Involvement of a guanine-nucleotide-binding component in membrane IgM-stimulated phosphoinositide breakdown

Cross-linking of membrane immunoglobulin, the B cell receptor for antigen, activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) by phospholipase C. This reaction yields two intracellular second messengers, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes an increase in cytoplasmic Ca2+. The experiments reported here demonstrate that activation of phospholipase C by membrane IgM (mIgM) involves a guanine nucleotide-dependent step. Saponin was used to permeabilize WEHI-231 B lymphoma cells and permit direct manipulation of nucleotide and Ca2+ concentrations. Very high levels of Ca2+ (greater than 100 microM) activated the phospholipase maximally without a requirement for cross-linking of mIgM. However, at much lower, physiologically relevant Ca2+ concentrations (100 to 500 nM), receptor-stimulated PtdInsP2 hydrolysis could be demonstrated. The ability of anti-IgM antibodies to activate phospholipase C in permeabilized WEHI-231 cells was greatly increased by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues (guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate), but not by guanosine diphosphate or guanosine diphosphate analogues or by a nonhydrolyzable analogue of adenosine triphosphate. This specificity for GTP analogues is consistent with the hypothesis that a GTP-binding regulatory protein analogous to those that couple receptors to adenylate cyclase is involved in the activation of phospholipase C by mIgM in WEHI-231 B lymphoma cells. In order to characterize this putative GTP-binding component, we examined the ability of pertussis toxin and cholera toxin to affect anti-IgM-stimulated inositol phosphate production. These bacterial toxins covalently modify and modulate the activity of various GTP-binding regulatory proteins and in some cell types can block receptor-stimulated PtdInsP2 breakdown. In WEHI-231 B lymphoma cells, neither toxin blocked signaling by mIgM. Thus mIgM appears to be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to both pertussis toxin and cholera toxin.     

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.     

Antigen receptor-induced cell cycle arrest in WEHI-231 B lymphoma cells depends on the duration of signaling before the G1 phase restriction point.

Stimulation of antigen receptors on WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-immunoglobulin M [IgM]) causes irreversible growth arrest. This may be a model for antigen-induced tolerance to self components in the immune system. Antigen receptor stimulation also causes inositol phospholipid hydrolysis, producing diacylglycerol, which activates protein kinase C, and inositol 1,4,5-trisphosphate, which causes release of calcium from intracellular stores. To better understand the nature of the antigen receptor-induced growth arrest of WEHI-231 cells, we have examined the basis for it. WEHI-231 cells in various phases of the cell cycle were isolated by centrifugal elutriation, and their response was evaluated following treatment with either anti-IgM or pharmacologic agents that raise intracellular free calcium levels and activate protein kinase C. Treatment with anti-IgM or the pharmacologic agents did not lengthen the cell cycle. Instead, growth inhibition was solely the result of arrest in the G1 phase. The efficiency of G1 arrest increased with the length of time during which the cells received signaling before reaching the G1 phase arrest point. Maximum efficiency of arrest was achieved after approximately one cell cycle of receptor signaling. These results imply that anti-IgM causes G1 arrest of WEHI-231 cells by slowly affecting components required for S phase progression, rather than by rapidly inhibiting such components or by rapidly activating a suicide mechanism. Antigen receptor stimulation was twice as effective as stimulation via the mimicking reagents phorbol dibutyrate and ionomycin. Thus, although the phosphoinositide second messengers diacylglycerol and calcium probably play roles in mediating the effects of anti-IgM on WEHI-231 cells, other second messengers may also be involved.     

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.     

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+.     

Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells

Phorbol esters which activate protein kinase C increased the percentage of membrane-bound protein kinase C activity in bovine adrenal chromaffin cells from less than 10 to 20-50% within 30 min. Permeabilization of chromaffin cells with digitonin in the absence of Ca2+ and phorbol esters caused virtually 100% of the protein kinase C activity to leave the cells within 1 h, which is consistent with protein kinase C being soluble and cytosolic. However, if cells were incubated for 15-30 min with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to permeabilization, 50-60% of the protein kinase C activity exited from the cells within 1 h of permeabilization. In cells not incubated with phorbol ester, permeabilization in the presence of 1-10 microM Ca2+ also decreased the rate at which protein kinase C exited from the cells. The slower release of protein kinase C caused by prior incubation of the cells with TPA or because of the presence of micromolar Ca2+ in permeabilized cells was associated with increased membrane-bound protein kinase C. The effects of TPA and permeabilization in the presence of micromolar Ca2+ were approximately additive. Active phorbol esters had different abilities to cause retention of protein kinase C in digitonin-treated cells. Dioctanoylglycerol, which activates protein kinase C in vitro and enhanced Ca2+-dependent secretion from permeabilized chromaffin cells similarly to TPA, also increased membrane-bound protein kinase C in intact cells, but had no effect on the retention of protein kinase C in permeabilized cells in the presence or absence of Ca2+. The different abilities of protein kinase C activators to cause retention of protein kinase C in subsequently permeabilized cells suggest differences in the reversibility of the binding. The mixed nicotinic-muscarinic agonist carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium, but not the muscarinic agonist muscarine, caused 3-10% of the total protein kinase C activity to become membrane-bound within 3 min in intact chromaffin cells. Thus, nicotinic stimulation of chromaffin cells may rapidly activate protein kinase C.     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.     

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.     

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.     

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells

Summary The effect of a reduction in protein kinase C activity on the metabolism of exogenous [³H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [³H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [³H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.Abbreviations IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - MG monoacylglycerol - PL phospholipid(s) - diC8 dioctanoylglycerol - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - monoC8 monooctanoylglycerol - PS phosphatidylserine - PDBu phorbol 12,13-dibutyrate     

Physiologic 1,2-diacylglycerol levels induce protein kinase C-independent translocation of a regulatory enzyme

Phorbol esters and 1,2-diacylglycerols have been used interchangeably to study protein kinase C action. This laboratory first suggested that 1,2-diacylglycerols may also act independent of protein kinase C using protein kinase C-"down-modulated" cells (Kolesnick, R. N., and Paley, A. E. (1987) J. Biol. Chem. 262, 9204-9210). Unfortunately, down-modulation was never complete. The present studies establish an in vitro system of enzyme translocation to resolve this issue. Choline phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme for phosphatidylcholine biosynthesis, was utilized. Cytidylyltransferase translocation from cytosol to membranes mediates phorbol ester-induced phosphatidylcholine synthesis in GH3 pituitary cells. In the present studies, 1,2-diacylglycerols similarly induced phosphatidylcholine synthesis and cytidylyltransferase translocation. 1,2-Diacylglycerol-induced phosphatidylcholine synthesis, however, was not concentration-dependent but proportional to the moles of 1,2-diacylglycerol added per cell, i.e. subject to surface dilution. For instance, at constant cell number (1.67 x 10(6)/sample) and 1,2-dioctanoylglycerol concentration (diC8; 20 micrograms/ml), 32Pi incorporation into phosphatidylcholine varied from 150 to 350% above control as the incubation volume increased from 0.3 to 1.2 ml. Hence, the effective diC8 concentrations 0.5-30 micrograms/ml are preferably referred to as doses and reported as 0.25-15 nmol/10(6) cells. These doses increased cellular 1,2-diacylglycerol levels within a few fold of basal (374 pmol/10(6) cells). In vitro, diC8 also induced translocation of purified cytidylyltransferase devoid of protein kinase C to microsomes. Translocation was again subject to surface dilution. Translocation occurred with the same ratio of diC8 to microsomal membrane as phosphatidylcholine synthesis in intact cells (1-10 nmol of diC8/10(6) cell membranes). Despite stimulating cytidylyltransferase translocation in intact cells, phorbol esters failed to stimulate translocation in vitro. Hence, 1,2-diacylglycerols are not always interchangeable with phorbol esters and at physiologic levels may stimulate enzyme translocation by an alternative mechanism to protein kinase C.     

Phorbol ester modulates serotonin-stimulated phosphoinositide breakdown in cultured vascular smooth muscle cells

Stimulation of cultured rabbit aortic vascular smooth muscle cells (VSMC) with serotonin (5HT) induced a rapid generation of inositol phosphates from receptor-mediated hydrolysis of inositol phospholipids. Pretreatment of these cells with 500ng/ml of pertussis toxin for 24h prior to addition of 5HT reduced 5HT-induced formation of inositol phosphates. Phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutyrate (PDBu), are known to activate protein kinase C (PKC), but their role on cultured VSMC stimulated by 5HT has not been defined. TPA exhibited a rapid inhibition of 5HT-stimulated phosphoinositide breakdown, although 4 alpha-phorbol-12,13-didecanoate (4 alpha PDD), an inactive phorbol ester, did not inhibit it. These data suggest that a guanine nucleotide inhibitory (Gi) protein couples 5HT receptor to phospholipase C and TPA modulates 5HT-stimulated hydrolysis of inositol phospholipids in cultured VSMC through activation of PKC.     

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.     

Regulation of B-lymphocyte activation, proliferation, and immunoglobulin secretion

Lymphocyte growth and differentiation are controlled by signals resulting from the interaction of antigen and cellular products, such as lymphokines, with specific cell membrane receptors. Resting B lymphocytes can be activated by low concentrations (1-5 micrograms/ml) of antibodies to membrane IgM, which is the B-lymphocyte receptor for antigen. The binding of anti-IgM to B cells causes a rapid increase in intracellular free calcium concentration ([Ca2+]i), in inositol phosphate concentration, and in protein kinase activity. Moreover, the effects of anti-IgM on B cells are mimicked by the combined use of calcium ionophores and phorbol esters. Since phorbol esters activate protein kinase c, this suggests that the increase in [Ca2+]i and in phosphatidylinositol metabolism stimulated by anti-IgM are critical events in B-cell activation. The entry into S phase of B cells stimulated with anti-IgM depends on the action of a T-cell-derived factor designated B-cell stimulatory factor (BSF)-1. This is a 20,000-Da protein which is a powerful inducer of class II major histocompatibility complex molecules. Although an important cofactor for B-cell proliferative responses to anti-IgM, its major locus of action is on resting B cells. B cells stimulated with anti-IgM and BSF-1 do not synthesize secretory IgM. However, if two additional T-cell-derived factors, B151-TRF and interleukin-2, are added to cultures, a substantial proportion of stimulated B cells produce secretory IgM. BSF-1 has also been shown to participate in the "switch" in Ig class expression. Resting B cells cultured with lipopolysaccharide will switch to IgG1 secretion in the presence of purified BSF-1.     

Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells

Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties.     

Analysis of the primary signals required for activation of the mitogenic pathway in murine thymocytes from protein phosphorylation patterns

It has been proposed that phorbol esters and the Ca2+ ionophore A23187 are effective comitogens for some species of lymphocytes because together they mimic the normal secondary signals for cell activation by mitogens that cause phosphatidylinositol 4,5-bisphosphate (PtdInsP2) breakdown (e.g., anti-TCR and anti-Thy-1 antibodies and Con A). To test whether activation of protein kinase C and an increase in [Ca2+]i account for the activation of the mitogenic pathway in murine thymocytes by the mitogens that cause PtdInsP2 breakdown, the two-dimensional phosphorylation patterns generated by the three classes of mitogens (protein kinase C activator, Ca2+ ionophore, and activator of PtdInsP2 breakdown) and by activators of cAMP-dependent kinases have been compared. From the phosphorylation patterns, by which each mitogen could be distinguished reproducibly, it was concluded that: 1) The phosphorylation patterns generated by the mitogens that activate PtdInsP2 breakdown are only slightly affected by the removal of extracellular Ca2+ under conditions that abolish the normal rise in [Ca2+]i and do not therefore depend on the activation of Ca2(+)-dependent protein kinases. In contrast, the phosphorylation pattern generated by A23187 is totally dependent on extracellular Ca2+. 2) Neither A23187 nor the mitogens that activate PtdInsP2 breakdown nor activators of cAMP-dependent kinases caused significant activation of protein kinase C assayed by phosphorylation of the diagnostic proteins 80b and 78a. Consistent with this conclusion, only the phorbol esters or oleoyl acyl glycerol caused translocation of protein kinase C activity from the cytosolic to the membrane fraction. 3) Neither A23187 nor the mitogens that cause PtdInsP2 breakdown activated cAMP-dependent kinases. Taken together the data imply that the mitogens that cause PtdInsP2 breakdown must generate an additional, independent primary mitogenic signal. It is suggested that this signal may be the activation of tyrosine kinases (e.g., p56lck) via the TCR and working hypotheses for effective combinations of primary mitogenic signals that will activate DNA synthesis are developed.     

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.     

Phorbol esters inhibit inositol phosphate and diacylglycerol formation in proliferating HL60 cells. Relationship to differentiation

Phorbol 12-myristate 13-acetate (PMA) induces the differentiation of the human promyelocytic cell line, HL60, towards adherent macrophage-like cells within 2 days. We have examined the early effects of PMA on inositol phosphates and on diacylglycerol production, two second messengers derived from inositol lipids. In proliferating HL60 cells, PMA induced a time- and concentration-dependent decrease in inositol phosphate levels. Maximal effects were seen after 1 h at 10 nM PMA. PMA also induced the translocation of protein kinase C from the cytosol to the membrane. Comparison between the differentiating effects of several phorbol esters and of 1-oleoyl-2-acetylglycerol with their ability to inhibit inositol phosphate formation suggests that the two effects are correlated.