Stimulation of ovarian cyclic guanosine 3',5'-monophosphate levels by the subunits of human chorionic gonadotropin

Injection of human chorionic gonadotropin (HCG) into the tail vein of superovulated rats resulted in a significant (P<0.01) increase in peripheral plasma progesterone without a concomitant increase in ovarian cyclic GMP (cGMP) levels. However, when equimolar quantities of α and β subunits of HCG were injected, a significant increase in plasma progesterone was accompanied by a concomitant and significant (P<0.01) increase in ovarian cGMP levels. The observation that these subunits increase ovarian cGMP levels without increasing cAMP suggests the possibility of cGMP involvement in steroidogenesis induced by subunits.     

Human chorionic gonadotropin affects tissue levels of progesterone and cyclic adenosine 3',5'-monophosphate in the metestrus rat uterus in vitro

The effect of human chorionic gonadotropin (hCG) on in vitro progesterone (P) level in the uterus was investigated by using short-term incubations of uterine tissue taken from 4-day cyclic rats at different stages of the estrous cycle. Control incubations resulted in a decrease of endogenous P content in uteri removed from rats in proestrus (PRO), estrus (EST), and metestrus (MET): -25% (n = 6), -60% (n = 6), and -45% (n = 8), respectively. The amount of P found after incubation of MET tissue in the presence of hCG was significantly higher (p less than 0.001) than that found after control incubations. The hCG effect was dose-dependent and was not observed with PRO or EST tissue. Although the mean P level found in MET tissue after incubation with 10 IU/ml hCG was not significantly different from the mean level found in unincubated tissue (1562 +/- 341 vs. 1470 +/- 174 pg/mg protein, n = 8), an obvious synthesis was observed in two experiments. It thus seems likely that observed hCG effect would involve a de novo P synthesis rather than a decrease of P catabolism. Furthermore, hCG induced a dose-dependent increase of cyclic adenosine 3', 5'-monophosphate (cAMP) uterine levels in MET tissue. N,O'-dibutyryl cyclic AMP [Bu)2 cAMP) at 5 mM induced a significative increase (p less than 0.01) of P uterine level in EST and MET tissue compared to control incubations, but had no effect on PRO tissue. Our results suggest the progressive maturation throughout the rat estrous cycle of a luteinizing hormone/hCG- and cAMP-dependent process able to regulate uterine P content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that arginine vasotocin inhibits human chorionic gonadotropin and cyclic adenosine 3',5' monophosphate stimulated ovarian steroidogenesis

Lyngbyatoxin A, isolated from a marine blue-green alga, and dihydroteleocidin B, a hydrogenated derivative of teleocidin, induce ornithine decarboxylase in mouse skin. In addition, dihydroteleocidin B was recently shown to be a potent tumor promoter in mouse skin. The present studies demonstrate that both lyngbyatoxin A and dihydroteleocidin B induce increased prostaglandin release and choline turnover in HeLa cells at concentrations of 6–20 ng/ml, with a time course similar to that of the potent phorbol ester tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. Thus these indole alkaloids, although structurally different from phorbol ester tumor promoters, share several properties with the latter compounds.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

Two novel stimuli of cyclic adenosine 3',5'-monophosphate (cAMP) in human lymphocytes.

Polystyrene latex particles (PLP) and zymosan particles (ZP), two commonly employed phagocytic stimuli, were noted to bind to purified human peripheral blood lymphocytes. This interaction was not accompained by ingestion but did lead to a marked increase in intracellular cyclic AMP. The cAMP response to PLP was proportional to the particle cell ratio which, in turn, correlated with the number of membrane-associated particles. After the addition of PLP to lymphocytes, the cAMP response occurred within 2 min, peaked between 4 and 15 min, and returned to baseline by 30 to 60 min. The cAMP response to ZP was similar in onset and duration to that seen with PLP but was less marked (2- to 4-fold vs 25- to 50-fold) and more variable in magnitude. This is probably a reflection of the smaller number of cells interacting with ZP. At high PLP to cell ratios almost all of the lymphocytes bound PLP but only 10 to 28% of the mixed lymphocyte population bound ZP. Two lines of evidence established conclusively that the cAMP response was taking place in the lymphocytes themselves rather than in contaminating cells. 1) When lymphocytes were purified additionally by filtration through a nylon wool column (99 to 100% lymphocytes), they were found to undergo a similar cAMP response to PLP. Since the nylon filtration procedure also removes almost all of the B cells, this further indicates that T cells are capable of undergoing the response. 2) Immunofluorescence studies with anti-cAMP antibody revealed an increase in intralymphocytic cAMP which was primarily adjacent to the site of PLP or ZP attachment. The likely explanation of this data is that PLP and ZP perturb the lymphocyte surface leading to regional activation of membrane-bound adenylate cyclase and subsequent cAMP accumulation. Although the physiologic significance of these observations remains to be determined, the results: 1) provide histologic confirmation for the concept of cAMP compartmentablization, 2) clarify conflicting results regarding the localization of cAMP accumulation during the phagocytosis of PLP by mixed leukocyte populations, and 3) suggest that this experimental system may allow an analysis of the mechanism by which perturbations of the lymphocyte surface modulate cAMP.     

Regulation of dimorphism in Histoplasma capsulatum by cyclic adenosine 3',5'-monophosphate.

During temperature-induced transition of the dimorphic pathogenic fungus Histoplasma capsulatum from the single yeast cell form to the multicellular mycelial form, there was an increase in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels as well as a striking accumulation of cAMP in the medium. cAMP levels also changed during the reverse mycelium-to-yeast transition.