Differences in LH receptor down-regulation between rat and mouse Leydig cells: effects of 3',5'-cyclic AMP and phorbol esters.

The role of cyclic AMP and phorbol esters in luteinizing hormone (LH) receptor down-regulation in Leydig cells has been studied. Dibutyryl cyclic AMP (db-cAMP) (0.01, 0.1 and 1 mM), forskolin (80 microM) and cholera toxin (1.19 nM) caused a 30-50% loss of [125I]hCG binding sites and an inhibition of receptor-[125I]hCG complex internalization in mouse tumour Leydig (MA10, MLTC-1) cells during 2 h. In contrast, db-cAMP had no effect on the level of binding sites or internalization of the hormone receptor complex in rat testis Leydig cells or a rat tumour (R2C) Leydig cell. Phorbol 12-myristate 13-acetate (PMA) at concentrations from 10(-9) to 10(-5) M had no effect on hormone binding or hormone-receptor complex internalization in any of the Leydig cells. In contrast a 2 h preincubation of MLTC-1 cells with 10(-7) M PMA caused a loss of subsequent LH-stimulated cyclic AMP and pregnenolone production. These results indicate that LH receptor down-regulation is mediated by cyclic AMP dependent kinase, but not protein kinase C, in mouse Leydig cells. No down-regulation of rat Leydig cell LH receptor occurs with either kinase.     

Beta1-adrenoceptors in rat anterior pituitary may be constitutively active. Inverse agonism of CGP 20712A on basal 3',5'-cyclic adenosine 5'-monophosphate levels

Catecholamines directly stimulate GH, ACTH, and prolactin secretion from rat anterior pituitary through the beta(2)-adrenoceptor (AR). We recently showed that gonadotrophs express the beta(1)-AR and that glucocorticoids drastically increase its mRNA expression level. The present investigation explores whether beta(1)-ARs are functionally coupled to adenylate cyclase. In anterior pituitary cell aggregates, the highly selective beta(1)-AR antagonists CGP 20712A and ICI 89,406-8a attenuated isoproterenol-stimulated cAMP accumulation, but no agonist action of norepinephrine could be detected. Remarkably, CGP 20712A inhibited basal cAMP levels by its own for at least 50%, an action that tended to be more effective in dexamethasone-supplemented medium. The latter effect was abolished by the beta-AR antagonist carvedilol, but not by other beta-AR antagonists. Pretreatment with pertussis toxin abolished the action of CGP 20712A on basal cAMP. CGP 20712A also attenuated isoproterenol-induced cAMP accumulation in the gonadotroph cell lines alphaT3-1 and LbetaT2, but not in the somatotroph precursor cell line GHFT and the folliculo-stellate cell line TtT/GF. However, in LbetaT2 cells CGP 20712A did not inhibit basal cAMP levels by its own. The present data suggest that beta(1)-AR in the anterior pituitary is positively coupled to adenylyl cyclase but is constitutively active in a pertussis toxin-sensitive manner. CGP 20712A may act as an inverse agonist with approximately 50% negative intrinsic activity, suggesting that the beta(1)-AR significantly contributes to basal adenylate cyclase activity in the pituitary.     

Effect of partially purified NSILA on adenylate cyclase, phosphodiesterase and 3',5'-cyclic AMP in fat cells.

Partially purified, non-suppressible, insulin-like material (NSILA-S) was studied with respect to its effect on the levels of 3',5'-cyclic adenosine monophosphate (cAMP) and its mechanism of action in the control of this nucleotide in rat fat cells. NSILA-S prevents the rise of cAMP in fat cells under the influence of isoproterenol with similar kinetics to insulin. A maximal effect is observed at about 70 ng/ml with a biological activity equivalent to 200 muU/ml of insulin. NSILA-S inhibits norepinephrine-stimulated adenylate cyclase activity in fat cell ghosts and partially purified plasma membrane preparations. At 10 mM Mg2+, the inhibition is characterized by an effect of Vmax without change in affinity towards ATP (apparent KM 30 muM). Similarly there is no observed change in affinity towards Mg2+. With respect to inhibition of norepinephrine-stimulated adenylate cyclase, the dose-response curve of NSILA-S is similar to that already found with intact cells. The effect of norepinephrine is inhibited throughout the dose-response range between 5 X 10(-7) and 5 X 10(-4) M. In contrast to previous observations with insulin in ghosts, NSILA-S inhibits the basal adenylate cyclase activity. Cyclic nucleotide phosphodiesterase activity in homogenates as measured at 1.0 muM substrate is increased by 90% after previous incubation of fat cells with NSILA-S. The study suggests that the anti-lipolytic effect of NSILA-S is mediated by a lowering of cAMP through inhibition of the adenylate cyclase and/or stimulation of the phosphodiesterase system.     

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads.

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.     

Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues

Homogeneous preparations of type I and type II regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinase (cAMP kinase) were utilized as antigens to obtain isozyme specific antisera. Injections of pure catalytic subunit (C) from the type I isozyme resulted in antisera that reacted with C subunit obtained from either isozyme type. Cross-reactivity of the antisera raised against isolated subunits of the kinase was assessed by immunodiffusion analysis and by measuring the cAMP binding and phosphotransferase activities of the subunits after immunoprecipitation. These antisera were used to localize subunits of type I and type II cAMP kinases in rat skeletal muscle, liver, and adrenal by using indirect immunofluorescence and immunoperoxidase techniques. Specificity of the immunofluorescence was shown by absorption of the antisera with pure homologous antigens. In skeletal muscle, both R and C subunits of the type I and type II cAMP kinases were localized in the area of the sarcoplasmic reticulum and in periodic crossbands. Specific fluorescence for these components was observed in both isotropic and anisotropic band regions of the sarcomere. Densitometric determinations of immunoperoxidase staining revealed a larger amount of RI, RII, and C subunits in the isotropic band than in the anisotropic band regions. In liver, C, RI, and RII subunits were distributed both in cytoplasmic and nuclear areas and along plasma membranes of hepatocytes; however, there were qualitative differences observed among these various subcellular sites. With each antiserum, fluorescence was blocked by prior absorption with homologous antigen. After treatment of rats with glucagon, dramatic changes in the relative distribution patterns of C and RII were noted in the nucleus. In the adrenal gland, RI, RII, and C subunits were localized in both cytoplasmic and nuclear areas, and an apparent redistribution of these subunits occurred after treatment of (dexamethasone-suppressed) rats with ACTH. The application of this immunocytochemical approach provides a tool for examining and monitoring the subcellular distribution of these components of cAMP kinase in biological systems.     

Effects of synthetic LH-RH and its analogs on rat anterior pituitary cyclic AMP and LH and FSH release

Synthetic LH-RH (1 μg/ml) in vitro increased cyclic AMP concentration of rat anterior pituitary within 1 min and HL release into the medium after 15 min incubation. The minimum effective dose of synthetic LH-RH on cyclic AMP formation was 10 ng/ml. Data obtained using synthetic analogs of LH-RH indicated that the N-terminal amino acid and the C-terminal carboamide group of synthetic LH-RH are necessary for biologic activity.     

Regulation of adenosine-3',5'-monophosphate levels in rat anterior pituitary by adrenal corticoids

The effect of adrenal cortical hormones on basal and prostaglandin-E2 (PGE2)-stimulated levels of cAMP was studied in incubated rat anterior pituitaries. It was found that oral administration of dexamethasone (DEX) at the concentration of 10 micrograms/mL of drinking saline to adrenalectomized (ADX) rats for four days decreases basal and PGE2-stimulated cAMP levels in the pituitary but not in the hypothalamus. Furthermore, a time-course study demonstrated that acute intraperitoneally (IP) DEX treatment had no effect; whereas, after one day of oral DEX, PGE2-stimulated levels, but not basal, were already reduced. The effect of DEX was also obtained with two natural rat adrenal hormones like corticosterone and deoxycorticosterone, whereas, progesterone and testosterone, which share with the corticoids a ring A-unsaturated-three-ketone structure but are devoid of corticoid activity, did not modify at physiological doses the levels of cAMP. More physiological validation of these results was given by experimental procedures that modified endogenous adrenal secretion. Thus, reduction of plasma corticoids by ADX significantly increased basal and PGE2-stimulated cAMP levels in the pituitary, whereas, increments of plasma corticosterone produced by ACTH had the reverse effect in intact rats. It is suggested that adrenal gland regulation of cAMP may reflect involvement of the nucleotide in the mechanism of action of corticoids at the pituitary level.     

Calcium requirement for stimulation of cyclic AMP accumulation in anterior pituitary gland by LH-RH.

Removal of Ca2+ from the incubation medium by addition of 2 mM ethylene glycol bis-(beta-aminoethyl ether)-N, N'tetraacetic acid (EGTA) leads to at least 75% inhibition of the luteinizing hormone-releasing hormone (LH-RH)-induced accululation of adenosine 3'5'-monophoshpate (cyclic AMP) in rat anterior pituitary gland in vitro. This inhibitory effect of EGTA is reversed by the addition of Ca2+. A half-maximal effect of Ca2+ on LH-RH--induced cyclic AMP accumulation is observed at 2-5 X 10-5 M free Ca2+. The LH-RH-induced LH and FSH release is completely dependent upon the presence of Ca2+ in the incubation medium, a half-maximal effect being measured at 1-2 X 10-4 M free Ca2+. The basal release release of LH is increased upon Ca2+ removal.     

Potentiation of carbon tetrachloride-induced liver damage by dibutylyl-3',5'-cyclic AMP in unstarved rats

It has been reported that carbon tetrachloride-induced liver damage is potentiated by starvation partly due to fat accumulation in the liver and a decrease in hepatic reduced glutathione concentration and that dibutylyl-3′,5′-cyclic AMP (DBcAMP) affects fuel metabolism and decreases hepatic reduced glutathione. We investigated the effects of DBcAMP on carbon tetrachloride-induced liver damage both in unstarved and starved rats. In unstarved rats, intraperitoneal administration of DBcAMP potentiated an increase in serum alanine aminotransferase activity and fatty vacuolization in the liver, both of which were induced by carbon tetrachloride. Hepatic reduced glutathione concentration was also reduced by DBcAMP, although the change was not significant. In contrast, the administration of DBcAMP in starved rats did not affect carbon tetrachloride-induced changes in serum alanine aminotransferase activity, histological alterations and hepatic reduced glutathione concentration. Administration of DBcAMP to control rats induced different responses in unstarved control rats compared with starved control rats: in unstarved rats, blood glucose concentration decreased but serum free fatty acid concentration increased, whereas in starved rats, blood glucose concentration increased and serum free fatty acid concentration decreased. It was suggested that DBcAMP potentiated carbon tetrachloride-induced liver damage in unstarved rats, probably due to hepatic fat accumulation and a decreased hepatic reduced glutathione concentration. The former could increase the affinity of the liver for carbon tetrachloride and the latter could accelerate carbon tetrachloride-induced lipid peroxidation. It was also suggested that DBcAMP failed to affect carbon tetrachloride-induced liver damage in starved rats, probably because starvation had already decreased hepatic glutathione concentration and DBcAMP had different effects on fuel metabolism compared with effects observed in unstarved rats.     

Evidence on the participation of the 3',5'-cyclic AMP pathway in the non-genomic action of 1,25-dihydroxy-vitamin D3 in cardiac muscle.

Several studies have suggested that vitamin D plays a role in cardiovascular function. It has been recently shown that in vitro treatment of vitamin D-deficient chick cardiac muscle with physiological concentrations of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) induces a rapid (1-10 min) increase of tissue 45Ca uptake which can be suppressed by Ca channel blockers. The hormone simultaneously stimulated heart microsomal membrane protein phosphorylation. Experiments were performed to investigate the existence of a relationship between these changes and to obtain information about the mechanism involved in 1,25(OH)2D3-induced modifications in cardiac protein phosphorylation. Dibutyryl cyclic AMP (10 microM) and forskolin (10 microM), known activators of the cAMP pathway, produced time courses of changes in 45Ca uptake by chick heart tissue similar to 1,25(OH)2D3 (10(-10) M). Analogously to the hormone, the effects of both compounds were abolished by nifedipine (30 microM) and verapamil (10 microM). In agreement with these observations, 1,25(OH)2D3 significantly increased (34-70%) heart muscle cAMP levels within 1-10 min of treatment. In addition, 1,25(OH)2D3 and forskolin caused similar changes in cardiac microsomal membrane protein phosphorylation (e.g. stimulation in 43 kDa and 55 kDa proteins). These changes were also evidenced by direct exposure of isolated heart microsomes to 1,25(OH)2D3, suggesting a direct membrane action of the hormone. The fast effects of 1,25(OH)2D3 on dihydropyridine-sensitive cardiac muscle Ca uptake could be reproduced in primary-cultured myocytes isolated from chick embryonic heart. Furthermore, the effects of the hormone could be suppressed by a specific protein kinase A inhibitor. These results suggest that 1,25(OH)2D3 affects heart cell calcium metabolism through regulation of Ca channel activity mediated by the cAMP pathway.     

Biological effects and catabolic metabolism of 3',5'-cyclic nucleotides and derivatives in rat adipose tissue and liver

Experiments on the catabolism of N⁶, O^2′-dibutyryl cyclic AMP and N⁶-monobutyryl cyclic AMP in rat liver and epididymal adipose tissue were described. During the regulation by the dibutyryl cyclic nucleotide of several parameters of the metabolism of epididymal adipose tissue adipocytes (lipolysis and glucose metabolism), neither of the butyryl groups was removed enzymatically, and nonenzymatic formation of butyric acid amounted to no more than 4 per cent over 2 hours of incubation. Therefore, intact adipocytes exhibited no deacylase activities. Furthermore, cyclic nucleotide phosphodiesterases, isolated from subcellular fractions derived from homogenates of liver and adipose tissue, which actively hydrolyzed cyclic AMP, GMP, IMP, UMP, and TMP, but not cyclic CMP, were sterically hindered by substituent groups on the N⁶ and O^2′ positions of cyclic AMP; that is, the enzyme was completely inactive against N⁶, O^2′-dibutyryl cyclic AMP and exhibited only a trace amount of activity against N⁶-monobutyryl cyclic AMP. However, mechanisms do exist in these tissues for the removal of one or both butyryl groups; subcellular fractions derived from tissue homogenates contained an N-acyl hydrolase active against N⁶, O^2′-dibutyryl and N⁶-monobutyrl cyclic AMP, and, possibly, also esterase activity against the former. The relationships of function (as lipolytic agents and substrates for cyclic nucleotide phosphodiesterases) to structure among a variety of 3′, 5′-cyclic nucleotides were investigated using adipocytes and partially purified enzymes. An incubation medium consisting of a sodium chloride-phosphate buffer devoid of Mg²⁺, Ca²⁺, and K⁺ permitted entry of all nucleotides into adipocytes. In such permeable cells, all purine and pyrimidine cyclic nucleotides, with the exception of the thymine analogs, stimulated lipolytic activity to various extents; however, no relationships, inverse or otherwise, existed between the potencies of the compounds as activators of lipase activity and their susceptibility to hydrolysis by the cyclic nucleotide phosphodiesterase. Combinations of saturating concentrations of cyclic nucleotides produced rates of lipolysis in cells that were similar to the rate produced by the more potent member of each pair when tested alone; only the lipolytically inactive thymine cyclic nucleotides inhibited the effects of other cyclic nucleotides upon lipolysis. Such results indicated that all active cyclic nucleotides stimulated the same lipolytic mechanism in adipose tissue.     

In vivo evidence for the intermediary role of 3',5'-cyclic AMP in parathyroid hormone-induced stimulation of 1alpha,25-dihydroxyvitamin D3 synthesis in rats.

Teh stimulatory effect of parathyroid hormone (PTH) on renal 1alpha-hydroxylation of 25-hydroxyvitamin D3 (25-OH-D3) was studied in thyro-parathyroidectomized (TPTX), vitamin D-deficient rats into which bovine PTH, theophylline, cAMP or dibutyryl cAMP (dbcAMP) was constantly infused. The accumulation in plasma of 1alpha,25-dihydroxy-vitamin D3 [1alpha,25-(OH)2-D3], produced from 25-OH-D3, was enhanced by infusion of either cAMP (0.9 MUMOL/H) OR DBCAMP (1 mumol/h) to a level similar to the maximum obtained by PTH (i95--7.5 U/h) infusion. A submaximal dose (1 U/h) of PTH caused a similar extent of stimulation, when infused with theophylline. When either 2 mumol/h of cAMP or 7.5 U/h of bovine PTH was infused starting 18 h after TPTX, the accumulation of 1alpha,25-(OH)2-D3 in plasma was similarly restored withing 6 h to the level found in the sham-operated animals. These results strongly support the concept that cAMP plays an important intermediary role in the stimulation of 1alpha,25-(OH)2-D3 production induced by both exogenous and endogenous PTH.