Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes

To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.     

Polymyxin B causes coordinate inhibition of phorbol ester-induced C-kinase activity and proliferation of B lymphocytes

Lymphocytes were found to be rich in phospholipid/Ca2+-dependent (C-kinase) activity. Addition of polymyxin B (PMB) to in vitro assays of endogenous and exogenous phosphorylation resulted in profound inhibition of C-kinase activity. The phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) directly activated C-kinase, leading to increased phosphorylation of the same substrates. TPA also stimulated proliferation of B cells as assessed by 3H-thymidine uptake, and PMB strongly inhibited this effect. This coordinate inhibition of TPA-induced phosphorylation and mitogenesis indicates that PMB is a potentially useful inhibitor of C-kinase activity, and that this enzyme may play an important role in mediating B cell responses.     

Response of hepatic ornithine decarboxylase and polyamine concentration to surgical stress in the rat: evidence for a permissive effect of catecholamines on glucocorticoid action

The effects of dibutyryl cAMP (Bt2cAMP) and phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate (PDD) on VIP/PHM-27 gene expression in human neuroblastoma cells in culture were investigated. Bt2cAMP and phorbol esters increased the VIP/PHM-27 mRNA level by about 9- and 4-fold, respectively. In the presence of both Bt2cAMP and phorbol esters, the VIP/PHM-27 mRNA level increased by about 36-fold. The intracellular cAMP level was essentially unaffected by phorbol esters. The VIP/PHM-27 gene dosage was unchanged by Bt2cAMP and phorbol esters. The results suggest that cAMP and phorbol esters synergistically induce the VIP/PHM-27 gene expression through independent pathways.     

Involvement of protein kinase C in the regulation of oocyte maturation in amphibians (Rana dybowskii)

Ovarian oocytes of Rana dybowskii, isolated early in the hibernation period (late autumn), failed to mature, i.e., germinal vesicle breakdown (GVBD), in response to progesterone during in vitro follicle culture. Oocytes collected during the middle hibernation period matured in response to progesterone, whereas those collected late during the hibernation period (close to the breeding season) underwent spontaneous maturation without added hormone (Kwon et al., '89). The maturational response (GVBD) of oocytes, collected at the three stages of hibernation, to protein kinase C (PKC) activation was investigated and compared to that of progesterone stimulation. A phorbol ester, phorbol 12-myristate 13-acetate (TPA) was used for PKC activation. TPA addition to cultured follicles collected during the early or middle period of hibernation induced oocyte GVBD. The incidence of maturation (% GVBD) induced by TPA varied markedly between animals. TPA (10 microM) induced oocyte maturation in the presence or absence of follicle cells. The time course of the TPA-induced maturation was similar to that of progesterone-stimulated maturation (ED50, 7-9 h). TPA also accelerated the onset of maturation of the follicular oocytes exhibiting spontaneous in vitro maturation. Both TPA- and progesterone-stimulated maturation was blocked by treatment with cycloheximide (1 microgram/2 ml), forskolin (9 microM) (an adenylate cyclase stimulator), and verapamil (0.27 mM) (a calcium transport blocker). Treatment of oocytes with a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (100 microM) or a PKC inactivator 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) (50 microM) likewise suppressed TPA- or progesterone-induced maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phorbol ester on tyrosine hydroxylase phosphorylation and activation in cultured bovine adrenal chromaffin cells

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused phosphorylation of phosphoproteins of 56-kDa which co-migrated with and had identical pI values to subunits of tyrosine hydroxylase. The phosphorylation was closely correlated with an increase of [3H]3,4-dihydroxyphenylalanine (DOPA) production which is a reflection of increased tyrosine hydroxylase activity. Only those phorbol esters which activate protein kinase C induced phosphorylation of the 56-kDa proteins and increased [3H]DOPA production. Neither TPA-induced phosphorylation of the 56-kDa proteins nor TPA-induced enhancement of [3H] DOPA production required extracellular Ca2+. TPA caused increases in phosphorylation of the 56-kDa proteins and increases in [3H]DOPA production over similar concentration ranges (10-1000 nM). TPA did not increase cellular cAMP. The data suggest that phorbol ester-induced phosphorylation of intracellular tyrosine hydroxylase, possibly by protein kinase C, results in increased tyrosine hydroxylase activity.     

Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation

Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction.     

Decreased incidence of gap junctions between Chinese hamster V-79 cells upon exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate

Previous studies have shown that metabolic cooperation between Chinese hamster V-79 cells is inhibited by the tumor-promoting phorbol esters, but not by those phorbol esters inactive in tumor promotion. Metabolic cooperation is believed to be mediated by gap junctions. Therefore, in the present study we have used freeze-fracture and quantitative morphological techniques to examine the effect of the most potent tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of the inactive analog, 4α-phorbol-12,13-didecanoate (4α-PDD) on the frequency of gap-junctional contacts between V-79 cells. In both control and in 4α-PDD treated cultures the junctions were frequent, though rather small in size. In contrast, in the TPA-treated cultures, gap junctions were few, and the area of membrane occupied by gap junctions was reduced more than 20-fold from that found for controls. These results suggest that the TPA-induced inhibition of metabolic cooperation between V-79 cells is a consequence of a decrease in the number of gap junctions.     

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.     

Phorbol Ester-Induced Upregulation of Angiotensin II Receptors in Neuronal Cultures Is Potentiated by a Calcium Ionophore

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.     

Phorbol ester-induced down-regulation of the 80-kDa myristoylated alanine-rich C-kinase substrate-related protein in Swiss 3T3 fibroblasts. Inhibition by staurosporine.

A polyclonal antiserum raised against an oligopeptide with an amino acid sequence corresponding to a sequence of the myristoylated alanine-rich C-kinase substrate (MARCKS) from mouse macrophages and rat brain recognizes the 80-kDa C-kinase substrate from Swiss 3T3 fibroblasts. Using this antiserum for quantitative determination of the 80-kDa MARCKS-related protein, we found that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid down-regulation of this protein in the fibroblasts. In accordance with earlier reports, TPA causes phosphorylation of the 80-kDa protein which can be inhibited by staurosporine. Staurosporine also suppresses the TPA-induced down-regulation, possibly indicating that the down-regulation of the MARCKS-related protein is dependent on its phosphorylation by protein kinase C.     

Specific binding and biological effects of tumor promoting phorbol esters on sponges

Sponges grown in the presence of 12-O-tetradecanoyl phorbol-13-acetate (TPA) show deep alterations of their structure and development. Their aquiferous system (flagellated cells and canals) is largely altered and the tissues show an unusually high cell density. This focalized effect of TPA on the aquiferous system seems specific and is reversible at low concentrations (100 ng/ml). A toxic, non-specific effect is also noted, particularly at high concentrations (5000 ng/ml). Using 3H-phorbol-12, 13-dibutyrate (3H-PDBu), we demonstrate a class of specific binding sites for phorbol esters in the homogenates of sponges. These binding sites have high affinity (Kd = 26.0 nM) for PDBu and at saturation about 20 pmoles of 3H-PDBu is bound per mg protein of sponge homogenates. The binding of 3H-PDBu was inhibited by other phorbol esters and their congeners, and there was a good correlation between their potency in binding inhibition and their tumor promoting activity. It is concluded that sponges have a class of specific saturable and high affinity receptors for phorbol esters and that there is a very high conservation of these receptors during evolution. Such specific binding may be responsible for subsequent biological effect of TPA on sponges.     

Phorbol esters stimulate phosphate accumulation synergistically with A23187 in cultured renal tubular cells

The effects of phorbol esters and diacylglycerol on phosphate accumulation in the cultured mouse kidney cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on the renal phosphate handling. 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated phosphate accumulation dose-dependently. TPA-induced phosphate accumulation was synergistically enhanced with A23187. 4 alpha-phorbol 12,13-didecanoate did not stimulate the phosphate accumulation, while 4 beta-phorbol 12,13-didecanoate stimulated it. Additionally, 1-oleoyl-2-acetyl-glycerol exhibited a stimulatory effect on phosphate accumulation. These data indicated that protein kinase C is one of possible regulators of phosphate transport at the renal tubules.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulates somatostatin release from isolated perfused rat stomach

Effect of TPA (12-O-tetradecanoyl phorbol-13-acetate), a potent tumor promoter, on immunoreactive somatostatin release was investigated using the isolated perfused rat stomach. TPA at the concentration as low as 20nM significantly stimulated the somatostatin release from isolated perfused rat stomach. The integrated net output of somatostatin induced by TPA was dose-dependent in a range of 5 - 50nM TPA. Since TPA is known to activate C-kinase specifically at a low dose (less than 20nM), these findings suggest that C-kinase system may be involved in the regulation of somatostatin release in rat stomach.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.