Decrease In Cyclic AMP Production Research Articles

Chapter 6 Regulation of renal salt and water transporters during vasopressin escape

Hyponatremia, defined as a serum sodium < 135 mmol/l, is one of the most commonly encountered and serious electrolyte disorders of clinical medicine. The predominant cause of hyponatremia is an inappropriate elevation of circulating vasopressin levels relative to serum osmolality or the 'syndrome of inappropriate antidiuretic hormone secretion' (SIADH). Fortunately, the degree of the hyponatremia is limited by a process that counters the water-retaining action of vasopressin, namely 'vasopressin escape'. Vasopressin escape is characterized by a sudden increase in urine volume with a decrease in urine osmolality independent of circulating vasopressin levels. Until recently, little was known about the molecular mechanisms underlying escape. In the 1980s, we developed an animal model for vasopressin escape in which male Sprague-Dawley rats were infused with dDAVP, a V2-receptor-selective agonist of vasopressin, while being fed a liquid diet. Rats drank a lot of water in order to get the calories they desired. Using this model, we demonstrated that the onset of vasopressin escape (increased urine volume coupled to decreased urine osmolality) coincided temporally with a marked decrease in renal aquaporin-2 (water channel) protein and mRNA expression in renal collecting ducts. This protein reduction was reversible and correlated to decreased water permeability of the collecting duct. Studies examining the mechanisms underlying AQP2 decrease revealed a decrease in V2-receptor mRNA expression and binding, as well as a decrease in cyclic AMP production in response to acute-dDAVP challenge in collecting duct suspensions from these escape animals. Additional studies showed an increase in sodium transporters of the distal tubule. These changes, hypothetically, might help to attenuate the hyponatremia. Future studies are needed to fully elucidate systemic, intra-organ, and cellular signaling responsible for the physiological phenomenon of vasopressin escape.

Evidence for cocaine and methylecgonidine stimulation of M(2) muscarinic receptors in cultured human embryonic lung cells.

1. Muscarinic cholinoceptor stimulation leads to an increase in guanylyl cyclase activity and to a decrease in adenylyl cyclase activity. This study examined the effects of cocaine and methylecgonidine (MEG) on muscarinic receptors by measurement of cyclic GMP and cyclic AMP content in cultured human embryonic lung (HEL299) cells which specifically express M(2) muscarinic receptors. 2. A concentration-dependent increase in cyclic GMP production was observed in HEL299 cells incubated with carbachol, cocaine, or MEG for 24 h. The increase in cyclic GMP content was 3.6 fold for 1 microM carbachol (P < 0.01), 3.1 fold for 1 microM cocaine (P < 0.01), and 7.8 fold for 1 microM MEG (P < 0.001), respectively. This increase in cyclic GMP content was significantly attenuated or abolished by the muscarinic receptor antagonist atropine or the M(2) blocker methoctramine. 3. In contrast, cocaine, MEG, and carbachol produced a significant inhibition of cyclic AMP production in HEL299 cells. Compared to the control, HEL299 cells treated with 1 microM cocaine decreased cyclic AMP production by 30%. MEG and carbachol at 1 microM decreased cyclic AMP production by 37 and 38%, respectively. Atropine or methoctramine at 1 or 10 microM significantly attenuated or abolished the cocaine-induced decrease in cyclic AMP production. However, the antagonists alone had neither an effect on cyclic GMP nor cyclic AMP production. Pretreatment of HEL299 cells with pertussis toxin prevented the cocaine-induced reduction of cyclic AMP production. 4. Western blot analysis showed that HEL299 cells specifically express M(2) muscarinic receptors without detectable M(1) and M(3). Incubation of HEL299 cells with cocaine, carbachol, and atropine did not alter the expression of M(2) protein levels. However, the inducible isoform of nitric oxide synthase (iNOS) was induced in the presence of cocaine or carbachol and this induction was significantly attenuated after addition of atropine or methoctramine. 5. The present data show that cocaine and MEG significantly affect cyclic GMP and cyclic AMP production in cultured HEL299 cells. Our results also show that these effects result from the drug-induced stimulation of M(2) muscarinic receptors accompanied with no alterations of receptor expression. However, the induction of iNOS by cocaine may result in the increase in cyclic GMP production.

Selective inhibition of adenylyl cyclase by octopamine via a human cloned alpha 2A-adrenoceptor.

1. In this study we have compared the abilities of the enantiomers of the structural isomers of the phenolamines, octopamine and synephrine, and the catecholamines, noradrenaline and adrenaline, to couple selectively a human cloned alpha 2A-adrenoceptor, stably expressed in a Chinese hamster ovary (CHO) cell line, to G-protein linked second messenger pathways mediating an increase and a decrease in cyclic AMP production. 2. The catecholamines couple the alpha 2A-adrenoceptor to both an increase and a decrease in the rate of cyclic AMP production. In the absence of pertussis toxin pretreatment both catecholamines tested showed a dose-dependent decrease with a maximum at 100 nM. After pertussis toxin pretreatment they both produced a dose-dependent increase in cyclic AMP production with a maximum at 10 microM. 3. The phenolamines, octopamine and synephrine were only able to couple the alpha 2A-adrenoceptor to a dose-dependent decrease in cyclic AMP production at concentrations up to 1 mM, with the synephrine isomers being more potent than the corresponding octopamine isomers. The meta-isomers of both phenolamines were more potent than the corresponding para-isomers and the (-)-enantiomers were more potent than the (+)-enantiomers. Thus, (-)-meta-synephrine [(-)-phenylephrine] was the most effective isomer tested with an observable decrease occurring between 100 nM and 1 microM. 4. The effects of octopamine and the catecholamines on the decrease in cyclic AMP production were additive at submaximal concentrations, whilst octopamine reduced the stimulant effect of submaximal concentrations of noradrenaline on cyclic AMP production after pertussis toxin pretreatment. 5. The time courses of the inhibitory effects of both meta-octopamine and noradrenaline were parallel and peaked after a 1 min exposure to the agonist. In contrast, the stimulant effects of noradrenaline after pertussis toxin pretreatment were of a much slower time course with a maximum effect occurring after a 5 min incubation period. 6. Since octopamine and synephrine occur naturally in, and are co-released with catecholamines from, mammalian tissues, the results of the present study suggest that the human cloned alpha 2A-adrenoceptor can be coupled selectively by different endogenous agonists to G-protein pathways mediating the regulation of adenylyl cyclase activity.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M 1 and M 2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M 2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M 2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.

Evaluation of central adrenergic receptor signal transmissions after an antidepressant administration to the rat

The effects of several antidepressants, amitrityline, citalopram, desipramine, fluoxetine, maprotiline, mianserin, nialamide, nomifensine, tranylcypromine and viloxazine, on the accumulation of cyclic AMP and inositol monophosphates were studied in rat cerebral cortical slices. The two enzymatic systems were stimulated either by adrenergic agonists or by forskolin. Cyclic AMP and inositol monophosphates (IPs) formed were determined by a double label method. In vitro all drugs, except inhibitors of monoamine oxidase, nialamide and tranylcypromine, inhibited α,-agonist-mediated production but did not modify the cyclic AMP accumulation. Otherwise, chronic desipramine but not citalopram administration decreased the accumulation of cyclic AMP (−39%) elicited by β-adrenoceptor agonists; no change was observed in inositol phosphate metabolism after administration of these two drugs. These data support previous investigations showing a decrease in cyclic AMP production after chronic treatment with norepinephrine uptake blockers but do not confirm the hypothesis of a modification of α 1-adrenoceptor-stimulated inositol phosphate metabolism.

Chronic ethanol intake inhibits both the vasoactive intestinal peptide binding and the associated cyclic AMP production in rat enterocytes

1. 1. Chronic ethanol intake during 6, 8, 10 or 12 weeks resulted in a decrease of 125I-vasoactive intestinal peptide (VIP) binding to rat enterocytes. 2. 2. Native peptide displaced 125I-VIP binding to enterocytes, exhibiting a IC 50 at about 4 nM native VIP in control and ethanol-treated animals. 3. 3. The number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. This reduction is observed in both the high-affinity and the low-affinity binding sites. 4. 4. Increasing concentrations of native VIP produced a similar cyclic AMP rise in enterocytes from control or ethanol-treated rats during 6 weeks. However, after 8 weeks of ethanol treatment, a significant decrease in cyclic AMP production stimulated by VIP was observed.

Studies of receptor‐mediated inhibition of 45Ca accumulation into synaptosomes

1. The effects of alpha 2-adrenoceptor and kappa-opiate receptor activation on 45Ca accumulation into rat cortical synaptosomes were examined. 2. Clonidine (1 microM) and U50488H (1 microM) significantly reduced 45Ca accumulation under both resting (5 mM K+) and depolarizing (15-30 mM K+) conditions. 3. The inhibitory effects of the agonists on 45Ca accumulation into synaptosomes were enhanced in the presence of the Na(+)-Ca2+ exchange inhibitor sodium orthovanadate (vanadate, 2 mM), and were not present in mitochondrial preparations. 4. When the agonists were used together, their inhibitory effects were not additive but were, in fact, attenuated. 5. In the presence of the alpha 2-adrenoceptor antagonist idazoxan (1 microM), the inhibitory effect of U50488H on 45Ca accumulation was enhanced. A similar increase in the inhibitory effectiveness of clonidine was observed in the presence of naloxone (20 microM). 6. When synaptosomes were pretreated with 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM), dibutyrylcyclic AMP (db-cyclic AMP, 10 microM) or 8-bromo-cyclic AMP (8Br-cyclic AMP, 10 microM), the inhibitory effects of clonidine and U50488H were abolished, suggesting that a decrease in cyclic AMP production is part of the receptor-effector coupling mechanism of both receptor systems. 7. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.05 microM) increased 45Ca accumulation but did not alter the inhibitory effects of clonidine or U50488H, thus showing that the effects of the agonists are not mediated by protein kinase C. 8. We conclude that alpha 2-adrenoceptor and Kappa-opiate receptor activation dramatically reduce 45Ca influx through Ca21 channels (e.g., by 50%), that there is a functional antagonism between the two receptor systems and that in both cases, the receptor effector mechanism involves a decrease in cyclic AMP production.

Characterisation of an octopamine-sensitive adenylate cyclase in haemocyte membrane fragments of the American cockroach Periplaneta americana L.

An octopamine-sensitive adenylate cyclase has been studied in a fragmented haemocyte-membrane preparation of the American cockroach, Periplaneta americana. The enzyme complex is similar to that of adenylate cyclases from other eukaryotic cells in having a requirement for magnesium ions and GTP and in displaying elevated prodution of cyclic AMP in the presence of sodium fluoride and forskolin. The preparation responds to GTP in a dose-dependent manner and a positive dose-dependent response to calmodulin is also reported. Octopamine and synephrine are the most potent stimulators of cyclic AMP production in the haemocyte-membrane preparation and additivity studies indicate that synephrine and another activator, tyramine, are interacting with the enzyme complex through the octopamine-receptor. The adrenergic agonists, naphazoline and clonidine increased cyclic AMP production and the formamidines, demethylchlordimeform and BTS 27271 also were effective in this regard. Hydroxymandelic acid caused a slight but statistically significant decrease in cyclic AMP production. The most potent antagonists of the octopamine-mediated response were mianserin > promethazine > phentolamine > gramine > cis- flupenthixol > dibenamine. Lineweaver-Burk plots indicate that the mianserin, promethazine and gramine-effects, as with earlier reports for phentolamine, result from competitive inhibition

The effect of calcium on the potentiation of LH-stimulated steroidogenesis and inhibition of LH-stimulated cyclic AMP production by LHRH agonist (ICI 118630) in rat Leydig cells

A luteinizing hormone-releasing hormone (LHRH) agonist (ICI 118630) potentiated the effects of luteinizing hormone (LH) and dibutyryl cyclic AMP on steroidogenesis during 4 h incubations with rat Leydig cells. LH-stimulated cyclic AMP levels were decreased by the addition of the LHRH agonist. The potentiation of the LH-increased steroidogenesis was dependent on Ca 2+; maximum effects required at least 2.5 mM Ca 2+ in the incubation medium. The calcium ionophore A23187 negated the potentiation in a dose-dependent manner (ED 50 = 0.2–0.3 μM), but had no effect on LH-induced steroidogenesis, despite a 90% decrease in cyclic AMP production. The latter decrease was found to be dependent on the Ca 2+ concentration. In the presence of the phosphodiesterase inhibitor methylisobutylxanthine (MIX), the ionophore A23187 induced a dose-dependent decrease in both LH and LH plus LHRH agonist-stimulated steroidogenesis and cyclic AMP production. The results obtained indicate that calcium, rather than cyclic AMP, is the mediator of the potentiating effects of LHRH agonist on LH-increased steroidogenesis in rat Leydig cells. The marked inhibition of the synergism in the presence of calcium ionophore A23187 suggests that Leydig cell calcium homeostasis must be intact for LHRH agonist action to occur. LHRH agonist causes a Ca 2+-dependent decrease in LH-stimulated cyclic AMP production.

Mepacrine blocks beta-adrenergic agonist-induced desensitization in astrocytoma cells.

C6 astrocytoma cells contain beta-adrenergic receptors coupled to adenylate cyclase. A 2-hr exposure to l-isoproterenol results in an 80% decrease in cyclic AMP production in response to a subsequent challenge by l-isoproterenol (desensitization). This loss in responsiveness is paralleled by a 20-30% decrease in the apparent number of beta-adrenergic receptors and by increased release of arachidonic aciid into the medium. The increased release of arachidonic acid is caused by the action of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and corresponds to increased turnover of methylated phospholipids. Mepacrine and tetracaine, both inhibitors of this phospholipase A2, are able to block l-isoproterenol-induced desensitization of cyclic AMP production and the decrease in beta-adrenergic receptors. Mellitin and phorbol ester, two activators of phospholipase A2, when preincubated with the cells cause a decreased cyclic AMP response of the cells to l-isoproterenol. These results suggest that the activation of phospholipase A2 in the local domain of the beta-adrenergic receptor may be involved in desensitization.

Effect of psychotropic drugs on RNA synthesis in brain cell nuclei

Changes in the distribution of biogenic amines in the brain were produced by intraperitoneal injection of psychotropic drugs — reserpine (80 μg/100 g) and chlorpromazine (300 μg/100 g) — during chronic experiments on rats. Under these conditions reserpine reduced the endogenous RNA polymerase activity of types I and II of the rat brain cell nuclei on average by 61 and 34%, and chlorpromazine did so by 32 and 38% respectively. In a cell-free system reserpine and chlorpromazine in concentrations of 0.1 and 1 mM had no effect on the RNA-systhesizing activity of isolated rat brain cell nuclei. It is suggested that the action of psychotropic drugs on the genetic apparatus may be mediated through, either a decrease in cyclic AMP production or inhibition of RNA-synthesizing activity.

Adenosine 3′:5′-cyclic monophosphate production and steroidogenesis by isolated rat adrenal glomerulosa cells. Effects of angiotensin II and [Sar1,Ala8]angiotensin II

Angiotensin II effects on cyclic AMP production and steroid output were studied in a sensitive preparation of isolated rat adrenal glomerulosa cells. With increasing concentrations of angiotensin II logarithmic dose-response curves for aldosterone and cyclic AMP production were similar. The minimum effective dose (0.2nm) for stimulation of aldosterone production also significantly (P<0.001) increased cyclic AMP output. For both aldosterone and cyclic AMP production, the peptide hormone concentration eliciting maximal response (0.2mum) and the ED(50) (median effective dose) values (1nm) were the same; this is consistent with cyclic AMP acting as an intracellular mediator for angiotensin II-stimulated aldosterone production by glomerulosa cells. The angiotensin II antagonist [Sar(1),Ala(8)]angiotensin II inhibited angiotensin II-stimulated corticosterone and aldosterone production in these cells. An equimolar concentration of antagonist halved the response to 20nm-angiotensin II, and complete inhibition was observed with 0.2mum-antagonist. In contrast, [Sar(1),Ala(8)]angiotensin II had no effect on maximally stimulated steroidogenesis induced by serotonin and a raised extracellular K(+) concentration. Increasing concentrations of [Sar(1),Ala(8)]angiotensin II alone decreased corticosterone and aldosterone outputs significantly (P<0.05) at concentrations of 20nm and 2nm of antagonist respectively. A significant (P<0.001) decrease in cyclic AMP production occurred with 2mum antagonist and this was comparable with the decrease in aldosterone production. It is concluded that [Sar(1),Ala(8)]angiotensin II can independently affect glomerulosa-cell steroidogenesis, possibly by modulating adenylate cyclase activity.