Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.     

Annexin V counteracts apoptosis while inducing Ca(2+) influx in human lymphocytic T cells

We have previously shown that when annexin V is present during the execution of a cell death program, apoptosis is delayed. This is reflected by the inhibition of DNA cleavage and of the release of apoptotic membrane particles, and by reduction of the proteolytic processing of caspase-3. Here, we have studied the mechanism(s) through which annexin V counteracts apoptosis in the human CEM T cell line. The degree of apoptosis inhibition was associated with an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). Reduction of the extracellular Ca(2+) concentration by EGTA abolished the anti-apoptotic effect, suggesting that annexin V favors Ca(2+) influx and that Ca(2+) acts as an inhibitor rather than an activator of apoptosis in CEM T cells. The effects on apoptosis and [Ca(2+)](i) of several modified annexins with different electrophysiological properties indicate that the N-terminal domain of annexin V is necessary for the Ca(2+)-dependent anti-apoptotic action of annexin V. These results suggest that annexin V regulates membrane Ca(2+) permeability and is protective against apoptosis by increasing [Ca(2+)](i) in CEM T cells.     

Dopamine-induced graded intracellular Ca2+ elevation via the Na+Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts

Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca(2+) influx from the extracellular space. Additionally, dopamine induces a massive [Na(+)](i) elevation via the Na(+)K(+)2Cl(-) cotransporter (NKCC). We have reasoned that Ca(2+)-entry is mediated by the Na(+)Ca(2+) exchanger (NCE) operating in the Ca(2+)-entry mode. To test this hypothesis, [Ca(2+)](i) and [Na(+)](i) were measured by using the fluorescent dyes Fura-2, Fluo-3, and SBFI. Inhibition of Na(+)-entry from the extracellular space by removal of extracellular Na(+) or inhibition of the NKCC by 10 microM bumetanide did not influence resting [Ca(2+)](i) but completely abolished the dopamine-induced [Ca(2+)](i) elevation. Simultaneous recordings of [Ca(2+)](i) and [Na(+)](i) revealed that the dopamine-induced [Na(+)](i) elevation preceded the [Ca(2+)](i) elevation. During dopamine stimulation, the generation of an outward Na(+) concentration gradient by removal of extracellular Na(+) boosted the [Ca(2+)](i) elevation. Furthermore, prolonging the dopamine-induced [Na(+)](i) rise by blocking the Na(+)/K(+)-ATPase reduced the recovery from [Ca(2+)](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na(+)](i), which reverses the NCE activity into the reverse mode causing a graded [Ca(2+)](i) elevation in the duct cells.     

Role of calcium and calmodulin in ciliary stimulation induced by acetylcholine

The goal of this work was to elucidate the molecular events underlying stimulation of ciliary beat frequency (CBF) induced by acetylcholine (ACh) in frog esophagus epithelium. ACh induces a profound increase in CBF and in intracellular Ca(2+) concentration ([Ca(2+)](i)) through M(1) and M(3) muscarinic receptors. The [Ca(2+)](i) slowly decays to the basal level, while CBF stabilizes at an elevated level. These results suggest that ACh triggers Ca(2+)-correlated and -uncorrelated modes of ciliary stimulation. ACh response is abolished by the phospholipase C (PLC) inhibitor U-73122 and by depletion of intracellular Ca(2+) stores but is unaffected by reduction of extracellular Ca(2+) concentration and by blockers of Ca(2+) influx. Therefore, ACh activates PLC and mobilizes Ca(2+) solely from intracellular stores. The calmodulin inhibitors W-7 and calmidazolium attenuate the ACh-induced increase in [Ca(2+)](i) but completely abolish the elevation in CBF. Therefore, elevation of [Ca(2+)](i) is necessary for CBF enhancement but does not lead directly to it. The combined effect of Ca(2+) elevation and of additional factors, presumably mobilized by Ca(2+)-calmodulin, results in a robust CBF enhancement.     

Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

Normalization of intracellular Ca(2+) induces a glucose-responsive state in glucose-unresponsive beta-cells

Although intracellular Ca(2+) in pancreatic beta-cells is the principal signal for insulin secretion, the effect of chronic elevation of the intracellular Ca(2+) concentration ([Ca(2+)](i)) on insulin secretion is poorly understood. We recently established two pancreatic beta-cell MIN6 cell lines that are glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14). In the present study we have determined the cause of the glucose unresponsiveness in MIN6-m14. Initially, elevated [Ca(2+)](i) was observed in MIN6-m14, but normalization of the [Ca(2+)](i) by nifedipine, a Ca(2+) channel blocker, markedly improved the intracellular Ca(2+) response to glucose and the glucose-induced insulin secretion. The expression of subunits of ATP-sensitive K(+) channels and voltage-dependent Ca(2+) channels were increased at both mRNA and protein levels in MIN6-m14 treated with nifedipine. As a consequence, the functional expression of these channels at the cell surface, both of which are decreased in MIN6-m14 without nifedipine treatment, were increased significantly. Contrariwise, Bay K8644, a Ca(2+) channel agonist, caused severe impairment of glucose-induced insulin secretion in glucose-responsive MIN6-m9 due to decreased expression of the channel subunits. Chronically elevated [Ca(2+)](i), therefore, is responsible for the glucose unresponsiveness of MIN6-m14. The present study also suggests normalization of [Ca(2+)](i) in pancreatic beta-cells as a therapeutic strategy in treatment of impaired insulin secretion.     

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.     

Release of Ca(2+) from intracellular stores and entry of extracellular Ca(2+) are involved in sea squirt sperm activation.

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores.     

Cadmium is acutely toxic for murine hepatocytes: effects on intracellular free Ca(2+) homeostasis

We studied cadmium toxicity in murine hepatocytes in vitro. Cadmium effects on intracellular free Ca(2+) concentration ([Ca(2+)](i)) were assayed, using a laser scanning confocal microscope with a fluorescent probe, Fluo-3/AM. The results showed that administration of cadmium chloride (CdCl(2), 5, 10, 25 microM) resulted in a dose-dependent decrease of hepatocyte viability and an elevated aspartate aminotransferase (AST) activity in the culture medium (p<0.05 for 25 microM CdCl(2) vs. control). Significant increases of lactate dehydrogenase (LDH) activities in 10 and 25 microM CdC1(2)-exposed groups were observed (p<0.05 and p<0.01, respectively). A greatly decreased albumin content and a more malondialdehyde (MDA) formation also occurred after CdC1(2) treatment. The Ca(2+) concentrations in the culture medium of CdCl(2)-exposed hepatocytes were significantly decreased, while [Ca(2+)](i) appeared to be significantly elevated (p<0.05 or p<0.01 vs. control). We found that in Ca(2+)-containing hydroxyethyl piperazine ethanesulfonic acid-buffered salt solution (HBSS) only, CdCl(2) elicited [Ca(2+)](i) increases, which comprised an initially slow ascent and a strong elevated phase. However, in Ca(2+)-containing HBSS with addition of 2-aminoethoxydiphenyl borane (2-APB), CdCl(2) caused a mild [Ca(2+)](i) elevation in the absence of an initial rise phase. Removal of extracellular Ca(2+) showed that CdCl(2) induced an initially slow [Ca(2+)](i) rise alone without being followed by a markedly elevated phase, but in a Ca(2+)-free HBSS with addition of 2-APB, CdCl(2) failed to elicit the [Ca(2+)](i) elevation. These results suggest that abnormal Ca(2+) homeostasis due to cadmium may be an important mechanism of the development of the toxic effect in murine hepatocytes. [Ca(2+)](i) elevation in acutely cadmium-exposed hepatocytes is closely related to the extracellular Ca(2+) entry and an excessive release of Ca(2+) from intracellular stores.     

Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells

Upon its genesis during apoptosis, ceramide promotes gross reorganization of the plasma membrane structure involving clustering of signalling molecules and an amplification of vesicle formation, fusion and trafficking. The annexins are a family of proteins, which in the presence of Ca(2+), bind to membranes containing negatively charged phospholipids. Here, we show that ceramide increases affinity of annexin A1-membrane interaction. In the physiologically relevant range of Ca(2+) concentrations, this leads to an increase in the Ca(2+)sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramide-specific antibody, we establish a direct interaction of annexin A1 with areas of the plasma membrane enriched in ceramide (ceramide platforms). In living cells, the intracellular dynamics of annexin A1 match those of plasmalemmal ceramide. Among proteins of the annexin family, the interaction with ceramide platforms is restricted to annexin A1 and is conveyed by its unique N-terminal domain. We demonstrate that intracellular Ca(2+)overload occurring at the conditions of cellular stress induces ceramide production. Using fluorescently tagged annexin A1 as a reporter for ceramide platforms and annexin A6 as a non-selective membrane marker, we visualize ceramide platforms for the first time in living cells and provide evidence for a ceramide-driven segregation and internalization of membrane-associated proteins.     

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Immediate cell signal induced by laminin in rat sertoli cells.

Rat Sertoli cells in primary culture have been studied for their ability to respond to extracellular matrix macromolecules by increases of [Ca(2+)](i). We observed that cells seeded on glass coverslips, loaded with the intracellular Ca(2+) indicator fura-2, responded to laminin, but not to fibronectin, with an immediate [Ca(2+)](i) raise, with a peak followed by a prolonged plateau. [Ca(2+)](i) increases were dependent upon Ca(2+) influx across the plasma membrane and Ca(2+) release from intracellular Ca(2+) pools. Ca(2+) influx was inhibited by extracellular Ca(2+) removal by EGTA, and by treatment with La(3+), or with the L-type voltage operated Ca(2+) channel blocker, nifedipine. Ca(2+) release from intracellular Ca(2+) storing organelles, was inhibited by the microsomal Ca(2+)-ATPase blocker thapsigargin. Responses were mimicked by synthetic peptides carrying the Arg-Gly-Asp adhesion sequence, but not by the control Arg-Gly-Glu-containing peptide, in which aspartic acid was replaced by glutamic acid. Laminin-dependent [Ca(2+)](i) increases were down-regulated by the follicle-stimulating hormone. However, this occurred only when cells were not subjected to homotypic cell-cell contact, and responded to the hormone with a significant [Ca(2+)](i) elevation. These results indicate that laminin may regulate Sertoli cells by intracellular signals that perturb Ca(2+) homeostasis. This role may be related to an effect exerted by the seminiferous epithelium basement membrane on the regulation of spermatogenesis.     

In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase

Adding the membrane-permeant oxidant tert-butylhydroperoxide (t-BOOH) to the incubation medium, in SH-SY5Y human neuroblastoma cells, induced a marked and progressive concentration-dependent (300, 500 and 1000 microM) increase of free radical production, as evaluated by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and of the intracellular Ca(2+) ion concentrations [Ca(2+)](i). The removal of extracellular Ca(2+) ions did not prevent t-BOOH-induced [Ca(2+)](i) elevation, whereas the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy) ethane-N,N, N',N'-tetraacetic acid (BAPTA) (10 microM) was shown to be effective. Both t-BOOH-induced free radical formation and the [Ca(2+)](i) increase were completely prevented by the peroxyl scavenger alpha-tocopherol (50 microM). t-BOOH induced a time-dependent SH-SY5Y cell injury, monitored by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (approximately 25% at 1 h, 50% at 3 h, 80% at 5 h) and by fluorescein diacetate (FDA)-propidium iodide (PI) fluorescent staining. The entity of t-BOOH-induced cell damage was the same both in the absence and in the presence of the intracellular Ca(2+) ion chelator BAPTA. By contrast, the peroxyl scavenger alpha-tocopherol (50 microM) completely prevented cell injury due to oxidative stress. Finally, superoxide dismutase (SOD) (500 ng/ml) caused a 30% reduction of t-BOOH-induced 2', 7'-dichlorofluorescein (DCF) fluorescence, whereas it did not modify the extent of cell injury produced by the oxidant. Collectively, the results of the present study demonstrated that in SH-SY5Y human neuroblastoma cells, the rise of [Ca(2+)](i) which occurs during oxidative stress is not involved in cell injury. Therefore, oxidative stress-induced cell death may be exclusively attributed to free radical overproduction.     

Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.     

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.     

Involvement of K(+) channels in the inhibitory effects of adenosine on anoxia-induced [Ca(2+) ](i) increase in cultured rat hippocampal CA1 neurons.

Changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in cultured hippocampal CA1 neurons isolated from newborn rats were measured by a confocal laser scanning microscope, using the Ca(2+) indicator Fluo-3. The results showed that exogenous adenosine (100 microM) significantly attenuated the increase of neuronal [Ca(2+)](i) induced by acute anoxia. This effect of adenosine could be suppressed by the adenosine A1 receptor antagonist 8-cyclopentyltheophylline. Moreover, potassium channel blockers, aminopyridine, and glipizide could also block the inhibitory role of adenosine, but tetraethylammonium had no effect. These results suggest that adenosine may activate 4-AP or ATP-sensitive potassium channels via an A1-receptor-mediated mechanism and consequently inhibit anoxia-induced [Ca(2+)](i) elevation in hippocampal neurons.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.     

Critical role of sodium in cytosolic [Ca2+] elevations in cultured hippocampal CA1 neurons during anoxic depolarization

Although the extent of ischemic brain damage is directly proportional to the duration of anoxic depolarization (AD), the mechanism of cytosolic [Ca(2+)] ([Ca(2+)](c)) elevation during AD is poorly understood. To address the mechanism in this study, [Ca(2+)](c) was monitored in cultured rat hippocampal CA1 neurons loaded with a Ca-sensitive dye, fura-2FF, and exposed to an AD-simulating medium containing (in mmol/L): K(+) 65, Na(+) 50, Ca(2+) 0.13, glutamate 0.1, and pH reduced to 6.6. Application of this medium promptly elevated [Ca(2+)](c) to about 30 micromol/L, but only if oxygen was removed, the respiratory chain was inhibited, or if the mitochondria were uncoupled. These high [Ca(2+)](c) elevations depended on external Ca(2+) and could not be prevented by inhibiting NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors, or gadolinium-sensitive channels. However, they could be prevented by removing external Na(+) or simultaneously inhibiting NMDA and AMPA/kainate receptors; 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), an inhibitor of plasmalemmal Na(+)/Ca(2+) exchanger, partly suppressed them. The data indicate that the [Ca(2+)](c) elevations to 30 micromol/L during AD result from Na(+) influx. Activation of either NMDA or AMPA/kainate channels provides adequate Na(+) influx to induce these [Ca(2+)](c) elevations, which are mediated by KB-R7943-sensitive and KB-R7943-resistant mechanisms.