Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels

Inhibition of the mitochondrial Na+/Ca2+ exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca2+ homeostasis. However, the Ca2+ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca2+ levels are modulated by CGP37157 (10 mu M) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca2+ homeostasis using cameleon-based mitochondrial Ca2+ and cytosolic Ca2+ ([Ca2+](i)) live imaging. We observed that NCLX-driven mitochondrial Ca2+ exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca-2](i) concomitant with a Ca2+ accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca2+ efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca2+](i) increase by blocking voltage-gated Ca2+ channels (VGCCs), whereas it did not induce depletion of ER Ca2+ stores. Moreover, mitochondrial Ca2+ overload was reduced as a consequence of diminished Ca2+ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca2+ overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca2+ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.

Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle

This study assessed the contribution of L-type Ca2+ channels and other Ca2+ entry pathways to Ca2+ store refilling in choroidal arteriolar smooth muscle. Voltage-clamp recordings were made from enzymatically isolated choroidal microvascular smooth muscle cells and from cells within vessel fragments (containing <10 cells) using the whole-cell perforated patch-clamp technique. Cell Ca2+ was estimated by fura-2 microfluorimetry. After Ca2+ store depletion with caffeine (10 mM), refilling was slower in cells held at -20 mV compared to -80 mV (refilling half-time was 38 +/- 10 and 20 +/- 6 s, respectively). To attempt faster refilling via L-type Ca2+ channels, depolarising steps from -60 to -20 mV were applied during a 30 s refilling period following caffeine depletion. Each step activated L-type Ca2+ currents and [Ca2+]i transients, but failed to accelerate refilling. At -80 mV and in 20 mM TEA, prolonged caffeine exposure produced a transient Ca2+-activated Cl- current (I(Cl)(Ca)) followed by a smaller sustained current. The sustained current was resistant to anthracene-9-carboxylic acid (1 mM; an I(Cl)(Ca) blocker) and to BAPTA AM, but was abolished by 1 microM nifedipine. This nifedipine-sensitive current reversed at +29 +/- 2 mV, which shifted to +7 +/- 5 mV in Ca2+-free solution. Cyclopiazonic acid (20 microM; an inhibitor of sarcoplasmic reticulum Ca2+-ATPase) also activated the nifedipine-sensitive sustained current. At -80 mV, a 5 s caffeine exposure emptied Ca2+ stores and elicited a transient I(Cl)(Ca). After 80 s refilling, another caffeine challenge produced a similar inward current. Nifedipine (1 microM) during refilling reduced the caffeine-activated I(Cl)(Ca) by 38 +/- 5 %. The effect was concentration dependent (1-3000 nM, EC50 64 nM). In Ca2+-free solution, store refilling was similarly depressed (by 46 +/- 6 %). Endothelin-1 (10 nM) applied at -80 mV increased [Ca2+]i, which subsided to a sustained 198 +/- 28 nM above basal. Cell Ca2+ was then lowered by 1 microM nifedipine (to 135 +/- 22 nM), which reversed on washout. These results show that L-type Ca2+ channels fail to contribute to Ca2+ store refilling in choroidal arteriolar smooth muscle. Instead, they refill via a novel non-selective store-operated cation conductance that is blocked by nifedipine.

Contribution of reverse Na+/Ca2+ exchange to spontaneous activity in interstitial cells of cajal in the rabbit urethra

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+-Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mM, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB-R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mM) and noradrenaline (10 microM) induced transient Ca2+-activated chloride currents (I(ClCa)) at -60 mV due to release of Ca2+ from ryanodine- and inositol trisphosphate (IP3)-sensitive Ca2+ stores, respectively, but these responses were not blocked by KB-R7943 or SEA0400 suggesting that neither drug blocked Ca2+-activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration-dependent fashion. Finally, single cell RT-PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+-Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca2+ signaling, and insulin secretion in the anx7(+/−) knockout mouse

The mammalian anx7 gene codes for a Ca2+-activated GTPase, which supports Ca2+/GTP-dependent secretion events and Ca2+ channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca2+ signaling in secreting pancreatic β cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the β cells. The nullizygous anx7 (−/−) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/−) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/−) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca2+ channel functions are normal. However, electrooptical recordings indicate that the (+/−) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP3)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP3 receptor expression and function in pancreatic islets. The profound increase in islets, β cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic β cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca2+ signaling through IP3-sensitive Ca2+ stores.

Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca2+ stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca2+-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca2+ entry and to test for a role of their respective binding partners in Ca2+ entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca2+ entry. In contrast, F2g enhanced the two forms of Ca2+ entry. We conclude that store depletion-activated Ca2+ entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca2+ from stores and activating Ca2+ influx in response to either increasing IP3 or decreasing luminal Ca2+.

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells1

Anoxia induces a rapid elevation of the cytosolic Ca2+ concentration ([Ca2+]cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca2+ release is important for gene activation and survival in O2-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca2+ to the anoxic [Ca2+]cyt elevation in maize cells. Imaging of intramitochondrial Ca2+ levels showed that a majority of mitochondria released their Ca2+ in response to anoxia and took up Ca2+ upon reoxygenation. We also investigated whether the mitochondrial Ca2+ release contributed to the increase in [Ca2+]cyt under anoxia. Analysis of the spatial association between anoxic [Ca2+]cyt changes and the distribution of mitochondrial and other intracellular Ca2+ stores revealed that the largest [Ca2+]cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca2+]cyt under aerobic conditions but prevented a [Ca2+]cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca2+]cyt and participate in the signaling of O2 deprivation.

Rat protease-activated receptor–1 (rPAR1) expression and characterization in Sf9 cells

Connue pour son rôle dans la cascade de coagulation, la thrombine, une protéase à sérine, peut également agir par l’intermédiaire de PAR1, un récepteur activé par protéase et couplé aux protéines G liant le GTP (GPCR). La thrombine se lie et clive l’extrémité N-terminale du PAR1 entre l’Arg41 et la Ser42, exposant une nouvelle extrémité terminale qui agit elle-même comme un ligand. La thrombine et une séquence peptidique de cinq acides aminés, composée des résidus Ser42 à Arg46, nommée PAR1-AP, déclenchent dans diverses cellules de mammifères une réponse intracellulaire comportant une composante calcique. Dans cette étude, le système d’expression par baculovirus dans les cellules Sf9 d'insecte nous a permis d'exprimer le récepteur PAR1 du rat à la surface de ces cellules et de réaliser son couplage fonctionnel à leur signalisation intracellulaire (modèle rPAR1-Sf9). La composante calcique de celle-ci, en réponse au PAR1-AP, a ensuite été étudiée en détail à l’aide de la sonde fluorescente Fura-2 et de plusieurs inhibiteurs agissant sur les canaux calciques ou d'autres éléments de la cascade de signalisation du calcium intracellulaire. Lorsque le milieu extracellulaire contient du calcium (Ca2+), la thrombine ou PAR1-AP déclenchent un signal calcique qui consiste en une augmentation rapide de [Ca2+]i suivi d’un plateau relativement soutenu, puis d'un retour lent vers le niveau de base initial. En l'absence de Ca2+ dans le milieu extracellulaire, l'augmentation initiale rapide de [Ca2+]i est suivie d'un retour rapide vers le [Ca2+]i de base. À l’aide d’inhibiteurs de canaux calciques, tels que le lanthane, la nifédipine et le D-600, l'entrée du calcium du milieu extracellulaire dans les cellules est inhibée, abolissant le plateau soutenu de [Ca2+]i. L’inhibition de la pompe Ca2+-ATPase par la thapsigargine supprime la réponse au PAR1-AP après épuisement des sites de stockage de Ca2+intracellulaire. Le TMB-8 agit de façon discordante quant à l’inhibition de la libération de Ca2+ des sites de stockage intracellulaires. La réponse à PAR1-AP n’est pas affectée par le D-609, un inhibiteur de la phospholipase β. L’inhibition de la protéine kinase C (PKC) par le bisindolylmaléimide induit des oscillations en présence de Ca2+ extracellulaire et atténue fortement le signal calcique en absence de Ca2+ extracellulaire. En présence de Ca2+ extracellulaire, l’activation de la PKC par le PBDu tronque le flux de [Ca2+]i tandis que la réponse calcique est abolie en absence de Ca2+ dans le milieu extracellulaire. Le H-89, un inhibiteur de la protéine kinase A (PKA), cause une prolongation de la durée du plateau de [Ca2+]i dans un milieu riche en calcium et la suppression de la réponse à PAR1-AP lorsque le milieu extracellulaire est dépourvu de Ca2+. Les résultats obtenus nous permettent de conclure que la PKC et possiblement la PKA jouent un rôle critique dans la mobilisation du Ca2+ induite par le PAR1-AP dans le modèle rPAR1-Sf9.

NAADP regulates human platelet function.

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca2+ transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y(1), P2Y(2) and P2X(4) receptors or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP analogue-induced Ca2+ transients. In neuronal-differentiated cells, P2Y(2), P2Y(6), P2X(2) and possibly P2X(2)/P2X(6) heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca2+](i). We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-beta S-induced proliferation in P19 cells was mediated by P2Y, and P2Y2 receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y, and P2Y2 receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca2+ stores. (C) 2008 ISDN. Published by Elsevier Ltd. All rights reserved.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Specific association of the gene product of PKD2 with the TRPC1 channel

The function(s) of the genes (PKD1 and PKD2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While PKD1 encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell–cell or cell–matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca2+ channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca2+ stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and in vitro. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2–TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca2+ entry in response to G protein-coupled receptor activation and/or store depletion.

Evidence of a role for cyclic ADP-ribose in long-term synaptic depression in hippocampus

Ca2+ released from presynaptic and postsynaptic intracellular stores plays important roles in activity-dependent synaptic plasticity, including long-term depression (LTD) of synaptic strength. At Schaffer collateral–CA1 synapses in the hippocampus, presynaptic ryanodine receptor-gated stores appear to mobilize some of the Ca2+ necessary to induce LTD. Cyclic ADP-ribose (cADPR) has recently been proposed as an endogenous activator of ryanodine receptors in sea urchin eggs and several mammalian cell types. Here, we provide evidence that cADPR-mediated signaling pathways play a key role in inducing LTD. We show that biochemical production of cGMP increases cADPR concentration in hippocampal slices in vitro, and that blockade of cGMP-dependent protein kinase, cADPR receptors, or ryanodine-sensitive Ca2+ stores each prevent the induction of LTD at Schaffer collateral–CA1 synapses. A lack of effect of postsynaptic infusion of either cADPR antagonist indicates a probable presynaptic site of action.

Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na+-dependent mechanism

Nicotine at very low doses (5–30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist, d-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca2+ influx through either the nicotinic receptors or the voltage-gated Ca2+ channels because the release was increased moderately when the extracellular solution was changed into a Ca2+-free solution that also contained Mg2+ (4 mM) and Cd2+ (200 μM). The release did not depend on Ca2+ release from the intraterminal Ca2+ stores either because fura-2 fluorimetry showed extremely low Ca2+ elevation (≈30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca2+] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na+ because substitution of extracellular NaCl with N-methyl-d-glucamine chloride completely blocked the release. The Na+-dependent mechanism was not via Na+ influx through the voltage-gated Na+ channels because the release was not affected by tetrodotoxin (1–50 μM) plus Cd2+ (200 μM). Thus, nicotine at very low concentrations induced LHRH release via a Na+-dependent, Ca2+-independent mechanism.

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36M3R cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.