Mobilization of intracellular calcium by substance p in a human astrocytoma cell line (U-373 MG)

Variations in intracellular free calcium concentration (Δ[Ca²⁺]_i) were measured in intact and isolated human astrocytoma cells (U373 MG) loaded with fura-2 acetoxymethylester. Microperfusion of 50 nM substance P (SP), applied for 1 s, increased [Ca²⁺]_i by 351 nM from a stable basal level of [Ca²⁺]_i of 26 nM. The peak Δ[Ca²⁺]_i induced by SP was dose dependent with a threshold of 10_-3 nM, an ED₅₀ of 1.3 nM and a maximal effect for concentrations of SP greater than 100 nM. The NKI receptor agonist, [Sar⁹Met(O₂)¹¹]SP, mimicked the effect of SP, while the NK₂ and NK₃ selective receptor agonists, [N1¹⁰]NKA(4-10) and senktide, respectively, had no effect. The Δ[Ca²⁺]_i induced by SP was unaffected by 100 μM cadmium or by removal of extracellular calcium ions. Caffeine up to 30 mM had no effect on [Ca²⁺]_i. In contrast, thapsigargin increased resting [Ca²⁺]_i by 92 nM and reduced the Δ[Ca²⁺]_i induced by SP. A pertussis treatment (500 ng/ml-24 h) did not modify the Δ[Ca²⁺]_i induced by SP. We conclude that SP, acting on a NK1 receptor, mobilizes cytosolic calcium from an intracellular calcium pool which can be partially depleted by thapsigargin. © 1994 Wiley-Liss, Inc.     

Calcium Signalling in Mouse Bergmann Glial Cells Mediated by α1-adrenoreceptors and H1 Histamine - Receptors

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20–25 days was determined using fura-2 Ca²⁺ microfluorimetry. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i), either individual cells were loaded with the Ca²⁺-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane-permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 μM) and histamine (10-100 μM) triggered a transient increase in [Ca²⁺]_i. The involvement of the α₁-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca²⁺]_i responses were blocked by the selective α¹-adreno-antagonist prazosin and were mimicked by the α¹-adreno-agonist phenylephrine. The monoamine-induced [Ca²⁺]_i signals were not affected by β- and α₂-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β- and α₂-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca²⁺]_i responses demonstrated specific sensitivity to only H₁ histamine receptor modulators. [Ca²⁺]_i responses to monoamines and histamine did not require the presence of extracellular Ca²⁺ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca²⁺]_i increase is due to release from intracellular pools. No [Ca²⁺]_i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with α₁ -adrenoreceptors and H₁ histamine receptors which induce the generation of intracellular [Ca²⁺]_i signals via activation of Ca²⁺ release from inositol-l,4,5-trisphosphate-sensitive intracellular stores.     

Effects of hypoxia induced by Na2S2O4 on intracellular calcium and resting potential of mouse glomus cells

Isolated and cultured glomus cells, obtained from mouse carotid bodies, were superfused with Ham's F-12 equilibrated with air (mean PO₂, 119 Torr; altitude 1350 m). [Ca²⁺]_o was 3.0 mM. In one experimental series, dual cell penetrations with microelectrodes measured intracellular calcium ([Ca²⁺]_i) and the resting potential (E_m). In another series, [Ca²⁺]_i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean E_m of −42.4 mV and [Ca²⁺]_i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential (E_Ca) was 137±0.74 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM, reduced pO₂ to 10–14 Torr. This effect was accompanied by cell depolarization to −19.1 mV. Hypoxia increased [Ca²⁺]_i to 231 nM when detected with Ca-sensitive microelectrodes, but only to 130.2 nM when measured with Indo-1/AM. Calcium increases were preceded by decreases in [Ca²⁺]_i, which also were more pronounced with microelectrode measurements. CoCl₂ 1 mM blocked the hypoxic [Ca²⁺]_i increase and exaggerated the decreases in [Ca²⁺]_i. Correlations between ΔE_m and Δ[Ca²⁺]_i during hypoxia were significant (p<0.05) in 19% of the cells. But, in 29% of them significance was at the p<0.1 level. In the rest (52%), there was no correlation between these parameters. Thus, voltage-gated calcium channels are rare in mouse glomus cells. Their activation by depolarization cannot explain the two to threefold increase in [Ca²⁺]_i seen during hypoxia. More likely, [Ca²⁺]_i increase may be due to hypoxic inactivation of a Ca–Mg ATPase transport system across the cell membrane. The blunting of hypoxic [Ca²⁺]_i increase, seen in Indo-1/AM experiments, is probably due to its solvent (DMSO), which also depresses hypoxic cell depolarization.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.     

Intracellular Ca2+ during metabolic activation of KATP channels in spontaneously active dorsal vagal neurons in medullary slices

Intracellular Ca²⁺ ([Ca²⁺]_i) and membrane properties were measured in fura-2 dialysed dorsal vagal neurons (DVN) spontaneously active at a frequency of 0.5–5 Hz. [Ca²⁺]_i increased by about 30 nm upon rising spike frequency by more than 200% due to 20–50 pA current pulses or 10 μm serotonin. It fell by 30 nm upon block of spiking by current-injection, tetrodotoxin or Ni²⁺ and also during hyperpolarization due to γ-aminobutyric acid or opening of adenosine triphosphate (ATP) -sensitive K⁺ (K_ATP) channels with diazoxide. K_ATP channel-mediated hyperpolarizations during anoxia or cyanide produced an initial [Ca²⁺]_i decrease which reversed into a secondary Ca²⁺ rise by less than 100 nm . Similar moderate rises of [Ca²⁺]_i were observed during block of aerobic metabolism under voltage-clamp as well as in intact cells, loaded with fura-2 AM. The magnitude of the metabolism-related [Ca²⁺]_i transients did not correlate with the amplitude of the K_ATP channel-mediated outward current. [Ca²⁺]_i did not change during diazoxide-induced or spontaneous activation of K_ATP outward current observed in 10% of cells after establishing whole-cell recording. Increasing [Ca²⁺]_i with cyclopiazonic acid did not activate K_ATP channels. [Ca²⁺]_i was not affected upon block of outward current with sulphonylureas, but these K_ATP channel blockers were effective to reverse inhibition of spike discharge and, thus, the initial [Ca²⁺]_i fall upon spontaneous or diazoxide-, anoxia- and cyanide-induced K_ATP channel activation. A sulphonylurea-sensitive hyperpolarization and [Ca²⁺]_i fall was also revealed in the early phase of iodoacetate-induced metabolic arrest, whereas after about 20 min, occurrence of a progressive depolarization led to an irreversible rise of [Ca²⁺]_i to more than 1 μm . The results indicate that K_ATP channel activity in DVN is not affected by physiological changes of intracellular Ca²⁺ and the lack of a major perturbance of Ca²⁺ homeostasis contributes to their high tolerance to anoxia.     

Spontaneous intracellular calcium oscillations in cortical astrocytes from a patient with intractable childhood epilepsy (Rasmussen's Encephalitis)

Many studies have demonstrated that astrocytes respond with fluctuations in intracellular calcium concentration ([Ca²⁺]_i) and membrane potential following the application of a number of ligands. Moreover, calcium (Ca²⁺) waves that spread through astrocytic syncitia have been described in numerous reports. We had the rare opportunity to study Ca²⁺ responses in astrocytes obtained from a patient diagnosed with Rasmussen's encephalitis, a rare form of intractable epilepsy. Using the ratiometric fluorescent indicator fura-2, we observed large spontaneous [Ca²⁺]_i oscillations. The mean time between initial rise in [Ca²⁺]_i and the return to baseline was 5.1 ± 0.19 minutes (SEM; n = 201) and [Ca²⁺]_i increased to a mean level of 271 ± 8 nM (SEM; n = 201) from a baseline of 136 ± 6 nM (SEM; n = 201). Removal of Ca²⁺ from the perfusion solution combined with the addition of the Ca²⁺ chelator EGTA (2 mM) completely but reversibly eliminated all oscillations suggesting the fluctuations were dependent on Ca²⁺ flux across the membrane. The percentage of cells undergoing spontaneous changes in [Ca²⁺]_i decreased over time in culture. At 10–11 days post-surgery, approximately 70% of the cells were exhibiting this behavior, and by day 23 transients were no longer observed. We did not observe comparable spontaneous [Ca²⁺]_i oscillations in rat cortical astrocytes. The potential that the spontaneous [Ca²⁺]_i oscillations observed may be a unique feature of epileptic tissues is discussed. GLIA 21:332–337, 1997. © 1997 Wiley-Liss, Inc.     

Properties of the Ryanodine-sensitive Release Channels that Underlie Caffeine-induced Ca2+ Mobilization from Intracellular Stores in Mammalian Sympathetic Neurons

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca²⁺ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [³ H]ryanodine binding and recording of Ca²⁺ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca²⁺ measurements showed that these cells possess caffeine-sensitive intracellular Ca²⁺ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca²⁺ showed a complex dependence on [Ca²⁺]_i. Transient elevations of [Ca²⁺]_i by 50–500 nM were facilitatory, but they became less facilitatory or depressing when [Ca²⁺]_i reached higher levels. The caffeine-induced Ca²⁺ release and its dependence on [Ca²⁺]_i was further examined by [³ H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (K_D= 10 nM) similar to that previously reported for brain microsomes. However, the density of [³H]ryanodine binding sites (B_max= 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [³ H]Ryanodine binding showed a sigmoidal dependence on [Ca²⁺] in the range 0.1–10 μM that was further increased by caffeine. Caffeine-dependent enhancement of [³ H]ryanodine binding increased and then decreased as [Ca²⁺] rose, with an optimum at [Ca²⁺] between 100 and 500 nM and a 50% decrease between 1 and 10 μM. At 100 μM [Ca²⁺], caffeine and ATP enhanced [³ H]ryanodine binding by 35 and 170% respectively, while binding was reduced by >90% with ruthenium red and MgCl₂. High-conductance (240 pS) Ca²⁺ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca²⁺ from the cytosolic side, and were blocked by Mg²⁺ and ruthenium red. Ryanodine (2 μM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca²⁺ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca²⁺ release. Our data also suggest that the release of Ca²⁺ by caffeine has a bell-shaped dependence on Ca²⁺ in the physiological range of cytoplasmic [Ca²⁺].     

P2U nucleotide receptor activation in rat glial cell line induces [Ca2+]i oscillations which depend on cytosolic pH

In single rat glioma cells, the signal transduction process activated by the UTP sensitive purinergic nucleotide receptor was studied by determining [Ca²⁺]_i by Fura-2 fluorescence and measuring pH by BCECF fluorescence to elucidate the control of [Ca²⁺]_i oscillations by intracellular pH. Addition of UTP for long time periods (some min) causes a [Ca²⁺]_i response composed of i) an initial large peak and a following sustained increase (160 s duration), and ii) subsequent regular [Ca²⁺]_i oscillations (amplitude 107 nM, frequency 1.5 oscillations per min). The maintenance of the [Ca²⁺]_i oscillations depends on the continued presence of agonist. The oscillations are abolished by reducing extracellular Ca²⁺ concentration. The interaction of UTP receptors and bradykinin receptors during the [Ca²⁺]_i oscillations was investigated because previous studies have already shown that the peptide causes comparable [Ca²⁺]_i oscillations. During [Ca²⁺]_i oscillations induced by UTP or bradykinin, long-term admission of both hormones (400–500 s) causes a large initial response superimposed on regular [Ca²⁺]_i oscillations. Short pulses (12 s) of the second agonist given in any phase of the oscillations induce large [Ca²⁺]_i peaks. In both cases, the following oscillations are not disturbed. The influence of cytosolic pH was studied by alkalinizing pH_i by application of NH₄Cl. [Ca²⁺]_i oscillations stop after addition of NH₄Cl. Recovery of NH₄Cl-induced alkalinization is reduced by furosemide. To the same degree, the interruption of [Ca²⁺]_i oscillations is significantly prolonged in the presence of furosemide. Thus cytosolic alkalinization suppresses hormone-induced [Ca²⁺]_i oscillations in rat glioma cells. The understanding of the molecular mechanism of this interference of pH should provide an important contribution for unravelling the function of cytosolic pH in cellular signal transduction. © 1996 Wiley-Liss, Inc.     

Developmental Loss of GABA- and Glycine-induced Depolarization and Ca2+ Transients in Embryonic Rat Dorsal Horn Neurons in Culture

More than 90% of dorsal horn neurons from embryonic day 15–16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) when maintained in culture for <1 week. This [Ca²⁺]_i response has previously been shown to be due to depolarization and subsequent Ca²⁺ entry through voltage-gated Ca²⁺ channels following activation of bicuculline-sensitive GABA_A receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca²⁺]_i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca²⁺]_i. The loss of the [Ca²⁺]_i response was not due to a change in the abundance or the properties of voltage-gated Ca²⁺ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca²⁺]_i transient in response to 30 mM K⁺. Nor was the loss of the [Ca²⁺]_i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca²⁺]_i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons earlyin development and play a role in postmitotic differentiation.     

Effects of the removal of extracellular Ca on [Ca]i responses to FCCP and acetate in carotid body glomus cells of adult rabbits

The effects of the removal of extracellular Ca²⁺ on the responses of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to acidic stimuli, a protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and an organic acid acetate, were examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2 microfluorometry. Application of FCCP (1 μM) induced an increase in [Ca²⁺]_i (mean±S.E.M., 108±14%). After withdrawal of the protonophore the increased [Ca²⁺]_i returned slowly to a resting level. The [Ca²⁺]_i response was attenuated by an inorganic Ca²⁺ channel antagonist Ni²⁺ (2 mM) by 81±4%, and by an L-type voltage-gated Ca²⁺ channel antagonist D600 (10 μM) by 53±13%. The removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i response in 71% of the tested cells (n=17), and depressed it by 68±6% in the rest. Recovery following stimulation with FCCP in the absence of Ca²⁺ reversibly produced a rapid and large rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/FCCP. The magnitude of a [Ca²⁺]_i rise after Ca²⁺-free/FCCP (285±28%, P<0.05) was larger than that of an increase in [Ca²⁺]_i induced by FCCP in the presence of Ca²⁺ and had a correlation with the intensity of the suppression of the [Ca²⁺]_i response by Ca²⁺ removal. A [Ca²⁺]_i rise after Ca²⁺-free/FCCP was inhibited mostly by D600. Similarly, recovery following exposure to acetate in the absence of Ca²⁺ caused a rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/acetate which was sensitive to D600. The magnitude of the [Ca²⁺]_i rise was larger than that of a change in [Ca²⁺]_i caused by acetate in the presence of Ca²⁺. These results suggest that FCCP-induced increase in [Ca²⁺]_i was, in most cells, due to Ca²⁺ influx via L-type voltage-gated Ca²⁺ channels and, in some cells, due to both Ca²⁺ influx and Ca²⁺ release from internal Ca²⁺ pool. The removal of extracellular Ca²⁺ might modify [Ca²⁺]_i responses to acidic stimuli, causing [Ca²⁺]_i rises after Ca²⁺-free/acidic stimuli which involve mostly L-type Ca²⁺ channels.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

Lipopolysaccharide regulates both serotonin- and thrombin-induced intracellular calcium mobilization in rat C6 glioma cells: Possible involvement of nitric oxide synthase-mediated pathway

To investigate the mechanisms by which lipopolysaccharide (LPS) affects Ca²⁺ signaling systems, we studied the effects of LPS on the serotonin (5-HT)- or thrombin-induced intracellular Ca²⁺ ([Ca²⁺]_i) increase in rat C6 glioma cells. Pretreatment of the cells with 1 μg/ml LPS for 24 hr significantly inhibited [Ca²⁺]_i increase induced by 10 μM 5-HT- or 0.5 U/ml thrombin. Its inhibitory effects were both dose- and time-dependent. Treatment with 1 mM dibutyryl cGMP (dbcGMP) for 30 min also significantly inhibited the 5-HT- and thrombin-induced [Ca²⁺]_i increase to approximately 60–70% of control. However, simultaneous pretreatment with LPS and dbcGMP did not show any synergistic inhibition. The simultaneous pretreatment with LPS and the potent cGMP-dependent protein kinase (PKG) inhibitors H-8 and KT5823 for 24 hr significantly antagonized the inhibitory effect of LPS. Pretreatment of the cells with 1 μg/ml LPS for 24 hr significantly enhanced cGMP accumulation, while dexamethasone and NMMA (NOS inhibitors) significantly attenuated the LPS-induced enhancement in cGMP accumulation. In addition, pretreatment of the cells with 100 nM dexamethasone for 24 hr significantly suppressed LPS-induced inducible nitric oxide synthase (iNOS; type II NOS, NOS-II) protein expression. These results indicate that LPS may inhibit both 5-HT- and thrombin-induced [Ca²⁺]_i increase via iNOS expression and PKG activation pathway in rat C6 glioma cells. J. Neurosci. Res. 51:517–525, 1998. © 1998 Wiley-Liss, Inc.     

Ca2+ influx into leech glial cells and neurones caused by pharmacologically distinct glutamate receptors

The effect of glutamatergic agonists on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leech Hirudo medicinalis was investigated by using iontophoretically injected fura-2. In physiological Ringer solution the [Ca²⁺]_i levels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64). In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca²⁺]_i which was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). This increase was caused by a Ca²⁺ influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca²⁺. The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist-induced [Ca²⁺]_i increase was abolished after replacing extracellular Na⁺ by organic cations or by mM amounts of Ni²⁺, whereas in glial cells the [Ca²⁺]_i increase was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca²⁺ influx is predominantly mediated by voltage-dependent Ca²⁺ channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutamate receptors.     

Effect of lead on intracellular free calcium ion concentration in a presynaptic neuronal model: F-NMR study of NG108-15 cells

The intracellular free calcium ion concentration ([Ca²⁺]_i) of the neuroblastoma × glioma hybrid cell line, NG108-15, was measured using the ¹⁹F-nuclear magnetic resonance divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (5F-BAPTA). The basal [Ca²⁺]_i was measured to be 106 ± 14 nM. Treatment with 5 μM lead (Pb) for 2 h produced a 2-fold increase in [Ca²⁺]_i to 200 ± 24 nM and a measurable intracellular free Pb²⁺ concentration ([Pb²⁺]_i) of 30 ± 10 pM. Intracellular free Zn²⁺ concentrations ([Zn²⁺]_i) were also observed in the presence of Pb. This represents the first direct demonstration that Pb elevates the [Ca²⁺]_i in neurons, thus providing evidence for a role of [Ca²⁺]_i in mediating the neurotoxicity of Pb.     

Involvement of Na+–Ca2+ Exchanger in Reperfusion-induced Delayed Cell Death of Cultured Rat Astrocytes

In some cells, Ca²⁺ depletion induces an increase in intracellular Ca²⁺ ([Ca²⁺]_i) after reperfusion with Ca²⁺-containing solution, but the mechanism for the reperfusion injury is not fully elucidated. Using an antisense strategy we studied the role of the Na⁺-Ca²⁺ exchanger in reperfusion injury in cultured rat astrocytes. When astrocytes were perfused in Ca²⁺-free medium for 15–60 min, a persistent increase in [Ca²⁺]_i was observed immediately after reperfusion with Ca²⁺-containing medium, and the number of surviving cells decreased 3–5 days latter. The increase in [Ca²⁺]_i was enhanced by low extracellular Na⁺ ([Na⁺]_o) during reperfusion and blocked by the inhibitors of the Na⁺-Ca²⁺ exchanger amiloride and 3,4-dichlorobenzamil, but not by the Ca²⁺ channel antagonists nifedipine, Cd²⁺ and Ni²⁺. Treatment of astrocytes with antisense, but not sense, oligodeoxynucleotide to the Na⁺-Ca²⁺ exchanger decreased Na⁺–Ca²⁺ exchanger protein level and exchange activity. The antisense oligomer attenuated reperfusion-induced increase in [Ca²⁺]ⁱ and cell toxicity. The Na⁺-Ca²⁺ exchange inhibitors 3,4-dichlorobenzamil and ascorbic acid protected astrocytes from reperfusion injury partially, while the stimulators sodium nitroprusside and 8-bromo-cyclic GMP and low [Na⁺]_o exacerbated the injury. Pretreatment of astrocytes with ouabain and monensin caused similar delayed glial cell death. These findings suggest that Ca²⁺ entry via the Na⁺–Ca²⁺ exchanger plays an important role in reperfusion-induced delayed glial cell death.     

Inhibition of noradrenaline stimulated increase in [Ca]i in cultured astrocytes by chronic treatment with a therapeutically relevant lithium concentration

Chronic treatment of mouse astrocytes in primary cultures with 1 mM lithium chloride for 7–14 days decreased the basal level of free cytosolic calcium concentration ([Ca²⁺]_i) from 50–70 nM to 70% of this value and reduced the increase in [Ca²⁺]_i caused by exposure to 1 μM noradrenaline (normally to 500–700 nM) by almost one half. A similar, but much smaller, response to serotonin was unaffected by chronic treatment with lithium. Acute exposure to lithium (30 min) had no effect on either basal or noradrenaline stimulated [Ca²⁺]_i The dependence on chronic, versus acute treatment suggests that this effect may be related to the therapeutic effect of lithium as a mood-stabilizing drug, which likewise requires chronic treatment. Since good evidence is found that noradrenaline increases [Ca²⁺]_i by activation of the phosphoinositol second messenger system the present findings are also consistent with literature data that lithium acts by interfering with this system.     

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

Neurotensin and neuromedin N elevate the cytosolic calcium concentration via transiently appearing neurotensin binding sites in cultured rat cortex cells

Through assessment of the changes in the intracellular free-calcium concentration ([Ca²⁺]_i), which was measured using the calcium sensitive dye, fura-2, the character of the neurotensin (NT) binding sites which appeared transiently during the early ontogenetic stage in the rat cerebral cortex was analyzed in primary cultures of cerebral cortex cells from neonatal rats. NT (1–1000 nM) elevated [Ca²⁺]_i of the cells even when extracellular calcium was chelated with 1 mM ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). These findings suggest that the transiently appearing NT-binding sites in the cortex are receptors for NT and that some of the NT-induced increase in [Ca²⁺]_i is due to mobilization from the intracellular calcium store. Further application of NT after 10 min washing caused an increase in [Ca²⁺]_i again. This is in contrast to the findings for cortical slices from adult rats and mRNA-injected oocytes; desensitization due to NT was of long duration and further application of NT failed to activate the neurons which had responded the first time to NT. These facts suggest that the character of the NT-binding sites in the cerebral cortex differs between neonatal and adult rats. In addition, we showed that neuromedin N had a similar property to NT as to mobilization of [Ca²⁺]_i and acted only on NT-responsive cells, suggesting the interaction between NT and neuromedin N at the postsynaptic level via the same receptor.