Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.     

Platelet-derived growth factor stimulates the release of protein kinase A from the cell membrane

The mitogenic action of growth factors involves the stimulation of intracellular protein kinases. In this report we have characterized the major protein kinase released from Balb/c 3T3 and normal rat kidney plasma membranes by the action of platelet-derived growth factor (PDGF). PDGF appears to stimulate the release of approximately 10 proteins, at least one of which is a kinase capable of phosphorylating proteins on Ser or Thr (as determined by the lability of the phosphate to alkali treatment). More than 90% of the Ser/Thr kinase activity was inhibited by PKI5-22, a specific peptide inhibitor of the cAMP-dependent protein kinase (PKA). We used immunoblotting to confirm that the kinase released in response to PDGF was PKA. cAMP also stimulated the release of PKA, and the set of protein substrates phosphorylated was similar following PDGF or cAMP stimulation. Interestingly, in the presence of a cAMP analogue ((Rp)-cAMPS), cAMP could not induce dissociation of PKA from the membranes, whereas stimulation by PDGF increased the level of PKA activation. Furthermore, unlike Swiss 3T3 cells, neither Balb/c 3T3 fibroblasts nor normal rat kidney cells accumulate cAMP in response to PDGF, yet the level of PKA in the cytosol of these intact cells increases in response to PDGF. Thus, it appears as though PDGF activation of the membrane-associated form of the PKA holoenzyme occurs by a mechanism independent of an elevation in cAMP levels.     

Regulation of 11beta-hydroxysteroid dehydrogenase type II in rat adrenocortical cells

The regulation of 11beta-hydroxysteroid dehydrogenase type II (11beta-HSD2) gene expression was studied in primary cultures of rat adrenocortical cells. The protein kinase A (PKA) pathway agonists forskolin, dibutyryl cAMP and ACTH caused a 5-10 fold increase in 11beta-HSD2 mRNA as determined by semiquantitative PCR. The effect of forskolin could be partially inhibited by the addition of the phorbol ester TPA, an activator of the protein kinase C (PKC) pathway. The increase in mRNA encoding 11beta-HSD2 was accompanied by increased synthesis of 11beta-HSD2 as measured by immunoprecipitation of labeled protein. It is concluded that both the PKA and PKC pathways are involved in the regulation of rat adrenal 11beta-HSD2 gene expression.     

Effects of ethanol on basal and prostaglandin E1-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells

Our recent studies determining the effect of cAMP-elevating agents forskolin and dibutyryl cAMP on ethanol-induced immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic cells in primary cultures suggested the possibility that both stimulatory and adaptive secretory responses of beta-EP neurons after ethanol exposure may involve the cAMP system. To determine further the role of cAMP, the effects of prostaglandin E1 (PGE1) on basal and ethanol-regulated IR-beta-EP secretion and cAMP productions were determined in primary cultures of hypothalamic cells. The results presented in this study show that a 50 mM dose of ethanol, which is within the EC50 dose of ethanol required to elevate IR-beta-EP release from hypothalamic cells, increased cellular levels of cAMP and elevated IR-beta-EP release simultaneously from the cultured neurons for a period of 6 hr. The cAMP and IR-beta-EP secretory responses developed desensitization to ethanol challenge after 24 hr of constant ethanol incubation. The cAMP-elevating agent PGE1 increased the cellular content of cAMP and IR-beta-EP release in a dose-dependent manner. The EC50 dose of PGE1 for elevation of IR-beta-EP and cAMP was approximately 0.5 microM. As with ethanol, chronic treatment with PGE1 desensitized the cAMP and IR-beta-EP responses of hypothalamic neurons to PGE1. Acute exposure to ethanol increased the PGE1-stimulated levels of cAMP and IR-beta-EP, whereas chronic exposure to ethanol resulted in diminished cAMP responses to PGE1. These data provide evidence that the cAMP system may be involved in controlling hypothalamic beta-EP secretion, as well in regulating the stimulatory and adaptive responses of beta-EP neurons to ethanol.     

Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.     

Regulatory mechanism of pepsinogen gene expression in monolayer cultured guinea pig gastric chief cells

We have investigated the effects of dibutyryl cAMP, forskolin, carbamylcholine chloride (carbachol), ionomycin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of guinea pig pepsinogen mRNA in monolayer cultured gastric chief cells. After exposure of the cells to each of these compounds for 4 to 24 hr, and at 48 hr after primary culture, total cellular RNA was isolated using acid guanidium-phenol-chloroform and then was reverse transcribed to cDNA. Obtained cDNA was amplified by polymerase chain reaction (PCR) using primers detecting guinea pig pepsinogen mRNA and human beta-actin mRNA as an internal standard. The PCR products were separated and quantified using capillary electrophoresis. Dibutyryl cAMP and forskolin significantly increased pepsinogen mRNA, but carbachol, ionomycin, and TPA failed to increase that. These findings suggested that pepsinogen gene expression was up-regulated by intracellular cAMP, but not by intracellular calcium or protein kinase C in guinea pig chief cells.     

Adrenaline enhances LPS-induced IL-10 synthesis: evidence for protein kinase A-mediated pathway

The aim of the present study was to investigate the role of cAMP in enhanced IL-10 synthesis in human mononuclear cells. Adrenaline is known to act via the alpha- and beta-adrenergic receptors which are coupled to adenylyl cyclase. The effects of cAMP elevation on IL-10 synthesis were studied at the protein level by ELISA and at the level of mRNA by RT/PCR. In this in vitro model adrenaline enhanced the LPS-induced synthesis of IL-10 with parallel suppression of TNF synthesis. These effects were demonstrated both at the protein level and the level of mRNA. To analyze the role of cAMP we antagonized this effect by application of (Rp)-cAMPS, a diastereomer of adenosine-3',5'-cyclic phosphorothioate, known to inhibit competitively the cAMP-induced activation of protein kinase A. Simultaneous addition of adrenaline and (Rp)-cAMPS led to a reversal of IL-10 synthesis to values induced by LPS stimulation alone. The kinetic analysis in LPS-stimulated mononuclear cells revealed a significant delay of IL-10 synthesis starting after 7 h compared with TNF synthesis which showed the first significant increase at 90 min. Finally, the combination of adrenaline and exogenous IL-10 led to a more pronounced suppression of TNF synthesis after LPS stimulation compared to suppression by IL-10 or adrenaline alone. The present results suggest the role of protein kinase A activation for adrenaline-induced IL-10 synthesis in human mononuclear cells. Additionally, based on the kinetic analysis and further experiments described in the literature, endogenous IL-10 could contribute to the adrenaline-induced suppression of TNF synthesis after prolonged incubation. These in vitro results could explain the suppression of TNF plasma concentration after parallel infusion of LPS and epinephrine compared to LPS infusion alone as has been demonstrated in a first human study.     

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA alpha-isoforms RIalpha, RIIalpha, and Calpha and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3',5'-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.     

Modulation of Ca2+ channels by cyclic nucleotide cross activation of opposing protein kinases in rabbit portal vein

Cyclic nucleotides are known to modify voltage-gated (L-type) Ca2+ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP- and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca2+ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 micromol/L), significantly increased (14% to 16%) peak Ba2+ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba2+ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba2+ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca2+ channel currents were significantly inhibited by the PKA blocker 8-Bromo-2'-O-monobutyryladenosine-3',5'-monophosphorothioate, Rp-isomer (Rp 8-Br-MP cAMPS, 30 micromol/L) and enhanced by the PKG inhibitor beta-Phenyl-1,N2-etheno-8-bromoguanosine-3',5'-monophosphorothioate, Rp-isomer (Rp-8-Br PET cGMPS, 10 nmol/L). In the presence of Rp 8-bromo PET cGMPS (10 to 100 nmol/L), both 8-bromo cAMP (0.1 mmol/L) and 8-bromo cGMP (0.1 mmol/L) enhanced Ba2+ currents (13% to 39%). The excitatory effect of 8-bromo cGMP was blocked by Rp 8-bromo MB-cAMPS. Both 8-bromo cAMP (0.05 mmol/L) and forskolin (10 micromol/L) elicited time-dependent effects, including an initial enhancement followed by suppression of Ba2+ currents. Ba2+ currents were also enhanced when cells were dialyzed with the catalytic subunit of PKA. This effect was reversed by the PKA blocker KT 5720 (200 nmol/L). Our results suggest that cAMP/PKA stimulation enhances and cGMP/PKG stimulation inhibits L-type Ca2+ channel activity in rabbit portal vein myocytes. Our results further suggest that both cAMP and cGMP have a primary action mediated by their own kinase as well as a secondary action mediated by the opposing kinase.     

Vasoactive intestinal peptide enhances its own expression in sympathetic neurons after injury

Neurons in the adult rat superior cervical sympathetic ganglion (SCG) dramatically increase their content of vasoactive intestinal peptide (VIP) and its mRNA after axotomy in vivo and after explantation. Because the VIP gene contains a functional cAMP response element, the effects of cAMP-elevating agents on VIP expression were examined. VIP, forskolin, or isoproterenol increased cAMP accumulation in explanted ganglia. Secretin, a peptide chemically related to VIP, or forskolin increased VIP levels above those seen in ganglia cultured in control medium, whereas treatment with VIP or secretin increased the level of peptide histidine isoleucine (PHI), a peptide coded for by the same mRNA that encodes VIP. VIP or forskolin also increased VIP-PHI mRNA. In contrast, isoproterenol did not alter levels of VIP, PHI, or VIP-PHI mRNA. Although VIP or forskolin increased cAMP levels in both dissociated neurons and in non-neuronal cells, isoproterenol significantly stimulated cAMP accumulation only in the latter. VIP6-28 was an effective antagonist of the actions of exogenous VIP on cAMP and VIP-PHI mRNA in neuron-enriched cultures. When adult SCG explants were cultured in defined medium, endogenous VIP immunoreactivity was released. When VIP6-28 was added to such cultures, it significantly inhibited the increase in VIP-PHI mRNA that normally occurs. These data indicate that VIP, or a closely related molecule, produced by adult neurons after injury can enhance the expression of VIP. Such a mechanism may prolong the period during which VIP is elevated after axonal damage. The possibility is also discussed that, because VIP is present in preganglionic neurons in normal animals, its release during periods of increased sympathetic nerve activity could alter VIP expression in the SCG.     

Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model

Current organ preservation strategies subject graft vasculature to severe hypoxia (PO2 approximately 20 Torr), potentially compromising vascular function and limiting successful transplantation. Previous work has shown that cAMP modulates endothelial cell (EC) antithrombogenicity, barrier function, and leukocyte/EC interactions, and that hypoxia suppresses EC cAMP levels. To explore the possible benefits of cAMP analogs/agonists in organ preservation, we used a rat heterotopic cardiac transplant model; dibutyryl cAMP added to preservation solutions was associated with a time- and dose-dependent increase in the duration of cold storage associated with successful graft function. Preservation was also enhanced by 8-bromo-cAMP, the Sp isomer of adenosine 3',5'monophosphorothioate, and types III (indolidan) and IV (rolipram) phosphodiesterase inhibitors. Neither butyrate alone nor 8-bromoadenosine were effective, and the cAMP-dependent protein kinase antagonist Rp isomer of adenosine 3',5'monophosphorothioate prevented preservation enhancement induced by 8-bromo-cAMP. Grafts stored with dibutyryl cAMP demonstrated a 5.5-fold increase in blood flow and a 3.2-fold decreased neutrophil infiltration after transplantation. To explore the role of cAMP in another cell type critical for vascular homeostasis, vascular smooth muscle cells were subjected to hypoxia, causing a time-dependent decline in cAMP levels. Although adenylate cyclase activity was unchanged, diminished oxygen tensions were associated with enhanced phosphodiesterase activity (59 and 30% increase in soluble types III and IV activity, respectively). These data suggest that hypoxia or graft ischemia disrupt vascular homeostasis, at least in part, by perturbing the cAMP second messenger pathway. Supplementation of this pathway provides a new approach for enhancing cardiac preservation, promoting myocardial function, and maintaining vascular homeostatic properties.     

Signaling role of PDE isozymes in pathobiology of glomerular mesangial cells. Studies in vitro and in vivo

Mesangial cells (MC) of renal glomeruli respond to immune-inflammatory injury by accelerated proliferation and generation of reactive oxygen metabolites (ROM). We studied in vivo and in vitro roles of cAMP-protein kinase A (PKA) signaling in modulation of these pathobiologic processes with focus on PDE isozymes. Mitogenic synthesis of DNA in mesangial cells grown in primary culture was blocked by forskolin and dibutyryl cyAMP. Incubation of MC with PDE-3 inhibitors, cilostamide and lixazinone, inhibited (> 50%) mitogenesis, whereas inhibitors of PDE-4, rolipram and denbufylline, caused little or no inhibition. Conversely, inhibitors of PDE-4 suppressed generation of ROM in MC, whereas inhibitors of PDE-3 had no effect. Incubation of mesangial cells with cilostamide or with rolipram increased in situ activity of PKA, and effects of the two inhibitors were additive. PDE inhibitors also decreased activity of mitogen-activated protein kinase. The efficacy of PDE isozyme inhibitors (IC50) to suppress mitogenesis or ROM generation paralleled IC50 for inhibition of cAMP hydrolysis by extracts from mesangial cells. Administration of lixazinone or lixazinone in combination with rolipram to rats with mesangial proliferative glomerulonephritis induced by antithymic serum suppressed proliferation of mesangial cells and also reduced other histopathologic manifestations of the disease. Based on these observations, we propose that in MC, a cAMP pool that is hydrolyzed by PDE-3 inhibits by negative crosstalk via activation of PKA, mitogen-activated protein kinase (MAPK) pathway, and mitogenesis; whereas cAMP pool linked to PDE-4 inhibits, also via activation of PKA, ROM generation in mesangial cells. Results also suggest that PDE isozyme inhibitors, in particular inhibitors of PDE-3, should be investigated for potential use for "signal transduction pharmacotherapy" of glomerulonephritis.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

Protein kinase A, protein kinase C, and Ca(2+)-regulated expression of 21-hydroxylase cytochrome P450 in H295R human adrenocortical cells

The physiological importance of adrenal 21-hydroxylase cytochrome P450 (CYP21) expression is clearly demonstrated by 21-hydroxylase deficiency, which results in adrenal hyperplasia and over-production of C19 steroids, leading to virilization. The mechanisms regulating normal expression of this key enzyme in human adrenocortical cells are ill defined. Herein we examine the role of the calcium, protein kinase C, and protein kinase A signaling pathways in the expression of CYP21 messenger ribonucleic acid (mRNA) using the H295R human adrenocortical cell model. Forskolin (10 mumol/L) treatment caused a progressive increase in CYP21 mRNA levels (maximum, 4-fold; P < 0.05) over 36 h of treatment, whereas angiotensin II (AII; 10 nmol/L) produced a smaller, biphasic rise (maximum, 1.8-fold at 12 h; P < 0.05). K+ (14 mmol/L) also induced a time-dependent (maximal, 1.5-fold at 12 h; P < 0.05) and dose-dependent (P < 0.05 12 mmol/L or above at 20 h) rise in CYP21 mRNA levels. The action of forskolin was reproduced by dibutyryl cAMP, confirming the involvement of cAMP in this response. The action of AII was greater than that of K+ or the calcium channel agonist BAYK8644, suggesting that AII action was not solely through the Ca2+ signaling pathway. The action of AII was reproduced and indeed exceeded by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA; 10 nmol/L; 5.5-fold increase; P < 0.05). The actions of forskolin alone were not significantly increased by combined treatment with AII, suggesting neither synergy nor attenuation of the effects of protein kinase A activation. This was further demonstrated at the level of mRNA and 21-hydroxylase activity by the observation that the effect of forskolin and TPA in combination did not exceed that of TPA alone. Inhibition of protein synthesis with cycloheximide blocked induction of CYP21 as well as type II 3 beta-hydroxysteroid dehydrogenase (3 beta HSDII) mRNA expression in response to AII, forskolin, and dibutyryl cAMP, but had no effect on 17 alpha-hydroxylase cytochrome P450 (CYP17) or cholesterol side-chain cleavage cytochrome P450 (CYP11A) mRNA. Together, these findings were remarkably similar to those of our previous studies regarding mechanisms regulating 3 beta HSDII expression and underline the existence of a subset of steroidogenic enzymes regulated positively (CYP21 and 3 beta HSDII) as opposed to negatively (CYP17 and CYP11A) by the protein kinase C signaling pathway. The additional finding of a small induction of CYP21 expression in response to increased Ca2+, as previously reported for CYP17, but not 3 beta HSDII, expression, also demonstrates that the mechanisms of control of CYP21 and 3 beta HSDII are not identical. This latter finding may also relate to how CYP21 as well as CYP17 expression continues in the zona reticularis after adrenarche, whereas 3 beta HSD expression declines.     

A hepatocyte-targeted conjugate capable of delivering biologically active colchicine in vitro

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. WHen the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effect were also inhibited by pretreatment with staurosporine or calphostin C. An addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+]i were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m3 mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.     

Inhibition of T lymphocyte activation by cAMP is associated with down-regulation of two parallel mitogen-activated protein kinase pathways, the extracellular signal-related kinase and c-Jun N-terminal kinase

The induction of T cell proliferation requires signals from the TCR and a co-receptor molecule, such as CD28, that activate parallel and partially cross-reactive signaling pathways. These pathways are disrupted by agonists that utilize adenylate cyclase and cAMP-dependent protein kinase A (PKA). We found that the adenylate cyclase activator, forskolin, inhibits anti-CD3-induced shift in Lck electrophoretic mobility, suggesting an intervention at the TCR-coupled phosphoinositide turnover that precedes the activation of PKC. The shift of Lck following direct PKC activation by 12-O-tetradecanoyl phorbol 13-acetate, which bypasses early receptor-triggered biochemical events, is insensitive to forskolin. Nevertheless, forskolin also inhibits PKC downstream events, such as c-jun expression, which is critical for the activation process of T cells. To further analyze potential cross points between positively and negatively regulating signaling pathways in T cells, we tested the effects of activators of the adenylate cyclase or PKA on two parallel mitogen-activated protein kinase signaling pathways mediated by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase. Using a PKC-specific inhibitor, GF109203X, or PKC-depleted T cells, we found that a large part of the anti-CD3-induced ERK activation is PKC dependent. Both PKC-dependent and -independent activation of ERK were sensitive to inhibition by forskolin or a cell-permeable cAMP analogue, dbcAMP. Furthermore, the effect of 12-O-tetradecanoyl phorbol 13-acetate and ionomycin, which synergized to fully activate c-Jun N-terminal kinase, was also sensitive to inhibition by forskolin. Our results suggest that PKA inhibits T cell activation by interfering with multiple events along the two signaling pathways operating downstream of the TCR and the CD28 co-receptor molecules.