Induction of phorbol ester responsiveness in conventional B cells after activation via surface Ig.

The signals required to induce S phase entry in murine splenic B cells were found to be altered by prolonged treatment with low doses of anti-Ig antibody. Whereas fresh splenic B cells are stimulated by the combination of a phorbol ester protein kinase C agonist plus a calcium ionophore, anti-Ig-treated splenic B cells were stimulated by phorbol ester alone, in the absence of a comitogen. The majority of these phorbol ester responsive B cells expressed CD5. The phorbol ester responses of anti-Ig-treated splenic B cells paralleled those previously reported for untreated peritoneal CD5+ B cells in a number of respects: responses were not idiosyncratic to phorbol esters but occurred with nonphorbol protein kinase C agonists; phorbol ester responses were enhanced by IL-4; and, phorbol ester responses occurred rapidly and were greater at 24 than at 48 h. However, the effect of agents that act to raise intracellular levels of cAMP distinguished between anti-Ig-treated splenic B cells and untreated peritoneal B cells in that the phorbol ester responses of the former were enhanced whereas the responses of the latter were inhibited. The present results add a functional dimension to the phenotypic similarity between splenic B cells treated with anti-Ig and resident peritoneal B cells that constitutively express CD5; however, some differences in behavior were noted.     

Peritoneal B cells respond to phorbol esters in the absence of co-mitogen

B cells obtained by irrigation of the peritoneal cavity differ from splenic B cells in signaling requirements for the initiation of DNA synthesis. Splenic B cells are stimulated to enter S phase by phorbol esters in conjunction with a second signal provided by calcium ionophore; however, splenic B cells are not stimulated by phorbol ester alone. In contrast, peritoneal B cells from NZB and BALB/c mice were stimulated to incorporate tritiated thymidine by each of the phorbol esters, PMA and phorbol dibutyrate, acting alone. Stimulation of peritoneal B cells was apparent when cells were cultured at lower than usual cell densities, and responses were unaffected by coculture with splenic B cells. Responding cells adhered to plastic petri dishes coated with anti-mouse IgM antibody, but were not completely removed by treatment with anti-Ly-1.2 antibody plus C. These results indicate that phorbol esters constitute a complete signal that stimulates some peritoneal B cells to enter S phase.     

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Effect of Age on Brain Cortical Protein Kinase C and Its Mediation of 5-Hydroxytryptamine Release

The effects of age on the activity and translocation of protein kinase C (PKC) and on the facilitation of 5-hydroxytryptamine (5-HT, serotonin) release induced by PKC activation with the phorbol ester phorbol 12-myristate 13-acetate were investigated. The activities of cortical PKC and its translocation in response to K+ depolarization and phorbol ester stimulation were reduced during aging in Fischer-344 rats. Parietal cortical brain slices from 6-, 12-, and 24-month-old animals were preloaded with [3H]5-HT and release was evoked by 65 mM K+ or the calcium ionophore A23187. 5-HT release induced by either K+ or A23187 was found to be reduced in 12- and 24-month-old as compared to 6-month-old animals. This decrease was not reversed by high extracellular Ca2+. Activation of PKC resulted in a facilitated transmitter release in tissue from 6- and 12-month-old animals but reduced [3H]5-HT release in slices from 24-month-old animals. These responses were prevented by the putative PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), but not by increasing extracellular or intracellular Ca2+. The results demonstrate an age-related change (1) in brain PKC activity and translocation and (2) in a physiological response to PKC stimulation. These results may have implications for other PKC-mediated functions that are altered during senescence.     

Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins

Cross-linking of sIg on primary B lymphocytes leads to increased nuclear DNA-binding activity specific for the tetradecanoyl phorbol acetate-response element (TRE), as judged by gel mobility shift assays. Stimulation of B cells to enter S phase of the cell cycle by treatment with the combination of phorbol ester plus calcium ionophore also stimulated nuclear TRE-binding activity within 2 h, with maximal expression at 4 h; however, phorbol ester and calcium ionophore were not as effective in stimulating binding activity when examined separately. Stimulated nuclear expression of TRE-binding activity appears to require protein synthesis. Fos- and Jun/AP-1-related proteins participate directly in the identified nucleoprotein complex, as shown by the ability of c-fos- and c-jun-specific antisera to either alter or completely abolish electrophoretic migration of the complex in native gels. Further, UV photo-cross-linking studies identified two major TRE-binding protein species, whose sizes correspond to TRE-binding proteins derived from HeLa cell nuclear extracts. The results suggest that in primary B cells nuclear TRE-binding activity represents a "downstream" signaling event that occurs subsequent to changes in protein kinase C activity and intracellular Ca2+ but that can be triggered "physiologically" through sIg.     

Effects of ethanol on protein kinase C activity induced by filamentous actin

Protein kinase C (PKC) can be activated by interaction with filamentous actin (F-actin) in the absence of membrane lipids (S.J. Slater, S.K. Milano, B.A. Stagliano, K.J. Gergich, J.P. Curry, F.J. Taddeo and C.D. Stubbs, Biochemistry 39 (2000) 271-280). Here, the effects of ethanol on the F-actin-induced activities of a panel of PKC isoforms consisting of 'conventional' (cPKC) alpha, betaI, gamma, 'novel' (nPKC) delta, epsilon and 'atypical' (aPKC) zeta were investigated using purified PKC and F-actin. Ethanol was found to inhibit the Ca2+- and phorbol ester-dependent activities of cPKCalpha and betaI, and the Ca2+- and phorbol ester-independent activity of cPKCgamma, whereas the activities of nPKCdelta, epsilon and aPKCzeta were unaffected. Although the activities of cPKCalpha and betaI induced by saturating levels of phorbol ester were inhibited by ethanol, the binding of these isozymes to F-actin was unaffected within the same phorbol ester concentration range. Conversely, within submaximal levels of phorbol ester, cPKCalpha and betaI activities were unaffected by ethanol whereas binding to F-actin was inhibited. The potency of the inhibition of F-actin-induced cPKCbetaI activity increased with n-alkanol chain length up to n-hexanol, after which it declined. The results indicate that PKC activities associated with F-actin, and therefore cellular processes involving the actin cytoskeleton, are potential targets for ethanol action. The effects of ethanol on these processes may differ according to the particular regulating PKC isoform, its intracellular localization and the presence of activators and cofactors.     

Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C

We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.     

Properties of a Protein Kinase C Activity in Synaptic Plasma Membrane and Postsynaptic Density Fractions Isolated from Canine Cerebral Cortex

Protein kinase C (PKC) activity (phosphorylation increased by addition of Ca2+/phosphatidylserine or Ca2+/phosphatidylserine/phorbol ester) was found in both a synaptic plasma membrane (SPM) and a postsynaptic density (PSD) fraction. The SPM fraction had as endogenous substrates 87K-, 60K-, 50K-, and 20K-Mr proteins, whereas the PSD fraction had only the 20K-Mr protein. The PKC activity was also detected using histone III-S as a substrate, in SPM but much less in PSD. Phosphorylations of histone and the endogenous substrates of PKC, assayed in the absence of Ca2+, were enhanced in the SPM prepared after treatment of brain homogenate with phorbol 12-myristate 13-acetate (TPA), but very little enhancement was found in PSD after such treatment. The SPM PKC activity (both for endogenous substrate proteins and for histone), which was enhanced by TPA treatment of brain homogenate, was inhibited by calcium (IC50, 3 x 10(-7) M). The phosphorylations of the 20K-Mr protein in PSD, and in SPM prepared with and without TPA treatment, were all inhibited by H-7. The 20K-Mr protein in the PSD fraction is also phosphorylated by a PSD Ca2+/calmodulin-dependent protein kinase II. The evidence indicates that both SPM and PSD fractions contain a PKC activity. Detergent treatment of SPM, to produce a purified PSD fraction, results in a PSD fraction that has lost most of the endogenous substrates, lost the TPA-induced enhanced activity assayed in the absence of Ca2+, and lost the inhibitory effect of low Ca2+ concentration.     

Interleukin-4 enhances peritoneal B cell stimulation induced by phorbol ester

The activation requirements of murine peritoneal B cells differ from those of conventional (splenic) B cells; in particular, peritoneal B cells are stimulated to enter S phase by phorbol ester, acting alone. This pathway was studied to assess the susceptibility of peritoneal B cells to regulation by T cell products. Three T cell supernatants enhanced phorbol myristate acetate (PMA)-induced peritoneal B cell stimulation. This enhancement was reproduced by recombinant interleukin 4 (IL-4), and IL-4-mediated enhancement was reversed by 11B11 anti-IL-4 antibody. Enhancement of S phase entry was dose dependent for IL-4 and required stimulatory concentrations of PMA. In addition, IL-4 in combination with PMA produced a marked increase in IgM secretion by peritoneal B cells cultured in vitro. Neither an enhancement of S phase entry nor an increase in IgM secretion was observed with splenic B cells similarly treated with IL-4 and PMA. These results suggest that IL-4 modulates the proliferative and differentiative responses of the unusual B cells that reside in the peritoneal cavities of normal mice.     

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.     

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.     

Human immune cells express ppMCH mRNA and functional MCHR1 receptor

The ubiquitously expressed Na(+)/H(+) exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor-mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype-specific dominant negative and constitutively active mutants or downregulation of PKC by isotype-specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKC(alpha)-specific effect. Elevation of cytoplasmic calcium by a Ca(2+) ionophore or thapsigargin causes a growth factor-independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKC(alpha) for the activation of the NHE, while calcium/calmodulin-dependent kinases are essential in thapsigargin induced stimulation of the NHE.     

Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes

Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non-phorbol ester tumor promoter (teleocidin) to replace TPA in induction of cell activation correlated with their ability to bind to and to activate PKC. In addition, the synergistic effect of Ca2+ ionophore and TPA was blocked by either a Ca2+ chelator (EGTA) or cAMP, which is thought to inhibit phosphatidylinositol metabolism. To determine whether the induction of this cytotoxic activity was mediated by a direct effect of Ca2+ ionophore and TPA on cytotoxic T (Tc) cells or was secondary to IL 2 secretion by activated helper T (Th) cells, we tested the effect of Ca2+ ionophore and TPA on isolated populations of cloned, alloantigen-specific Th and Tc cells. Both agents induced cell proliferation and IL 2 production by Th cells, but not by Tc cells. Activation of mixed clones of Th and Tc cells, but not of Tc cells alone, resulted in cytotoxic activity, an effect that could be blocked by anti-IL 2 receptor antibodies. The results thus demonstrate that an increased concentration of intracellular Ca2+ in conjunction with PKC activation can bypass the signal provided by antigen-receptor interaction on Th cells, but does not substitute for IL 2 in activating cytotoxicity by isolated Tc cells.     

Calcium/phospholipid-dependent protein kinases in rat Sertoli cells: regulation of androgen receptor messenger ribonucleic acid

The possibility that Sertoli cell responses to testosterone are modulated by the calcium/phospholipid-dependent protein kinase (protein kinase C; PKC) was examined in rat Sertoli cells in culture. Both soluble and particulate cell fractions showed low constitutive phosphotransferase activity. Incubation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) was associated with a transient induction in both cell fractions of calcium/phosphatidylserine-dependent PKC activity, which was elevated from 15 min to 1 h. Consistent with this, mRNAs for the calcium/phospholipid-dependent isomeric forms of PKC (alpha, beta, and gamma) were detected. The expression levels of mRNAs for PKCalpha and PKCbeta were also up-regulated (2.5- to 3-fold) by TPA (10(-7) M), but these effects were much slower (peaking after 12 h) than those on phosphotransferase activity. In the presence of TPA (10(-7) M), expression of androgen receptor (AR) mRNA showed a transient time-dependent down-regulation ( approximately 70%), in which the nadir was reached after 6 h and baseline expression was again obtained after 12 h. The regulatory effect of PKC activation on AR mRNA was confirmed by the absence of response to a biologically inactive phorbol ester. A concentration-dependent decrease (half-maximal effect at approximately 10(-8) M TPA) of AR mRNA was also observed. These data suggest that Sertoli cell responses to testosterone may be inhibited by a transiently active PKC with a wide intracellular distribution.     

Pharmacomechanical coupling in rat vas deferens: effects of agents that modulate intracellular release of calcium and protein kinase C activation.

The effects of agents that modulate intracellular release of calcium and protein kinase C (PKC) activation on noradrenaline (NA)-induced contractions of epididymal vas deferens in calcium-free/EGTA (1 mM) medium were investigated. NA (100 microM) or methoxamine (100 microM) evoked repeatable contractions. Clonidine (100-300 microM) was ineffective. The contractions to NA were reduced by procaine (1-10 mM) but not by thapsigargin (0.1-30 microM), ryanodine (1-30 microM) or TMB-8 (1-30 microM). Contractions to cumulative additions of NA (1-100 microM) were enhanced in the presence of cyclopiazonic acid (10 & 30 microM) but not ryanodine (10 & 30 microM). Sequential contractions to NA were not blocked by PKC inhibitors, calphostin C (1 microM) or Ro 31-8220 (1-30 microM) but were reduced by H-7 (1-30 microM), a broad spectrum protein kinase inhibitor. Although RT-PCR experiments detected mRNA for some Ca2+-dependent/DAG-activated and Ca2+-independent/DAG-activated PKC isoforms in epididymal vas deferens, the PKC activators, phorbol 12, 13-dibutyrate (100 microM) or phorbol 12-myristate 13-acetate (100 microM) failed to activate the tissues in calcium-free medium but enhanced subsequent contractions to NA. These results indicate a limited role for intracellular calcium stores and phorbol ester/DAG-sensitive PKC isoforms in NA-induced contraction of epididymal rat vas deferens in calcium-free medium. The results suggest that pharmacomechanical coupling triggered by NA may involve the sensitization of contractile myofilaments to Ca2+ or a Ca2+-independent mechanism. The possible involvement of Ca2+-independent/DAG-insensitive PKC isoforms and agonist-dependent but PKC-independent sensitization pathway is discussed.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells.

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca2+ release from internal stores and a subsequent sustained Ca2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca2+ inflow as inferred with Mn2+ quench method was blocked by Ni2+ and a receptor-operated Ca2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKC beta, whereas longer treatment was needed for down-regulation of PKC alpha. Taken together, it was suggested that the BK-induced initial Ca2+ peak and the sustained Ca2+ inflow through the activation of a receptor-operated Ca2+ channel, are differentially regulated by PKC isozymes alpha and beta, respectively, in osteoblast-like MC3T3-E1 cells.     

Activation of human T lymphocytes by bryostatin

The immunologic effects of bryostatin (Bryo), a PKC activator with antineoplastic activity, were assessed and compared to PMA. Bryo induced IL-2R expression on CD4+ and CD8+ human T lymphocytes with a dose response comparable to PMA. However, Bryo induced only a marginal proliferative response as compared with the vigorous response induced by PMA. Bryo mediated functional receptor expression because the proliferative response was enhanced by addition of rIL-2. Furthermore, the proliferative response was inhibited by the relatively specific Ca+, phospholipid-dependent protein kinase (PKC) inhibitor, H-7, indicating a role of PKC in Bryo-induced activation. Addition of the calcium ionophore, ionomycin, to Bryo-stimulated lymphocytes resulted in the production and secretion of IL-2 with a concomitant proliferative response. This effect of the calcium ionophore could be inhibited by cyclosporine with identical results obtained in PMA-stimulated cultures. A most intriguing finding was that Bryo could effectively antagonize PMA-induced T cell proliferation. Although this mechanism of inhibition is unclear, a discussion with respect to differential effects on potential intracellular PKC isoforms is provided. These studies indicated that Bryo has potent immunopotentiating properties that share some similar effects of the phorbol ester, PMA, but offers the additional property of modulating other phorbol ester effects on proliferation.