Ba2+ differentially inhibits the Rb+ efflux promoting and the vasorelaxant effects of levcromakalim and minoxidil sulfate in rat isolated aorta

The K⁺ channel openers activate ATP-sensitive K⁺ channels (K_ATP) in vascular smooth muscle and induce relaxation. In this study, the relationship between these two effects was examined in rings of rat aorta using levcromakalim and minoxidil sulfate as the openers and Ba²⁺ as the K⁺ channel blocker; K⁺ channel opening was assessed by determining the rate constant of ⁸⁶Rb⁺ efflux from the preparation.Ba²⁺ inhibited the ⁸⁶Rb⁺ efflux stimulated by levcromakalim in a noncompetitive manner with an IC₅₀ value of 29 M and a Hill-coefficient of 1.2. At concentrations > 300 M, Ba²⁺ increased the tension of rat aortic rings concentration-dependently. Levcromakalim relaxed contractions to Ba²⁺ (0.5 and 1 mM) with potencies similar to those determined against KCl (25 mM) or noradrenaline as spasmogens (EC₅₀ values 15–40 nM). The vasorelaxant effect against Ba²⁺ was inhibited by the K_ATP channel blockers, glibenclamide and tedisamil, and abolished in depolarizing medium (55 mM KCl). At 3 mM Ba²⁺, levcromakalim was still able to transiently induce complete relaxation; however, within 1 h oscillations in tension developed, leading to a stable level of only 15% relaxation. A similar level of relaxation was achieved against 10 mM Ba²⁺ whereas the combination of 0.5 mM Ba²⁺ and 3 M tedisamil blocked the relaxant effect of levcromakalim completely. With minoxidil sulfate as the K_ATP channel opener the results of the ⁸⁶Rb⁺ efflux and tension experiments were similar to those obtained with levcromakalim.It is concluded that Ba²⁺ is more potent in inhibiting the K⁺ channel opening than the vasorelaxant effects of the openers. On the basis of the ⁸⁶Rb⁺ efflux experiments it is estimated that at least 97% of the channels opened by the activators can be blocked without major effects on vasorelaxation suggesting a dissociation between the two effects. However, if the block is pushed to extremes ( 99.95%) the vasorelaxant effect of the openers is also abolished suggesting a link between both effects. This paradoxon remains to be solved.     

Multiple conformational equilibria of cyclic octapeptide,cyclo (L-Pro-Sar)4 in solution

Cyclo (l -Pro-Sar)₄ is asynthetic cyclic octapeptide composed of only N -substituted amino acids. The conformation of this peptide in different solvents was examined by ¹H-and ¹³C-n.m.r. spectroscopy, ¹H-n.m.r. data of this cyclic peptide demonstrated that multiple conformational equilibria take place in solution and they vary with solvent polarity. Three conformers are interconverting with each other in the nonpolar chloroform (CDCl₃); one C₄-symmetric conformer (49%) and two C₂-symmetric conformers (37% and 14%). While three C₂-symmetric (59%, 19%, and 18%) and one asymmetric conformer (4%) are detected to coexist in acetonitrile (CD₃CN), one largely populated C₂-symmetric one (97%) is favored in the polar dimethyl sulfoxide (Me₂SO-d ₆). N.m.r. measurement employing various strategy predicted the occurrence of trans-cis-cis-cis-trans-cis-cis-cis (two Sar-Pro bonds: trans) (tccctccc) peptide bond sequence in a predominant C₂-symmetric conformer of Me₂SO-d ₆ solution. In the same way, tccctccc and ctttcttt (two Sar-Pro bonds: cis) peptide unit arrangement was proposed for the first and the secondly populated conformer in CD₃CN, respectively. In CDCl₃ a conformer having tctctctc (four Sar-Pro bonds: trans) C₄-symmetric peptide bond sequence was deduced.     

Differences in alkaline earth stimulation of neurotransmitter release from isolated brain synaptosomes

Summary In the presence of 15 or 55 mM K⁺ buffer (calcium-deficient), pulses of the alkaline earths (Ca²⁺, Sr²⁺, Ba²⁺) stimulated secretion of previously accumulated [¹⁴C]GABA and [³H]norepinephrine (NE). For a 1 mM alkaline earth pulse in the presence of 15 mM K⁺, both GABA and NE release were ordered Ba²⁺>Sr²⁺>Ca²⁺. On the other hand, in 55 mM K⁺ the ordering was Ba²⁺>Sr²⁺Ca²⁺. The change in ordering resulted from an increase in Ca-dependent release at higher K⁺. In fact, Ba-dependent release was smaller in 55 mM K⁺ solution than in 15 mM K⁺ solution.D-600 (10 M), an antagonist of calcium influx, decreased both Ca- and Ba-dependent release of [¹⁴C]-GABA and [³H]NE in 15 mM K⁺ buffer. D-600 inhibited Ca-dependent release 60–70% whereas Ba-dependent release was inhibited only 30–45%.A CaCl₂ (1 mM), BaCl₂ (1 mM) or KCl (30 mM) pulse in 15 mM K⁺ buffer increased both [¹⁴C]GABA and endogenous GABA release. The Ba²⁺ pulse released more [¹⁴C] and endogenous GABA than did the Ca²⁺ pulse. The 30 mM KCl pulse released less [¹⁴C] and endogenous GABA than did either the Ba²⁺ or the Ca²⁺ pulse. However, the specific activity of the K-induced release was significantly higher than the specific activity of either Ca- or Ba-dependent GABA release.In substituting for Ca²⁺, Ba²⁺ may gain intracellular access to stimulus-secretion coupling processes via permeation at both Ca²⁺ and K⁺ ionophores.Supported by: NINCDS Grant 08597 and an NSF Predoctoral Fellowship to J. W. Haycock. C. W. Cotman is the recipient of a Research Scientist Award from the National Institute on Drug Abuse     

Effects of Rb1 from the Panax notoginseng saponins on arrhythmia and Ca2+ movement in rat heart cells

We determined the antiarrhythmic effect of Rb₁ (from Panax notoginseng) on different arrhythmia models and its effect on cytoplasmic Ca²⁺ concentrations ([Ca²⁺]i) in rat heart cells with fura-2 fluorescence. At doses of 30 and 50 mg/Kg, Rb₁ produced an antirrhythmic effect on BaCl₂-induced ventricular tachyarrhythmia in rats, atrial fibrillation induced by CaCl₂-ACh, and ventricular fibrillation induced by chloroform in mice. The rest [Ca²⁺]i was 108 ± 10.7 nM in the freshly isolated rat heart cells. Sixty mM KCl caused an increase in [Ca²⁺]i to 379.4 ± 77 nM. Rb₁ from 0.1 to 0.8 mM significantly reduced this increase in a concentration-dependent manner. mM Rb₁ (0.4 mM) also completely inhibited the increase in [Ca²⁺]i induced by 1 μM isoprenaline. The radioligand binding study in rat heart cells showed that Rb₁ did not change the B_max and K_d values of [³H]-dihydroalprenolol binding. These data suggest Rb₁ can inhibit Ca²⁺ entry through voltage-dependent and receptor-linked Ca²⁺ channels, and that this is related to its antiarrhythmic effect. Drug Dev. Res. 39:179–183. © 1997 Wiley-Liss, Inc.     

Loss of Ca2+-mediated ion transport during colitis correlates with reduced ion transport responses to a Ca2+-activated K+ channel opener

Background and purpose:

Epithelial surface hydration is critical for proper gut function. However, colonic tissues from individuals with inflammatory bowel disease or animals with colitis are hyporesponsive to Cl⁻ secretagogues. The Cl⁻ secretory responses to the muscarinic receptor agonist bethanechol are virtually absent in colons of mice with dextran sodium sulphate (DSS)-induced colitis. Our aim was to define the mechanism underlying this cholinergic hyporesponsiveness.

Experimental approach:

Colitis was induced by 4% DSS water, given orally. Epithelial ion transport was measured in Ussing chambers. Colonic crypts were isolated and processed for mRNA expression via RT-PCR and protein expression via immunoblotting and immunolocalization.

Key results:

Expression of muscarinic M₃ receptors in colonic epithelium was not decreased during colitis. Short-circuit current (I_SC) responses to other Ca²⁺-dependent secretagogues (histamine, thapsigargin, cyclopiazonic acid and calcium ionophore) were either absent or severely attenuated in colonic tissue from DSS-treated mice. mRNA levels of several ion transport molecules (a Ca²⁺-regulated Cl⁻ channel, the intermediate-conductance Ca²⁺-activated K⁺ channel, the cystic fibrosis transmembrane conductance regulator, the Na⁺/K⁺-ATPase pump or the Na⁺/K⁺/2Cl⁻ co-transporter) were not reduced in colonic crypts from DSS-treated mice. However, protein expression of Na⁺/K⁺-ATPase α1 subunits was decreased twofold during colitis. Activation of Ca²⁺-activated K⁺ channels increased I_SC significantly less in DSS colons compared with control, as did the protein kinase C activator, phorbol 12-myristate 13-acetate.

Conclusions and implications:

Decreased Na⁺/K⁺-ATPase expression probably contributes to overall epithelial hyporesponsiveness during colitis, while dysfunctional K⁺ channels may account, at least partially, for lack of epithelial secretory responses to Ca²⁺-mediated secretagogues.     

Effect of capsaicin on membrane currents in cultured vascular smooth muscle cells of rat aorta

The application of capsaicin (1 μM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K⁺ or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba²⁺ inward current (I_Ba) through the voltage-dependent L-type Ca²⁺ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in I_Ba. Capsaicin did not alter the overall shape of current-voltage relationship of I_Ba. However, capsaicin (3 μM) shifted the quasi-steady-state inactivation curve of I_Ba to more negative membrane potential by about 5 mV. These effects of capsaicin on I_Ba were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K⁺ currents. These results suggest that inhibition of the voltage-dependent L-type Ca²⁺ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage-dependent K⁺ channels might produced an increase in cell excitability.     

G02 AND Gi3 PROTEINS MEDIATE THE ACTION OF SOMATOSTATIN ON MEMBRANE Ca2+ AND K+ CURRENTS IN OVINE PITUITARY SOMATOTROPHS

1. Growth hormone (GH) secretion from the anterior pituitary gland is mainly regulated by hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIF). Somatostatin reduces both spontaneous and GHRH-stimulated GH secretion. 2. Exocytosis of GH is mainly determined by the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is regulated by the influx of Ca²⁺ via membrane Ca²⁺ channels. Somatostatin reduces the influx of Ca²⁺ through two separate mechanisms, namely a direct action on Ca²⁺ channels and an indirect action on membrane potentials through the activation of K⁺ channels. 3. In the present experiments, somatotroph-enriched cells were obtained from the ovine pituitary gland by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the effect of SRIF (10 nmol/L) on Ca²⁺ or K⁺ currents. 4. A significant reduction in Ca²⁺ currents and an increase in K⁺ currents was obtained in response to local application of SRIF (10 nmol/L), but vehicle application had no effect. The responses of Ca²⁺ and K⁺ currents to SRIF were reversible after removal of SRIF. 5. Dialysis of GTP-λ-s (200 μmol/L) abolished the recovery phase of K⁺ current response to SRIF after its removal, whereas GDP-β-s (200 μmol/L) totally blocked the response. Pretreatment of the cells with pertussis toxin (100 nmol/L) overnight abolished the Ca²⁺ current response to SRIF. 6. Intracellular dialysis of antibodies to αo, α_{1_3}, ai_1-2 and ai₃summits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-ai_1-3 or anti-@aLi₃ antibodies significantly attenuated the increase in the K⁺ current in response to 10 nmol/L SRIF, whereas neither anti-αo nor anti-αi_2 antibodies diminished the effect of SRIF on the K⁺ current. 8. Dialysis of anti-αo antibodies significantly attenuated the reduction in the Ca²⁺ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-αi-2 nor anti-αi3 antibody dialysis diminished the effect of SRIF on the Ca²⁺ current. 9. Dialysis of the ao common antisense oligonucleotides (ASm) but not the αi₃ AS significantly diminished the inhibitory effect of SRIF on the Ca²⁺ current. This effect of ao ASm dialysis occurred at 12 h incubation after dialysis, reaching a maximal level at 48 h and partially recovering at 72 h incubation. Antisense oligonucleotides specific for αo₁ (αo₁ AS) or αo₂(α0₂ AS) were dialysed into somatotrophs and only αo₂ AS significantly attenuated the inhibition of SRIF on the Ca²⁺ current. 10. It is concluded that the G_i3 protein mediates the effect of SRIF on the K⁺ current and that the G02 protein mediates the effect of SRIF on the Ca²⁺ current in primary cultured ovine somatotrophs.     

Pharmacological properties of Ca2+-activated K+ currents of ramified murine brain macrophages

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I_K,Ca) were investigated in ramified murine brain macrophages. In order to induce I_K,Ca the intracellular concentration of nominal free Ca²⁺ was adjusted to 1μM. The Ca²⁺-activated K⁺ current of brain macrophages did not show any voltage dependence at test potentials between –120 and +30mV. A tenfold change in extracellular K⁺ concentration shifted the reversal potential of I_K,Ca by 51mV. The bee venom toxin apamin applied at concentrations of up to 1μM did not affect I_K,Ca. Ca²⁺-activated K⁺ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I_K,Ca at concentrations between 1 and 50nM. Tetraethylammonium (TEA) blocked 8.0% of I_K,Ca at a concentration of 1mM, whereas 31.4% of current was blocked by 10mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2mM for their ability to block I_K,Ca. La³⁺ reduced I_K,Ca by 72.8%, whereas Cd²⁺ decreased I_K,Ca by 17.4%; in contrast, Ni²⁺ did not have any effect on I_K,Ca. Ba²⁺ applied at a concentration of 1mM reduced I_K,Ca voltage-dependently at hyperpolarizing potentials. Received: 17 January / Accepted: 5 May 1997     

Actions of hydrogen sulphide on ion transport across rat distal colon

Background and purpose:

The aim of this study was to identify the actions of H₂S on ion transport across rat distal colon.

Experimental approach:

Changes in short-circuit current (Isc) induced by the H₂S-donor, NaHS, were measured in Ussing chambers. Cytosolic Ca²⁺ concentration was evaluated using fura-2.

Key results:

NaHS concentration-dependently induced a change in Isc, that was only partially inhibited by the neurotoxin, tetrodotoxin. Lower concentrations (≤10⁻³ mol·L⁻¹) of NaHS induced a monophasic increase in Isc, whereas higher concentrations induced an additional, secondary fall of Isc, before a third phase when Isc rose again. Blockers of H₂S-producing enzymes (expression demonstrated immunohistochemically) decreased basal Isc, suggesting that endogenous production of H₂S contributes to spontaneous anion secretion. The positive Isc phases induced by NaHS were due to Cl⁻ secretion as shown by anion substitution and transport inhibitor experiments, whereas the transient negative Isc induced by higher concentrations of the H₂S-donor was inhibited by mucosal tetrapentylammonium suggesting a transient K⁺ secretion. When applied from the serosal side, glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and tetrapentylammonium, a blocker of Ca²⁺-dependent K⁺ channels, suppressed NaHS-induced Cl⁻ secretion suggesting different types of K⁺ channels are stimulated by the H₂S-donor. NaHS-induced increase in cytosolic Ca²⁺ concentration was confirmed in isolated, fura-2-loaded colonic crypts. This response was not dependent on extracellular Ca²⁺, but was inhibited by blockers of intracellular Ca²⁺ channels present on Ca²⁺ storage organelles.

Conclusions and implications:

H₂S induces colonic ion secretion by stimulation of apical as well as basolateral epithelial K⁺ channels.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Characteristics and possible mechanisms of low - Na+ induced contractions in rat aorta

The influence of reducing external Na⁺ concentration ([Na⁺]^ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na⁺]_ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na⁺]_ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10^–6 M. Elimination of the endogenous catecholamine participation using either phentolamine 10^–5 M or guanethidine 3.10^–6 M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10^–5 M, an antagonist of the voltage operated Ca²⁺ channels (25–30% residual tension for all the substitutes). Large reductions in [Na⁺]_ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65–75 %, sucrose replacement) and insensitive to D600 (all the substitutes). The Na⁺/K⁺ ATPase inhibitor ouabain (10^–4 M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10^–6 M control.Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na⁺]_ex dependence. Phenylephrine 10^–6 M failed to further contract the ouabain 10^–4 M precontracted rings.We conclude that relatively small reductions in [Na⁺]_ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na⁺/Ca²⁺ exchange. Larger reductions in [Na⁺]_ex appear to cause contraction through muscular Na⁺/Ca²⁺ exchange.     

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

Summary: Glibenclamide, glipizide and glibornuride showed dual effects in guinea-pig isolated trachea. The drugs antagonized the relaxant response to the K⁺ channel opener cromakalim (order of effectiveness: glibenclamide > glipizide > glibornuride) and at concentrations of 1-1000 μM produced airway smooth muscle relaxation (order of potency: glibenclamide > glipizide = glibornuride). Gliclazide, tolbutamide and chlorpropamide did not antagonize cromakalim nor did the two latter drugs produce tracheal relaxation. The sulphonylureas and cromakalim were compared as airway relaxants against a panel of different spasmogens. The order of tissue responsiveness for the sulphonylureas was: spontaneous tone = LTD₄ > PGF_2α = histamine = 30 mM K⁺ > carbachol and for cromakalim: spontaneous tone = LTD₄ = PGF_2α = histamine > carbachol > 30 mM K⁺. Glibenclamide, but not cromakalim, relaxed contractions induced by 124 mM K⁺. Phentolamine and Ba²⁺, which are reported blockers of ATP-regulated K⁺ channels, failed to influence sulphonylurea-induced airway smooth muscle relaxation. Glibenclamide reversed tracheal relaxation produced by cromakalim, whereas cromakalim failed to reverse relaxation induced by glibenclamide. The mechanism for the additional property of sulphonylureas to relax airway smooth muscle is unclear, but the results do not support a role for involvement of cromakalim-sensitive K⁺ channels.     

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⁺.     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

Role of ion channels in sepsis-induced atrial tachyarrhythmias in guinea pigs

BACKGROUND AND PURPOSE

Supraventricular tachyarrhythmias, including atrial fibrillation, are occasionally observed in patients suffering from sepsis. Modulation of cardiac ion channel function and expression by sepsis may have a role in the genesis of tachyarrhythmias.

EXPERIMENTAL APPROACH

Sepsis was induced by LPS (i.p.; 300 µg·kg⁻¹) in guinea pigs. Membrane potentials and ionic currents were measured in atrial myocytes isolated from guinea pigs 10 h after LPS, using whole cell patch-clamp methods.

KEY RESULTS

In atrial cells from LPS-treated animals, action potential duration (APD) was significantly shortened. It was associated with a reduced L-type Ca²⁺ current and an increased delayed rectifier K⁺ current. These electrophysiological changes were eliminated when N^G-nitro-l-arginine methyl ester (l-NAME) or S-ethylisothiourea was given together with LPS. In atrial tissues from LPS-treated animals, Ca²⁺ channel subunits (Ca_v1.2 and Ca_v1.3) decreased and delayed rectifier K⁺ channel subunits (K_v11.1 and K_v7.1) increased. However, L-NAME treatment did not substantially reverse such changes in atrial expression in LPS-treated animals, with the exception that K_v11.1 subunits returned to control levels. After LPS injection, inducible NOS in atrial tissues was up-regulated, and atrial NO production clearly increased.

CONCLUSIONS AND IMPLICATIONS

In atrial myocytes from guinea pigs with sepsis, APD was significantly shortened. This may reflect nitration of the ion channels which would alter channel functions, rather than changes in atrial expression of the channels. Shortening of APD could serve as one of the mechanisms underlying atrial tachyarrhythmia in sepsis.     

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Background and purpose:

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl⁻ channels (CaCCs) and large conductance, calcium-gated K⁺ channels (K_Ca1.1). The goal of the present study was to ascertain whether blockers of K_Ca1.1 inhibited calcium-activated Cl⁻ currents (I_ClCa) and if the pharmacological overlap between K_Ca1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K⁺ channels.

Experimental approaches:

Whole-cell Cl⁻ and K⁺ currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca²⁺].

Key results:

The selective K_Ca1.1 blocker paxilline (1 µM) inhibited I_ClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I_ClCa by ∼20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca²⁺-binding domain of murine K_Ca1.1 had no effect on I_ClCa while inhibiting spontaneous K_Ca1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K⁺ channels (K_Ca2.1 and K_Ca2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on I_ClCa.

Conclusions and implications:

These data show that the selective K_Ca1.1 blockers also reduce I_ClCa considerably. However, the pharmacological overlap that exists between CaCCs and K_Ca1.1 does not extend to the calcium-binding domain or to other calcium-gated K⁺ channels.     

Activators of potassium channels enhance calcium influx into endothelial cells as a consequence of potassium currents

Summary Ca²⁺ influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca²⁺ influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K⁺ channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca²⁺]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 mol/l) and cromakalim (1 mol/l) elicited outward currents carried by K⁺ and significantly prolonged the Ca²⁺-dependent K⁺ currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca²⁺ indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1–1 mol/l elicited long-lasting increases in [Ca²⁺)_i from 100 ± 10 to 550 ± 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K⁺. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca²⁺]_i when applied during the decline of [Ca²⁺]_i after the initial peak. Cicletanine elicited K⁺ currents in resting and attenuated K⁺ currents in bradykinin-stimulated cells. It elevated [Ca²⁺]_i even in the absence of extracellular Ca²⁺ and in K⁺-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K⁺ channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca²⁺]_i secondary to their activation of K⁺ channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca²⁺ influx. In this way, the two drugs may promote Ca²⁺-dependent formation of endothelium-derived relaxing factor. Send offprint requests to A. Lückhoff at the above address     

An active metabolite of carbamazepine, carbamazepine-10,11-epoxide, inhibits ion channel-mediated catecholamine secretion in cultured bovine adrenal medullary cells

 We have recently reported inhibitory effects of carbamazepine (CBZ) on ion channel-mediated secretion of catecholamines in bovine adrenal medullary cells. Here, we report the effects of carbamazepine-10,11-epoxide (CBZ-E), an active metabolite of CBZ, and carbamazepine-10,11-diol (CBZ-D), a non-active metabolite, on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured adrenal medullary cells. CBZ-E, but not CBZ-D inhibited ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion induced by carbachol or veratridine with a half-maximal inhibitory concentration (IC₅₀) of 0.26 or 0.68 μg/ml, respectively. CBZ-E also inhibited high K⁺-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion (IC₅₀ = 0.3 μg/ml), but CBZ-D did not. These findings suggest that CBZ-E, but not CBZ-D, attenuates catecholamine secretion by inhibiting nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na⁺ channels and voltage-dependent Ca²⁺ channels in the cells. This inhibition of CBZ-E as well as CBZ may be related to the clinical effects in neuropsychiatric disorders. Received: 13 May 1997 /Final version: 4 August 1997     

人参皂甙Rb1降低细胞内Ca2+作用的机制

使用荧光探针Fura-2/AM,采用双波长荧光分光光度法观察到,人参皂甙Rb₁(10,50,100μmol·L^-1)能剂量依赖性减少新生鼠脑细胞内钙浓度,并能增加由硫酸亚铁及半胱氨酸所降低的膜流动性,Rb₁(10μmol·L^-1)能使离体大鼠尾动脉去甲肾上腺素量—效曲线右移,最大效应降低;Rb₁(10,100μmol·L^-1)能降低离体鼠基底动脉5-HT所引起的收缩。使用全细胞膜片钳技术发现人参皂甙Rb₁(50,100μmol·L^-1)对钙电流无明显影响;Rb₁在低剂量能增加大鼠突触体Na⁺-K⁺ATPase及Ca²⁺-Mg²⁺ATPase活性。从而揭示Rb₁降低胞内钙含量可能通过增加ATP酶活性而产生。     

Opposite effects of tolbutamide and diazoxide on 86Rb+ fluxes and membrane potential in pancreatic B cells

The effects of tolbutamide and diazoxide on ⁸⁶Rb⁺ fluxes, ⁴⁵Ca²⁺ uptake, insulin release and B cell membrane potential have been studied in rat or mouse islets. In the presence of 3 mM glucose, tolbutamide rapidly and reversibly decreased Rb⁺ efflux from perifused islets and depolarised B cells. The effect on Rb⁺ efflux was paradoxically more marked with 20 than 100 μg/ml tolbutamide, at least in the presence of extracellular calcium. Addition of tolbutamide to a medium containing 6 mM glucose and calcium increased Rb⁺ efflux transiently with 20 μg/ml and permanently with 100 μg/ml. The drug also inhibited Rb⁺ influx in islet cells, but had little effect on Rb⁺ net uptake. Diazoxide rapidly, steadily and reversibly increased Rb⁺ efflux in a dose-dependent manner (20–100 μg/ml). When 20 μg/ml tolbutamide and diazoxide were combined in the presence of 3mM glucose, only a slight decrease in Rb⁺ efflux was observed. The depolarisation of B cells normally produced by tolbutamide was markedly reduced and the electrical activity completely suppressed by diazoxide. In the presence of 10 mM glucose, diazoxide increased Rb⁺ efflux from the islets and hyperpolarised B cells. Tolbutamide, tetraethylammonium and quinine reversed the increase in Rb⁺ efflux, the inhibition of Ca²⁺ uptake and the suppression of insulin release produced by diazoxide. Tolbutamide rapidly reversed the hyperpolarisation and restored electrical activity. It is suggested that the stimulation and inhibition of insulin release by tolbutamide and diazoxide are due to their respective ability to decrease and to increase the K permeability of the B cell membrane. This change in K permeability leads either to depolarisation and stimulation of Ca²⁺ influx or to hyperpolarisation and inhibition of Ca²⁺ influx.