Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration

A rapid, transient rise in the free cytosolic Ca2+ concentration ([Ca2+]i) is one of the earliest events in neutrophil activation and is assumed to be involved in many of the subsequent cellular reactions. Both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space contribute to the rise in [Ca2+]i. In an attempt to assess the relative importance of these pools and the sequences leading to the rise in [Ca2+]i, we have studied the time course of changes in [Ca2+]i after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet-activating factor (PAF) using the Ca2+ indicators quin-2 and fura-2. We observed a time lag of 1-3 s between stimulation and rise in [Ca2+]i. This lag depends on the agonist concentration but is independent of extracellular Ca2+. Thus Ca2+ release from intracellular stores is rate limiting for the rise in [Ca2+]i. After this, cation channels in the plasma membrane (measured with the patch clamp method) are opened. These non-selective channels, which also pass Ca2+, are activated by the initial rise in [Ca2+]i, but by neither fMLP nor inositol 1,4,5-trisphosphate (IP3) directly.     

Oscillations of intracellular Ca2+ in mammalian cardiac muscle

Contraction of cardiac muscle depends on a transient rise of intracellular calcium concentration ([Ca2+]i) which is initiated by the action potential. It has, however, also been suggested that [Ca2+]i can fluctuate in the absence of changes in membrane potential. The evidence for this is indirect and comes from observations of (1) fluctuations of contractile force in intact cells, (2) spontaneous cellular movements, and (3) spontaneous contractions in cells which have been skinned to remove the surface membrane. The fluctuations in force are particularly prominent when the cell is Ca2+-loaded, and have been attributed to a Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. In these conditions of Ca2+-loading the normal cardiac contraction is followed by an aftercontraction which has been attributed to the synchronization of the fluctuations. The rise of [Ca2+]i which is thought to underlie the aftercontraction also produces a transient inward current. This current, which probably results from a Ca2+-activated nonspecific cation conductance, has been implicated in the genesis of various cardiac arrhythmias. However, despite the potential importance of such fluctuations of [Ca2+]i their existence has, so far, only been inferred from tension measurements. Here we present direct measurements of such oscillations of [Ca2+]i.     

Quantification of Ca2+-activated K+ channels under hormonal control in pig pancreas acinar cells

Ca2+- and voltage-activated K+ channels are found in many electrically excitable cells and have an important role in regulating electrical activity. Recently, the large K+ channel has been found in the baso-lateral plasma membranes of salivary gland acinar cells, where it may be important in the regulation of salt transport. Using patch-clamp methods to record single-channel currents from excised fragments of baso-lateral acinar cell membranes in combination with current recordings from isolated single acinar cells and two- and three-cell clusters, we have now for the first time characterized the K+ channels quantitatively. In pig pancreatic acini there are 25-60 K+ channels per cell with a maximal single channel conductance of about 200 pS. We have quantified the relationship between internal ionized Ca2+ concentration [( Ca2+]i) membrane potential and open-state probability (p) of the K+ channel. By comparing curves obtained from excised patches relating membrane potential to p, at different levels of [Ca2+]i, with similar curves obtained from intact cells, [Ca2+]i in resting acinar cells was found to be between 10(-8) and 10(-7) M. In microelectrode experiments acetylcholine (ACh), gastrin-cholecystokinin (CCK) as well as bombesin peptides evoked Ca2+-dependent opening of the K+ conductance pathway, resulting in membrane hyperpolarization. The large K+ channel, which is under strict dual control by internal Ca2+ and voltage, may provide a crucial link between hormone-evoked increase in internal Ca2+ concentration and the resulting NaCl-rich fluid secretion.     

Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator

The physiological actions of Ca2+ as a trigger and second messenger depend on the maintenance of large inward resting Ca2+ gradients across the cell plasma membrane. An ATP-fuelled Ca-pump, originally discovered and still best characterized in human red cells, is now believed to mediate resting Ca2+ extrusion in most animal cells. However, even in red cells, the truly physiological pump-leak turnover rate and cytoplasmic free Ca2+ level are unknown. Previous estimates were only very imprecise upper limits because normal intact red cells have a minute total pool of exchangeable Ca of less than 1 mumol 1 cells; Ca fluxes could not be measured without artificially increasing that pool with ionophores or disrupting the membrane to incorporate Ca buffers. Both procedures leave the membrane considerably leakier than in intact cells. Here, we have increased the exchangeable Ca pool by non-disruptively loading a Ca-chelator into intact cells, using intracellular hydrolysis of a membrane-permeant ester. The trapped chelator made the free cytoplasmic calcium concentration, [Ca2+]i, an easily defined function of directly measurable total cell Ca. We were then able to establish the physiological steady-state [Ca2+]i and pump-leak turnover rate of fresh cells suspended in their own plasma. If [Ca2+]i was lowered below the normal resting level, the Ca pump rate decreased according to the square of [Ca2+]i, and the inward Ca leak increased. The increase in leak did not develop if the cells were depleted of ATP and ADP.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Phytohaemagglutinin activation of T cells through the sheep red blood cell receptor

Expression of receptors for sheep red blood cells and the ability to proliferate in response to phytohaemagglutinin (PHA) are the traditional properties of human T cells, but the function of the sheep red cell receptor (the T11 antigen) is controversial and the mechanism of PHA-induced mitogenesis unclear. Mitogenesis involves a complex series of cell-mediated and factor-dependent interactions, but a rise in intracellular free calcium concentration, [Ca2+]i, seems to be an important primary event in T-cell activation. We have now investigated the effects of three monoclonal antibodies, previously shown to inhibit mitogen-induced proliferation, on T-cell [Ca2+]i. We find that anti-LFA-2 and OKT11, which react with the sheep red cell receptor, have no effect on [Ca2+]i, nor do they inhibit the rise in [Ca2+]i induced by concanavalin A (Con A) or the mitogenic anti-T3 monoclonal antibody UCHT1 (ref. 11). They do, however, block PHA-induced Ca2+ mobilization. Anti-LFA-1, which reacts with the lymphocyte function-associated antigen, has no effect on intracellular Ca2+. These studies suggest that the sheep red blood cell receptor is an activation pathway for T cells and that the effects of PHA are mediated through this pathway.     

Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas.

Exocrine gland cells secrete Cl(-)-rich fluid when stimulated by neurotransmitters or hormones. This is generally ascribed to a rise in cytosolic Ca2+ concentration ([Ca2+]i), which leads to activation of Ca2(+)-dependent ion channels. A precise understanding of Cl- secretion from these cells has been hampered by a lack of knowledge about the spatial distribution of the Ca2+ signal and of the Ca2(+)-dependent ion channels in the secreting epithelial cells. We have now used the whole-cell patch-clamp method and digital imaging of [Ca2+]i to examine the response of rat pancreatic acinar cells to acetylcholine. We found a polarization of [Ca2+]i elevation and ion channel activation, and suggest that this comprises a novel 'push-pull' mechanism for unidirectional Cl- secretion. This mechanism would represent a role for cytosolic Ca2+ gradients in cellular function. The cytosolic [Ca2+]i gradients and oscillations of many other cells could have similar roles.     

Effects of ATP and vanadate on calcium efflux from barnacle muscle fibres

Calcium ions carry the inward current during depolarization of barnacle muscle fibres and are involved in the contraction process. Intracellular ionized calcium ([Ca2+]i) in barnacle muscle, as in other cells, is kept at a very low concentration, against a large electrochemical gradient. This large gradient is maintained by Ca2+ extrusion mechanisms. When [Ca2+]i is below the contraction threshold, Ca2+ efflux from giant barnacle muscle fibres is, largely, both ATP dependent and external Na+ (Na+0) dependent (see also refs 5,6). When [Ca2+]i is raised to the level expected during muscle contraction (2-5 muM), most of the Ca2+ efflux from perfused fibres is Na0 dependent; as in squid axons, this Na+0-dependent Ca2+ efflux is ATP independent. Orthovanadate is an inhibitor of (Na+ + K+) ATPase and the red cell Ca2+-ATpase. We report here that vanadate inhibits ATP-promoted, Na+0-dependent Ca2+ efflux from barnacle muscle fibres perfused with low [Ca2+]i (0.2-0.5 microM), but has little effect on the Na+0-dependent, ATP-independent Ca2+ efflux from fibres with a high [Ca]i (2-5 microM). Nevertheless, ATP depletion or vanadate treatment of high [Ca2+]i fibres causes an approximately 50-fold increase of Ca2+ efflux into Ca2+-containing lithium seawater. These results demonstrate that both vanadate and ATP affect Ca2+ extrusion, including the Na+0-dependent Ca2+ efflux (Na-Ca exchange), in barnacle muscle.     

Depletion of intracellular calcium stores activates a calcium current in mast cells.

In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.     

Ca2+ signals induced from calcium stores in pancreatic islet β cells

In single rat pancreatic β cells,using fura-2 microfluorometry to measure [Ca2+]i response upon different stimuli,the ways of calcium regulation have been studied.When the extracellular calcium concentration was 2.5 mmol/L,either 60 mmol/L KCl,20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]i.Such increase in [Ca2+]i was absent when the same stimuli were applied under zero extracellular calcium.These results indicate that the increase of [Ca2+]i is induced by the activation of voltage-dependent calcium channels in β cells.The manifold forms of [Ca2+]i change induced by glucose imply that the effects of glucose are complex.5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+]i ,which is independent of the external calcium,suggesting that [Ca2+]i can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in β cells.The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s).It is concluded that there are multiple calcium stores in rat pancreatic β cells.     

Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients

Calcium is transported across the surface membrane of both nerve and muscle by a Na+-dependent mechanism, usually termed the Na:Ca exchange. It is well established from experiments on rod outer segments that one net positive charge enters the cell for every Ca2+ ion extruded by the exchange, which is generally interpreted to imply an exchange stoichiometry of 3 Na+:1 Ca2+. We have measured the currents associated with the operation of the exchange in both forward and reversed modes in isolated rod outer segments and we find that the reversed mode, in which Ca2+ enters the cell in exchange for Na+, depends strongly on the presence of external K+. The ability of changes in external K+ concentration ([K+]o) to perturb the equilibrium level of [Ca2+]i indicates that K+ is co-transported with calcium. From an examination of the relative changes of [Ca2+]o, [Na+]o, [K+]o and membrane potential required to maintain the exchange at equilibrium, we conclude that the exchange stoichiometry is 4 Na+:1 Ca2+, 1 K+ and we propose that the exchange should be renamed the Na:Ca, K exchange. Harnessing the outward K+ gradient should allow the exchange to maintain a Ca2+ efflux down to levels of internal [Ca2+] that are considerably lower than would be possible with a 3 Na+:1 Ca2+ exchange.     

Antigen-dependent increase in cytosolic free calcium in specific human T-lymphocyte clones

Calcium has been implicated as an intracellular messenger in the cellular response to various external stimuli. Exposure of lymphocytes to various mitogens and lectins results in rapid transmembrane calcium fluxes and increased cytoplasmic calcium concentrations ([Ca2+]i). It is not clear, however, whether the mechanisms by which these non-physiological stimuli activate cells are related to those involved in antigen-specific activation. We have now used antigen-specific T-cell clones to study changes in [Ca2+]i associated with specific activation and show here that these cells respond specifically in the presence of antigen and antigen-presenting cells (APC) with increased [Ca2+]i and that this increased [Ca2+]i shows the same genetic restrictions as are seen in the proliferation assay. The kinetics of the [Ca2+]i response to antigen indicate that antigen undergoes a time-dependent processing step as a prerequisite for recognition by T cells, as has been shown for T-cell proliferative responses, but that the [Ca2+]i response to processed antigen is extremely rapid. The close correlation between changes in [Ca2+]i and cell activation resulting in proliferation suggests that Ca2+ may act as an intracellular messenger in antigen-specific responses.     

Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels

It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement

Cytosolic free calcium ([Ca2+]cyt) is a ubiquitous signalling component in plant cells. Numerous stimuli trigger sustained or transient elevations of [Ca2+]cyt that evoke downstream stimulus-specific responses. Generation of [Ca2+]cyt signals is effected through stimulus-induced opening of Ca2+-permeable ion channels that catalyse a flux of Ca2+ into the cytosol from extracellular or intracellular stores. Many classes of Ca2+ current have been characterized electrophysiologically in plant membranes. However, the identity of the ion channels that underlie these currents has until now remained obscure. Here we show that the TPC1 ('two-pore channel 1') gene of Arabidopsis thaliana encodes a class of Ca2+-dependent Ca2+-release channel that is known from numerous electrophysiological studies as the slow vacuolar channel. Slow vacuolar channels are ubiquitous in plant vacuoles, where they form the dominant conductance at micromolar [Ca2+]cyt. We show that a tpc1 knockout mutant lacks functional slow vacuolar channel activity and is defective in both abscisic acid-induced repression of germination and in the response of stomata to extracellular calcium. These studies unequivocally demonstrate a critical role of intracellular Ca2+-release channels in the physiological processes of plants.     

Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH

There is now good evidence that cytoplasmic pH (pHi) may have an important role in the metabolic activation of quiescent cells. In particular, growth stimulation of mammalian fibroblasts leads to a rapid increase in pHi (refs 3-6), due to activation of a Na+/H+ exchanger in the plasma membrane, and this alkalinization is necessary for the initiation of DNA synthesis. However, the mechanism by which mitogens activate the Na+/H+ exchanger to raise pHi is not known, although an increase in cytoplasmic free Ca2+ ([Ca2+]i) has been postulated as the primary trigger. We now present data suggesting that the Na+/H+ exchanger is set in motion through protein kinase C, a phospholipid- and Ca2+-dependent enzyme normally activated by diacylglycerol produced from inositol phospholipids in response to external stimuli. Using newly developed pH microelectrodes and fluorimetric techniques, we show that a tumour promoting phorbol ester and synthetic diacylglycerol, both potent activators of kinase C (refs 12-15), mimic the action of mitogens in rapidly elevating pHi in different cell types. Furthermore, we demonstrate that, contrary to previous views, an early rise in [Ca2+]i is not essential for the activation of Na+/H+ exchange and the resultant increase in pHi. Finally, we suggest that an alkaline pHi shift, mediated by Na+/H+ exchange, may be a common signal in the action of those hormones which elicit the breakdown of inositol phospholipids.     

Caffeine induces a transient inward current in cultured cardiac cells

Electrical excitation of cardiac muscle may sometimes be due to initiation of inward current by the presence of Ca2+ ions at the inner surface of the cell membrane. During digitalis toxicity and other conditions that abnormally augment cellular Ca2+ stores, premature release of Ca2+ from the sarcoplasmic reticulum leads to a transient inward current, which is large enough to initiate premature beats and is accompanied by a transient contractile response. This inward current may be mediated either by electrogenic sodium-calcium exchange or by specific Ca2+-activated cation channels that have recently been characterized in tissue cultures of cardiac myocytes. An obvious question raised by these observations is whether release of the sequestered Ca2+ stores during each normal beat exerts a similar influence on membrane potential. To explore this, chick embryonic myocardial cell aggregates were voltage-clamped during abrupt exposure to caffeine, which is known to release Ca2+ from the sarcoplasmic reticulum. The speed of the perfusion system and the relative absence of diffusion barriers in the tissue-cultured cells allowed the effects of caffeine-induced Ca2+ release to be studied on a time scale comparable to that of a single normal beat. We report here that abrupt exposure of the cells to caffeine produced a transient inward current having similar features to that of digitalis toxicity, and which was both large enough and rapid enough to potentially contribute to the action potential.     

Identification of Na-Ca exchange current in single cardiac myocytes

In cardiac muscle the exchange of intracellular Ca2+ for extracellular Na+ is an important transport mechanism for regulation of the intracellular free Ca2+ concentration [( Ca]i) and hence the contractile strength of the heart. Due to its stoichiometry of greater than or equal to 3:1 Na+/Ca2+ (refs 3,5), Na-Ca exchange is supposed to generate a current across the cell membrane. It is thought that such a current may contribute to cardiac action potential and physiological or pathological pacemaker activity. Although the occurrence of Na-Ca exchange is well documented, a membrane current generated by this transport has not been identified unequivocally. Previous attempts to detect such a current in multicellular preparations, for example, by measuring small current differences after varying the extracellular ionic composition, although providing evidence, did not rule out other possible interpretations. Here we demonstrate that a transient rise in [Ca]i caused by release of Ca from sarcoplasmic reticulum (SR) generates a membrane current in cardiac myocytes. The dependence of this current on the transmembrane gradients for Na+ and Ca2+ and on membrane potential meets the criteria for a current produced by electrogenic Na-Ca exchange. Cyclic activation of this current by release of Ca from the SR can cause maintained spontaneous activity, suggesting that Na-Ca exchange contributes to certain forms of cardiac pacemaking.     

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.     

Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes

Compelling evidence has existed for more than a decade for a sodium/calcium (Na-Ca) exchange mechanism in the surface membrane of mammalian heart muscle cells which exchanges about three sodium ions for each calcium ion. Although it is known that cardiac muscle contraction is regulated by a transient increase in intracellular calcium ([Ca2+]i) triggered by the action potential, the contribution of the Na-Ca exchanger to the [Ca2+]i transient and to calcium extrusion during rest is unclear. To clarify these questions, changes in [Ca2+]i were measured with indo-1 in single cardiac myocytes which were voltage clamped and dialysed with a physiological level of sodium. We find that Ca entry through the Na-Ca exchanger is too slow to affect markedly the rate of rise of the normal [Ca2+]i transient. On repolarization, Ca extrusion by the exchanger causes [Ca2+]i to decline with a time constant of 0.5 s at -80 mV. The rate of decline can be slowed e-fold with a 77-mV depolarization. Calcium extrusion by the exchanger can account for about 15% of the rate of decline of the [Ca2+]i transient (the remainder being calcium resequestration by the sarcoplasmic reticulum (SR]. The ability of the cell to extrude calcium was greatly reduced on inhibiting the exchanger by removing external sodium, which itself led to an increase in resting [Ca2+]i. This finding is in contrast to the suggestion that calcium extrusion at rest is mediated mainly by a sarcolemmal Ca-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)