K+ channel openers,cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery

Summary The effects of K⁺ channel openers, cromakalim and an acetoxyl derivative of KRN 2391 (Ki 4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca²⁺]_i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP₃) production induced by the thromboxane A₂ analogue, U46619, in canine coronary arteries. Upon single dose applications of U46619 at 300 nmol/l, phasic and tonic increases in [Ca²⁺]_i and force were seen, which were almost abolished by cromakalim (10 mol/l) and Ki4032 (100 mol/l).In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction. Cromakalim (0.01–10 mol/l) and Ki4032 (0.1–100 mol/l) concentration-dependently inhibited the increases in [Ca²⁺]_i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K⁺ channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/l KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K⁺ channel openers inhibits the production of IP₃ and Ca ²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.Correspondence to T. Yanagisawa at the above address     

Modulation of intracellular calcium by potassium channel openers in vascular muscle

Summary We investigated two putative K⁺ channel openers, pinacidil and BRL34915 (cromakalim), and demonstrated their vasorelaxant effectiveness on rat artery contractions induced by K⁺, tetraethylammonium (TEA), or norepinephrine. The K⁺ channel opener-induced decrease in tension was rapid, even when tension was stimulated by 100 mmol/l K⁺. Measurements of intracellular free Ca⁺⁺ (activity) by ultra-high sensitivity digital imaging microscopy was carried out by briefly loaded fura2 (fluorescence ratio) quantitation in isolated, contracting cells of rat azygos vein. Submicron resolution was achieved by measuring cytoplasmic Ca⁺⁺-sensitive fluorescence at each pixel, and size and intensity of areas with high Ca⁺⁺ concentrations, called hot spots, were determined by a computer-generated, 3 algorithm. Hot spots, which most likely represent the sites of Ca⁺⁺ release and re-uptake by Ca⁺⁺-regulatory organelles, increased in size and intensity upon addition of K⁺ or norepinephrine, reaching an early peak prior to the whole cell average peak in cytoplasmic Ca⁺⁺ activity. Both norepinephrine and K⁺-induced stimulation resulted in Ca⁺⁺ activity increases that were primarily due to Ca⁺⁺ release from storage sites. Reduction of free Ca⁺⁺ activity to resting or lower levels occurred upon addition of pinacidil or cromakalim. Intracellular Ca⁺⁺ decreases due to K⁺ channel openers appeared abruptly beginning at the central portions of the cells, resulting in a pronounced early drop in central Ca⁺⁺ activity while elevated Ca⁺⁺ levels persisted at the periphery. While this late stage residual of peripheral Ca⁺⁺ appears to be a significant step in the vascular muscle relaxant action of both K⁺ channel opener drugs, the level of Ca⁺⁺ at peripheral sites was greater in response to pinacidil than to cromakalim. The results of this study suggest that in addition to increasing K⁺ conductance, pinacidil and cromakalim cause 1) decreased Ca⁺⁺ activity in central regions of the myocytes, and 2) a shift in Ca⁺⁺ distribution to primarily subsarcolemmal sites. These observations lead us to hypothesize separate control of peripheral and central Ca⁺⁺ activity within a vascular muscle cell, with Ca⁺⁺ redistribution that can be altered by vasorelaxants. We suggest that intracellular Ca⁺⁺ redistribution may contribute the membrane potential-independent part of the vasorelaxant action of the K⁺ channel openers.This study was supported by NIH grants HL38537 and HL38645, and Eli Lilly Co. P.E. was supported by the Swiss Foundation of Cardiology and by the SNF 32-029 975.90     

cAMP- and diacylglycerol-mediated pathways elevate cytosolic free calcium concentration via dihydropyridine-sensitive, ω-conotoxin-insensitive calcium channels in normal rat anterior pituitary cells

Summary The effect of 8-bromocyclic AMP (8-Br-cAMP) and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, on cytosolic free calcium concentration ([Ca²⁺]_i) in normal rat anterior pituitary cells was examined. [Ca²⁺]_i was monitored directly by the fluorescent indicator fura-2. 8-Br-cAMP as well as PMA elevated [Ca²⁺]_i in a concentration-dependent manner. Forskolin (10 mol/l), which activates adenylate cyclase, and 1-oleoyl-2-acetyl-glycerol (10 mol/l), another activator of protein kinase C, also increased [Ca²⁺]_i. Both the 8-Br-cAMP (2 mmol/l)- and the PMA (100 nmol/l)-induced increase in [Ca²⁺]_i was dependent on the presence of extracellular calcium and could be inhibited by the calcium channel blockers Mg²⁺ and nifedipine, but not by onotoxin (100 nmol/l). The half-maximally inhibitory concentrations of Mg²⁺ and nifedipine were about 12 mmol/l and 160 nmol/l, respectively, for the [Ca²⁺]_i response to 8-Br-cAMP (2 mmol/l), and were about 6 mmol/l and 400 nmol/1, respectively, for the PMA (100 nmol/1)-induced increase in [Ca²⁺]_i. The sodium channel blocker tetrodotoxin (5 mol/l) had no influence on the effect of 8-Br-cAMP (2 mmol/l) or PMA (100 nmol/l) on [Ca²⁺]_i. After pretreatment for 3 min with PMA (100 nmol/l), the subsequent K⁺ (100 mmol/l)- or arachidonic acid (3 mol/l)-induced increase in [Ca²⁺]_i was decreased by about 50%. By contrast, pretreatment (3 min) with 8-Br-cAMP (2–10 mmol/1) markedly enhanced the subsequent [Ca²⁺]_i response to K⁺ (100 mmol/l), and left the effect of arachidonic acid (3 mol/l) on [Ca²⁺]_i unimpaired. These data indicate that both cAMP- and diacylglycerol-mediated pathways increase [Ca²⁺ _i in normal rat anterior pituitary cells via an influx of extracellular Ca²⁺ through dihydropyridine-sensitive, -conotoxin-insensitive voltage-dependent calcium channels. These second messengers may thus be involved in Ca²⁺ channel activation by hypothalamic releasing hormones. Effects of cAMP- or diacylglycerol-induced pathways on anterior pituitary function may not be independent of but be mediated also by changes in [Ca²⁺]_i. However, substantial differences appear to exist in how cAMP and diacylglycerol influence voltage-dependent calcium channels.     

Effects of cinnarizine on calcium and pressure-dependent potassium currents in guinea pig vestibular hair cells

In vestibular hair cells, K⁺ currents induced by rises in hydrostatic pressure have recently been demonstrated. These currents are inhibited by charybdotoxin, a blocker of Ca²⁺-dependent K⁺ channels. On the other hand, cinnarizine is a blocker of voltage-gated Ca²⁺ currents in hair cells and is used as a drug in conditions with vestibular vertigo. Our aim was to test in patch-clamp experiments (conventional whole-cell mode) whether cinnarizine, by reducing Ca²⁺ influx, inhibited Ca²⁺ and pressure-sensitive K⁺ currents in vestibular type-II hair cells of guinea pigs. A quantitatively similar inhibition of K⁺ currents was evoked by extracellular Ca²⁺ removal, cinnarizine (0.5 M), and the L-type Ca²⁺ channel blocker nifedipine (3 M). Cinnarizine abrogated increases of K⁺ currents induced by increases in the hydrostatic pressure (from 0.2 to 0.5 cm H₂O). At a higher concentration (1 M), cinnarizine elicited K⁺ current inhibitions larger than those elicited by Ca²⁺ removal. Moreover, it reduced K⁺ currents in the absence of Ca²⁺, in contrast to nifedipine. However, charybdotoxin abolished these effects of cinnarizine. We thus conclude that cinnarizine inhibits, by two mechanisms, pressure-induced currents that are sensitive to charybdotoxin and Ca²⁺. It reduces Ca²⁺ influx and exerts a Ca²⁺-independent inhibition, with a lower IC₅₀ than that required for Ca²⁺ channel blockade. These two actions may importantly contribute to its therapeutic effects.P. Düwel and T. Haasler contributed equally to this work.     

Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

BACKGROUND AND PURPOSE

Controlling vascular tone involves K⁺ efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K_Ca2.3 and K_Ca3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.

EXPERIMENTAL APPROACH

Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K_Ca2.3 and K_Ca3.1 expression by immunohistochemistry and RT-PCR. K_Ca currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K_Ca3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.

KEY RESULTS

K_Ca3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K_Ca2.3 and K_Ca3.1 mRNA expression. With 200 nM [Ca²⁺]_i, the K_Ca2.1-2.3/K_Ca3.1 opener NS309 increased whole-cell currents. CyPPA, a K_Ca2.2/K_Ca2.3 opener, was without effect. With 1 µM [Ca²⁺]_i, the K_Ca3.1 inhibitor TRAM-34 reduced currents whereas apamin (K_Ca2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K_Ca3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K_Ca3.1 and the large-conductance K_Ca channel, K_Ca1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.

CONCLUSION AND IMPLICATIONS

Astrocytes express functional K_Ca3.1 channels, and these contribute to neurovascular coupling.

LINKED ARTICLES

This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3     

Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells

Summary The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca²⁺]_i) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 M) enhanced the increase in [Ca²⁺]_i elicited by bradykinin (3 nM) and also caused an increase in resting [Ca²⁺]_i when given alone. This increase in resting [Ca²⁺]_i was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N^G-nitro-l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca²⁺]_i and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B₂-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B₂-receptors with a subsequent increase in [Ca²⁺]_i and nitric oxide formation. Send offprint requests to R. Busse at the above address     

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

BRL 38227 (levcromakalim)-induced hyperpolarization reduces the sensitivity to Ca2+ of contractile elements in canine coronary artery

Summary Potassium (K⁺) channel openers decrease intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) by hyperpolarizing the membrane and deactivating the Ca²⁺-channels. To examine whether the hyperpolarization produced by K⁺-channel openers has other effects on the mechanical activity of vascular smooth muscle, we investigated the effects of levcromakalim (BRL 38227) on membrane potential, [Ca²⁺]_i, as measured with fura-2, and force of contraction induced by 30 mmol/l KCl-physiological salt solution (PSS), in canine coronary arteries. BRL 38227 hyperpolarized the membrane and reduced increases in [Ca²⁺]_i and in contractile force induced by 30 mmol/l KCl-PSS. The [Ca²⁺]_i-contractile force curve, determined in the presence of BRL 38227, was located to the right of the control curve determined by decreasing extracellular Ca²⁺ concentrations ([Ca²⁺]_o) in 30 mmol/l KCl-PSS. The [Ca²⁺ _i-contractile force curve, determined by decreasing extracellular K⁺ concentrations ([K⁺]_o), was also located to the right of that determined by decreasing [Ca²⁺]_o in 30 mmol/l KCl-PSS. The effect of BRL 38227, a reduction in the Ca ²⁺-sensitivity of contractile elements, was antagonized by the ATP-sensitive K⁺-channel blocker, glibenclamide (10^–6 or 10^–5 mol/1). These results suggest that the membrane hyperpolarization induced by BRL 38227, or the repolarization caused by reducing ([K⁺]_o), decreases the Ca²⁺-sensitivity of contractile elements of vascular smooth muscle.Correspondence to T. Yanagisawa at the above address     

The mechanism of honokiol-induced and magnolol-induced inhibition on muscle contraction and Ca2+ mobilization in rat uterus

The effects of honokiol and magnolol extracted from the Magnolia officinalis on muscular contractile responses and intracellular Ca²⁺ mobilization were investigated in the non-pregnant rat uterus. Honokiol and magnolol (1–100 mol/l) were observed to inhibit spontaneous and uterotonic agonists (carbachol, PGF₂, and oxytocin)-, high K⁺-, and Ca²⁺ channel activator (Bay K 8644)-induced uterine contractions in a concentration-dependent manner. The inhibition rate of honokiol on spontaneous contractions appeared to be slower than that of magnolol-induced response. The time periods that were required for honokiol and magnolol, at 100 mol/l, to abolish 50% spontaneous contractions were approximately 6 min. Furthermore, honokiol and magnolol at 10 mol/l also blocked the Ca²⁺-dependent oscillatory contractions. Consistently, the increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by PGF₂ and high K⁺ were suppressed by both honokiol and magnolol at 10 mol/l. After washout of these treatments, the rise in [Ca²⁺]_i induced by PGF₂ and high K⁺ was still partially abolished. In conclusion, the inhibitory effects of honokiol and magnolol on uterine contraction may be mediated by blockade of external Ca²⁺ influx, leading to a decrease in [Ca²⁺]_i. Honokiol and magnolol may be considered as putative Ca²⁺ channel blockers and be of potential value in the treatment of gynecological dysfunctions associated with uterine muscular spasm and dysmenorrhea.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery

The rank order of potency of a series of benzopyran and cyanoguanidine K⁺ channel openers (KCOs) for causing relaxation of the PGF₂-precontracted porcine coronary artery was determined. Glyburide, an inhibitor of K_ATP channels, showed an apparent competitive inhibition of the vasorelaxant activity of the KCOs. The pA₂ values of glyburide when cromakalim and CGP 14877 (P1060) were used as vasorelaxants were 7.66 and 7.83, respectively. Charybdotoxin (40 nM), an inhibitor of BK_Ca channels, also caused a significant inhibition of the cromakalim mediated relaxation of the porcine coronary artery. In order to clarify the site of action of these KCOs, we identified a K⁺ channel current in single porcine coronary arterial cells and measured channel activity in the presence of these compounds. The prominent K⁺ ion current in these cells had characteristics typical of the conventional large Ca²⁺-activated K⁺ channel BK_Ca present in other smooth muscle cells. Using symmetrical K⁺ concentrations, the channel had a conductance of 214 pS and was found to be sensitive to [Ca²⁺]_i and membrane potential. The KCOs were found to reversibly increase the open probability (P_o) of the channel without changing channel conductance. The potency of the KCOs to increase K⁺ channel opening was similar to the potency of these compounds to cause coronary artery relaxation. These results indicate that the porcine coronary artery contains the BK_Ca channel and that this channel, along with other types of K⁺ channels (K_ATP), mediate the vasorelaxant effects of K⁺ channel openers.     

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K+

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Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

Levcromakalim-induced modulation of membrane potassium currents,intracellular calcium and mechanical activity in rat mesenteric artery

In freshly-dispersed cells from rat mesenteric artery, levcromakalim (1 and 10 M) induced a non-inactivating potassium current (I_KCO), an event which was associated with increased current noise. I_KCO was fully inhibited in the presence of 10 M glibenclamide. Stationary fluctuation analysis of the current noise associated with I_KCO induced by levcromakalim at a holding potential of –10 mV indicated that the unitary conductance of the underlying K-channels was 10.2 pS at 0 mV under the quasi-physiological conditions of the experiment.In isolated arterioles both levcromakalim (10 nM - 10 M) and nifedipine (10 nM - 10 M) each elicted full, concentration-dependent, parallel reductions of the increases in [Ca²⁺]_i (assessed using fura-2) and tension induced by 10 M noradrenaline. However, the effects of both drugs on KCl-induced increases in tension and in [Ca²⁺]_i, did not follow a simple relationship. Levcromakalim relaxed KCl- and noradrenaline-induced sustained contractions with a similar potency. This was in contrast to nifedipine which was approximately 20 times more potent against KCl-induced contractions.It is concluded that levcromakalim relaxes rat mesenteric arterioles primarily by the opening of a small conductance, glibenclamide-sensitive K-channel. An additional action of levcromakalim is suggested by its relative inability to suppress the increase in [Ca²⁺]_i produced by 30 mM K⁺-PSS. Correspondence to: A. H. Weston at the above address     

Rhynchophylline-induced vasodilation in human mesenteric artery is mainly due to blockage of L-type calcium channels in vascular smooth muscle cells

Rhynchophylline (Rhy) is a pharmacologically active substance isolated from Uncaria rhynchophylla which has been used to treat cardiovascular diseases and has drawn considerable attention in recent years for its antihypertensive activities. We investigated the actions of Rhy on endothelium-denuded human mesenteric artery by tension measurement and its actions on high conductance Ca²⁺-activated K⁺ channels (BK_Ca) currents and calcium currents (I_Ca) in freshly isolated smooth muscle cells using perforated patch clamp technique. Intracellular Ca²⁺ level was measured in Fura-2-loaded cells. Rhy inhibited both the KCl and BayK-evoked mesenteric artery constrictions in a dose-dependent manner. K⁺ channel blockers (TEA, glibenclamide, IbTX, and 4-AP) did not affect the vasorelaxing effect of Rhy. Rhy inhibited L-type voltage-gated Ca²⁺ current (I_Ca,L) but had no significant effect on macroscopic BK_Ca current. Rhy preincubation markedly reduced the elevation of [Ca²⁺]_i level induced by KCl depolarization. Caffeine-stimulated [Ca²⁺]_i elevation was also decreased to some extent by pretreatment with Rhy for 20 min. Our results show that Rhy relaxes smooth muscles of human mesenteric resistance arteries, mainly due to inhibition of Ca²⁺ influx by blockage of L-type Ca²⁺ channels and thereby the decrease in [Ca²⁺]_i.     

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium

BACKGROUND AND PURPOSE

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium.

EXPERIMENTAL APPROACH

Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123.

KEY RESULTS

NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na⁺-K⁺-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²⁺-dependent) basolateral K⁺ channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²⁺]_i, an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²⁺. Initial falls in [Ca²⁺]_i were not mediated by a sequestration of Ca²⁺ in intracellular Ca²⁺ storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²⁺]_i were inhibited by 2′,4′-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺-exchanger, and attenuated in Na⁺-free buffer, suggesting a transient stimulation of Ca²⁺ outflow by this transporter, directly demonstrated by Mn²⁺ quenching experiments.

CONCLUSIONS AND IMPLICATIONS

ATP-sensitive and Ca²⁺-dependent basolateral K⁺ conductances, the basolateral Na⁺-K⁺-pump as well as Ca²⁺ transporters were involved in the action of H₂S in regulating colonic ion secretion.     

Enhancement of muscle blood cell flux and pO2 by cromakalim (BRL 34915) and other compounds enhancing membrane K+ conductance,but not by Ca2+ antagonists or hydralazine,in an animal model of occlusive arterial disease

Summary The effect of Ca²⁺ antagonists, hydralazine and agents which enhance membrane K⁺ conductance (cromakalim, pinacidil and nicorandil) in smooth muscle cells, was compared on normal and hypoxic skeletal muscle blood cell flux and pO₂. The K⁺ conductance enhancers and verapamil, diltiazem and nifedipine increased blood cell flux in normally perfused muscle. At equieffective blood pressure lowering dosages, the Ca²⁺ antagonists produced greater increases than the K⁺ channel openers. Hydralazine did not elevate blood cell flux in the normal muscle. In hypoxic skeletal muscle, the K⁺ conductance enhancers produced a marked increase in blood cell flux and in tissue oxygen tension, indicating that they had increased the nutritive blood flow in the muscle. The Ca²⁺ antagonists and hydralazine either did not change hypoxic muscle blood cell flux and pO₂ or reduced them. The dissimilarity in the activity of the compounds may be due to differences in their site of action in the vascular bed. Ca²⁺ antagonists and hydralazine are known to reduce arteriolar vessel resistance and do not increase blood flow in hypoxic skeletal muscle. The positive effect of cromakalim, pinacidil and nicorandil may be due to relaxant activity on larger arterial blood vessels including collaterals. This effect could be related to their ability to enhance membrane K²⁺ conductance in vascular smooth muscle cells.Send offprint requests to: D. Angersbach     

Differential Vasorelaxant Effects of K+-Channel Openers and Ca2+-Channel Blockers on Canine Isolated Arteries

Abstract— The vasorelaxant effects of the K⁺-channel openers, pinacidil and cromakalim, were compared with those of the Ca²⁺-channel blockers, verapamil and KB-2796 (1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)piperazine dihydrochloride), in canine isolated coronary, renal, basilar and mesenteric arteries precontracted with U46619, a thromboxane A₂ mimetic. The relaxation induced by pinacidil and cromakalim was greater in coronary than in other arteries, the magnitude of relaxation being in the order of coronary > renal > basilar > mesenteric arteries. The relaxant responses to both drugs were inhibited by glibenclamide, a blocker of ATP-sensitive K⁺ channels. The relaxation induced by verapamil and KB-2796, in contrast, was greater in basilar than in other arteries, the magnitude of relaxation being in the order of basilar > coronary > renal and mesenteric arteries. In fura-2-loaded, U46619-stimulated arteries, pinacidil and cromakalim produced a greater reduction in intracellular Ca²⁺ concentration and muscle tension in coronary than in mesenteric arteries, while verapamil and KB-2796 reduced these values more potently in basilar than in mesenteric arteries. These results suggest that K⁺-channel openers exhibit a vasorelaxant selectivity for coronary arteries, whereas Ca²⁺-channel blockers exhibit such selectivity for cerebral arteries. The selective vasorelaxant action induced by these drugs appears to correspond, in part, to their effects on the concentration of intracellular Ca²⁺.     

Direct interference of HIV protease inhibitors with pancreatic β-cell function

The aim of the present study was to evaluate whether HIV protease inhibitors directly interfere with stimulus-secretion coupling in pancreatic -cells. Insulin secretion was determined by a radioimmunoassay (RIA), cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) with the fluorescence dye fura-2 and whole-cell membrane currents with the patch-clamp technique.Glucose-induced insulin secretion was inhibited in a concentration-dependent manner by ritonavir and nelfinavir but not by indinavir. Ritonavir and nelfinavir lowered [Ca²⁺]_c in the presence of a stimulatory glucose concentration whereas indinavir again had no effect. Ritonavir and nelfinavir completely inhibited the effect of tolbutamide, which normally increases [Ca²⁺]_c by blocking K_ATP channels. This observation points to an action of both drugs on K_ATP channels or a step distal to these channels in stimulus-secretion coupling. Ritonavir was used to further evaluate the direct effects of HIV protease inhibitors on -cell ion channel currents. Unexpectedly, ritonavir inhibited neither the whole-cell K_ATP current nor the whole-cell L-type Ca²⁺ current. Tolbutamide almost completely suppressed the K_ATP current in the presence of ritonavir excluding that ritonavir alters the tolbutamide sensitivity of the K_ATP channel. Ritonavir increased the length and decreased the frequency of glucose-induced action potentials. This effect can be attributed to inhibition of voltage-dependent K⁺ currents. Intracellular stores seem not to be involved in the ritonavir-induced lowering of [Ca²⁺]_c.In conclusion, different HIV protease inhibitors surprisingly reveal distinct effects on insulin secretion. Ritonavir inhibits insulin secretion by lowering [Ca²⁺]_c but this effect is evidently independent of the opening of K_ATP channels or the closure of voltage-dependent Ca²⁺ channels, which are commonly considered to play a key role in stimulus-secretion coupling.Abbreviations [Ca²⁺]_c Cytosolic Ca²⁺ concentration - RIA Radioimmunoassay     

(-)-Epigallocatechin Gallate Inhibits the Pacemaker Activity of Interstitial Cells of Cajal of Mouse Small Intestine

The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at 30℃ and Ca²⁺ image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive K⁺ channel blocker and TEA, a Ca²⁺-activated K⁺ channel blocker. Also, we found that EGCG inhibited the spontaneous [Ca²⁺]_i oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced [Ca²⁺]_i oscillations by cAMP-, cGMP-, ATP-sensitive K+ channel-independent manner.