Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β-Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., ( R,S )-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca²⁺]_i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Regulation of motility in bovine brain endothelial cells

Scatter factor (SF) is a fibroblast-derived cytokine which stimulates motility of epithelial and vascular endothelial cells. We used a quantitative assay based on migration of cells from microcarrier beads to flat surfaces to study the regulation of motility in bovine brain endothelial cells (BBEC). Peptide growth factors (EGF, ECGF, basic FGF) did not stimulate migration. Tumor promoting phorbol esters (PMA, PDD) markedly stimulated migration, while inactive phorbol esters (4a-PDD, phorbol-13,20-diacetate) did not affect migration. Both SF- and PMA-stimulated migration were inhibited by 1) TGF-beta; 2) protein kinase inhibitors (e.g., staurosporine, K-252a); 3) activators of the adenylate cyclase signaling pathway (e.g., dibutyryl cyclic AMP, theophylline); 4) cycloheximide; and 5) anti-cytoskeleton agents (e.g., cytochalasin B, colcemid). However, PMA and SF pathways were distinguishable: 1) PMA induced additional migration at saturating SF concentrations; 2) the onset of migration-stimulation was immediate for PMA and delayed for SF; and 3) down-modulation of protein kinase C (PKC) ablated PMA but not SF responsiveness. Assessment of PKC by (3H)-phorbol ester (PDBu) binding and by immunoblot showed 1) scatter factor does not cause significant redistribution or down-modulation of PDBu binding or alpha-PKC; and 2) PDBu mediates redistribution and down-modulation of both binding and alpha-PKC. These findings suggest two pathways for BBEC motility: a PKC-dependent pathway and an SF-stimulated/PKC-independent pathway.     

Differential regulation of microglial NO production by protein kinase C inhibitors

Nitric oxide (NO) produced by microglia has been implicated in the pathogenesis of various central nervous system diseases; however, the intracellular signal pathways for the production of NO are not well known. Protein kinase C (PKC) plays a key role in a variety of signal transduction processes. To elucidate how PKC regulates microglial NO production, we examined the effects of PKC inhibitors on lipopolysaccharide (LPS)-stimulated NO production by primary cultured rat microglia. Staurosporine, a non-selective PKC inhibitor, increased LPS-induced production of NO at 0.1-10 nM range of concentration. Protein kinase A (PKA) inhibitor, H89, did not affect LPS-induced NO production, suggesting that staurosporine effect is not mediated by inhibition of PKA. However, other two PKC inhibitors, whose specificities for PKC isoforms were different, G?6976 and Ro-32-0432, exhibited different effects on NO production from staurosporine; the former inhibited and the latter showed no effect. Interestingly, an activator of PKC, phorbol 12-myristate 13-acetate (PMA) also increased LPS-induced production of NO at 1-10 nM range of concentration, suggesting that prolonged incubation with PMA caused down-regulation of PKC. These results indicate that the inhibition or down-regulation of some PKC isoforms causes the enhancement of NO production. The different effects of PKC inhibitors on the NO production suggest that the different PKC isoforms play different roles in regulation of NO production in microglia.     

Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling.

Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA, 100nM) for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO), 1 microM) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG) (50 microM) also elevated beta-receptor responses, but 4 beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure (12 seconds) to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 microM) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). Elevation of cyclic AMP by FMLP was insensitive to H7. PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalised compartments, or the capacity of ISO to induce beta-receptor internalisation. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation in the presence of PMA were not elevated by PMA. These findings indicate that PMA exerts a potentiating effect on neutrophil adenylate cyclase responses through protein kinase C activation. FMLP elevation of neutrophil cyclic AMP in the absence of other stimuli, appears however, to be insensitive to protein kinase inhibition.     

Loss of Protein Kinase C-αβ in Brain of Heroin Addicts and Morphine-Dependent Rats

Abstract: The biochemical status of human brain protein kinase C (PKC)-αβ during opiate dependence was studied by means of immunoblotting techniques in postmortem brain of heroin addicts who had died by opiate overdose. In the frontal cortex, a marked decrease (53%, p < 0.05) in the immunoreactivity of PKC-αβ was found in heroin addicts compared with matched controls. The loss of PKC-αβ in the brain of human addicts paralleled that observed in the frontal cortex of rats after chronic treatment with morphine (10–100 mg/kg i.p. for 5 days) (PKC-αβ decreased by 34%, p < 0.05). Chronic treatment with naloxone (1 mg/kg i.p. every 12 h for 5 days) did not alter PKC-αβ immunoreactivity in the rat brain. However, in morphine-dependent rats, naloxone-precipitated withdrawal induced a rapid and strong behavioral reaction with a concomitant up-regulation of PKC-αβ immunoreactivity to control values. These results indicated that the decrease of brain PKC-αβ induced by heroin/morphine is a μ-opioid receptor-mediated effect. The chronic administration of opiates has been associated with a marked sensitization of the adenylyl cyclase/cyclic AMP system, although this phenomenon is not exclusive of the opioid system but the general cellular adaptation to chronic inhibition of adenylyl cyclase. In this context, chronic treatment of rats with other inhibitory agonists (e.g., clonidine, 1 mg/kg i.p. every 12 h for 14 days) acting through receptors (e.g., α₂-adrenoceptors) also coupled to adenylyl cyclase did not alter brain PKC-αβ immunoreactivity. Together these findings suggest that the brain PKC system might play a major role in opiate addiction.     

Multiple non-specific effects of sphingosine on adenylate cyclase and cyclic AMP accumulation in S49 lymphoma cells preclude its use as a specific inhibitor of protein kinase C.

Recent studies with phorbol esters have suggested that protein kinase C (PKC) may play a role in the regulation of adenylate cyclase in mammalian cells. Since D-sphingosine has been reported to specifically inhibit PKC in many cell types, we evaluated its effects on stimulation of cyclic AMP accumulation by adrenaline in S49 lymphoma cells. We found sphingosine to have multiple non-specific effects which could not be explained by an inhibition of PKC. These effects included: (i) inhibition by sphingosine (50 microM) of adrenaline-stimulated cyclic AMP accumulation and sphingosine permeation of the cells which rendered them leaky to ATP; (iii) sphingosine (20 microMs) augmentation of adrenaline-stimulated cyclic AMP accumulation; (iii) inhibition by sphingosine of adrenaline-stimulated adenylate cyclase in isolated membranes by up to 95%; and (iv) sphingosine (20 microM) inhibition of cellular mechanisms for the elimination of cyclic AMP. These results demonstrate the importance of evaluating the non-specific effects of sphingosine before concluding that its actions are the consequences of a specific inhibition of PKC.     

Role of G Protein βγ Subunits in Muscarinic Receptor-Induced Stimulation and Inhibition of Adenylyl Cyclase Activity in Rat Olfactory Bulb

Abstract: In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and G_s-stimulated adenylyl cyclase activities and inhibiting the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein βγ subunits by examining whether the muscarinic responses were reproduced by the addition of βγ subunits of transducin (βγ_t) and blocked by putative βγ scavengers. Membrane incubation with βγ_t caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, βγ_t potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by βγ synergistically with activated G_s. In addition, βγ_t inhibited the Ca²⁺/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two βγ scavengers, the GDP-bound form of the α subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by βγ subunits likely acting on distinct isoforms of adenylyl cyclase.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.     

Pituitary Adenylate Cyclase-Activating Polypeptide: Control of Rat Pineal Cyclic AMP and Melatonin but Not Cyclic GMP

Abstract: In this study, the effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic nucleotide accumulation and melatonin (MT) production in dispersed rat pinealocytes were measured. Treatment with PACAP (10⁻⁷ M ) increased MT production 2.5-fold. PACAP (10⁻⁷ M ) also increased cyclic AMP accumulation four- to fivefold; this effect was potentiated two- to three-fold by α₁-adrenergic activation. This potentiation appears to involve protein kinase C (PKC) because α₁-adrenergic activation is known to translocate PKC and the PACAP-stimulated cyclic AMP accumulation was potentiated ninefold by a PKC activator, 4β-phorbol 12-myristate 13-acetate (PMA). Phenylephrine and PMA also potentiated the PACAP-stimulated MT accumulation. These results indicate that cyclic AMP is one second messenger of PACAP in the pineal gland and that the effects of PACAP on cyclic AMP and MT production can be potentiated by an α₁-adrenergic → PKC mechanism. In addition to these findings, it was observed that PACAP treatment with or without phenylephrine or PMA did not alter cyclic GMP accumulation. This indicates that PACAP is the first ligand identified that increases cyclic AMP accumulation in the pineal gland without increasing cyclic GMP accumulation. That PACAP fails to activate the vasoactive intestinal peptide/cyclic GMP pathway suggests that the vasoactive intestinal peptide receptors present in the pineal may be distinct from the type II PACAP receptors.     

Inhibition of receptor-mediated stimulation of cyclic AMP accumulation in neuroblastoma-hybrid NCB-20 cells by a phorbol ester

Activation of protein kinase C by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), modulates responsiveness of the cyclase system in many cell types. In the neuroblastoma-hybrid cell line NCB-20, PMA causes a reduction in receptor-mediated accumulation of cyclic AMP. The reduction in receptor responses by PMA occurs within 3 min and is still apparent at 40 min. This occurs in a concentration-dependent manner with an EC50 for PMA of approx. 30 nM. Accumulations of cyclic AMP that are elicited by prostaglandin E2, vasoactive intestinal peptide or 2-chloroadenosine are decreased in the presence of PMA. Accumulations of cyclic AMP that are elicited by forskolin in the absence of a receptor agonist are unaffected by the presence of PMA. Inhibition of cyclic AMP generation by dopamine is not diminished by PMA suggesting the receptor input through the inhibitory Ni-guanyl nucleotide binding protein is still functional after PMA treatment. The generalized inhibition of receptor-mediated responses by PMA could be due to a protein kinase C-mediated phosphorylation of the stimulatory Ns-guanyl nucleotide binding protein, but other mechanisms are possible.     

Nerve Growth Factor Amplifies Cyclic AMP Production in the HT4 Neuronal Cell Line

Abstract: There has been considerable interest and controversy in the relationship between nerve growth factor (NGF) and the cyclic AMP (cAMP) second messenger system. We have used a novel, neuronal cell line (HT4) to investigate the effect of NGF on the adenylyl cyclase signaling system. Treatment of cells with NGF (100 ng/ml 15 min) amplified cAMP accumulation (≈75%) in response to activation of adenosine A₂ receptors (5 min) with 5′-N-ethylcarboxamidoadenosine or activation of adenylyl cyclase directly with forskolin. Basal cAMP accumulation was not altered by NGF. This amplification appears to be mediated by activation of protein kinase C (PKC) because (1) it was mimicked by activators (phorbol esters and a diacylglycerol analogue) of PKC, (2) the effects of NGF and phorbol ester on cAMP accumulation were not additive, (3) NGF amplification of cAMP accumulation was abolished by down-regulation of PKC, (4) NGF increased cytosolic PKC activity, and (5) inhibitors of PKC blocked the NGF-induced amplification of cAMP accumulation. Although NGF-induced amplification of cAMP accumulation was dependent upon PKC, mechanisms other than the classic activation pathway (i.e., hydrolysis of inositol phospholipids or the production of diacylglycerol) appeared to mediate PKC activation by NGF. The tyrosine kinase inhibitor, lavendustin A, blocked NGF-mediated amplification of cAMP accumulation, suggesting a novel interaction between a tyrosine kinase and protein kinase C.     

Effects of protein kinase C activation on human platelet cyclic AMP metabolism

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).     

Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells

Activation of protein kinase C (PKC) bu phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat live cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by β-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was loner than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.     

LOW CONCENTRATIONS OF LITHIUM INHIBIT THE SYNTHESIS OF CYCLIC AMP AND CYCLIC GMP IN THE RAT PINEAL GLAND

Abstract— Lithium chloride (2 m m ) significantly inhibited the increases in cyclic AMP and in cyclic GMP caused by norepinephrine or high concentrations of potassium in intact rat pineal glands. Adenylyl cyclase activity in homogenates and its stimulation by isoproterenol, a β-adrenergic agonist, were also inhibited. Lithium reduced the apparent V _max of isoproterenol-stimulated adenylyl cyclase activity without significantly affecting the apparent affinity for isoproterenol. There was no effect on the binding of the antagonist [³H]dihydroalprenolol to the β-adrenergic receptors, nor on the competition for binding sites by isoproterenol. Inhibition of adenylyl cyclase activity by lithium was inversely related to the magnesium concentration in the reaction mixture. There was no differential effect of lithium on adenylyl cyclase activity from supersensitive vs subsensitive glands. Lithium may inhibit cyclic nucleotide synthesis by interfering with the role of divalent cations.     

Protein kinase C enhances growth hormone releasing factor (1-40)-stimulated cyclic AMP levels in anterior pituitary. Actions of somatostatin and pertussis toxin

The brain peptide human growth hormone releasing factor (1-40) (GRF), which stimulates adenylate cyclase activity in the anterior pituitary, is the predominant hormone signal for pituitary growth hormone (GH) release. Activators of protein kinase C such as teleocidin and 4 beta-phorbol 12-myristate 13-acetate (PMA) double the cyclic AMP accumulation induced by GRF, with no apparent effect on GRF potency; an inactive 4-alpha-PMA has no such action in cultured anterior pituitary cells. This PMA potentiation can be measured as early as 60 s, is maximal by 15 min, and wanes such that by 3-4 h there is no such amplifying effect of PMA. PMA, phorbol 12,13-dibutyrate, and teleocidin ED50 values for potentiating GRF activity are similar to those obtained for direct protein kinase C activation. The major inhibitory peptide somatostatin reduced both GRF- and GRF + PMA-stimulated cyclic AMP accumulation. Pertussis toxin totally blocked this somatostatin action without affecting the degree of maximal GRF potentiation achieved with PMA. Thus, the pertussis toxin target(s) are required for somatostatin inhibition of the cyclic AMP generating system, but may not be involved in the PMA potentiation of GRF-stimulated cyclic AMP accumulation.     

Diminished Noradrenergic Stimulation Reduces the Activity of Rolipram-Sensitive, High-Affinity Cyclic AMP Phosphodiesterase in Rat Cerebral Cortex

Abstract: The present study examined the in vivo regulation of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of cyclic AMP, formed by stimulation of β-adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of cyclic AMP formed under these conditions was inhibited by the PDE4-selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6-hydroxydopamine (6-OHDA; 200 µg) decreased the rate constant of cyclic AMP hydrolysis and increased the cyclic AMP half-life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6-OHDA infusion, respectively. This was followed by the development of supersensitivity of β-adrenergic receptor-linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6-OHDA (300 µg) was used, the reduction in the rate constant of cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6-OHDA treatment, continuous blockade of β-adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of β-adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor-effector system in the brain.