Length dependence of staircase potentiation: interactions with caffeine and dantrolene sodium

In skeletal muscle, there is a length dependence of staircase potentiation for which the mechanism is unclear. In this study we tested the hypothesis that abolition of this length dependence by caffeine is effected by a mechanism independent of enhanced Ca2+ release. To test this hypothesis we have used caffeine, which abolishes length dependence of potentiation, and dantrolene sodium, which inhibits Ca2+ release. In situ isometric twitch contractions of rat gastrocnemius muscle before and after 20 s of repetitive stimulation at 5 Hz were analyzed at optimal length (Lo), Lo - 10%, and Lo + 10%. Potentiation was observed to be length dependent, with an increase in developed tension (DT) of 78 +/- 12, 51 +/- 5, and 34 +/- 9% (mean +/- SEM), at Lo - 10%, Lo, and Lo + 10%, respectively. Caffeine diminished the length dependence of activation and suppressed the length dependence of staircase potentiation, giving increases in DT of 65+/-13, 53 +/- 11, and 45 +/- 12% for Lo - 10%, Lo, and Lo + 10%, respectively. Dantrolene administered after caffeine did not reverse this effect. Dantrolene alone depressed the potentiation response, but did not affect the length dependence of staircase potentiation, with increases in DT of 58 +/- 17, 26 +/- 8, and 18 +/- 7%, respectively. This study confirms that there is a length dependence of staircase potentiation in mammalian skeletal muscle which is suppressed by caffeine. Since dantrolene did not alter this suppression of the length dependence of potentiation by caffeine, it is apparently not directly modulated by Ca2+ availability in the myoplasm.     

Effects of pH on the length-dependent twitch potentiation in skeletal muscle.

When muscle is elongated, there is a length dependence of twitch potentiation and an increased Ca(2+) sensitivity of the myofilaments. Changes in the charge potential of myofilaments, induced by a decrease in pH, are known to abolish the length dependence of Ca(2+) sensitivity. This study was aimed at testing the hypothesis that a decrease in pH, and the concomitant loss of length dependence of Ca(2+) sensitivity, depresses the length dependence of staircase potentiation. In vitro, isometric twitch contractions of fiber bundles dissected from the mouse extensor digitorum longus, performed before and after 10 s of 10-Hz stimulation (i.e., the staircase potentiation protocol) were analyzed at five different lengths, ranging from optimal length for maximal force production (L(o); = 12 +/- 0.7 mm) to L(o) + 1.2 mm (L(o) + 10%). These measurements were made at an extracellular pH of 6.6, 7.4, and 7.8 (pH changes induced by altering the CO(2) concentration of the bath solution). At pH 7.4 and 7.8, the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased fiber bundle length (r(2) = 0.95 and r(2) = 0.99, respectively). At pH 6.6, the length dependence of potentiation was abolished, and the slope of the length-potentiation relationship was not different from zero (r(2) = 0.05). The results of this study indicate that length dependence of potentiation in intact skeletal muscle is abolished by lowering the pH. Because decreasing the pH decreases Ca(2+) sensitivity and changes the charge potential of the filaments, the mechanism of length-dependent potentiation may be closely related to the length dependence of Ca(2+) sensitivity, and changes in the charge potential of the myofilaments may be important in regulating this relationship.     

Length-dependent twitch contractile characteristics of skeletal muscle

The length dependence of force development of mammalian skeletal muscles was evaluated during twitch, double-pulse, and tetanic contractions, and the relation between muscle length and the time-dependent characteristics of twitch and double-pulse contractions were determined. In situ isometric contractions of the rat gastrocnemius muscle were analyzed at seven different lengths, based on a reference length at which the maximal response to double-pulse contractions occurred (Lopt-2P). Twitch and double-pulse contractions were analyzed for developed tension (DT), contraction time (tC), average rate of force development (DT-tC(-1)), half-relaxation time (t50%R), peak rate of relaxation (DT x dtmin(-1)), and 90%-relaxation time (t90%R). Considering the length at which maximal tetanic DT occurred to be the optimal length (Lo-TET), the peak DT for twitch contractions and double-pulse contractions was observed at Lo-TET + 0.75 mm (p < 0.05) and Lo-TET + 0.1 mm (p > 0.05), respectively. When measured at the length for which maximal twitch and double-pulse contractions were obtained, tetanic DT was 95.2 +/- 3 and 99.0 +/- 2% of the maximal value, respectively. These observations suggest that double-pulse contractions are more suitable for setting length for experimental studies than twitch contractions. Twitch and double-pulse contraction tC were 15.53 +/- 1.14 and 25.0 +/- 0.6 ms, respectively, at Lopt-2P, and increased above Lopt-2P and decreased below Lopt-2P. Twitch t50%R was 12.18 +/- 0.90 ms at Lopt-2P, and increased above Lopt-2P and below Lopt-2P. Corresponding changes for double-pulse contractions were greater. Stretching the muscle leads to slower twitch contractions and double-pulse contractions, but the mechanisms of this change in time course remain unclear.     

The shape of the force-elbow angle relationship for maximal voluntary contractions and sub-maximal electrically induced contractions in human elbow flexors

The force-length relationship is a basic property of skeletal muscle. Knowledge of this relationship is necessary for most analyses of human movement, and in simulation models predicting movement control strategies. Studies on animal muscles have shown that force-length relationships for sub-maximal contractions are not related through a simple scaling procedure to the relationship for maximal contractions. Furthermore, potentiation might produce a shift of sub-maximal relative to maximal force-length relationships. In this study, we tested the hypothesis that human force-elbow angle relationships for sub-maximal unpotentiated contractions are shifted to larger elbow angles (i.e. larger muscle lengths) compared to the relationship for maximal voluntary contractions (MVC), and that this shift is reduced, or even abolished, for sub-maximal potentiated contractions. Force-elbow angle relationships (48-160 degrees) were obtained from healthy subjects (n=13). At each of nine tested elbow angles, the test set consisted of a single twitch (ST(pre)) and a doublet twitch (DT(pre)) stimulation of m. biceps brachii, followed by an MVC, followed by another single twitch (ST(post)) and a doublet twitch (DT(post)) stimulation. The single and doublet twitches induced sub-maximal contractions. The force-elbow angle relationships for the pre-MVC (unpotentiated) twitch contractions were shifted to larger angles compared to those obtained for MVC. The force-elbow angle relationships for the post-MVC (potentiated) twitch contractions were shifted to smaller angles compared to those obtained for the unpotentiated twitch contractions. These results support the idea that the shift to larger muscle lengths for the sub-maximal, unpotentiated force-length relationships relative to the relationship for maximal contractions may be caused by a length-dependent Ca(2+) sensitivity that may be offset, at least in part, by potentiation.     

Effects of caffeine on the mechanical properties of developing rat heart ventricles

The effects of caffeine on isometric contractions of right-ventricular strips during the postnatal development of the rat heart were studied. Caffeine (2-10 mM) had a positive inotropic effect on ventricular strips of 3-15-days-old rats but a negative inotropic effect on the muscles of rats older than 22 days. Rest-twitch potentiation was most prominent in the muscles of 3-15-days-old rats but weakened clearly after that age. The potentiation was eliminated by 5 and 10 mM caffeine. An abrupt increase in frequency from 0.2 to 2 Hz caused a positive tension-staircase in 3-15-days-old rats but a negative staircase in older rats, the latter effect being eliminated by caffeine. It is suggested that the observed changes during the third postnatal week are due to a shift from extracellular to intracellular Ca2+ as the main source of this cation.     

Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

Sarcomere length-dependence of activity-dependent twitch potentiation in mouse skeletal muscle

Background  

It has been reported that potentiation of a skeletal muscle twitch response is proportional to muscle length with a negative slope during staircase, and a positive slope during posttetanic potentiation. This study was done to directly compare staircase and posttetanic responses with measurement of sarcomere length to compare their length-dependence.     

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+.     

Caffeine potentiates low frequency skeletal muscle force in habitual and nonhabitual caffeine consumers.

The mechanism of action underlying the ergogenic effect of caffeine is still unclear. Caffeine increases the force of muscular contraction during low-frequency stimulation by potentiating calcium release from the sarcoplasmic reticulum. Studies have also suggested an enhancement of lipid oxidation and glycogen sparing as potential mechanisms. Given that several studies have found an ergogenic effect of caffeine with no apparent metabolic effects, it is likely that a direct effect upon muscle is important. Twelve healthy male subjects were classified as habitual (n = 6) or nonhabitual (n = 6) caffeine consumers based on a 4-day diet record analysis, with a mean caffeine consumption of 771 and 14 mg/day for each group, respectively. Subjects were randomly allocated to receive caffeine (6 mg/kg) and placebo (citrate) in a double-blind, cross-over fashion approximately 100 min before a 2-min tetanic stimulation of the common peroneal nerve in a custom-made dynamometer (2 trials each of 20 and 40 Hz). Tetanic torque was measured every 30 s during and at 1, 5, and 15 min after the stimulation protocol. Maximal voluntary contraction strength and peak twitch torque were measured before and after the stimulation protocol. Caffeine potentiated the force of contraction during the final minute of the 20-Hz stimulation (P<0.05) with no effect of habituation. There was no effect of caffeine on 40-Hz stimulation strength nor was there an effect on maximal voluntary contraction or peak twitch torque. These data support the hypothesis that some of the ergogenic effect of caffeine in endurance exercise performance occurs directly at the skeletal muscle level.     

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.     

Interaction between the sarcoplasmic reticulum and mitochondria in the control of contractile calcium in guinea-pig atria--does it exist?

The inhibitory action of caffeine on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) and interference with mitochondrial (Ca2+) fluxes by a mitochondrial uncoupler protonfore CCCP were utilized to define a calcium pool responsible for potentiation of post-rest twitch tension in guinea-pig atria. The Ca2+ fluxes were assessed by means of 45Ca2+. Caffeine and CCCP when applied separately did not affect post-rat 45Ca2+ content. Yet, when they were applied together it was markedly reduced to the resting level. It is concluded that a possible source of contractile Ca2+ may be located in mitochondria and an eventual shift of Ca2+ between mitochondria and the SR seems to be a plausible assumption.     

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.     

Verapamil and zero Ca2+ alter responses of cat muscle to halothane and caffeine

Strips of soleus (slow twitch, oxidative) and gracilis (fast-twitch, glycolytic) muscle were obtained from 27 anesthetized cats and mounted in organ baths filled with oxygenated Krebs-Ringer solution (37 degrees C). The responses to caffeine, halothane (1%), caffeine in the presence of halothane, and electrical stimulation in the presence of halothane were examined in the two fiber types. These responses were compared with those observed in paired strips of muscle that had been treated with verapamil (10 or 28 microM), a slow calcium (Ca2+) channel blocker, with zero Ca2+, or with zero Ca2+ where magnesium (3.7 mM Ca2+) was added to replace the Ca2+. Halothane-induced contractures in the soleus were blocked by verapamil and zero Ca2+. Caffeine-induced contractures and tetanic contractions were attenuated in zero Ca2+ and by verapamil in both fiber types. Halothane overcame verapamil-induced reductions of caffeine contractures and tetanic contractions in both fiber types. In contrast, halothane did not overcome zero Ca2+-induced reductions in caffeine contractures or tetanic contractions in either fiber type. Furthermore, the addition of Mg2+ to the zero Ca2+ did not restore the responses. The findings with verapamil indicate that in cat muscle, both halothane- and caffeine-induced contractures and tetanic contractions are dependent on the influx of extracellular Ca2+. This extracellular Ca2+ may enter through the slow Ca2+ channels. However, because halothane in combination with caffeine or electrical stimulation overcame the effects of verapamil, there may be other sites involved.     

Effects of caffeine, verapamil, lithium, and KB-R7943 on mechanics and energetics of rat myocardial bigeminies

We examined the effects of pharmacological alteration of Ca2+ sources on mechanical and energetic properties of paired-pulse ("bigeminic") contractions. The fraction of heat release that is related to pressure development and pressure-independent heat release were measured during isovolumic contractions in arterially perfused rat ventricles. The heat released by regular and bigeminic contractions showed two brief pressure-independent components (H1 and H2) and a pressure-dependent component (H3). We used the ratio of active heat (Ha') to pressure-time integral (PtI) and the ratio of H3 to PtI to estimate the energetic cost of muscle contraction (overall economy) and pressure maintenance (contractile economy), respectively. Neither of these ratios was affected by stimulation pattern. Caffeine (an inhibitor of sarcoplasmic reticulum function) significantly decreased mechanical responses and increased the energetic cost of contraction (delta = 101 +/- 12.6%). Verapamil (an L-type Ca2+ channel blocker) decreased pressure maintenance of extrasystolic (delta = 43.4 +/- 3.7%) and postextrasystolic (delta = 37.5 +/- 3.5%) contractions without affecting postextrasystolic potentiation, suggesting that a verapamil-insensitive fraction is responsible for potentiation. The verapamil-insensitive fraction was further studied in the presence of lithium (45 mM) and KB-R7943 (5 microM), inhibitors of the Na+/Ca2+ exchanger. Both agents decreased all mechanical responses, including postextrasystolic potentiation (delta = 67.3 +/- 3.3%), without altering overall or contractile economies, suggesting an association of the verapamil-insensitive Ca2+ fraction to the sarcolemmal Na+/Ca2+ exchanger. The effect of the inhibitors of the Na+/Ca2+ exchanger on potentiation suggests an increased participation of extracellular Ca2+ (and, thus, a redistribution of the relative participation of the Ca2+ pools) during bigeminic contractions in rat myocardium.     

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.     

Rat atrial muscle responses with caffeine: dose-response, force frequency, and postrest contractions.

Caffeine has been reported to have a positive and (or) a negative inotropic effect on cardiac muscle. In this study, the force-frequency and postrest characteristics of rat atrium were studied in the presence of caffeine (1.0-10 mM) to see if the interval between beats affected the response of cardiac muscle to caffeine. When stimulation frequency was 0.5 or 2.0 Hz, there was a positive followed by a negative inotropic response with 1, 5, or 10 mM caffeine. Incomplete relaxation occurred under these circumstances, giving rise to contracture. At low frequency of stimulation (0.1 Hz) caffeine had only a negative inotropic effect, and this effect was greater with 1 mM caffeine than with 5 mM caffeine. In the absence of caffeine, when stimulation at 0.5 or 3 Hz was interrupted, a pause of 2-20 s resulted in potentiation. When caffeine was present (2.0 mM), postrest potentiation was severely attenuated, but the steady-state contraction amplitude within the range 0.5-3.0 Hz was not different. These results are consistent with the hypothesis that caffeine induces a leak of Ca2+ from the sarcoplasmic reticulum, and this Ca2+ is extruded from the cell, possibly by Na+/Ca2+ exchange. Sarcoplasmic reticular uptake of Ca2+ and the translocation to release sites appear not to be affected by caffeine within 1-5 mM concentrations.     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.     

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.     

Effect of caffeine and glycerin on the Ca transport system of sarcoplasmic reticulum fragments from frog skeletal muscles

Parameters of the Ca2+-ion transport system by a fragmented sarcoplasmic reticulum isolated from phasic and tonic frog skeletal muscles were investigated under the action of caffeine or caffeine in combination with glycerol. No changes were observed in the Ca-transport system of a light fraction of the sarcoplasmic reticulum under the influence of caffeine and caffeine-glycerol combination. Caffeine reduced the value of Ca/ATP and enhanced the outflux of Ca2+-ions from membrane fragments of the caffeine-sensitive sarcoplasmic reticulum fraction of both the muscles; the combined effect of caffeine and glycerol was analogous to the action of caffeine applied alone. It is concluded that the potentiation of muscle contraction in the presence of glycerol is not due to the excess of Ca-release from the sarcoplasmic reticulum caused by this agent.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of caffeine on the intracellular [Ca2+] transient, membrane currents, and contraction

The effects of caffeine on tension, membrane potential, membrane currents, and intracellular [Ca2+], measured as the light emitted by the Ca2+-activated photoprotein aequorin, were studied in canine cardiac Purkinje fibers. An initial, transient, positive inotropic effect of caffeine was accompanied by a transient increase in the second component of the aequorin signal (L2) but not the first (L1). In the steady state, 4 or 10 mM caffeine always decreased twitch tension and greatly reduced both L1 and L2. At a concentration of 2 mM, caffeine usually reduced but occasionally increased the steady state twitch tension. However, 2 mM caffeine always reduced both L1 and L2. Caffeine eliminated the diastolic oscillations of intracellular [Ca2+] induced by high extracellular [Ca2+]. In voltage-clamp experiments, 10 mM caffeine reduced the transient outward current and the peak tension elicited by step depolarization from a holding potential of -45 mV. In the presence of 20 mM Cs+, 10 mM caffeine reduced slow inward current. However, the time course of this reduction was far slower than that in tension and light observed in separate experiments. The simplest explanation of the results is that caffeine inhibits the sequestration of Ca2+ by the sarcoplasmic reticulum. The results also suggest that in Purkinje fibers caffeine increases the sensitivity of the myofilaments to Ca2+.