The origin of (+)-tubocurarine resistance in dystrophic mice.

Intracellular recording, twitch responses and radio-ligand binding techniques were used to study the causes of resistance to (+)-tubocurarine (curare) of extensor digitorum longus (EDL) muscles from dystrophic mice (129 ReJ/strain). The indirectly evoked twitch response of muscles from dystrophic mice was more resistant to block by curare than the twitch response of muscles from normal littermates. The IC50 (concentration producing 50% inhibition of stimulus-evoked contractions) values for the curare block of muscle twitch were 0.78 +/- 0.03 microM and 1.32 +/- 0.05 microM (mean +/- 95% confidence limits) for muscles from normal and dystrophic mice, respectively. There was no difference between muscles from normal and dystrophic mice in the number of alpha-bungarotoxin binding sites per endplate. The amplitudes of both spontaneous miniature endplate potentials (m.e.p.ps) in unblocked preparations and of evoked endplate potentials (e.p.ps) in 1.91 microM curare were greater in muscles from dystrophic mice than in muscles from normal mice. The ratio dystrophic/normal was greater for the e.p.p. amplitudes than for the m.e.p.p. amplitudes. The quantum content of e.p.ps in magnesium-blocked and in cut-fibre preparations was greater in muscles from dystrophic mice than in muscles from normal littermates. Calculation of the binomial parameters n and p in the cut-fibre preparations indicated that this increased quantum content was caused by an increase in the value of p. It is concluded that at least part of the increased resistance to curare of the indirectly evoked twitch response of muscles from dystrophic mice is due to an increase in the quantum content of e.p.ps in these muscles.

The relaxant action of nicorandil in guinea-pig isolated trachealis.

Nicorandil (1-1000 mumol l-1) caused concentration-dependent relaxation of guinea-pig isolated trachealis. Propranolol (1 mumol l-1) did not modify the relaxant action of nicorandil but antagonized isoprenaline. Among K+-channel inhibitors tested, apamin (0.1 mumol l-1) and procaine (5 mmol l-1) did not modify the relaxant action of nicorandil. In contrast, tetraethylammonium (TEA, 8 mmol l-1) caused five fold antagonism. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Nicorandil relaxed the K+-depolarized tissue though its concentration-effect curve was shifted markedly to the right. In tissues in which tone was induced by histamine, methylene blue (100 mumol l-1) antagonized nicorandil and sodium nitroprusside but did not modify the relaxant action of aminophylline. Intracellular electrophysiological recording showed that nicorandil (1 mumol l-1) could evoke some relaxation in the absence of electrical changes. Higher concentrations (10-1000 mumol l-1) reduced the amplitude and frequency of spontaneous electrical slow waves. Nicorandil also caused concentration-dependent hyperpolarization and relaxation. When the hyperpolarization was sufficiently pronounced slow wave activity was abolished. TEA (8 mmol l-1) induced slow waves which were surmounted by a spike potential. TEA slightly reduced the maximal hyperpolarization induced by nicorandil and increased the time required for nicorandil to abolish slow wave discharge. Procaine (5 mmol l-1) induced slow waves of relatively low frequency. Sometimes these were surmounted by a spike potential Procaine markedly reduced the hyperpolarization induced by nicorandil and increased the time required for abolition of slow waves. In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, nicorandil (1000 mumol l-1) increased the efflux rate constant, whereas isoprenaline (1 mumol l-1) was without effect. It is concluded that nicorandil-induced relaxation does not involve the activation of beta-adrenoceptors but is partly attributable to the formation of nitric oxide from the nitrate moiety in its molecular structure. Nicorandil can evoke relaxation in the absence of membrane potential change but towards the upper end of its effective concentration range, nicorandil increases membrane K+ conductance and thereby evokes hyperpolarization of trachealis cells. The K+ channels opened by nicorandil are permeable to 86Rb, insensitive to apamin and TEA but may be inhibited by procaine.

Inhibitory responses to nicotine and transmural stimulation in hyoscine-treated guinea-pig isolated trachealis: an electrical and mechanical study.

Guinea-pig isolated trachealis muscle treated with hyoscine (1 microM) exhibited mechanical tone which could be suppressed by transmural stimulation and, in a concentration-dependent manner, by nicotine (10-1000 microM). Hexamethonium (500 microM) did not itself cause tone changes, antagonized effects of nicotine but did not antagonize those of isoprenaline. Tetrodotoxin (0.3 microM) did not itself cause tone changes, did not modify the action of isoprenaline but antagonized the effects of nicotine and very markedly reduced responses to transmural electrical stimulation. Guanethidine (50 microM) did not itself cause tone changes, potentiated the action of isoprenaline, antagonized effects of nicotine and reduced responses to transmural electrical stimulation. Propranolol (1 microM) did not itself cause tone changes, antagonized effects of both isoprenaline and nicotine and reduced responses to transmural electrical stimulation. Propranolol (10 microM) caused greater antagonism of isoprenaline but did not further antagonize nicotine or further reduce responses to electrical stimulation. Intracellular electrophysiological recording from hyoscine-treated trachealis showed that 10 microM nicotine caused little or no mechanical or electrical change. Higher concentrations (100 microM and 1 mM) evoked relaxation which was often though not invariably accompanied by transient hyperpolarization and transient inhibition of electrical slow waves in the impaled cell. Hexamethonium (500 microM), tetrodotoxin (0.3 microM), guanethidine (50 microM) and propranolol (1 microM) each suppressed the electrical or mechanical changes evoked by nicotine (100 microM). However, nicotine (1 mM) tested in the presence of propranolol (1 microM), caused relaxation which could be accompanied by slow wave suppression but not by change in resting membrane potential. Transmural stimulation of hyoscine-treated trachea with single pulses of supramaximal voltage and 0.5 ms duration evoked neither relaxation nor membrane potential changes. Stimulation with similar pulses in trains of 5 s duration evoked relaxation which was dependent on pulse frequency. In many cells this relaxation was not accompanied by membrane potential change. In other cells suppression of slow waves occurred. At high pulse frequencies (greater than 16 Hz) this was generally accompanied by membrane hyperpolarization. In tissue treated with hyoscine and propranolol (both 1 microM), transmural stimulation with pulse trains as described above always evoked relaxation but no membrane potential changes were observed. 10 It is concluded that nicotine and transmural stimulation can excite intramural noradrenergic nerves in guinea-pig trachea and thereby evoke relaxation. The membrane potential changes (slow wave suppression and hyperpolarization) are similar to those evoked by the administration of agonists at beta-adrenoceptors. Nicotine and transmural stimulation also excite non-adrenergic non-cholinergic inhibitory (NANCI) nerves. The relaxation evoked by the NANCI neurotransmitter is accompanied by little, if any, membrane potential change.

Electrophysiological and other aspects of the relaxant action of isoprenaline in guinea-pig isolated trachealis.

In guinea-pig isolated trachealis isoprenaline (0.001-0.1 mumol l-1) caused concentration-dependent relaxation. Propranolol (1 mumol l-1) antagonized the effects of isoprenaline by more than 100 fold but did not modify the relaxant action of sodium nitrite. The tracheal relaxant actions of isoprenaline and ATP were unaffected by apamin (0.1 mumol l-1) but apamin profoundly antagonized the effects of noradrenaline and ATP on guinea-pig isolated taenia caeci. Tetraethylammonium (TEA; 8 mmol l-1) and procaine (5 mmol l-1) each evoked tracheal spasm but neither agent antagonized the isoprenaline-evoked relaxation of the trachealis. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Both isoprenaline and sodium nitrite relaxed the K+-depolarized tissue though concentration-effect curves for both relaxants were moved to the right compared to those obtained in non-depolarized tissues. The maximal effect of sodium nitrite was markedly reduced. Intracellular electrophysiological recording showed that isoprenaline (0.01-1 mumol l-1) caused hyperpolarization and reduced or abolished slow wave discharge in trachealis muscle. These effects were accompanied by relaxation. Propranolol (1 mumol l-1) virtually abolished both the electrical and mechanical responses to isoprenaline (0.1 mumol l-1). Apamin (0.1 mumol l-1) did not alter the spontaneous electrical activity of trachealis cells or their electrical and mechanical responses to isoprenaline (0.1 mumol l-1). TEA (8 mmol l-1) caused depolarization and often increased slow wave amplitude and induced spike discharge. Isoprenaline (0.01 mumol l-1) failed to hyperpolarize TEA-treated trachealis cells. Higher concentrations of isoprenaline suppressed TEA-induced spasm, caused hyperpolarization and thereby increased slow wave or spike amplitude. Slow wave or spike frequency decreased as the hyperpolarization progressed but abolition of slow waves or spikes sometimes required more than 4 min exposure to isoprenaline. Procaine (5 mmol l-1) increased the amplitude of slow waves and induced spike discharge. Procaine markedly reduced the hyperpolarization induced by isoprenaline (0.1 and 1 mumol l-1) but had little effect on isoprenaline-induced relaxation. It is concluded that isoprenaline activates beta-adrenoceptors in guinea-pig trachealis and thereby evokes relaxation and hyperpolarization of the smooth muscle. The hyperpolarization does not involve the opening of apamin-sensitive K+-channels and it probably plays a supportive rather than a crucial role in the process by which isoprenaline-induced relaxation is achieved.

Electrical and mechanical effects of BRL34915 in guinea-pig isolated trachealis.

BRL34915 (0.1-10 microM) suppressed the spontaneous tone of guinea-pig isolated trachealis in a concentration-dependent manner. BRL34915 was not antagonized by propranolol (1 microM). In trachea where spontaneous tone was suppressed by indomethacin (2.8 microM) but subsequently restored to the same level with acetylcholine or histamine, the relaxant potency of BRL34915 was reduced. In Krebs solution containing K+ (120 mM), isolated trachealis muscle developed near-maximal tension. The relaxant effects of BRL34915 were virtually abolished in this medium. Concentration-effect curves for KCl, acetylcholine and histamine were constructed in tissues treated with indomethacin (2.8 microM). BRL34915 (10 microM) depressed the foot of the concentration-effect curve for KCl and caused minor rightward shifts in the concentration-effect curves of acetylcholine and histamine. Four K+-channel inhibitors were tested. Apamin (0.1 microM) did not modify the action of BRL34915. Tetraethylammonium (8 mM) had little effect but procaine (5 mM) and 4-aminopyridine (5 mM) each significantly inhibited the relaxant action of BRL34915. Intracellular electrophysiological recording showed that BRL34915 (0.1 microM) caused very minor relaxation and little, if any, electrical change. Higher concentrations (1-10 microM) evoked relaxation, suppression of spontaneous electrical slow waves and marked hyperpolarization of the trachealis cells. In the presence of TEA (8 mM) or procaine (5 mM) the hyperpolarization induced by BRL34915 was significantly reduced. In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 microM) was unaffected either by BRL34915 (10 microM) or by nicorandil (1 mM). In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, BRL34915 (1 and 10 microM) increased the efflux rate constant. It is concluded that BRL34915 evokes relaxation of the trachealis by a mechanism that involves neither beta-adrenoceptor activation nor direct reduction of the sensitivity of the intracellular contractile machinery to cytosolic free Ca2+. The action of BRL34915 may depend on the opening of K+ channels in the plasma membrane which are permeable to 86Rb+. The opening of these channels, or the effects of their opening, may be reduced by K+-channel inhibitors such as 4-aminopyridine, procaine and TEA but not by apamin.

Mechanical and electrical aspects of the relaxant action of aminophylline in guinea-pig isolated trachealis.

Aminophylline (1-1000 mumol l-1) suppressed the spontaneous tone of guinea-pig isolated trachealis in a concentration-dependent manner. In Krebs solution containing acetylcholine (1 mmol l-1), histamine (200 mumol l-1) or K+ (120 mmol l-1) isolated trachealis muscle developed near-maximal tension. The log concentration-effect curve for aminophylline was shifted 20 fold, 3 fold and 4 fold to the right, respectively, in the presence of these spasmogens. Three K+-channel inhibitors were tested: tetraethylammonium (TEA, 8 mmol l-1) did not modify the action of aminophylline, procaine (5 mmol l-1) shifted the log concentration-effect curve for aminophylline 2 fold to the left and 4-aminopyridine (5 mmol l-1) shifted the curve 2.5 fold to the right. Intracellular electrophysiological recording showed that aminophylline 10 mumol l-1 could cause relaxation in the absence of electrical changes. Higher concentrations of aminophylline (100-1000 mumol l-1) suppressed spontaneous slow waves and hyperpolarized the trachealis cells. In the presence of procaine (5 mmol l-1) or TEA (8 mmol l-1), the hyperpolarization induced by aminophylline (1000 mumol l-1) was significantly reduced but its relaxant effect was unchanged. In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 mumol l-1) was unaffected either by aminophylline (1000 mumol l-1) or by isoprenaline (1 mumol l-1). In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, aminophylline (100-1000 mumol l-1) was without effect whereas nicorandil (100 and 1000 mumol l-1) increased the efflux rate constant. It is concluded that aminophylline does not directly reduce the sensitivity of the contractile proteins to cytosolic Ca2+. In low concentration (1-10 mumol l-1) its relaxant action is not accompanied by membrane potential change but towards the upper end of its effective concentration range, aminophylline evokes hyperpolarization. This hyperpolarization may involve the opening of K+-channels which are inhibited by procaine and (to a lesser extent) by TEA. These K+-channels may be impermeable to 86Rb+.