ATP inhibits the hypoxia response in type I cells of rat carotid bodies

Summary During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of type I cells dissociated from rat carotid bodies using a Ca(2+) imaging technique. ATP did not affect the resting [Ca(2+)](i) but strongly suppressed the hypoxia-induced [Ca(2+)](i) elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2-methylthioATP > ATP > ADP > alpha, beta-methylene ATP > UTP, implicating the involvement of P2Y(1) receptors. Simultaneous measurements of membrane potential and [Ca(2+)](i) show that ATP inhibited the hypoxia-induced Ca(2+) signal by reversing the hypoxia-triggered depolarization. However, ATP did not oppose the hypoxia-mediated inhibition of the oxygen-sensitive TASK-like K(+) background current. Neither the inhibition of the large-conductance Ca(2+)-activated K(+) (maxi-K) channels nor the removal of extracellular Na(+) could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK-like K(+), maxi-K or Na(+) channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the oxygen sensing type I (glomus) cells of rat carotid bodies via reduction of a background TASK-like K+ current

Pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice are prone to sudden neonatal death and have reduced respiratory response to hypoxia. Here we found that PACAP-38 elevated cytosolic [Ca(2+)] ([Ca(2+)](i)) in the oxygen sensing type I cells but not the glial-like type II (sustentacular) cells of the rat carotid body. This action of PACAP could not be mimicked by vasoactive intestinal peptide but was abolished by PACAP 6-38, implicating the involvement of PAC(1) receptors. H89, a protein kinase A (PKA) inhibitor attenuated the PACAP response. Simultaneous measurement of membrane potential and [Ca(2+)](i) showed that the PACAP-mediated [Ca(2+)](i) rise was accompanied by depolarization and action potential firing. Ni(2+), a blocker of voltage-gated Ca(2+) channels (VGCC) or the removal of extracellular Ca(2+) reversibly inhibited the PACAP-mediated [Ca(2+)](i) rise. In the presence of tetraethylammonium (TEA) and 4-aminopyridine (4-AP), PACAP reduced a background K(+) current. Anandamide, a blocker of TWIK-related acid-sensitive K(+) (TASK)-like K(+) channel, occluded the inhibitory action of PACAP on K(+) current. We conclude that PACAP, acting via the PAC(1) receptors coupled PKA pathway inhibits a TASK-like K(+) current and causes depolarization and VGCC activation. This stimulatory action of PACAP in carotid type I cells can partly account for the role of PACAP in respiratory disorders.     

Oxygen sensing by the carotid body chemoreceptors.

Carotid bodies are sensory organs that detect changes in arterial blood oxygen, and the ensuing reflexes are critical for maintaining homeostasis during hypoxemia. During the past decade, tremendous progress has been made toward understanding the cellular mechanisms underlying oxygen sensing at the carotid body. The purpose of this minireview is to highlight some recent concepts on sensory transduction and transmission at the carotid body. A bulk of evidence suggests that glomus (type I) cells are the initial site of transduction and that they release transmitters in response to hypoxia, which causes depolarization of nearby afferent nerve endings, leading to an increase in sensory discharge. There are two main hypotheses to explain the transduction process that triggers transmitter release. One hypothesis assumes that a biochemical event associated with a heme protein triggers the transduction cascade. The other hypothesis suggests that a K(+) channel protein is the oxygen sensor and that inhibition of this channel by hypoxia leading to depolarization is a seminal event in transduction. Although there is body of evidence supporting and questioning each of these, this review will try to point out that the truth lies somewhere in an interrelation between the two. Several transmitters have been identified in glomus cells, and they are released in response to hypoxia. However, their precise roles in sensory transmission remain uncertain. It is hoped that future studies involving transgenic animals with targeted disruption of genes encoding transmitters and their receptors may resolve some of the key issues surrounding the sensory transmission at the carotid body. Further studies are necessary to identify whether a single sensor or multiple oxygen sensors are needed for the transduction process.     

Granule matrix property and rapid "kiss-and-run" exocytosis contribute to the different kinetics of catecholamine release from carotid glomus and adrenal chromaffin cells at matched quantal size

Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ~100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ~200-250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca(2+) ([Ca(2+)](i)) to ~0.5 μmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca(2+)](i) triggers a form of rapid "kiss-and-run" exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).     

Inhibition of H2 histamine receptor-mediated cation channel opening by protein kinase C in human promyelocytic cells

Histamine, through H(2) receptors, triggers a prominent rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in addition to an elevation of cAMP level in HL-60 promyelocytes. Here we show that the histamine-induced [Ca(2+)](i) rise was due to influx of Ca(2+) from the extracellular space, probably through nonselective cation channels, as incubation of the cells with SKF 96365 abolished the histamine-induced [Ca(2+)](i) rise, Na(+) influx, and membrane depolarization. The Ca(2+) influx was specifically inhibited by pretreatment of the cells with PMA or extracellular ATP with 50% inhibitory concentrations of 0.12 +/- 0.03 nM and 185 +/- 17 microM, respectively. Western blot analysis of protein kinase C (PKC) isoforms revealed that PMA (< or =1 nM) and ATP (300 microM) caused selective translocation of PKC-delta to the particulate/membrane fraction. Costimulation of the cells with histamine and SKF 96365 partially reduced histamine-induced granulocytic differentiation, which was evaluated by looking at the extent of fMet-Leu-Phe-induced [Ca(2+)](i) rise and superoxide generation. In conclusion, nonselective cation channels are opened by stimulation of the H(2) receptor, and the channels are at least in part involved in the induction of histamine-mediated differentiation processes. Both effects of histamine were selectively inhibited probably by the delta isoform of PKC in HL-60 cells.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. Calcium ions (Ca(2+)) play an important role in the differentiation and proliferation of hMSCs. We have demonstrated that spontaneous [Ca(2+)](i) oscillations occur without agonist stimulation in hMSCs. However, the precise mechanism of its generation remains unclear. In this study, we investigated the mechanism and role of spontaneous [Ca(2+)](i) oscillations in hMSCs and found that IP(3)-induced Ca(2+) release is essential for spontaneous [Ca(2+)](i) oscillations. We also found that an ATP autocrine/paracrine signaling pathway is involved in the oscillations. In this pathway, an ATP is secreted via a hemi-gap-junction channel; it stimulates the P(2)Y(1) receptors, resulting in the activation of PLC-beta to produce IP(3). We were able to pharmacologically block this pathway, and thereby to completely halt the [Ca(2+)](i) oscillations. Furthermore, we found that [Ca(2+)](i) oscillations were associated with NFAT translocation into the nucleus in undifferentiated hMSCs. Once the ATP autocrine/paracrine signaling pathway was blocked, it was not possible to detect the nuclear translocation of NFAT, indicating that the activation of NFAT is closely linked to [Ca(2+)](i) oscillations. As the hMSCs differentiated to adipocytes, the [Ca(2+)](i) oscillations disappeared and the translocation of NFAT ceased. These results provide new insight into the molecular and physiological mechanism of [Ca(2+)](i) oscillations in undifferentiated hMSCs.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

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.     

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.     

Does AMP-activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2-sensing cells?

Specialized O2-sensing cells exhibit a particularly low threshold to regulation by O2 supply and function to maintain arterial pO2 within physiological limits. For example, hypoxic pulmonary vasoconstriction optimizes ventilation-perfusion matching in the lung, whereas carotid body excitation elicits corrective cardio-respiratory reflexes. It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O2-sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca2+ signaling mechanisms remains elusive, and investigation of previous hypotheses has generated contrary data and failed to unite the field. We propose that a rise in the cellular AMP/ATP ratio activates AMP-activated protein kinase and thereby evokes Ca2+ signals in O2-sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with alpha1 beta2 gamma1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-alpha1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMP-activated protein kinase-alpha1 to be located throughout the cytoplasm of pulmonary arterial myocytes. In contrast, it was targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMP-activated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca2+ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca2+ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca2+ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.     

Ca2+-induced Ca2+ release from the endoplasmic reticulum amplifies the Ca2+ signal mediated by activation of voltage-gated L-type Ca2+ channels in pancreatic beta-cells

Stimulus-secretion coupling in pancreatic beta-cells involves membrane depolarization and Ca(2+) entry through voltage-gated L-type Ca(2+) channels, which is one determinant of increases in the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)). We investigated how the endoplasmic reticulum (ER)-associated Ca(2+) apparatus further modifies this Ca(2+) signal. When fura-2-loaded mouse beta-cells were depolarized by KCl in the presence of 3 mm glucose, [Ca(2+)](i) increased to a peak in two phases. The second phase of the [Ca(2+)](i) increase was abolished when ER Ca(2+) stores were depleted by thapsigargin. The steady-state [Ca(2+)](i) measured at 300 s of depolarization was higher in control cells compared with cells in which the ER Ca(2+) pools were depleted. The amount of Ca(2+) presented to the cytoplasm during depolarization as estimated from the integral of the increment in [Ca(2+)](i) over time (integralDelta[Ca(2+)](i).dt) was approximately 30% higher compared with that in the Ca(2+) pool-depleted cells. neo-thapsigargin, an inactive analog, did not affect [Ca(2+)](i) response. Using Sr(2+) in the extracellular medium and exploiting the differences in the fluorescence properties of Ca(2+)- and Sr(2+)-bound fluo-3, we found that the incoming Sr(2+) triggered Ca(2+) release from the ER. Depolarization-induced [Ca(2+)](i) response was not altered by, an inhibitor of phosphatidylinositol-specific phospholipase C, suggesting that stimulation of the enzyme by Ca(2+) is not essential for amplification of Ca(2+) signaling. [Ca(2+)](i) response was enhanced when cells were depolarized in the presence of 3 mm glucose, forskolin, and caffeine, suggesting involvement of ryanodine receptors in the amplification process. Pretreatment with ryanodine (100 microm) diminished the second phase of the depolarization-induced increase in [Ca(2+)](i). We conclude that Ca(2+) entry through L-type voltage-gated Ca(2+) channels triggers Ca(2+) release from the ER and that such a process amplifies depolarization-induced Ca(2+) signaling in beta-cells.     

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.     

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

Parallel oscillations of intracellular calcium activity and mitochondrial membrane potential in mouse pancreatic B-cells

Insulin secretion in normal B-cells is pulsatile, a consequence of oscillations in the cell membrane potential (MP) and cytosolic calcium activity ([Ca(2+)](c)). We simultaneously monitored glucose-induced changes in [Ca(2+)](c) and in the mitochondrial membrane potential DeltaPsi, as a measure for ATP generation. Increasing the glucose concentration from 0.5 to 15 mM led to the well-known hyperpolarization of DeltaPsi and ATP-dependent lowering of [Ca(2+)](c). However, as soon as [Ca(2+)](c) rose due to the opening of voltage-dependent Ca(2+) channels, DeltaPsi depolarized and thereafter oscillations in [Ca(2+)](c) were parallel to oscillations in DeltaPsi. A depolarization or oscillations of DeltaPsi cannot be evoked by a substimulatory glucose concentration, but Ca(2+) influx provoked by 30 mM KCl was followed by a depolarization of DeltaPsi. The following feedback loop is suggested: Glucose metabolism via mitochondrial ATP production and closure of K(+)(ATP) channels induces an increase in [Ca(2+)](c). The rise in [Ca(2+)](c) in turn decreases ATP synthesis by depolarizing DeltaPsi, thus transiently terminating Ca(2+) influx.