Stimulatory Effect Of Phorbol 12-myristate 13-acetate Research Articles

Phorbol-12-myristate 13-Acetate Acting through Protein Kinase Cε Induces Translocator Protein (18-kDa) Tspo Gene Expression

Translocator protein (TSPO) is an 18-kDa cholesterol-binding protein that is expressed at high levels in steroid synthesizing and several cancer cells where it is involved in steroidogenesis and cell proliferation, respectively. The factors regulating Tspo expression are unknown. We analyzed Tspo transcriptional responses to the tumor promoter, phorbol-12-myristate 13-acetate (PMA), in cells with varying TSPO levels. PMA induced Tspo promoter activity and Tspo mRNA levels in TSPO-poor nonsteroidogenic cells (NIH-3T3 fibroblasts and COS-7 kidney) but not in TSPO-rich steroidogenic cells (MA-10 Leydig) with high basal Tspo transcriptional activity. The stimulatory effect of PMA was mediated by an 805-515-bp region upstream of the transcription start site. Electrophoretic mobility shift assay (EMSA) revealed that PMA induced binding of c-jun and GA-binding protein transcription factor (GABP-alpha) to their respective activator protein 1 (AP1) and v-ets erythroblastosis virus E26 oncogene homologue (Ets) sites in this region. Protein kinase C (PKC)-specific inhibitors blocked PMA induction of Tspo promoter activity with an inhibition profile suggestive of involvement of PKCepsilon. PKCepsilon expression correlated with TSPO content in the three cell lines. In NIH-3T3 cells, PKCepsilon overexpression induced Tspo promoter activity and mRNA levels and enhanced PMA-induced up regulation of c-jun and TSPO. In MA-10 cells, a PKCepsilon-specific translocation inhibitor peptide reduced basal Tspo promoter activity. PKCepsilon siRNA pool reduced PKCepsilon and TSPO levels in MA-10 cells indicating a role for PKCepsilon in regulating TSPO expression. Taken together, these data suggest that elevated TSPO expression in steroidogenic cells may be due to high constitutive expression of PKCepsilon that renders them unresponsive to further induction while PMA activation of PKCepsilon drives inducible TSPO expression in nonsteroidogenic cells, likely through AP1 and Ets.

Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase Cδ-Tyr311 Phosphorylation in Cardiomyocyte Caveolae

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCdelta to caveolae and increase PKCdelta-Tyr(311) phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase.

The present study describes events postulated to be involved in the regulated mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lines stably transfected either with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR(tr)), we show that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), caused a rapid time- and dose-dependent increase in GHBP secretion, which, as expected, was matched by a corresponding decrease in cell-surface GHR. Furthermore, PMA equally enhanced GHBP release from CHO/hGHR(tr) cells, suggesting that the cytoplasmic domain of hGHR is not essential for PMA-induced shedding. PMA is known to specifically activate protein kinase C and, indeed, the stimulatory effects of PMA in both cell lines were completely inhibited by the protein kinase inhibitor, staurosporine (100 nM), suggesting that activation of protein kinase C (PKC) may mediate PMA-induced GHBP shedding. Since proteolytic cleavage of several cell-surface proteins was shown to be stimulated by modulators of PKC activity and inhibited by metalloprotease inhibitors, we studied the effects of two hydroxamic acid-based inhibitors of zinc-dependent metalloproteases, BB-3103 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with both these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-dependent manner, with IC(50) values of approximately 0.41 microM for BB-3103 and approximately 0.97 microM for Ro31-9790. In addition, these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator, N-ethylmaleimide (NEM), with IC(50) values of approximately 0.32 microM and approximately 0.58 microM for BB-3103 and Ro31-9790 respectively. It was of interest to find out that Ro31-9790 acted not only to modulate PMA- or NEM-induced shedding processes, but also markedly reduced the spontaneous, time-dependent accumulation of GHBP released from CHO/hGHR cells growing in serum-containing medium. Taken together, these results suggest that one or more zinc-dependent metalloprotease(s), acting at the cell surface, may be involved in GHBP secretase activity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch mechanism and/or PKC-dependent phosphorylation. In the long run, specific inhibitors of these processes could be applied in the regulation of GHBP levels and, thus, of GH availability and/or activity.

Ultracytochemical study on the localization of superoxide producing sites in stimulated rat neutrophils.

Superoxide anion production in neutrophils plays an important role in the microbicidal defense system in the body. In this study, isolated rat neutrophils were stimulated experimentally and examined by electron microscopy to determine the site of superoxide production and its subsequent translocation during different cell stimulation time periods. Blood and peritoneal neutrophils were incubated for periods of 5, 10, and 15 min with phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and combinations of PMA and cytochalasin B (CB) and fMLP and CB. Ultracytochemical detection of O(2)(-) was performed with the 3, 3'-diaminobenzidine-manganese (DAB/Mn) cytochemical method and cationized ferritin (CF) particles were added to stimulation media to monitor endocytotic events that occurred during neutrophil stimulation. Unstimulated neutrophils were devoid of O(2)(-) activity in cytoplasmic granules and at the plasma membrane surface. After 5 min stimulations with PMA, PMA + CB, or fMLP + CB, electron-dense DAB/Mn reaction product was detected in small, centrally located tubular compartments within the neutrophils. CF particles which were added to the stimulation media became internalized in endocytotic vesicles after 5 min stimulation; these vesicles were devoid of O(2)(-) activity. At 10 min stimulation with PMA, O(2)(-)-positive granules subsequently fused with each other and translocated to sub-plasma membrane regions where they either contacted the plasma membrane or fused with CF-containing endocytotic vesicles. Little reaction product was observed on the surface of the neutrophils. Spectrophotometric comparison of the stimulatory effects of PMA, fMLP, and fMLP + CB revealed different rates and yields of O(2)(-) production. Results from this study suggest that the O(2)(-)-producing sites of rat neutrophils originate intracellularly and translocate to the plasma membrane surface following stimulation with PMA, PMA + CB, and fMLP + CB, but not with fMLP or CB alone. Furthermore, these compartments appear to possess the ability to fuse with endocytotic vesicles, a process that may be linked to intracellular microbicidal activity in circulating and tissue neutrophils.

Effect of phorbol ester on the regulation of [formula omitted] receptors

Granulosa cells have been used to study the regulation of LH hCG receptor protein and mRNA expression. Phorbol 12-myristate 13-acetate (PMA) dose-dependently attenuates the increases in LH hCG receptor mRNA and protein induced by FSH and forskolin (FSK). The presence of PMA caused a decrease in cAMP production stimulated by FSH and FSK. These results suggest that PMA-mediated decreases in cAMP are a major factor in PMA-mediated decreases in LH hCG receptor mRNA. On the other hand, in the presence of 8-Br-cAMP, PMA significantly increased LH hCG receptor mRNA and protein, with maximal stimulation between PMA concentrations of 3 to 30 nM (1.5 fold) with 8-Br-cAMP. These findings suggest that activation of protein kinase C by PMA attenuates the increase in cAMP accumulation induced by FSH but enhances the effect of cAMP on LH hCG receptor expression, and that the inhibitory and stimulatory effects of PMA on LH hCG receptor content are correlated with regulation of LH hCG receptor mRNA levels. Since the half-life study revealed no change in the stability of the LH hCG receptor mRNA following PMA treatment, a change in the rate of LH hCG receptor gene transcription must be responsible for the change in the LH hCG receptor mRNA levels.

Inhibition of Protein Kinase C-Dependent Noradrenaline Release by Wortmannin in PC12 Cells

Wortmannin is a specific phosphatidylinositol 3-kinase inhibitor, and it inhibits secretion in many cell types. Previously we reported that high K+and external ATP stimulated a Ca2+influx and [3H]noradrenaline ([3H]NA) release from rat pheochromocytoma cell line 12 (PC12) cells in the presence of extracellular CaCl2. Addition of phorbol 12-myristate 13-acetate (PMA) stimulated [3H]NA release by itself and enhanced the maximal responses of high K+and ATP. In this study, we investigated the effects of wortmannin on NA release from PC12 cells. Wortmannin inhibited the [3H]NA release induced by high K+and ATP, and the stimulatory effects of PMA, in a dose-dependent manner. Wortmannin caused 50% inhibition of high K+-induced [3H]NA release at a concentration of 2.78 ± 0.68 μM(n= 5). The increased cytosolic free Ca2+concentrations ([Ca2+]i), induced by ATP, were not inhibited by wortmannin. Wortmannin inhibited PMA-induced phosphorylation of a 80-kDa protein in the cytosol fraction of PC12 cells. Calphostin C, a specific protein kinase C inhibitor, also inhibited high K+-, ATP-, and PMA-induced NA release, and the phosphorylation of the 80-kDa protein induced by PMA. Mastoparan, an amphiphilic tetradecapeptide from wasp venom, stimulated NA release in the presence or absence of extracellular CaCl2. Neither wortmannin nor calphostin C inhibited the NA release induced by mastoparan. These findings suggest that wortmannin inhibits the PKC-dependent pathway, not [Ca2+]imobilization, resulting in the inhibition of NA release from PC12 cells.

Adhesion-induced generation of oxygen free radical from human alveolar macrophages and its mechanisms

Background : Neutrophils or monocytes separated in vitro by the adherence to plastic surface are known to be activated by surface adherence itself and subsequent experimental data might be altered by surface adherence. In the process of surface adherence, adhesion molecules have a clear role in intracellular signal pathway of cellular activation. Human alveolar macrophages(HAM) are frequently purified by the adherence procedure after bronchoalveolar lavage. But the experimental data of many reports about alveolar macrophages have ignored the possibility of adhesion-induced cellular activation. Method : Bronchoalveolar lavage was performed in the person whose lung of either side was confirmed to be normal by chest CT. With the measurement of hydrogen peroxide release from adherent HAM to plastic surface and non-adherent HAM with or without additional stimulation of phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP), we observed the effect of the adherence to plastic surface. We also evaluated the effect of various biological surfaces on adhesion-induced activation of HAM. Then, to define the intracellular pathway of signal transduction, pretreatment with cycloheximide, pertussis toxin and anti-CD11/CD18 monoclonal antibody was done and we measured hydrogen peroxide in the culture supernatant of HAM. Results : 1) The adherence itself to plastic surface directly stimulated hydrogen peroxide release from human alveolar macrophages and chemical stimuli such as phorbol myristate acetate(PMA) or N-formyl-methionyl-leucyl-phenylalanine(fMLP) colud not increase hydrogen peroxide release in these adherent macrophages which is already activated. 2) PMA activated human alveolar macrophages irrespective of the state of adhesion. However, fMLP stimulated the release of hydrogen peroxide from the adherent macrophages, but not from the non-adherent macrophages. 3) HAM adherent to A549 cell(type II alveolar epithelium-like human cell line) monolayer released more hydrogen peroxide in response to both PMA and fMLP. This adherence-dependent effect of fMLP was blocked by pretreatment of macrophages with cycloheximide, pertussis toxin and anti-CD18 monoclonal antibody, Conclusion : These results suggest that the stimulatory effect of PMA and fMLP can not be found in adherent macrophage because of the activation of human alveolar macrophage by the adherence to plastic surface and the cells adhered to biologic surface such as alveolar epithelial cells are appropriately responsive to these stimuli. It is also likely that the effect of fMLP on the adherent macrophage requires new protein synthesis via G protein pathway and is dependent on the adhesion between alveolar macrophages and alveolar epithelial cells by virtue of CD11/CD18 adhesion molecules.

Modulation of the Stress-Induced Synthesis of Stress Proteins by a Phorbol Ester and Okadaic Acid1

The expression of alphaB crystallin, hsp27, and hsp70 in C6 cells increased when they were exposed to arsenite (50 microM for 1 h) or heat (42 degrees C for 30 min), as detected by specific immunoassays, Western blot analysis, and Northern blot analysis. When cells were exposed to arsenite in the presence of 0.1 microM phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, or 0.2 microM okadaic acid, an inhibitor of phosphoserine/phosphothreonine protein phosphatases, expression of alphaB crystallin was markedly enhanced. The induction of hsp27 and hosp70 expression was also stimulated to a considerable extent in the same cells. The stimulatory effect of PMA was further enhanced in the presence of okadaic acid, but it was strongly inhibited in the presence of 0.5 microM staurosporine, an inhibitor of protein kinase C. PMA and okadaic acid also stimulated the response to heat stress of the expression of alphaB crystallin, but they barely stimulated the response to heat stress of hsp27. The extent of stimulation of the arsenite-induced responses by PMA and okadaic acid was greater when the concentration of arsenite (i.e. the magnitude of the stress) was relatively low (25-50 microM). The arsenite-induced release of arachidonic acid from cells was also stimulated in the presence of PMA and/or akadaic acid, and the stimulatory effects of PMA and okadaic acid on the arsenite-induced accumulation of alphaB crystallin and hsp27 were strongly suppressed by quinacrine, an inhibitor of phospholipase A2. These results suggest that the stimulatory effects of PMA and okadaicacid on the stress responses are cuased, in part, by the increased metabolic activity of the arachidonic acid cascade, as a consequence of the activation of phospholipase A.

Structure and Regulation of the Gene Encoding the Neuron-specific Protein Kinase C Substrate Neurogranin (RC3 Protein)

A 13-kilobase pair genomic DNA encoding a 78-amino acid brain-specific calmodulin-binding protein kinase C (PKC) substrate, neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The Ng/RC3 gene is composed of four exons and three introns, with the protein-coding region located in the first and second exons. This gene was found to have multiple transcriptional start sites clustered within 20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the proximal upstream region of the start sites. The promoter activity was characterized by transfection of 293 cells with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene. A minimal construct containing bp +11 to +256 was nearly as active as that covering bp -1508 to +256, whereas a shorter one covering bp +40 to +256 had a greatly reduced activity. Between bp +11 and +40 lies a 12-nucleotide sequence (CCCCGCCCACCC) containing overlapping binding sites for AP2 (CCGCCCACCC) and SP1 (CCCGCC); this region may be important for conferring the basal transcriptional activity of the Ng/RC3 gene. The expression of a Ng/RC3-luciferase fusion construct (-1508/+256) in transfected 293 cells was stimulated by phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic acid, vitamin D, retinoic acid, or thyroxines T3 and T4. PMA caused a 2-4-fold stimulation of all the reporter gene constructs ranging from +11/+256 to -1508/+256. The stimulatory effects of PMA could be magnified by cotransfection with both Ca(2+)-dependent and -independent phorbol ester-binding PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon cDNAs, but not by non-phorbol ester-binding PKC-zeta cDNA. The Ng/RC3 and PKC-gamma genes have a similar expression pattern in the brain during development. These two genes share at least four conserved sequence segments 1.5 kilobase pair upstream from their transcriptional start sites and a gross similarity in that they possess several AT-rich segments within bp -550 to -950. A near homogeneous 20-kDa DNA-binding protein purified from rat brain was able to bind to these AT-rich regions of both Ng/RC3 and PKC-gamma genes with footprints containing ATTA, ATAA, and AATA sequences.

Stimulatory and Inhibitory Effects of a Phorbol Ester on Chloride Secretion by Rat Epididymal Epithelium1

The effects of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by rat cauda epididymal epithelium were studied through use of the short-circuit current (Isc) technique. PMA alone could stimulate the Isc in a dose-dependent manner. The PMA-induced Isc was blocked by the Cl channel blocker, diphenylamine-2-carboxylate, but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. PMA also exerted an inhibitory effect on the subsequent Ca(2+)-activated Isc. ATP or ionomycin-induced Isc was significantly reduced by treatment with PMA (5-10 min). Both stimulatory and inhibitory effects of PMA could be mimicked by a diacylglycerol analog, 1,2-dioctanoyl-sn-glycerol, but not an inactive analog of PMA, 4 alpha-phorbol 12,13-didecanote (4 alpha D). Down-regulation of protein PKC by prolonged treatment of epididymal cells with PMA (12 h) diminished both stimulatory and inhibitory effects of PMA on Isc. These results suggest that the dual effect of PMA on Isc was mediated by PKC. However, the PKC inhibitor, calphostin C, could block the inhibitory effect of PMA on ATP-induced Isc but not the stimulatory effect of PMA alone on Isc. The stimulatory effect of PMA was apparent only when PMA was applied to the apical aspect; in contrast, the inhibitory effect of PMA on ATP-induced Isc was readily seen with application of PMA to either side of the epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Tannin inhibition of protein kinase C in airway epithelium.

Tannin, a polydisperse polyphenol extracted from cotton bracts (CBE), has been implicated in the pathogenesis of byssinosis, a lung disease of mill workers. CBE tannin inhibits chloride secretion in airway epithelial cells by means of an unknown mechanism(s). Activation of protein kinase C (PKC) by PMA (phorbol 12-myristate 13-acetate) in airway cells increases chloride secretion. The effect of tannin on this PKC pathway was examined, using canine tracheal epithelium mounted in Ussing chambers. PMA addition (10 nM) to the mucosal bath resulted in a 0.36 +/- 0.07 microEq/cm2.h (mean +/- SEM, n = 20) increase in short-circuit current (Isc) and a 0.38 +/- 0.17 microEq/cm2.h increase in net chloride secretion (Jnet). The inactive 4 alpha-phorbol had no effect. Tannin addition to the mucosal bath produced a dose-dependent decrease in Isc and Jnet. In tissues pretreated with 2-50 micrograms/ml tannin, and subsequently stimulated with PMA, tannin inhibited PMA stimulation of chloride secretion beginning at a tannin concentration of 10 micrograms/ml (0.09 +/- 0.05 microEq/cm2.h [n = 10] increase in Isc and 0.08 +/- 0.03 microEq/cm2.h increase in Jnet with PMA after tannin pretreatment). At 50 micrograms/ml tannin, the stimulatory effect of PMA was completely abolished. The known PKC inhibitor, H-7 (20 microM), inhibited PMA stimulation, while chelerythrine (2 microM) had not effect on PMA-stimulated Isc and Jnet, and calphostin C was toxic to the airway epithelium. In membrane fragments, 2.5 micrograms/ml tannin inhibited the rate of histone III phosphorylation by PMA from 32.1 +/- 4.4 nmol/mg protein per min to 20.1 +/- 2.7 nmol/mg protein per min (n = 7). In bovine airway cells, tannin pretreatment (2.5 micrograms/ml) decreased the cytosolic activity of PKC but had no effect on PKC translocation to the membrane. We conclude that tannin inhibits chloride secretion in airway epithelial cells in part by inhibiting PKC.

The zinc chelator 1,10-phenanthroline enhances the stimulatory effects of protein kinase C activators and staurosporine, but not sphingosine and H2O2, on phospholipase D activity in NIH 3T3 fibroblasts

Protein kinase C (PKC), an enzyme which is believed to mediate the stimulatory effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on phospholipase D (PLD) activity, has a zinc-dependent structure required for phorbol ester binding. Accordingly, zinc or zinc chelators would be expected to promote or inhibit, respectively, the stimulatory effects of PMA on PLD-mediated phospholipid hydrolysis. Instead, treatment of [14C]choline- and [14C]ethanolamine-labelled NIH 3T3 fibroblasts with the high-affinity zinc chelator 1,10-phenanthroline (0.2-1 mM) for 20-30 min was found to enhance the stimulatory effects of PMA on PLD-mediated hydrolysis of phosphatidylcholine and phosphatidylethanolamine. In [14C]palmitic acid-labelled fibroblasts, in the presence of ethanol, phenanthroline also enhanced the stimulatory effect of PMA on the synthesis of phosphatidylethanol, a marker of PLD activity. Addition of zinc (250 microM) to phenanthroline-treated fibroblasts reversed the stimulatory effects of the chelator. The potentiating effects of phenanthroline were also partially reversed by cadmium, whereas iron, lead, copper, magnesium and calcium were without effects. Of the other activators of PLD tested, phenanthroline also enhanced the stimulatory effects of platelet-derived growth factor and staurosporine, but not that of sphingosine and H2O2, on the hydrolysis of both phospholipids. These results suggest that regulation of PLD by PKC activators and staurosporine involves a common intermediate step, which is inhibited by a chelatable cellular pool of zinc.

Field stimulation-induced noradrenaline release from guinea-pig atria is modulated by prejunctional alpha 2-adrenoceptors and protein kinase C.

Guinea-pig left atria were loaded with 10 microCi 7-[3H]noradrenaline, and noradrenaline release from sympathetic nerve endings was then elicited by refractory period field stimulation. When one pulse of 0.2 ms duration was applied during each refractory period, the resulting transmitter release was halved by 3 x 10(-7) mol/l of the alpha 2-adrenoceptor agonist clonidine and increased about 2.5-fold by either 3 x 10(-7) mol/l of the alpha 2-adrenoceptor antagonist idazoxan, 5 x 10(-3) mol/l of the potassium channel blocker tetraethylammonium chloride (TEA) or 3 x 10(-7) mol/l phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC). Phorbol-12-myristate-13-acetate-4-O-methylether, a compound which does not stimulate PKC, was ineffective. The stimulatory effect of PMA was antagonized by 7 x 10(-5) mol/l of the PKC inhibitor polymyxin B. No significant transmitter release was observed when either PMA or TEA was applied together with 10(-7) mol/l of the sodium channel blocker tetrodotoxin. Combinations of either idazoxan and TEA or PMA and TEA caused greater increases of the noradrenaline release than any individual drug given alone. Thus, different mechanisms of action seem to mediate the increase of noradrenaline release by action potential prolongation on the hand and activation of PKC or inhibition of alpha 2-adrenoceptors on the other hand. In contrast, the effects of idazoxan and PMA were not additive, which suggests a common mechanism of action. In atria pretreated for 10 min with 10(-4) mol/l N-ethylmaleimide, an alkylating agent which inactivates Gi-proteins neither idazoxan nor PMA caused a significant increase of the stimulation-induced transmitter release, while TEA was still effective. When a train of four pulses, lasting 0.05 ms each, was applied during each refractory period, the resulting transmitter release was not modified by idazoxan or PMA, but was significantly increased by TEA. From these results, a scheme is proposed which links the regulation of noradrenaline release by prejunctional alpha 2-adrenoceptors and protein kinase C via an influence on a common inhibitory Gi-protein.

Role of protein kinase-C in regulation of insulin-like growth factor-binding protein-1 production by HepG2 cells.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a liver-derived protein that modulates the mitogenic actions of the insulin-like growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in vivo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of protein kinase-C (PKC) on HepG2 cell IGFBP-1 production. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 production in a time- and dose-dependent manner, with maximal stimulation occurring at 10-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than 10-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin did not inhibit PMA stimulation. The transient PKC activator diC8 had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein.

Second Messenger Pathways Mediating Chicken Luteinizing Hormone Secretion from Dispersed Pituitary Cells1

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)

The Stimulation of El-4 Cells to Produce Interleukin-2 and its Potential Use in Immunocytotoxicity Testing

The ability of EL-4 thymoma cells to produce interleukin-2 (IL-2) following exposure to phorbol-12-myristate 13-acetate (PMA) and Concanavalin A (Con A) has been studied in vitro using medium containing either 10% or 1% fetal calf serum (FCS). The potent stimulatory effect of PMA on IL-2 production by EL-4 cells has been confirmed by measuring 3H-thymidine incorporation by the IL-2-dependent T cell line, CTLL-2, in the presence of conditioned medium (CM) from stimulated cultures. EL-4 cells produced several times more IL-2 when cultured in medium containing 10% FCS than when only 1% FCS was present. Added together, PMA and Con A acted synergistically in some EL-4 cell cultures. The ability of E:-4 cells to produce IL-2 was maintained after further incubation without stimulants. CM with IL-2 activity from stimulated EL-4 cells could prove useful in immunotoxicity testing.

Effect of phorbol myristate acetate on secretion of parathyroid hormone

The influence of phorbol myristate acetate (PMA), an activator of protein kinase c, on the secretion of parathyroid hormone from collagenase-dispersed bovine parathyroid cells was tested. The cells were incubated at low (0.5 mM) or high (2.0 mM) concentrations of calcium in the medium, and the hormone secreted into the medium was measured by a radioimmunoassay that recognizes both intact and C-terminal fragments of hormone. At low calcium, the secretory rate averaged 32 +/- 3.8 ng.h-1.(10(5) cells)-1. The addition of 1.6 microM PMA did not affect secretion. At high calcium there was a significant suppression of secretion by 38% to 19.8 +/- 3 ng.h-1.(10(5) cells)-1. The addition of 1.6 microM PMA significantly stimulated hormone secretion to 35.8 +/- 8 ng.h-1.(10(5) cells)-1, a rate indistinguishable from low calcium. This stimulatory effect of PMA at high calcium was seen at PMA concentrations as low as 1.6 nM, did not occur with a biologically inactive 4 alpha-isomer of phorbol ester, and was independent of changes in cellular adenosine 3',5'-cyclic monophosphate levels. Examination of 32P-labeled phosphoproteins by two-dimensional gel electrophoresis revealed acidic proteins of approximately 20,000 and 100,000 Da that were phosphorylated at low and high calcium + 1.6 microM PMA but not at high calcium alone. The protein kinase c activity associated with the membrane fraction of parathyroid cells significantly decreased 40% when the cells were incubated at high vs. low calcium. The data suggest that calcium may regulate parathyroid hormone secretion through changes in protein kinase c activity of the membrane fraction of the cell and protein phosphorylation.

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.

Dual Effect of Phorbol Myristate Acetate (PMA) on Murine Thymocyte Cultures

4β-phorbol 2-myristate 13-acetate (PMA), a potent tumor promoter, was found to have a dual effect on interleukin 1-(IL 1) stimulated murine thymocyte cultures. In the absence of 2-mercaptoethanol (2-ME) or other sulfhydryl containing compounds, PMA inhibited 3H-thymidine incorporation, while at the same concentrations but in the presence of sulfhydryl agents, PMA exerted a potent stimulatory effect. The other sulfhydryl compounds giving effects resembling 2-ME were dithiothreitol and cysteine, while glutathione and various oxygen radical scavengers (catalase, superoxide dismutase, histidine, α-tocopherol or mannitol) had no such effect. 2-ME enhanced the response of thymocytes incubated with PMA even when added 24 h after initiation of the cultures, but not at the 48 h interval. Harvesting the thymocyte cultures at various intervals showed that the inhibitory effect of PMA was already fully pronounced 48 h after initiation of the cultures. On the other hand, the stimulatory effect of PMA on cultures activated with IL 1 in the presence of 2-ME took place mainly during the last 24 h of the 72-h culture period. The modulation of the effect of PMA on thymocyte activation by 2-ME or similar agents may account for the apparent discrepancies in the literature concerning the action of PMA in lymphocyte cultures.

Tumor-promoting phorbol esters stimulate C3b and C3b' receptor-mediated phagocytosis in cultured human monocytes.

Monocytes were isolated in high yield (approximately 80%) and purity (greater than 90%) by Percoll gradient centrifugation and incubated in Teflon culture vessels. Using this culture method, we routinely recovered 80% of the cells originally placed into culture. Studies of the C3b and C3b' receptors of these monocytes showed that the function of both receptors could be dramatically altered by treating the cells with tumor-promoting phorbol esters. Both C3b and C3b' receptors of human monocytes efficiently mediate attachment of erythrocytes coated with the corresponding ligands, but do not promote their ingestion. However, monocytes treated with phorbol myristate acetate (PMA) or phorbol didecanoate ingest C3b- and C3b'-coated erythrocytes. Phorbol esters that are inactive as tumor promoters do not stimulate C3 receptor-mediated phagocytosis. The ability of monocytes to respond to PMA by activation of C3 receptors is developmentally regulated. Freshly isolated monocytes do not take up C3b- or C3b'-coated erythrocytes in response to PMA, but after 3 d of culture they show strong PMA-stimulated uptake. The stimulatory effect of PMA on monocyte C3b and C3b' receptor function occurs within minutes, is stable for hours, is cycloheximide insensitive, and can be inhibited with colchicine. Several lines of evidence indicates that phagocytosis of C3b or C3b'-coated erythrocytes is specifically mediated by the monocytes' C3b and C3b' receptors. First, erythrocytes attached to monocytes with concanavalin A are not ingested when the monocytes are treated with PMA. Second, monocytes plated on IgG-bearing substrates lose Fc receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b- or C3b'-coated erythrocytes after PMA treatment. Third, PMA-treated monocytes plated on C3b-coated surfaces lose C3b receptor activity on their nonadherent surfaces but retain the capacity to ingest C3b'-coated erythrocytes. Conversely, PMA-treated monocytes plated on C3b'-coated surfaces show reduced C3b' receptors activity on their nonadherent surfaces but retain the capacity to ingest C3b-coated erythrocytes.