Reduction In Cyclic AMP Accumulation Research Articles

Melatonin modulates rat myotube-acetylcholine receptors by inhibiting calmodulin

Melatonin, the pineal gland hormone, modulates α-bungarotoxin sensitive nicotinic acetylcholine receptors in sympathetic nerve terminals, cerebellum and chick retina imposing a diurnal variation in functional responses [Markus, R.P., Zago, W.M., Carneiro, R.C., 1996. Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 279, 18–22; Markus, R.P., Santos, J.M., Zago, W., Reno, L.A., 2003. Melatonin nocturnal surge modulates nicotinic receptors and nicotine-induced [ 3HI] glutamate release in rat cerebellum slices. J. Pharmacol. Exp. Ther. 305, 525-530; Sampaio, L.F.S., Hamassaki-Britto, D.E., Markus, R.P., 2005. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells. Braz. J. Med. Biol. Res. 38, 603–613]. Here we show that in rat myotubes forskolin and melatonin reduced the number of nicotinic acetylcholine receptors expressed in plasma membrane. In addition, these cells expressed melatonin MT1 receptors, which are known to be coupled to G i-protein. However, the pharmacological profile of melatonin analogs regarding the reduction in cyclic AMP accumulation and number of nicotinic acetylcholine receptors did not point to a mechanism mediated by activation of G i-protein coupled receptors. On the other hand, calmidazolium, a classical inhibitor of calmodulin, reduced in a similar manner both effects. Considering that one isoform of adenylyl cyclase present in rat myotubes is regulated by Ca 2+/calmodulin, we propose that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation.

Effects of melatonin in isolated rat papillary muscle

Melatonin ( N-acetyl-5-methoxytryptamine), the principal hormone of the vertebral pineal gland, elicits several neurobiological effects. However, the effects of melatonin on cardiac muscle are still unknown. The first goal of the study was to investigate the role of melatonin on myocardial contractility in isolated rat papillary muscle using dose-response curves to melatonin, to isoproterenol and calcium either in the presence or in the absence of melatonin (0.3 nM). Response curves to isoproterenol were additionally performed in the presence of melatonin plus the specific receptor antagonist N-acetyltryptamine (10 μM); the adenylate-cyclase stimulator forskolin (10 μM) was also used. Melatonin has no direct inotropic effect in isolated rat papillary muscle but counteracts isoproterenol but not [Ca 2+] effects. In fact, the EC 50 for isoproterenol was significantly higher in the presence than in the absence of melatonin ( p<0.001). This anti-adrenergic action occurs through an interaction to a specific cardiac receptor. Forskolin-stimulated adenylate cyclase induced an increase of contractile force (+118±25%) which was reduced in the presence of melatonin (+26±10%; p<0.01). In conclusion, we found that melatonin possess anti-adrenergic effect in isolated rat papillary muscle. This phenomenon was abolished in the presence of its receptor antagonist N-acetyltryptamine demonstrating that melatonin operates through a specific cardiac receptor. The reduction of contractility increase, induced by forskolin-stimulated adenylate cyclase, shows that melatonin may act through a reduction of cyclic AMP accumulation.

Atrial natriuretic peptide reduces cyclic AMP by activating cyclic GMP-stimulated phosphodiesterase in vascular endothelial cells.

Bovine aortic endothelial cells contain cyclic GMP-stimulated phosphodiesterase (PDE) regulating intracellular cyclic AMP and cyclic GMP levels. To investigate the roles of this PDE isoform for cyclic AMP hydrolysis in intact endothelial cells (EC), we used an adenine prelabeling method to determine cyclic AMP accumulation in response to agents that might produce effects that increase cyclic GMP levels. Atrial natriuretic peptide (ANP), which dramatically increased cyclic GMP accumulation, reduced cyclic AMP in cultured EC from bovine aorta with or without addition of L-isoproterenol (L-ISO), whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP had no effect. The reduction in cyclic AMP by ANP was dose dependent (> or = 1.0 nM) and rapid (significant reduction was induced in < or = 15 s) and was abolished when EC were preincubated with 3-isobutyl-1-methylxanthine (IBMX) and HL-725, both nonselective PDE inhibitors. ANP had no effects on adenylate cyclase activity, nor any direct effects on the activities of partially purified cyclic GMP-stimulated PDE isoform or cyclic AMP-specific isoform. However, cyclic AMP hydrolyzing activities of EC were enhanced when EC were pretreated with 0.1 microM ANP. ANP activates cyclic GMP-stimulated PDE and induces reduction of cyclic AMP accumulation in intact EC, which may modify cyclic GMP-dependent endothelial function involved in ANP.

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 μM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. B max of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 μM VIP. No significant change was observed in receptor affinity, in B max of the low-affinity receptor, in ED 50, or in ED 100 of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 μM, 3 h) caused 24% reduction in [ 125I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 μM, 3 h).

Acute desensitization of lymphocyte beta-adrenergic-stimulated adenylate cyclase in old age and Alzheimer's disease

The effect of prior incubation with a single concentration of isoproterenol (10 −4 M) for 2 hours at 37°C on isoproterenol-stimulated cyclic AMP accumulation in intact lymphocytes from young, old and subjects with Alzheimer's disease was studied. In lymphocytes from all three subject groups prior incubation of cells with isoproterenol resulted in a significant reduction of cyclic AMP accumulation upon subsequent stimulation with isoproterenol.

Repression by dexamethasone of epinephrine-induced modulation of the fatty acyl-CoA desaturase system in Tetrahymena microsomes.

Epinephrine modulation of the fatty acyl-CoA desaturase activities in Tetrahymena microsomes, namely the stimulation of delta 9 desaturase activity with a decreased activity of delta 12 desaturase, was markedly inhibited by dexamethasone administered 30 min prior to epinephrine addition. This effect by dexamethasone was dose-dependent. With epinephrine or dexamethasone prior to epinephrine administration, the activities of delta 9-terminal and delta 12-terminal components were found to change in parallel with those of delta 9 and delta 12 desaturase activities respectively. Furthermore, addition of dexamethasone as early as 30 min before epinephrine repressed the epinephrine-mediated stimulation of cyclic AMP production and of adenylate cyclase activity. Therefore, it is suggested that dexamethasone may inhibit the modulation by epinephrine of the terminal component activity, which plays a crucial role in microsomal fatty acyl-CoA desaturase system, through the reduction of cyclic AMP accumulation by decreased responsiveness to epinephrine of Tetrahymena cells.

Inhibitory effect and mode of action of somatostatin on lipolysis in chicken adipocytes

The effect of somatostatin on lipolysis was investigated utilizing isolated chicken adipocytes. Somatostatin-14 and -28 inhibited basal lipolysis. This ability to suppress glycerol release (used as an index of lipolysis) was emphasized in presence of stimulated lipolysis. Concentration of 1 ng/ml somatostatin-14 (0.625 nM) and somatostatin-28 (0.312 nM) was found to inhibit completely the glycerol release induced by concentrations of glucagon up to 2 ng/ml (0.58 nM). The percentage of inhibition was dose-dependent. The antilipolytic effect of somatostatin-14 was also observed during ACTH and aminophylline-stimulated lipolysis. Among the mechanisms which could account for the inhibition, a possible competitive effect of somatostatin-14 with 125I-labelled glucagon binding to adipocyte membranes was excluded. The small inhibiting effect of somatostatin-14 on glycerol release prompted by dibutyryl cyclic AMP, together with the significant inhibiting effect on aminophylline-stimulated lipolysis argued for a reduction of cyclic AMP accumulation. The increase of cyclic AMP levels induced by glucagon was substantially reduced in presence of somatostatin-14. It was concluded that in chicken adipocytes somatostatin inhibited the rate of lipolysis and that reduction on cyclic AMP could be responsible, at least in part, for the antilipolytic effect.

Cyclic Adenosine 3′:5′-Monophosphate Metabolism in Normal and SV40-transformed WI-38 Cells

Abstract Intact SV40-transformed WI-38 cells (VA13-2RA) responded to catecholamines, prostaglandins, and adenosine with much more dramatic increases in cyclic adenosine 3':5'-monophosphate (cyclic AMP) than did normal WI-38 cells. Further, the responses of VA13-2RA to the catecholamines were independent of the cell population density, whereas the responses of WI-38 were diminished as they approached confluence. Time course studies likewise showed clear differences between VA13-2RA and its normal counterpart, in that the maximal intracellular cyclic AMP levels in response to prostaglandin E1 (PGE1) were reached much later in the transformed than in the normal cells (20 min versus 5 min). Further, the rapid decline in cellular cyclic AMP levels seen in WI-38 (as in most cell systems) was much attenuated in VA13-2RA. Basal cyclic AMP levels were lower in the transformed than in the normal cells, as has been the case with many other systems. Theophylline appeared to have two distinct effects in WI-38, namely, synergism (with prostaglandins), as expressed by increased cellular cyclic AMP levels, and reduction of cyclic AMP accumulation in the media. On the other hand, theophylline had only small (or in some cases no) effects on cyclic AMP accumulation in VA13-2RA. Adenylate cyclase activities in cell-free preparations of WI-38 and VA13-2RA cells differed in several respects. Although homogenates of WI-38 were more active than VA13-2RA preparations in the presence of PGE1 (10 µm) and NaF (10 mm), VA13-2RA activity exceeded WI-38 activity by more than 3-fold in the presence of epinephrine and, in fact, cyclic AMP accumulation in homogenates of the transformed cells was significantly greater in the presence of epinephrine than with NaF. Finally, WI-38 homogenates showed much greater cyclic nucleotide 3':5'-monophosphate phosphodiesterase activity than VA13-2RA preparations (131 ± 8 versus 7.1 ± 5.4 nmoles of cyclic AMP destroyed per mg of protein per 30 min).