Inhibition by phorbol esters of antiimmunoglobulin-induced calcium signalling and B-cell activation

The inhibitory effect of phorbol dibutyrate (PDB) on B-cell stimulation was evaluated using a model in which activation is induced by modest doses of antiimmunoglobulin antibody (anti-Ig) and progression to DNA synthesis is induced by cytochalasin. PDB preferentially inhibited anti-Ig-induced activation and did so during brief (2 hr) preincubation with anti-Ig. Activation was inhibited whether PDB was added before or shortly after anti-Ig. Since activation for cytochalasin responsiveness appears to be mediated by Ca2+, the effect of PDB on the anti-Ig-induced rise in intracellular Ca2+ was evaluated. PDB (and other phorbol esters that activate protein kinase C) inhibited the rise in Ca2+ normally associated with anti-Ig treatment; moreover, PDB reversed an established anti-Ig-induced Ca2+ response. These data suggest that phorbol esters inhibit B-cell activation by interfering with the elevated levels of intracellular Ca2+ produced by cross-linking of surface immunoglobulin by anti-Ig. This could represent a "feedback inhibition" type of response, but it remains to be seen if this occurs under physiological conditions of protein kinase C activation.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.     

Phorbol esters and dioctanoylglycerol block anti-IgM-stimulated phosphoinositide hydrolysis in the murine B cell lymphoma WEHI-231

Cross-linking of membrane IgM (mIgM) on both normal resting B cells and on the murine B cell lymphoma WEHI-231 activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), which results in the generation of two second-messengers: inositol trisphosphate (InsP3), which can cause the release of Ca2+ from intracellular stores, and diacylglycerol (DG), which activates protein kinase C. In examining the effects of exogenous activation of protein kinase C on WEHI-231 cells, we found that phorbol esters blocked some of the biologic effects of anti-IgM on WEHI-231 cells. The mechanism of this effect was investigated. Phorbol ester treatment of WEHI-231 cells blocked the ability of anti-IgM to stimulate production of inositol phosphates and accumulation of phosphatidic acid, the phosphorylated product of DG. Phorbol esters also blocked the ability of anti-IgM to cause an increase in intracellular Ca2+. Thus, it is clear that phorbol esters block anti-IgM-stimulated PtdInsP2 hydrolysis in WEHI-231 cells. In addition, a synthetic DG, dioctanoylglycerol (diC8), also blocked anti-IgM-stimulated inositol phosphate production and the anti-IgM-stimulated rise in cytoplasmic Ca2+. The ability of phorbol esters and diC8 to block mIgM-mediated signaling may reflect a feedback inhibition mechanism by which activated protein kinase C limits the magnitude and duration of receptor signaling.     

IL-4 promotes anti-Ig-mediated protein kinase C translocation and reverses phorbol ester-mediated protein kinase C down-regulation in murine B cells

Co-stimulation of B lymphocytes with IL-4 plus nonmitogenic concentrations of anti-Ig antibodies, or protein kinase C (PKC) activators, drives resting B cells into DNA synthesis. Although cross-linking of the sIg receptors provokes the generation of the intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, the molecular mechanism utilized by IL-4R in murine B cells has not, as yet, been defined. In human B cells IL-4 has been shown to induce a transient rise in IP3 followed by a sustained elevation of cAMP. However, in murine B cells, IL-4 does not induce the release of IP3, Ca2+ mobilization, PKC translocation, or indeed modify signaling via the phosphoinositide pathway induced by ligation of sIg receptors. We now present evidence that, in murine B cells, IL-4 synergizes with nonmitogenic concentrations of anti-Ig to provoke translocation of PKC from the cytosol to membranes. In addition, the lymphokine up-regulates PKC levels and activity and prevents phorbol ester-induced PKC down-regulation in B cells. We therefore propose that (unknown) signals generated via IL-4R potentiate and/or prolong sIg-induced PKC activation. These observations may therefore provide a biochemical basis for explaining how IL-4 and anti-Ig synergize to induce B cell activation.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.     

Increased plasma membrane permeability to Ca2+ in anti-Ig-stimulated B lymphocytes is dependent on activation of phosphoinositide hydrolysis

The biochemical basis of Ca2+ mobilization after anti-Ig binding to B cell Ag-R has been further characterized by flow cytometric analysis of indo-1-loaded B cells. The ability to distinguish intracellular Ca2+ release from extracellular Ca2+ influx by using an extracellular calcium depletion-repletion approach has allowed us to study the relationship between the mobilization of Ca2+ from these sources. Studies involving manipulation of the Ca2+ gradient across the plasma membrane indicate that a significant portion of the Ca2+ mobilization response is preserved even when the normal inwardly directed Ca2+ gradient is reversed. In the presence of an extracellular calcium concentration ([Ca2+]o) of 10 microM, the response to anti-Ig is not blocked by the organic Ca2+ channel blockers. This response is not reduced by further depletion of [Ca2+]o by EGTA Ca2+-binding buffers. Thus, the Ca2+ response that occurs when [Ca2+]o less than or equal to 10 microM represents intracellular calcium release. Analysis of B cells stimulated with anti-Ig in low Ca2+ medium ([Ca2+]o = less than 10 microM) followed by repletion of [Ca2+]o to 1 to 5 mM reveals that a significant increase in permeability of the plasma membrane to Ca2+ develops in the stimulated cells. The resultant Ca2+ influx is nimodipine (20 microM) sensitive. Both intracellular Ca2+ release and Ca2+ influx are reduced in parallel as the concentration of anti-Ig stimulus is decreased, suggesting that Ca2+ influx may be coupled to the release of intracellular stores. Neomycin blocks anti-Ig-stimulated formation of inositol trisphosphate, which mediates release of Ca2+ from the endoplasmic reticulum. It also blocks the anti-Ig-induced release of intracellular Ca2+ stores as well as Ca2+ influx, indicating that both responses may be dependent upon phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Role of phosphoinositide-derived second messengers in mediating anti-IgM-induced growth arrest of WEHI-231 B lymphoma cells

Anti-IgM irreversibly inhibits the growth of WEHI-231 B lymphoma cells and induces phosphoinositide hydrolysis--producing diacylglycerol, which activates protein kinase C, inositol 1,4,5-trisphosphate, which induces the release of calcium from intracellular storage sites into the cytoplasm, and other inositol polyphosphates. The roles of two of the possible second messengers, cytoplasmic free calcium and diacylglycerol, in mediating the action of anti-IgM on WEHI-231 cells were assessed by elevating [Ca2+]i with ionomycin and by activating protein kinase C with phorbol 12,13-dibutyrate (PdBu). The combination of 250 nM ionomycin and 4 to 7 nM PdBu was found to cause growth arrest and cell volume decrease responses in WEHI-231 cells which were similar to those caused by anti-IgM, although clearly slower. Both anti-IgM and the combination of mimicking reagents induced growth arrest of WEHI-231 cells in the G1 phase of the cell cycle. In both cases, this growth arrest was mitigated by addition of bacterial LPS. Moreover, 250 nM ionomycin plus 4 to 7 nM PdBu did not inhibit the growth of two other murine B lymphoma cell lines, each of which did exhibit increased phosphoinositide hydrolysis but not growth arrest in response to anti-Ig. Taken together, these results suggest that ionomycin and PdBu, at the concentrations used, did not inhibit WEHI-231 growth by general toxicity, but rather by mimicking the effects of the natural second messengers generated from Ag receptor cross-linking. Thus, the phosphoinositide-derived second messengers Ca2+i and diacylglycerol are capable of playing important roles in mediating the action of anti-IgM on WEHI-231 B lymphoma cells. However, the response of WEHI-231 cells to anti-IgM could not be fully reproduced with ionomycin and phorbol diester. These results suggest that another second messenger induced by anti-IgM may also play an important role in mediating the growth arrest of these cells.     

B lymphocyte receptors and polyphosphoinositide degradation

Resting B lymphocytes can be activated and induced to proliferate by antibodies against their antigen receptors (anti-lg). We demonstrate an early increase in the level of [3H]inositol trisphosphate in [3H]inositol-labeled murine B cells, which suggests breakdown of phosphatidylinositol bisphosphate by phospholipase C. In line with this, the level of [3H]1,2-diacylglycerol was also elevated after incubation of [3H]arachidonic-acid-labeled B cells with anti-Ig. Anti-lg also caused a rapid increase in the level of cytosolic Ca2+ in B cells. In contrast, two other polyclonal B cell activators, lipopolysaccharide and phorbol myristate acetate, failed to induce any of these effects. Our results suggest that anti-lg may induce B cell growth via phosphoinositide degradation and Ca2+ mobilization, and that phorbol myristate acetate, and possibly lipopolysaccharide, bypass these initial events.     

Mechanisms of T cell activation by the calcium ionophore ionomycin

We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.     

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.     

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells.

Bombesin-related peptides stimulate a rapid increase in polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells. These peptides generate an increase in the efflux of 45Ca2+ from pre-labelled cells, a response consistent with an inositol trisphosphate-mediated mobilization of intracellular Ca2+. The bombesin-stimulated release of cellular 45Ca2+ is inhibited by tumour-promoting phorbol esters (e.g. 12-O-tetradecanoylphorbol 13-acetate, TPA). Although there are several possible sites of action at which this effect might occur, our results indicate that TPA induces an uncoupling of bombesin-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) without decreasing cellular binding of bombesin. In cultured cells, protein kinase C can be down-modulated by a prolonged incubation of the cells with phorbol esters. Such pretreatment greatly decreased the inhibitory effect of TPA on bombesin-stimulated PIP2 hydrolysis, suggesting that this action of the phorbol ester is mediated via protein kinase C. Since diacylglycerol is an endogenous activator of protein kinase C and a direct product of PIP2 hydrolysis, these results suggest that protein kinase C inhibition of polyphosphoinositide hydrolysis may function as a negative-feedback pathway. Cells in which protein kinase C has been down-modulated show elevated basal and bombesin-stimulated production of inositol phosphates, providing evidence that such a feedback loop limits polyphosphoinositide turnover in both unstimulated and mitogen-stimulated cells.     

Transmembrane signaling during interleukin 1-dependent T cell activation. Interactions of signal 1- and signal 2-type mediators with the phosphoinositide-dependent signal transduction mechanism

The murine T lymphoma line, LBRM-33 1A5, requires synergistic signals delivered by phytohemagglutinin (PHA) and interleukin 1 (IL1) for activation to high level interleukin 2 production. The activation signals provided by PHA and IL1 were replaced by the Ca2+ ionophore, ionomycin, and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), respectively. These observations supported a two-signal model for T cell activation involving increases in intracellular Ca2+ concentration ([Ca2+]i) (signal 1) and activation of protein kinase C (signal 2) as necessary and sufficient events. However, biochemical analyses revealed that additional signals were involved in the activation of LBRM-33 cells by both receptor-dependent and -independent mediators. Both signal 1-type mediators, PHA and ionomycin, exerted pleiotropic effects at the concentrations required for synergy with signal 2-type mediators (IL1, TPA). Within 1-2 min of addition, PHA stimulated phospholipid turnover, including hydrolysis of phosphatidylinositol 4,5-bisphosphate, Ca2+ mobilization, and protein kinase C activation. The [Ca2+]i increase induced by PHA was due to influx from both intracellular and extracellular Ca2+ pools. Similarly, ionomycin increased phospholipid turnover, [Ca2+]i, and directly affected protein kinase C activity in LBRM-33 cells. In contrast, the signal 2-type mediators, TPA and IL1, appeared to act by distinct intracellular mechanisms. TPA induced a protracted association of cellular protein kinase C with the plasma membrane, consistent with the two-signal activation model. Furthermore, acute TPA treatment inhibited PHA-stimulated inositol phosphate release and Ca2+ mobilization, suggesting that this mediator partially antagonized signal 1 delivery. IL1, in contrast, neither activated protein kinase C directly nor did it positively modulate the coupling of signal 1-type mediators to [Ca2+]i or protein kinase C via the phosphoinositide pathway. The intracellular signal delivered by IL1 is, therefore, generated through a mechanism distinct from or distal to the activation of protein kinase C. These studies indicate that the two-signal hypothesis, in its simplest form, is inadequate to explain the signals required for the initiation of IL1-dependent T cell activation.     

Anti-Ig-stimulated B lymphoblasts can be restimulated via their surface Ig

Engaging AgR (surface Ig) on B lymphocytes leads to rapid inositol phosphate turnover and elevation of intracellular [Ca2+]. Continuous receptor occupancy (greater than 18 h) by anti-Ig leads to transit of most B lymphocytes from G0 to G1 stage of the cell cycle (blast transformation); a fraction of cells continue into S phase but do not proliferate continuously in the absence of growth factors. Prolonged exposure to ligand can induce receptor desensitization of some receptors. We therefore investigated whether such desensitization occurs in B cells activated by insolubilized anti-Ig. Resting B cells and anti-Ig-activated blasts were examined for their potential to elevate [Ca2+]i, maintain viability, and synthesize DNA in response to reexposure to anti-Ig. B cells and anti-Ig blasts had similar basal [Ca2+]i levels. Anti-Ig blasts retained the capacity to increase [Ca2+]i in response to anti-Ig; the magnitude of the increase was equal to or greater than that observed with resting B cells and occurred in more than 90% of cells. Isolated anti-Ig blasts subcultured in the presence of T cell-derived growth factors for 3 to 5 days responded to restimulation by anti-Ig with an increase in [Ca2+]i similar to that observed in freshly isolated blasts. The B cell and B lymphoblast ion channels were found to be permeable to Ca2+ but impermeable to Mn2+. Finally, blasts restimulated by anti-Ig retained viability and incorporated low levels of [3H]thymidine for 24 h. These results suggest that AgR on activated B lymphocytes can remain functionally coupled to intracellular signaling pathways and can participate in immune responses subsequent to initial activation.     

BSF1 induces membrane protein phosphorylation but not phosphoinositide metabolism, Ca2+ mobilization, protein kinase C translocation, or membrane depolarization in resting murine B lymphocytes

The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction.     

Receptor-mediated activation of human B lymphocytes in a nonphosphotyrosine-dependent manner.

The B cell AgR regulates two signal transduction pathways: the tyrosine kinase and the phosphatidylinositol (PtdIns) pathways. Stimulation of B cells with Ag or anti-Ig antibody results in a rapid increase in tyrosine phosphorylation of multiple substrates. The AgR also mediates the activation of phospholipase C-gamma 1 (PLC-gamma 1) thus producing the second messengers, inositol trisphosphate and diacylglycerol. Although the detailed relationship between these two signaling pathways remains unclear, it has recently become apparent that PLC-gamma 1 might be a target for the AgR-associated protein tyrosine kinase. To address the question of whether tyrosine kinase activity is essential for B cell activation, we studied early biochemical changes and later cellular events induced by ligation of the purinoceptor (P2R). Ligation of ATP to its receptor on B cells has been previously shown to elicit increases in cytosolic free Ca2+ and inositol phosphate production as well as induction of c-fos mRNA expression and increased expression of IL-2 and transferrin receptors. We show here that ATP in a wide range of concentrations did not increase protein tyrosine kinase activity. In contrast with the AgR, P2R did not mediate tyrosine phosphorylation of PLC-gamma 1, thus suggesting that it may use another phosphoinositide-specific PLC that does not require phosphorylation on tyrosine residues for its activation. The results were supported by experiments with a specific tyrosine kinase inhibitor, tyrphostin AG-126. Preincubation with this inhibitor blocked AgR but not P2R-mediated inositol phosphate production, cytosolic free Ca2+ changes, and IL-2 and transferrin receptor expression. The results indicate that the PtdIns pathway may be sufficient to induce activation of B cells and that the tyrosine phosphorylation pathway is not necessary for nonantigenic B cell activation.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

Ethanol-induced phospholipase C activation is inhibited by phorbol esters in isolated hepatocytes.

Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.