Showing papers on "Cyclase published in 1973"

Octopamine-Sensitive Adenylate Cyclase: Evidence for a Biological Role of Octopamine in Nervous Tissue

Abstract: An adenylate cyclase that is activated specifically by very low concentrations of octopamine has been identified both in homogenates and in intact cells of the thoracic ganglia of an insect nervous system. This enzyme appears to be distinct from two other adenylate cyclases present in the same tissue, which are activated by dopamine and by 5-hydroxytryptamine, respectively. The data raise the possibility of a role of octopamine-sensitive adenylate cyclase in the physiology of synaptic transmission.

Regulation of Cyclic Nucleotide Concentrations in Photoreceptors: An ATP-Dependent Stimulation of Cyclic Nucleotide Phosphodiesterase by Light

Abstract: Regulation of cyclic nucleotide concentrations in rod outer segments (Rana pipiens) has been further examined. The present studies show that illumination markedly diminishes the concentration of cyclic nucleotides in suspensions of photoreceptor membranes, but the locus of regulation is cyclic nucleotide phosphodiesterase (EC 3.1.4.c) (light-stimulated) and not adenylate cyclase. There is a marked disproportionality between bleaching of rhodopsin and stimulation of phosphodiesterase. Bleaching only 0.6% of the rhodopsin produces half the stimulation produced by bleaching 100% of the rhodopsin. The process of activation of phosphodiesterase by light is in two steps, a light-dependent step followed by an ATP-dependent step. Illumination (in the absence of ATP) produces a trypsin-resistant, heat-labile, macromolecular stimulator. In the presence of 0.75 mM ATP (GTP or ITP) this stimulator produces a greater than 5-fold increases in the Vmax of photoreceptor phosphodiesterase without changing the Km. At physiological substrate concentrations (10-7 M) the rate of hydrolysis of cyclic GMP is 23 times greater than that of cyclic AMP. The light-produced stimulator appears unique to the photoreceptor membranes and does not activate phosphodiesterase in other tissues.

The modulating influence of cyclic nucleotides upon lymphocyte-mediated cytotoxicity.

Abstract: 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.

Adrenergic-adenosine 3',5'-monophosphate regulation of serotonin N-acetyltransferase activity and the temporal relationship of serotonin N-acetyltransferase activity synthesis of 3H-N-acetylserotonin and 3H-melatonin in the cultured rat pineal gland.

Abstract: The regulation of serotonin N-acetyltransferase actiyity has been studied in cultured pineal glands. Addition of l-norepinephrine (NE) to cultures produces a 10- to 30-fold increase in enzyme activity after six hours of treatment. There is a plateau in enzyme activity for another six hours followed by a spontaneous decrease in enzyme activity to base-line values. NE can stimulate the enzyme over a range of 10-4 to 10-9 M concentratioin. Exposure to NE does not have to be constant for a long-term response. Exposure for as little as 15 minutes to NE produces about a 50% maximal response at six hours. The initial increase in N-acetyltransferase activity is blocked by cycloheximide. Cycloheximide treatment after a gland has been stimulated with NE does not cause a precipitous fall in enzyme activity but does prevent a further increase. Aliphatic amines. indoleamines. an imidazolamine and tyramine are ineffective in stimulating the enzyme. The relative potency of the compounds that stimulate enzyme activity is: l-NE DL-isoproterenol > l-epinephrine > DL-octopamine > d-norepinephrine > 3,4-dihydroxyphenylamine > l-3,4-dihydroxyphenylalanine. The stimulation of enzyme activity is blocked by propranolol and is enhanced by phentolamine. This indicates that the receptor involved is a beta adrenergic receptor and that the response to beta adrenergic stimulation can be influenced by an alpha adrenergic mechanism. The effects of NE on N-acetyltransferase activity are mimicked by dibutyryl cyclic adenosine monophosphate (AMP), which is more effective than theophylline. Cyclic AMP is only slightly effective. The effects of dibutyryl cyclic AMP are blocked by cycloheximide but not by cyclic AMP. The stimulation of N-acetvltransferase by either NE or dibutyryl cyclic AMP is coincident in time and magnitude with the stimulation of the total production of 3H-N-acetylserotonin and 3H-melatonin by glands incubated with 3H-tryptophan. This study describes a striking number of similarities between the factors regulating pineal adenyl cyclase, cyclic AMP, radiolabeled melatonin production from radiolabeled tryptophan, and serotonin N-acetyl-transferase activity in cultured pineal glands. The findings add support to the hypothesis that melatonin production is regulated by the amount of N-acetylserotonin made available for O-methylation and that N-acetylserotonin production by serotonin N-acetyl-transferase is regulated by an adrenergic-cyclic AMP mechanism.

Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclase.

Abstract: Sixty-two spontaneous mutations have been characterized which reduce the level of expression of catabolite-sensitive operons. These mutations appear to affect either the crp (catabolite gene activator protein) or cya (adenyl cyclase) loci. No new loci have been discovered. Deletions of the cya gene do not remove an essential function. φ80 transducing phage for the cya gene have been used to do recombination and complementation studies on cya mutants.

Adenylate-cyclase receptors for adrenergic neurotransmitters in rat cerebral cortex.

Abstract: The response of adenylate cyclase systems to putative neurotransmitter agents has been studied in cell-free preparations from various brain regions of young adult rats. Homogenates of cerebral cortex contained adenylate cyclase systems which were responsive to norepinephrine and dopamine. A large proportion of this catecholamine-sensitive adenylate cyclase activity was concentrated in the crude mitochondrial fraction. Adenylate cyclase activity in this fraction was also responsive to epinephrine and isoproterenol, showed very small stimulation with histamine and serotonin, and was unresponsive to acetylcholine and 4-aminobutyric acid. The natural l-forms of the catecholamines were much more active than the d-forms. Adrenocorticotrophic hormone, glucagon, thyroid stimulating hormone and luteinizing hormone were without significant effect in the cerebral preparation. Basal values for adenylate cyclase activity and the degree of stimulation by the catecholamines were critically dependent upon appropriate concentrations of ATP, an ATP-regenerating system, H+, Mg2+ and Ca2+ in the assay medium. Addition of the membrane phospholipids, phosphatidylserine and phosphatidylinositol, enhanced stimulation by the catecholamines. Concentrations of norepinephrine or dopamine as low as 0.005 mM produced significant increases in adenylate cyclase activity. The maximum response to norepinephrine was two to three times that with dopamine. The response of cerebral adenylate cyclase to 0.05 mM norepinephrine was strongly blocked by concentrations (0.1 mM) of the β-adrenergic blocking agents, propranolol and dichloroisoproterenol, which had no effect on the response to 0.05 mM dopamine. Haloperidol eliminated the dopamine response at very low concentration (0.01 mM). The data suggest that norepinephrine and dopamine activate adenylate cyclase systems in rat cerebral cortex by reacting with specific receptors which have the properties of β-adrenergic and dopaminergic receptors, respectively.

Mechanism of the Lower Esophageal Sphincter Relaxation ACTION OF PROSTAGLANDIN E1 AND THEOPHYLLINE

Abstract: A B S T R A C T The intravenous injection of prostaglandin E1 (PGE1) causes a dose-dependent relaxation of the lower esophageal sphincter (LES) in the intact, lightly anesthetized opossum. The action of PGE1 is not inhibited by the drugs that produce muscarinic or nicotinic cholinergic antagonism or alpha and beta adrenergic antagonism in the doses that inhibited the action of respective agonists. Moreover, this action is not affected by exogenous gastrin pentapeptide. The action of PGE1 on the LES is mimicked by isoproterenol, theophylline ethylenediamine, and dibutyryl cyclic AMP. Both theophylline, a phosphodiesterase inhibitor, and isoproterenol, an adenyl cyclase stimulator, added to the action of PGE1. On the other hand, adenyl cyclase inhibitor nicotinic acid, as well as phosphodiesterase stimulator, imidazole inhibited its action. Further, both nicotinic acid and imidazole inhibited the degree of LES relaxation produced by esophageal distension. These studies suggest that intracellular cyclic AMP may act as the "second messenger" in the regulation of the lower esophageal sphincter relaxation.

Activation of Renal Cortical Adenylate Cyclase by Circulating Immunoreactive Parathyroid Hormone Fragments

Abstract: Three distinct immunoreactive species of parathyroid hormone (PTH) are present in human serum. One has an estimated mol wt of 9,500 and probably represents glandular hormone, the second 7,000-7,500 mol wt, and the third 4,500-5,000 mol wt. In order to assess the biological activity of these circulating forms of PTH, we determined their ability to activate renal cortical adenylate cyclase. The 9,500 mol wt and 4,500-5,000 mol wt fractions produced four- to sixfold increases in cyclic 3′,5′-AMP accumulation above control; the 7,000-7,500 mol wt fraction was inactive. None of the fragments had any effects on phosphodiesterase activity. Antiserum to bovine PTH did not block the activation of adenylate cyclase by either the gragments or bovine PTH. The data suggest that a large proportion of circulating immunoreactive human PTH is biologically active and that the biologically and immunologically active sites of the hormone are distinct.

Effects of Cholera Toxin on In Vitro Models of Immediate and Delayed Hypersensitivity. FURTHER EVIDENCE FOR THE ROLE OF CYCLIC ADENOSINE 3′,5′-MONOPHOSPHATE

Abstract: Cholera enterotoxin inhibits the antigen-induced. IgE-mediated release of histamine from human leukocytes and the lysis of allogeneic mastocytoma cells by splenic lymphocytes from specifically immunized mice. This effect requires a prolonged preincubation time of the toxin with the lymphocyte/leukocyte preparations: a demonstrable inhibition requires about 30 min of pre-incubation and the toxin activity is still increasing at 90-180 min. Cholera enterotoxin also stimulates adenyl cyclase and leads to increased levels of cyclic AMP in the lymphocyte/leukocyte preparations. The concentration of toxin required for both cyclic AMP accumulation and inhibition of the biologic responses is about the same (ca. 1 ng/ml), and the time course of cyclic AMP accumulation parallels the development of inhibitory activity. Both activities, inhibition of the in vitro hypersensitivity reactions and cyclic AMP accumulation, are blocked by cholera antitoxin and by a toxoid prepared from the toxin (choleragenoid). These are specific antagonists in that they do not block the inhibiting activity or rise in cyclic AMP levels caused by other adenyl cyclase stimulators. Because cholera enterotoxin has no known activity other than the stimulation of adenyl cyclase and because of its unusual time course and the availability of specific antagonists, this data considerably strengthens the hypothesis that the cyclic AMP system influences the expression of these two forms of hypersensitivity phenomena.

Influence of neuroleptic drugs and apomorphine on dopamine-sensitive adenylate cyclase of retina.

Abstract: THE NEUROLEPTIC drugs (major tranquilizers) selectively antagonize a number of behavioral and physiological actions of dopamine, and are believed to exert this effect through a specific postsynaptic blockade of ‘dopamine receptors’ (HORNYKIEWICZ, 1971 ; A N D ~ N et al., 1970; ERNST, 1969; RANDRUP and MUNKVAD, 1970; YEH et al., 1969). Apomorphine mimics the effects of dopamine in several systems and is thought to act by a direct stimulation of the ‘dopamine receptor’ (ANDBN et al., 1967; ERNST, 1969; GOLDBERG, 1968). The effects of the neuroleptic drugs and of apomorphine on the metabolism of dopamine ( A N D ~ N et al., 1967, 1970; NYBACK and SEDVALL, 1968; CHERAMY et al., 1970), as well as their physiological effects on the extrapyramidal system (HAASE and JANSSEN, 1965; D ~ ~ B Y et al., 1971), further suggest that the dopamine receptor can be defined pharmacologically in terms of the actions of these compounds. The biochemical nature of the receptor with which dopamine and these agents interact has not been defined. The effects of catecholamines in a number of peripheral tissues are mediated through activation of adenylate cyclase, and there is evidence indicating that the 13-adrenergic receptor is part of or closely associated with this enzyme system (ROBISON, BUTCHER and SUTHERLAND, 1971). Several lines of evidence suggest that the adenylate cyclase system similarly may mediate the central actions of catecholamines as neurotransmitters (KAKIUCHI and RALL, 1968; SIGGINS et al., 1969; CHASIN eta/., 1971 ; SEEDS and GILMAN, 1971 ; BURKARD, 1972). We have reported previously (BROWN and MAKMAN, 1972) that dopamine, the predominant catecholamine serving as a neurotransmitter in the retina (HAGGENDAL and MALMFORS, 1965; NICHOLS et al., 1967; KRAMER et al., 1971), activates retinal adenylate cyclase and increases the concentration of cyclic AMP in intact isolated retinas. Our finding that dopamine was more potent than epinephrine or norepinephrine as an activator of this enzyme suggested that the receptor associated with the retinal adenylate cyclase was different from the p-adrenergic receptor and indeed might have the pharmacological properties of a specific dopamine receptor. Adenylate cyclase activity was assayed as described (BROWN and MAKMAN, 1972) by the incubation of [a-23P]ATP with homogenates of bovine retina for 5 min at 30°, followed by separation of labelled cyclic AMP by thin layer chromatography and determination of radioactivity. The concentration of cyclic AMP in intact bovine retinas was measured following incubation of the isolated tissue by a modification (BROWN and MAKMAN, 1972; SHERLINE et al., 1972) of the method of GILMAN (1970). We have examined aand 8-adrenergic blocking agents and the neuroleptic drugs, haloperidol, chlorpromazine and fluphenazine, for their capacity to inhibit activation of the adenylate cyclase of calf retina by dopamine. Figure 1 illustrates the inhibition by these agents of the response to 20 p~ dopamine. Propranolol, a potent 8-adrenergic blocking agent, failed to produce greater than a 50% inhibition even at concentrations 25 times that of dopamine. In contrast, haloperidol, chlorpromazine and fluphenazine each inhibited almost completely the activation by equimolar concentrations of dopamine, and caused a 50% blockade when used in concentrations approximately 1/50 that of the catecholamine. Even at equimolar concentrations chlorpromazine sulphoxide, the pharmacologically inactive metabolite of chlorpromazine, did not inhibit the response to 20 p~ dopamine. Haloperidol and the phenothiazines inhibited neither basal adenylate cyclase activity nor activity in the presence of NaF (a nonspecific activator) at the concentrations required to produce complete inhibition of the response to dopamine. Phentolamine and ergotamine also inhibited activation by dopamine of the retinal adenylate cyclase. Phentolamine, an a-adrenergic blocking agent, inhibited at concentrations 50-100 times those necessary for blockade with the neuropletic drugs, and the blockade observed with ergotamine may be unrelated to its a-adrenergic receptor blocking properties since several other ergot alkaloids, none of which are a-adrenergic blocking agents, were equally effective in inhibiting activation by dopamine (OPLER, BROWN and MAKMAN, in preparation). The relative effectiveness of the a-adrenergic blocking agents on the retinal adenylate cyclase is consistent with the influence of these agents in other systems responsive to dopamine. Thus a-adrenergic blocking agents do not block the behavioral and vasodilatory actions of dopamine which are blocked by neuroleptic drugs (ANDBN et al., 1966; MUNKVAD and UDSEN, 1963; YEH et al., 1969), and are less potent than the phenothiazine neuroleptics or than

Phosphodiesterase in Tongue Epithelium: Activation by Bitter Taste Stimuli

Abstract: THE role of cyclic nucleotides in stimulus-response coupling at the cellular level has received much attention1. The level of adenosine cyclic 3′,5′-monophosphate (cAMP) is dependent on its rate of synthesis, catalysed by adenyl cyclase, and on its rate of destruction, catalysed by phosphodiesterase. Although most investigators have measured the regulation of cAMP levels by alterations in adenyl cyclase activity, others have noted the presence of high levels of phosphodiesterase in certain sensory receptors and have suggested that this enzyme may be of special importance in such cells2. We studied the phosphodiesterase activity in tongue epithelium to determine whether unusually high levels of this enzyme might also be associated with taste receptors, and to explore the possibility that taste stimuli might affect phosphodiesterase activity, thereby altering the intracellular cAMP concentrations.

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

Abstract: 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',5 -Monophosphate and Neutrophil Function COMPARISON WITH OTHER COMPOUNDS WHICH STIMULATE LEUKOCYTE ADENYL CYCLASE

Abstract: A B S T R A C T 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 prostaglandinE1 (PGE1) 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 PGE1 did prevent its effects; (c) PGE1, 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 cyclohexi

Inhibition of adenosine cyclic 3', 5'-monophosphate accumulation in fat cells by adenosine, N6-(phenylisopropyl) adenosine, and related compounds.

Abstract: The hypothesis that adenosine may serve as a physiologica1 feedback regulator of adenylate cyclase in rat white fat cells was examined. Adenosine (0.2 µM), when added to incubated fat cells, caused 50% inhibition of the increase in adenosine cyclic 39,59-monophosphate accumulation due to 1.5 µM norepinephrine in the presence and absence of methylxanthines. The onset of adenosine inhibition was rapid and, if added 1 min after activators of adenylate cyclase, it reduced cyclic AMP accumulation during the next minute. Adenosine 59-monophosphate and adenosine 59-triphosphate were less effective than adenosine as inhibitors of cyclic AMP accumulation. Of a variety of nucleosides only N 6 -(phenylisopropyl)adenosine was more effective than adenosine in inhibiting cyclic AMP accumulation. Under conditions in which adenosine markedly reduced the small increase in cyclic AMP accumulation due to norepinephrine alone, it did not reduce lipolysis. In contrast, insulin reduced lipolysis but had a smaller effect on cyclic AMP accumulation. Adenosine inhibited lipolysis in the presence of insulin but produced no greater effect on cyclic AMP accumulation than was seen with adenosine alone. Drugs such as dipyridamole or papaverine did not affect cyclic AMP accumulation due to norepinephrine or block the inhibitory action of adenosine. Inhibition of cyclic AMP accumulation by adenosine was demonstrable also when cells were incubated in calcium-free buffer containing 0.25 mM ethylene glycol bis(β-aminoethyl ether)- N,N 9-tetraacetic acid. The addition of adenosine deaminase to incubated fat cells increased basal lipolysis and cyclic AMP and potentiated the norepinephrine-induced increase in cyclic AMP. The present results suggest that adenosine or related compounds may serve a physiologically important function as feedback regulators of adenylate cyclase.

On the mechanism of adenyl cyclase inhibition by adenosine.

Abstract: Adenosine inhibited basal and fluoride- or hormone-stimulated adenyl cyclase activities from various sources. Inhibition was usually noted at 0.01 mM adenosine and was 40–75% at 1 mM. The effect ap...