Diacylglycerol increases cytosolic free Ca2+ concentration in rat pituitary cells. Relationship to thyrotropin-releasing hormone action

To elucidate possible functions of elevation of endogenous diacylglycerol induced by thyrotropin-releasing hormone in pituitary cells, we have studied the actions of two synthetic diacylglycerols, sn-1-oleoyl-2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (DiC8), on cytosolic free calcium concentration ([Ca2+]i) in GH4C1 cells. OAG induced an immediate increase in [Ca2+]i which gradually reached a peak that was twice the basal level after the first min; [Ca2+]i then returned to remain at basal level after 3 min. The increase in [Ca2+]i was dependent on the concentration of OAG added with two apparent potencies; half-maximal actions on [Ca2+]i were observed at 70 nM and greater than 20 microM. The increase in [Ca2+]i induced by OAG was blocked completely by chelating extracellular calcium, or by pretreatment with calcium channel blockers. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which itself induces a rise in [Ca2+]i in these cells that is similar in time course, magnitude, and drug sensitivity to that of OAG, blocked completely the actions of subsequent exposure to OAG. Analogous results were obtained using DiC8, although DiC8 induced a transient inhibition to 75% of basal levels of [Ca2+]i after the initial increase in [Ca2+]i, and DiC8 was less potent than OAG. These data indicated that diacylglycerols induce influx of extracellular calcium in these cells, possibly by activation of voltage-dependent Ca2+ channels. Furthermore, diacylglycerols and phorbol esters appear to utilize a common pathway in eliciting these actions on [Ca2+]i, possibly involving activation of a protein kinase C. These actions of diacylglycerol provide a pathway by which thyrotropin-releasing hormone may act to enhance calcium channel activity.     

Intracellular free calcium regulates the onset of the respiratory burst of human neutrophils activated by phorbol myristate acetate.

Thapsigargin was used to study the regulation of different static calcium level ([Ca2+]i) on the respiratory hurst of human neutrophils stimulated with phorbol myristate acetate (PMA). The result showed that the onset time of the respiratory hurst was obviously reduced by elevation of static [Ca2+]i but is still much longer than that stimulated with N-formylmethionylleucylphenylalanine (fMLP). To find the reason, the onset times of the respiratory burst stimulated with fMLP, 1,2-dioctanoyl-sn-glycerol (DiC8), and PMA were determined at different static [Ca2+]i. It turns out that although DiC8 was unable to induce the respiratory burst at low [Ca2+], the onset time of DiC8-stimulated response at high [Ca2+]i was almost the same as that stimulated with fMLP. The study revealed that the fast onset of the fMLP-stimulated respiratory burst in comparison with PMA-stimulated response is not only due to the transient rise of [Ca2+]i, but is also due to the higher efficiency of diacylglycerol (DAG) in activating protein kinase c (PKC). The determining step in governing the onset of a respiratory burst is the activation of PKC.     

Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells

Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.     

Physiologic activation of protein kinase C limits IL-2 secretion

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.     

Activation of Na+/H+ exchange and Ca2+ mobilization start simultaneously in thrombin-stimulated platelets. Evidence that platelet shape change disturbs early rises of BCECF fluorescence which causes an underestimation of actual cytosolic alkalinization.

Although an increase in cytosolic pH (pHi) caused by Na+/H+ exchange enhances Ca2+ mobilization in platelets stimulated by low concentrations of thrombin [Siffert & Akkerman (1987) Nature (London) 325, 456-458], studies using fluorescent indicators for pHi (BCECF) and [Ca2+]i (fura2) suggest that Ca2+ is mobilized while the cytosolic pH decreases. Several lines of evidence indicate that the initial fall in BCECF fluorescence is not due to cytosolic acidification but is caused by a platelet shape change. (1) Pulse stimulation of platelets by successive addition of hirudin (4 unit/ml) and thrombin (0.2 unit/ml) induced a shape change of 43 +/- 8% and a fall in BCECF fluorescence, which both remained unchanged when Na+/H+ exchange was inhibited by ethylisopropylamiloride (EIPA, 100 microM). (2) Increasing the thrombin concentration to 0.4 unit/ml doubled the shape change and the fall in BCECF fluorescence, but again EIPA had no effect on these responses. (3) Treating platelets with 2 microM-ADP induced shape change and a decline in BCECF fluorescence that was unaffected by EIPA. (4) A second addition of thrombin to platelets that had already undergone shape change induced an immediate increase in BCECF fluorescence without a prior decrease. (5) Activation of protein kinase C by 1,2-dioctanoyl-sn-glycerol (DiC8) neither induced shape change nor a decline in BCECF fluorescence; in contrast BCECF fluorescence rapidly increased indicating an immediate cytosolic alkalinization. Concurrent analysis of [Ca2+]i under conditions in which shape change did not interfere with BCECF fluorescence showed that cytosolic alkalinization and Ca2+ mobilization started almost simultaneously. These observations suggest that cytosolic alkalinization is not preceded by a fall in pHi and can support Ca2+ mobilization induced by weak agonists.     

Dual actions of phorbol esters on cytosolic free Ca2+ concentrations and reconstitution with ionomycin of acute thyrotropin-releasing hormone responses

We have used phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), to study the actions of protein kinase C (a TPA receptor) on cytosolic free Ca2+ concentrations [( Ca2+]i) and hormone secretion in rat pituitary cells (GH cells), and to elucidate the role of diacylglycerol (a protein kinase C activator) in thyrotropin-releasing hormone (TRH) action. TPA had a dual action on [Ca2+]i, inducing a stimulatory phase from 300 (basal) to 420 nM, which was interrupted in 30-60 s by an inhibitory phase which transiently lowered [Ca2+]i to 240 nM and rose in 3-10 min to yield the stimulatory phase. TPA-mediated changes in [Ca2+]i were induced by other phorbol esters and mezerein but not by phorbol or activators of kinases different from protein kinase C. Both phases of TPA action on [Ca2+]i were abolished by 5-min pretreatment with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (1.33 mM) or Ca2+ channel antagonists (verapamil or nifedipine). TPA also enhanced the rate of sustained hormone secretion without inducing a burst of hormone release (unlike TRH). Also, stimulation of secretion by TPA was not inhibited by Ca2+ channel antagonists and was resistant (10%) to EGTA. Simultaneous addition of TPA with the ionophore ionomycin (100 nM) reconstituted a TRH-like spike, nadir and plateau of [Ca2+]i. Ionomycin generated the spike in [Ca2+]i by releasing TRH-sensitive Ca2+ stores, while TPA induced the nadir (inhibitory phase), and a nifedipine/verapamil-sensitive plateau of [Ca2+]i (stimulatory phase). Concurrent (but not separate) addition of ionomycin and TPA also reconstituted a TRH-like burst of hormone secretion. These and previous results indicate that activation of protein kinase C by TPA or diacylglycerol (which is elevated by TRH) and a simultaneous spike in [Ca2+]i are required for burst secretion. Diacylglycerol may also mediate the TRH-induced nadir and plateau of [Ca2+]i; the latter process contributes to Ca2+-dependent stimulation of steady secretion by TRH.     

Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes

We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.     

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.     

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

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.     

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.     

Thimerosal induces calcium mobilization, fructose 2,6-bisphosphate synthesis and cytoplasmic alkalinization in rat thymus lymphocytes

The effect of thimerosal on intracellular calcium ([Ca2+]i), pH (pHi) and fructose 2,6-bisphosphate (Fru 2,6-P2) in thymus lymphocytes was investigated. The effect of thimerosal on cell growth was also examined. Thimerosal produced a dose-dependent increase in [Ca2+]i, pHi and in the level of fructose 2,6-bisphosphate. Thimerosal was, however, unable to produce cell proliferation and inhibited [3H]thymidine incorporation when cells were challenged with PHA and costimulator. In the absence of external calcium, thimerosal produced only a slight increase in [Ca2+]i. In Na(+)-containing buffer, thimerosal induced an initial acidification (0.05 +/- 0.01 pH units), followed by an alkalinization of 0.08 pH units/min, whereas in Na(+)-free media, pHi decreased 0.2 +/- 0.02 units and this acidification was maintained for more than 40 min. When external calcium was removed the initial acidification was unchanged and no further increase in pHi was observed. Polymyxin B, an inhibitor of protein kinase C, did not modify the initial thimerosal-induced acidification although pH returned to basal levels after 10 min. It was concluded that alkalinization induced by thimerosal is probably due to activation of the Na+/H+ exchanger and that changes in internal Ca2+, pH and metabolic rate are not sufficient to induce cellular proliferation. The mechanism by which thimerosal inhibits thymocyte proliferation remains to be clarified.     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Interactions between calcium and protein kinase C in the control of signaling and secretion in pituitary gonadotrophs.

Single pituitary gonadotrophs exhibit episodes of spontaneous fluctuations in cytoplasmic calcium concentration [( Ca2+]i) due to entry through voltage-sensitive calcium channels (VSCC) and show prominent agonist-induced oscillations in [Ca2+]i that are generated by periodic release of intracellular Ca2+. Gonadotropin releasing hormone (GnRH) elicited three types of Ca2+ responses: at low doses, subthreshold, with an increase in basal [Ca2+]i; at intermediate doses, oscillatory, with dose-dependent modulation of spiking frequency; and at high doses, biphasic, without oscillations. Elevation of [Ca2+]i or activation of protein kinase C (PKC) did not influence the frequency of agonist-induced [Ca2+]i spikes but caused dose-dependent reductions in amplitude for all types of Ca2+ response. Stimulation of transient Ca2+ spikes by GnRH was followed by inhibition of the spontaneous fluctuations. GnRH also reduced the ability of high extracellular K+ to promote Ca2+ influx through VSCC. Activation of PKC by phorbol esters stimulated Ca2+ influx in quiescent cells but inhibited influx when VSCC were already activated, either spontaneously or by high K+. In contrast to their biphasic actions on [Ca2+]i, phorbol esters exerted only stimulatory actions on gonadotropin release, even when Ca2+ influx was concomitantly reduced. However, pituitary cells had to be primed with an appropriate [Ca2+]i level before exocytosis could be amplified by PKC. In PKC-depleted cells, all actions of phorbol esters on Ca2+ entry and amplitude modulation, and on LH release, were abolished. GnRH-induced LH secretion was also significantly reduced, especially the plateau phase of the response. These data indicate that Ca2+ and PKC serve as interacting signals during the cascade of cellular events triggered by agonist stimulation, in which Ca2+ turns cell responses on or off, and PKC amplifies the positive and negative effects of Ca2+.     

Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.     

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.     

Correlation of intracellular and extracellular calcium ion concentrations with synergy between 1,2-dioctanoyl-sn-glycerol and ionomycin in platelet arachidonic acid mobilization

The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites. This hypothesis was confirmed by simultaneous measurements of [Ca2+]i increases, and the production of the arachidonic acid metabolites. With the aequorin method, the optimal concentrations of [Ca2+]i fell to between 10 microM and 20 microM, and with the fura-2 method, it fell to between 800 nM and 1800 nM. Direct measurements of [14C]arachidonic acid release suggested that the DiC8-potentiated production of arachidonic acid metabolites induced by ionomycin was attributable to increased arachidonic acid release. Since ionomycin and DiC8 induced relatively low levels of phosphatidic acid production, an indicator of phospholipase C activation, it was suggested that the increased arachidonic acid release was largely dependent upon phospholipase A2. Synergy between DiC8 and ionomycin was also observed with aggregation and serotonin release. Aggregation was induced by lower concentrations of ionomycin, and appeared to be more dependent upon extracellular Ca2+, while serotonin release required higher concentrations of ionomycin, and variations in extracellular Ca2+ affected the response minimally. These findings suggest that the mechanisms underlying the synergy between protein kinase C activation and Ca2+ mobilization differ among the three functions evaluated in this study.     

Unidirectional, heterologous desensitization of the pertussis toxin receptor by the CD3/TCR complex.

Prolonged exposure of many types of receptors to their cognate agonists can lead to a progressive lack of responsiveness. When this occurs after stimulation by the primary agonist for a given receptor it is termed homologous desensitization, and heterologous desensitization when to an agonist binding to a different type of receptor. Pertussis toxin (PTx) is a potent mitogen for human T lymphocytes. We have previously identified the human T cell PTx receptor (PTx-R) as a 43-kDa plasma membrane protein that, when stimulated, leads to the production of the intracellular second messengers, inositol-1,4,5-triphosphate, 1,2-sn-diacylglycerol, and elevated cytosolic calcium. The PTx-R appears to require the co-expression of the CD3/TCR complex because mutant cells that lack the AgR, but express the PTx-R, fail to respond to PTx. In this report, we have investigated the relationship between these two receptor systems. Activation of the PTx-R with submaximal concentrations of PTx did not affect the ability of an anti-CD3 antibody combined with rabbit anti-mIg to stimulate increases in intracellular free calcium concentration [Ca2+]i or diacylglycerol in human peripheral blood T cells. However, treatment with soluble anti-CD3 mAb, which lead to only a modest increase in [Ca2+]i, completely inhibited the effect of PTx. The cells were not refractory to further stimulation of the AgR because cross-linking with rabbit anti-mIg resulted in the standard maximal stimulation. This effect could be observed within 1 min of treatment with anti-CD3 mAb, and persisted for at least 1 h. The effect was not caused by production of either diacylglycerol (leading to activation of PK-C) or an increase in [Ca2+]i by anti-CD3 mAb because the effect could not be mimicked by either phorbol esters or a calcium ionophore. Pretreatment of either resting T lymphoblasts or PBL with anti-CD3 mAb also prevented enhanced [3H]TdR incorporation stimulated by PTx. These observations suggest a model in which T cells can regulate amplification of a non-AgR stimulatory pathway by heterologous desensitization.     

Changes in sphingolipid levels induced by epidermal growth factor in osteoblastic cells. Effects of these metabolites on cytosolic calcium levels

Sphingolipids mediate a number of cellular functions in a variety of cell systems. The role they play in osteoblast signaling is yet unknown. This study investigated the effects of epidermal growth factor (EGF) on the levels of ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P) in rat calvariae osteoblastic cells, and whether these metabolites mediated cytosolic calcium ([Ca2+]i) mobilization in these cells. EGF significantly (P<0.05) increased the levels of all three sphingolipids, and the phorbol ester PMA partially inhibited these effects. SPH and S1P markedly increased [Ca2+]i levels, with thapsigargin (depletes [Ca2+]i pools) decreasing the response by 60%. Verapamil (calcium channel blocker) only inhibited ceramide's effects on [Ca2+]i. Furthermore, SPH enhanced the EGF' induced increase in [Ca2+]i. This study demonstrates that ceramide, SPH and S1P mediate [Ca2+]i mobilization in rat calvarial osteoblastic cells, and that EGF induces changes in the levels of these metabolites with PKC playing an important role in the mechanisms regulating these events.     

Cytosolic free calcium spiking affected by intracellular pH change

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.