Identification of Kainate Receptor-Mediated Intracellular Calcium Increases in Cultured Rat Cerebellar Granule Cells

Abstract: The functional expression of the kainate subtype of glutamate receptor (GluR) has been investigated in cultured rat cerebellar granule cells using single cell intracellular calcium ([Ca²⁺]_i) measurements. Both AMPA- and kainate-induced [Ca²⁺]_i increases could be blocked completely by the AMPA receptor-selective antagonist LY300168 (50 µ M ). However, following treatment with concanavalin A, an inhibitor of kainate receptor desensitisation, 30% of cells showed a kainate-induced [Ca²⁺]_i rise of >100 n M in the presence of LY300168. Responses to 30 µ M kainate in the presence of LY300168 were virtually abolished by the AMPA and GluR5 kainate receptor competitive antagonist LY293558 (100 µ M ). These results demonstrate the presence of functional kainate receptors on cultured cerebellar granule cells, and suggest that the GluR5 subtype of kainate receptor plays a significant role in kainate receptor-mediated [Ca²⁺]_i increases.     

Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca²⁺]_i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca²⁺]_i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca²⁺]_i after a second addition of LPA. The LPA-induced increase in [Ca²⁺]_i required extracellular calcium. However, studies with Cd²⁺, Ni²⁺, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca²⁺]_i, because this increase was inhibited by NMDA receptor antagonists and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg²⁺ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

Presynaptic GABAB Receptor Modulation of Glutamate Exocytosis from Rat Cerebrocortical Nerve Terminals: Receptor Decoupling by Protein Kinase C

Abstract: GABA and the GABA_B receptor agonist (−)-baclofen inhibited 4-aminopyridine (4AP)- and KCl-evoked, Ca²⁺-dependent glutamate release from rat cerebrocortical synaptosomes. The GABA_B receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (−)-Baclofen-mediated inhibition of glutamate release was insensitive to 2 µg/ml pertussis toxin. As determined by examining the mechanism of GABA_B receptor modulation of glutamate release, (−)-baclofen caused a significant reduction in 4AP-evoked Ca²⁺ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP-mediated depolarization; thus, the inhibition of Ca²⁺ influx could not be attributed to GABA_B receptor activation causing a decrease in synaptosomal excitability. Ionomycin-mediated glutamate release was not affected by (−)-baclofen, indicating that GABA_B receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABA_B receptors to voltage-dependent Ca²⁺ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (−)-baclofen-mediated inhibition of 4AP-evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine-tune transmitter release.     

Involvement of Protein Kinase C in Ca2+-Signaling Pathways to Activation of AP-1 DNA-Binding Activity Evoked via NMDA- and Voltage-Gated Ca2+ Channels

Abstract: Stimulation of cultured cerebellar granule cells with N -methyl- d -aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein-1 (AP-1) DNA-binding activity, which can be monitored by an increase in 12- O -tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-binding activity, in concert with c- fos induction. For this increase in TRE-binding activity, Ca²⁺ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca²⁺ chelator, BAPTA-AM, abolished this increase. Close correspondence between the dose-response curves of ⁴⁵Ca²⁺ uptake and TRE-binding activity by NMDA or KA suggested that Ca²⁺ influx not only triggered sequential activation of Ca²⁺-signaling processes leading to the increase in TRE-binding activity, but also controlled its increased level. Stimulation of non-NMDA receptors by KA mainly caused Ca²⁺ influx through voltage-gated Ca²⁺ channels, whereas stimulation of NMDA receptors caused Ca²⁺ influx through NMDA-gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE-binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down-regulation of PKC inhibited the increase in TRE-binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca²⁺-signaling pathways for the increase in TRE-binding activity coupled with the activation of NMDA- and non-NMDA receptors.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Astroglial Gap Junction Communication Is Increased by Treatment with Either Glutamate or High K+ Concentration

Abstract: Astroglia are extensively coupled by gap junctions and form a functional syncytium. Astroglial gap junctions are thought to be involved in the spatial buffering of K⁺ in vivo and in the Ca²⁺ waves seen on glutamate receptor activation. The conductivity of gap junctions is regulated by several second messengers, with up-regulation by cyclic AMP and down-regulation through activation of protein kinase C, decreases in intracellular pH, or increases in the free cytosolic Ca²⁺ concentration. The results presented here indicate that dye coupling of astroglia is significantly up-regulated by membrane depolarization, both by increases in the extracellular K⁺ concentration and directly by ionophores. Furthermore, glutamate, kainate, and quisqualate, which depolarize astroglial cells through activation of ionotropic receptors, also increase dye coupling in astroglia. The effect of kainate and quisqualate was reversed by 6-cyano-7-nitroquinoxaline-2,3-dione, an inhibitor of the ionotropic glutamate receptor. A dose-dependent decrease in dye coupling was seen when the cells were injected with increasing concentrations of Ca²⁺. However, if the cells were simultaneously depolarized, the inhibitory effect of Ca²⁺ on gap junctional conductance was reversed. Significant increases over basal coupling was attained when the cells were injected with Ca²⁺ if they were treated with kainate or K⁺. These data suggest that ligands that depolarize astroglia enhance gap junction communication between astroglia and that this enhancement may be important in maintaining communication between astroglia in the face of elevated Ca²⁺ levels.     

Differential fading of inhibitory and excitatory B2 bradykinin receptor responses in rat sympathetic neurons: a role for protein kinase C

Through inhibitory and excitatory effects on sympathetic neurons, B₂ bradykinin receptors contribute to protective and noxious cardiovascular mechanisms. Presynaptic inhibition of sympathetic transmitter release involves an inhibition of Ca_V2 channels, neuronal excitation an inhibition of K_V7 channels. To investigate which of these mechanisms prevail over time, the respective currents were determined. The inhibition of Ca²⁺ currents by bradykinin reached a maximum of 50%, started to fade within the first minute, and became attenuated significantly after ≥ 4 min. The inhibition of K⁺ currents reached a maximum of 85%, started to fade after > 3 min, and became attenuated significantly after ≥ 7 min. Blocking Ca²⁺-independent protein kinase C (PKC) enhanced the inhibition of Ca²⁺ currents by bradykinin and delayed its fading, left the inhibition of K⁺ currents and its fading unaltered, and enhanced the reduction of noradrenaline release and slowed its fading. Conversely, direct activation of PKC abolished the inhibition of noradrenaline release and largely attenuated the inhibition of Ca²⁺ currents. These results show that the inhibitory effects of bradykinin in sympathetic neurons are outweighed over time by its excitatory actions because of more rapid, PKC-dependent fading of the inhibitory response.     

Dinucleotide Receptor Modulation by Protein Kinases (Protein Kinases A and C) and Protein Phosphatases in Rat Brain Synaptic Terminals

Abstract: The diadenosine polyphosphates, diadenosine tetraphosphate and diadenosine pentaphosphate (Ap₅A), can activate an ionotropic dinucleotide receptor that induces Ca²⁺ transients into synaptosomes prepared from rat brain. This receptor, also termed the P₄ purinoceptor, is sensitive only to adenine dinucleotides and is insensitive to ATP. Studies on the modulatory role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases on the response of diadenosine polyphosphate receptors were performed by measuring the changes in the intracellular Ca²⁺ levels with fura-2. Activation and inhibition of PKA were carried out by means of forskolin and the PKA inhibitory peptide (PKA-IP), respectively. The Ap₅A response was inhibited by forksolin to 35% of control values, but PKA-IP induced an increase of 37%. The effect of PKC activation was similar to that observed for PKA. PKC stimulation with phorbol 12,13-dibutyrate produced an inhibition of 67%, whereas the PKC inhibitors staurosporine and PKC inhibitory peptide enhanced the responses elicited by Ap₅A to 40% in both cases. Protein phosphatase inhibitors diminished the responses elicited by Ap₅A to 17% in the case of okadaic acid, to 50% for microcystin, and to 45% in the case of cyclosporin A. Thus, the activity of dinucleotide receptors in rat brain synaptosomes appears to be modulated by phosphorylation/dephosphorylation. These processes could be of physiological significance in the control of transmitter release from neurons that are postsynaptic to nerves that release diadenosine polyphosphates.     

Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT₃ receptor, a ligand-gated ion channel. The 5-HT₃ receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K⁺ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca²⁺ concentration ([Ca²⁺]_i), K⁺-induced depolarization resulted in variable but rapid increases in apparent [Ca²⁺]_i among the individual terminals, with some synaptosomes displaying large transient [Ca²⁺]_i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca²⁺]_i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT₃ receptors induced slow increases in [Ca²⁺]_i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca²⁺ entry and inhibited by millimolar external Mg²⁺. We conclude that significant increases in brain nerve terminal Ca²⁺, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

Tachykinins Potentiate N-Methyl-D-Aspartate Responses in Acutely Isolated Neurons from the Dorsal Horn

Abstract: Substance P and neurokinin A both potentiated N -methyl- d -aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca²⁺]_i in these cells. Substance P, but not neurokinin A, increased [Ca²⁺]_i in a subpopulation of neurons. The increase in [Ca²⁺]_i was found to be due to Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Substance P and neurokinin A also potentiated the increase in [Ca²⁺]_i produced by NMDA, but not by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 m M K⁺. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca²⁺]_i can be dissociated from their effects on NMDA receptors.     

A2A Adenosine Receptors Inhibit ATP-Induced Ca2+ Influx in PC12 Cells by Involving Protein Kinase A

Abstract: The regulatory role of A_2A adenosine receptors in P₂ purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2- p -(2-carboxyethyl)-phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A_2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca²⁺ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca²⁺]_i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp -adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A_2A adenosine receptors leads to inhibition of P₂ purinoceptor-mediated Ca²⁺ influx through ATP-gated cation channels and involves protein kinase A.     

N-Methyl-d-Aspartate- and Non-N-Methyl-d-Aspartate-Evoked Adenosine Release from Rat Cortical Slices: Distinct Purinergic Sources and Mechanisms of Release

Abstract: Excitatory amino acids, acting at both N methyl- d -aspartate (NMDA) and non-NMDA receptors, release the inhibitory neuromodulator adenosine from superfused rat cortical slices. This study was initiated to investigate the possible purinergic sources and mechanisms of release for the adenosine release evoked by NMDA and non-NMDA receptor activation. Inhibition of the bidirectional nucleo-side transporter with dipyridamole greatly enhanced adenosine release evoked by glutamate, NMDA, kainate, and ( RS -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Inhibition of ecto -5'-nucleotidase with α,β-methylene ADP and GMP had no effect on either kainateor AMPA-evoked adenosine release, but it decreased glutamate- and NMDA-evoked adenosine release by 23 and 68%, respectively. A similar inhibition of NMDA-evoked adenosine release was observed with α,β-methylene ADP alone, indicating that the inhibitory effect was not due to the reported competitive inhibition of NMDA receptors by GMP. Finally, NMDA-evoked adenosine release, but not kainate- or AMPA-evoked release, was Ca²⁺ dependent. These results indicate that activation of non-NMDA receptors releases adenosine per se in a Ca²⁺-independent manner. In contrast, NMDA receptor activation releases primarily a nucleotide that is subsequently converted extracellularly to adenosine; in this case, release is Ca²⁺ dependent. Although neither NMDA- nor non-NMDA-evoked adenosine release occurs via the nucleoside transporter, this transporter does appear to be a major route for removal of adenosine from the extracellular space.     

Stimulation of Muscarinic Receptors Induces Expression of Individual fos and jun Genes Through Different Transduction Pathways

Abstract: The transduction pathways coupling muscarinic receptors to induction of fos and jun genes were investigated in neuroblastoma SH-SY5Y cells. Stimulation with carbachol induced expression of c- fos , fosB , c- jun , junB , and junD . This effect was abolished by pretreatment with atropine, indicating an involvement of muscarinic receptors. These genes were also induced by activation of protein kinase C with phorbol ester or by elevating the intracellular Ca²⁺ concentration with a Ca²⁺ ionophore. The Ca²⁺ effect was inhibited by KN-62, suggesting an induction through Ca²⁺/calmodulin-dependent kinase II. Inhibition of protein kinase C with GF109203X suppressed the carbachol-stimulated increase in mRNA levels of c- fos , fosB , and junB by ∼70% but had only minor effects on the expression of c- jun and junD . On the other hand, preincubation with KN-62 attenuated the carbachol-induced increase in c- jun and junD expression by 70% but had no effect on c- fos , fosB , and junB mRNA levels. Simultaneous inhibition of both protein kinase C and Ca²⁺/calmodulin-dependent kinase II completely abolished the carbachol-stimulated expression of c- jun and junD , but c- fos , fosB , and junB were still expressed to a certain extent under this condition. Comparison of the inhibitory effects of GF109203X and Gö 6976 suggests the involvement of classical protein kinase C isozymes in muscarinic receptor-stimulated expression of fos and jun genes. These results demonstrate that the muscarinic receptor-induced expression of individual fos and jun genes is regulated via different pathways, primarily protein kinase C or Ca²⁺/calmodulin-dependent kinase II.     

M1 Muscarinic Receptors Stimulate Rapid and Prolonged Phases of Neuronal Nitric Oxide Synthase Activity: Involvement of Different Calcium Pools

Abstract: This study shows that activation of M₁ muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)-K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca²⁺ concentration and nNOS activity. We examined the relationship between receptor-mediated increases in intracellular Ca²⁺ concentration and activation of nNOS over both short and long intervals using guanosine 3',5'-cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca²⁺ from intracellular stores in both the CHO M₁/nNOS transfected cells and in neuroblastoma (N1E-115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M₁ muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l -[³H]arginine to l -[³H]citrulline as well as cGMP formation following stimulation of M₁ muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca²⁺ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca²⁺.     

The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation

Abstract: The metabotropic glutamate receptor mGluR5, but not the closely related mGluR1, is expressed in cultured astrocytes, and this expression is up-regulated by specific growth factors. We investigated the capability and underlying mechanisms of mGluR5 to induce oscillatory responses of intracellular calcium concentration ([Ca²⁺]_i) in cultured rat astrocytes. Single-cell [Ca²⁺]_i recordings indicated that an mGluR-selective agonist, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate (1 S ,3 R -ACPD), elicits [Ca²⁺]_i oscillations in good agreement with the growth factor-induced up-regulation of mGluR5 in cultured astrocytes. A protein kinase C (PKC) inhibitor, bisindolylmaleimide I, converted a 1 S ,3 R -ACPD-mediated oscillatory response into a nonoscillatory response. In addition, the PKC activator phorbol 12-myristate 13-acetate completely abolished the [Ca²⁺]_i increase. These and other pharmacological properties of 1 S ,3 R -ACPD-induced [Ca²⁺]_i oscillations correlate well with those of the cloned mGluR5 characterized in heterologous expression systems. Furthermore, the potential involvement of protein phosphatases in [Ca²⁺]_i oscillations is suggested. The present study demonstrates that mGluR5 is capable of inducing [Ca²⁺]_i oscillations in cultured astrocytes and that phosphorylation/dephosphorylation of mGluR5 is critical in [Ca²⁺]_i oscillations, analogous to the cloned mGluR5 expressed in heterologous cell lines.     

Ca2+-Dependent Inactivation of P-Type Calcium Channels in Nerve Terminals

Abstract: Rapid Ca²⁺ signals evoked by K⁺ depolarization of rat cerebral cortical synaptosomes were measured by dual-channel Ca²⁺ spectrofluorometry coupled to a stopped-flow device. Kinetic analysis of the signal rise phase at various extracellular Ca²⁺ concentrations revealed that the responsible voltage-dependent Ca²⁺ channels, previously identified as P-type Ca²⁺ channels, inactivate owing to the rise in intracellular Ca²⁺ levels. At millimolar extracellular Ca²⁺ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca²⁺, thus limiting the Ca²⁺ signal amplitudes to 500–600 n M. A slower, probably voltage-dependent regulation appears to be effective at lower Ca²⁺ influx rates, leading to submaximal Ca²⁺ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca²⁺ levels appears to be responsible for the different inhibition characteristics of Cd²⁺ versus ω-agatoxin IVa.     

Presynaptic Modulation of Cholecystokinin Release by Protein Kinase C in the Rat Hippocampus

Abstract: The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4β-phorbol 12,13-dibutyrate (β-PDBu) dose dependently (5–5,000 n M ) increased CCK-8 release in a strictly Ca²⁺-dependent way. This effect was observed only when synaptosomes were stimulated with the K⁺_A channel blocker 4-aminopyridine (4-AP; 1 m M ) but not with KCI (10–30 m M ). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by α-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. β-PDBu (50–100 n M ) also stimulated the 4-AP-evoked Ca²⁺-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of colocalizing neurotransmitters via modulation of presynaptic K⁺ channels in rat hippocampus.