Extracellular ATP triggers superoxide production in human neutrophils

The effects of ATP on the concentration of cytosolic calcium [( Ca2+]i) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system in human neutrophils. Addition of ATP to cytochalasin B-treated neutrophils resulted in two sequential increase in [Ca2+]i: an initial phase presumably related to the mobilization of Ca2+ from intracellular stores and a second phase dependent upon the presence of extracellular Ca2+. The second phase was associated with an increase in the rate of O2- production, which also required the presence of extracellular Ca2+. The results suggest that increased Ca2+ influx may act to trigger a cascade of Ca2+-sensitive events, leading to stimulated O2-production.     

Human neutrophils have a novel purinergic P2-type receptor linked to calcium mobilization

Stimulation of suspensions of fura-2-loaded human neutrophils with ATP resulted in an elevation in cytosolic free calcium concentration ([Ca2+]i) from a basal value of 0.1 microM to a transient peak of 1 microM. The response is due to Ca2+ release from intracellular stores and influx of extracellular Ca2+. Release from intracellular stores is shown by the response in the absence of extracellular Ca2+. The greater and more maintained response in the presence of extracellular Ca2+ is indicative of stimulated Ca2+ entry and a stimulated influx pathway was confirmed by using Mn2+ as a surrogate for Ca2+. A variety of purinergic agonists were used to characterize the pharmacology of this [Ca2+]i response. Their rank order of potency was ATP greater than adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) greater than ADP much greater than 2-methylthioadenosine 5'-triphosphate (2Me-SATP), where ATP had an EC50 value of 3 microM and 2MeSATP had an EC50 value of 1000 microM. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S), adenylyl (alpha,beta-methylene)- diphosphonate (AMPCPP) and adenosine were inactive at 1 mM. These results suggest that neutrophils have a novel type of purinergic P2 receptor that is neither P2x nor P2y.     

Endothelin evoked Ca2+ transients and oscillations in A10 vascular smooth muscle cells

Endothelin (200 nM) evoked a rapid rise in [Ca2+]i which was then followed by a maintained elevation of [Ca2+]i. The initial transient can be explained by the release of stored Ca2+ whilst the maintained plateau is likely to be an influx of Ca2+ as it was partially inhibited by nifedipine (5 microM) and the remaining component abolished by the removal of extracellular Ca2+. Vasopressin (1 nM) evoked a similar response which also showed a nifedipine insensitive component to it's plateau phase. Endothelin also evoked oscillations in [Ca2+]i; these where characterised by a rapid rising phase followed by a slower decline, with no 'pacemaker' rise in [Ca2+]i preceding the rising phase. The oscillations were inhibited by the addition of 5 microM nifedipine or the removal of extracellular Ca2+ suggesting they are at least in part dependent on voltage gated Ca2+ entry.     

Extracellular nucleotides mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms

We have studied the effects of extracellular nucleotides on the cytosolic free calcium concentration [( Ca2+]i) in J774 macrophages using quin2 and indo-1 as indicator dyes. Micromolar quantities of ATP induced a biphasic increase in [Ca2+]i: a rapid and transient increase (peak I) which was due to mobilization of Ca2+ from intracellular stores and a second more sustained elevation (peak II) due to influx of extracellular Ca2+. The sustained peak II elevation had two components, a "low threshold" (1 microM ATP) response which saturated at 10-50 microM ATP and a "high threshold" response, apparent at [ATP] greater than 100 microM. The latter component was not seen with nucleotides other than ATP and correlated with an ATP-induced generalized increase in plasma membrane permeability. A variant J774 cell line was isolated which does not demonstrate this ATP-induced increase in plasma membrane permeability; nevertheless, it demonstrated both the release of Ca2+ from intracellular stores and the low threshold component of the Ca2+ influx across the plasma membrane in response to nucleoside di- and triphosphates. Several lines of evidence indicate that the fully ionized (i.e. free acid) forms of nucleoside di- and triphosphates were the ligands that mediated these increases in [Ca2+]i. These data show that extracellular nucleotides mediate Ca2+ fluxes by two distinct mechanisms in J774 cells. In one, the rise in [Ca2+]i is due to release of Ca2+ from intracellular stores and Ca2+ influx across the plasma membrane. This response is elicited preferentially by the free acid forms of purine and pyrimidine nucleoside di- and triphosphates. In the other, the rise in [Ca2+]i reflects a more generalized increase in plasma membrane permeability and is elicited by ATP4- only.     

Calcium oscillations induced by lindane in peritoneal macrophages of mice: control by the maturation stage of the macrophage

Mouse resident peritoneal macrophages loaded with Fluo-3 were examined for changes in cytosolic calcium concentration ([Ca2+]i) after stimulation with gamma-hexachlorocyclohexane (Lindane or gamma-HCCH). These studies, realized on macrophage populations, or single cells, by digital imaging microscopy, sought to determine the role of calcium influx on cyclical changes according to maturation stages of macrophages. Single cell analysis of [Ca2+]i changes in macrophages, after gamma-HCCH exposure in 600 microM extracellular calcium, demonstrated that: 1) these [Ca2+]i variations were asynchronous oscillations with the same frequency (1.7 min-1), and 2) these [Ca2+]i variations in macrophages were not at the same [Ca2+]i level. This heterogeneity could be correlated to a cell size partition of the macrophage population (10.1 +/- 0.44 and 11.45 +/- 0.43 microns). In the presence of 100 microM calcium, gamma-HCCH induced a calcium influx into the two subpopulations, but the calcium oscillations appeared only in small macrophages. In the largest ones, [Ca2+]i slowly decreased back down to the basal level. The cell size variation could be correlated to a phenotypic heterogeneity, linked to the differenciation stage of the cell. Peroxydase activity showed that small macrophages were in fact exudate macrophages and the largest ones were resident macrophages. Inhibition of the oscillatory patterns by a decrease in the extracellular calcium concentration ([Ca2+]ext) or by lanthanum chloride (LaCl3) addition is indicative of the important role of calcium influx in the triggering of oscillations. The calcium influx was transient and induced inositol phosphate (InsP3) production in macrophages. The maintainance of these calcium oscillations depended on calcium mobilization from intracellular calcium stores by InsP3, since neomycin and 8-(diethylamino) octyl 3,4,5-trimethoxybenzoate (TMB-8) abolished the oscillations. gamma-HCCH induced a transient calcium entry which triggered phospholipase C activation and the associated [Ca2+]i oscillations. However, we showed that differences in cell responses were observed in relationship with the differentiation stage of the mouse peritoneal macrophages, and with the extracellular calcium concentration.     

Video imaging of cytosolic Ca2+ in pancreatic beta-cells stimulated by glucose, carbachol, and ATP.

In order to define the differences in the distribution of cytosolic free Ca2+ ([Ca2+]i) in pancreatic beta-cells stimulated with the fuel secretagogue glucose or the Ca(2+)-mobilizing agents carbachol and ATP, we applied digital video imaging to beta-cells loaded with fura-2.83% of the cells responded to glucose with an increase in [Ca2+]i after a latency of 117 +/- 24 s (mean +/- S.E., 85 cells). Of these cells, 16% showed slow wave oscillations (frequency 0.35/min). In order to assess the relationship between membrane potential and the distribution of the [Ca2+]i rise, digital image analysis and perforated patch-clamp methods were applied simultaneously. The system used allowed sufficient temporal resolution to visualize a subplasmalemmal Ca2+ transient due to a single glucose-induced action potential. Glucose could also elicit a slow depolarization which did not cause Ca2+ influx until the appearance of the first of a train of action potentials. [Ca2+]i rose progressively during spike firing. Inhibition of Ca2+ influx by EGTA abolished the glucose-induced rise in [Ca2+]i. In contrast, the peak amplitude of the [Ca2+]i response to carbachol was not significantly different in normal or in Ca(2+)-deprived medium. Occasionally, the increase of the [Ca2+]i rise was polarized to one area of the cell different from the subplasmalemmal rise caused by glucose. The amplitude of the response and the number of responding cells were significantly increased when carbachol was applied after the addition of high glucose (11.2 mM). ATP also raised [Ca2+]i and promoted both Ca2+ mobilization and Ca2+ influx. The intracellular distribution of [Ca2+]i was homogeneous during the onset of the response. A polarity in the [Ca2+]i distribution could be detected either in the descending phase of the peak or in subsequent peaks during [Ca2+]i oscillations caused by ATP. In the absence of extracellular Ca2+, the sequential application of ATP and carbachol revealed that carbachol was still able to raise [Ca2+]i after exhaustion of the ATP response. This may be due to desensitization to the former agonist, since the response occurred in the same area of the cell. These results reveal subtle differences in [Ca2+]i distribution following membrane depolarization with glucose or the application of Ca(2+)-mobilizing agonists.     

Selective activation of Ca2+ influx by extracellular ATP in a pancreatic beta-cell line (HIT)

The action of exogenous ATP on cytoplasmic free Ca2+ ([Ca2+]i) was studied in insulin secreting cells using fura-2. Stimulation of clonal pancreatic beta-cells (HIT) with ATP (range 2-20 microM) evoked a sustained elevation in [Ca2+]i. ATP selectively promoted Ca2+ influx and not Ca2+ mobilization since (1) the effect required external Ca1+ and (2) was observed in cells in which internal stores were depleted with ionomycin (3) the rate of Mn2+ influx, measured as the quenching of the fura-2 signal, was accelerated by ATP. The action of ATP was unaffected by the voltage-sensitive Ca2+ channel blockers nifedipine and verapamil as well as by a depolarizing concentration of K+. The effect on [Ca2+]i was highly specific for ATP since AMP, ADP, adenosine 5'-[gamma-thio]triphosphate, adenosine 5'-[beta, gamma-methylene]triphosphate, GTP and adenosine were ineffective. In normal pancreatic islet cells, both exogenous ATP (range 0.2-2 microM) and ADP induced a transient Ca2+ elevation that did not require external Ca2+. The nucleotide specificity of the effect on [Ca2+]i suggests that ATP activates P2 gamma purinergic receptors in normal beta-cells. Thus, ATP evokes a Ca2+ signal in clonal HIT cells and normal islet cells by different transducing systems involving distinct purinoreceptors. A novel mechanism for increasing [Ca2+]i by extracellular ATP is reported in HIT cells, since the nucleotide specificity and the selective activation of Ca2+ influx without mobilization of internal Ca2+ stores cannot be explained by mechanisms already described in other cell systems.     

Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells

Fura-2 fluorescence in single rat basophilic leukemia cells was monitored to study the rise in intracellular free ionized calcium ([Ca2+]i) produced by aggregation of immunoglobulin E receptors. Repetitive transient increases in [Ca2+]i were induced by antigen stimulation and were measured using digital video imaging microscopy at high time resolution. The [Ca2+]i oscillations were not dependent upon changes in the membrane potential of the cells and were observed in cells stimulated with antigen either with or without extracellular Ca2+. Transient oscillations in [Ca2+]i were also observed when calcium influx was blocked with La3+. These results suggested that during antigen stimulation of cells under normal physiological conditions, release of Ca2+ from intracellular stores makes an important contribution to the initial increase in [Ca2+]i. Oscillations in [Ca2+]i are not induced by elevating [Ca2+]i with the calcium ionophore ionomycin. Mitochondrial calcium buffering is not required for [Ca2+]i oscillations to occur. The results show that rat basophilic leukemia cells have significant stores of calcium and that release of calcium from these stores can participate in both the initial rise and the oscillations in [Ca2+]i.     

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.     

Studies on the increase in cytosolic free calcium induced by epidermal growth factor, serum, and nucleotides in individual A431 cells

The response of cytosolic calcium [Ca2+]i to epidermal growth factor (EGF), fetal calf serum, and nucleotides was determined in individual A431 cells, using the fluorescent probe fura-2 and quantitative digital video fluorescence microscopy. In the presence of 1 mM external Ca2+, EGF caused a rapid rise in [Ca2+]i, followed by a slower and variable decrease. The cells responded after a lag that varied from 10 to 30 seconds, and there was considerable cell-to-cell variation in extent of the rise in [Ca2+]i. A second challenge with EGF gave negative results. No response was obtained in nominally Ca2+-free medium supplemented with 100 microM EGTA. Somewhat similar results were obtained with fetal calf serum except that a rise in [Ca2+]i was observed both in the presence and absence of external Ca2+. The A431 cells responded to external ATP with a rise in [Ca2+]i in less than 10 seconds, both in Ca2+-containing and Ca2+-free media. A coverslip with attached cells was mounted on a small chamber, allowing complete change of medium in 2 seconds. A nearly full response was obtained with only 10 seconds of contact of cells with ATP-containing medium. After washing out ATP, there was little or no response to a second addition given 100 seconds after the first. However, a second response was obtained when the concentration of agonist was increased 10-20-fold. These data favor the idea of receptor desensitization. Both homologous and heterologous receptor desensitization was observed. A transient rise in [Ca2+]i was also noted with UTP, while ITP and CTP were inactive.     

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.     

Inositol tetrakisphosphate isomers and elevation of cytosolic Ca2+ in vasopressin-stimulated insulin-secreting RINm5F cells.

Signal generation during the stimulation of insulin secretion by arginine vasopressin (AVP) was investigated in RINm5F cells. AVP (0.1 microM) caused a biphasic cytosolic Ca2+ ([Ca2+]i) rise, namely a rapid transient marked elevation after stimulation followed by a series of oscillations. In the absence of extracellular Ca2+, the sustained oscillations were abolished, while the initial [Ca2+]i transient was only partly decreased, indicating that the former are due to Ca2+ influx and the latter due mainly to mobilization from internal Ca2+ stores. AVP also evoked a transient depolarization of the average membrane potential. AVP-induced Ca2+ influx during the sustained phase, which was strictly dependent on receptor occupancy, was attenuated by membrane hyperpolarization with diazoxide. However, blockade of Ca2+ channels of the L- or T-type was ineffective. AVP stimulated production of diacylglycerol and inositol phosphates; for the latter both [3H] inositol labeling and mass determinations were performed. A transient increase in Ins(1,4,5)P3 was followed by a marked enhancement of Ins(1,3,4,5)P4 (8-fold) peaking at 15 s and gradually returning to basal values. Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 exhibited the most long-lasting augmentation (4- and 1.7-fold, respectively), and therefore correlated best with the period of sustained [Ca2+]i oscillations. InsP5 and InsP6 were not elevated. The effects of AVP, including the stimulation of insulin secretion from perifused cells, were obliterated by a V1 receptor antagonist. In conclusion, AVP induces protracted [Ca2+]i elevation in RINm5F cells which is associated with long-lasting increases in InsP4 isomers. The accumulation of InsP4 isomers reflects receptor occupancy and accelerated metabolism of the inositol phosphates. Activation of second messenger-operated Ca2+ channels is not necessarily implicated because of the attenuating effect of membrane hyperpolarization.     

Receptor specific for certain nucleotides stimulates inositol phosphate metabolism and Ca2+ fluxes in A431 cells

We have recently reported that extracellular ATP induces a transient rise in cytosolic free Ca2+ [( Ca2+]i) in individual human epidermoid carcinoma A431 cells (Gonzalez et al: Journal of Cellular Physiology 135:269-276, 1988). We have now studied nucleotide specificity and desensitization for several early responses. Extracellular ATP (5-100 microM) caused the rapid formation of inositol trisphosphate and later its metabolites, inositol bisphosphate and inositol monophosphate. ATP also induced the efflux of 45Ca2+ from pre-loaded cells. In addition, an increase in the rate of influx of 45Ca2+ stimulated by extracellular ATP was detected. Based on measurements of 45Ca2+ efflux and influx, desensitization studies, and chlortetracycline fluorimetry, we conclude that ATP mobilizes Ca2+ from internal stores and also stimulates entry across the plasma membrane. These effects were also displayed by UTP and to a lesser extent by ITP, while other nucleoside triphosphates as well as ADP, AMP, and adenosine, were inactive. Furthermore, desensitization of the response to ATP and UTP was seen after prolonged exposure to either nucleotide. This was specific for the nucleotide receptor since a response to bradykinin was not affected by the ATP pretreatment, although pretreatment with phorbol ester inhibited responses to both the nucleotides and bradykinin. Quantitative data on rate of recovery from the desensitized state and the response of desensitized cells to greatly elevated levels of ATP are presented. Extracellular ATP stimulated another early change previously reported for epidermal growth factor, namely, the phosphorylation of an 81-kDa cytoskeletal protein. The stimulation of these events involves an ATP receptor whose properties differ from other ATP receptors that have been described.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium

Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.     

Extracellularly applied ATP alters the calcium flux through dihydropyridine-sensitive channels in cultured chick myotubes

Extracellularly applied ATP mediates a biphasic calcium signal in cultured chick myotubes. A rapid and transient increase in cytosolic calcium was independent of extracellular calcium while a second signal, slower in onset and decay, was absent without extracellular calcium. In depolarized myotubes, the cytosolic [Ca2+] was increased more than ten times above baseline level. Addition of ATP to the incubation medium immediately increased the rate of return of cytosolic Ca2+ levels to baseline. The ATP effect was half-maximal at about 10 microM ATP and was mimicked by ATP S. This ATP-sensitive calcium influx was also rapidly stopped by addition of dihydropyridines such as PN 200-110, suggesting that it is the voltage operated Ca2+-channel that was inactivated by ATP.     

Spatial and temporal characteristics of the increase in intracellular Ca2+ induced in cytotoxic T lymphocytes by cellular antigen

The increase in intracellular Ca2+ concentration [( Ca2+]i) in cytolytic T lymphocytes in response to target cell binding was investigated by ratio image fluorescence microscopy. Ca2+ mobilization from intracellular stores occurred at a site distal to target cell contact and was transient. Extracellular Ca2+ influx resulted in an increase in [Ca2+]i that was prolonged and distributed more proximal to the target cell. Oscillations in [Ca2+]i were observed after target cell contact, although the periodicity was dependent on the presence of extracellular Ca2+. The Ag-specific reorientation of cytoplasmic granules occurred well after [Ca2+]i had begun to decline to a resting level, but was dependent on extracellular Ca2+. These studies indicate that the Ag-stimulated increase in [Ca2+]i exhibits considerable spatial and temporal variation, and that these characteristics are altered by the availability of extracellular Ca2+. The results also suggest that these changes in [Ca2+]i may play a role in the cytoplasmic events that accompany T cell-mediated cytolysis.     

Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria

The effects of membrane potential on resting and bradykinin-stimulated changes in [Ca2+]i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic [Ca2+]i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient [Ca2+]i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in [Ca2+]i was determined by varying [K+]o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular [K+] (52 mV change/10-fold change in [K+]o). The observed decrease in resting and bradykinin-stimulated changes in [Ca2+]i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in [Ca2+]i in confluent endothelial cell monolayers. These [Ca2+]i oscillations between the plateau and resting [Ca2+]i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying [Ca2+]i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.