急、慢性乙醇处理后大鼠伏核内cAMP反应元件结合蛋白磷酸化的变化

采用免疫组织化学的方法,检测急性、慢性乙醇作用及戒断后大鼠伏核内cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)磷酸化的变化。结果显示,急性腹腔注射乙醇后15min,伏核内磷酸化CREB(Phospho-CREB,p-CREB)蛋白明显增加,30min后达高峰,至1和6h后仍明显高于对照组。而慢性饮乙醇溶液显著降低大鼠伏核内P—CREB蛋白含量,在撤除乙醇后24、72h时,伏核内p—CREB蛋白含量仍明显较低,戒断后7d,恢复到正常水平。结果表明,急性乙醇处理增加伏核内CREB磷酸化作用,而慢性乙醇作用则降低伏核内CREB磷酸化作用,这可能是乙醇依赖的分子机制之一。     

Tyrosine Kinase Phosphorylation of GABAA Receptor α1, β2 and γ2 Subunits Following Chronic Intermittent Ethanol (CIE) Exposure of Cultured Cortical Neurons of Mice

There is evidence that many of the GABA_A receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol’s regulation of GABA_A receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the β₂- and γ₂- subunits and no effect on α₁-subunit of the GABA_A receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA_A receptor subunits (α₁, β₂, and γ₂) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques. We observed that there was an up-regulation in the tyrosine kinase phosphorylation of the GABA_A receptor β₂- and γ₂-subunits following CIE exposure, and no effect on α₁-subunit in the cultured cortical neurons of mice. These CIE changes, unlike CE, were not reverted back to the control level following ethanol withdrawal even after 7 days. Acute exposure of ethanol did not cause any change in the tyrosine kinase regulation of the GABA_A receptor subunits. In conclusion, the CIE exposure, unlike chronic/acute ethanol exposure, regulates the tyrosine kinase phosphorylation of the selective population of GABA_A receptors in a long lasting manner.     

Association of protein kinase C with GABA(A) receptors containing alpha1 and alpha4 subunits in the cerebral cortex: selective effects of chronic ethanol consumption

Previous studies have suggested that protein kinase C (PKC) isoforms differentially influence the sensitivity of gamma-aminobutyric acid(A) (GABA(A) ) receptor responses in brain. Both PKCgamma and PKCepsilon knock-out mice exhibit altered ethanol potentiation of GABA(A) receptor mediated Cl(-) flux. Furthermore, chronic ethanol consumption alters GABA(A) receptor function and receptor subunit peptide expression by mechanisms that are not yet understood. The present study explored the possibility that PKC isoforms are directly associated with GABA(A) receptors, and this association is influenced by chronic ethanol exposure. GABA(A) receptors containing alpha1 or alpha4 subunits were immunoprecipitated from solubilized protein derived from the membrane fraction of rat cerebral cortex using selective antibodies. Immunoprecipitated receptors were screened by western blot analysis for the presence of PKCdelta, gamma and epsilon isoforms. We found pronounced labeling of PKCgamma but not PKCdelta or PKCepsilon in the alpha1 and alpha4 subunit immunoprecipitates. Immunoprecipitation with PKCgamma, but not with IgG antibody also yielded GABA(A) receptor alpha1 and alpha4 subunits in the immunoprecipitate. The association of PKCgamma with alpha1-containing receptors was decreased 44 +/- 11% after chronic ethanol consumption. In contrast, PKCgamma associated with alpha4-containing receptors was increased 32 +/- 7% after chronic ethanol consumption. These results suggest that PKCgamma may be involved in GABA(A) receptor adaptations following chronic ethanol consumption.     

Effect of Chronic Administration of Ethanol on the Regulation of Tyrosine Kinase Phosphorylation of the GABA<Subscript>A</Subscript> Receptor Subunits in the Rat Brain

One of the many pharmacological targets of ethanol is the GABA inhibitory system, and chronic ethanol (CE) is known to alter the polypeptide levels of the GABA_A receptor subunits in rat brain regions. In the present study, we investigated the regulation of the tyrosine kinase phosphorylation of the GABA_A receptor α₁-, β₂- and γ₂-subunits in the rat cerebellum, cerebral cortex and hippocampus following chronic administration of ethanol to the rats. We observed either down-regulation or no change in the tyrosine kinase phosphorylation of the α₁ subunit, whereas there was an up-regulation or no change in the case of β₂- and γ₂-subunits of the GABA_A receptors depending on the brain region following chronic administration of ethanol to the rats. These changes reverted back to the control level following 48 h of ethanol-withdrawal. These results suggest that tyrosine kinase phosphorylation of GABA_A receptors may play a significant role in ethanol dependence.     

Acute and Chronic Ethanol Consumption Effects on the Immunolabeling of Gq/11α Subunit Protein and Phospholipase C Isozymes in the Rat Brain

Abstract: The goal of this investigation was to examine whether postreceptor sites [G_q/11 protein and phospholipase C (PLC) isozymes] of the phosphoinositide signal transduction system are involved in neuroadaptational mechanisms in the brain during chronic ethanol consumption. It was observed that acute ethanol treatment has no effect on the immunolabeling of PLC-β₁, -γ₁, and -δ₁ and the α subunit of G_q/11 protein in the rat cortex as determined by western blotting using specific monoclonal antibodies. On the other hand, chronic ethanol consumption (15 days) resulted in a significant decrease in the immunolabeling of PLC-β₁, whereas under identical conditions, the immunolabeling of PLC-γ₁ and -δ₁ isozymes was not significantly altered. The decreased immunolabeling of PLC-β₁ during chronic ethanol consumption was not altered by 24 h of withdrawal after 15 days of ethanol consumption. The immunolabeling of the α subunit of G_q/11 protein was significantly decreased after 15 days of ethanol consumption but had returned to normal levels after 24 h of ethanol withdrawal. Also, chronic ethanol treatment resulted in a significant decrease in phosphatidylinositol 4,5-bisphosphate-specific PLC activity, which remained the same after 24 h of ethanol withdrawal. These results suggest that decreased PLC activity during ethanol consumption and its withdrawal may be due to decreased protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme but not the PLC-γ₁ or -δ₁ isozyme in the rat cortex. It is possible that changes in the protein levels of the G_q/11 protein-coupled PLC-β₁ isozyme and in PLC activity in the brain may be involved in the cellular adaptation to chronic ethanol exposure.     

Differential signaling and hypertrophic responses in cyclically stretched vs endothelin-1 stimulated neonatal rat cardiomyocytes

Numerous neurohumoral factors such as endothelin (ET)-1 and angiotensin (Ang) II as well as the stretch stimulus act concertedly in the in vivo overloaded heart in inducing hypertrophy and failure. The primary culture of rat neonatal cardiomyocytes is the only in vitro model that allows the comparative analysis of growth responses and signaling events in response to different stimuli. In the present study, we examined stretched rat cardiomyocytes grown on flexible bottomed cultured plates for hypertrophic growth responses (protein synthesis, protein/DNA ratio, and cell volume), F-actin filaments rearrangement (by confocal, laser scanning microscopy), and for signaling events (activation of phospholipase C [PLC-β, protein kinase C [PKC], mitogenactivated protein [MAP] kinases] and compared these responses with ET-1 (10⁻⁸ M)-stimulated cells. Cyclic stretch for 48 h induced hypertrophic growth in cardiomyocytes indicated by increases in the rate of protein synthesis, cell volume, and diameter, which were less pronounced in comparison to stimulation by ET-1. During cyclic stretch, we observed disoriented F-actin, particularly stress-fibers whereas during ET-1 stimulation, F-actins rearranged clearly in alignment with sarcomeres and fibers. The upstream part of signaling by cyclic stretch did not follow the PLCβ-PKC cascade, which, in contrast, was strongly activated during ET-1 stimulation. Cyclic stretch and, to greater extent, ET-1 stimulated downstream signaling through ERK, p38 MAP kinase, and JNK pathways, but the, involvement of tyrosine kinase and PI3 kinase-Akt signaling during cyclic stretch could not be proven. Taken together, our results demonstrate that both cyclic stretch and ET-1 induce hypertrophic responses in cardiomyocytes with different effects on organization of F-actin stress fibers in case of stretch. Furthermore, on the short-term basis, cyclical stretch, unlike ET-1, mediates its hypertrophic response not through activation of PLC-β and PKC but more likely through integrin-linked pathways, which both lead to downstream activation of the MAP kinase family.     

Ethanol Alters Glutamate but Not Adenosine Uptake in Rat Astrocytes: Evidence for Protein Kinase C Involvement

Glutamate is the primary excitatory neurotransmitter in brain. By stimulating neuronal activity, glutamate increases cellular energy utilization, enhances ATP hydrolysis and promotes the formation of adenosine. Adenosine has receptor-mediated effects that reduce or oppose the excitatory effects of glutamate. As a possible mechanism for ethanol's ability to inhibit excitatory effects of glutamate and enhance inhibitory effects of adenosine, we tested the hypothesis that ethanol promotes [³H]glutamate uptake and inhibits [³H]adenosine uptake. Using primary cultures of rat astrocytes, we found that acute treatment with ethanol (50 mM, 30 min) inhibited [³H]glutamate uptake and reduced protein kinase C (PKC)-induced stimulation of [³H]glutamate uptake. Prolonged treatment (50 mM, 3 day) with ethanol, however, increased both [³H]glutamate uptake and PKC activity. Contrary to other cell types, neither acute or chronic ethanol exposure affected [³H]adenosine uptake in astrocytes. These data indicate that in rat cortical astrocytes ethanol affects [³H]glutamate uptake but not [³H]adenosine uptake by affecting PKC modulation of transporter activity.     

A kinetic model of phospholipase C-γ1 linking structure-based insights to dynamics of enzyme autoinhibition and activation

Phospholipase C-γ1 (PLC-γ1) is a receptor-proximal enzyme that promotes signal transduction through PKC in mammalian cells. Because of the complexity of PLC-γ1 regulation, a two-state (inactive/active) model does not account for the intricacy of activation and inactivation steps at the plasma membrane. Here, we introduce a structure-based kinetic model of PLC-γ1, considering interactions of its regulatory Src homology 2 (SH2) domains and perturbation of those dynamics upon phosphorylation of Tyr783, a hallmark of activation. For PLC-γ1 phosphorylation to dramatically enhance enzyme activation as observed, we found that high intramolecular affinity of the C-terminal SH2 (cSH2) domain–pTyr783 interaction is critical, but this affinity need not outcompete the autoinhibitory interaction of the cSH2 domain. Under conditions for which steady-state PLC-γ1 activity is sensitive to the rate of Tyr783 phosphorylation, maintenance of the active state is surprisingly insensitive to the phosphorylation rate, since pTyr783 is well protected by the cSH2 domain while the enzyme is active. In contrast, maintenance of enzyme activity is sensitive to the rate of PLC-γ1 membrane (re)binding. Accordingly, we found that hypothetical PLC-γ1 mutations that either weaken autoinhibition or strengthen membrane binding influence the activation kinetics differently, which could inform the characterization of oncogenic variants. Finally, we used this newly informed kinetic scheme to refine a spatial model of PLC/PKC polarization during chemotaxis. The refined model showed improved stability of the polarized pattern while corroborating previous qualitative predictions. As demonstrated here for PLC-γ1, this approach may be adapted to model the dynamics of other receptor- and membrane-proximal enzymes.     

Ethanol inhibits palmitoylation of G protein G alpha(s)

Neurobiological actions of ethanol have been linked to perturbations in cyclic AMP (cAMP)-dependent signaling processes. Chronic ethanol exposure leads to desensitization of cAMP production in response to physiological ligands (heterologous desensitization). Ethanol-induced alterations in neuronal expression of G proteins G(s) and G(i) have been invoked as a cause of heterologous desensitization. However, effects of ethanol on G protein expression vary considerably among different experimental protocols, various brain regions and diverse neuronal cell types. Dynamic palmitoylation of G protein alpha subunits is critical for membrane localization and protein-protein interactions, and represents a regulatory feature of G protein function. We studied the effect of ethanol on G alpha(s) palmitoylation. In NG108-15 rat neuroblastoma x glioma hybrid cells, acute exposure to pharmacologically relevant concentrations of ethanol (25-100 mm) inhibited basal and prostaglandin E1-stimulated incorporation of palmitate into G alpha(s). Exposure of NG108-15 cells to ethanol for 72 h induced a shift in G alpha(s) to its non-palmitoylated state, coincident with an inhibition of prostaglandin E1-induced cAMP production. Both parameters were restored following 24 h of ethanol withdrawal. Chronic ethanol exposure also induced the depalmitoylation of G alpha(s) in human embryonic kidney (HEK)293 cells that overexpress wild-type G alpha(s) and caused heterologous desensitization of adenylyl cyclase. By contrast, HEK293 cells that express a non-palmitoylated mutant of G alpha(s) were insensitive to heterologous desensitization after chronic ethanol exposure. In summary, the findings identify a novel effect of ethanol on post-translational lipid modification of G alpha(s), and represent a mechanism by which ethanol might affect adenylyl cyclase activity.     

Bcl-2 attenuates anticancer agents-induced apoptosis by sustained activation of Akt/protein kinase B in U937 cells

Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family contributes to resistance to anticancer therapeutic drugs. Thus, this protein represent attractive target for novel anticancer agents. In the present study, we determined the effect of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-γ1 degradation and Akt activation during the various anticancer agents-induced apoptosis. Treatment with chrysin for 12 h produced morphological features of apoptosis in U937 cells, which was associated with caspase-3 activation and PLC-γ1 degradation. Induction of apoptosis was also accompanied by down-regulation of XIAP and inactivation of Akt. Chrysin-induced caspase-3 activation, PLC-γ1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit ceramide-, and Akt specific inhibitor (SH-6)-induced apoptosis by sustained Akt activation. Thus, our findings imply that some of the biological functions of Bcl-2 may be attributed to their ability to inhibit anticancer agents-induced apoptosis through the sustained Akt activation.     

Chronic ethanol consumption enhances internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex

The molecular mechanisms that underlie ethanol dependence involve alterations in the functional properties and subunit expression of GABAA receptors. Chronic ethanol exposure decreases GABAA receptor alpha1 subunits and increases alpha4 subunit levels in cerebral cortical membranes. This study explored the effect of chronic ethanol exposure on internalization of GABAA/benzodiazepine receptors. Chronic ethanol exposure increased alpha1 subunit levels by 46 +/- 12% and [3H]flunitrazepam binding by 35 +/- 9% in the clathrin-coated vesicle (CCV) fraction. There was a corresponding 34 +/- 8% decrease in alpha1 peptide expression and 37 +/- 6% decrease in [3H]flunitrazepam binding in the synaptic fraction. Chronic ethanol consumption also increased the alpha1 subunit immunoprecipitate in the cytosolic fraction (77 +/- 22%), measured by western blot analysis. Moreover, co-immunoprecipitation of both clathrin and adaptin-alpha with alpha1 subunits was increased in the cytosolic fraction, suggesting that alpha1 subunit endocytosis is enhanced by chronic ethanol consumption. In contrast, alpha4 subunit peptide levels were not altered in the CCV fraction despite a 39 +/- 13% increase in peptide levels in the synaptic fraction of cortex. Moreover, acute ethanol exposure did not alter alpha1 subunit peptide expression or [3H]flunitrazepam binding in the synaptic or CCV fractions. These results suggest that chronic ethanol consumption selectively increases internalization of alpha1 subunit-containing GABAA receptors in cerebral cortex.     

Ethanol and prostaglandin E1: Biochemical and behavioral interactions

Low concentrations of ethanol enhanced prostaglandin (PG) E₁-stimulated adenosine-3′, 5′-cyclic monophosphate (cAMP) accumulation in human platelets and in rat brain slices. Ethanol also potentiated platelet synthesis of PGE₁ from dihomo-gamma-linolenic acid. These interactions may derive from the fluidizing effects of ethanol on lipid-containing cell membranes, and suggest a possible role for PGE₁ as a mediator of certain acute effects of ethanol. The derivative possibility that “down regulation” of PGE₁ systems is involved in the development of ethanol dependence is supported by data showing that PGE₁ administered to mice following chronic exposure to ethanol reduced withdrawal syndrome intensity.     

Specific protein phosphorylation during cyclic AMP-mediated morphological reversion of transformed cells

Treatment of transformed Chinese hamster ovary cells with dibutyryl cAMP or other agents that elevate cAMP results in the acquisition of growth and morphology characteristic of normal fibroblasts. The role of specific protein phosphorylation in this process of morphological reversion has been examined using metabolic labelling of Chinese hamster ovary (CHO) cells with ³²P-orthophosphate in the presence or absence of N⁶O^2′-dibutyryladenosine 3′:5′-cyclic monophosphoric acid (Bt₂cAMP). Analysis of labelled cultures by SDS gel electrophoresis and radioautography demonstrate dramatic changes in the phosphorylation of only 2 cellular proteins during reverse transformation. A 55,000 dalton protein (pp55) was phosphorylated and a 20,000 dalton protein (pp20) was dephosphorylated. The time course of these events was consistent with the kinetics of morphological reversion. The lower molecular weight species, pp20, was dephosphorylated within 15–30 minutes, prior to all morphological changes except membrane tranquilization. The higher molecular weight protein, pp55, was maximally phosphorylated over 1–2 hours following addition of Bt₂cAMP, paralleling early stages in the establishment of fibroblastic form. The phosphorylated forms of pp20 and pp55 were both extracted from cellular cytoskeletons by 0.5% Triton X-100, but analysis of ³⁵S-methioninelabelled cultures suggested that unphosphorylated pp 20 may be bound to the cytoskeleton. Since pp20 was found to comigrate with the 20,000 dalton myosin light chain, it is possible that dephosphorylation of CHO cell myosin induced by cAMP may alter its interaction with actin microfilaments and modulate the assembly of stress fibers during morphological reversion.     

Protein Kinase C in Rat Brain Is Altered by Developmental Lead Exposure

The absence of learning-related redistribution of hippocampal protein kinase C (PKC) has been correlated with impairment of learning performance induced by developmental lead (Pb) exposure. This study was designed to examine whether the properties of brain PKC are altered by chronic Pb exposure during development. Two-tenth percent Pb acetate was administered to pregnant and lactating dams and then administered to weanlings in drinking water until postnatal day (PN) 56. Effects of Pb on translocation of PKC were studied in brain slices prepared from hippocampus. When the slices were treated with 0.33 M phorbol-12, 13-dibutyrate (PDBu) for 15 min, a significant increase in PKC activity was observed in the membrane fraction of hippocampal slices from Pb-exposed rats, suggesting that chronic Pb exposure potentiates PDBu-activated PKC translocation. Data obtained from saturation binding assays in the frontal cortices of Pb-exposed rats showed a decrease in the dissociation constant (K_D) in both membrane and cytosolic PKC. A decrease in the total binding sites (B_max) of [³H]PDBu binding was only observed in membrane PKC. Furthermore, developmental Pb exposure decreased PKC-, but not PKC-, -II, and - in the membrane fraction of the hippocampus and the frontal cortex. These results indicate that chronic Pb exposure during development increases phorbol ester binding affinity, enhances phorbol ester-induced translocation of PKC, and down-regulates membrane PKC, mainly PKC-.     

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC₅₀ values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCα/βII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of ∼70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.     

Protein kinase a dependent phosphorylation activates mg2+ efflux in the basolateral region of the liver

Isolated hepatocytes in physiological [Na⁺] ₀ tightly maintain [Mg²⁺] _i . Upon β-adrenergic stimulation or in the presence of permeable cAMP, hepatocytes release 5–10% (1–3 mM Mg²⁺) of their total Mg²⁺ content. However, isolated basolateral liver plasma membranes (bLPM), release Mg²⁺ in the presence of [Na⁺] _o even in the absence of catecholamine stimulation. The data indicate that a physiological brake for Mg²⁺ efflux is present in the hepatocyte and is removed upon cellular signaling. In contrast, this regulation “brake” is absent in purified bLPM thus rendering them fully active. The present study was carried out to reconstruct the missing regulatory component. Activation of Mg²⁺ extrusion in intact cells is consistent with cAMP dependent phosphorylation of the transporter or a regulatory protein. Treatment of bLPM with a non-specific phosphatase such as alkaline phosphatase (AP), decreased Mg²⁺ efflux by 70% compared to untreated bLPM. When AP-treated bLPM were loaded with protein kinase A (PKA), and stimulated with permeable cAMP, Mg²⁺ transport fully recovered. These data suggest that phosphorylation of the Na⁺/Mg²⁺ exchanger or a nearby protein activates the Mg²⁺ transport mechanism in hepatocytes.     

In vivo nicotine treatment regulates mesocorticolimbic CREB and ERK signaling in C57Bl/6J mice

The extracellular regulated kinase (ERK) pathway was studied to determine its role in neuronal plasticity related to the development of nicotine dependence. Levels and phosphorylation state of ERK, cAMP response element binding protein (CREB) and proline-rich/Ca2+-activated tyrosine kinase (PYK2), and levels of tyrosine hydroxylase (TH), were determined using western blotting. C57Bl/6J mice received acute or chronic nicotine (200 microg/mL) in their drinking water or were withdrawn from nicotine for 24 h following chronic exposure. CREB phosphorylation was reduced in the nucleus accumbens following chronic nicotine, consistent with previous reports that decreased accumbens CREB activity increases drug reinforcement. In contrast, CREB phosphorylation was increased in the prefrontal cortex following chronic nicotine exposure and in the ventral tegmental area during nicotine withdrawal. In addition, total and phosphorylated ERK decreased in the amygdala following chronic nicotine exposure, but ERK phosphorylation increased in the prefrontal cortex. TH levels increased in both the amygdala and prefrontal cortex, supporting the hypothesis that increased catecholaminergic tone contributes to nicotine reinforcement. Overall, these results support a role for ERK and CREB activity in neural plasticity associated with nicotine dependence.     

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.