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We studied the antagonism between aminophylline and two calcium channel blockers, nifedipine and verapamil, and its effect on the resting membrane potential of rat diaphragm fibers in vitro at 25 degrees C. Aminophylline hyperpolarizes the fibers in a dose-dependent manner, and the maximum effect is reached with 1 mM of the drug, approximately 9 mV compared with normal values. Both nifedipine and verapamil (1-5 microM) decreased the amount of hyperpolarization induced by aminophylline, and this is partially reversed when the xanthine concentration in the bath is increased. From the Hill equation we obtained a value of 2 for the slope, suggesting that two molecules of aminophylline bind to the receptor. Nifedipine modifies the affinity and the intrinsic activity of aminophylline, whereas verapamil reduces its intrinsic activity. The effect of nifedipine and verapamil is explained on the basis of the changed action of aminophylline on its site as a result of the interaction of the calcium channel blockers with their interdependent receptors.

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Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.

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We studied the generation of calcium action potentials (Ca APs) in innervated and denervated fibres of the extensor digitorum longus of the rat in a tetraethylammonium (TEA) sulphate solution plus 3-4 diaminopiridine (3-4 DAP). The main results are the following: (1) more than 90% of the innervated fibres were capable of developing well-sustained Ca APs that were blocked by Cd or nifedipine; (2) the incidence of Ca APs obtained from the denervated fibres was substantially lower than in the control preparations; (3) no relation was found between the appearance of Ca APs in the denervated fibres and the resting membrane potential (Vm), stimulus duration (500-2000 ms) or holding potential (-80, -100 mV); (4) The percentage of denervated fibres that exhibited Ca APs was increased significantly with the following procedures. First, by raising the external Ca concentration to 14 mM; second, by depleting the intracellular K concentration by overnight exposure of the muscles to a free K-Cs solution; (c) and third, by incubating the muscles in 500 nM apamin, a venom that inhibits the K conductance activated by Ca. Several factors may be involved in the lower incidence of Ca APs obtained in denervated fibres: (1) a diminished Ca current due to a reduction in the driving force as a result of an increment in the intracellular Ca concentration; (2) a persistence of a shunting K conductance that is not inhibited by TEA and 3-4 DAP; (3) a shift in the voltage dependence of the activation and inactivation parameters of the Ca current or the appearance of a new type of Ca channel with a different kinetics.

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We studied the effect of aminophylline (0.1-1 mM) on the mechanical and electrical activity of rat diaphragm bundles in vitro (25 degrees C). The main findings are the following. 1) Aminophylline potentiates the twitch tension. The tetanus tension is not modified, although the rate of decay is decreased. 2) The relation between K contracture tension and resting membrane potential (Vm) is shifted toward more negative values of the Vm in the aminophylline-containing solution. 3) The mechanical threshold measured by direct visualization of the fiber is also lowered in the fibers equilibrated in aminophylline. These effects are reversible by washing out the preparation with normal solution. 4) The shift in the relation between tension and Vm induced by aminophylline is reversed by 1 microM nifedipine. 5) The generation of action potential is not modified by aminophylline. We suggest that the shift in the mechanical threshold is the principal factor involved in the potentiating effect of aminophylline. We speculate that theophylline acts to augment tension by enhancing calcium release from the sarcoplasmic reticulum and that nifedipine blocks this release (E. Rios, and G. Brum. Nature Lond. 325: 717-720, 1987).

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In this work we studied the effect of ruthenium red (RR) on the mechanical and electrical properties of rat diaphragm bundles in vitro (30 degrees C). Two concentrations of RR were used: 5 and 10 microM. We measured: 1) twitches, tetanus and caffeine contracture; 2) relation between mechanical tension and resting membrane potential (Vm); 3) contraction threshold by visualization of the contraction around the stimulated area of the fiber. The main finding are the following: a) RR potentiates the twitch tension. The tetanic tension is not affected and the time course of the caffeine contracture is shortened in the RR containing solutions; b) the relationship between mechanical tension and resting membrane potential (Vm) is shifted toward more negative values of Vm in RR; c) the mechanical threshold is lowered about 7 mV in the presence of RR; d) the rates of depolarization and repolarization of the action potential are decreased in the test solutions. We suggested that the shift in the mechanical threshold and the prolongation of the action potential are the main factors involved in the potentiating effect of RR. The mechanism by which RR shifts the mechanical threshold is not known.

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We studied the effect of aminophylline and theophylline (0.1-2 mM) on the resting membrane potential (Vm) of rat diaphragm fibers in vitro (25 degrees C). The main findings are the following. 1) Aminophylline and theophylline hyperpolarize the fibers in a dose-dependent manner. This effect is present with 0.1 and 0.25 mM of aminophylline and theophylline, respectively, and the maximum effect is reached with 1 mM of the drug (approximately 5-8 mV in comparison to the normal values). This effect is reversible by washing out the preparation with normal solution. 2) Dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP, 2 mM) produces a similar increment in the Vm. 3) The hyperpolarizing action observed in the presence of aminophylline, theophylline, and DBcAMP is suppressed by 5 X 10(-4) M ouabain or by lowering the bath temperature to 5 degrees C. These results suggest that the xanthines may directly or indirectly stimulate a Na-K pump. Two possibilities may be considered: 1) an electrogenic effect of the Na-K pump and 2) a reduction in the extracellular K+ concentration in the solution contacting the external side of the cell as a consequence of the activity of the Na-K pump. Alternative mechanisms such as a reduction in Na permeability or an increment in K permeability might collaborate in the hyperpolarizing effect of the drugs tested.

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The aim of this work was to study the electrical and mechanical properties of small bundles of rat diaphragm muscle treated with two blockers of the delayed potassium rectification channels: 3,4-diaminopyridine (3,4-DAP, 2.5 mM) and tetraethylammonium (TEA, 20 mM). Twitch tension was significantly potentiated by TEA and 3,4-DAP (39% and 59% respectively). Maximal tetanic tension was not affected by both drugs. The voltage dependence of the tension vs the resting membrane potential was shifted to lower values in TEA and 3,4-DAP. 3,4-DAP increased the caffeine contracture tension (2.5-10 mM) and lowered the caffeine contracture threshold. The duration of the action potential (measured at the level of -40 mV) was increased by TEA and 3,4-DAP solutions. This change was a consequence of the decrease in the rate of repolarization of the action potential. In addition, TEA reduced the amplitude and the rate of rise of the action potential. We suggested that the increment in the duration of the action potential and the shift of the mechanical threshold to more negative values of membrane potential might be the factors involved in the twitch potentiation induced by the TEA and 3,4-DAP solutions.

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The effects of verapamil (0.01-0.1 mM) and Ca-free solutions (0 Ca, 3 mM MgCl2, 5 mM EGTA) upon mechanical and electrical properties of a fast twitch mammalian skeletal muscle (extensor digitorum longus) have been studied. Ca-free saline reduces twitch and tetanus tension. The response to single pulses was not affected in the presence of verapamil, but as the rate of stimulation increased, peak tensions were lower and a gradual decrease of tension was observed: frequency-dependent effect. Caffeine (30 mM) contracture was significantly reduced by both solutions. The fibers in Ca-free saline were depolarized (6 mV) and +dV/dt, -dV/dt and overshoot of the AP's were reduced. On the other hand, verapamil did not affect Vm. The presence of 0.1 mM verapamil hindered the ability of the fibers to generate AP's at high rates of stimulation. Lower concentration of verapamil (0.01) did not affect the repetitive electrical activity, but tetanic tension was decreased. These findings support partially the hypothesis about the dependence of mammalian skeletal muscle from external Ca. Verapamil would have several mechanisms of action: a) Ca-channel blocker action, b) local anaesthetic effect, and c) reduction of Ca release from the sarcoplasmic reticulum.

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The effects of verapamil (0.01-0.1 mM) and Ca-free solutions (0 Ca, 3 mM MgCl2, 5 mM EGTA) upon mechanical and electrical properties of a fast twitch mammalian skeletal muscle (extensor digitorum longus) have been studied. Ca-free saline reduces twitch and tetanus tension. The response to single pulses was not affected in the presence of verapamil, but as the rate of stimulation increased, peak tensions were lower and a gradual decrease of tension was observed: frequency-dependent effect. Caffeine (30 mM) contracture was significantly reduced by both solutions. The fibers in Ca-free saline were depolarized (6 mV) and +dV/dt, -dV/dt and overshoot of the APs were reduced. On the other hand, verapamil did not affect Vm. The presence of 0.1 mM verapamil hindered the ability of the fibers to generate APs at high rates of stimulation. Lower concentration of verapamil (0.01) did not affect the repetitive electrical activity, but tetanic tension was decreased. These findings support partially the hypothesis about the dependence of mammalian skeletal muscle from external Ca. Verapamil would have several mechanisms of action: a) Ca-channel blocker action, b) local anaesthetic effect, and c) reduction of Ca release from the sarcoplasmic reticulum.

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