Effect of deuterium oxide (D2O) on the IgE-mediated Ca2+ influx, arachidonic acid and histamine release in rat basophilic leukemia cells

Deuterium oxide (D2O), which is known to stimulate microtubule aggregation, enhanced the IgE-mediated 45Ca2+ influx, (14C)-arachidonic acid and histamine release in rat basophilic leukemia cells (RBL-2H3) in the same dose-dependent manner (up to 90% (v/v]. We compared the interaction between D2O and a variety of groups of pharmacological agents. A microtubule depolymerizing agent, demecolcine, which inhibited the IgE-mediated (14C)-arachidonic acid and histamine release without affecting 45Ca2+ influx, was counteracted by 45% D2O. Taxol, a microtubule stabilizing agent, which had an inhibitory effect on the above three steps, was also reversed by 45% D2O. These results would support the previous data on the interaction between D2O and microtubules and would further suggest that the status of microtubule aggregation may be related to the secretory process. Calmodulin inhibitors (W-7, trifluoperazine) blocked the IgE-mediated 45Ca2+ influx, (14C)-arachidonic acid and histamine release in the same dose-dependent manner, but were counteracted by 45% D2O. In contrast, the effects of proteinase inhibitors (TPCK, TLCK), an adenylate cyclase inhibitor (ddAdo), a phosphodiesterase inhibitor (aminophylline), a phospholipid methylation inhibitor (DZA + Hcy) and microfilament blockers (cytochalasin B and D) were not counteracted by 45% D2O. These results would suggest that D2O may be associated with calmodulin directly or indirectly possibly through some relationship between calmodulin and microtubules.     

Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils.

We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.     

Effect of atropine, ouabain, antimycin A, and A23187 on "trigger Ca2+ pool" in exocrine pancreas

45Ca2+ fluxes have been analyzed in dispersed acinar cells prepared from rat pancreas. Sudden addition of carbamylcholine (CCh) to 45Ca2+-preloaded acinar cells at quasi-steady state for 45Ca2+ resulted in a quick 45Ca2+ release followed by a slower 45Ca2+ reuptake with net accumulation of 45Ca2+. Subsequent sudden addition of atropine caused a further transient increase in cellular 45Ca2+ followed by a slow decrease to a steady-state value. 45Ca2+ release could not be evoked a second time by pancreozymin when prestimulated with CCh. However, if CCh stimulation was abolished by an interposed step of atropine, restimulation by cholecystokinin-pancreozymin was possible. Addition of A23187 or antimycin A to cells induced a fast decrease in cellular 45Ca2+. This effect was not additive to the CCh effect. In ouabain-pretreated cells, the CCh-induced sudden loss of cellular 45Ca2+ was blocked by 60%. The following slow reuptake of 45Ca2+ was blocked completely. Subsequent addition of atropine caused a fast uptake of cellular 45Ca2+ with no secondary decline. The data are consistent with the following model: acetylcholine releases Ca2+ from a cellular "trigger pool" into the cytosol located in or near the cell membrane. Then Ca2+ is extruded from the cell via Ca2+ pumps partly by a Na+-dependent Ca2+ transport system (quick phase of 45Ca2+ release). Subsequently, due to increased Ca2+ permeability of the plasma membrane as induced by acetylcholine, Ca2+ influx occurs and Ca2+ is taken up from the cytosol into intracellular Ca2+ pools (slow 45Ca2+ reuptake phase). Atropine causes refilling of the trigger Ca2+ pool and return of the increased Ca2+ permeability of the plasma membrane back to the unstimulated state.     

Sequestration of depolarization-induced Ca2+ loads by mitochondria and Ca2+ efflux via mitochondrial Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We used fura-2 microfluorometry to investigate the role of mitochondria in regulating the increase in the cytosolic Ca2+ concentration ([Ca]in) and the mechanism(s) underlying the subsequent Ca2+ efflux from mitochondria in bovine adrenal chromaffin cells. The rate of [Ca]in decay during and following stimulation with 100 mM KCl depolarization was markedly increased when the mitochondrial Na+/Ca2+ exchanger was inhibited by clonazepam or CGP-37157(CGP). In contrast, the addition of gramicidin, which increased the cytosolic Na+ concentration, following KCl depolarization caused a secondary increase in [Ca]in. This secondary increase in [Ca]in was prevented by the addition of clonazepam or CGP, and by the removal of external Na+. The subsequent removal of clonazepam or CGP, or the delayed addition of Na+ caused a slow increase in [Ca]in. A protonophore (FCCP) applied following KCl depolarization also caused a robust, secondary increase in [Ca]in, which was insensitive to blocking by clonazepam or CGP. Neither gramicidin nor FCCP altered the [Ca]in decay when applied following stimulation with histamine or caffeine, which mobilized Ca2+ from intracellular stores. These results suggest that the large [Ca]in increase induced by Ca2+ influx, but not by intracellular Ca2+ release, is buffered by mitochondria, and that the mitochondrial Na+/Ca2+ exchanger makes a major contribution to the subsequent Ca2+ efflux from mitochondria.     

Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors

Previous studies suggest that acetylcholine (ACh) is a transmitter released from taste cells as well as a transmitter in cholinergic efferent neurons innervating taste buds. However, the physiological effects on taste cells have not been established. I examined effects of ACh on taste-receptor cells by monitoring [Ca2+]i. ACh increased [Ca2+]i in both rat and mudpuppy taste cells. Atropine blocked the ACh response, but D-tubocurarine did not. U73122, a phospholipase C inhibitor, and thapsigargin, a Ca2+-ATPase inhibitor that depletes intracellular Ca2+ stores, blocked the ACh response. These results suggest that ACh binds to M1/M3/M5-like subtypes of muscarinic ACh receptors, causing an increase in inositol 1,4,5-trisphosphate and subsequent release of Ca2+ from the intracellular stores. A long incubation with ACh induced a transient response followed by a sustained phase of [Ca2+]i increase. In Ca2+-free solution, the sustained phases disappeared, suggesting that Ca2+ influx is involved in the sustained phase. Depletion of Ca2+ stores by thapsigargin alone induced Ca2+ influx. These findings suggest that Ca2+ store-operated channels may be present in taste cells and that they may participate in the sustained phase of [Ca2+]i increase. Immunocytochemical experiments indicated that the M1 subtype of muscarinic receptors is present in both rat and mudpuppy taste cells.     

Effect of thromboxane A2 synthetase inhibitor on immediate-type hypersensitivity reactions

The effect of the thromboxane (TX) A2 synthetase inhibitor, OKY-046, on human leukocyte histamine release and bronchial hypersensitivity in asthmatic subjects was evaluated. It was found that OKY-046 inhibited IgE- and Ca2+ ionophore A23187-mediated leukocyte histamine release in a dose-dependent fashion (IC50: 1.0 and 3.0 X 10(-3) M, respectively) and that OKY-046 could diminish bronchial hypersensitivity, determined by leukotriene D4 inhalation, following a 2-week oral medication. These data suggest that the TXA2 synthetase inhibitor can produce favorable effects upon the course of immediate-type hypersensitivity reactions.     

Prostaglandin E2 sensitizes primary sensory neurons to histamine

1. Histamine is able to elicit a dose-dependent rise in intracellular Ca2+ in a proportion of rat dorsal root ganglion (DRG) neurons. Pre-treatment with prostaglandin (PGE2) prior to a histamine challenge increases the proportion of neurons responding to low concentrations of histamine (10-100 microM). 2. The dose-response curve for histamine is shifted to the left by approximately two orders of magnitude following 45 s pre-treatment with 1 microM PGE2. 3. The phospholipase C (PLC) inhibitor 1-[6-[[17-beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) completely blocked the response to histamine (100 microM) in non-sensitized cells but, after PGE2 pre-treatment, this inhibitor reduced the proportion of cells responding to histamine by approximately a half. Removal of extracellular Ca2+ blocked the response in the remaining cells so that, in this subgroup of histamine sensitive neurons, the PGE2 sensitization is the result of activation of a Ca influx pathway. 4. The sensitization is dependent on an increase in cAMP as it is mimicked by pre-treatment with 8-bromo cyclic AMP (8-Br-cAMP) and by forskolin stimulation of adenylyl cyclase activity. It is inhibited by THFA (tetrahydrofuryl adenine) an inhibitor of adenylyl cyclase. The sensitization is also blocked by pre-treatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), an inhibitor of protein kinase A. We conclude that the PGE2 sensitization of DRG neurons to histamine is dependent on activation of the cAMP-protein kinase A cascade.     

Calcium mediation of cholinergic-stimulated amylase release from mouse parotid gland.

Amylase release from mouse parotid fragments was stimulated independently by cholinergic and beta-adrenergic agents. The cholinergic agonist, carbachol, significantly increased release of amylase only in Ca2+ containing medium whereas isoproterenol-stimulated amylase release was unaffected by Ca2+ removal. The ionophore, A23187, mimicked the effect of cholinergic stimulation when Ca2+ was present in the medium. Uptake of 45Ca2+ into tissue fragments was enhanced by carbachol and A23187 but not by isoproterenol; atropine blocked the effect of carbachol. Diphenylhydantoin (DPH) and verapamil partially inhibited carbachol-stimulated amylase release and 45Ca2+ uptake, whereas diazoxide potentiated these effects; in all cases there was good parallelism between 45Ca2+ uptake and amylase release. It was concluded that the primary step in the release of amylase from mouse parotid gland in response to cholinergic agents is an increased influx of Ca2+.     

Characterization of platelet cytoplasmic Ca2+ mobilization in patients with congenital cyclo-oxygenase deficiency and with defective platelet aggregation to A23187

Agonist-induced platelet cytoplasmic Ca2+ concentrations ([Ca2+]i) in patients with congenital cyclo-oxygenase deficiency (A) and with impaired aggregation to A23187 (B) were measured with aequorin in the presence or absence of extracellular Ca2+. The influence of TMB-8 or ONO3708 on agonist-induced [Ca2+]i in those platelets was also investigated. In Patient 1, there was a single aequorin luminescence peak in response to arachidonate, which was a thromboxane A2(TXA2) independent Ca2+ influx. The luminescence peak due to the formation of TXA2 was not detectable. The A23187-induced [Ca2+] i was decreased in the presence of extracellular Ca2+, but was within normal limits in the absence of extracellular Ca2+. A thrombin or STA2-induced elevation of [Ca2+] i was always within normal limits under any conditions. These results suggest that cyclo-oxygenase activity (CO activity) contributes to the A23187-induced Ca2+ influx, but does not contribute to the Ca2+ release from intracellular stores, and that the thrombin or STA2-induced Ca2+ influx and release do not depend on the CO activity. In Patient 2, the time lag from the addition of A23187 to the aequorin luminescence peak was found both in the presence and absence of extracellular Ca2+, which was more obvious in the latter. This A23187-induced elevation of [Ca2+] i disappeared after treatment of the platelets with TMB-8 in the absence of extracellular Ca2+, which is rarely seen in normal platelets. The most striking finding was that the thrombin-induced rise in [Ca2+] i in the absence of extracellular Ca2+ was not detectable. These findings might be closely related to abnormal platelet function in this patient.     

Role of the cytoskeleton in Ca2+ release from the intracellular Ca store of rat peritoneal mast cells

In order to study the role of the cytoskeleton in histamine release from mast cells, the effects of cytochalasin D, cholchicine and vinblastine on Ca²⁺ release from the intracellular Ca store induced by compound 48/80 were investigated by means of a video-intensified microscopy system. When the quin 2-loaded mast cells were stimulated by 0.35 g/ml of compound 48/80, a rapid increase in intracellular Ca²⁺ was observed. At concentrations higher than 10^–6 M, both colchicine and vinblastine pretreatments significantly inhibited the increase in intracellular Ca²⁺ concentrations caused by compound 48/80, although cytochalasin D had no effect. When permeabilized mast cells were exposed to potassium-antimonate solution, microtubules became attached to the endoplasmic reticulum, where many dots of Ca-antimonate were observed; in some areas, the microtubules interconnected the endoplasmic reticulum and granules in the mast cells. From the results of the present study, it was assumed that microtubules play some important role in the processes leading to Ca²⁺ release from the intracellular Ca store.     

Intracellular Ca2+ buffering potentiates an 86Rb+ permeability response in human lymphocytes

The effects of antibodies against immunoglobulin delta-heavy chains (anti-delta) on intracellular free Ca2+ concentrations, [Ca2+]i, and 86Rb+ influx in human neoplastic B-cells were tested in vitro. When preloading the cells with high concentrations of the fluorescent Ca2+ chelator quin 2 and subsequently stimulating in EGTA medium, the anti-delta induced rise in [Ca2+]i was strongly reduced or blocked. Nevertheless, 86Rb+ influx, also induced by anti-delta, was potentiated. In fact, in a population of cells in which anti-delta increased [Ca2+]i, but not 86Rb+ influx under standard conditions, the combination of quin-2 preloading and subsequent extracellular Ca2+ chelation by EGTA revealed an anti-delta induced 86Rb+ influx. Most of this influx was ouabain resistant, suggesting only a minor contribution from the Na+/K+ pump. Based on the Ca2+ buffer effect of quin 2 we suggest that the Ca2+ effect on 86Rb+ (K+ analogue) permeability is not mediated by increased [Ca2+]i but rather by the Ca2+ release per se from the plasma membrane.     

FMLP-induced enzyme release from neutrophils: a role for intracellular calcium

The ability of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) to stimulate beta-glucuronidase release and 45Ca2+ release from rabbit neutrophils was studied. FMLP stimulated enzyme release from cytochalasin B-treated cells either in the presence or the absence of extracellular calcium. Depletion of cell calcium, by exposure to either ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or the calcium ionophore A23187, blocked the ability of FMLP to stimulate enzyme release and 45Ca2+ release in the absence of extracellular calcium. The ability of A23187 to lower the 45Ca2+ content of neutrophils, to block FMLP-stimulated 45Ca2+ release, and to inhibit FMLP-stimulated enzyme release in the absence of calcium was dose dependent over the same concentration range (10(-8) to 10(-6) M A23187) for all three actions. In contrast, FMLP stimulated enzyme release from A23187-treated cells, provided that extracellular calcium was present. This secretory response was normal as judged by cell ultrastructure and FMLP dose-response relationships. It is concluded that A23187 depletes a pool of intracellular calcium usually released by FMLP and that release of calcium from this pool is necessary for initiation of enzyme secretion in the absence of extracellular calcium.     

The K+ channel iKCA1 potentiates Ca2+ influx and degranulation in human lung mast cells

BACKGROUND: Human lung and blood-derived mast cells express a Ca2+-activated K+ channel (KCA) that has electrophysiological properties resembling the intermediate conductance KCA (iKCA1). This channel is predicted to enhance IgE-dependent mast cell responses. OBJECTIVE: To confirm the identity of this channel as iKCA1 in human lung mast cells and to examine the effect of an iKCA1 opener, 1-ethyl-2-benzimidazolinone (1-EBIO), on Ca2+ influx and degranulation after IgE-dependent activation. METHODS: iKCA1 expression was examined by using RT-PCR. Ion currents were measured by using the patch clamp technique in human peripheral blood-derived mast cells, freshly isolated human lung mast cells (HLMCs), and long-term cultured HLMCs (LTHLMCs). Currents were manipulated with the specific iKCA1 opener 1-EBIO and the iKCA1 blockers clotrimazole and TRAM-34. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells, and histamine release was measured by radioenzymatic assay. RESULTS: Both fresh HLMCs and LTHLMCs expressed iKCA1 mRNA. The iKCA1 opener 1-EBIO induced iKCA1 currents in 89% of human peripheral blood-derived mast cells, 12% of fresh HLMCs, and 67% of LTHLMCs, which were blocked by the iKCA1 blockers clotrimazole and TRAM-34. After cell activation with a suboptimal concentration of anti-IgE, 1-EBIO enhanced the IgE-dependent rise in cytosolic-free Ca2+ and potentiated IgE-dependent histamine release. CONCLUSION: Opening of iKCA1 enhances IgE-dependent Ca2+ influx and histamine release in HLMCs. Inhibition of iKCA1 may provide a novel approach to the treatment of mast cell-mediated disease.     

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.     

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle

Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca2+, which is known to cause damage during electrical stimulation, is a causative factor in anoxia-induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4-week-old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O2, 5% CO2), anoxia (95% N2, 5% CO2) or varying degrees of reduced oxygenation. At varying extracellular Ca2+ concentrations ([Ca2+]o), 45Ca influx and total cellular Ca2+ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mM Ca2+, 15-75 min of exposure to anoxia increased 45Ca influx by 46-129% (P<0.001) and Ca2+ content by 20-50% (P<0.001). Mg2+ (11.2 mM) reduced the anoxia-induced increase in 45Ca influx by 43% (P<0.001). In muscles incubated at 20 and 5% O2, 45Ca influx was also stimulated (P<0.001). Increasing [Ca2+]o to 5 mM induced a progressive increase in both 45Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca2+ content and LDH release increased markedly and showed a significant correlation (r2=0.55, P<0.001). In conclusion, anoxia or incubation at 20 or 5% O2 leads to an increased influx of 45Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca2+. The loss of cell membrane integrity further increases Ca2+ influx, which may elicit a self-amplifying process of cell membrane leakage.     

Sphingosine-1-phosphate-induced intracellular Ca2+ mobilization in human endothelial cells.

The authors have studied the effect of sphingosine-1-phosphate (S1P) on Ca2+ release from intracellular stores in cultured human umbilical vein endothelial cells (HUVECs). In the presence of extracellular Ca2+, S1P increased intracellular Ca2+ concentration ([Ca2+]i) and this increase was partially inhibited by La3+ (1 microM), indicating that S1P induces Ca2+ influx from extracellular pool and Ca2+ release from intracellular stores. S1P increased [Ca2+]i concentration dependently in Ca2+-free extracellular solution. The Hill coefficient (1.7) and EC50 (420 nM) was obtained from the concentration-response relationship. When caffeine depleted Ca2+ store in the presence of ryanodine, S1P did not induce intracellular Ca2+ release. Furthermore, the Ca2+-induced Ca2+ release inhibitors ruthenium red or dantrolene completely inhibited S1P-induced intracellular Ca2+ release. S1P-induced intracellular Ca2+ release was inhibited by the phospholipase C (PLC) inhibitors neomycin and U73312, or the inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker aminoethoxybiphenyl borane (2-APB). In contrast, S1P-induced intracellular Ca2+ release was not inhibited by the mitochondrial Ca2+ uptake inhibitor CCCP or the mitochondrial Ca2+ release inhibitor cyclosporin A. These results show that S1P mobilizes Ca2+ from intracellular stores primarily via Ca2+-induced and IP3-induced Ca2+ release and this Ca2+ mobilization is independent of mitochondrial Ca2+ stores.     

Regulation of eosinophil adhesion by lysophosphatidylcholine via a non-store-operated Ca2+ channel

We examined the mechanism by which lysophosphatidylcholine (LPC) regulates beta2-integrin-mediated adhesion of eosiniophils. Eosinophils were isolated from blood of mildly atopic volunteers by negative immunomagnetic selection. beta2-integrin-dependent adhesion of eosinophils to plated bovine serum albumin (BSA) was measured by residual eosinophil peroxidase activity. LPC caused maximal adhesion of eosinophils to plated BSA at 4 microM. Lysophosphatidylinositol, which has a similar molecular shape, mimicked the effect of LPC on eosinophil adhesion, while neither lysophosphatidylserine nor lysophosphatidylethanolamine had any effect. Phosphatidylethanolamine, a lipid that has a molecular orientation that is the inverse of LPC, blocked eosinophil adhesion caused by LPC. Unlike platelet-activating factor, a G-protein-coupled receptor agonist, LPC did not cause Ca2+-store depletion, but caused increased Ca2+ influx upon addition of Ca2+ to extracellular medium. This influx was not inhibited by U73122, a phospholipase C inhibitor, demonstrating independence from the G protein-activated phospholipase C pathway. Ca2+ influx was inhibited by either preincubation of phosphotidylethanolamine or La3+, a broad spectrum blocker of cation channels. LPC induced up-regulation of the active conformation of CD11b, which was blocked by preincubation with phosphatidylethanolamine. These data suggest that LPC causes a non-store-operated Ca2+ influx into eosinophils, which subsequently activates CD11b/CD18 to promote eosinophil adhesion.     

Cyclic AMP inhibits chemotactic-peptide-induced but not Ca2+-ionophore- or tetradecanoylphorbol-acetate-induced enzyme secretion in guinea pig neutrophils

cAMP-increasing agents such as prostaglandin El, dibutyryl cAMP and 8-bromo-cAMP inhibited N-acetyl-beta-D-glucosaminidase secretion induced by the chemotactic peptide formylmethionyl-leucyl-phenylalanine in guinea pig neutrophils. However, they could not inhibit but rather potentiated the secretion significantly when secretion was induced by the Ca2+ ionophore A23187. Similarly, these agents did not inhibit tetradecanoylphorbol acetate (TPA)-induced secretion. Moreover, the secretion induced by combined treatment with Ca2+ ionophore A23187 and TPA was hardly blocked by cAMP-increasing agents. These results strongly suggest that cAMP inhibits the secretion through suppression of a signal transduction from receptor activation to events such as Ca2+ mobilization and protein kinase C activation, probably of the inositol phospholipid metabolism.     

C-terminal modulator controls Ca2+-dependent gating of Ca(v)1.4 L-type Ca2+ channels

Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Ca(v)1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and alpha1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Ca(v)1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human alpha1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Ca(v)1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.     

Protein kinase Cdelta functions downstream of Ca2+ mobilization in FcepsilonRI signaling to degranulation in mast cells

BACKGROUND: Mobilization of Ca 2+ plays an important role in the degranulation of mast cells. Although events upstream of Ca 2+ mobilization in the regulation of degranulation are relatively well characterized, the downstream mediators of Ca 2+ remain largely unknown. OBJECTIVE: We sought to characterize the downstream signaling mechanism by which Ca 2+ mobilization mediates degranulation in antigen-stimulated mast cells. METHODS: The effect of various inhibitors was examined in the antigen-induced or Ca 2+ ionophore A23187-induced degranulation process, and the effect of inhibitors on histamine release was tested in the mouse model of asthma. RESULTS: The delta isoform of protein kinase C (PKC) functions downstream of Ca 2+ in the signaling pathway from FcepsilonRI to degranulation in RBL-2H3 mast cells. Stimulation of cells with either antigen or the Ca 2+ ionophore A23187 induced a rapid translocation of PKC-delta from the cytosol to the cellular membranes, and either treatment with the PKC-delta-specific inhibitor rottlerin or infection with an adenovirus encoding a dominant negative mutant of PKC-delta markedly inhibited degranulation induced with antigen or A23187. Furthermore, both the translocation of PKC-delta and degranulation induced by A23187 were inhibited by prevention of the accumulation of reactive oxygen species normally elicited by the ionophore. Finally, intraperitoneal injection of rottlerin prevented the increase in the concentration of histamine in bronchoalveolar lavage fluid induced by means of antigen challenge in a mouse model of allergic asthma. conclusion: PKC-delta plays an essential downstream mediatory role in the degranulation elicited by Ca 2+ mobilization, and reactive oxygen species mediate the activation of PKC-delta by Ca 2+ in the regulation of degranulation.