Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.     

Glucagon and vasopressin interactions on Ca2+ movements in isolated hepatocytes.

The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.     

Calcium gradients in conifer pollen tubes; dynamic properties differ from those seen in angiosperms

Pollen tubes are an established model system for examining polarized cell growth. The focus here is on pollen tubes of the conifer Norway spruce (Picea abies, Pinaceae); examining the relationship between cytosolic free Ca2+, tip elongation, and intracellular motility. Conifer pollen tubes show important differences from their angiosperm counterparts; they grow more slowly and their organelles move in an unusual fountain pattern, as opposed to reverse fountain, in the tip. Ratiometric ion imaging of growing pollen tubes, microinjected with fura-2-dextran, reveals a tip-focused [Ca2+]i gradient extending from 450 nM at the extreme apex to 225 nM at the base of the tip clear zone. Injection of 5,5' dibromo-BAPTA does not dissipate the apical gradient, but stops cell elongation and uniquely causes rapid, transient increases of apical free Ca2+. The [Ca2+]i gradient is, however, dissipated by reversible perfusion of extracellular caffeine. When the basal cytosolic free Ca2+ concentration falls below 150 nM, again a large increase in apical [Ca2+]i occurs. An external source of calcium is not required for germination but significantly enhances elongation. However, both germination and elongation are significantly inhibited by the inclusion of calcium channels blockers, including lanthanum, gadolinium, or verapamil. Modulation of intracellular calcium also affects organelle position and motility. Extracellular perfusion of lanthanides reversibly depletes the apical [Ca2+]i gradient, altering organelle positioning in the tip. Later, during recovery from lanthanide perfusion, organelle motility switches direction to a reverse fountain. When taken together these data show a unique interplay in Picea abies pollen tubes between intracellular calcium and the motile processes controlling cellular organization.     

Agonist-dependent patterns of cytosolic Ca2+ changes in single bovine adrenal chromaffin cells: relationship to catecholamine release.

The patterns of agonist-induced elevations of cytosolic free Ca2+ ([Ca2+]i) were characterized and compared by the use of single adrenal chromaffin cells. Initial histamine- or angiotensin II (AII)-induced elevations of [Ca2+]i were equal in magnitude (peaks 329 +/- 20 [SE] and 338 +/- 46 nM, respectively). These initial increases of [Ca2+]i were transient, insensitive to either Gd3+ or removing external Ca2+, and were primarily the result of Ca2+ release from intracellular stores. After the initial peak(s) of [Ca2+]i, a second phase of moderately elevated [Ca2+]i was observed, and this response was sensitive to either Gd3+ or removing external Ca2+, supporting a role for Ca2+ entry. In most cases, the second phase of elevated [Ca2+]i was sustained during histamine stimulation but transient during AII stimulation. Maintenance of the second phase was a property of the agonist rather than of the particular cell being stimulated. Thus, individual cells exposed sequentially to histamine and AII displayed distinct patterns of [Ca2+]i changes to each agonist, regardless of the order of addition. Histamine also stimulated twice as much [3H]catecholamine release as AII, and release was completely dependent on external Ca2+. Therefore, the ability of histamine and AII to sustain (or promote) Ca2+ entry appears to underlie their efficacy as secretagogues. These data provide evidence linking agonist-dependent patterns of [Ca2+]i changes in single cells with agonist-dependent functional responses.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa

Previous work has shown that hyphal elongation in the fungus Neurospora crassa requires a tip-high cytosolic Ca2+ gradient. The source of the Ca2+ appears to be intracellular stores as there is no net transplasma membrane Ca2+ flux at the elongating hyphal tip and modification of ion fluxes across the plasma membrane using voltage clamp is without effect on tip growth. To decode the internal mechanisms which generate and maintain the tip-high Ca2+ gradient we first identified calcium regulators which affect hyphal growth and morphology, then determined how they modify cytosolic [Ca2+] and the actin cytoskeleton using fluorescent dyes and confocal microscopy. Cyclopiazonic acid (a known inhibitor of the endoplasmic reticulum calcium ATPase) inhibits growth and increases cytoplasmic [Ca2+] in the basal region 10-25 microm behind the hyphal tip. 2-APB (2-aminoethoxydiphenyl borate, an inhibitor of IP3-induced Ca2+ release) inhibits hyphal elongation and dissipates the tip-high Ca2 gradient 0-10 microm from the tip. Microinjections of the IP3 receptor agonists adenophostin A and IP3 (but not control microinjections of the biologically inactive L-IP3) transiently inhibited growth and induced subapical branches. IP3 microinjections, but not L-IP3, lowered tip-localized [Ca2+] and increased basal [Ca2+]. Even though their effect on [Ca2+] gradients was different, both cyclopiazonic acid and 2-APB disrupted similarly the normal actin pattern at the hyphal apex. Conversely, disruption of actin with latrunculin B dissipated tip-localized Ca2+. We conclude that the tip-high Ca2+ gradient is generated internally by Ca2+ sequestration into endoplasmic reticulum behind the tip and Ca2+ release via an IP3 receptor from tip-localized vesicles whose location is maintained by the actin cytoskeleton.     

Elevation of the cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal in alfalfa.

In root hairs of alfalfa (Medicago sativa), the requirement of Ca(2+) for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 microm behind the growing tip, 0.1 microM NodRm-IV(C16:2,S) increased the cytosolic free [Ca2+] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca(2+) concentrations below cytosolic values if the free EGTA concentration remained low (相似文献   

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.     

Modulation by external Ca2+ and nicardipine of Ca2+ influx and cytosolic concentration in human erythrocytes

Variations of Ca2+ influx (evaluated by the initial rate of 45Ca2+ uptake) and cytosolic free Ca2+ concentration ([Ca2+]i, measured with fura-2) were investigated in human erythrocytes. When external Ca2+ concentration ([Ca2+]o) rose from 1 to 2 mM, the initial rate of Ca2+ influx nearly doubled whereas [Ca2+]i increased only by 15%. Nicardipine dose-dependently decreased both initial rate of Ca2+ influx and [Ca2+]i (up to 53 and 18%. respectively at 10(-6) M). The less marked changes in [Ca2+]i than in Ca2+ influx indicate a partial adjustment of the Ca2+ extruding-pump activity to of Ca2+ influx. In vivo administration of nicardipine reduced [Ca2+]i only when its initial value exceeded 80 nM and prevented the rise in [Ca2+]i induced by the increase in [Ca2+]o. Our results indicate that nicardipine may reduce Ca2+ influx in human erythrocytes and participate in the control of [Ca2+]i when elevated.     

Imaging of the Yellow Cameleon 3.6 indicator reveals that elevations in cytosolic Ca2+ follow oscillating increases in growth in root hairs of Arabidopsis

In tip-growing cells, the tip-high Ca(2+) gradient is thought to regulate the activity of components of the growth machinery, including the cytoskeleton, Ca(2+)-dependent regulatory proteins, and the secretory apparatus. In pollen tubes, both the Ca(2+) gradient and cell elongation show oscillatory behavior, reinforcing the link between the two. We report that in growing root hairs of Arabidopsis (Arabidopsis thaliana), an oscillating tip-focused Ca(2+) gradient can be resolved through imaging of a cytosolically expressed Yellow Cameleon 3.6 fluorescence resonance energy transfer-based Ca(2+) sensor. Both elongation of the root hairs and the associated tip-focused Ca(2+) gradient show a similar dynamic character, oscillating with a frequency of 2 to 4 min(-1). Cross-correlation analysis indicates that the Ca(2+) oscillations lag the growth oscillations by 5.3 +/- 0.3 s. However, growth never completely stops, even during the slow cycle of an oscillation, and the concomitant tip Ca(2+) level is always slightly elevated compared with the resting Ca(2+) concentration along the distal shaft, behind the growing tip. Artificially increasing Ca(2+) using the Ca(2+) ionophore A23187 leads to immediate cessation of elongation and thickening of the apical cell wall. In contrast, dissipating the Ca(2+) gradient using either the Ca(2+) channel blocker La(3+) or the Ca(2+) chelator EGTA is accompanied by an increase in the rate of cell expansion and eventual bursting of the root hair tip. These observations are consistent with a model in which the maximal oscillatory increase in cytosolic Ca(2+) is triggered by cell expansion associated with tip growth and plays a role in the subsequent restriction of growth.     

Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration: studies on Ca2+ channels in rat parotid cells

1. Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration [( Ca2+]i)were studied using quin2. 2. Nicardipine (NIC), diltiazem (DIL) and verapamil (VER) had no effect on the rise in [Ca2+]i evoked by carbachol. Methoxamine-elevated [Ca2+]i was inhibited by VER but not by NIC and DIL. 3. All Ca2+ antagonists tested produced a decline of [Ca2+]i elevated by isoproterenol to the resting level. 4. The addition of 30 mM K+ gradually elevated [Ca2+]i in normal and Ca2+-free media, but it did not increase 45Ca2+ uptake into cells. BAY K 8644 did not increase [Ca2+]i. 5. We suggest that voltage-sensitive Ca2+ channels are lacking and that at least 2 distinct receptor-operated Ca2+ channels exist in rat parotid cells.     

Recording of intracellular Ca2+ from smooth muscle cells by sub-micron tip, double-barrelled CA2+-selective microelectrodes

Novel, double-barrelled Ca2+-selective microelectrodes with tip diameters of approximately 0.1 micron were constructed by using Simon's neutral Ca2+ ligand (ETH 1001). Concentric micropipettes were utilized for the first time for Ca2+-selective microelectrodes in which the Ca2+ ligand was incorporated into a protruding inner pipette, surrounded by an outer reference electrode. In addition, they were made from high resistance aluminosilicate glass tubing (Corning Code 1724). These Ca2+-selective electrodes had linear responses from pCa 3 to pCa 7 in the presence of constant [K+]. They provided on-line observation of changes in intracellular [Ca2+] and in the resting membrane potential in single smooth muscle cells isolated from toad stomach. The mean concentration of intracellular Ca2+ in resting cells was 163.6 +/- 20 nM (+/- SEM, n = 16). Doubling the intracellular Ca2+ level by exposure of cells to elevated [K+] was sufficient to cause shortening.     

Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana

The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip-focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of Arabidopsis. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip-focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre-treated with microtubule antagonists, using the caged-calcium ionophore Br-A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.     

Effect of calcium chelators on the Ca2+-dependent luminescence of aequorin.

The luminescence of aequorin, a useful tool for studying intracellular Ca2+, was recently found to be inhibited by the free EDTA and EGTA that are present in calcium buffers. In the present study we have examined the effect of the free forms of various chelators in the calibration of [Ca2+] with aequorin. Free EDTA and EGTA in low-ionic-strength solutions strongly inhibited the Ca2+-triggered luminescence of aequorin, causing large errors in the calibration of [Ca2+] (approx. 2 pCa units), whereas in solutions containing 150mM-KCl, errors were relatively small (0.2-0.3 pCa units). Citric acid in low-ionic-strength solutions and [(carbamoylmethyl)imino]diacetic acid in high-ionic-strength solutions showed no inhibition and did not cause detectable error in the calibration of [Ca2+], indicating that they are better chelators than EDTA and EGTA for use with aequorin.     

Ca2+ dynamics in rat pancreatic AR-42J and AR-IP cells.

Spatiotemporal change of the cytosolic free Ca2+ concentration ([Ca2+]i) in response to a variety of secretagogues was examined in rat pancreatoma AR-42J and AR-IP cells by microspectroflurometry and digital imaging microscopy after loading with fura-2. In the presence of external Ca2+, carbachol, CCK-OP (cholecystokinin-octapeptide), gastrin, norepinephrine or high K+ evoked a large transient increase in [Ca2+]i in AR-42J cells which declined to a sustained level before slowly declining towards the resting level. In the absence of external Ca2+, a transient increase in [Ca2+]i were evoked by all the ligands except for high K+ stimulation, which declined rapidly towards the resting level. The [Ca2+]i increase caused by carbachol and high K+ treatment was inhibited by muscarinic receptor antagonist, atropine, and by L-type Ca2+ channel blocker, nifedipine, respectively. The transient [Ca2+]i increase induced by gastrin stimulation was not blocked by Ca2+ channel blocker, lanthanum. In the AR-IP cells, which are non-differentiated pancreatoma cell line, all stimulations including high K+ treatment have failed to evoke [Ca2+]i response. These intracellular Ca2+ mobilizations in response to ligands in AR-42J cells were displayed by digital imaging microscopy. From these results we conclude that AR-42J cells has an alpha-adrenergic receptor, in addition to muscarinic acetylcholine receptor, CCK-OP receptor, gastrin receptor and voltage dependent Ca2+ channel. In marked contrast, AR-IP cells have neither any hormone receptor for the above ligands nor voltage dependent Ca2+ channel.     

(Ca2+ + Mg2+)-ATPase activity associated with the maintenance of a Ca2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca2+]. The effect of hypothyroidism

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.     

Ca2+]i independent mitogenesis in cultured human fibroblasts revealed by single cell microfluorimetry

A transient rise in intracellular free Ca2+ concentration ([Ca2+]i) has been implicated in mitogenic induction of cell division. Individual human foreskin fibroblasts in confluent cultures examined with the Ca2+ indicator Fura-2 and a fluorescence microscope-imaging system had a basal [Ca2+]i which varied markedly from cell-to-cell. A transient serum-induced rise in [Ca2+]i was demonstrated the magnitude of which was directly correlated with the basal [Ca2+]i level. In contrast to serum-induced increase in [Ca2+]i, exposure to an elevated level of extracellular Ca2+, which is at least equally mitogenic for fibroblasts, did not alter the basal [Ca2+]i of single subconfluent cells or confluent cells. Elevated extracellular Ca2+ does not exert its mitogenicity via a transient rise in [Ca2+]i.     

Synaptosomal [Ca2+]i as influenced by Na+/Ca2+ exchange and K+ depolarization.

The modulation of the intrasynaptosomal concentration of Ca2+, [Ca2+]i, by Na+/Ca2+ exchange was studied using Indo-1 fluorescence. The electrochemical gradient of Na+ was manipulated by substituting Li+ or choline for Na+ in the external medium and, then, the influx of 45Ca2+ and the [Ca2+]i were measured. It was found that the increase in [Ca2+]i induced by K+ depolarization is lower if the value of [Ca2+]i has been previously raised by Na+/Ca2+ exchange, suggesting that Ca2+ entering by Na+/Ca2+ exchange reduces the Ca2+ entering by voltage-dependent calcium channels. Our results show that a value of [Ca2+]i of about 650 nM induced by Na+/Ca2+ exchange reduces by 50% the Ca2+ entering due to K+ depolarization and no Ca2+ enters through the channels if the [Ca2+]i is previously raised above about 800 nM. Furthermore, predepolarization of the synaptosomes in a Ca-free medium also inhibits by at least 40% the [Ca2+]i rise through Ca2+ channels. Thus, the results suggest that both predepolarization and [Ca2+]i rise due to Na+/Ca2+ exchange decrease the Ca2+ entering by voltage-sensitive Ca2+ channels. The Ca2+ entering by Na+/Ca2+ exchange might contribute to the regulation of neurotransmitter release. Our results also show that the presence of Li+ in the external medium decreases the buffering capacity of synaptosomes, probably by releasing Ca2+ from mitochondria by Li+/Ca2+ exchange.     

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

Overshooting cytosolic Ca2+ signals evoked by capacitative Ca2+ entry result from delayed stimulation of a plasma membrane Ca2+ pump

The effect of capacitative Ca2+ entry on cytosolic free Ca2+ concentration ([Ca2+]c) was examined in calf pulmonary artery endothelial cells treated with thapsigargin. Restoration of extracellular Ca2+ evoked an overshoot in [Ca2+]c: the initial rate of Ca2+ influx was 12.4 +/- 0.5 nM/s as [Ca2+]c rose monoexponentially (time constant, tau = 36 +/- 2 s) to a peak (322 +/- 16 nM) before declining to 109 +/- 14 nM after 2000 s. Rates of Ca2+ removal from the cytosol were measured throughout the overshoot by recording the monoexponential decrease in [Ca2+]c after rapid removal of extracellular Ca2+. The time constant for recovery (tau rec decreased from 54 +/- 4 s when Ca2+ was removed after 10 s to its limiting value of 8.8 +/- 1.0 s when it was removed after 2000 s. The time dependence of the changes in tau rec indicate that an increase in [Ca2+]c is followed by a delayed (tau = 408 s) stimulation of Ca2+ removal, which fully reverses (tau approximately 185 s) after Ca2+ entry ceases. Numerical simulation indicated that the changes in Ca2+ removal were largely responsible for the overshooting pattern of [Ca2+]c. Because prolonged (30 min) Ca2+ entry did not increase the total 45Ca2+ content of the cells, an increased rate of Ca2+ extrusion across the plasma membrane most likely mediates the Ca2+ removal, and since it persists in the absence of extracellular Na+, it probably results from stimulation of a plasma membrane Ca2+ pump. We conclude that delayed stimulation of a plasma membrane Ca2+ pump by capacitative Ca2+ entry may protect cells from excessive increases in [Ca2+]c and contribute to oscillatory changes in [Ca2+]c.