In vitro responses of cat skeletal muscle to halothane and caffeine

Strips of soleus (100% type I) and gracilis (90% type II) muscle were obtained from anesthetized cats and mounted in organ baths filled with aerated Krebs-Ringer solution (37 degrees C). The contractile patterns in response to electrical stimulation (0.1 Hz, 25 V, 5 ms), caffeine, halothane, and caffeine in the presence of halothane were examined in the two fiber types. The ability of 25 microM dantrolene to alter the contractile patterns was also evaluated. In vitro contractile properties in response to electrical stimulation were similar to properties observed in situ, except that twitch tension in soleus muscle was significantly less in vitro than in situ. In the presence of halothane, type I soleus muscle developed a rapid contracture. The contracture was blocked by pretreatment with dantrolene and was reversed by addition of dantrolene at the peak of the response. Halothane-induced contractures were not observed at any time in type II gracilis. Type I soleus was also significantly more sensitive both to caffeine alone and to caffeine in the presence of halothane than was type II gracilis. In both fiber types, halothane increased the sensitivity of the muscles to caffeine. Dantrolene attenuated caffeine-induced contractures in both fiber types, but the attenuating effect was less in the presence of halothane. The findings of a halothane-induced contracture in the cat soleus and differential sensitivities of the two muscle fiber types to caffeine indicate that further studies in these two muscles may be useful for delineating the mechanisms inducing contracture in muscle from individuals susceptible to malignant hyperthermia.     

Twitch potentiation and caffeine contractures in isolated rat soleus muscle

1. Electrically-evoked twitch and tetanic tension were measured in isolated rat soleus muscle after exposure to caffeine. 2. Between 0.01 and 2.5 mM caffeine twitch tension was potentiated, reaching a peak of 150% of Resting Tension at 0.5 mM. 3. Biphasic Tension development with relaxation was observed at 2.5 mM caffeine with maximal contractures (110% tetanic tension) occurring at 20 mM. 4. Creatine phosphate and ATP stores were maintained throughout the period of tension development and relaxation. 5. In contrast with amphibian muscle, the isolated soleus is very sensitive to low doses of caffeine and produces biphasic caffeine contractures which relax in the presence of caffeine.     

Effects of modulators of sarcoplasmic Ca2+ release on the development of skeletal muscle fatigue.

The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.     

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation.

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

Caffeine and length dependence of staircase potentiation in skeletal muscle

Skeletal muscle sensitivity to Ca2+ is greater at long lengths, and this results in an optimal length for twitch contractions that is longer than optimal length for tetanic contractions. Caffeine abolishes this length dependence of Ca2+ sensitivity. Muscle length (ML) also affects the degree of staircase potentiation. Since staircase potentiation is apparently caused by an increased Ca2+ sensitivity of the myofilaments, we tested the hypothesis that caffeine depresses the length dependence of staircase potentiation. In situ isometric twitch contractions of rat gastrocnemius muscle before and after 10 s of 10-Hz stimulation were analyzed at seven different lengths to evaluate the length dependence of staircase potentiation. In the absence of caffeine, length dependence of Ca2+ sensitivity was observed, and the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased length (DT = 1.47 - 0.05 ML, r2 = 0.95, where DT is developed tension). Length dependence of Ca2+ sensitivity was decreased by caffeine when caffeine was administered in amounts estimated to result in 0.5 and 0.75 mM concentrations. Furthermore, the negative slope of the relationship between staircase potentiation and muscle length was diminished at the lower caffeine dose, and the slope was not different from zero after the higher dose (DT = 1.53 - 0.009 ML, r2 = 0.43). Our study shows that length dependence of Ca2+ sensitivity in intact skeletal muscle is diminished by caffeine. Caffeine also suppressed the length dependence of staircase potentiation, suggesting that the mechanism of this length dependence may be closely related to the mechanism for length dependence of Ca2+ sensitivity.     

Effects of calcium antagonists on contraction of a holothurian muscle

Contractions evoked by acetylcholine or by tetanic stimulation differed from caffeine contractures, in muscle strips isolated from the longitudinal muscle bands of the body wall of Isostichopus badionotus (Selenka), an aspidochirote holothurian. Tetanic contractions and responses to acetylcholine remained reproducible for hours in sea water or artificial sea water. Caffeine contractures declined rapidly during a series of repetitions, carried out in a bath medium which had a calcium content equivalent to that of sea water. Manganese, lathanum, and dantrolene have been used as calcium antagonists, with the objective of differentiating the calcium stores used in reproducible contractions from the calcium stores used in rapidly extinguishing contractures. Muscle strips were pretreated with an ionophore (X-537A) to confer reproducibility in a series of caffeine contractures, before use of calcium antagonists. For reproducible caffeine contractures, the order of effectiveness of calcium antagonists was lanthanum greater than manganese greater than dantrolene. The order of reversibility was manganese greater than dantrolene greater than lanthanum. For acetylcholine contractions, or tetanic contractions, the order of effectiveness of calcium antagonists was lanthanum greater than manganese and the order of reversibility was manganese greater than lanthanum. Dantrolene reversibility enhanced contractions.     

Potentiation of the halothane-cooling contractures of mammalian muscles by denervation

Denervation potentiated the cooling-induced contractures and the halothane-cooling contractures of isolated extensor digitorum longus and soleus muscles of the mouse. These effects were more striking in extensor digitorum longus than in soleus muscles. Significant increases in the peak amplitudes of the halothane-cooling contractures of both muscles and of the cooling contractures of soleus muscle were observed within 2 and 7 days of denervation. The potentiation of the contractures persisted for 90 days, the period of this study. Denervation (greater than 2 days) endowed extensor digitorum longus with the ability to generate cooling contractures in the absence of halothane. The rate of tension development of cooling-induced contractures in the absence or presence of halothane was significantly greater in denervated (2-90 days) than in innervated muscles. Denervation also reduced the effectiveness of procaine in inhibiting the halothane-cooling contractures. It is proposed that the potentiation of cooling-induced contractures in denervated muscles results primarily from an increase in the rate of efflux and in the quantity of Ca2+ released from the sarcoplasmic reticulum, upon cooling and (or) when challenged with halothane.     

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive inotropism in mammalian skeletal muscle in vitro during and after fatigue

We tested the hypothesis that positive inotropic factors decrease fatigue and improve recovery from fatigue in mammalian skeletal muscle in vitro. To induce fatigue, we stimulated mouse soleus and extensor digitorum longus (EDL) to perform isometric tetanic contractions (50 impulses x s(-1) for 0.5 s) at 6 contractions x min(-1) for 60 min in soleus and 3 contractions x min(-1) for 20 min in EDL. Muscles were submerged in Krebs-Henseleit bicarbonate solution (Krebs) at 27 degrees C gassed with 95% nitrogen - 5% carbon dioxide (anoxia). Before and for 67 min after the fatigue period, muscles contracted at 0.6 contractions x min(-1) in 95% oxygen - 5% carbon dioxide (hyperoxia). We added a permeable cAMP analog (N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate at 10(-3) mol x L(-1) (dcAMP)), caffeine (2 x 10(-3) mol x L(-1), or Krebs as vehicle control at 25 min before, during, or at the end of the fatigue period. In soleus and EDL, both challenges added before fatigue significantly increased developed force but only caffeine increased developed force when added during the fatigue period. At the end of fatigue, the decrease in force in challenged muscles was equal to or greater than in controls so that the force remaining was the same or less than in controls. EDL challenged with dcAMP or caffeine at any time recovered more force than controls. In soleus, caffeine improved recovery except when added before fatigue. With dcAMP added to soleus, recovery was better after challenges at 10 min and the end of the fatigue period. Thus, increased intracellular concentrations of cAMP and (or) Ca2+ did not decrease fatigue in either muscle but improved recovery from fatigue in EDL and, in some conditions, in soleus.     

Effects of caffeine at different temperatures on contractile properties of slow-twitch and fast-twitch rat muscles

The slow-twitch soleus muscle (SOL) exhibits decreased twitch tension (cold depression) in response to a decreased temperature, whereas the fast-twitch extensor digitorum longus (EDL) muscle shows enhanced twitch tension (cold potentiation). On the other hand, the slow-twitch SOL muscle is more sensitive to twitch potentiation and contractures evoked by caffeine than the fast-twitch EDL muscle. In order to reveal the effects of these counteracting conditions (temperature and caffeine), we have studied the combined effects of temperature changes on the potentiation effects of caffeine in modulating muscle contractions and contractures in both muscles. Isolated muscles, bathed in a Tyrode solution containing 0.1-60 mM caffeine, were stimulated directly and isometric single twitches, fused tetanic contractions and contractures were recorded at 35 degrees C and 20 degrees C. Our results showed that twitches and tetani of both SOL and EDL were potentiated and prolonged in the presence of 0.3-10 mM caffeine. Despite the cold depression, the extent of potentiation of the twitch tension by caffeine in the SOL muscle at 20 degrees C was by 10-15 % higher than that at 35 degrees C, while no significant difference was noted in the EDL muscle between both temperatures. Since the increase of twitch tension was significantly higher than potentiation of tetani in both muscles, the twitch-tetanus ratio was enhanced. Higher concentrations of caffeine induced contractures in both muscles; the contracture threshold was, however, lower in the SOL than in the EDL muscle at both temperatures. Furthermore, the maximal tension was achieved at lower caffeine concentrations in the SOL muscle at both 35 degrees C and 20 degrees C compared to the EDL muscle. These effects of caffeine were rapidly and completely reversed in both muscles when the test solution was replaced by the Tyrode solution. The results have indicated that the potentiation effect of caffeine is both time- and temperature-dependent process that is more pronounced in the slow-twitch SOL than in the fast-twitch EDL muscles.     

Effects of caffeine on the electrical and mechanical activity of guinea-pig ureter smooth muscle

The effects of caffeine on the electrical and mechanical activity of the guinea-pig ureter smooth muscle were studied. Under untreated conditions caffeine mainly showed inhibitory action on the ureter, inhibiting the evoked action potentials and phasic contractions as well as potassium contracture. Caffeine was also found to suppress the low-Na contracture of Na-loaded ureter muscle. It is established that Na-loaded tissue is able to generate transient contracture in response to caffeine application at 37 degrees C. These caffeine contractures could be evoked under completely removed [Ca2+]0 and in the presence of high doses of Ca-channel blockers (nifedipine, diltiazem, Mn ions) and could be reversibly blocked by tetracaine, procaine and benzocaine. Caffeine contractures could also be produced by the ureter muscle placed in isotonic K-solution. Cooling significantly potentiated low-Na, potassium and caffeine contractures of the ureter muscle. Filling of the store is totally dependent on the entry of Ca ions from the extracellular Ca2+ store sites which sequester Ca ions entering the cell on either Na-Ca exchange or via voltage operated Ca channels.     

Studies of the halothane-cooling contractures of skeletal muscle

The characteristics of transient contractures elicited by rapid cooling of frog or mouse muscles perfused in vitro with solutions equilibrated with 0.5-2.0% halothane are reviewed. The data indicate that these halothane-cooling contractures are dose dependent and reproducible, and their amplitude is larger in muscles containing predominantly slow-twitch type fibers, such as the mouse soleus, than in muscles in which fast-twitch fibers predominate, such as the mouse extensor digitorum longus. The halothane-cooling contractures are potentiated in muscles exposed to succinylcholine. The effects of Ca2+-free solutions, of the local anesthetics procaine, procainamide, and lidocaine, and of the muscle relaxant dantrolene on the halothane-cooling contractures are consistent with the proposal that the halothane-cooling contractures result from synergistic effects of halothane and low temperature on Ca sequestration by the sarcoplasmic reticulum. Preliminary results from skinned rabbit muscle fibers support this proposal. The halothane concentrations required for the halothane-cooling contractures of isolated frog or mouse muscles are comparable with those observed in serum of patients during general anesthesia. Accordingly, fascicles dissected from muscle biopsies of patients under halothane anesthesia for programmed surgery develop large contractures when rapidly cooled. The amplitude of these halothane-cooling contractures declined with the time of perfusion of the muscle fascicles in vitro with halothane-free physiological solutions. It is suggested that the halothane-cooling contractures could be used as a simple experimental model for the investigation of the effects of halothane on Ca homeostasis and contractility in skeletal muscle and for study of drugs of potential use in the management of the contractures associated with the halothane-induced malignant hyperthermia syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the actions of 4-aminopyridine, caffeine and quinine on the toad isolated rectus abdominis muscle

1. 4-Aminopyridine (4-AP)-induced contractures have been compared with those evoked by caffeine and quinine on the toad rectus abdominis muscle. 2. All three compounds produced slowly-developing sustained contractures. The time to half maximal contracture and relaxation was significantly longer for 4-AP than for caffeine or quinine. 3. Verapamil and manganese inhibited 4-AP, caffeine and quinine-induced contractures. 4. Ca2+-free-EGTA Ringer and procaine severely inhibited caffeine and quinine responses, but 4-AP contractures were relatively unaffected. 5. In depolarizing (100 mM K+) Ringer solution, caffeine and quinine responses were reduced to 6-9% of their controls. 4-AP responses were reduced by about 25%. 6. It is concluded that in the toad rectus muscle, 4-AP-induced contractures differ from those produced by caffeine and quinine, and appear to rely mainly on the release of intracellular located Ca2+, while caffeine and quinine are considered to act predominantly on plasma membrane sites.     

Influence of tenotomy on posttetanic responses of the rat fast and slow muscle

The effect of two weeks of tenotomy on posttetanic isometric contractile responses of the rat fast: Extensor digitorum longus and slow: soleus muscles was studied in experiments on isolated muscle preparations. Direct tetanic stimulation (100 impulses, 50 Hz) increased the force of contractions by 20-25% (p < 0.05) of both, control and tenotomized fast muscles. Identical to above tetanic stimulation of control, slow muscle resulted in posttetanic depression, a decrease in the amplitude of contractile responses. Tenotomized slow muscles did not develop posttetanic depression. Caffeine (4 mM) increased and dandrolene (10 microM) decreased the force of unitary and tetanic contractions of control and tenotomized muscles. Neither drug, however, affected development of posttetanic phenomena in ether fast or slow muscles. The fact that in extensor digitorum longus, posttetanic potentiation is preserved for at least forty days of tenotomy but disappears after only 2 weeks of denervation suggests important role of neurotrophic influences in regulation of posttetanic responses of fast muscles.     

Increased cAMP as a positive inotropic factor for mammalian skeletal muscle in vitro

To test the hypothesis that an increased cAMP concentration improves skeletal muscle force development, we stimulated mouse soleus and extensor digitorum longus (EDL) in the presence of isoproterenol (1 x 10(-5) mol.L-1), a beta-adrenergic agonist, or N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dcAMP) (1 x 10(-3) mol.L-1), a membrane-permeable cAMP analogue. Drugs used in the challenges were dissolved in Krebs-Henseleit bicarbonate buffer (Krebs) at 27 degrees C and gassed with 95% O2 - 5% CO2. Stimulation at 50 impulses.s-1 for 0.5 s produced an isometric tetanic contraction. Over 25 min of contractions at 0.6 contractions.min-1, developed force increased significantly with the addition of isoproterenol (soleus, 2.5% +/- 1.1%; EDL, 13.8% +/- 2.0%) or dcAMP (soleus, 2.3% +/- 0.5%; EDL, 10.9% +/- 1.9%) as compared with vehicle controls (cont) with Krebs added (soleus, 0.0% +/- 0.2%; EDL, -2.5% +/- 0.7%). To investigate the role of Ca2+ availability, we amplified or attenuated sarcolemmal L-type Ca2+ channels with Bay K 8644 (Bay K) (5.6 x 10(-6) mol.L-1) or diltiazem hydrochloride (dilt) (10(-4) mol.L-1), respectively. Ca2+ release from the sarcoplasmic reticulum was increased with caffeine (2 x 10(-3) mol.L-1) or decreased with dantrolene sodium (dant) (4.2 x 10(-7) mol.L-1). With Ca2+availability modified, dcAMP addition in soleus significantly increased force development above control (cont, 2.3% +/- 0.4%; Bay K, 4.0% +/- 1.0%; dilt, 52.3% +/- 3.6%; caffeine, 2.3% +/- 0.7%; dant, 6.0% +/- 2.0%; dilt + dant, 55.0% +/- 23.0%). In EDL, the addition of dcAMP also increased force development above control (cont, 13.7% +/- 1.9%; Bay K, 17.0% +/- 4.0%; dilt, 170.0% +/- 40.0%; caffeine, 23.0% +/- 4.0%; dant, 72.0% +/- 10.0%; dilt + dant, 54.0% +/- 14.0%). Thus, a positive inotropic effect of cAMP existed in both fast- and slow-twitch mammalian skeletal muscle with both normal and altered Ca2+ flux into the sarcoplasm.     

The effect of menadione on sarcoplasmic reticulum Ca2+ and contractions of single guinea-pig cardiomyocytes.

We investigated the effect of 2-methyl-1,4-naphtoquinone (Menadione) on sarcoplasmic reticulum (SR) Ca2+ content and electrically stimulated contractions (ESCs) of single isolated myocytes of guinea-pig ventricular myocardium. The contractures initiated by means of microinjections of caffeine into the close vicinity of the cell were used as an indirect index of the SR Ca2+ content. Superfusion of the cells for 45 min with Menadione resulted in gradual disappearance of contractile responses to caffeine, prolongation of time to peak amplitude of ESCs by 48 +/- 15% and complete inhibition of postrest and postextrasystolic potentiation. These results are consistent with those of Floreani and Carpenedo (7) who found that Menadione strongly inhibits the SR Ca2+ ATPase. Despite depletion of the SR Ca2+ the amplitude of ESCs did not change which suggests that contractions were initiated in the cells treated with Menadione by Ca2+ derived from the sources other than the SR.     

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.