Differential effects of fentanyl and morphine on intracellular Ca2+ transients and contraction in rat ventricular myocytes

BACKGROUND: Our objective was to elucidate the direct effects of fentanyl and morphine on cardiac excitation-contraction coupling using individual, field-stimulated rat ventricular myocytes. METHODS: Freshly isolated myocytes were loaded with fura-2 and field stimulated (0.3 Hz) at 28 degrees C. Amplitude and timing of intracellular Ca2+ concentration (at a 340:380 ratio) and myocyte shortening (video edge detection) were monitored simultaneously in individual cells. Real time Ca2+ uptake into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. RESULTS: The authors studied 120 cells from 30 rat hearts. Fentanyl (30-1,000 nM) caused dose-dependent decreases in peak intracellular Ca2+ concentration and shortening, whereas morphine (3-100 microM) decreased shortening without a concomitant decrease in the Ca2+ transient. Fentanyl prolonged the time to peak and to 50% recovery for shortening and the Ca2+ transient, whereas morphine only prolonged the timing parameters for shortening. Morphine (100 microM), but not fentanyl (1 microM), decreased the amount of Ca2+ released from intracellular stores in response to caffeine in intact cells, and it inhibited the rate of Ca2+ uptake in isolated sarcoplasmic reticulum vesicles. Fentanyl and morphine both caused a downward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on the Ca2+ transient. CONCLUSIONS: Fentanyl and morphine directly depress cardiac excitation-contraction coupling at the cellular level. Fentanyl depresses myocardial contractility by decreasing the availability of intracellular Ca2+ and myofilament Ca2+ sensitivity. In contrast, morphine depresses myocardial contractility primarily by decreasing myofilament Ca2+ sensitivity.     

Activation of Ca2+-dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose precursors

Cultured dorsal root ganglion neurons were voltage clamped at -90 mV to study the effects of intracellular application of nicotinamide adenine dinucleotide (betaNAD+), intracellular flash photolysis of caged 3',5'-cyclic guanosine monophosphate (cGMP), and metabotropic glutamate receptor activation. The activation of metabotropic glutamate receptors evoked inward Ca2+-dependent currents in most cells. This was mimicked both by intracellular flash photolysis of the caged axial isomer of cGMP [P-1-(2-nitrophenyl)ethyl cGMP] and intracellular application of betaNAD+. Whole cell Ca2+-activated inward currents were used as a physiological index of raised intracellular Ca2+ levels. Extracellular application of 10 microM glutamate evoked the activation of Ca2+-dependent inward currents, thus reflecting a rise in intracellular Ca2+ levels. Similar inward currents were also activated after isolation of metabotropic glutamate receptor activation by application of 10 microM glutamate in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione and 20 microM dizocilpine maleate (MK 801), or by extracellular application of 10 microM trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid. Intracellular photorelease of cGMP, from its caged axial isomer, in the presence of betaNAD+ was also able to evoke similar Ca2+-dependent inward currents. Intracellular application of betaNAD+ alone produced a concentration-dependent effect on inward current activity. Responses to both metabotropic glutamate receptor activation and cGMP were suppressed by intracellular ryanodine, chelation of intracellular Ca2+ by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, and depletion of intracellular Ca2+ stores, but were insensitive to the removal of extracellular Ca2+. Therefore both cGMP, possibly via a mechanism that involves betaNAD+ and/or cyclic ADP-ribose, and glutamate can mobilize intracellular Ca2+ from ryanodine-sensitive stores in sensory neurons.     

CHEB, a convulsant barbiturate, evokes calcium-dependent spontaneous glutamate release from rat cerebrocortical synaptosomes

CHEB [5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid] is a potent convulsant barbiturate that causes direct neuronal excitation by an unknown mechanism. We have analyzed the effects of CHEB on the release of endogenous glutamate from rat cerebrocortical synaptosomes using an on-line enzyme-coupled fluorimetric assay. CHEB evoked spontaneous Ca(2+)-dependent glutamate release with an EC50 = 14.2 microM and an Emax = 3.2 mumol/min/mg. The non-convulsant barbiturates pentobarbital and phenobarbital evoked significantly less glutamate release at high concentrations. CHEB (30 microM) increased intrasynaptosomal [Ca2+] by 58 +/- 4 nM (p < 0.01; n = 4) above baseline compared to an increase of 5 +/- 4 nM (NS; n = 4) produced by pentobarbital (30 microM). CHEB-evoked glutamate release was inhibited by pentobarbital, phenobarbital, EGTA, CoCl2/CdCl2 and flunarizine, but not by local anesthetics, tetrodotoxin, nitrendipine or omega-conotoxin GVIA. These results demonstrate that CHEB acts as a potent and effective secretogogue for glutamate by a pre-synaptic mechanism that does not require activation of Na+ channels or of L-type or N-type Ca2+ channels. Stimulation of spontaneous glutamate release may contribute to the convulsant properties of CHEB.     

Halothane and isoflurane alter calcium dynamics in rat cerebrocortical synaptosomes

An increase in synaptosomal Ca2+ triggers neurotransmitter release and volatile anesthetics have been shown to inhibit neurotransmitter release by inhibition of Ca2+ entry. We have examined the effect of isoflurane and halothane on the kinetics of increase and decrease of Ca2+ in rat cerebrocortical synaptosomes ([Ca2+]in). We have also used specific Ca2+ antagonists to examine the role of L-, N-, and P-type Ca2+ channels. Synaptosomal [Ca2+]in was measured spectrofluorometrically using fura-2 as a Ca2+ reporter; Ca2+ transients were initiated by depolarization with 40 mM KCl. We found that < or = 1 minimum alveolar anesthetic concentration halothane and isoflurane decreased peak [Ca2+]in by approximately 40%, that both anesthetics decreased the rate of [Ca2+]in increase and decrease, that specific voltage-dependent calcium channel antagonists had little effect on peak or plateau [Ca2+]in, and that the volatile anesthetics increased the permeability of synaptosomal membranes to Ca2+. These results suggest that the volatile anesthetics, at clinically relevant concentrations, can alter Ca2+ homeostasis in the synapse. IMPLICATIONS: Clinically relevant concentrations of halothane and isoflurane markedly depress K+-evoked increases in rat cerebrocortical synaptosomal calcium (Ca2+) unrelated to L-, N-, and P-type voltage-dependent calcium channels and increase the Ca2+ permeability of the synaptosomal membrane. These changes in Ca2+ dynamics could have profound effects on Ca2+ signaling in the synapse.     

Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate

We monitored simultaneously the changes in the intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i) in individual neurons from primary cultures of cerebellar granule cells loaded with sodium-binding benzofuran isophthalate and fluo-3. An application of glutamate (50 microM) in Mg(2+)-free medium containing 10 microM glycine evoked [Na+]i and [Ca2+]i increases that exceeded 60 mM and 1 microM, respectively. The kinetics of [Na+]i and [Ca2+]i decreases after the termination of the glutamate pulse were different. [Na+]i failed to decrease immediately after glutamate withdrawal and the delay in the onset of [Na+]i decrease after the glutamate pulse termination was proportional to the glutamate dose, the glutamate pulse duration, and the extent of [Ca2+]i elevation elicited by glutamate. The kinetics of [Ca2+]i decrease were biphasic, with the first phase occurring immediately after glutamate withdrawal and the second phase being correlated in time with a [Na+]i value lower than 15-20 mM. These results were interpreted to indicate that the glutamate-evoked calcium influx may lead to sodium homeostasis destabilization. The delay in the restoration of the sodium gradient may in turn prolong the neuronal exposure to toxic [Ca2+]i values, due to the decrease in the efficiency of the Na+/Ca2+ exchanger to extrude calcium. The glutamate effects on [Na+]i and [Ca2+]i were potentiated by glycine. Glycine (10 microM) added alone also evoked [Na+]i and [Ca2+]i increases; this effect was inhibited by a competitive inhibitor of the N-methyl-D-aspartate receptor, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, indicating an involvement of endogenous glutamate.     

Dynamic regulation of intracellular Ca2+ concentration in aortic endothelial cells

In non-excitable cells, a Ca2+ entry pathway is opened after the depletion of intracellular Ca2+ store sites. We have tried to estimate the sensitivity of this pathway to Ca2+ release using bovine aortic endothelial cells. Single application of a high concentration (30 microM) of ATP released almost all stored Ca2+ in Ca(2+)-free extracellular solution, whereas a low concentration of ATP (30 nM) produced a partial (57.3 +/- 3.0%) release of Ca2+. By 10 min of Ca2+ re-perfusion, the Ca2+ store site was reloaded to 97.1% of its initial filling state. When thapsigargin was applied to this cell in Mn2+ solution, Mn(2+)-induced quenching of fura-2 dye started when 19.3 +/- 5.3% of Ca2+ release, produced by 30 nM ATP, had occurred. Therefore, Ca2+ release required for Mn2+ entry was estimated as 11.1 +/- 3.0% of stored Ca2+. These results indicate that intracellular Ca2+ concentration is controlled dynamically by simultaneously occurring Ca2+ release and entry in bovine aortic endothelial cells.     

Chromaffin granules release calcium on contact with annexin VI: implications for exocytosis

Chromaffin granules represent a substantial and exchangeable intracellular calcium pool which is thought to be regulated by a sodium/calcium exchange protein and also by a putative inositol trisphosphate-activated calcium channel. A family of calcium-binding proteins, called the annexins, has been shown to bind to chromaffin granules. We have therefore investigated the possible involvement of these proteins in the regulation of chromaffin granule sequestered calcium. Annexin VI (A-VI) produced a concentration-dependent release of 45Ca2+ from chromaffin granules; half-maximal release occurred at 1 microM A-VI, with near-maximum release being at 20 microM A-VI. The A-VI-induced release of 45Ca2+ was rapid, being essentially complete by our first time point of 7 s, and corresponded to 40% of the total sequestered 45Ca2+. A-VI-induced release occurred at extravesicular Ca2+ concentrations ranging from a pCa2+ of 4.12 to 6.86 and also appeared specific to this protein since neither annexin I nor annexin II (tetramer) could evoke any 45Ca2+ release. Given the predominant localization of A-VI to the apical plasmalemma, these results suggest that this protein could participate in the secretory event by mediating the localized release of Ca2+ at sites of contact between the chromaffin granule and plasma membrane.     

The novel insulinotropic mechanism of pimobendan: direct enhancement of the exocytotic process of insulin secretory granules by increased Ca2+ sensitivity in beta-cells

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.     

Mapping of a carboxyl-terminal active site of parathyroid hormone by calcium-imaging

We recently showed that the C-terminal fragment PTH (52-84) effectively increases intracellular free calcium ([Ca2+]i) in a subset of growth plate chondrocytes not activated by the N-terminal PTH fragment (1-34). Here we characterize the active site on C-terminal PTH (52-84) with respect to calcium (Ca2+)-signaling and the mechanism involved by using synthetic PTH-subfragments in digital CCD ratio-imaging experiments. Our results show amino acids 73-76 to be the core region for increasing [Ca2+]i. Ryanodine (1 microM), caffeine (10 mM), lithium (2 mM), or cyclopiazonic acid (2-5 microM), agents that interfere with intracellular Ca2+ release, all failed to block PTH (52-84) induced [Ca2+]i increases. Depletion of extracellular calcium ([Ca2+]o) blocked PTH (52-84) induced [Ca2+]i increases, indicating a transmembrane Ca2+ influx. In contrast to voltage-gated and Ca2+ release activated Ca2+ influx, PTH (52-84) evoked Ca2+ influx was not blocked by nickel (1 mM). We conclude that PTH amino acids 73-76 are essential for activation of a nickel-insensitive Ca2+ influx pathway in growth plate chondrocytes that is likely to be of relevance for matrix calcification, a key step in endochondral bone formation.     

Alterations in the Ha-ras-1 and the p53 pathway genes in the progression of N-methyl-N-nitrosourea-induced rat mammary tumors

Glutamate is the most prominent excitatory neurotransmitter in the retina and brain. It has become clear that the physiology of many glial cells, including retinal Müller cells, is modified by a host of neurotransmitters, including glutamate. The experiments presented here demonstrate that Müller cells isolated from the tiger salamander retina have metabotropic glutamate receptors that, when activated, lead to the release of calcium ions (Ca2+) from intracellular stores. The Ca2+-sensitive fluorescent dye, Fura-2, and video imaging microscopy were used to monitor changes in cytosolic calcium ion concentration ([Ca2+]i) evoked by glutamate (30-50 microM), (1S,3R)-ACPD (50-200 microM), quisqualate (10-50 microM), and L-AP4 (5-100 microM). Bath application of each of these metabotropic receptor agonists in the absence of extracellular Ca2+ resulted in an increase in [Ca2+]i that often began in the distal end of the cell and occurred later in the endfoot. This wavelike increase in [Ca2+]i is reminiscent of the Ca2+ waves evoked in these cells by other Ca2+ releasing agents such as ryanodine and caffeine. Extracellular application ofATP also evoked increases in [Ca2+] in Müller cells. The presence on Müller cells of receptors for retinal neurotransmitters, such as glutamate and ATP, demonstrates that these glial cells can respond to changes in the retinal extracellular environment and hence neuronal activity. Since Müller cells span almost all layers of the retina, they are likely to be exposed to most retinal neurotransmitters. The Ca2+ waves evoked in Müller cells by neurotransmitters could represent a form of signaling from the outer retinal layers to the inner ones.     

Modulation of multiple potassium currents by metabotropic glutamate receptors in neurons of the hypothalamic supraoptic nucleus

We studied the effects of activation of the metabotropic glutamate receptors on intrinsic currents of magnocellular n urons of the supraoptic nucleus (SON) with whole cell patch-clamp and conventional intracellular recordings in coronal slices (400 micron) of the rat hypothalamus. Trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 10-100 microM), a broad-spectrum metabotropic glutamate receptor agonist, evoked an inward current (18.7 +/- 3.45 pA) or a slow depolarization (7.35 +/- 4.73 mV) and a 10-30% decrease in whole cell conductance in approximately 50% of the magnocellular neurons recorded at resting membrane potential. The decrease in conductance and the inward current were caused largely by the attenuation of a resting potassium conductance because they were reduced by the replacement of intracellular potassium with an equimolar concentration of cesium or by the addition of potassium channel blockers to the extracellular medium. In some cells, trans-ACPD still elicited a small inward current after blockade of potassium currents, which was abolished by the calcium channel blocker, CdCl2. Trans-ACPD also reduced voltage-gated and Ca2+-activated K+ currents in these cells. Trans-ACPD reduced the transient outward current (IA) by 20-70% and/or the IA-mediated delay to spike generation in approximately 60% of magnocellular neurons tested. The cells that showed a reduction of IA generally also showed a 20-60% reduction in a voltage-gated, sustained outward current. Finally, trans-ACPD attenuated the Ca2+-dependent outward current responsible for the afterhyperpolarization (IAHP) in approximately 60% of cells tested. This often revealed an underlying inward current thought to be responsible for the depolarizing afterpotential seen in some magnocellular neurons. (RS)-3,5-dihydroxyphenylglycine, a group I receptor-selective agonist, mimicked the effects of trans-ACPD on the resting and voltage-gated K+ currents. (RS)-alpha-methyl-4-carboxyphenylglycine, a group I/II metabotropic glutamate receptor antagonist, blocked these effects. A group II receptor agonist, 2S,1'S,2'S-2carboxycyclopropylglycine and a group III receptor agonist, (+)-2-amino-4-phosphonobutyric acid, had no effect on the resting or voltage-gated K+ currents, indicating that the reduction of K+ currents was mediated by group I receptors. About 80% of the SON cells that were labeled immunohistochemically for vasopressin responded to metabotropic glutamate receptor activation, whereas only 33% of labeled oxytocin cells responded, suggesting that metabotropic receptors are expressed preferentially in vasopressinergic neurons. These data indicate that activation of the group I metabotropic glutamate receptors leads to an increase in the postsynaptic excitability of magnocellular neurons by blocking resting K+ currents as well as by reducing voltage-gated and Ca2+-activated K+ currents.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [3H]cyclohexyladenosine (CHA) to the cellular fractions and P2 subfractions of the goldfish brain was studied. The A1 receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P2), 30 mM K+ stimulated glutamate, taurine and GABA release in a Ca(2+)-dependent fashion, whereas the aspartate release was Ca(2+)-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 microM) inhibited K(+)-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K+ (30 mM) stimulated Ca2+ influx (46.8 +/- 6.8%) and this increase was completely abolished by pretreatment with 100 nM omega-conotoxin. Pretreatment with 100 microM R-PIA or 100 microM CHA, reduced the evoked increase of intra-synaptosomal Ca2+ concentration, respectively by 37.7 +/- 4.3% and 39.7 +/- 9.0%. A possible correlation between presynaptic A1 receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002

Angiotensin II (AngII) is coupled to several important intracellular signaling pathways, and increases intracellular Ca2+. In vascular smooth muscle (VSM) cells, AngII is known to activate enzymes such as tyrosine protein kinase (Tyr-PK), phospholipase C (PLC), protein kinase C (PKC), and phophatidylinositol-3-kinase (PI-3-K). A non-receptor Tyr-PK, pp60(c-src), and PKC have been reported to stimulate the Ca2+ channels in VSM cells. However, less is known about AngII action on the voltage-gated Ca2+ channels. The Ca2+-channel currents of a cultured rat aortic smooth muscle cell line, A7r5, were recorded using whole-cell voltage clamp. Application of 50 nM AngII significantly increased the amplitude of Ba2+ currents through the voltage-gated Ca2+ channels (IBa) by 34. 5+/-9.1% (n=10) within 1 min. In the presence of lavendustin-A (5 microM), a selective inhibitor of Tyr-PK, AngII failed to stimulate IBa (n=5). AngII stimulation of IBa was also prevented by (5 microM) LY-294002, an inhibitor of PI-3-K (n=5). In contrast, H-7 (30 microM), an inhibitor of PKC, did not prevent the effect of AngII on IBa (n=6). These results suggest that AngII may stimulate the Ca2+ channels of VSM cells through Tyr-PK and PI-3-K under conditions that probably exclude participation of PK-C.     

Depletion of intracellular calcium stores facilitates the influx of extracellular calcium in platelet derived growth factor stimulated A172 glioblastoma cells

Calcium signaling in non-excitable cells is the consequence of calcium release from intracellular stores, at times followed by entry of extracellular calcium through the plasma membrane. To study whether entry of calcium depends upon the level of saturation of intracellular stores, we measured calcium channel opening in the plasma membrane of single confluent A172 glioblastoma cells stimulated with platelet derived growth factor (PDGF) and/or bradykinin (BK). We monitored the entry of extracellular calcium by measuring manganese quenching of Indo-1 fluorescence. PDGF raised intracellular calcium concentration ([Ca2+]i) after a dose-dependent delay (tdel) and then opened calcium channels after a dose-independent delay (tch). At higher doses (> 3 nM), BK increased [Ca2+]i after a tdel approximately 0 s, and tch decreased inversely with both dose and peak [Ca2+]i. Experiments with thapsigargin (TG), BK, and PDGF indicated that BK and PDGF share intracellular Ca2+ pools that are sensitive to TG. When these stores were depleted by treatment with BK and intracellular BAPTA, tdel did not change, but tch fell to almost 0 s in PDGF stimulated cells, indicating that depletion of calcium stores affects calcium channel opening in the plasma membrane. Our data support the capacitative model for calcium channel opening and the steady-state model describing quantal Ca2+ release from intracellular stores.     

Prolonged hormone secretion from neuroendocrine cells of Aplysia is independent of extracellular calcium

The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co(2+)-substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.     

ATP stimulation of Ca2+ -dependent plasminogen release from cultured microglia

1. ATP (10-100 microM), but not glutamate (100 microM), stimulated the release of plasminogen from microglia in a concentration-dependent manner during a 10 min stimulation. However, neither ATP (100 microM) nor glutamate (100 microM) stimulated the release of NO. A one hour pretreatment with BAPTA-AM (200 microM), which is metabolized in the cytosol to BAPTA (an intracellular Ca2+ chelator), completely inhibited the plasminogen release evoked by ATP (100 microM). The Ca2+ ionophore A23187 induced plasminogen release in a concentration-dependent manner (0.3 microM to 10 microM). 2. ATP induced a transient increase in the intracellular calcium concentration ([Ca2+]i) in a concentration-dependent manner which was very similar to the ATP-evoked plasminogen release, whereas glutamate (100 microM) had no effect on [Ca2+]i (70 out of 70 cells) in microglial cells. A second application of ATP (100 microM) stimulated an increase in [Ca2+]i similar to that of the first application (21 out of 21 cells). 3. The ATP-evoked increase in [Ca2+]i was totally dependent on extracellular Ca2+, 2-Methylthio ATP was active (7 out of 7 cells), but alpha,beta-methylene ATP was inactive (7 out of 7 cells) at inducing an increase in [Ca2+]i. Suramin (100 microM) was shown not to inhibit the ATP-evoked increase in [Ca2+]i (20 out of 20 cells). 2'- and 3'-O-(4-Benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), a selective agonist of P2X7 receptors, evoked a long-lasting increase in [Ca2+]i even at 1 microM, a concentration at which ATP did not evoke the increase. One hour pretreatment with adenosine 5'-triphosphate-2', 3'-dialdehyde (oxidized ATP, 100 microM), a selective antagonist of P2X7 receptors, blocked the increase in [Ca2+]i induced by ATP (10 and 100 microM). 4. These data suggest that ATP may transit information from neurones to microglia, resulting in an increase in [Ca2+]i via the ionotropic P2X7 receptor which stimulates the release of plasminogen from the microglia.     

Distribution and activation of intracellular Ca2+ stores in cultured olfactory bulb neurons

The presence and distribution of intracellular Ca2+ release pathways in olfactory bulb neurons were studied in dissociated cell cultures. Histochemical techniques and imaging of Ca2+ fluxes were used to identify two major intracellular Ca2+ release mechanisms: inositol 1, 4,5-triphosphate receptor (IP3R)-mediated release, and ryanodine receptor-mediated release. Cultured neurons were identified by immunocytochemistry for the neuron-specificmarker beta-tubulin III. Morphometric analyses and immunocytochemistry for glutamic acid-decarboxylase revealed a heterogeneous population of cultured neurons with phenotypes corresponding to both projection (mitral/tufted) and intrinsic (periglomerular/granule) neurons of the in vivo olfactory bulb. Immunocytochemistry for the IP3R, and labeling with fluorescent-tagged ryanodine, revealed that, irrespective of cell type, almost all cultured neurons express IP3R and ryanodine binding sites in both somata and dendrites. Functional imaging revealed that intracellular Ca2+ fluxes can be generated in the absence of external Ca2+, using agonists specific to each of the intracellular release pathways. Local pressure application of glutamate or quisqualate evoked Ca2+ fluxes in both somata and dendrites in nominally Ca2+ free extracellular solutions, suggesting the presence of IP3-dependent Ca2+ release. These fluxes were blocked by preincubation with thapsigargin and persisted in the presence of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Local application of caffeine, a ryanodine receptor agonist, also evoked intracellular Ca2+ fluxes in the absence of extracellular Ca2+. These Ca2+ fluxes were suppressed by preincubation with ryanodine. In all neurons, both IP3- and ryanodine-dependent release pathways coexisted, suggesting that they interact to modulate intracellular Ca2+ concentrations.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

Different effects of endothelin-3 on the Ca2+ discharge induced by agonists and Ca(2+)-ATPase inhibitors in human platelets

1. The present study demonstrates that endothelin-3 (ET-3), previously shown to attenuate thrombin-evoked aggregation of human platelets, delayed the dose-dependent aggregatory response to thapsigargin (Tg). As this Ca(2+)-ATPase inhibitor induces platelet activation in part through the depletion of internal Ca(2+)-stores, we examined the influence of ET-3 on Ca2+ discharge from internal pools. 2. Cytosolic Ca2+ concentration was evaluated with Fura-2 in the absence of Ca2+ influx. Platelet preincubation for 15 min with 5 x 10(-7) M ET-3 decreased the Ca2+ release evoked by thrombin and U46619, a thromboxane-mimetic. However, ET-3 did not affect Ca2+ movements induced by 1 microM ADP. Addition of Tg (0.5 to 5 microM) to resting platelets induced a cytosolic [Ca2+] rise with concentration-dependent increase of the initial rate and decrease of the time to reach the peak. ET-3 slowed down these dose-dependent effects with a more marked influence on the responses induced by low concentrations of Tg. 3. ET-3 did not modify the Ca2+ response to another Ca(2+)-ATPase inhibitor, 2,5-di-(tert-butyl)-1,4-benzohydroquinone(tBuBHQ). The thromboxane A2 receptor antagonist, SQ 29548, reduced by 53% the calcium signal evoked by 1 microM Tg, which became similar to that induced by 15 microM tBuBHQ. Under these conditions, the ET-3 effects were suppressed. A subsequent addition of thrombin induced a substantial further Ca2+ increase which was again sensitive to ET-3. 4. ET-3 attenuates Ca2+ mobilization from an internal pool dependent on the stimulation of thrombin and thromboxane A2 receptors and insensitive to the direct effect of Ca2+-ATPase inhibitors. The small but significant inhibitory effect of ET-3 leads us to propose that endothelin-3 acts as a modulator of platelet activation.     

Aluminum potentiates glutamate-induced calcium accumulation and iron-induced oxygen free radical formation in primary neuronal cultures

Aluminum is a neurotoxic metal that may be involved in the progression of neurodegenerative diseases, including Alzheimer disease and amyotrophic lateral sclerosis (ALS). Although the mechanism of action is not known, aluminum has been shown to alter Ca2+ flux and homeostasis, and facilitate peroxidation of membrane lipids. Since abnormal increases of intracellular Ca2+ and oxygen free radicals have both been implicated in pathways leading to neurodegeneration, we examined the effect of aluminum on these parameters in vitro using primary cultures of cerebellar granule cells. Exposure to glutamate (1-300 microM) caused a concentration-dependent uptake of 45Ca in granule cells to a maximum of 280% of basal. Pretreatment with AlCl3 (1-1000 microM) had no effect on 45Ca accumulation, but increased the uptake induced by glutamate. Similarly, AlCl3 had no effect on intracellular free Ca2+ levels measured using fluorescent probe fura-2, but potentiated the increase induced by glutamate. The production of reactive oxygen species (ROS) was examined using the fluorescent probe dichlorofluorescin. By itself, AlCl3 had little effect on ROS production. However, AlCl3 pretreatment potentiated the ROS production induced by 50 microM Fe2+. These results suggest that aluminum may facilitate increases in intracellular Ca2+ and ROS, and potentially contribute to neurotoxicity induced by other neurotoxicants.