Sensitivity of Ca-dependent slow action potentials to methacholine is induced by phosphodiesterase inhibitors in embryonic chick ventricles

Choline esters fail to depress developed tension or the maximum upstroke velocity (Vmax) of slow action potentials in embryonic chick ventricles, but they inhibit the stimulatory effect of the phosphodiesterase inhibitor methylisobutylxanthine (MIX). The mechanism by which the choline ester methacholine (MCh) counteracts the effects of MIX was examined in ventricular myocardium obtained from 7-day-old embryonic chicks. Four possible hypotheses were 1) that the physiological response to MCh is mediated by cyclic GMP, the production of which is potentiated by MIX; 2) that MCh acts by a mechanism independent of cyclic nucleotides; 3) that the binding of MIX to adenosine receptors induces sensitivity to MCh; or 4) that MCh acts by depressing basal cyclic AMP levels. Interactions between MCh, Angiotensin II and a nonmethylxanthine phosphodiesterase inhibitor (Ro 7-2956) were assessed by measuring tissue levels of cyclic nucleotides and the Vmax of slow action potentials. MCh significantly reduced the basal cyclic AMP level of embryonic chick ventricles, despite having no physiological effect. The results favor the final hypothesis and imply that MCh effects are mediated by inhibition of adenylate cyclase activity. The physiological response of the myocardium to a reduction in basal adenylate cyclase activity appears to be dependent on the initial tissue level of cyclic AMP.     

Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter

These studies were performed in vitro to investigate the nature of the second messenger for lower esophageal sphincter (LES) smooth muscle relaxation in response to electrical field stimulation (EFS) and vasoactive intestinal polypeptide (VIP). It was seen that VIP, permeant derivatives of the cyclic nucleotide 8-bromo cyclic GMP (BrcGMP) and 8-bromo cyclic AMP (8-BrcAMP), the guanylate cyclase stimulant sodium nitroprusside (SNP), the adenylate cyclase stimulant forskolin, M&B 22,948 (cGMP phosphodiesterase inhibitor) and SK&F 94,120 (cAMP phosphodiesterase inhibitor) caused dose-dependent and tetrocotoxin resistant fall in LES tension. Guanylate cyclase inhibitor methylene blue (MB) (3 x 10(-5) M), caused significant antagonism of fall in LES tension by SNP without modifying the inhibitory response of forskolin. The possible adenylate cyclase inhibitor N-ethylmaleimide (NEM) (1 x 10(-4) M), on the other hand, caused significant antagonism of fall in LES tension by forskolin without any effect on that caused by SNP. The inhibitory responses of 8-BrcGMP and 8-BrcAMP were not modified by MB or NEM. NEM (1 x 10(-4) M) and MB (3 x 10(-5) M) caused significant inhibition of the fall in LES tension with EFS. NEM also caused inhibition of fall in LES tension by VIP. Furthermore, SK&F 94,120 and not M&B 22,948 caused significant potentiation of fall in LES tension by EFS. From these results we conclude that: 1) cAMP and cGMP may act as second messengers for LES relaxation with EFS and VIP, and 2) VIP may act primarily via cAMP system and remains a strong possibility for one of the inhibitory neurotransmitters in the LES.     

Influence of a phosphodiesterase inhibitor on the chronotropic effects of glucagon and norepinephrine in fetal mouse hearts

Fetal mouse hearts develop tachycardia in response both to norepinephrine and to glucagon, but although adenylate cyclase is stimulated and adenosine 3':5'-monophosphate (cyclic AMP) elevated by norepinephrine, no measurable changes are produced by glucagon. To test further the possible independence of glucagon chronotropy from the cyclic AMP system, the effects of a phosphodiesterase inhibitor were evaluated. The dose-response curve to norepinephrine was shifted to the left by the phosphodiesterase inhibitor 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro7-2956), but the dose-response curve to glucagon was unaltered. Thus, 10(-6) M norepinephrine produced an increase of 40 +/- 5 beats/min in hearts pretreated with Ro7-2956, as compared to an increase of 22 +/- 3 in control hearts (P less than .01). In contrast, 10(-6) M glucagon produced a rate increase of 25 +/- 4 beats/min in treated hearts vs. 26 +/- 4 beats/min in controls. These data are compatible with the hypothesis that adenylate cyclase and cyclic AMP are involved in the chronotropic response of the fetal mouse heart to norepinephrine but not to glucagon.     

Specific inhibition of isoproterenol-stimulated cyclic AMP accumulation by M2 muscarinic receptors in rat intestinal smooth muscle.

The ability of oxotremorine-M to inhibit cyclic AMP accumulation in the presence of a variety of adenylate cyclase activators was studied in slices from the longitudinal muscle of the rat ileum. Oxotremorine-M was found to inhibit forskolin- and isoproterenol-stimulated cyclic AMP accumulation maximally by 17 and 32%, respectively, but not the stimulation due to other activators of adenylate cyclase. Inhibition of cyclic AMP accumulation by oxotremorine-M was unaffected by tetrodotoxin and was completely reversed by atropine. AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidynyl]acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one) an M2-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant (KB) of 0.20 microM, consistent with the dissociation constants for binding at the M2 muscarinic receptor site (KD = 0.092 microM) and inhibition of adenylate cyclase activity (KB = 0.13 microM). Hexahydrosiladifenidol, an M3-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant of 0.67 microM, again consistent with the dissociation constant for binding at the M2 site (KD = 0.83 microM). The agreement between the estimates of the dissociation constants of muscarinic antagonists for binding and for inhibition of cyclic AMP accumulation suggest that oxotremorine-M inhibition of isoproterenol-stimulated cyclic AMP accumulation in slices of rat intestinal smooth muscle is mediated by the M2 receptor.     

Measurement of adenylate cyclase activity in the right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure

A highly sensitive fluorometric assay technique was adopted in order to examine the adenylate cyclase activity in the minute right ventricular endomyocardial biopsy samples from patients with chronic congestive heart failure (n = 10). Norepinephrine (10(-4) M) and adenosine (10(-3) M) were incubated for 30 min with 10 microl of membrane preparation (1-2 mg protein/mg) to analyze the extent of the receptor-coupled adenylate cyclase activity. Forskolin (10(-4) M) stimulation was used to estimate the maximum adenylate cyclase activity (pmol/mg protein/min, mean +/- SE). The new microanalytical cyclic AMP assay involves four steps: enzymatic destruction of noncyclic adenine nucleotides and phosphorylated metabolites, conversion of cyclic AMP to ATP, amplification of ATP by enzymatic cycling, and fluorometric measurement of NADPH, which is generated in proportion to initial cyclic AMP levels. Basal and forskolin-stimulated maximum adenylate cyclase activities were 75 +/- 8 and 123 +/- 15, respectively. Norepinephrine increased the adenylate cyclase activity to 107 +/- 14, while adenosine tended to decrease it to 65 +/- 7. In addition, elimination of adenosine by adenosine deaminase (10 U/ml) slightly increased the adenylate cyclase activity to 82 +/- 9. These results indicate that the adenylate cyclase activity can be measured in minute endomyocardial biopsy samples. Use of this new approach shows promise of becoming a new and potentially important way to predict the efficacy of pharmacological treatment.     

Inhibition of bradykinin stimulation of renal medullary prostaglandin E2 synthesis by phosphodiesterase inhibitors

Effects of phosphodiesterase inhibitors DL-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro-20) and 1-methyl-3-isobutylxanthine (MIX) on prostaglandin E2 (PGE2) synthesis were examined using rabbit renal inner medullary slices incubated in Krebs' buffer with or without 1 mM RO-20 or 2 mM MIX. Basal and bradykinin-mediated PGE2 synthesis were inhibited in a dose-dependent, reversible manner by both RO-20 and MIX. Arachidonic acid-mediated increases in PGE2 synthesis were not inhibited. By contrast, 1 mM aspirin completely inhibited PGE2 synthesis. Phosphodiesterase inhibitors increased slice cyclic AMP content more than 6-fold. However, this elevation in tissue cyclic AMP content did not appear to be the cause of decreased PGE2 synthesis. Exogenous 3 mM cyclic AMP and 3 mM dibutyryl cyclic AMP did not alter PGE2 synthesis. 2',5'-Dideoxyadenosine, an inhibitor of adenylate cyclase, prevented RO-20 and MIX-mediated increases in cyclic AMP but had no effect on PGE2 synthesis. Exogenous 3 mM cyclic GMP and dibutyryl cyclic GMP did not alter PGE2 synthesis. Neither RO-20 nor MIX had a direct effect on PG endoperoxide synthetase. These results indicate MIX and RO-20 inhibit renal medullary PGE2 production by limiting the availability of arachidonic acid. These effects of MIX and RO-20 on PGE2 synthesis are not secondary to their effects on cyclic nucleotide phosphodiesterase.     

Adenosine receptors on human leukocytes. IV. Characterization of an A1/Ri receptor.

Adenosine (10(-9)-10(-6) mol/l) and R-phenylisopropyladenosine (10(-9)-10(-7) mol/l) partially inhibited the intracellular accumulation of cyclic AMP induced by isoproterenol, prostaglandin E1, histamine and 5'-N-ethylcarboxamidoadenosine in lymphocytes. In contrast, S-phenylisopropyladenosine, which is a poor agonist of the adenosine A1/Ri receptor, had essentially no inhibitory effect. 8-Phenyltheophylline, in low concentrations that do not inhibit cyclic AMP phosphodiesterase, completely blocked the inhibitory effect of R-phenylisopropyladenosine on the increase in cyclic AMP induced by prostaglandin E1. R-Phenylisopropyladenosine (10(-8)-10(-6) mol/l) also inhibited the cyclic AMP accumulation in lymphocytes induced by forskolin (10(-5) mol/l), which activates adenylate cyclase through direct interaction with the enzyme. We also investigated the presence of the adenosine A1/Ri receptor on human polymorphonuclear leukocytes. R-Phenylisopropyladenosine (3 x 10(-9)-10(-7) mol/l) abolished the stimulating effects of prostaglandin and forskolin on cyclic AMP accumulation in polymorphonuclear leukocytes. This effect was blocked by 8-phenyltheophylline and was not observed with the stereoisomer S-phenylisopropyladenosine. The results support the existence of an A1/Ri receptor that regulates cyclic AMP metabolism of human lymphocytes and polymorphonuclear leukocytes.     

Evidence for Delayed Development of the Glucagon Receptor of Adenylate Cyclase in the Fetal and Neonatal Rat Heart

The effects, in vivo, of epinephrine, glucagon, and dibutyryl cyclic adenosine 3',5'-monophosphate (cyclic AMP) on the glycogen content of rat heart and liver and, in vitro, upon adenylate cyclase activity in homogenates of rat heart and liver were determined during the latter third of gestation and the neonatal period. Hepatic glycogen was depleted by epinephrine, glucagon, and dibutyryl cyclic adenosine 3',5'-monophosphate, but myocardial glycogen was depleted only by epinephrine and dibutyryl cyclic AMP in the neonates. Hepatic adenylate cyclase activity was augmented by both epinephrine (10(-5) M) and glucagon (10(-5) M), and myocardial cyclase was increased only by epinephrine in tissue obtained from 16, 18, and 20 day fetal rats. Myocardial adenylate cyclase responsiveness to glucagon was present in tissue obtained from rats 4 wk of age and older. It is concluded that in contrast to hepatic adenylate cyclase, myocardial adenylate cyclase in the rat is not responsive to glucagon during gestation and that responsiveness to glucagon and the associated ability of glucagon to deplete myocardial glycogen do not develop until well after birth.     

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.     

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.     

Effect of chilling on platelet cyclic adenosine 3:5-monophosphate and adenylate cyclase activity

Exposure of platelets to 1 C led to a transient increase in cyclic AMP levels (determined either by a protein binding method or by radioimmunoassay) within five to ten minutes reaching a maximum 10 to 15 minutes after chilling was begun and returning subsequently to baseline values. Addition of EDTA to the platelet suspension medium prevented this increase. Rewarming at 37 C produced a sudden reduction in platelet cyclic AMP. To determine whether the cold-induced increase in cyclic AMP was due to a transient stimulation of platelet adenylate cyclase or a rapid inhibition of phosphodiesterase, these enzymes were assayed in ruptured platelet suspensions. Platelet adenylate cyclase activity was found to possess certain characteristics similar to those of the enzyme derived from other sources but there was a marked potentiation of fluoride-stimulated adenylate cyclase activity by 0.001 M EDTA. This effect was limited to low EDTA concentrations. Exposure of platelets to 1 C for up to 60 minutes did not increase adenylate cyclase activity but lowered it substantially compared with controls kept at room temperature. Phosphodiesterase activity at 1 C was depressed sooner and to a greater extent than was adenylate cyclase. The transient rise in cyclic AMP levels in chilled platelets appears to be due to a disproportionate reduction of cyclic nucleotide phosphodiesterase activity.     

Somatostatin desensitization: loss of the ability of somatostatin to inhibit cyclic AMP accumulation and adrenocorticotropin hormone release

Addition of somatostatin (SRIF) inhibits corticotropin-releasing factor- and forskolin-stimulated cyclic AMP accumulation and adrenocorticotropin hormone secretion from mouse anterior pituitary tumor cells (AtT-20/D16-16). However, prior exposure of these cells to SRIF reduced the potency of SRIF to inhibit both corticotropin-releasing factor- and forskolin-stimulated cyclic AMP accumulation and adrenocorticotropin hormone release. This SRIF desensitization is time- and concentration-dependent and reversible. Cross-desensitization to SRIF analogs also occurred whereas SRIF pretreatment did not affect the inhibition by SRIF of 8-bromo-cyclic AMP-stimulated adrenocorticotropin hormone release or did it affect basal cyclic AMP levels, protein content or phosphodiesterase activity. These data indicate that SRIF can regulate the sensitivity of its own receptor and that SRIF desensitization may involve either a down-regulation of SRIF receptors or an uncoupling of these inhibitory receptors from adenylate cyclase.     

Interaction of prostaglandins and histamine with enzymes of cyclic AMP metabolism from guinea pig gastric mucosa.

Prostaglandins (PGE1, PGE2, PGA1) and histamine have opposing effects on gastric HCl secretion, but we found that both stimulate adenylate cyclase activity in cell-free membrane preparations of guinea pig gastric fundic mucosa. The stimulatory effect of prostaglandins was found in this study to be specific and dose-dependent over a concentration range from 10(-7) to 10(-4) M. In similar preparations from antral regions of guinea pig gastric mucosa, the adenylate cyclase was stimulated only by PGE1, PGE2, and PGA1 and not by histamine. Maximum stimulating doses of PGE1, PGE2, or PGA1, and of histamine had an additive effect on the adenylate cyclase activity from fundic gastric mucosa. Metiamide, a histamine H2-receptor antagonist, inhibited the stimulation of fundic mucosa adenylate cyclase by histamine but did not interfere with the stimulation by prostaglandins. Cyclic AMP phosphodiesterase activity of guinea pig gastric mucosa was unaffected by PGE1 and PGE2 or by histamine, and was slightly depressed by PGA1. These results indicate that histamine and prostaglandins stimulate two different adenylate cyclase systems both present in guinea pig gastric mucosa tissue. Therefore, the known inhibitory effect of prostaglandins on gastric acid secretion is not related to the interference with the stimulation of the histamine H2-receptor-sensitive adenylate cyclase complex by histamine nor do prostaglandins accelerate cyclic AMP breakdown by cyclic AMP phosphodiesterase to reduce cyclic AMP levels.     

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.     

Regulation of endothelial cell cyclic nucleotide metabolism by prostacyclin.

An analysis of prostaglandin-stimulated adenosine 3',5'-cyclic monophosphate (cyclic AMP) accumulation in cultured human umbilical vein endothelial cells showed prostacyclin (PGI2) to be the most potent agonist followed by prostaglandin (PG)H2, which was more potent than PGE2, while PGD2 was essentially inactive. The endothelial cells studied apparently have a high rate of cyclic AMP phosphodiesterase activity because significant PGI2-mediated increases in cyclic AMP could not be shown in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (MIX). Endoperoxide PGH2-stimulation of cyclic AMP accumulation was inhibited 75--80% by the prostacyclin synthetase inhibitors 12-hydroperoxyeicosatetraenoic acid or 9,11-azoprosta-5,13-dienoic acid. These data indicate that the PGH2-stimulation is due primarily to conversion to PGI2. The beta-adrenergic agonist L-isoproterenol stimulated cyclic AMP accumulation in the endothelial cells. This accumulation was completely blocked by propranolol. However, stimulation of cyclic AMP accumulation by the beta-adrenergic agent did not equal that induced by PGI2. Furthermore, the PGI2 response could not be blocked by propranolol. Thrombin-stimulated PGI2 biosynthesis was attenuated by PGE1 or isoproterenol in the presence of MIX. MIX alone was less effective than a combination of PGE1 or isoproterenol plus MIX. These data suggest two potential effects of PGI2 biosynthesis by endothelial cells: first, the PGI2 can elevate cyclic AMP in platelets, and second, endothelial cell cyclic AMP can be elevated as well, so that subsequent PGI2 synthesis will be attenuated.     

Stimulation of osmotic water flow in toad bladder by prostaglandin E1. Evidence for different compartments of cyclic AMP.

The effect of prostaglandin E1 (PGE1) on osmotic water flow across toad bladder and cyclic AMP content of the mucosal epithelial cells has been determined under basal conditions and in the presence of either theophylline or antidiuretic hormone (ADH); Under basal conditions and with PGE1 concentrations from 10(-8) to 10(-5) M no evidence of stimulation of water flow was observed, and with 10(-7) M PGE1 a significant inhibition was foundmcyclic AMP content under control conditions was 8 pmol/mg protein. It was 9 at 10(-8) M PGE1, 13 at 10(-7) M, 16 at 10(-6) M, and 23 at 10(-5) M. In the presence of theophylline, 10(-8) and 10(-7) M PGE1 inhibited the theophylline-induced water flow as expected. In contrast, 10(-6) and 10(-5) M PGE1 enhanced the rate of water flow. Theophylline increased cyclic AMP content from 8 to 18 pmol/mg protein. PGE1 in the presence of theophylline caused marked increases in cyclic AMP content; The content was 23 at 10(-7) M, 41 at 10(-6) M, and 130 at 10(-5) M; Thus PGE1 stimulates theophylline-induced water flow at cyclic AMP concentrations somewhere between 23 and 41 pmol/mg. Further evidence along these lines was obtained from experiments in which the effects of PGE1 on ADH-induced water flow were studied. Inhibitory effects of PGE1 were not observed at concentrations of PGE1 which raised the level of intracellular cyclic AMP to 30 pmol/mg protein or higher. These results were obtained despite the fact that all four concentrations of PGE1 tested were found capable of inhibiting ADH-induced water flow under appropriate conditions or, in other words, were inhibiting the adenylate cyclase controlling water flow, Thus the increase in cyclic AMP content in response to PGE1 is not derived from this enzyme. Thus the stimulation of water flow by PGE1 in the presence of theophylline is thought to be caused by cyclic AMP spilling over from one compartment to the water flow compartment. No evidence was obtained to directly suggest spillover into the sodium transport compartment. Furthermore evidence is discussed to suggest that most of the cyclic AMP generated in the tissue does not originate from the enzyme controlling sodium transport. As cyclic AMP-stimulated water flow and sodium transport are thought to occur in one cell type, the granular cells, distinct pools of cyclic AMP are thought to be present in one and the same cell type. Thus one pool controls water flow and one controls sodium transport. With high concentrations of PGE1 in the presence of theophylline or high concentrations of ADH, the adenylate cyclase responsible for water flow is inhibited; However, PGE1 can stimulate a tissue adenylate cyclase to sufficiently high levels that cyclic AMP spills over into the "water flow compartment" and thus stimulates water flow.     

Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes.

Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation.     

Stimulation of acetylcholine release from myenteric neurons of guinea pig small intestine by forskolin and cyclic AMP

Forskolin, an activator of adenylate cyclase, was used to examine the regulation of [3H]acetylcholine (ACh) release by cyclic AMP (cAMP)-related mechanisms in myenteric plexus-longitudinal muscle preparations of guinea pig small intestine. Forskolin evoked a dose-related increase in [3H]ACh release. Both dibutyryl-cAMP and 8-Br-cAMP significantly elevated [3H]ACh secretion. In the presence of phosphodiesterase inhibitors (theophylline and 3-isobutyl-1-methylxanthine), the basal [3H]ACh output was increased. There was a significantly greater stimulation when forskolin was used to incite endogenous cAMP synthesis and phosphodiesterase inhibitors were simultaneously applied to prevent cAMP breakdown. The enhancement of forskolin-stimulated release by theophylline or 3-isobutyl-1-methylxanthine strongly implicates a synergistic interaction between the two. These findings suggest that forskolin acts to increase ACh release by a modulation of endogenous cAMP and further support a cAMP-mediated mechanism in the secretion of ACh from myenteric cholinergic neurons.     

Dissociation between muscarinic receptor-mediated inhibition of adenylate cyclase and autoreceptor inhibition of [3H] acetylcholine release in rat hippocampus

Activation of muscarinic cholinergic receptors (mAChRs) in the central nervous system reduces the catalytic activity of membrane-bound adenylate cyclase and attenuates depolarization-dependent release of acetylcholine (ACh). Inasmuch as reports have indicated that these mAChR-mediated responses exhibit pharmacological profiles similar to the M2 subclass of mAChR, the present studies were undertaken to ascertain whether attenuation of presynaptic adenylate cyclase activity [and concurrent reduction of intraneuronal cyclic AMP (cAMP) levels] underlies mAChR-mediated autoinhibition of electrically evoked ACh release. In [3H]choline-prelabeled rat hippocampal slices, the mAChR agonists oxotremorine (EC50 = 15 microM) and carbachol (EC50 = 80 microM) caused atropine-reversible inhibition of [3H]ACh release up to a maximum of 80% reduction. The rank order of potency for antagonist reversal of this inhibitory action (N-methylatropine = atropine greater than scopolamine much greater than pirenzepine) was generally consistent with an M2 mAChR-mediated response although pirenzepine was ineffective up to 1 mM. Under these assay conditions, forskolin (1-10 microM) and 8-bromo-cAMP (30-300 microM) enhanced electrically evoked [3H]ACh release maximally by 50 to 60%; however, neither agent significantly reversed mAChR agonist-induced inhibition of [3H]ACh release. Additional studies were undertaken to determine the consequences of chemically uncoupling mAChR from their G protein-adenylate cyclase effector system in this tissue. Whereas brief pretreatment with the sulfhydryl alkylating agent N-ethylmaleimide (30 microM) or pertussis toxin (1 microgram/ml) markedly attenuated carbachol inhibition of adenylate cyclase activity in hippocampal tissue, there was no concurrent reduction of carbachol-inhibited [3H] ACh release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Histamine and Its Methyl Derivatives on Cyclic AMP Metabolism in Gastric Mucosa and Its Blockade by an H2 Receptor Antagonist

In a cell-free system prepared from guinea pig gastric mucosa, histamine and Nalpha-methyl-histamine produced dose-dependent stimulation of cyclic AMP formation and 1,4-methylhistamine had a minimal stimulatory effect. N-methyl-N'-(2-[5-methylimidazole-4-yl-methylthio]-ethyl) -thiourea (metiamide), a new H2 receptor inhibitor, selectively blocked the stimulation of adenylate cyclase by histamine and its active methyl derivative but had no substantial effect on the basal adenylate cyclase activity or adenylate cyclase stimulated by sodium fluoride. Metiamide inhibited the histamine stimulation of adenylate cyclase at 1/100 the concentration of the histamine. Histamine, its methyl derivatives, and metiamide did not influence the activity of cyclic AMP phosphodiesterase from gastric mucosa. Therefore, histamine stimulates gastric mucosal adenylate cyclase via interaction with the H2 receptor without influencing cyclic AMP breakdown, and N-methylation of histamine on the side chain preserves or even increases its stimulating ability. On the other hand, N-methylation in the ring nearly abolishes the ability of histamine to interact with the H2 receptor.