86Rb Efflux Research Articles

Potassium channel opening properties of thiazide diuretics in isolated guinea pig resistance arteries.

We examined the role of K+ channels in mediating the acute vascular actions of hydrochlorothiazide, indapamide, cicletanine, and cromakalim, studying the effect of K+ channel blockers on drug-induced relaxation and drug-induced 86Rb efflux in guinea pig mesenteric arteries. Cromakalim-induced relaxation was unaffected by charybdotoxin, apamin, or phencyclidine (PCP) but was reduced by 75% (with 30 microM cromakalim) by glibenclamide (p < 0.001). Cromakalim increased 86Rb efflux from guinea pig vessels, an effect that was abolished by glibenclamide. Hydrochlorothiazide and cicletanine-induced relaxations have been shown to be inhibited by charybdotoxin by unaffected by glibenclamide, apamin, or PCP. Hydrochlorothiazide and cicletanine increased 86Rb efflux from guinea pig mesenteric arteries. These increases were abolished by charybdotoxin. Indapamide-induced relaxation was not affected by incubation with any of the K+ channel blockers. Indapamide did not alter basal 86Rb efflux. The results suggest that in guinea pig mesenteric arteries indapamide-induced relaxation is not mediated by an action on K+ channels. Cromakalim-induced effects are mediated by KATP. Large conductance KCa mediates the hydrochlorothiazide and cicletanine-induced vascular effects in part.

Effect of minoxidil sulfate and pinacidil on single potassium channel current in cultured human outer root sheath cells and dermal papilla cells

Minoxidil sulfate and pinacidil are K channel openers and are considered to promote hair growth. However, there have been no studies on the single channel current of isolated cells from hair follicles. Therefore, we characterized the single K channel current of outer root sheath cells and dermal papilla cells and the effect of K channel openers on K currents by patch clamp. We also carried out 86Rb efflux studies to observe macroscopic K channel currents. In physiological saline, these two cells showed two types of K channels, large and small conductance Ca 2+-activated K channels, both intact cell-attached and excised inside-out patches. In symmetrical 150 mM K solution, unitary conductances were 246 and 70 pS, respectively. Intracellular ATP (up to 5 mM) or glibenclamide (20 nM), a specific ATP-sensitive K channel blocker, did not block these channels. Minoxidil sulfate (5 μg/ml) or pinacidil (10 μM) did not open these two types of K channels or increase 86Rb efflux. These results suggest that minoxidil sulfate or pinacidil did not activate K channel current in hair follicles, and that the drug effect on hair growth might be mediated by other mechanisms such as increased blood flow.

Adenosine triphosphate activates ion permeabilities in biliary epithelial cells

Background/Aims: The biliary epithelium contributes to bile formation through absorption and secretion of fluid and electrolytes. The effects of extracellular nucleotides on membrane ion transport were assessed in isolated bile duct cells from rats and Mz-ChA-1 cells from a human cholangiocarcinoma. Methods: The rates of efflux of 125I and 86Rb were used to assess membrane CI− and K+ permeabilities, respectively. Patch clamp recordings of whole cell currents were used to evaluate the properties of adenosine triphosphate (ATP)-activated currents. Results: Purinergic receptor agonists ATP and uridine triphosphate stimulated 125I and 86Rb efflux about twofold above basal levels. The effects were reproduced by a nonhydrolyzable analogue of ATP (adenosine 5′-O-[3-thiophosphate]) and were unaffected by an adenosine receptor blocker xanthine amine congener. 125I efflux was also stimulated by adenosine and its receptor agonists 5′-N-ethylcar-boxamidoadenosine, N6-(2-phenylisopropyl)adenosine; these effects were inhibited by xanthine amine congener, suggesting a separate adenosine receptor. ATP, adenosine 5′-O-(3-thiophosphate), and uridine triphosphate each stimulated release of Ca2+ from intracellular stores, whereas adenosine had no effect. In whole cell recordings of Mz-ChA-1 cells, ATP activated an early transient outward current consistent with a K+ conductance and a later, sustained inward current consistent with a CI− conductance. Conclusions: Biliary cells possess at least two classes of nucleotide receptors that modulate membrane ion permeability through Ca2+-dependent and -independent pathways, and ATP may be involved in the regulation of biliary secretion.

Stimulation of total CO2 flux by 10% CO2 in rabbit CCD: role of an apical Sch-28080- and Ba-sensitive mechanism.

These studies examine the effect of ambient PCO2 on net bicarbonate (total CO2) absorption by the in vitro perfused cortical collecting duct (CCD) from K-replete rabbits and the mechanism responsible for this effect. Exposure to 10% CO2 increased net bicarbonate flux (total CO2 flux, JtCO2) by 1.8-fold (P < 0.005), and this effect was inhibited by luminal 10 microM Sch-28080, an H-K-adenosinetriphosphatase (H-K-ATPase) inhibitor. In contrast, exposure to 10% CO2 significantly decreased Rb efflux, and this decrement in Rb efflux was blocked by luminal 2 mM Ba, a K channel blocker. Thus transepithelial tracer Rb flux did not increase upon exposure to 10% CO2 as we have observed in this segment under K-restricted conditions. The observation that 10% CO2 increased net bicarbonate absorption without a change in absorptive Rb flux suggested that 10% CO2 increased apical K recycling. To test this hypothesis, we examined whether luminal Ba inhibited the stimulation of luminal acidification induced by 10% CO2. If apical K exit were necessary for full activation of proton secretion, then inhibiting K exit should indirectly affect the stimulation of JtCO2 by 10% CO2. In fact, the effect of 10% CO2 on JtCO2 in the presence of 2 mM luminal Ba was quantitatively indistinguishable from the effect of 10% CO2 on JtCO2 in the presence of 10 microM luminal Sch-28080.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume- and catecholamine-dependent regulation of Na/H antiporter and unidirectional potassium fluxes in Salmo trutta red blood cells

β-Adrenergic- and volume-dependent regulation of 22Na influx and 86Rb influx and efflux in erythrocytes of brown trout (Salmo trutta m. lacustris) were studied. Norepinephrine (10-6 mol·1-1) increased the rate of 22Na influx 10-to 20-fold via the activation of a Na/H exchanger (ethyl isopropyl amiloride inhibited component of 22Na influx). Unlike carp erythrocytes the activity of the Na, K-pump (ouabain-inhibited 86Rb influx) was only slightly (25–35%) increased by norepinephrine. The norepinephrine-induced increment of Na, K-pump activity was completely abolished by ethyl isopropyl amiloride thus indicating that this effect was mediated by Na/H exchanger-induced increase of intracellular Na+ concentration. Cell shrinkage in hyperosmotic media resulted in a several-fold activation of the Na/H exchanger. Cell swelling in hypotonic media increased both the rate of K, Cl-cotransport [((dihydroindenyl)oxy)alcanaic acidsensitive components of 86Rb influxe and efflux] and passive permeability (leakage) of erythrocyte membranes for Na+ and K+. No volume-dependent regulation of Na, K, 2Cl-cotransport (bumetanide-sensitive components of 86Rb fluxes) was found. It may be concluded that the regulation of monovalent cation transport in erythrocytes of fast-moving (carnivorous) brown trout differs essentially from that in slowly moving (herbivorous) carp.

The role of sulfhydryl groups in d-aspartate and rubidium release from neonatal rat primary astrocyte cultures

We have recently demonstrated that both methylmercury (MeHg) and mercuric chloride (MC) induce d-aspartate release from neonatal rat primary astrocyte cultures maintained in isotonic conditions [1,3,21,22]. In the present study, we compare several other sulfhydryl-(-SH) selective alkylating reagents [methyl methanethiosulfonate (MMTS), N-ehtylmaleimide (NEM), and iodoacetamide (IA)] in isotonic, as well as hypotonic conditions to discern the functional importance of -SH groups in [ 3H] d-aspartate and 86rubidium ( 86Rb) release from astrocytes. Treatment of astrocytes (5 min) in isotonic buffer with the hydrophobic reagent NEM (10 μM) caused a marked increase in 86Rb release but had no effect of [ 3H] d-aspartate release. Neither IA-, nor MMTS-treatment (both at 10 μM) induced increase in [ 3H] d-aspartate of 86Rb release in isotonic buffer. In hypotonic condition (−50 mM Na +), astrocytes were most sensitive to MC exposure (5 μM), exhibiting an increase in both [ 3H] d-aspartate and 86Rb efflux. The hydrophobic compounds MMTS and NEM, and the hydrophilic -SH modifying reagent, IA, attenuated the hypotonic-induced efflux of [ 3H] d-aspartate, in the absence of an effect of 86Rb release. These observations are consistent with a critical role for -SH groups both in basal (i.e. isotonic) and hypotonic-induced release of d-aspartate and Rb from astrocytes. Lack of uniformity of these effects may be attributed to site-specificity, related to the physicochemical properties of these -SH alkylating reagents.

The effect of Ganglioside on Oxidation-induced Permeability Changes in Lens and in Epithelial Cells of Lens and Retina

Several recent reports have indicated that ganglioside treatment both in vivo and in vitro has a protective effect on the loss of membrane permeability resulting from inhibition of transport enzyme(s) in different experimental models. In this study we have investigated the effect of monosialoganglioside on oxidation-induced changes in organ-cultured rabbit lenses and in cultured dog lens epithelium and human retinal pigment epithelial cells. Exposure of organ-cultured lenses to 0·5 mM hydrogen peroxide for 1 hr increased the efflux of 86Rb from intact lenses and loss of myoinositol from the capsule epithelium. Pretreatment of the lenses with monosialoganglioside significantly reduced the efflux rate of 86Rb and loss of myoinositol. Monosialoganglioside also prevented morphological changes induced by 0·1 mM hydrogen peroxide in dog lens epithelium and loss of cell viability caused by docosahexaenoic acid in dog lens epithelium and in human retinal pigment epithelial cells. In contrast to the protective effect of monosialoganglioside on permeability and morphological changes in cultured cells, it had no effect against single-strand breaks of DNA in dog lens epithelium resulting from exposure to hydrogen peroxide. X-ray and UV-B radiation. Although the molecular mechanisms by which monosialoganglioside prevents permeability and morphological changes induced by hydrogen peroxide and docosahexaenoic acid are not known, it appears that this ganglioside serves as a membrane stabilizer rather than as a free-radical scavenger.

Cytochrome P-450 arachidonate metabolites affect ion fluxes in rabbit medullary thick ascending limb.

The medullary thick ascending limb of Henle's loop (mTALH) of the rabbit metabolizes arachidonic acid (AA) via a cytochrome P-450 (P-450) monooxygenase pathway to several products, of which the principal are 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 1,20-eicosatetraenedioic acid (20-COOH-AA). To understand their mechanism of action on alkali cation metabolism in mTALH cells, we have compared their effects with those of ouabain and furosemide. Incubation of rabbit isolated mTALH cells with either 1 mM ouabain or furosemide decreased K+ content from a control of 1,015 +/- 51 peq/micrograms protein to 717 +/- 41 and to 548 +/- 48 peq/micrograms protein, respectively, whereas they had opposite effects on Na+ content; from a control of 138 +/- 22 peq/micrograms protein, ouabain increased Na+ content to 357 +/- 37 peq/micrograms protein, and furosemide decreased it to 64 +/- 23 peq/micrograms protein. Preincubation with either 20-HETE (1 microM) or 20-COOH-AA (1 microM) decreased Na+ and K+, resembling furosemide in their effects on Na+ and K+ content. In other experiments we used monensin-treated cells to determine 86Rb uptake under conditions in which Na+ entry into the cell was not rate limiting. Under these conditions ouabain still inhibited 86Rb uptake, and the effect of AA was blocked. A major action of AA metabolites on Na(+)-K(+)-adenosinetriphosphatase was thereby excluded. Furthermore, AA metabolites did not inhibit Ba(2+)-sensitive 86Rb efflux, indicating that they do not act through K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of the stimulation of insulin release by saturated fatty acids. A study of palmitate effects in mouse beta-cells.

The mechanisms by which fatty acids increase insulin release are not known. In this study, mouse islets were used as a model and palmitate as a reference compound to study in vitro how saturated fatty acids influence pancreatic beta-cells. Palmitate (625 microM) was bound to albumin. It did not affect basal insulin release (3 mM glucose) but increased the release induced by 10-15 mM glucose. This effect was dependent on the concentration of free rather than total palmitate. It was reversible and abolished by epinephrine, diazoxide, nimodipine, or omission of extracellular Ca. Bromopalmitate and methyl palmoxirate, two inhibitors of fatty acid oxidation, were ineffective alone, and only bromopalmitate partially inhibited the effects of palmitate on insulin release. The increase in insulin release produced by palmitate could not be ascribed to a blockade of ATP-sensitive K(+)-channels because the fatty acid only barely decreased 86Rb efflux and did not depolarize beta-cells in 3 mM glucose. The small effect on 86Rb efflux might be attributed to a slight rise in the ATP/ADP ratio. No such rise occurred when palmitate was tested in 15 mM glucose, and the fatty acid consistently accelerated 86Rb efflux under these conditions. Measurements of beta-cell membrane potential (intracellular microelectrodes) and of free cytoplasmic calcium (Cai2+) in beta-cells (Fura 2 technique) showed that palmitate increases Ca2+ influx; it also caused a very small mobilization of intracellular Ca. The persistence of this stimulation of Ca2+ influx in the presence of diazoxide and high K+ suggests that palmitate might act on Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that Prostaglandin E2 Can Block Calcium‐Activated 86Rb Efflux from Rat Brain Synaptosomes via a Protein Kinase C‐Dependent Mechanism

The effects of prostaglandin E2 (PGE2) on 86Rb efflux from rat brain synaptosomes were studied to explore its role in nerve ending potassium (K+) channel modulation. A selective dose-dependent inhibition of the calcium-activated charybdotoxin-sensitive component of efflux was found upon application of PGE2. No significant effect was seen on basal and voltage-dependent components over the concentration range of 10(-8) to 10(-5) M. The protein kinase C (PKC) inhibitors H-7 (10 microM) and staurosporine (100 nM), as well as prolonged preincubation (90 min) with 4 beta-phorbol 12,13-dibutyrate, which has been reported to down-regulate PKC, abolished the PGE2-induced inhibition, whereas HA1004 (10 microM) and Rp-3',5'-cyclic phosphorothioate (100 nM), which are relatively more selective for protein kinase A than PKC, did not. 4 beta-Phorbol 12,13-dibutyrate (100 nM), an activator of PKC, produced a similar inhibition of the Ca(2+)-dependent component of 86Rb efflux but also had no effect on the basal and voltage-dependent components. These data suggest that PGE2 can inhibit rat brain nerve ending calcium-activated 86Rb efflux, and this inhibition may involve PKC activation.

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.

A pyridothiadiazine (BPDZ 44) as a new and potent activator of ATP-sensitive K + channels

The present study was undertaken to characterize the effects of [3-(1',2'-dimethyl-propyl)amino-4 H-pyrido[4,3- e][1,2,4]thiadiazine 1,1-dioxide] (BPDZ 44), a new pyridothiadiazine derivative, on ionic and secretory events in rat pancreatic islets. The drug increased the rate of 86Rb outflow regardless of the extracellular glucose concentration. The effects of BPDZ 44 on 86Rb outflow persisted in the absence of extracellular Ca 2+ but were abolished by glibenclamide. BPDZ 44 markedly decreased 45Ca outflow and insulin output from islets perifused in the presence of 16.7 mM glucose and extracellular Ca 2+. The drug did not affect the increase in 45Ca outflow mediated by K + depolarization. Lastly, in single B-cells, BPDZ 44 inhibited the glucose but not the KCl-induced rise in cytosolic Ca 2+ concentration ([Ca 2+] i). These data suggest that BPDZ 44 inhibits the insulin releasing process by activating ATP-sensitive K + channels. This K + channel activation will lead to a decrease in Ca 2+ influx and reduction in [Ca 2+] i.

Involvement of calmodulin in Ca 2+-activated K + efflux in human colonic cell line, HT29-19A

The receptor-mediated agonist, neurotensin (NT) stimulated Ba 2+- and charybdotoxin-sensitive 86Rb (K +) efflux in the HT29-19A colonic cell line. Efflux was also stimulated by ionomycin and thapsigargin and could be abolished by incubation with the intracellular Ca 2+ chelator, BAPTA. Together, these data suggest a rise in [Ca 2+] i is prerequisite for activation of K + efflux in these cells. Comparison of the temporal profiles for NT-induced increases in [Ca 2+] i and 86Rb efflux, however, failed to show a direct relationship between these parameters. The NT-stimulated increase in [Ca 2+] i was transient, returning to baseline within 4–5 min, while efflux was sustained over a much longer period (>12 min). Ca 2+-activated 86Rb efflux was inhibited by pretreatment with calmodulin (CaM) antagonist, W7. W7 had no effect on basal efflux, but reduced both NT- and IM-activated efflux up to 80%, with a K i of 38 μM. Other CaM antagonists inhibited efflux with an order of potency (TFP ≈ W8 > W7 ⪢ W5) consistent with inhibition of a CaM-dependent process. Inhibition by W7 was not abolished by ouabain or bumetanide, indicating its effects are not mediated by action upon K + uptake processes. W7 did not inhibit NT-stimulated 125I efflux but significantly reduced efflux stimulated by the Ca 2+ ionophore, ionomycin. NT-stimulated 86Rb + efflux was localized to the basolateral membrane of HT29-19A monolayers grown on permeable supports. These data are consistent with the involvement of CaM in mediating Ca 2+-dependent activation of K + conductance in HT29-19A colonocytes.

Activation of osmotically-activated potassium transporters after injection of mRNA from A6 cells in Xenopus oocytes

The different potassium pathways, under iso-osmotic or hypo-osmotic conditions, were examined in Xenopus oocytes that were micro-injected with mRNA from A6 cells. Hypo-osmotically stimulated 86Rb (K +) effluxes could be measured from intact oocytes 1–4 days after injection of 25 ng of poly (A) + RNA isolated from A6 cells. 86Rb (K +) effluxes were 2.2 times higher from oocytes micro-injected with 25 ng of poly(A) + RNA, than from water injected control oocytes. Water-injected oocytes themselves, however, were 7-fold more responsive to a hypo-osmotic shock than non-injected Xenopus oocytes. There was no significant effect of the different K + transport blockers tested (TEA, bumetanide, glybenclamide or quinidine) on the endogenous 86Rb (K +) effluxes from non-injected oocytes in either iso- or hypo-osmotic media. The 86Rb (K +) effluxes from water-and mRNA-injected oocytes in hypo-osmotic media were both inhibited by TEA. In mRNA-injected oocytes the increase in 86Rb (K +) transport following a medium dilution was also inhibited in the presence of glybenclamide or bumetanide. The present study reports that the activation of hypo-osmotically-activated potassium transporters in the oocytes of Xenopus laevis, after injection of mRNA from A6 cells differs quantitatively and in part qualitatively (glybenclamide-sensitivity) from the endogenous K + pathways of non-injected and of water-injected Xenopus oocytes.

In vitro stimulation of insulin release by SL 84.0418, a new α2-adrenoceptor antagonist

SL 84.0418 (2-(4,5-dihydro-1 H-imidazol-2-yl)-1,2,4,5-tetrahydro-2-propyl-Pyrrolo[3,2,1- hi]-indole hydrochloride) is a novel α 2-adrenoceptor antagonist which possesses anti-hyperglycaemic properties in vivo. In the present in vitro study, we tested its effects in insulin release from isolated mouse islets. In the presence of 15 mM glucose, SL 84.0418 produced a concentration-dependent (0.1–100 μM) increase of insulin release with a slightly higher potency than tolbutamide. SL 84.0418 antagonized the inhibition of glucose-induced insulin release caused either by the α 2-adrenoceptor agonist clonidine or by diazoxide, a selective opener of ATP-sensitive K + channels in the β-cell membrane. Its potency was greater on the inhibition by clonidine than on that by diazoxide, complete antagonism of the inhibition being achieved by 0.9 μM and 6 μM SL 84.0418 respectively. When α 2-adrenoceptors were blocked by the antagonist idazoxan, the low concentrations of SL 84.0418 (0.1-0.3 μM) no longer increased insulin release, whereas the effect of higher concentrations (⩾1 μM) was not affected. SL 84.0418 (⩾1 μM) inhibited 86Rb efflux from islets perifused with a medium containing 3 mM glucose, i.e. under conditions where many ATP-sensitive K + channels are open. It also reduced the acceleration of 86Rb efflux that diazoxide caused in the presence of 6 mM glucose. Moreover, SL 84.0418 directly inhibited ATP-sensitive K + currents measured in single β-cells by the whole-cell mode of the patch-clamp technique. It is concluded that two mechanisms contribute to the stimulation of insulin release induced by SL 84.0418 in vitro: the small increase produced by low concentrations of the drug may be due to blockade of α 2-adrenoceptors and the large increase produced by higher concentrations may result from blockade of ATP-sensitive K + channels.

Potassium efflux from infant and adult rat choroid plexuses: Effects of CSF anion substitution, N-ethylmaleimide and C1 transport inhibitors

To analyze interdependent transport of K and Cl, we investigated Rb (K) efflux from in vitro choroid plexus (CP) in isotonic artificial CSF (aCSF) medium containing anions or agents that alter KCl transport. Lateral ventricle CP was loaded with 86Rb for release to enable calculation of the efflux rate coefficient, k. With Cl as the main anion in control aCSF, the k value for 86Rb (K) in CP of 1 week infant rats (0.177 min −1) was 19% lower than in adults (0.218 min −1) ( P < 0.005). Replacing CSF Cl with NO 3 or SCN, respectively, reduced k for K in infant CP by 73% and 43%; similar anion selectivity was observed in adult tissues ( P < 0.05). N-Ethylmaleimide (NEM), which stimulates KCI cotransport, significantly enhanced K efflux in infants and adults. In adult CP, the KCl cotransport inhibitor, furosemide (1 mM), decreased K efflux by 23% or 65%, respectively, when aCSF had Cl or NO 3 as the main anion. In infant rat CP, 0.1 mM bumetanide (another KCl cotransport inhibitor), reduced k for K by 65%, whereas the Cl channel blocker diphenylamine car☐ylate (1 mM) did not significantly alter K efflux. The collective findings for rat CP indicate a substantial component of K efflux that is associated with Cl concentration and the Cl transport protein sensitive to loop diuretics and NEM. The Cl-dependent K efflux is present in infants.

Action of KC-399, a Newly Synthesized Potassium Channel Opener, on Mechanical Activity and 86Rb Efflux in Rat Aorta

To clarify the characteristics of KC-399, a newly synthesized potassium channel opener, we investigated the effects of KC-399 and lemakalim on the contractions induced by norepinephrine (NE 1 microM) and K+ (30 and 90 mM) and on 86Rb efflux in rat thoracic aorta. KC-399 (0.01-10 nM) and lemakalim (0.001-10 microM) induced relaxation in aortic rings precontracted with 30 mM K+ or NE, but not with 90 mM K+. The vasorelaxant effect of KC-399 was almost 500 times more potent than that of lemakalim. The vasorelaxation with KC-399 developed more slowly and was more resistant to washout than that induced by lemakalim. Glibenclamide (0.1-1 microM), a blocker of ATP-sensitive K-channels, produced concentration-dependent inhibition of the relaxant action of KC-399 in aorta treated with 30 mM K+. KC-399 (3-100 nM) and lemakalim (0.3-10 microM) stimulated 86Rb efflux in rat aorta; the potency of KC-399 was approximately 100 times greater than that of lemakalim. The effects of KC-399 on 86Rb efflux persisted after an 18-min washout period, but those of lemakalim did not. The stimulatory effects of KC-399 (10 and 30 nM) and lemakalim (1 and 3 microM) on 86Rb efflux were also significantly reduced by glibenclamide (1 microM). These results suggest that KC-399 is a potent and long-lasting vasodilator in vitro and that opening of the ATP-sensitive K+ channel may be involved in its mechanism of action.

Effect of cromakalim and pinacidil on 86Rb efflux from guinea pig urinary bladder smooth muscle.

86Rb efflux assay was used to investigate the effect of cromakalim, pinacidil and P1075 in guinea pig urinary bladder strips. The type of K channel opened by cromakalim and pinacidil was determined using specific K channel blockers through 86Rb efflux assay in detrusor strips. Cromakalim, pinacidil and P1075 all three potassium channel openers (PCOs) evoked a concentration-dependent increase in 86Rb efflux in bladder strips. This increase in isotope release was inhibited by pretreatment of bladder with 10 and 30 microM glibenclamide suggesting that both cromakalim and pinacidil interact with ATP-sensitive K channels (KATP). Further, an increase in basal 86Rb efflux was observed when 2-deoxy-D-glucose was substituted for glucose together with 0.24 micrograms/ml of oligomycin (known to lower intracellular ATP) indicating the presence of KATP channels in bladder smooth muscle. Charybdotoxin and apamin, blockers of large and small conductance Ca(2+)-dependent K channels, were found not to be involved in the action of these PCOs in bladder strips. alpha-Dendrotoxin, known to block voltage-dependent K channels, slightly reduced pinacidil-induced 86Rb release without affecting cromakalim-induced 86Rb efflux. The present studies show that ATP-sensitive K channels are present in guinea pig urinary bladder and that cromakalim and pinacidil act by opening these ATP-sensitive K channels in detrusor strips.

Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium.

Inhibition of Na(+)-K(+)-ATPase activates Na(+)-K(+)-2Cl- cotransporter activity in cultured ciliary epithelium. Am. J. Physiol. 266 (Cell Physiol. 35): C198-C205, 1994.--86Rb uptake experiments were conducted to measure Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl- cotransporter activity in a cell line derived from rabbit nonpigmented ciliary epithelium. The presence of a Na(+)-K(+)-2Cl- cotransporter was supported by the observation of a bumetanide-sensitive 86Rb uptake component that was dependent on the extracellular concentration of both sodium and chloride. Potassium influx mediated by the Na(+)-K(+)-2Cl- cotransporter and Na(+)-K(+)-ATPase accounted for approximately 46 and 33% of total potassium uptake, respectively, whereas both ouabain- and bumetanide-resistant uptake accounted for 9%. Inhibition of the Na(+)-K(+)-ATPase had a stimulatory effect on Na(+)-K(+)-2Cl- cotransporter activity, which was dependent on the extent and duration of Na(+)-K(+)-ATPase inhibition. Ouabain treatment stimulated the potassium (86Rb) efflux rate and reduced intracellular potassium ([K]i). Potassium channel blockers suppressed the ouabain-activated potassium efflux and inhibited the ouabain-induced activation of the Na(+)-K(+)-2Cl- cotransporter. We conclude that Na(+)-K(+)-ATPase inhibition leads to the opening of potassium channels, which exacerbates the depletion of cellular potassium; Na(+)-K(+)-2Cl- cotransporter stimulation caused by the fall of [K]i overrides the tendency of increased cellular sodium to inhibit the cotransporter.

Propranolol antagonizes coronary artery relaxation by a potassium channel opener

Coronary artery preparations from cattle hearts responded with stable contractions to the thromboxane A 2 analogue, U 46619. These contractions were progressively reduced by increasing concentrations of the prototypical potassium channel opener pinacidil (3.8 × 10 −8 to 1.1 × 10 −4 M). Pinacidil-induced relaxations were antagonized significantly by d, l-propranolol (1.2×10 −6 to 1.2×10 −5 M). Forskolin-induced relaxations of coronary preparations were also antagonized by d, 1-propranolol, but those to nitroprusside were not. d-Propranolol also antagonized relaxations to pinacidil but only when used in higher concentrations than the l-isomer. Nadolol and metoprolol, two other beta receptor antagonists with differing profiles of action, also antagonized to some extent the vasodilator action of pinacidil. The known potassium channel antagonist, glibenclamide, shifted the concentration-relaxation curve for pinacidil to the right, but d,l-propranol produced an additional antagonistic effect in the presence of glibenclamide. Relaxations of contracted tracheal ring preparations of guinea pig by pinacidil, however, were not antagonized by d, l-propranolol, suggesting specificity for vascular tissue. Isoproterenol increased significantly the cyclic AMP levels in coronary tissue, but pinacidil had no such effect, ruling out an adrenergic component to pinacidil action. Pinacidil increased the efflux of 86Rb in isolated coronary preparations, and this effect was blunted by propranolol. It is concluded that beta receptor antagonists inhibit relaxations to a potassium channel opener by a mechanism independent of beta adrenergic receptors and that this effect may have therapeutic implications.