Receptor-induced Ca2 Research Articles

Altered Development of CD8 + T Cell Lineages in Mice Deficient for the Tec Kinases Itk and Rlk

Mutations affecting the Tec kinases Itk and Rlk decrease T cell receptor-induced Ca(2+) mobilization and Erk kinase activation and impair both positive and negative thymic selection. Itk(-/-) and Rlk(-/-)Itk(-/-) mice also have decreased CD4:8 T cell ratios, suggestive of altered CD4:8 lineage commitment. Nonetheless, we find that CD8 single-positive (SP) thymocytes and peripheral CD8(+) T cells in these mice do not resemble conventional CD8(+) T cells. Instead, these cells express memory markers, rapidly produce interferon-gamma, and can be selected on hematopoietically derived cells, similar to MHC class Ib-restricted "innate-type" lymphocytes. Itk deficiency also greatly increases the number of cells selected by MHC class Ib. Expression of a hypersensitive Erk2 mutant partially corrects the CD8(+) T cell phenotypes in Itk(-/-) mice, arguing that altered signaling permits development of this innate-type CD8(+) cell population. Our results suggest that Tec kinases differentially regulate development of conventional versus nonconventional lymphocytes.

The SRC family kinase LYN redirects B cell receptor signaling in human SLP65-deficient B cell lymphoma cells

SLP65 represents a critical component in (pre-) B cell receptor signal transduction but is compromised in a subset of pre-B cell-derived acute lymphoblastic leukemia. Based on these findings, we investigated (i.) whether SLP65-deficiency also occurs in mature B cell-derived lymphoma and (ii.) whether SLP65-deficient B cell lymphoma cells use an alternative B cell receptor signaling pathway in the absence of SLP65. Indeed, expression of SLP65 protein was also missing in a fraction of B cell lymphoma cases. While SLP65 is essential for B cell receptor-induced Ca2+ mobilization in normal B cells, B cell receptor engagement in SLP65-deficient as compared to SLP65-reconstituted B cell lymphoma cells resulted in an accelerated yet shortlived Ca2+-signal. B cell receptor engagement of SLP65-deficient lymphoma cells involves SRC kinase activation, which is critical for B cell receptor-dependent Ca2+-mobilisation in the absence but not in the presence of SLP65. As shown by RNA interference, the SRC kinase LYN is required for B cell receptor-induced Ca2+ release in SLP65-deficient B cell lymphoma cells but dispensable after SLP65-reconstitution. B cell receptor engagement in SLP65-deficient B cell lymphoma cells also resulted in tyrosine-phosphorylation of the proliferation- and survival-related MAPK1 and STAT5 molecules, which was sensitive to silencing of the SRC kinase LYN. Inhibition of SRC kinase activity resulted in growth arrest and cell death specifically in SLP65-deficient lymphoma cells. These findings indicate that LYN can short-circuit conventional B cell receptor signaling in SLP65-deficient B cell lymphoma cells and thereby promote activation of survival and proliferation-related molecules.

STIM1 has a plasma membrane role in the activation of store-operated Ca(2+) channels.

Receptor-induced Ca(2+) signals are key to the function of all cells and involve release of Ca(2+) from endoplasmic reticulum (ER) stores, triggering Ca(2+) entry through plasma membrane (PM) "store-operated channels" (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca(2+)-binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca(2+) sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca(2+) sensor, STIM1 functions within the PM to control operation of the Ca(2+) entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca(2+) release-activated Ca(2+) (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current (I(CRAC)) into a constitutively active, Ca(2+) store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties. Last, Ab externally applied to the STIM1 N-terminal EF hand blocks both I(CRAC) in hematopoietic cells and SOC-mediated Ca(2+) entry in HEK293 cells, revealing that STIM1 has an important functional presence within the PM. The results reveal that, in addition to being an ER Ca(2+) sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca(2+)-regulated processes including secretion, contraction, metabolism, cell division, and apoptosis.

The Vav Family: At the Crossroads of Signaling Pathways

The Vav family of Rho-guanine nucleotide exchange factors (GEFs) is thought to control a diverse array of signaling pathways emanating from antigen receptors in lymphocytes, although the exact mechanism by which Vav exerts its function is only beginning to emerge. Vav proteins are modular and contain the Dbl-homology domain, typical of all known Rho-GEFs, in addition to several other structural domains characteristic of proteins involved in signal transduction. Recently, our laboratory generated mice congenitally lacking all three Vav isoforms, providing genetic evidence that the Vav family is critical and nonredundant in T- and B-lymphocyte development and function and is essential in the formation of the adaptive immune system. These experiments also demonstrated that Vav proteins are indispensable for both T-cell receptor- and B-cell receptor-induced Ca++ fluxes. However, detailed analyses of Vav-deficient mice revealed unexpected complexity of Vav involvement in cellular activation. Notably, we observed lineage-specific Vav regulation of mitogen-activated protein kinase signaling, in which Vav was required in T-cells, but not in B-cells. Moreover, the three Vav proteins appear to function specifically in distinct signaling pathways emanating from activating receptors of natural killer cells that trigger natural cytotoxicity.

Phosphotidylinositol 4,5-Bisphosphate Signals Underlie Receptor-Specific Gq/11-Mediated Modulation of N-Type Ca2+Channels

Modulation of voltage-gated Ca2+ channels via G-protein-coupled receptors is a prime mechanism regulating neurotransmitter release and synaptic plasticity. Despite extensive studies, the molecular mechanism underlying Gq/11-mediated modulation remains unclear. We found cloned and native N-type Ca2+ channels to be regulated by phosphatidylinositol [correction] 4,5-bisphosphate (PIP2). In inside-out oocyte patches, PIP2 greatly attenuated or reversed the observed rundown of expressed channels. In sympathetic neurons, muscarinic M1 ACh receptor suppression of the Ca2+ current (ICa) was temporally correlated with PIP2 hydrolysis, blunted by PIP2 in whole-cell pipettes, attenuated by expression of PIP2-sequestering proteins, and became irreversible when PIP2 synthesis was blocked. We also probed mechanisms of receptor specificity. Although bradykinin also induced PIP2 hydrolysis, it did not inhibit ICa. However, bradykinin receptors became nearly as effective as M1 receptors when PIP2 synthesis, IP3 receptors, or the activity of neuronal Ca2+ sensor-1 were blocked, suggesting that bradykinin receptor-induced intracellular Ca2+ increases stimulate PIP2 synthesis, compensating for PIP2 hydrolysis. We suggest that differential use of PIP2 signals underlies specificity of Gq/11-coupled receptor actions on the channels

Toward a consensus on the operation of receptor-induced calcium entry signals.

Receptor-induced Ca2+ signals involve both Ca2+ release from intracellular stores and extracellular Ca2+ entry across the plasma membrane. The channels mediating Ca2+ entry and the mechanisms controlling their function remain largely a mystery. Here we critically assess current views on the Ca2+ entry process and consider certain modifications to the widely held hypothesis that Ca2+ store emptying is the fundamental trigger for receptor-induced Ca2+ entry channels. Under physiological conditions, receptor-induced store depletion may be quite limited. A number of distinct channel activities appear to mediate receptor-induced Ca2+ entry, and their activation is observed to occur through quite diverse coupling processes.

Positive feedback regulation of PLCgamma1/Ca(2+) signaling by PKCtheta in restimulated T cells via a Tec kinase-dependent pathway.

PKCtheta plays an essential role in activation of mature T cells. Here, we report that the TCR/CD28-induced tyrosine phosphorylation and activation of PLCgamma1 was significantly impaired in PKCtheta (-/-) primary, restimulated T cells. Consistent with this finding, receptor-induced Ca(2+) mobilization, NF-AT DNA-binding activity and the membrane translocation of PKCalpha, a PLCgamma1-dependent conventional PKC, were also markedly reduced in the same cells. Moreover, a dominant-negative PLCgamma1 mutant blocked the PKCtheta-induced activation of an AP-1 reporter gene in Jurkat and primary cells. Regulation of PLCgamma1 signaling by PKCtheta required the tyrosine kinase Tec since a dominant-negative Tec mutant blocked PKCtheta-induced AP-1 (but not NF-kappaB) activation. In addition, wild-type Tec, but not Itk or Rlk, potently activated AP-1. Furthermore, Tec was found to constitutively associate with PKCtheta, an interaction that like AP-1 activation required the pleckstrin-homology domain of Tec. These findings define a novel PKCtheta-initiated pathway that regulates Ca(2+) signaling and AP-1 activation via Tec and PLCgamma1. Moreover, they identify Tec as a key point downstream of PKCtheta, where TCR- and PKCtheta-induced signaling pathways, leading to AP-1 versus NF-kappaB activation, diverge in T cells.

Non-voltage-gated L-type Ca2+ Channels in Human T Cells: PHARMACOLOGY AND MOLECULAR CHARACTERIZATION OF THE MAJOR α PORE-FORMING AND AUXILIARY β-SUBUNITS

In T lymphocytes, engagement of the antigen receptor leads to a biphasic Ca2+ flux consisting of a mobilization of Ca2+ from intracellular stores followed by a lower but sustained elevation that is dependent on extracellular Ca2+. The prolonged Ca2+ flux is required for activation of transcription factors and for subsequent activation of the T cell. Ca2+ influx requires as yet unidentified Ca2+ channels, which potentially play a role in T cell activation. Here we present evidence that human T cells express a non-voltage-gated Ca2+ channel related to L-type voltage-gated Ca2+ channels. Drugs that block classical L-type channels inhibited the initial phase of the antigen receptor-induced Ca2+ flux and could also inhibit the sustained phase of the Ca2+ signal suggesting a role for the L-type Ca2+ channel in antigen receptor signaling. T cells expressed transcripts for the alpha(1) 1.2 and alpha(1) 1.3 pore-forming subunits of L-type voltage-gated Ca2+ channels and transcripts for all four known beta-subunits including several potential new splice variants. Jurkat T leukemia cells expressed a small amount of full-length alpha(1)1.2 protein but the dominant form was a truncated protein identical in size to a truncated alpha(1) 1.2 protein known to be expressed in B lymphocytes. They further expressed a truncated form of the alpha(1) 1.3 subunit and auxiliary beta1- and beta3-subunit proteins. Our data strongly suggest that functional but non-voltage-gated L-type Ca2+ channels are expressed at the plasma membrane in T cells and play a role in the antigen receptor-mediated Ca2+ flux in these cells.

Real-time analysis of phospholipase C activity during different patterns of receptor-induced Ca 2+ responses in HEK293 cells

[Ca 2+] i oscillations can either depend on oscillatory inositol-1,4,5-trisphosphate (InsP 3) formation by phospholipase C (PLC) or rely on local feedback mechanisms involving the InsP 3 receptor. To assess the PLC activity underlying carbachol-induced [Ca 2+] i oscillations in single HEK293 cells, we co-imaged [Ca 2+] i with fluorescent fusion proteins of protein kinase C (PKC) isotypes and the PH domain of PLC-δ1 (PLC-δ1(PH)). The translocation of PKCα-YFP in single cells followed two discrete patterns. Upon maximally effective agonist concentrations, a fast association and delayed dissociation ( k on> k off) was the predominant pattern. The delayed dissociation has been linked to diacylglycerol formation. Upon stimulation with submaximally effective agonist concentrations as well as during regenerative [Ca 2+] i waves, we mainly observed short translocations with k on≈ k off. Translocation time courses and efficiencies of the diacylglycerol-sensing PKCϵ-CFP and the InsP 3/phosphatidylinositol-4,5-bisphosphate-sensing YFP-PLC-δ1(PH) were closely correlated. Significant PLC activity was only detectable upon strong receptor stimulation, which typically failed to trigger [Ca 2+] i oscillations. During [Ca 2+] i oscillations induced by submaximal receptor stimulation, YFP-PLC-δ1(PH) did not translocate, whereas a fluorescent PKCϵ fusion protein has been reported to exhibit a slow, non-oscillatory accumulation at the plasma membrane. We conclude that carbachol-induced [Ca 2+] i oscillations in HEK293 cells develop at low levels of presumably non-oscillatory PLC activity.

Essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex.

Many receptor and nonreceptor tyrosine kinases activate phosphoinositide 3-kinases (PI3Ks). To assess the role of the delta isoform of the p110 catalytic subunit of PI3Ks, we derived enzyme-deficient mice. The mice are viable but have decreased numbers of mature B cells, a block in pro-B-cell differentiation, and a B1 B-cell deficiency. Both immunoglobulin M receptor-induced Ca(2+) flux and proliferation in response to B-cell mitogens are attenuated. Immunoglobulin levels are decreased substantially. The ability to respond to T-cell-independent antigens is markedly reduced, and the ability to respond to T-cell-dependent antigens is completely eliminated. Germinal center formation in the spleen in response to antigen stimulation is disrupted. These results define a nonredundant signaling pathway(s) utilizing the delta isoform of p110 PI3K for the development and function of B cells.

Ca2+-Mobilizing Endothelin-A Receptors Inhibit Voltage-Gated Ca2+ Influx through Gi/oSignaling Pathway in Pituitary Lactotrophs

In excitable cells, receptor-induced Ca(2+) release from intracellular stores is usually accompanied by sustained depolarization of cells and facilitated voltage-gated Ca(2+) influx (VGCI). In quiescent pituitary lactotrophs, however, endothelin-1 (ET-1) induced rapid Ca(2+) release without triggering Ca(2+) influx. Furthermore, in spontaneously firing and depolarized lactotrophs, the Ca(2+)-mobilizing action of ET-1 was followed by inhibition of spontaneous VGCI caused by prolonged cell hyperpolarization and abolition of action potential-driven Ca(2+) influx. Agonist-induced depolarization of cells and enhancement of VGCI upon Ca(2+) mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin (PTX). Activation of adenylyl cyclase by forskolin and addition of cell-permeable 8-bromo-cAMP did not affect ET-1-induced sustained inhibition of VGCI, suggesting that the cAMP-protein kinase A signaling pathway does not mediate the inhibitory action of ET-1 on VGCI. Consistent with the role of PTX-sensitive K(+) channels in ET-1-induced hyperpolarization of control cells, but not PTX-treated cells, ET-1 decreased the cell input resistance and activated a 5 mM Cs(+)-sensitive K(+) current. In the presence of Cs(+), ET-1 stimulated VGCI in a manner comparable with that observed in PTX-treated cells, whereas E-4031, a specific blocker of ether-a-go-go-related gene-like K(+) channels, was ineffective. Similar effects of PTX and Cs(+) were also observed in GH(3) immortalized cells transiently expressing ET(A) receptors. These results indicate that signaling of ET(A) receptors through the G(i/o) pathway in lactotrophs and the subsequent activation of inward rectifier K(+) channels provide an effective and adenylyl cyclase-independent mechanism for a prolonged uncoupling of Ca(2+) mobilization and influx pathways.

Presence of a population of CD20+, CD38- B lymphocytes with defective proliferative responsiveness in the synovial compartment of patients with rheumatoid arthritis.

To provide a comprehensive understanding of the humoral immune response that takes place at the site of inflammation in rheumatoid arthritis (RA), we studied the functional properties of synovial B cells. In particular, the response to various modes of mitogen stimulation was investigated. Purified synovial fluid (SF) B cells were cultured in the presence of CD40 ligand (CD40L)-expressing fibroblasts and cytokines, activated T cells, or phorbol myristate acetate (PMA)/ionomycin. Proliferation was determined by 3H-thymidine incorporation. Release of intracellular calcium was studied by flow cytometry. The inflamed joints of RA patients contained a population of CD20+,CD38- B cells with dramatically impaired mitogen responsiveness. Although the Ig-producing capacity was intact, these cells failed to proliferate in response to (a) CD40 in the presence of interleukin-2 (IL-2) and IL-10, (b) activated T cells, or (c) stimulation via the B cell receptor. Moreover, SF CD20+,CD38- B cells revealed a defective B cell receptor-induced Ca2+ influx, reminiscent of anergic B cells. Release of intracellular Ca2+ by ionomycin in the presence of the protein kinase C activator PMA did not restore the proliferative capacity. These findings indicate blockades in the proximal and distal intermediates involved in mitogen signaling. SF CD20+,CD38- B cells have functionally impaired proliferative responsiveness. The capacity of these cells to respond to activation by the production of Ig supports the notion that these cells might serve as Ig-producing effector cells and, as such, play a role in the pathophysiology of RA.

Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

Receptor-induced Ca(2+) signaling in cultured myenteric neurons.

We studied the effect of excitatory neurotransmitters (10(-5) M) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of cultured myenteric neurons. ACh evoked a response in 48.6% of the neurons. This response consisted of a fast and a slow component, respectively mediated by nicotinic and muscarinic receptors, as revealed by specific agonists and antagonists. Substance P evoked a [Ca(2+)](i) rise in 68.2% of the neurons, which was highly dependent on Ca(2+) release from intracellular stores, since after thapsigargin (5 microM) pretreatment only 8% responded. The responses to serotonin, present in 90.7%, were completely blocked by ondansetron (10(-5) M), a 5-HT(3) receptor antagonist. Specific agonists of other serotonin receptors were not able to induce a [Ca(2+)](i) rise. Removing extracellular Ca(2+) abolished all serotonin and fast ACh responses, whereas substance P and slow ACh responses were more persistent. We conclude that ACh-induced signaling involves both nicotinic and muscarinic receptors responsible for a fast and a more delayed component, respectively. Substance P-induced signaling requires functional intracellular Ca(2+) stores, and the 5-HT(3) receptor mediates the serotonin-induced Ca(2+) signaling in cultured myenteric neurons.

Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea‐pig gastric pylorus

1. Intracellular recordings made in single bundle strips of a visceral smooth muscle revealed rhythmic spontaneous membrane depolarizations termed slow waves (SWs). These exhibited 'pacemaker' and 'regenerative' components composed of summations of more elementary events termed spontaneous transient depolarizations (STDs). 2. STDs and SWs persisted in the presence of tetrodotoxin, nifedipine and ryanodine, and upon brief exposure to Ca2+-free Cd2+-containing solutions; they were enhanced by ACh and blocked by BAPTA AM, cyclopiazonic acid and caffeine. 3. SWs were also inhibited in heparin-loaded strips. SWs were observed over a wide range of membrane potentials (e.g. -80 to -45 mV) with increased frequencies at more depolarized potentials. 4. Regular spontaneous SW activity in this preparation began after 1-3 h superfusion of the tissue with physiological saline following the dissection procedure. Membrane depolarization applied before the onset of this activity induced bursts of STD-like events (termed the 'initial' response) which, when larger than threshold levels initiated regenerative responses. The combined initial-regenerative waveform was termed the SW-like action potential. 5. Voltage-induced responses exhibited large variable latencies (typical range 0.3-4 s), refractory periods of approximately 11 s and a pharmacology that was indistinguishable from those of STDs and spontaneous SWs. 6. The data indicate that SWs arise through more elementary inositol 1,4,5-trisphosphate (IP3) receptor-induced Ca2+ release events which rhythmically synchronize to trigger regenerative Ca2+ release and induce inward current across the plasmalemma. The finding that action potentials, which were indistinguishable from SWs, could be evoked by depolarization suggests that membrane potential modulates IP3 production. Voltage feedback on intracellular IP3-sensitive Ca2+ release is likely to have a major influence on the generation and propagation of SWs.

Bradyzide, a potent non-peptide B(2) bradykinin receptor antagonist with long-lasting oral activity in animal models of inflammatory hyperalgesia.

Bradyzide is from a novel class of rodent-selective non-peptide B(2) bradykinin antagonists (1-(2-Nitrophenyl)thiosemicarbazides). Bradyzide has high affinity for the rodent B(2) receptor, displacing [(3)H]-bradykinin binding in NG108-15 cells and in Cos-7 cells expressing the rat receptor with K(I) values of 0.51+/-0.18 nM (n=3) and 0.89+/-0.27 nM (n=3), respectively. Bradyzide is a competitive antagonist, inhibiting B(2) receptor-induced (45)Ca efflux from NG108-15 cells with a pK(B) of 8.0+/-0.16 (n=5) and a Schild slope of 1.05. In the rat spinal cord and tail preparation, bradyzide inhibits bradykinin-induced ventral root depolarizations (IC(50) value; 1.6+/-0.05 nM (n=3)). Bradyzide is much less potent at the human than at the rodent B(2) receptor, displacing [(3)H]-bradykinin binding in human fibroblasts and in Cos-7 cells expressing the human B(2) receptor with K(I) values of 393+/-90 nM (n=3) and 772+/-144 nM (n=3), respectively. Bradyzide inhibits bradykinin-induced [(3)H]-inositol trisphosphate (IP(3)) formation with IC(50) values of 11.6+/-1.4 nM (n=3) at the rat and 2.4+/-0.3 microM (n=3) at the human receptor. Bradyzide does not interact with a range of other receptors, including human and rat B(1) bradykinin receptors. Bradyzide is orally available and blocks bradykinin-induced hypotension and plasma extravasation. Bradyzide shows long-lasting oral activity in rodent models of inflammatory hyperalgesia, reversing Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in the rat knee joint (ED(50), 0.84 micromol kg(-1); duration of action >4 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal effect exceeding that of the non-steroidal anti-inflammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. In summary, bradyzide is a potent, orally active, antagonist of the B(2) bradykinin receptor, with selectivity for the rodent over the human receptor. British Journal of Pharmacology (2000) 129, 77 - 86

Selective inhibition of inositol 1,4,5-trisphosphate-induced Ca 2+ release in the CA1 region of the hippocampus in the ischemic gerbil

We examined the effect of ischemia on inositol 1,4,5-trisphosphate receptor-induced Ca 2+ release by functional and morphological approaches, using the gerbil model after 6-h unilateral occlusion of the common carotid artery. Autoradiographic study revealed that the basal uptake of 45Ca 2+ into the endoplasmic reticulum and caffeine-induced 45Ca 2+ release from the endoplasmic reticulum were normal in the presence of ATP in each ischemic brain region, whereas inositol 1,4,5-trisphosphate receptor-induced 45Ca 2+ release from the endoplasmic reticulum was inhibited only in the CA1 region of the hippocampus on the ischemic side. In moderately ischemic gerbils, electron microscopic study demonstrated aggregation of swollen endoplasmic reticulum in the CA1 region of the hippocampus, so that abundant endoplasmic reticulum assembled in close contact to form endoplasmic reticulum cisternal stacks. In severely ischemic gerbils, immunohistochemical analysis of the hippocampus showed loss of type 1 inositol 1,4,5-trisphosphate receptor protein with preservation of immunoreactivity for type 2 and 3 inositol 1,4,5-trisphosphate receptor proteins, which was confirmed by western blot analysis. Such selective inhibition of inositol 1,4,5-trisphosphate receptor-induced Ca 2+ release and the loss of type 1 inositol 1,4,5-trisphosphate receptor in the CA1 region of the hippocampus in cerebral ischemia may be associated with its region-specific vulnerability to ischemia.

Cutting Edge: Role of the Inositol Phosphatase SHIP in B Cell Receptor-Induced Ca2+ Oscillatory Response

AbstractSrc homology-2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP) is a recently identified protein that has been implicated as an important signaling molecule. Although SHIP has been shown to participate in the FcγRIIB-mediated inhibitory signal, the functional role of SHIP in activation responses by immunoreceptor tyrosine-based activation motif-bearing receptors such as B cell receptor (BCR) remains unclear. Indeed, it has been proposed that SHIP serves as a linking molecule for the regulation of the extracellular signal-regulated kinase pathway in BCR signaling, because SHIP associates with Shc. We now report that SHIP-deficient DT40 B cells display enhanced Ca2+ mobilization in response to BCR ligation, whereas extracellular signal-regulated kinase activation is unaffected. This Ca2+ enhancement is due to a sustained intracellular Ca2+ increase or to long-lasting Ca2+ oscillations by loss of SHIP, as revealed by single-cell Ca2+ imaging analysis. These results demonstrate the importance of SHIP in B cell activation by the modulation of Ca2+ mobilization.

Chronic imipramine administration amplifies the serotonin2A receptor-induced intracellular Ca2+ mobilization in C6 glioma cells through a calmodulin-dependent pathway.

In the present study, we examined whether chronic exposure of C6BU-1 cells to 100 nM of several different types of antidepressants directly influences serotonin2A (5-HT2A) receptor-stimulated intracellular Ca2+ mobilization. Imipramine, desipramine, clomipramine, and maprotiline amplified the 5-HT response at 48, but not at 2, h. Imipramine increased the maximum response to 5-HT without altering the EC50 of the dose-response curve. This effect was time dependent and cycloheximide blocked the maximal induction, suggesting an essential role for protein synthesis in this process. Previous exposure of the cells to thrombin or isoproterenol did not influence 5-HT2A receptor function and pretreatment with imipramine did not alter the thrombin- or bradykinin-induced Ca2+ mobilization, which indicates that the effects of imipramine appear to be specific to the 5-HT2A receptor. The effect of imipramine was potently suppressed by a calmodulin antagonist, W-13, in a dose-dependent manner. Furthermore, this amplified 5-HT response was blocked by KN-93, but not by H-7. Taken together, these results suggest that imipramine has a modulatory effect on the 5-HT2A receptor-coupled intracellular Ca2+ in C6 cells through a calmodulin-dependent pathway, possibly involving Ca2+/calmodulin kinase activation.

Fenspiride inhibits histamine-induced responses in a lung epithelial cell line.

Using the human lung epithelial WI26VA4 cell line, we investigated the capacity of fenspiride, an anti-inflammatory drug with anti-bronchoconstrictor properties, to interfere with histamine-induced intracellular Ca2+ increase and eicosanoid formation. Histamine and a histamine H1 receptor agonist elicited a rapid and transient intracellular Ca2+ increase (0-60 s) in fluo 3-loaded WI26VA4 cells. This response was antagonized by the histamine H1 receptor antagonist, diphenhydramine, the histamine H2 receptor antagonist, cimetidine, having no effect. Fenspiride (10(-7)-10(-5) M) inhibited the histamine H1 receptor-induced Ca2+ increase. In addition, histamine induced a biphasic increase in arachidonic acid release. The initial rise (0-30 s), a rapid and transient arachidonic acid release, was responsible for the histamine-induced intracellular Ca2+ increase. In the second phase release (15-60 min), a sustained arachidonic acid release appeared to be associated with the formation of cyclooxygenase and lipoxygenase metabolites. Fenspiride (10(-5) M) abolished both phases of histamine-induced arachidonic acid release. These results suggest that anti-inflammatory and antibronchoconstrictor properties of fenspiride may result from the inhibition of these effects of histamine.