Cyclic AMP stimulates dephosphorylation of specific proteins in intact S49 mouse lymphoma cells

Two-dimensional gel electrophoresis of proteins labeled with ³²P₁ in S49 mouse lymphoma cells revealed five phosphoproteins that were rapidly and reversibly dephosphorylated in response to elevation of cyclic AMP (cAMP). Under basal conditions, labeling of at least two of these proteins was limited by slow turnover of protein-bound phosphate. The rapid cAMP-mediated dephosphorylation of these species was attributable, therefore, to stimulation of a specific phosphoprotein phosphatase.     

Impact de l'échantillonnage sur la mésure des nucléotides adényliques (ATP,ADP, AMP) du microplancton

The influence of sampling and sample treatment upon adenylic nucleotide (ATP, ADP, AMP) content of microplankton is studied. Changes in light conditions during nigh-sampling and extracting do not induce significant variations, in the adenylic nucleotide content of microplankton or in energy charge values.The contribution of zooplankton (size up to 200 µm) to microplankton adenosine values can be neglected for inshore surface waters and traditional sample volumes (about one liter). This result can been explained by the low density of zooplankton in such a small sample volume and by differences in efficiency of the extracting method used.

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Impact de l'échantillonnage sur la mésure des nucléotides adényliques (ATP, ADP, AMP) du microplancton

     

Expression of A and B Types of Monoamine Oxidase in Differentiated Neuroblastoma Hybrid Cells

The total activities of monoamine oxidase (MAO) and the ratio of type B/type A activities were determined in mouse neuroblastoma N1E-115 cells, and in NX31T and NG108-15 hybrid cells derived from mouse neuroblastoma X rat sympathetic ganglion hybrid or mouse neuroblastoma X rat glioma hybrid cells. N1E-115 and NX31T cells possessed type A activities exclusively, although NG108-15 cells showed both type A (65-90%) and type B (10-35%) MAO activities. The activity of type A MAO in NX31T and N1E-115 cells was relatively constant during culturing periods in the presence or absence of dibutyryl cyclic AMP (Bt2cAMP), whereas total MAO activity and the ratio of type B MAO/type A MAO in NG108-15 cells increased as a function of culture periods. Prostaglandin E1 (PGE1) and theophylline, the best known combination to increase intracellular cyclic AMP content of NG108-15 cells, caused similar increases of MAO and of the type B/type A ratio in NG108-15 cells. The results suggest that MAO activity and expression of MAO B activity are regulated in NG108-15 cells in a cyclic AMP-dependent manner.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Glucocorticoids Potentiate the Prostaglandin E1-Mediated Cyclic AMP Formation by a Cultured Murine Neuroblastoma Clone

The effect of steroid hormones on the prostaglandin E1 (PGE1)-mediated cyclic AMP formation by murine neuroblastoma clone N1E-115 was studied. Dexamethasone at submicromolar concentrations and corticosterone at micromolar concentrations (steroids with glucocorticoid activity) were able to modify the PGE1-mediated response whereas testosterone, progesterone, and estradiol each at 10 microM had no effect. Glucocorticoids added to the culture medium of N1E-115 cells produced an increase in the maximal response to PGE1 only after long-term (greater than or equal to 4 h) incubation with the hormone. Inhibitors of protein and RNA synthesis blocked this effect of glucocorticoids. Basal activity of adenylate cyclase in treated cells was twofold higher than that in control cells, and this enzyme seemed to be the primary target for the hormone action, since the activity of 3':5'-cyclic AMP phosphodiesterase and the binding of [3H]PGE1 to its receptors were not altered by glucocorticoid treatment. Our results indicate that glucocorticoids modulate receptor-mediated responses in cells of neural origin through a mechanism that involves induction of protein synthesis.     

Stimulatory effects of human pancreatic polypeptide on rat pancreatic acini

The effect of human pancreatic polypeptide (HPP) on rat pancreatic acini has been studied. It was found that HPP stimulated amylase and lipase release from the acini. The secretory response of acini to HPP was dose-dependent in a sigmoidal fashion. Between 10(-9) M and 10(-8) M concentration of HPP there was a slow increase of enzyme release to about 40-60% over basal release. At concentrations of HPP above 10(-8) M there was a rapid increase of enzyme release, amounting to 4-6 times over basal release at 10(-6) M concentration of HPP. The potency of HPP compared to other secretagogues at 10(-7) M concentration was 45% of CCK, 60% of carbachol and 75% of secretin. HPP did not inhibit the effect of CCK, secretin and carbachol on amylase release. The amylase release stimulated by HPP was accompanied by an increase in 45Ca2+ efflux. Atropine or dibutyryl cyclic GMP did not influence the effect of HPP. It is concluded that HPP stimulates the release of enzymes from rat pancreatic acini and that Ca2+ may be a mediator for this secretion.     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Thiols and pancreatic beta-cell function: a review

In pancreatic islets insulin secretion in response to a variety of stimulators is sensitive to the redox state of extracellular and intracellular thiols. In this connection variations of plasma glutathione (GSH) may also be of importance. In the process of stimulus-secretion coupling, membrane thiols play an important role. One major localization of critical thiols appears to be related to the influx of calcium through the voltage-dependent channel. Other transmembranal ion movements and the cAMP system seem to be less sensitive to thiol oxidation than calcium influx via voltage-dependent Ca channels.     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of gonadotropin releasing hormone.     

Suppression of cyclic AMP-induced cell excitation in Dictyostelium discoideum by inhibitors of eicosanoid oxidation

Abstract Receptor-mediated stimulation of Dictyostelium cells by the aggregative chemoattractant cyclic AMP leads to a complex excitatory response resulting in chemotaxis and the synthesis and release of cyclic AMP as the relayed chemotactic signal. However, the mechanism of this stimulus-response coupling is not well understood. In this study, we show that a number of compounds, best known as inhibitors of cyclooxygenase activity in mammalian cells, prevent cyclic AMP receptor-mediated cell excitation and cyclic AMP accumulation in aggregation-competent Dictyostelium cells. These observations suggest that some eicosanoid-like compound(s) may be involved in stimulus-response coupling in this organism, as is the case in higher eukaryotic cells.