Effect of polymyxin-B on T-lymphocyte protein synthesis

The role of protein kinase-C (PK-C) protein phosphorylation on the mitogen triggered responses of T-lymphocytes was examined by observing the effect of polymyxin-B (an inhibitor of PK-C) on mitogen induced protein and DNA synthesis. Polymyxin-B inhibited 3H-thymidine incorporation by PHA activated T-lymphocytes over a range of PHA concentrations. 3H-leucine incorporation by PHA activated T-lymphocytes was inhibited by polymyxin-B in a dose dependent manner. A partially purified PK-C fraction from polymyxin-B treated PHA activated T-lymphocytes demonstrated less than 25% of the phosphorylating activity of untreated lymphocytes. These results suggest that protein synthesis during the T-lymphocyte activation process is dependent on PK-C activity.     

Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.     

Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport

Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre-replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis.     

Role of Ca2+ influx in bombesin-induced mitogenesis in Swiss 3T3 fibroblasts

In Swiss 3T3 fibroblasts a peptide mitogen bombesin, which acts through the phospholipase C-protein kinase C signaling pathway, stimulates DNA synthesis in a manner strictly dependent on the medium calcium concentration: [3H]thymidine incorporation into DNA in the presence of a saturating concentration of bombesin (10(-8) M) is 4-fold greater at 3.0 mM extracellular calcium as compared with a value obtained at 0.03 mM calcium. In the present study we attempted to identify the site and the mechanism of action of Ca2+ influx along the bombesin-induced mitogenic signaling pathway, by comparing bombesin effects at 0.03 and 3.0 mM of medium calcium. Bombesin induces the same extent of increases in [3H]inositol phosphates after 1 min, and comparable sustained increases in the cellular content of 1,2-diacylglycerol for up to 4 h, at either 0.03 or 3.0 mM calcium. Bombesin induces the same extent of phosphorylation of MARCKS protein, the major cellular substrate for protein kinase C, irrespective of the medium calcium concentration for at least 4 h. Moreover, diverse cellular responses elicited by bombesin, including c-fos expression, activation of microtubule-associated protein 2 kinase and S6 kinase, glucose uptake, and protein synthesis but not the release of arachidonic acid and its metabolites, are induced similarly at either 0.03 or 3.0 mM calcium. Down-regulation of cellular protein kinase C nearly completely abolishes bombesin effects on c-fos expression, S6 kinase activation, glucose uptake, and DNA synthesis. These results suggest that the target of Ca2+ influx in bombesin-induced mitogenic signaling pathway is not located along the phospholipase C-protein kinase C signal transduction system including cellular events in early G1 phase that exist downstream to protein kinase C action.     

Studies on the mechanism by which prolactin regulates protein, RNA, and DNA synthesis in Nb2 node lymphoma cells

Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C.     

Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis

The tumor-promoting agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibenzoate inhibited the increased accumulation of [32P]phosphatidylinositol (PI) induced in mouse spleen lymphocytes by mitogenic lectins in the presence of [32P]orthophosphate. Similar inhibition of [32P]PI levels by TPA was seen in human tonsil T-lymphocytes stimulated with phytohemagglutinin. Only co-mitogenic phorbol esters prevented the [32P]PI accumulation during early mitogenesis. No increased 32P-labelling due to mitogen or decreases due to TPA was observed when cells were equilibrated with [32P]orthophosphate for 24 h prior to stimulation with mitogen, from which it is concluded that the total concentrations of phosphatidylcholine (PC) and PI are unaffected by mitogen or co-mitogen. The [32P]PI elevation but not the [32P]PC elevation was proportional to T-cell mitogenic potency for the lectins concanavalin A, divalent succinyl concanavalin A and phytohemagglutinin, and was prevented in each case by 5 X 10(-8) M TPA. Escherichia coli lipopolysaccharide did not give increased 32P incorporation into PI or PC, and TPA had no effect on 32P labelled phospholipid levels in the presence of this B-cell mitogen. The results indicate that the phosphatidylinositol response is not an invariable correlate of T-cell mitogenesis by polyclonal mitogens.     

Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C

The proliferation of human skin fibroblasts in culture was examined using a [3H]thymidine incorporation assay. Histamine inhibited thymidine incorporation with an IC50 of about 0.2 microM. This effect was blocked by the H1 receptor antagonist mepyramine but not by the H2 receptor antagonist cimetidine. Protein kinase C activators, including several phorbol esters and mezerine, also inhibited thymidine incorporation. The IC50 for beta-phorbol 12,13-didecanoate was less than 0.1 nM. The alpha-isomer of this compound was inactive. Long-term treatment of cells with the beta-isomer eliminated the ability of both histamine and phorbol ester to inhibit thymidine incorporation, presumably due to downregulation of protein kinase C. Our results suggest that histamine H1 receptors are linked to activation of protein kinase C and that activation of this enzyme leads to an inhibition of cell proliferation.     

Extracellular ATP and ADP stimulate proliferation of porcine aortic smooth muscle cells.

The mitogenic effect of extracellular ATP on porcine aortic smooth muscle cells (SMC) was examined. Stimulation of [3H]thymidine incorporation by ATP was dose-dependent; the maximal effect was obtained at 100 microM. ATP acted synergistically with insulin, IGF-1, EGF, PDGF, and various other mitogens. Incorporation of [3H]thymidine was correlated with the fraction of [3H]thymidine-labeled nuclei and changes in cell counts. The stimulation of proliferation was also determined by measurement of cellular DNA using bisbenzamide and by following the increase of mitochondrial dehydrogenase protein. The effect of ATP was not due to hydrolysis to adenosine, which shows synergism with ATP. ATP acted as a competence factor. The mitogenic effect of ATP, but not adenosine, was further increased by lysophosphatidate, phosphatidic acid, or norepinephrine. The inhibitor of adenosine deaminase, EHNA, stimulated the effect of adenosine but not ATP. The adenosine receptor antagonist theophylline depressed adenosine-induced mitogenesis. ADP and the non-hydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate (AMP-PNP) were equally mitogenic. Thus extracellular ATP stimulated mitogenesis of SMC via P2Y purinoceptors. The mechanism of ATP acting as a mitogen in SMC was further explored. Extracellular ATP stimulated the release of [3H]arachidonic acid (AA) and prostaglandin E2 (PGE2) into the medium, and enhanced cAMP accumulation in a dose-dependent fashion similar to ATP-induced [3H]thymidine incorporation. Inhibitors of the arachidonic acid metabolism pathway, quinacrine and indomethacin, partially inhibited the mitogenic effect of ATP but not of adenosine. Pertussis toxin inhibited ATP-stimulated DNA synthesis, AA release, PGE2 formation, and cAMP accumulation. Down-regulation of protein kinase C (PKC) by long-term exposure to phorbol dibutyrate (PDBu) partially prevented stimulation of DNA synthesis and activation of the AA pathway by ATP. The PKC inhibitor, staurosporine, antagonized mitogenesis stimulated by ATP. No synergistic effect was found when PDBu and ATP were added together. Therefore, a dual mechanism, including both arachidonic acid metabolism and PKC, is involved in ATP-mediated mitogenesis in SMC. In addition, ATP acted synergistically with angiotensin II, phospholipase C, serotonin, or carbachol to stimulate DNA synthesis. Finally, the possible physiological significance of ATP as a mitogen in SMC was further studied. The effect of endothelin and heparin, which are released from endothelial cells, on ATP-dependent mitogenesis was investigated. Extracellular ATP acted synergistically with endothelin to stimulate a greater extent of [3H]thymidine incorporation than was seen with PDGF plus endothelin. Heparin, believed to have a regulatory role, partially inhibited the stimulation of DNA synthesis caused both by ATP and PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of activation of protein kinase C on the agonist-induced stimulation and inhibition of cyclic AMP formation in intact human platelets.

Jakobs, Bauer & Watanabe [(1985) Eur. J. Biochem. 151, 425-430] reported that treatment of platelets with phorbol 12-myristate 13-acetate (PMA) prevented GTP- and agonist-induced inhibition of adenylate cyclase in membranes from the platelets. This was attributed to the phosphorylation of the inhibitory guanine nucleotide-binding protein (Gi) by protein kinase C. In the present study, the effects of PMA on cyclic [3H]AMP formation and protein phosphorylation were studied in intact human platelets labelled with [3H]adenine and [32P]Pi. Incubation mixtures contained indomethacin to block prostaglandin synthesis, phosphocreatine and creatine kinase to remove ADP released from the platelets, and 3-isobutyl-1-methylxanthine to inhibit cyclic AMP phosphodiesterases. Under these conditions, PMA partially inhibited the initial formation of cyclic [3H]AMP induced by prostaglandin E1 (PGE1), but later enhanced cyclic [3H]AMP accumulation by blocking the slow decrease in activation of adenylate cyclase that follows addition of PGE1. PMA had more marked and exclusively inhibitory effects on cyclic [3H]AMP formation induced by prostaglandin D2 and also inhibited the action of forskolin. Adrenaline, high thrombin concentrations and, in the absence of phosphocreatine and creatine kinase, ADP inhibited cyclic [3H]AMP formation induced by PGE1. The actions of adrenaline and thrombin were attenuated by PMA, but that of ADP was little affected, suggesting differences in the mechanisms by which these agonists inhibit adenylate cyclase. sn-1,2-Dioctanoylglycerol (diC8) had effects similar to those of PMA. The actions of increasing concentrations of PMA or diC8 on the modulation of cyclic [3H]AMP formation by PGE1 or adrenaline correlated with intracellular protein kinase C activity, as determined by 32P incorporation into the 47 kDa substrate of the enzyme. Parallel increases in phosphorylation of 20 kDa and 39-41 kDa proteins were also observed. Platelet-activating factor, [Arg8]vasopressin and low thrombin concentrations, all of which inhibit adenylate cyclase in isolated platelet membranes, did not affect cyclic [3H]AMP formation in intact platelets. However, the activation of protein kinase C by these agonists was insufficient to account for their failure to inhibit cyclic [3H]AMP formation. Moreover, high thrombin concentrations simultaneously activated protein kinase C and inhibited cyclic [3H]AMP formation. The results show that, in the intact platelet, the predominant effects of activation of protein kinase C on adenylate cyclase activity are inhibitory, suggesting actions additional to inactivation of Gi.     

Involvement of phosphatidylcholine-specific phospholipase C in thromboxane A2-induced activation of mitogen-activated protein kinase in astrocytoma cells

Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in 1321N1 human astrocytoma cells. 9,11-Epithio-11,12-methano-TXA2 (STA2), a TXA2 receptor agonist, induced Ca2+ mobilization and phosphoinositide hydrolysis in a concentration-dependent manner. These responses were inhibited by treatment with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C, or by culturing in 0.5% fetal calf serum containing 0.5 mM dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) for 2 days. However, the dbcAMP treatment augmented the TXA2 receptor-mediated phosphorylation of mitogen-activated protein kinase (MAPK). These results were confirmed by a functional MAPK assay measuring the incorporation of 32P into the MAPK substrate peptide. The TXA2 receptor-mediated MAPK activation was inhibited by SQ29548, a TXA2 receptor antagonist, and GF109203X, an inhibitor of protein kinase C. Although U73122 did not inhibit or only slightly inhibited the activation of MAPK, D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, potently attenuated the activation in a concentration-dependent manner. Furthermore, STA2 accelerated the release of [3H]choline metabolites from the cells prelabeled with [3H]choline chloride. This release was inhibited by treatment with D-609. These results suggest that phosphatidylcholine-specific phospholipase C and protein kinase C, but not phosphatidylinositol-specific phospholipase C, are involved in TXA2 receptor-mediated MAPK activation in 1321N1 human astrocytoma cells.     

Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [3H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [3H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.     

Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes

Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes.     

Synergistic effect of phorbol myristate acetate on bacterial lipopolysaccharide induced rat splenocyte proliferation

1. Bacterial lipopolysaccharide (LPS) stimulated [3H]TdR incorporation of rat splenocytes in a concentration dependent manner. 2. Phorbol 12-myristate 13-acetate (PMA) alone has little effect on rat splenocyte proliferation but it exerted a marked synergistic effect on LPS-induced [3H]TdR incorporation when added at the first few hours of incubation with LPS. Minimal synergistic effect of PMA was observed if it was added later than 4 hr after LPS application. 3. Both LPS-stimulated and PMA synergized incorporation of [3H]TdR in rat splenocytes were inhibited by H-7, a protein kinase C inhibitor. 4. The results support the notion that the activation of protein kinase C is a necessary but insufficient cellular signal in the initiation of proliferative response of rat splenocytes by LPS.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9- or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/- 7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/- 10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/- 31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14,15-EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c-P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.     

Thyrotropin-releasing hormone and phorbol esters stimulate sphingomyelin synthesis in GH3 pituitary cells. Evidence for involvement of protein kinase C

Previous studies demonstrated that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 14525-14530). Since phosphatidylcholine may serve as the precursor for sphingomyelin synthesis, studies were performed to assess the effect of protein kinase C on sphingomyelin synthesis. The potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated time- and concentration-dependent incorporation of 32Pi into the head group of sphingomyelin in cells short term labeled with 32Pi and resuspended in medium without radiolabel. TPA (10(-7) M) increased incorporation at a rate 1.4-fold of control after 2 h; EC50 congruent to 2 x 10(-9) M TPA. This correlated closely to TPA-induced phosphatidylcholine synthesis; EC50 congruent to 9 x 10(-10) M TPA. TRH (10(-7) M), which activates protein kinase C via a receptor-mediated mechanism, similarly stimulated 32Pi incorporation into sphingomyelin at a rate 1.5-fold of control; EC50 congruent to 5 x 10(-10) M TRH. This correlated closely with TRH-induced phosphatidylcholine and phosphatidylinositol synthesis; EC50 congruent to 2 x 10(-10) and 1.5 x 10(-10) M TRH, respectively. In cells short term labeled with [3H]palmitate, TRH induced a time- and concentration-dependent reduction in the level of [3H]ceramide and a quantitative increase in the level of [3H]sphingomyelin. Compositional analysis of the incorporated [3H]palmitate revealed that TRH increased radiolabel into both the sphingoid base and the fatty acid moieties of sphingomyelin. Similarly, TRH increased incorporation of [3H] serine into sphingomyelin to 145 +/- 8% of control after 3 h. TPA also stimulated these events. Like the effect of TRH on phosphatidylcholine synthesis, TRH-induced sphingomyelin synthesis was abolished in cells "down-modulated" for protein kinase C. In contrast, TRH-induced phosphatidylinositol synthesis still occurred in these cells. These studies suggest that protein kinase C stimulates coordinate synthesis of phosphatidylcholine and sphingomyelin. This is the first report of stimulation of sphingomyelin synthesis via a cell surface receptor.     

Transduction of signals in the activation of T lymphocytes: relation to leukemia

The biochemical events initiated by mitogen in T lymphocytes are the subject of this paper. Following interaction of the mitogen with its receptors, a transmembrane 'trigger-type' signal is propagated which has both positive and negative correlates. The negative signal occurs with high mitogen concentrations and is associated with membrane freezing, microtubular aggregation, receptor capping, adenylate cyclase activation, and cellular cyclic AMP increases. The positive signal occurs with optimal mitogen concentrations and is associated with changes in membrane permeability and transport with influx of calcium and potassium ion and efflux of sodium, in transport processes for glucose, amino acids, and nucleosides, and in a collected series of early membrane lipid changes which can be considered essential for the positive signal. These lipid changes include the uptake of arachidonic acid and other fatty acids, choline, phosphate and other molecules, their incorporation into membrane phospholipids, particularly phosphatidylinositol (PI), and a turnover of PI with the production of inositol triphosphate, which can be related to calcium mobilization and diacylglycerol which activates a cytoplasmic protein kinase C. A key event associated with mitogen action is arachidonic acid release. Arachidonic acid may give rise to prostaglandins and thromboxanes as part of negative components of the signal through effects on the adenylate cyclase/cyclic AMP system. Arachidonic acid gives rise to eicosanoids like 5-, 11-, possibly 12- and 15-hydroxyperoxy and hydroxy eicosatetraenoic acids and leukotrienes B4 and C4. The activation of the 5-lipoxygenase, a critical calcium-dependent step, leads via the production of 5-HPETE and 5-HETE to the activation of membrane and soluble guanylate cyclase and the production of cyclic GMP. Cyclic GMP appears to be essential for mitogen activation and is associated with cyclic GMP-dependent protein kinase activation and the phosphorylation of a number of substrates. Calcium ion influx is clearly central to mitogen action. Calcium through its influx and mobilization from cellular stores is thought to contribute directly and indirectly through the action of calmodulin and protein kinase C to the activation of a number of enzymatic processes involved in the positive signal including phospholipase C, diglyceride kinase and lipase, 5-lipoxygenase, and guanylate cyclase. Cyclic GMP and calcium ion both participate in nuclear processes leading to RNA and protein synthesis. Interleukin 2 is associated with midcycle increases in cyclic GMP and entry into DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crosstalk of sterol-dependent and non-sterol-dependent signaling in human monocytes after in vitro addition of LDL

The aim of the present study was to investigate low density lipoprotein (LDL)-induced, non-sterol-dependent signaling and its possible role in cholesterol balance. LDL in 10 microg ml(-1) concentration could induce inositol trisphosphate (IP3) and Ca2+ signal generation through a pertussis toxin (PT) sensitive G protein in human monocytes. The increase in [Ca2+]i was derived from the intracellular pools. LDL also induced activation and translocation of protein kinase C (PKC) into the cell membrane, by processes, which were significantly inhibited in the first 20 min by preincubation with PT and PKC-inhibitor H-7. The PKC-activating phorbol-12-myristate-13-acetate (PMA), differently from LDL, enhanced the LDL-receptor (LDL-R)-mediated binding and degradation of [125I]LDL, but inhibited endogenous cholesterol synthesis, and both effects were inhibited by H-7. The LDL-induced inhibition of binding and degradation of [125I]LDL was not affected by H-7, whereas decreased cholesterol synthesis was counteracted by H-7. These results suggest the existence of a non-sterol-dependent signal pathway of LDL-Rs, by which endogenous cholesterol synthesis, that is, the [14C]acetate incorporation, is regulated through PKC activation.