Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

Protein kinase C differentially inhibits muscarinic receptor operated Ca2+ release and entry in human salivary cells

We investigated the effects of phorbol myristate acetate on muscarinic receptor-induced Ca2+ release from intracellular stores and extracellular entry in a human salivary duct cell line, HSG-PA. Phorbol myristate acetate (approximately 10(-7) M) blocked both Ca2+ release and Ca2+ entry induced by the muscarinic agonist carbachol. This blockade was the result of the activation of protein kinase C since 4 alpha-phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit Ca2+ mobilization responses to carbachol. Importantly, at lower phorbol myristate acetate concentrations (approximately 10(-9) M), carbachol-induced Ca2+ release was blocked, but carbachol-induced Ca2+ entry was maintained. These results show that carbachol-induced Ca2+ entry does not occur via an intracellular store and that protein kinase C plays a role in a feedback control mechanism for muscarinic-induced Ca2+ mobilization at different levels.     

Rapid calcium release from the isolated sarcoplasmic reticulum is triggered via the attached transverse tubular system

Rapid replacement of 0.15 M K gluconate with 0.15 M choline Cl led to multiphasic Ca2+ release from a heavy fraction of rabbit skeletal muscle microsomes. Following the initial lag period (0-50 ms), about 15 nmol of Ca2+/mg of protein was rapidly released with first-order rate constants k = 60-140 s-1. Subsequently, a larger amount of Ca2+ (up to 56 nmol/mg) was released at a slower rate (k = 0.8-1.5 s-1). The Ca2+ released in both rapid and slow phases was reaccumulated within 60 s. In agreement with a previous report (Caswell, A. H., Lau, Y. H., Garcia, M., and Brunschwig, J-P. (1979) J. Biol. Chem. 254, 202-208), French press treatment of the tubule/sarcoplasmic reticulum (SR) complex results in dissociation of transverse tubular membrane (T-tubules) from SR. Subsequent incubation with 0.4 M potassium cacodylate results in the reassociation of the complex, as shown by sucrose density-gradient sedimentation. Upon T-tubule dissociation, both rapid and slow Ca2+ release was inhibited. Upon reassociation, the rapid Ca2+ release was completely restored and the slow phase partially restored. The results indicate that the T-tubule associated with SR plays a crucial role in triggering rapid Ca2+ release induced by ionic replacement. Other types of Ca2+ release, e.g. those induced by Ca2+ alone or with drugs such as caffeine and quercetin, are unaffected by T-tubule dissociation, and hence produced by direct stimulation of the SR membrane.     

Involvement of protein kinase C in prostaglandin E2-induced catecholamine release from cultured bovine adrenal chromaffin cells

We recently reported that prostaglandin (PG) E2 stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain induced a gradual secretion of catecholamines from the cells (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). Here we examined the involvement of two signal pathways, Ca2+ mobilization and protein kinase C activation resulting from phosphoinositide metabolism, in the PGE2-induced catecholamine release. Either the Ca2+ ionophore ionomycin or 12-O-tetradecanoylphorbol 13-acetate (TPA) could enhance the release in the presence of ouabain, and ionomycin-induced release was additive to PGE2-induced release, but TPA-induced release was not additive. PGE2 dose-dependently stimulated the formation of diacylglycerol and caused the translocation of 4% of the total protein kinase C activity to become membrane-bound within 5 min. These effects were specific for PGE2 and PGE1 among PGs tested (PGE2 = PGE1 greater than PGF2 alpha greater than PGD2). Furthermore, the phosphoinositide-specific phospholipase C inhibitor neomycin inhibited PGE2-induced accumulation of inositol phosphates, diacylglycerol formation, translocation of protein kinase C, and also stimulation of catecholamine release. Both PGE2- and TPA-induced release were inhibited by the depletion of protein kinase C caused by prolonged exposure to TPA, but ionomycin-induced release was not inhibited. We recently found that the amiloride-sensitive Na+, H+-antiport participates in PGE2-evoked catecholamine release (Tanaka, T., Yokohama, H., Negishi, M., Hayashi, H., Ito, S., and Hayaishi, O. (1990) J. Neurochem. 54, 86-95). In agreement with our recent report, PGE2 and TPA induced a sustained increase in intracellular pH that was abolished by the protein kinase C inhibitor staurosporine but not by the calmodulin inhibitor W-7. Ionomycin also induced a marked increase in intracellular pH, but this increase was abolished by W-7 but not by staurosporine. These results demonstrate that PGE2-induced activation of the Na+, H(+)-antiport and catecholamine release in the presence of ouabain are mediated by activation of protein kinase C, rather than by Ca2+ mobilization, resulting from phosphoinositide metabolism.     

Thermodynamic changes in the binding of Ca2+ to a mutant human lysozyme (D86/92). Enthalpy-entropy compensation observed upon Ca2+ binding to proteins.

The thermodynamic change in the binding of Ca2+ to a mutant human lysozyme having an engineered Ca2+ binding site (Kuroki, R., Taniyama, Y., Seko, C., Nakamura, H., Kikuchi, M., and Ikehara, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6903-6907) was analyzed by calorimetry and interpreted in terms of structural information obtained from x-ray crystallography. It was found that the enthalpic contribution for the Ca2+ binding reaction was small, driven primarily by entropy release (10 kcal/mol). This release of entropy was also observed in some organic chelators. Moreover, through the information of the tertiary structures of the apo- and holomutant lysozyme, it was confirmed that the entropy release (10 kcal/mol) upon the binding of Ca2+ arises primarily from the release of bound water molecules hydrating the free Ca2+. Previous studies of Ca2+ binding to proteins have involved significant changes in protein conformation. They can now be reevaluated to determine the contribution of conformational changes to Ca2+ binding. After removing the thermodynamic contribution of Ca2+ binding itself, it is found that upon the binding of Ca2+ the enthalpy change is negative but is almost compensated by the negative entropy change. The negative change in both enthalpy and entropy is characteristic of values seen in the thermodynamic change upon the folding of proteins.     

Genistein inhibits lysosomal enzyme release by suppressing Ca2+ influx in HL-60 granulocytes

The tyrosine kinase inhibitor genistein (5-200 microM) suppressed Ca(2+)-dependent fMLP (1 microM) and ATP (100 microM)-induced release of the lysosomal enzyme, beta-glucuronidase from neutrophil-like HL-60 granulocytes. Agonist-induced Ca2+ mobilization resulted from the release of intracellular Ca2+ stores and the influx of extracellular Ca2+. Genistein (200 microM) suppressed fMLP (1 microM) and ATP (100 microM)-induced Ca2+ mobilization, by 30-40%. Ca2+ release from intracellular stores was unaffected by genistein, however, genistein abolished agonist-induced Ca2+ (Mn2+) influx. Consistent with these findings, genistein (200 microM) or removal of extracellular Ca2+ (EGTA 1 mM), inhibited Ca(2+)-dependent agonist-induced beta-glucuronidase release by similar extents (about 50%). In the absence of extracellular Ca2+, genistein had a small additional inhibitory effect on fMLP and ATP-induced beta-glucuronidase release, suggesting an additional inhibitory site of action. Genistein also abolished store-operated (thapsigargin-induced) Ca2+ (Mn2+) influx. Neither fMLP nor ATP increased the rate of Mn2+ influx induced by thapsigargin (0.5 microM). These data indicate that agonist-induced Ca2+ influx and store-operated Ca2+ influx occur via the same genistein-sensitive pathway. Activation of this pathway supports approximately 50% of lysosomal enzyme release induced by either fMLP or ATP from HL-60 granulocytes.     

Evidence for an alteration in the microsomal Ca2+ release mechanism in senescent rat parotid acinar cells

Previously, we have shown that Ca2+ mobilization following an alpha 1-adrenergic receptor stimulus is reduced in parotid acinar cells from senescent rats as a result of an altered ability of inositol 1,4,5-trisphosphate (IP3) to induce Ca2+ release from a non-mitochondrial, intracellular Ca2+ store (Ishikawa, Y., et al. Biochim. Biophys. Acta 968, 203-210). We have used this model to examine the IP3-induced Ca2+ release mechanism in these cells. 45Ca2+ efflux, after exposure to (-) epinephrine, from cells of young adult (3-6 months) rats was approx. 2-fold that observed from cells from older animals (approx. 24 months) either in the presence or absence of extracellular Ca2+. Similarly, cytosolic Ca2+ levels were greater in cells of young adult rats under these same incubation conditions. However, microsomal membrane preparations, from both age groups displayed similar IP3 binding sites (Kd approximately 90 nM, Bmax approximately 850 fmol/mg protein) and ATP-dependent Ca2+ transport ability (approx. 8 nmol/mg protein.min -1). These data suggest that there is an alteration in the IP3-induced Ca2+ release mechanism in microsomal membranes of parotid glands from senescent rats which may account for the decreased Ca2+ release seen after agonist stimulation of this tissue.     

Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats

Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 microM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 microM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+,Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels.     

Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673-21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were approximately 10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.     

Multiple transduction mechanisms are likely involved in calcium-mediated exocrine secretory events in rat parotid cells

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L. & Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).     

Inhibition of translational initiation in eukaryotic cells by calcium ionophore

Ca2+ has been recently reported to be required for high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). In the present investigation low concentrations of the Ca2+ ionophores, A23187 and ionomycin, were found to rapidly suppress the Ca2+-dependent component of protein synthesis in GH3 cells. More ionophore was required to inhibit amino acid incorporation into protein as extracellular Ca2+ was increased. Pre-existing inhibitions of protein synthesis produced by low concentrations of ionophore at low extracellular Ca2+ concentrations were reversed by adjustment to high extracellular Ca2+. Treatment with ionophore reduced the cellular contents of polysomes and 43 S preinitiation complex to values equivalent to those found for Ca2+-depleted cells. Average ribosomal transit times were unaffected by ionophore, and treated cells retained the ability to accumulate polysomes when incubated with cycloheximide. Cell types, such as HeLa and Chinese hamster ovary, that normally display only a modest Ca2+-dependent component of protein synthesis, manifested a strong underlying Ca2+ dependence in amino acid incorporation and polysome formation following treatment with low concentrations of ionophore. Protein synthesis in GH3 or HeLa cells during recovery from heat shock and arsenite treatment was not affected by cellular Ca2+ depletion or ionophore treatment. On the basis of these results, Ca2+ ionophore is proposed to inhibit Ca2+-dependent translational initiation through facilitating the mobilization of sequestered intracellular Ca2+.     

Spatial and temporal aspects of Ca2+ signaling mediated by P2Y receptors in cultured rat hippocampal astrocytes

ATP produces a variety of Ca2+ responses in astrocytes. To address the complex spatio-temporal Ca2+ signals, we analyzed the ATP-evoked increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured rat hippocampal astrocytes using fura-2 or fluo-3 based Ca2+ imaging techniques. ATP at less than 10 nM produced elementary Ca2+ release event "puffs" in a manner independent of extracellular Ca2+. Stimulation with higher ATP concentrations (3 or 10 micro M) resulted in global Ca2+ responses such as intercellular Ca2+ wave. These Ca2+ responses were mainly mediated by metabotropic P2Y receptors. ATP acting on both P2Y1 and P2Y2 receptors produced a transient Ca2+ release by inositol 1,4,5-trisphosphate (InsP3). When cells were stimulated with ATP much longer, the transient [Ca2+]i elevation was followed by sustained Ca2+ entry from the extracellular space. This sustained rise in [Ca2+]i was inhibited by Zn2+ (<10 micro M), an inhibitor of capacitative Ca2+ entry (CCE). CCE induced by cyclopiazonic acid or thapsigargin and Ca2+ entry evoked by ATP share the same pharmacological profile in astrocytes. Taken together, the hierarchical Ca2+ responses to ATP were observed in hippocampal astrocytes, i.e., puffs, global Ca2+ release by InsP3, and CCE in response to depletion of InsP3-sensitive Ca2+ stores. It should be noted that these Ca2+ signals and their modulation by Zn2+ could occur in the hippocampus in situ since both ATP and Zn2+ are rich in the hippocampus and could be released by excitatory stimulation.     

Serotonin secretion from human platelets may be modified by Ca2+-activated, phospholipid-dependent myosin phosphorylation

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), which has been identified as a potent inhibitor of protein kinase C in vitro (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, in press), enhanced serotonin release from human platelets that was induced by the 12-O-tetradecanoyl phorbol 13-acetate and correspondingly decreased incorporation of radioactive phosphate into a 20,000-dalton protein. H-7 did not affect the protein phosphorylation or the serotonin secretion in unstimulated platelets. A phosphopeptide with a molecular weight of 20,000 has previously been identified as a light chain (LC20) of platelet myosin and both protein kinase C and Ca2+-calmodulin-dependent myosin light-chain kinase have been shown to be involved in its phosphorylation. Two-dimensional peptide mapping following tryptic hydrolysis revealed that H-7 selectively inhibited the protein kinase C-catalyzed phosphorylation of myosin light chain. This pharmacological evidence suggests that Ca2+-activated, phospholipid-dependent myosin light-chain phosphorylation may play an inhibitory role in the release reaction.     

Inhibition of alpha 1-adrenergic responsiveness in intact cells by a new, irreversible receptor antagonist

A novel alpha 1-adrenoreceptor antagonist, 1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo [2.2.2] octa-2,5-dienylcarbonyl) piperazine, was synthesized and shown to potently block alpha 1-adrenoceptor-induced Ca2+ mobilization in intact rat parotid acinar cells. Irreversible inhibition was complete in less than 5 min. This alkylating prazosin derivative blocked Ca2+ release (IC50 approximately 5 X 10(-10)M) and [3H]-prazosin membrane binding (IC50 approximately 3 X 10(-10)M) in a concentration dependent fashion and increased the EC50 of epinephrine for Ca2+ efflux by approximately 35 fold. The agent however had no effect on muscarinic receptor-induced Ca2+ mobilization, or beta-adrenoreceptor-induced protein secretion, from cells. These findings suggest that this irreversible alpha 1-adrenoreceptor antagonist will be a valuable tool in probing alpha 1-adrenoreceptor function and metabolism in intact cells.     

Relationship between secretagogue-induced Ca2+ release and inositol polyphosphate production in permeabilized pancreatic acinar cells

We have previously shown that inositol trisphosphate (IP3) releases Ca2+ from a nonmitochondrial pool of permeabilized rat pancreatic acinar cells (Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1984) Nature 306, 67-69). This pool was later identified as endoplasmic reticulum (Streb, H., Bayerdorffer, E., Haase, W., Irvine, R. F., and Schulz, I. (1984) J. Membr. Biol. 81, 241-253). As IP3 is produced by hydrolysis of phosphatidylinositol bisphosphate on activation of many "Ca2+-mobilizing receptors," our observation supported the proposal that IP3 functions as a second messenger to release Ca2+ from the endoplasmic reticulum. We have here used the same preparation of permeabilized acinar cells to study the relationship of secretagogue-induced Ca2+ release and IP3 production. We show that: 1) secretagogue-induced Ca2+ release in permeabilized cells is accompanied by a parallel production of inositol trisphosphate. 2) When the secretagogue-induced increase in intracellular free Ca2+ concentration was abolished by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering, secretagogue-induced IP3 production was unimpaired. 3) When secretagogue-induced IP3 production was reduced by inhibiting phospholipase C with neomycin, secretagogue-induced Ca2+ release was also abolished. 4) When the IP3 breakdown was reduced either by lowering the free Mg2+ concentration of the incubation medium or by adding 2.3-diphosphoglyceric acid, the rise in IP3 and the release of Ca2+ induced by secretagogues were both increased. These results further support the role of IP3 as a second messenger to induce Ca2+ mobilization.     

Evidence that fertilization activates starfish eggs by sequential activation of a Src-like kinase and phospholipase cgamma

Recent evidence has indicated a requirement for a Src family kinase in initiating Ca(2+) release at fertilization in starfish eggs (Giusti, A. F., Carroll, D. J., Abassi, Y. A., Terasaki, M., Foltz, K. R., and Jaffe, L. A. (1999) J. Biol. Chem. 274, 29318-29322). We now show that injection of Src protein into starfish eggs initiates Ca(2+) release and DNA synthesis, as occur at fertilization. These responses depend on the phosphorylation state of the Src protein; only the kinase active form is effective. Like Ca(2+) release at fertilization, the Ca(2+) release in response to Src protein injection is inhibited by prior injection of the SH2 domains of phospholipase Cgamma. These findings support the conclusion that in starfish, sperm-egg interaction causes egg activation by sequential activation of a Src-like kinase and phospholipase Cgamma. Injection of the SH2 domain of Src, which inhibits Ca(2+) release at fertilization, does not inhibit Ca(2+) release caused by Src protein injection. This indicates that the requirement for a Src SH2 domain interaction is upstream of Src activation in the pathway leading to Ca(2+) release at fertilization.     

Epidermal growth factor increases c-myc mRNA without eliciting phosphoinositide turnover, protein kinase C activation, or calcium ion mobilization in Swiss 3T3 fibroblasts

Incubation of quiescent cultures of Swiss 3T3 cells with epidermal growth factor (EGF) caused an increase in c-myc mRNA. Under these conditions, EGF did not induce phosphoinositide turnover, formation of diacylglycerol, formation of inositol tris-, bis-, and monophosphates, protein kinase C activation, or Ca2+ mobilization. Although it has been reported that both protein kinase C and Ca2+ may be responsible for the platelet-derived growth factor- and fibroblast growth factor-induced increases in c-myc mRNA in Swiss 3T3 cells (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., & Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192), these results indicate that neither protein kinase C nor Ca2+ is involved in the EGF-induced increase in c-myc mRNA, and that an unidentified system may be involved in this reaction.     

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.