Calcium influx in skeletal muscle at rest,during activity,and during potassium contracture

Calcium influx in the sartorius muscle of the frog (Rana pipiens) has been estimated from the rate of entry of Ca⁴⁵. In the unstimulated preparation it is about equal to what has been reported for squid giant axons, but that per impulse is at least 30 times greater than in nerve fibers. The enhanced twitch when NO^-₂ replaces Cl^- in Ringer's is associated with at least a 60 per cent increase in influx during activity, whereas this anion substitution does not affect the passive influx significantly. Calcium entry during potassium contracture is even more markedly augmented than during electrical stimulation, but only at the beginning of the contracture; thus, when a brief Ca⁴⁵ exposure precedes excess K⁺ application, C⁴⁵ uptake is increased three- to fivefold over the controls not subjected to K⁺, whereas when C⁴⁵ and K⁺ are added together, no measurable increase in Ca⁴⁵ uptake occurs. These findings are in keeping with the brevity of potassium contracture in "fast (twitch)" fibers such as in sartorius muscle.     

Effects of Ca2+ and other divalent cations on K+-evoked force production of slow muscle fibers from Rana esculenta and Rana pipiens

Summary Slow muscle fibers were dissected from cruralis muscles of Rana esculenta and Rana pipiens. Isometric contractures were evoked by application of K⁺-rich Ringer's containing Ca²⁺, Ni²⁺, Co²⁺, Mn²⁺ or Mg²⁺. High (7.2 mmol/liter) external Ca²⁺ concentration raised, 0 Ca²⁺ lowered the K⁺ threshold. Replacing Ca²⁺ by Ni²⁺ or Co²⁺ had an effect similar to that of high Ca²⁺ Ringer's. In Mg²⁺ Ringer's the K⁺ concentration-response curve was flattened. These effects were observed already after short exposure times in both species of slow fibers. When Ca²⁺ was removed for long periods of time the slow fibers of R. esculenta lost their contractile response to application of high K⁺ concentrations much more quickly than those of R. pipiens, while the response to caffeine (20 mmol/liter) was maintained. Upon readmission of Ca²⁺ contractile ability was quickly restored in the slow fibers of both R. esculenta and R. pipiens, but the effects of Ni²⁺ (or Co²⁺, Mn²⁺ and Mg²⁺) were much larger in R. esculenta than in R. pipiens slow fibers. It is concluded that divalent cations have two different sites of action in slow muscle fibers. K⁺ threshold seems to be affected through binding to sites at the membrane surface; these sites bind Ni²⁺ and Co²⁺ more firmly than Ca²⁺. The second site is presumably the voltage sensor in the transverse tubular membrane, which controls force production, and where Ca²⁺ is the most effective species of the divalent cations examined.We are grateful to Mrs. S. Pelvay for technical assistance.     

Slow fibers in the frog cruralis muscle

Extrafusal slow fibers have been identified by electron microscopy in a muscle fiber bundle isolated from the anterior head (m. cruralis) of the triceps femoris of Rana pipiens. Light microscopic examination of this muscle revealed two equally numerous fiber types, one Fibrillenstruktur-like (twitch) and the other structureless (slow). A comparison of force developed by tetanic stimulation and by K⁺ depolarization also suggested approximately equal numbers of twitch and slow fibers.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

Electrophoretic and functional identification of two troponin C isoforms in toad skeletal muscle fibers

The differential sensitivity of frog twitch and slow-tonic fibers to Ca²⁺ and Sr²⁺ suggests that these two fiber types express different troponin C (TnC) isoforms. To date, only one TnC isoform from anurans (resembling the mammalian fast-twitch isoform) has been isolated and characterized. In this study, we examined the possibility that anuran striated muscle contains more than one TnC isoform. Toward this end, we determined the TnC isoform composition of 198 single fibers from the rectus abdominis of the cane toad (a mixed slow-tonic and twitch muscle) and of toad cardiac muscle using a method that enables the identification of TnC isoforms on the basis of the effect of Ca²⁺ on their electrophoretic mobility. The fibers were typed according to their myosin heavy chain (MHC) isoform composition. The data indicate that striated muscle of the cane toad contains two TnC isoforms, one of which (TnC-t) is present in all fibers displaying only twitch MHC isoforms and the other of which (TnC-T/c) is present in fibers displaying the tonic MHC isoform and in cardiac muscle. For a subpopulation of 15 fibers, the TnC isoform composition was also compared with Ca²⁺ and Sr²⁺ activation characteristics. Fibers containing the TnC-T/c isoform were 3-fold more sensitive to Ca²⁺, 40-fold more sensitive to Sr²⁺, and responded to a 4.6-fold broader range of [Ca²⁺] than did fibers containing the TnC-t isoform. The Ca²⁺ activation properties of toad fibers containing the TnC-T/c isoform appear to be consistent with the previously reported physiological characteristics of amphibian slow-tonic muscle fibers. myofibrillar proteins; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; alanine SDS-PAGE; hybrid fibers; Ca²⁺-binding proteins; single fiber; muscle protein polymorphism; fiber type     

Quinine and caffeine effects on 45Ca movements in frog sartorius muscle

1 mM caffeine, which produces only twitch potentiation and not contracture in frog sartorius muscle, increases both the uptake and release of ⁴⁵Ca in this muscle by about 50 %, thus acting like higher, contracture-producing concentrations but less intensely. Quinine increases the rate of release of ⁴⁵Ca from frog sartorius but not from the Achilles tendon. The thresholds for the quinine effect on ⁴⁵Ca release and contracture tension are about 0.1 and 0.5 mM, respectively, at pH 7.1. Quinine (2 mM) also doubles the uptake of ⁴⁵Ca by normally polarized muscle. However, there are variable effects of quinine upon ⁴⁵Ca uptake in potassium-depolarized muscle. Quinine (2 mM), increases the Ca, Na, and water content of muscle while decreasing the K content. Both caffeine (1 mM) and quinine (2 mM) act to release ⁴⁵Ca from muscles that have been washed in Ringer''s solution from which Ca was omitted and to which EDTA (5 mM) was added. These results, correlated with those of others, indicate that a basic effect of caffeine and quinine on muscle is to directly release activator Ca²⁺ from the sarcoplasmic reticulum in proportion to the drug concentration. The drugs may also enhance the depolarization-induced Ca release caused by extra K⁺ or an action potential. In respect to the myoplasmic Ca²⁺ released by direct action of the drugs, a relatively high concentration is required to activate even only threshold contracture, but a much lower concentration, added to that released during excitation-contraction coupling, is associated with the condition causing considerable twitch potentiation.     

Effect of yttrium on calcium-dependent processes in vertebrate myocardium

Inoptopic effect of yttrium acetate (Y³⁺) on myocardium of the marsh frog Rana ridibunda and its effect on ion transport across the inner mitochondrial membrane (IMM) of rat heart was studied. Y³⁺ was found to decrease the rate of heart contractions and to stimulate ion transport in the rat heart mitochondria in media with 10 mM glutamate and 2 mM malate. Presence of Y³⁺ induced inhibition of energy-dependent Ca²⁺ transport into mitochondria, which was expressed as a marked decrease of their swelling in the media containing 125 mM NH₄NO₃ and Ca²⁺ or 25 mM potassium acetate, 100 mM sucrose and Ca²⁺. It is suggested that the Y³⁺-induced decrease in rat muscle contractions is determined not only by direct suppressing effect of Y³⁺ on potential-modulated Ca²⁺-channels of pacemaker and contractile cardiomyocytes (CM), but also by its indirect effect on Ca²⁺-carrier in IMM. The data confirming that Y³⁺ activates energy-dependent K⁺ transport catalyzed by mitochondrial uniporter and blocks Ca²⁺-channels in the mitochondrial membrane are important for more complete understanding of mechanisms of the Y³⁺ action on vertebrates and human CM.     

Proctolin and octopamine actions on the contractile systems of insect leg muscles

1. The pentapeptide proctolin produced contractions of the coxal depressor muscle of the cockroach, Periplaneta americana.2. The contraction was dependent upon extracellular calcium and the contraction was completely blocked by a Ca-free EGTA saline.3. Caffeine elicited transient contractions which were unaffected by manganese treatment.4. When the muscle was pre-treated with the conditioning solution with different K⁺ concentrations (1–100 mM), the amplitude of proctolin-induced contractions was reduced in the low K⁺ saline as well as in the high K⁺ saline.5. The results suggest that voltage sensitive calcium channels account for the proctolin-induced contractions.6. Octopamine (OA) reduced the contractions resulting from brief applications of elevated K⁺ concentration and of caffeine.7. The effect of OA on the response to elevated K⁺ concentrations was blocked by the α-adrenergic blocker, phentolamine.     

Urea and salt effects on enzymes from estivating and non-estivating amphibians

The effects of urea, cations (K⁺, NH₄, Na⁺, Cs⁺, Li⁺), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle, were analyzed in two anuran amphibians, an estivating species, the spadefoot toadScaphiopus couchii, and a semi-aquatic species, the leopard frogRana pipiens. Urea, which accumulates naturally to levels of 200–300 mM during estivation in toads, had only minor effects on the V_max, kinetic constants and pH curves of PK from either species and no effects on PFK V_max or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the V_max of toad PFK and of PK from both species and altered the K_m values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the V_max of PK from either species was reduced by more than 80% by the addition of 200 mM NH₄Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the K_m values for substrates of toad lactate dehydrogenase and strongly reduced the V_max of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes. Rather, we found that urea is largely a non-perturbing solute for anuran enzymes (I₅₀ values were>1 M for both PK and PFK in both species) and we propose that its accumulation in high concentrations during estivation helps to minimize the increase in cellular ionic strength that would otherwise occur during desiccation and to alleviate the accompanying negative effects of high salt on individual enzyme activities and overall metabolic regulation.Abbreviations PFK 6-phosphofructo-1-kinase - PK pyruvate kinase     

Role of acetylcholine in Ca<Superscript>2+</Superscript>-dependent regulation of functional activity of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis> myocardium

To study role of acetylcholine (ACh) in Ca²⁺-dependent regulation of rhythm and strength of cardiac contractions in the frog Rana temporaria, we studied in parallel experiments the ACh chrono- and inotropic effects on the background of action of blockers of the potential-controlled Ca²⁺-channels, ryanodine and muscarine receptors. The obtained results indicate participation of acetylcholine in the Ca²⁺-dependent regulation of the rhythm and strength of the frog cardiac contractions.     

Metabolic suppression in anoxic frog muscle

Microcalorimetry is the only direct method for measuring moment-to-moment changes in whole-cell metabolism (as heat output) during anoxia. We have adapted this methodology, in conjunction with standard muscle isolation techniques, to monitor metabolic transitions in isolated frog (Rana temporaria) sartorius muscle during anoxia and recovery (reoxygenation). Anoxia (sustained 1 h, following 2 h progressive hypoxia) suppressed muscle heat output to 20% of the stable normoxic level. This effect was fully reversible upon reoxygenation. Metabolite profiles were consistent with other anoxia-tolerant vertebrates – most notably, adenosine triphosphate (ATP) content during anoxia and reoxygenation remained unchanged from normoxia (pre-anoxic control). In addition, the concentration of K⁺ ions ([K⁺]) in interstitial dialysates remained stable (2–3 mM) throughout anoxia and recovery. Interstitial [lactate⁻] increased slightly, in accord with anaerobiosis supporting suppressed metabolic rates during anoxia. The degree of anoxic suppression of metabolism observed is similar to other vertebrate models of anoxia tolerance. Furthermore, stable ATP concentrations and interstitial [K⁺] in the isolated tissue suggests that intrinsic mechanisms suppress metabolism in a manner that coordinates ATP supply and demand and avoids the severe ion imbalances that are characteristic of hypoxia-sensitive systems. Accepted: 15 January 1998     

Contractile activation in the retractor unguis muscle of the cockroach Periplaneta americana

1. The K⁺-induced contracture consists of a phasic and a sustained component. Both were eliminated in Ca²⁺-free saline, but the sustained component recovered on the addition of Ca²⁺ to the muscle.2. Procaine mainly inhibited the phasic component. 3. Unlike the sustained component, the phasic component was inhibited by nifedipine in a concentration dependent manner.4. Divalent cations such as Mn²⁺, Co²⁺ and Ni²⁺ markedly increased the sustained component at low concentrations, but decreased it at high concentrations. The cations also modified the phasic component differentially, but to a lesser extent. High concentration abolished the phasic component.5. Ouabain markedly enhanced the sustained component.6. Caffeine contracture was a phasic type. Its duration and amplitude were augmented by pre-soaking the muscle in Na⁺-reduced salines. Immediate pre-treatment with caffeine eliminated the phasic component of the 160mM K⁺-induced contracture.7. These results suggest that a Na-Ca exchange mechanism may play a role in excitation-contraction coupling in insect muscle. Calcium ions flowing into the cell upon membrane depolarization may specifically activate the phasic component by way of a calcium-induced calcium releasing mechanism.     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.     

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K−Cl cotransport

Pathways of K⁺ movement across the erythrocyte membrane of frog Rana temporaria were studied using ⁸⁶Rb as a tracer. The K⁺ influx was significantly blocked by 0.1 mmol·l^-1 ouabain (by 30%) and 1 mmol·l^-1 furosemide (by 56%) in the red cells incubated in saline at physiological K⁺ concentration (2.7 mmol·l^-1). Ouabain and furosemide had an additive effect on K⁺ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K⁺ influx saturated as f(K⁺)_e with apparent K _m values for external K _e ⁺ concentration of 0.96±0.11 and 4.6±0.5 mmol·l^-1 and V _max of 0.89±0.04 and 2.8±0.4 mmol·l cells^-1·h^-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of K _e ⁺ medium concentration. Total K⁺ influx was significantly reduced when frog erythrocytes were incubated in NO _- ³ medium. Furosemide did not affect K⁺ transport in frog red cells in NO ₃ ^- media. At the same K _e ⁺ concentration the ouabain-furosemide-insensitive K⁺ influx in Cl^- medium was significantly greater than that in NO _- ³ medium. We found no inhibitory effect of 1 mmol·l^-1 furosemide on Na⁺ influx in frog red cells in Cl^- medium. K⁺ loss from the frog erythrocytes in a K⁺-free medium was significantly reduced (mean 58%) after replacement of Cl^- with NO _- ³ . Furosemide (0.5 mmol·l^-1) did not produce any significant reduction in the K⁺ loss in both media. The Cl^--dependent component of K⁺ loss from frog red cells was 5.7±1.2 mmol·l^-1·h^-1. These results indicate that about two-thirds of the total K⁺ influx in frog erythrocytes is mediated by a K–Cl cotransport which is only partially blocked by furosemide.Abbreviations DMSO dimethyl sulphoxide - K _e ⁺ external concentration of K⁺ - K _m apparent Michaelis constant for external - K⁺ K _e ⁺ at V _max/2 - RBC red blood cell(s) - V _max maximal velocity of the unidirectional K⁺ influx - TRIS tris(hydroxymethyl)aminomethane     

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers

Summary Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca²⁺ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca²⁺ exceeds 0.3 to 0.4 m, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg²⁺ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca²⁺ concentration dependence of Ca uptake (K _0.5, 0.5 to 0.8 m; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.     

Electrophysiological studies of the effect of the neuropeptide proctolin on the hyperneural muscle of Periplaneta americana (L.)

The myotropic neuropeptide proctolin is, in additional to its action on proctodaeum and on some other systems, highly effective on the hyperneural muscle of Periplaneta americana and evokes long-term contractions. During this proctolin response the input resistance (R_input) increases by about 25% accompanied by only slight depolarization. These processes require extracellular Ca²⁺ but are still present in Na⁺-free solution.Junction potentials evoked by threshold stimulation of the nerve are not affected by proctolin. Synaptic processes do not seem to be important for the proctolin action on hyperneural muscle. It is more likely that the whole membrane of the muscle fibre serves as target for proctolin. Proctolin reduces the threshold for neurally evoked muscle contractions, the only available route of excitation since the muscle fibres themselves are not electrically excitable.The K⁺-channel blocker 4-aminopyridine may evoke contraction as well as proctolin, but this is only a transitory response. In contrast to proctolin, 4-aminopyridine is still effective after blocking the Ca²⁺-channels by Co²⁺, but the response is smaller. Therefore proctolin seems to be primarily effective via Ca²⁺-channels, whereas 4-aminopyridine exerts its effects via K⁺-channels. The decrease in membrane conductance produced by proctolin could result from a Ca²⁺-dependent reduction of the K⁺-outward current.     

Inhibitory effects of HgCl2 on excitation-secretion coupling at the motor nerve terminal and excitation-contraction coupling in the muscle cell

Summary 1. Indirect and direct twitch (0.1-Hz) stimulation of the rat phrenic nerve-diaphragm disclosed that the inhibitory effect of HgCl₂, 3.7 × 10^–5 M, on the neuromuscular transmission and in the muscle cell, was accelerated by 10-sec periods of 50-Hz tetanic stimulation every 10 min. This activity-dependent enhancement suggested an inhibitory mechanism of HgCl₂ related to the development of fatigue, like membrane depolarization or decreased excitability, decreased availability of transmitter, or interference with the factors controlling excitation-secretion coupling of the nerve terminal, i.e. (Ca²⁺)₀ or (Ca²⁺)_i, and excitation-contraction coupling in the muscle cell, i.e., (Ca²⁺)_i.2. During both indirect and direct stimulation, HgCl₂-induced inhibition was enhanced markedly by pretreatment with caffeine, which releases Ca²⁺ from endoplasmic and sarcoplasmic reticulum in the nerve terminal and muscle cell, respectively. This caffeine-induced enhancement was completely antagonized by dantrolene, which inhibits the caffeine-induced release. However, dantrolene alone did not antagonize the HgCl₂-induced inhibition.3. Since caffeine depletes the intracellular Ca²⁺ stores of the smooth endoplasmic reticulum, HgCl₂ probably inhibits by binding to SH groups of transport proteins conveying the messenger function of (Ca²⁺)_i. In the muscle cell this leads to inhibition of contraction. In the nerve terminal, an additional enhancement of the HgCl₂-induced inhibition, by inhibiting reuptake of choline by TEA and tetanic stimulation, suggested that HgCl₂ inhibited a (Ca²⁺)_i signal necessary for this limiting factor in resynthesis of acetylcholine.4. The (Ca²⁺)₀ signal necessary for stimulus-induced release of acetylcholine was not affected by HgCl₂. Hyperpolarization in K⁺-free solution antagonized the inhibitory effect of HgCl₂ at indirect stimulation, and Ca²⁺-free solution enhanced the inhibitory effect at direct stimulation. K⁺ depolarization, membrane electric field increase with high Ca²⁺, membrane stabilization with lidocaine, and half-threshold stimulation, did not change the inhibitory effect of HgCl CH₃HgCl, 1.85 × 10^–5 M, disclosed a synergistic interaction with caffeine during direct, but not during indirect, stimulation.     

A comparative analysis of the contracture responses induced by acetylcholine and choline in the twitch and tonic fibers of frog skeletal muscles

A comparative analysis of the contractile responses induced by acetylcholine and replacement of the external Na⁺ ions with choline ions in the isolated twitch and tonic fibers of frog skeletal muscles was performed. The effects of extracellular Ca²⁺ concentration and several pharmacological agents modulating the activity of various systems maintaining Ca²⁺ level in the myoplasm (dantrolene, cresol, d-tubocurarine, and tetrodotoxin) were studied. It has been found that a voltage-dependent Ca²⁺ release from the sarcoplasmic reticulum depot is the main mechanism inducing the acetylcholine contracture in the fibers of both types. However, the twitch and tonic fibers differ in the properties of the α-isoform and(or) the ratio of α- to β-isoforms of ryanodine-sensitive channels. In the fibers of both types, the replacement of over 25% of Na⁺ ions with choline induces long-term contracture responses, which are also mediated by activation of acetylcholine receptors. It is assumed that an additional mechanism—accumulation of choline ions in the myoplasm and their direct action on the ryanodine-sensitive channels—is involved in the development of such contractile responses.     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.     

Regulatory mechanism of contraction in the proboscis retractor muscle of a sipunculid worm,Phascolosoma scolops

Summary Regulatory mechanism of contraction in the proboscis retractor muscle of Phascolosoma scolops was studied by physiological measurements and cytochemical electron microscopy. The magnitude of K⁺-contracture was dependent on external Ca²⁺ concentration and the contracture disappeared in Ca²⁺-free solution. The K⁺-contracture was suppressed by application of procaine and Mn²⁺. Caffeine induced contracture even when external Ca²⁺ was absent. Ultrastructural observations of the retractor muscle cells showed the presence of a large number of vesicles (subsarcolemmal vesicles), corresponding to the sarcoplasmic reticulum in vertebrate skeletal muscle, underneath the plasma membrane. For the cytochemical electron microscopy, the muscle fibers were fixed with 1% OsO₄ solution containing 2% K-pyroantimonate. In the relaxed fibers, pyroantimonate precipitates were localized along the inner surface of plasma membrane and in the subsarcolemmal vesicles. In the contracting fibers, the precipitates were uniformly distributed in the myoplasm. The X-ray microanalysis revealed that the precipitates contained Ca. These results suggest that the contractile system is activated by the influx of extracellular Ca²⁺ as well as by the release of Ca²⁺ from the intracellular structures such as the inner surface of the plasma membrane and subsarcolemmal vesicles.