EGF enhances Ca(2+) mobilization and capacitative Ca(2+) entry in mouse mammary epithelial cells

Effects of epidermal growth factor (EGF) on the intracellular Ca(2+) ([Ca(2+)](i)) responses to nucleotides, Ca(2+) release from thapsigargin-sensitive stores and capacitative Ca(2+) entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti-PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S-phase of cell cycle. EGF treatment apparently increased the number of PCNA-stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca(2+)](i) response. EGF treatment for 1-2 days in culture, however, enhanced [Ca(2+)](i) responses including [Ca(2+)](i) increase by ATP, UTP and other nucelotides, Ca(2+) release from thapsigargin-sensitive stores, as well as capacitative Ca(2+) entry. Genistein, a tyrosine kinase inhibitor, prevented EGF-induced cell proliferation and the [Ca(2+) ](i) responses in a dose-dependent manner. These results indicate that EGF treatment enhances Ca(2+) mobilization and capacitative Ca(2+) entry, well correlated with cellular proliferation in mammary epithelial cells.     

Direct coupling between arachidonic acid-induced Ca2+ release and Ca2+ entry in HEK293 cells

Arachidonic acid (AA) modulates intracellular Ca2+ signaling via Ca2+ release or/and Ca2+ entry. However, the mechanism underlies either process is unknown; nor is it clear as to whether the two processes are mechanistically linked. By using Fura2/AM, we found that AA induced mobilization of internal Ca2+ store and an increment in Ca2+, Mn2+ and Ba2+ influx in HEK293 cells. The AA-mediated Ca2+ signaling was not due to AA metabolites, and insensitive to capacitative Ca2+ entry inhibitors. Interestingly, isotetrandrine and Gd3+ inhibited both AA-induced Ca2+ release and Ca2+ entry in a concentration-dependent manner without affecting Ca2+ discharge caused by carbachol, caffeine, or thapsigargin. Additionally, similar pattern of inhibition was observed with tetracaine treatment. More importantly, the three compounds exhibited almost equal potent inhibition of AA-initiated Ca2+ release as well as Ca2+ influx. Therefore, this study, for the first time, provides evidence for a direct coupling between AA-mediated Ca2+ release and Ca2+ entry.     

Developmental changes in capacitative Ca(2+) entry in mouse mammary epithelial cells

Developmental changes in capacitative Ca(2+) entry and Ca(2+) release from intracellular stores were measured using fura-2 fluorescence method during the pregnancy period (day 3-;18) in mouse mammary epithelial cells. Ca(2+) release was identified with the transient intracellular Ca(2+) ([Ca(2+)](i)) increase induced by thapsigargin addition in a Ca(2+)-free solution. Capacitative Ca(2+) entry was measured by the transient [Ca(2+)](i) increase induced by re-addition of extracellular Ca(2+) after depletion of Ca(2+) stores by thapsigargin. The capacitative Ca(2+) entry was greatest at the early stage of pregnancy (i.e. day 3 of pregnancy) and decreased as pregnancy progressed, while Ca(2+) release remained unchanged throughout the developmental stages. These findings indicate that in contrast to Ca(2+) release, a close correlation exists between capacitative Ca(2+) entry and pregnancy-induced development in mammary epithelial cells.     

Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Activation of muscarinic receptors reduces store-operated Ca2+ entry in HEK293 cells

In many cell types membrane receptors for hormones or neurotransmitters activate a signal transduction pathway which releases Ca2+ from intracellular Ca2+ stores by the second messenger inositol 1,4,5-trisphosphate. As a consequence store-operated Ca2+ entry (SOCE) becomes activated. In the present study we addressed the question if receptor/agonist binding can modulate Ca2+ entry by mechanisms different from the store-operated one. Therefore SOCE was examined in HEK293 cells microscopically with the fura-2 technique and with patch clamp. We found that maximally preactivated SOCE could, concentration dependently, be reduced up to 80% by the muscarinic agonist acetylcholine when the cytoplasmic Ca2+ concentration was used as a measure. Muscarinic receptors seem to mediate this decrease since atropine blocked the effect completely and cell types without muscarinic receptors (BHK21, CHO) did not show acetylcholine-induced decrease of Ca2+ entry. Moreover expression of muscarinic receptor subtypes M1 and M3 in BHK21 cells established the muscarinic decrease of SOCE. Electrical measurements revealed that the membrane potential of HEK293 cells did not show any response to ACh, excluding that changes of driving forces are responsible for the block of Ca2+ entry. In contrast the electrical current which is responsible for SOCE in HEK293 cells (Ca2+ release-activated Ca2+ current (I(CRAC)) was inhibited (maximally 55%) by 10 microM ACh. From these data we conclude that in HEK293 cells a muscarinic signal transduction pathway exists which decreases the cytoplasmic Ca2+ concentration by an inhibition of I(CRAC). This mechanism may serve as a modulator of Ca2+ entry preventing a Ca2+ overload of the cytoplasm after Ca2+ store depletion.     

A mechanism for both capacitative Ca(2+) entry and excitation-contraction coupled Ca(2+) release by the sarcoplasmic reticulum of skeletal muscle cells

We have previously established that L6 skeletal muscle cell cultures display capacitative calcium entry (CCE), a phenomenon established with other cells in which Ca(2+) uptake from outside cells increases when the endoplasmic reticulum (sarcoplasmic reticulum in muscle, or SR) store is decreased. Evidence for CCE rested on the use of thapsigargin (Tg), an inhibitor of the SR CaATPase and consequently transport of Ca(2+) from cytosol to SR, and measurements of cytosolic Ca(2+). When Ca(2+) is added to Ca(2+)-free cells in the presence of Tg, the measured cytosolic Ca(2+) rises. This has been universally interpreted to mean that as SR Ca(2+) is depleted, exogenous Ca(2+) crosses the plasma membrane, but accumulates in the cytosol due to CaATPase inhibition. Our goal in the present study was to examine CCE in more detail by measuring Ca(2+) in both the SR lumen and the cytosol using established fluorescent dye techniques for both. Surprisingly, direct measurement of SR Ca(2+) in the presence of Tg showed an increase in luminal Ca(2+) concentration in response to added exogenous Ca(2+). While we were able to reproduce the conventional demonstration of CCE-an increase of Ca(2+) in the cytosol in the presence of thapsigargin-we found that this process was inhibited by the prior addition of ryanodine (Ry), which inhibits the SR Ca(2+) release channel, the ryanodine receptor (RyR). This was also unexpected if Ca(2+) enters the cytosol first. When Ca(2+) was added prior to Ry, the later was unable to exert any inhibition. This implies a competitive interaction between Ca(2+) and Ry at the RyR. In addition, we found a further paradox: we had previously found Ry to be an uncompetitive inhibitor of Ca(2+) transport through the RyR during excitation-contraction coupling. We also found here that high concentrations of Ca(2+) inhibited its own uptake, a known feature of the RyR. We confirmed that Ca(2+) enters the cells through the dihydropyridine receptor (DHPR, also known as the L-channel) by demonstrating inhibition by diltiazem. A previous suggestion to the contrary had used Mn(2+) in place of direct Ca(2+) measurements; we showed that Mn(2+) was not inhibited by diltiazem and was not capacitative, and thus not an appropriate probe of Ca(2+) flow in muscle cells. Our findings are entirely explained by a new model whereby Ca(2+) enters the SR from the extracellular space directly through a combined channel formed from the DHPR and the RyR. These are known to be in close proximity in skeletal muscle. Ca(2+) subsequently appears in the cytosol by egress through a separate, unoccupied RyR, explaining Ry inhibition. We suggest that upon excitation, the DHPR, in response to the electrical field of the plasma membrane, shifts to an erstwhile-unoccupied receptor, and Ca(2+) is released from the now open RyR to trigger contraction. We discuss how this model also resolves existing paradoxes in the literature, and its implications for other cell types.     

Neurotransmitter release from bovine adrenal chromaffin cells is modulated by capacitative Ca(2+)entry driven by depleted internal Ca(2+)stores

Two potential mechanisms by which the intracellular Ca(2 stores might modulate catecholamine release from bovine adrenal chromaffin cells were investigated: (i) that the cytosolic Ca(2+)transient caused by Ca(2+)release from the intracellular stores recruits additional chromaffin granules to a readily releasable pool that results in augmented catecholamine release when this is subsequently evoked, and (ii) that the Ca(2+)influx that follows depletion of intracellular stores (i.e. store-operated Ca(2+)entry) triggers release per se thereby augmenting evoked catecholamine release. When histamine or caffeine were applied in Ca(2+)-free perfusion media, a transient elevation of intracellular free Ca(2+)occurred owing to mobilization of Ca(2+)from the stores. When Ca(2+)was later readmitted to the perfusing fluid there followed a prompt and maintained rise in intracellular Ca(2+)concentrations of magnitude related to the degree of store mobilization. In parallel experiments, increased catecholamine secretion was measured under the conditions when Ca(2+)influx following store-mobilization occurred. Furthermore, the size of the catecholamine release increment correlated with the degree of Ca(2+)influx. Store-operated Ca(2+)entry evoked by mobilization with histamine and/or caffeine did not augment nicotine-evoked secretion per se; that is, it augmented evoked catecholamine release only to the extent that it increased basal catecholamine release. The nicotine-evoked catecholamine release was sensitive to cytosolic BAPTA, which, at the concentration used (50 microM BAPTA-AM), reduced release by approximately 25%. However, the increment in basal catecholamine release which followed Ca(2+)influx triggered by Ca(2+)store mobilization was not reduced by intracellular BAPTA. This finding is inconsistent with the hypothesis that the elevated cytosolic Ca(2+)from store mobilization recruits additional vesicles of catecholamine to the sub-plasmalemmal release sites to augment subsequently evoked secretion. This position is supported by the observation that histamine (10 microM) in Ca(2+)-free medium caused a pronounced elevation of cytosolic free Ca(2+), but this caused no greater catecholamine release when Ca(2+)was re-introduced than did prior exposure to Ca(2+)-free medium alone, which caused no elevation of cytosolic free Ca(2+). It is concluded that intracellular Ca(2+)stores can modulate secretion of catecholamines from bovine chromaffin cells by permitting Ca(2+)influx through a store-operated entry pathway. The results do not support the notion that the Ca(2+)released from intracellular stores plays a significant role in the recruitment of vesicles into the ready-release pool under the experimental conditions reported here.     

Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 microm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 microm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 microm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 microm Gd3+, as well as 30-100 microm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 microm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2)]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 microm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+) entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.     

Store depletion by caffeine/ryanodine activates capacitative Ca(2+) entry in nonexcitable A549 cells

Capacitative Ca(2+) entry is essential for refilling intracellular Ca(2+) stores and is thought to be regulated primarily by inositol 1, 4,5-trisphosphate (IP(3))-sensitive stores in nonexcitable cells. In nonexcitable A549 cells, the application of caffeine or ryanodine induces Ca(2+) release in the absence of extracellular Ca(2+) similar to that induced by thapsigargin (Tg), and Ca(2+) entry occurs upon the readdition of extracellular Ca(2+). The channels thus activated are also permeable to Mn(2+). The channels responsible for this effect appear to be activated by the depletion of caffeine/ryanodine-sensitive stores per se, as evidenced by the activation even in the absence of increased intracellular Ca(2+) concentration. Tg pretreatment abrogates the response to caffeine/ryanodine, whereas Tg application subsequent to caffeine/ryanodine treatment induces further Ca(2+) release. The response to caffeine/ryanodine is also abolished by initial ATP application, whereas ATP added subsequent to caffeine/ryanodine induces additional Ca(2+) release. RT-PCR analyses showed the expression of a type 1 ryanodine receptor, two human homologues of transient receptor potential protein (hTrp1 and hTrp6), as well as all three types of the IP(3) receptor. These results suggest that in A549 cells, (i) capacitative Ca(2+) entry can also be regulated by caffeine/ryanodine-sensitive stores, and (ii) the RyR-gated stores interact functionally with those sensitive to IP(3), probably via Ca(2+)-induced Ca(2+) release.     

Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca2+ entry pathways

Receptor-activated Ca(2+) entry is usually thought to occur via capacitative or store-operated Ca(2+) channels. However, at physiological levels of stimulation, where Ca(2+) store depletion is only transient and/or partial, evidence has suggested that an arachidonic acid-dependent noncapacitative Ca(2+) entry is responsible. Recently, we have described a novel arachidonate-regulated Ca(2+)-selective (ARC) conductance that is entirely distinct from store-operated conductances in the same cell. We now show that these ARC channels are indeed specifically activated by low agonist concentrations and provide the predominant route of Ca(2+) entry under these conditions. We further demonstrate that sustained elevations in cytosolic Ca(2+), such as those resulting from activation of store-operated Ca(2+) entry by high agonist concentrations, inhibit the ARC channels. This explains earlier failures to detect the presence of this noncapacitative pathway in experiments where store-operated entry had already been fully activated. The result is that the respective activities of ARC and store-operated Ca(2+) channels display a unique reciprocal regulation that is related to the specific nature of the [Ca(2+)](i) signals generated at different agonist concentrations. Importantly, these data show that at physiologically relevant levels of stimulation, it is the noncapacitative ARC channels that provide the predominant route for the agonist-activated entry of Ca(2+).     

Relation between phosphatidylserine exposure and store-operated Ca(2+) entry in stimulated cells

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.     

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.     

Activation of a capacitative Ca(2+) entry pathway by store depletion in cultured hippocampal neurones

Intracellular Ca(2+) ([Ca(2+)](i)) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca(2+), the cholinergic agonist carbachol (200 microM) and the sarcoendoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (0.4 microM) both transiently elevated [Ca(2+)](i). A subsequent addition of Ca(2+) into the bathing medium caused a second [Ca(2+)](i) change which was blocked by lanthanum (50 microM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca(2+) entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca(2+) entry in excitable cells.     

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.     

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.     

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.     

Role of capacitative Ca2+ entry in bronchial contraction and remodeling.

Asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and airway obstruction by bronchospasm and bronchial wall thickening due to smooth muscle hypertrophy. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) may serve as a shared signal transduction element that causes bronchial constriction and bronchial wall thickening in asthma. In this study, we examined whether capacitative Ca2+ entry (CCE) induced by depletion of intracellular Ca2+ stores was involved in agonist-mediated bronchial constriction and bronchial smooth muscle cell (BSMC) proliferation. In isolated bronchial rings, acetylcholine (ACh) induced a transient contraction in the absence of extracellular Ca2+ because of Ca2+ release from intracellular Ca2+ stores. Restoration of extracellular Ca2+ in the presence of atropine, an M-receptor blocker, induced a further contraction that was apparently caused by a rise in [Ca2+]cyt due to CCE. In single BSMC, amplitudes of the store depletion-activated currents (I(SOC)) and CCE were both enhanced when the cells proliferate, whereas chelation of extracellular Ca2+ with EGTA significantly inhibited the cell growth in the presence of serum. Furthermore, the mRNA expression of TRPC1, a transient receptor potential channel gene, was much greater in proliferating BSMC than in growth-arrested cells. Blockade of the store-operated Ca2+ channels by Ni2+ decreased I(SOC) and CCE and markedly attenuated BSMC proliferation. These results suggest that upregulated TRPC1 expression, increased I(SOC), enhanced CCE, and elevated [Ca2+]cyt may play important roles in mediating bronchial constriction and BSMC 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.     

Interaction between store-operated and arachidonate-activated calcium entry

A ubiquitous pathway for cellular Ca(2+) influx involves 'store-operated channels' that respond to depletion of intracellular Ca(2+) pools via an as yet unknown mechanism. Due to its wide-spread expression, store-operated Ca(2+) entry (SOCE) has been considered a principal route for Ca(2+) influx. However, recent evidence has suggested that alternative pathways, activated for example by lipid metabolites, are responsible for physiological Ca(2+) influx. It is not clear if these messenger-activated Ca(2+) entry routes exist in all cells and what interaction they have with SOCE. In the present study we demonstrate that HEK-293 cells and Saos-2 cells express an arachidonic acid (AA)-activated Ca(2+) influx pathway that is distinct from SOCE on the basis of sensitivity to pharmacological blockers and depletion of cellular cholesterol. We examined the functional interaction between SOCE and the arachidonate-triggered Ca(2+) influx (denoted non-SOCE). Both Ca(2+) entry routes could underlie substantial long-lasting Ca(2+) elevations. However, the two pathways could not operate simultaneously. With cells that had an on-going SOCE response, addition of arachidonate gave two profound effects. Firstly, it rapidly inhibited SOCE. Secondly, the mode of Ca(2+) influx switched to the non-SOCE mechanism. Addition of arachidonate to na?ve cells resulted in rapid activation of the non-SOCE pathway. However, this Ca(2+) entry route was very slowly engaged if the SOCE pathway was already operative. These data indicate that the SOCE and arachidonate-activated non-SOCE pathways interact in an inhibitory manner. We probed the plausible mechanisms by which these two pathways may communicate.