Inhibition by protein kinase C of the 86Rb+ efflux and vasorelaxation induced by P1075, a KATP channel opener, in rat isolated aorta

In rat aortic rings, P1075, an opener of ATP-dependent potassium channels (K_ATP channels), produces relaxation and ⁸⁶Rb⁺ efflux from preloaded tissues; the increase in ⁸⁶Rb⁺ efflux qualitatively reflects K_ATP channel opening. In this study we have investigated the effects of protein kinase C modulation on the ⁸⁶Rb⁺ efflux stimulating, the vasorelaxant and the binding properties of P1075. Phorbol 12,13-dibutyrate (PDBu), a direct activator of protein kinase C, inhibited the ⁸⁶Rb⁺ efflux produced by P1075 with an IC₅₀ value of 20±2nM. Phorbol 12-myristate 13-acetate (PMA), another stimulator of protein kinase C, was 150 times weaker in this respect whereas 4α-PDBu, the inactive stereoisomer of PDBu, was ineffective. Staurosporine (300nM), an inhibitor of protein kinase C, induced a small but significant increase of P1075-induced tracer efflux and partially reversed the inhibitory effect of PDBu on P1075-stimulated tracer efflux. The vasorelaxant effect of P1075 was inhibited only to a moderate degree by PDBu at concentrations which inhibited P1075-induced ⁸⁶Rb⁺ efflux to >90%; however, in the presence of PDBu, the relaxation kinetics of P1075 were increasingly slowed. The vasorelaxant effect of P1075 in the presence of PDBu was still sensitive to inhibition by glibenclamide (100nM), the standard inhibitor of the K_ATP channel openers. Specific binding of [³H]-P1075 to rat aortic rings was unaffected by PDBu and PMA even in the micromolar concentration range. The data show that stimulation of protein kinase C inhibits the K⁺ channel opening effect of P1075 in rat aorta and suggest that protein kinase C may exert a weak tonic inhibition on the K_ATP channels in this vessel under quasiphysiological conditions. At concentrations of PDBu which essentially abolished P1075-induced tracer efflux, the glibenclamide-sensitive vasorelaxant effect of P1075 was slowed down but not prevented; this supports earlier suggestions that K⁺ channel openers are also able to relax smooth muscle cells by a mechanism independent of K_ATP channel opening. Received: 11 March 1997 / Accepted: 12 May 1997     

Interaction of the diuretics torasemide and U-37883A with the KATP channel in rat isolated aorta

In this study we have investigated the interaction of the loop diuretics torasemide and furosemide and of the eukalemic diuretic U-37883A (4-morpholinocarboximidine-N–1-adamantyl-N’-cyclohexylhydrochloride) with the ATP-sensitive K⁺ channel (K_ATP channel) in rat aortic rings. Torasemide contains a sulphonylurea group which might enable the compound to interfere with K_ATP channels; this group is lacking in furosemide. U-37883A blocks several types of K_ATP channels. The interaction with the vascular K_ATP channel was probed in binding studies, ⁸⁶Rb⁺ efflux experiments and vasorelaxation assays. Torasemide inhibited the binding of the K_ATP channel inhibitor [³H]glibenclamide and of the opener [³H]P1075 with IC₅₀ values of 19 and 45 μM, respectively; furosemide and U-37883A were inactive or interfered with binding in a nonspecific way. In ⁸⁶Rb⁺ efflux experiments, the loop diuretics, at μM concentrations, inhibited basal tracer efflux to 50% whereas U-37883A had no effect. P1075-stimulated ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was inhibited by U-37883A and torasemide with IC₅₀ values of 0.06 and 130 μM, respectively; furosemide induced only a small (23%) inhibition. In experiments measuring isometric force, torasemide and furosemide partially relaxed endothelium-denuded aortic rings precontracted with noradrenaline or KCl with EC₅₀ values between 6 and 10 μM. The vasorelaxant effect of P1075 was inhibited in a noncompetitive manner by torasemide (300 μM) but unaffected by furosemide. U-37883A increased noradrenaline-induced force and inhibited the vasorelaxant effect of P1075 in an apparently competitive manner with an inhibition constant of 0.4 μM. The data show that torasemide interferes specifically with the binding of the K_ATP channel modulators [³H]glibenclamide and [³H]P1075 and with the K_ATP channel opening and vasorelaxant effects of P1075 whereas furosemide is inactive. This suggests that the interaction of torasemide with the vascular K_ATP channel is due to the sulphonylurea group present in torasemide. U-37883A, which does not inhibit P1075 binding, is one of the most potent blockers of P1075-induced ⁸⁶Rb⁺ efflux yet described but is relatively weak as an inhibitor of P1075-mediated vasorelaxation. The opposite vascular actions of torasemide and U-37883A are expected to contribute to the renal effects of these drugs. Received: 28 January 1998 / Accepted: 20 April 1998     

Binding and effects of P1075, an opener of ATP-sensitive K+ channels, in the aorta from streptozotocin-treated diabetic rats

Diabetes mellitus is associated with major vascular complications. It was the aim of this study to examine the function of the ATP-sensitive K⁺ channel (K_ATP channel) in aortic rings prepared from diabetic rats and from age-matched controls. Diabetes was induced by injection of streptozotocin (60mg/kg i.p.) and the animals were sacrificed 10 weeks after treatment. The binding of the K_ATP channel opener, P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-3-pyridylguanidine), as well as the vasorelaxant and ⁸⁶Rb⁺ efflux stimulating effects of the drug were measured. ATP channel opener P1075 against noradrenaline was shifted rightwards by a factor of 1.3 and the maximum relaxation was reduced from 81 to 71% of initial tension (P<0.01). However, specific binding of ³H-P1075 was increased by 20% without a change in affinity, indicating that the number of binding sites for the opener was increased as a consequence of diabetes. In addition, P1075-induced ⁸⁶Rb⁺ efflux, a qualitative measure of K_ATP channel opening, was augmented by 50%. + channel opening response to P1075 is markedly increased; however, the vasorelaxant effect to the K_ATP channel opener is slightly impaired. A possible explanation of these findings is that the vasorelaxant mechanisms (which are in part independent of plasmalemmal K_ATP channel opening) may be altered; alternatively, the link between membrane potential and smooth muscle tone may be changed in this model of insulin-dependent diabetes mellitus. Received: 11 February 1997 / Accepted: 9 May 1997     

Alteration of binding sites for [3H]P1075 and [3H]glibenclamide in renovascular hypertensive rat aorta

Aim: The alterations of the binding sites for ATP-sensitive K channel (KATP) openers and blockers in aortic strips were investigated in hypertensive rats. Methods: Radioligand binding techniques were used to compare the specific binding properties of [3H]P1075 and [3H]glibenclamide (Gli) in normotensive (NWR) and reno-vascular hypertensive rat (RVHR) aortic strips. Results: The KD values of [^3H]P1075 binding were increased by 1.5-fold, while the Bmax values were unchanged in RVHR. The IC50 values of P1075 and pinacidil (Pin) for displacing the [^3H]P1075 binding in RVHR were increased by 1.8-and 1.7-fold, respectively. The kinetic processes of association and dissociation of [^3H]P1075 binding were slower in RVHR. Glibenclamide pretreatment slowed down the kinetic processes of the association and dissociation of [3H]P1075 binding in NWR, but failed to alter the kinetic processes of [^3H]P1075 binding in RVHR. The IC50 values of Gli for displacing the [^3H]Gli binding at high-affinity sites were increased by 3-fold, while those at low-affinity sites remained to be unchanged in RVHR. The kinetic processes of association of [^3H]Gli binding were decreased and those of the dissociation were accelerated in RVHR. The treatment with Pin slowed down the association kinetic processes but accelerated the process of the dissociation of [^3H]Gli binding in NWR, but did not alter the kinetics of [3H]Gli binding in RVHR. Conclusion: The affinity of binding sites for [3H]p 1075 and of high-affinity binding sites for [^3H]Gli are decreased, and the negative allosteric interactions between the two binding sites are impaired in RVHR aorta.     

Binding of KATP channel modulators in rat cardiac membranes

1. The binding of [³H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K⁺ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
2. Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [³H]-P1075 binding with an EC₅₀ value of 100 μM and a Hill coefficient of 1.4.
3. In saturation experiments [³H]-P1075 binding was homogeneous with a K_D value of 6±1 nM and a binding capacity (B_max) of 33±3 fmol mg⁻¹ protein.
4. Upon addition of an excess of unlabelled P1075, the [³H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min⁻¹. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM⁻¹ min⁻¹. From the kinetic experiments the K_D value was calculated as 4.7±0.6 nM.
5. Openers of the ATP-sensitive K⁺ channel belonging to different structural classes inhibited specific [³H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
6. Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [³H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC₅₀ value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC₅₀ value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
7. It is concluded that binding studies with [³H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [³H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR_2A) and vascular smooth muscle cells (SUR_2B), differs from that expected for SUR_2A and SUR_2B.

     

Potassium diet as a determinant for the renal response to systemic potassium channel modulation in anesthetized rats

The role of potassium intake in the response of kidney function and plasma renin activity (PRA) to systemic application of U37883A (4-morpholinecarboximidine-N-1-adamantyl-N’-cyclohexyl-hydrochloride), a putative blocker of ATP-sensitive potassium channels (K_ATP), and P1075 (N-cyano-N’-(1,1-dimethylpropyl)-N’’-pyridylguanidine), an opener of K_ATP channels, was studied in the anesthetized rat. It was found that under normal potassium diet (0.7% K), U37883A (15 mg/kg, i.v.) increased urinary flow rate (UV) and sodium excretion (UNaV), decreased urinary potassium excretion (UKV), and significantly diminished heart rate (HR) without affecting mean arterial blood pressure (MAP) or glomerular filtration rate (GFR). P1075 (10 μg/kg, i.v.) lowered UV, UNaV and UKV, at least in part due to the fall in MAP and GFR.PRA was diminished by U37883A and increased by P1075.Variation in potassium diet (0.04 or 2% K) left the response in MAP, HR or GFR to both potassium channel modulators essentially unchanged. The reduction in renal excretion rates to P1075 also appeared unaffected, further supporting a predominant role of the change in MAP and GFR in this response. Variation in potassium diet, however, elicited the following alterations: (1) under both low and high potassium diet U37883A did no longer cause a significant natriuresis; (2) U37883A elicited a significant kaliuresis under high potassium diet, whereas potassium excretion remained essentially unchanged on very low levels under low potassium diet; (3) the increase in PRA to P1075 was blunted under low potassium diet. Additional experiments provided evidence that P1075 releases renin from freshly isolated juxtaglomerular cells of rats on normal but not on low potassium diet. In summary, systemic potassium channel modulation employing U37883A or P1075, respectively, exerts distinct effects on blood pressure and heart rate independent of potassium diet. In contrast, potassium diet appears to be a determinant for the concomitant reponses in plasma renin activity and renal sodium and potassium excretion. Received: 3 December 1997 / Accepted: 23 April 1998     

Interaction of a novel dihydropyridine K+ channel opener, A-312110, with recombinant sulphonylurea receptors and KATP channels: comparison with the cyanoguanidine P1075

1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. 2. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with K(i) values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.     

Binding of [3H]-P1075, an opener of ATP-sensitive K+ channels, to rat glomerular preparations

ATP-sensitive K⁺ channels (K_ATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [³H]-P1075 ([³H]-N-cyano-N-(1,1-dimethylpropyl)-N-3-pyridylguanidine), a selective opener of K_ATP channels, in rat glomerular preparations.Equilibrium (saturation, competition) and kinetic experiments indicated that [³H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg^–1 glomerular protein. The association rate constant of the complex was 6,5×10⁷ M^–1 min^–1; dissociation occurred with a half-time of 6.2 min. Specific [³H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [³H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [³H]-P1075 binding was inhibited by representatives of the different classes of K_ATP channel openers and by sulphonylureatype blockers with inhibition constants similar to those obtained in rat aortic rings.It is concluded that rat glomerular preparations possess specific binding sites for K_ATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 m^–2) is much higher than that of K_ATP channels in smooth muscle.     

Effects of the potassium channel openers KRN4884 and levcromakalim on the contraction of rat aorta induced by A23187, compared with nifedipine

We examined the different vasodilatory effects of the K⁺ channel openers levcromakalim and 5-amino-N2-[2-(2-chlorophenyl)ethyl]-N-cyano-3-pyridinecarboxamidine (KRN4884), and the Ca²⁺ channel blocker nifedipine in the rat aorta. KRN4884 (10^–10-10^–5 M) and nifedipine (10^–10–10^–5 M) produced concentration-dependent relaxation in the rat aorta precontracted by 25 mM KCl. The K⁺ channel blocker glibenclamide (1 M) inhibited the relaxation induced by KRN4884 but did not influence nifedipine-induced relaxation. KRN 4884 had almost no effect on contraction induced by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl. These results indicate that KRN4884 is a K+ channel opener. We investigated the relaxant effects of KRN4884 (10^–10-10^–5 M), levcromakalim (10^–9-10^–5 M) and nifedipine (10^–9-10^–5 M) on A23187 (1 M)-induced contraction. KRN4884 and levcromakalim had a potent relaxant effect but nifedipine only a weak effect on the smooth muscle contracted by A23187. Glibenclamide (1 M) inhibited the relaxation induced by KRN4884 and levcromakalim, but did not influence the nifedipine-induced relaxation. KRN 4884 (1 M) produced a larger relaxation of A23187-induced contraction but had little effect on the increase in intracellular [Ca²⁺] induced by A23187. These results suggest that KRN4884 is a specific K⁺ channel opener and its vasodilating mechanisms involve not only deactivation of Ca²⁺ channels but also a decrease in the Ca²⁺ sensitivity of contractile elements.     

Activators of protein kinase A induce a glibenclamide-sensitive 86Rb+ efflux in rat isolated aorta

The effect of activators of protein kinase A on membrane K⁺ permeability and the interaction of these compounds with cromakalim, an opener of ATP-sensitive K⁺ channels (K_ATP channels), were investigated. Membrane K⁺ permeability was assessed by measuring ⁸⁶Rb⁺ efflux from rings of rat aorta. Forskolin, an activator of adenylate cyclase, and isobutylmethylxanthine (IBMX), a nonselective phosphodiesterase inhibitor, induced small, concentration-dependent increases in tracer efflux up to 20-40% over the basal level. The effect of forskolin was abolished by the K⁺ channel blocker tedisamil (1 μM) and partially inhibited by glibenclamide (1 μM), a relatively selective blocker of K_ATP channels. Further studies were conducted in the presence of 35mM KCl in the bath in order to increase the size of the ⁸⁶Rb⁺ efflux stimulated by forskolin and IBMX. At high concentrations, these compounds produced a biphasic effect with a peak increase being followed by a lower plateau value. Glibenclamide inhibited the ⁸⁶Rb⁺ efflux response to forskolin and IBMX by 50-80%. The K⁺ channel blockers tedisamil (1 μM), Ba²⁺ (1mM) and tetraethylammonium (10mM) also reduced the peak response to forskolin by about 50% and abolished or greatly inhibited the plateau response. In addition to the small effect on basal ⁸⁶Rb⁺ efflux, forskolin (0.3 μM) increased cromakalim-induced ⁸⁶Rb⁺ efflux 3.4 times. At higher concentrations, however, a concentration-dependent inhibition was observed with an IC₅₀ value of 7.6 ±0.4 μM. 1,9-dideoxyforskolin, which does not increase cAMP, increased neither basal nor cromakalim-induced ⁸⁶Rb⁺ efflux; however, it inhibited cromakalim-stimulated tracer efflux with an IC₅₀ value of 22 ±2 μM. It is concluded that forskolin and IBMX, probably by increasing intracellular cAMP levels, induce a ⁸⁶Rb⁺ efflux from rat aorta, the major part of which is glibenclamide-sensitive and may pass through K_ATP channels. In addition, low concentrations of forskolin greatly facilitate the K_ATP channel opening effect of cromakalim whereas high concentrations block the channel; this blocking effect of forskolin is unrelated to the cAMP elevating action. Received: 25 September 1996 / Accepted: 20 December 1996     

Comparison of cromakalim-induced relaxation of potassium precontracted rabbit,cat, and rat isolated cerebral arteries

Summary The effects of cromakalim were investigated in KCl-precontracted cat, rabbit, and rat isolated cerebral arteries with intact endothelium. Potassium induced contraction of all cerebral arteries studied, but exhibited marked vessel and species variation with no spasm to 20 or 30 mmol/l KCl in the rat basilar artery or 20 mmol/l KCl in the rabbit middle cerebral artery. On sustained tension to 20 mmol/l KCl, cromakalim induced concentration-related relaxation in the rabbit basilar artery and the cat basilar and middle cerebral arteries with Hill coefficients greater than unity. Cromakalim was more potent in the rabbit basilar artery precontracted with 20 or 30 mmol/KCl than in the rabbit middle cerebral artery or the cat basilar or middle cerebral artery. Elevation of the KCl concentration to 50 mmol/l inhibited cromakalim-induced relaxation and produced a decrease in the Hill coefficient. Preincubation of cerebral arteries with glibenclamide (100 nmol/l–1 mol/1) produced concentration-related inhibition of the cromakalim-induced relaxation in the rabbit basilar, cat basilar, and cat middle cerebral arteries precontracted with 20 mmol/l KCl. The degree of rightward shift of concentration-effect curves by glibenclamide was calculated at the EC₂₅, EC₅₀, and EC₇₅ levels. A good correlation was observed between the shifts at the EC₅₀ and EC₅₀ levels. However, the shift in concentration — effect curves for cromakalim produced at the EC₂₅ level was markedly less than the-EC₅₀ or EC₇₅ levels in the presence of 1 mol/1 glibenclamide. The pA ₂ values for glibenclamide calculated at the EC₅₀ level were 6.6 ± 0.09, 7.1 ± 0.1, and 6.5 ± 0.5 in the rabbit basilar, cat basilar, and cat middle cerebral artery, respectively. The slope of the Schild regression for the inhibitory effect of glibenclamide in the rabbit basilar artery was significantly greater than unity but did not differ from unity in cat cerebral arteries. Glibenclamide (1 mol/l) produced a similar degree of inhibition of the cromakalim-induced relaxation in the 30 mmol/l KCl precontracted rabbit middle cerebral artery and in the rabbit basilar artery exposed to 20 mmol/l KCl. In contrast, tolbutamide 10 mol/l was essentially inactive against the cromakalim-induced relaxation in all vessels studied. It is concluded that cromakalim produces concentration-dependent relaxations of rabbit and cat isolated cerebral arteries by a mechanism that is similar to that identified in peripheral vasculature and visceral smooth muscle. In this study we were unable to demonstrate effects of cromakalim on the KCl precontracted rat basilar artery, possibly due to the low sensitivity of this preparation to KCl. Send offprint requests to M. Wahl at the above address     

Electrocardiographic interactions between pinacidil,a potassium channel opener and class I antiarrhythmic agents in guinea-pig isolated perfused heart.

1. Drugs that shorten action potential duration could decrease the Na-channel blocking effect of class I antiarrhythmic agents by reducing the availability of Na channel in the inactivated state. 2. This hypothesis was tested in guinea-pig perfused heart, measuring the surface ECG effects of three class I drugs endowed with different binding kinetics (15 microM mexiletine, 10 microM quinidine and 3 microM flecainide) in the presence of increasing concentrations of pinacidil (10 microM, 30 microM, 50 microM), a potassium channel opener that shortens action potential duration. 3. The ECG parameters measured were: the QRS interval, i.e. the intraventricular conduction time; the JT interval, which reflects the duration of ventricular repolarization; the ratio between JT peak (the time from the end of QRS and the peak of T wave) and JT interval, which quantifies changes in the morphology of the T wave. 4. At the concentrations tested all the antiarrhythmic drugs widened the QRS complex by 55-60%. Flecainide did not significantly change JT interval, but quinidine prolonged and mexiletine shortened it. Mexiletine also decreased the JTpeak/JT ratio. Pinacidil by itself decreased the JT interval and the JT peak/JT ratio in a dose-dependent way, but did not affect QRS duration. 5. In the presence of fixed antiarrhythmic drug concentrations, however, pinacidil decreased the QRS prolongation induced by mexiletine (-17%) and quinidine (-8%), but not that induced by flecainide: this effect was already maximal at the lower concentration tested (10 microM) and there was no relationship between pinacidil-induced JT shortening and QRS changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Geissoschizine methyl ether,an indole alkaloid extracted from Uncariae Ramulus et Uncus,is a potent vasorelaxant of isolated rat aorta

Effects of geissoschizine methyl ether, an indole alkaloid isolated from the hook of Uncariae Ramulus et Uncus, on vascular responses were examined using isolated strips of rat aorta. Geissoschizine methyl ether (10(-7)-10(-4) M) relaxed norepinephrine (5x10(-8) M)-induced contraction in a dose-dependent manner. The potency (50% efficacy concentration, EC(50)=0.744 microM) was approximately 14 times greater than that (EC(50)=10.6 microM) of hirsutine, one of the indole alkaloids isolated from Uncariae Ramulus et Uncus that demonstrates a vasorelaxant effect by Ca(2+)-channel blocking. The vasorelaxant effect of geissoschizine methyl ether found at the lower concentrations (10(-7)-3x10(-6) M) on the norepinephrine-induced contraction was abolished by pretreatment with N(G)-nitro-L-arginine (10(-4) M), an inhibitor of nitric oxide synthesis, or by denuding aortas of endothelium, while the effects at the higher concentrations (10(-5)-10(-4) M) were not completely prevented by either N(G)-nitro-L-arginine and deendothelialization. Furthermore, geissoschizine methyl ether did not relax high K(+)-, Ca(2+)- and a Ca(2+)-channel agonist Bay K8644-induced contractions at the lower concentrations that markedly relaxed the norepinephrine-induced contractions, while the higher concentrations of geissoschizine methyl ether relaxed the high K(+)-, Ca(2+)- and Bay K8644-induced contractions. These results suggest that the vasorelaxant effect of geissoschizine methyl ether is composed of two different mechanisms: endothelial dependency with nitric oxide and endothelial independency with voltage-dependent Ca(2+)-channel blocking.     

Mg2+ and ATP dependence of KATP channel modulator binding to the recombinant sulphonylurea receptor,SUR2B

1. The binding of modulators of the ATP-sensitive K⁺ channel (K_ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K_ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K_ATP channel opener, [³H]-P1075.
2. Binding of [³H]-P1075 required the presence of Mg²⁺ and ATP. MgATP activated binding with EC₅₀ values of 10 and 3 μM at free Mg²⁺ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg²⁺, binding was lower than at 3 μM Mg²⁺.
3. [³H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg²⁺ concentrations of 3 μM and 1 mM, gave K_D values of 1.8 and 3.4 nM and B_MAX values of 876 and 698 fmol mg⁻¹, respectively.
4. In competition experiments, openers inhibited [³H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
5. Glibenclamide inhibited [³H]-P1075 binding with K_i values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg²⁺, respectively. Glibenclamide enhanced the dissociation of the [³H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg²⁺ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [³H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

     

KR-31378, a novel benzopyran analog, attenuates hypoxia-induced cell death via mitochondrial KATP channel and protein kinase C-epsilon in heart-derived H9c2 cells

A novel compound KR-31378 [(2S,3S,4R)-N'-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methly-2-dimethoxy-methly-2H-benzo-pyran-4-yl)-N-benzylguanidine] has been demonstrated as an anti-ischemic agent in rat heart and brain. Here, we report the effects of this compound on hypoxia-induced cell death and possible signaling pathways in heart-derived H9c2 cells. Treatment with KR-31378 (3-30 microM) 1 h before and during hypoxia significantly reduced hypoxia-induced cell death in a concentration-dependent manner. In addition, increase in hypoxia-induced transferase UTP nick end labeling (TUNEL)-positive cells was reduced by KR-31378, suggesting its antiapoptotic potential in H9c2 cells. The protective effect conferred by KR-31378 (10 microM) was abolished by cotreatment with 5-hydroxydecanoate (5HD), a specific blocker of the mitochondrial KATP (mtKATP) channel, but not by HMR-1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea), a specific blocker of the sarcolemmal KATP channel. We observed that the treatment with KR-31378 could increase the expression of protein kinase C (PKC)-epsilon protein, but not other PKC isotypes (-alpha, -beta, -delta, -zeta), in the particulate fraction. This increased level of PKC-epsilon was sustained during the hypoxic period up to 8 h. In addition, our results showed that treatment with KR-31378 induced the expression of PKC-epsilon mRNA as early as 15 min after the treatment. A specific inhibitor for PKC-epsilon isoform, epsilonV1-2, completely blocked the protective effect of KR-31378 against hypoxia-induced cell death. In conclusion, our results suggest that KR-31378 can protect cultured H9c2 cells from hypoxia-induced death via the mtKATP channel and PKC-epsilon.     

Comparative studies of ZD0947, a novel ATP-sensitive K<Superscript>+</Superscript> channel opener,on guinea pig detrusor and aortic smooth muscles

The effects of ZD0947, a novel urinary bladder selective ATP-sensitive potassium channel (K_ATP channel) opener, on carbachol-induced contractions of isolated guinea pig urinary bladder strips were investigated to compare its ability to relax norepinephrine-induced contraction of the aorta. Electrophysiological techniques were also utilized to compare the effects of ZD0947 on membrane currents between guinea pig detrusor and aortic myocytes. ZD0947 caused a significant reduction of the carbachol-induced contractile activity, demonstrating a biphasic relaxation (the first and second components). Although glibenclamide antagonized the effects of two components for the ZD0947-induced relaxation, gliclazide, a selective sulphonylurea receptor 1 (SUR1) antagonist, reduced the effects of the first component but not the second component of the ZD0947-induced relaxation. ZD0947 also reduced the norepinephrine-induced contraction of the aorta. ZD0947 reduced electrical excitability of detrusor smooth muscles, inhibiting spike discharges and also hyperpolarizing the membrane as measured with microelectrodes. In conventional whole-cell configuration, ZD0947 caused a glibenclamide-sensitive K⁺ current (i.e., K_ATP current) at a holding potential of −60 mV in guinea pig detrusor and aortic myocytes. The current density of ZD0947-induced K_ATP currents in guinea pig detrusor myocytes was significantly larger than that in aortic smooth muscle cells. These results show that ZD0947 caused a significant relaxation through the activation of K_ATP channels in detrusor muscle.     

KR-31762, a novel KATP channel opener, exerts cardioprotective effects by opening SarcKATP channels in rat models of ischemia/reperfusion-induced heart injury

The cardioprotective effects of KR-31762, a newly synthesized K⁺ _ATP opener, were evaluated in rat models of ischemia/reperfusion (I/R) heart injury. In isolated rat hearts subjected to 30-min global ischemia followed by 30-min reperfusion, KR-31762 (3 and 10 μM) significantly increased the left ventricular developed pressure (LVDP) and double product (heart rate × LVDP) after 30-min referfusion in a concentration-dependent manner, while decreasing the left ventricular end-diastolic pressure (LVEDP). KR-31762 also significantly increased the time to contracture (TTC) during ischemic period (20.0, 22.4 and 26.4 min for control, 3 and 10 μM, respectively), while decreasing the release of lactate dehydrogenase (LDH) from the heart during 30 min reperfusion (30.4, 14.3 and 19.7 U/g heart weight, respectively). All these parameters except LDH release were reversed by glyburide (1 μM), a nonselective blocker of K⁺ _ATP channel, but not by 5-hydroxydecanoate, a selective blocker of mitoK⁺ _ATP channel. In anesthetized rats subjected to 45-min occlusion of left anterior descending coronary artery followed by 90-min reperfusion, KR-31762 significantly decreased the infarct size (60.8, 40.5 and 37.8% for control, 0.3 and 1.0 mg/kg, iv, respectively). KR-31762 slightly relaxed the isolated rat aorta precontracted with methoxamine (IC₅₀: 23.5 μM). These results suggest that KR-31762 exerts potent cardioprotective effects through the opening of sarcolemmal K_ATP channel in rat hearts with the minimal vasorelaxant effects.     

Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.     

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

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

Influence of depolarization on vasorelaxant potency and efficacy of Ca2+ entry blockers, K+ channel openers, nitrate derivatives, salbutamol and papaverine in rat aortic rings

We examined the effects of various KCl concentrations on the actions of some vasodilators belonging to different pharmacological classes in rat aortic rings. In some experiments, tissues were precontracted with noradrenaline after blocking voltage-dependent channels to assess the effects of depolarisation unaccompanied by the entry of extracellular Ca²⁺ into the cytosol. Concentration/response curves for the vasorelaxant effect of calcium entry blockers (e.g. diltiazem), K⁺ channel openers (e.g. aprikalim), nitrate derivatives (e.g. nitroglycerin), a β₂-adrenergic agonist (salbutamol) and papaverine were obtained by using endothelium-denuded rat aortic rings precontracted with KCl (20–60 mM) to determine the potencies and efficacies of the drugs. The efficacies and potencies of calcium entry inhibitors were virtually independent of the [KCl]. A reduction in the potency (up to 18-fold) of papaverine occurred without changes in efficacy when the [KCl] was raised from 20 to 60 mM. The decline in potency was even greater for nitrate-like compounds. The potency of K⁺ channel openers in aortic rings precontracted with 30 mM KCl decreased by three- to sixfold compared with those precontracted with 20 mM KCl. With the exception of pinacidil, the efficacy of these agents already started to decline in preparations precontracted with 25 mM KCl and was virtually zero in preparations precontracted with 60 mM KCl. In contrast to other K⁺ channel openers, the vasorelaxant action of pinacidil was relatively resistant to glibenclamide. Salbutamol produced only a slight relaxation even in preparations precontracted with 20 mM KCl. In nitrendipine-pretreated, noradrenaline-precontracted aortic rings, the vasorelaxant effects of aprikalim, but not those of linsidomine or papaverine, declined when the [KCl] of the bathing medium was increased. In conclusion, the vasorelaxant potency and efficacy of calcium entry blockers is independent of the [KCl] used to precontract rat aortic rings, and thus, of the degree of membrane depolarisation. In contrast, increasing the [KCl] strongly reduces the potency and the efficacy of K⁺ channel openers not only in this preparation but also in noradrenaline-precontracted rings in which the entry of extracellular Ca²⁺ was prevented with nitrendipine. This indicates that, with the exception of pinacidil, the vasorelaxant activity of K⁺ channel openers depends on the degree of membrane depolarisation. Finally, the vasorelaxant potency and efficacy of nitrate-like compounds and papaverine are independent of depolarisation per se but they are markedly affected by the influx of Ca²⁺ accompanying elevated [KCl]. Thus, the degree of vessel depolarisation should be taken into consideration when attempting to compare potencies and efficacies among vasorelaxant agents. Received: 4 May 1998 / Accepted: 6 July 1998