Adenylate and guanylate cylase system function in human hyperplastic adrenals in Itsenko-Cushing disease

The content of cyclic nucleotides and the activity of adenylate and guanylate cyclases as well as of cAMP phosphodiesterase in the human hyperplastic adrenals were determined after one- and two-step bilateral adrenalectomy for Itsenko-Cushing's disease. A decrease in cGMP concentration and a corresponding increase in cAMP/cGMP correlation were seen in the 2nd hyperplastic adrenal comparatively to those in the 1st one. An enhanced basal activity of adenylate cyclase and its lowered sensitivity to ACTH were found in the 1st and the 2nd adrenals. These changes correlate with a rise in the blood ACTH concentration in patients after the ablation of one adrenal. It is suggested that the augmented adenylate cyclase activity leads to an increased activation of steroidogenesis enzymes in the rest adrenal, ensuring the elevated rate of corticosteroid secretion. The nature of changes in guanylate cyclase activity is contrary to that of adenylate cyclase; namely, guanylate cyclase basal activity of the 2nd hyperplastic adrenal was shown to be lower than that of the 1st one. The cAMP phosphodiesterase activity in the 1st and the 2nd adrenals remained unchanged.     

Effects of cyclic nucleotides on deoxyribonucleic acid synthesis in hypophysectomized rat cartilage: stimulation of thymidine incorporation and potentiation of the action of somatomedin by analogs of adenosine 3',5'-monophosphate or a cyclic nucleotide phosphodiesterase inhibitor

The actions of cyclic nucleotides on basal and somatomedin-stimulated thymidine incorporation into DNA by costal cartilage from hypophysectomized rats were investigated. Three analogs of cAMP (dibutyryl, 8-bromo, and 8-dimethylamino derivatives, which are alternate activators of cAMP-dependent protein kinase and resistant to degradation by cAMP phosphodiesterase but represent a wide difference in potency as phosphodiesterase inhibitors) in range of concentrations from about 10(-5) to 3 X 10(-4) M enhanced basal and somatomedin-stimulated thymidine incorporation. Each cAMP analog at optimal concentration produced combined effects with a suboptimal concentration of somatomedin which were additive or greater. cAMP itself, 5'-AMP, adenosine, 8-Br-5'-AMP, 8-Br-AMPT, and cGMP at concentrations from 10(-7)--10(-3) M or dibutyryl cGMP at concentrations from 10(-10)--10(-3) M did not reproduce the effects of the cAMP analogs. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) at concentrations of 100 or 500 microM also potentiated the effects of somatomedin. At 100- or 500-microM concentrations, the phosphodiesterase inhibitor increased cartilage levels of cAMP and cGMP. These results suggest a role for cAMP in DNA synthesis in rat cartilage. However, they fail to support the hypothesis that all effects of somatomedin on that process are mediated by cAMP, since stimulation of thymidine incorporation by the hormone can be demonstrated in cartilage maximally stimulated by analogs of cAMP.     

Effect of thymic immunomodulators on the system of cyclic nucleotides of splenic T-lymphocytes after BCG vaccination

The experiments on guinea pigs was performed to study the effects of thymogen , thymalin and vilosene on the activity of cAMP, cGMP, adenylate cyclase, guanylate cyclase, cAMP- and cGMP-phosphodiesterases in T lymphocytes during BCG vaccination. It was shown that the disease was accompanied by increased activity of enzymes of anabolism and catabolism of cyclic nucleotides. Thymogen and thymalin mainly activated the synthesis of cGMP, thus causing the shift of cAMP/cGMP value below the reference level. At the same time vilosene increased this value in the process of a repeated BCG injection.     

Stimulation of glucose transport in rat cardiac myocytes by guanosine 3',5'-monophosphate

Glucose transport in isolated rat cardiomyocytes is stimulated by insulin, catecholamines, and anoxia approximately 2- to 3-fold over basal rates. The molecular mechanisms controlling these responses are unknown. In our search for possible cellular mediators of glucose transport stimulation, we examined the effects of a number of nucleotides on 3-O-methylglucose transport in heart cells. The nucleotides and/or permeable analogs (monosuccinyl, 8-bromo, and dibutyryl derivatives) included cUMP, cIMP, cCMP, cAMP, and cGMP at concentrations ranging from 10 nM to 1 mM. Of all the nucleotides tested only cGMP analogs induced a significant stimulation of transport at concentrations as low as 100 nM. This effect was observed in both the 8-bromo- and dibutyryl derivatives and with 1 mM cGMP itself. The effect was concentration dependent for both analogs and produced a maximal response equivalent to that of 100 nM insulin. This insulinomimetic effect of cGMP was examined in more detail in order to evaluate its role as a potential mediator of this response. Agents that are known to stimulate guanylate cyclase in the heart produced a clear stimulation of transport when added to cardiomyocytes. These include insulin, aminophylline, histamine, beta-estradiol, and biotin-nitrophenyl ester. Methylene blue, an inhibitor of guanylate cyclase, blocked the insulin response when added to cells before insulin, but was ineffective when added after insulin. In addition, agents that raise intracellular cGMP levels by inhibiting cyclic nucleotide phosphodiesterases were also examined for effects on glucose transport. Out of several phosphodiesterase inhibitors tested, only Zaprinast (which selectively increases cGMP in heart) stimulated transport in a concentration-dependent manner to within 80% of the maximal insulin effect. These results are consistent with the notion that cGMP may be involved in glucose transport stimulation.     

Rapid changes in specific estrogen binding elicited by cGMP or cAMP in cytosol from human endometrial cells.

Addition of cGMP to cytosol of human endometrium or to cells of the endometrial cancer line HEC-1 produced severalfold increases in specific estrogen binding (EB) levels. This effect was maximal with 1 microM cGMP in the presence of 0.1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor) during incubations with [3H]estradiol. In contrast, cAMP decreased EB levels under similar conditions. The effects of cyclic nucleotides on EB levels were complete in less than 15 min in the presence of Mg2+, Mn2+, or Ca2+. The EB sites generated by the addition of cGMP during labeling of cytosol with 10 nM [3H]estradiol were found to sediment in the 8S and 4S regions of low-salt glycerol gradients. No changes in EB levels were observed when cyclic nucleotides were added to cytosol depleted of ATP by preincubation at 4 degrees C for 3 hr, but responsiveness was restored by addition of exogenous ATP. The ATP requirement and the pattern of dependence of cyclic nucleotide actions on divalent cation concentrations suggest that cGMP and cAMP effects may be mediated by kinases and may involve phosphorylations. Another possibility is that the cyclic nucleotides interact allosterically with the binder in the presence of ATP. Addition of sodium molybdate, ATP, and GTP to homogenates of endometrial tissue or HEC-1 cells produces increases in EB levels similar to those obtained by the addition of cGMP. However, these compounds are much less active when added to cytoplasm or cytosol. On the basis of these and other observations, it is hypothesized that molybdate, ATP, and GTP affect EB levels primarily by increasing cGMP concentrations through processes involving a plasma membrane-bound guanylate cyclase.     

Duodenal mucosa and extracellular cyclic nucleotide pattern in coeliac disease.

This study measured the values of cyclic nucleotides and adenylate and guanylate cyclase activities in duodenal mucosa homogenates to verify if they played a part in coeliac disease. Nine controls, 13 patients who did not receive treatment and nine patients who received treatment were studied. Cyclase activity assays were performed under basal conditions and in the presence of gliadin derived peptides. Duodenal mucosa cyclic nucleotide values and adenylate cyclase activity were significantly higher in patients who did not receive treatment than in those who did and in controls, whereas guanylate cyclase activity was similar in all groups. Gliadin derived peptides did not affect guanylate cyclase activity, but significantly increased adenylate cyclase activity in homogenates from patients who did not receive treatment. As extracellular cyclic nucleotide concentrations could reflect changes in their intracellular metabolism, plasma and urine cyclic nucleotide values were also measured in 25 controls and in 55 patients studied at different stages of their disease. Extracellular cyclic nucleotides were considerably high in patients who were not healed and became normal after about one year of treatment. These data suggest that cyclic nucleotides may participate in the pathophysiological processes of coeliac disease.     

Role of cyclic nucleotides in rapid platelet adhesion to collagen

Adhesion of human platelets to type I collagen under arterial flow conditions is extremely fast, being mediated primarily by the alpha 2 beta 1 integrin (glycoprotein Ia/IIa). We have investigated the involvement of cyclic nucleotides in platelet adhesion to soluble native collagen immobilized on Sepharose beads using a new microadhesion assay under arterial flow conditions. To prevent platelet stimulation by thromboxanes and adenosine diphosphate (ADP), experiments were performed with aspirin-treated platelets in the presence of ADP-removing enzyme systems such as creatine phosphate/creatine phosphokinase or apyrase. Rapid reciprocal changes in platelet adenosine 3'5'-cyclic monophosphate (cAMP) and guanosine 3'5'-cyclic monophosphate (cGMP) occurred during adhesion. cAMP levels in adherent platelets were 2.4-fold lower than in effluent platelets or in static controls, whereas cGMP levels were increased 2.4-fold. These results suggest that contact between platelets and collagen stimulates guanylate cyclase and inhibits adenylate cyclase. This occurs in the absence of the platelet release reaction. We also studied short-term effects of agents that regulate cyclic nucleotide synthesis, prostaglandin E1 (PGE1) and sodium nitroprusside (SNP). After only 3.8 seconds at 10 to 30 dyne/cm2, PGE1 (10 mumol/L) increased cAMP 16.4- fold, whereas SNP (50 mumol/L) increased cGMP ninefold and caused a 3.2- fold increase in cAMP. Both PGE1 and SNP rapidly (< 5 seconds) inhibited platelet adhesion in a dose-dependent manner that was correlated with the increase in cyclic nucleotides. Our data suggest that cAMP and cGMP play a regulatory role in the initial phases of platelet adhesion to collagen mediated by the alpha 2 beta 1 integrin receptor.     

Insulin increases cyclic nucleotide content in human vascular smooth muscle cells: a mechanism potentially involved in insulin-induced modulation of vascular tone

Summary It has been suggested that insulin exerts a vasodilating effect, but the mechanisms involved are not completely understood. Since cyclic nucleotides mediate the vasodilation induced by endogenous substances, such as prostacyclin and nitric oxide, we aimed to investigate the influence of insulin (concentration range 240–960 pmol/l) on both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) content in human vascular smooth muscle cells. Insulin dose-dependently increased both nucleotides (cAMP: from 0.7±0.1 to 2.6±0.4 pmol/10⁶ cells, p=0.0001; cGMP: from 1.3±0.2 to 3.4±0.7 pmol/10⁶ cells, p=0.033). This increase is receptor-mediated, since it was blunted when cells were preincubated with the tyrosine kinase inhibitor genistein. The effect of insulin remained significant (p=0.0001) when preincubation with the phosphodiesterase inhibitor theophylline prevented cyclic nucleotide catabolism. The increase of cGMP was blunted when the cells were preincubated with the guanylate cyclase inhibitor methylene blue, and with the nitric oxide-synthase inhibitor N^G-monomethyl-l-arginine. At all the concentrations tested, insulin potentiated the increase of cAMP induced by the stable prostacyclin analogue Iloprost (p=0.0001), whereas only at 1920 pmol/l did it potentiate the cGMP increase induced by glyceryltrinitrate (p=0.05). This study demonstrates that the vasodilating effects exerted by insulin may at least in part be attributable to an increase of both cGMP and cAMP via a receptor-mediated activation of adenylate and guanylate cyclases in human vascular smooth muscle cells and that the insulin effect on cGMP is mediated by nitric oxide.Abbreviations cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - PDE phosphodiesterases - NO nitric oxide - hVSMC human vascular smooth muscle cells - l-NMMA N^G-monomethyl-l-arginine - GTN glyceryltrinitrate - BSA bovine serum albumin - NIDDM non-insulin-dependent diabetes mellitus - MEM minimal essential medium - RIA radioimmunoassay     

Cyclic GMP phosphodiesterase and guanylate cyclase activities in rabbit ovaries and the effect of in-vivo stimulation with LH

The activities of guanylate cyclase and cyclic GMP (cGMP) phosphodiesterase, enzymes that are responsible for maintaining tissue levels of cGMP, were determined in the ovaries of rabbits killed without treatment or 4 h after administration of LH. Ovarian activities of the two enzymes were determined in the 100 000 g supernatant fraction (cytosol) and the resulting pellet (particulate fraction). Significant phosphodiesterase and cyclase activities were detected in both the cytosol and particulate fractions. Administration of LH had no significant effect on phosphodiesterase activity in either of the tissue fractions. On the other hand, LH caused a significant drop in guanylate cyclase activity in the cytosol and particulate fractions. This drop in the cyclase activity may be the cause of the decreased rabbit ovarian concentrations of cGMP that we have previously observed after LH stimulation.     

Regulation of hepatic nuclear guanylate cyclase.

In immunohistochemical studies of rat liver tissue slices and purified nuclei, adenosine 3':5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP) immunofluorescence on the nuclear membrane are sequentially increased after glucagon administration. An explanation for the increased cGMP immunofluorescence was sought in experiments in which guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2]activity of hepatic subcellular fractions was determined. The results showed that a nuclear guanylate cyclase exists which can be distinguished from the soluble and crude particulate guanylate cyclases. The activity of the nuclear enzyme was increased by 35% in nuclei isolated from rats 30 min after glucagon injection, the time at which maximal nuclear membrane cGMP immunofluorescence is observed. Because glucagon altered both cAMP location and levels prior to the observed changes in nuclear cGMP metabolism, the hypothesis that cAMP acted as the second messenger was tested. In vitro incubation of nuclei isolated from control rats with 10(-5) M cAMP produced a 25% increase in nuclear guanylate cyclase activity. With nuclei isolated from glucagon-treated rats, no significant increase in enzyme activity was observed; this indicates that maximal stimulation of nuclear guanylate cyclase by cAMP occurred at levels that are obtained in vivo after glucagon administration. These findings suggest that hepatic nuclear cGMP content may be regulated by a specific organelle guanylate cyclase and that cAMP may be one of the determinants of this enzyme's activity.     

Immunocytology on microwave-fixed cells reveals rapid and agonist-specific changes in subcellular accumulation patterns for cAMP or cGMP.

We developed a method for cAMP and cGMP immunocytology based upon fixation by microwave irradiation. Fixation by microwave irradiation prevented three problems found with other fixation methods: nucleotide loss from cells, nucleotide diffusion within cells, and chemical modification of immunologic epitopes. Six agonists (four that stimulate adenylate cyclase and two that stimulate guanylate cyclase) produced cAMP or cGMP accumulation patterns that were agonist-specific, dose-dependent, detectable at physiologic concentrations of hormone, and time-dependent within 15 sec to 30 min. cAMP accumulation after 1 mM forskolin was greatest in the nucleus. Isoproterenol, prostaglandin E2, or calcitonin caused initial accumulation of cAMP along the plasma membrane, but later accumulation was greater in the cytoplasm. With calcitonin the later accumulation of cAMP was selectively perinuclear and along the nuclear membrane. Sodium nitroprusside stimulated cGMP accumulation diffusely throughout the cytoplasm. Atrial natriuretic peptide initiated cGMP accumulation near the plasma membrane, and cGMP accumulation moved from there into the cytoplasm. In conclusion, microwave irradiation preserved cell structure and allowed visualization of expected as well as unsuspected changes in intracellular accumulation patterns of cAMP and cGMP.     

Insulin influences the nitric oxide cyclic nucleotide pathway in cultured human smooth muscle cells from corpus cavernosum by rapidly activating a constitutive nitric oxide synthase

AIMS: We have evaluated, in cultured human cavernosal smooth muscle cells, the expression and activity of calcium-dependent constitutive nitric oxide synthase (cNOS) and the ability of insulin to induce nitric oxide (NO) production and to increase intracellular cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). METHODS: cNOS mRNA was detected by RT-PCR amplification, cNOS protein by immunofluorescence, cNOS activity as l-[3H]-citrulline production from l-[3H]-arginine and cyclic nucleotides by radioimmunoassay. RESULTS: cNOS mRNA and cNOS protein were found in cultured cells; cNOS activity was increased by 5-min exposure to 1 micro mol/l calcium ionophore ionomycin (from 0.1094+/-0.0229 to 0.2685+/-0.0560 pmol/min per mg cell protein, P=0.011) and to 2 nmol/l insulin (from 0.1214+/-0.0149 to 0.2045+/-0.0290 pmol/min per mg cell protein, P=0.041). Insulin increased both cGMP and cAMP in a dose- and time-dependent manner (i.e. with 2 nmol/l insulin, cGMP rose from 2.71+/-0.10 to 6.80+/-0.40 pmol/10(6) cells at 30 min, P=0.0001; cAMP from 1.26+/-0.06 to 3.02+/-0.30 pmol/10(6) cells at 60 min, P=0.0001). NOS inhibitor N(G)-monomethyl-l-arginine and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 blunted these effects of insulin. The action of insulin on cyclic nucleotides persisted in the presence of phosphodiesterase inhibition, guanylate cyclase activation by NO donors and adenylate cyclase activation by Iloprost or forskolin. CONCLUSION: Human cavernosal smooth muscle cells, by expressing cNOS activity, are a source of NO and not only its target; in these cells, insulin rapidly activates cNOS through a PI 3-kinase pathway, with a consequent increase of both cyclic nucleotides, thus directly influencing the mechanisms involved in penile vascular tone and interplaying with classical haemodynamic mediators.     

Cyclic nucleotides and the regulation of canine gastric acid secretion

The response of the cyclic nucleotide system (cAMP, cGMP, adenylate cyclase, guanylate cyclase, and specific phosphodiesterases) to two gastric acid secretagogues, histamine and acetylcholine, and two secretory inhibitors, prostaglandin E₂ and secretin, was studiedin vivo andin vitro in canine gastric fundic mucosa. Histamine and acetylcholinein vivo failed to stimulate cAMP but significantly increased cGMP;in vitro they affected neither adenylate cyclase nor guanylate cyclase. Prostaglandin E₂ and secretin, however, increased cAMPin vivo and significantly stimulated adenylate cyclasein vitro. Specific phosphodiesterases were unaffected by these compounds. The changes, while not specifically localized to the acid-producing cells, are consistent with the suggestion that the control of canine gastric acid secretion may be mediated by changes in mucosal cAMP and cGMP.Presented in part at Digestive Diseases Week, May 25, 1977, Toronto, Canada.This study was supported in part by a grant from the John A. Hartford Foundation, Inc.     

Adenosine-mediated stimulation of bone cell adenylate cyclase activity.

Adenosine rapidly stimulated adenylate cyclase activity but did not modify cyclic AMP degradation when added to a particulate fraction prepared from isolated bone cells. The effect of adenosine was one-half maximal at 5-10 micronM, and was not mimicked by 5' AMP, inosine, guanosine, uridine, adenine, or ribose. Basal and adenosine-stimulated adenylate cyclase activites were directly proportional to the concentration of particulate protein in the assay system. Theophylline decreased the degree to which adenosine stimulated adenylate cyclase activity, whereas another phosphodiesterase inhibitor, RO-20-1724, failed to iiinfluence the effect of adenosine. Adenosine itself, and not a metabolite of adenosine is the stimulator of adenylate cyclase, since it was neither phosphorylated nor deaminated appreciably by the particulate fraction. The particulate fraction did not convert substrate ATP to adenosine in amounts sufficient to enhance adenylate cyclase. The stimulatory effect of adenosine was maximal at 1.2 mM Mg2+, declined with increases in the Mg2+ concentration, and was replaced by inhibition at 20 mM Mg2+. At 2.4 mM Mg2+, basal adenylate cyclase activity peaked at 1.1 mM ATP, and was inhibited by higher ATP concentrations. The magnitude of adenosine stimulation was greater at inhibitory concentrations of ATP than at concentrations which yielded maximum activity. The results suggest that the previously demonstrated ability of adenosine to increase cyclic 3'5' AMP levels in intact bone cells stems from its effect on adenylate cyclase. Adenosine may act by modifying the regulatory nfluence of free Mg2+, uncomplexed ATP, (or both), on adenylate cyclase. Theophylline appears to interfere with the action of adenosine by a mechanism which is distinct from its capacity to inhibit cyclic AMP phosphodiesterase activity. (Endocrinology 99:901,1976)     

Sodium nitroprusside inhibition of parathyroid hormone release is not mediated through cyclic GMP

Sodium nitroprusside effected a rapid, dose-dependent increase in intracellular cGMP accumulation in freshly dispersed bovine parathyroid cells. The effect was half-maximal between 10(-4) and 3 X 10(-4)M, maximal at 3 X 10(-3)M nitroprusside and could be amplified (approximately 50%) by the addition of methylisobutylxanthine (4 X 10(-4)M). The dose-response characteristics were similar to those previously described for the inhibition of cAMP accumulation and PTH release by this agent. Neither dibutyryl cGMP (10(-3)M) nor 8'-bromo-cGMP (10(-3)M) mimicked the inhibitory effect of nitroprusside on cAMP accumulation or PTH release. Dose-dependent stimulation of guanylate cyclase was found in a particulate preparation of parathyroid cells; activity was maximal at 10(-4)M nitroprusside while higher concentrations appeared to inhibit the enzyme. Nitroprusside significantly reduced both (-)isoproterenol and guanine nucleotide-stimulated adenylate cyclase activity in the particulate preparation with maximum inhibition between 10(-3)-10(-2)M. cGMP concentrations as high as 10(-4)M did not affect agonist-stimulated cAMP synthesis. Thus, although the kinetic and dose-response characteristics of the nitroprusside effect on cGMP suggest a linkage to its previously described effects on cAMP and PTH secretion, no direct evidence was found to indicate a causal relationship between the two. Rather it would appear that the effects on the adenylate and guanylate cyclase enzymes occur in parallel, possibly the result of some common primary perturbation of cellular physiology.     

Role of cyclic nucleotides in fetal hemoglobin induction in cultured CD34+ cells

OBJECTIVE: In vivo, several drugs have been shown to increase fetal hemoglobin (HbF), including 5-azacytidine (AZA), sodium butyrate (SB), and hydroxyurea (HU). Studies in K562 cells suggest that cyclic guanosine monophosphate (cGMP) is required for HbF induction; however, the role of cyclic nucleotides in HbF induction in primary erythroid cultures has not been established. METHODS: CD34-selected peripheral blood monocytes cultured in a semi-solid serum-free system that mimics in vivo F-cell production are utilized to explore the role of cyclic adenosine monophosphate (cAMP) and cGMP in HbF induction in response to HU, AZA, and SB. RESULTS: In serum-free CD34 cultures, HU, SB, and AZA all markedly stimulate FNRBC production up to 30-fold, associated with induction of gamma-globin mRNA and total HbF protein. Guanylate cyclase inhibition results in only minimal blunting of HbF induction by each agent. In contrast, adenylate cyclase inhibition markedly reduces HU, SB, and AZA-mediated FNRBC induction and gamma-globin mRNA induction. The adenylate cyclase activator forskolin modestly induces FNRBC production and augments the action of standard induction agents. HU, AZA, and SB, however, fail to significantly stimulate adenylate cyclase themselves. CONCLUSIONS: In human CD34(+) cultures, cAMP production is required for full induction of HbF by HU, SB, and AZA, while perturbation of cGMP production has only minimal effects. These findings are in marked contrast to data in K562 cells where cGMP production is critical for HbF induction while cAMP stimulation blunts HbF response, and suggest that these agents may share a common induction pathway.     

Forskolin increases cellular cyclic adenosine monophosphate content and parathyroid hormone release in dispersed bovine parathyroid cells

We studied the effects of the positive-inotropic and hypotensive agent forskolin on parathyroid hormone (PTH) release, cAMP accumulation, and adenylate cyclase activity in dispersed bovine parathyroid cells. Forskolin stimulated PTH release 1.7 to 3.7-fold at 10(-5) mol/L and increased cAMP content maximally 37.6-fold at 10(-4) mol/L. Forskolin induced a maximal increase in cAMP accumulation by 5 minutes of incubation. The rate of PTH release in forskolin-treated cells also reached maximal levels at five to ten minutes of incubation. There was a dose-dependent increase in PTH release and cAMP content over the range of 10(-7) to 10(-4) M forskolin with half-maximal stimulation at 10(-7) to 10(-6) mol/L for PTH release and between 10(-5) and 10(-4) mol/L for cAMP content. PTH release and cAMP accumulation were inhibited by increasing extracellular calcium concentrations in both control cells and those incubated with 10(-5) mol/L forskolin. Forskolin stimulated adenylate cyclase activity in lysates from parathyroid cells 15.7-fold with half-maximal activation between 10(-6) and 10(-5) mol/L forskolin. Forskolin-stimulated (10(-5) mol/L) and basal adenylate cyclase activities declined in parallel with increasing calcium concentrations. In contrast, phosphodiesterase activity was unaffected by forskolin at 10(-6) to 10(-4) mol/L. Forskolin, therefore, by directly activating adenylate cyclase, enhances cAMP accumulation and PTH release in dispersed bovine parathyroid cells. As with other agents activating cAMP accumulation in this system, these effects are inhibited by elevated calcium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinergic control of cyclic nucleotide metabolism in human thyroid cells

In the presence of Ro 20-1724, a selective inhibitor of cyclic nucleotide phosphodiesterase, carbamylcholine increases cAMP and cGMP levels in human thyroid cells in primary culture. The increase of cAMP exhibited at concentrations of carbamylcholine between 10 fM and 10 pM, is dose- and time-dependent, it is maximum after 30 min and is abolished after 60 min. At higher carbamylcholine concentration (10 microM), cAMP increases rapidly, becoming maximum after 15 min, but returns to unstimulated values after 30 min. The increase of cGMP is also dose-dependent (0.1 nM-10 microM); it reaches the maximum after 30 min and returns to unstimulated values after 120 min. A significant increase of phosphodiesterase activity is observed at 10 microM carbamylcholine. Atropine, a muscarinic receptor antagonist, blocks carbamylcholine effects on both cAMP and cGMP production without affecting the thyrotropin-induced cAMP accumulation. Hexamethonium, a nicotinic receptor antagonist does not affect the cholinergic effects. In the presence of Ro 20-1724, 10 microM carbamylcholine significantly inhibits the effect of thyrotropin on cAMP production, while the combined addition of low doses of carbamylcholine and thyrotropin (0.1 nM and 10 pM, respectively) results in an additive effect on cAMP levels. Inhibition of thyrotropin activity on cAMP production, similar to that exerted by 10 microM carbamylcholine is produced by increasing free intracellular calcium; this inhibition is relieved by using a calmodulin-sensitive phosphodiesterase inhibitor, M and B 22948 at 50 microM dose. High concentrations (10 microM) of carbamylcholine increase the adenylate cyclase activity, without any significant effect on the thyrotropin-induced activation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide metabolism in compensatory renal hypertrophy and neonatal kidney growth.

Cyclic nucleotide metabolism was investigated in growing kidneys of rats during compensatory hypertrophy and during neonatal development. After unilateral nephrectomy a mild and short-lasting decrease in cyclic 3':5" adenosine monophosphate (cAMP) was observed in the hypertrophying kidney. In contrast, cyclic 3':5' guanosine monophosphate (cGMP) showed a sharp decline to 20% of control at 15 min and a rapid rise to 200-300% above base-line at 1-72 hr. The alterations in renal tissue levels of cGMP were associated with parallel changes in the soluble, 100,000 X g supernatant guanylate cyclase activity [GTP pyrophosphate-lyase (cyclizing): EC 4.6.1.2]. No change was observed in total cGMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase; EC 3.1.4.17). In the rapidly growing kidney of newborn rats cAMP levels were 983 +/- 65 and 833 +/- 42 pmol/g of kidney at 4 and 7 days after birth, and increased to adult levels (1518 +/- 57 pmol/g) at 21 days whereas cGMP levels were 59.8 +/- 6.8 and 92.5 +/- 13.9 pmol/g at 4 and 7 days and decreased to adult levels (36 +/- 1.5) at 21 days. The results indicate that compensatory renal hypertrophy and neonatal kidney growth are associated with changes in cAMP and cGMP metabolism.     

Role of nitric oxide/cyclic GMP and cyclic AMP in beta3 adrenoceptor-chronotropic response

In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects.