Alpha-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes. Similarity of receptor with alpha-2 adrenergic receptors

The present communication shows the effects of several alpha-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 micrograms/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the alpha-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 microM methoxamine; 10 microM methyl norepinephrine; 10 microM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of alpha-adrenergic blocking drugs on prevention of the inhibitor effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the alpha-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to alpha-2 adrenergic receptors.     

α-adrenergic inhibition of cyclic AMP accumulation in hamster adipocytes similarity of receptor with α-2 adrenergic receptors

The present communication shows the effects of several α-adrenergic agonists and antagonists on cyclic AMP levels in hamster epididymal adipocytes. In response to ACTH (30 mU/ml) in combination with 1-methyl-3-isobutylxanthine (0.10 mM) or adenosine deaminase (1.0 μg/ml), cyclic AMP levels increased to a maximum by 10 min and this level was maintained for another 20 min. Elevated cyclic AMP levels were partially suppressed by the α-adrenergic agents clonidine, methoxamine, methyl norepinephrine and phenylephrine. The lowest effective concentration of each of these agonists required to suppress cyclic AMP levels was 10 nM clonidine; 3 μM methoxamine; 10 μM methyl norepinephrine; 10 μM phenylephrine. Clonidine and methoxamine suppressed cyclic AMP levels by nearly 65% while phenylephrine and methyl norepinephrine caused only a 30% decline. Studies of the relative potencies of α-adrenergic blocking drugs on prevention of the inhibitory effect of clonidine on cyclic AMP levels disclosed that phentolamine and yohimbine were more potent blockers of clonidine action than phenoxybenzamine and prazosin. The rank order of potencies of agonists at causing suppression of cyclic AMP levels and the rank order of potencies of antagonists of clonidine action suggest similarity of the α-adrenergic receptors present on hamster adipocytes, which affect cyclic AMP accumulation to α-2 adrenergic receptors.     

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin?phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha₁ adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ?prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha₂ adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha₁ and alpha₂ adrenoceptors but about 90% of the binding sites were alpha₂. These data support the hypothesis that alpha₂ effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha₁ adrenoceptor activation.     

α-Adrenergic inhibition of cyclic AMP accumulation in epithelial cells isolated from rat small intestine

The present work shows that α-adrenergic agonists induce the suppression of basal and hormone-stimulated cyclic AMP levels in rat intestinal epithelial cells. Epinephrine (100 μM) suppresses by 35% the cyclic AMP levels evoked by the vasoactive intestinal peptide (VIP). The adrenergic agent induces a similar percentage of inhibition at 15, 30 and 37°C. Addition of epinephrine 20 min prior to, on 5 or 20 min after VIP yields the same magnitude of inhibition as when performed together with the stimulus. The α-adrenergic agent does not alter the K_0.5 of VIP in stimulating cyclic AMP production but reduces its efficacy. Epinephrine also suppresses prostaglandin E₁- and E₂-stimulated cyclic AMP levels by about 35%. The lowest effective concentration of epinephrine required to suppress VIP-stimulated cyclic AMP levels is 0.1 μM, half-maximal (K_0.5) and maximal effects being observed at 5 and 100 μM, respectively. Norepinephrine has the same efficacy but a slightly lower potency (K_0.5 = 18 μM) than epinephrine. Phenylephrine acts as a partial agonist of very low potency; clonidine has very little intrinsic activity and antagonizes the inhibition by epinephrine. The inhibition of VIP-stimulated cyclic AMP levels is observed in the absence of any blocking agents. It is not affected by the β blocker propranolol, but is completely reversed with α blockers with the following order of potency: dihydroergotamine>yohimbine>phentolamine. Yohimbine is much more potent than prazosin, which only partially reverses the inhibition induced by epinephrine. It is concluded that α-adrenoreceptors of the α₂ subtype mediate the suppression of VIP-stimulated cyclic AMP levels in intestinal epithelial cells. This effect is likely to be due to the inhibition of adenylate cyclase within intact cells as epinephrine is able to reduce adenylate cyclase activity of intestinal epithelial cell plasma membranes.     

Abrogation by prostaglandin F2alpha of LH-stimulated cyclic AMP accumulation in isolated rat corpora lutea of pregnancy.

Corpora lutea explanted from rats on the sixth day of pregnancy responded to luteinizing hormone (LH; 5 μg/ml) in vitro with a two- to five-fold increase in cellular cyclic AMP (cAMP) concentration. The maximal cAMP level was reached within 60 min and maintained to the end of the 2 hr-incubation. On incubation with prostaglandin F_2α (PGF_2α) in addition to LH, this rise in cAMP accumulation was prevented. For significant suppression, 1.4 × 10^?5M PGF_2α was required. In the absence of LH, PGF_2α (4.2 × 10^?5M) caused no change in cellular cAMP. Addition of PGF_2α (4.2 × 10^?5M) to the incubation medium after the maximal response to LH was attained, caused the cAMP concentration to return to its basal level within 15 min. This abrogation of LH-stimulated cAMP accumulation represents the earliest and hence possibly the triggering event in PGF_2α-induced luteolysis.     

Cholinergic inhibition of catecholamine-stimulable cyclic AMP accumulation in murine atria.

Carbachol antagonizes isoproterenol-stimulable cyclic AMP accumulation in mouse atria by direct activation of cardiac muscarinic receptors. Inhibition by carbachol occurs rapidly and is completely reversed when the drug is removed. Neither nitroprusside nor 8-bromo-cyclic GMP mimics the actions of carbachol and low concentrations of carbachol block cyclic AMP accumulation without increasing the intracellular cyclic GMP content. Carbachol does not block cyclic AMP accumulation by activating phosphodiesterase since it is fully effective in the face of marked phosphodiesterase inhibition, nor does it appear to inhibit the catalytic activity of adenylate cyclase since it does not decrease either basal or cholera toxin-stimulated cyclic AMP accumulation. The interaction between carbachol and isoproterenol is not competitive, since cholinergic inhibition cannot be surmounted by increasing concentrations of isoproterenol. The site of muscarinic action therefore appears to involve the mechanisms coupling the hormone-receptor complex to adenylate cyclase. This site is distinct from that of cholera toxin action since there is no antagonism between the effects of cholera toxin and carbachol on cyclic AMP metabolism in the atrium.     

Alcohol potentiation of isoproterenol-stimulated cyclic AMP accumulation in rat parotid

The ability of beta-adrenergic agonists to elevate rat parotid cyclic AMP concentrations is potentiated greatly by certain organic solvents. Propanol was found to be more effective than other tested solvents. Propanol stimulated adenylate cyclase and inhibited low Km cyclic AMP phosphodiesterase activities however the magnitude of effect upon these enzyme activities probably does not account for the potentiation of cyclic AMP accumulation observed in intact cells.     

Somatostatin and alpha 2-adrenergic agonists selectively inhibit vasopressin-induced cyclic AMP accumulation in MDCK cells

The effect of somatostatin and alpha 2-adrenergic agonists on cyclic AMP accumulation was examined in MDCK cells, grown in defined medium. These hormones inhibited vasopressin-induced cyclic AMP formation, without affecting either the basal or the glucagon- and prostaglandin E2-stimulated level. Pretreating the cells with pertussis toxin, or incubating them with MnCl2 at a low concentration reversed the effect of somatostatin and alpha 2-agonists. These results suggest that somatostatin and norepinephrine could selectively modulate the renal effect of vasopressin, via the inhibitory regulatory subunit (Ni) of adenylate cyclase.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

Depolarization-evoked accumulation of cyclic AMP in brain slices: inhibition by exogenous adenosine deaminase

In guinea pig cerebral cortical slices labeled during a prior incubation with radioactive adenine, electrical stimulation or the presence of depolarizing agents such as veratridine, ouabain, and high concentrations of K⁺ elicit a marked accumulation of radioactive cyclic AMP. This accumulation is reduced in all cases by the presence of theophylline, a compound that antagonizes the stimulatory effects of adenosine on cyclic AMP accumulation in brain slices. Exogenous adenosine deaminase also reduced the accumulation of cyclic AMP elicited by electrical stimulation, veratridine, and high concentrations of K⁺. Thus, adenosine formed in neuronal compartments under depolarizing conditions appears to be released into the extracellular medium as a prerequisite to stimulation of the cyclic AMP-generating system. Adenosine deaminase does not prevent the reduction in levels of ATP under depolarizing conditions, nor does it antagonize the accumulation of cyclic AMP elicited by a combination and norepinephrine. Adenosine deaminase does not, however, prevent the accumulations of cyclic AMP elicited by the depolarizing agent, ouabain.     

Prostaglandin F2 alpha inhibition of epinephrine stimulated cyclic AMP and progesterone production by rat corpora lutea of various ages

Epinephrine can mimic the stimulatory effects of LH in vitro on cyclic AMP (cAMP) and progesterone production by isolated rat corpora lutea. The aim of the present study was to test whether the effects of epinephrine in vitro on the rat corpus luteum, as with LH, can be inhibited by prostaglandin F2 alpha (PGF2 alpha). The stimulatory effect of epinephrine on tissue levels of cAMP in 1-day-old corpora lutea was not inhibited by PGF2 alpha. A dose-dependent inhibition by PGF2 alpha (0.5-50 microM) was seen for 3-day-old corpora lutea and this inhibition could not be overcome by higher concentrations of epinephrine (0.165-165 microM). The stimulation by epinephrine on progesterone production was inhibited by PGF2 alpha (5 microM) in 3- and 5-day-old, but not in 1-day-old corpora lutea. Thus, PGF2 alpha can inhibit the stimulatory effect of epinephrine in 3- and 5-day-old corpora lutea, but not in the newly formed corpora lutea (1-day-old) and PGF2 alpha shows in this respect the same age dependent inhibitory pattern as in relation to LH stimulation.     

Islet cyclic AMP levels are not lowered during alpha 2-adrenergic inhibition of insulin release

Epinephrine-induced changes in insulin release and cyclic AMP levels were measured simultaneously in isolated rat islets. Forskolin was used to enhance islet cyclic AMP levels. Forskolin (30 microM) stimulated adenylate cyclase activity 10-fold in islet homogenates and raised cyclic AMP levels 5-fold in intact islets (both at low and high glucose). Insulin release was enhanced by forskolin only at high glucose. Epinephrine (0.1 microM) inhibited glucose- and forskolin-induced insulin release to basal rates. At the same time epinephrine potentiated forskolin-elevated cyclic AMP levels. In contrast epinephrine attenuated forskolin-stimulated adenylate cyclase activity in islet homogenates. At low glucose, both alpha 2- and beta-adrenergic blockade counteracted the epinephrine potentiation, each by 50%. At high glucose the effect was mainly beta-adrenergic in nature. The actions of epinephrine in the presence of a beta-blocker were mimicked by the alpha 2-agonist clonidine. Despite the variations in cyclic AMP levels stimulated insulin release was always inhibited by activation of alpha 2-receptors. Finally, insulin release stimulated by exogenous cyclic AMP was abolished by epinephrine. These results suggest that epinephrine inhibits insulin release at a step distal to the generation of cyclic AMP.     

Pertussis toxin does not inhibit alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cyclic AMP accumulation in rat pinealocytes

The hypothesis that Gi might be involved in the alpha 1-adrenergic, protein kinase C (PKC)-mediated amplification of beta-adrenergic cyclic AMP stimulation in rat pinealocytes was investigated. Treatment of pinealocytes with a high concentration of pertussis toxin (500 ng/ml, 18 h) almost completely (approximately 95%) inactivated two cell membrane G-proteins (kDa 40.7 and 39.8) judged by back ADP-ribosylation of pinealocyte membrane proteins. However, this treatment failed to inhibit either the beta-adrenergic (isoprenaline, ISO 10(-6) M), alpha 1-plus beta-adrenergic (noradrenaline, NA 10(-5) M) or beta-adrenergic plus 12-O-tetradecanoylphorbol 13-acetate (TPA 10(-7) M) induced stimulation of cyclic AMP or cyclic GMP. These results suggest that alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cyclic AMP and cyclic GMP does not involve a pertussis toxin-sensitive G-protein.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Ba++ stimulates accumulation of cyclic AMP in rat pancreatic islets.

In pancreatic islets prelabelled with (³H) adenine, Ba⁺⁺ augmented (³H) cyclic AMP in 1–10 min incubations. 3-isobutyl-l-methylxanthine markedly enhanced and prolonged the Ba⁺⁺-induced nucleotide as well as the insulin response. In the presence of the methyl xanthine 1.6 mM Ba⁺⁺ was a maximally and 0.4 mM a submaximally effective concentration both for the stimulation of (³H) cyclic AMP and insulin. A 5-fold excess of Ca⁺⁺ partly inhibited the Ba⁺⁺-induced nucleotide and — more profoundly — the insulin response. Increasing Mg⁺⁺ from 2 to 10 mM was also inhibitory. Stimulation by Ba⁺⁺ was observed in the absence as well as in the presence of D-glucose. It is concluded that the insulinotropic action of Ba⁺⁺ is at least partly mediated by cyclic AMP.     

Interactions between catecholamines,methyl xanthines and adenosine in regulation of cyclic AMP accumulation in hamster adipocytes

In the presence of either methyl xanthines or adenosine deaminase, isoproterenol elicited large dramatic increases in accumulation of cyclic AMP. In contrast, cyclic AMP accumulation in response to epinephrine or norepinephrine was not potentiated by either methyl xanthines or by adenosine deaminase. Blocking the alpha adrenergic activity of norepinephrine and epinephrine with phentolamine established synergism between these catecholamines and methyl xanthines and adenosine deaminase. The activity of the particulate phosphodiesterase was not influenced by norepinephrine suggesting that the lack of synergism between the catecholamines norepinephrine and epinephrine and methyl xanthines is unrelated to this enzyme. The data are interpreted to suggest that the alpha adrenergic activity of catecholamines prevents the potentiation of cyclic AMP accumulation that occurs when the action of endogenously produced adenosine is interfered with, either by its degradation with adenosine deaminase or by receptor blockade with methyl xanthine. Because a major action of adenosine on fat cells is to inhibit adenylate cyclase it is suggested that alpha adrenergic receptor activation limits the extent to which the enzyme adenylate cyclase can be activated in a fashion similar to that of adenosine.