Paul Basset

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.

Transcriptional Induction of Stromelysin-3 in Mesodermal Cells Is Mediated by an Upstream CCAAT/Enhancer-binding Protein Element Associated with a DNase I-hypersensitive Site

Stromelysin-3 (ST3) is a matrix metalloproteinase whose synthesis is markedly increased in stromal fibroblasts of most invasive human carcinomas. In the present study, we have investigated the molecular mechanisms by which high levels of ST3 expression can be induced. In contrast to the early and transient induction of interstitial collagenase by 12-O-tetradecanoylphorbol-13-acetate (TPA), the fibroblastic induction of ST3 was found to be delayed and to require protein neosynthesis. We demonstrated that this induction is transcriptional and does not result from changes in RNA stability. By looking next to promoter regions accessible to DNase I upon gene induction, we have identified two distal elements and have characterized their role in the transcriptional regulation of ST3. The first one is a TPA-responsive element that controls the base-line ST3 promoter activity but is not required for its activation. We demonstrate that ST3 gene induction is actually mediated by the second element, a C/EBP-binding site, by showing: (i) that this element becomes accessible in cells induced to express ST3, (ii) that endogenous C/EBPβ binds to the ST3 promoter, and (iii) that this binding leads to ST3 transcriptional activation. Our study provides new insights into the regulation of ST3 and suggests an additional role for C/EBP transcription factors in tissue remodeling processes associated with this MMP.

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.

Neuronal and glial localization of guanylate cyclase. Immunohistochemical evidence in cultured cells

A study has been carried out on the localization of guanylate cyclase employing cultured brain cells. Guanylate cyclase has been found to be located in neurons as well as in glial cells. This has been supported by the immunohistochemical as well as biochemical data.

Antigenic conservation of brain guanylate cyclase during evolution

A comparative study of brain guanylate cyclase from different animal species (including man, bird, fish and amphibian) has been performed using a specific antibody directed against soluble rat brain guanylate cyclase. Analyses were performed on supernatant fractions by the double-immunodiffusion test, by the protein blotting technique after SDS-polyacrylamide gel electrophoresis and by analytical isoelectric focusing on agarose allowing specific immunodetection of isoelectric patterns. Membrane-bound guanylate cyclase from rat brain and soluble guanylate cyclase from several rat tissues cross-reacted with the antibody. All the brain enzymes tested were found to be identical by double-immunodiffusion. The electrophoretic and isoelectrophoretic profiles of the different brain guanylate cyclases were found to exhibit many common features with some differences between mammalian and non-mammalian enzymes. In human brain, guanylate cyclase has been localized in glial and neuronal cells by immunohistochemistry. The results demonstrate that guanylate cyclase has been well conserved during the course of evolution and are consistent with the involvement of guanylate cyclase and cyclic GMP in basic cellular function.

Choline acetyltransferase aggregates from human placenta and rat brain

Choline acetyltransferase (EC 2.3.1.6., ChAcT) is the enzyme which synthesizes acetylcholine (ACh) from choline and acetyl-CoA. Animal species may have one or more forms of ChAcT showing different pH values, ranging from pI 5-8.5 [l-4]. In rat [4] and human [2] brains the presence of an acidic form of ChAcT (pI<7.1 and 5 S, respectively) was thought of being artifactual and possibly due to binding of the enzyme with other proteins. Moreover, in the rat brain [4], three basic forms of ChAcT were shown to have different affinities for membranes depending on the ionic concentration of the medium and/or pH. ChAcT has been purified from different animal sources in several laboratories [5-141 and some authors have claimed to have obtained a monospecific antibody to their purified preparation [ 15,161. There is however much debate concerning the validity of such antibodies [ 17,181 and the enzymatic forms of ChAcT [ 111. Two bands of proteins have been obtained on SDS-polyacrylamide gels, [I 1,12,19] one of which is not pure enzyme in the opinion of the same authors. Others [ 16,201 have found several bands on SDS-~gels and have explained this result as due to different subunits and/or polymers of ChAcT. Our group is presently involved in the purification of ChAcT from human placenta (preliminary reports have already appeared [2 1,221) and in the course of

Guanylate Cyclase Activators Hemin and Sodium Nitroprusside Stimulate the Growth of Transformed Cells in Serum-Free Medium

Hemin and sodium nitroprusside, which strongly activate purified guanylate cycclase in vitro, were also found to stimulate leukemic, glioma and neuroblastoma cells to divide in serum-free medium. The involvement of guanylate cyclase activation in the proliferative response to hemin and sodium nitroprusside is discussed.