Presence Of TPA Research Articles

In Vitro Clonal Analysis of Mouse Neural Crest Development

Analysis of lineage segregation during mammalian neural crest development has not been sufficiently performed due to technical difficulties. In the present study, therefore, we established a clonal culture system of mouse neural crest cells in order to analyze developmental potentials of individual neural crest cells and their patterns of lineage segregation. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and cholera toxin (CT) were applied to culture medium to trigger melanogenic differentiation of mouse neural crest cells. Three morphologically distinct types of clones were observed. (1) "Pigmented clones" consisted of melanocytes only, suggesting that the clone-forming cells were committed to the melanogenic lineage. These clones were observed only in the presence of TPA and CT. The proportion of this type of clone (8%) was much lower than that of the equivalent type of clone in quail trunk neural crest (40-60%; Sieber-Blum and Cohen, 1980, Dev. Biol. 80, 96-106). It therefore appears that the segregation pattern to the melanogenic lineage during mouse neural crest development in vitro differs quantitatively from that in the quail. (2) "Mixed clones" consisted of pigmented and unpigmented cells. Like pigmented clones, they were observed only in the presence of TPA and CT. The clones contained up to four types of cells: melanocytes, S100-positive cells (Schwann cells or melanogenic precursor cells), serotonin (5-HT)-positive autonomic neuron-like cells, and substance P (SP)-immunoreactive sensory neuron-like cells. Thus, at least some mixed clone-forming cells are pluripotent. (3) Two classes of "unpigmented clones" were observed that consisted of unpigmented cells only. These clones developed in the presence and absence of TPA and CT. Unpigmented clones in one class contained up to three types of cells as well as other, as yet unidentified cells: S100-, 5-HT-, and SP-positive cells. This observation suggests that at least some of these clones originate from cells with a partially restricted developmental potential. Clones in another class consisted of S100- or SP-positive cells only. These clones might be derived from cells restricted to the SP-positive neuronal cell or melanocyte/Schwann cell lineage. The present data indicate that at initiation of migration, the mouse neural crest of the trunk region is a heterogeneous population of cells containing pluripotent cells, cells with a restricted developmental potential, and cells apparently committed to the melanogenic cell lineage.

Follicle-stimulating hormone and human chorionic gonadotropin induced changes in granulosa cell glycosyl-phosphatidylinositol concentration.

In the present investigation, a hCG sensitive glycosyl-phosphatidylinositol (GPI) was isolated from cultured rat granulosa cells obtained from the ovaries of diethylstilbestrol (DES) implanted immature rats. The inositol-phosphoglycan (IPG) moiety of the GPI-lipid contains galactose, glucosamine, and myoinositol as demonstrated by metabolic labelling of granulosa cells for different time periods (5-96 h) with [3H]galactose, [3H]glucosamine, or [3H]myoinositol and treatment of the purified [3H]GPI with phosphatidylinositol-specific phospholipase C. Labelling equilibrium of the GPI-lipid was achieved after 24 h ([3H]galactose and [3H]myoinositol) or 72 h ([3H]glucosamine) incubation, whereas incorporation of other labelled carbohydrates tested ([3H]galactosamine, [3H]mannose, and [3H]sorbitol) was negligible throughout the time period studied. The glucosamine C-1 appears to be linked through a glycosidic bond to the myoinositol molecule of the IPG moiety as revealed by the generation of phosphatidylinositol (PtdIns) after nitrous acid deamination of dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) glycosyl-phosphatidylinositol. To investigate the fatty acid composition of the diacylglycerol (DAG) backbone of the GPI, granulosa cells were also labelled (5-72 hr) with [14C]linoleate, [3H]myristate, [3H]oleate, [3H]palmitate, or [3H]stearate and the radioactivity associated with the purified glycosyl-phosphatidylinositol determined. Incorporation of [3H]palmitate and [3H]myristate into the GPI-lipid peaked after 8 h and 24 h of labelling, respectively, and both fatty acids were partially released after PLA2 treatment of the dual labelled ([3H]glucosamine/[14C]palmitate or [3H]glucosamine/[14C]myristate) GPI. In parallel experiments no significant incorporation of labelled stearate, oleate, or linoleic acid into the DAG backbone of the glycosylphosphatidylinositol could be detected. Granulosa cells were also labelled with [3H]glucosamine in the presence of FSH (30 ng/ml), cholera toxin (1 microgram/ml), or the membrane permeable cAMP analog (but)2cAMP (1 mM). Time related increases in GPI-labelling were apparent after 48 h and reached a maximum level (3-, 5-, and 7-fold for FSH, CT, and (but)2cAMP, respectively) after 72 h in culture. In another set of experiments, granulosa cells were labelled for 72 h with [3H]glucosamine in the presence of (but)2cAMP (1 mM), TPA (10(-7) M), or combination thereof. The effect of treatment with the membrane permeable cAMP analog on GPI labelling was prevented in the presence of TPA, whereas no differences in [3H]GPI content could be observed in untreated granulosa cells or cells cultured in the presence of the protein kinase C-activating phorbol ester alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible involvement of the base exchange enzymes in the phospholipid metabolism in LAN-2 cells

The incorporation of [14C] choline, [14C] ethanolamine, and [14C] serine by LAN-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no differences between TPA-exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. These results suggest that a choline base exchange enzyme was stimulated in TPA exposed cells. In addition, the enhanced incorporation of serine by TPA into its corresponding phospholipid implies the stimulation of the serine base exchange enzyme which is responsible for phosphatidylserine synthesis in mammals. These observations suggest a

Potentiation of miniature endplate potential frequency by ATP in Xenopus tadpoles.

1. Extracellular application of ATP (1 mM), a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiated the spontaneous secretion of acetylcholine (ACh) but had no effect on the amplitude and decay time constant of miniature endplate potentials (m.e.p.ps) at neuromuscular synapses in Xenopus tadpoles. 2. alpha,beta-Methylene ATP (0.3 mM) and GTP (1 mM) were also effective in increasing m.e.p.p. frequency. On the other hand, ADP, AMP and adenosine (all at 1 mM) decreased m.e.p.p. frequency. 3. Unlike the transient effect of ATP analogue and GTP on m.e.p.p. frequency, the phorbol ester TPA (2 microM) which is a protein kinase C activator, increased m.e.p.p. frequency consistently and the effects lasted as long as the presence of TPA. 4. Staurosporine (0.5 microM) and H-7 (10 microM), which are protein kinase C inhibitors, each decreased the basal level of m.e.p.p. frequency and markedly inhibited the effects of both ATP and TPA. 5. These results suggest that there is a basal activity of cytosolic protein kinases in the nerve terminals of Xenopus tadpoles and the effect of ATP is probably mediated by the binding of membrane surface purinoceptors which in turn activates cytosolic protein kinases and increases ACh release.

Different regulatory patterns of M 1 and M 2 muscarinic receptor subtype RNA in SH-SY5Y human neuroblastoma induced by phorbol ester or DMSO

The human neuroblastoma cells SH-SY5Y were treated with the differentiation agents 12- O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethyl sulfoxide (DMSO) and the muscarinic receptor subtype M 1 and M 2 RNA levels analyzed. After a decrease induced by both agents, the M 1 level gradually returned to normal in the presence of TPA but remained minimal with DMSO. As for M 2, several phases were observed with TPA, while DMSO caused a drastic increase. The data obtained with TPA were tentatively correlated with the amounts of immunoreactive PKCα. In conclusion, our results reveal: (a) differential regulation of M 1 and M 2 muscarinic receptor subtypes by either treatment; (b) opposing effects of TPA and DMSO on both subtypes.

Stimulatory and inhibitory effects of 1,25-dihydroxyvitamin D 3 on thymocyte mitogenesis induced by phorbol ester and calcium ionophore

Mouse medullary thymocytes have specific receptors for 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3). The mitogenic stimulation of these cells by phytohemagglutinin in the presence or absence of the phorbol ester TPA is inhibited by 1,25(OH) 2D 3. The calcium ionophore A23187 did not reverse the inhibition by 1,25(OH) 2D 3 of phytohemagglutinin. Stimulation of thymocytes with either TPA or A23187 alone did not result in proliferation. Co-stimulation of the thymocytes with TPA and A23187 induces cell proliferation. 1,25(OH) 2D 3 markedly enhanced the TPA and A23187-induced cell proliferation even when added 4 h after the initiation of the culture. In contrast, DNA synthesis by thymocytes incubated for 4 h in the presence of TPA and A23187 and then cultured in medium containing 1,25(OH) 2D 3 but in the absence of both TPA and A23187, was inhibited by 1,25(OH) 2D 3. The extent of inhibition was comparable to the inhibition of lectin-induced stimulation by the hormone. Using monoclonal antibodies to neutralize IL-2 and block IL-2 receptors we showed that 1,25(OH) 2D 3 enhanced the IL-2-independent component of the A23187- and TPA-induced mitogenesis. In conclusion: (1) The nature and presence of the mitogenic signal determines whether 1,25(OH) 2D 3 enhances or inhibits thymocyte stimulation. (2) Both stimulatory and inhibitory actions of 1,25(OH) 2D 3 seem to take place at points distal to the initial increase in intracellular calcium or activation of protein kinase C.

Regulation of cell division of mature B cells by ionomycin and phorbol ester.

The growth of a human B lymphoma cell line B104, an experimental model for mature B cells, was inhibited by ionomycin but not 12-O-tetradecanoylphorbol-13-acetate (TPA). Ionomycin inhibited B104 cells from entering into the M phase of the cell cycle without affecting DNA synthesis. The inhibition of cell division of B104 cells by ionomycin occurred within 24 h after stimulation. Because such a mode of action resembles that of anti-IgM antibodies, signals transduced by Ca2+ may be responsible for the inhibition of cell division of B104 cells by anti-IgM antibodies. Indeed, EGTA suppressed the inhibition of cell division of B104 cells caused not only by ionomycin, but also by anti-IgM antibody. Although TPA itself did not have any ability to promote the growth of B104 cells, it could cancel the inhibition of cell division of B104 cells by ionomycin and increase the proportion of B104 cells entering into the M phase of the cell cycle. Staphylococcus aureus Cowan I causes the greatest proliferation of normal human peripheral blood B cells during the period from 48 to 72 h after stimulation. When ionomycin was added to S. aureus Cowan I-stimulated peripheral blood B cells at 48 h of culture, it inhibited cell division during this period without affecting DNA synthesis. In the presence of TPA, this activity of ionomycin was suppressed, and the proportion of M-phase cells increased. These results suggest that cell division of mature B cells is regulated by the signals mediated by Ca2+ and protein kinase C in a mode quite different from that of regulation of DNA synthesis.

Protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate modulates messenger ribonucleic acid levels for two of the regulatory subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinases (RII beta and RI alpha) via multiple and distinct mechanisms.

Messenger RNAs (mRNA) for two of the regulatory subunits of cAMP-dependent protein kinases (PKA), RII beta and RI alpha, are transiently (maximal levels at 6 h) stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in cultured rat Sertoli cells in a time- and concentration-dependent manner. Whereas TPA (10(-7) M) stimulated RII beta mRNA 11 +/- 2.8 fold (mean +/- SEM), mRNA levels for RI alpha increased only 2.5 +/- 0.6-fold (mean +/- SEM). No effects of TPA on the other subunits of PKA (RII alpha, C alpha) were observed. TPA-dependent accumulation of mRNAs for RII beta and RI alpha was observed to the same extent in nucleus and cytoplasm. We have previously shown that mRNA levels for all the PKA subunits are increased by cAMP, particularly that of RII beta (greater than 50-fold). TPA modulated the stimulatory effects of cAMP on RII beta and RI alpha mRNAs in opposite directions. Whereas treatment with both 8-CPTcAMP and TPA gave an additive effect on RI alpha mRNA, TPA reduced the cAMP-dependent increase in RII beta mRNA. Although the mRNA for RII beta had returned to basal levels after 24 h of incubation with TPA, the presence of TPA still inhibited cAMP-dependent induction of mRNA for RII beta. In contrast, similar TPA treatment did not influence the subsequent cAMP-dependent stimulation of RI alpha mRNA. Preincubation with 8-CPTcAMP did not influence TPA-dependent stimulation of mRNAs for either RII beta or RI alpha. TPA induction of RII beta mRNA was completely blocked by cycloheximide (an inhibitor of protein synthesis), whereas that of RI alpha was not. The inhibitory effect of TPA on cAMP stimulation of RII beta mRNA was independent of ongoing protein synthesis. These results indicate that TPA induction of mRNAs for RI alpha and RII beta involves multiple and distinct mechanisms. The stimulatory effect of TPA on RI alpha mRNA levels and the inhibitory effect of TPA on cAMP-stimulated RII beta mRNA expression are probably mediated through stable factors, whereas proteins with rapid turnover or factors induced by TPA are involved in the stimulatory effect of TPA on RII beta mRNA.

Effect of a phorbol ester on immunoreactive endothelin-1 release from cultured porcine aortic endothelial cells.

This study was designed to examine how protein kinase C (PKC) regulates the release of endothelin-1 (ET-1) from cultured porcine aortic endothelial cells. We measured the release of immunoreactive (IR)-ET-1 from cells cultured for up to 72 h in the presence or absence of a phorbol ester TPA. The release of IR-ET-1 from control cells (no TPA) increased according to time for up to 72 h. In the presence of TPA, the release of IR-ET-1 from the cells was higher than the control level for up to 8 h, but was lower thereafter and reached a plateau after 48 h. TPA dose-dependently stimulated IR-ET-1 release during incubation for 4 h, but suppressed it after incubation for 72 h. Stimulation of PKC by diacylglycerol mimicked the early (4 h) action of TPA. On the other hand, pretreatment of cells with TPA to downregulate PKC significantly suppressed basal and thrombin- or FCS-stimulated IR-ET-1 release. These findings suggest that the activation of PKC is related to the stimulation of ET-1 release and that down-regulation of PKC leads to the suppression of ET-1 release from cultured endothelial cells.

Radiomimetic cell cycle delay induced by tetranodecanoyl phorbol acetate is enhanced by caffeine and by the protein kinase inhibitor 2-aminopurine.

The tumour promoter and protein kinase C agonist, 12-O-tetranodecanoyl-phorbol-13-acetate (TPA), has been reported to show a radiomimetic action because it transiently delays the passage of HeLa cells through the G2 phase, as do ionizing radiation and other DNA damaging agents. Caffeine is known to override the G2 delay imposed by DNA damage; it is shown here that caffeine does not override the radiomimetic delay imposed by TPA in HeLa, but instead enhances it, without affecting G2 progression in control cells. Most of the other agents which more specifically affect some of the diverse range of caffeine targets either do not affect G2 progression after TPA, or delay G2 progression in control cells and exert a further delay in the presence of TPA. The exception is 2-aminopurine, a protein kinase inhibitor which has been shown to have an action similar to that of caffeine is allowing progression of the cell cycle to mitosis after the inhibition of DNA synthesis, without affecting normal cycle progression through G2. This agent, like caffeine, also has the contrary action of retarding cycle progression after TPA. It is concluded that the G2 delays induced by ionizing radiation and by TPA operate by different mechanisms, which are modulated in opposite senses by mechanisms involving protein kinase inhibition.

Potassium transfer assay for cell communication: effects of phorbol esters, retinoic acid, and furosemide

Using a mass culture assay for the contact-dependent transfer of potassium among cells with intrinsic differences in ability to concentrate it, we have investigated the ability of several drugs to influence this form of cell communication. We concentrated on 12-O-tetradecanoylphorbol-13-acetate (TPA), which is known to interfere with gap junction-mediated communication and ion transport in several other systems, and compared its effects with those of its inactive derivative, 4-O-methyl-TPA. We found that the communication between mouse BALB/c 3T3 cells and human diploid fibroblasts was reduced in the presence of TPA but not O-methyl-TPA and that this inhibition was not obscured by small but measurable influences of TPA on steady-state content and transport of 86Rb+. We confirmed these findings using an autoradiographic assay for transfer of uridine derivatives among cells in contact. We also showed that retinoic acid had no effect on communication in the ion transfer assay but that furosemide, an inhibitor of Na(+)-K(+)-2Cl- cotransport, stimulated ion transfer dramatically both in the presence and absence of TPA. These results indicate both the promise and the limitations of the potassium transfer assay for identifying potential modulators of gap junction-mediated cell communication.

The level of substrate ornithine can alter polyamine‐dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

Although the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decarboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady-state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12-O-tetradecanoylphorbol-beta-acetate (TPA) in the presence of serum (Wu and Byus: (Biochem. Biophys. Acta 804:89-99, 1984); Wu et al.: (Cancer Res. 41:3384-3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum-free conditions for 2-3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum-containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum-restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4-5-fold increase in intracellular steady-state ornithine levels and by a 6-8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984] the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3-5-fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA-induced DNA synthesis was further stimulated more than twofold in a dose-dependent manner. Insulin (10(-10)-10(-8) M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady-state level of ornithine and putrescine in comparison with TPA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of A T lymphocyte mutant defective in the protein kinase C signal transduction pathway

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.

Different Mechanisms of β‐Adrenoceptor Down‐Regulation by Chronic Imipramine and Electroconvulsive Treatment: Possible Role for Protein Kinase C

The aim of this study was to find out how protein kinase C (PKC) is involved in down-regulation of the beta-adrenoceptor in cortical slices of rats subjected to antidepressant treatments. The responses of the cyclic AMP generating system to forskolin, isoproterenol, and noradrenaline were tested in the absence and presence of a PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). The antidepressive treatments applied were chronic administration of imipramine and electroconvulsive shock. The potentiating effect of the phorbol ester on cyclic AMP response to isoproterenol was retained in imipramine-treated animals and even accentuated in rats subjected to electroconvulsive treatment; the TPA effect on noradrenaline-induced cyclic AMP response was blunted in rats receiving imipramine, but augmented in those receiving electroconvulsive treatment. In imipramine-treated rats the beta-down-regulation was still evident in the presence of TPA; after electroconvulsive treatment the phorbol ester-induced potentiation was so high that no significant beta-down-regulation could be observed. No procedure affected the response to forskolin. The beta-down-regulation that develops during chronic imipramine treatment differs from that caused by chronic electroconvulsive treatment; in both cases it is not related to the direct effect on adenylate cyclase.

Antimitogenic Effects of Dexamethasone in Chemically Transformed Mouse Fibroblasts

Dexamethasone (a synthetic glucocorticoid) inhibited the entry into the S-phase of quiescent chemically transformed mouse fibroblasts (BP-A31) stimulated with 12-O-tetradecanoyl 13-acetate (TPA; a protein kinase-C activator) or with basic fibroblast growth factor. The basal rate of DNA synthesis was also strongly reduced by dexamethasone. In contrast, the mitogenic activity of insulin (acting via the insulin-like growth factor-I receptor) was little or not at all affected by dexamethasone. The antimitogenic activity of dexamethasone was enhanced when the steroid was included in the culture medium 24 h before the addition of mitogens. The effects of dexamethasone were glucocorticoid specific, partially reversed by the antiglucocorticoid RU 486, and prevented by cycloheximide (suggesting the involvement of glucocorticoid-induced protein synthesis in the antimitogenic activity of dexamethasone). Under the conditions of exponential growth in serum-free medium as well as in the presence of TPA, dexamethasone arrested the proliferation of sparsely seeded cells after a delay of 24-48 h. The BP-A31 cells are known to be constitutively competent and express at quiescence certain genes related to the G0/G1 transition in the original nontransformed A31 cell line. Of the transcripts corresponding to these genes, dexamethasone caused a rapid elimination of the JE mRNA, coding for a protein of the family of cytokines. The cell content of c-jun mRNA was also strongly reduced in the cells incubated at quiescence with dexamethasone (in the absence of mitogen). The presence of TPA along with dexamethasone prevented the elimination of c-jun, but not of JE mRNA. Short (30-min; together with the inducers) or long (24-h) treatment of the cells with dexamethasone did not prevent the induction of the c-fos gene expression by either TPA or basic fibroblast growth factor, indicating that dexamethasone does not interfere with mitogenic signal transduction. We conclude that in TPA-stimulated cells, the antiproliferative effect of dexamethasone is not due to interference with the expression of the c-jun gene, but may be related to the decreased level of the JE cytokine mRNA as well as to the synthesis of growth inhibitory protein(s).

Down-Regulation of Messenger Ribonucleic Acid and Protein Levels for Estrogen Receptors by Phorbol Ester and Calcium in MCF-7 Cells*

Treatment of MCF-7 cells, a human mammary carcinoma cell line, with phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] or calcium ionophore (A23187) was associated with striking effects on levels of estrogen receptor (ER) mRNA, specific binding of 17 beta-[3H]estradiol [( 3H]E2), and immunoreactive ER. TPA (10(-7) M) caused a time-dependent reduction of ER mRNA which was below the level of detection after 9 h. Similar effects of TPA appeared at levels of specific binding of [3H]E2 as well as immunoreactive ER. In contrast, TPA induced an increase in mRNA for beta-actin. Incubation of MCF-7 cells with increasing concentrations of TPA (10(-11)-10(-7) M) was associated with biphasic effects on ER mRNA and proteins. Levels of immunoreactive progesterone receptors (PR) were induced by E2 (10(-9) M) in a time-dependent manner. In the presence of TPA (10(-7) M), where ER levels were suppressed, no induction of PR was observed. Removal of TPA (10(-7) M) after 10 h (ER mRNA) or 22 h (ER proteins) of treatment was associated with a continued suppression of both mRNA and protein levels during the entire incubation period (48 h). Treatment with A23187 (2 x 10(-7) M) also caused a time-dependent down-regulation of levels of ER mRNA and proteins. These effects occurred somewhat more slowly than those of TPA. Levels of beta-actin mRNA were not changed by this treatment. These results indicate that changes in estrogen sensitivity are mediated by calcium-dependent protein kinases in human mammary carcinoma MCF-7 cells.

NADPH-oxidase activation by protein kinase C-isotypes

The cell free activation of NADPH-Oxidase in membranes of mouse peritoneal macrophages by purified PKC-isotypes was investigated. Unstimulated intrinsic activity of PKC-isotypes showed little dependence on Ca 2+ for activation of the oxidase. In the presence of TPA, the activation of the oxidase was greatly enhanced, and α-, and γ-subtypes were strongly Ca 2+ dependent in this system. β-, δ- and ϵ-subtypes were active both in the presence and absence of free Ca 2+ ions. The results suggest that at resting Ca 2+ levels certain PKC-isotypes can activate NADPH-oxidase.

Phorbol ester-induced inhibition of proliferation of Daudi Burkitt's lymphoma cells by impairment of cytokinesis

The phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA) exerts a dose-dependent effect on Daudi cell proliferation. A low concentration has a slight mitogenic effect but higher concentrations inhibit proliferation. The inhibitory effect is associated with increases in cell size, macromolecular content, and incorporation of precursors into RNA and protein. Cell cycle analysis indicates that TPA at 1–10 n M leads to an apparent accumulation of cells in G 2 M phase. However, within this population a significant proportion of cells undergo nuclear division but fail to carry out cytokinesis, giving rise to cells with two or more nuclei. Consistent with this, DNA synthesis continues in cells which cease to divide in the presence of TPA. The ability of the phorbol ester to inhibit proliferation can thus be attributed mainly to an inhibition of cytokinesis rather than DNA replication

Insulin-Like, Antilipolytic Effect of 12-O-Tetradecanoyl Phorbol 13-Acetate in Rat White Adipocytes

Previous experimental data documenting an insulin like-effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA), a specific activator of protein kinase C, on glucose transport in adipocytes prompted us to test the hypothesis that TPA might display another insulin-like effect, i.e., antagonize catecholamine-induce lipolysis. TPA (100 nM) led to a decrease of both free fatty acid (41%) and glycerol (58%) release due to 1 microM norepinephrine stimulation in isolated rat adipocytes. TPA also diminished the antilipolytic effect of insulin (5 ng.ml-1) in the presence of 1 microM norepinephrine. Thus, the residual lipolysis with insulin was 25% for free fatty acids and 24% for glycerol release. In the presence of TPA, these values increased to 50% and 45%, respectively. TPA (100 nM) addition to isolated adipocytes induced protein kinase C translocation from the cytosol to the membrane fraction. In control cells, 94.7 +/- 2.9% of the enzyme was found in the cytosol, with the rest found in the membrane. At 10 min after TPA (100 nM) addition, the corresponding value was 43.6 +/- 17.4%, with the rest in the membrane (n = 6, P less than 0.05). These findings indicate that protein kinase C might be involved not only in the insulin action on glucose transport, but also in the mechanism of insulin's antilipolytic action.

A selective effect of protein kinase C activators on noradrenaline release compared with subsequent contraction in canine isolated saphenous veins

1. Effects of protein kinase C (PKC) activators and inhibitors on both tritium overflow and subsequent contraction evoked by transmural nerve stimulation (TNS) were investigated in canine saphenous veins prelabelled with [3H]-noradrenaline. 2. Activation of PKC by stepwise increasing concentrations (0.01 nM-1 microM) of 12-O-tetradecanoylphorbol 13-acetate (TPA), phorbol 12,13-dibutyrate (PDBu) or mezerein caused a significant and concentration-dependent enhancement of the tritium overflow evoked by TNS, while the activators failed to affect the corresponding contraction except with the highest concentration of PDBu when the contraction was significantly reduced. Phorbol, which is inactive on PKC, had no effects on the tritium overflow and contraction induced by TNS. 3. PKC inhibitors, polymyxin B (1 and 10 microM) and the isoquinolinesulphonamide, H-7 (1 microM), inhibited significantly the phorbol ester-potentiated tritium overflow evoked by TNS with no effects on the contraction. H-7 and the related inhibitor H-8 at 10 microM reduced significantly both responses to TNS in the presence of TPA, while they suppressed only the TNS-induced contraction in the absence of TPA. 4. None of the PKC activators or inhibitors affected the spontaneous tritium overflow. 5. PDBu (0.01 and 0.1 microM) elevated resting tension of the veins more effectively than TPA and mezerein. 6. These results suggest that PKC may modulate electrically stimulated noradrenaline release from adrenergic nerve endings of the canine saphenous veins and the PKC activators may act more selectively on presynaptic than postsynaptic sites, but have no apparent effect on postjunctional noradrenergic mechanisms.