The antilipolytic action of insulin on adrenocorticotrophin-stimulated rat adipocytes. The roles of adenosine 3',5'-monophosphate and the protein kinase dependent on adenosine 3',5'-monophosphate

The extent to which a fall in cellular cyclic AMP could account for the antilipolytic action in rat epididymal adipocytes incubated with adrenocorticotrophic hormone was studied. The antilipolytic effect, measured by suppression of glycerol release, was always associated with a decrease in cyclic AMP, but the magnitude of the fall was modified by several factors. For example, it was greater when the cAMP level was high, as when it is at its peak after hormone stimulation, or when cell concentrations are low. Glucose did not modify appreciably the insulin effect on the nucleotide level. The inhibitory effects of insulin on corticotrophin-stimulated lipolysis and cyclic AMP levels were detectable at the concentrations of 1 microU/ml and were biphasic, with maximal effects at 10-100 microU/ml. Protein kinase activity ratio was similarly affected. Activity of cyclic-AMP-dependent protein kinase conformed closely to the level of cyclic AMP. There was no indication that insulin modified the sensitivity of the kinase to cyclic AMP. Insulin did not alter the relationship of cellular cyclic AMP levels to glycerol when adipocytes were incubated with various concentrations of corticotrophin. This was true, irrespective of whether measurements were made when cyclic AMP was on the upward rise after hormone stimulation, or on the decline. The curves obtained with and without insulin were superimposable. It is concluded that the inhibitory action of insulin on lipolysis in fat cells can be fully accounted for by a decrease in cyclic AMP.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Effect of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate on RNA release from isolated nuclei.

The addition of physiological concentrations of either cAMP or cGMP stimulated the release of RNA from isolated prelabeled rat liver nuclei to a fortified cytosol in a cell-free system. The released RNA was shown to be primarily mRNA by its binding to oligo(dT)-cellulose and its sedimentation profile. Treatment of rats with cAMP or cGMP 30 min prior to the preparation of cyclic nucleotides on the cell-free system. Cyclic nucleotides stimulation of RNA release occurred in systems prepared from resting rat liver, Novikoff hepatoma, and Morris hepatoma 5123D, but not the 18-h regenerating liver. The response of the cell-free system to added cyclic nucleotides reflected the in vivo concentration of these substances in the tissues from which the system was prepared. Those with high in vivo levels were not stimulated while those with lower levels did respond to added cyclic nucleotides. Neither cAMP nor cGMP had an appreciable effect on rRNA release.     

Effects of hydralazine on guanosine cyclic 3', 5'-monophosphate levels in rat aorta

The mechanism of the vasodilator effect of hydralazine on isolated rat aorta was studied. Results demonstrated that the vasodilator effect of hydralazine was greater on intact aortas than on endothelium-denuded preparations, particularly at low concentrations of between 0.1 mM and 0.5 mM. In addition, hydralazine did not have any effect on cyclic GMP levels. We also found that methylene blue, an inhibitor of guanylate cyclase, completely abolished the vasorelaxant action of nitroglycerin but not that of hydralazine. These results indicate that the vasodilator effect of hydralazine was not due to elevating the cyclic GMP levels. On the other hand, hydralazine significantly inhibited both the contractions induced by norepinephrine and/or high-potassium. In conclusion, a part of the vasodilator effect of hydralazine seems to depend on the integrity of the vascular endothelium. However, this vasodilator effect was not associated with any elevation in cyclic GMP level. Thus, the direct vasodilator action of hydralazine may be related to its interference with the movement and/or translocation of calcium across the cell membrane.     

Effect of adrenergic agents on alpha-amylase release and adenosine 3',5'-monophosphate accumulation in rat parotid tissue slices.

The role of cyclic AMP in stimulus-secretion coupling with investigated in rat parotid tissue slices in vitro. Isoproterenol and norepinephrine stimulated a rapid intracellular accumulation of cyclic AMP, which reached a maximum level of 20-30 times the control value by 5 to 10 min after addition of the drug. Isoproterenol was approximately ten times more potent in stimulating both alpha-amylase release and cyclic AMP accumulation than were norepinephrine and epinephrine, which had nearly equal effects on these two parameters. Salbutamol and phenylephrine were less effectivema parallel order of potency and sensitivity was observed for the stimulation of adenylate cyclase activity in a washed particulate fractionmthe results suggest that these drugs are acting on a parotid acinar cell through a beta1-adrenergic mechanismmat the lowest concentrations tested, each of the adrenergic agonists stimulated significant alpha-anylase release with no detectable stimulation of cyclic AMP accumulationmeven in the presence of theophylline, phenylephrine at several concentrations increased alpha-amylase release without a detectable increase in cyclic AMP levels. However, phenylephrine did stimulate adenylate cyclase. These data suggest that, under certain conditions, large increases in the intra-cellular concentration of cyclic AMP may not be necessary for stimulation of alpha-amylase release by adrenergic agonists. Also consistent with this idea was the observation that stimulation of cyclic AMP accumulation by isoproterenol was much more sensitive to inhibition by propranolol than was the stimulation of alpha-amylase release by isoproterenol. Stimulation of alpha-amylase release by phenylephrine was only partially blocked by either alpha- or beta-adrenergic blocking agents, whereas stimulation of adenylate cyclase by phenylephrine was blocked by propranolol and not by phentolaminemphenoxybenzamine and phentolamine potentiated the effects of norepinephrine and isoproterenol on both cyclic AMP accumulation and alpha-amylase release by N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate; These observations may indicate a non-specific action of phenoxybenzamine, and demonstrate the need for caution in interpreting evidence obtained using alpha-adrenergic blocking agents as tools for investigation of alpha- and beta-adrenergic antagonism.