The ability of calcium to change cyclic AMP from a stimulator to an inhibitor to thymic lymphoblast proliferation

Calcium is a major regulator of thymic lymphoblast proliferation in vivo and in vitro. The proliferative activity of the lymphoblasts in thymic lymphocyte (thymocyte) populations in vitro is both constant and low in the presence of calcium concentrations between 0 and 1.0 mM, but higher concentrations increase proliferation by an endogenous cyclic AMP-mediated promotion of the initiation of DNA synthesis. Lower concentrations (10^?7 to 10^?5 M) of exogenous cyclic AMP (but not 5′-AMP) stimulate lymphoblast proliferation in a low-calcium (0.5 mM) medium, but higher concentrations do not. However, all exogenous cyclic AMP concentrations between 10^?7 and 10^?3 M (but again not 5′-AMP) block the stimulation of lymphoblast proliferation in a high-calcium (1.5 mM) medium. Exogenous cyclic AMP does not prevent calcium from “activating” lymphoblasts, but it reversibly blocks the reaction responsible for the initiation of DNA synthesis in these stimulated cells. Finally, cyclic AMP's inhibitory action, in contrast to its stimulatory action in low-calcium medium, is not specific for the cyclic nucleotide since a low, non-mitogenic concentration of cyclic GMP also prevents calcium from stimulating DNA synthesis and cell proliferation.     

Second messenger pathways mediating chicken luteinizing hormone secretion from dispersed pituitary cells.

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissimilar effects of 1-oleoyl-2-acetylglycerol and phorbol 12-myristate 13-acetate on fatty acid synthesis in isolated rat-liver cells

Exogenous 1-oleoyl-2-acetylglycerol (OAG) is known to mimic the action of tumour-promoting phorbol esters in various cell types. However, in isolated rat hepatocytes OAG depressed the rate of de novo fatty acid synthesis and the activity of the key enzyme acetyl-CoA carboxylase (EC 6.4.1.2), in contrast to the pronounced stimulation of both parameters by phorbol 12-myristate 13-acetate (PMA). The inhibition by OAG appeared to be dose- and time-dependent. On the other hand, medium-chain 1,2-diacylglycerols like 1,2-dioctanoyl-sn-glycerol did mimic the stimulatory action of PMA. The anomalous effect of OAG may well be explained by its metabolic breakdown leading to liberation of oleate and subsequent inhibition of acetyl-CoA carboxylase activity by endogenously formed oleoyl-CoA. The stimulatory effects of both PMA and medium-chain diacylglycerols are likely to be mediated by protein kinase C.     

Mitochondrial reactive oxygen species and calcium uptake regulate activation of phagocytic NADPH oxidase

Production of superoxide (O(2)(·-)) by NADPH oxidases contributes to the development of hypertension and atherosclerosis. Factors responsible for activation of NADPH oxidases are not well understood; interestingly, cardiovascular disease is associated with both altered NADPH oxidase activity and age-associated mitochondrial dysfunction. We hypothesized that mitochondrial dysfunction may contribute to activation of NADPH oxidase. The effect of mitochondrial inhibitors on phagocytic NADPH oxidase in human lymphoblasts and whole blood was measured at the basal state and upon PKC-dependent stimulation with PMA using extracellular 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium or mitochondria-targeted 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethylpiperidine spin probes and electron spin resonance (ESR). Intracellular cytosolic calcium [Ca(2+)](i) was measured spectrofluorometrically using fura-2 AM. Incubation of lymphoblasts with the mitochondrial inhibitors rotenone, antimycin A, CCCP, or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter) did not significantly change basal activity of NADPH oxidase. In contrast, preincubation with the mitochondrial inhibitors prior to PMA stimulation of lymphoblasts resulted in two- to three-fold increase of NADPH oxidase activity compared with stimulation with PMA alone. Most notably, the intracellular Ca(2+)-chelating agent BAPTA-AM abolished the effect of mitochondrial inhibitors on NADPH oxidase activity. Cytosolic Ca(2+) measurements with fura-2 AM showed that the mitochondrial inhibitors increased [Ca(2+)](i), while BAPTA-AM abolished the increase in [Ca(2+)](i). Furthermore, depletion of cellular Ca(2+) with thapsigargin attenuated CCCP- and antimycin A-mediated activation of NADPH oxidase in the presence of PMA by 42% and 31%, correspondingly. Our data suggest that mitochondria regulate PKC-dependent activation of phagocytic NADPH oxidase. In summary, increased mitochondrial O(2)(·-) and impaired buffering of cytosolic Ca(2+) by dysfunctional mitochondria result in enhanced NADPH oxidase activity, which may contribute to the development of cardiovascular diseases.     

The possible role of protein kinase C and phospholipids in the regulation of steroid production in rat Leydig cells

We have studied the possible involvement of the activation of calcium-dependent phospholipid-activated protein kinase (PK-C) in the stimulatory action of LHRH on Leydig cells, using 4 beta-phorbol-12-myristate-13-acetate (PMA) and phospholipase C (PL-C). LHRH agonist (LHRH-A) and PL-C had a large synergistic effect on LH-stimulated steroid production, whereas PMA inhibited the effect of LH. However, PMA always caused an increase in steroid production stimulated by various doses of dibutyryl cAMP. LH and PMA stimulated the phosphorylation of 17 and 33 kDa proteins, whereas LHRH-A and PL-C had no effect. Of all effectors used, LH had the most pronounced effect on the synthesis of 14, 27 and 30 kDa proteins. The present results suggest that the mechanisms of action of LHRH-A and PL-C on steroid production in Leydig cells may be similar and different from PMA, and may involve stimulation of a specific type of PK-C or hydrolysis of a specific pool of phospholipids.     

Phorbol esters inhibit the fMet-Leu-Phe- and leukotriene B4-stimulated calcium mobilization and enzyme secretion in rabbit neutrophils

The tumor co-promoter phorbol 12, myristate 13, acetate (PMA) has previously been shown to stimulate several of the characteristic functions (aggregation, degranulation, and the oxidative burst) of polymorphonuclear leukocytes (neutrophils). We describe here a novel feature of the action of PMA on neutrophils, namely its ability to inhibit the chemotactic factor-induced increased in the enzyme secretion and in the intracellular concentration of free calcium. The inhibition is maximal within 3 min of the addition of PMA and is concentration-dependent (IC50 = 8.5 ng/ml). The site of action of PMA is distal to the binding of the chemotactic factors. PMA inhibits both the release of intracellular calcium and the permeability changes to calcium induced by chemotactic factors, but does not affect the stimulation of the rate of influx of sodium produced by the same agents. The PMA analog 4 alpha-phorbol 12, 13-didecanoate, which lack tumorigenicity and the ability to activate the calcium- and phospholipid-dependent protein kinase (protein kinase C), does not inhibit any of the above fMet-Leu-Phe-stimulated neutrophil functions. The present results thus demonstrate that phorbol esters, either directly or indirectly, possibly through the activation of protein kinase C, inhibit the signal(s) responsible for the stimulated mobilization of calcium in rabbit neutrophils.     

Stimulation of adipocyte phospholipid methyltransferase activity by phorbol 12-myristate 13-acetate. Differential regulation of phospholipid methyltransferase and lipolysis.

The present studies demonstrate that treatment of rat adipocytes with the phorbol ester phorbol 12-myristate 13-acetate (PMA) causes a dose-dependent stimulation of phospholipid methyltransferase (PLMT) activity. The stimulatory effect of PMA was not additive with that of isoprenaline or forskolin. The sensitivity of stimulated PLMT activity to inhibition by insulin, however, was decreased in the presence of PMA. The inhibitory effect of a maximal concentration of insulin on PLMT was unchanged in the presence of PMA. In contrast with the effects on PLMT, the lipolytic response of adipocytes to isoprenaline and the anti-lipolytic response to insulin were unaffected by PMA. These data suggest that PLMT is, whereas hormone-sensitive lipase is not, an intracellular target for the action of PMA. The lack of effect of PMA on lipolysis suggests that PLMT and hormone-sensitive lipase can be regulated by separate mechanisms. Furthermore, phorbol esters do not interfere in the regulatory pathway whereby insulin inhibits PMLT or lipolysis.     

Inhibitory effect of the intrauterine infusion of phorbol 12-myristate 13-acetate and 1-oleoyl-2-acetylglycerol on the decidual cell reaction in rats

In rats, prostaglandins (PGs) have an essential role in the decidual cell reaction (DCR), but their mechanism of action at the cellular level within the endometrium is at present uncertain. To test the hypothesis that both protein kinase C activation and calcium mobilization mediate the action of PGs within the endometrium during decidualization, the phorbol ester phorbol 12-myristate 13-acetate (PMA) or the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG), activators of protein kinase C in vitro, and the calcium ionophore A23187, which causes calcium mobilization, were infused, alone or combined, into the uterine lumen of rats sensitized for the DCR. The results obtained indicate that both PMA and OAG have an inhibitory effect on the DCR in rats. The calcium ionophore A23187, although having no apparent effect by itself, had a synergistic effect with PMA, but not with OAG, in inhibiting the DCR. The intrauterine infusion of PMA and/or A23187 had no effect on the increase in endometrial vascular permeability (EVP), which precedes the DCR. The inhibitory effect of PMA or PMA plus A23187 on decidualization is probably not mediated by a decrease in uterine PG synthesis, as assessed by the measurement of uterine prostaglandin E concentrations at various times during the intraluminal infusion. These data suggest that activation of protein kinase C can modulate the DCR.     

Attenuation of hen granulosa cell steroidogenesis by a phorbol ester and 1-oleoyl-2-acetylglycerol

A series of studies was conducted to evaluate the effects of phorbol esters and a diacylglycerol analog on basal and hormone-stimulated steroidogenesis in granulosa cells from the largest preovulatory follicle of the domestic hen. Agents that previously have been shown to activate protein kinase C, such as the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and the synthetic diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), suppressed luteinizing hormone (LH)-induced progesterone (PMA at levels of 10 and 100 ng/tube; OAG at levels of 10 and 25 micrograms/tube), and androgen (10 and 100 ng PMA; 25 micrograms OAG) production, but had no effect on basal levels of either steroid. Furthermore, PMA decreased the ability of vasoactive intestinal peptide to induce steroidogenesis, suggesting that protein kinase C activation may generally modulate the activity of hormones that act via the adenylyl cyclase/cyclic 3',5'-adenosine monophosphate (cAMP) second messenger system. In further support of this proposal was the finding that PMA and OAG decreased the production of cAMP in response to LH, and attenuated the steroidogenic response in granulosa cells exposed to 10 mM 8-bromo-cAMP. By contrast, the induction of calcium mobilization using a calcium ionophore (A23187; 0.5-2.0 microM) stimulated progesterone and androgen production without increasing intracellular levels of cAMP, and this stimulatory effect on steroidogenesis was not inhibited by the presence of 100 ng PMA/tube. From these data, we suggest that the activation of protein kinase C in granulosa cells of the hen may provide a physiological mechanism by which receptor-mediated steroidogenesis, involving the adenylyl cyclase second messenger system, is modulated.     

Opposing effects of PKCalpha and PKCepsilon on basolateral membrane dynamics in intestinal epithelia

PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKCepsilon followed by late activation of the conventional isoform PKCalpha in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKCepsilon was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKCalpha was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKCalpha on basolateral endocytosis. Inhibition of PKC by the selective conventional PKC inhibitor G?-6976 (5 microM) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKCepsilon-selective agonist carbachol (100 microM) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by G?-6850 but not by G?-6976, implicating PKCepsilon as the key isoform responsible for actin disruption. The Ca2+ agonist thapsigargin (5 microM) induced early activation of PKC when added simultaneously with PMA. This early activation of PKCalpha blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKCalpha stabilizes F-actin and thereby opposes the effect of PKCepsilon on membrane remodeling in T84 cells.     

Effects of mitogenic agents upon glucocorticoid action in human tonsillar T-lymphocytes

The treatment of human tonsillar T-lymphocytes with 4-phorbol 12-myristate 13-acetate (PMA), resulted in about two fold increase in glucocorticoid receptor (GR) number, without any significant change in the receptor affinity. This increase disappeared in the presence of cycloheximide.Alone, PMA and calcium inophore A23187 did not affect, but together stimulated, like phytohaemagglutinin (PHA), leucine and, in particular, thymidine incorporation. PMA enhanced slightly the stimulatory effect of PHA. Alone, these agents failed to alter the suppressive effect of dexamethasone on thymidine and leucine incorporation; however, PMA-A23187 and PMA-PHA combinations appeared to antagonize the supression by dexamethasone.     

Signal transduction of somatostatin in human B lymphoblasts

Somatostatin (SST) and somatostatin receptors (SSTR) are widely distributed in lymphoid tissues. Here, we report on the stimulatory effects of SST in Epstein-Barr virus-immortalized B lymphoblasts. By RT-PCR, we demonstrated the exclusive expression of the somatostatin receptor isoform 2A (SSTR2A) in B lymphoblasts. Addition of SST rapidly increased the cytosolic free calcium concentration [Ca(2+)](i) maximally by about 200 nM, with an EC(50) of 1.3 nM, and stimulated the formation of inositol phosphates. Furthermore, SST increased binding of guanosine 5'-O-(3-thiotriphosphate) by 50% above basal. These effects were partly inhibited by pertussis toxin (PTX), which indicates the involvement of PTX-sensitive G proteins. We provide further evidence that Galpha(16,) a PTX-insensitive G protein confined to lymphohematopoietic cells, is involved in the otherwise unusual coupling of SSTR2A to phospholipase C activation. In addition, SST activated extracellular regulated kinases and induced a 3.5-fold stimulation of DNA synthesis and a 4.4-fold stimulation of B lymphoblast proliferation, which was accompanied by an enhanced immunoglobulin formation. Thus SST exerts a growth factor-like activity on human B lymphoblasts.     

Cooperative effects of ethanol and protein kinase C activators on phospholipase-D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts.

In a previous study, ethanol was shown to enhance the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), a prominent activator of protein kinase C (PKC), on phospholipase-D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts (Kiss et al. (1991) Eur. J. Biochem. 197, 785-790). Here, the mechanism and possible significance of ethanol-stimulated PtdEtn hydrolysis was further studied. In [14C]ethanolamine-labeled NIH 3T3 fibroblasts, 10 mM ethanol enhanced PMA-induced hydrolysis of PtdEtn 1.5-2.0-fold during a 2.5-15-min incubation period. Other alcohols, including glycerol, methanol, and 1-propanol, also enhanced PMA-induced PtdEtn hydrolysis. Of the other PLD activators tested, ethanol potentiated the PKC-dependent stimulatory effect of bombesin but failed to alter the apparently PKC-independent stimulatory effect of serum. Pretreatment of [14C]ethanolamine-labeled fibroblasts with 200 mM ethanol for 20 min resulted in increased (approx. 2-fold) hydrolysis of [14C]PtdEtn in isolated membranes. In membranes from ethanol-treated, but not from untreated, cells, PMA further enhanced (approx. 1.5-fold) the production of [14C]ethanolamine. Ethanol exerted none of the above stimulatory effects on phosphatidylcholine hydrolysis. These results suggest that the specific stimulatory action of ethanol on PLD-mediated PtdEtn hydrolysis can occur in vivo and may involve increased binding of a regulatory PKC-isoform to membranes.     

Growth factor production and requirements during the proliferative response of human T lymphocytes to anti-CD3 monoclonal antibody

Anti-CD3 was administered with three different accessory stimuli to purified populations of human T cells. Sepharose conjugated anti-CD3, monocytes, and PMA each could induce the p55 component of the IL-2R as well as responsiveness to exogenous IL-2. Sepharose anti-CD3 did not induce IL-2, although the levels of IL-2 protein and mRNA were 10 to 30 times higher with PMA than with monocytes. Despite these differences in IL-2 production, the amount of DNA synthesis and the number of lymphoblasts were comparable when monocytes or PMA were used as the accessory stimulus, and the responses were equally sensitive to inhibition by an anti-IL-2R antibody. To pursue the functional relevance of the "supraoptimal" levels of IL-2 that are induced by PMA, anti-CD3-induced lymphoblasts were isolated free of monocytes and challenged with lymphokines. It could be shown that 1) the small amounts of IL-2 in the monocyte-T cell conditioned medium would drive DNA synthesis, but that 2) higher levels of IL-2 (20 to 100 U/ml) were needed to induce IFN-gamma, as well as the mRNA for IL-4 and the p55 IL-2R. We suggest that the capacity to produce high levels of IL-2, as seen with PMA, is required under physiologic conditions for two reasons: to up-regulate the IL-2R when small amounts of Ag rather than large amounts of anti-CD3 are ligands for the T cell, or to induce the release of lymphokines like IL-4 and IFN-gamma from sensitized lymphoblasts.     

Modulation of stimulatory action of follicle stimulating hormone (FSH) and inhibitory action of epidermal growth factor (EGF) on aromatase activity in Sertoli cells by calcium

Aromatization of testosterone by cultured Sertoli cells isolated from immature rats was stimulated more than 7-fold by follicle stimulating hormone (FSH) or dcAMP. The effects of FSH and dcAMP could be partly inhibited by epidermal growth factor (EGF) in a dose-dependent manner (ID500.5 nM). The phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) could also inhibit aromatase activity in a fashion similar to EGF. When 3 mM EGTA was present in the culture medium, the inhibitory effect of EGF was abolished but the stimulatory effect of FSH or dcAMP was magnified. These results suggest that EGF exerts a negative control on aromatase via calcium and protein kinase C. The abolishment of the inhibitory effect of EGF and the enhancement of the stimulatory effect of FSH or dcAMP by a calcium deficiency may be an indication that growth factors produced by Sertoli cells negatively controls FSH-induced responses in an autocrine fashion.     

Steroidogenesis in Fundulus heteroclitus. IV. Dichotomous effects of a phorbol ester on ovarian steroid production and oocyte maturation.

The possible role of protein kinase C (PKC) activation in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) on ovarian steroidogenesis and oocyte maturation was investigated. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), alone slightly increased basal 17 alpha-hydroxy,20 beta-dihydroprogesterone (DHP) and 17 beta-estradiol (E2) synthesis and significantly stimulated germinal vesicle breakdown (GVBD). Addition of FPE promoted synthesis of DHP, testosterone (T), and E2, and initiated GVBD. Phorbol ester inhibited FPE-induced steroidogenesis but increased the number of oocytes that underwent GVBD. Phorbol ester also markedly impeded induction of steroidogenesis by dibutyryl cAMP and differentially affected the conversion of 25-hydroxycholesterol, pregnenolone, or progesterone to DHP, T, and E2: DHP production was not affected; T production diminished; and E2 synthesis increased (T aromatization also increased). These results suggest an inhibitory role for the PKC pathway on FPE-induced ovarian steroid production, with PMA appearing to affect various steroidogenic steps. The stimulatory action of PMA on oocyte maturation seems to be independent of follicular steroid production since aminoglutethimide, an inhibitor of steroidogenesis, did not block PMA-induced GVBD. Moreover, PMA had a marked stimulatory effect on GVBD in denuded oocytes. Thus, in contrast to the inhibitory role found for the PKC pathway on ovarian follicular steroidogenesis, activation of PKC in the oocyte may serve as a signal-transducing mechanism leading to GVBD.     

Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation.

The possibility that the intracellular signals generated upon phosphoinositide hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass-harvested and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTA/AM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1) Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1 nM PMA has a stimulatory effect while 1 microM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non-tumor-promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose-dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous and heterologous desensitization of one of the pathways of the alpha 1-adrenergic action. Effects of epinephrine, vasopressin, angiotensin II and phorbol 12-myristate 13-acetate

Activation of protein kinase C blocks the alpha 1-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or epinephrine + propranolol markedly diminished the alpha 1-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the alpha 1-adrenergic responsiveness was assayed in buffer containing calcium no effect of the preincubation with vasopressin, angiotensin II or PMA was observed. Preincubation with epinephrine diminished the alpha 1-adrenergic responsiveness of the cells. In hepatocytes from hypothyroid rats the preincubation with the activators of protein kinase C (vasopressin, angiotensin II, phorbol 12-myristate 13-acetate and epinephrine) reduced markedly the alpha 1-adrenergic responsiveness of the cells, whereas in identical experiments using cells from adrenalectomized rats only the preincubation with epinephrine diminished the responsiveness. It is concluded that activation of protein kinase C induces desensitization of the alpha 1-adrenergic action in hepatocytes and that the calcium-independent pathway of the alpha 1-adrenergic action (predominant in cells from hypothyroid animals) resensitizes more slowly than the calcium-dependent pathway (predominant in cells from adrenalectomized rats). Epinephrine in addition to inducing this type of desensitization (through protein kinase C) leads to a further refractoriness of the cells towards alpha 1-adrenergic agonists.     

A novel succinyl dipeptide stimulates directed cell migration by modulating protein kinase C activity

In determining the mechanism of the chemokinetic action of the thiol protease inhibitor, E-64, in endothelial cell monolayers subjected to wounding, we synthesized succinyl-leucyl-agmatine (SLA), an analogue of E-64 that lacked the epoxy group and protease inhibitory effect. We observed that this analogue retained its chemokinetic effect on wounded endothelial cells. Its stimulatory action on endothelial cell polarization response to wounding was rapid and associated with directed cell migration. Furthermore, its effect on cellular polarization was blocked by protein kinase C (PKC) inhibitors and mimicked by pharmacologic agents that stimulated PKC activity. To determine if SLA's chemokinetic action was mediated by protein kinase C activation, we compared the effects of SLA and the tumor promoter phorbol myristate acetate (PMA) on the translocation of PKC activity in endothelial cells. We observed that both SLA and PMA induced the translocation of PKC activity from the cytosolic to the particulate fraction of the cells. We also observed that both SLA and PMA induced the phosphorylation of two proteins (Mr 23.4 and 36.5 kDa) in intact 32P-labeled cells. Thus, SLA stimulates the endothelial cell locomotor response to wounding by stimulating PKC activity.     

Interaction of insulin,glucocorticoids, and protein kinase C in the regulation of insulin-like growth factor-binding protein-1 production by H4IIE rat hepatoma cells

A sensitive RIA was used to examine regulation of IGFBP-1 in H4IIE rat hepatoma cells. IGFBP-1 was stimulated up to tenfold by dexamethasone and corticosterone, and this stimulation was abolished by RU486. The effect of dexamethasone increased with time in culture. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 up to fourfold with a maximal effect in short-term culture. Dexamethasone and PMA were additive in stimulating IGFBP-1. Under basal conditions IGFBP-1 production was linearly related to cell density: however, stimulation by dexamethasone was greatest in confluent cells, and PMA had a greater effect in sparse cultures. Insulin inhibited IGFBP-1 up to 80%, and this effect diminished with time in culture but was unaffected by cell density. Dexamethasone was stimulatory in the presence of a maximal inhibitory concentration of insulin, and insulin was inhibitory in the presence of maximal dexamethasone from 3–48 h in culture, regardless of cell density. PMA abolished the inhibitory action of insulin on IGFBP-1 secretion and mRNA expression during incubation periods of less than 4 h and not during longer incubations. PMA did not influence the stability of IGFBP-1 mRNA. We conclude that, in rat H4IIE cells, dexamethasone and PMA stimulate IGFBP-1 by independent mechanisms and speculate that when protein kinase C is activated the inhibitory action of insulin is blocked. © 1996 Wiley-Liss, Inc.