Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.     

Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry

Transient receptor potential (TRP) cation channels are a critical pathway for Ca2+ entry during pulmonary artery (PA) smooth muscle contraction. However, whether canonical TRP (TRPC) subunits and which TRP channel isoforms are involved in store depletion-induced pulmonary vasoconstriction in vivo remain unclear. This study was designed to test whether overexpression of the human TRPC1 gene (hTRPC1) in rat PA enhances pulmonary vasoconstriction due to store depletion-mediated Ca2+ influx. The hTRPC1 was infected into rat PA rings with an adenoviral vector. RT-PCR and Western blot analyses confirmed the mRNA and protein expression of hTRPC1 in the arterial rings. The amplitude of active tension induced by 40 mM K+ (40K) in PA rings infected with an empty adenoviral vector (647 +/- 88 mg/mg) was similar to that in PA rings infected with hTRPC1 (703 +/- 123 mg/mg, P = 0.3). However, the active tension due to capacitative Ca2+ entry (CCE) induced by cyclopiazonic acid was significantly enhanced in PA rings overexpressing hTRPC1 (91 +/- 13% of 40K-induced contraction) compared with rings infected with an empty adenoviral vector (61 +/- 14%, P < 0.001). Endothelial expression of hTRPC1 was not involved since the CCE-induced vasoconstriction was also enhanced in endothelium-denuded PA rings infected with the adenoviral vector carrying hTRPC1. These observations demonstrate that hTRPC1 is an important Ca(2+)-permeable channel that mediates pulmonary vasoconstriction when PA smooth muscle cell intracellular Ca2+ stores are depleted.     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry     

Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry

Endothelial nitric-oxide synthase (eNOS), a Ca(2+)/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through its N-myristoylation, the significance of membrane association in locating eNOS near sources of Ca(2+) entry is uncertain. To assess the Ca(2+) source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca(2+) sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca(2+) concentration, demonstrating preferential sensitivity to Ca(2+) originating from intracellular pools (MHA) or from capacitative Ca(2+) entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca(2+) influx via capacitative Ca(2+) entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca(2+) release. When eNOS activation by CCE was compared with that generated by an equal rise in [Ca(2+)](i) due to the Ca(2+) ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca(2+) influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.     

The effect of oxidative stress on Ca2+ release and capacitative Ca2+ entry in vascular endothelial cells

Oxidative stress imposed by the accumulation of oxygen free radicals (reactive oxygen species, ROS) has profound effects on Ca2+ homeostasis in the vascular endothelium, leading to endothelial dysfunctions and the development of cardiovascular pathologies. We tested the effect of the oxidant and ROS generator tert-butyl-hydroperoxide (tBuOOH) on Ca2+ signaling in single cultured calf pulmonary artery endothelial (CPAE) cells loaded with the fluorescent Ca2+ indicator indo-1. Acute brief (5 min) exposures to tBuOOH had no effect on basal cytosolic free Ca2+ ([Ca2+](i)), agonist (ATP)-induced Ca2+ release from the endoplasmic reticulum (ER) and on Ca(2+) store depletion-dependent capacitative Ca2+ entry (CCE). Prolonged (60 min) exposure to tBuOOH did not affect intracellular Ca2+ release, but caused a profound inhibition of CCE. After 120 min of treatment with tBuOOH not only was CCE further reduced, but also ATP-induced Ca2+ release due to a slow depletion of the stores that resulted from CCE inhibition. The antioxidant Trolox (synthetic vitamin E analog) prevented the inhibition of CCE by tBuOOH and attenuated the increase of [ROS](i), indicating that inhibition of CCE was due to the oxidant effects of tBuOOH. The data suggest that in vascular endothelial cells oxidative stress primarily affects Ca2+ influx in response to Ca2+ loss from internal stores. [Ca2+](i) is an important signal for the production and release of endothelium-derived factors such as nitric oxide (NO). Since CCE is the preferential Ca2+ source for NO synthase activation, the finding that oxidative stress inhibits CCE may explain how oxidative stress contributes to endothelial dysfunction-related cardiovascular pathologies.     

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.     

Fendiline increases [Ca2+]i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca2+ followed by capacitative Ca2+ entry

The effect of fendiline, a documented inhibitor of L-type Ca2+ channels and calmodulin, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca2+ probe. Fendiline at 5-100 microM significantly increased [Ca2+]i concentration-dependently. The [Ca2+]i rise consisted of an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca2+ influx and internal Ca2+ release. In Ca(2+)-free medium, pretreatment with 50 microM fendiline nearly abolished the [Ca2+]i rise induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor, and vice versa, pretreatment with thapsigargin prevented fendiline from releasing internal Ca2+. This indicates that the internal Ca2+ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 microM, fendiline caused Mn2+ quench of fura-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La3+ by 50%, implying that fendiline-induced Ca2+ influx has two components separable by La3+. Consistently, 0.1 mM La3+ pretreatment suppressed fendiline-induced [Ca2+]i rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 50-100 microM fendiline in Ca(2+)-free medium. However, 50-100 microM fendiline inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 inhibited 50 microM fendiline-induced internal Ca2+ release by 48%, but inhibition of phospholipase C with 2 microM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca2+]i in MDCK cells by releasing internal Ca2+ in a manner independent of inositol-1,4,5-trisphosphate (IP3), followed by external Ca2+ influx.     

Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (I(SOC)) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I(SOC), was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I(SOC) and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I(SOC) and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+](cyt).     

Upregulated TRP and enhanced capacitative Ca(2+) entry in human pulmonary artery myocytes during proliferation

A rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through plasmalemmal Ca(2+) channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), a mechanism involved in maintaining Ca(2+) influx and refilling intracellular Ca(2+) stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca(2+) channels that are activated by Ca(2+) store depletion. In this study, we examined whether CCE, activity of store-operated Ca(2+) channels, and human TRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca(2+) and depletion of intracellularly stored Ca(2+) inhibited PASMC growth in media containing serum and growth factors. Resting [Ca(2+)](cyt) as well as the increases in [Ca(2+)](cyt) due to Ca(2+) release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca(2+) stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca(2+)](cyt) and intracellularly stored [Ca(2+)] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially via hTRP1-encoded Ca(2+)-permeable channels, may be an important mechanism required to maintain the elevated [Ca(2+)](cyt) and stored [Ca(2+)] in human PASMC during proliferation.     

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca²⁺ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La³⁺; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca²⁺ influx, which is not inhibited by 2-APB or La³⁺, indicating that AA activates a second Ca²⁺ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca]_i overload is to cause delayed cell death in astrocytes.     

Mitochondrial localization as a determinant of capacitative Ca2+ entry in HeLa cells

Whether different subsets of mitochondria play distinct roles in shaping intracellular Ca2+ signals is presently unresolved. Here, we determine the role of mitochondria located beneath the plasma membrane in controlling (a) Ca2+ release from the endoplasmic reticulum (ER) and (b) capacitative Ca2+ entry. By over-expression of the dynactin subunit dynamitin, and consequent inhibition of the fission factor, dynamin-related protein (Drp-1), mitochondria were relocalised from the plasma membrane towards the nuclear periphery in HeLa cells. The impact of these changes on free calcium concentration in the cytosol ([Ca2+]c), mitochondria ([Ca2+]m) and ER ([Ca2+]ER) was then monitored with specifically-targeted aequorins. Whilst dynamitin over-expression increased the number of close contacts between the ER and mitochondria by >2.5-fold, assessed using organelle-targeted GFP variants, histamine-induced changes in organellar [Ca2+] were unaffected. By contrast, Ca2+ influx elicited significantly smaller increases in [Ca2+]c and [Ca2+]m in dynamitin-expressing than in control cells. These data suggest that the strategic localisation of a subset of mitochondria beneath the plasma membrane is required for normal Ca2+ influx, but that the transfer of Ca2+ ions between the ER and mitochondria is relatively insensitive to gross changes in the spatial relationship between these two organelles.     

Reduced store-operated Ca2+ entry in pulmonary endothelial cells from chronically hypoxic rats

Chronic hypoxia (CH)-induced pulmonary hypertension may influence basal endothelial cell (EC) intracellular Ca(2+) concentration ([Ca(2+)](i)). We hypothesized that CH decreases EC [Ca(2+)](i) associated with membrane depolarization and reduced Ca(2+) entry. To test this hypothesis, we assessed 1) basal endothelial Ca(2+) in pressurized pulmonary arteries and freshly isolated ECs, 2) EC membrane potential (E(m)), 3) store-operated Ca(2+) current (I(SOC)), and 4) store-operated Ca(2+) (SOC) entry in arteries from control and CH rats. We found that basal EC Ca(2+) was significantly lower in pressurized pulmonary arteries and freshly isolated ECs from CH rats compared with controls. Similarly, ECs in intact arteries from CH rats were depolarized compared with controls, although no differences were observed between groups in isolated cells. I(SOC) activation by 1 muM thapsigargin displayed diminished inward current and a reversal potential closer to 0 mV in cells from CH rats compared with controls. In addition, SOC entry determined by fura 2 fluorescence and Mn(2+) quenching revealed a parallel reduction in Ca(2+) entry following CH. We conclude that differences in the magnitude of SOC entry exist between freshly dispersed ECs from CH and control rats and correlates with the decrease in basal EC [Ca(2+)](i). In contrast, basal EC Ca(2+) influx is unaffected and membrane depolarization is limited to intact arteries, suggesting that E(m) may not play a major role in determining basal EC [Ca(2+)](i) following CH.     

Time-dependent modulation of capacitative Ca2+ entry signals by plasma membrane Ca2+ pump in endothelium

In vascular endothelial cells, depletion of intracellularCa²⁺ stores elicited capacitativeCa²⁺ entry (CCE) that resulted inbiphasic changes of intracellular Ca²⁺ concentration([Ca²⁺]_i)with a rapid initial peak of[Ca²⁺]_ifollowed by a gradual decrease to a sustained plateau level. Weinvestigated the rates of Ca²⁺entry, removal, and sequestration during activation of CCE and theirrespective contributions to the biphasic changes of[Ca²⁺]_i.Ca²⁺ buffering by mitochondria,removal byNa⁺/Ca²⁺exchange, and a fixed electrical driving force forCa²⁺ (voltage-clamp experiments)had little effect on the CCE signal. The rates of entry ofMn²⁺ andBa²⁺, used as unidirectionalsubstitutes for Ca²⁺ entry throughthe CCE pathway, were constant and did not follow the concomitantchanges of[Ca²⁺]_i.Pharmacological inhibition of the plasma membraneCa²⁺ pump, however, abolished thesecondary decay phase of the CCE transient. The disparity between thebiphasic changes of[Ca²⁺]_iand the constant rate of Ca²⁺entry during CCE was the result of a delayed,Ca²⁺-dependent activation of thepump. These results suggest an important modulatory role of the plasmamembrane Ca²⁺ pump in the netcellular gain of Ca²⁺ during CCE.

     

Hypoxia/reoxygenation stimulates Ca2+-dependent ICAM-1 mRNA expression in human aortic endothelial cells

Increased endothelial ICAM-1 expression is found in normal aging and in atherosclerosis and is related to the chronic effects of oxidative stress. We examined the Ca(2+)-dependence of ICAM-1 mRNA expression in human aortic endothelial cells (HAEC) exposed to hypoxia/reoxygenation (H/R) as a model of oxidative stress. HAEC were exposed to glucose-free hypoxia (95% N(2)/5% CO(2)) for 60 min and were then reoxygenated (21% O(2)/5% CO(2)) and observed for up to 6h. Reactive oxygen species (ROS) generation was measured by dichlorofluorescein fluorescence and ICAM-1 mRNA was assessed by Northern blot. Upon reoxygenation after hypoxia, ROS production occurred in HAEC and was inhibited by diphenyleneiodonium and by polyethylene glycol-catalase, suggesting the involvement of NADPH oxidase-derived hydrogen peroxide. Hypoxia alone did not increase either ROS production or ICAM-1 mRNA levels, but a 2.5-fold increase in ICAM-1 mRNA was noted by 30 min of reoxygenation. This was not observed in Ca(2+)-free buffer or in cells treated with diphenyleneiodonium. Thus, H/R upregulates ICAM-1 mRNA in HAEC by a Ca(2+)- and ROS-dependent mechanism. Characterizing the signaling pathways involved in H/R-induced adhesion molecule expression may result in a better understanding of the vascular biology of normal aging and the pathobiology of atherosclerosis.     

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.     

Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes

Depletion of Ca²⁺ stores inthe sarcoplasmic reticulum (SR) activates extracellularCa²⁺ influx via capacitativeCa²⁺ entry (CCE). Here, CCE levelsin proliferating and growth-arrested human pulmonary artery smoothmuscle cells (PASMCs) were compared by digital imaging fluorescencemicroscopy. Resting cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)in proliferating PASMCs was twofold higher than that in growth-arrestedcells. Cyclopiazonic acid (CPA; 10 µM), which inhibits SRCa²⁺-ATPase and depletes inositol1,4,5-trisphosphate-sensitiveCa²⁺ stores, transiently increased[Ca²⁺]_cytin the absence of extracellularCa²⁺. The addition of 1.8 mMCa²⁺ to the extracellular solutionin the presence of CPA induced large increases in[Ca²⁺]_cyt,indicative of CCE. The CPA-induced SRCa²⁺ release in proliferatingPASMCs was twofold higher than that in growth-arrested cells, whereasthe transient rise of[Ca²⁺]_cytdue to CCE was fivefold greater in proliferating cells. CCE wasinsensitive to nifedipine but was significantly inhibited by 50 mMK⁺, which reduces the drivingforce for Ca²⁺ influx, and by 0.5 mM Ni²⁺, a putative blocker ofstore-operated Ca²⁺ channels.These data show that augmented CCE is associated with proliferation ofhuman PASMCs and may be involved in stimulating and maintaining cell growth.

     

Chronic hypoxia potentiates capacitative Ca2+ entry in type-I cortical astrocytes

Prolonged hypoxia exerts profound effects on cell function, and has been associated with increased production of amyloid beta peptides (A beta Ps) of Alzheimer's disease. Here, we have investigated the effects of chronic hypoxia (2.5% O2, 24 h) on capacitative Ca2+ entry (CCE) in primary cultures of rat type-I cortical astrocytes, and compared results with those obtained in astrocytes exposed to A beta Ps. Chronic hypoxia caused a marked enhancement of CCE that was observed after intracellular Ca2+ stores were depleted by bradykinin application or by exposure to thapsigargin (1 microM). Exposure of cells for 24 h to 1 microM A beta P(1-40) did not alter CCE. Enhancement of CCE was not attributable to cell hyperpolarization, as chronically hypoxic cells were significantly depolarized as compared with controls. Mitochondrial inhibition [by FCCP (10 microM) and oligomycin (2.5 microg/mL)] suppressed CCE in all three cell groups, but more importantly there were no significant differences in the magnitude of CCE in the three astrocyte groups under these conditions. Similarly, the antioxidants melatonin and Trolox abolished the enhancement of CCE in hypoxic cells. Our results indicate that chronic hypoxia augments CCE in cortical type-I astrocytes, a finding which is not mimicked by A beta P(1-40) and appears to be dependent on altered mitochondrial function.     

Positive regulation of capacitative Ca2+ entry by intracellular Ca2+ in Xenopus oocytes expressing rat TRP4

We have investigated the role of intracellular Ca2+ in the opening of capacitative Ca2+ entry (CCE) channels formed with rat TRP4 (rTRP4) using Xenopus oocytes. In rTRP4-expressing oocytes pretreated with thapsigargin, perfusion with A23187, a Ca2+ ionophore, significantly potentiated the delayed phase of the CCE-mediated Cl- current response evoked by extracellular perfusion with Ca2+, without affecting the transient phase of CCE response. In control oocytes, the potentiation of delayed CCE response by A23187 was not significant. Using cut-open recording in combination with artificial intracellular perfusion of oocytes, CCE-mediated Cl- response was recorded at controlled cytosolic Ca2+ concentrations. Intracellular perfusion with a Ca2+ free solution containing 10 mM EGTA abolished most of the CCE responses of both non-injected and rTRP4-expressing oocytes. The native CCE response was not fully recovered by subsequent increases in the intracellular Ca2+ concentration up to 300 nM. However, CCE response of the rTRP4-expressing oocytes was restored at an internal Ca2+ concentration of 110 nM. Blockade of endogenous Cl- channels with anion channel blocker isolated Ca2+ current flowing through CCE channels and clarified the difference in the sensitivity to an internal Ca2+ concentration. These findings indicate that recombinant CCE channels formed with rTRP4 are positively regulated by cytosolic Ca2+ at higher sensitivity compared to oocyte-endogenous CCE channels.     

Suppression of EGF-induced cell proliferation by the blockade of Ca2+ mobilization and capacitative Ca2+ entry in mouse mammary epithelial cells

The role of intracellular Ca2+ stores and capacitative Ca2+ entry on EGF-induced cell proliferation was investigated in mouse mammary epithelial cells. We have previously demonstrated that EGF enhances Ca2+ mobilization (release of Ca2+ from intracellular Ca2+ stores) and capacitative Ca2+ entry correlated with cell proliferation in mouse mammary epithelial cells. To confirm their role on EGF-induced cell cycle progression, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), a reversible inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and SK&F 96365, a blocker of capacitative Ca2+ entry, on mitotic activity induced by EGF. Mitotic activity was examined using an antibody to PCNA for immunocytochemistry. SK&F 96365 inhibited capacitative Ca2+ entry in a dose-dependent manner (I50: 1-5 microM). SK&F 96365 also inhibited EGF-induced cell proliferation in the same range of concentration (I50: 1-5 microM). DBHQ suppressed [Ca2+]i response to UTP and thus depleted completely Ca2+ stores at 5 microM. DBHQ also inhibited EGF-induced cell proliferation at an I50 value of approximately 10 microM. The removal of these inhibitors from the culture medium increased the reduced mitotic activity reversibly. Using a fluorescent assay of DNA binding of ethidium bromide, no dead cells were detected in any of the cultures. These results indicate that the inhibitory effects of SK&F 96365 and DBHQ on cell proliferation were due to the inhibition of capacitative Ca2+ entry and Ca2+ mobilization suggesting the importance of capacitative Ca2+ entry and Ca2+ mobilization in the control of EGF-induced cell cycle progression in mouse mammary epithelial cells.