. The Na-sensitive Ca efflux was partially inhibited by external La(3+) (1 mM), but not by high concentrations of Mg(2+), Ca(2+), Mn(2+) or Co(2+). It was stimulated by NEM and ethacrynic acid, which was considered to be due to an indirect effect upon the sarcoplasmic reticulum.7. It is concluded that Ca movements in these single crustacean muscle fibres involve Na gradients and a Ca: Ca exchange and from the relative insensitivity of Ca movements to various inhibitors, in contrast to the Mg system, it seems that there may be two independent modes for divalent cation transport in this preparation.

1. The relative isometric tension-pCa relationship has been determined for isolated bundles of barnacle myofibrils under a variety of ionic conditions using [Ca(2+)]-buffered solutions which also contained an ATP regenerating system (creatine phosphate and creatine kinase).2. The results are in better agreement with the ;consecutive' scheme of reaction rather than with the ;independent' alternative (Ashley & Moisescu, 1972) for the co-operative action of two Ca(2+) ions in the process of tension activation in crustacean skeletal muscle.3. Variations in the pH of the activating solutions did have a marked effect on the relative tension-Ca curve, although no effect was observed on the absolute maximum value for isometric tension. A shift in pH by 0.5 u. in the range 6.6-7.6 shifted the Ca(2+)-activation curve by 0.5 log u. towards lower free Ca(2+) concentrations.4. Changes in the free Mg(2+) concentration of the activating solutions in the millimolar range produced a pronounced shift of the relative tension-pCa curve along the pCa axis. Increasing [Mg(2+)] from 1 to 5 mM shifted the curve by about 0.7 log u. to higher free Ca(2+) concentrations, without significantly modifying its steepness.5. Changes in the MgATP concentration of the activating solutions in the range of 1-13 mM had no significant effect on the relative tension-pCa relationship.6. Varying the K(+) concentration in the activating solutions was also observed to have a marked effect upon the tension-pCa relationship in barnacle. An increase in the K(+) concentration from 90 to 170 mM shifted the curve by some 0.6 log u. towards higher free Ca(2+) concentrations.7. Cooling the standard activating solutions from room temperature to +4 degrees C made no apparent difference to the relative tension-pCa relationship, but decreased significantly the absolute tension responses.8. The results presented show that tonicity by itself has a marked effect upon the absolute steady-state tension levels in isolated bundles of myofibrils.9. Maximum isometric tension in this preparation was not simply related to ionic strength, or to the monovalent cation concentration, but it depended, as well, upon the anionic composition of the activating solution. In addition, a change in ionic strength of 25 mM over the range of 245-270 mM did not appear to modify the relative tension-pCa relationship.10. The effect of the physiologically occurring cations H(+), K(+), Mg(2+) upon the relative isometric tension-pCa relationship can be accounted for on the basis of a model of competitive inhibition between these cations and Ca(2+) for the functional unit for tension. This inhibitory effect appears to involve at least one H(+), one Mg(2+) and two K(+) per each Ca(2+) ion participating in the activation process of the functional unit for tension.

1. In single striated muscle fibres from the barnacle Balanus nubilus tension development following axial injection of caffeine (50 mM in 150 mM-KCl, pH 7.1) was used as an index of releasable Ca. It was shown that fibres incubated in 0 Ca (Na replaced) salines for up to 400 min gave ca. 15% of the control tension response. Inclusion of 1 mM-La in the 0 Ca (Na) saline significantly reversed this decline.2. Estimates of the total fibre calcium assayed under the same experimental conditions indicated a 67% loss of fibre Ca in 0 Ca, and only a 37% loss in La-0 Ca media. No correction was made for the loss of calcium from the extracellular space.3. Experiments with (45)Ca indicated that the efflux of calcium from this preparation was inhibited by 1 mM-La externally and that this effect was still significant even in the presence of 0 Ca (Na) salines. The caffeine-stimulated efflux of Ca was also reduced by ca. 70% in the presence of 1 mM La saline externally.4. The influx and efflux of (14)C caffeine were shown to be rapid, and apparently passive. The diffusion coefficient for caffeine following intracellular injection was 2.4 +/- 0.2 x 10(-6) cm(2) sec(-1) at 18-22 degrees C.5. There was no significant loss of (140)La over a period of 2 hr following axial micro-injection into the fibres.6. In (140)La uptake experiments there was a progressive increase in the La space over 10.5 hr, in contrast to the results with [(3)H]inulin, whose uptake saturated in ca. 1.5 hr. The probability of surface binding and the precipitation of La salts in the extensive extracellular space was suggested as an explanation.7. It is concluded that internal Ca within the sarcoplasmic reticulum, and not cleft or extracellular Ca is the most significant source for these caffeine-induced contractions. Fluxes across the surface membrane can however alter the internal Ca stores over longer periods of time.

1. In the large single muscle fibres from the barnacle Balanus nubilus, the total fibre Mg concentration was estimated as 15.1 m-mole/kg wet wt., of which about 3-3.5 m-mole/kg wet wt. was extracellular. The diffusible Mg, measured by internal sampling, was 11.5 m-mole/kg wet wt., of which at least half may be complexed to larger diffusible molecules. The free ionized Mg level was estimated as < 5 m-mole/kg wet wt.2. The loss of [(28)Mg]MgCl(2) from both Maia and Balanus muscle fibres following axial micro-injection approximated to first-order kinetics. The maximum rate constant for the loss was 1.51 +/- 0.20 (S.E.) x 10(-5) sec(-1) for Balanus (sixty-seven fibres) and 1.06 +/- 0.46 (S.E.) x 10(-5) sec(-1) for Maia (seven fibres) at 20-25 degrees C.3. The calculated Mg efflux was in the range 6-12 p-mole/cm(2).sec based on this rate constant, assuming isotopic equilibration internally and that the surface area of the fibres approximated to that of a simple cylinder. If account was taken of the area of the cleft system the efflux was reduced by about fifteen times.4. The diffusion coefficient for injected (28)Mg was estimated as 2-3 x 10(-6) cm(2) sec(-1), about half the value in free solution. Injections of 2 M-MgCl(2) or 200 mM-EDTA subsequent to the injection of the isotope caused about a 30% reduction in the tracer efflux.5. External application of salines containing 100 mM-Ca or Mg caused a rapid but reversible inhibition of the magnesium efflux. Similar effects were observed with salines containing 32 mM-Co or Mn chlorides or 1-2 mM-La or Gd chlorides. Polyarginine (200 mug/ml.) had no effect.6. The Mg efflux had a Q(10) of 3-4 over the temperature range of about 5-20 degrees C. It was irreversibly inhibited by the sulphydryl reagent NEM (1 mM), but PCMBS (0.2-2 mM) had no effect. Contractile agents (5 mM caffeine or 200 mM-K salines) and a variety of inhibitors of ion movement or active transport had no appreciable effect on the Mg efflux. Lowering the pH of the saline from 7 to 5 produced a 70% reduction in the efflux which was reversible over short periods of application.7. Replacement of external Na, but not Ca or Mg, with Li, choline or sucrose caused a rapid and partially reversible reduction of the Mg efflux, but increasing the internal Na by micro-injection in zero Na salines had no consistent effect. It is suggested that the extrusion of Mg from these muscle cells is largely dependent upon the inward movement of Na.

1. The efflux of calcium, as the isotope (45)Ca, has been investigated from single muscle fibres from the barnacle Balanus nubilus and from the crab Maia squinado.2. If the isotope was initially injected with sufficient calcium (5-65 mM) to cause a contraction, the efflux did not follow first order kinetics. There was an early rapid phase which reached a peak after 5-10 min and then declined slowly over a period of 50-150 min to a low residual value.3. Injection of the isotope with the calcium-binding agent EGTA, so that the injected free calcium concentration was ca. 2 x 10(-8)M, abolished the initial rapid loss of calcium. The efflux rose to give a steady value after 10-15 min and its magnitude was similar to the value of the residual efflux.4. The rate constant for the low residual loss was ca. 7 x 10(-4) min(-1) for Maia and ca. 17 x 10(-4) min(-1) for Balanus. The rate constant predicted a calcium efflux of 0.4 p-mole/cm(2).sec for Maia and 1-2 p-mole/cm(2).sec for Balanus at 16-25 degrees C based on the total fibre calcium concentration.5. The residual calcium efflux was not affected by 0.5 mM ouabain or 0 potassium salines applied externally. It was stimulated, some 10-15 times in Maia and to a lesser extent in Balanus, by salines containing 600 mM potassium or 2-5 mM caffeine. The increased efflux was associated with a brisk contraction.6. External application of salines containing 20, 40 or 60 mM potassium or 0.5 mM caffeine in Maia produced some stimulation of the residual efflux but no visible contraction.7. Pre-treatment of Maia fibres with 40 mM potassium or 0.5 mM caffeine salines abolished the ability of the fibres to respond to higher concentrations of these agents. A depletion of a releasable calcium fraction by these subthreshold stimuli could explain this phenomenon.8. Electrical stimulation, the injection of 50 mM calcium chloride or 50 mM caffeine produced an elevated calcium efflux which was associated with a contraction.9. Intracellular injections of EGTA only lowered the residual efflux by up to half its initial value. This suggests that calcium can be released rapidly within these muscle fibres and that the sarcoplasmic calcium concentration is not much altered from its normal value by the injection.10. The experiments suggest that in Maia, changes in the calcium efflux reflect in magnitude, but not in time course, the internal calcium changes which can be observed with the calcium-sensitive protein, aequorin.

1. The calcium-sensitive photoprotein aequorin has been used to follow the rapid changes in intracellular calcium concentration that occur during the contraction of single muscle fibres from the barnacle Balanus nubilus, Darwin.2. The transient change in calcium-mediated light emission (calcium transient) and the changes in membrane potential and tension were recorded simultaneously, thus permitting an examination of the relationships between the chemical, electrical, and mechanical events of excitation-contraction coupling.3. With short-duration stimuli (< 200 msec), the calcium transient shows an S-shaped rising phase reaching a maximum soon after the cessation of the stimulus pulse. During membrane repolarization the calcium transient begins an exponential falling phase which has a time constant of 50-80 msec at 11-12 degrees C.4. The shape of the calcium transient resembles the first derivative of the rising phase of the isometric tension response, thus suggesting that calcium controls the rate of tension development.5. There is no detectable increase of the light emission above resting values, during the falling phase of isometric tension.6. A plot of the calcium transient area (lumen x sec) versus peak isometric force (g. cm(-2)) is linear over, at least, a range of forces from ca. 50-400 g. cm(-2).7. When the fibre is capable of producing an active membrane response following the intracellular injection of potassium citrate, the onset and cessation of the calcium transient follow closely the onset and cessation of the active membrane response. Tension responses under these conditions are much suppressed, suggesting that excitation-contraction coupling may be partially blocked between calcium release and the development of tension.8. Hypertonic salines (1 M sucrose or 1 M glycerol) cause little change in the membrane response, but greatly suppress the calcium transient and completely abolish the tension responses. These effects are readily reversible when normal saline is reintroduced, suggesting that excitation-contraction coupling may be temporarily blocked between the membrane response and calcium release.9. If the stimulus is prolonged (> 250-300 msec), the calcium transient falls slowly from its maximum value despite continued membrane depolarization, suggesting a time-dependent change in the ratio of the rate of release of calcium to the rate of calcium binding. The results from brief tetanic stimulation also support this suggestion.