Purification and characterization of human liver dehydroepiandrosterone sulphotransferase.

A BAL17 B lymphoma cell line bearing mu and delta chains on its surface behaves in a similar manner to normal mature B cells in terms of initial biochemical transmembrane signalling [Mizuguchi, Beaven, Ohara & Paul (1986) J. Immunol. 137, 2162-2167; Mizuguchi, Yong-Yong, Nakabayashi, Huang, Beaven, Chused & Paul (1987) J. Immunol. 139, 1054-1059]. Therefore the effects of protease inhibitors on increases in inositol phospholipid metabolism and intracellular free calcium concentration ([Ca2+]i) were examined. We show that the serine protease inhibitors Tos-Phe-CH2Cl (1-chloro-4-phenyl-3-L-tosylamidobutan-2-one-, TPCK) and Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one; TLCK) inhibit anti-IgM-mediated accumulation of inositol phosphates in a dose-dependent manner. InsP3 production induced by anti-IgM is also inhibited by pretreatment with Tos-Lys-CH2Cl or Tos-Phe-CH2Cl. Tos-Lys-CH2Cl- Tos-Phe-CH2Cl-mediated inhibition is not overcome by high concentrations of anti-IgM. Moreover, anti-IgM-mediated increases in [Ca2+]i are inhibited by pretreatment of the cells with these inhibitors. However, increases in inositol phospholipid metabolism caused by NaF, an activator of guanine-nucleotide-binding proteins (G-proteins), are approx. 10-fold more resistant to Tos-Lys-CH2Cl and Tos-Phe-CH2Cl inhibition compared with anti-IgM-induced changes. Furthermore, NaF-induced increases in [Ca2+]i are not inhibited by Tos-Lys-CH2Cl or Tos-Phe-CH2Cl pretreatment, suggesting that the inhibitors act at a step proximal to phospholipase C activation. The Tos-Lys-CH2Cl or Tos-Phe-CH2Cl treatment does not change the membrane IgM density as measured by flow cytometry, indicating that the active site of the inhibitors is distal to the membrane IgM molecule. These results indicate that serine proteases may be involved in coupling the receptor cross-linkage to G-protein.     

Inhibitory and stimulatory effects of phorbol ester on vasopressin-induced cellular responses in cultured rat aortic smooth muscle cells

In rat aortic smooth muscle cells, vasopressin (AVP) induces prostacyclin (PGI2) production, probably as the consequence of phospholipase C activation. Our study analyzes the effects of phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC) activation on AVP-induced inositol 1,4,5-trisphosphate formation, cytosolic free Ca2+ concentration [( Ca2+]c), and PGI2 production. PMA rapidly decreased PKC activity in the cytosol of smooth muscle cells, while increasing it transiently in the membranes with a maximum around 20 min. Prior exposure of the cells to PMA resulted in a transient inhibition of both AVP-induced inositol 1,4,5-trisphosphate formation and [Ca2+]c rise. This was inversely correlated with membraneous PKC activity and partially reversed by the PKC inhibitor staurosporine. In contrast, pretreating the cells with PMA markedly potentiated A23187 or AVP-induced PGI2 production. Under those conditions, AVP-induced PGI2 production did not correlate either with PMA-induced membranous PKC activity or with AVP-induced PLC activation. However, this potentiating effect of PMA was reversed by staurosporine and was not mimicked by the 4 alpha-phorbol, an inactive analogue of PMA. Thus, the possibility is raised that, while inhibiting AVP-induced PLC activation, PMA-induced PKC activation increases the Ca2+ sensitivity of the cellular signaling system leading to PGI2 production.     

Phorbol myristate acetate inhibits increases in membrane fluidity induced by anti-IgM in B cells

Anti-IgM or anti-IgD stimulates B cells to induce increases in inositol phospholipid metabolism and intracellular free calcium concentration [( Ca2+]i). Anti-IgM also causes increases in membrane fluidity that occur more promptly than those in [Ca2+]i in resting B cells as well as BAL17 B lymphoma cells. However, other B cell activators such as LPS or PMA did not induce the membrane fluidity changes. Furthermore, sodium fluoride, which is considered to be an activator of the guanine nucleotide-binding protein, caused increases in membrane fluidity as well as increased [Ca2+]i or inositol phospholipid metabolism. Anti-IgM- or sodium fluoride-induced increases in membrane fluidity were inhibited by 20-min pretreatment of cells with PMA, but not by 24-h pretreatment. These results indicate that membrane fluidity changes are closely associated with increased [Ca2+]i after cross-linkage of membrane Ig and are regulated by protein kinase C in B cells.     

Modulation of Ca2+ mobilization by protein kinase C in rat submandibular acinar cells

The effects of protein kinase C (PKC) activation and inhibition on the inositol 1,4,5-trisphosphate (IP3) and cytosolic Ca2+ ([Ca2+]i) responses of rat submandibular acinar cells were investigated. IP3 formation in response to acetylcholine (ACh) was not affected by the PKC activator phorbol 12-myristate 13-acetate (PMA), nor by the PKC inhibitor calphostin C (CaC). The ACh-elicited initial increase in [Ca2+]i in the absence of extracellular Ca2+ was not changed by short-term (0.5 min) exposure to PMA, but significantly reduced by long-term (30 min) exposure to PMA, and also by pre-exposure to the PKC inhibitors CaC and chelerythrine chloride (ChC). After ACh stimulation, subsequent exposure to ionomycin caused a significantly (258%) larger [Ca2+]i increase in CaC-treated cells than in control cells. However, pre-exposure to CaC for 30 min did not alter the Ca2+ release induced by ionomycin alone. These results suggest that the reduction of the initial [Ca2+]i increase is due to an inhibition of the Ca2+ release mechanism and not to store shrinkage. The thapsigargin (TG)-induced increase in [Ca2+]i was significantly reduced by short-term (0.5 min), but not by long-term (30 min) exposure to PMA, nor by pre-exposure to ChC or CaC. Subsequent exposure to ionomycin after TG resulted in a significantly (70%) larger [Ca2+]i increase in PMA-treated cells than in control cells, suggesting that activation of PKC slows down the Ca2+ efflux or passive leak seen in the presence of TG. Taken together, these results indicate that inhibition of PKC reduces the IP3-induced Ca2+ release and activation of PKC reduces the Ca2+ efflux seen after inhibition of the endoplasmic Ca2+-ATPase in submandibular acinar cells.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.     

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.     

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

Since secretion from intact bovine adrenal chromaffin cells in response to depolarization by nicotine is triggered by a rise in the concentration of intracellular Ca2+ ([Ca2+]i) to about 200-300 nM above basal, it has been assumed that the failure of the inositol 1,4,5-trisphosphate (InsP3)-mobilizing muscarinic agonists to induce secretion reflects the fact that the 50 nM rise in [Ca2+]i they elicit is insufficient to trigger the exocytotic machinery. A recent report, however, has demonstrated that some of the nicotine-induced rise in [Ca2+]i could originate from the InsP3-releasable Ca2+ store. The role of this Ca2+ store in secretion from bovine adrenal chromaffin cells is therefore unclear. In order to investigate in more detail the role of the InsP3-sensitive Ca2+ store in secretion from these cells, we have used a combination of an InsP3-mobilizing muscarinic agonist and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from the InsP3-sensitive store and, additionally, that the Ca2+ response to TG was composed of two distinct, temporally separated, components: a) a slow (1 min) increase in [Ca2+]i to approximately 50 nM above basal that was independent of extracellular Ca2+ and b) the maintenance of this level at a new steady-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin-evoked acetylcholine release via inositol trisphosphate-dependent elevation in free calcium in neuroblastoma x glioma hybrid NG108-15 cells

The mechanism underlying the bradykinin (BK)-induced increase of acetylcholine (ACh) release was studied in neuroblastoma x glioma hybrid NG108-15 cells and their synapses formed onto mouse muscle cells. External application of BK or iontophoretic injection of extrinsic inositol 1,4,5-trisphosphate (InsP3) into the cytoplasm of NG108-15 cells produced membrane hyperpolarization in the hybrid cells and an increase in the frequency of miniature end-plate potentials (MEPPs) in paired myotubes. Ba2+ blocked the hyperpolarization in response to BK, but facilitation of MEPPs was still observed. InsP3-dependent facilitation of MEPPs was also observed in cells where the InsP3 injections produced no detectable hyperpolarization or even depolarization. Real-time quantitative monitoring of intracellular free Ca2+ concentration [( Ca2+]i) with fura-2 in single NG108-15 cells showed that BK application or InsP3 injection induced an elevation of [Ca2+]i which coincided in time with membrane hyperpolarization recorded from the same cell. The [Ca2+]i rise produced by InsP3 injection started from the single site of injection and that produced by BK began from a deep compartment of the cytoplasm of the NG108-15 cells. The BK- and InsP3-evoked facilitation of MEPPs and the [Ca2+]i rise were relatively independent of extracellular Ca2+. These findings suggest that the BK-induced ACh release results not from membrane potential changes but from a transient InsP3-dependent elevation of [Ca2+]i.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

Cross-linking membrane IgM induces production of inositol trisphosphate and inositol tetrakisphosphate in WEHI-231 B lymphoma cells

The addition of anti-IgM to the immature B lymphoma cell line WEHI-231 resulted in breakdown of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). These reactions have recently been demonstrated in mature resting B cells stimulated with anti-IgM, as well. In addition to Ins(1,4,5)P3, inositol tetrakisphosphate (InsP4) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) were rapidly generated in WEHI-231 cells upon stimulation of the antigen receptor with anti-IgM. These two inositol polyphosphates are probably generated from Ins(1,4,5)P3 by phosphorylation to yield InsP4 and removal of the 5-phosphate from InsP4 to yield Ins(1,3,4)P3. It is possible that these inositol polyphosphates play a second messenger role in mediating the biologic effects of antigen-receptor signaling. It had previously been shown that anti-IgM also causes an increase in cytoplasmic free calcium. Therefore, the relationship between Ca2+ elevation and phosphoinositide breakdown was investigated. Although elevation of cytoplasmic Ca2+ with ionophores can trigger phosphoinositide breakdown, this required levels of Ca2+ well beyond those normally seen in response to anti-IgM. Thus, the Ca2+ elevation seen in response to anti-IgM cannot be the event controlling phosphoinositide breakdown. WEHI-231 cells have been shown to have a calcium storage compartment that releases Ca2+ in the presence of Ins(1,4,5)P3; therefore, it is likely that anti-IgM stimulates phosphoinositide breakdown as a primary event and this leads to the elevation of cytoplasmic Ca2+.     

B cell activation. VII. Independent and synergistic effects of mobilized calcium and diacylglycerol on membrane potential and I-A expression

Although cross-linking of murine B cell membrane Ig (mIg) has been shown to induce a rapid increase in intracellular free calcium [Ca++)i), both the source and the function of the Ca++ in lymphocyte activation is unclear. Toward elucidation of its function, we investigated the relationship between the initial (Ca++)i response and other cell physiologic changes that occur early after mIg cross-linking, apparently as a linear cascade, leading to increased membrane I-A expression. Results suggest that the (Ca++)i response results from polyphosphoinositol hydrolysis induced by mIg cross-linking. The (Ca++)i response cannot be induced by activation of protein kinase C (PKC) with phorbol diesters (e.g., PMA) or synthetic diacylglycerol (DAG), suggesting that this response precedes the PKC activation. However, inhibition of phosphatidylinositol turnover by exposure of cells to dbcAMP during anti-Ig stimulation significantly inhibits the (Ca++)i response, suggesting that phosphatidylinositol turnover may be causally related to Ca++ mobilization. The ability of exogenous phospholipase C to induce the (Ca++)i response also supports this conclusion. Of the products of mono- and poly-phosphatidylinositol hydrolysis, the inositol phosphates (InsP, InsP2, InsP3) are implicated as promoters of Ca++ mobilization, because exogenous synthetic diacylglycerol is without effect on (Ca++)i. In light of recent evidence obtained with other systems, we suggest that InsP3 is responsible for mIg cross-linking-induced Ca++ mobilization from intracellular stores in B lymphocytes. Both depolarization and increased I-A expression are induced by increasing (Ca++)i with the Ca++ ionophores A23187 and ionomycin. These events can also be induced by the activation of PKC with high doses of PMA. When suboptimal doses of both A23187 and PMA are present, these reagents synergize in the induction of depolarization. This suggests that one role for the initial rise in (Ca++)i is to act with the DAG liberated from PtdIns turnover, possibly by enhancing translocation of cytosolic PKC to the plasma membrane, and thereby promote changes in ion transport that are apparent as a decrease in the membrane potential.     

Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Intracellular calcium responses in bovine oocytes induced by spermatozoa and by reagents

The objectives of the present study were to clarify and compare the characteristics of the transient rises in intracellular calcium concentrations ([Ca2+]i) induced either by spermatozoa or by stimulation with artificial activators in bovine oocytes. These transient rises in [Ca2+]i in oocytes matured in vitro were recorded with Ca2+ imaging using the Ca2+ indicator fura-2. During fertilization, a series of transient rises in [Ca2+]i was observed. The first Ca2+ response peaked at a concentration of 521 +/- 39 nM (n = 20) and lasted for 4 min, while the subsequent Ca2+ responses were significantly smaller and shorter, with a peak of 368 +/- 13 nM (n = 23) and a duration of 2 min. Injection of inositol 1,4,5- triphosphate (InsP3) into unfertilized oocytes caused a transient rise in [Ca2+]i in a dose-dependent manner. The maximum response was induced by 20 nA x 1 sec injection of InsP3. Thimerosal, a sulfhydryl reagent, induced the repetitive transient rises in [Ca2+]i. The peak and the duration of the rises in [Ca2+]i induced by InsP3 or thimerosal were smaller and shorter, respectively, than those of the first rise induced by spermatozoa. Ethanol and Ca2+ ionophore IA23187, which are general parthenogenetic activators of unfertilized oocytes, each induced a single transient rise in [Ca2+]i. The duration of the rise in [Ca2+]i by ethanol or Ca2+ ionophore was significantly longer than that by spermatozoa at fertilization, although the peaks were smaller. These results clarified the characteristics of the rises in [Ca2+]i induced by spermatozoa and by several artificial reagents, and showed that the first rise in [Ca2+]i induced by spermatozoa had a higher peak [Ca2+]i and a longer duration compared with each the subsequent rises in [Ca2+]i and the rises in [Ca2+]i induced by artificial reagents. These indicate that a mode like as the first rise in [Ca2+]i induced by spermatozoa is an effective trigger for artificial activation of oocytes.     

Evidence for an inhibitory effect of protein kinase C on G-protein-mediated repetitive calcium transients in hamster eggs.

Hamster eggs undergo repetitive increases in cytoplasmic free calcium concentration ([Ca2+]i) at fertilization or after injecting guanosine-5'-0-(3-thiotriphosphate) (GTP[S]). We report the effects of protein kinase C (PKC) agonists and antagonists on these repetitive [Ca2+]i transients as measured by their associated membrane potential hyperpolarizing responses (HRs). Iontophoretic injection of GTP[S] into unfertilized eggs caused a series of repetitive HRs that declined in amplitude with time. Continuous injection of inositol 1,4,5-trisphosphate (InsP3) also caused a series of repetitive HRs, but these HRs declined in amplitude less markedly. GTP[S]-induced HRs were inhibited by the PKC agonists phorbol 12-myristate 13-acetate (TPA) (100 nM) and 1,2-dioctanoyl-glycerol (diC8) (250 microM). Conversely the PKC inhibitor sphingosine (10 microM) enhanced the number of large HRs after GTP[S] injection. TPA or sphingosine did not alter InsP3-induced HRs. We suggest that G-protein-mediated InsP3 production causes repetitive [Ca2+]i transients but that GTP[S] injection stimulates a negative feedback loop involving PKC. Adding TPA (100 nM) before insemination caused a reduction in the frequency of HRs at fertilization, but neither TPA nor sphingosine affected the frequency or size of HRs when they were added after the start of fertilization. Fertilizing sperm may stimulate G-protein-mediated InsP3 production in a way that precludes feedback inhibition by PKC.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.     

The biphasic stimulation of insulin secretion by bombesin involves both cytosolic free calcium and protein kinase C.

Members of the bombesin family of peptides potently stimulate insulin release by HIT-T15 cells, a clonal pancreatic cell line. The response to bombesin consists of a large burst in secretion during the first 30 s, followed by a smaller elevation of the secretory rate, which persists for 90 min. The aim of this study was to identify the intracellular messengers involved in this biphasic secretory response. Addition of 100 nM-bombesin to cells for 20 s increased the cellular accumulation of [3H]diacylglycerol (DAG) by 40% and that of [3H]inositol monophosphate (InsP), bisphosphate (InsP2) and trisphosphate (InsP3) by 40%, 300%, and 800%, respectively. In contrast, cyclic AMP concentrations were unaffected. Bombesin stimulation of [3H]InsP3 formation was detected at 2 s, before the secretory response, which was not measurable until 5 s. Furthermore, the potency of bombesin to stimulate [3H]InsP3 generation (ED50 = 14 +/- 9 nM) agreed with its potency to stimulate insulin release (ED50 = 6 +/- 2 nM). Consistent with its effects on [3H]InsP3 formation, bombesin raised the intracellular free Ca2+ concentration [( Ca2+]i) from a basal value of 0.28 +/- 0.01 microM to a peak of 1.3 +/- 0.1 microM by 20 s. Chelation of extracellular Ca2+ did not abolish either the secretory response to bombesin or the rise in [Ca2+]i, showing that Ca2+ influx was not required. Although the Ca2+ ionophore ionomycin (100 nM) mimicked the [Ca2+]i response to bombesin, it did not stimulate secretion. However, pretreating cells with ionomycin decreased the effects of bombesin on both [Ca2+]i and insulin release, suggesting that elevation of [Ca2+]i was instrumental in the secretory response to this peptide. To determine the role of the DAG produced upon bombesin stimulation, we examined the effects of another activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA did not affect [Ca2+]i, but it increased insulin secretion after a 2 min lag. However, an immediate increase in secretion was observed when ionomycin was added simultaneously with TPA. These data indicate that the initial secretory burst induced by bombesin results from the synergistic action of the high [Ca2+]i produced by InsP3 and DAG-activated protein kinase C. However, activation of protein kinase C alone appears to be sufficient for a sustained secretory response.     

Inositol trisphosphate mediates thyrotropin-releasing hormone mobilization of nonmitochondrial calcium in rat mammotropic pituitary cells

Thyrotropin-releasing hormone (TRH) stimulation of prolactin secretion from GH3 cells, cloned rat pituitary tumor cells, is associated with 1) hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate (InsP3) and 2) elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i), caused in part by mobilization of cellular calcium. We demonstrate, in intact cells, that TRH mobilizes calcium and, in permeabilized cells, that InsP3 releases calcium from a nonmitochondrial pool(s). In intact cells, TRH caused a loss of 16 +/- 2.7% of cell-associated 45Ca which was not inhibited by depleting the mitochondrial calcium pool with uncoupling agents. Similarly, TRH caused an elevation of [Ca2+]i from 127 +/- 6.3 nM to 375 +/- 54 nM, as monitored with Quin 2, which was not inhibited by depleting mitochondrial calcium. Saponin-permeabilized cells accumulated Ca2+ in an ATP-dependent manner into a nonmitochondrial pool, which exhibited a high affinity for Ca2+ and a small capacity, and into a mitochondrial pool which had a lower affinity for Ca2+ but was not saturated under the conditions tested. Permeabilized cells buffered free Ca2+ to 129 +/- 9.2 nM when incubated in a cytosol-like solution initially containing 200 to 1000 nM free Ca2+. InsP3, but not other inositol sugars, released calcium from the nonmitochondrial pool(s); half-maximal effect occurred at approximately 1 microM InsP3. Ca2+ release was followed by reuptake into a nonmitochondrial pool(s). These data suggest that InsP3 serves as an intracellular mediator (or second messenger) of TRH action to mobilize calcium from a nonmitochondrial pool(s) leading to an elevation of [Ca2+]i and then to prolactin secretion.     

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.