Desensitization Of Protein Kinase Research Articles

Mechanism‐Based Approaches for the Reversal of Drug Neurobehavioral Teratogenicity

Understanding the mechanism of neurobehavioral teratogenicity is the primary prerequisite for reversal of the defect. Progress in such studies can be best achieved if the investigation focuses on behaviors related to a specific brain region and innervation. Our model focused on teratogen-induced deficits in hippocampus-related eight-arm and Morris maze behaviors. Different "cholinergic" teratogens, mainly heroin, induced both pre- and postsynaptic hyperactivity in the hippocampal cholinergic innervation that terminated in desensitization of Protein Kinase C (PKC) isoforms to cholinergic receptor stimulation. Understanding this mechanism enabled its reversal with a pharmacological therapy-nicotine infusion. Studies by others provided similar findings by targeting the deficits respective to the model investigated. Consistently, destruction of the A10-septal dopaminergic pathways that downregulate the septohippocampal cholinergic innervation ameliorated maze performance. Grafting of embryonic differentiated cholinergic cells or neural progenitors similarly reversed the biochemical/molecular alterations and the resulting deficits. Reversal therapies offer a model for the understanding of neurobehavioral teratogenicity and, clinically, offer a model for potential treatment of these deficits. Whereas neural progenitor grafting appears to be the most effective treatment, pharmacological reversal with nicotine infusion seems to possess the most feasible and immediate therapy for neurobehavioral birth defects produced by various teratogens, including drugs. This is true even though the effect of pharmacological therapies is diffuse, affecting multiple areas of the brain. "Everybody is talking about the weather but nobody does anything about it." (Mark Twain).

Neurotensin modulates the amplitude and frequency of voltage-activated Ca2+ currents in frog pituitary melanotrophs: implication of the inositol triphosphate/protein kinase C pathway.

Many excitatory neurotransmitters and neuropeptides regulate the activity of neuronal and endocrine cells by modulating voltage-operated Ca2+ channels. Paradoxically, however, excitatory neuromediators that provoke mobilization of intracellular calcium from inositol trisphosphate (IP3)-sensitive stores usually inhibit voltage-gated Ca2+ currents. We have recently demonstrated that neurotensin (NT) stimulates the electrical and secretory activities of frog pituitary melanotrophs, and increases intracellular calcium concentration in these cells. In the present study, we have investigated the effects of NT on Ca2+ currents in cultured frog melanotrophs by using the perforated patch-clamp technique. Frog neurotensin (f NT) reduced the amplitude and facilitated the inactivation of both L- and N-type Ca2+ currents. Application of the membrane-permeant Ca2+ chelator BAPTA-AM, the sarcoendoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, or the IP3 receptor antagonist 2-APB suppressed the reduction of Ca2+ currents induced by f NT. Incubation of melanotrophs with the diacylglycerol analogue PMA, which causes desensitization of protein kinase C (PKC), or with the PKC inhibitors chelerythrine and calphostin C, reduced the inhibitory effect of f NT. The NT-induced action potential waveforms, applied as voltage-clamp commands, decreased the amplitude of Ca2+ currents, and enhanced Ca2+ influx by increasing the Ca2+ spike frequency. Altogether, these data indicate that the inhibitory effect of f NT on Ca2+ currents results from activation of the IP3/PKC pathway. The observation that NT controls Ca2+ signalling through both amplitude and frequency modulations of Ca2+ currents suggests that NT might induce spacial and temporal changes of intracellular Ca2+ concentration leading to stimulation of exocytosis.

Delta Opiate Receptors Account for the Castration-Induced Unmasking of Gonadotropin-Releasing Hormone Binding Sites in the Rat Pituitary

Under control incubation conditions, gonadotropin-releasing hormone (GnRH) binds only a fraction of its receptors in rat-cultivated pituitary cells. Unmasking of the remaining receptors, which have been termed ‘cryptic’, requires drug- or peptide-induced protein kinase activation. Spontaneous masking however is not observed on pituitary cells sampled from castrated male rats, suggesting the presence of an intrinsic unmasking factor. Many endogenous factors could theoretically account for the effect. Here we attempted to identify the factor involved by taking advantage of their differential dependency upon second messengers and transduction cascades. Spontaneous unmasking of GnRH binding was found reversed by pertussis toxin (PTX), an inhibitor of α_i and α_o subunits of heterotrimeric G proteins, and by U73122, a phospholipase C (PLC) inhibitor. In contrast, desensitization of protein kinase C (PKC) or inhibition of tyrosine kinase by herbimycin were ineffective. Among endogenous pituitary factors able to unmask GnRH receptors in pituitary cells from normal male rats, as EGF, NPY or opiate peptides, only the latter were found to correspond to this transduction profile. In an attempt to characterize the pharmacology of opiate effects, naloxone (10 µM), a poorly selective opiate antagonist, restored masking of GnRH binding in cells from castrates. Only the δ antagonist naltrindole (1 µM) was able to mimick the action of naloxone. Conversely, when tested on cells from intact animals, morphine (10 µM), as well as dslet (1 µM) and met-ENK (10 nM), preferential δ agonists, but not dago and β-endorphin or U50488 H and dynorphin, respectively µ and ĸ agonists, were able to suppress masking. Among opioid peptides endogenous to the pituitary, only met-ENK was able to unmask cryptic receptors, an effect antagonized by naltrindole. We conclude that an opiate δ receptor subtype is endogenously activated in the pituitary of castrated male rats to prevent masking of GnRH binding.

Synergistic stimulation of interleukin 6 release and gene expression by phorbol esters and interleukin 1 beta in rat cortical astrocytes: role of protein kinase C activation and blockade.

The involvement of protein kinase C and its interaction with interleukin 1 beta in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C and by the desensitization of protein kinase C. Interleukin 1 beta increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1 beta stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1 beta (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurospine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y enhances LHRH binding to rat gonadotrophs in primary culture

Incubation of dispersed adenohypophyseal cells from intact male rats with Neuropeptide Y (NPY) or Peptide YY (YY) at 21°C increased maximal 125I LHRH a binding (Bmax) by about 50%. In presence of 10 −7 M NPY, Bmax calculated from saturation isotherm curves was 15.3 ± 1.9 fmolex × mg −1 proteins, as compared to 10 ± 1 fmoles × mg −1 in control incubates. The increase was dose dependent with an EC 50 of 6.3 ± 1.8 10 −10 M NPY. Preincubation of the cells with pertussis toxin (PT, 15 ng/ml) for 24 h abolished the effect, suggesting coupling of NPY receptors to Gα o or Gα i proteins. NPY 10 −7 M inhibited basal and Forskolin 10 −5 M stimulated intracellular cyclic AMP formation by 31.9 ± 3.4% and 30.6 ± 2.3% respectively. Desensitization of protein kinase C by overnight preincubation of the cells with 10 −6 M phorbol ester (PMA) did not interfere with the effect of NPY. In contrast, W7, a calmodulin inhibitor, as well as H7, a protein kinase C inhibitor with a relatively wide spectrum, suppressed the effect of NPY with IC 50 of 1.4 ± 0.6 10 −6 M and 2.2 ± 0.5 10 −5 M, respectively. Taken together, these results suggest that NPY is able to control unmasking of a cryptic LHRH receptor pool in pituitary cells by a process dependent upon both GTP binding proteins and calmodulin dependent protein kinase.

Angiotensin II potentiates agonist-induced 3',5'-cyclic adenosine monophosphate production by cultured bovine adrenal cells through protein kinase C and calmodulin pathways.

Interactions between signal transducing systems may be important in the integrated control of cellular processes in basal and hormonally regulated cells. The cultured bovine adrenal fasciculata cell provides a model to study the interactions between the cAMP and calcium-sensitive phospholipid dependent protein kinase C. In this study, angiotensin II (A-II) and phorbol ester (PMA) potentiated the stimulatory actions of ACTH in a dose-dependent manner on cAMP production. At maximal concentrations, A-II and PMA also potentiated the effects of cholera toxin and forskolin on cAMP production. Both staurosporine, a protein kinase C inhibitor, and desensitization of protein kinase C by a 24-h pretreatment with PMA blunted the effect of PMA, but only partially inhibited (34%) the effect of A-II. Neither nifedipine, a specific calcium channel antagonist, nor pretreatment of cells with pertussis toxin modified the amplifying effects of A-II or PMA. In contrast, trifluoperazine, a calmodulin inhibitor, reduced the potentiating effect of A-II by about 35%, but association with staurosporine blunted its effects. Moreover, the steroidogenic effects of ACTH plus A-II were more than additive, but this synergism was blunted in the presence of both inhibitors. In conclusion, PMA and A-II potentiated agonist-induced cAMP production by bovine adrenal fasciculata cells. The data suggest that the effects of PMA were mediated exclusively by protein kinase C, whereas those of A-II were mediated by both protein kinase C and calmodulin.

Inhibition of cyclic AMP accumulation in intact NCB-20 cells as a direct result of elevation of cytosolic Ca2+.

Earlier studies established that adenylyl cyclase in NCB-20 cell plasma membranes is inhibited by concentrations of Ca2+ that are achieved in intact cells. The present studies were undertaken to prove that agents such as bradykinin and ATP, which elevate the cytosolic Ca2+ concentration ([Ca2+]i) from internal stores in NCB-20 cells, could inhibit cyclic AMP (cAMP) accumulation as a result of their mobilization of [Ca2+]i and not by other mechanisms. Both bradykinin and ATP transiently inhibited [3H]cAMP accumulation in parallel with their transient mobilization of [Ca2+]i. The [Ca2+]i rise stimulated by bradykinin could be blocked by treatment with thapsigargin; this thapsigargin treatment precluded the inhibition of cAMP accumulation mediated by bradykinin (and ATP). A rapid rise in [Ca2+]i, as elicited by bradykinin, rather than the slow rise evoked by thapsigargin was required for inhibition of [3H]cAMP accumulation. Desensitization of protein kinase C did not modify the inhibitory action of bradykinin on [3H]cAMP. Effects of Ca2+ on phosphodiesterase were also excluded in the present studies. The accumulated data are consistent with the hypothesis that hormonal mobilization of [Ca2+]i leads directly to the inhibition of cAMP accumulation in these cells and presumably in other cells that express the Ca(2+)-inhibitable form of adenylyl cyclase.

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.

Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages

Activation of the superoxide-generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester-and zymosan-induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na +/H + antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide-generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.

Vasopressin and phorbol ester-stimulated phosphatidylcholine metabolism in mesangial cells.

We have studied the effects of the vasoactive agents phorbol 12-myristate 13-acetate (PMA) and vasopressin (VP) on phosphatidylcholine metabolism in cultured rat glomerular mesangial cells. PMA and VP stimulate the incorporation of [3H]choline into phosphatidylcholine and the release of [3H]choline into the culture medium. VP, but not PMA, also increases the release of phosphorylcholine into the medium. This suggests that PMA specifically stimulates phospholipase D, whereas VP stimulates phospholipases C and D. Experiments were also conducted to look for production of phosphatidic acid and diacylglycerol, products of phospholipase D- and C-mediated breakdown of phosphatidylcholine. Treatment of cells prelabeled with [3H]myristic acid for 2.5 min with PMA or VP increases the content of [3H]myristic acid in diacylglycerol and phosphatidic acid. A dual labeling study ([3H]myristic acid and [14C]arachidonic acid) suggests that phosphatidylcholine is an important source of diacylglycerol in cells treated with VP and PMA. When PMA or VP are added to [3H]myristic acid-labeled cells in the presence of ethanol, increased labeling of phosphatidylethanol is seen as early as 2.5 min. Desensitization of protein kinase C by overnight treatment of cells with PMA blocked subsequent VP-stimulated formation of phosphatidylethanol and release of [3H]choline. When cells were simultaneously treated with VP and PMA, additive effects on phosphatidylethanol formation and [3H]choline release were observed.

Chronic stimulation of D 2 dopamine receptors specifically inhibits calcium but not potassium currents in rat lactotrophs

The present study examines the effect of chronic dopamine treatment, known to inhibit prolactin release from anterior pituitary, on two Ca 2+ and K + currents in cultured rat lactotrophs. K + and Ca + currents were recorded using the whole-cell mode of the patch-clamp technique. The two types of voltage-dependent Ca 2+ currents are called SD and FD (slowly deactivating and fast deactivating current component, respectively) and the two types of voltage-dependent K + currents,I AandI K. All current types were isolated by tail current analysis. The amplitude of both normalized calcium components depended on the length of the culture(n = 48) while normalized amplitudes of both potassium currents remained constant(n = 9). Incubation of cells during 72 h with 50 μM of Actinomycin D, an inhibitor of mRNA synthesis, suggested that this increase in Ca 2+ currents involved the synthesis of proteins. Long-lasting D 2 receptor stimulation (8 days; 10 nM RU 24213) prevented this selective effect through activation of a pertussis toxin-sensitive G protein. We also examined whether cyclic adenosine-3′,5′-cyclic-monophosphate (cyclic AMP) or Ca 2+/phospholipid-dependent protein kinase (protein kinase C) could affect this development of channel activity. The increase of SD Ca 2+ current was enhanced during 3 days of treatment (between day 1 and day 4) with a membrane-permeant analogue of cyclic AMP, 8-bromo-adenosine-3′,5′-cyclic-monophosphate (8-Br-cyclic AMP; 1 mM): a36.6 ± 3.9% increase in SD component amplitude in control cells(n = 18), compared to55.6 ± 7.7% (n = 9) in 8-Br-cyclic AMP-treated cells. Desensitization of protein kinase C by prolonged exposure to phorbol esters increased SD Ca 2+ current amplitude by only11.4 ± 3.5% during a similar period(n = 11). Moreover, down-regulation of protein kinase C considerably reduced the long-term effects of RU 24213, whereas chronic elevation of cyclic AMP did not. These data reveal that chronic treatment of dopamine can prevent Ca 2+ current development activity via a pertussis toxin-sensitive G protein(s) pathway but cyclic AMP independent process. A reduction of protein kinase C activity could mediate, in part, this long-lasting effect of dopamine. These findings may represent an aspect of the mechanism by which chronic dopamine tonically inhibits prolactin release.

The involvement of protein kinase C, calcium, and 5-lipoxygenase in the production of tumor necrosis factor by a cloned interleukin-3 dependent cell line with natural cytotoxic activity

The cloned interleukin-3 dependent cell line, M1-A5 was studied to determine whether protein kinase C, calcium mobilization, and 5-lipoxygenase activity were involved in the signal transduction pathways required for the production of TNF. TNF release was stimulated by 10 ng/ml phorbol myristate acetate (PMA), 2 μM calcium ionophore A23187, and 1 μg/ml lipopolysaccharide (LPS) with synergism seen between PMA and A23187. All signals were blocked by phloretin and the PMA signal was blocked by H-7, both drugs acting as protein kinase C inhibitors. Desensitization of protein kinase C by PMA (1 μg/ml for 24 h) provided evidence that both PMA- and LPS-stimulated TNF production were protein kinase C-dependent while A23187-stimulated TNF production was not. Both the calcium chelator, EGTA, and the intracellular calcium antagonist, TMB-8, inhibited TNF production stimulated by all agents, indicating that TNF stimulation by all agents was calcium dependent. Finally, the 5-lipoxygenase inhibitors, ketaconazole and L-656,224, but not the cyclo-oxygenase inhibitor ASA, inhibited TNF stimulated by all agents. These findings indicate that, although TNF production by M1-A5 cells can be stimulated either by a calcium/protein kinase C- or by a calcium-dependent signal, there is a convergence of signals at the level of 5-lipoxygenase activation.

Interleukin 1 induces beta-endorphin secretion via Fos and Jun in AtT-20 pituitary cells.

Previous work had shown that interleukin 1 (IL-1), after a long period of treatment, stimulates beta-endorphin release and potentiates the effects of secretagogues in AtT-20 cells, a mouse anterior pituitary cell line. Treatment of AtT-20 cells with IL-1 induced a transient and early stimulation of mRNA expression by both immediate-early protooncogenes Fos and Jun (mouse c-fos and c-jun). The effect appeared within 30 min, and returned to basal levels after 2 hr. Desensitization of protein kinase C by phorbol ester pretreatment had no effect on the ability of IL-1 to induce Fos and Jun mRNA expression. Somatostatin, an inhibitor of cAMP and beta-endorphin secretion, did not reduce the IL-1 effect on Fos and Jun mRNA expression. Addition to AtT-20 cells of antisense oligonucleotides to Fos and Jun abolished the secretion induced by IL-1. These results indicate that immediate-early signals Fos and Jun are involved in IL-1-induced beta-endorphin secretion in AtT-20 cells.

Interleukin 1 induces early protein phosphorylation and requires only a short exposure for late induced secretion of beta-endorphin in a mouse pituitary cell line.

Previous work has shown that prolonged pretreatment of a mouse anterior pituitary cell line, AtT-20 cells, with the cytokine interleukin 1 (IL-1) stimulates beta-endorphin release and potentiates the secretion induced by many secretagogues. Desensitization of protein kinase C (PKC) by pretreatment with phorbol ester [phorbol 12-tetradecanoate 13-acetate (TPA)] for 8 hr abolished the secretion induced by TPA as well as the enhancement of TPA-induced beta-endorphin release produced by IL-1. Desensitization of PKC only partly abolished the potentiating effects of IL-1 on corticotropin-releasing factor-induced beta-endorphin secretion. In contrast, IL-1-induced beta-endorphin release was independent of PKC. We observed that treatment of AtT-20 cells with IL-1 markedly phosphorylated 19-, 20-, and 60-kDa proteins within minutes, presumably by early activation of protein kinases. Prolonged treatment with TPA, which was shown to desensitize an 87-kDa protein (a substrate for PKC), had no effect on IL-1-induced phosphorylation of 20-, 60-, and 87-kDa proteins, indicating that the phosphorylation of these proteins does not involve PKC. IL-1 does not generate cAMP in AtT-20 cells, suggesting that a cAMP-dependent protein kinase is also not involved. Prolonged treatment with IL-1 abolishes the capacity of cytokine to induce the phosphorylation of 20- and 60-kDa proteins. The presence of IL-1 was required initially only for a short time to induce late secretion in AtT-20 cells. These observations indicate that once IL-1 generates an early signal, its presence is no longer necessary for the subsequent secretion of beta-endorphin.

Homologous and heterologous desensitization mediated by vasopressin in smooth muscle cells

Prolonged exposure of A-10 cells to Arginine Vasopressin (AVP) resulted in the following responses: (a) loss of vasopressin receptors from the cell surface (30–40%), (b) increased basal levels of inositol and inositol monophosphate, (c) decreased inositol di- and trisphosphate production and decreased intracellular calcium release in response to a second challenge with AVP, (d) attenuation of AVP-mediated inhibition of isoproterenol-stimulated cAMP and ANF-stimulated cGMP accumulation and (e) attenuation of thrombin and ATP-mediated increase in inositol di- and trisphosphate accumulation and intracellular calcium release. All the above responses depended on the time of exposure of the cells to AVP with the responses being attenuated as early as 5–10 min of exposure to AVP. The desentization also depende on the concentration of AVP used with 50% of maximal desentization for each response being observed at 5 nM of AVP. This concenttation of AVP corresponded well with the K d of vasopressin for binding to these sites. Desensitization of protein kinase C (PKC) by prolonged exposure of the cells to PDBu or addition of the PKC inhibitor staurosporin during pretreatment with AVP did not prevent AVP-mediated desensitization, suggesting that PKC may not be involved in AVP-mediated desentisization in smooth muscle cells. It is concluded that AVP induced both homologous and heterologous desensitization of phosphatidylinostiol turnover and calcium release in smooth muscle cells. The desensitization processes did not appear to be mediated by protein kinase C. The possibility that the locus of the heterologous desensitization may be at the level of substrates such as PI, PIP and PIP 2 is discussed.

Augmentation of neurotransmitter receptor-stimulated cyclic AMP accumulation in rat brain: differentiation between the effects of baclofen and phorbol esters

The augmentation of isoproterenol or vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in rat brain slices by the GABA B agonist baclofen was compared to that mediated by tumor-promoting phorbol esters. The protein kinase C inhibitor H7 and desensitization of protein kinase C reduced the cyclic AMP augmenting effect of the phorbol ester, but not baclofen. Incubation of brain slices in the presence of both baclofen and a phorbol ester amplified the cyclic AMP response to isoproterenol or VIP to a greater degree than that found with either baclofen or the phorbol ester alone, with the increased augmentation appearing to be additive, These findings indicate that although stimulation of GABA B receptors or protein kinase C activation by phorbol esters have similar effects on transmitter-stimulated cyclic AMP production in brain, these augmenting actions appear to be independently mediated.