Inhibited Phorbol 12-myristate 13-acetate Research Articles

Phorbol Myristate Acetate Inhibition of Phospholipase C Activation by Carbachol in Slices of Rat Brain Cortex Is a Delayed and Indirect Effect

We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.

Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.

The effect of staurosporine on drug-induced, topoisomerase II-mediated DNA cleavage in human leukemia cells.

Phorbol-12-myristate 13-acetate (PMA), a stimulator of protein kinase C, dramatically decreased topoisomerase II-reactive drug-induced DNA cleavage in HL-60 human leukemia cells. The effect of staurosporine, an inhibitor of protein kinase C, on drug-induced, topoisomerase II-mediated DNA cleavage was quantified in the same cells. Staurosporine decreased the magnitude of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)- and etoposide-induced DNA cleavage in a dose- and time-dependent fashion. Measurement of several parameters of cell proliferation revealed no clear and uniform correlation between staurosporine's inhibition of these parameters and its effects on drug-induced DNA cleavage. A direct comparison with PMA's effects on drug-induced DNA cleavage showed that whereas PMA's inhibition of etoposide-induced cleavage was much greater than its inhibition of m-AMSA-induced cleavage, the magnitude of staurosporine's effect on the cleavage produced by the two topoisomerase II-reactive drugs was similar. Thus, although PMA stimulates protein kinase C and staurosporine inhibits this enzyme, it is unlikely that the actions of either on topoisomerase II-reactive, drug-induced DNA cleavage are mediated directly via protein kinase C. Furthermore, it is likely that the mechanisms by which PMA and staurosporine inhibit topoisomerase II-reactive drug-induced cleavage are different.

Induction of chronic human immunodeficiency virus infection is blocked in vitro by a methylphosphonate oligodeoxynucleoside targeted to a U3 enhancer element

Oligodeoxynucleosides with internucleoside methylphosphonate linkages complementary to regions within U3 of human immunodeficiency virus type 1 were evaluated for their ability to block phorbol myristate acetate upregulation of virus in chronically infected promonocytic and T-lymphoblastoid cell lines. One such oligomer, targeted to an NF-kappa B enhancer element, inhibited phorbol myristate acetate induction of viral replication and tat-mediated trans activation of the human immunodeficiency virus long terminal repeat. The effect of this construct is contrasted with classical antisense methylphosphonate-derivatized oligomers complementary to initiation codon and splice acceptor sites of human immunodeficiency virus structural and regulatory genes. Its activity suggests a novel application of the modified oligonucleotide strategy in the blockade of viral induction from latently infected cells.

Low levels of interleukin‐4 and high levels of transforming growth factor β in rheumatoid synovitis

Since interleukin-4 (IL-4) displays agonistic effects on both T and B cells, we studied whether this lymphokine is involved in rheumatoid synovitis, a disease characterized by intense T cell infiltration and B cell stimulation. Rheumatoid arthritis synovial fluids (RA SF) contained no (less than 15 pg/ml) or very low amounts (less than 25 pg/ml) of IL-4, as measured by a sensitive and specific enzyme-linked immunosorbent assay. No IL-4 was produced by unstimulated rheumatoid synovial membrane. RA SF were found to inhibit phorbol myristate acetate (PMA)-dependent proliferation of normal peripheral blood lymphocytes (PBL). An inhibitory fraction with an apparent molecular weight of 150 kd was isolated by gel filtration. The inhibitory fraction strongly blocked the proliferation of PBL induced by PMA, PMA + IL-2, or PMA + IL-4. However, this fraction was less effective in blocking the proliferation of PBL induced by PMA + IL-2 + IL-4. High levels of transforming growth factor beta (TGF beta) were found in these RA SF, and an anti-TGF beta antibody was able to partially reduce the inhibitory activity. RA SF were found to inhibit phytohemagglutinin-induced IL-4 production by PBL. These data indicate that IL-4, similar to other T cell lymphokines, cannot be detected in RA SF and that RA SF contains an inhibitory activity, related in part to TGF beta, which blocks mitogen-induced proliferation of PBL, at least in part through an inhibition of T cell-derived lymphokine release.

Regulation of angiotensin II binding sites in neuronal cultures by protein kinase C

The radioligand binding of 125I-angiotensin II (ANG II) and calcium phospholipid-dependent protein kinase C (PKC) activity were measured to study the specificity and mechanisms of PKC involvement in the regulation of ANG II-specific binding site expression in neuronal cultures prepared from the brains of 1-day-old rats. Previously, PKC-activating phorbol esters were shown to increase the specific binding of 125I-ANG II in neuronal cultures. However, phorbol esters have many biological effects, which may nonspecifically act to increase 125I-ANG II-specific binding. In the present study, mezerein and teleocidin A, two activators of PKC that are chemically unrelated to phorbol esters, increased the specific binding of 125I-ANG II in a dose- and time-dependent manner with 50% effective dose (ED50) values of 32 and 79 nM, respectively. The PKC antagonist H-7 dose dependently inhibited phorbol 12-myristate 13-acetate (TPA)-stimulated increases in 125I-ANG II binding, whereas downregulation of PKC activity by chronic phorbol ester incubations of 24 and 48 h prevented TPA-stimulated increases in 125I-ANG II-specific binding. TPA (0.8 microM), mezerein (0.76 microM), and teleocidin A (0.5 microM) all caused a rapid translocation of PKC activity from the cytosol to the particulate fraction by 15 min. Temporally, the maximal stimulation of PKC translocation by mezerein, teleocidin A, and TPA preceded their ability to stimulate maximal 125I-ANG II-specific binding. Taken together, these results suggest that PKC is directly involved in the stimulation of ANG II-specific binding site expression and that translocation of PKC is a prerequisite for the increased expression of ANG II binding sites.

Effects of Polymyxin B on Superoxide Anion Release and Priming in Human Polymorphonuclear Leukocytes

We studied the effect of a potent inhibitor of protein kinase C, polymyxin B (PMXB), on superoxide anion (O2-) release by human polymorphonuclear leukocytes (PMNL). PMXB was compared with another inhibitor of protein kinase C, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine (H-7). Both PMXB and H-7 inhibited phorbol myristate acetate (PMA)-stimulated O2- release. Formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated O2- release by cytochalasin B-treated PMNL was not inhibited significantly by either PMXB or H-7. 1-Oleoyl-2-acetyl-glycerol (OAG,25-100 microM) stimulated PMNL to release O2- with a long lag-time (8-10 min). Although H-7 inhibited OAG-stimulated O2- release, PMXB augmented the OAG-stimulated response by increasing rate and reducing lag time. The augmenting effect of PMXB was evident only when added after stimulation by OAG, with maximum effect observed at 3 min after addition of OAG. The augmenting effect was also seen with PMXB immobilized on agarose beads. PMXB did not affect the respiratory burst response to 1,2-dioctanoylglycerol. PMXB-augmented, OAG-stimulated O2- release was inhibited by the addition of H-7 before OAG. In contrast to the effect on O2- release, OAG-stimulated protein phosphorylation was inhibited similarly by either PMXB or H-7, when these agents were added 3 min after stimulation by OAG. These results suggested that initial activation of protein kinase C by OAG is essential for O2- release, but that PMXB acts in a manner independent from protein kinase C to augment OAG-stimulated O2- release. When priming by OAG for enhanced O2- release (as opposed to direct stimulation of O2- release) in FMLP-stimulated PMNL was examined, PMXB inhibited O2- release in OAG-primed PMNL, suggesting that protein kinase C is involved in priming of PMNL by OAG.

Phorbol myristate acetate inhibits acidification by turtle urinary bladder

The effects of phorbol myristate acetate (PMA) on acid secretion by the turtle urinary bladder were examined to evaluate the importance of protein phosphorylation in modulating the distal acidification system. In HCO3-free PO4 buffer 0.2 mM mucosal PMA inhibited reverse short-circuit current (RSCC) by 42%. The inhibition of RSCC was dose dependent, and RSCC approached zero at high concentrations of PMA. PMA also inhibited acid secretion measured titrimetrically but had no effect on RSCC from bladders in which proton secretion was selectively inhibited by an adverse pH gradient or serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The inhibition by PMA was duplicated by 1-oleoyl-2-acetyl-rac-glycerol, but not by an inactive phorbol ester. The PMA inhibition was much more potent with mucosal compared with serosal application. The PMA inhibition caused a significant 0.15 pH unit reduction of carbonic anhydrase (CA) cell cytoplasmic pH and a change in the CA cell morphology. Unlike the inhibition of proton transport induced by acetazolamide, the PMA inhibition was neither reversed nor prevented by sodium azide. We conclude that PMA decreases basal acid secretion and blocks the stimulatory effects of CO2 by an azide-insensitive mechanism distinct from that of acetazolamide. In view of recent findings that PMA stimulates HCO3 secretion by the turtle bladder, these results suggest that kinase C-mediated protein phosphorylation may be a central event in the transition from the secretion of acid to the secretion of base.

Neutrophil Membrane Sulfhydryl Groups Are Involved in Stimulated Neutrophil Adherence to Endothelium

We investigated the role of membrane sulfhydryl groups in adherence of stimulated polymorphonuclear neutrophils to cultured endothelial cells. Treatment of neutrophils with p-chloromercuriphenyl sulfonate (PCMPS), a slowly penetrating sulfhydryl reagent, inhibited phorbol myristate acetate (PMA)- or calcium ionophore A23187-stimulated adherence to cultured human or bovine endothelial cells. At concentrations which completely blocked PMA-stimulated adherence, PCMPS did not cause release of lactic dehydrogenase, inhibit PMA-mediated degranulation or hydrogen peroxide production, or prevent the PMA-induced increased surface expression of CD11b/CD 18 (Mac-1). Coincubation with a competing reduced sulfhydryl compound protected neutrophils from inhibition of PMA-stimulated adherence by PCMPS, whereas coincubation with an oxidized sulfhydryl compound did not. Monobromotrimethylammoniobimane, a nonpenetrating sulfhydryl reagent that is structurally unrelated to PCMPS, also inhibited stimulated neutrophil adherence to endothelium cultures. We conclude that stimulated neutrophil adherence to endothelium involves neutrophil membrane protein sulfhydryl groups.

Effects of phorbol esters and various growth factors on prostaglandin E2 synthesis by cultured porcine thyroid cells

The effects of phorbol esters and various growth factors on prostaglandin E2 (PGE2) synthesis by cultured porcine thyroid cells were examined. Phorbol 12-myristate, 13-acetate (PMA), phorbol 12, 13-dibutylate, epidermal growth factor (EGF) and fetal bovine serum (FBS) stimulated PGE2 production in a dose-related fashion. PMA stimulated PGE2 production over fifty-fold with a dose of 10(-7) M compared with controls during 4h incubation. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were around 1 X 10(-9) M and 5 X 10(-10) M respectively. FBS also clearly stimulated it about ten-fold with a concentration of 20%. Thyroid stimulating hormone (TSH) and inactive phorbol, however, did not stimulate PGE2 production. The release of radioactivity from [3H]-arachidonic acid (A.A.) prelabeled cells was also stimulated by PMA, EGF and FBS. These results indicate that PMA, EGF and FBS are potent stimulators of PGE2 production, associated with the activity to stimulate A.A. release in thyroid cells. Secondly, in order to elucidate some mechanism(s) of the stimulation by these factors of PGE2 production, effects of various agents such as EGTA, Ca2+-ionophore (A-23187), cycloheximide, hydrocortisone and para-bromophenacylbromide (p-BPB) were examined on PGE2 production and/or [3H]-A.A. release by thyroid cells. Chelation of Ca2+ by EGTA, treatment of cells with hydrocortisone, an inducer of lipocortin which may inhibit phospholipase A2 (PLA2) activity, or inhibition of PLA2 activity by p-BPB clearly inhibited PGE2 production and/or [3H]-A.A. release induced by PMA, EGF and FBS. Cycloheximide also blocked the PMA-or EGF-stimulated [3H]-A.A. release and PGE2 synthesis. On the other hand, Ca2+-ionophore (A-23187) potentiated PMA- or EGF-stimulation. Furthermore, the Ca2+-ionophore alone-induced stimulation was clearly inhibited by the treatment with hydrocortisone or p-BPB. These data indicate that an increase of Ca2+ in cytosol is important to PLA2 activation, and also that PLA2 may be activated by PMA, EGF and FBS in thyroid cells. The inhibition of PMA- or EGF-induced [3H]-A.A. release and PGE2 synthesis by cycloheximide suggests that both factors-induced stimulation may be sensitive to regulation by short-lived protein(s). Finally, other growth factors such as insulin (0.1-10 micrograms/ml), insulin like growth factor I (IGF-1) (10(-9)-10(-7) M) and interleukin 1 alpha (1-100 ng/ml) also stimulated PGE2 production by thyroid cells in the presence or absence of EGF (10(-10)-10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Sphingolipids inhibit insulin and phorbol ester stimulated uptake of 2-deoxyglucose

Studies are presented demonstrating inhibition of both insulin and phorbol myristate acetate stimulated uptake of 2-deoxyglucose uptake by 3T3-L1 fibroblasts. Greatest inhibition of uptake was seen with sphinganine while sphingosine was also potent in this regard. Ceramide inhibited phorbol myristate acetate but not insulin stimulation of uptake. It is suggested that sphingolipid inhibition of glucose transport relates to the previously demonstrated effect of corticosteroids to increase membrane sphingomyelin and inhibit glucose transport.

Reversal by protein kinase C inhibitor of suppressive actions of phorbol-12-myristate-13-acetate on polyphosphoinositide metabolism and cytosolic Ca 2+ mobilization in thrombin-stimulated human platelets

In the presence of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C in vitro, phorbol-12-myristate-13-acetate (PMA) did not suppress the thrombin-induced increase of cytosolic Ca2+ concentration in human platelets. The H-7 reversal of the inhibitory action of PMA was also observed in thrombin-induced polyphosphoinositide breakdown by phospholipase C. These results provide additional support to the developing theory that the inhibition of PMA on Ca2+ mobilization and phosphoinositide turnover may be mediated by protein kinase C activation.

Polymyxin B inhibits phorbol 12-myristate 13-acetate, but not chemotactic factor, induced effects in rabbit neutrophils

The addition of the amphipathic polycationic antibiotic polymyxin B to a suspension of rabbit neutrophils results in inhibition of the agonist (secretion of secondary granules) and antagonist (inhibition of chemotactic factor induced degranulation) properties of phorbol 12-myristate 13-acetate. On the other hand, polymyxin B does not inhibit the degranulation of the neutrophils that is induced by chemotactic factors. These results imply that the role of protein kinase C in the initiation of neutrophil functions in response to the addition of chemotactic factors is less critical than previously thought. In addition, the reversal of the inhibitory properties of phorbol esters by polymyxin B indicates that the former are mediated by the ability of the tumor promoters to activate protein kinase C. These results thus strengthen the hypothesis that protein kinase C plays important roles in the regulation (as contrasted to initiation) of neutrophil functions.

Suppression of phorbol myristate acetate-triggering of macrophage H2O2 release by sarcoma 180 originating factor.

A high molecular weight proteinaceous factor in the cell extract of sarcoma 180 (S-180) was found to inhibit phorbol myristate acetate (PMA)-triggering of macrophage H2O2 release. This factor (S-180 factor) was stable at 56 C for 1 hr and resistant to ultraviolet-irradiation. The S-180 factor inhibited the specific binding of PMA to macrophages and this was accompanied by a parallel reduction of PMA-triggered H2O2 release. S-180 factor preferentially depressed macrophage H2O2 release in response to phorbol diesters including PMA, 4 beta-phorbol 12 13 beta,13 alpha-diacetate, 4 beta-phorbol 12 beta,13 alpha-didecanoate, 4 beta-phorbol 12 beta,13 alpha-dibenzoate, and 4-omicron-methyl-PMA rather than the H2O2 release triggered by wheat germ agglutinin or by phagocytosis of latex particles. The S-180 factor failed to affect the PMA-elicited macrophage cell spreading and macrophage phagocytic activity against latex beads with or without PMA-mediated stimulation. A similar inhibitory factor was found in the extracts of some other murine tumor cells (Ehrlich carcinoma and thymic leukemia) and normal cells (liver, spleen, and peritoneal exudate cells).

Inhibition of vasopressin-stimulated water flow in toad bladder by phorbol myristate acetate, dioctanoylglycerol, and RHC-80267. Evidence for modulation of action of vasopressin by protein kinase C.

The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium-phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C.

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma.

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.

Mechanism of action of phorbol myristate acetate on human natural killer cell activity

We have investigated the kinetics of inhibition and regeneration of human natural killer (NK) cell-mediated lysis of K562, a human erythroleukemia cell line, by the potent tumor-promoting agent phorbol-12-myristate-13-acetate (PMA). It is shown that PMA inhibits NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner whether the compound is present throughout the 4-hr cytotoxic assay or the effector cells (EC) are pretreated with PMA. Pretreatment of the target cells (TC) with PMA produced a different profile of NK activity suggesting that PMA inhibition of NK-CMC is primarily due to the inactivation of EC. PMA-induced inhibition of NK-CMC does not affect TC binding and is not circumvented by compounds that enhance intracellular levels of cyclic guanosine monophosphate (cGMP) or calcium. Furthermore, and contrary to a recent report, PMA-induced inhibition of NK-CMC is independent of monocytes. Finally, kinetic studies revealed that PMA-induced inhibition of NK-CMC occurs rapidly and is fully reversible provided that “regenerated EC” are thoroughly washed, prior to the cytotoxic assay, to rid the cell suspension of residual PMA. The potential implications of these results to the currently accepted theory of TC destruction by NK cells, the stimulus-secretion model, are discussed.