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.     

Functioning of the electromechanical connection in the course of the contracture contraction

The effects of calcium release blocker dantrolene was tested on electrically evoked twitches and on contractures induced by potassium depolarization, by acetylcholine or caffeine. It was shown that the first: developmental, stage of potassium or acetylcholine contracture is inhibited by dantrolene and is not influenced by calcium free medium, therefore we may interpret it as based on a "voltage-dependent Ca release" (VDCR) mechanism of activation, whereas depolarization directly opens the rhyanodin receptor calcium channels. On the contrary, the next stage: the long-lasting plateau of contracture, is directly dependent on external Ca2+ and inhibited by dantrolene, and therefore can be described as "calcium induced Ca-release" (CICR) activation mechanism. In this case stored calcium is also released by rhyanodine receptors, although by means of entering the extracellular Ca2+. Finally, the last stage of low amplitude is not influenced by dantrolene nor by calcium-free medium. Therefore the activation of contraction on this stage is not based on the Ca2+ release through the rhyanodin receptor calcium channels.     

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.     

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.     

Changes in the electromechanical activity in the course of tetanic contraction

The functioning of excitation-contraction coupling during tetanic contraction was investigated on frog skeletal muscle. The effect of the calcium release blocker dantrolene was tested on electrically evoked twitches and tetanic contractions. It was shown that the first: developmental stage of tetanus is inhibited by dantrolene as well as a twitch contraction, and does not influenced by calcium-free medium. This substantiates it as based on "voltage dependent Ca-release" (VDCR) mechanism of activation, when depolarization directly opens the rhyanodin receptor calcium channels. The next stage: the long lasting plateau of tetanic contraction, is directly dependent on external Ca2+ entry and also inhibited by dantrolene, and therefore may be described as "calcium-induced Ca-release" (CICR) activation mechanism. It is proposed that such change in ECC mechanism taking place during tetanic contraction, can occur also in conditions of natural muscle activity, because of its rhythmical nature.     

Excitatory effects of cholinergic,adrenergic and glutaminergic agonists on a buccal muscle of Aplysia

The anterior extrinsic protractors in the buccal mass of Aplysia are symmetrical sheets of branching bundles of muscle fibers which form an electrical syncytium. The addition of potassium or cholinergic, adrenergic, and glutaminergic agonists to the sea water bathing medium produces contracture of the muscle. Strychnine and cholinergic or adrenergic antagonists all block contractures produced by cholinergic and adrenergic agonists but not those produced by potassium or glutamate. Iontophoretic application through microelectrodes of acetylcholine or dopamine anywhere on the muscle surface produced a graded depolarization of the membrane. By contrast, glutamate produces depolarization only at discrete membrane sites. Endogeneous contractions often appeared spontaneously or could be induced by drug exposure. ATP inhibits endogeneous contractions.     

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.     

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.     

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.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

Effect of Some Calcium Antagonists on Muscarinic Receptor-Mediated Cyclic GMP Formation

Several calcium antagonists were screened for their effect on muscarinic acetylcholine receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). Mn2+, Ni2+, and verapamil rapidly antagonized the response noncompetitively, with the following order of potency: verapamil greater than Mn2+ greater than Ni2+. The effects of Mn2+ and Ni2+, but not those of verapamil, were largely reversed by increasing extracellular calcium concentration. Additional effects of these agents included displacement of [3H]quinuclidinyl benzilate binding by verapamil and elevation of cyclic GMP levels by Mn2+ and Ni2+ in the absence of agonists. These results are in support of the hypothesis that receptor-mediated cyclic GMP formation by these cells is dependent upon entry of calcium into the cell and demonstrate the complexity of the effects of calcium antagonists.     

Agonist interactions at the calcium pools in skinned and unskinned canine tracheal smooth muscle

We studied the functionally discrete calcium sources used by acetylcholine, 5-hydroxytryptamine, histamine and high K+ in the dog tracheal smooth muscle. The extracellular calcium dependence of their responses was assessed by altering the calcium and by pretreatment with the calcium antagonist, nifedipine. The intracellular calcium pool was assessed by studying the interactions between caffeine and the agonists in both skinned and unskinned preparations. The extent of overlap for the different calcium pools between the various agonists was determined by studying the dose-response relationships of these agents before and after pretreatment with another agonist, i.e., the conditioning agonist, in zero calcium conditions. The rank order of sensitivity to calcium removal and to nifedipine was histamine greater than KCl greater than 5-hydroxytryptamine greater than acetylcholine. Caffeine-induced atenuation of the agonist responses was predominantly through physiological antagonism. However, the caffeine responses in unskinned fibres were augmented by pretreatment with the agonists through both nifedipine-sensitive (as with KCl) and -insensitive (as with acetylcholine) mechanisms. The responses to acetylcholine and caffeine were inhibited by theophylline and forskolin. In the skinned muscle fibres, the pCa-tension relationship suggested high calcium sensitivity, a significant caffeine-sensitive calcium pool, and no evidence of calcium release by exogenous inositol trisphosphate. The results are consistent with multiple extracellular and intracellular calcium sources for the agonist responses. We observed considerable overlap of the calcium sources used by these agonists. Of the four agonists studied, histamine appeared to inhibit the release and sequestration of calcium utilized by the other agonists most effectively.     

Effect of different calcium modulators on motilin-induced contractions of the rabbit duodenum. Comparison with acetylcholine

Motilin and acetylcholine (ACh) have a direct contractile effect on rabbit small intestinal smooth muscle. To explore the role of calcium influx in these contractions, we studied the effect of extracellular calcium concentration and of calcium antagonists on the response of longitudinal muscle preparations from rabbit duodenum. Motilin- (10(-7) M) and ACh- (10(-4) M)-induced contractions were abolished in Ca2+-depleted medium. ACh (10(-4) M) or motilin (10(-8) and 10(-7) M) increased the contractile response to added Ca2+ to 130 +/- 6%, 129 +/- 10% and 145 +/- 5% of the maximal response to Ca2+ added alone (10 mM in a cumulative concentration response curve). The sensitivity to Ca2+ was greater in the presence of ACh and motilin (EC50 = 1.0 and 1.1 mM Ca2+) than in the absence of any agonist (1.7 mM). In cumulative concentration response (CCR) curves for motilin and ACh, pD2'-values were 7.0 and 6.6 for diltiazem, 8.4 and 7.8 for verapamil (two calcium entry blockers), 5.6 and 5.2 for TMB-8 (an inhibitor of intracellular calcium), 5.3 and 5.2 for TFP (a calmodulin-antagonist). All CCR-curves showed metactoid-like action of the antagonistic drugs. We conclude that ACh and motilin cause calcium to enter the smooth muscle cell. They are probably operating via separate channels, and use a mechanism which differs from K+-induced influx. Intracellular calcium stores appear to play a minor role in these contractions.     

Effects of cobalt, magnesium, and cadmium on contraction of rat soleus muscle.

The effects on isometric tension of three divalent ions that block calcium channels, magnesium, cobalt, and cadmium, were tested in small bundles of rat soleus fibers. Cobalt, at a concentration of 2 or 6 mM, reversibly depressed twitch and tetanic tension and the depression was much greater in solutions containing no added calcium ions. Magnesium caused much less depression of tension than cobalt. The depression of tension was not accompanied by membrane depolarization or a reduction in the amplitude of action potentials. A reduction caused by 6 mM cobalt in the amplitude of 40 or 80 mM potassium contractures was not accompanied by a comparable reduction in tension during 200 mM potassium contractures, and could be explained by a shift in the potassium contracture tension-voltage curve to more positive potentials (by +7 mV on average). Similar effects were not seen with 2 or 6 mM magnesium. At a concentration of 20 mM, both cobalt and magnesium depressed twitch and tetanic tension, cobalt having greater effect than magnesium. Both ions shifted the potassium contracture tension-voltage curve to the right by +5 to +10 mV, caused a small depression of maximum tension, and slowed the time course of potassium contractures. Cadmium (3 mM) depressed twitch, tetanic, and potassium contracture tension by more than 6 mM cobalt, but experiments were complicated by the gradual appearance of large contractures that became even larger, and sometimes oscillatory, when the solution containing cadmium was washed out. It was concluded that divalent cations affect both activation and inactivation of tension in a manner that cannot be completely explained by a change in surface charge.     

Effect of various agents on the cytoplasmic calcium concentration in cultured human muscle cells

1. We determined the cytoplasmic Ca2+ concentration ([Ca2+]i) in cultured human muscle cells using the fluorescent indicator Quin-2. 2. The [Ca2+]i was dependent on the external Ca2+ concentration. Acetylcholine in the presence of external Ca2+ caused a transient increase in [Ca2+]i. Inhibition by nifedipine indicated that this response was mediated through activated voltage-operated channels. In nominally Ca2(+)-free buffer acetylcholine did not markedly increase [Ca2+]i. Therefore, the increase in [Ca2+]i as a response to depolarization is mainly due to influx of external Ca2+. 3. Various concentrations of caffeine did not influence the [Ca2+]i. Dantrolene decreased [Ca2+]i, both in the presence and absence of external Ca2+. The reduction probably resulted from an action of dantrolene on the intracellular Ca2+ stores, since dantrolene did not influence 45Ca2+ influx or efflux and caffeine partially counteracted the reduction.     

Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.     

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.     

Tension in Isolated Frog Muscle Fibers Induced by Hypertonic Solutions

The effect of hypertonic solutions on the tension of isolated twitch muscle fibers of the frog has been investigated. Increased tonicity up to about 1.7 times normal (1.7 T) caused a very small, graded, maintained tension increase. Above about 1.7 T a large, transient contracture response was superimposed on the small tension. The contracture response was graded with tonicity and reached a maximum at 2.5 T of 108 ± 25 mN·mm² a third of the maximum tetanic tension in isotonic solution. Contracture tension developed with a delay which decreased with increased tonicity. The contracture threshold was lower and the delay shorter in small fibers than in large. Contractures were obtained equally well in depolarized as in polarized fibers. They were completely suppressed by 0.1–0.5 mM tetracaine. The possible mechanism responsible for the tension-inducing effect of hypertonic solutions is discussed in terms of the close similarity between the properties of these contractures and those caused by caffeine, and it is suggested that the effect is due to a release of calcium from internal stores.     

Paradoxical electromechanical effect of lanthanum ions in cardiac muscle cells.

Although lanthanum ions (La+++) block calcium influx in cardiac cells, they may paradoxically accentuate the sodium-free contracture. We have therefore studied the effects of La+++ on the zero sodium response in chick embryonic myocardial cell aggregates. Zero sodium alone causes: (a) A maintained contracture; (b) Asynchronous localized contractions that are selectively inhibited by caffeine or ryanodine, and presumably reflect release of calcium from the sarcoplasmic reticulum; (c) A nonspecific conductance increase that is ascribable to calcium-activated ion channels. Addition of La+++ potentiates the sodium-free contracture, and causes similar potentiation of the localized contractions and the conductance increase. All three phenomena occur 5-10-fold faster in 1 mM La+++ than in sodium-free fluid alone. In contrast, when La+++ is combined with caffeine or ryanodine, the zero sodium response is suppressed. We conclude that the paradoxical effect of La+++ on the contracture is not due to calcium influx, but to enhancement, or disinhibition of intracellular calcium release. Relaxation of normal myocardium may involve control of spontaneous calcium release by lanthanum- and sodium-sensitive calcium transport across the surface membrane.     

Blockade of K+ contractures in skeletal muscle by opioid drugs: a nonstereospecific effect

The effect of several opioid drugs was tested on the K+ contractures in frog's skeletal muscle. These contractures are produced by the entrance of extracellular Ca2+ ions via the voltage-dependent, slow Ca2+ channels located in the T tubules. Morphine and other opioid agonists in concentrations ranging from 10(-10) to 10(-5) M inhibited K+ contractures. The stereoisomers, dextrorphan and levorphanol, were found to have identical potency in inhibiting high K+ contractures, suggesting that this was a nonstereospecific blockade of voltage-dependent calcium channels by the opioid drugs despite the low effective drug concentrations. In agreement with this conclusion it was found that the inhibition of K+ contractures by the opioids was not antagonized by naloxone. It also was observed using a sucrose gap apparatus that these opioid drugs in concentrations used to block the high K+ contractures did not reduce the K+-induced membrane depolarization. Raising the bathing solution Ca2+ concentration from 1.08 to 5 mM produced a reversal of the opioid-induced block of K+ contractures. Finally it was shown that while opioids completely blocked K+ contractures, they did not produce any effect on caffeine contractures showing that opioids do not deplete intracellular Ca2+ stores or inhibit the release of Ca2+ from intracellular sarcoplasmic reticulum stores. It was concluded that several opioid drugs in very low concentrations block K+ contractures in frog's skeletal muscle by a nonstereospecific block of voltage-dependent slow calcium channels.