Pertussis toxin treatment blocks the inhibition of somatostatin and increases the stimulation by forskolin of cyclic AMP accumulation and adrenocorticotropin secretion from mouse anterior pituitary tumor cells

Somatostatin (SRIF) inhibits stimulated cyclic AMP accumulation and adrenocorticotropin (ACTH) release from mouse anterior pituitary tumor cells (AtT-20/D16-16). In order to determine whether guanine nucleotide inhibitory proteins (Ni) mediate these effects, AtT-20 cells were treated with pertussis toxin, an agent that inactivates Ni. Pertussis toxin catalyses the ADP-ribosylation of a 41,000 MW protein in membranes of AtT-20 cells. Pretreatment with pertussis toxin prevents the subsequent ability of toxin to catalyse the labeling of Ni. This effect is dependent on the time of pretreatment and is not reversible. The inhibition of SRIF of forskolin-stimulated cyclic AMP accumulation and ACTH release is prevented by pertussis toxin treatment. The blockade is dependent on the time and concentration of toxin used and is not reversible. Pertussis toxin treatment prevents SRIF from inhibiting corticotropin releasing factor and cholera toxin-stimulated cyclic AMP synthesis. The inhibition of K+ and 8-bromocyclic AMP-stimulated ACTH release by SRIF is attenuated partially by toxin treatment. The ability of forskolin and cholera toxin to stimulate cyclic AMP formation and ACTH release is enhanced by treatment of AtT-20 cells with pertussis toxin. The increased cyclic AMP response to forskolin is prevented by cycloheximide. The data indicate that Ni mediates the inhibition by SRIF of cyclic AMP formation and the ACTH release that results from adenylate cyclase stimulation.     

Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL.

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.     

Serum-induced sensitization of cyclic AMP accumulation in C62B rat glioma cells.

Exposure of C62B rat glioma cells to fresh medium containing fetal bovine serum induced a sensitization of the subsequent ability of isoproterenol and forskolin to stimulate cyclic AMP accumulation, compared to cells exposed to fresh medium without serum. Isoproterenol stimulation was typically increased by 2- to 4-fold and forskolin stimulation by 3- to 5-fold. Sensitization occurred rapidly, was rapidly reversible and appeared to result from an increase in maximal stimulation. A commercial preparation of albumin, purified chromatographically so as to retain bound lipids and other factors, was able to mimic the effect of serum. In contrast to the effects of serum, exposure of cells to phorbol 12-myristate, 13-acetate induced little or no change in forskolin stimulation but a marked desensitization of isoproterenol stimulation that was due primarily to a decrease in potency. Neither the protein kinase C inhibitor staurosporine or overnight exposure to phorbol 12-myristate, 13-acetate to down-regulate protein kinase C prevented serum-induced sensitization. Pertussis toxin almost completely blocked serum-induced sensitization, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in mediating the effects of serum. Sensitization was poorly retained in membrane adenylate cyclase assays. Studies with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, direct assays of cyclic AMP degradation by intact cells and assays of phosphodiesterase activity in cell lysates all indicated that degradation of cyclic AMP was decreased in serum-pretreated cells. Thus, both increased cyclic AMP synthesis and decreased cyclic AMP degradation may contribute to sensitization in these cells.     

Influence of pertussis toxin on parathyroid hormone stimulated cyclic AMP production and phosphate transport in opossum kidney cells

There is evidence that guanine nucleotide-sensitive (G) proteins intervene in the activation of adenylate cyclase by parathyroid hormone (PTH). Furthermore, recent studies suggest that G proteins may be involved in the activation by PTH of phospholipase C, with subsequent elevation of diacylglycerol, inositol trisphosphate, and intracellular calcium. Since G proteins may be involved in both transduction systems postulated to mediate the actions of PTH, the present studies were performed to evaluate the influence of pertussis toxin, which prevents receptor-mediated activation of G proteins, on the effects of PTH in opossum kidney (OK) cells. In OK cell membranes, pertussis toxin catalyzed the adenosine diphosphate (ADP) ribosylation of a protein with a molecular weight of 41 kd on SDS-PAGE. Cholera toxin catalyzed the ribosylation of two proteins of molecular weight 52 and 45 kd. Pretreatment of the cells with pertussis toxin abolished the labelling of this 41 kd protein, confirming the access of the toxin into the cells and the presence of pertussis toxin-sensitive substrates. The ribosylation of the cholera toxin substrates was unaffected by pertussis toxin pretreatment of the cells. Treatment of OK cells with pertussis toxin did not change the basal levels of cyclic AMP, but increased the levels of cyclic AMP in response to bPTH 1-34 from 355 +/- 17 to 449 +/- 20 pmoles cyclic AMP per 5 minutes per culture. These results were consistent with the inactivation of an inhibitory G protein. Furthermore, PTH-stimulated cyclic AMP generation was inhibited by norepinephrine from 362 +/- 10 to 228 +/- 18 pmole cyclic AMP per 5 minutes per culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of adenylate cyclase attenuates adenosine receptor-mediated relaxation in coronary artery.

This study was designed to evaluate whether the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) would attenuate the relaxation produced by adenosine analogs in order to provide functional evidence in support of the working hypothesis that adenosine receptor-mediated relaxation of coronary artery involves adenylate cyclase. Rings from porcine left anterior descending coronary artery were mounted in organ chambers for measurement of isometric force. Rings contracted with KCl (30 mM) relaxed in a concentration-dependent manner to 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), isoproterenol, sodium nitroprusside (SNP) and forskolin. Treatment of coronary rings with DDA (50 microM) significantly attenuated the relaxation produced by CAD, NECA, forskolin and isoproterenol, but had no effect on the relaxation response to SNP. The nucleoside transport inhibitor dilazep (10 microM) completely reversed the inhibitory effect of DDA on the relaxation produced by forskolin and CAD, whereas dilazep only partially reversed the DDA inhibition of NECA-induced relaxation. In a membrane preparation from porcine coronary artery CAD, but not NECA, increased cyclic AMP production in a GTP-dependent manner. DDA significantly decreased basal cyclic AMP production and also decreased CAD-, forskolin-, GTP- and NaF-stimulated cyclic AMP production. These results provide functional and biochemical evidence in support of the working hypothesis that adenosine receptor-mediated coronary relaxation involves adenylate cyclase. Furthermore, the results from this study suggest that the signaling mechanisms responsible for adenosine receptor-mediated coronary relaxation are more complicated than a single receptor coupled with adenylate cyclase because 1) dilazep completely reversed the inhibitory effect of DDA on the CAD relaxation but not the NECA relaxation, and 2) NECA did not increase cyclic AMP production.     

Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms.

The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation.     

The dopamine-1 agonist, SKF 82526, stimulates phospholipase-C activity independent of adenylate cyclase

Dopamine-1 (DA-1) receptors have been found in renal tubular membranes which stimulate both adenylate cyclase and phospholipase-C activity. In renal cortical plasma membrane preparations the DA-1 agonist SKF 82526, forskolin and NaF stimulated adenylate cyclase activity. 2',5'-dideoxyadenosine inhibited basal and DA-1 agonist stimulated adenylate cyclase activity. Forskolin, NaF, dibutyryl-cyclic AMP and 2',5'-dideoxyadenosine had no effect on basal or DA-1 agonist stimulated phospholipase-C activity in these membranes. These studies indicate that DA-1 agonist stimulates adenylate cyclase and phospholipase-C activities independently. Phospholipase-C activity was also increased by the nonhydrolyzable GTP analog, guanosine-5'-O-(3-thiophosphate). When DA-1 agonist and guanosine-5'-O-(3-thiophosphate) were added together there was a slight but significant increase in phospholipase-C activity. This increase was inhibited in the presence of guanosine-5'-O-(2-thiodiphosphate). DA-1 stimulated phospholipase-C activity was found to be insensitive to both cholera and pertussis toxins. The present studies indicate a cyclic AMP independent transduction pathway for DA-1 receptor mediated through a guanine nucleotide regulatory protein associated phospholipase-C.     

Mechanisms of beta-adrenoceptor mediated increase in delayed rectifier potassium current

Experiments were carried out in single ventricular cells of the guinea-pig heart. Isoproterenol, forskolin, intracellularly applied cyclic AMP and 3-isobutyl-1-methylxanthine increased the delayed rectifier potassium current (IK). The effect of isoproterenol was abolished by intracellularly applied guanosine 5'-O-(3-thio-triphosphate). These results indicate that isoproterenol stimulates beta-adrenoceptors to activate adenylate cyclase by mediation through the stimulatory GTP-binding protein, and causes an increase in intracellular cyclic AMP levels. Then IK is probably increased by phosphorylation of the IK-channel protein by cyclic AMP-dependent protein kinase.     

Effects of Cholera Enterotoxin on Adenosine 3′,5′-Monophosphate and Neutrophil Function. COMPARISON WITH OTHER COMPOUNDS WHICH STIMULATE LEUKOCYTE ADENYL CYCLASE

Cholera enterotoxin caused a delayed accumulation of adenosine 3',5'-monophosphate (cyclic AMP) in human leukocytes, associated with an increase in leukocyte adenyl cyclase activity. The action of cholera enterotoxin contrasted with that of other agents which stimulate adenyl cyclase: (a) the effects of the toxin were delayed in onset, while prostaglandin-E(1) (PGE(1)) and isoproterenol acted rapidly; (b) removal of the soluble toxin from the extracellular medium did not abolish its effects on cyclic AMP and inhibition of antigenic histamine release, while removal of PGE(1) did prevent its effects; (c) PGE(1), but not cholera enterotoxin, stimulated adenyl cyclase activity when added directly to broken cell preparations. Binding of the toxin to leukocytes was rapid and irreversible, and was followed by a gradual increase in cyclic AMP which was not prevented by cycloheximide.Cholera enterotoxin caused accumulation of cyclic AMP in purified human neutrophils as well as mono-nuclear cells, but did not prevent the extrusion of lysosomal hydrolases from phagocytic cells. The toxin only slightly inhibited the ability of human neutrophils to kill Candida albicans. Thus these results with the toxin cast doubt on previous proposals that cyclic AMP regulates these two functions of neutrophils. The unique action of cholera enterotoxin on cyclic AMP production provides a potentially useful pharmacologic tool, in addition to methylxanthines and dibutyryl cyclic AMP, for testing hypotheses relating cyclic AMP to altered function of leukocytes and, perhaps, of other mammalian cells.     

3,3′,5-Triiodothyronine Administration In Vivo Modulates the Hormone-sensitive Adenylate Cyclase System of Rat Hepatocytes

The ability of 10 muM epinephrine or isoproterenol to stimulate cyclic AMP accumulation was decreased in hepatocytes isolated from hyperthyroid (triiodothyronine treated) as compared to euthyroid rats. In the presence of methylisobutylxanthine, epinephrine or isoproterenol-stimulated cyclic AMP accumulation was approximately 65% lower in hyperthyroid as compared with euthyroid rat hepatocytes. The ability of glucagon to stimulate a cyclic AMP response was also decreased in the hyperthyroid state, when assayed in either the absence or presence of a methyl xanthine. The character of the catecholamine-stimulated cyclic AMP response was beta adrenergic in both the hyperand euthyroid states. No evidence for an alpha(2) adrenergic mediated component of catecholamine action on cyclic AMP levels was noted. Cyclic AMP phosphodiesterase activity of hepatocyte homogenates was not altered in the hyperthyroid state. Hormone-stimulated, guanine nucleotide- and fluoride-activatable adenylate cyclase activity was reduced in subcellular fractions obtained from hyperthyroid as compared with euthyroid rat hepatocytes. Beta adrenergic receptor binding was reduced approximately 35% and glucagon receptor binding reduced approximately 50% in the hyperthyroid as compared with euthyroid rat hepatocyte membrane fractions. The status of the regulatory components of adenylate cyclase were examined by in vitro treatment of subcellular fractions with cholera toxin. The ability of cholera toxin to modulate adenylate cyclase was not altered by hyperthyroidism. Cholera toxin catalyzed AD[(32)P]ribosylation of hyperthyroid and euthyroid rat hepatocyte proteins separated electrophoretically displayed nearly identical autoradiograms. Studies of the reconstitution of adenylate cyclase activity of S49 mouse lymphoma cyc(-) mutant membranes by detergent extracts from rat hepatocyte membranes, indicated that hyperthyroidism was associated with a reduced capacity of regulatory components to confer fluoride, but not guanine nucleotide activatability to catalytic cyclase. Thyroid hormones regulate the hormone-sensitive adenylate cyclase system of rat hepatocytes at several distinct loci of the system.     

THE MODULATING INFLUENCE OF CYCLIC NUCLEOTIDES UPON LYMPHOCYTE-MEDIATED CYTOTOXICITY

The capacity of allosensitized thymus-derived lymphocytes to destroy target cells bearing donor alloantigens is modulated by the cellular levels of cyclic AMP and cyclic GMP. Increases in the cyclic AMP levels of attacking lymphocytes by stimulation with prostaglandin E₁, isoproterenol, and cholera toxin inhibit lymphocyte-mediated cytotoxicity; whereas, depletion of cyclic AMP with imidazole enhances cytotoxicity. The augmentation of cytotoxicity produced by cholinergic stimulation with carbamylcholine is not associated with alterations in cyclic AMP levels and is duplicated by 8-bromo-cyclic GMP. The effects of activators of adenylate cyclase, cholinomimetic agents, and 8-bromocyclic GMP are upon the attacking and not the target cells and occur at the time of initial interaction of attacking and target cells. Indeed, the level of cyclic nucleotide (cyclic AMP and cyclic GMP) at the time of initial cell-to-cell interaction determines the extent of cytotoxicity.     

Pharmacological characterization of muscarinic receptors mediating inhibition of adenylate cyclase activity in the rat retina

Incubation of the rat retina with acetylcholine resulted in about a 20 to 30% decrease of basal cyclic AMP accumulation. Oxotremorine, arecoline, [4-hydroxy-2-butynyl]trimethylammonium chloride, m-chlorocarbanilate and carbachol also inhibited cyclic AMP accumulation. Nicotine had no effect. The response was blocked by atropine and pirenzepine but not gallamine. Intraocular injection of pertussis toxin 72 hr before testing also blocked the response to acetylcholine. The presence of forskolin exaggerated the response to acetylcholine. Intraocular injection of the cholinotoxin AF64A resulted in apparent supersensitivity of the response to acetylcholine. Our results suggest that rat retina contains muscarinic M1 receptors that are coupled negatively to adenylate cyclase.     

Desensitization of the beta-2 adrenoceptor-mediated vasodilation in rat aorta after prolonged treatment with the beta-2 adrenoceptor agonist clenbuterol

Administration to rats of the selective beta-2 adrenoceptor agonist (+/-)-clenbuterol (CLEN) (0.3 mg.kg-1 s.c., twice daily for 14 days) decreased the relaxant responses to the beta adrenoceptor agonist (-)-isoproterenol (IS) and to CLEN in KCl-contracted aortic rings. The treatment did not modify the vasodilation induced by forskolin (a direct activator of the catalytic subunit of the adenylate cyclase), 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), adenosine or acetylcholine. IS increased (cAMP) cyclic AMP levels dose-dependently in rat aorta, and this effect was reduced markedly in arteries from CLEN-treated rats. By contrast, the treatment did not modify the forskolin-induced cAMP production. The contractile response to (-)-norepinephrine (NE) was inhibited in the presence of IS or CLEN in control aortic rings. However, this modulatory effect was not seen in arteries from CLEN-treated rats. Preincubation of the arteries with either cholera toxin (an activator of the stimulatory guanine nucleotide binding protein, Gs) or forskolin reduced NE-induced vasoconstriction to the same extent in aortic rings from both control and CLEN-treated rats. The chronotropic response to NE in rat atria (beta-1-mediated) was not affected by the treatment. These results suggest that prolonged administration of CLEN to rats induced desensitization of beta-2 adrenoceptor-mediated vascular relaxation by alterations at the level of the beta-2 adrenergic receptor, but not in the mechanisms related to Gs, adenylate cyclase or in those distal to cAMP production.     

Modulation of cyclic AMP production by signal transduction pathways in preglomerular microvessels and microvascular smooth muscle cells

Cyclic AMP affects microvascular smooth muscle contraction and growth. Therefore, it is important to elucidate mechanisms regulating cyclic AMP production in microvascular smooth muscle. In this study, we determined whether several signal transduction pathways regulate receptor-induced cyclic AMP in isolated preglomerular microvessels and microvascular smooth muscle cells. Preglomerular microvessels were incubated with isoproterenol (beta-adrenoceptor agonist) and with and without U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), 1-butanol (phospholipase D inhibitor), CGP77675 (c-src inhibitor), HA1077 (Rho kinase inhibitor), Y27632 (Rho kinase inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), dipenyleneiodonium (NADPH oxidase inhibitor), or Tempol (superoxide dismutase mimetic). Cultured preglomerular microvascular smooth muscle cells were incubated with isoproterenol or forskolin (direct activator of adenylyl cyclase) and with or without U73122, C(2)-ceramide (phospholipase D inhibitor), or PP1 [src family inhibitor, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine]. All studies were conducted with 3-isobutyl-1-methylxanthine (broad-spectrum phosphodiesterase inhibitor) to eliminate changes in cyclic AMP degradation. In microvessels isoproterenol-induced cyclic AMP was not affected by Y27632, HA1007, LY294002, dipenylene-iodonium, or Tempol; was increased by U73122 and GF109203X; and was decreased by 1-butanol and CGP77675. In cells, U73122 increased and C(2)-ceramide and PP1 decreased isoproterenol-induced cyclic AMP. Forskolin-induced cyclic AMP was not altered. These results indicate that receptor-mediated activation of adenylyl cyclase is 1) not modulated by Rho kinase, phosphatidylinositol-3-kinase, NADPH oxidase, or superoxide; 2) is attenuated by phospholipase C and protein kinase C; and 3) is augmented by phospholipase D and src. Phospholipase C, phospholipase D, and src modulate receptor-induced cyclic AMP by affecting beta-adrenoreceptor/G protein/adenylyl cyclase coupling rather than by directly affecting adenylyl cyclase activity.     

Atrial natriuretic factor reduces cyclic adenosine monophosphate content of human fibroblasts by enhancing phosphodiesterase activity

Radioligand binding studies disclosed one class of high affinity atrial natriuretic factor (ANF) receptors on human fibroblast membranes (Kd = 66 pM; maximum number of binding sites [Bmax] = 7,000 sites/cell). ANF increased cellular cyclic guanosine monophosphate (cGMP) content and suppressed isoproterenol- and PGE1-elevated, but not basal, cAMP content. Pertussis toxin pretreatment, which maximally ADP-ribosylated Gi, the guanine nucleotide-binding protein that couples inhibitory receptors to adenylate cyclase and blocks receptor-mediated inhibition of adenylate cyclase, did not interfere with ANF suppression of isoproterenol- or PGE1-elevated cellular cAMP content. Preliminary incubation of fibroblasts with 8-bromo cGMP or phosphodiesterase inhibitors, including 3-isobutyl-1-methylxanthine, Ro 20-1724, and cilostamide, however, prevented the ANF suppression of cAMP. MB 22948, an inhibitor that is partially selective for cGMP phosphodiesterase, did not block the effect of ANF. We conclude that in these cells, unlike other systems, ANF reduces cAMP content by activating a phosphodiesterase rather than by inhibiting adenylate cyclase.     

Cholera toxin and pertussis toxin stimulate prostaglandin E2 synthesis in a murine macrophage cell line

When RAW264.7 murine macrophages were incubated with cholera toxin or pertussis toxin, prostaglandin E2 (PGE2) synthesis was enhanced markedly. Cholera toxin and pertussis toxin added together synergistically stimulated PGE2 synthesis. Cholera toxin and pertussis toxin also stimulated cyclic AMP (cAMP) accumulation. However, PGE2 synthesis was independent of increases in cAMP, as neither forskolin nor isoproterenol, which increased cAMP accumulation, nor dibutyryl-cAMP had any effect on PGE2 synthesis. In intact cells, cholera toxin and pertussis toxin stimulated phospholipase A2 to enhance metabolism of phosphatidylinositol to lysophosphatidylinositol and glycerophosphoinositol, with time courses similar to their stimulation of PGE2 synthesis. Cholera toxin catalyzed ADP-ribosylation of proteins of Mr 45,000 and 49,000 in intact cells, whereas an additional substrate of Mr 41,000 was observed in vitro. Preincubation of intact cells with pertussis toxin blocked subsequent in vitro labeling of the Mr 41,000 protein by cholera toxin, suggesting that the same protein was ADP-ribosylated by both toxins. Western blot analysis using specific antisera against Gi, Go and Gs revealed that the Mr 41,000 substrate was bound by the anti-Gi and anti-Go but not anti-Gs. The present data suggest that guanine nucleotide binding regulatory proteins are involved in the regulation of arachidonic acid metabolism to PGE2 in RAW264.7 cells. Furthermore, the possibility is raised that phospholipase A2 is regulated by both stimulatory and inhibitory guanine nucleotide binding proteins.     

ATP receptor regulation of adenylate cyclase and protein kinase C activity in cultured renal LLC-PK1 cells.

In cultured intact LLC-PK1 renal epithelial cells, a nonhydrolyzable ATP analogue, ATP gamma S, inhibits AVP-stimulated cAMP formation. In LLC-PK1 membranes, several ATP analogues inhibit basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in a dose-dependent manner. The rank order potency of inhibition by ATP analogues suggests that a P2y type of ATP receptor is involved in this inhibition. The compound ATP gamma S inhibits agonist-stimulated adenylate cyclase activity in solubilized and in isobutylmethylxanthine (IBMX) and quinacrine pretreated membranes, suggesting that ATP gamma S inhibition occurs independent of AVP and A1 adenosine receptors and of phospholipase A2 activity. The ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity is not affected by pertussis toxin but is attenuated by GDP beta S, suggesting a possible role for a pertussis toxin insensitive G protein in the inhibition. Exposure of intact LLC-PK cells to ATP gamma S results in a significant increase in protein kinase C activity. However, neither of two protein kinase C inhibitors (staurosporine and H-7) prevents ATP gamma S inhibition of AVP-stimulated adenylate cyclase activity, suggesting that this inhibition occurs by a protein kinase C independent mechanism. These findings suggest the presence of functional P2y purinoceptors coupled to two signal transduction pathways in cultured renal epithelial cells. The effect of P2y purinoceptors to inhibit AVP-stimulated adenylate cyclase activity may be mediated, at least in part, by a pertussis toxin insensitive G protein.     

Adenosine and muscarinic cholinergic receptors attenuate cyclic AMP accumulation by different mechanisms in 1321N1 astrocytoma cells

An adenosine receptor has been characterized to unambiguously demonstrate that the inhibitory guanine nucleotide regulatory protein, Gi, of 1321N1 human astrocytoma cells is fully capable of functionally coupling to adenylate cyclase. Adenosine receptor agonists attenuated cyclic AMP accumulation by 35 to 75% with the order of potency of N6(R-phenylisopropyl)-adenosine greater than adenosine = 2-chloroadenosine greater than N6-methyladenosine = N6-benzyladenosine. 3-Isobutyl-1-methylxanthine competitively antagonized the effect of adenosine receptor agonists. Adenylate cyclase activity measured in cell-free preparations from 1321N1 cells was inhibited by N6(R-phenylisopropyl)-adenosine. Pretreatment of 1321N1 cells with pertussis toxin blocked both adenosine receptor-mediated inhibition of adenylate cyclase activity and attenuation of cyclic AMP accumulation. In contrast to the effects on responses to adenosine receptor agonists, 3-isobutyl-1-methylxanthine noncompetitively antagonized muscarinic receptor-mediated attenuation of cyclic AMP accumulation and pertussis toxin had no effect. These data are consistent with the ideas that Gi is fully functional in 1321N1 cells and links inhibitory adenosine receptors to adenylate cyclase, and that the muscarinic receptor of these cells couples to the phosphoinositide response system, but is incapable of functionally coupling through Gi to inhibit adenylate cyclase.     

Sensitization of neuronal A2A adenosine receptors after persistent D2 dopamine receptor activation

Acute activation of Galpha(i/o)-coupled D2 dopamine receptors inhibits A2A adenosine receptor stimulation of adenylate cyclase. This antagonistic interaction between D2 dopamine and A2A adenosine receptors has been well documented; however, the effects of persistent activation of D2 dopamine receptors on subsequent A2A adenosine receptor signaling have not been explored. The present study investigated the effects of short-term (3-h) and long-term (18-h) activation of D2L dopamine receptors on subsequent A2A adenosine receptor stimulation of adenylate cyclase in CAD-D2L and NS20Y-D2L neuroblastoma cells. Short- and long-term activation of D2L dopamine receptors markedly increased 5'-N-methylcarboxamidoadenosine (MECA)-stimulated cyclic AMP accumulation 1.4-fold and 1.7-fold, respectively. D2L receptor-induced sensitization of A2A-stimulated cyclic AMP accumulation was blocked by the D2 antagonist spiperone and pertussis toxin pretreatment. In addition, persistent activation of A2A adenosine receptors resulted in 50% desensitization of subsequent MECA-stimulated cyclic AMP accumulation; however, MECA-induced desensitization of A2A adenosine receptors did not prevent completely quinpirole-induced sensitization of adenylate cyclase. These studies revealed a novel mode of regulation between D2L dopamine and A2A adenosine receptors and suggest a cooperative interaction in the regulation of cyclic AMP signaling.     

Bacterial toxins affect early events of T lymphocyte activation.

The effects of pertussis toxin and cholera toxin on early events of T lymphocyte activation were examined in the T lymphocyte cell line, Jurkat. Pertussis toxin treatment of these T cells increased inositol phosphates production and led to increases in intracellular free calcium concentration. These effects were produced by the isolated B (binding) subunit of pertussis toxin, alone. Inositol phosphates production resulting from perturbation of the T cell antigen receptor-CD3 complex by MAb was not affected by pertussis toxin treatment but was markedly inhibited by cholera toxin. This effect of cholera toxin paralleled elevations in cAMP content. However, forskolin, in concentrations equipotent for cAMP production, was a weaker inhibitor of inositol phosphates production. Cholera toxin inhibition of inositol phosphates production did not result from inhibition of baseline incorporation of inositol into phosphoinositide substrates of phospholipase C. These studies underline the complexity of toxin effects on cellular systems and suggest that other approaches will be required to implicate guanine nucleotide-binding regulatory proteins in control of the early events of T lymphocyte activation. However, the data presented here provide a molecular basis for the clinical observations of lymphocytosis and the in vitro observations of lymphocyte mitogenesis after pertussis toxin stimulation.