Channel Opener Cromakalim Research Articles

Effects of potassium channel openers on Na + and K + currents in rabbit sinus node and atrial myocytes

The effects of the potassium channel openers (KCOs) Cromakalim and Lemakalim on rabbit sinoatrial and atrial myocytes were examined by means of the whole-cell patch-clamp technique. Lemakalim (up to 100 μM) had no effect on potassium current in sinoatrial cells. Both Lemakalim and Cromakalim (100 μM) displayed a two-fold action on atrial myocytes: (1) they increased an outwardly rectifying conductance at potentials positive to E K and, (2) they markedly decreased a TTX-sensitive Na + current active in the voltage range −50 −30 mV. This novel action on TTX-sensitive currents is of particular since these two benzopyrans have been thought to specifically target potassium channels.

CROMAKALIM SUPPRESSES HYPERTONIC SALINE‐INDUCED RENAL VASOCONSTRICTION IN ANAESTHETIZED DOGS

1. Intrarenal arterial infusion of hypertonic saline (HS) transiently increased and then gradually reduced renal blood flow (RBF) in anaesthetized dogs. Glomerular filtration rate (GFR) but not filtration fraction decreased at the end of the infusion. 2. In the presence of a potassium channel opener cromakalim (0.3 microgram/kg per min), HS infusion failed to reduce RBF; the initial increase in RBF was maintained throughout the infusion. Since cromakalim also prevented the decrease in GFR, HS infusion lowered filtration fraction. 3. The results suggest that cromakalim inhibits both pre-and postglomerular vasoconstriction induced by HS infusion.

Zeneca ZD6169 and its analogs from a novel series of anilide tertiary carbinols: in vitro KATP channel opening activity in bladder detrusor.

The potassium (K+) channel opening activity of Zeneca ZD6169 and one of its pyridylsulfonyl analogs from the anilide tertiary carbinol series was ascertained. Their mechanoinhibitory effects on the myogenic activity of the guinea pig bladder detrusor muscle were measured in a set of functional assays. Elevating the K+ concentration in the tissue bath from 15 to 80 mmol/l increased the IC50 value of ZD6169 from 1.61 +/- 0.22 223 +/- 37 mumol/l. This result suggests that ZD6169 may act as a K+ channel opener. Similar to the prototypic ATP-sensitive K+ (KATP) channel opener cromakalim, the K+ channel openers from the anilide tertiary carbinol series displayed stereoselective mechanoinhibitory activity only in the test protocol in which the detrusor was stimulated with 15 mmol/l KCl. Being the active enantiomer, ZD6169 has an activity more than 30-fold higher than the less active enantiomer. ZD6169 at 10 mumol/l hyperpolarized the guinea pig detrusor membrane potential by 6.1 +/- 1.2 mV and increased the whole cell KATP current in isolated guinea pig smooth muscle cells by 34.9 +/- 7.9 pA. This is comparable to the increase of 26.8 +/- 5.0 pA obtained with 10 mumol/l of lemakalim, the active enantiomer of cromakalim. The K+ channel opening activity of ZD6169 and the pyridylsulfonyl analog was competitively antagonized by the KATP channel blocker glibenclamide in the guinea pig detrusor with a pA2 value of 7.2. This activity, however, was unaffected by blockers of small and large conductance Ca-dependent K+ channels, such as apamin and charybdotoxin, respectively. The present study showed that Zeneca ZD6169 and its analog from the anilide tertiary carbinol series are K+ channel openers that activate KATP channels in vitro to relax bladder detrusors.

Calcitonin gene-related peptide mediated neurogenic vasorelaxation in the isolated canine lingual artery.

The nature of neurogenic relaxation was investigated in ring preparations of canine lingual artery. In all experiments, the preparations were previously treated with guanethidine (5 x 10(-6) M) to block neurogenic constrictor responses. In the presence of norepinephrine (10(-5) M) to induce tone, electrical stimulation (10 V, 4 to 16 Hz, for 45 sec) produced relaxation of the rings in an endothelium-independent fashion. The relaxant response in endothelium-denuded rings was not changed by propranolol (10(-5) M), and atropine (10(-5) M) did not affect the relaxation elicited by electrical stimulation in endothelium-intact rings. NG-monomethyl-L-arginine (10(-4) M) or NG-nitro-L-arginine methyl ester (10(-4) M), a nitric oxide (NO) synthase inhibitor, had no effect on the electrical stimulation-induced relaxation of endothelium-denuded rings. Human calcitonin gene-related peptide (CGRP)-(8 - 37) (2 x 10(-8) M), a CGRP1-receptor antagonist, inhibited neurogenic relaxation of endothelium-denuded rings; substance P (10(-6) M) failed to mimic the observed effect of electrical stimulation. The demonstrated effect of electrical stimulation was inhibited by glibenclamide (10(-5) M), but not tetraethylammonium (2 x 10(-4) M); glibenclamide abolished the relaxation in response to exogenous CGRP or the ATP-sensitive K+ channel opener cromakalim (10(-6) M) in endothelium-denuded rings. Moreover, tetrodotoxin (3.13 x 10(-6) M) inhibited the relaxation of endothelium-denuded rings induced by electrical stimulation. The relaxation was selectively inhibited when endogenous CGRP had been depleted from perivascular nerves by capsaicin (10(-6) M). These results suggest that CGRP, but not NO, released from non-adrenergic non-cholinergic nerves by electrical stimulation produces relaxation of canine lingual artery that is mediated by activation of CGRP1 receptors.

Protective Effect of the Nitrate Ester ITF 296 Against

The activity of ITF 296 against methacholine-induced myocardial ischemia was investigated in anesthetized rats in comparison with the organic nitrates nitroglycerin (NTG) and isosorbide dinitrate (ISDN), the K(+)-channel openers nicorandil and cromakalim, the Ca(2+)-channel blocker amlodipine, and the vasodilator dipyridamole. Given as i.v. boluses, ITF 296 (0.1-100 micrograms/kg) dose-dependently prevented methacholine-induced ST-segment elevation without affecting mean arterial blood pressure or heart rate to a significant extent. In this situation, ITF 296 was 10- to 30-fold more potent than the other coronary vasodilators tested. The protective effect of ITF 296 and ISDN against myocardial ischemia was also observed after oral administration, demonstrating good absorption and a limited first-pass metabolism of the two drugs. The anti-ischemic action of ITF 296 at 1 and 10 micrograms/kg/min was well maintained during 2 h of continuous i.v. infusion, whereas the effect of NTG and ISDN was attenuated or abolished by the end of the infusion. The new nitrate ester ITF 296 has a potent and long-lasting cardioprotective action in the anesthetized rat. The anti-ischemic effect displayed by this compound is probably mediated by an improvement of myocardial blood supply caused by pronounced coronary dilatation, whereas the contribution of systemic vasodilation is not important. Moreover, the maintenance of the anti-ischemic action during continuous i.v. infusion suggests a reduced "tolerance" development for ITF 296 compared with other nitrovasodilators.

Ventricular Overdrive Pacing-Induced Preconditioning and No-Flow Ischemia-Induced Preconditioning in Isolated Working Rat Hearts

To examine preconditioning induced by short periods of ventricular overdrive pacing (VOP) as compared with that induced by no-flow ischemia, we subjected isolated working rat heart to 10-min coronary artery occlusion (test ischemia) followed by 3-min reperfusion after three intermittent periods of VOP (10 Hz) or 5-min no-flow ischemia, respectively. In the nonpreconditioned group, coronary occlusion decreased aortic flow (AF) from 46.6 +/- 2.4 to 13.7 +/- 1.7 ml/min and increased left ventricular end-diastolic pressure (LVEDP) from 0.53 +/- 0.05 to 2.02 +/- 0.07 kPa. Preconditioning by VOP or no-flow ischemia significantly increased AF to 25.1 +/- 2.3 ml/min (p < 0.001) and to 27.3 +/- 1.4 ml/min (p < 0.001) and decreased LVEDP to 1.38 +/- 0.1 kPa (p < 0.001) and to 1.65 +/- 0.05 kPa (p < 0.05), respectively, after test ischemia. Glibenclamide 10(-7) M which blocked the anti-ischemic effect of the ATP-sensitive K(+)-channel (KATP) opener cromakalim, inhibited VOP-induced protection (AF 20.3 +/- 2.3 ml/min; LVEDP 1.82 +/- 0.15 kPa), but did not affect no-flow ischemia-induced preconditioning [AF 26.6 +/- 2.4 ml/min (p < 0.001), LVEDP 1.60 +/- 0.07 kPa (p < 0.01)]. VOP and no-flow ischemia precondition heart, however their cardioprotective mechanisms may be different in terms of KATP activation in rats.

Dilating effect of perivascularly applied potassium channel openers cromakalim and pinacidil in rat and cat pial arteries in situ

The aim was to test the vasodilator effect of perivascularly applied potassium channel openers cromakalim and pinacidil in pial arteries of cat and rat. Using an open cranial window technique the diameter of pial arteries in the parietal cortex of rat and cat was recorded with an image splitting method. Employing micropuncture techniques, test compounds were dissolved in inert cerebrospinal fluid and infused into the perivascular space of individual arteries. Cromakalim (7 x 10(-18)-7 x 10(-13) M) induced concentration dependent dilatation of feline pial arteries with a maximum effect of 26.8(SEM 3.2)% at 7 x 10(-15) M. In rat arteries, cromakalim had a maximum effect of 32.1(4.97)% at 10(-15) M, while pinacidil (10(-10)-10(7) M) exerted a maximum dilatation of 29.5(3.3)% at 10(-8) M. The latter is consistent with previous findings in feline pial arteries. The sulphonylurea glibenclamide (10(-8)-10(-6) M) had no effect on the resting diameter of rat and cat pial arteries, indicating that their resting tone is not influenced by mechanisms involving ATP sensitive potassium channels. However, glibenclamide reduced the dilating effect of cromakalim and pinacidil in rat and cat in a dose dependent manner. A comparison with previously published data obtained in isolated middle cerebral and basilar arteries showed that potassium channel openers of the benzopyrane and cyanoguanidine type are much more potent in pial arteries than in peripheral arteries in situ or in isolated arteries of the circle of Willis and of peripheral vascular beds.

Synergistic action of vasodilators that increase cGMP and cAMP in the hamster cremaster microcirculation.

Compounds such as endothelium derived nitric oxide (NO) and prostacyclin (prostaglandin I2) which increase cGMP and cAMP inhibit platelet activation in a synergistic manner. The aim of this study was to examine whether these compounds also interact synergistically in the control of smooth muscle tone. Vascular diameters in the cremaster of 49 anaesthetised hamsters (465 arterioles) were studied during superfusion with compounds raising cAMP (isoprenaline and prostacyclin) and cGMP (sodium nitroprusside) alone or in combination. (1) The isoprenaline induced maximum dilator response was significantly attenuated, from 86.1(SEM 0.7)% to 37.1(0.2)%, after inhibition of NO-synthase with NG-nitro-L-arginine (L-NNA, 30 microM). Superfusion with sodium nitroprusside (30 nM, dilatation alone: 6.7%), which was used to substitute for endothelium derived NO, restored the attenuated isoprenaline response. The combined effects of isoprenaline and sodium nitroprusside were supra-additive. Virtually identical results were obtained when prostacyclin, another cAMP raising compound, was used instead of isoprenaline. The K+ channel opener cromakalim (100 nM) which acts cGMP independently was without effect on the prostacyclin induced dilator response. (2) The sodium nitroprusside induced maximum dilator response was attenuated from 80.9(0.25)% to 70.1(0.4)%, after indomethacin (3 microM) and restored by simultaneous application of prostacyclin (1 nM, dilatation alone: 1.4%) but not of cromakalim. Again, the combined effects were supra-additive, suggesting a synergistic action of these compounds. (3) Although indomethacin or L-NNA alone decreased the resting diameter by approximately 9.5%, the simultaneous application of both inhibitors failed to decrease the resting diameter further (10.0%, p = 0.97). Vasodilators increasing cGMP and cAMP act synergistically in vivo. Continuous release of NO and prostaglandins by the endothelium may therefore not only modulate the efficacy of such cyclic nucleotide increasing vasodilators but also interact synergistically in controlling basal vascular tone.

Inhibition by Cromakalim, Pinacidil, Terbutaline, Theophylline and Verapamil of Non-cholinergic Nerve-mediated Contractions of Guinea-pig Isolated Bronchi

Summary: Contractions induced by electrical field stimulation of sensory non-cholinergic excitatory nerves in guinea-pig isolated bronchi are due to the release of substance P (SP) and related tachykinins. Release of such neuropeptides are thought to play a pathophysiological role in asthma. Two K + channel openers cromakalim ( pD 2=6.45; E max=95%) and pinacidil ( pD 2=6.06; E max=87%) were shown to concentration-dependently inhibit non-cholinergic nerve-mediated contractions in guinea-pig bronchi in vitro. Cromakalim ( pD 2=6.27; E max=25%) and pinacidil ( pD 2=6.03; E max=25%) each had a much lower inhibitory efficacy against contractions induced by exogenously applied SP but the same potency as found against contractile responses to non-cholinergic neurostimulation. Also the β 2-adrenoceptor agonist terbutaline ( pD 2=8.29; E max=83%), the xanthine derivative theophylline ( pD 2=4.19; E max=100%) and the Ca 2+ blocker verapamil ( pD 2=5.55; E max=100%) suppressed responses to non-cholinergic neurostimulation. Terbutaline ( pD 2=6.32; E max=74%), theophylline ( pD 2=3.25; E max=71%) and verapamil ( pD 2=4.01; E max=100%) had a 10-100-fold lower inhibitory potency against SP-induced contractions but each drug showed about the same efficacy as found against nerve-mediated contractions. Glibenclamide (1 μM) reversed the inhibitory effects of cromakalim and pinacidil on neurally-mediated contractions but did not influence the effects of terbutaline, theophylline and verapamil. The results demonstrate that cromakalim, pinacidil, terbutaline, theophylline and verapamil inhibit non-cholinergic excitatory neurotransmission in guinea-pig bronchi and suggest that they act preferentially at a pre-junctional site.

Role of ATP-sensitive K+ channels in antinociception induced by R-PIA, an adenosine A1 receptor agonist.

The influence of several K+ channel-acting drugs on antinociception induced by the adenosine A1 receptor agonist (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) was evaluated with a tail flick test in mice. The subcutaneous administration of R-PIA (0.5-8 mg/kg) induced a dose-dependent antinociceptive effect. The ATP-sensitive K+ (KATP) channel blocker gliquidone (2-8 micrograms/mouse, i.c.v.) produced a dose-dependent displacement to the right of the R-PIA dose-response line, whereas the KATP channel opener cromakalim (32 micrograms/mouse, i.c.v.) shifted it to the left. Several KATP channel blockers dose-dependently antagonized the antinociceptive effect of R-PIA, the order of potency being gliquidone > glipizide > glibenclamide (i.e., the same order of potency shown by these drugs in blocking KATP channels in neurons). In contrast, the K+ channel blockers 4-aminopyridine and tetraethylammonium did not antagonize the effect of R-PIA. These data suggest that antinociception produced by adenosine A1 receptor agonists is mediated by the opening of ATP-sensitive K+ channels. The present results, together with those of previous studies, further support a role for K+ channel opening in the antinociceptive effect of agonists of receptors coupled to Gi/Go proteins.

Effect of potassium on the action of the KATP modulators cromakalim, pinacidil, or glibenclamide on arrhythmias in isolated perfused rat heart subjected to regional ischaemia

The ATP sensitive potassium channel openers cromakalim (n = 10) and pinacidil (n = 10), and a blocker of this channel, glibenclamide (n = 10), were studied in isolated perfused rat hearts subjected to regional ischaemia at varying concentrations (2 to 8 mM) of external potassium ([K+]o). Hearts were isolated and perfused on a Langendorff apparatus. Vehicle (0.1% DMSO), cromakalim (10 microM), pinacidil (10 microM), or glibenclamide (10 microM) were given 10 min before ischaemia. The left coronary artery was then occluded for 15 min and reperfused for 5 min. No agent caused more than a 10% change in heart rate. Both cromakalim and pinacidil increased (30%), and glibenclamide decreased (30%) coronary flow at 4 and 6 mM [K+]o. In the vehicle group, increases in [K+]o produced concentration dependent reductions in arrhythmia scores by decreasing ventricular fibrillation. No concentration dependent effects of [K+]o on ischaemic ventricular tachycardia was observed. Under ischaemic conditions, potassium channel openers and glibenclamide more markedly reduced ischaemic ventricular tachycardia and fibrillation relative to the effects of increased [K+]o. Ischaemic ventricular fibrillation was inversely related to changes in [K+]o, whereas effects on ventricular tachycardia were all-or-none. Neither potassium channel openers nor glibenclamide elicited significant proarrhythmic activity despite variations in [K+]o. These data suggest that both potassium channel openers and glibenclamide display potential antiarrhythmic activity through their ability to abolish two distinct arrhythmogenic mechanisms during ischaemia. It is also suggested that the underlying mechanisms of ventricular tachycardia and fibrillation are coupled during ischaemia in the rat.

Effects of the potassium channel openers cromakalim and pinacidil on catecholamine secretion and calcium mobilization in cultured bovine adrenal chromaffin cells

The effects of two K + channel openers, cromakalim and pinacidil, on voltage-dependent and receptor-mediated catecholamine secretion and Ca 2+ mobilization in bovine adrenal chromaffin cells were studied to determine the role of membrane K + channels in the regulation of a Ca 2+-dependent secretory process. Both cromakalim and pinacidil stimulated the efflux of 86Rb (used to monitor K + permeability) from preloaded cells. Cromakalim and pinacidil did not affect the catecholamine secretion induced by excessive depolarization with 56 mM K +, but inhibited that induced by moderate depolarization with 31 mM K + in a concentration-dependent manner (1 μM–100 μM). The 31 mM K +-induced 45Ca 2+ influx and increase in intracellular free Ca 2+ concentration [Ca 2+]i were also inhibited by these agents at similar concentrations to those for inhibition of catecholamine secretion. Cromakalim and pinacidil inhibited catecholamine secretion, 45Ca 2+ influx and increase in (Ca 2+]i induced by stimulation of nicotinic acetylcholine (ACh) receptors with carbamylcholine. Furthermore, both cromakalim and pinacidil inhibited the increase in [Ca 2+]i induced by carbamylcholine in the absence of extracellular Ca 2+, which is thought to be mediated by muscarinic ACh receptors. On the other hand, they did not affect catecholamine secretion induced by Bay-K 8644, Ba 2+, A23187, histamine or bradykinin. These results indicate that the K + channel openers, cromakalim and pinacidil, selectively inhibit catecholamine secretion induced by moderate depolarization or by nicotinic ACh receptor stimulation by inhibiting Ca 2+ influx and increase in [Ca 2+]i. Furthermore, the results suggest that these K + channel openers-sensitive membrane K + channels are involved in the regulation of catecholamine secretion mainly indirectly through effects on the voltage-dependent membrane Ca 2+ channels.

Prejunctional effects of cromakalim, nicorandil and pinacidil on noradrenergic transmission in rat isolated mesenteric artery

The present study investigated the effects of cromakalim, nicorandil and pinacidil on resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. The efflux of radioactivity evoked by field stimulation of peri-arterial sympathetic nerves (pulses at 2 Hz frequency in trains of 60 s duration) was taken as an index of transmitter noradrenaline release. Cromakalim (1-100 microM) and nicorandil (1-1000 microM) produced minor effects on resting and S-I effluxes of radioactivity, but these did not exhibit concentration-dependency. Pinacidil (1-1000 microM) produced concentration-dependent increases, in both resting and S-I effluxes of radioactivity. With 1000 microM pinacidil, resting and S-I effluxes were increased to approximately 348% and 358% of their respective control values. The effects of pinacidil on resting and S-I effluxes were unaltered when the neuronal amine transport system was inhibited by desipramine (1 microM). Inhibition of monoamine oxidase with pargyline (100 microM) treatment markedly reduced the enhancement of resting efflux by 1000 microM pinacidil but did not alter its effect on S-I efflux. It is proposed that the enhanced resting efflux produced by pinacidil without pargyline treatment consists of deaminated [3H]-noradrenaline metabolites formed from [3H]-noradrenaline displaced from transmitter storage vesicles by pinacidil. The enhancement of S-I efflux by pinacidil does not appear to involve disruption of alpha 2-adrenoceptor auto-inhibition of transmitter release since equi-effective concentrations of phentolamine (1 microM) and pinacidil (1000 microM) produced additive effects on S-I efflux, whereas increasing the concentration of phentolamine from 1 to 2M produced no further increases in S-I efflux. In conclusion, this study has provided no evidence of a prejunctional inhibitory effect of the potassium channel openers cromakalim, nicorandil and pinacidil on transmitter noradrenaline release. However, the findings with pinacidil suggest that, in high concentrations, pinacidil displaces noradrenaline from transmitter stores, such that deaminated noradrenaline metabolites are released from the nerve terminals. Furthermore, pinacidil enhances S-I transmitter noradrenaline release, possibly by blocking neuronal potassium channels.

Effect of glibenclamide on membrane response to metabolic inhibition in smooth muscle of rat portal vein.

Vascular smooth muscle tone is dependent on oxidative metabolism, a phenomenon of potential importance for the metabolic regulation of blood flow to tissues. The response of the rat portal vein to inhibition of cell respiration by cyanide (0.1-1 mM) is a reduction of its spontaneous myogenic activity. The trains of action potentials triggering phasic contractions are reduced in duration, while the frequency of trains is often somewhat increased as the resting membrane potential in the intervals between spike trains is less negative by 6.5 mV. Glibenclamide (10(-7) M) did not affect the resting membrane potential or spontaneous mechanical activity of oxygenated portal veins, but partly restored the depressed myogenic activity in the presence of cyanide (0.5 mM). The spike trains were longer, while the membrane was depolarized by 3 mV compared with the effects of cyanide alone. Inhibition of both oxidative and glycolytic metabolism by 2 mM NaCN in a medium where glucose was replaced by beta-hydroxybutyrate caused a hyperpolarization which was abolished by 10(-7) M glibenclamide. The relaxing effect of the K+ channel opener cromakalim (5 x 10(-9) to 6.25 x 10(-7) M) was partly antagonized by glibenclamide. Basal cytosolic [Ca2+] was increased by cyanide, while the Ca2+ transients associated with phasic contractions were reduced in duration. This latter effect was partially reversed by glibenclamide. The effect of cyanide on high-K+ contractures, which are associated with sustained membrane depolarization and not dependent on repetitive spike activity, was not influenced by 10(-7) M glibenclamide. The effects of inhibited cell respiration on spontaneous electrical activity seem to reflect a depolarizing drive caused by inhibited active ion exchange mechanisms, modified by a repolarizing drive, possibly from ATP-regulated K+ channels, causing reduced duration of the spike trains. While glibenclamide affects spontaneous activity at all levels of oxidative blockade, glibenclamide-sensitive hyperpolarization is seen only when both oxidative and glycolytic metabolism is inhibited.

Effects of ATP-sensitive K+ channel openers on pacemaker activity in isolated single rabbit sino-atrial node cells.

Electrophysiologic effects of ATP-sensitive K+ channel openers (cromakalim, pinacidil, and nicorandil) in single rabbit sino-atrial (SA) node cells were examined by a whole-cell voltage- and current-clamp technique. Cromakalim (> 30 microM), pinacidil (> 1 microM), and nicorandil (> 500 microM) caused a negative chronotropic effect. At low concentrations, the maximum diastolic potential was hyperpolarized and the maximum rate of depolarization was enhanced, but at high concentrations, both were reversed and action potential amplitude (APA) was decreased significantly. Pinacidil (> 30 microM) and nicorandil (> 3 microM) prolonged AP duration (APD), but cromakalim did not affect it. In whole-cell voltage-clamp experiments, cromakalim (100 microM), pinacidil (> 30 microM), and nicorandil (> 300 microM) inhibited the Ca2+ current significantly but had little or no effect on the delayed rectifying K+ current and the hyperpolarization-activated inward current. Glibenclamide (1 microM) did not antagonize the effects of K+ channel openers on Ca2+ current and APs. These results indicate that the K+ channel openers have direct inhibitory actions on spontaneous APs and Ca2+ current in rabbit SA node cells (but K+ current was unaffected), resulting in decreased pacemaker activity.

Cromakalim does not protect against skeletal muscle fatigue in an anaesthetized rat model of acute hindlimb ischaemia

The effects of the potassium (K +) channel opener, cromakalim, on skeletal muscle performance were studied in a model of acute hindlimb ischaemia in the anesthetized rat. Twitch contractions to direct electrical stimulation of the extensor digitorum and tibialis anterior skeletal muscles were recorded following administration of cromakalim (10–100 μg kg −1 i.v.) under normal and reduced whole limb blood flow. With normal blood flow, twitch responses (0.5 and 1 Hz) of the hindlimb skeletal muscles were sustained for > 30 min. Controlled adjustment of the perfusion pressure in the contralateral hindlimb to 45, 30 or 0 mm Hg 0 by partial or total occlusion of the abdominal aorta produced a pressure-related fall in flow to the working hindlimb, and a corresponding increase in the rate of muscle fatigue. Cromakalim (10–100 μg kg −1 i.v.) produced a dose-dependent reduction in mean carotid arterial blood pressure, femoral arterial pressure and hindlimb vascular resistance together with an increase in iliac artery blood flow and heart rate, but did not attenuate skeletal muscle fatigue under the different conditions of muscle work and ischaemia employed. A similar profile was observed with levcromakalim (15 μg kg −1 i.v), the active enantiomer of cromakalim. These results demonstrate that in the direct muscle-stimulated hindlimb of the anaesthetized rat, the K + channel opener cromakalim does not prevent acute ischaemia-induced skeletal muscle fatigue. The previous observation that K + channel openers improve nutritive blood flow in a chronic model of rat hindlimb ischaemia is not reflected by an improvement in muscle function in the present study.

Effects of Antidiabetic Sulphonylureas, Cromakalim and their Interaction in Guinea-pig Isolated Tracheal Smooth Muscle

Summary: Glibenclamide, glipizide and glibornuride showed dual effects in guinea-pig isolated trachea. The drugs antagonized the relaxant response to the K + channel opener cromakalim (order of effectiveness: glibenclamide > glipizide > glibornuride) and at concentrations of 1-1000 μM produced airway smooth muscle relaxation (order of potency: glibenclamide > glipizide = glibornuride). Gliclazide, tolbutamide and chlorpropamide did not antagonize cromakalim nor did the two latter drugs produce tracheal relaxation. The sulphonylureas and cromakalim were compared as airway relaxants against a panel of different spasmogens. The order of tissue responsiveness for the sulphonylureas was: spontaneous tone = LTD 4 > PGF 2α = histamine = 30 mM K + > carbachol and for cromakalim: spontaneous tone = LTD 4 = PGF 2α = histamine > carbachol > 30 mM K +. Glibenclamide, but not cromakalim, relaxed contractions induced by 124 mM K +. Phentolamine and Ba 2+, which are reported blockers of ATP-regulated K + channels, failed to influence sulphonylurea-induced airway smooth muscle relaxation. Glibenclamide reversed tracheal relaxation produced by cromakalim, whereas cromakalim failed to reverse relaxation induced by glibenclamide. The mechanism for the additional property of sulphonylureas to relax airway smooth muscle is unclear, but the results do not support a role for involvement of cromakalim-sensitive K + channels.

Combined administration of an IK(ATP) activator and Ito blocker increases coronary flow independently of effects on heart rate, QT interval, and ischaemia-induced ventricular fibrillation in rats.

The effects of combined administration of the ATP-dependent K+ channel opener cromakalim and the transient outward K+ current (Ito) blocker tedisamil on ventricular tachyarrhythmias, QT interval, and coronary flow were investigated in isolated rat heart (n = 12/group) subjected to 30-min regional ischaemia. When administered alone, beginning 5 min before onset of ischaemia and continuously thereafter, neither cromakalim (10 microM) nor tedisamil (3 microM) had any significant influence on the incidence of VF, although tedisamil abolished sustained VF (SVF, defined as VF lasting > 2 min). Cromakalim did not affect tedisamil's ability to abolish SVF when administered concurrently with tedisamil and had no effect on SVF when administered alone; incidences of SVF were 64, 0 (p < 0.05), 66, and 0% (p < 0.05) in control, tedisamil, cromakalim, and combined treatment groups, respectively. Occurrence of SVF was related to the width of the ventricular complex at 50% of repolarisation (QT50). Pretreatment with tedisamil widened QT50 (p < 0.05), and this effect was not attenuated by combined treatment with cromakalim. Cromakalim increased coronary flow (14 +/- 0.4 in controls versus 21 +/- 0.6 ml/min/g in cromakalim-treated hearts, p < 0.05). This effect was not reduced by coadministration of tedisamil (20 +/- 0.9 ml/min/g in hearts cotreated with both drugs). Tedisamil alone had no significant effect on coronary flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct pathways to refill ACh-sensitive internal Ca2+ stores in canine airway smooth muscle

The ability of extracellular Ca2+ to refill internal Ca2+ stores of canine tracheal smooth muscle after a prior depletion by acetylcholine (ACh) was assessed using a novel sarcoplasmic reticulum (SR) Ca2+ pump inhibitor, cyclopiazonic acid (CPA). The transient contraction induced by ACh in a medium free of Ca2+ was used as an index for the content of agonist-sensitive intracellular Ca2+ stores. CPA inhibited in a concentration-dependent manner the refilling of the stores occurring during high KCl stimulation, and this inhibitory effect was independent of the external Ca2+ concentration. On the other hand, CPA was less effective in inhibiting the refilling occurring during prolonged ACh stimulation, especially when external Ca2+ concentration was raised. At 5.0 mM external Ca2+ or when 0.1 microM BAY 8644 was present in the medium, CPA was ineffective in inhibiting the refilling occurring during prolonged ACh stimulation. The maximum ACh-induced contraction in Ca(2+)-containing medium was independent of the extent of internal store Ca2+ load in the absence of L-type Ca2+ channel blocker but was highly dependent on the extent of internal Ca2+ load in the presence of the Ca2+ channel blocker. Hyperpolarization of the plasma membrane with the K+ channel opener cromakalim reduced the amplitude of ACh tonic contraction. Subsequent addition of nifedipine further reduced ACh tonic contraction. It is concluded that two different pathways for external Ca2+ are used to refill ACh-sensitive internal stores. One involves active Ca2+ uptake via a CPA-sensitive Ca2+ pump, and the other involves a CPA-insensitive pathway whose nature remains to be determined.

The discovery of a novel calcium channel blocker related to the structure of potassium channel opener cromakalim

During our studies aimed at the identification of novel analogs of the potassium channel opener cromakalim ( 3), we serendipitously observed pyranoquinoline 4 to possess pure calcium channel blocking activity. The results of the studies conducted to confirm the calcium channel blocking mechanism of 4 are reported in this paper.