Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells

Neutrophil-like HL-60 cells reacted to N -formyl- l -Methionyl- l -Leucyl- l -P henylalanine (f MLP) with a rise in the intracellular calcium concentration ([Ca2]i), NADPH oxidase activation, and increased superoxide anion (O2-) production. [Ca2+]i mobilization and superoxide production were largely dependent on extracellular calcium (Ca2+]e) and a capacitative calcium entry. The monomeric G-protein, Rac-1, regulates NADPH oxidase activity. We tested the effect of removal of Ca2+]e on Rac-1 plasma membrane sequestration and activation of NADPH oxidase using immunodetection and a double labelling fluorescent method. Results showed that Rac-1 activation is mediated via a pertussis toxin (PTX)-sensitive heteromeric G-protein pathway, and that Rac-1 membrane sequestration was preceded by [Ca2+]i mobilization following entry of Ca2+ e. Therefore, we propose that O2- production is dependent on activation of PTX-sensitive G-proteins and sequestration of Rac-1 in the plasma membrane, following entry of Ca2+ e.     

Capacitative Ca2+ entry involves Ca2+ influx factor in rat glioma C6 cells.

Capacitative Ca2+ entry exists in rat glioma C6 cells; however, how the information of depletion of Ca2+ in intracellular stores transmits to the plasma membrane is unknown. In the present study, we examined whether Ca2+ influx factor (CIF) causes capacitative Ca2+ entry in C6 cells. CIF was extracted from non-treated (Non-CIF), bombesin-treated (BBS-CIF) and thapsigargin-treated (TG-CIF) C6 cells by a reverse-phase silica cartridge. The addition of BBS-CIF and TG-CIF gradually increased cytoplasmic Ca2+ concentration ([Ca2+]i) but Non-CIF did not increase [Ca2+]i. Neither BBS-CIF nor TG-CIF elevated [Ca2+]i in the absence of extracellular Ca2+. Gd3+ inhibited the increase in [Ca2+]i induced by BBS-CIF and TG-CIF. Genistein abolished an elevation of [Ca2+]i induced by BBS-CIF and TG-CIF. BBS-CIF and TG-CIF did not increase inositol 1,4,5-trisphosphate accumulation. The results suggest that capacitative Ca2+ entry is caused by CIF in rat glioma C6 cells.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

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.     

Dynamic regulation of [Ca2+]i by plasma membrane Ca(2+)-ATPase and Na+/Ca2+ exchange during capacitative Ca2+ entry in bovine vascular endothelial cells.

The dynamic regulation of Ca2+ extrusion by the plasma membrane Ca(2+)-ATPase (PMCA) and Na+/Ca2+ exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]i elicited by activation of capacitative Ca2+ entry (CCE) served to characterize kinetic parameters of these Ca2+ extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca(2+)- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]i has been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+ and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+ extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+ removal system. The contribution by the low-affinity NCX to Ca2+ clearance became apparent at [Ca2+]i > approximately 150 nM under conditions of submaximal activation of the PMCA.     

Bradykinin-induced Ca2+ entry, release, and refilling of intracellular Ca2+ stores. Relationships revealed by image analysis of individual human fibroblasts.

Lys-Bradykinin (BK), a mitogen for human foreskin fibroblasts (HSWP cells) (Owen, N. E., and Villereal, M. L. (1983) Cell 32, 979-985), elicits a rapid, transient elevation of intracellular free Ca2+ concentration ([Ca2+]i) in these cells. We have used image analysis of fura-2-loaded HSWP cells to examine the BK-induced [Ca2+]i changes in individual cells. BK-stimulated Ca2+ entry and release of intracellular Ca2+ stores can be distinguished by stimulating cells in the presence or absence of extracellular Ca2+, or by inhibiting Ca2+ entry with 5 mM NiCl2. BK-sensitive intracellular Ca2+ stores can be depleted by exposure of the cells to BK in Ca(2+)-free medium; refilling of the stores requires extracellular Ca2+. A component of BK-stimulated Ca2+ entry persists after removal of agonist, but inactivates with a t1/2 of approximately 5 min. Although previous studies have attributed the Ca2+ entry which persists after agonist removal to a "capacitative Ca2+ entry" pathway activated by the depletion of the intracellular Ca2+ stores, we find that a large component of this BK-stimulated Ca2+ entry is not due to capacitative Ca2+ entry since (1) ionomycin can deplete the BK-sensitive intracellular Ca2+ stores without appreciably stimulating Ca2+ entry and without inhibiting the BK-stimulated Ca2+ entry and (2) this Ca2+ entry pathway inactivates at a time when the Ca2+ pools are still empty and a capacitance entry pathway should still be open. On the other hand, refilling of the intracellular Ca2+ stores can occur after the noncapacitative Ca2+ entry component has inactivated or when it is inhibited by Ni2+; in these cases refilling occurs without a detectable elevation of [Ca2+]i suggesting that refilling of internal Ca2+ pools might occur by a capacitative route.     

Characterization of a 1,25(OH)2-vitamin D3-responsive capacitative Ca2+ entry pathway in rat osteoblast-like cells

We investigated the existence of a capacitative Ca2+ entry (CCE) pathway in ROS 17/2.8 osteoblast-like cells and its responsiveness to 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3]. Depletion of inner Ca2+ stores with thapsigargin or 1,25(OH)2D3 in the absence of extracellular Ca2+ transiently elevated cytosolic Ca2+ ([Ca2+]i); after recovery of basal values, Ca2+ re-addition to the medium markedly increased Ca2+ entry, reflecting pre-activation of a CCE pathway. Recovery of the Ca2+ overshoot that followed the induced CCE was mainly mediated by the plasma membrane Ca2+-ATPase. Addition of 1,25(OH)2D3 to the declining phase of the thapsigargin-induced CCE did not modify further [Ca2+]i, indicating that steroid activation of CCE was dependent on store depletion. Pre-treatment with 1 microM Gd3+ inhibited 30% both thapsigargin- and 1,25(OH)2D3-stimulated CCE, whereas 2.5 microM Gd3+ was required for maximal inhibition ( approximately 85%). The activated CCE was permeable to both Mn2+ and Sr2+. Mn2+ entry sensitivity to Gd3+ was the same as that of the CCE. However, 1-microM Gd3+ completely prevented capacitative Sr2+ influx, whereas subsequent Ca2+ re-addition was reduced only 30%. These results suggest that in ROS 17/2.8 cells CCE induced by thapsigargin or 1,25(OH)2D3 is contributed by at least two cation entry pathways: a Ca2+/Mn2+ permeable route insensitive to very low micromolar (1 microM) Gd3+ accounting for most of the CCE and a minor Ca2+/Sr2+/Mn2+ permeable route highly sensitive to 1 microM Gd3+. The Ca2+-mobilizing agonist ATP also stimulated CCE resembling the Ca2+/Sr2+/Mn2+ permeable entry activated by 1,25(OH)2D3. The data demonstrates for the first time, the presence of a hormone-responsive CCE pathway in an osteoblast cell model, raising the possibility that it could be an alternative Ca2+ influx route through which osteotropic agents influence osteoblast Ca2+ homeostasis. Copyright Wiley-Liss, Inc.     

The role of extracellular Ca2+ in the response of the hepatocyte to Ca2+-dependent hormones

The influence of extracellular Ca2+ on hormone-mediated increases of cytosolic free Ca2+ [( Ca2+]i) and phosphorylase activity was studied in isolated hepatocytes. In the presence of 1.3 mM extracellular Ca2+, the stimulation of phosphorylase activity produced by vasopressin or phenylephrine was maintained for 20-30 min. In contrast, the change in [Ca2+]i under these conditions was more transient and declined within 3-4 min to steady state values only 70 +/- 8 nM above the resting [Ca2+]i. Removal of the hormone from its receptor with specific antagonists caused a decline in [Ca2+]i back to the original resting values. Subsequent addition of a second hormone elicited a further Ca2+ transient. If the antagonist was omitted, the second hormone addition did not increase [Ca2+]i indicating that the labile intracellular Ca2+ pool remains depleted during receptor occupation. When extracellular Ca2+ was omitted, both the changes of [Ca2+]i and phosphorylase a caused by vasopressin were transient and returned exactly to resting values within 3-4 min. The subsequent readdition of Ca2+ to these cells produced a further increase of [Ca2+]i and phosphorylase activity which was larger than the changes observed upon Ca2+ addition to untreated cells. This reactivation of phosphorylase showed saturation kinetics with respect to extracellular [Ca2+], was maximally stimulated within 1 min of vasopressin addition and was inhibited by high concentration of diltiazem. We conclude that entry of extracellular Ca2+ into the cell is required in order to obtain a sustained hormonal stimulation of phosphorylase activity and is responsible for the maintenance of a small steady state elevation of [Ca2+]i.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [( Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.     

Changes in luminal flow rate modulate basal and bradykinin-stimulated cell [Ca2+] in aortic endothelium.

Hemodynamic forces influence many endothelial cell functions. The coupling between hemodynamic forces and cell function could be mediated by mechano-sensitive ion channels present in the plasma membrane of endothelial cells. Because one of these channels is permeable to Ca2+, we tested whether hemodynamic forces influence endothelial cell Ca2+ ([Ca2+]i). Bovine aortic endothelial cells were grown inside cylindrical glass tubes, loaded with fura-2, and perfused at different pressures and flow rates on the stage of a fluorescence microscope. Decreasing flow from 110 to 2 ml.min-1 raised [Ca2+]i from 57 +/- 11 to 186 +/- 29 nM (mean +/- SEM, p less than 0.01) by increasing the entry of extracellular Ca2+ into the cytoplasm. Increasing flow from 2 to 110 ml.min-1 transiently decreased [Ca2+]i from 62 +/- 3 to 33 +/- 5 nM (p less than 0.01) apparently due to reduced Ca2+ entry and concomitant extrusion by the plasma membrane Ca(2+)-ATPase. The rise in [Ca2+]i induced by bradykinin was magnified during a decrease in flow; in control cells, 10(-7) M bradykinin increased [Ca2+]i by 162 +/- 26 nM, whereas [Ca2+]i increased 350 +/- 67 nM (p less than 0.05) in cells previously exposed to 110 ml.min-1. These observations suggest that flow-induced changes in [Ca2+]i might be a signal-transduction mechanism for endothelial functions responsive to hemodynamic forces and may also modulate the magnitude of hormonally mediated increases in [Ca2+]i.     

Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells

It is generally thought that receptor-operated Ca2+ entry is related to store-operated or capacitative Ca2+ entry mechanism. Recent evidence suggests that non-capacitative Ca2+ entry pathways are also involved in receptor activated Ca2+ influx in many different kinds of cells. In this study, we studied whether alpha1-adrenoreceptor (alpha1-AR)-activated Ca2+ entry is coupled to both capacitative and non-capacitative pathways in A10 vascular smooth muscle cells by fura-2 fluorescence probe and conventional whole-cell patch clamp techniques. We found that both thapsigargin (TG) and phenylephrine (Phe) induced transient increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in Ca2+-free medium, and subsequent addition of Ca2+ evoked a sustained [Ca2+]i rise. When the membrane potential was held at -60 mV, both TG and Phe activated inward currents, which were inhibited by GdCl3(Gd3+), 0Na+/0Ca2+ solution and 1-{beta[3-(4-mehtoxyphenyl)propoxy]-4-methoxypheneth-yl}-1H- imidazole hydro-chloride (SK&F96365), but not by nifedipine. When Ca2+ store was depleted by TG in Ca2+-free solution, Phe failed to further evoke [Ca2+]i rise. However, when capacitative Ca2+ entry was activated by TG in the medium containing Ca2+, 10 microM Phe further increased [Ca2+]i. At the same concentration, TG activated an inward cation current, subsequent addition of Phe also further induced an inward cation current. Furthermore, the amplitudes of [Ca2+]i increase and current density induced by Phe in the presence of TG were less than that induced by Phe alone. Our results suggest that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca2+ influx induced by activation of alpha1-AR in A10 vascular smooth muscle cells.     

Dose-dependency in spatial dynamics of [Ca2+]c in adrenal chromaffin cells

The spatial dynamics of cytosolic Ca2+ concentration, [Ca2+]c, in guinea pig adrenal chromaffin cells was monitored by a digital image analysing technique using Fura-2. When a freshly isolated cluster of cells was stimulated with lower concentrations of carbachol (CCh; 0.3-1 microM), the [Ca2+]c began to increase in the region beneath the plasma membrane facing the extracellular environment. The [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCh at a higher concentration (100 microM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. These results are compatible with the view that the receptor activation with a physiological concentration of secretagogue accelerates Ca2+ entry, and that stimulation with a higher concentration of the secretagogue induces small transient Ca2+ release from intracellular stores and predominant continuous Ca2+ entry.     

Regulation of intracellular Ca2+ oscillation in AR42J cells.

Recordings of [Ca2+]i in single AR42J cells loaded with Fura 2 were used to study regulation of [Ca2+]i oscillation. Continuous stimulation with the cholecystokinin analogue, (t-butyloxycarbonyl-Tyr-(SO3)-norleucine-Gly-Trp-Nle-Asp-2-phenylethyl ester) or carbachol evoked long lasting oscillation in [Ca2+]i. Removal of CCK-JMV-180 after brief stimulation did not abruptly stop the oscillation. Rather, removal of CCK-JMV-180 resulted in time-dependent reduction in amplitude with little change in frequency of oscillation. The patterns of [Ca2+]i oscillation were affected by activation of protein kinase C and protein kinase A. However, down-regulation of protein kinase C activity did not prevent stimulation of [Ca2+]i oscillation. Hence, we conclude that an active protein kinase C pathway is not crucial for [Ca2+]i oscillation in this cell line. Variation in extracellular Ca2+ concentration (Ca2+out) was used to further characterize the oscillation. Reducing Ca2+out to approximately 10 microM resulted in a time dependent inhibition of [Ca2+]i oscillation. Subsequent step increases in Ca2+out up to 2-3 mM resulted in increased amplitude and frequency of oscillation. Further increase in Ca2+out or an increase in plasma membrane permeability to Ca2+, brought about by an increase in pHo, resulted in increased amplitude, decreased frequency, and modified shape of the [Ca2+]i spikes. These observations point to the existence of regulatory mechanisms controlling the duration of Ca2+ release and entry during [Ca2+]i oscillation.     

Evidence for electrogenic Na+/Ca2+ exchange in Limulus ventral photoreceptors

The Ca2+ indicator photoprotein, aequorin, was used to estimate and monitor intracellular Ca2+ levels in Limulus ventral photoreceptors during procedures designed to affect Na+/Ca2+ exchange. Dark levels of [Ca2+]i were estimated at 0.66 +/- 0.09 microM. Removal of extracellular Na+ caused [Ca2+]i to rise transiently from an estimated 0.5-0.6 microM in a typical cell to approximately 21 microM; [Ca2+]i approached a plateau level in 0-Na+ saline of approximately 5.5 microM; restoration of normal [Na+]o lowered [Ca2+]i to baseline with a time course of 1 log10 unit per 9 s. The apparent rate of Nao+-dependent [Ca2+]i decline decreased with decreasing [Ca2+]i. Reintroduction of Ca2+ to 0-Na+, 0-Ca2+ saline in a typical cell caused a transient rise in [Ca2+]i from an estimated 0.36 microM (or lower) to approximately 16.5 microM. This was followed by a decline in [Ca2+]i approaching a plateau of approximately 5 microM; subsequent removal of Cao2+ caused [Ca2+]i to decline slowly (1 log unit in approximately 110 s). Intracellular injection of Na+ in the absence of extracellular Na+ caused a transient rise in [Ca2+]i in the presence of normal [Ca2+]o; in 0-Ca2+ saline, however, no such rise in [Ca2+]i was detected. Under constant voltage clamp (-80 mV) inward currents were measured after the addition of Nao+ to 0-Na+ 0-Ca2+ saline and outward currents were measured after the addition of Cao2+ to 0-Na+ 0-Ca2+ saline. The results suggest the presence of an electrogenic Na+/Ca2+ exchange process in the plasma membrane of Limulus ventral photoreceptors that can operate in forward (Nao+-dependent Ca2+ extrusion) or reverse (Nai+-dependent Ca2+ influx) directions.     

Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.