K-channel Opening Research Articles

The effect of amphotericin B on the K-channel activity of MDCK cells

By using the whole-cell patch technique, it is shown that the total outward current is increased, as a function of time, after the addition of amphotericin B to the bathing solution. The whole-cell current is shown to be primarily a K-channel current by the blockage of this current upon application of TEA to the bathing solution. Single K-channel studies, using the outside-out patch-clamp technique, reveal that the single K-channel opening probability increases by a factor of six after the addition of amphotericin B. In addition, single K-channel voltage dependent studies, using the inside-out patch-clamp technique, demonstrate that this increase in opening probability is due to an increase in the amplitude of P o (v) . In contrast to the present belief that amphotericin B simply creates pores in a cell's membrane, these results suggest that amphotericin B can also influence the function of the cell's K-channel proteins.

Effects of rubidium on responses to potassium channel openers in rat isolated aorta.

1. In a physiological salt solution (PSS) in which potassium (K) was replaced by rubidium (Rb), segments of rat aorta precontracted with 20 mM RbCl were fully relaxed by K-channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were inhibited by glibenclamide. 2. Segments of rat aorta bathed in normal PSS and precontracted with 20 mM KCl were also relaxed by these K-channel openers with an order of potency levcromakalim > cromakalim > aprikalim > RP 49356. These relaxations were glibenclamide-sensitive. However, the absolute potencies of the K-channel openers were approximately four times greater in normal PSS than in RbPSS. 3. In RbPSS, minoxidil sulphate relaxed segments of aorta precontracted with 20 mM RbCl by approximately 20% whereas in normal PSS it fully relaxed those contracted with 20 mM KCl. 4. In RbPSS, levcromakalim-induced relaxation of aortic segments precontracted with 20 mM RbCl was initially well-maintained but then faded by approximately 60% of the initial relaxation to a new, stable level. Subsequent exposure to RP 49356 or to higher concentrations of levcromakalim was without further relaxant effect. Similar changes were observed when RP 49356 was the initial relaxant and tissues were exposed to either RP 49356 or levcromakalim. In normal PSS, levcromakalim- or RP 49356-induced relaxation of contractions produced by 20 mM KCl was well-maintained. 5. In RbPSS, minoxidil sulphate-induced relaxation of aortic segments precontracted with 20 mM RbCl was well-maintained. Subsequent exposure to either levcromakalim or to RP 49356 produced further tissue relaxation. 6. In RbPSS, levcromakalim produced no detectable increase in either 86Rb- or 42K-efflux from rat aortic strips. In normal PSS, a significant increase in the exchange of both isotopes was detected.7. Levcromakalim hyperpolarized segments of rat aorta bathed both in normal PSS and after depolarization by the addition of 20 mM KCI. Exposure to RbPSS depolarized the tissue and under these conditions, levcromakalim had no effect on membrane potential.8. In Rb- and normal PSS, levcromakalim produced a similar degree of inhibition of the refilling of then or adrenaline-sensitive Ca store.9. It is concluded that millimolar concentrations of Rb inhibit the plasmalemmal ATP-sensitive K-channels (KATP) which are the target of the K-channel openers. The relaxant actions of the K-channel openers in both normal and Rb-PSS and the inhibition of these effects by glibenclamide may reflect a functional interaction between these agents at ATP-binding sites associated with both KATP and with intracellular structures including Ca stores.

Effects of P1060 and aprikalim on whole‐cell currents in rat portal vein; inhibition by glibenclamide and phentolamine

1 Smooth muscle cells of the rat portal vein were dispersed by enzymatic treatment and recordings of whole-cell currents were made by the voltage-clamp technique. The effects of the potassium (K) channel openers, P1060 (0.3-10 microM) and aprikalim (3-30 microM) on these currents were investigated. Antagonism of these agents by glibenclamide and phentolamine was also studied. 2 When cells were clamped at -10 mV, P1060 (1 microM) and aprikalim (3 microM) each induced a slowly-developing K-current (IKCO), the noise of which gradually increased. The rate of onset of IKCO was greater for P1060 than for aprikalim. Current-voltage plots showed that P1060 and aprikalim each caused an approximately 25 mV negative shift of the reversal potential at zero current. 3 P1060 (1 microM) and aprikalim (3 microM) each inhibited the slowly activating, slowly inactivating delayed rectifier current, ITO. 4 Addition of MgATP (5 mM) to the recording pipette inhibited the generation of IKCO by P1060 (1 microM) and reduced the accompanying inhibition of ITO. 5 Stationary fluctuation analysis of the current noise associated with IKCO induced by P1060 (1 microM) or aprikalim (3 microM) at a holding potential of -10 mV indicated that the unitary conductance of the underlying K-channels was 10.5 pS at 0 mV under the quasi-physiological conditions of the experiment. 6 In the absence of K-channel openers, neither phentolamine (30-100 microM) nor glibenclamide (1 microM) affected the magnitude of control non-inactivating currents. However, phentolamine (30-100 microM), but not glibenclamide (1 microM) inhibited the control delayed rectifier current ITO. 7. After induction of IKCO by P1060 (1 microM) or aprikalim (3 microM), subsequent exposure to glibenclamide(1 microM) or phentolamine (30 microM) inhibited this current. After aprikalim-induced reduction of ITO had developed, subsequent exposure to glibenclamide was able partially to reverse the inhibition of ITO whereas phentolamine was without effect. Pre-exposure to glibenclamide (1 microM) prevented both the generation of IKCO by aprikalim (3 microM) and the inhibitory effect of this agent on ITO.8. It is concluded that P1060 and aprikalim each induce the current IKCO by opening the same small conductance, ATP-sensitive K-channel (KATP), an effect which can be inhibited by glibenclamide orphentolamine. The opening of KATP by both P1060 and aprikalim probably involves competition between these agents and ATP for the ATP-control site associated with the channel. Inhibition of the delayed rectifier current, ITO, by P1060 and aprikalim was glibenclamide-sensitive and may be caused by the induction of a state of run-down in the channel which underlies this current.

Mechanism of insulin secretion by midaglizole

Midaglizole was introduced as a hypoglycemic agent, but its insulin releasing mechanism remains unknown. In the present study, the effect of midaglizole upon the B cell function of the pancreas was investigated, using an in situ local circulation of the canine pancreas. The graded doses of midaglizole (0.2, 1.0 and 2.0 mg/kg) revealed a dose-related response of plasma insulin. The administration of yohimbine, a classical α 2-antagonist (1.11 mg/kg) revealed a similar increase in plasma insulin to that with midaglizole of equimolar amount. During the clonidine infusion midaglizole did not elicit any significant rise in plasma insulin, whereas yohimbine increased plasma insulin significantly. During glucagon infusion plasma insulin increased following midaglizole infusion but not by yohimbine. The simultaneous administration of diazoxide (K-channel opener) suppressed the midaglizole-induced insulin secretion. These results obtained in the present experiments revealed a different mechanism of insulin releasing action of midaglizole from that of yohimbine. Furthermore, the finding with diazoxide administration suggests that midaglizole stimulates insulin release through an interaction of K +-channel of the pancreatic B-cell.

Mechanism of Action of KRN2391 in Canine Coronary Vascular Bed

The present studies were performed to clarify the mechanism of action of KRN2391 in various sizes of canine coronary artery. We used the responses of isolated large and small coronary arteries and the changes in coronary blood flow (CBF) as indicators reflecting the responses of conductive arteries and resistive arterioles, respectively. In isolated small coronary artery, the effect of KRN2391 (10(-8)-10(-5) M) was antagonized by either methylene blue or glibenclamide. In isolated large coronary artery, the vasorelaxant effect of KRN2391 (10(-8)-10(-5) M) and nicorandil (10(-7)-10(-4) M) were antagonized by methylene blue (10(-5) M) but not by glibenclamide (10(-6) M). The relaxant effect of cromakalim was antagonized by glibenclamide but not by methylene blue in isolated large coronary artery. Intracoronary arterial injection of KRN2391, nicorandil or cromakalim produced an increase in CBF dose-dependently. Glibenclamide (5 mg/kg, i.v.) attenuated the increase in CBF caused by KRN2391, nicorandil and cromakalim. ED20, the dose that produced an increase in CBF by 20 ml/min, increased about 5-fold for KRN2391 and nicorandil and about 12-fold for cromakalim after administration of glibenclamide. These results suggest that the mechanism of action of KRN2391 and nicorandil depends on the segment of coronary arteries; i.e., they show a nitrate action alone in large coronary artery, and a K-channel opening action in addition to a nitrate action as the size of the coronary artery decreases.

Characteristics of the contractile response of rabbit aorta produced by cromakalim in calcium-free solution.

1 The effect of potassium channel opening compounds has been investigated in the smooth muscle of rabbit aorta under Ca-free conditions. Examination of the characteristics of the response has been performed using cromakalim as the prototype compound. 2 In order of potency, Ro 31-6930, cromakalim, minoxidil sulphate and pinacidil each produced a contraction in rabbit aortic strips bathed in Ca-free MOPS-buffered physiological salt solution (PSS). In contrast, forskolin, glyceryl trinitrate and nifedipine each failed to increase tension under identical conditions. Cromakalim also evoked contraction of bovine trachealis muscle bathed in Ca-free PSS. 3. The contractile response to cromakalim in rabbit aortic strips was of delayed onset (15-20 min) and reached a plateau after approximately 120 min (1.8 g maximum with 1 microM cromakalim). No cromakalim-induced tension changes were observed in either 1 mM or 2.5 mM Ca-containing PSS. 4. Raising the [KCl] of the Ca-free PSS to 65.9 mM fully inhibited the cromakalim-induced contraction in rabbit aortic strips. In addition, pretreatment of aortic strips with the sulphonylurea glibenclamide antagonized the subsequent mechanical response to cromakalim. 5. In Ca-free PSS, cromakalim (1 microM) stimulated 42K-efflux with a time-course corresponding to the contractile event. Glibenclamide (1 microM) inhibited this cromakalim-induced 42K-efflux. 6. In sharp microelectrode studies in bovine trachealis, cromakalim (10 microM) produced a sustained membrane hyperpolarization in normal PSS. In contrast, the cromakalim-induced hyperpolarization in Ca-free PSS was not sustained. The fading of the hyperpolarization was temporally correlated with the increase in tension under these experimental conditions. 7. It is concluded that the K-channel opener-induced smooth muscle contractile response revealed in Ca-free PSS is the consequence of K-channel opening. The nature of the detailed mechanism which underlies this contractile phenomenon remains to be determined.

Potassium channel openers: pharmacological and clinical aspects.

Opening of plasmalemmal K+ channels leads to cellular hyperpolarization which, in excitable tissues possessing voltage-dependent Ca2+ channels, prevents the opening of such channels and thus prevents excitation. In the last few years, an increasing number of compounds have been identified which elicit their effects by opening K+ channels, preferentially in smooth muscle, but also in other excitable tissues. These include the novel benzpyrans, cromakalim and bimakalim, the thioformamide aprikalim, and also well known antihypertensives such as minoxidil sulphate, diazoxide and pinacidil. After a short overview of the various families of K+ channel openers (KCOs), their basic pharmacological properties, including inhibition by the sulfonyl ureas (such as glibenclamide) are presented. The actual discussion concerning the type of K+ channel(s) opened by these compounds and their mechanism(s) of vasorelaxation will be reported. The therapeutic potential of these compounds in the cardiovascular field (as antihypertensives and, in particular, as anti-ischemic agents in heart and skeletal muscle), and in asthma (where they reverse established airway hyperreactivity) will also be discussed. Improved tissue selectivity may be the essential pre-requisite for true clinical success of this class of compounds.

Characterization of potassium currents modulated by BRL 38227 in rat portal vein.

1. Smooth muscle cells of the rat portal vein were dispersed by enzymatic treatment and recordings of whole-cell membrane potassium currents were made by the voltage-clamp technique. In isolated cells by use of combined voltage- and current-clamp the effect of BRL 38227 on membrane potential and ionic currents was also studied. 2. BRL 38227 (0.1 to 10 microM) induced a non-inactivating potassium current (IKCO) which developed slowly (900 s to 300 s, respectively) to its full size. These effects of BRL 38227 were reversible. 3. In addition to its K-channel opening properties, BRL 38227 (1 to 10 microM) inhibited the amplitude and changed the activation and inactivation characteristics of a slowly-inactivating, calcium influx-independent, outward potassium current (I(TO)). 4. Application of stationary fluctuation analysis to IKCO, showed a mean single channel current of 0.65 pA at -10 mV under a quasi-physiological potassium gradient. 5. In a combined voltage-clamp/current-clamp configuration, BRL 38227 (1 microM) induced a mean hyperpolarization of 22 mV. 6. The induction of IKCO by BRL 38227 and the associated hyperpolarization were suppressed by glibenclamide (1 to 10 microM) in a concentration-dependent manner. Glibenclamide (1 microM) had no effect on the inhibition of I(TO) by BRL 38227 (1 microM).

Identification of a specific binding site for K+ channel openers in rat aorta

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)

Spasmolytic action of nicorandil in canine conductive coronary arteries in vivo is not modified by glibenclamide.

The spasmolytic effects of nicorandil, cromakalim, and nitroglycerin on coronary arteries were investigated by angiographic technique in anesthetized dogs. With intracoronary arterial (i.a.) U 46619, a thromboxane A2 mimetic, the diameter of coronary arteries decreased in a sustained manner by 36.1 +/- 1.6% from control levels (coronary spasm). With a successive i.a. injection of nicorandil (300 micrograms), cromakalim (30 micrograms), or nitroglycerin (3 micrograms), the diameter recovered control levels (102.9 +/- 3.9, 96.8 +/- 5.6, and 100.1 +/- 4.3%, respectively). In dogs treated intravenously (i.v.) with glibenclamide, a pharmacologic antagonist of K-channel openers, the spasmolytic effect of cromakalim was significantly reduced, whereas the activity of nicorandil or nitroglycerin remained unaffected. We also investigated a possible modification by glibenclamide of the increase in coronary blood flow (CBF) induced by i.a. nicorandil and cromakalim in anesthetized dogs. The dose-dependent blood flow responses to cromakalim and nicorandil were significantly attenuated by glibenclamide, whereas the response to nitroglycerin remained unaffected. These results suggest that the spasmolytic effect of nicorandil on canine conductive coronary vessels is not mediated by K-channel opening but by a nitroglycerin-like action and that the dilatation of resistive coronary vessels induced by nicorandil may be largely due to its action as a K-channel opener.

S-8-5 - Comparison of the effects of nicorandil and KRN 2391 as a hybrid between K-channel openers and nitrates on coronary vasculatures and venous return with those of cromakalim and nitroglycerin

S-8-5 - Comparison of the effects of nicorandil and KRN 2391 as a hybrid between K-channel openers and nitrates on coronary vasculatures and venous return with those of cromakalim and nitroglycerin

S-8-1 - Physiological and clinical significance of ATP-sensitive potassium channels (KATP) and K-channel openers

S-8-1 - Physiological and clinical significance of ATP-sensitive potassium channels (KATP) and K-channel openers

O-431 - Effects of Y-26763, an active metabolite of a new K-channel opener Y-27152, on renal function in anesthetized dogs

O-431 - Effects of Y-26763, an active metabolite of a new K-channel opener Y-27152, on renal function in anesthetized dogs

Comparison of the effects of several potassium‐channel openers on rat bladder and rat portal vein in vitro

1. The ability of several K-channel openers to inhibit KCl-induced contractions of rat bladder detrusor and spontaneous mechanical activity in rat portal vein was examined. 2. Lemakalim, pinacidil, Ro 31-6930, RP 49356, P1060 and S 0121 dose-dependently relaxed rat detrusor, precontracted with 20 mM KCl. With the exception of pinacidil, concentrations of these agents below 30 microM did not inhibit 80 mM KCl-included contractions. Pinacidil (10 microM) produced a small, but significant (P < 0.05) relaxation of 80 mM KCl-induced mechanical activity. Minoxidil sulphate and BRL 38226 produced some relaxation of 20 mM but not 80 mM KCl-induced contractions. 3. Glibenclamide (0.3-3 microM) antagonized the relaxant effects of lemakalim, pinacidil, Ro 31-6930, RP 49356, P1060 and S 0121 in a competitive manner (pA2 values 6.3-6.6). The effects of minoxidil sulphate and BRL 38226 were fully antagonized by 3 microM glibenclamide. 4. Lemakalim, pinacidil, S 0121, BRL 38226 and minoxidil sulphate were each approximately 8 times more potent as inhibitors of the spontaneous contractions of rat portal vein than KCl-induced contractions of the rat detrusor. Minoxidil sulphate was approximately 30 times more potent in the rat portal vein than in the bladder. This may indicate that either minoxidil sulphate is acting at different recognition sites in these two tissues, or that this compound has an additional mechanism of action in the portal vein. 5. With the exception of minoxidil sulphate, all the compounds tested stimulated 86Rb efflux and 42K efflux from preloaded rat detrusor strips. The stimulated 86Rb efflux was qualitatively but not quantitatively similar to the stimulated 42K efflux. Minoxidil sulphate stimulated 42K efflux from rat portal vein but not from rat bladder. 6. It is concluded that all the compounds tested cause relaxation of rat detrusor predominantly by Kchannel opening. Selectivity for bladder rather than vascular smooth muscle was not shown by any compound.

P-388 - Sustained increase in coronary blood flow by a new K-channel opener, Y-27152, in conscious dogs.

P-388 - Sustained increase in coronary blood flow by a new K-channel opener, Y-27152, in conscious dogs.

O-226 - Long lasting antihypertensive effects of a new K-channel opener, Y-27152, in spontaneously hypertensive rats (SHRs) and renal hypertensive dogs (RHDs) in conscious states.

O-226 - Long lasting antihypertensive effects of a new K-channel opener, Y-27152, in spontaneously hypertensive rats (SHRs) and renal hypertensive dogs (RHDs) in conscious states.

P-389 - Anti-spasmodic action of Y-26763, an active metabolite ol a new K-channel opener Y-27152, in isolated coronary arteries.

P-389 - Anti-spasmodic action of Y-26763, an active metabolite ol a new K-channel opener Y-27152, in isolated coronary arteries.

P-412 - K-channel opening actions of Y-26763, an active metabolite of a new antihypertensive agent Y-27152.

P-412 - K-channel opening actions of Y-26763, an active metabolite of a new antihypertensive agent Y-27152.

Nicorandil increases coronary blood flow predominantly by K-channel opening mechanism

Nicorandil has a hybrid property between nitrates and potassium (K)-channel openers. In order to clarify which mechanism of action is responsible for its effect in increasing coronary blood flow, we investigated how this effect was antagonized by glibenclamide, which was recently found to behave as a pharmacologic antagonist of K-channel openers. Cromakalim, one of the most specific K-channel openers currently available, and nitroglycerin were used as reference drugs. In isolated, blood-perfused papillary muscle preparations of dogs, intraarterial injections of nicorandil and cromakalim increased (coronary) blood flow, and at high doses a negative inotropic effect and ventricular fibrillation occurred. Dose-response curves for the increase in coronary blood flow produced by nicorandil or cromakalim were shifted to the right in a parallel manner and to similar extents by glibenclamide given intravenously to support dogs. Schild analysis yielded pA2 values of 6.08 and 6.34 for glibenclamide versus nicorandil and cromakalim, respectively. Nitroglycerin injected intraarterially produced only an increase in coronary blood flow. This effect was not affected by glibenclamide. These results indicate that the effect of nicorandil in increasing coronary blood flow, like that of cromakalim, is predominantly due to its mechanism of action as a K-channel opener. The negative inotropy and ventricular fibrillation seen with high doses of nicroandil and cromakalim were also antagonized by glibenclamide, indicating that these effects are also due to K-channel opening.

K+-Channel Openers for Relaxation of Isolated Penile Erectile Tissue from Rabbit

The effects of the K+-channel openers (KCOs) cromakalim (BRL 34915) and pinacidil were investigated and compared with those of papaverine on isolated corpus cavernosum from rabbit. Preparations were mounted in organ baths and isometric tension was recorded. Spontaneous contractile activity was effectively abolished by the KCOs tested, cromakalim being the most potent of them. The KCOs concentration-dependently and effectively depressed electrically induced contractions and also contractions induced by exogenously applied noradrenaline and by low (≤20mM) concentrations of K+. Cromakalim was three to four times more potent than pinacidil. Pinacidil and cromakalim were shown to cause increases in the efflux of 86Rb from preloaded cavernous tissue. Papaverine also effectively depressed spontaneous contractile activity, and contractions evoked by electrical stimulation and noradrenaline. It had a potency 19 to 36 times lower than that of cromakalim. However, papaverine did not increase 86Rb efflux from preloaded tissue. The results show that cromakalim and pinacidil effectively relax penile erectile tissue, probably by the opening of K+-channels and subsequent hyperpolarization. Further investigations on human material seems motivated in order to elucidate if the principle of K+-channel opening offers any therapeutic advantages to other drugs in the diagnosis and treatment of penile erectile dysfunction.