Cyclase

About: Cyclase is a research topic. Over the lifetime, 10162 publications have been published within this topic receiving 388566 citations.

Forskolin activation of adenylate cyclase in vivo stimulates nerve regeneration

Abstract: The previous demonstration of an increase and redistribution of adenylate cyclase activity in injured peripheral nerve1 suggests that an increase in neuronal cyclic AMP concentration could play a role in peripheral nerve regeneration. We report our finding that accumulating adenylate cyclase activity was translated into a twofold increase in cyclic AMP concentration in the regenerating nerve stump, coincident with the initiation and elongation of regenerative nerve sprouts. We sought to magnify the role of cyclic AMP in regeneration by using forskolin, a robust activator of adenylate cyclase2, to produce an additional increase in neuronal cyclic AMP in situ. Forskolin in vitro produced an approximately 40-fold greater elevation in neuronal cyclic AMP than an equimolar (10−5) concentration of isoprenaline. Moreover, the elevated cyclic AMP concentration persisted for at least 60 min in the continued presence of forskolin. Daily injection of forskolin into the dorsal lymph sac of Rana pipiens, or delivery of forskolin through an implanted osmotic pump produced a sustained 40% increase in the rate of sensory nerve regeneration in freeze-lesioned sciatic nerves. We conclude that an increase in cyclic AMP concentration and, presumably, the activation of appropriate protein kinases stimulates regenerative nerve growth following trauma.

Functional Characterization of the Recombinant Human 5-Hydroxytryptamine7(a) Receptor Isoform Coupled to Adenylate Cyclase Stimulation

Abstract: Functional characterization of the recombinant human 5-hydroxytryptamine7(a) (h5-HT7(a)) receptor isoform was performed using stably transfected LM(tk−) cells. Expression levels of the h5-HT7(a) receptor determined from saturation studies using either a labeled agonist ([3H]5-HT) or antagonist ([3H]LSD) were very similar (Bmax = 160–190 fmol/mg protein), suggesting that all receptors may exist in the high affinity (G protein-coupled) state. In intact cells, 5-HT produced a concentration-dependent elevation of intracellular cAMP levels ([cAMP]i) with an EC50 value of 80 nM and a maximal response of 5-fold increase above basal levels. The rank order of agonist potencies in the second messenger assay paralleled their rank order of binding affinities: 5-carboxamidotryptamine > 5-hydroxytryptamine ≥ 5-methoxytryptamine > 8-hydroxy N,N-dipropyl aminotetralin > sumatriptan. Agonist potencies (EC50 values) to stimulate [cAMP]i were more than 25-fold lower relative to their respective binding affinities ( K i values) obtained in [3H]5-HT competition assays. In contrast, antagonist potencies ( K b values) to block 5-HT-stimulated [cAMP]i were in close agreement with their corresponding K i values. These data may indicate low efficiency of receptor-effector coupling to adenylate cyclase stimulation. Pretreatment of stably transfected cells with cholera toxin abolished the 5-HT-mediated elevation of [cAMP]i, indicating that the 5-HT7(a) subtype directly interacts with Gαs protein(s) to activate adenylate cyclase(s). Clonal cell lines stably expressing h5-HT7 receptor isoforms will serve as valuable cellular models to study their function and regulation, as well as assist in the development of selective 5-HT7 receptor agents to uncover the biological roles and potential therapeutic applications of this novel receptor subtype.

Modification of calmodulin function by enzymatic carboxyl methylation

Abstract: Calmodulin is a ubiquitous calcium-binding protein that regulates a variety of enzymes such as adenylate cyclase, cyclic nucleotide phosphodiesterase, ATPase and protein kinase (for review see ref. 1). So far, no enzymatic modification of calmodulin has been shown to affect its function. Another ubiquitous protein, the enzyme protein-carboxyl methylase (PCM), modifies proteins post-translationally by methylating their free carboxyl groups and thus neutralizing negative charges2–6. This enzyme is one of three elements of the protein-carboxyl methylation system; the two others are the substrates, the methyl acceptor proteins (MAP) and the demethylating enzyme, protein methylesterase (PME)7. We report here that calmodulin is an exceptionally good substrate for PCM and that the enzymatic post-translational methylation of calmodulin inhibits its stimulatory effect on cyclic nucleotide phosphodiesterase. Furthermore, we present evidence that carboxyl methylation of calmodulin occurs in intact cells.

Modulation by monoamines of somatostatin-sensitive adenylate cyclase on neuronal and glial cells from the mouse brain in primary cultures

Abstract: : Primary cultures of mouse embryonic neuronal or glial cells from the cerebral cortex, striatum, and mesencephalon were used to identify and determine the cellular localization of somatostatin receptors coupled to an adenylate cyclase. Somatostatin inhibited basal adenylate cyclase activity on neuronal but not on glial crude membranes in the three structures examined. The somatostatin-inhibitory effect on neuronal crude membranes was still observed in the presence of (—)-isoproterenol, 3,4-dihydroxyphenylethylamine (dopamine, DA), or 5-hydroxytryptamine (5-HT, serotonin) used at a concentration (10−5M) inducing maximal adenylate cyclase activation. In addition, in most cases biogenic amines modified the pattern of the somatostatin-inhibitory effect, triggering either an increase in the peptide apparent affinity for its receptors or an increase in the maximal reduction of adenylate cyclase activity or both. However, 5-HT did not modify the somatostatin-inhibitory response on striatal and cortical neuronal crude membranes. The changes in somatostatin-inhibitory responses were interpreted as a colocalization of the amine and the peptide receptors on subtypes of neuronal cell populations. Finally, somatostatin was shown to inhibit adenylate cyclase activity following its activation by (—)-isoproterenol on glial crude membranes of the striatum and the mesencephalon but not on those of the cerebral cortex.

Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium.

Abstract: Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cells containing normal activities of the phosphotransferase system enzymes. In contrast, phosphoenolpyruvate could not overcome the inhibitory effect of this sugar in strains deficient for enzyme I or HPr. Although the in vivo sensitivity of adenylate cyclase to inhibition correlated with sensitivity of carbohydrate permease function to inhibition in most strains studied, a few mutant strains were isolated in which sensitivity of carbohydrate uptake to inhibition was lost and sensitivity of adenylate cyclase to regulation was retained. These results are consistent with the conclusions that adenylate cyclase and the carbohydrate permeases were regulated by a common mechanism involving phosphorylation of a cellular constituent by the phosphotransferase system, but that bacterial cells possess mechanisms for selectively uncoupling carbohydrate transport from regulation.