Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages.

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.     

Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.     

Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes

To investigate the mechanisms by which inositol phosphates regulate cytosolic free Ca2+ concentration ([Ca2+]c), we injected Xenopus oocytes with inositol phosphates and measured Ca2+-activated Cl- currents as an assay of [Ca2+]c. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) injection (0.1-10.0 pmol) induced an initial transient Cl- current (I1) followed by a second more prolonged Cl- current (I2). Both currents were Ca2+-dependent, but the source of Ca2+ was different. Release of intracellular Ca2+ stores produced I1, whereas influx of extracellular Ca2+ produced I2; Ca2+-free bathing media and inorganic calcium channel blockers (Mn2+, Co2+) did not alter I1 but completely and reversibly inhibited I2. Injection of the Ins(1,4,5)P3 metabolite, inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) (0.2-10.0 pmol) generated a Ca2+-dependent Cl- current with superimposed current oscillations that resulted from release of intracellular Ca2+, not Ca2+ influx. Injection of the Ins(1,3,4,5)P4 metabolite, inositol 1,3,4-trisphosphate (10.0 pmol), or the synthetic inositol trisphosphate isomer, inositol 2,4,5-trisphosphate (1.0-10.0 pmol), mimicked the effect of Ins(1,4,5)P3, stimulating an I1 resulting from release of intracellular Ca2+ and an I2 resulting from influx of extracellular Ca2+. The results indicate that several inositol trisphosphate isomers stimulate both release of intracellular Ca2+ and influx of extracellular Ca2+. Ins(1,3,4,5)P4 also stimulated release of intracellular Ca2+, but it was neither sufficient nor required for Ca2+ influx.     

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.     

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.     

Antigen-stimulated metabolism of inositol phospholipids in the cloned murine mast-cell line MC9.

Cells of the murine mast-cell clone MC9 grown in suspension culture were sensitized with an anti-DNP (dinitrophenol) IgE and subsequently prelabelled by incubating with [32P]Pi. Stimulation of these cells with DNP-BSA (bovine serum albumin) caused marked decreases in [32P]polyphosphoinositides (but not [32P]phosphatidylinositol) with concomitant appearance of [32P]phosphatidic acid. Whereas phosphatidylinositol monophosphate levels returned to baseline values after prolonged stimulation, phosphatidylinositol bisphosphate levels remained depressed. Stimulation of sensitized MC9 cells with DNP-BSA increased rates of incorporation of [32P]Pi into other phospholipids in the order: phosphatidylcholine greater than phosphatidylinositol greater than phosphatidylethanolamine. In sensitized cells prelabelled with [3H]inositol, release of inositol monophosphate, inositol bisphosphate and inositol trisphosphate, was observed after stimulation with DNP-BSA. When Li+ was added to inhibit the phosphatase activity that hydrolysed the phosphomonoester bonds in the sugar phosphates, greater increases were observed in all three inositol phosphates, particularly in inositol trisphosphate. The IgE-stimulated release of inositol trisphosphate was independent of the presence of extracellular Ca2+. In addition, the Ca2+ ionophore A23187 caused neither the decrease in [32P]polyphosphoinositides nor the stimulation of the release of inositol phosphates. These results demonstrate that stimulation of the MC9 cell via its receptor for IgE causes increased phospholipid turnover, with effects on polyphosphoinositides predominating. These data support the hypothesis that hapten cross-bridging of IgE receptors stimulates phospholipase C activity, which may be an early event in stimulus-secretion coupling of mast cells. The results with the Ca2+ ionophore A23187 indicate that an increase in intracellular Ca2+ alone is not sufficient for activation of this enzyme.     

Cholinergic Stimulation of Inositol Phosphate Formation in Bovine Adrenal Chromaffin Cells: Distinct Nicotinic and Muscarinic Mechanisms

The ability of cholinergic agonists to activate phospholipase C in bovine adrenal chromaffin cells was examined by assaying the production of inositol phosphates in cells prelabeled with [3H]inositol. We found that both nicotinic and muscarinic agonists increased the accumulation of [3H]inositol phosphates (mainly inositol monophosphate) and that the effects mediated by the two types of receptors were independent of each other. The production of inositol phosphates by nicotinic stimulation required extracellular Ca2+ and was maximal at 0.2 mM Ca2+. Increasing extracellular Ca2+ from 0.22 to 2.2 mM increased the sensitivity of inositol phosphates formation to stimulation by submaximal concentrations of 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) but did not enhance the response to muscarine. Elevated K+ also stimulated Ca2+-dependent [3H]inositol phosphate production, presumably by a non-receptor-mediated mechanism. The Ca2+ channel antagonists D600 and nifedipine inhibited the effects of DMPP and elevated K+ to a greater extent than that of muscarine. Ca2+ (0.3-10 microM) directly stimulated the release of inositol phosphates from digitonin-permeabilized cells that had been prelabeled with [3H]inositol. Thus, cholinergic stimulation of bovine adrenal chromaffin cells results in the activation of phospholipase C by distinct muscarinic and nicotinic mechanisms. Nicotinic receptor stimulation and elevated K+ probably increased the accumulation of inositol phosphates through Ca2+ influx and a rise in cytosolic Ca2+. Because Ba2+ caused catecholamine secretion but did not enhance the formation of inositol phosphates, phospholipase C activation is not required for exocytosis. However, diglyceride and myo-inositol 1,4,5-trisphosphate produced during cholinergic stimulation of chromaffin cells may modulate secretion and other cellular processes by activating protein kinase C and/or releasing Ca2+ from intracellular stores.     

Is vacuole the richest store of IP3-mobilizable calcium in plant cells?

Inositol trisphosphate is known to mobilize calcium from internal stores in plant cells. However, with the exception of the vacuole, the largest plant cell compartment, organelles responsive to inositol trisphosphate have not been extensively identified. In this way, we have separated membrane vesicles from the same carrot microsomal fraction and identified them, both by marker enzyme activities and electron microscopy. These correspond to pure plasma membrane, pure tonoplast and mixed mitochondria, endoplasmic reticulum, Golgi membrane fractions. All the fractions accumulated calcium in a ATP-dependent manner and were tightly sealed. Inositol trisphosphate-dependent calcium releases were accurately measured only in fractions corresponding functionally and structurally to tonoplast, the vacuolar membrane. The process was dose-dependent and fairly specific for inositol trisphosphate. While highly significant, approximately 40% of the mobile calcium only may be released from tonoplast vesicles by inositol trisphosphate which remained basically intact during the release experiments. From these results it is concluded that the vacuole is the richest store of calcium directly mobilizable by inositol trisphosphate in plant cells, but inositol trisphosphate is not able to release the overall mobile vacuolar calcium.     

Calcium mediated neurohumoral inhibition of chicken enterocyte Na influx: role of phosphatidylinositol metabolites.

Carbachol (CCH), serotonin (5HT), divalent ionophore A23187, cAMP, and certain neuropeptides, i.e. substance P (SP), inhibit the initial rate of uptake (influx) of 22Na into isolated chicken villus enterocytes. All these agents also increase cytosolic Ca. However, the increases stimulated by CCH, 5HT, and cAMP are not blocked by chelation of extracellular Ca, whereas those of A23187 and SP are. Only CCH and 5HT stimulate hydrolysis of membrane phosphoinositides to form inositol phosphates. CCH and 5HT also stimulate incorporation of [32P]-PO4 into membrane polyphosphoinositides. These studies suggest that at least three mechanisms exist to increase cytosolic Ca in chicken enterocytes and thereby inhibit Na influx. Certain neurohumoral agents such as SP open a plasma membrane permeability for Ca, permitting extracellular Ca to enter the cell down its electrochemical gradient. These agents do not stimulate phosphatidylinositol breakdown. CCH and 5HT stimulate phosphatidylinositol breakdown and via the formation of inositol trisphosphate release Ca from intracellular stores. A third mechanism exists for cAMP which mobilizes Ca from intracellular stores, but does not involve the metabolism of membrane phosphatidylinositols.     

Bradykinin-induced changes in inositol trisphosphate mass in MDCK cells

Bradykinin produces increases in cytosolic calcium in MDCK cells. We have extracted and separated Inositol 1,4,5 trisphosphate by HPLC and after-acid hydrolysis and conversion to the hexatrifluoro-acetyl derivative quantitated by negative ion chemical ionization mass spectrometry the mass of inositol trisphosphate in MDCK cells. Bradykinin causes an increase in the mass of Inositol trisphosphate from basal levels of 152 pmoles/mg cell protein to 537 pmoles/mg cell protein by 10 secs of stimulation. We conclude that bradykinin stimulates PLC hydrolysis of PIP2 with rapid release of IP3 in sufficient amount to account for the increase in cytosolic Ca++.     

alpha Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells

alpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system.     

Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate.

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.     

Nerve Growth Factor Potentiates Bradykinin-Induced Calcium Influx and Release in PC12 Cells

To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PC12 cells. Short-term (1 h) treatment with NGF increased the potency of bradykinin to raise intracellular calcium by about 10-fold, whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term potentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol 1,4,5-trisphosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself.     

Role of inositol trisphosphate as a second messenger in signal transduction processes: An essay

This essay attempts to summarize some of the best evidence for the role of inositol trisphosphate as a second messenger in signal transduction processes. The following aspects are addressed in the essay: (a) The synthesis of inositol trisphosphate and other inositol lipids, (b) Receptor-phosphatidylinositol bisphosphate phospholipase C coupling and the N-ras protooncogene, (c) Inositol trisphosphate and intracellular calcium, (d) Cell growth and oncogenes, (e) Receptors linked to the phosphatidylinositol cycle, (f) Phototransduction and (g) Interactions between inositol trisphosphate and other second messengers.Abbreviations Cyclic AMP Adenosine 3,5-cyclic monophosphate - Cyclic GMP Guanosine 3,5-cyclic monophosphate - DG sn, 1,2-Diacylglycerol - EGF Epidermal growth factor - GDP Guanosine diphosphate - GTP Guanosine triphosphate - IP Inositol 1-monophosphate - IP₂ Inositol 1,4-diphosphate - IP₃ Inositol 1,4,5-trisphosphate - PA Phosphatidic acid - PDGF Platelet-derived growth factor - PI Phosphatidylinositol - PIP Phosphatidylinositol 4-monophosphate - PIP₂ Phosphatidylinositol 4,5-bisphosphate - PIP₃ Phosphatidylinositol 3,4,5-trisphosphate - PLC Phospholipase C     

Formation of second messengers in response to activation of ion channels in excitable cells

1. Depolarization of excitable cells of the central nervous system results in the formation of the second messengers cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerides. 2. Depolarization-evoked accumulation of cyclic AMP in brain preparations can be accounted for mainly by the release of adenosine, which subsequently interacts with stimulatory adenosine receptor linked to adenylate cyclase. 3. Depolarization-evoked formation of cyclic GMP in brain preparations is linked to activation of voltage-dependent calcium channels, presumably leading to activation of guanylate cyclase by calcium ions. 4. In brain slices depolarization-evoked stimulation of phosphoinositide breakdown and subsequent formation of inositol phosphates and diacylglycerides are linked to activation of voltage-dependent calcium channels, which are sensitive to dihydropyridines, presumably leading to activation of phospholipase(s) C by calcium ions. 5. In the synaptoneurosome preparation depolarization-evoked stimulation of phosphoinositide breakdown does not involve activation of dihydropyridine-sensitive calcium channels and, instead, appears to be regulated primarily by the intracellular concentration of sodium ions. Thus, agents that induce increases in intracellular sodium--such as toxins that open or delay inactivation of voltage-dependent sodium channels; ouabain, an inhibitor of Na+/K+ ATPase that transports sodium outward and a sodium ionophore--all stimulate phosphoinositide breakdown. Mechanistically, increases in intracellular sodium either might directly affect phospholipase(s) C or might lead to influx of calcium ions through Na+/Ca2+ transporters. 6. Depolarization-evoked stimulation of cyclic AMP formation and phosphoinositide breakdown can exhibit potentiative interactions with responses to receptor agonists, thereby providing mechanisms for modulation of receptor responses by neuronal activity. 7. Since all these second messengers can induce phosphorylation of ion channels through the activation of specific kinases, it is proposed that depolarization-evoked formation of second messengers represents a putative feedback mechanism to regulate ion fluxes in excitable cells.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.     

Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration.

The accumulation of inositol phosphates in myo-[3H]inositol-labelled human neutrophils stimulated with the chemotactic peptide fMet-Leu-Phe was measured. The challenge with the chemotactic peptide caused the generation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3). The formation of the three inositol phosphates followed a differential time course: InsP3 accumulated very rapidly and transiently, whereas InsP increased steadily for more than 2 min. Inositol phosphate formation was only partially decreased by procedures which prevented the fMet-Leu-Phe-dependent increase of cytosolic free Ca2+ concentration.     

Age-related impairment in rat parotid cell alpha 1-adrenergic action at the level of inositol trisphosphate responsiveness

Alpha 1-Adrenergic-stimulated calcium efflux from rat parotid cell aggregates declines approx. 40% between 3 and 24 months of age, with the bulk of the reduction occurring between 12 and 24 months. Intracellular free calcium levels following alpha 1-adrenoceptor stimulation are also reduced about 40% between 3 and 24 months. No significant age differences in stimulation of inositol mono-, bis- or trisphosphate production are observed. However, the ability of inositol trisphosphate to directly stimulate calcium efflux is reduced by about 50% with increasing age. Concentrations of this inositol phosphate required for maximal calcium release do not change between 3 and 24 months. Differences in response are not due to a reduction in uptake of inositol trisphosphate into older cells, but suggest an age-related defect in the ability of inositol trisphosphate to liberate calcium from intracellular stores. Such dysfunction may be at least partially responsible for impaired alpha 1-adrenergic responsiveness during aging.