Inhibition of hepatic alpha 1-adrenergic effects and binding by phorbol myristate acetate

Treatment of isolated hepatocytes with the tumor-promoting agent, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and dose-dependent, non-competitive inhibition of alpha 1-adrenergic responses, including the activation of phosphorylase, increase in Ca2+ efflux, increase in free cytosolic Ca2+, and release of myo-inositol-1,4,5-P3. The actions of [8-arginine] vasopressin (AVP) on liver cells were also inhibited by PMA, but the inhibition could be overcome by high AVP concentrations. No significant inhibition of beta-adrenergic and glucagon-mediated activation of phosphorylase was induced by PMA and no inhibitory or synergistic effects of PMA were observed on the dose-dependent activation of phosphorylase by the Ca2+ ionophore A23187. In radioligand binding studies, PMA did not directly interfere with [3H]prazosin specific binding, the displacement of [3H]prazosin by (-)-norepinephrine nor with [3H]AVP specific binding to purified liver plasma membranes. Plasma membranes prepared from livers perfused with PMA exhibited a 30-44% reduction in [3H]prazosin binding capacity. Under identical conditions [3H]AVP binding was unchanged. The alpha 1-receptors remaining in membranes from PMA-treated livers had equivalent affinities for [3H]prazosin and (-)-norepinephrine, and were unaffected in terms of coupling to guanine nucleotide-regulating proteins as indicated by the ability of guanosine 5'-(beta, gamma-imido)triphosphate to promote the conversion of the remaining alpha 1-receptors into a low affinity state. These data indicate that tumor promoters are potent antagonists of alpha 1-adrenergic and vasopressin (low dose) responses in liver. It is proposed that PMA acting via protein kinase C (which presumably mediates the action of PMA) exerts its inhibitory action on alpha 1-adrenergic responses at the alpha 1-adrenergic receptor itself and also at a site close to or before myo-inositol-1,4,5-P3 release.     

Modulation of human neutrophil LTA hydrolase activity by phorbol myristate acetate

The phorbol ester, phorbol 12-myristate 13-acetate enhanced leukotriene B4 production stimulated by formyl-methionyl-leucyl-phenylalanine and arachidonic acid and reduced the production of the all-trans isomers of LTB4 by human neutrophils. Production of 5-hydroxyeicosatetraenoic acid was unaffected. These observations are consistent with a stimulatory effect of phorbol ester on LTA hydrolase, the enzyme which catalyses the conversion of LTA4 to LTB4. We demonstrate that a protein of the same molecular weight as LTA hydrolase is phosphorylated upon stimulation of neutrophils with PMA. These data suggest that the activity of LTA hydrolase may be regulated by protein kinase C-dependent phosphorylation.     

Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetate in human skin fibroblasts

Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.     

Modulation of the activity of the platelet-derived growth factor receptor by phorbol myristate acetate

Phorbol myristate acetate (PMA) weakly activates Na+/H+ exchange in NR-6 cells. Simultaneously, PMA blocks the activation of Na+/H+ exchange by platelet-derived growth factor or by serum. Phorbol esters that do not activate protein kinase C do not show this metabolic response. We conclude that activation of Na+/H+ exchange by platelet-derived growth factor or serum does not require the intermediate activation of protein kinase C. We postulate from this and previous observations that a major role of protein kinase C is to act as an inhibitor of the activity of cell surface receptors, in particular mitogen receptors.     

Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism

Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.     

Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and beta-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate.

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.     

Modulation of platelet-activating-factor-induced calcium influx and intracellular calcium release in platelets by phorbol esters.

1. The rate of 45Ca2+ efflux from prelabelled rat islets of Langerhans was stimulated by carbachol in a dose-dependent manner. 2. Significant stimulation occurred in the presence of 0.2 microM-carbachol; the response was half-maximal at 3-5 microM and was maximal at 20 microM. 3. Stimulation of 45Ca2+ efflux by carbachol was not dependent on the presence of extracellular Ca2+ and was enhanced in Ca2+-depleted medium. 4. Stimulation of 45Ca2+ efflux by 5 microM-carbachol occurred independently of any change in [3H]arachidonic acid release in prelabelled islets, and probably reflected generation of inositol trisphosphate in the cells. 5. The amphipathic peptide melittin failed to increase islet-cell 45Ca2+ efflux at a concentration of 1 microgram/ml, and caused only a modest increase at 10 micrograms/ml. 6. Despite its failure to increase 45Ca2+ efflux, melittin at 1 microgram/ml caused a marked enhancement of 3H release from islets that had been prelabelled with [3H]arachidonic acid. 7. The stimulation of 3H efflux caused by melittin correlated with a dose-dependent increase in the unesterified [3H]arachidonic acid content of prelabelled islets and with a corresponding decrease in the extent of labelling of islet phospholipids. 8. Combined addition of melittin (1 microgram/ml) and 5 microM-carbachol to perifused islets failed to augment 45Ca2+ efflux relative to that elicited by carbachol alone. 9. The data indicate that melittin promotes an increase in arachidonic acid availability in intact rat islets. They do not, however, support the proposal that this can either directly reproduce or subsequently modify the extent of intracellular Ca2+ mobilization induced by agents that cause an increase in inositol trisphosphate.     

Contrasting effects of phorbol dibutyrate and phorbol myristate acetate in rabbit aorta

In rat hepatocytes, active phorbol esters inhibited the alpha 1-adrenergic stimulation of phosphatidylinositol labeling with the expected potency order: phorbol myristate acetate (PMA) greater than phorbol dibutyrate (PDB). In contrast, in rabbit aorta the alpha 1-adrenergic action was inhibited dose-dependently by PDB but not by PMA. Similarly PDB (but not PMA) induced a strong contraction in rabbit aorta. The phorbol ester-induced contraction developed slowly, was dose-dependent and independent of extracellular calcium. These effects of PDB in rabbit aorta were neither inhibited by the protein kinase inhibitor H-7 nor mimicked by the synthetic diacylglycerol, OAG. Our results raise some doubts on the mechanism(s) through which the actions of PDB take place in rabbit aorta.     

The cartilage-resorbing protein catabolin is made by synovial fibroblasts and its production is increased by phorbol myristate acetate.

Pig synovial fibroblasts were shown to produce a protein that caused live cartilage to resorb its proteoglycan matrix in vitro. Fibroblasts were obtained either from synovial tissue digest or by allowing them to grow out of explants. The population derived from the digests was homogeneous and free of macrophage-like cells after two passages, but was still producing the cartilage-resorbing protein after seven passages. The active protein was found to have Mr 20,000 on gell filtration, and pI 4.8 on isoelectric focussing in polyacrylamide gel. It was indistinguishable from a protein with the same activity from pig mononuclear leucocytes, which has been called catabolin. Production of the protein was increased if the synovial fibroblasts were cultured with the tumour promoter phorbol 12-myristate 13-acetate. Fibroblasts from other sources (joint capsule and peritoneum) also apparently made the protein. The possibility that catabolin is the same as interleukin-1 is discussed: if they are, then the results suggest that fibroblasts can make an interleukin-1-life protein.     

Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate

A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.     

Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate

Cultured microvascular endothelial cells isolated from fenestrated capillaries have been shown to express many properties of their in vivo differentiated phenotype, yet they contain very few diaphragmed fenestrae. We show here that treatment of capillary endothelial cells with the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, induces more than a fivefold increase in the frequency of fenestrae per micron 2 of cell surface, as determined from a quantitative evaluation on freeze-fracture replicas. In quick-frozen, deep-etched preparations, the endothelial fenestrae appeared to be bridged by a diaphragm composed of radial fibers interweaving in a central mesh, as previously observed in vivo. These results indicate that diaphragmed fenestrae are inducible structures, and provide an opportunity to study them in vitro.     

Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types

In washed human platelets and in HL60 granulocytes phorbol myristate acetate (PMA, 1-2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL60 cells maintained a maximal response at PMA concentrations between 30-300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast cells. However, in the mast cell PMA concentrations above 10nM reduced maximum histamine release in a dose-dependent manner.     

Modulation by phorbol myristate acetate of arachidonic acid release and leukotriene synthesis by human polymorphonuclear leukocytes stimulated with A23187

Phorbol myristate acetate augmented the release of 3H-AA and the synthesis of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human polymorphonuclear leukocytes stimulated by A23187. PMA alone had no effect. Enhancement of the response to A23187 was not seen when the inactive phorbol ester 4-alpha phorbol didecanoate was added with A23187. These data are consistent with the hypothesis that activation of protein kinase C enhances AA release and metabolism in stimulated polymorphonuclear leukocytes.     

Role of circulating granulocytes in sheep lung injury produced by phorbol myristate acetate

Phorbol myristate acetate (PMA) and endotoxin cause pulmonary granulocyte sequestration and alteration in lung fluid and solute exchange in awake sheep that are felt to be analogous to the adult respiratory distress syndrome in humans. The basic hypothesis that PMA causes lung injury by activating circulating granulocytes has never been tested. The effects of infused PMA on lung mechanics and the cellular constituents of lung lymph have also not been reported. We therefore characterized the effects of intravenous PMA, 5 micrograms/kg, on lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, blood and lymph leukocyte counts, and plasma and lymph cyclooxygenase products of arachidonate metabolism in 10 awake sheep with normal granulocyte counts and after granulocyte depletion with hydroxyurea. PMA significantly altered lung mechanics from base line in both nongranulocyte depleted and granulocyte-depleted sheep. Dynamic compliance decreased by over 50% and resistance to airflow across the lungs increased over threefold acutely following PMA infusion in both sets of experiments. Changes in lung mechanics, pulmonary hemodynamics, lung fluid and solute exchange, pulmonary gas exchange, and plasma and lymph arachidonate metabolites were not significantly affected by greater than 99% depletion of circulating granulocytes. We conclude that the lung injury caused by PMA in chronically instrumented awake sheep probably is not a result of activation of circulating granulocytes.     

Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate

The development of LPS tolerance has been suggested to be mediated by an inhibition of cytokine synthesis. Here we have studied serum IL-6 and TNF levels in mice after LPS administration. Repeated administration of LPS (35 micrograms daily for 4 days) to mice induced a refractoriness (tolerance) to subsequent administrations of LPS in terms of induction of circulating IL-6 and TNF. To investigate the mechanism by which LPS down-regulates its own induction of cytokine synthesis and the relationship between IL-6 and TNF production, we attempted to revert the inhibition of IL-6 and TNF production using agents like PMA or IFN-gamma, previously reported to activate macrophage production of cytokines. Pretreatment with PMA (4 micrograms, 10 min before LPS) partially restored IL-6 production in LPS-tolerant mice given 2 micrograms LPS. On the other hand, PMA did not restore TNF induction in LPS-tolerant mice, even when administered with high doses of LPS (up to 200 micrograms). A similar reversal of LPS resistance to IL-6, but not TNF, induction by PMA was observed in genetically LPS-resistant C3H/HeJ mice. IFN-gamma also restored, although to a lesser extent than PMA, IL-6 production. However, unlike PMA, IFN-gamma could also partially restore TNF production in LPS-tolerant mice, although only when LPS was administered at high doses. By contrast with PMA, IFN-gamma was clearly more active in restoring TNF synthesis than that of IL-6. Similar results were obtained in genetically LPS-unresponsive C3H/HeJ mice. These data suggest that different mechanisms are implicated in the inhibition of IL-6 and TNF synthesis in LPS-tolerant mice and that part of this inhibition can be overcome by PMA or IFN-gamma.