Inositol trisphosphate and ryanodine receptors share a common functional Ca2+ pool in cerebellar Purkinje neurons

Changes in the intracellular free calcium concentration ([Ca2+]i) control many important processes in excitable and nonexcitable cells. In cerebellar Purkinje neurons, increases in [Ca2+]i modulate excitability by turning on calcium-activated potassium and chloride conductances, and modifying the synaptic efficacy of inhibitory and excitatory inputs to the cell. Calcium release from the intracellular stores plays an important role in the regulation of [Ca2+]i. Purkinje neurons contain both inositol trisphosphate (InsP3) and ryanodine (Ry) receptors. With the exception of the dendritic spines, where only InsP3 receptors are found, InsP3 and Ry receptors are present in the entire cell. The distribution of the two calcium release channels, however, is not uniform, and it has been suggested that InsP3 and Ry receptors use separate Ca2+ pools. The functional properties of InsP3 and Ry Ca2+ pools were investigated by flash photolysis and single-cell microspectrofluorimetry. It was found that depletion of ryanodine-sensitive Ca2+ stores renders InsP3 incapable of releasing more Ca2+ from the stores. Abolishing calcium-induced calcium release by blocking ryanodine receptors with ruthenium red did not have a significant effect on InsP3-evoked Ca2+ release. It is concluded that InsP3 receptors use the same functional Ca2+ pool as that utilized by Ry receptors in Purkinje neurons.     

Synergistic release of Ca2+ from IP3-sensitive stores evoked by synaptic activation of mGluRs paired with backpropagating action potentials

Increases in postsynaptic [Ca2+]i can result from Ca2+ entry through ligand-gated channels or voltage-gated Ca2+ channels, or through release from intracellular stores. Most attention has focused on entry through the N-methyl-D-aspartate (NMDA) receptor in causing [Ca2+]i increases since this pathway requires both presynaptic stimulation and postsynaptic depolarization, making it a central component in models of synaptic plasticity. Here, we report that repetitive synaptic activation of metabotropic glutamate receptors (mGluRs), paired with backpropagating action potentials, causes large, wave-like increases in [Ca2+]i predominantly in restricted regions of the proximal apical dendrites and soma of hippocampal CA1 pyramidal neurons. [Ca2+]i changes of several micromolars can be reached by regenerative release caused by the synergistic effect of mGluR-generated inositol 1,4,5-trisphosphate (IP3) and spike-evoked Ca2+ entry acting on the IP3 receptor.     

Modifications of Ca2+ mobilization and noradrenaline release by S-nitroso-cysteine in PC12 cells

The effects of nitrogen monoxide (NO)-related compounds on cytosolic free Ca2+ concentrations ([Ca2+]i) and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. The addition of S-nitroso-cysteine (SNC) stimulated [Ca2+]i increases from an intracellular Ca2+ pool continuously in a concentration-dependent manner. Other NO donors, which stimulate cyclic GMP accumulation, did not cause [Ca2+]i increases. After treatment with 0.2 mM SNC, transient increases in [Ca2+]i from the Ca2+ pool induced by caffeine were completely abolished. The addition of N-ethylmaleimide (NEM) caused sustained [Ca2+]i increases from the intracellular Ca2+ pool. Furthermore, caffeine did not stimulate further [Ca2+]i increases in PC12 cells pretreated with NEM. These findings suggest that SNC and NEM predominantly interact with a caffeine-sensitive Ca2+ pool. The addition of dithiothreitol (DTT) to 0.4 mM SNC-stimulated cells reduced [Ca2+]i to basal levels, and the addition of DTT to NEM-stimulated cells locked [Ca2+]i at high levels. The stimulatory effects of SNC but not NEM were not abolished by pretreatment with DTT. These findings suggest that modification of the oxidation status of the sulfhydryl groups on the caffeine-sensitive receptors by SNC or NEM regulates Ca2+ channel activity in a reversible manner. SNC did not stimulate NA release by itself but did inhibit ionomycin-stimulated NA release. In contrast, NEM stimulated NA release in the absence of extracellular CaCl2 and further enhanced ionomycin-stimulated NA release. Ca2+ mobilization by SNC from the caffeine-sensitive pool was not a sufficient factor, and other factors stimulating NA release may be negatively regulated by SNC.     

The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization.

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.     

Supralinear Ca2+ signaling by cooperative and mobile Ca2+ buffering in Purkinje neurons

Endogenous high-affinity Ca2+ buffering and its roles were investigated in mouse cerebellar Purkinje cells with the use of a low-affinity Ca2+ indicator and a high-affinity caged Ca2+ compound. Increases in the cytosolic Ca2+ concentration ([Ca2+]i) were markedly facilitated during repetitive depolarization, resulting in the generation of steep micromolar Ca2+ gradients along dendrites. Such supralinear Ca2+ responses were attributed to the saturation of a large concentration (0.36 mM) of a mobile, high-affinity (dissociation constant, 0.37 microM) Ca2+ buffer with cooperative Ca2+ binding sites, resembling calbindin-D28K, and to an immobile, low-affinity Ca2+ buffer. These data suggest that the high-affinity Ca2+ buffer operates as the neuronal computational element that enables efficient coincidence detection of the Ca2+ signal and that facilitates spatiotemporal integration of the Ca2+ signal at submicromolar [Ca2+]i.     

High K+ elevates cytosolic free Ca concentration due to mobilization from internal storage sites in rat parotid cells

1. Effects of high K+ on cytosolic free Ca concentration ([Ca2+]i) in rat parotid cells were studied using quin2. 2. High K+ elevated [Ca2+]i in a dose-dependent manner in normal and Ca-free media. The elevation of [Ca2+]i induced by high K+ was less in the latter medium. 3. High K+ depolarized the membrane in a dose-dependent manner in normal and Ca-free media. 4. Although monensin increased [Ca2+]i, high K+ did not affect 22Na uptake into cells. 5. After treatment with oligomycin, high K+ but not carbachol raised [Ca2+]i. 6. We suggest that high K+ increases [Ca2+]i due to mobilizing Ca2+ from the intracellular storage site which does not need energy.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of maprotiline on [Ca2+]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

VPAC receptor modulation of neuroexcitability in intracardiac neurons: dependence on intracellular calcium mobilization and synergistic enhancement by PAC1 receptor activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been found within mammalian intracardiac ganglia, but the cellular effects of these neuropeptides remain poorly understood. Fluorometric calcium imaging and whole cell patch clamp recordings were used to examine the effects of PACAP and VIP on [Ca2+]i and neuroexcitability, respectively, in intracardiac neurons of neonatal rats. PACAP and VIP evoked rapid increases in [Ca2+]i that exhibited both transient and sustained components. Pharmacological experiments using PAC1 and VPAC receptor-selective antagonists demonstrated that the elevations in [Ca2+]i result from the activation of VPAC receptors. The transient increases in [Ca2+]i were shown to be the product of Ca2+ mobilization from caffeine/ryanodine-sensitive intracellular stores and were not due to inositol 1,4,5-trisphosphate-mediated calcium release. In contrast, the sustained [Ca2+]i elevations were dependent on extracellular Ca2+ and were blocked by the transient receptor channel antagonist, 2-aminoethoxydiphenyl borate, which suggests that they are due to Ca2+ entry via store-operated channels. In addition to elevating [Ca2+]i, both PACAP and VIP depolarized intracardiac neurons, and PACAP was further shown to augment action potential firing in these cells. Depolarization of intracardiac neurons by the neuropeptides was dependent on activation of VPAC receptors and the concomitant increases in [Ca2+]i. Although activation of PAC1 receptors alone had no direct effects on neuroexcitability, PAC1 receptor stimulation potentiated the VPAC receptor-induced depolarizations. Furthermore, enhanced action potential firing was only observed upon concurrent stimulation of PAC1 and VPAC receptors, which indicates that these receptors act synergistically to enhance neuroexcitability in intracardiac neurons.     

Intracellular Ca2+ stores in neurons. Identification and functional aspects.

Various aspects of the rapidly exchanging intracellular Ca2+ stores of neurons and nerve cells are reviewed: their multiplicity, with separate sensitivity to either the second messenger, inositol 1,4,5-trisphosphate, or ryanodine-caffeine (the latter stores are probably activated via Ca(2+)-induced Ca2+ release); their control of the plasma membrane Ca2+ permeability, via the activation of a peculiar type of cation channels; their ability to sustain localized heterogeneities of the [Ca2+]i that could be of physiological key-importance. Finally, the molecular composition of these stores is discussed. They are shown (by high resolution immunocytochemistry and subcellular fractionation) to express: i) a Ca2+ ATPase responsible for the accumulation of the cation; ii) Ca2+ binding protein(s) of low affinity and high capacity to keep Ca2+ stored; and iii) a Ca2+ channel, activated by either one of the mechanisms mentioned above, to release Ca2+ to the cytosol. Results obtained in Purkinje neurons document the heterogeneity of the stores and the strategical distribution of the corresponding organelles (calciosomes; specialized portions of the ER) within the cell body, dendrites and dendritic spines.     

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.     

Effects of ryanodine on intracellular Ca2+ transients in mammalian cardiac muscle

We observed the effects of ryanodine on the aequorin luminescence, membrane potential, and contraction of canine cardiac Purkinje fibers and ferret ventricular muscle. In canine Purkinje fibers, ryanodine (10 nM to 1 microM) abolished the spontaneous spatiotemporal fluctuations in [Ca2+] that occur as a result of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) during exposure to low-Na+ solutions. Ryanodine strongly reduced the twitch and both components of the intracellular aequorin luminescence signal (L1 and L2), which normally accompanies contraction. The small luminescence signals that remained in ryanodine could be abolished by a Ca2+ channel blocker (nitrendipine, 10 microM). The plateau phase of the action potential was reduced by nitrendipine in the presence of ryanodine, which suggests that Ca2+ current was not blocked by ryanodine. In ferret ventricular tissue, ryanodine (1 microM) prolonged the action potential and reduced the peak amplitudes of both the aequorin transient and the twitch, while greatly prolonging the time-to-peak of both signals. Increases in extracellular [Ca2+] restored the peak amplitudes of the twitch and the aequorin luminescence, but did not restore the normal time-to-peak. The results show that in both tissues, the negative inotropic effect of ryanodine is due to the reduction of the intracellular [Ca2+] transient. Inasmuch as neither Ca2+ entry via surface membrane Ca2+ channels nor Na+-Ca2+ exchange appears to be blocked by ryanodine, the most probable cause of reduction of the [Ca2+] transient is an inhibition of Ca2+ release by the SR.     

Nordihydroguaiaretic acid-induced Ca2+ handling and cytotoxicity in human prostate cancer cells

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells was investigated. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. NDGA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 30 microM. The Ca2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca2+ partly decreased the NDGA-induced [Ca2+]i increase, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and intracellular Ca2+ release. NDGA-induced Ca2+ influx was independently confirmed by measuring NDGA-induced Mn2+ -coupled quench of fura-2 fluorescence. The NDGA-induced Ca2+ influx was not affected by L-type Ca2+ channel blockers. In Ca2+ -free medium, the NDGA-induced [Ca2+]i increase was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca2+]i increase. NDGA-induced intracellular Ca2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20-50 microM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca2+]i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca2+]i increase via releasing stored Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. NDGA also caused cytotoxicity at higher concentrations.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).     

Effect of celecoxib on Ca2+ fluxes and proliferation in MDCK renal tubular cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular CaCa2+ concentration ([Ca2+]i) and proliferation was examined by using the Ca(2 +)-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (> or =1 micro M) caused an increase of [CaCa2+]i in a concentration-dependent manner. Celecoxib-induced [CaCa2+]i increase was partly reduced by removal of extracellular CaCa2+. Celecoxib-induced CaCa2+ influx was independently suggested by MnCa2+ influx-induced fura-2 fluorescence quench. In Ca(2 +)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2 +)-ATPase, caused a monophasic [CaCa2+]i increase, after which celecoxib only induced a tiny [CaCa2+]i increase; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [CaCa2+]i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [CaCa2+]i increases. Overnight incubation with 1 or 10 micro M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [CaCa2+]i increase in renal tubular cells by stimulating both extracellular CaCa2+ influx and intracellular CaCa2+ release and is highly toxic to renal tubular cells in vitro.     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.