R.C. Small

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

1 BRL34915 (0.1–10 μM) suppressed the spontaneous tone of guinea‐pig isolated trachealis in a concentration‐dependent manner. BRL34915 was not antagonized by propranolol (1 μM). 2 In trachea where spontaneous tone was suppressed by indomethacin (2.8 μM) but subsequently restored to the same level with acetylcholine or histamine, the relaxant potency of BRL34915 was reduced. 3 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. 4 Concentration‐effect curves for KCl, acetylcholine and histamine were constructed in tissues treated with indomethacin (2.8 μM). BRL34915 (10 μM) depressed the foot of the concentration‐effect curve for KCl and caused minor rightward shifts in the concentration‐effect curves of acetylcholine and histamine. 5 Four K+‐channel inhibitors were tested. Apamin (0.1 μM) 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. 6 Intracellular electrophysiological recording showed that BRL34915 (0.1 μM) caused very minor relaxation and little, if any, electrical change. Higher concentrations (1–10 μM) 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. 7 In trachealis skinned of its plasma membranes, tension development induced by Ca2+ (20 μM) was unaffected either by BRL34915 (10 μM) or by nicorandil (1 mM). 8 In studies of the efflux of 86Rb+ from muscle‐rich strips of trachea, BRL34915 (1 and 10 μM) increased the effux rate constant. 9 It is concluded that BRL34915 evokes relaxation of the trachealis by a mechanism that involves neither β‐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.

Evidence that the mechanism of the inhibitory action of pinacidil in rat and guinea‐pig smooth muscle differs from that of glyceryl trinitrate

1 The effects of pinacidil have been compared with those of glyceryl trinitrate (GTN) using the aorta and portal vein of the rat and the trachealis and taenia caeci of the guinea‐pig. 2 In aorta, both pinacidil and GTN inhibited responses to noradrenaline and showed some selective inhibition of contractions to 20 mm K+. Responses to 80 mm K+ were little affected. 3 In trachealis, both pinacidil and GTN inhibited spontaneous tone and selectively relaxed spasms to 20 mm K+. Responses to 80 mm K+ were unaffected. 4 In portal vein, pinacidil completely inhibited spontaneous electrical and mechanical activity. GTN reduced the amplitude of tension waves and extracellularly‐recorded discharges, but increased the frequency of spontaneous electrical and mechanical activity. 5 In portal vein, pinacidil inhibited contractions to noradrenaline and selectively inhibited responses to 20 mm K+. GTN had little inhibitory effect on responses to either noradrenaline or K+. 6 In portal veins loaded with 86Rb as a K+‐marker, pinacidil significantly increased the 86Rb efflux rate coefficient whilst GTN had no effect on 86Rb exchange. 7 In taenia caeci, both pinacidil and GTN inhibited the spontaneous tone of the preparation. These inhibitory effects were not antagonized by apamin. 8 It is concluded that pinacidil and GTN do not share a common relaxant mechanism. Evidence has been obtained that pinacidil exerts its inhibitory effects by the opening of apamin‐insensitive, 86Rb‐permeable K+ channels.

Antagonism of Ca2+ and other actions of verapamil in guinea-pig isolated trachealis

1 In trachealis bathed by a K+‐rich, Ca2+‐free physiological salt solution, calcium chloride (CaCl2) at 0.01 to 10 mmol l−1 evoked concentration‐dependent spasm. Verapamil (0.1 to 10 μmol l−1) was an effective antagonist of CaCl2. 2 Spasm evoked by acetylcholine, histamine, potassium chloride (KCl) and tetraethylammonium (TEA) was studied in trachealis bathed by normal Krebs solution. Verapamil (0.1 to 10 μmol l−1) markedly suppressed spasm evoked by KCl and TEA. In contrast the actions of acetylcholine and histamine were much less affected by verapamil. 3 Spasm evoked by prostaglandin E2 was studied in trachealis bathed by Krebs solution containing indomethacin (2.8 μmol l−1). Verapamil (0.1 to 10 μmol l−1) had little or no effect against prostaglandin E2‐induced spasm. 4 Verapamil (0.1 to 10 μmol l−1) had relatively little effect on the tone of trachealis bathed by normal Krebs solution. In contrast bathing in Krebs solution lacking CaCl2 caused almost complete tone loss. 5 Extracellular electrophysiological recording showed that verapamil (10 μmol l−1) suppressed not only TEA‐evoked spasm but also TEA‐evoked slow waves and spike potentials. Verapamil also abolished the transient period of slow wave activity associated with the spasm evoked by KCl. 6 Intracellular electrophysiological recording showed that TEA‐induced spike activity was resistant to tetrodotoxin (3 μmol l−1). However, verapamil (10 μmol l−1) abolished the tetrodotoxin‐resistant spikes without increasing the resting membrane potential. 7 It is concluded that verapamil suppresses TEA‐ or KCl‐induced spasm, slow waves or spikes by inhibition of Ca2+ influx. Spasm evoked by acetylcholine, histamine and prostaglandin E2 depends on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to verapamil. The failure of verapamil markedly to depress tissue tone is consistent with the proposal that tone results from the activity of endogenous prostaglandins.

Evidence that the spasmogenic action of tetraethylammonium in guinea-pig trachealis is both direct and dependent on the cellular influx of calcium ion.

1 Tetraethylammonium (TEA, 1–8 mmol/l) evoked spasm of guinea‐pig trachealis which was unaffected by atropine (1 μmol/l), mepyramine (1 μmol/l) or tetrodotoxin (3 μmol/l). 2 The spasm evoked by TEA was markedly suppressed in Ca2+‐free Krebs solution while that evoked by acetylcholine was much less affected. 3 Extracellular electrical recording showed that exposure to Ca2+‐free Krebs solution suppressed both spontaneous electrical slow wave activity of the trachea and the spasm and slow waves induced by TEA. These effects were reversible. 4 TEA (2 and 8 mmol/1) increased the lanthanum‐resistant calcium fraction of trachea. 5 It is concluded that TEA acts directly on the smooth muscle of guinea‐pig trachea, that the spasm and electrical slow waves evoked are Ca2+‐dependent and that the cellular influx of Ca2+ is increased.

Cromakalim‐induced relaxation of guinea‐pig isolated trachealis: antagonism by glibenclamide and by phentolamine

1 Tested against the spontaneous tone of guinea‐pig isolated trachealis, cromakalim (0.1–100 μm), isoprenaline (1 nm‐1 μm) and theophylline (1 μm‐1 mm) each produced concentration‐dependent relaxation. 2 Glibenclamide (0.1–10 μm) did not itself alter the spontaneous tone of the trachea nor did it modify the relaxant actions of isoprenaline or theophylline. In contrast, glibenclamide (0.1 and 1 μm) caused a concentration‐dependent rightward shift of the log concentration‐effect curve of cromakalim. Glibenclamide (10 μm) reduced the slope of the log concentration‐effect curve of cromakalim and moved the foot of the curve back towards the control position. 3 Phentolamine (1, 10 and 100 μm) did not itself alter the spontaneous tone of the trachea nor did it modify the relaxant actions of isoprenaline or theophylline. In contrast phentolamine caused concentration‐dependent depression of the log concentration‐effect curve of cromakalim. 4 Neither prazosin (1 μm) nor yohimbine (10 μm) modified the spontaneous tone of the trachea. Prazosin and yohimbine each failed to antagonise the effects of cromakalim, isoprenaline and theophylline. 5 Intracellular electrophysiological recording showed that glibenclamide (1 μm) and phentolamine (100 μm) caused minor change in the resting membrane potential of trachealis cells. Slow wave activity was slightly depressed by these agents. In contrast tetraethylammonium (TEA; 8 mm) caused marked depolarisation, and promoted the conversion of slow waves into regenerative action potentials. These electrical changes were accompanied by tonic tension development. 6 Phentolamine (100 μm) and glibenclamide (1 μm) reduced and reversed both the relaxation and the hyperpolarisation induced by cromakalim (10 μm). 7 It is concluded that glibenclamide and phentolamine each provide selective antagonism of the relaxant action of cromakalim in guinea‐pig trachealis. These agents also inhibit the plasmalemmal hyperpolarisation induced by cromakalim. The effect of phentolamine is unrelated to the blockade of α1 or α2‐adrenoceptors. If either glibenclamide or phentolamine act to block the K+ channels opened by cromakalim, then such channels are not identical to those which endow the trachealis plasmalemma with its powerful rectifying behaviour.

The relaxant action of BRL 34915 in rat uterus

1 BRL 34915 (0.04‐1.3 μm) caused concentration‐dependent inhibition of spontaneous phasic spasms of the isolated uterus of the term pregnant rat and this effect was not antagonized by propranolol. Spasms evoked by low concentrations of KCl (< 20 mm) were inhibited by BRL 34915 but those evoked by higher concentrations (> 40 mm) were unaffected. 2 In experiments using extracellular electrical recording, BRL 34915 (10 μm) selectively inhibited oxytocin‐induced phasic spasms and the associated spike activity but had little effect on the tonic component of the spasms. BRL 34915, as an inhibitor of phasic spasms to oxytocin (0.2 nm), was antagonized by procaine (0.3 and 1 mm). 3 BRL 34915 (10 μm) did not inhibit Ca2+‐induced spasm of saponin‐skinned thin myometrial strips. 4 Intracellular microelectrode recording from myometrial strips showed that BRL 34915 (10 μm) inhibited action potentials and phasic spasms in the presence of oxytocin (0.2 nm) and produced a hyperpolarization of 5 mV. 5 In single myometrial cells under current or voltage clamp, BRL 34915 (10 μm) had no effect on action potentials and inward current in Ca2+‐ or Ba2+‐containing media in the presence of tetraethylammonium, 4‐aminopyridine and caesium chloride. In the absence of these K+‐channel inhibitors, BRL 34915 had no effect on resting membrane potential, membrane resistance, action potentials, inward current or outward current. 6 BRL 34915 (1 or 10 μm) had no effect on 86Rb efflux from myometrial strips. 86Rb efflux was increased by oxytocin (0.2 and 20 nm). 7 The relaxant profile of BRL 34915 in the rat uterus is similar to that described for other smooth muscles where an action to open membrane K+‐channels has been proposed. BRL 34915 inhibited spike production but produced only a small hyperpolarization without a detectable increase in 86Rb efflux. Membrane resistance and transmembrane currents were unaffected. These results suggest that in the uterus the effects of BRL 34915 may be restricted to K+‐channels involved in the production of pacemaker activity.

Some features of the spasmogenic actions of acetylcholine and histamine in guinea‐pig isolated trachealis

1 Intracellular electrophysiological recording showed that acetylcholine (1 μmol l−1) and histamine (2 μmol l−1) depolarized trachealis cells and often increased the frequency of slow waves. Higher concentrations of these agents caused greater depolarization and abolition of slow waves. Marked depolarization was often associated with the appearance of electrical ‘noise’. These electrical phenomena were accompanied by tonic tension development in a contiguous segment of trachea. 2 Electrical ‘noise’ and tension evoked by high concentrations of acetylcholine or histamine could be dissipated by washing the agonist from the tissue. Acetylcholine‐induced ‘noise’ was resistant to tetrodotoxin (3 μmol l−1) and to hexamethonium (1 mmol l−1). 3 Neither acetylcholine (10–1,000 μmoll−1) nor histamine (2–200 μmol l−1) increased the lanthanum‐resistant calcium fraction of muscle‐containing strips of trachea. 4 It is concluded that, while developing tension under the influence of acetylcholine or histamine, trachealis cells depolarize markedly but there is relatively little cellular influx of Ca2+.

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

1 In guinea‐pig isolated trachealis isoprenaline (0.001‐0.1 μmol 1−1) caused concentration‐dependent relaxation. Propranolol (1μmol 1−1) antagonized the effects of isoprenaline by more than 100 fold but did not modify the relaxant action of sodium nitrite. 2 The tracheal relaxant actions of isoprenaline and ATP were unaffected by apamin (0.1 μmol 1−1) but apamin profoundly antagonized the effects of noradrenaline and ATP on guinea‐pig isolated taenia caeci. 3 Tetraethylammonium (TEA; 8 mmol 1−1) and procaine (5 mmol 1−1) each evoked tracheal spasm but neither agent antagonized the isoprenaline‐evoked relaxation of the trachealis. 4 Trachealis exposed to K+‐rich (120 mmol 1−1) 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. 5 Intracellular electrophysiological recording showed that isoprenaline (0.01–1 μmol 1−1) caused hyperpolarization and reduced or abolished slow wave discharge in trachealis muscle. These effects were accompanied by relaxation. Propranolol (1 μmol 1−1) virtually abolished both the electrical and mechanical responses to isoprenaline (0.1 μmol 1−1). 6 Apamin (0.1 μmol 1−1) did not alter the spontaneous electrical activity of trachealis cells or their electrical and mechanical responses to isoprenaline (0.1 μmol 1−1). 7 TEA (8 mmol 1−1) caused depolarization and often increased slow wave amplitude and induced spike discharge. Isoprenaline (0.01 μmol 1−1) 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. 8 Procaine (5 mmol 1−1) increased the amplitude of slow waves and induced spike discharge. Procaine markedly reduced the hyperpolarization induced by isoprenaline (0.1 and 1 μmol 1−1) but had little effect on isoprenaline‐induced relaxation. 9 It is concluded that isoprenaline activates β‐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.

Guinea‐pig isolated trachealis: the effects of charybdotoxin on mechanical activity, membrane potential changes and the activity of plasmalemmal K+‐channels

1 A study has been made, in guinea‐pig isolated trachealis, of the effects of charybdotoxin in modulating (a) the activity of large conductance K+‐channels, (b) the spontaneous electrical activity of intact cells and (c) the mechanical effects of some bronchodilator drugs. 2 Single smooth muscle cells were isolated from guinea‐pig trachealis by enzymic digestion and were studied by the patch clamp recording technique. Recordings were made from outside‐out plasmalemmal patches when the medium bathing the external surface of the patches contained 1.2 mm Ca2+ and 6 mm K+ while that bathing the cytosolic surface contained 0.1 μm Ca2+ and 140 mm K+. Charybdotoxin (100 nm), applied to the external surface of patches held at 0 mV, abolished the unitary currents associated with the opening of large conductance K+‐channels. 3 Opened segments of guinea‐pig trachea were used for the simultaneous recording of membrane potential and tension changes. In these experiments charybdotoxin (100 nm) caused the conversion of spontaneous electrical slow waves into spike‐like action potentials. This effect was accompanied by a very small reduction in resting membrane potential. 4 Tissue bath recording showed that charybdotoxin (100 nm) increased the spontaneous mechanical tone of the tissue, antagonized (2.8 fold in each case) the relaxant actions of isoprenaline and theophylline but did not antagonize the relaxant actions of cromakalim or RP 49356. 5 It is concluded that charybdotoxin is an effective inhibitor of large conductance K+‐channels in guinea‐pig trachealis cells. The ability of charybdotoxin to convert spontaneous slow waves into spike‐like action potentials suggests that the large, charybdotoxin‐sensitive, K+‐channels play an important role in determining the strong outward rectifying behaviour of the cells. The ability of charybdotoxin to antagonize isoprenaline and theophylline, but not to antagonize cromakalim and RP 49356, suggests that opening of the large conductance, charybdotoxin‐sensitive K+‐channel is implicated in the action of the former but not the latter pair of bronchodilator drugs.

The spasmogenic action of potassium chloride in guinea‐pig trachealis

1 Tissue bath experiments showed that potassium chloride (KCl) at 10–40 mmol l−1 evoked spasm of guinea‐pig trachealis which was unaffected by atropine (1 μmol l−1), mepyramine (1 μmol l−1), tetrodotoxin (3 μmol l−1) or indomethacin (2.8 μmol l−1). 2 Spasm evoked by KCl was depressed in Ca2+‐free Krebs solution or by exposure of tissues to LaCl3 (0.25–1 mmol l1). 3 Extracellular electrical; recording showed that the spasm evoked by KCl 10 mmol l−1 was associated with promotion of electrical slow wave activity. Higher concentrations of KCl abolished slow wave activity but caused further tension development. 4 Intracellular recording confirmed the ability of KCl 10 mmol l−1 transiently to promote slow wave activity in individual trachealis cells. This action was associated with depolarization and tension development. Higher concentrations of KCl evoked further tension development but slow waves were suppressed as the depolarization evoked by KCl increased. 5 KCl (10–40 mmol 1−1) increased the lanthanum‐resistant calcium fraction of muscle‐containing strips of trachea. 6 It is concluded that KCl acts directly on the smooth muscle of guinea‐pig trachea. The spasmogenic action is associated with transient promotion of slow wave activity and a fall in resting membrane potential. The spasm involves the cellular influx of Ca2+ and is dependent on the presence of Ca2+ in the extracellular fluid.