Katharine Bray

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.

The action of diazoxide and minoxidil sulphate on rat blood vessels: a comparison with cromakalim

1 The actions of diazoxide and minoxidil sulphate have been compared with those of cromakalim in rat aorta and portal vein. 2 Diazoxide and minoxidil sulphate hyperpolarized the rat portal vein in a similar manner to cromakalim. 3 Cromakalim, diazoxide and minoxidil sulphate increased 42K and 86Rb efflux from rat portal vein, although minoxidil sulphate had only a small effect on 86Rb efflux. 4 Cromakalim, diazoxide and minoxidil sulphate increased 42K efflux from rat aorta but only cromakalim and diazoxide increased 86Rb efflux from this tissue. 5 Glibenclamide inhibited the relaxant actions of cromakalim, diazoxide and minoxidil sulphate on rat aorta and the increase in 42K efflux produced by these agents in this tissue. 6 Diazoxide relaxed an 80 mm KCl‐induced contraction of rat aorta, whilst cromakalim and minoxidil sulphate were without effect. 7 Cromakalim, diazoxide and minoxidil sulphate had no effect on cyclic AMP or cyclic GMP concentrations in rat aorta. 8 It is concluded that diazoxide and minoxidil sulphate like cromakalim exhibit K+ channel opening properties in vascular smooth muscle. Diazoxide exerts an additional inhibitory action not related to the production of cyclic AMP or cyclic GMP. The action of minoxidil sulphate may be primarily located at a K+ channel which is relatively impermeable to 86Rb.

Differences between the effects of cromakalim and nifedipine on agonist-induced responses in rabbit aorta

1 The effects of cromakalim on endothelium‐denuded rabbit aortic strips were compared with those of the calcium (Ca2+) entry blocking agent, nifedipine. 2 Pre‐incubation with cromakalim or nifedipine had no significant effect on the initial phasic component of noradrenaline (NA)‐induced responses. 3 Cromakalim (0.3–10 μm), but not nifedipine, inhibited the maintained tonic contractions produced by NA. The effects of cromakalim were antagonized by raising extracellular [K+] or by glibenclamide. 4 Nifedipine inhibited contractions produced by KCl (40 mm) whereas cromakalim had no effect. 5 In Ca2+‐free physiological salt solution (PSS), cromakalim produced a significant inhibition of both the refilling of and the release of Ca2+ from NA‐releasable Ca2+ stores, whereas nifedipine was ineffective. 6 In tissues preloaded with 42K+ cromakalim (0.3–10 μm) produced a concentration‐dependent increase in the 42K+ efflux rate coefficient. NA (0.3 μm) also produced an increase in the rate of efflux of 42K+, an effect which was not antagonized by nifedipine (0.3 μm). 7 When microelectrodes were used, cromakalim (1–10 μm) produced a maintained concentration‐dependent membrane hyperpolarization. However, low concentrations of cromakalim (<1 μm) which relaxed the aorta had no effect on membrane potential. NA had no significant effect on membrane potential. 9 It is concluded that the ability of cromakalim to relax NA‐induced contractions in rabbit aorta is not exerted by the indirect closure of nifedipine‐sensitive Ca2+ channels. Instead, cromakalim may exert a direct inhibitory action on Ca2+ uptake into and release from Ca2+ stores and additionally inhibit the pathway through which Ca2+ passes from the extracellular fluid to intracellular Ca2+ stores.

The potassium channel opening action of pinacidil; studies using biochemical, ion flux and microelectrode techniques

SummaryIn rat aorta and rat portal vein, (−)- and (+)-pinacidil each produced a concentration-dependent inhibition of tension development. Although the (−) isomer was the more potent, concentration effect curves for each isomer were steep with similar slopes. In rat portal vein, tetraethylammonium and procaine antagonised the relaxant effect of (±)-pinacidil, whereas 3,4-diamino-pyridine was without effect. Intracellular microelectrode recording in rat portal vein showed that low concentrations of (±)-pinacidil reduced the duration of multispike electrical complexes. In both rat aorta and rat portal vein, higher concentrations of (±)-pinacidil hyperpolarised the membrane towards the potassium equilibrium potential. (±)-Pinacidil increased 86Rb efflux from rat aorta and rat portal vein in a concentration dependent manner. In a separate study, (±)-pinacidil increased 42K efflux from rat portal vein. (±)-Pinacidil had no effect on cyclic GMP or cyclic AMP levels in rat aorta. It is concluded that pinacidil opens 86Rb-permeable potassium channels in rat aorta and rat portal vein. This mechanism is independent of cyclic nucleotide changes and may be responsible for the antihypertensive effect of pinacidil.

Differential inhibition by tedisamil (KC 8857) and glibenclamide of the responses to cromakalim and minoxidil sulphate in rat isolated aorta

SummaryThe effects of the K+ channel blockers tedisamil and glibenclamide on cromakalim- and minoxidil sulphate-induced 42K+ and 86Rb+ efflux and vasorelaxation in rat aorta, were investigated. In aortic strips preloaded with 42K+ or 86Rb+, cromakalim (1 μmol/l) induced increases in tracer efflux, which were concentration-dependently inhibited by tedisamil with similar potencies (pD2 ≈ 7.3) but different amplitudes (maximum inhibition of 86Rb+ efflux to 0% of control, 42K+ efflux to 10 ± 1%). The 42K+ efflux elicited by a low concentration of cromakalim (100 nmol/l) was, however, fully inhibited by tedisamil. The tracer effluxes induced by minoxidil sulphate were fully inhibited by tedisamil and glibenclamide (300 nM).Cromakalim and minoxidil sulphate, produced a concentration-dependent inhibition of noradrenaline (100 nmol/l)-induced tone, with pD2 values of ≈7.3. Tedisamil (300 nmol/1) and glibenclamide (300 nmol/l), which inhibited cromakalim- and minoxidil sulphate-induced 42K+ and 86Rb+ efflux by ≥80%, produced 2-fold and 40-fold shifts in the concentration-relaxation curve for cromakalim, and 3.5-fold and 2200-fold shifts in the concentration-relaxation curve for minoxidil sulphate, respectively. Similar shifts of the cromakalim concentration-relaxation curve in the presence of tedisamil and glibenclamide were also observed when the tissues were precontracted with potassium chloride (25 mmol/l).The results show that tedisamil and glibenclamide inhibit the cromakalim- and minoxidil sulphate-induced tracer effluxes with similar potencies whereas they differ greatly in their ability to inhibit the vasorelaxant effects of the two K+ channel openers. This suggests that the opening of 42K+/86Rb+ permeable K+ channels in the plasma membrane cannot fully explain the vasorelaxant effects of the two drugs. The mechanism(s) of vasorelaxation of cromakalim and minoxidil sulphate, which is not due to the opening of plasmalemmal K+ channels, is sensitive to inhibition by glibenclamide but comparatively insensitive to inhibition by tedisamil.

Differences in the K+‐channels opened by cromakalim, acetylcholine and substance P in rat aorta and porcine coronary artery

1 The effects of acetylcholine and substance P on the efflux of 86Rb+ and 42K+ from rat aorta and pig coronary artery, respectively, were compared with those of the K+ channel opening agent, cromakalim. 2 In rat aorta preloaded with 86Rb+ and/or 42K+, acetylcholine produced transient, concentration‐dependent increases in the efflux rate coefficients of these tracers (maximum ≅ 35%). These effects were abolished by endothelial cell removal. 3 Donor/acceptor experiments with rat aorta suggested that at least some of the efflux of 86Rb+ seen in the presence of acetylcholine was not derived from the endothelium, but came from the smooth muscle itself. 4 Acetylcholine (10 μm)‐induced 86Rb+ efflux was reduced by tetraethylammonium (TEA, 10 mm) to 33% and ouabain (300 μm) to 54% of control. Preincubation with Ba2+ (100 μm) did not significantly inhibit acetylcholine‐induced efflux. 5 Acetylcholine‐induced 42K+/86Rb+ efflux was unaffected by preincubation with glibenclamide (10 μm). In contrast, the 42K+/86Rb+ efflux induced by cromakalim was inhibited by glibenclamide (50 nm) by 50%. 6 Acetylcholine (0.3–10 μm)‐induced inhibition of phenylephrine (1 μm)‐induced tone was abolished by endothelial cell removal but unaffected by glibenclamide. Cromakalim‐induced relaxations were endothelium‐independent and were inhibited by glibenclamide in a concentration‐dependent manner. 7 lG‐monomethyl l‐arginine (l‐NMMA, 250 μm) produced a significant (37 ± 14%) inhibition of acetylcholine‐induced 86Rb+ efflux whereas DG‐monomethyl l‐arginine was without effect. In the tissue bath l‐NMMA inhibited relaxations produced by acetylcholine (0.3–10 μm), but was without effect on responses to cromakalim. 8 In the pig coronary artery, substance P induced an endothelium‐dependent efflux of 86Rb+ and 42K+, which was unaffected by preincubation with glibenclamide (10 μm) or l‐NMMA (250 μm). 9 The present study shows that acetylcholine and substance P each open K+‐channels in arterial smooth muscle. However, the insensitivity of the stimulated 86Rb+/42K+ efflux to inhibition by glibenclamide suggests that the K+‐channel opened by these agents is different from the K+‐channel opened by cromakalim. In addition, the inability of l‐NMMA to inhibit fully the acetylcholine‐ and substance P‐stimulated 86Rb+ efflux suggests that in rat aorta and pig coronary artery the endothelium‐derived hyperpolarizing factor(s) (EDHF) is different from endothelium‐derived relaxing factor (EDRF).

The contribution of Rb-permeable potassium channels to the relaxant and membrane hyperpolarizing actions of cromakalim, RP49356 and diazoxide in bovine tracheal smooth muscle

1 Cromakalim (1 and 10 μm), RP49356 (5 and 50 μm) and diazoxide (100 and 300 μm) produced full relaxation of smooth muscle strips pre‐contracted with 25 mm KCl. These agents caused membrane hyperpolarization and increased 42K and 86Rb efflux. The time taken to achieve the maximum change in each of these parameters (tmax) was less for the higher concentration levels of cromakalim, RP49356 and diazoxide than for the lower concentration levels. 2 Calculation of permeability (P) changes showed that cromakalim (1 and 10 μm) produced a greater rise in PK than PRb, although the PRb:PK ratio was simiiar at both concentration levels. Similarly RP49356 produced a greater change in PK than PRb. However, in contrast to cromakalim, this difference was more marked at the higher concentration (50 μm) and was reflected by a differential effect of the two concentrations of RP49356 on the PRb:PK ratio. Diazoxide (100 and 300 μm) produced similar changes in PK and PRb. 3 For cromakalim (1 and 10 μm) the tmax for the electrical and mechanical effects and also the profile of change in these parameters corresponded to changes in both PK and PRb. For RP49356 (5 μm), changes in tension and membrane potential were related to both changes in PK and PRb, whereas at 50 μm these responses more closely corresponded to changes in PK. For diazoxide (100 and 300 μm) the electrical and mechanical effects corresponded to changes in both PK and PRb. 4 The results show that changes in 42K and 86Rb efflux induced by cromakalim, RP49356 and diazoxide are good indicators of changes in membrane PK and PRb evoked by these agents. Furthermore, it is concluded that the K channels involved in the mechanical and electrical effects of cromakalim are represented by the opening of a single population through which Rb can pass less easily than K, whilst the K channels associated with actions of diazoxide are equally permeable to both K and Rb. In contrast, the relaxant and membrane hyperpolarizing actions of RP49356 may involve the opening of more than one group of K channels which differ in their permeability to Rb.

The Potassium Channel Openers: A New Class of Vasorelaxants

Cromakalim, pinacidil, nicorandil, diazoxide and RP-49356 belong to the class of drugs termed potassium channel openers. In rat portal vein diazoxide, like cromakalim, abolished spontaneous mechanical and electrical activity and in rat aorta caused an increase in 86Rb efflux and inhibited KCl(20 mM)-induced contractions. However, in contrast to cromakalim, diazoxide (greater than 100 microM) also inhibited mechanical responses evoked by 80 mM KCl in rat aorta suggesting that it possesses pharmacological properties in addition to K channel opening. Since glibenclamide can attenuate the effects of cromakalim and diazoxide in vascular tissues, it is possible that a channel resembling the ATP-sensitive K channel found in pancreatic beta-cells may be involved in the vasorelaxant effects of these agents. However, differences exist in the order of potency of cromakalim and diazoxide for producing smooth muscle relaxation and for decreasing insulin secretion in pancreatic beta-cells. Furthermore galanin (which opens ATP-sensitive K channels in beta-cells) increases mechanical activity in rat portal vein. It is anticipated that new chemical developments will produce K channel opening molecules with greater potency and tissue selectivity.

Some degree of overlap exists between the K+-channels opened by cromakalim and those opened by minoxidil sulphate in rat isolated aorta

SummaryThe effects of the K+ channel opening drugs minoxidil sulphate and cromakalim, on 42K+ and 86Rb+ efflux and on vasorelaxation in rat isolated aorta, were compared. In rat aortic rings precontracted with noradrenaline (100 nmol/l), minoxidil sulphate and cromakalim concentration-dependently inhibited induced tension by up to 90%, with pD2 values of 7.35±0.1 and 7.17±0.1, respectively. Glibenclamide (300 nmol/l), produced 2200- and 19-fold rightward shifts in the concentration-relaxation curves to minoxidil sulphate and cromakalim, respectively, without an effect on the maximum relaxation.Both minoxidil sulphate and cromakalim increased the efflux of 42K+ and 86Rb+ from aorta in a concentration-dependent manner, with midpoints in the µmol/l range; the maximum efflux induced by minoxidil sulphate being approximately one tenth of that induced by cromakalim. The ratio of stimulated 86Rb+/42K+ efflux increased from 0.22 to 0.48 with increasing cromakalim concentrations, but was approximately constant (≈0.39) when the minoxidil sulphate concentration was varied. In the presence of minoxidil sulphate, the effects of cromakalim on 42K+ and 86Rb+ efflux were inhibited in a concentration-dependent manner, by up to 60%. In the continuing presence of cromakalim (300 nmol/l), minoxidil sulphate (10 µmol/l)-induced increases in 42K+ and 86Rb+ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 µmol/l) inhibited the 86Rb+ efflux stimulated by additional superfusion of cromakalim (1 µmol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 µmol/l)- and cromakalim (1 µmol/l)-induced increases in 42K+ and 86Rb+ efflux in a concentration-dependent manner with IC50 values of approximately 80 nmol/l.In conclusion, the efflux data suggest that considerable overlap exists between the channels opened by minoxidil sulphate and those opened by cromakalim in rat aorta. Minoxidil sulphate has a weak efficacy as a K+ channel opener, and may act to open a homogeneous population of K+ channels. In contrast, the actions of cromakalim (≥1 µmol/l) are associated with large increases in tracer efflux, which are probably mediated via a heterogeneous population of K+ channels. However, only a small proprtion of this induced efflux appears to be required for relaxation. The differential inhibition by glibenclamide of the vasorelaxant effects of minoxidil sulphate and cromakalim may result from (a) the partial agonist properties of minoxidil sulphate in opening K+ channels and/or (b) additional mechanisms of vasorelaxation, which differ in their sensitivity to glibenclamide.

TEDISAMIL (KC 8857) DIFFERENTIALLY INHIBITS THE 86RB+ EFFLUX-STIMULATING AND VASORELAXANT PROPERTIES OF CROMAKALIM

Tedisamil, a blocker of cardiac K+ channels, potently inhibited cromakalim-induced 86Rb+ efflux from rat aorta with a pIC50 = 7.3, a value similar to that obtained with the sulphonylurea glibenclamide. However, tedisamil was approximately 30 times less potent than glibenclamide in inhibiting the vasorelaxant effects of cromakalim. The data suggest that tedisamil can dissociate between the efflux-inducing and vasorelaxant effects of cromakalim and may therefore prove to be an important tool in elucidating the mechanism of action of this vasorelaxant.