Acid secretagogues induce Ca2+ mobilization coupled to K+ conductance activation in rat parietal cells in tissue culture

Intracellular recordings from cultured parietal cells of the rat gastric fundus showed that carbachol, pentagastrin, histamine (in the presence of isobutylmethylxanthine; IBMX) and dibutyryl cyclic AMP induced hyperpolarizing responses which were sensitive to a K+ channel blocker, quinine. The Ca2+ ionophore, ionomycin, also induced a quinine-sensitive hyperpolarization. Deprivation of extracellular Ca2+ preferentially inhibited the hyperpolarizing responses to histamine (plus IBMX) and to dibutyryl cyclic AMP. Caffeine, oxalate and dantrolene sodium, which are known to affect Ca2+ transport in the endoplasmic reticulum, selectively inhibited the carbachol response. Mitochondrial inhibitors (KCN and carbonylcyanide p-trifluoromethoxyphenylhydrazone) preferentially suppressed the gastrin response. Cytosolic Ca2+ measurements with fura-2 indicated that significant increases in the intracellular concentration of free Ca2+ were induced not only by Ca2+-mediated acid secretagogues (carbachol and gastrin), but also by a cyclic AMP-mediated secretagogue (histamine plus IBMX). Dibutyryl cyclic AMP also increased cytosolic Ca2+ ions. It is concluded that stimulation of receptors to histamine, carbachol and gastrin gives rise to mobilization of Ca2+ ions into the cytoplasm from the different sources, thereby stimulating Ca2+-activated K+ channels in cultured rat parietal cells.     

Ion channels and regulation of intracellular calcium in vascular endothelial cells

Endothelial cells in vivo form an interface between flowing blood and vascular tissue, responding to humoral and physical stimuli to secrete relaxing and contracting factors that contribute to vascular homeostasis and tone. The activation of endothelial cell-surface receptors by vasoactive agents is coupled to an elevation in cytosolic Ca2+, which is caused by Ca2+ entry via ion channels in the plasma membrane and by Ca2+ release from intracellular stores. Ca2+ entry may occur via four different mechanisms: 1) a receptor-mediated channel coupled to second messengers; 2) a Ca2+ leak channel dependent on the electrochemical gradient for Ca2+; 3) a stretch-activated nonselective cation channel; and 4) internal Na+-dependent Ca2+ entry (Na+-Ca2+ exchange). The rate of Ca2+ entry through these ion pathways can be modulated by the resting membrane potential. Membrane potential may be regulated by at least two types of K channels: inwardly rectifying K channels activated upon hyperpolarization or shear stress; and a Ca2+-activated K channel activated upon depolarization, which may function to repolarize the agonist-stimulated endothelial cell. After agonist stimulation, cytosolic Ca2+ increases in a biphasic manner, with an initial peak due to inositol 1,4,5-trisphosphate-mediated Ca2+ release from intracellular stores, followed by a sustained plateau that is dependent on the presence of [Ca2+]o and on membrane potential. The delay in agonist-activated Ca2+ influx is consistent with the coupling of receptor activation to Ca2+ entry via a second messenger. Oscillations in [Ca2+]i, which may involve both Ca2+ entry and release, have been observed in isolated and confluent endothelial cell monolayers stimulated by histamine and bradykinin. Receptor-mediated Ca2+ entry, release, and refilling of intracellular stores follows a cycle that involves the plasma membrane.     

Prostaglandin Induces Ca2+ Influx and Cyclic GMP Formation in Mouse Neuroblastoma × Rat Glioma Hybrid NG108–15 Cells in Culture

Various prostaglandins (PGs) (10 nM-30 microM) were added to NG108-15 cells in culture, and changes in the levels of intracellular cyclic GMP and Ca2+ were investigated. Exposure of the cells to PGF2 alpha, PGD2, and PGE2 (10 microM) transiently increased the cyclic GMP content 7.5-, 3.9-, and 3.1-fold, respectively. Furthermore, the increased levels of cyclic GMP correlated well with the rise in cytosolic free Ca2+ concentrations induced by the PGs. Other PGs (10 microM), including metabolites and synthetic analogs, which had no effect on intracellular Ca2+, failed to increase the cyclic GMP content in the cells. When extracellular Ca2+ was depleted from the culture medium, the PG-induced increase in cyclic GMP level was almost completely abolished. In addition, treatment of the cells with quin 2 tetraacetoxymethyl ester dose-dependently inhibited the PG-induced cyclic GMP formation. The increase in cyclic GMP content caused by treatment of the cells with a high K+ level (50 mM) was completely blocked by voltage-dependent Ca2+ entry blockers, such as verapamil (10 microM), nifedipine (1 microM), and diltiazem (100 microM); however, the PG (10 microM)-induced increase in cyclic GMP content was not affected by such Ca2+ entry blockers. These findings indicate that PG-induced cyclic GMP formation may require the rise in intracellular Ca2+ level and that the voltage-dependent Ca2+ channels may not be involved in the PG-induced rise in Ca2+ content.     

The role of K+ in the regulation of the increase in intracellular Ca2+ mediated by the T lymphocyte antigen receptor

The regulation of the increase in intracellular calcium ([Ca2+]i) occurring in cytolytic T lymphocytes (CTLs) upon their interaction with antigen was examined. This [Ca2+]i increase and lytic function were insensitive to verapamil, a Ca channel blocker. An antigen-independent increase in [Ca2+]i was not induced by depolarization of CTLs with excess extracellular K+, suggesting that Ca2+ influx is not mediated by the ubiquitous voltage-gated Ca channel. The antigen-induced [Ca2+]i increase was inhibited by prior membrane hyperpolarization with valinomycin. Hyperpolarization occurred under normal circumstances in CTLs exposed to antigen-receptor-specific antibodies. This potential change was Ca2+-dependent and inhibited by K channel blockade. Conversely, K channel blockade augmented the antigen-specific [Ca2+]i increase while markedly decreasing the K+ efflux associated with CTL lytic function. Therefore, either membrane potential or intracellular K+ regulates the antigen-specific [Ca2+]i increase in CTLs.     

C1q induces chemotaxis and K+ conductance activation coupled to increased cytosolic Ca2+ in mouse fibroblasts

Cultured mouse fibroblasts (L cells) respond to whole C with a slow hyperpolarization. Among the C components tested, C1q was found to be most effective. In contrast, the cell did not respond to C1, in which the collagen-like region of the C1q molecule is masked. The C1q-induced hyperpolarizing response was inhibited by collagen or C1q-specific antisera. Human diploid skin fibroblasts (Flow 1,000 cells) also exhibited similar membrane potential changes in response to whole C or C1q. After repeated applications of C1q, the cell membrane became unresponsive (desensitized). The treatment of L cells with pronase E inhibited the C1q-induced response, whereas the response to ATP, which is known to interact to its own receptor, was still preserved. The reversal potential of C responses was close to the K+ equilibrium potential. The hyperpolarizing response was inhibited by a blocker of Ca2+-activated K+ channels in fibroblasts (quinine), by deprivation of extracellular Ca2+ or by a Ca2+ channel blocker (nifedipine). By means of Ca2+-selective microelectrodes, the cytosolic free Ca2+ concentration was found to increase from 126 to 206 nM upon stimulation of L cells with C1q. Using an agarose-well method, L cells were observed to migrate predominantly toward C1q or whole C. It is concluded that the fibroblasts have the C1q receptor sensitive to pronase E and that activation of C1q receptors gives rise to Ca2+ influx, triggering an increase in the cytosolic free Ca2+ ions, which in turn induces a hyperpolarizing response as a result of the stimulation of Ca2+-activated K+ channels and initiates chemotaxis to C1q.     

Depolarization of macrophage polykaryons in the absence of external sodium induces a cyclic stimulation of a calcium-activated potassium conductance

Macrophage polykaryons associated with the foreign body granuloma display several electrophysiological properties when studied with intracellular microelectrodes. One of the most evident properties is the slow hyperpolarization (2-5 s long, 10-60 mV amplitude), due to transient openings of Ca2+-dependent K+ channels, that is similar to those observed in macrophages. How this oscillation of membrane potential is triggered is not well known and the only way to repeatedly activate it under experimental control is through the intracellular injection of Ca2+. Although this technique is important for understanding the properties of the K+ channels, no information has been obtained about the way Ca2+ levels are raised and controlled in the cytosol. Slow hyperpolarizations can also be triggered by electrical stimulation, but reproducibility is low with cells bathed in physiological solutions. We then decided to investigate the effect of depolarization on the electrophysiological properties of macrophage polykaryons exposed to bathing solutions of several ionic compositions. We show in this paper that cell membrane depolarization induced by a long current pulse can trigger several patterns of membrane potential changes and that, in the absence of extracellular Na+, repetitive oscillations of decaying amplitudes are observed in almost all the cells. They are very similar to the slow hyperpolarizations, are dependent on the presence of extracellular Ca2+, and are blocked by quinine and D-600. Whole-cell patch clamp recording under voltage clamp conditions showed an outward current that oscillates and that also exhibits decaying amplitudes. The data presented here indicate that these oscillations are a consequence of the cyclic opening of the Ca2+-activated K+ channels and support the hypothesis that favors the participation of Ca2+ channels and of the Ca2+/Na+ exchange system in their triggering. These two mechanisms are not enough to explain either why the K+ channels close or why the membrane potential returns to the original level at the end of each cycle. The possibility of using these oscillations as a model to study the slow hyperpolarization is discussed.     

Inhibition of excessive neuronal apoptosis by the calcium antagonist amlodipine and antioxidants in cerebellar granule cells

Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age-induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage-sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10-100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N-methyl-D-aspartate receptor activation. Amlodipine also inhibited free radical-induced damage to lipid constituents of the membrane in a dose-dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose- and time-dependent manners by antioxidants (U-78439G, alpha-tocopherol, and melatonin), nitric oxide synthase inhibitors (N-nitro-L-arginine and N-nitro-D-arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage-sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.     

Receptor-mediated calcium influx in PC12 cells. ATP and bradykinin activate two independent pathways

In the neurosecretory cell line PC12 the cytosolic free Ca2+ concentration, [Ca2+]i, and membrane potential were affected by both external ATP and the nonapeptide bradykinin, BK. The latter caused a rapid and large release of Ca2+ from intracellular stores (Ca2+ redistribution) and, in the presence of external Ca2+, a long lasting, but moderate Ca2+ influx, which was insensitive to dihydropyridine blockers. On the contrary, ATP evoked a [Ca2+]i rise which rapidly inactivated. At least three different mechanisms accounted for the ATP-induced increase in [Ca2+]i: less than 20% of the total response was due to intracellular Ca2+ redistribution, consistent with a small increase in inositol 1,4,5-trisphosphate level; the rest (over 80%) was equally accounted for by ATP-activated cation channels and voltage-gated Ca2+ channels. ATP and BK (the latter after K+ channel blockade) caused plasma membrane depolarization. With both agonists the inward current was carried by both Na+ and Ca2+, although the BK-activated current appeared to be more selective for Ca2+. Channels triggered by ATP and BK differed not only in their cation selectivity, but also in modulation by both [Ca2+]i and drugs such as the phorbol ester phorbol 12-myristate 13-acetate and the new antagonist of ligand-activated Ca2+ influx, SK&F 96365.     

Estrogen and the Ca2+-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell

In this study, we have systematically evaluated the signaling mechanisms underlying stimulated nitric oxide (NO) synthesis by estrogen (E2) and other vasoactive agents at the level of a single endothelium-derived cell. To do so, we have characterized and contrasted rapid E2-evoked NO synthesis with that of ATP using single-cell microfluorimetry and patch-clamp recordings to monitor stimulated changes in cellular NO synthesis (via 4-amino-5-methylamino-2',7'-difluorofluorescein), Ca2+ transients (via Fluo-3), and membrane hyperpolarization in cultured human EA.hy926 cells. E2-evoked NO synthesis in single cells (EC50 approximately 0.3 nM) was blocked by the E2 receptor antagonist ICI 182,780 and the NO synthase inhibitor N(omega)-nitro-l-arginine methyl ester. Although both E2 and ATP stimulated comparable Ca2+ transients, E2-induced NO synthesis was insensitive to intracellular BAPTA-AM or removal of external Ca2+. In contrast, ATP-evoked NO production was abolished by either one of these treatments. ATP-evoked hyperpolarizations ( approximately 20 mV) and NO production were both inhibited by the respective small-conductance and intermediate-conductance calcium- activated K+ channel blockers apamin and charybdotoxin. E2 minimally affected membrane potential, and stimulated NO synthesis was insensitive to calcium-activated K+ channel blockers. Exposure to either the phosphatidylinositol 3-kinase inhibitor LY-294002 or the MAP kinase inhibitor PD-98059 abolished the NO response to E2, but not that to ATP. Finally, the NO response evoked by a combined stimulus of E2 plus ATP was similar to that of ATP alone. In conclusion, our data directly demonstrate that an individual human EA.hy926 cell contains at least two distinct mechanisms for stimulated NO synthesis that depend on either calcium or protein kinase signaling events.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.     

Intracellular calcium and the control of neuronal pacemaker activity

Pacemaker activity of the Aplysia bursting pacemaker neuron R-15 was analyzed. It was shown that the free intracellular Ca2+ concentration, as measured by arsenazo III, increases during the depolarizing phase of the pacemaker cycle and declines throughout the hyperpolarizing phase that follows. This increase in Ca2+ results from the activation of voltage-dependent Ca2+ channels that open during the depolarizing phase of the cycle. The extracellular K+ concentration also increases during the depolarizing phase of the cycle and is correlated with an outward K+ current that opposes the inward current carried by Ca2+ ions. The increase in internal Ca2+ is sufficient to activate a K+ conductance that depends on the magnitude of the change in internal Ca2+ and on membrane potential, which is responsible for the hyperpolarizing phase of the cycle. It is proposed that the membrane oscillation depends on three separate but linked systems, which include a voltage-dependent Ca2+ channel, the internal Ca2+ concentration, and a Ca2+-activated K+ channel.     

Thyrotropin-releasing hormone activates KCa channels in gastric smooth muscle cells via intracellular Ca2+ release

Thyrotropin-releasing hormone (TRH) is released in high concentrations into gastric juice, but its direct effect on gastric smooth muscles has not been studied yet. We undertook studies on TRH effect on gastric smooth muscle using contraction and patch clamp methods. TRH was found to inhibit both acetylcholine- and BaCl2-induced contractions of gastric strips. TRH, applied to single cells, inhibited the voltage-dependent Ca2+ currents and activated the whole-cell K+ currents. The TRH-induced changes in K+ currents and membrane potential were effectively abolished by inhibitors of either intracellular Ca2+ release channels or phospholipase C. Neither activators, nor blockers of protein kinase C could affect the action of TRH on K+ currents. In conclusion, TRH activates K+ channels via inositol-1,4,5-trisphosphate-induced release of Ca2+ in the direction to the plasma membrane, which in turn leads to stimulation of the Ca2+-sensitive K+ conductance, membrane hyperpolarization and relaxation. The data imply that TRH may act physiologically as a local modulator of gastric smooth muscle tone.     

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.     

Possible participation of calmodulin in stimulation of leucine transport by concanavalin A in human lymphocytes

Stimulation of leucine uptake by addition of concanavalin A, mediated by increase of intracellular free Ca2+ concentration [( Ca2+]), in lymphocytes (Mitsumoto, Y., Sato, K. and Mohri, T. (1988) Biochim. Biophys. Acta 968, 353-358) was abolished by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and chlorpromazine, which inhibited membrane hyperpolarization induced by the mitogen. Quinine (0.5-1 mM) completely inhibited the concanavalin A-induced hyperpolarization and extensively inhibited the induced stimulation of leucine uptake. Based on these results, we suggest that the stimulation of leucine uptake by concanavalin A is largely due to activation of the Ca2+-dependent K+ channel which reinforces negative potential of the plasma membrane and is regulated by calmodulin.     

Factors responsible for oscillations of membrane potential recorded with tight-seal-patch electrodes in mouse fibroblasts

Summary In giant fibroblastic L cells, penetration of a conventional microelectrode brought about marked decreases in the membrane potential and input resistance measured with a patch electrode under tight-seal whole-cell configuration, and repeated hyperpolarizations were often observed upon penetration. Therefore, the question arose whether such leakage artifact is a causal factor for generation of the membrane potential oscillation even in giant L cells. During whole-cell recordings, however, regular potential oscillations were observed in the cells that had not been impaled with a conventional microelectrode, as far as the Ca²⁺ buffer was not strong in the pipette solution. Oscillatory changes in the intracellular potential were detected by extracellular recordings with a tight-seal patch electrode in the cell-attached configuration. Thus, the potential oscillation occurs even in the absence of penetration-induced leakage or without rupture of the patch membrane. Withdrawal of a micropipette from one cell was often found to induce marked cell damage and elicit oscillatory hyperpolarizations in a neighboring cell with a certain time lag. The longer the distance between the injured and recorded cells, the greater was the time lag. Application of the cell lysate on the cell surface also gave rise to oscillatory hyperpolarizations. After repeated applications of the lysate, the membrane became unresponsive (desensitized), suggesting the involvement of receptors for the lysate factor. The lysates of different cell species (mouse lymphoma L5178Y cells or human epithelial Intestine 407 cells) produced similar effects. The effective component was heat stable and distinct from ATP. Lysate-induced hyperpolarizations were inhibited by deprivation of extracellular Ca²⁺ and by application of a Ca²⁺ channel blocker (nifedipine) or a K⁺ channel blocker (quinine) in the same manner as spontaneous oscillatory hyperpolarizations. It is concluded that the mouse fibroblast exhibits membrane potential oscillations, when the cell was activated, presumably via receptor systems, by some diffusible factors released from damaged cells.     

Cytosolic calcium and membrane conductance in response to platelet-derived growth factor and bradykinin stimulation in single human fibroblasts

Bradykinin (BK) and platelet-derived growth factor (PDGF) act as mitogens and stimulate phosphatidylinositol (PI) turnover in human fibroblasts. By coupling whole-cell electrophysiological measurements with cytosolic Ca2+ determinations using fura-2 microfluorimetry, we have studied the changes in cytosolic calcium and in membrane conductance in single cells following stimulation with BK or PDGF. Both agonists produce variable patterns of response which include: single transient, sustained pulsations, damped oscillations, no response. In all cases, there is a very good temporal correlation between increases in intracellular Ca2+ and membrane current. The cytosolic calcium elevation appears to be insensitive to membrane potential changes, indicating that Ca2+ is released from an intracellular source. The Ca2(+)-activated current is not blocked by 1 microM apamin or by 0.5 mM (+)-tubocurarine; it is instead strongly reduced by 5 mM tetraethylammonium (TEA). We can conclude that BK and PDGF induce very similar early responses in human fibroblasts, and that the variable pattern of response does not depend on the particular mitogen used. The membrane currents are due to a kind of Ca2(+)-activated K+ channels which, according to their voltage-dependence and specific blockers, belong to the "maxi K+" class.     

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Calcium-activated potassium channels in human platelets

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.     

Multiple acute effects on the membrane potential of PC12 cells produced by nerve growth factor (NGF)

We studied whether nerve growth factor (NGF) can affect the membrane potential and conductance of PC12 cells. We demonstrate that NGF depolarizes the membrane of PC12 cells within a minute and by using transfected NIH 3T3-Trk and -p75 cells we show that both the high affinity NGF receptor p140(trk) and the low affinity NGF receptor or p75(NGF) may be involved in the depolarization. Tyrosine kinase inhibitor, K252a, partially inhibited the depolarization, but two agents affecting intracellular calcium movements, Xestospongin C (XeC) and thapsigargin, did not. The early depolarization was eliminated in Na+ free solutions and under this condition, a 'prolonged' (> 2 min) hyperpolarization was observed in PC12 cells in response to NGF. This hyperpolarization was also induced in PC12 cells by epidermal growth factor (EGF). Voltage clamp experiments showed that NGF produced a late (> 2 min) increase in membrane conductance. The Ca2+-dependent BK-type channel blocker, iberiotoxin, and the general Ca2+-dependent K+ channel blocker, TEA, attenuated or eliminated the hyperpolarization produced by NGF in sodium free media. Under pretreatment with the non-selective cation channel blockers La3+ and Gd3+, NGF hyperpolarized the membrane of PC12 cells. These results suggest that three different currents are implicated in rapid NGF-induced membrane voltage changes, namely an acutely activated Na+ current, Ca2+-dependent potassium currents and non-selective cation currents.     

Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.