Barnacle Muscle Fibers Research Articles

Charges fixes du protoplasme des fibres musculaires de balane; effet de la longueur des sarcomères

The thermodynamically effective charge density (phi X) of myoplasm was measured on barnacle muscle fibers. A small section of a muscle cell formed a liquid junction between two electrolyte solutions and the electrical potential between the solutions permitted to evaluate phi X. If the sarcomere length was kept constant (Ls = 12.5 micron), phi X increased from 0.068 to 0.090 equiv./L when the water content was reduced from 75 to 65 g/100 g wet weight. This increment of X resulted from an increase of X when the concentration of macromolecules is raised in the sample of cytoplasm. But it also indicates that the counterions of the fixed charges concur less to the activity of the counterions when X is increased by lowering the water content. If the water content is maintained constant, phi X increased from 0.068 to 0.084 equiv./L for sarcomere lengths equal to 9.6 and 11.5 micron but phi X remained constant for Ls greater than 12 micron. Thus, when the length of the sarcomere is greater than 12 micron the contribution of the counterions of the fixed charges to the activity of the counterion is larger than for Ls = 9.6 micron.

Stimulation by octopamine of the sodium efflux in barnacle muscle fibres

1. The behaviour of the ouabain-insensitive Na efflux in barnacle muscle fibres towards dl-octopamine has been investigated. 2. It is found that these fibres are quite often sensitive to external application of octopamine. A concentration as low as 10 −9 M is effective. 3. The kinetic results indicate that the stimulatory response develops within 5–10 min of exposure of the fibre to octopamine and is transitory in nature. 4. The response to octopamine is greater in size in the presence of l-isoamyl-3-isobutylxanthine (IAX) than in the presence of l-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX). But neither IAX nor PMX stops the response from decaying. 5. The response to octopamine is highly dependent on the presence of external Ca 2+. 6. The provisional conclusion is that the barnacle muscle fibre may be a useful preparation for studying the hormonal function of octopamine.

The importance of external magnesium in the sensitivity of the sodium efflux to injected cyclic AMP: The barnacle muscle fibre as a preparation

1. 1. The dependence of the response of the Na efflux in ouabain-poisoned barnacle muscle fibres on the external Mg 2+ concentration has been investigated. 2. 2. External Mg 2+ suppresses stimulation by injected cAMP of the ouabain-insensitive Na efflux over a wide concentration range. The response of fibres suspended in 50 mM Mg 2+-ASW is rather small. 3. 3. By contrast, the response of fibres suspended in 50 mM Mg 2+-ASW containing 1-propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine to the injection of cAMP is quite large. 4. 4. The response of fibres suspended in 50 mM Mg 2+-ASW to the injection of catalytic subunit of cAMP-protein kinase is also large. 5. 5. These results give support to the view that a persistent catalytic reaction is a condition which reduces the suppressive action of external Mg 2+.

The nature of the increased sensitivity to injected GTP of the sodium efflux in barnacle muscle fibers pre-exposed to aldosterone

1. 1. A study has been made of the increased sensitivity to injected GTP of the sodium efflux in barnacle muscle fibers pre-exposed to aldosterone and of the problem whether or not aldosterone acts by raising the internal ATP level. 2. 2. The results indicate that increased sensitivity to injected GTP develops fully some 8 hr following external application of 10 −6M aldosterone. 3. 3. Neither actinomycin D nor cycloheximide abolishes this extrasensitivity. This is also true of colchicine and cytochalasin B. 4. 4. The magnitude of the sustained response to injected “dialyzed” cholera toxin or cAMP-protein kinase catalytic subunit is practically the same as that of unexposed fibers. 5. 5. Internal ATP levels in pre-exposed fibers are higher than in unexposed fibers, even in the presence of cycloheximide. Injection of ADP (0.1 M) raises the ATP levels and reduces the ArP levels, more so in unexposed fibers. 6. 6. The suggestion is made that extrasensitivity of pre-exposed fibers to injected guanine nucleotides represents a post-translational phenomenon which might involve delay in the reassociation of R 2 with C (of cAMP-PK).

Sensitivity to injected cholera toxin of the sodium efflux in single barnacle muscle fibers

1. A study has been made of the effect of microinjected cholera toxin (CT) on the Na efflux in single barnacle muscle fibers. Characteristically, injected CT causes sustained stimulation of the ouabain-insensitive Na efflux but only after a lag phase. An effect is seen with as little as a 10 −7 M-solution of CT. Sustained stimulation after a lag phase is also seen following injection of subunit A fragment. 2. Enrichment of fibers with NAD + fails to enhance the response to CT. 3. Prior injection of GTP or its non-hydrolyzeable analogue, Gpp(NH)p, markedly reduces the response to CT, whilst prior injection of CT reduces the response to guanine nucleotides. 4. Evidence is also brought forward that omission of external Ca 2+ reversibly reduces the response to CT and that pre- or postinjection of EGTA markedly reduces the response to CT. In addition, fibers preinjected with CT show increased aequorin light emission. 5. Whereas verapamil and Cd 2+ are ineffective, both Mg 2+ and trace metals, e.g. Fe and Zn, reverse the response to CT following injection. 6. Prior injection of protein kirtase inhibitor reduces the response to CT. 7. As for calmodulin inhibitors, e.g. chlorpromazine, imipramine and mepacrine, they are effective in reducing the response to CT but not calmodulin antibody (IgG). 8. Collectively, the above results are compatible with the view that sustained stimulation of the ouabain-insensitive Na efflux by injected CT is due to persistent activation of adenylate cyclase by the toxin and that a fall in myoplasmic pCa facilitates or augments this activation mechanism.

Freezing point and melting point of barnacle muscle fibers.

The freezing point and the melting point of myoplasm were measured with two experimental models. In all samples, a supercooled stage was reached by lowering the temperature of the sample to approximately - 7 degrees C, and the freezing of the sample was mechanically induced. The freezing process was associated with a phase transition in the interstices between the contractile filaments. In intact muscle fibers, the freezing point showed a structural component (0.43 degrees C), and the melting point indicated that the intracellular and the extracellular compartments are isotonic. When the sample of myoplasm, previously inserted in a cylindrical cavity was incubated in an electrolyte solution, the freezing point showed a structural component similar to that of the intact muscle fiber, but the melting point was lower than the freezing and the melting points of the embedding solution. This was interpreted as evidence that the counterions around the contractile filaments occupied a nonnegligible fraction of the intracellular compartment.

Further observations of aldosterone response in barnacle muscle fibers.

Work with single muscle fibers from the barnacle Balanus nubilus has revealed that these fibers can be rendered sensitive to external application of aldosterone by preexposing the barnacle in vivo to the steroid for 16 h. Experiments investigating the dose-response relationship, glucocorticoid sensitivity of the preparation, and the role of the mitochondria in the aldosterone response in this preparation are presented. It is demonstrated that saturation kinetics may be seen in the dose-response curve if care is taken to isolate only fibers from animals that are in midmolt cycle. Data is presented that demonstrated a specific mineralocorticoid response to aldosterone in this preparation rather than a combined mineralocorticoid-glucocorticoid response, as has been demonstrated in other preparations. Experiments investigating the role of the mitochondrial inhibitors and substrates suggest that aldosterone modulates Na efflux in barnacle muscle fibers by altering mitochondrial function, presumably at the level of succinic hydrogenase.

Intracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation.

The ion transport mechanism that regulates intracellular pH (pHi) in giant barnacle muscle fibers was studied by measuring pHi and unidirectional Na+ fluxes in internally dialyzed fibers. The overall process normally results in a net acid extrusion from the cell, presumably by a membrane transport mechanism that exchanges external Na+ and HCO-3 for internal Cl- and possibly H+. However, we found that net transport can be reversed either by lowering [HCO-3]o and pHo or by reducing [Na+]o. This reversal (acid uptake) required external Cl-, was stimulated by raising [Na+]i, and was blocked by SITS. When the transporter was operating in the net forward direction (acid extrusion), we found a unidirectional Na+ influx of approximately 60 pmol . cm-2 . s-1, which required external HCO-3 and internal Cl- and was stimulated by cyclic AMP and blocked by SITS or DIDS. These properties of the Na+ influx are all shared with the net acid extrusion process. We also found that under conditions of net forward transport, the pHi-regulating system mediated a unidirectional Na+ efflux, which was significantly smaller than the simultaneous Na+ influx. These data are consistent with a reversible transport mechanism which, even when operating in the net forward direction, mediates a small amount of reversed transport. We also found that the ouabain-sensitive Na+ efflux was sharply inhibited by acidic pHi, being totally absent at pHi values below approximately 6.8.

Effects of calcium on membrane potential and sodium influx in barnacle muscle fibers.

The influence of internal and external Ca2+ on membrane potential and 22Na influx were tested in internally perfused giant barnacle muscle fibers. The fibers depolarized by about 2-3 mV, and Na+ influx increased when external Ca2+ was removed. These effects were inhibited and reversed by adding 2 mM La3+ externally but not by tetrodotoxin (TTX). Ca2+ channel blockers did not prevent the depolarization. Increasing internal free Ca2+ ([Ca2+]i) from 10(-7) to 10(-5) M also stimulated Na+ influx and depolarized the fibers by a few millivolts. Neither external La3+ nor TTX prevented the effects of raising [Ca2+]i; however, internal tetrabutylammonium ions depolarized the fibers and attenuated the internal Ca2+-dependent effects. These data are consistent with the idea that removal of external Ca2+ activates a La3+-sensitive channel that is permeable to Na+; raising [Ca2+]i activates a La2+-insensitive, Na+-permeable channel that may be similar to the internal Ca2+-activated nonselective cation channels observed in cardiac muscle. The results demonstrate that all Na+ (and Ca2+) fluxes that do not contribute to Na-Ca exchange must be carefully identified before the exchange stoichiometry can be determined from Na+ and Ca2+ flux measurements.

Sugar transport in giant barnacle muscle fibres.

The kinetics of 3-O-methylglucose transport in the giant muscle cells of Balanus nubilus have been studied both in intact fibres and in fibres subjected to intracellular solute control using internal dialysis. 3-O-methylglucose is not metabolized by barnacle muscle and at equilibrium the 3-O-methylglucose space of the tissue does not differ significantly from the water content of the muscle. These results indicate that 3-O-methylglucose transfer in barnacle muscle is mediated by a passive process. 3-O-methylglucose transport is facilitated by a saturable, symmetric transfer mechanism inhibited by cis but not trans sugars and by low concentrations of phloretin and cytochalasin B. The kinetic constants for uptake and exit are identical. These features indicate that sugar transport in barnacle muscle is mediated by a limited number of membrane transport sites. The number of sugar-displaceable cytochalasin B binding sites in barnacle muscle is 3 X 10(13) cm-2. Indirect kinetic estimates indicate that the number of sugar transport sites is in the order of 1.6 X 10(12) cm-2. This passive, facilitated, selective, saturable transport system is consistent with both symmetric mobile carrier (one-site) and symmetric simultaneous carrier (two-site) models for transport.

Concerning the stimulation by injected cyclic AMP of the sodium efflux in barnacle muscle fibres and its transient nature

1. 1. The efflux of 22Na in ouabain-poisoned barnacle muscle fibres and its transitory response to injected cAMP has been studied by using a new xanthine derivative, 1-propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine (PMX). 2. 2. Injection of PMX prior to cAMP fails to significantly alter the behaviour of the ouabain-insensitive Na efflux towards the nucleotide. By contrast, injection of PMX following peak stimulation by injected cAMP stops the rate constant for 22Na efflux from falling. This effect of PMX is not mimicked by injected HEPES. 3. 3(a). Injection of Mg 2+ following PMX brings about almost complete reversal of the sustained stimulatory response, (b). Injection of trace metals, e.g. Fe and Zn, following PMX brings about complete reversal of the sustained stimulatory response, (c). Injection of R I or R II subunits following PMX brings about partial reversal of the sustained stimulatory response. 4. 4. Partial reversal is also seen with externally applied imipramine (50 μM). 5. 5. These results support the view that the transitory nature of the response of the ouabain-insensitive Na efflux to injected cAMP is due to high phosphodiesterase activity in these fibres and that the major portion of the response itself is due to activation by cAMP of cAMP-dependent protein kinase.

Increased sensitivity to injected 5′-guanylylimidodiphosphate of the sodium efflux in barnacle muscle fibres preexposed to aldosterone

1. A study has been made of the response to injected Gpp(NH)p of the ouabain-insensitive Na efflux in barnacle muscle fibres preexposcd to aldosterone. 2. The response to injected Gpp(NH)p is not only greater in size than in unexposed fibres but also sustained. 3. Injection of MgCl 2 following peak stimulation causes a partial reversal of the response. 4. Injection of ATPNa 2 (and 5′-App(NH(p) leads to a sustained stimulatory response which is not significantly greater than that seen in unexposed fibres. 5. MgCl 2 injection causes complete reversal of this response. 6. The response of preexposed fibres to injected CaCl 2 in varying concentration and to injected cholera toxin is not significantly different from that seen in unexposed fibres. 7. This is also true of Gpp(NH)p when it is injected after peak stimulation by cholera toxin. 8. Prior application of verapamil (10 −4 M) drastically reduces the response to injected Gpp(NH)p. 9. The residual response is sustained but markedly reduced by injected Mg 2+. Fe or Zn. 10. Injection of PKI following Gpp(NH)p reduces the response, provided PK.I is also injected before Gpp(NH)p. By contrast, injection of R ll subunits causes a partial reversal if injected only once. 11. Imipramine and trifluoperazine. when applied externally (5 × 10 −5 M), cause almost complete reversal of the response. 12. The suggestion is made that the response to injected Gpp(NH)p is mainly due to activation of Ca 2+ -channels resulting in activation of the calmodulin/Ca-dependent form of adenylate cyclase and that the primary site of aldosterone action is at the level of the calmodulin form of adenylate cyclase.

Some observations on the behaviour of the sodium efflux in barnacle muscle fibres towards lithium ions

1. 1. The behaviour of the Na efflux towards Li + was studied using single barnacle muscle fibres as a preparation. 2. 2. It is found that the Na efflux into Li +-ASW (artificial seawater) is reduced and that this effect is not fully reversed by returning back to Na +-ASW. 3. 3. Preinjection of 100 mM-EGTA reduces the magnitude of the fall of the Na efflux into Li +-ASW. 4. 4(a). The remaining Na efflux into Li +-ASW is further reduced by external application of 10 −4 M-ouabain. (b) The remaining Na efflux in ouabain-poisoned fibres is reduced by replacing Na e by Li +. However, some fibres show a rise rather than a fall. 5. 5. Fibres loaded with NaCl (by injection) show a prompt and sustained stimulation of the Na efflux when Na e is replaced by Li +. A similar but less pronounced response is often seen with ouabain-poisoned fibres. 6. 6. Injection of LiCl (e.g. a 2 M-solution), causes a 20% fall in Na efflux. Subsequent replacement of Na e by Li + fails to bring about a fall in the remaining efflux. 7. 7. Itis concluded that the Na efflux in these fibres consists of a Na-Na exchange diffusion component which is not mediated by the Na-K pump and that its operation is interrupted by injecting Li +. The relative size of this component is about one-fifth and not one-half of the Na efflux.

Some aspects of sodium efflux from single barnacle muscle fibres

1. 1. This mini-review attempts to summarize information about the efflux of 22Na from single barnacle muscle fibres, based on the use of the microinjection technique. 2. 2. The view is put forward that the Na efflux consists of three components: an ouabain-sensitive component, an ouabain-insensitive component (representing secondary active transport), and an Na-Na exchange diffusion component. 3. 3. Evidence is brought forward which supports the view that the ouabain-insensitive Na efflux is divisible operationally into 3 phases: (i) the cyclic nucleotide-sensitive phase, (ii) the Ca i-sensitive phase, and (iii) the pH e-sensitive phase. 4. 4. It is shown how the barnacle muscle fibre preparation has yielded information about the validity of the cAMP-protein kinase hypothesis and how it can be used to shed some light on the post-translational mechanism of aldosterone action.

Some quantitative aspects of myoplasmic ATPMg and total internal ATP and ArP levels in resting barnacle muscle fibres

1. 1. Flash height recorded following the injection of firefly into the external calibration medium depends on the concentration of K-glutamate used, e.g. 120 mM K glutamate reduces flash height by ∼20%. 2. 2. Suspending the fibre in air instead of artificial sea water (ASW) or replacement of NaCl in the bathing medium with Na-glutamate fails to alter flash height. 3. 3. Firefly preparations from DuPont, Packard and SAI give similar myoplasmic ATPMg values viz. 1.1 mM. 4. 4. Analysis of 36 fibres shows the following: myoplasmic ATP = 1.03 ± 0.06 mM; total ATP (firefly method) = 5.26 ± 0.12 mmol/kg water; total ATP (enzymic fluorimetry) = 6.27 ± 0.13 mmol/kg water and ArP = 20.76 ± 0.59 mmol/kg water. 5. 5. Measurement of ATPMg in samples of myoplasmic aspirate gives a value that is greater than that obtained in situ. 6. 6. Iodoacetate, whether applied externally or internally, reduces resting luminescence in a dose-dependent manner. It also reduces myoplasmic ATP and total ATP. 7. 7. 2-Deoxy-d-glucose fails to reduce myoplasmic ATP but reduces total ATP. 8. 8. Diethylpyrocarbonate, whether applied externally or internally, reduces myoplasmic ATP. It also causes a slow decline in ArP but little change in total ATP. 9. 9. Injection of l-arginine causes a fall in resting luminescence in some fibres while in others it causes a prompt transitory rise. Injection of l-arginine also causes a fall in total ATP. 10. 10. Collectively, these results suggest that the immediate buffering system in the myoplasm is ArP and that ATP supplied by glycolysis lies in a compartment, presumably the interfibrillar space, which is inaccessible to injected firefly.

Some further observations on the stimulation by high external potassium of the sodium efflux in barnacle muscle fibers.

A study has been made in single barnacle muscle fibers of the mechanism underlying the stimulatory response of the ouabain-insensitive Na efflux to high K0. Fibers poisoned with ouabain and preinjected with GTPNa2 show a biphasic stimulatory response to 100 mM K0 which is practically abolished by exposure of these fibers to verapamil. Prior or post-injection of cAMP-protein kinase type II regulatory subunits reduces the size of the response to 100 mM K0. Prior injection of cAMP-protein kinase catalytic subunits (CSU) attenuates the response to 100 mM K0 in fibers showing moderate sensitivity to CSU. In fibers showing extreme sensitivity to injected CSU, elevation of K0 partially reverses the sustained stimulatory response to CSU. Prior injection of Mg2+ leads to almost complete abolition of the response to 100 mM K0. External and internal application of F- reduces the response to 100 mM-K0. Injection of KF following decay of the response to high K0 results in a sustained stimulatory response. The drugs, chlorpromazine, trifluoperazine, imipramine and diphenylhydantoin are able to appreciably reduce the response to 100 mM K0. Taken together, these observations support the idea that the stimulatory response to high K0 is primarily due to activation by newly formed cAMP of cAMP-protein kinase, and that the transitory nature of the response and variable sensitivity of these fibers to high K0 is closely linked to the activity of a putative Mg2+-dependent protein phosphatase.

The influence of injected cyclic AMP protein kinase catalytic subunit on the sodium efflux in barnacle muscle fibres.

1. A study has been made of the response of the Na efflux in single barnacle muscle fibres to the injection of cyclic AMP protein kinase catalytic subunit (CSU).2. (i) Injected CSU stimulates the Na efflux in unpoisoned and ouabain-poisoned fibres. (ii) Stimulation in both situations is always sustained, and (iii) stimulation of the ouabain-insensitive Na efflux is observed with as low a concentration as 10(-7) M-CSU.3. (i) The response to injected CSU depends on [Ca](o) but is unaffected by prior external application of verapamil. (ii) An effect fails to occur in fibres cooled to 0 degrees C.4. (i) Injection of 5'-guanylylimidodiphosphate (Gpp(NH)p) following peak stimulation by CSU causes a further sustained stimulatory response. (ii) Injection of CSU following peak stimulation by Gpp(NH)p causes a further sustained stimulatory response. (iii) Injection of undialysed cholera toxin following CSU causes little or no effect, whereas injection of dialysed cholera toxin causes a stimulatory effect. Injection of CSU following undialysed cholera toxin causes little or no effect, whereas its injection after dialysed cholera toxin causes a fairly appreciable effect.5. (i) The response to CSU is reduced by prior injection of protein kinase type 2 regulatory subunit (R(II)) but not when R(II) is injected following peak response to CSU. (ii) Injection of protein kinase inhibitor partially reverses the response to CSU.6. (i) Injection of Mg(2+) fully reverses the response to CSU. (ii) Fe and Zn cause only partial reversal.7. (i) External imipramine but not trifluoperazine reduces the response to CSU. (ii) Diphenylhydantoin is ineffective.8. These results support the view that CSU behaves as a modulator of the ouabain-insensitive Na efflux and that Mg(2+), Fe and Zn are inhibitors of the catalytic reaction.

Water in barnacle muscle. IV. Factors contributing to reduced self-diffusion

The relative self-diffusion coefficients D/Do, of water in various solutions, in fresh barnacle muscle fibers, and in membrane-damaged fibers equilibrated with several media have been estimated from NMR relaxation rates in the presence of applied field gradients. A model has been developed to account for the contributions to the observed reduction in D/Do from small organic solutes, and from the hydration and obstruction effect of both soluble macromolecules and myofilament proteins. Intracellular ions do not affect D/Do, but all tested organic solutes do. Solute effects are additive. When artificially combined in the proportions found in barnacle muscle ultracentrifugate (measured D/Do = 0.77), organic acids, small nitrogenous solutes, and proteins give D/Do = 0.77. After correcting the D/Do measured in fibers for this value, we calculate the myofilament hydration, Hm, in fresh muscle to be 0.65 g H2O/g macromolecule. Only in membrane-damaged fibers, highly swollen by salt-rich media, was this significantly increased. Because our earlier NMR relaxation measurements indicate only 0.07 g H2O bound/g myofilament protein, we conclude that the "hydration" water measured by reduction of D/Do cannot be described by stationary layers of water molecules; instead, we propose that nonpolar groups on the proteins cause extensive, hydrophobically-induced interactions among a large fraction of solvent molecules, slowing their translational motion.

The effect of pH on the rate of relaxation of isolated barnacle myofibrillar bundles

CO 2-induced acidosis in barnacle muscle fibres prolongs the relaxation phase of the electrically stimulated contraction (Ashley, C.C., Franciolini, F., Lea, T.J. and Lignon, J. (1979) J. Physiol. 296, 71P). In order to test if this effect is due to a direct action of H + on the relaxation kinetics of the myofilaments, isolated myofibrillar bundles were contracted and relaxed in Ca 2+ buffer solutions at pH 6.0 and 7.1, in the presence of 20 mM caffeine to inactivate the sarcoplasmic reticulum. At pH 7.1, the relaxation half-time was reduced from 1.5 to 0.3 s as the EGTA concentration in the relaxing solution was progressively increased from 0.3 to 50 mM. The resulting curve was shifted in the direction of increasing EGTA concentration by lowering the pH to 6.0. This effect could be explained by the reduction in affinity of Ca 2+ for EGTA at pH 6.0, since relaxation half-times for a given relaxing p Ca (calculated from the contaminating Ca 2+ concentrations in the relaxing solutions) were shorter (by about 40%) at pH 6.0 compared with 7.1. However, similar experiments using the new Ca 2+-chelating agent 1,2-bis( o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA), which is much less pH sensitive than EGTA, indicated that there was no significant difference between relaxation half-times at pH 6.0 and 7.1 for a given relaxing p Ca. It is concluded that because no prolongation of relaxation of the myofibrils was observed on lowering the pH from 7.1 to 6.0, the effect of CO 2 on the relaxation of intact muscle fibres is probably due to a modification of sarcoplasmic reticulum activity.

Sarcoplasmic Ca2+ transients during the contractile cycle of single barnacle muscle fibres: measurements with arsenazo III-injected fibres.

Single muscle fibres from Balanus nubilus were injected with the metallochromic Ca2+ indicator arsenazo III; multi-wavelength microspectrophotometry was used to monitor changes in the absorption spectrum of such fibres during isometric tension transients elicited by electrical stimulation. Differential absorbance changes, recorded in the 660-720 nm region of the spectrum, were characterized by rapid, nearly linear increases during constant current depolarizing pulses (25-200 ms); upon termination of electrical stimuli the increased differential absorbance described an exponential (k = 0.6-2.0 s-1) decay toward baseline levels. The maximal increases in these absorbance signals preceded the peaks of the tension transients by 300-400 ms at 15-17 degrees C. Calibration of these optical transients indicated that the initial velocity of the rising phase corresponded to a 0.03 microM ms-1 increase in sarcoplasmic free Ca2+. Appreciable tension was not generated until the apparent peak magnitude of the free Ca2+ transient exceeded 2 microM; a further three-fold increase (from 2 to 6 micrometers) in the maximal value of the free Ca2+ transient was accompanied by a 20-fold increase in the magnitude of the tension transient.