Journal of cyclic nucleotide and protein phosphorylation research最新文献

Isoproterenol, prostaglandin E1 and forskolin-stimulated cyclic AMP accumulation were compared in intact lymphocytes obtained from nine monozygotic and nine sib pairs matched for age and sex. Heritability was calculated by three different methods, two based on the intraclass correlation coefficients and one based directly on the variances. Only for forskolin is a significant proportion of variance (0.68-0.91) attributable to genetic factors, suggesting that forskolin-stimulated activity may prove to be a valuable genetic marker in studies of human pathology. Neither basal nor isoproterenol and prostaglandin E1-stimulated activity show significant heritability in intact human lymphocytes. The individual differences observed in levels of beta-adrenergic and prostaglandin stimulated receptor activity in human lymphocytes are, therefore, most likely due to environmental factors.

Helical strips of rat aorta contracted with norepinephrine (10 nM) respond to ultraviolet (UV) radiation (340-360 nm) with a transient decrease in tension. The UV radiation-dependent relaxation is completely reversible and endothelial cell-independent. Although decreased tension is associated with a rise in tissue cGMP content, the cAMP level is unchanged after UV radiation. A significant inhibition of phosphorylase a formation which occurs coincidently with the rise in cGMP and decline in tension is observed with UV radiation. The effects of UV radiation on cGMP, phosphorylase a formation and tension were blocked by methylene blue. Relaxation and inhibition of phosphorylase a formation in response to UV radiation were also partially reversed with higher concentrations of norepinephrine (100 nM). Our results suggest that cGMP may mediate UV radiation-dependent reactions by reducing cytoplasmic Ca2+.

Pretreatment of either intact wild type S49 lymphoma cells (WT) or the uncoupled variants, cyc-, H21a, or UNC with epinephrine results in the redistribution of 20-30% of the beta-adrenergic receptors into a light vesicle fraction in sucrose gradients. Since the variants are uncoupled with respect to hormonal stimulation of adenylate cyclase, it appears that productive interaction with Gs is not required for the sequestration of beta-adrenergic receptors. Characterization of the epinephrine-induced redistribution of the beta-adrenergic receptor has revealed the following: The EC50 for the redistribution in WT cells was between 100 and 200 nM. Pretreatment of WT cells with 50 nM epinephrine for 30 min induced only a slight redistribution of receptors in sucrose gradients but produced a significant desensitization of adenylate cyclase. The desensitization was characterized by an increase in the Kact of epinephrine stimulation of adenylate cyclase while the Vmax was unaltered. Pretreatment with 10 microM epinephrine resulted in a significant decrease in the Vmax (50%) of epinephrine stimulation of adenylate cyclase and a 3-fold increase in Kact in the heavy vesicles. The beta-receptors in the light vesicle fraction of WT were uncoupled from adenylate cyclase and displayed low affinity for epinephrine binding, comparable to the cyc-. The "desensitized" receptor in the light vesicle fractions of cyc- was capable of stimulating adenylate following reconstitution with cholate extracts of WT membranes containing Gs. The molecular weight of the photolabeled beta-receptor in the light vesicle fractions (65,000 +/- 2,000) was not significantly different from the Mr 65,000 polypeptide photolabeled in the heavy fractions. The Mr 55,000 beta-receptor polypeptide was not detected in the light vesicles. Our results suggest first that the redistribution of the beta-receptor into light vesicles may follow an earlier stage of desensitization, and second that the beta-receptor in light vesicles while sequestered from Gs is capable of activating adenylate cyclase.

A phosphorylation occurs at two sites in chicken gizzard myosin light chain kinase that appears to be catalyzed by an autophosphorylation reaction. This reaction is inhibited by approximately 75% in the presence of Ca2+-calmodulin, but is unaffected by the cAMP-dependent protein kinase inhibitor. Whereas the catalytic subunit of cAMP-dependent protein kinase phosphorylates myosin light chain kinase at only serine residues, the non cAMP-dependent phosphorylation occurs at both serine and threonine residues. One, if not both, of these latter sites are distinct from the sites recognized by the catalytic subunit of cAMP-dependent protein kinase. Consequently, there must be at least three and possibly four sites in myosin light chain kinase capable of incorporating phosphate, either in response to catalytic subunit or by autophosphorylation.

Previous reports have described hormone-stimulated release of radiolabeled guanine nucleotides from prelabeled membranes. In this paper, we report the inhibition by GDP of both hormone-independent and hormone-specific stimulation of [3H]Gpp(NH)p release from human platelet membranes. This inhibition was shown to be concentration specific and suggests a more complex mechanism of [3H]Gpp(NH)p release than was previously thought. A model is proposed in which guanosin triphosphates cause dissociation of the subunits of the guanine nucleotide binding protein, whereas guanosine diphosphate prevents this dissociation resulting respectively in stimulation or inhibition of the release of bound [3H]Gpp(NH)p.

Forskolin stimulated adenylate cyclase activity 55-fold in crude rat pancreatic plasma membranes. Dose-response curves were better fitted by a two-component model with apparent Ka for forskolin of 0.8 microM and 85 microM corresponding, respectively, to 15% and 85% of total activity. Gpp (NH)p alone or the combined presence of GTP plus a hormone (secretin, VIP or CCK-8) potentiated activation through the high affinity forskolin component. These results are in favour of a dual mode of action of forskolin: a high affinity component related to the stimulatory guanine nucleotide-binding regulatory subunit, and a low affinity component more closely related to the catalytic subunit of the enzyme. In dispersed rat pancreatic acini, forskolin increased cyclic AMP levels 26-fold and potentiated the increase induced by secretin, VIP, and CCK-8. It also stimulated the phosphorylation of three particulate proteins (Mr = 21K, 25K and 33K). In terms of secretion, it raised amylase secretion by 60%, a weak effect comparable to that exerted by VIP but much lower than that of secretin or CCK-8. Forskolin did, however, potentiate the secretory effect of CCK-8 (a hormone inducing a redistribution of cellular calcium) while being without influence on the secretory effects of secretin and VIP.

Prostaglandin D2, 2-chloroadenosine, forskolin and combinations of these agents increase cyclic AMP-levels in intact human platelets. The inhibition of activated cyclic AMP-generating systems by 1) alpha2-adrenergic receptor-mediated hormonal input (norepinephrine), 2) a P-site agent (2',5'-dideoxyadenosine) and 3) a divalent cation (calcium) were examined: 1) Norepinephrine produces non-competitive inhibition of both forskolin and prostaglandin D2(PGD2)-stimulated cyclic AMP-accumulation in intact human platelets. The Ki values for norepinephrine versus forskolin, PGD2 and 2-chloroadenosine are similar in magnitude, while the Ki versus a forskolin-PGD2 combination is approximately 10-fold greater. Onset of inhibition by norepinephrine of the PGD2-response is several fold faster than for the forskolin-response. When platelets stimulated by the forskolin and PGD2 combination are exposed to norepinephrine, there is a transient increase in levels of cyclic AMP due to the potentiation of a minor beta-adrenergic component. This stimulation is followed by inhibition. 2) 2',5'-Dideoxyadenosine produces a non-competitive inhibition of the forskolin-response with a Ki of 110 microM. The inhibition of the PGD2-response by 2',5'-dideoxyadenosine is competitive with a Ki of 6-13 microM, while inhibition of the forskolin-PGD2 response has a Ki of 30 microM. Onset of inhibition by 2',5'-dideoxyadenosine is identical for forskolin or PGD2-stimulated platelets. There is a lag period for inhibition of platelets stimulated with the forskolin-PGD2 combination. The PGD2-forskolin combination appears to stabilize the cyclic AMP-generating system of platelets against inhibition by either norepinephrine or 2',5'-dideoxyadenosine. 3) Calcium ions cause a similar inhibition of cyclic AMP-generation in intact platelets, regardless of the type of stimulation.

Two isoenzymes (type I and type II) of cAMP-dependent protein kinases were found in GH3 cytosol, isozyme type II activity being the predominant form (approximately 90%). Photoaffinity labeling of GH3 cell extracts with 8-N3-[32P]cAMP revealed three cAMP-binding proteins exhibiting molecular weights of 53,000, 51,000 and 48,000, respectively. The latter represents the regulatory subunit of type I isoenzyme whereas the 53,000 and 51,000 cAMP-binding proteins correspond to two different molecular forms of the type II isoenzyme regulatory subunit which are phosphorylated by a cAMP-dependent mechanism.

In order to facilitate the in situ localization of cyclic nucleotides, a large supply of high-titer anti-sera for immunohistologic analyses would be valuable. Therefore, monoclonal antibodies (Mab) directed at 2'-0-succinyl cyclic GMP (s-cGMP) or 2'-0-succinyl cyclic AMP (s-cAMP) were generated. Immunized mice developed polyclonal antibodies of high titer to either cyclic nucleotide as reflected in an enzyme-linked immunosorbent assay (ELISA) employed to monitor these anti-sera. With the use of standard hybridoma technology and ELISA to monitor anti-cyclic nucleotide antibody production from positive clones, several Mab-producing clones were grown to large volume in tissue culture and ascites fluid. The anti-cGMP and anti-cAMP anti-sera, which demonstrated significant ELISA titers at greater than 1:2,000,000 dilutions, are monospecific and can be employed for radioimmunoassays, ELISA, or immunohistologic localization of cyclic nucleotides in situ.

The regulation of adenylate cyclase by muscarinic cholinergic receptors has been studied in rat heart membranes. In the presence of isoproterenol the K0.5 for Mg2+-mediated activation of adenylate cyclase was 0.1 mM; the addition of the muscarinic receptor agonist oxotremorine increased the K0.5 for Mg2+, and activation by Mg2+ no longer followed mass action kinetics for a single site interaction. The extent of oxotremorine-mediated attenuation of adenylate cyclase exhibited a Mg2+ concentration dependence: in short-time assays at 0.25 mM free Mg2+ the attenuation of enzyme activity was 55 percent, whereas at 20mM Mg2+, only 20 percent inhibition was observed. The increase in the apparent K0.5 for Mg2+ in the presence of oxotremorine was less in 20 min assays than in 4 min assays. The effects of oxotremorine on the rates of activation and deactivation of adenylate cyclase also were examined. Oxotremorine increased the rate of deactivation of adenylate cyclase by two-fold. At low free Mg2+ concentrations, oxotremorine also decreased the rate of activation of adenylate cyclase in the presence of either 0.1 uM GTP gamma S or 1 uM GTP. An oxotremorine-mediated decrease in the rate of activation of adenylate cyclase activity was not detectable in the presence of greater than 3 mM Mg2+.