Circadian variation of adrenocortical cyclic nucleotides (cyclic AMP and cyclic GMP) in hypophysectomized rats

Summary The existence of a circadian variation in the adrenocortical concentrations of cyclic AMP and cyclic GMP in male adult Wistar rats examined 10 days after hypophysectomy is demonstrated. The results suggest that the circadian variations of adrenocortical cyclic nucleotides observed previously in intact rats might not entirely depend upon pituitary corticotrophin.Acknowledgment. Thanks are due to Prof. J. M. Racadot and Dr Y. Drouet for their assistance with the histological examinations.     

Circadian variation of adrenocortical cyclic nucleotides (cyclic AMP and cyclic GMP) in hypophysectomized rats

The existence of a circadian variation in the adrenocortical concentrations of cyclic AMP and cyclic GMP in male adult Wistar rats examined 10 days after hypophysectomy is demonstrated. The results suggest that the circadian variations of adrenocortical cyclic nucleotides observed previously in intact rats might not entirely depend upon pituitary corticotrophin.     

A simplified assay for cyclic AMP using protein kinase binding

Summary The protein kinase binding assay for cAMP was modified by substitution of adsorption by QAE cellulose for the membrane filtration. This modification obviates the variation of recovery of cAMP with the volume of buffer used to wash the filter. The assay is reproducible and technically simpler than those currently employed.Acknowledgment. This work was partially supported by grants HL-16583 and HL-18827 from the National Heart and Lung Institute.     

Specific inhibition of a calcium dependent activation of brain cyclic AMP phosphodiesterase activity by vinblastine

Summary Vinblastine selectively inhibits the activation of brain cyclic AMP phosphodiesterase activity by Ca⁺⁺-protein activator (50% inhibition by 2×10^–5 M). This inhibitory effect was reversed by excessive amounts of the activator, whereas large quantities of Ca⁺⁺ caused only a slight suppression of the vinblastine effect. This result of vinblastine suggests a new site of its action and also suggests the possible role of protein activator, phosphodiesterase proteins or cyclic nucleotides in the previously known effects of vinblastine in vivo and in vitro.Our thanks to Drs A. R. Rhoads, C. B. Klee and R. A. Fergusson for analyzing PA preparations by gel electrophoresis and gel electrofocusing, and to Drs A. A. Suran and D. Soifer for their valuable critiques to this work. This work was partly supported by Biochemical Research Support Grant No. 5SO7RRO5361 to K.W. and Graduate Training Grant No. 5TO2GMO500002 to W.L.W. from NIH.     

Specific inhibition of a calcium dependent activation of brain cyclic AMP phosphodiesterase activity by vinblastine.

Vinblastine selectively inhibits the activation of brain cyclic AMP phosphodiesterase activity by Ca++-protein activator (50% inhibition by 2 x 10(-5) M). This inhibitory effect was reversed by excessive amounts of the activator, whereas large quantities of Ca++ caused only a slight suppression of the vinblastine effect. This result of vinblastine suggests a new site of its action and also suggests the possible role of protein activator, phosphodiesterase proteins or cyclic nucleotides in the previously known effects of vinblastine in vivo and in vitro.     

Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB

p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.     

Microinjection of synthetic protein kinase inhibitor into single barnacle muscle fibers before and after cyclic AMP

Single muscle fibers from the barnacle Balanus nubilus have been used as a preparation to see if a synthetic 20-residue PKI (5-24)-peptide is able to block or reverse the stimulatory response of the ouabain-insensitive Na efflux to injected cAMP. The results obtained show that this peptide behaves as a powerful inhibitor of the cAMP-mediated response and is also able to partially reverse the sustained stimulation of the Na efflux observed in ouabain-poisoned fibers following the injection of subunit A of cholera toxin.     

Microinjection of synthetic protein kinase inhibitor into single barnacle muscle fibers before and after cyclic AMP

Summary Single muscle fibers from the barnacleBalanus nubilus have been used as a preparation to see if a synthetic 20-residue PKI (5-24)-peptide is able to block or reverse the stimulatory response of the ouabain-insensitive Na efflux to injected cAMP. The results obtained show that this peptide behaves as a powerful inhibitor of the cAMP-mediated response and is also able to partially reverse the sustained stimulation of the Na efflux observed in ouabain-poisoned fibers following the injection of subunit A of cholera toxin.Warmest thanks to Prof. E. H. Fischer and Kurt Diltz for a sample of sPK1. This work received support from NSF, the Graduate School Research Committee and from NIH through a General Research Support Grant to the University of Wisconsin Medical School.     

Conversion of mammalian cyclic GMP-dependent protein kinase into modulator-dependent protein kinase (type II) in vitro

The spontaneous conversion of mammalian cyclic GMP-dependent protein kinase (G-PK) into modulator-dependent protein kinase (type II) (M-PKII) in the absence of cGMP or histone was observed in vitro. The findings, together with similarity in substrate protein specificity, suggest that M-PKII is the catalytic subunit of mammalian G-PK.     

Separation of modulator-dependent protein kinase (type I) from cyclic GMP-dependent protein kinase in mouse testes

A new type of enzyme, modulator-dependent protein kinase (type I) (M-PKI), was successfully isolated from the cytosol fraction of mouse testes. It was eluted slightly after the peak of cyclic GMP-dependent protein kinase (G-PK) by Sephadex G-200 gel filtration. Unlike either cyclic AMP-dependent protein Kinase (A-PK) or G-PK, its maximal activity depended exclusively on the presence of crude protein kinase modulators (PKM) or partially purified stimulatory modulator (PKMs).     

Intracellular cyclic AMP in vesicular stomatitis or Senda? virus infected cells (author's transl)

In KB cells, MRC5 and adult skin fibroblasts infected by low doses of Senda? virus, intracellular cyclic AMP levels rose and fell in the first hours following infection, then remained lower than basal level during at least 2 days in KB cells and adult skin fibroblasts. When compared to other viruses or cAMP inducers previously described, this effect appeared specific of Senda? virus. Mechanisms and roles of cAMP variations are discussed. VSV-infected KB cells showed slightly decreased cAMP levels during the first hours following infection.     

Cyclic AMP and cyclic GMP in rats paw edema by prostaglandins

Summary Experimental results indicate that rat's paw oedema development by PGs is inhibited by 35 AMP and increased by 35 GMP. This research confirms the opposite effect to that of cyclic nucleotides. The respective behaviour of 35 AMP and 35GMP in their primary oedema-induced activity is discussed.We gratefully acknowledge the technical assistence of Mr.Sauro Pellegrini.     

Inverse regulation of spore germination and growth by cyclic AMP inStreptomyces hygroscopicus

Summary The cyclic AMP level in germinating spores ofStreptomyces hygroscopicus rises to a maximum at outgrowth of germ tubes. Exogenous cyclic AMP results in an inverse effect on germination speed and growth.