Possible involvement of ATP-sensitive K+ channels in the relaxant response of dog middle cerebral artery to cromakalim

To determine the functions of ATP-sensitive K+ (KATP) channels in cerebral arterial smooth muscle, the effects of cromakalim, an opener of these channels, on tension and 86Rb efflux were investigated in endothelium-removed strips of dog middle cerebral arteries (MCAs). Cromakalim relaxed the strips that were precontracted with 20.9 mM K+ with a small maximum response. The relaxant responses to cromakalim were competitively antagonized by glibenclamide, a blocker of KATP channels. In strips precontracted with 65.9 mM K+, cromakalim failed to relax the strips. The addition of cromakalim to a resting strip caused a dose-dependent relaxation. In the resting strips of MCAs preloaded with 86Rb, cromakalim did not increase the 86Rb efflux. With 42K as the tracer ion, cromakalim still had no effect on the efflux from the resting strips. On the other hand, cromakalim increased the 86Rb and 42K efflux from the strips of dog coronary arteries (CAs). In 20.9 mM K(+)-contracted strips of MCAs, cromakalim significantly decreased the 86Rb efflux. However, after the inactivation of Ca(++)-activated K+ channels by the addition of 1 x 10(-7) M nifedipine to the 20.9 mM K(+)-contracted strips of MCAs, cromakalim produced a small but significant increase in the 86Rb efflux. Similarly, when the resting strips of MCAs were placed in the Ca(++)-free 12 mM-Mg(+)+ solution, cromakalim increased the 86Rb efflux. In 65.9 mM K(+)-contracted strips, cromakalim increased the 86Rb efflux from both arteries. However, the extent of the increase in 86Rb efflux was significantly smaller in the MCA than in the CA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of diazoxide, cromakalim and pinacidil on adrenergic neurotransmission and 86Rb+ efflux in rat brain cortical slices.

The effects of diazoxide, cromakalim and pinacidil on depolarization-evoked tritium overflow from the rat brain cortical slices preloaded with [3H]noradrenaline were studied. Diazoxide inhibited both transmural nerve stimulation (TNS)- and 25 mM K(+)-evoked tritium overflows more potently than cromakalim. Diazoxide effects were only partially antagonized and cromakalim ones were totally reversed by 1 microM glibenclamide. Diazoxide, but not cromakalim, reduced the 45 mM K(+)-evoked tritium overflow, which was not antagonized by glibenclamide. Both diazoxide and cromakalim stimulated 86Rb+ efflux to a similar extent, the effects being completely abolished by glibenclamide. Glibenclamide (> or = 3 microM) by itself enhanced the TNS-evoked tritium overflow. Pinacidil increased both TNS- and K+ (25 and 45 mM)-evoked tritium overflows with little effect on 86Rb+ efflux. Pinacidil-induced increase in the TNS-evoked tritium overflow was still observed in the presence of cocaine or hydrocortisone. Pinacidil failed to affect the inhibitory action of xylazine on the TNS-evoked tritium overflow, whereas phentolamine attenuated it. These results indicate that ATP-sensitive K+ channels are present in the adrenergic nerve endings of rat brain. These channels seem to be pharmacologically different from those reported for vascular smooth muscles and pancreatic beta-cells.     

Alterations by glyburide of effects of BRL 34915 and P 1060 on contraction, 86Rb efflux and the maxi-K+ channel in rat portal vein

Effects of the K+ channel blocking agent, glyburide, on the actions of two K+ channel openers, BRL 34915 (cromakalim) and P 1060 (Leo), a potent pinacidil derivative (N-(t-butyl)-N"-cyano-N'-3-pyridyl-guanidine), were ascertained. Tension responses and 86Rb fluxes in rat portal vein strips and single channel electrophysiological recordings in enzymatically dissociated rat portal vein cells were obtained. Glyburide (0.3 microM) increased spontaneous contractile activity and caused concentration-dependent shifts in the relaxation responses to BRL 34915 and P 1060. Increases in 86Rb efflux were obtained only at much higher concentrations of BRL 34915 or P 1060, and these increases were blocked only at higher concentrations of glyburide (5.0 microM). BRL 34915 and P 1060 specifically increase the open-state probability of the Ca+(+)-activated K+ (maxi-K+) channel, and these actions are blocked by glyburide and also by charybdotoxin. Changes in single channel activity and contractile responsiveness occur at similar concentrations of agonists and antagonists. Thus, the membrane channel in rat portal vein affected by glyburide, BRL 34915 and P 1060 appears to be the Ca+(+)-activated maxi-K+ channel (that does not show ATP dependence under the conditions of these experiments). Concentrations of agonists and antagonists effective on maxi-K+ channel activity correspond to those affecting contractile responsiveness and are lower than those eliciting changes in 86Rb flux.     

The effects of the putative potassium channel activator WAY-120,491 on 86Rb efflux from the rabbit aorta

WAY-120,491 [(-)-(3S-trans)-2-[3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl]-2,3-dihydro-1H-isoindol-1-one] is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.     

In vitro and in vivo comparison of two K+ channel openers, diazoxide and cromakalim, and their inhibition by glibenclamide

Diazoxide caused an increase in 86Rb+ efflux from the rat aorta and portal vein and inhibited spontaneous activity of the latter at concentrations 100 times higher than the K+ channel opener cromakalim. In the rabbit aorta both drugs inhibited vasoconstrictor responses to angiotensin II, noradrenaline and low concentrations (less than or equal to 30 mM) of KCl in a similar manner, the antivasoconstrictor activities being abolished in vessels depolarized with greater than or equal to 35 mM K+. In vivo cromakalim was about 100 times more potent than diazoxide at lowering blood pressure in rats. Diazoxide (30 mg/kg) caused a more than 2-fold increase in plasma glucose in rats and prevented any return toward base line within 1.5 hr after a glucose load. Cromakalim had minimal effects upon glucose homeostasis at equihypotensive doses. Glibenclamide, a potent blocker of ATP-dependent K+ channels, inhibited the stimulation by cromakalim and diazoxide of 86Rb+ efflux from the portal vein and aorta (IC50 approximately 0.1 microM), antagonized their vasorelaxant effects in vitro and in vivo (20-30 mg/kg i.v.) and reversed the diazoxide-induced changes in plasma glucose and insulin levels. These results provide evidence that diazoxide, like cromakalim, is able to open 86Rb+-permeable K+ channels in vascular smooth muscle. This action is likely to be responsible for the in vitro and in vivo vasodilator activity of these two drugs. However, there would seem to be pharmacological differences between the K+ channels affected by these drugs in vascular smooth muscle and the (ATP-sensitive) K+ channels of pancreatic beta-cells, which are thought to be responsible for the effects of diazoxide on plasma glucose.     

Effects of K+ channel blockers and cromakalim (BRL 34915) on the mechanical activity of guinea pig detrusor smooth muscle.

There is strong evidence that cromakalim (BRL 34915) relaxes smooth muscle by opening cell membrane K+ channels. The aim of this study was to use relatively selective K+ channel blockers to investigate 1) the K+ channel type(s) opened by cromakalim in guinea pig detrusor and 2) the role of different K+ channel types in the control of basal tension. Cromakalim produced a concentration-related relaxation (IC50 = 0.50 +/- 0.03 microM, n = 42) of 15 mM K(+)-evoked mechanical activity. The ATP-sensitive K+ channel blocker glyburide (0.3-3 microM) antagonized the effects of cromakalim in an apparently competitive manner (pA2 = 6.76). Charybdotoxin and iberiatoxin (3-30 nM), blockers of the large conductance, Ca(++)-activated K+ channel, appeared to functionally antagonize cromakalim. Apamin (1 microM) and leiurotoxin I (0.3 microM), blockers of the small conductance, Ca(++)-activated K+ channel, and noxiustoxin (0.3 microM), a blocker of squid axon delayed rectifer K+ channels, all failed to antagonize cromakalim. Cumulative administration of charybdotoxin and iberiatoxin produced marked, concentration-related stimulation of mechanical activity per se whereas glyburide, noxiustoxin, apamin and leiurotoxin I had no effect. Apamin and leiurotoxin I did stimulate mechanical activity to a small extent when administered noncumulatively, however. The results suggest that cromakalim opens ATP-sensitive K+ channels in detrusor and suggest that cromakalim does not open CA(++)-activated K+ channels and noxiustoxin-sensitive, delayed rectifier K+ channels. The marked stimulatory effects of charybdotoxin and iberiatoxin per se suggest an important role for large conductance, Ca(++)-activated K+ channels in the control of basal tension and, presumably, membrane potential in detrusor smooth muscle cells.     

Relaxant effects of beta-carbolines on rat aortic rings.

The effects of two beta-carbolines, methyl 6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylate (DMCM) and ethyl beta-carboline-3-carboxylate (beta CCE) were assayed on rat aortic rings precontracted with different agonists. The beta-carbolines tested induced a concentration-dependent (2-200 microM) relaxation of aortic rings precontracted with 30 mM KCl. This relaxation was not modified by the removal of the rat aortic endothelium. Contractions elicited by the activation of either voltage-gated calcium channels (0.05 microM BAY K 8644) or receptor-operated calcium channels (0.1 microM norepinephrine), as well as contractions produced by the entry of calcium as a lipid-soluble complex (10 microM A23187), were also reduced by DMCM and by beta CCE. In addition, whereas DMCM did not modify calmodulin activity, both beta-carbolines inhibited in a concentration-dependent manner (0.6-200 microM) the rat aortic cyclic nucleotide phosphodiesterase activity. Moreover, DMCM as well as beta CCE potentiated the relaxation of K(+)-contracted aortic rings induced by the stimulation of either adenylyl cyclase with forskolin (0.1-1 microM) or guanylyl cyclase with sodium nitroprusside (0.1-100 nM). The intracellular rat aortic levels of cyclic AMP measured in the presence of 0.1 microM forskolin were increased by 100% in the presence of DMCM. On the other hand, 6 microM DMCM potentiated the relaxation induced by nifedipine in K(+)-contracted aortic rings, whereas the K+ channel blocker 10 mM tetraethylammonium did not modify the relaxation elicited by DMCM in the norepinephrine-contracted preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chiral recognition of pinacidil and its 3-pyridyl isomer by canine cardiac and smooth muscle: antagonism by sulfonylureas

Pinacidil, a potassium channel opener (PCO), relaxes vascular smooth muscle by increasing potassium ion membrane conductance, thereby causing membrane hyperpolarization. PCOs also act on cardiac muscle to decrease action potential duration (APD) selectively. To examine the enantiomeric selectivity of pinacidil, the stereoisomers of pinacidil (a 4-pyridylcyanoguanidine) and its 3-pyridyl isomer (LY222675) were synthesized and studied in canine Purkinje fibers and cephalic veins. The (-)-enantiomers of both pinacidil and LY222675 were more potent in relaxing phenylephrine-contracted cephalic veins and decreasing APD than were their corresponding (+)-enantiomers. The EC50 values for (-)-pinacidil and (-)-LY222675 in relaxing cephalic veins were 0.44 and 0.09 microM, respectively. In decreasing APD, the EC50 values were 3.2 microM for (-)-pinacidil and 0.43 microM for (-)-LY222675. The eudismic ratio was greater for the 3-pyridyl isomer than for pinacidil in both cardiac (71 vs. 22) and vascular (53 vs. 17) tissues. (-)-LY222675 and (-)-pinacidil (0.1-30 microM) also increased 86Rb efflux from cephalic veins to a greater extent than did their respective optical antipodes. The antidiabetic sulfonylurea, glyburide (1-30 microM), shifted the vascular concentration-response curve of (-)-pinacidil to the right by a similar extent at each inhibitor concentration. Glipizide also antagonized the response to (-)-pinacidil, but was about 1/10 as potent with a maximal shift occurring at 10 and 30 microM. Glyburide antagonized the vascular relaxant effects of 0.3 microM (-)-LY222675 (EC50, 2.3 microM) and reversed the decrease in APD caused by 3 microM (-)-LY222675 (EC50, 1.9 microM). Nitroprusside did not alter 86Rb efflux, and vascular relaxation induced by sodium nitroprusside was unaffected by sulfonylureas. Thus, the enantiomers of the 3-pyridyl isomer of pinacidil demonstrate enhanced stereospecificity in both canine cardiac and vascular tissues compared to the enantiomers of pinacidil. However, the relative selectivity of pinacidil and its 3-pyridyl isomer for cardiac and vascular smooth muscle remains unaltered. Sulfonylureas antagonize the more potent enantiomers in both tissues, supporting the involvement of an ATP-sensitive potassium channel in the action of PCOs; however, antagonism in canine vascular smooth muscle by sulfonylureas does not resemble classical competitive antagonism.     

Effects of putative K+ channel activator BRL-34915 on arterial contraction and 86Rb efflux

Segments of thoracic aorta, mesenteric and tail arteries from male Wistar rats were used for the determination of isometric relaxation responses to BRL-34915 [chromakalin or (+/-)-6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl )-2H-benzo- [b]-pyran-3-ol] after contraction with half-maximal (ED50) values of norepinephrine or high K(+)-physiological salt solution. Contiguous segments were incubated in the presence of 86Rb and used for study of the effects of BRL-34915 on 86Rb efflux. BRL-34915 produced a dose-dependent relaxation of norepinephrine- or K(+)-induced active stress that was essentially complete (100%) in the aorta and mesenteric artery but only partial (30-40%) in the tail artery. The ED50 value of BRL-34915 for relaxation responses was about 0.1 to 0.3 microM. BRL-34915 had no significant sustained effect on 86Rb efflux from the tail artery or mesenteric artery branches but produced a dose-dependent sustained increase in efflux from thoracic aorta and portal vein. Responses reached a peak effect in 2 to 4 min after exposure but subsequently declined over a slower time course. Efflux responses to BRL-34915 had peak values that averaged 100 to 150% above basal levels with an ED50 value of about 1 to 3 microM. This suggests a dissociation of the effects of BRL-34915 in relaxation and efflux experiments. Treatment with 30 microM BRL-34915 decreased both the initial and sustained components of the subsequent 86Rb efflux response to 10 microM norepinephrine in both thoracic aorta and tail artery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-ischemic effects of the potassium channel activators pinacidil and cromakalim and the reversal of these effects with the potassium channel blocker glyburide

The direct cardioprotective efficacy of the potassium channel activators pinacidil and cromakalim was determined in isolated globally ischemic rat hearts. Isolated buffer-perfused rat hearts were subjected to 25 min of ischemia followed by 30 min of reperfusion. These hearts were pretreated with 1 to 100 microM pinacidil, 1 to 7 microM cromakalim or vehicle. Pinacidil resulted in significant improvements in reperfusion function and cardiac compliance, though it did not significantly reduce lactate dehydrogenase release at any concentration. The protective effects of pinacidil were greatest at a 10 microns concentration and were slightly diminished at higher concentrations (30 and 100 microns). Although not affecting the severity of ischemia alone, 10 microM glyburide (potassium channel blocker) completely reversed the protective effects of pinacidil on reperfusion function and compliance. Cromakalim (7 microM) resulted in a greater than 50% improvement in reperfusion function and compliance and unlike pinacidil significantly reduced lactate dehydrogenase release by approximately 50%. At 1 microM, glyburide alone did not significantly affect the severity of ischemia but reversed the protective effects of cromakalim. Not only did glyburide reverse the protective effects of cromakalim, it resulted in a worsening of ischemia compared to vehicle, an effect not seen with glyburide alone. Thus, both pinacidil and cromakalim appear to have direct cardioprotective efficacy, though some differences between them may be possible. The mechanism of their protective effects appears to be via potassium channel opening as the potassium channel blocker glyburide reverses the protective effect of these compounds. Intracellular electrophysiological studies showed that ischemia-induced depolarization was reversed with cromakalim, which increased the resting potential nearly back to preischemic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tacrine on insulin secretion and 86Rb+ and 45Ca++ efflux from rat pancreatic islets.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine), a drug that has attained interest because of its ability to alleviate symptoms in Alzheimer's type of dementia, was found to stimulate insulin secretion from isolated rat pancreatic islets. The insulinotropic effect of the drug was observed at 8.3 mM but not at 3.3 mM glucose and was dependent on extracellular Ca++. From perifused 86Rb(+)-prelabeled islets, tacrine inhibited the fractional efflux of 86Rb+ at 3.3 mM glucose, but stimulated 86Rb+ efflux at 8.3 mM glucose. These effects persisted in the absence of extracellular Ca++. Tacrine also stimulated 45Ca++ efflux from perifused 45Ca(++)-prelabeled islets at 8.3 mM but had no effect on 45Ca++ efflux at 3.3 mM glucose or in the absence of extracellular Ca++. It is concluded that tacrine potentiates glucose-stimulated insulin secretion by a mechanism that is dependent on extracellular Ca++ and involves an increased Ca++ influx. The increased Ca++ influx is either secondary to a decreased K+ permeability induced by an inhibition of ATP-dependent K+ channels and/or due to a direct effect of tacrine on glucose-activated Ca++ channels.     

Similarities between the effects of pinacidil and diazoxide on ionic and secretory events in rat pancreatic islets

The present study aimed at comparing the effects of pinacidil, a putative K+ channel opener, and diazoxide on ionic and secretory events in rat pancreatic islets. Pinacidil and diazoxide provoked a dose-dependent increase in 86Rb outflow from pancreatic islets perifused in the presence of glucose. Both drugs inhibited the glucose- and tolbutamide-induced increase in 45Ca outflow and insulin release whereas failing to affect the ionic and secretory responses to K+ depolarization. Pinacidil and diazoxide, in contrast to quinine, failed to affect 86Rb outflow from pancreatic islets stimulated by the Ca++-ionophore A23187. Pinacidil as well as diazoxide abolished the glucose-induced increase in [Ca++]i but did not modify the rise in [Ca++]i provoked by KCl. Lastly, both drugs were shown to stimulate an ouabain-resistant modality of 86Rb inflow into the islet cells. The close similarities between the ionic and secretory events mediated by pinacidil and diazoxide suggest that pinacidil could interfere with the same target site as diazoxide; namely the beta-cell ATP-sensitive K+ channel.     

Mechanism of hypoxic K loss in rabbit ventricle.

Although a critical factor causing lethal ischemic ventricular arrhythmias, net cellular K loss during myocardial ischemia and hypoxia is poorly understood. We investigated whether selective activation of ATP-sensitive K (KATP) channels causes net cellular K loss by examining the effects of the KATP channel agonist cromakalim on unidirectional K efflux, total tissue K content, and action potential duration (APD) in isolated arterially perfused rabbit interventricular septa. Despite increasing unidirectional K efflux and shortening APD to a comparable degree as hypoxia, cromakalim failed to induce net tissue K loss, ruling out activation of KATP channels as the primary cause of hypoxic K loss. Next, we evaluated a novel hypothesis about the mechanism of hypoxic K loss, namely that net K loss is a passive reflection of intracellular Na gain during hypoxia or ischemia. When the major pathways promoting Na influx were inhibited, net K loss during hypoxia was almost completely eliminated. These findings show that altered Na fluxes are the primary cause of net K loss during hypoxia, and presumably also in ischemia. Given its previously defined role during hypoxia and ischemia in promoting intracellular Ca overload and reperfusion injury, this newly defined role of intracellular Na accumulation as a primary cause of cellular K loss identifies it as a central pathogenetic factor in these settings.     

Paradoxical inhibitory effect of cromakalim on 86Rb outflow from pancreatic islet cells

Cromakalim appears to be the most potent pharmacologic agent belonging to the new class of smooth muscle relaxants: the "K+ channel openers." The present study aimed at characterizing the effects of cromakalim on 86Rb outflow, 45Ca outflow and insulin release from prelabeled and perifused rat pancreatic islets. Cromakalim provoked a concentration-dependent reduction in 86Rb outflow. This inhibitory effect was attenuated in islets exposed throughout to glibenclamide or to a Ca+(+)-free medium. In islets exposed to glucose and extracellular Ca++, cromakalim induced a dose-dependent reduction in 45Ca outflow. The drug also inhibited the increase in 45Ca outflow mediated by K+ depolarization. Lastly, cromakalim elicited a concentration-dependent inhibition of insulin release from islets perifused in the presence of glucose and extracellular Ca++. The present data suggest that the paradoxical inhibitory effect of cromakalim on 86Rb outflow probably reflects the capacity of the drug to reduce the activity of the ATP-sensitive K+ channels and to indirectly inhibit the Ca+(+)-activated K+ channels. Furthermore, the cromakalim-induced changes in 45Ca outflow are compatible with an inhibitory effect of the drug on the voltage-dependent Ca++ channels.     

Mechanism of relaxation induced by K+ and nicotine in dog duodenal longitudinal muscle.

Dog duodenal longitudinal muscle strips precontracted with bradykinin responded to K+ (10 mM) with a transient relaxation, which was abolished by tetrodotoxin and oxyhemoglobin, but not influenced by atropine, ouabain and apamin. The induced relaxation was suppressed by treatment with 10(-5) M NG-nitro-L-arginine (L-NNA) a nitric oxide synthesis inhibitor, but not by the D-enantiomer. The inhibitory effect was antagonized by L- but not D-arginine. High concentrations (20 mM or higher) of K+ produced a relaxation followed by a sustained contraction; nicardipine abolished the contraction, but did not alter the relaxation. Nicotine produced a contraction, which was converted to a relaxation by atropine. The relaxant response was abolished by tetrodotoxin, hexamethonium and oxyhemoglobin, but was unaffected by timolol and phentolamine. L-NNA suppressed the relaxation, and L-arginine reversed the inhibition. The addition of K+ (20 mM) increased the content of cyclic GMP in the strips, the effect being prevented by tetrodotoxin and L-NNA. These findings suggest that K+ selectively stimulates the nonadrenergic inhibitory nerve, whereas nicotine stimulates both the excitatory cholinergic and inhibitory nerves. Nitric oxide released from the inhibitory nerve appears to transmit information to duodenal smooth muscle by increasing the production of cyclic GMP.     

The effects of levcromakalim and pinacidil on the human internal mammary artery

Summary— The present study was undertaken to examine the effects of pinacidil and levcromakalim, two potassium channel openers, on human internal mammary artery (HIMA) obtained from patients undergoing coronary artery bypass surgery, and to clarify the contribution of different K⁺ channel subtypes in pinacidil and levcromakalim action in this blood vessel. Pinacidil and levcromakalim induced a concentration-dependent relaxation of the precontracted arterial segments (pEC₅₀ = 5.77 ± 0.05 and 6.89 ± 0.03, respectively). 4-Aminopyridine (3 mM), a non-selective blocker of K⁺ channels, induced significant shifts to the right of the concentration-response curves for pinacidil and levcromakalim. Tetraethylammonium (6 mM), charybdotoxin (0.4 μM) and apamin (0.1 μM), blockers of Ca²⁺-sensitive K⁺ channels, had no effect on the pinacidil- and levcromakalim-evoked relaxation. Glibenclamide (0.1–10 μM), a selective blocker of adenosine triphosphate (ATP)-sensitive K⁺ channels, competitively antagonized the response to levcromakalim (pK_B = 7.92 ± 0.07). In contrast, glibenclamide, in significantly higher concentrations (3–30 μM), non-competitively antagonized the response to pinacidil. High concentrations of pinacidil (> 10 μM) relaxed arterial rings bathed by a medium containing 100 mM K⁺ with maximum response 83 ± 6%. Under the same conditions, the maximum levcromakalim-induced relaxation on HIMA was almost abolished (15 ± 2%). It is concluded that pinacidil and levcromakalim do not relax the HIMA through the same subtype of K⁺ channel. ATP-sensitive K⁺ channels are probably involved in levcromakalim- but not in a pinacidil-induced relaxation in the HIMA. In addition, in pinacidil-induced relaxation of the HIMA, K⁺ channel-independent mechanisms seem to be involved.     

ATP-dependent K+ channels modulate vasoconstrictor responses to severe hypoxia in isolated ferret lungs.

In normo- and hypoglycemic ferret lungs, the pulmonary vascular response to severe hypoxia (PiO2 less than or equal to 10 mmHg) is characterized by an initial intense vasoconstriction followed by marked vasodilation, whereas in hyperglycemic lungs, vasodilation is minimal, causing vasoconstriction to be sustained. In contrast, the response to moderate hypoxia is characterized by a slowly developing sustained vasoconstriction which is unaffected by glucose concentration. To determine the role of ATP-dependent K+ (KATP) channels in these responses, we examined the effects of cromakalim, which opens KATP channels, and glibenclamide, which closes them. During steady-state vasoconstriction induced in isolated ferret lungs by moderate hypoxia, cromakalim caused dose-dependent vasodilation (EC50 = 7 x 10(-7) M) which was reversed by glibenclamide (IC50 = 8 x 10(-7) M), indicating that KATP channels were present and capable of modulating vascular tone. During severe hypoxia in hypoglycemic lungs [( glucose] less than 1 mM), glibenclamide markedly inhibited the secondary vasodilation. Raising perfusate glucose concentration to 14 +/- 0.4 mM had the same effect. As a result, initial vasoconstrictor responses were well sustained. However, neither glibenclamide nor hyperglycemia affected vasoconstrictor responses to moderate hypoxia or KCl, indicating that effects during severe hypoxia were not due to nonspecific potentiation of vasoconstriction. These findings suggest that in the ferret lung (a) severe hypoxia decreased ATP concentration and thereby opened KATP channels, resulting in increased K+ efflux, hyperpolarization, vasodilation, and reversal of the initial vasoconstrictor response; and (b) hyperglycemia prevented this sequence of events.     

Gastrointestinal, selective airways and urinary bladder relaxant effects of Hyoscyamus niger are mediated through dual blockade of muscarinic receptors and Ca2+ channels

This study describes the spasmolytic, antidiarrhoeal, antisecretory, bronchodilatory and urinary bladder relaxant properties of Hyoscyamus niger to rationalize some of its medicinal uses. The crude extract of H. niger seeds (Hn.Cr) caused a complete concentration-dependent relaxation of spontaneous contractions of rabbit jejunum, similar to that caused by verapamil, whereas atropine produced partial inhibition. Hn.Cr inhibited contractions induced by carbachol (1 microM) and K(+) (80 mM) in a pattern similar to that of dicyclomine, but different from verapamil and atropine. Hn.Cr shifted the Ca(2+) concentration-response curves to the right, similar to that caused by verapamil and dicyclomine, suggesting a Ca(2+) channel-blocking mechanism in addition to an anticholinergic effect. In the guinea-pig ileum, Hn.Cr produced a rightward parallel shift of the acetylcholine curves, followed by a non-parallel shift with suppression of the maximum response at a higher concentration, similar to that caused by dicyclomine, but different from that of verapamil and atropine. Hn.Cr exhibited antidiarrhoeal and antisecretory effects against castor oil-induced diarrhoea and intestinal fluid accumulation in mice. In guinea-pig trachea and rabbit urinary bladder tissues, Hn.Cr caused relaxation of carbachol (1 microM) and K(+) (80 mM) induced contractions at around 10 and 25 times lower concentrations than in gut, respectively, and shifted carbachol curves to the right. Only the organic fractions of the extract had a Ca(2+) antagonist effect, whereas both organic and aqueous fractions had anticholinergic effect. A constituent, beta-sitosterol exhibited Ca(2+) channel-blocking action. These results suggest that the antispasmodic effect of H. niger is mediated through a combination of anticholinergic and Ca(2+) antagonist mechanisms. The relaxant effects of Hn.Cr occur at much lower concentrations in the trachea and bladder. This study offers explanations for the medicinal use of H. niger in treating gastrointestinal and respiratory disorders and bladder hyperactivity.     

Insulinotropic effect of new glibenclamide isosteres

The aim of the present study was to characterize the effects of BM 208 (N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]-N'-cyano- N"- cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene), two newly synthesized isosteres of glibenclamide, on ionic and secretory events in rat pancreatic islet cells. Both compounds inhibited 86Rb (42K substitute) outflow from rat pancreatic islets perifused throughout at low (2.8 mM) D-glucose concentration. In excised inside-out membrane patches, BM 208 and BM 225 reduced the frequency of KATP+ channel openings. The inhibition of 86Rb outflow induced by BM 208 and BM 225 coincided with an increase in 45Ca outflow. The latter phenomenon was abolished in islets exposed to Ca2+-free media. Both isosteres of glibenclamide increased the [Ca2+]i in single pancreatic islet cells. This effect was counteracted by verapamil, a Ca2+ entry blocker. In islets exposed to 2.8 mM glucose and extracellular Ca2+, BM 208 and BM 225 stimulated insulin output. The secretory capacity of BM 225 was more marked than that of BM 208, but the time courses of the cationic and secretory responses exhibited obvious dissociations. These data suggest that the secretory capacity of BM 208 and BM 225 results, at least in part, from the inhibition of ATP-sensitive K+ channels with subsequent increase in Ca2+ inflow. The dissociation between cationic and secretory variables further suggests that the modifications in Ca2+ handling are not solely attributable to a primary inhibition of the ATP-sensitive K+ channels.