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.     

Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.     

Glucose stimulates Ca2+ influx and insulin secretion in 2-week-old beta-cells lacking ATP-sensitive K+ channels

In adult beta-cells glucose-induced insulin secretion involves two mechanisms (a) a K(ATP) channel-dependent Ca(2+) influx and rise of cytosolic [Ca(2+)](c) and (b) a K(ATP) channel-independent amplification of secretion without further increase of [Ca(2+)](c). Mice lacking the high affinity sulfonylurea receptor (Sur1KO), and thus K(ATP) channels, have been developed as a model of congenital hyperinsulinism. Here, we compared [Ca(2+)](c) and insulin secretion in overnight cultured islets from 2-week-old normal and Sur1KO mice. Control islets proved functionally mature: the magnitude and biphasic kinetics of [Ca(2+)](c) and insulin secretion changes induced by glucose, and operation of the amplifying pathway, were similar to adult islets. Sur1KO islets perifused with 1 mm glucose showed elevation of both basal [Ca(2+)](c) and insulin secretion. Stimulation with 15 mm glucose produced a transient drop of [Ca(2+)](c) followed by an overshoot and a sustained elevation, accompanied by a monophasic, 6-fold increase in insulin secretion. Glucose also increased insulin secretion when [Ca(2+)](c) was clamped by KCl. When Sur1KO islets were cultured in 5 instead of 10 mm glucose, [Ca(2+)](c) and insulin secretion were unexpectedly low in 1 mm glucose and increased following a biphasic time course upon stimulation by 15 mm glucose. This K(ATP) channel-independent first phase [Ca(2+)](c) rise was attributed to a Na(+)-, Cl(-)-, and Na(+)-pump-independent depolarization of beta-cells, leading to Ca(2+) influx through voltage-dependent calcium channels. Glucose indeed depolarized Sur1KO islets under these conditions. It is suggested that unidentified potassium channels are sensitive to glucose and subserve the acute and long-term metabolic control of [Ca(2+)](c) in beta-cells without functional K(ATP) channels.     

cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells

Calcium is a crucial regulator of many physiological processes such as cell growth, division, differentiation, cell death and apoptosis. In this study, we examined the effect of cGMP on agonist-induced [Ca(2+)](i) transient in isolated rat aortic endothelial cells. 100 microM ATP was applied to the cells bathed in a Ca(2+)-free physiological solution to induce a [Ca(2+)](i) transient that was caused by Ca(2+) release from intracellular stores. cGMP, which was applied after [Ca(2+)](i) reached its peak level, accelerated the falling phase of [Ca(2+)](i) transient. Pre-treatment of the cells with CPA abolished the accelerating effect of cGMP on the falling phase of [Ca(2+)](i) transient. The effect of cGMP was reversed by KT5823, a highly specific inhibitor of protein kinase G. Taken together, these data suggest that cGMP may reduce [Ca(2+)](i) level by promoting Ca(2+) uptake through sarcoplasmic/endoplasmic reticulum ATPase and that the effect of cGMP may be mediated by protein kinase G.     

VEGF and ATP act by different mechanisms to increase microvascular permeability and endothelial [Ca(2+)](i)

Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.     

Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway

Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.     

ATP induces intracellular calcium increases and actin cytoskeleton disaggregation via P2x receptors

The consequences of purinoceptor activation on calcium signalling, inositol phosphate metabolism, protein secretion and the actin cytoskeleton were demonstrated in the WRK-1 cell line. Extracellular ATP was used as a secretagogue to induce a rise in intracellular Ca(2+) concentration ([Ca(2+)](i)), acting via P2x purinergic receptors, which causes actin skeleton disaggregation and protein secretion. ATP bound specifically to purinergic receptors, with Ki of 0.8 microM. The magnitude order for binding of different nucleotides was alpha beta-Met-ATP >or= dATPalphaS > ATP >or= ADP > UTP > AMP > suramin. No increase in inositol phosphates (IPs) was observed after ATP application suggesting that the purinergic sites in WRK-1 cells are not of a P2y type. ATP (1-100 microM) caused a concentration-dependent increase in [Ca(2+)](i)(EC(50)= 30 microM). The responses were reproducible without any desensitization over several applications. The response to ATP was abolished when extracellular calcium ([Ca(2+)](e)) was reduced to 100 nM. A non-specific purinergic antagonist, suramin, reversibly inhibited the ATP-response suggesting that ATP is able to bind to P2x purinergic sites to trigger Ca(2+) entry and increase of [Ca(2+)](i). ATP induced a concentration-dependent disaggregation of actin and exocytotic release of proteins both, which were dependent upon [Ca(2+)](e). Similarly, alpha,beta-Met-ATP, a potent P2x agonist also stimulated Ca(2+) mobilization, actin network destructuration, and protein release. In the isolated rat neurohypophysial nerve terminals, ATP was shown to act as a physiological stimulus for vasopressin release via Ca(2+) entry through a P2x receptor [6]. Here, we show that in these nerve terminals, ATP is also able to induce actin disaggregation by a Ca(2+) dependent mechanism. Thus, actin cytoskeleton alterations induced by ATP through activation of P2x receptors could be a prelude to exocytosis.     

Altered Ca2+ handling of smooth muscle cells in aorta of apolipoprotein E-deficient mice before development of atherosclerotic lesions

To study the effect of hypercholesterolemia on vascular smooth muscle cell (VSMC) function, atherosclerosis-prone but plaque-free endothelium-denuded aortic rings (width 2mm) from C57Bl6 Wild Type (WT) and apolipoprotein E-deficient (apoE(-/-)) mice (age 4 months) were mounted in a myograph and loaded with Fura-2 AM to simultaneously measure free Ca(2+) ([Ca(2+)](i)) and force development. In comparison with WT, apoE(-/-) mice displayed higher basal [Ca(2+)](i). Moreover, the time constant of the second phase of the biphasic high K(+)-induced [Ca(2+)](i) response was significantly increased in apoE(-/-) compared to WT mice. This phase was abolished by treatment with cyclopiazonic acid (CPA), depleting sarcoplasmic reticulum (SR). Further investigation of SR dependent [Ca(2+)](i) handling with CPA and caffeine revealed no alteration of maximal SERCA or ryanodine receptor function. Inositol (1,4,5)-triphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) release was, however, significantly increased in apoE(-/-) mice compared to WT mice as established with phenylephrine and ATP. In Ca(2+)-free conditions the ATP-induced [Ca(2+)](i) was not altered. The ATP-induced store-operated Ca(2+) entry was, however, significantly increased in apoE(-/-) compared to WT mice. The results demonstrate that basal [Ca(2+)](i) levels and IP(3)R-mediated store-operated [Ca(2+)](i) release over the plasma membrane were elevated in hypercholesterolemic but plaque-free apoE(-/-) mice.     

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.     

Differences between mouse and rat pancreatic islets: succinate responsiveness,malic enzyme,and anaplerosis

Succinic acid methyl esters are potent insulin secretagogues in rat pancreatic islets, but they do not stimulate insulin release in mouse islets. Unlike rat and human islets, mouse islets lack malic enzyme and, therefore, are unable to form pyruvate from succinate-derived malate for net synthesis of acetyl-CoA. Dimethyl-[2,3-(14)C]succinate is metabolized in the citric acid cycle in mouse islets to the same extent as in rat islets, indicating that endogenous acetyl-CoA condenses with oxaloacetate derived from succinate. However, without malic enzyme, the net synthesis from succinate of the citric acid cycle intermediates citrate, isocitrate, and alpha-ketoglutarate cannot occur. Glucose and other nutrients that augment alpha-ketoglutarate formation are secretagogues in mouse islets with potencies similar to those in rat islets. All cycle intermediates can be net-synthesized from alpha-ketoglutarate. Rotenone, an inhibitor of site I of the electron transport chain, inhibits methyl succinate-induced insulin release in rat islets even though succinate oxidation forms ATP at sites II and III of the respiratory chain. Thus generating ATP, NADH, and anaplerosis of succinyl-CoA plus the four-carbon dicarboxylic acids of the cycle and its metabolism in the citric acid cycle is insufficient for a fuel to be insulinotropic; it must additionally promote anaplerosis of alpha-ketoglutarate or two intermediates interconvertible with alpha-ketoglutarate, citrate, and isocitrate.     

Effect of hydrogen peroxide and superoxide anions on cytosolic Ca2+: comparison of endothelial cells from large-sized and small-sized arteries

We compared the Ca(2+) responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H(2)O(2)) caused an increase in cytosolic Ca(2+) levels ([Ca(2+)](i)) in both cell types. The [Ca(2+)](i) rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP(3)) receptors. Removal of Ca(2+) from the bath also decreased the [Ca(2+)](i) rises in response to H(2)O(2). In addition, treatment of endothelial cells with H(2)O(2) reduced the [Ca(2+)](i) responses to subsequent challenge of ATP. The decreased [Ca(2+)](i) responses to ATP were resulted from a pre-depletion of intracellular Ca(2+) stores by H(2)O(2). Interestingly, we also found that Ca(2+) store depletion was more sensitive to H(2)O(2) treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca(2+)](i) rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H(2)O(2) but not by hydroxyl radical. H(2)O(2) contribution in HX-XO-induced [Ca(2+)](i) rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H(2)O(2) could induce store Ca(2+) release via phospholipase C-IP(3) pathway in endothelial cells. Resultant emptying of intracellular Ca(2+) stores contributed to the reduced [Ca(2+)](i) responses to subsequent ATP challenge. The [Ca(2+)](i) responses were more sensitive to H(2)O(2) in endothelial cells of small-sized arteries than those of large-sized arteries.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

Role of intracellular stores in the regulation of rhythmical [Ca2+]i changes in interstitial cells of Cajal from rabbit portal vein

Interstitial cells of Cajal (ICCs) freshly isolated from rabbit portal vein and loaded with the Ca(2+)-sensitive indicator fluo-3 revealed rhythmical [Ca(2+)](i) changes occurring at 0.02-0.1 Hz. Each increase in [Ca(2+)](i) originated from a discrete central region of the ICC and propagated as a [Ca(2+)](i) wave towards the cell periphery, but usually became attenuated before reaching the ends of the cell. In about 40% of ICCs each rhythmical change in [Ca(2+)](i) consisted of an initial [Ca(2+)](i) increase (phase 1) followed by a faster rise in [Ca(2+)](i) (phase 2) and then a decrease in [Ca(2+)](i) (phase 3); the frequency correlated with the rate of rise of [Ca(2+)](i) during phase 1, but not with the peak amplitude. Rhythmical [Ca(2+)](i) changes persisted in nicardipine, but were abolished in Ca(2+)-free solution as well as by SK&F96365, cyclopiazonic acid, thapsigargin, 2-APB, xestospongin C or ryanodine. Intracellular Ca(2+) stores visualised with the low-affinity Ca(2+) indicator fluo-3FF were found to be enriched with ryanodine receptors (RyRs) detected with BODIPY TR-X ryanodine. Rhythmical [Ca(2+)](i) changes originated from a perinuclear S/ER element showing the highest RyR density. Immunostaining with anti-TRPC3,6,7 antibodies revealed the expression of these channel proteins in the ICC plasmalemma. This suggests that these rhythmical [Ca(2+)](i) changes, a key element of ICC pacemaking activity, result from S/ER Ca(2+) release which is mediated via RyRs and IP(3) receptors and is modulated by the activity of S/ER-Ca(2+)-ATPase and TRP channels but not by L-type Ca(2+) channels.     

P2Y-receptor regulates size of endothelial cells in an intracellular Ca2+ dependent manner

The effects of P2 receptor agonists on cell size and intracellular calcium levels, [Ca(2+)](i), was investigated using cultured endothelial cells isolated from the caudal artery of male Wistar rats. Cell size and [Ca(2+)](i) were measured using a phase-contrast and fluorescent confocal microscopic image analyzer and a Calcium Green fluorescence probe. P2Y receptor agonists, 2-methylthio ATP (2meS-ATP), ADP, UTP and ATP decreased the cell size and increased [Ca(2+)](i) in endothelial cells from rat caudal artery. However, alpha,beta-methylene ATP, a P2X receptor agonist, did not induce these responses. The decrease in size and the increase in [Ca(2+)](i), by 2meS-ATP were blocked by PPADS (P2-antagonist), suramin (P2-antagonist), thapsigargin (Ca(2+) pump inhibitor) and U-73122 (phospholipase C inhibitor). The present results show that activation of P2Y receptors, not P2X receptors, induces a decrease in cell size and an increase in [Ca(2+)](i), and the pharmacological properties of these two responses are the same. We concluded that the size of endothelial cells is regulated by P2Y receptors via intracelluar Ca(2+) derived from Ca(2+) stores.     

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.     

Glucose induces synchronous mitochondrial calcium oscillations in intact pancreatic islets

Mitochondria shape Ca(2+) signaling and exocytosis by taking up calcium during cell activation. In addition, mitochondrial Ca(2+) ([Ca(2+)](M)) stimulates respiration and ATP synthesis. Insulin secretion by pancreatic beta-cells is coded mainly by oscillations of cytosolic Ca(2+) ([Ca(2+)](C)), but mitochondria are also important in excitation-secretion coupling. Here, we have monitored [Ca(2+)](M) in single beta-cells within intact mouse islets by imaging bioluminescence of targeted aequorins. We find an increase of [Ca(2+)](M) in islet-cells in response to stimuli that induce either Ca(2+) entry, such as extracellular glucose, tolbutamide or high K(+), or Ca(2+) mobilization from the intracellular stores, such as ATP or carbamylcholine. Many cells responded to glucose with synchronous [Ca(2+)](M) oscillations, indicating that mitochondrial function is coordinated at the whole islet level. Mitochondrial Ca(2+) uptake in permeabilized beta-cells increased exponentially with increasing [Ca(2+)], and, particularly, it became much faster at [Ca(2+)](C)>2 microM. Since the bulk [Ca(2+)](C) signals during stimulation with glucose are smaller than 2 microM, mitochondrial Ca(2+) uptake could be not uniform, but to take place preferentially from high [Ca(2+)](C) microdomains formed near the mouth of the plasma membrane Ca(2+) channels. Measurements of mitochondrial NAD(P)H fluorescence in stimulated islets indicated that the [Ca(2+)](M) changes evidenced here activated mitochondrial dehydrogenases and therefore they may modulate the function of beta-cell mitochondria. Diazoxide, an activator of K(ATP), did not modify mitochondrial Ca(2+) uptake.     

Invited review: significance of spatial and temporal heterogeneity of calcium transients in smooth muscle.

The multiplicity of mechanisms involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca(2+)](i). Heterogeneity in [Ca(2+)](i) regulation is reflected by the presence of spontaneous, localized [Ca(2+)](i) transients (Ca(2+) sparks) representing Ca(2+) release through ryanodine receptor (RyR) channels. Ca(2+) sparks display variable spatial Ca(2+) distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca(2+)](i) that occasionally trigger propagating [Ca(2+)](i) waves. Ca(2+) sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca(2+)](i) oscillations that originate from foci with high spark incidence and also represent Ca(2+) release through RyR channels. With increasing agonist concentration, the peak of regional [Ca(2+)](i) oscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca(2+)](i), representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca(2+)](i) oscillations lies in the establishment of a global [Ca(2+)](i) level for slower Ca(2+)-dependent physiological processes.     

ATP regulation of type 1 inositol 1,4,5-trisphosphate receptor channel gating by allosteric tuning of Ca(2+) activation.

Inositol 1,4,5-trisphosphate (InsP(3)) mobilizes intracellular Ca(2+) by binding to its receptor (InsP(3)R), an endoplasmic reticulum-localized Ca(2+) release channel. Patch clamp electrophysiology of Xenopus oocyte nuclei was used to study the effects of cytoplasmic ATP concentration on the cytoplasmic Ca(2+) ([Ca(2+)](i)) dependence of single type 1 InsP(3)R channels in native endoplasmic reticulum membrane. Cytoplasmic ATP free-acid ([ATP](i)), but not the MgATP complex, activated gating of the InsP(3)-liganded InsP(3)R, by stabilizing open channel state(s) and destabilizing the closed state(s). Activation was associated with a reduction of the half-maximal activating [Ca(2+)](i) from 500 +/- 50 nM in 0 [ATP](i) to 29 +/- 4 nM in 9.5 mM [ATP](i), with apparent ATP affinity = 0.27 +/- 0.04 mM, similar to in vivo concentrations. In contrast, ATP was without effect on maximum open probability or the Hill coefficient for Ca(2+) activation. Thus, ATP enhances gating of the InsP(3)R by allosteric regulation of the Ca(2+) sensitivity of the Ca(2+) activation sites of the channel. By regulating the Ca(2+)-induced Ca(2+) release properties of the InsP(3)R, ATP may play an important role in shaping cytoplasmic Ca(2+) signals, possibly linking cell metabolic state to important Ca(2+)-dependent processes.     

Platelet hyperactivity and abnormal Ca(2+) homeostasis in diabetes mellitus

We sought to determine the mechanisms for hyperactivity and abnormal platelet Ca(2+) homeostasis in diabetes. The glycosylated Hb (HbA(1c)) level was used as an index of glycemic control. Human platelets were loaded with Ca- green-fura red, and cytosolic Ca(2+) ([Ca(2+)](i)) and aggregation were simultaneously measured. In the first series of experiments, the platelets from diabetic and normal subjects were compared for the ability to release Ca(2+) or to promote Ca(2+) influx. A potent and relatively specific inhibitor of Na(+)/Ca(2+) exchange, 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), increased the second phase of thrombin-induced Ca(2+) response, suggesting that the Na(+)/Ca(2+) exchanger works in the forward mode to mediate Ca(2+) efflux. In contrast, in the platelets from diabetics, CB-DMB decreased the Ca(2+) response, indicating that the Na(+)/Ca(2+) exchanger works in the reverse mode to mediate Ca(2+) influx. In the second series of experiments we evaluated the direct effect of hyperglycemia on platelets in vitro. We found that thrombin- and collagen-induced increases in [Ca(2+)](i) and aggregation were not acutely affected by high glucose concentrations of 45 mM. However, when the platelet-rich plasma was incubated with a high glucose concentration at 37 degrees C for 24 h, the second phase after thrombin activation was inhibited by CB-DMB. In addition, collagen-stimulated [Ca(2+)](i) response and aggregation were also increased. Thus in diabetes the direction and activity of the Na(+)/Ca(2+) exchanger is changed, which may be one of the mechanisms for the increased platelet [Ca(2+)](i) and hyperactivity. Prolonged hyperglycemia in vitro can induce similar changes, suggesting hyperglycemia per se may be the factor responsible for the platelet hyperactivity in diabetes.