Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Extracellular ATP induces hyperpolarization and motility stimulation of ciliary cells.

Cellular membrane potential and ciliary motility were examined in tissues cultures prepared from frog palate and esophagus epithelia. Addition of micromolar concentrations of extracellular ATP caused membrane hyperpolarization and enhanced the beat frequency. These two effects of ATP were 1) dose dependent, reaching a maximum at 10 microM ATP; 2) dependent on the presence of extracellular Ca2+ or Mg2+; 3) insensitive to inhibitors of voltage-gated calcium channels; 4) abolished after depleting the intracellular Ca2+ stores with thapsigargin; 5) attenuated by quinidine (1 mM), Cs+ (5-20 mM), and replacement of extracellular Na+ by K+; 6) insensitive to charybdotoxin (5-20 nM), TEA (1-20 microM), and apamin (0.1-1 microM); 7) independent of initial membrane potential; and 8) unaffected by amiloride. In addition, extracellular ATP induced an appreciable rise in intracellular Ca2+. Addition of thapsigargin caused an initial enhancement of the ciliary beat frequency and membrane hyperpolarization. These results strongly suggest the involvement of calcium-dependent potassium channels in the response to ATP. The results show that moderate hyperpolarization is closely associated with a sustained enhancement of ciliary beating by extracellular ATP.     

Lymphocyte membrane potential and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements

A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (psi) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of psi than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte psi was calculated to be -70 mV. T and B lymphocytes maintain a constant psi as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (EK) is shown to result from Na+ permeability. At [K+]o values greater than 10 mM, lymphocytes behave as K+ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca2+-sensitive K+ channel by pharmacologic manipulations. Hence, T cells depolarized by 4-aminopyridine (4-AP, 10 mM) were observed to return to resting psi by A23187-induced elevation of [Ca2+]i. Cells depolarized by quinine (100 microM) were unaffected by A23187. The Ca2+-activated channel does not contribute to resting psi in T cells since it may be selectively blocked by quinine (20 microM) or modulated by calmodulin antagonists (5 microM trifluperazine) without affecting resting psi.     

Involvement of K(+)-channel opening in endothelin-1 induced suppression of spontaneous contractions in the guinea pig taenia coli.

Effects of porcine-human endothelin-1 on mechanical as well as electrical activities and on intracellular free Ca2+ levels in the guinea pig taenia coli were compared with those of nifedipine, a voltage-dependent Ca2+ channel blocker. Endothelin-1 (0.1-100 nM) caused a concentration-dependent suppression of spontaneous contractions but did not significantly affect the sustained contraction evoked by 40 mM KCl. However, nifedipine (0.1-100 nM) inhibited both types of contractions in a concentration-dependent manner. In electrophysiological studies, endothelin-1 (30 nM) or nifedipine (30 nM) eliminated spontaneous spike discharges. Endothelin-1 produced hyperpolarization, while nifedipine did not change the resting membrane potential. The endothelin-1 induced suppression of spontaneous contractions was dose-dependently antagonized by apamin (0.01-10 nM), an inhibitor of a small conductance Ca(2+)-dependent K+ channel, and D-tubocurarine (10-100 microM), an inhibitor of Ca(2+)-dependent K+ channel, but was unaffected by 4-aminopyridine (0.01-1 mM), an inhibitor of a voltage-dependent K+ channel. In the study with fura 2 excited at 340 nm, endothelin-1 abolished, from the tissue, the fluorescence signals that were coupled with spontaneous contraction. It is suggested that the inhibitory action of endothelin-1 on spontaneous contraction may be caused by hyperpolarization of the membrane that reduces the spontaneous generation of spike discharge coupled normally to an increase in the intracellular free Ca2+ levels in the guinea pig taenia coli. The hyperpolarization may be caused by activating apamin-sensitive Ca(2+)-dependent K+ channels.     

Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells

The effects of glucose, diazoxide, K+, and tolbutamide on the activity of K+ channels, membrane potential, and cytoplasmic free Ca2+ concentration were investigated in beta-cells from the Uppsala colony of obese hyperglycemic mice. With [K+]e = [K+]i = 146 mM, it was demonstrated that the dominating channel at the resting potential is a K+ channel with a single-channel conductance of about 65 picosiemens and a reversal potential of about +70 mV (pipette potential). This channel is characterized by complex kinetics with openings grouped in bursts. The channel was completely inhibited by 20 mM glucose in intact cells or by intracellularly applied Mg-ATP (1 mM). The number of active channels was markedly reduced already by 5 mM glucose. However, the single channel current of the channels remaining active was unaffected, indicating no major depolarization. To evoke a substantial depolarization of the membrane and thereby action potentials, a total block in channel activity was necessary. This could be achieved either by increasing the concentration of glucose to 20 mM or by combining 5 mM glucose with 100 microM tolbutamide. In both cases, the effect was counteracted by the hyperglycemic sulfonamide diazoxide. The effects on single channel activity were paralleled by changes in membrane potential and cytoplasmic free Ca2+ concentration, also when the latter measurements were performed at room temperature. The transient increase in the number of active channels and the resulting hyperpolarization observed after raising the glucose concentration to 20 mM probably reflected a drop in cytoplasmic ATP concentration. It is suggested that ATP works as a key regulator of the beta-cell membrane potential and thereby the opening of voltage-activated Ca2+ channels.     

K(Ca) channel blockers reveal hyperpolarization and relaxation to K+ in rat isolated mesenteric artery

Raising extracellular K+ concentration ([K+](o)) around mesenteric resistance arteries reverses depolarization and contraction to phenylephrine. As smooth muscle depolarizes and intracellular Ca(2+) and tension increase, this effect of K+ is suppressed, whereas efflux of cellular K+ through Ca(2+)-activated K+ (K(Ca)) channels is increased. We investigated whether K+ efflux through K(Ca) suppresses the action of exogenous K+ and whether it prestimulates smooth muscle Na(+)-K(+)-ATPase. Under isometric conditions, 10.8 mM [K+](o) had no effect on arteries contracted >10 mN, unless 100 nM iberiotoxin (IbTX), 100 nM charybdotoxin (ChTX), and/or 50 nM apamin were present. Simultaneous measurements of membrane potential and tension showed that phenylephrine depolarized and contracted arteries to -32.2 +/- 2.3 mV and 13.8 +/- 1.6 mN (n = 5) after blockade of K(Ca), but 10.8 mM K+ reversed fully the responses (107.6 +/- 8.6 and 98.8 +/- 0.6%, respectively). Under isobaric conditions and preconstriction with phenylephrine, 10.7 mM [K+](o) reversed contraction at both 50 mmHg (77.0 +/- 8.5%, n = 9) and 80 mmHg (83.7 +/- 5.5%, n = 5). However, in four additional vessels at 80 mmHg, raising K+ failed to reverse contraction unless ChTX was present. Increases in isometric and decreases in isobaric tension with phenylephrine were augmented by either ChTX or ouabain (100 microM), whereas neither inhibitor altered tension under resting conditions. Inhibition of cellular K+ efflux facilitates hyperpolarization and relaxation to exogenous K+, possibly by indirectly reducing the background activation of Na(+)-K(+)-ATPase.     

Properties of smooth muscle hyperpolarization and relaxation to K+ in the rat isolated mesenteric artery

Smooth muscle membrane potential and tension in rat isolated small mesenteric arteries (inner diameter 100-200 microm) were measured simultaneously to investigate whether the intensity of smooth muscle stimulation and the endothelium influence responses to exogenous K+. Variable smooth muscle depolarization and contraction were stimulated by titration with 0.1-10 microM phenylephrine. Raising external K+ to 10.8 mM evoked correlated, sustained hyperpolarization and relaxation, both of which were inhibited as the smooth muscle depolarized and contracted to around -38 mV and 10 mN, respectively. At these higher levels of stimulation, raising the K+ concentration to 13.8 mM still hyperpolarized and relaxed the smooth muscle. Relaxation to endothelium-derived hyperpolarizing factor, released by ACh, was not altered by the level of stimulation. In endothelium-denuded arteries, the concentration-relaxation curve to K+ was shifted to the right but was not depressed. In denuded arteries, relaxation to K+ was unaffected by the extent of prior stimulation and was blocked with 0.1 mM ouabain but not with 30 microM Ba2+. The ability of K+ to stimulate simultaneous hyperpolarization and relaxation in the mesenteric artery is consistent with a role as an endothelium-derived hyperpolarizing factor activating inwardly rectifying K+ channels on the endothelium and Na+-K+-ATPase on the smooth muscle cells.     

Mechanisms of coronary artery depolarization by uridine triphosphate

We sought to define the basic mechanisms by which pyrimidine nucleotides constrict rat coronary resistance arteries. Uridine triphosphate (UTP) caused a dose-dependent constriction in coronary arteries stripped of endothelium. UTP also depolarized and increased cytosolic Ca2+ in coronary smooth muscle cells. Nisoldipine, an antagonist of voltage-operated Ca2+ channels, blocked the rise in cytosolic Ca2+ and reduced UTP-induced vasoconstriction by approximately 75% which suggests a prominent role for depolarization in this constrictor response. The ionic basis of UTP-induced depolarization was subsequently explored in coronary smooth muscle cells using whole-cell patch-clamp electrophysiology. In the absence of K+ and with CsCl in the pipette, UTP (40 microM) activated a sustained inwardly rectifying current (-0.66 +/- 0.10 pA/pF at -60 mV). A 100 mM reduction in bath Na+ shifted the reversal potential of this current (from -2 +/- 1 to -28 +/- 4 mV) and reduced the magnitude (from -2.26 +/- 0.61 to -0.51 +/- 0.11 pA/pF). In addition to activating a depolarizing cation current, UTP inhibited hyperpolarizing outward currents. Specifically, UTP inhibited ATP-sensitive and voltage-dependent K+ currents yet had no effect on inwardly rectifying and Ca2+-activated K+ channels. This study indicates that electromechanical coupling is integral to pyrimidine-induced constriction in coronary resistance arteries.     

Ionic events induced by epidermal growth factor. Evidence that hyperpolarization and stimulated cation influx play a role in the stimulation of cell growth.

Charybdotoxin, a blocker of K+ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca2+ channels, were employed in mouse NIH-3T3 fibroblasts overexpressing the epidermal growth factor (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell growth. The [Ca2+]i response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (greater than 3 x 10(-8) M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na+ and Ca2+. Pretreatment with 10 microM SC38249 induced only minor changes of the intracellular Ca2+ release by EGF (the process responsible for the initial phase of the [Ca2+]i and membrane potential responses) and blocked the persistent, second phase [Ca2+]i and the hyperpolarization responses, both dependent on Ca2+ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and growth of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, growth stimulated by EGF was inhibited, moderately (-20%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca2+]i sustained by Ca2+ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Vasopressin-stimulated Ca2+ influx in rat hepatocytes is inhibited in high-K+ medium.

We examined the effects of K+ substitution for Na+ on the response of hepatocytes to vasopressin, and on the hepatocyte plasma-membrane potential. (1) High K+ (114 mM) had no effect on the initial increase in phosphorylase a activity in response to vasopressin, but abolished the ability of the hormone to maintain increased activity beyond 10 min. With increasing concentrations a decrease in the vasopressin response was first observed at 30-50 mM-K+. (2) High K+ (114 mM) had no effect on basal 45Ca2+ influx, but abolished the ability of vasopressin to stimulate influx. This effect was also first observed at a concentration of 30-50 mM-K+. (3) Increasing K+ had little effect on the plasma-membrane potential until a concentration of 40 mM was reached. With further increases in concentration the plasma membrane was progressively depolarized. (4) Replacement of Na+ with N-methyl-D-glucamine+ depolarized the plasma membrane to a much smaller extent than did replacement with K+, and was also much less effective in inhibiting the vasopressin response. (5) The plasma-membrane potential was restored to near the control value by resuspending cells in normal-K+ medium after exposure to high-K+ medium. The effects of vasopressin on phosphorylase activity were also restored. (6) We conclude that the Ca2+ channels responsible for vasopressin-stimulated Ca2+ influx are closed by depolarization of the plasma membrane.     

A depolarization can trigger Ca2+ uptake and the acrosome reaction when preceded by a hyperpolarization in L. pictus sea urchin sperm.

The acrosome reaction (AR) is an exocytotic event that allows sperm to recognize and fuse with the egg. In the sea urchin sperm this reaction is triggered by the outer investment of the egg, the jelly, which induces ionic movements leading to increases in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi), a K(+)-dependent transient hyperpolarization which may involve K+ channels, and a depolarization which depends on external Ca2+. The present paper explores the role of the hyperpolarization in the triggering of the acrosome reaction. The artificial hyperpolarization of Lytechinus pictus sperm with valinomycin in K(+)-free seawater raised the pHi, caused a small increase in 45Ca2+ uptake, and triggered some AR. When the cells were depolarized with KCl (30 mM) 40-60 sec after the induced hyperpolarization, the pHi decreased and there was a significant increase in 45Ca2+ uptake, [Ca2+]i, and the AR. This waiting time was necessary in order to allow the pHi change required for the AR to occur. Thus, the jelly-induced hyperpolarization may lead to the intracellular alkalinization required to trigger the AR, and, on its own or via pHi, may regulate Ca2+ transport systems involved in this process. Because of the key role played by K+ in the triggering of the AR, the presence and characteristics of ion channels in L. pictus isolated sperm plasma membranes are being explored. Planar lipid bilayers into which these membranes were incorporated by fusion displayed 85 pS single channel transitions which were cation selective.     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

Modulation of the activity of Ca(2+)-activated K+ channels by internal Mg2+ in cultured kidney cells vero.

The inside-out mode of the patch-clamp method was used to study the effects of internal Mg2+ on single large-conductance (193+/-7 pS) Ca(2+)-activated K+ channels in cultured kidney cells. In the absence of Ca2+, Mg2+ (1 to 10 mM) did not activate the channels but modified the activating effect of Ca2+ ions: it decreased the Hill coefficient (n), reduced the apparent dissociation constant (K0.5), and modified the channel open and closed times. K0.5 was found to be a voltage-dependent parameter. In the absence of Mg2+, it averaged 600 microM at -20 mV and 27 microM at +30 mV (22 degrees C, pH 6.8). Mg2+ at saturating concentrations (5 to 10 mM) decreased K0.5 to 50 microM at -20 mV and to 15 microM at +30 mV. Irrespective of the membrane potential, K0.5 tended to its limit value of about 12.6 microM. Thus, the effects of membrane depolarization and Mg2+ exhibited a non-additive, competitive relationship. Mg2+ perturbed the exponential shape of the voltage dependences of K0.5. The Hill coefficient characterizing the interaction of Ca2+ ions with the channels was found to be voltage-dependent. In the absence of Mg2+, it increased rather sharply from approx. 2 to 3.5 when the membrane potential was raised from -10 to 0 mV. Mg2+ increased n in a dose-dependent manner; however, about a twofold increase of n occurred within a narrow concentration range (2 to 3 mM). The action of Mg2+ on n was, apparently, voltage-independent, and the effects of Mg2+ and voltage on n were seemingly additive.     

Dynamics of ionic activities in the apoplast of the sub-stomatal cavity of intact Vicia faba leaves during stomatal closure evoked by ABA and darkness

Stomatal movement is accomplished by changes in the ionic content within guard cells as well as in the cell wall of the surrounding stomatal pore. In this study, the sub-stomatal apoplastic activities of K+, Cl-, Ca2+ and H+ were continuously monitored by inserting ion-selective micro-electrodes through the open stomata of intact Vicia faba leaves. In light-adapted leaves, the mean activities were 2.59 mM (K+), 1.26 mM (Cl-), 64 microM (Ca2+) and 89 microM (H+). Stomatal closure was investigated through exposure to abscisic acid (ABA), sudden darkness or both. Feeding the leaves with ABA through the cut petiole initially resulted in peaks after 9-10 min, in which Ca2+ and H+ activities transiently decreased, and Cl- and K+ activities transiently increased. Thereafter, Ca2+, H+ and Cl- activities completely recovered, while K+ activity approached an elevated level of around 10 mM within 20 min. Similar responses were observed following sudden darkness, with the difference that Cl- and Ca2+ activities recovered more slowly. Addition of ABA to dark-adapted leaves evoked responses of Cl- and Ca2+ similar to those observed in the light. K+ activity, starting from its elevated level, responded to ABA with a transient increase peaking around 16 mM, but then returned to its dark level. During stomatal closure, membrane potential changes in mesophyll cells showed no correlation with the K+ kinetics in the sub-stomatal cavity. We thus conclude that the increase in K+ activity mainly resulted from K+ release by the guard cells, indicating apoplastic compartmentation. Based on the close correlation between Cl- and Ca2+ changes, we suggest that anion channels are activated by a rise in cytosolic free Ca2+, a process which activates depolarization-activated K+ release channels.     

Transient and sustained effects of hormones and calcium on membrane potential in a bone cell clone

Measurements were made of the electrophysiological and cAMP response to changes in extracellular [Ca2+] and to hormone application in a bone cell clone. Both transient and long-term electrophysiological responses were studied. An increase in extracellular [Ca2+] usually resulted in a transient hyperpolarization of about 60-sec duration. In addition, increases in extracellular [Ca2+] from 0.9 to 1.8 mM and from 1.8 to 3.6 mM resulted in long-term hyperpolarization and increased potential fluctuations. Increasing bathing [Ca2+] until the membrane potential reached the K+ equilibrium level resulted in a significant decrease in fluctuations. Addition to the bathing medium of quinine, a putative blocker of the Ca2+-dependent K+ channel, resulted in long-term depolarization of the mean membrane potential, and a long-term decrease in potential fluctuations. Addition of Mg2+, a mild antagonist of Ca2+ entry into the cell, produced transient depolarization and reduction of potential fluctuations. These effects suggest that the potential fluctuations reflect cytoplasmic [Ca2+] fluctuations via Ca2+-dependent K+ membrane channels. Under an extracellular [Ca2+] of 1.8 mM, the application of prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone produced no significant effect on mean membrane potential or on the sustained potential fluctuations, but PGE2 did significantly raise intracellular cAMP. Under an increased bathing [Ca2+], significant changes in mean potential and fluctuations did occur in response to PGE2, but not in response to the other hormones, while the PGE2 effect on cAMP was not greatly changed. Hyperpolarizing transients of about 30-sec duration occurred in response to all of the hormones, particularly at an extracellular [Ca2+] of 3.6 mM. Thus, there are both transient and long-term electrophysiological responses to hormone application, with only the long-term response correlated with the production of cAMP. These electrophysiological responses may represent separate transient and long-term calcium transport responses to hormone application.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Hyperpolarization contributes to vascular hyporeactivity in rats with lipopolysaccharide-induced endotoxic shock

We have examined the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) in endothelial-denuded strips of rat thoracic aorta ex vivo. The injection of rats with LPS caused a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine. The membrane potential recording showed that endotoxemia caused a hyperpolarization when compared to the control. This hyperpolarization was fully restored by methylene blue (MB; 10 microM) and partially reversed by Nomega-nitro-L-arginine methyl ester (L-NAME; 0.3 mM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), tetraethylammonium (TEA; 10 mM), charybdotoxin (CTX; 0.1 microM), or glibenclamide (GB; 10 microM), however, this hyperpolarization was not significantly affected by apamin (0.1 microM), 4-aminopyridine (4-AP; 1 mM), or Ba2+ (50 microM). In addition, the basal tension of the tissues obtained from endotoxemic rats was enhanced by the following order: MB > or = ODQ > TEA > or = L-NAME > or = CTX > GB; whereas apamin, 4-AP or Ba2+ had no significant effects on these tissues. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Our electrophysiological results further confirmed previous studies showing that in addition to nitric oxide, the large conductance Ca2+-activated K+-channels and ATP-sensitive K+-channels are, most likely, responsible for endotoxin-mediated hyporeactivity to vasoconstrictor agents in vascular smooth muscle.     

Role of Hyperpolarization Attained by Linoleic Acid in Chick Myoblast Fusion

Our previous report has suggested that hyperpolarization generated by reciprocal activation of calcium-activated potassium (K(Ca)) channels and stretch-activated channels induces calcium influx that triggers myoblast fusion. Here we show that linoleic acid is involved in the process of generating hyperpolarization in cultured chick myoblasts and hence in promotion of the cell fusion. Linoleic acid dramatically hyperpolarized the membrane potential from -14 +/- 3 to -58 +/- 5 mV within 10 min. This effect was partially blocked by 1 mM tetraethylammonium (TEA) or 30 nM charybdotoxin, a selective K(Ca) channel inhibitor, and completely abolished by 10 mM TEA. Single-channel recordings revealed that linoleic acid activates TEA-resistant potassium channels as well as K(Ca) channels. Furthermore, linoleic acid induced calcium influx from extracellular solution, and this effect was partially blocked by 1 mM TEA and completely prevented at 10 mM, similar to the effect of TEA on linoleic acid-mediated hyperpolarization. Since the valinomycin-mediated hyperpolarization promoted calcium influx, hyperpolarization itself appears capable of inducing calcium influx. In addition, gadolinium prevented the valinomycin-mediated increase in intracellular calcium level under hypotonic conditions, revealing the involvement of stretch-activated channels in calcium influx. Furthermore, linoleic acid stimulated myoblast fusion, and this stimulatory effect could completely be prevented by 10 mM TEA. These results suggest that linoleic acid induces hyperpolarization of membrane potential by activation of potassium channels, which induces calcium influx through stretch-activated channels, and thereby triggers myoblast fusion.