Effect of prostaglandin E2 on cyclic AMP levels in limb cells of mouse mutant brachypodism.

Mouse embryo limb cells carrying either the brachypodism (bpH/bpH) mutation or its wild-type (+/+) allele were tested for their ability to accumulate cyclic AMP in response to prostaglandin E2 (PGE2) between Embryonic Days E12 and E14. Mutant cells exhibited a precocious increase in cyclic AMP. In the absence of PGE2 but in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX), the brachypodism cells accumulated a significantly lower amount of cyclic AMP by Day E14. Limb cells carrying the bpH mutation may provide a useful experimental system to study the PGE2-cyclic AMP-cartilage differentiation interrelationship.     

Glucocorticoids Potentiate the Prostaglandin E1-Mediated Cyclic AMP Formation by a Cultured Murine Neuroblastoma Clone

The effect of steroid hormones on the prostaglandin E1 (PGE1)-mediated cyclic AMP formation by murine neuroblastoma clone N1E-115 was studied. Dexamethasone at submicromolar concentrations and corticosterone at micromolar concentrations (steroids with glucocorticoid activity) were able to modify the PGE1-mediated response whereas testosterone, progesterone, and estradiol each at 10 microM had no effect. Glucocorticoids added to the culture medium of N1E-115 cells produced an increase in the maximal response to PGE1 only after long-term (greater than or equal to 4 h) incubation with the hormone. Inhibitors of protein and RNA synthesis blocked this effect of glucocorticoids. Basal activity of adenylate cyclase in treated cells was twofold higher than that in control cells, and this enzyme seemed to be the primary target for the hormone action, since the activity of 3':5'-cyclic AMP phosphodiesterase and the binding of [3H]PGE1 to its receptors were not altered by glucocorticoid treatment. Our results indicate that glucocorticoids modulate receptor-mediated responses in cells of neural origin through a mechanism that involves induction of protein synthesis.     

Regulation of Cyclic AMP by the μ-Opioid Receptor in Human Neuroblastoma SH-SY5Y Cells

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple Neurite Formation in Neuroblastoma Cell Lines by Griseolic Acid, a Potent Inhibitor of Cyclic Nucleotide Phosphodiesterases

The morphological change of several neuroblastoma cell lines induced by griseolic acid, a novel and potent inhibitor of cyclic nucleotide phosphodiesterase (PDE), was examined. In the cell lines tested, Neuro-2a (a murine neuroblastoma cell line) showed dose-dependent (1 microM-1 mM) neurite extension. Griseolic acid markedly increased the intracellular cyclic AMP level of Neuro-2a cells, suppressed DNA synthesis (82% at 1 mM), and induced multipolar (multiple-neurite-bearing)-type neuritogenesis. A similar type of neurite outgrowth was induced by 8-bromo-cyclic AMP, which also elevated the intracellular cyclic AMP concentration. In contrast, when Neuro-2a cells were treated with retinoic acid, neurite formation was of the monopolar (single-neurite-bearing) type. Papaverine and theophylline, which have been frequently used as PDE inhibitors, failed to induce these morphological changes up to 1 mM, probably owing to the lesser potency of these compounds as compared with griseolic acid on the inhibition of PDE. Retinoic acid, theophylline, and papaverine were ineffective at elevating the intracellular cyclic AMP level. These results suggest that multipolar-type cell shape change in Neuro-2a cells is correlated with the accumulation of intracellular cyclic AMP and that griseolic acid is a useful compound to induce neuroblastoma cells into terminal differentiation.     

Inhibition of adenylate cyclase in human blood platelets by 9-substituted adenine derivatives.

A series of 9-substituted adenine derivatives inhibited adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) of a particulate preparation of human blood platelets. A 3--6 fold elevation of adenylate cyclase activity by prostaglandin E1 (PGE1) was inhibited in a concentration-related manner by 9-(tetrahydro-5-methyl-2-furyl) adenine (SQ 22,538), 9-(tetrahydro-2-furyl) adenine (SQ 22,536), 9-cyclopentyladenine (SQ 22,534), 9-furfuryladenine (sQ 4647) and 9-benzyladenine (SQ 218611). The I50 values ranged from 21 microM for SQ 22,538 to 140 microM for SQ 21,611. These same adenine derivatives reversed the inhibition by PGE1 of ADP-induced aggregation and the PGE1-stimulated elevation of adenosine 3':5'-monophosphate (cyclic AMP). The reversal of platelet aggregation inhibition by SQ 22,536 and SQ 4647 was concentration-related with I50 values of 30 microM in each case, whereas SQ 22,534 and SQ 21,611 reversed inhibition by 30% at 100 microM. SQ 22,536, SQ 22,534 and SQ 21,611 also blocked the increase in cyclic AMP levels in a concentration-related manner with I50 values of 1, 4 and 60 microM, respectively. SQ 4647 inhibited the elevation of cyclic AMP by more than 85% at 1000 microM. The adenine derivatives had no effect on platelet aggregation or on cyclic AMP levels in the absence of PGE1. These results provide additional evidence that the inhibition of platelet aggregation by PGE1 is mediated by cyclic AMP.     

An Assessment of the Role of Opioid Receptors in the Response to Cannabimimetic Drugs

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.     

Effect of thioacetamide on cytochrome P-450 synthesis in rat liver.

Both calcitonin and prostaglandin E2 (PGE2) stimulate adenylate cyclase activity in the human breast cancer cell line (T 47D). The maximum cyclic AMP response to calcitonin exceeds that of PGE2. When maximal concentrations of the two hormones were added simultaneously to the cells, the amount of cyclic AMP generated was less than that seen with calcitonin alone. When cells were treated with the protein toxin of Bordetella pertussis (islet-activating protein; IAP) which inactivates the inhibitory regulatory component (Ni) of adenylate cyclase, there was no change in basal or calcitonin-responsive adenylate cyclase in intact cells. However, the PGE2 response was augmented at all dose levels, and this effect was dependent on the concentration of IAP. Moreover, in cells pretreated with IAP, simultaneous addition of PGE2 and calcitonin resulted in additivity rather than in inhibition of cyclic AMP production. The additivity of the response to calcitonin and PGE2 after IAP treatment implies activation of separate pools of adenylate cyclase catalytic subunit by the two hormones. These data are consistent with a model in which calcitonin acts on adenylate cyclase in T 47D cells through stimulatory regulatory components alone, whereas PGE2 acts on the same cells through both stimulatory and inhibitory components. The Ni input can limit the maximum effect of PGE2 and is capable of limiting calcitonin effects when the two agonists are used simultaneously.     

Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E1 or E2 (PGE1 or PGE2) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10(-9) M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.     

Potentiation of PGE2-mediated cAMP production during neuronal differentiation of human neuroblastoma SK-N-BE(2)C cells

The prostaglandin-evoked cAMP production was studied in human neuroblastoma SK-N-BE(2)C cells during neuronal differentiation induced by all-trans retinoic acid. The incubation with 5 microM all-trans retinoic acid for 4-6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E(2) (PGE(2))-induced cAMP production was dramatically increased, whereas forskolin- and AlF-induced cAMP productions were not changed. The increase reached maximum after 4-days of incubation with all-trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half-maximal effective concentration of the PGE(2)-induced cAMP production. In addition, the binding of [(3)H]PGE(2) to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE(1) = PGE(2) > PGD(2) = PGF(2alpha) = PGI(2)) in cAMP production did not change during the differentiation, suggesting that mainly E-prostanoid (EP) receptors were involved. Butaprost, an EP(2) receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE(2) upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP(2) mRNA level was about seven times higher in differentiated cells, while the dopamine beta-hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP(2) receptor results in an increase in the PGE(2)-evoked cAMP production in SK-N-BE(2)C cells during neuronal differentiation.     

Decreased Cyclic AMP Degradation in NG 108-15 Neuroblastoma × Glioma Hybrid Cells and S49 Lymphoma Cells Chronically Treated with Drugs that Inhibit Adenylate Cyclase

The increase in hormone-stimulated cyclic AMP accumulation observed in a variety of intact cells after chronic pretreatment with drugs that inhibit adenylate cyclase activity has been attributed to an increase in adenylate cyclase activity following withdrawal of the inhibitory drug. In NG 108-15 mouse neuroblastoma X rat glioma hybrid cells (NG cells) chronically treated with the muscarinic cholinergic agonist carbachol, we have found a significant decrease in the apparent degradation rate constant for cyclic AMP, in addition to an increase in the prostaglandin E1 (PGE1)-stimulated cyclic AMP synthesis rate in intact cells. In carbachol-pretreated NG cells that were stimulated with a maximally effective dose of PGE1, and that accumulated steady-state cyclic AMP concentrations fourfold or more higher than in control cells, the apparent rate constant for degradation was about 53% lower than the value for control cells. In carbachol-pretreated cells stimulated with a submaximal dose of PGE1 to yield a steady-state cyclic AMP concentration comparable to control cells, the apparent rate constant was 31% lower than the value for control cells. In S49 mouse lymphoma cells (S49 cells) chronically treated with an analog of the inhibitory agonist somatostatin, the first-order rate constant for cyclic AMP degradation in intact cells following isoproterenol stimulation was 29% lower than the value for control cells. Despite these changes in the kinetics of cyclic AMP degradation in intact NG cells and S49 cells, there was either no change or a minimal change (less than 10%) in phosphodiesterase activities assayed in extracts of cells chronically exposed to inhibitory drugs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary granulocyte influx and impaired alveolar macrophage adenylyl cyclase responsiveness in developing respiratory distress

Alveolar macrophages have recently been postulated as being involved in the aetiology of adult respiratory distress syndrome (ARDS). To evaluate their role, basal cyclic AMP levels and responsiveness of adenylyl cyclase alveolar macrophages were determined at four intermediate stages of developing respiratory distress in piglets using a protocol with repeated lung lavage. Examination of alveolar cells recovered from the subsequent lavages reveals an influx of granulocytes (neutrophils and eosinophils) within 1 h of two intensive lung lavages. During the developing respiratory distress the basal cyclic AMPlevel of alveolar macrophages increases and adenylyl cyclase responsiveness to prostaglandin E(2) (PGE(2)) and isoprelanaline diminishes. The previously observed impairment of macrophage activity can then be explained at a subcellular level.     

Studies of the Regulation of Basal Adenylate Cyclase Activity by Membrane Polyunsaturated Fatty Acids in Cultured Neuroblastoma

The role of membrane polyunsaturated fatty acids (PUFAs) in the regulation of basal adenylate cyclase activity was examined in intact N1E-115 neuroblastoma cells. Addition of linoleic acid (50 microM) to the culture medium for 48 h resulted in a significant increase in phospholipid PUFA content and in a two- to fivefold increase in basal accumulation of cyclic AMP (cAMP). Both phenomena were reversed on removal of linoleate from the medium. PUFA enrichment stimulated cell proliferation by approximately 20% without altering the relative proportion of cellular protein. The supplemented cells synthesized significantly larger amounts of prostaglandin (PG) E and D than did the controls; however, blockade of PG synthesis by indomethacin or ibuprofen did not alter cAMP formation. Supplemented cells contained higher levels of malondialdehyde (MDA) than did controls, and MDA formation was reduced by coculture with alpha-tocopherol; however, its inclusion in the medium did not affect cAMP accumulation. Linoleate-supplemented cells responded to cyclase-activating agonists to the same extent as did control cells. Responses to inhibitory agonists (e.g., isoproterenol and carbamylcholine) were altered, but not to a sufficient extent to account for the PUFA-dependent increases in basal adenylate cyclase activity.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Specific Receptor-Mediated Inhibition of Cyclic AMP Synthesis by Dopamine in a Neuroblastoma × Brain Hybrid Cell Line NCB-20

Abstract: Dopamine and dopamine receptor agonists were found to inhibit adenylate cyclase activity dose-de-β ndently in a neuroblastoma × Chinese hamster brain explant hybrid cell line NCB-20. Apomorphine (with an IC₅₀ value of 10 n M ) was the most effective inhibitor, followed by 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-naphthaline (ADTN), dopamine, and N -dipropyldopa-mine. The inhibition was potently reversed by sulpiride, butaclamol, and flupenthixol in a stereospecific manner, but was unaffected by yohimbine, except at high concentrations. Clonidine also inhibited adenylate cyclase activity in these cells and this was reversed by the α₂-adrenoreceptor antagonist yohimbine, but not by sulpiride. [ d -Ala², d -Leu⁵]Enkephalin inhibited adenylate cyclase activity in NCB-20 cells at nanomolar concentrations; this was reversed by naloxone. All three inhibitory neurotransmitters were able to reverse the stimulation of cyclic AMP synthesis by serotonin or prostaglandin E₁The dopamine receptor that modulates cyclic AMP synthesis in NCB-20 cells is pharmacologically quite distinct from a high-affinity spiperone binding site identified in these cells, but shows the pharmacologic specificity of the D₂ receptor previously described in mammalian brain.     

Cyclic AMP response to prostaglandin E1 in mononuclear cells from peripheral blood and synovial fluid of patients with rheumatoid arthritis.

The cyclic AMP response to prostaglandin E1 (PGE1) was studied in peripheral blood (PB) and synovial fluid (SF) mononuclear cells from patients with rheumatoid arthritis (RA). The PGE1 induced accumulation of cyclic AMP was consistently (7 of 8 patients) less in cell suspensions derived from SF than in suspensions of equivalent numbers of mononuclear cells obtained simultaneously from PB. The high PB/SF cyclic AMP ratio was seen most clearly at the lowest concentration (10(-6)M) of PGE1 tested. There was no correlation between the patients' therapy and cyclic AMP response to PGE1. The high PB/SF cyclic AMP ratio was not accounted for by the presence of platelets in PB cell suspensions.     

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.     

Prostaglandin deficiency promotes sensitization of adenylyl cyclase

Inhibition of prostaglandin synthesis by the drug indomethacin suppresses the synthesis of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), and leads to a metabolic state comparable to type II diabetes. It was of interest whether prostaglandin-deficiency likewise causes sensitization of adenylyl cyclase, as this has been reported for the diabetic state. In liver plasma membranes of indomethacin-treated male rats, basal and forskolin-stimulated cyclic AMP synthesis remained unchanged when compared to untreated control rats. In control rats, stimulation of cyclic AMP synthesis by fluoride (2.2-fold) or glucagon (3.5-fold) was much lower than stimulation by forskolin (6.6-fold). In contrast, in indomethacin-treated rats, stimulation of cAMP synthesis by fluoride (4.6-fold) or glucagon (5.2-fold) nearly matched the stimulation by forskolin (6.4-fold). The level of alpha1-adrenergic receptors was slightly reduced, from 450 to 320 fmol/mg protein, by the indomethacin treatment. Independent of the treatment by indomethacin, stimulation of cyclic AMP synthesis by adrenaline failed, in agreement with the low density of adrenergic beta-receptors. In conclusion, PGE deficiency sensitizes adenylyl cyclase in rat liver for G protein-coupled receptors (glucagon) and also for fluoride.     

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.