Elevation Of Cytoplasmic Free Ca2 Research Articles

CADP-ribose potentiates cytosolic Ca2+ elevation and Ca2+ entry via L-type voltage-activated Ca2+ channels in NG108-15 neuronal cells

The effects of cADP-ribose (cADPR), a metabolite of beta-NAD(+), on the elevation of cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) influx through voltage-activated Ca(2+) channels (VACCs) were studied in NG108-15 neuroblastomaxglioma hybrid cells. NG108-15 cells were pre-loaded with fura-2 and whole-cell patch-clamped. Application of cADPR through patch pipettes did not by itself trigger any [Ca(2+)](i) rise at the resting membrane potential. A rise in [Ca(2+)](i) was evoked upon sustained membrane depolarization, and was significantly larger in cADPR-infused cells than in non-infused cells. This potentiation in the [Ca(2+)](i) elevation was reproduced by infusion of beta-NAD(+), and was blocked by 8-bromo-cADPR and antagonized by external application of ryanodine or by pretreatment of cells with FK506. Nicotinamide inhibited beta-NAD(+)-induced, but not cADPR-elicited, potentiation. [Ca(2+)](i) increases or Ca(2+) influx, measured by Mn(2+) quenching, elicited by the same protocol of depolarization was blocked completely by nifedipine but not by omega-conotoxin. Ca(2+) influx in cADPR- or beta-NAD(+)-infused cells was steeper and greater than that in control cells, and was inhibited partly by ryanodine. In contrast, ryanodine accelerated Ca(2+) influx in non-infused cells. These results show that cADPR amplifies both depolarization-induced [Ca(2+)](i) increase and Ca(2+) influx through L-type VACCs. These results suggest that cADPR functions on ryanodine receptors as a direct agonist and also interacts with L-type VACCs as an indirect agonist, i.e. via a retrograde signal.

Cooling inhibits exocytosis in single mouse pancreatic B-cells by suppression of granule mobilization.

1. The mechanisms by which cooling inhibits insulin secretion were investigated by capacitance measurements of exocytosis in single mouse pancreatic B-cells maintained in short-term tissue culture. 2. A reduction of the bath temperature from 34 to 24 degrees C produced a gradual inhibition of exocytosis. Inhibition of exocytosis was use dependent rather than time dependent. The steady-state inhibition amounted to 90%, which was paralleled by a 30% reduction of the peak Ca2+ current. 3. The Q10 values (between 27 and 37 degrees C) for inhibition of exocytosis and the peak Ca2+ current amplitude were determined as > 5 and 1.6, respectively. From the temperature dependence of exocytosis, an energy of activation was estimated as 145 kJ mol-1. 4. Suppression of exocytosis was not the result of a reduction of Ca2+ influx. When the Ca2+ currents were blocked by 30% (comparable to that produced by cooling) by using a low concentration of Co2+, exocytosis was reduced by < 25%. 5. Elevation of cytoplasmic free Ca2+, by photorelease of 'caged' Ca2+ from Ca(2+)-nitrophenyl-EGTA preloaded into the B-cell, was equally effective at eliciting exocytosis at 24 and 34 degrees C. 6. Cooling produced 70% inhibition of exocytosis evoked by infusion of Ca2+ through the recording electrode. Omission of either MgATP or cAMP from the electrode solution resulted in a comparable reduction of exocytosis. Cooling had no additional inhibitory effect when exocytosis was already suppressed by removal of cytoplasmic MgATP. 7. Our data indicate that exocytosis of granules already docked beneath the membrane is little affected by cooling in the B-cell. Instead, the high overall temperature sensitivity of insulin secretion arises because the replenishment of the readily releasable pool is temperature dependent.

Agonist- and CPA-induced elevation of cytoplasmic free Ca2+ in intact valvular endothelium from rabbits.

Intracellular Ca2+ signals of intact endothelium from rabbit aortic or pulmonic valves loaded with fura 2 were studied using imaging fluorescence microscopy. Agonists such as ATP or carbachol and inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase such as cyclopiazonic acid (CPA), thapsigargin, and 2', 5'-di(tert-butyl)-1, 4,-benzohydroquinone (BHQ) induced an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) that was maintained above the prestimulated levels as long as extracellular Ca2+ was present, indicating that the maintained [Ca2+]i increase is dependent on the entry of extracellular Ca2+. The voltage-gated channel was not found to contribute to [Ca2+]i increase. SK&F-96365, a receptor-operated cation channel (ROC) blocker, and 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), a postulated phospholipase C inhibitor, were shown to effectively block ROC, since they greatly reduced the maintained [Ca2+]i increase caused by ATP, but not that caused by CPA, which was blocked by Ni2+. To further investigate the Ca2+ influx involved in this process, divalent cation entry was measured as Mn2+ quenching of fura 2 fluorescence at 360-nm excitation wavelength. At rest, fluorescence quenching at 360 nm by Mn2+ was observed, which was inhibited by Ni2+ but not by NCDC, indicating Mn2+ entry through the leak pathway. The quenching was enhanced following ATP stimulation, and this enhancement was abolished by pretreatment with NCDC. In contrast, the rate of Mn2+ quenching was unaffected by the application of CPA. These results demonstrate that ATP stimulates divalent cation influx that is not related to the Ca2+ content of ER. Abolition of Ca2+ uptake into ER was postulated to increase the effectiveness of the Ca2+ leak in raising [Ca2+]i.

Thyrotropin-releasing hormone stimulation of phosphoinositide hydrolysis desensitizes. Evidence against mediation by protein kinase C or calcium.

Previous reports have provided conflicting evidence as to whether the response to TRH desensitizes. Here we show that TRH stimulation of phosphoinositide (PPI) hydrolysis, measured as inositol phosphate accumulation in the presence of LiCl, desensitizes in rat pituitary GH3 cells and in rat glioma C6 cells stably transfected with mouse pituitary TRH receptor complementary DNA. In GH3 cells, the rate of stimulation by 1000 nM TRH of PPI hydrolysis was maximal initially and then decreased by 44 +/- 13% after 20 min. In an experimental paradigm in which PPI hydrolysis was measured by adding 20 mM LiCl at different times after TRH, desensitizations caused by 3, 10, and 1000 nM TRH were 33 +/- 5%, 41 +/- 6%, and 69 +/- 2%, respectively. In transfected C6 cells, TRH-induced desensitization of 76 +/- 9% was found. In GH3 cells, 1 microM phorbol myristate acetate (PMA), an activator of protein kinase C, inhibited the initial response to TRH by 75 +/- 6% and preexposure to PMA and TRH decreased the rate of PPI hydrolysis by 98 +/- 1% after 60 min. One hundred micromolar H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine), an inhibitor of protein kinases, abolished the effect of PMA but did not inhibit TRH-induced desensitization. Elevation of cytoplasmic free Ca2+ by K+ depolarization increased TRH stimulation of PPI hydrolysis. We conclude that TRH stimulation of PPI hydrolysis acutely desensitizes and that this effect is not specific to pituitary cells. TRH-induced desensitization, moreover, does not appear to be mediated by protein kinase C or by elevation of cytoplasmic free Ca2+.

Thyrotropin-Releasing Hormone (TRH) and Phorbol Myristate Acetate Decrease TRH Receptor Messenger RNA in Rat Pituitary GH3Cells: Evidence That Protein Kinase-C Mediates the TRH Effect

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.

Receptor Density Determines Secretory Response Patterns Mediate by Inositol Lipid-Derived Second Messengers: Comparison of Thyrotropin-Releasing Hormone and Carbamylcholine actions in Thyroid-Stimulating Hormone-Secreting Mouse Pituitary Tumor Cells

Abstract Signal transduction by thyrotropin-releasing hormone (TRH) and carbamylcholine (CCH) in some cells is mediated by inositol lipid hydrolysis forming the second messengers, inositol 1,4,5-trisphosphate (I-1,4,5-P3) and 1,2-diacylglycerol, and causing elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i). In mouse thyrotropic tumor (TtT) cells, maximally effective doses of TRH caused biphasic stimulation of thyroid-stimulating hormone (TSH) secretion, whereas CCH stimulated monophasic sustained TSH secretion without a burst phase. TRH, at maximally effective doses, stimulated a rapid marked increase in I-1,4,5-P3 which was associated with a rapid elevation of [Ca2+]i to approximately 1000 nM, whereas maximally effective doses of CCH caused little increase in I-1,4,5-P3 and no burst elevation of [Ca2+]i. Both TRH and CCH caused sustained modest (to 210-280 nM) elevations of [Ca2+]i which were inhibited by voltage-sensitive channel-blocking agents and stimulated sustained hydrolysis of inositol lipids. CCH-like responses were observed when TtT cells were stimulated by low doses of TRH. In TtT cells prepared from five tumors, the ratio of the number of TRH receptors to muscarinic receptors ranged from 10 to 40:1. Lastly, CCH-like responses were observed with maximally effective doses of TRH when the TRH receptor number was down-regulated to a level similar to that of muscarinic receptors. These data suggest that the kinetic pattern of stimulated TSH secretion caused by secretagogues that use the inositol lipid signal transduction pathway is determined by the density of receptors. In particular, there appears to be a minimal number of receptor-ligand complexes which is required to generate rapidly sufficient I-1,4,5-P3 to release intracellular Ca2+ and cause a secretory burst.

Thyrotropin-releasing hormone stimulation of prolactin secretion is coordinately but not synergistically regulated by an elevation of cytoplasmic calcium and 1,2-diacylglycerol.

The precise roles of the calcium and lipid pathways in TRH-stimulated PRL secretion from rat pituitary (GH3) cells are controversial. In particular, it is debated whether elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i) is sufficient to cause burst secretion (0-2 min) or whether an increase in 1,2-diacylglycerol must accompany the Ca2+ elevation. In this study, the effects of TRH, which elevates 1,2-diacylglycerol, on [Ca2+]i and stimulation of burst secretion were compared with those of depolarization by high extracellular K+, which does not increase 1,2-diacylglycerol. A maximal concentration of TRH (1 microM) and depolarization by 17.5 mM K+ caused elevation of [Ca2+]i from the resting level of 140 +/- 20 nM to 470 +/- 70 nM and 514 +/- 60 nM, respectively, and stimulated burst secretion from 0.6 +/- 0.2 ng/10(6) cells/min to 3.3 +/- 0.8 and 3.1 +/- 0.4 ng/10(6) cells/min, respectively, when a small component of TRH-stimulated secretion that is independent of elevation of [Ca2+]i was subtracted. A detailed comparison of multiple levels to which [Ca2+]i was elevated (up to 600 nM) and the degree of stimulation of burst phase secretion demonstrated the same positive linear correlation (correlation coefficient = 0.96) for TRH and K+ depolarization. Hence, elevation of [Ca2+]i is sufficient to cause burst secretion irrespective of elevation of 1,2-diacylglycerol. Optimal stimulation by TRH of sustained secretion of PRL did not depend on elevation of [Ca2+]i; sustained PRL secretion stimulated by 10 nM TRH was 2.6 +/- 0.4 and 2.7 +/- 0.2 ng/10(6) cells/min in control cells and arachidonic acid-pretreated cells in which [Ca2+]i was not elevated, respectively. The data from this and previous studies demonstrate that elevation of [Ca2+]i and 1,2-diacylglycerol may act coordinately, but not synergistically, to mediate TRH stimulation of PRL secretion from GH3 cells.

Inositol trisphosphate mediates thyrotropin-releasing hormone mobilization of nonmitochondrial calcium in rat mammotropic pituitary cells.

Thyrotropin-releasing hormone (TRH) stimulation of prolactin secretion from GH3 cells, cloned rat pituitary tumor cells, is associated with 1) hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate (InsP3) and 2) elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i), caused in part by mobilization of cellular calcium. We demonstrate, in intact cells, that TRH mobilizes calcium and, in permeabilized cells, that InsP3 releases calcium from a nonmitochondrial pool(s). In intact cells, TRH caused a loss of 16 +/- 2.7% of cell-associated 45Ca which was not inhibited by depleting the mitochondrial calcium pool with uncoupling agents. Similarly, TRH caused an elevation of [Ca2+]i from 127 +/- 6.3 nM to 375 +/- 54 nM, as monitored with Quin 2, which was not inhibited by depleting mitochondrial calcium. Saponin-permeabilized cells accumulated Ca2+ in an ATP-dependent manner into a nonmitochondrial pool, which exhibited a high affinity for Ca2+ and a small capacity, and into a mitochondrial pool which had a lower affinity for Ca2+ but was not saturated under the conditions tested. Permeabilized cells buffered free Ca2+ to 129 +/- 9.2 nM when incubated in a cytosol-like solution initially containing 200 to 1000 nM free Ca2+. InsP3, but not other inositol sugars, released calcium from the nonmitochondrial pool(s); half-maximal effect occurred at approximately 1 microM InsP3. Ca2+ release was followed by reuptake into a nonmitochondrial pool(s). These data suggest that InsP3 serves as an intracellular mediator (or second messenger) of TRH action to mobilize calcium from a nonmitochondrial pool(s) leading to an elevation of [Ca2+]i and then to prolactin secretion.