Monoamine Oxidase and Cerebral Uptake of Dopaminergic Drugs

The brain uptake of amines that do not enter the brain or enter it poorly was promoted by noncompetitive inhibitors of monoamine oxidase, as shown by behavioral and chemical criteria. Mice pretreated with water or enzyme inhibitors other than those mentioned were placid after receiving dopamine (3,4-dihydroxyphenethylamine). Mice pretreated with monoamine oxidase inhibitors (nialamide or iproniazid) showed upon treatment with dopamine the brisk motor responses characteristic of treatment with its precursor, L-dopa (3,4-dihydroxyphenylalanine). After receiving dopamine, intact nialamide-pretreated mice showed marked increases of brain dopamine, in contrast to water-pretreated test mice or water-treated controls. In unilaterally caudectomized, nialamide-pretreated mice, dopamine induced marked lateral curving of the body toward the lesion followed by running in that direction. Noradrenaline or adrenaline induced curving in caudectomized mice, whereas intact ones remained placid.These catecholamines are bound and inactivated by monoamine oxidase. The cerebral uptakes of chemicals that are bound but not inactivated by monoamine oxidase were thereafter tested. Nialamide induced increased behavioral responses to apomorphine and to N-propyl noraporphine, increased cerebral concentrations of both, and a deep coloration of the brain from methylene blue (bound by monoamine oxidase) but not Evans blue (bound by albumin). Even large doses of nialamide, however, failed to affect the behavioral responses to oxotremorine, which has cholinergic rather than adrenergic or dopaminergic properties. Mitochondrial monoamine oxidase seems therefore to play a specific regulatory role in the transport of substances that it binds, either to inactivate or to release them.     

Dopaminergic stimulation of cyclic AMP accumulation and parathyroid hormone release from dispersed bovine parathyroid cells

The effects of dopaminergic agonists and antagonists have been studied in dispersed bovine parathyroid cells. Dopaminergic agonists caused a transient 20- to 40-fold increase in cellular cyclic AMP and a 2- to 3-fold increase in parathyroid hormone release. Dose-response relationships were similar for cyclic AMP accumulation and hormone release, whether studied by increasing agonist concentration or by increasing concentration of antagonist with constant agonist. The effects on the dopamine receptor could be differentiated from those of the previously characterized beta-adrenergic receptor by specific inhibitors. These results appear to represent proof with a homogeneous cell population that dopaminergic receptors linked to adenylate cyclase can regulate a secretory process mediated by cyclic AMP. This system should be useful in further studies on dopamine receptors and should provide a valid tool for determining interactions of radiolabeled ligands with such receptors.     

Differences in rates of tyrosine aminotransferase deinduction with cyclic AMP and glucocorticoids.

As an indirect test of the possibility that cyclic AMP produces its stimulatory effects on the synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) by stabilizing its mRNA, the kinetics of decline in the rate of synthesis of this enzyme was measured in rat hepatoma cells following inducer removal or addition of RNA synthesis inhibitors. In all cases in which cells were previously exposed to cyclic AMP, the decline in enzyme synthesis after removal of inducer or addition of inhibitors of RNA synthesis was 4 to 5 times more rapid than in cells exposed to a glucocorticoid (dexamethasone) which also induces the aminotransferase. Thus, it seems unlikely that cyclic AMP had been acting by stabilizing the mRNA that directs the synthesis of the aminotransferase. Possible explanations for these paradoxical results are suggested. A more rapid decline in enzyme synthesis was also seen in cells induced with both cyclic AMP and glucocorticoid, suggesting direct interaction between the two inducers.     

Opposing effects of dopaminergic to cholinergic compounds on a cerebral dopamine-activated adenylate cyclase.

In contrast to antipsychosis drugs which inhibit the dopamine-activated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of caudate nucleus, dopaminergic drugs for treatment of Parkinson's disease stimulate this cyclase. Stimulants and inhibitors of cholinergic neurons inhibited this adenylate cyclase activity competitively and specifically. Thus, the mechanism by which dopaminergic medications ameliorate the effects of Parkinson's disease includes activation of the dopamine-sensitive adenylate cyclase. Excessive activation might be present during the psychotic episodes seen in patients with parkinsonism who are overtreated. The enzymatic effects of the drugs that affect cholinergic mechanisms seem to be generally in keeping with the pharmacological reciprocity between psychoses and extrapyramidal function, except for the anticholinergic ones which inhibited this cyclase although they can be hallucinogenic.     

Responses to adenine nucleotides and related compounds of isolated dog cerebral, coronary and mesenteric arteries

In helically cut strips of dog cerebral, coronary and mesenteric arteries contracted with prostaglandin F2 alpha, adenosine, AMP, ADP, ATP and cyclic AMP produced dose-related, persistent relaxation. On the basis of ED50S, the relaxant effect seen in cerebral arteries was in the order of AMP, ADP, ATP greater than adenosine, cyclic AMP greater than aderine, dibutyryl cyclic AMP greater than inosine. On the basis of maximum relaxations, the effect was in the order of adenine, dibutyryl cyclic AMP greater than or equal to adenosine, AMP, ADP, ATP greater than cyclic AMP, inosine. The relaxant responses to adenosine, AMP, ADP, ATP and cyclic AMP were attenuated by treatment with aminophylline, whereas the relaxations induced by dibutyryl cyclic AMP, adenine and inosine were not altered. It is concluded that adenosine, AMP, ADP and ATP, but not adenine and inosine, appear to share receptors underlying arterial relaxations, and the cyclic AMP-induced relaxation does not derive from increments in cellular cyclic AMP. Treatment with aspirin did not alter the relaxant effect of ADP, adenosine and adenine, suggesting that the release of vasoactive prostaglandins is not involved.     

Deficient production of cyclic AMP: pharmacologic evidence of an important cause of contractile dysfunction in patients with end-stage heart failure

We studied the effects of different classes of inotropic drugs on human working myocardium in vitro that was isolated from the hearts of patients with end-stage heart failure, and compared the responses to these drugs with those noted in muscles from nonfailing control hearts. Although peak isometric force generated in response to increased extracellular calcium reached control levels in the muscles from patients with heart failure, the time course of contraction and rate of relaxation were greatly prolonged. The inotropic effectiveness of the beta-adrenergic agonist isoproterenol and the phosphodiesterase inhibitors milrinone, caffeine, and isobutylmethylxanthine was markedly reduced in muscles from the patients with heart failure. In contrast, the effectiveness of inotropic stimulation with acetylstrophanthidin and the adenylate cyclase activator forskolin was preserved. After a minimally effective dose of forskolin was given to elevate intracellular cyclic AMP levels, the inotropic responses of muscles from the failing hearts to phosphodiesterase inhibitors were markedly potentiated. These data indicate that an abnormality in cyclic AMP production may be a fundamental defect present in patients with end-stage heart failure that can markedly diminish the effectiveness of agents that depend on generation of this nucleotide for production of a positive inotropic effect.     

A possible role of cyclic AMP in mediating the effects of thyrotropin-releasing hormone on prolactin release and on prolactin and growth hormone synthesis in pituitary cells in culture.

Thyrotropin-releasing hormone (TRH) has 3 effects on clonal strains of rat pituitary cells in culture (GH-cells). Two long-term effects of TRH on GH-cells, which are measurable after 3 h or longer, have been previously reported; these are an increase in prolactin synthesis and a decrease in growth hormone production. We report here that TRH also stimulates the rapid release of stored intracellular prolactin. We have investigated the role of cyclic AMP as a possible mediator of the effects of TRH on GH-cells. Cyclic AMP concentrations are higher in cells treated with TRH compared with paired controls; a maximum difference of greater than 150% of control values is detected at 15 min if the incubation is performed in serum-free medium in the presence of 1 mM theophylline. The concentration of TRH required to give half-maximum increases in both prolactin release and cyclic AMP accumulation is 0.3 nM; half-maximal increases in prolactin synthesis occur at 3 nM TRH. Exogenous cyclic AMP (1 mM) causes only a slight increase in prolactin release; 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (1 mM) do not cause significant release. Phosphodiesterase inhibitors (0.3 mM theophylline, 0.03 mM isobutyl-methylxanthine) increase prolactin release but their effects on hormone synthesis are more complicated. Isobutylmethylxanthine, 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (0.4 MM) increase prolactin synthesis, but do not significantly affect growth hormone synthesis. Theophylline increases the synthesis of both hormones. Dibutyryl cyclic AMP (0.5 mM or more) increases prolactin release and both growth hormone and prolactin synthesis, but equivalent amounts of sodium butyrate have the same effects. We conclude that in GH-cells under carefully defined experimental conditions: 1) TRH causes an increase in intracellular cyclic AMP concentrations; 2) the increase in endogenous cyclic AMP and the effects of phosphodiesterase inhibitors are consistent with a model with cyclic AMP as a mediator of the effects of TRH on prolactin release; however, they do not prove this model, because the interpretation of these results depends on assumptions which may not all be valid; and 3) none of the analogs of cyclic AMP or the phosphodiesterase inhibitors tested mimic the decrease in growth hormone production caused by TRH.     

Gonadotropin-induced regulation of luteinizing hormone receptors and desensitization of testicular 3'':5''-cyclic AMP and testosterone responses.

Administration of human chorionic gonadotropin (hCG) to male rats was followed by dose-related changes in luteinizing hormone (LH) receptors in the testis. After treatment with a low dose of hCG (10international units), the number of LH receptors increased slightly over the first 24 hr, then fell to about 30% of the control value. These changes occurred with occupancy of only 8% of the available receptors, and were initially accompanied by increased basal testosterone production in vitro with no change in basal 3'5'-cyclic AMP production. During stimulation with hCG in vitro, such testes showed a transient decrease in cyclic AMP response on the first day after gonadotropin treatment and no change in testosterone response. By contrast, a 20-fold higher dose of hCG caused more rapid and complete loss of LH receptors, with major and transient occupancy of receptors at 24 hr and marked elevations of basal cyclic AMP and testosterone production in vitro. The initial occupancy of receptors was accompanied by a rapid fall in the cyclic AMP response to hCG in vitro, and was followed by marked receptor loss and inhibition of the cyclic AMP response for up to 5 days. The testosterone response to hCG in vitro was completely inhibited for about 3 days, then rose to the control level at 5 days, when only a small proportion of the original receptor sites and cyclic AMP response had begun to return. Such complete recovery of the steroidogenic response when only a fraction of the receptor population had returned was consistent with the presence of receptor reserve or "spare" receptors in the testis. These studies have demonstrated that negative regulation of LH receptors by exogenous gonadotropin is accompanied by consequent changes in cyclic AMP and testosterone responses to hCG in vitro. Hormone induced desensitization of interstitial cell responses was initially related to occupancy of LH receptors and later to a protracted loss of receptor sites.     

Parathyroid hormone-induced calcium efflux from isolated renal cortical tubules: evidence for cyclic AMP mediation.

Effects of parathyroid hormone (PTH) upon cyclic AMP and calcium efflux in isolated renal cortical tubules from hamsters were investigated. PTH caused a rapid rise in cyclic AMP levels, temporally preceding an increase in calcium efflux. Increases in both cyclic AMP levels and calcium efflux were noted over an identical PTH concentration range 0.007--0.7 U/ml). Other peptide hormones tested which had no effect upon cyclic AMP levels did not enhance efflux of calcium. The phosphodiesterase inhibitor methyl isobutylxanthine (MIX) was utilized in other studies to potentiate the cyclic AMP response, and produce a range of cyclic AMP concentrations in response to PTH. In these experiments a range of calcium efflux responses was noted which closely paralleled changes in cyclic AMP. Direct addition of cyclic AMP or dibutyryl cyclic AMP to isolated renal tubules caused increased efflux of calcium, while addition of 5'-AMP did not. These results indicate a role for cyclic AMP as a mediator of PTH-induced calcium efflux in this system and suggest that cyclic AMP may mediate the action of this hormone in enhancing renal conservation of calcium in vivo.     

Adenylate cyclase and acetylcholine release regulated by separate serotonin receptors of somatic cell hybrids.

Serotonin activates adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of NCB-20 neuroblastoma--brain hybrid cells with an activation constant of 530 nM, but has little or no effect on cellular cyclic AMP or cyclic GMP content of NIE-115 neuroblastoma or NG108-15 hybrid cells. In homogenates of NCB-20 hybrid cells, lysergic acid diethylamide stimulates adenylate cyclase activity (Kact = 12 nM) and partially inhibits (Ki = 10 nM) the stimulation of adenylate cyclase activity by serotonin. No desensitization was detected of serotonin receptors coupled to adenylate cyclase. Serotonin also depolarizes NCB-20, NG108-15, and NIE-115 cells and increases acetylcholine release. Serotonin receptors mediating depolarizing responses desensitize rapidly and reversibly, and the depolarizing effects of serotonin are neither mimicked nor inhibited by lysergic acid diethylamide. These results indicate that (i) NCB-20 cells possess at least two species of serotonin receptors, which independently regulate cellular functions, (ii) activation of adenylate cyclase does not directly affect membrane potential or acetylcholine release, and (iii) serotonin-dependent cell depolarization does not affect cyclic AMP or cyclic GMP synthesis in the cell lines tested.     

Cyclooxygenase products and cyclic nucleotide levels with infusion of arachidonic acid, PGI2, PGE2, PGF2, and 6-keto-PGF1 in an isolated dog lung

Arachidonic acid (AA) was infused into the pulmonary artery of an isolated dog lung perfused with a physiologic salt solution. This led to elevations in pulmonary cyclic AMP and prostaglandins (PGs) including PGE2, PGF2 alpha, TXB2 (a metabolite of TXA2), and 6-keto-PGF1 alpha (a metabolite of PGI2). The elevations were prevented by PG synthesis inhibitors. A dose of PGI2 comparable to that produced from AA led to elevations in cyclic AMP. These elevations were not reduced by PG synthesis inhibitors; this indicated that the inhibitors did not reduce cyclic AMP except by inhibiting metabolism of AA. The PGE2 led to lesser elevations in cyclic AMP than did PGI2; PGF2 alpha and 6-keto-PGF1 alpha did not increase cyclic AMP. Levels of cyclic AMP were not elevated. We conclude that some of the elevation in cyclic AMP from AA was most likely from production of PGs since elevations in both were prevented by the inhibitors. However, the possibility remains that AA metabolites other than PGs also contributed to elevations in cyclic AMP. We also conclude that PGI2 most likely accounted for some of the cyclic AMP elevation from AA since PGI2 could be readily produced in amounts that elevate cyclic AMP. However, the possibility remains that PGE2, the less consistent cyclic AMP stimulators (l.e., PGF2 alpha and 6-keto-PGF1 alpha), TXA2 or TXB2, or PGs not measured in this study also contributed to the elevations in cyclic AMP from AA.     

alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

We determined the effect of alpha-adrenergic receptor stimulation on cyclic adenosine monophosphate (cyclic AMP) concentrations in isolated myocytes derived from adult rat hearts and in isolated perfused rat hearts. Activation of alpha-adrenergic receptors with either phenylephrine (10(-8) M to 10(-6) M) or epinephrine (10(-8) M to 10(-6) M) plus propranolol (10(-6) M) resulted in a reduction in cyclic AMP levels in isolated myocytes. The action of phenylephrine was antagonized by phentolamine (10(-6) M). Phenylephrine (10(-5)M attenuated cyclic AMP generation in response to isoproterenol (10(-8) M and 10(-5) M). However, this effect of phenylephrine was not antagonized by phentolamine. Elevation of cyclic AMP concentrations produced by glucagon and by theophylline in isolated myocytes was attenuated by phenylephrine and by epinephrine plus propranolol and the attenuation was antagonized by phentolamine. In isolated perfused rat hearts epinephrine (10(-6) M), when given with propranolol, diminished the rate of development of tension and also reduced tissue levels of cyclic AMP. Epinephrine alone, as well as isoproterenol, increased contractility and myocardial cyclic AMP concentrations as expected. These results indicate that catecholamines may increase or decrease cyclic AMP levels in rat myocardium, depending on the intensity of stimulation of receptor types. Increases are mediated by beta-adrenergic receptors, whereas decreases appear to by mediated by alpha-adrenergic receptors.     

Stimulation of Adenosine 3',5'-monophosphate formation in incubated rat hypothalamus by estrogenic compounds: relationship to biologic potency and blockade by anti-estrogens.

Several estrogenic compounds were examined for their abilities to stimulate the formation of adenosine 3',5'-monophosphate (cyclic AMP) in incubated hypothalami from immature (27-28 day old) female rats. 17-beta estradiol (E2) and diethylstilbestrol (DES) significantly increased the formation of cyclic AMP while estrone and estriol elicited small but not statistically significant increases in cyclic AMP formation. 17-alpha estradiol was ineffective in increasing cyclic AMP formation. The anti-estrogenic agent clomiphene was found to effectively antagonize the formation of cyclic AMP elicited by E2 and DES, while having no stimulatory effects on cyclic AMP formation itself. These data indicate that the binding of estrogenic compounds to cytoplasmic sites (receptors) in the hypothalamus is required for subsequent stimulation of cyclic AMP formations.     

Changes in histamine and cyclic nucleotide levels after bronchoprovocation in patients with extrinsic asthma.

The contents of histamine and cyclic nucleotides were studied in leukocytes before and after a bronchoprovocation test on patients with extrinsic asthma. The bronchospastic response correlated significantly with the reduction of the amount of histamine in leukocytes isolated after the provocation. The leukocytes cyclic AMP and cyclic GMP contents were slightly reduced after the provocation, while the plasma level of cyclic AMP was markedly increased. The beta-adrenoceptor response was tested on leukocytes, before the provocation. A normal elevation of the cyclic AMP content was obtained when the leukocytes were stimulated with isoprenaline, but after the provocation, isoprenaline had no significant effect. Only when a phosphodiesterase inhibitor was present did isoprenaline increase the cyclic AMP level in the postprovocation tests. It is suggested that the mediator release from the immunologic target cells is of importance for the bronchospastic response and that a desensitization of the beta-adrenoceptors of these cells is evident after bronchoprovocation.     

Differential effect of pertussis toxin on adenosine and muscarinic inhibition of cyclic AMP accumulation in canine ventricular myocytes.

Cyclic AMP regulation by muscarinic and adenosine receptors was investigated in isolated canine ventricular myocytes. Both the muscarinic receptor agonist, carbachol, and the adenosine receptor agonist, phenylisopropyladenosine, decreased isoproterenol-stimulated cyclic AMP accumulation in a concentration-dependent manner. Carbachol was more potent than phenylisopropyladenosine and had a greater inhibitory effect. At 10(-6) M, carbachol reduced isoproterenol-stimulated cyclic AMP by 73 +/- 5% while 10(-3) M phenylisopropyladenosine was required to decrease cyclic AMP accumulation by 54 +/- 8%. Pretreatment of myocytes with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, Gi, completely abolished the effect of phenylisopropyladenosine to reduce cyclic AMP stimulation. In comparison, pertussis toxin treatment blunted the response to carbachol and shifted the dose-effect curve to the right but did not eliminate the inhibitory action of carbachol. In toxin-treated myocytes, 10(-3) M carbachol produced a 26 +/- 6% reduction of isoproterenol-induced cyclic AMP accumulation. This pertussis toxin-insensitive action of carbachol was antagonized by atropine and pirenzepine and was prevented when either of two different phosphodiesterase inhibitors. RO-20-1724 or isobutylmethylxanthine, was included in the incubation medium. The results indicate that adenosine receptor-mediated inhibition of hormone-stimulated cyclic AMP accumulation in ventricular myocytes occurs by a single, Gi-dependent mechanism while muscarinic inhibition appears to involve both Gi-dependent and Gi-independent mechanisms. The Gi-independent mechanism may reflect enhanced phosphodiesterase activity which results from the activation of muscarinic receptors.     

Inhibition of prolactin release and blockade of adenohypophyseal cell cyclic AMP accumulation are two dissociable effects of dopaminergic and non-dopaminergic drugs

The secretion of PRL by the anterior pituitary gland is under a tonic inhibitory control exerted by dopamine (DA). However, the mechanism(s) involved in the inhibition of PRL secretion is not clearly defined. Several recently published papers supported the hypothesis that DA inhibits the release of PRL through blockade of the pituitary adenylate cyclase-cyclic AMP system. We have recently demonstrated that sodium ions are essential for dopaminergic inhibitory action on PRL secretion. The present paper reports the effects, in the presence or in the absence of Na+, of either DA, bromocriptine, apomorphine or 2 anticalmodulin drugs, penfluridol and W-7, on cyclic AMP accumulation by rat adenohypophyseal cells in primary culture. Studies with dopaminergic agonists show that in the presence of Na+ inhibition of both PRL and cyclic AMP is obtained at 15 and 30 min, while in the absence of the ion a dissociation exists between the inhibition of PRL release which is completely abolished, and that of cyclic AMP content which is still present. Dose-response studies done in the presence of Na+ show the existence of a good correlation between hormone and nucleotide effects of dopaminergic agonists while, in the absence of Na+, a dissociation is observed between the inhibition of PRL release, which is completely suppressed, and that of cyclic AMP accumulation which is slightly or not at all decreased. The inhibitory effects of penfluridol after 15 and 30 min of incubation were not suppressed by Na+ removal, although its hormonal actions were slightly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin resistance, a result of reduced synthesis of prostaglandylinositol cyclic phosphate, a mediator of insulin action?

Reduced ability or failure to stimulate cyclic adenosinemonophosphate (AMP) synthesis on a second addition of hormone 30 min after a first stimulation was taken as an indirect indication of the synthesis of the cyclic AMP antagonist prostaglandylinositol cyclic phosphate (cyclic PIP). In diabetic rats, because of an increased possibility of restimulating cyclic AMP synthesis, the formation of cyclic PIP should be reduced. Additionally, severalfold increased basal cyclic AMP synthesis can be observed in diabetic hepatocytes in comparison with controls. Upon measuring cyclic PIP levels after hormonal stimulation in all organs of diabetic rats, it was found that stimulation of cyclic PIP synthesis by insulin decreased gradually in a time-dependent manner. Plasma membranes were prepared from diabetic Ksj db/db mice and from spontaneously hypertensive rats (SHR), and in a subsequent assay for cyclic PIP synthetase, an up to 60% decrease of enzyme activity was found. Cyclic PIP synthetase can be completely inhibited by preincubation with protein kinase A. It is most likely that this serine phosphorylation reaction by which the enzyme is inhibited also in vivo is a result of increased cyclic AMP levels. The addition of 10^–5–10^–4 M sulfonylureas to the enzyme assay of liver plasma membrane causes full inhibition, and the addition of 10^–5–5×10^–4 M biguanides, a two- to fourfold activation of the enzyme. Activation of cyclic PIP synthetase by biguanides can also be demonstrated in intact cells. It is a fast reaction and additive with respect to the activation by fluoride or guanylyl-imidodiphosphate (GMP-PNP), and it is most likely the effect with which the biguanides produce the correcting changes in metabolism. Furthermore, antihypertensive drugs like captopril, guanethidine, and dihydralazine also activate cyclic PIP synthetase. In contrast to the activation by the biguanides, this effect is not additive to the activation by fluoride. It appears that essential hypertension and type 2 diabetes are connected with or may be the result of a reduction in synthesis of the intracellular messenger cyclic PIP, whose synthesis is stimulated by hormones like insulin and noradrenaline (α-adrenergic action). Received: 31 July 1996 / Accepted in revised form: 4 September 1997     

Amphibian oocyte maturation and protein synthesis: Related inhibition by cyclic AMP, theophylline, and papaverine

Two inhibitors of cyclic AMP phosphodiesterase (3':5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17), theophylline and papaverine, inhibit the maturation of Xenopus laevis oocytes induced by four different stimuli: human chorionic gonadotropin, progesterone, testosterone, and lanthanum ions. Addition of 1 mM cyclic AMP to the medium delays maturation by approximately 2 hr. Papaverine, theophylline, and cyclic AMP inhibit amino acid incorporation into oocyte proteins by 50% or more but do not inhibit amino acid uptake. The capacity of theophylline to block maturation and protein synthesis is reversed in a parallel fashion by addition of 1-5 mM calcium ion to the medium. Addition of papaverine, theophylline, and cycloheximide to oocytes at different times after hormonal treatment shows that the step sensitive to blockage by the three drugs is coincident and precedes germinal vesicle breakdown by about 1.5 hr. Theophylline and papaverine do not increase endogenous cyclic AMP levels in oocytes but do block the decrease of cyclic AMP levels observed 3 hr after progesterone treatment. Both drugs inhibit oocyte cyclic AMP phosphodiesterase measured in vivo and severely inhibit the stimulus of calcium uptake caused by progesterone and human chorionic gonadotropin. These results suggest that cyclic AMP, theophylline, and papaverine may block oocyte maturation by inhibiting protein synthesis, possibly via a cyclic AMP-dependent protein kinase as shown in reticulocytes [Datta, A., De Haro, C., Sierra, J. & Ochoa, S. (1977) Proc. Natl. Acad. Sci., USA 74, 1463-1467].     

Cyclic AMP levels in isolated frog skin epithelium: effects of phosphodiesterase inhibitors, oxytocin and catecholamines.

Direct measurements of cyclic AMP were performed in the isolated epithelium of frog skin. Phosphodiesterase inhibitors (methylxanthines, papaverine) and activators of adenylyl cyclase (oxytocin, catecholamines) significantly increased the cyclic AMP content. Propranolol completely blocked the generation of cAMP induced by beta-adrenergic agonists but had little or no effect on that induced by oxytocin. Phentolamine enhanced the cAMP production by adrenalin and noradrenalin. At supramaximal concentrations, oxytocin and isoproterenol produced similar increments in cAMP, while exposure to both agents roughly doubled the increase in cAMP. The results suggest the presence of independent receptors for oxytocin and catecholamines in frog skin, with additive effects on cAMP generation.     

Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells.

A novel variant of the S49 mouse lymphoma has been selected from wild-type cells by growth in medium containing the beta-adrenergic agonist terbutaline and inhibitors of cyclic nucleotide phosphodiesterase. In contrast to the situation in the wild-type clone, synthesis of adenosine 3':5'-monophosphate (cyclic AMP) is not stimulated by beta-adrenergic agonists or by prostaglandin E1 either in intact variant cells or in membrane preparations of such clones. However, basal and NaF-stimulated activities of adenylate cyclase [ATP pyrophosphate-lyase (cyclizine), EC 4.6.1.1] are normal, enzyme activity is stimulated by guanyl-5'-yl imidodiphosphate [Gpp(NH)p], and intact cells accumulate cyclic AMP when exposed to cholera toxin. Furthermore, variant cell membranes possess ligand-binding activity consistent with the conclusion that a normal or an excessive number of beta-adrenergic receptors is present. Thus, interaction between the hormone-binding and the catalytic moieties of the adenylate cyclase system is lost. This variant phenotype, designated as uncoupled (UNC), has been stable for more than 100 generations without exposure to the drugs used for selection. Such cells should be useful for the elucidation of methanisms of transmission of information from hormone receptors to adenylate cyclase.