Phorbol ester-induced stimulation of prostaglandin biosynthesis in human amnion cells

Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production by human amnion cells, with maximum stimulations of 10.8-fold and 5.9-fold, respectively. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester analogue, had little or no effect on PGE2 production by amnion cells. PMA and phorbol 12,13-dibutyrate (10(-7) M) induced a maximal increase in the rate of PGE2 biosynthesis within 15 min of treatment. These results suggest that there is an active protein kinase C present in amnion cells that is linked to arachidonic acid release and/or metabolism.     

Reversibility of phorbol diester-induced macrophage spreading

Phorbol 12-myristate 13-acetate and phorbol 12, 13-dibutyrate induced spreading of mouse macrophages with 50% effective concentrations of 3 nM and 35 nM, respectively. Macrophages treated with 100 or 1000 nM phorbol 12, 13- dibutyrate showed a time related decrease in spreading after washout. Spreading induced by 1, 10, or 100 nM phorbol 12-myristate 13-acetate was irreversible; however, washed phorbol 12,13-dibutyrate-treated cells respread after a second exposure to this compound. Washout of ³[H]phorbol diesters corroborated these observations in that 5% of ³H-phorbol 12-myristate 13-acetate and only 0.1% ³[H]phorbol, 12,13-dibutyrate remained associated with washed cells. Since phorbol 12-myristate 13 acetate is much more lipophilic than phorbol 12,13-dibutyrate, the reversibility of phorbol diester-induced macrophage spreading may depend upon the lipophilicity of the derivative utilized.Abbreviations DMEM Dulbecco's Minimal Essential Medium - PDA phorbol 12,13-diacetate - PDBu phorbol 12, 13-dibutyrate - PMA phorbol 12 myristate, 13 acetate - 4PDD phorbol 12, 13 didecanoate     

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

Acute effects of tumor-promoting phorbol esters on hepatic intermediary metabolism

In hepatocytes isolated from meal-fed rats, phorbol 12-myristate 13-acetate as well as phorbol 12,13-didecanoate stimulated de novo fatty acid synthesis in a dose-dependent manner. Moreover, phorbol 12-myristate 13-acetate inhibited ketogenesis from exogenous oleate, but slightly enhanced oleate esterification. The stimulation of esterification was more pronounced with endogenously synthesized fatty acids. In hepatocytes from 24h-starved rats a moderate stimulation of gluconeogenesis and ureogenesis was observed with glutamine as substrate. It is concluded that tumor-promoting phorbol esters mimick the short-term effects of insulin on hepatic fatty acid metabolism.     

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.     

Effect of phorbol 12-myristate 13-acetate and its analogue 4 alpha-phorbol 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.     

Phorbol esters induce synthesis of thromboplastin activity in human monocytes.

12-O-Tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-diacetate and phorbol 12,13-didecanoate were all potent inducers of thromboplastin activity in human monocytes in vitro, whereas 4 alpha-phorbol 12,13-didecanoate and 4 alpha-phorbol had no such effect. A concomitant increase in titrable apoprotein III antigen was found (apoprotein III is the protein component of thromboplastin). The increase was inhibited by cycloheximide and actinomycin D and partly by alpha-amanitin. The increase of thromboplastin activity was therefore most likely due to synthesis de novo of apoprotein III. The response was approximately halved in the absence of serum or Ca2+. Retinol had a weak inhibitory effect, and retinoic acid was inhibitory only at concentrations that also induced signs of cytotoxicity. TPA caused an initial rise in monocyte cyclic AMP concentration of about 90-120 min duration. No increase in 45Ca2+ influx was induced over 2 h. Good correlation exists between induction of apoprotein III synthesis in monocytes in vitro and mouse skin-tumour promotion in vivo by the various phorbol derivatives. Substances inactive in tumour promotion do not induce the synthesis of apoprotein III. General activating and cytotoxic effects of TPA were monitored by determining release of lysozyme, beta-glucuronidase and lactate dehydrogenase.     

Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).     

Pertussis toxin and H-7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide-primed macrophages. Eicosanoid release from lipopolysaccharide-primed macrophages

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1-2 micrograms/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 microM) or with phorbol 12-myristate 13-acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12-myristate 13-acetate. The presence of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12-myristate 13-acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis-toxin-sensitive, guanine-nucleotide-binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide-activated macrophages in response to A23187 and phorbol 12-myristate 13-acetate.     

Inhibition of denuded mouse oocyte meiotic maturation by tumor-promoting phorbol esters and its reversal by retinoids

Two tumor-promoting phorbol esters, phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA), when added to the culture medium of denuded mouse oocytes prevent their spontaneous meiotic maturation, whereas phorbol 13-acetate, which is inactive as a tumor promoter, does not inhibit this process. Retinoids appear to antagonize this inhibitory action of tumor promoters. However, the inhibitory effect of forskolin on meiotic maturation is not prevented, but is potentiated by retinal. These data indirectly suggest a role for calcium and/or phospholipids in the regulation of meiotic maturation. They also suggest that forskolin and phorbol esters mediate their effects through different pathways.     

Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.     

Stimulation of erythrophagocytosis in mouse peritoneal macrophages by chondroitin sulfates: correlation with effect of phorbol esters

Resident macrophages which were harvested from the mouse peritoneal cavity showed the attachment activity to opsonized erythrocytes (OE) without the treatment of chondroitin sulfates (CSA) or phorbol esters. Phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-diacetate) rapidly activated an opsonin-dependent erythrophagocytosis in resident macrophages, whereas CSA slowly activated it in vivo and in vitro. An additive effect of phorbol esters was observed in macrophages which were cultured with CSA in vitro or stimulated by the intraperitoneal injection of CSA for 1 or 2 day(s). In the case of macrophages stimulated by the intraperitoneal injection of CSA for 3 or 4 days the erythrophagocytic activity was at very high level and the additive effect of phorbol esters vanished. These results indicate that CSA plays a role in the induction of opsonin-dependent ingestion activity of resident macrophages.     

Specific binding of phorbol ester tumor promoters to mouse skin

The tumor promoter 20-³H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K_D = 2.4 × 10^?8 M). The potencies of phorbol esters for inhibiting binding of ³H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K_I = 0.74 nM; phorbol 12,13-didecanoate, K_I = 16 nM; phorbol 12,13-dibenzoate, K_I = 82 nM; mezerein, K_I = 98 nM; phorbol 12,13-diacetate, K_I = 3 μM; phorbol 12,13,20-triacetate, K_I = 5.6 μM; phorbol 13-acetate, K_I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, ³H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the ³H-PDBu binding activity mediates phorbol ester tumor promotion. ³H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE₂, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.     

Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate

Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 microM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethanolamine. Exposure of cells to phorbol 12-myristate 13-acetate induces an increase of the activity of the enzymes CTP: ethanolaminephosphate cytidylyltransferase and ethanolaminephosphotransferase. These effects are accompanied by a decrease of the pool size of ethanolaminephosphate and CDPethanolamine and an increase of the level of diacylglycerols after 30 min of incubation in the presence of phorbol 12-myristate 13-acetate. Upon prolonged incubation, the CDPethanolamine and diacylglycerol pools are restored to the level found in untreated cells. These results indicate that stimulation of phosphatidylethanolamine synthesis by phorbol 12-myristate 13-acetate is probably exerted at the level of CTP : ethanolaminephosphate cytidylytransferase, although there may be an additional effect on the subsequent step of phosphatidylethanolamine synthesis, the formation of phosphatidylethanolamines from CDPethanolamine and diacylglycerols.     

Phorbol-ester stimulated lysis of weak and nonspecific target cells by cytotoxic T lymphocytes

We have investigated the effects of short-term incubation of cloned and in vivo-produced cytotoxic T lymphocytes (CTL) with phorbol esters on their lytic activity against weak and nonspecific targets. These experiments demonstrate that 4 beta-phorbol-12-myristate, 13-acetate (PMA), 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate, but not the 4 alpha-phorbol-12,13 didecanoate esters stimulate the lytic apparatus. The stimulation is specific for the CTL rather than the target and appears to be nearly instantaneous in action. This rapid stimulation of the CTL lytic process is consistent with previously reported effects of phorbol esters associated with T cell activation in other functional assays.     

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.     

Phorbol12-Myristate13-Acetate Relieves the Inhibitory Effect of A23187 on Erythroid Differentiation of K562 Cells Induced by Dimethylsulfoxide

The differentiating effect of DMSO on K562 cells was studied against the background of pretreatment of the cells by the modulators of activities of protein kinase C and Ca signaling, phorbol 12-myristate 13-acetate, and ionophore A23187. The 2-hour pretreatment of K562 cells with A23187 (1 M), rather than phorbol 12-myristate 13-acetate (0.1 M), led to inhibition of the differentiating effect of DMSO (0.1%) during the subsequent four days of incubation. When the cells were pretreated jointly with A23187 and phorbol 12-myristate 13-acetate, the DMSO-induced erythroid differentiation was restored. Analysis of the DNA-degrading effect of the reagents used on K562 cells by fluorescent dyes under the same conditions suggests that this activity is induced in the presence of DMSO in the cells pretreated with a combination of phorbol 12-myristate 13-acetate and A23187 or without pretreatment.