Inhibitor Of Endoplasmic Reticulum Ca2 Research Articles

The role of mitochondria in the regulation of calcium influx into Jurkat cells.

In electrically nonexcitable cells the activity of the plasma membrane calcium channels is controlled by events occurring in mitochondria, as well as in the lumen of the endoplasmic reticulum. Thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, produces the release of calcium from the endoplasmic reticulum and thus, activation of store-operated calcium channels in the plasma membrane. However, thapsigargin failed to produce significant activation of the channels in Jurkat cells that had been pretreated with mitochondria-directed agents: an uncoupler (carbonyl cyanide m-chlorophenylhydrazone) and oligomycin. This is in spite of the fact that Jurkat cells pretreated with carbonyl cyanide m-chlorophenylhydrazone plus oligomycin are otherwise energetically competent, due to a high rate of glycolysis and the inhibition of mitochondrial F1Fo-ATPase by oligomycin. The pool of intracellular ATP was found not to be influenced by the pretreatments of cells with oligomycin or with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone. In the control cells, we found that the ATP pool amounted to 23.2 +/- 1.9 nmoles per 107 cells (n = 4). In cells pretreated with oligomycin the level of ATP was 21.8 +/- 1.9 nmoles per 107 cells (n = 4), and in cells pretreated with both oligomycin and an uncoupler the level of ATP was 22.1 +/- 0.2 nmoles per 107 cells (n = 3). Moreover, in cells pretreated with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone and suspended in a nominally calcium-free medium, thapsigargin produces transient increases in cytosolic calcium identical to those in the control cells. Thus, this pretreatment does not modify either the content of intracellular calcium stores and/or the activity of calcium ATPase in the plasma membrane. Similar results were obtained when Jurkat cells were challenged by myxothiazol, a potent inhibitor of mitochondrial cytochrome bc1 oxidoreductase. Thapsigargin, although producing calcium release from intracellular stores, was ineffective in triggering the activation of calcium channels in the plasma membrane in the case of cells pretreated with myxothiazol and oligomycin. Our results suggest that coupled mitochondria participate directly in the control of calcium channel activity in the plasma membrane of Jurkat cells. When the mitochondrial protonmotive force is collapsed, either by carbonyl cyanide m-chlorophenylhydrazone or myxothiazol, the channel remains inactive even under conditions of empty intracellular calcium stores.

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells.

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.

Calcium release from internal stores is required for the generation of spontaneous hyperpolarizations in dopaminergic neurons of neonatal rats.

We recently have demonstrated the existence of spontaneous hyperpolarizations in midbrain dopaminergic neurons of neonatal but not adult rats. These events are mediated by the opening of apamin-sensitive K(+) channels after a rise in the intracellular concentration of Ca(2+). They are resistant to tetrodotoxin in most cases and are probably endogenous (i.e., not synaptically activated). Here their mechanism was investigated. Cyclopiazonic acid (10 microM), a specific inhibitor of endoplasmic reticulum Ca(2+) ATPases, reversibly abolished the events. Caffeine, which promotes Ca(2+) release from intracellular stores, had concentration-dependent effects. At 1 mM, it markedly and steadily increased the frequency and the amplitude of the hyperpolarizations. At 10 mM, it induced a transient increase in their frequency followed by their cessation. All these effects were quickly reversible. Ryanodine (10 microM), which decreases the conductance of Ca(2+) release channels, irreversibly blocked the spontaneous hyperpolarizations. Dantrolene (100 microM), a blocker of Ca(2+) release from sarcoplasmic reticulum of striated muscle, did not affect the events. On the other hand, Cd(2+) (100-300 microM), a broad antagonist of membrane voltage-gated Ca(2+) channels, significantly reduced the amplitude and the frequency of the hyperpolarizations. However, when the frequency of the events was increased by 1 mM caffeine, Cd(2+) affected them to a smaller extent, whereas cyclopiazonic acid still abolished them. We conclude that internal stores are the major source of Ca(2+) ions that induce the K(+) channel openings underlying the spontaneous hyperpolarizations of these neurons.

The Effect of GHRP‐6 on the Intracellular Na+ Concentration of Rat Pituitary Cells in Primary Culture

The objective of the present study was to further investigate the ionic mechanism of the action of GHRP-6 on male rat pituitary cells in culture. A synthetic hexapeptide, GHRP-6 stimulates the secretion of growth hormone both in vivo and in vitro. It is generally accepted that Ca2+ and protein kinase C but not cAMP are involved in the signal transduction pathway of the action of GHRP-6. Ca2+-influx through voltage-gated Ca2+ channels and mobilization of internal stored Ca2+ are thought to be responsible for an increase in cytosolic Ca2+ concentration. For activation of the voltage-gated Ca2+ channels, however, it is not determined whether the membrane Na+ permeability plays a role. To answer this question, we measured intracellular Na+ concentration of the pituitary cells with ion imaging technique. We found that GHRP-6 increased [Na+]i; the Na+ response depended on the presence of extracellular Na+ and was blocked by Gd3+, known as a blocker of nonselective cation channels but not by tetrodotoxin, a blocker of the voltage-gated Na+ channel; thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, had no effect on the response; Ca2+ chelating agent, BAPTA had no inhibitory effect on the response; ouabain, an inhibitor of Na+-K+ ATPase, did not block the rise in [Na+]i induced by GHRP-6; somatostatin, which hyperpolarizes the cells by activating K+ channels, suppressed the response. These data clearly showed that GHRP-6 increased [Na+]i in the rat pituitary cells including somatotrophs. The rise in [Na+]i is likely to be due to an increase in the membrane Na+ permeability which should depolarize the cells, thereby activating the voltage-gated Ca2+ channels. This process leads to an influx of Ca2+ and subsequent increase in [Ca2+]i which results in an exocytotic release of GH.

Functional characterization of thapsigargin and agonist-insensitive acidic Ca2+stores in Drosophila melanogaster S2 cell lines

The role of acidic intracellular calcium stores in calcium homeostasis was investigated in theDrosophila Schneider cell line 2 (S2) by means of free cytosolic calcium ([Ca2+]i) and intracellular pH (pHi) imaging together with measurements of total calcium concentrations within intracellular compartments. Both a weak base (NH4Cl, 15 mM) and a Na+/H+ionophore (monensin, 10 μM) evoked cytosolic alkalinization followed by Ca2+release from acidic intracellular Ca2+stores. Pretreatment of S2 cells with either thapsigargin (1 μM), an inhibitor of endoplasmic reticulum Ca2+-ATPases, or with the Ca2+ionophore ionomycin (10 μM) was without effect on the amplitude of Ca2+release evoked by alkalinization. Application of the cholinergic agonist carbamylcholine (100 μM) to transfected S2-DM1 cells expressing a Drosophila muscarinic acetylcholine receptor (DM1) emptied the InsP3-sensitive Ca2+store but failed to affect the amplitude of alkalinization-evoked Ca2+release. Glycyl-L-phenylalanine-β-naphthylamide (200 μM), a weak hydrophobic base known to permeabilize lysosomes by osmotic swelling, triggered Ca2+release from internal stores, while application of brefeldin A (10 μM), an antibiotic which disperses the Golgi complex, resulted in a smaller increase in [Ca2+]i. These results suggest that the alkali-evoked calcium release is largely attributable to lysosomes, a conclusion that was confirmed by direct measurements of total calcium content of S2 organelles. Lysosomes and endoplasmic reticulum were the only organelles found to have concentrations of total calcium significantly higher than the cytosol. However, NH4Cl (15 mM) reduced the level of total calcium only in lysosomes. Depletion of acidic Ca2+stores did not elicit depletion-operated Ca2+entry. They were refilled upon re-exposure of cells to normal saline ([Ca2+]o= 2 mM), but not by thapsigargin-induced [Ca2+]ielevation in Ca2+-free saline.

ATP-induced inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells: lack of involvement of known purinoceptor-coupled signaling pathways.

P2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca2+]i, activation of phospholipases C (PLC) and A2 (PLA2), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na+,K+,Cl- cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier ( approximately 50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca2+]i that was abolished by a chelator of Ca2+i, BAPTA. However, neither BAPTA nor the Ca2+ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca2+-pump, thapsigargin, modified ATP-induced inhibition of Na+,K+, Cl- cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4, 5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na+,K+, Cl- cotransport activity. Inhibitors of PLA2 (AACOCF3), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na+,K+,Cl- cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na+ channels (amiloride), Cl- channels (NPPB) and Na+/H+ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na+,K+,Cl- cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na+,K+,Cl- cotransport and suggest the presence of a novel P2-receptor-coupled signaling mechanism.

Nitric Oxide Is Involved in Apoptosis Induced by Thapsigargin in Rat Mesangial Cells

The purpose of this study was to examine the mechanisms of thapsigargin-induced apoptosis in rat glomerular mesangial cells and the possible involvement of nitric oxide (NO) in this process. In mesangial cell monolayers incubated for 12 h in a medium without growth factors and with 10^–6 M thapsigargin, a known specific inhibitor of endoplasmic reticulum Ca²⁺-ATPase, a high percentage of cells showed typical nuclear features of apoptosis, assessed either by staining with propidium iodide (23 vs. 9% in control conditions) or by terminal desoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL; 17 vs. 5% in control conditions). When cells were maintained in a medium containing 10% fetal calf serum (FCS) or in a free-calcium medium, the thapsigargin-induced apoptosis rate was very low. In rat mesangial cells treatment with thapsigargin decreased the expression of BCL-2 protein and bcl-2 mRNA, whereas it did not alter the levels of BAX protein or bax mRNA. When mesangial cells were incubated with thapsigargin in the absence of FCS, we detected a significant increase in nitrite production (3.78 ± 0.96 vs. 1.76 ± 0.44 μmol/well). Furthermore, the treatment with the NO synthesis inhibitor L-NAME (10^–4 M) induced a significant decrease in the number of apoptotic cells (9%), whereas incubation with the NO donor SIN-1 (10^–5 M) induced a marked increase in the rate of apoptosis (29%). Western and Northern blot analysis of macrophage-type inducible NO synthase (iNOS) demonstrated that thapsigargin treatment induces the expression of the iNOS protein and iNOS mRNA. Treatment with L-NAME prevented the thapsigargin-induced BCL-2 decrease, whereas incubation with SIN-1 potentiated the effect of thapsigargin on BCL-2. Double labelling by immunohistochemistry for iNOS and TUNEL revealed that the same cells that suffered apoptosis were positive for iNOS. In summary, our results indicate that thapsigargin is able to enhance the apoptosis rate of rat mesangial cells by a mechanism that is mediated by an increase in cytosolic free calcium. Increased iNOS expression, and hence increased NO production, seems to be involved in this effect.

Polarized Ca2+ release in saponin-permeabilized parotid acinar cells evoked by flash photolysis of 'caged' inositol 1,4,5-trisphosphate.

In exocrine acinar cells, agonist stimulation results in a polarized Ca2+ signal, termed the 'Ca2+ wave', that propagates from the apical pole towards the basolateral region. We attempted to detect the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ wave in saponin-permeabilized rat parotid acinar cells using a digital imaging system. The permeabilized acinar cells were labelled with the membrane-bound Ca2+ indicator Calcium Green C18 to detect changes in Ca2+ concentration adjacent to the membrane of intracellular organelles. Application of InsP3 was made by the photolysis of InsP3 P4(5)-1-(2-nitrophenyl)ethyl ester (caged InsP3) to expose simultaneously all regions of the permeabilized acinar cells to InsP3. The increase in fluorescence ratio following the photolysis of 0.5 microM caged InsP3 started at the apical region of the acinar cells within 0.1 s and spread towards the basolateral region, indicating that Ca2+ release from intracellular Ca2+ stores was initially evoked at the apical region. Pretreatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pumps, failed to prevent the InsP3-induced Ca2+ wave, suggesting that the generation of the Ca2+ wave is not attributed to the polarized distribution of the Ca2+ pumps. The photolysis of a high concentration (10 microM) of caged InsP3 caused a homogeneous increase in the fluorescence ratio throughout the cells, indicating that all regions of intracellular Ca2+ stores similarly responded to the high concentration of InsP3. The present study is the first demonstration of the InsP3-induced Ca2+ wave in permeabilized exocrine acinar cells. The result provides fresh evidence that the apical region contains elements of intracellular Ca2+ stores particularly sensitive to InsP3 and that the Ca2+ wave results from a polarized distribution of InsP3-sensitive Ca2+ stores.

PROPIONIC ACID STIMULATES SUPEROXIDE GENERATION IN HUMAN NEUTROPHILS

Short-chain carboxylic acids are the metabolic by-products of pathogenic anaerobic bacteria and are found at sites of infection in millimolar quantities. We previously reported that propionic acid, one of the short-chain carboxylic acids, induces an increase in intracellular Ca2+([Ca2+]i) in human neutrophils. Here we investigate the effect of propionic acid on superoxide generation in human neutrophils. Propionic acid (10mm) induced inositol 1,4,5-trisphosphate (IP3) formation and a rapidly transient increase in [Ca2+]i, but not superoxide generation, whereas 1μmformylmethionyl-leucyl-phenylalanine (fMLP), a widely used neutrophil-stimulating bacterial peptide, stimulated not only IP3formation and Ca2+mobilization but also superoxide generation. The IP3level induced by propionic acid was slightly lower than that induced by fMLP. The transient increase in [Ca2+]iinduced by propionic acid immediately returned to the basal level, whereas a sustained increase in [Ca2+]i, which was higher than the basal level, following a transient increase in [Ca2+]iwas induced by fMLP. The peak level induced by propionic acid was lower than that with fMLP. In the absence of extracellular Ca2+, thapsigargin, a potent inhibitor of endoplasmic reticulum Ca2+-ATPase, induced an increase in [Ca2+]ieven after propionic acid stimulation, but not after fMLP. The Ca2+ionophore A23187 and thapsigargin induced superoxide generation by themselves. Propionic acid enhanced the superoxide generating effect of A23187 and thapsigargin. These results suggest that Ca2+mobilization induced by propionic acid is much weaker than that with fMLP, and propionic acid is able to generate superoxide in the presence of a Ca2+ionophore and a Ca2+influx activator.

Molecular Cloning and Functional Characterization of a Novel Receptor-activated TRP Ca2+ Channel from Mouse Brain

Characterization of mammalian homologues of Drosophila TRP proteins, which induce light-activated Ca2+ conductance in photoreceptors, has been an important clue to understand molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. We have here isolated cDNA that encodes a novel TRP homologue, TRP5, predominantly expressed in the brain. Recombinant expression of the TRP5 cDNA in human embryonic kidney cells dramatically potentiated extracellular Ca2+-dependent rises of intracellular Ca2+ concentration ([Ca2+]i) evoked by ATP. These [Ca2+]i transients were inhibited by SK&F96365, a blocker of receptor-activated Ca2+ entry, and by La3+. Expression of the TRP5 cDNA, however, did not significantly affect [Ca2+]i transients induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPases. ATP stimulation of TRP5-transfected cells pretreated with thapsigargin to deplete internal Ca2+ stores caused intact extracellular Ca2+-dependent [Ca2+]i transients, whereas ATP suppressed [Ca2+]i in thapsigargin-pretreated control cells. Furthermore, in ATP-stimulated, TRP5-expressing cells, there was no significant correlation between Ca2+ release from the internal Ca2+ store and influx of extracellular Ca2+. Whole-cell mode of patch-clamp recording from TRP5-expressing cells demonstrated that ATP application induced a large inward current in the presence of extracellular Ca2+. Omission of Ca2+ from intrapipette solution abolished the current in TRP5-expressing cells, whereas 10 nM intrapipette Ca2+ was sufficient to support TRP5 activity triggered by ATP receptor stimulation. Permeability ratios estimated from the zero-current potentials of this current were PCa:PNa:PCs = 14.3:1. 5:1. Our findings suggest that TRP5 directs the formation of a Ca2+-selective ion channel activated by receptor stimulation through a pathway that involves Ca2+ but not depletion of Ca2+ store in mammalian cells.

Calcium and protein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells.

Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inhibits VSMC tone, migration, adhesion, and proliferation, we examined the effects of high glucose on IL-1beta-induced NO release from VSMCs in culture. Confluent smooth muscle cells, preincubated with either 5 mmol/L (mM) or 20 mmol/L (mM) glucose for 48 hours, were stimulated with IL-1beta. Nitrite was measured in the culture medium after 24 hours. IL-1beta-induced a 15-fold increase in NO production in normal glucose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1beta. High glucose (20 mmol/L (mM)) inhibited IL-1beta-evoked NO production by approximately 50%. IL-1beta-stimulated [3H] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was reflected in diminished cellular levels of NOS protein. To assess the role of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting of the membranes revealed a glucose-induced increase in the PKC betaII isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high-glucose effect on IL-1beta-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediated inhibition of cytokine-induced NO release was determined by treatment with BAPTA, an intracellular chelator of calcium. BAPTA partially reversed the inhibitory effects of glucose. Increasing intracellular calcium by A23187, an ionophore or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, significantly decreased IL-1beta-induced NO release and NOS expression. These results indicate that glucose-induced inhibition of IL-1beta-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.

Energy turnover of vascular endothelial cells.

Two noninvasive methods, calorimetry and 31P nuclear magnetic resonance (NMR), were used to further define energy-consuming and energy-providing reactions in endothelial cells. With 31P-NMR, cellular ATP content was measured; with calorimetry, heat flux as a result of ATP turnover was measured. For these measurements, pig aortic endothelial cells were cultured on microcarrier beads and perfused in a column at constant flow rate. Pig aortic endothelial cells synthesize ATP mainly through glycolysis and, as determined by NMR, contain no phosphocreatine. In such a system, calorimetry-measured heat flux reflects rate of cellular ATP turnover. By use of inhibitors of ATP-dependent processes, the following changes in basal heat flux (231 +/- 65.5 microW/mg protein) were obtained: 18% for 2,3-butanedione monoxime (inhibitor of actomyosin-ATPase), 17% for wortmannin (inhibitor of myosin light chain kinase), 10% for cytochalasin D (inhibitor of actin polymerization), 23% for cycloheximide (inhibitor of protein synthesis), 11% for thapsigargin (inhibitor of endoplasmic reticulum Ca(2+)-ATPase), and 6% for bafilomycin A1 (inhibitor of lysosomal H(+)-ATPase). Cytochalasin D, 2,3-butanedione monoxime, wortmannin, and thapsigargin caused changes in F-actin distribution, as revealed by rhodamine-phalloidin cytochemistry. In a separate experimental series, when cells were perfused with a medium containing no glucose, heat flux decreased by 40% while cellular ATP remained unchanged. Inhibition of glycolysis with 2-deoxy-D-glucose decreased heat flux by 73%, and ATP was no longer visible with 31P-NMR. Despite this massive ATP depletion, which was maintained for 3 h, cells fully recovered heat flux and ATP when 2-deoxy-D-glucose was removed. The results, together with previously published data for Na(+)-K(+)-ATPase [M. L. H. Gruwel, C. Alves, and J. Schrader. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H351-H358, 1995], demonstrate that > 70% of total ATP-consuming processes of endothelial cells can be attributed to specific cellular processes. Actomyosin-ATPase (18%) and protein synthesis (23%) comprise the largest fraction. At least three-fourths of ATP synthesized is provided by glycolysis. Endothelial cells exhibit the remarkable ability to coordinate downregulation of ATP synthesis and consumption when glycolysis is inhibited.

Extracellular ATP Activates Calcium Signaling, Ion, and Fluid Transport in Retinal Pigment Epithelium

The presence of receptors for ATP has not been established in any native preparation of retinal neurons or glia. In the present study, we used conventional electrophysiological and [Ca2+]in fluorescence imaging techniques to investigate the effects of ATP added to Ringer's solution perfusing the retinal-facing (apical) membrane of freshly isolated monolayers of bovine retinal pigment epithelium (RPE). ATP (or UTP) produced large, biphasic voltage and resistance changes with a Kd of approximately 5 microM for ATP and approximately 1 microM for UTP. Electrical and pharmacological evidence indicates that the first and second phases of the response are attributable to an increase in basolateral membrane Cl conductance and a decrease in apical membrane K conductance, respectively. The ATP-induced responses were not affected by adenosine, but were reduced by the P2-purinoceptor blocker suramin. ATP also produced a large, transient increase in [Ca2+]in that was blocked by cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca2+-ATPases. The calcium buffer BAPTA attenuated the voltage effects of ATP. We also found that apical DIDS significantly inhibited the ATP-evoked [Ca2+]in and electrical responses, suggesting that DIDS blocked the purinoceptor. Measurements of fluid movement across the RPE using the capacitance probe technique demonstrated a significant increase in fluid absorption by apical UTP. These data indicate the presence of metabotropic P2Y/P2U-purinoceptors at the RPE apical membrane and implicate extracellular ATP in vivo as a retinal signaling molecule that could help regulate the hydration and chemical composition of the subretinal space.

The Mechanism of Rabbit Platelet Aggregation Induced by 2,5-Di-(tert-butyl)-l,4-benzohydroquinone, an Inhibitor of Endoplasmic Reticulum Ca2+-ATPase

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), an inhibitor of endoplasmic reticulum Ca2+-ATPase, caused aggregation accompanied by a marked increase in intracellular Ca2+ concentrations ([Ca2+]i) in washed rabbit platelets. Treatment of platelets with SK&F96365 or removal of extracellular Ca2+ inhibited both Ca2+ influx and platelet aggregation induced by tBuBHQ. These responses of platelets to tBuBHQ were also inhibited by genistein. Western blots using antibody against phosphorylated amino acid residues revealed that tBuBHQ activated tyrosine kinase independently of extracellular Ca2+. These results suggest that tBuBHQ induces Ca2+ influx into platelets probably through activation of tyrosine kinase, resulting in platelet aggregation.

Nerve growth factor inhibits apoptosis induced by S-100 binding in neuronal PC12 cells

When grown for seven days in a medium containing nerve growth factor (100 ng/ml), 10% horse serum and 5% fetal bovine serum PC12 cells stopped dividing, extended neurites and assumed a neuronal phenotype. Withdrawal of nerve growth factor from these cells resulted in loss of neurites and apoptotic changes in many cells. The apoptotic changes were exacerbated if the cells were also exposed to 1–2 μM S-100, a calcium binding protein purified from bovine brain. After exposure to S-100, the PC12 cells underwent characteristic apoptotic changes. Within 2 h neurites retracted, the cell body shrunk and submembranous accumulation of condensed cytoplasmic material was observed. DNA ladders were present after 24–48 h and 60% of the cells became hypodiploid after 72 h. S-100 induced apoptosis by binding to specific sites (K d=189 nM) on PC12 cells and this caused a rise in [Ca 2+] i due to a transmembrane capacitative flux followed by the depletion of internal stores. This increase was reversed if 5 μM nifedipine, a specific L-type Ca 2+ channel inhibitor, was added to the medium after S-100 and completely abolished if the cells were pretreated with 5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+-ATPase. The presence of nerve growth factor in the culture medium completely blocked the apoptotic changes induced by S-100, probably due to interaction of nerve growth factor and S-100 at the same binding sites. These data indicate that nerve growth factor not only prevents apoptosis during cell development, but also apoptosis induced by endogenous substances such as S-100.

Lowering glucose depletes a thapsigargin-sensitive calcium pool and inhibits transcription of the S14 gene.

S14 is a nuclear protein that is rapidly and synergistically induced by glucose and thyroid hormone, and the level of it's messenger RNA correlates with hepatocyte and adipocyte lipogenesis. We previously reported that the calcium ionophore A23187 markedly inhibits the carbohydrate response of the S14 gene without inhibiting glucose metabolism. Because the calcium ionophore not only increased intracellular cytosolic free calcium but also depletes intracellular calcium stores, we examined which of these two possibilities accounts for the regulation of S14 gene transcription. We found that increasing cytosolic calcium with arginine vasopressin is insufficient to inhibit S14 gene transcription. Furthermore, reduction of intracellular calcium by addition of EGTA to medium containing A23187 leads to further inhibition of S14 transcription. Measurement of intracellular free calcium in indo-1-loaded hepatocytes showed no significant changes induced by high glucose. These results suggested that depletion of an intracellular pool of calcium by A23187 causes the inhibition of S14 transcription. Addition of thapsigargin, which depletes intracellular calcium pools by inhibition of endoplasmic reticulum Ca2+-ATPase, led to significant inhibition of glucose-regulated S14 transcription. Lastly, continuous incubation in 5.5 mM glucose depletes the thapsigargin-sensitive calcium pool. These studies imply that the ability of glucose to induce S14 transcription is related to a thapsigargin-sensitive calcium pool, and depletion of this pool by lowering glucose inhibits S14 transcription.

Roles of calcium and kinases in regulation of thrombin-stimulated preproendothelin-1 transcription.

The release of the vasoactive peptide endothelin-1 (ET-1) is Ca2+ dependent after thrombin stimulation; however, little is known about the pathways involved. We studied the importance of Ca(2+)-dependent signal transduction pathways on preproET-1 mRNA induction in human endothelial cells. Thrombin-mediated preproET-1 mRNA induction was inhibited after clamping of cytosolic free CA2+ concentration ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Chelation of extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also had a significant inhibitory effect on the induction of preproET-1 mRNA. The Ca2+ ionophore A23187 induced constitutive as well as thrombin-stimulated preproET-1 mRNA expression. Mobilization of Ca2+ stores into the cytosol by inhibition of endoplasmic reticulum Ca(2+)-adenosinetriphosphatase with thapsigargin was effective also in inducing preproET-1 mRNA. Calmodulin antagonists W-7 and calmidazolium, as well as Ca2+/calmodulin-dependent kinase II inhibitor KN-62, significantly reduced thrombin-induced preproET-1 mRNA. Inhibition by cyclosporin A of the Ca(2+)-calmodulin-dependent phosphatase calcineurin potentiated constitutive preproET-1 mRNA. These data suggest that, in human endothelial cells, thrombin-mediated preproET-1 gene induction is regulated by a stimulatory Ca2+/calmodulin kinase II-dependent pathway.

Ca2+ clearance mechanisms in isolated rat adrenal chromaffin cells.

1. Intracellular Ca2+ clearance mechanisms were studied in rat adrenal chromaffin cells, by measuring slow tail currents through small-conductance Ca(2+)-activated K+ channels and using indo-1 photometry following depolarization-induced Ca2+ loading. 2. Following several-hundred millisecond depolarizations, [Ca2+]i decayed in three phases. An initial fast decay was followed by a long-lasting, low plateau, then [Ca2+]i returned to the resting level slowly. 3. Replacement of external Na+ moderately slowed [Ca2+]i decay, indicating a contribution of plasma membrane Na(+)-Ca2+ exchange. 4. Raising external pH or application of extracellular Eosin of La3+ prolonged slow tail currents, indicating a contribution of plasma membrane Ca(2+)-ATPase to Ca2+ clearance. 5. Ca(2+)-induced Ca2+ release from caffeine-sensitive stores occurred during depolarization. 6. Inhibition of endoplasmic reticulum Ca(2+)-ATPase had little effect on Ca2+ clearance. 7. Slow tail currents and [Ca2+]i decay following 0.2 - 2 s depolarizations were much prolonged by mitochondrial inhibition with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or Ruthenium Red, which abolished the initial rapid decay and plateau of [Ca2+]i. 8. In conclusion, mitochondrial Ca2+ uptake plays a major role in Ca2+ clearance by rapidly and reversibly sequestering Ca2+ during depolarization-evoked Ca2+ loads.

Ca 2+] i oscillations induced by bradykinin in rat glioma cells associated with Ca 2+ store-dependent Ca 2+ influx are controlled by cell volume and by membrane potential

Long-term superfusion with bradykinin causes oscillations of cytosolic Ca 2+ activity ([Ca 2+] i) in Fura-2 loaded rat glioma cells. The [Ca 2+] i rise is associated with synchronous plasma membrane hyperpolarization oscillating with a frequency of 8–18 per min. The initial large transient [Ca 2+] , rise, induced immediately with bradykinin admission results from InsP 3-mediated Ca 2+ release, whereas the subsequent oscillations depend mainly on Ca 2+ influx, as demonstrated: (i) by blockade of [Ca 2+] i oscillations by reduction of [Ca 2+] ex, or addition of Ca 2+ channel blockers; and (ii) evidence from Mn 2+ quench experiments. Suppression of [Ca 2+] i oscillations with high K + depolarization and with block of Ca 2+-dependent K + channels proves that membrane hyperpolarization is required for Ca 2+ influx during the oscillation. Ca 2+ release from intracellular stores by inhibitors of endoplasmic reticulum Ca 2+ ATPase attenuates or blocks the [Ca 2+] i Oscillations. This suggests that bradykinin-induced Ca 2+ influx is controlled by the filling state of the stores. The [Ca 2+] i oscillations are suppressed by hypertonic medium and enhanced by hypotonic medium. Cell swelling enhances Ca 2+ influx. We propose the following model for generation of the oscillations in the glial cell line: InsP 3-induced Ca 2+ release from internal stores periodically evokes Ca 2+ influx through Ca 2+-permeable cation channels. Hype rpolarization of the plasma membrane due to the activation of Ca 2+-dependent K + channels enhances the Ca 2+ influx. The concomitant K + efflux could lead to cell shrinkage which suppresses Ca 2+ influx. Cell volume and membrane potential probably serve as feedback regulators during the [Ca 2+] i oscillations.

Thapsigargin‐ and cyclopiazonic acid‐induced endothelium‐dependent hyperpolarization in rat mesenteric artery

1. The present study was designed to determine whether putative, selective inhibitors of the Ca(2+)-pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium-dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique. 2. In tissues with endothelium, TSG (10(-8)-10(-5) M) caused sustained hyperpolarization in a concentration-dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10(-5) M) also hyperpolarized the smooth muscle membrane, an effect that was endothelium-dependent and long-lasting. 3. The hyperpolarizing responses to these agents were not affected by indomethacin or NG-nitro-L-arginine (L-NOARG). 4. In Ca(2+)-free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response. 5. In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium-dependent relaxations. L-NOARG significantly inhibited the relaxations to these agents, but about 40-60% of the total relaxation was resistant to L-NOARG. The L-NOARG-resistant relaxations were abolished by potassium depolarization. 6. These results indicate that TSG and CPA can cause endothelium-dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium-derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium-dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca(2+)-pump ATPase activity.