J. Zwiller

Evidence for phosphorylation of rat brain guanylate cyclase by cyclic AMP-dependent protein kinase

Abstract Direct phosphorylation of purified rat brain guanylate cyclase by cyclic AMP-dependent protein kinase is demonstrated. In the presence of [γ- 32 P]ATP, 32 P was incorporated into the protein to the extent of 0.8 to 0.9 mol/mol of guanylate cyclase. The presence of 32 P in the guanylate cyclase molecule was demonstrated by gel-filtration and by autoradiography after gel electrophoresis. The phosphorylation was accompanied by an increase in enzyme activity, characterized by an increase of V M . These results suggest that the activity of guanylate cyclase may be regulated in vivo by phosphorylation.

Rat brain guanylate cyclase. Purification, amphiphilic properties and immunological characterization

Soluble guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) has been purified to apparent homogeneity from rat brain by chromatography on Blue-Sepharose CL-6B, precipitation with (NH4)2SO4, preparative isoelectric focusing and gel-filtration on Ultrogel AcA-34. On sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis the purified enzyme showed a single band with an apparent molecular weight 59 000, when stored in buffer without glycerol and 2-mercaptoethanol. Purified enzyme has been found to be very unstable; inactivation can however be partially reversed by an endogenous heat-stable activator fraction. A monospecific antiserum obtained by immunization of rabbits was found to precipitate guanylate cyclase. This antibody also reacted with membrane-bound enzyme, indicating a close similarity to the soluble enzyme. Metal divalent cations were in general found to be strong inhibitors of the enzyme activity, though Ca2+ had no effect. ATP, CTP or UTP were shown to be competitive inhibitors of purified guanylate cyclase. Sodium nitroprusside increased cyclic GMP formation by the purified enzyme. Lysophosphatidylcholine and oleic acid, at low concentration, activated guanylate cyclase. Other unsaturated fatty acids, particularly arachidonic acid, dramatically inhibited the enzyme activity. Lipids may regulate the enzyme activity by binding to an apolar domain, as suggested by charge-shift electrophoresis. The mechanism by which guanylate cyclase is regulated in the cell appears to be a complex phenomenon. It may occur through oxidative reductive processes, and/or depend on other effectors, such as triphospho-nucleotides, divalent cations and lipid microenvironment.

Separation from protein kinase C: a calcium-independent TPA-activated phosphorylating system

A calcium‐independent but 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐ or diacylglycerol‐activated phospholipid‐dependent phosphorylating activity has been separated from protein kinase C. This has been made possible by employing calcium‐dependent hydrophobic interaction chromatography. The material bound to phenyl‐Sepharose in the presence of calcium at low ionic strength was eluted with EGTA and was protein kinase C. While the unbound material passing through the phenyl‐Sepharose column showed no appreciable protein kinase C activity, instead it had a high phosphorylating activity manifested in the absence of calcium and in the presence of TPA plus phospholipid. The identification of this phosphorylating activity, distinct from protein kinase C, leads to important clues to cellular responses monitored by TPA in the absence of calcium.

CYCLIC GMP IN A NEUROBLASTOMA CLONE: POSSIBLE INVOLVEMENT IN MORPHOLOGICAL DIFFERENTIATION INDUCED BY DIBUTYRYL CYCLIC AMP

Abstract— DBcAMP induces morphological differentiation of mouse neuroblastoma cells grown in culture. DBcGMP or 8‐Br cyclic GMP when added alone also induces a discrete morphological differentiation. When analogues of cyclic GMP were added together with dBcAMP, neurite outgrowth was strikingly enhanced as compared to the effect of dBcAMP alone. Intracellular concentrations of cyclic GMP were increased during dBcAMP treatment and cyclic AMP levels were increased during 8‐Br cyclic GMP treatment. Both treatments produced an increased protein kinase activity, supporting the possibility that not only cyclic AMP but also cyclic GMP may be involved in the differentiation process.

Activation of Brain Guanylate Cyclase by Phospholipase A2

Abstract: Various pure snake venom phospholipases A2 were used for studying their effect on guanylate cyclase activity. All the phospholipases A2 tested were found to activate guanylate cyclase from a rat brain homogenate. It was shown that particulate guanylate cyclase was especially affected. Intact glial cells incubated in presence of phospholipase A2 showed also an increased guanylate cyclase activity, demonstrating that the phospholipase effect, observed in disrupted cells, occurs also at the cellular level. These results suggest that in intact cells membrane‐bound phospholipase A2 activity could be involved in the modulation of the cellular cyclic GMP content.

Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons

The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(1-24) in the presence of phosphodiesterase inhibitor isobutylmethylxanthine resulted in a sustained increase in cyclic AMP while rise in cyclic GMP level was transient. The values obtained for half-maximal stimulation were 0.5 microM and 0.03 nM for cyclic AMP and cyclic GMP respectively. Concomitantly, ACTH(1-24) stimulated guanylate cyclase activity (half-maximal stimulation at 0.02 nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action of ACTH in neurons.

Vulnerability to opiate intake in maternally deprived rats: implication of MeCP2 and of histone acetylation

We previously showed that maternal deprivation predisposes male rats to anxiety, accompanied with an increase in their opiate consumption. In the present report, we searched for brain epigenetic mechanisms that possibly underlie this increase. For that, we examined the expression of the methyl‐CpG‐binding protein MeCP2 and of the histone deacetylases HDAC2 and HDAC3, as well as the acetylation status of histone H3 and H4 in mesolimbic structures of adult maternally deprived rats, using immunohistochemistry and Western blot analysis. A long‐lasting increase in MeCP2 expression was found throughout the striatum of deprived rats. Enhanced HDAC2 expression and increased nuclear HDAC activity in the nucleus accumbens of deprived rats were associated with lower acetylation levels of histone H3 and H4. Treatment for 3 weeks with the HDAC inhibitor sodium valproate abolished HDAC activation together with the decrease in the acetylation levels of histone H4, and was accompanied with normalized oral morphine consumption. The data indicate that epigenetic mechanisms induced by early adverse environment memorize life experience to trigger greater opiate vulnerability during adult life. They suggest that sodium valproate may lessen vulnerability to opiate intake, particularly in subgroups of individuals subjected to adverse postnatal environments.

Opposite effects of arachidonic acid and of its hydroperoxides on brain soluble guanylate cyclase activity

Purified soluble guanylate cyclase from rat brain was found to be directly activated by arachidonic acid. Arachidonic acid hydroperoxides, obtained by the action of lipoxygenase, were inhibitory. An inhibition of soluble guanylate cyclase and a decrease of cyclic GMP content were also observed in pure neuronal cultures treated with lipoxygenase. The results suggest that lipoxygenase products are not involved in the production of cyclic GMP in neurons. This is in contrast with other tissues for which it is established that hormone-induced guanylate cyclase activation is regulated by lipoxygenase-catalysed metabolism of polyunsaturated fatty acids.

Guanylate cyclase activators hemin and sodium nitroprusside stimulate cell growth in serum-free medium

Abstract Hemin and sodium nitroprusside, which strongly activate purified rat brain guanylate cyclase in vitro, were also found to stimulate glioma C6 and neuroblastoma M1 and N1E-115 cells to divide in serum-free medium. Hemin and sodium nitroprusside each stimulate C6 cell growth to a comparable extent. Sodium nitroprusside was less potent than hemin for inducing growth of neuroblastoma cells. Moreover, both agents when added together caused a synergic cell growth enhancement which is comparable to the synergism observed in their guanylate cyclase stimulation in vitro. These results suggest that activation of guanylate cyclase may play a role in the proliferative response to these compounds.

Nature and Immunochemical Characteristics of a Ca2+/Calmodulin Kinase Activity Endowed in a Highly Insoluble Protein Purified from Adult Rat Brain

Using sequential extraction procedure of proteins from adult rat forebrain, a protein of Mr 52,000, insoluble in neutral detergents, capable of binding calmodulin in the presence of Ca2+, was isolated. Antibodies to this antigen had the capacity to inhibit the Ca2+/calmodulin‐dependent kinase activity associated with this protein. This protein (52K) (in many respects identical to the major protein of postsynaptic densities) shares by itself the Ca2+/calmodulin‐dependent kinase activity, thus differing from soluble Ca2+/calmodulin‐dependent kinases isolated by others. Despite its insolubility in most detergents, the 52K protein is not particularly rich in hydrophobic amino acids. Its richness in cysteine and proline residues suggests that the active conformation of the enzyme is sustained by numerous disulfide bridges.