Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Muscarinic Cholinoceptor-Stimulated Synthesis and Degradation of Inositol 1,4,5-Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [³H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 m M ) caused an immediate increase in level of Ins(1,4,5)P₃ (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP₅ and InsP₆ was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P₃ and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P₃ metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P₃ 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidylinositol 4-phosphate cannot be excluded.     

Chronic Activation of Muscarinic and Metabotropic Glutamate Receptors Down-Regulates Type I Inositol 1,4,5-Trisphosphate Receptor Expression in Cerebellar Granule Cells

Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP₃) over extended incubation periods, and consequently to affect the level of InsP₃ receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 m M ) evoked a biphasic accumulation of InsP₃, a rapid three- to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP₃ receptor, it was demonstrated that >50% down-regulation of type I InsP₃ receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 m M CCh. Over 24 h, 1 m M CCh caused an ∼85% decrease in type I InsP₃ receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP₃ receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1 S ,3 R -dicarboxylic acid (200 µ M ), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP₃ receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP₃ production in cerebellar granule cells is the down-regulation of InsP₃ receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.     

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα_16gust44 and Gα_15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP₃)-dependent Ca²⁺ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca²⁺ concentration, we used sweet and umami compounds as well as other InsP₃-generating ligands in FURA-2-based Ca²⁺ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca²⁺ response of HEK293 cells to the InsP₃-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca²⁺ store of the endoplasmic reticulum (ER) and independent of extracellular Ca²⁺. The function of CIB1 on InsP₃-evoked Ca²⁺ release from the ER is most likely mediated by its interaction with the InsP₃ receptor. Thus, CIB1 seems to be an inhibitor of InsP₃-dependent Ca²⁺ release in vivo .     

Involvement of Calcium Influx in Muscarinic Cholinergic Regulation of Phospholipase C in Cerebellar Granule Cells

Abstract: Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca²⁺ concentration ([Ca²⁺]_c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2–3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca²⁺]_c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca²⁺]_c and agonist-stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol-stimulated inositol phosphate accumulation was achieved using 20 m M KCl, which increased [Ca²⁺]_c from ∼20 to ∼75 n M , indicating the involvement of relatively low threshold Ca²⁺ channels and the high sensitivity of the relevant PLC to small changes in [Ca²⁺]_c. By contrast, increases in [Ca²⁺]_c induced by the Ca²⁺ ionophore ionomycin were associated with more modest and less potent effects on agonist-stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein-regulated PLC and voltage-stimulated elevations of [Ca²⁺]_c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.     

Histamine Evokes Greater Increases in Phosphatidylinositol Metabolism and Catecholamine Secretion in Epinephrine-Containing than in Norepinephrine-Containing Chromaffin Cells

Abstract: Chromaffin cells have H₁ histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol-containing phospholipids and stimulates catecholamine secretion from Chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP₁) and catecholamine secretion in purified cultures of norepinephrine-containing and epinephrine-containing bovine Chromaffin cells. Histamine-stimulated InsP, accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP¹ accumulation in the two Chromaffin cell subtypes. Histamine-stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K⁺, bradykinin, phorbol 12, 13-dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H₁ receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H₁ receptors on epinephrine cells may account for the greater effects of histamine on InsP₁ accumulation and catecholamine secretion in these cells.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.     

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum

Adenosine A_2A, cannabinoid CB₁ and metabotropic glutamate 5 (mGlu₅) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB₁R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A_2ARs. Such a permissive effect of A_2ARs towards CB₁Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A_2ARs. The selective mGlu₅R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A_2AR blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A_2ARs regulates the synaptic effects of CB₁Rs and that A_2ARs may control CB₁ effects also indirectly, namely through mGlu₅Rs.     

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⁺.     

N-Methyl-d-Aspartate-Sensitive Glutamate Receptors Induce Calcium-Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N -methyl-d-aspartate (NMDA) induced a dose-dependent release of [³H]arachidonic acid ([³H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [³H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A₂ (PLA₂) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA₂ rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [³H]AA release was dependent on the presence of extracellular Ca²⁺ and was mimicked by the Ca²⁺ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [³H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA₂ that is mediated by Ca²⁺ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.     

Type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors promote the differentiation of granule cell precursors in the postnatal cerebellum

During postnatal development of the cerebellum, granule cell precursors (GCPs) proliferate in the external granular layer (EGL), exit the cell cycle, differentiate, and migrate from the EGL to the internal granular layer. In the present study, we report that type 2 and 3 inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R2 and IP₃R3) regulate the differentiation of GCPs after postnatal day 12 (P12). 5-Bromodeoxyuridine labeling experiments revealed that in mutant mice lacking both of these receptors (double mutants) a greater number of GCPs remain undifferentiated after P12. Consequently, the EGL of the double mutants is thicker than that of control mice at this age and thereafter. In addition, granule cells remain in the EGL of the double mutants at P21, an age when migration has concluded in wild-type mice. Whereas differentiation of GCPs was reduced in the double mutants, the absence of IP₃R2 and IP₃R3 did not affect the doubling time of GCPs. We conclude that intracellular calcium release via IP₃R2s and IP₃R3s promotes the differentiation of GCPs within a specific interval of postnatal development in the cerebellum.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

Native Serotonin 5-HT3 Receptors Expressed in Xenopus Oocytes Differ from Homopentameric 5-HT3 Receptors

Abstract: Efficacies of the 5-hydroxytryptamine (serotonin) 5-HT₃ receptor (5-HT₃R) agonists 2-methyl-5-HT, dopamine, and m -chlorophenylbiguanide on 5-HT₃R native to N1E-115 cells and on homopentameric 5-HT₃R expressed in Xenopus oocytes were determined relative to that of 5-HT. Efficacies of 2-methyl-5-HT and dopamine on 5-HT₃R native to differentiated N1E-115 cells are high (54 and 36%) as compared with their efficacies on homopentameric 5-HT₃R-A_L and 5-HT₃R-A_s receptors expressed in oocytes (4–8%). m -Chlorophenylbiguanide does not distinguish between 5-HT₃R in N1E-115 cells and in oocytes. The distinct pharmacological profile of 5-HT₃R native to differentiated N1E-115 cells is conserved when poly(A)⁺ mRNA from these cells is expressed in oocytes. The results indicate that, apart from the known 5-HT₃R subunits, N1E-115 cells express additional proteins involved in 5-HT₃R function.     

Functional Wild-Type and Carboxy-Terminal-Tagged Rat Substance P Receptors Expressed in Baculovirus-Infected Insect Sf9 Cells

Abstract: The rat substance P (SP) receptor (SPR) was expressed in insect Sf9 cells by infection with recombinant baculovirus. The receptor bound SP with high affinity ( K _D = 360 p M ) and had a rank order of affinity of SP > neurokinin A > neurokinin B. Ligand activation of the receptor resulted in an increase in both inositol lipid hydrolysis and intracellular Ca²⁺ concentration ([Ca²⁺]_i). However, high-level expression of the receptor, in the absence of ligand, was correlated with increased basal turnover of inositol lipids and an elevated rate of Ca²⁺ influx. These results demonstrate that the Sf9 cells provide a suitable environment for the high-level expression of a functionally active SPR. Two carboxy-terminal epitope-tagged receptors (SPR-KT3 = SPR-TPPPEPET, COOH; SPR-Glu = SPR-EEEEYMPME, COOH) were also expressed. The affinity of the KT3-tagged receptor for ligand was similar to that of the wild-type receptor ( K _D = 405 p M ), and that of the Glu-tagged receptor was slightly lower ( K _D = 1,082 p M ). The high-affinity SP binding site of all three receptors was sensitive to guanosine 5'- O -(3-thiotriphosphate) pretreatment. The maximal signal-transducing ability of the epitope-tagged receptors was comparable to that of the wild-type receptor ([Ca²⁺]_i rise as a percentage of wild-type: SPR-KT3, 80–100%; SPR-Glu, 88–100%). These data show that heterologous expression in the baculovirus system results in high expression of functional wild-type and tagged receptors.     

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Agonists stimulate cannabinoid 1 receptor (CB₁R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization – likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB₁) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB₁R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB₁Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB₁R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of β-arrestin-2 recruitment to mutant CB₁R suggests that putative carboxy-terminal phosphorylation sites mediate β-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB₁R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB₁Rs.     

Depolarization and Activation of Dihydropyridine-Sensitive Ca2+ Channels Stimulate Inositol Phosphate Accumulation in Photoreceptor-Enriched Chick Retinal Cell Cultures

Abstract: Elevated concentrations of extracellular K⁺ increased inositol phosphate accumulation in primary cultures of chick retinal photoreceptors and multipolar neurons. K⁺-evoked stimulation of inositol phosphate accumulation was greater in photoreceptor-enriched cell cultures than in cultures where multipolar neurons were the predominant cell type. Destroying multipolar neurons, but not photoreceptors, with kainic acid and N -methyl- d -aspartate did not reduce the K⁺-evoked stimulation of inositol phosphate accumulation. Both of these observations indicate that the observed effects occur in photoreceptor cells. The K⁺-evoked stimulation of inositol phosphate accumulation was blocked by omitting Ca²⁺ from the incubation medium or by adding the dihydropyridine-sensitive Ca²⁺-channel antagonists, nitrendipine and nifedipine. Bay K 8644, a dihydropyridine agonist, stimulated inositol phosphate accumulation and enhanced the effect of K⁺. ω-Conotoxin GVIA, an inhibitor of N-type Ca²⁺ channels, had no significant effect on K⁺-stimulated inositol phosphate accumulation. Pretreatment with pertussis toxin neither blocked K⁺-evoked inositol phosphate accumulation nor altered the inhibitory effect of nifedipine. K⁺-evoked inositol phosphate accumulation appears to reflect activation of phosphatidylinositol-specific phospholipase C, as it is inhibited by U-73122. These results indicate that Ca²⁺ influx through voltage-gated, dihydropyridine-sensitive channels activates phospholipase C in photoreceptor inner segments and/or synaptic terminals.     

Signal Flows from Two Phospholipase C-Linked Receptors Are Independent in PC12 Cells

Abstract: Bradykinin (BK) receptor and P₂-purinergic receptor are known to be coupled to phospholipase C (PLC) in PC12 cells. To study the interaction between these two PLC-linked receptors, the presence of both receptors on individual cells was demonstrated by sequential Ca²⁺ spikes caused by BK and ATP in a single fura-2-loaded cell. BK- and ATP-induced catecholamine (CA) secretions were desensitized within 5 min. However, in the sequential experiment, the BK-induced homologous desensitization of CA secretion did not block the ATP-induced secretion, and vice versa. Each agonist-induced an increase in inositol 1,4,5-trisphosphate (IP₃) production and intracellular free Ca²⁺ concentration also led to homologous desensitization. However, there was no heterologous desensitization between the two agonists. When the cells were treated with both BK and ATP simultaneously, the amounts of CA secretion, IP₃ production, internal Ca²⁺ mobilization, and Ca²⁺ influx were all additive. We also found that both IP₃-induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx from extracellular space were able to release [³H]norepinephrine, and the secretion induced by both agonists was exactly additive in the absence or presence of extracellular Ca²⁺. The data suggest that the CA secretions caused by BK or ATP may have separate secretory pathways even though they activate identical second messenger pathways.