Early events in thymocyte activation. I. Stimulation of protein synthesis by a thymus-dependent human serum factor.

Human serum contains a thymus-dependent factor that raises cyclic AMP levels in thymocytes. We found that this factor stimulates protein synthesis in thymocytes cultured in vitro. This activity of serum factor is thymus-dependent, because it is absent in sera from thymectomized donors; furthermore, this effect is predominantly found on precursors of mature T cells. Incubation of thymocytes with other agents that increase cyclic AMP, induces an increase in protein synthesis similar to that observed with serum factor. Most likely, the increase in protein synthesis is one of the events following stimulation of adenylate cyclase in thymocytes that leads to cell differentiation.     

Serum catecholamines desensitize beta-adrenergic receptors of cultured C6 glioma cells

C6 glioma cells grown in medium containing fetal bovine serum have a decreased beta-adrenergic receptor number and beta-receptor-stimulated cyclic AMP accumulation as compared to cells grown in a serum-free, defined medium. The decreased number of receptors and decreased cAMP accumulation are attributable to a suppression of receptor binding and response by serum as opposed to increases produced by growth in the defined medium. Serum, when added to cells grown in the absence of serum, stimulated cellular cyclic AMP levels to 2-3 times basal levels. This direct stimulatory effect was blocked by incubation of the cells with the beta-adrenergic antagonist propranolol and was partially reversed by dialysis of the serum. In contrast, addition of serum to cells that have been grown with serum fails to stimulate cyclic AMP accumulation. The decrease in receptors following growth in serum can be mimicked by growing cells in serum-free medium in the presence of beta-adrenergic agonists such as isoproterenol or norepinephrine. Radioenzymatic assays indicate that fetal bovine serum contains approximately 0.3 nM norepinephrine and lower concentrations of epinephrine. It thus appears that growth of C6 cells in serum-containing media desensitizes the beta-adrenergic receptor/cyclic AMP system of these cells. This desensitized state appears to result primarily from the action of catecholamines present in serum. These data indicate that retained catecholamines are one component in serum that can modify expression of beta-adrenergic receptors and hormonal response of cultured glioma cells.     

Sera from both normal and thymectomized animals produce elevations of cyclic AMP in thymocytes.

Cyclic AMP content in mouse thymocytes has been measured after incubation either with sera or serum fractions from normal or thymectomized (Tx) mice and pigs or with a synthetic circulating pig thymic factor. Sera from both Tx and normal pigs and mice induced an increase in cyclic AMP in mouse thymocytes, whereas the synthetic pig thymic factor did not. It is concluded that the increase in cyclic AMP in mouse thymocytes should be used with caution for the evaluation of circulating thymic hormone levels.     

Early events in thymocyte activation. II. Changes in nonhistone chromatin proteins induced by a thymus-dependent human serum factor.

Previously, it has been shown that a human thymus-dependent serum factor (SF), isolated from peripheral blood and acting on precursors of mature T lymphocytes, induces an increase in the synthesis of cyclic AMP and proteins in thymocytes. We have now investigated the action of SF on the incorporation of 3H-leucine and 32P-orthophosphate into nuclear proteins of thymocytes after 15 to 240 min of culture. SF induced a rapid increase in the synthesis and phosphorylation of nuclear proteins, especially in the phosphorylated nonhistone chromatin proteins (P-NHCP). Electrophoretic patterns in polyacrylamide gels of the P-NHCP fractions, extracted from the chromatin of the stimulated cells, showed that proteins with m.w. higher than 50 x 10(3) were synthesized to a larger extent as compared with unstimulated cells. These data suggest that SF acts specifically on the synthesis of P-NHCP and may in this way control DNA-template activity.     

Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells.

The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.     

Short term and long term effects of beta-adrenergic effectors and cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle

The effects of short term stimulation of beta-adrenergic receptors and elevations in intracellular cyclic AMP on nitrendipine-sensitive voltage-dependent Ca2+ channels of skeletal muscle cells in vitro has been studied using both the 45Ca2+ flux technique and [3H] nitrendipine-binding experiments. Isoproterenol increased the nitrendipine-sensitive 45Ca2+ influx under depolarizing conditions. The effects of isoproterenol were additive to those of depolarization and were antagonized by alprenolol. Half-maximal inhibition of 45Ca2+ influx induced both by depolarization and by isoproterenol occurred at a nitrendipine concentration of 1 nM. Treatments that resulted in an increased level of intracellular cyclic AMP, such as treatment with 1-methyl-3-isobutylxanthine, theophylline, dibutyryl cyclic AMP, or 8-bromocyclic AMP also resulted in an increased rate of 45Ca2+ entry via nitrendipine-sensitive Ca2+ channel. In contrast, long term treatment of myotubes in culture with isoproterenol and other compounds that increased intracellular cyclic AMP led to a large increase in the number of nitrendipine receptors. This increase was accompanied by a 4-10-fold decrease in the affinity of the receptors for nitrendipine. Alprenolol inhibited the long term effects of isoproterenol. In vivo treatment of 7-day-old chicks with reserpine and alprenolol produced a decrease in the number of skeletal muscle nitrendipine receptors. This decrease in receptor number was accompanied by an increase in the affinity of nitrendipine for its receptor by a factor of 4 to 5. These effects on the nitrendipine receptor were prevented by simultaneous injection of isoproterenol. The results are discussed in relation to the role of beta-adrenergic receptors and intracellular cyclic AMP in the regulation of skeletal muscle Ca2+ channels.     

A role for adrenaline and calmodulin in modulating cyclic AMP levels during the lactogenic cycle

The effect on lactose production of several external modulators of intracellular cyclic AMP was studied in rat mammary gland tissue slices and explants. Adrenaline, a beta-adrenergic receptor effector, forskolin, a direct adenylate cyclase activator and fluphenazine, a calmodulin inhibitor, all produced an increase in the intracellular level of cyclic AMP and a concomitant inhibition of lactose production. These results suggest a role for adrenaline and calmodulin in modulating cyclic AMP levels in mammary tissue during the lactogenic cycle.     

Effect of monoamine receptor agonists and antagonists on cyclic AMP accumulation in human cerebral cortex slices.

In human cerebral cortex slices noradrenaline, isoproterenol (a beta-adrenergic agonist), dopamine, apomorphine (a dopaminergic agonist), and serotonin stimulated cyclic AMP formation: noradrenaline greater than or equal to isoproterenol greater than dopamine = apomorphine = serotonin. Clonidine (and alpha-adrenergic agonist) was ineffective in stimulating cyclic AMP formation in temporal cortex slices. The stimulatory effect of noradrenaline and isoproterenol was blocked by propranolol (a beta-adrenergic blocker) but not by phentolamine (an alpha-adrenergic blocker). Pimozide (a selective dopaminergic antagonist) inhibited the increase of cyclic AMP formation induced by dopamine or apomorphine but not that induced by noradrenaline, isoproterenol, or serotonin. Neither propranolol or phentolamine had any effect on dopamine- or serotonin-stimulated cyclic AMP formation. Chlorpromazine blocked the increase of cyclic AMP formation induced by noradrenaline, dopamine or serotonin, while cyproheptadine, a putative central serotonergic antagonist, was ineffective. These observations suggest that there may be at least two monoamine-sensitive adenylate cyclases in human cerebral cortex which have the characteristics of a beta-adrenergic and a dopaminergic receptor, respectively, and also possibly a serotonergic receptor.     

Phosphorylation of the mammalian beta-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein

In some systems, such as the turkey erythrocyte, agonist-promoted phosphorylation of the beta-adrenergic receptor appears to be associated with desensitization of the adenylate cyclase system. This process can be partially mimicked by cyclic AMP analogs. Accordingly, we have investigated the phosphorylation of the pure mammalian beta-adrenergic receptor by the pure catalytic subunit of the cyclic AMP-dependent protein kinase. The beta-adrenergic receptor, purified from hamster lung to apparent homogeneity, contains a single polypeptide of Mr approximately 64,000. The receptor can be phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase (approximately 2 mol of phosphate (on serine residues) per mol). Isoproterenol, a beta-agonist, promoted a 2-3-fold increase in the rate of receptor phosphorylation which was blocked by the beta-antagonists propranolol and alprenolol. High performance liquid chromatographic tryptic peptide mapping reveals two major phosphorylation sites. Phosphorylated receptor can be completely dephosphorylated by a high molecular weight phosphoprotein phosphatase. The rate of receptor dephosphorylation is enhanced 2-3-fold by isoproterenol and this effect is blocked by alprenolol. The functional significance of receptor phosphorylation was examined using ligand binding and reconstitution techniques. While the binding of isoproterenol and alprenolol to the receptor was unaffected by phosphorylation, the ability of the receptor to interact with the stimulatory guanine nucleotide regulatory protein, as assessed by isoproterenol-promoted GTPase activity, was decreased 24 +/- 1% (mean +/- S.E., p less than 0.001, n = 17). The quantitative extent of receptor phosphorylation and functional impairment are virtually identical to those previously observed when intact turkey erythrocytes were incubated with cyclic AMP. These data provide a direct demonstration of regulation of the function of the isolated beta-adrenergic receptor by cyclic AMP-dependent protein kinase.     

Characterization and Regulation of β1-Adrenergic Receptors in a Human Neuroepithelioma Cell Line

Intact human neuroepithelioma SK-N-MC cells bound the beta-adrenergic antagonist (-)-[3H]-CGP 12177 with a KD of 0.13 nM and a Bmax of 17,500 sites/cell. When the cells were exposed to beta-adrenergic agonists, they accumulated cyclic AMP in the following order of potency: isoproterenol much greater than norepinephrine greater than epinephrine, which is indicative of a beta 1-subtype receptor. Membranes prepared from the cells bound (-)-3-[125I]iodocyanopindolol with a KD of 11.5 pM. Inhibition of agonist-stimulated cyclic AMP production and competition binding experiments indicated that the beta 1-selective antagonists CGP 20712A and ICI 89,406 were much more potent than the beta 2-selective antagonist ICI 118,551. Analysis of the displacement curves indicated that the cells contained only beta 1-adrenergic receptors. Northern blot analysis of SK-N-MC mRNA using cDNA probes for the beta 1- and beta 2-adrenergic receptors revealed the presence of a very strong beta 1-adrenergic receptor mRNA signal, while under the same conditions no beta 2-adrenergic receptor mRNA was observed. Thus, SK-N-MC cells appear to express a pure population of beta 1-adrenergic receptors. When the cells were exposed to isoproterenol, there was no observable desensitization during the first hour. After longer exposure, desensitization slowly occurred and the receptors slowly down-regulated to 50% of control levels by 24 h. Other agents that elevate cyclic AMP levels, such as forskolin, cholera toxin, and cyclic AMP analogues, caused no or little substantial receptor loss.     

Adenovirus type 12 transformation involves loss of beta-adrenergic receptors and isoproterenol responsiveness.

The responsiveness of a growth-regulated rat 3Y1 cell line and five clones of 3Y1 cells transformed by the highly oncogenic human adenovirus type 12 to the catecholamine hormone (-)-isoproterenol was studied. The untransformed cells contained beta-adrenergic receptors characterized by specific binding of the beta-adrenergic receptor antagonist (-)-[3H]dihydroalprenolol, a 9- to 12-fold increase in cyclic AMP production in intact cells after incubation with 10 microM (-)-isoproterenol, and significantly increased adenylate cyclase (ATP pyrophosphatelyase [cyclizing], EC 4.6.1.1) activity in the presence of the hormone. In contrast, (-)-isoproterenol (10 to 100 microM) had no apparent effect on cyclic AMP production or the basal adenylate cyclase activity in the transformed cell lines. Binding studies revealed that untransformed cells contained approximately 19,400 beta-adrenergic receptor sites per cell. Three transformed cell clones tested showed a three- to fourfold loss of beta-adrenergic receptors.     

Effects of crp mutations on adenosine 3'',5''-monophosphate metabolism in Salmonella typhimurium.

Wild-type Salmonella typhimurium could not grow with exogenous cyclic adenosine 3',5'-monophosphate (AMP) as the sole source of phosphate, but mutants capable of cyclic AMP utilization could be isolated provided the parental strain contained a functional cyclic AMP phosphodiesterase.All cyclic AMP-utilizing mutants had the growth and fermentation properties of cyclic AMP receptor protein (crp) mutants, and some lacked cyclic AMP binding activity in vitro. The genetic defect in each such mutant was due to a single point mutation, which was co-transducible with cysG. crp mutants isolated by alternative procedures also exhibited the capacity to utilize cyclic AMP. crp mutants synthesized cyclic AMP at increased rates and contained enhanced cellular cyclic AMP levels relative to the parental strains, regardless of whether or not cyclic AMP phosphodiesterase was active. Moreover, adenylate cyclase activity in vivo was less sensitive to regulation by glucose, possibly because the enzyme II complexes of the phosphotransferase system, responsible for glucose transport and phosphorylation, could not be induced to maximal levels. This possibility was strengthened by the observation that enzyme II activity (measured both in vitro by sugar phosphorylation and in vivo by sugar transport and chemotaxis) was inducible in the parental strain but not in crp mutants. The results suggest that the cyclic AMP receptor protein regulates cyclic AMP metabolism as well as catabolic enzyme synthesis.     

Mechanism of Neurotransmitter Release Elicited by the Preferential α1-Adrenergic Receptor Agonist Phenylephrine in Superfused Superior Cervical Ganglion Cells in Culture

Phenylephrine increased [3H]norepinephrine efflux and accumulation of cyclic AMP in cultured rat superior cervical ganglion cells superfused with Tyrode's solution. The purpose of this study was to determine the mechanism and relationship between these two events. Electrical stimulation (1-2 Hz), potassium chloride (50 mM), and the preferential alpha 1-adrenergic receptor agonist phenylephrine (1-100 microM) increased fractional tritium efflux, whereas methoxamine, cirazoline, and amidephrine were relatively ineffective. Phenylephrine, but not methoxamine and cirazoline, also increased cyclic AMP accumulation. Phenylephrine-induced tritium efflux was not altered by alpha- and beta-adrenergic receptor antagonists or by removal of extracellular calcium. Phenylephrine-induced cyclic AMP accumulation was blocked by the beta-adrenergic receptor antagonists propranolol and atenolol. Forskolin (10 microM) and the nonhydrolyzable cyclic AMP analogue 8-(4-chlorophenylthio)cyclic AMP (100 microM) had minimal effect on tritium efflux. However, phenylephrine-evoked increase in tritium efflux was dose dependently attenuated by the neuronal uptake blocker cocaine, and phenylephrine dose-dependently inhibited the incorporation of [3H]norepinephrine into neuronal stores. We conclude that the increase in tritium efflux induced by phenylephrine is independent of cyclic AMP accumulation and appears to be mediated by uptake of phenylephrine via the neuronal carrier-mediated amine transport process, which in turn promotes efflux of the adrenergic transmitter from its storage sites.     

Islet cyclic AMP levels are not lowered during alpha 2-adrenergic inhibition of insulin release

Epinephrine-induced changes in insulin release and cyclic AMP levels were measured simultaneously in isolated rat islets. Forskolin was used to enhance islet cyclic AMP levels. Forskolin (30 microM) stimulated adenylate cyclase activity 10-fold in islet homogenates and raised cyclic AMP levels 5-fold in intact islets (both at low and high glucose). Insulin release was enhanced by forskolin only at high glucose. Epinephrine (0.1 microM) inhibited glucose- and forskolin-induced insulin release to basal rates. At the same time epinephrine potentiated forskolin-elevated cyclic AMP levels. In contrast epinephrine attenuated forskolin-stimulated adenylate cyclase activity in islet homogenates. At low glucose, both alpha 2- and beta-adrenergic blockade counteracted the epinephrine potentiation, each by 50%. At high glucose the effect was mainly beta-adrenergic in nature. The actions of epinephrine in the presence of a beta-blocker were mimicked by the alpha 2-agonist clonidine. Despite the variations in cyclic AMP levels stimulated insulin release was always inhibited by activation of alpha 2-receptors. Finally, insulin release stimulated by exogenous cyclic AMP was abolished by epinephrine. These results suggest that epinephrine inhibits insulin release at a step distal to the generation of cyclic AMP.     

Regulation of cyclic AMP accumulation in lymphoid cells

We have examined several features of the regulation of cyclic AMP accumulation in lymphoid cells isolated from peripheral blood of human subjects and in the murine T-lymphoma cell line, S49, S49 cells are unique because of the availability of variant clones with lesions in the pathway of cyclic AMP generation and response. We found that human lymphoid cells prepared at 4 degrees C showed substantially greater cyclic AMP accumulation in response to histamine and the beta-adrenergic agonist isoproterenol than did cells prepared at ambient temperature. The muscarinic cholinergic agonist carbamylcholine and peptide hormone somatostatin failed to inhibit cyclic AMP accumulation in human lymphoid cells and treatment with pertussis toxin (which blocks function of Gi, the guanine nucleotide binding protein that mediates inhibition of adenylate cyclase) only minimally increased cyclic AMP levels in these cells. Thus the Gi component of adenylate cyclase appears to play only a small role in modulating cyclic AMP levels in this mixed population of lymphoid cells. Incubation of whole blood with isoproterenol desensitized human lymphocytes to subsequent stimulation with beta agonist. This desensitization was associated with a redistribution of beta-adrenergic receptors such that a substantial portion of the receptors in intact cells could no longer bind a hydrophilic antagonist. Wild-type S49 lymphoma cells showed a similar redistribution of beta-adrenergic receptors after a few minutes' incubation with agonist. Based on studies in S49 variants, this redistribution is independent of components distal to receptors in the adenylate cyclase/cyclic AMP pathway. By contrast, a more slowly developing, agonist-mediated down-regulation of beta-adrenergic receptors was blunted in variants with defective interaction between receptors and Gs, the guanine nucleotide binding protein that mediates stimulation of adenylate cyclase. Unlike results in human lymphoid cells, S49 cells show a prominent inhibition of cyclic AMP accumulation mediated by Gi; this inhibition is promoted by somatostatin and blocked by pertussis toxin. Inhibition by Gi is unable to account for the marked decrease in ability of the diterpene forskolin to maximally stimulate adenylate cyclase in S49 variants having defective Gs. These results emphasize that both Gs and Gi component are important in modulating cyclic AMP accumulation and receptors linked to adenylate cyclase in S49 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Mediation by adenosine 3':5'-monophosphate.

The effects of growth hormone-dependent serum factors on amino acid transport and on cartilage cyclic AMP levels in embryonic chicken cartilage were studied in vitro. Cartilages incubated in medium containing rat serum showed a significantly greater uptake of alpha-amino [1-14C] isobutyrate or [1-14C] cycloleucine than control cartilages incubated in medium alone.Normal rat serum (5%) added to the incubation medium also caused an increase in cartilage cyclic AMP content (from as little as 23% to as much as 109%). The factors in serum which increase cartilage cyclic AMP and amino acid uptake are growth hormone dependent, since neither growth hormone itself nor serum from hypophysectomized rats restores these serum factors. Studies comparing the ability of sera with varying amounts of growth hormone-dependent factors to stimulate amino-aminoisobutyrate transport and to increase cartilage cyclic AMP show a striking linear correlation between the two effects (r=0.977). Theophylline and prostaglandin E1, WHICH RAISE CARTILAGE CYCLIC AMP also increase amino-aminoisobutyrate transport. Exogenous cyclic AMP, N6-monobutyryl cyclic AMP and n6, 02'-dibutyryl cyclic AMP increase cartilage amino-aminoisobutyrate transport. The data are compatible with the thesis that growth hormone-dependent serum factors increase cartilage amino acid transport by elevating cartilage cyclic AMP.     

Further studies of hormone-sensitive sodium and potassium transport in red cells from developing chick embryos

Sodium and potassium transport in the definitive series of chick embryo red cells changes significantly, both qualitatively and quantitatively, during maturation. Sodium efflux and potassium influx consist of three parts: a ouabain-sensitive, a furosemide-sensitive, and a ouabain-furosemide-insensitive component. In chick red cells of most ages, the ouabain-sensitive and furosemide-sensitive parts of the cation fluxes do not overlap. Cation transport in the more mature red cells is increased significantly by epinephrine, whereas cation transport in red cells from younger embryos is stimulated much less. This is a beta-adrenergic effect of epinephrine and is mediated by cyclic AMP. The relative lack of response in younger embryos is not due to the absence of beta-adrenergic receptor or the lack of production of cyclic AMP. Ouabain has no effect on the hormone-sensitive sodium or potassium transport. On the other hand, furosemide nearly completely abolishes the effect of epinephrine. In addition, there is a good correlation between furosemide-sensitive components of both sodium and potassium transport and the epinephrine-sensitive component. Furosemide has no effect on cyclic AMP levels in the presence or absence of epinephrine. This suggests that furosemide may act directly on the cation transport system. In the red cells from younger embryos, furosemide-sensitive units are present but cannot be fully activated by epinephrine. Therefore, the lack of the hormone effect on cation movements in these cells is consistent with the view that the appropriate units are present, but do not respond fully to intracellular cyclic AMP levels.     

Down-regulation of gonadotropin and beta-adrenergic receptors by hormones and cyclic AMP

Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.     

Adenosine 3',5'cyclic monophosphate in adipose tissue of diabetic rats.

Normal male rats were made chronically diabetic by injection of alloxan or acutely diabetic by injection of anti-insulin serum. The concentration of cyclic AMP in epididymal adipose tissue was increased approximately 2 1/2-fold 24 h after alloxan administration and up to 7-fold 72 h post-alloxan. Treatment of alloxan-diabetic rats with insulin for 4 h completely suppressed lipolysis but only partially suppressed cyclic AMP levels; 6 h following insulin treatment cyclic AMP levels were normal. When segments of the epididymal fat bodies were incubated in vitro the high cyclic AMP levels were not maintained but instead decreased spontaneously. Addition of insulin to the incubation media decreased lipolysis in tissues of diabetic rats to levels measured in tissues of normal rats and accelerated the decline in cyclic AMP levels but did not return cyclic AMP levels to normal. Rats rendered acutely insulin deficient by injection of anti-insulin serum showed increased plasma glucose and free fatty acid levels and increased adipose tissue free fatty acid, and cyclic AMP levels 30 min following injection of the antiserum. Plasma glucagon levels increased but not until 2 h following anti-insulin serum, thereby excluding the possibility that an increment in plasma glucagon is the primary stimulus for the acceleration of lipolysis in diabetes. These data are consistent with the view that control of adipose tissue cyclic AMP levels in situ is an important physiologic action of insulin.     

Effect of adrenergic agents on alpha-amylase release and adenosine 3',5'-monophosphate accumulation in rat parotid tissue slices.

The role of cyclic AMP in stimulus-secretion coupling with investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20-30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both alpha-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effectivema parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fractionmthe results suggest that these drugs are acting on a parotid acinar cell through a beta1-adrenergic mechanismmat the lowest concentrations tested, each of the adrenergic agonists stimulated significant alpha-anylase release with no detectable stimulation of cyclic AMP accumulationmeven in the presence of theophylline, phenylephrine at several concentrations increased alpha-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intra-cellular concentration of cyclic AMP may not be necessary for stimulation of alpha-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of alpha-amylase release by isoproterenol. Stimulation of alpha-amylase release by phenylephrine was only partially blocked by either alpha- or beta-adrenergic blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolaminemphenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and alpha-amylase release by N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate; These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using alpha-adrenergic blocking agents as tools for investigation of alpha- and beta-adrenergic antagonism.