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The synthesis of N-cyano-N'-[1,1-dimethyl-[2,2,3,3-3H]propyl]-N"-(3- pyridinyl)guanidine, [3H]-15, is described. The utility of this tritiated radioligand in characterizing the interactions of potassium channel openers and blockers with their receptors is demonstrated. Potassium channel openers of the pinacidil, cromakalim, aprikalim, diazoxide, and minoxidil types, as well as KATP channel blockers of the glibenclamide and eosine types, are all capable of displacing [3H]-15 from its receptor. The results indicate that all of these compounds interact with the same target protein, but that several different allosterically coupled receptor binding sites are probably involved. The highly significant correlation between the ability of the structurally diverse potassium channel openers to inhibit [3H]-15 binding and to relax vascular smooth muscle is consistent with their receptor binding sites being closely associated with the potassium channel protein which is the functional target of this class of drugs.

P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine], an analogue of the K+ channel opener pinacidil, was shown to be a K+ channel opener in rat aorta, based on (i) its ability to stimulate 86Rb+ efflux, (ii) its ability to relax contractions in response to noradrenaline under normal conditions (5 mM KCl) but not under depolarized conditions (55 mM KCl), and (iii) the sensitivity of these effects to inhibition by the sulfonylurea glibenclamide. In these assays, P1075 was approximately 20 times more potent than cromakalim. Using a tritiated derivative, [3H] P1075, specific binding could not be detected in microsomal preparations from various tissues. However, in rat aortic strips specific binding of [3H]P1075 has been observed and was reduced by lowering the temperature or by decreasing intracellular ATP levels via metabolic inhibition. Specific [3H]P1075 binding was influenced neither by depolarization (55 mM KCl) nor by lowering the pH from 7.4 to 6.0. Binding was inhibited by representatives from all major families of K+ channel openers, with potencies that correlated well with the potencies obtained in 86Rb+ efflux and relaxation studies. However, stimulation of 86Rb+ efflux occurred at 40 times higher concentrations than did binding (and vasorelaxation). Of the various inhibitors of the K+ channel openers tested, only the sulfonylureas inhibited [3H] P1075 binding with the same rank order of potencies as that required for inhibition of P1075-induced 86Rb+ efflux, although at higher concentrations. The results show that binding of [3H] P1075 is independent of membrane potential but decreases concomitantly with the intracellular ATP level. The excellent correlation between the potencies of the openers and sulfonylurea blockers in binding assays and functional studies suggests that the 'drug receptor' labeled by [3H]P1075 in rat isolated aorta is of functional relevance. However, the fact that binding of the openers occurred at concentrations considerably lower than those required for K+ channel opening and that binding of the sulfonylureas was only reflected at concentrations higher than those needed to block the channel requires complex models to link binding and effect, possibly involving two agonist binding sites coupled by negative cooperativity.

The K+ channel openers, including cromakalim, pinacidil, minoxidil sulfate, diazoxide, and nicorandil, form a chemically heterogeneous group of compounds, which relax smooth muscle by opening plasmalemmal K+ channels. At present it is not known whether these drugs elicit their effects by binding to the same target, presumably the K+ channel. In order to address this question, a binding assay for K+ channel openers has been developed in vascular smooth muscle. The novel tritiated K+ channel opener, [3H]P1075, an analogue of pinacidil, binds with high affinity (KD = 6 +/- 1 nM) to endothelium-denuded rings of rat aorta. Inhibition studies indicate that the different families of K+ channel openers bind to a common target. Evidence is presented to suggest that the binding site for the sulfonylurea, glibenclamide, the major blocker of the K+ channel openers, is coupled in a negative allosteric manner to the binding site(s) for the openers. The binding assay described here may open the way to the biochemical characterization of the drug receptor for the K+ channel openers.

The K+ channel openers (KCOs) form a structurally heterogeneous group of compounds which relax smooth muscle by opening K+ channels in the plasmalemma. At present it is not known whether these drugs open the same K+ channel in smooth muscle and, if so, whether they bind to the same site of this channel. To address these questions we present the first binding study with KCOs in a smooth muscle preparation. In intact rat aortic strips, the novel tritiated KCO, 3H-P1075 (N-cyano-N'-(1,1-dimethyl[2,2,3,3(3)H]propyl)-N"-3-pyridylguanidine++ +), a potent pinacidil analogue, showed saturable specific binding of high affinity (KD = 6 +/- 1 nM, Bmax = 23 +/- 3 fmol/mg tissue wet weight). Specific binding of 3H-P1075 was inhibited stereospecifically by representatives from all major families of K+ channel openers, with a rank order of potency that correlated well with the potencies for vasorelaxation in rat aorta. The sulfonylurea glibenclamide, a relatively specific blocker of ATP-sensitive K+ channels and an inhibitor of the effects of KCOs, also inhibited 3H-P1075 binding and increased the rate of dissociation of 3H-P1075 from the tissue in a concentration-dependent manner. Lowering temperature (from 37 degrees C to 2 degrees C) and decreasing intracellular ATP levels by metabolic poisoning, diminished specific 3H-P1075 binding by reducing Bmax. However, depolarization (KCl = 55 mM) or lowering pH from 7.4 to 6.0 did not influence binding. The data demonstrate the existence of a specific binding site for the KCO 3H-P10756 in rat isolated aorta, which seems to be of functional relevance.(ABSTRACT TRUNCATED AT 250 WORDS)

The 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 mumol/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 (approximately 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 mumol/l)-induced increases in 42K+ and 86Rb+ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 mumol/l) inhibited the 86Rb+ efflux stimulated by additional superfusion of cromakalim (1 mumol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 mumol/l)- and cromakalim (1 mumol/l)-induced increases in 42K+ and 86Rb+ efflux in a concentration-dependent manner with IC50 values of approximately 80 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

1. Cromakalim (1 and 10 microM), RP49356 (5 and 50 microM) and diazoxide (100 and 300 microM) 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 cormakalim (1 and 10 microM) produced a greater rise in PK than PRb, although the PRb:PK ratio was similar 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 microM) and was reflected by a differential effect of the two concentrations of RP49356 on the PRb:PK ratio. Diazoxide (100 and 300 microM) produced similar changes in PK and PRb. 3. For cromakalim (1 and 10 microM) 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 microM), changes in tension and membrane potential were related to both changes in PK and PRb, whereas at 50 microM these responses more closely corresponded to changes in PK. For diazoxide (100 and 300 microM) 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.

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 approximately 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 microM)-induced 86Rb+ efflux was reduced by tetraethylammonium (TEA, 10 mM) to 33% and ouabain (300 microM) to 54% of control. Preincubation with Ba2+ (100 microM) did not significantly inhibit acetylcholine-induced efflux. 5. Acetylcholine-induced 42K+/86Rb+ efflux was unaffected by preincubation with glibenclamide (10 microM). In contrast, the 42K+/86Rb+ efflux induced by cromakalim was inhibited by glibenclamide (50 nM) by 50%. 6. Acetylcholine (0.3-10 microM)-induced inhibition of phenylephrine (1 microM)-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 microM) 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 microM), 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 microM) or L-NMMA (250 microM). 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).

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.

In 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.