Direct Effects of Chronic Ethanol Exposure on β-Adrenergic and Adenosine-Sensitive Adenylate Cyclase Activities and Cyclic AMP Content in Primary Cerebellar Cultures

The direct effects of chronic ethanol exposure on adenylate cyclase activity and cyclic AMP content were investigated in primary cerebellar cultures. By morphological criteria these cultures mainly contain granule cells with some astrocytes, and each cell type appears to contain both beta-adrenergic and adenosine-sensitive adenylate cyclase systems. Chronic treatment of the primary cerebellar cultures with 120 mM ethanol for 6 days caused a reduction in the stimulation of cyclic AMP content by isoproterenol and by the adenosine analogue 2-chloroadenosine. Kinetic analysis indicated that the chronic ethanol treatment decreased maximal activation of adenylate cyclase, as well as increased the EC50 values for norepinephrine and 2-chloroadenosine. Activation of norepinephrine-stimulated adenylate cyclase activity by in vitro ethanol was significantly enhanced after the chronic ethanol exposure. However, the chronic treatment did not alter activation of the 2-chloroadenosine-stimulated enzyme by in vitro ethanol. A similar difference in the response to in vitro ethanol after the chronic treatment was observed when cyclic AMP content of the intact cells was measured. The present data indicate that chronic ethanol exposure causes a selective increase in the sensitivity of adenylate cyclase to ethanol in some brain cells and a more generalized desensitization of receptor-stimulated cyclic AMP production.     

Increased brain Na, K-ATPase activity of rats under stress

The activities of Na, K- and Mg-dependent ATPases were measured in crude synaptosomal fractions isolated from the rat brain gray matter. Prolonged (6 h) exposure to emotional painful stress stimulated Na, K-ATPase activity by 40% without affecting that of Mg-ATPase. Preliminary injection of the free radical scavenger ionol presented Na, K-ATPase activation, thus suggesting the involvement of lipid peroxidation initiated in brain tissues under stress in acceleration of NA-pump function. However, model studies with lipid peroxidation induced in vitro by an ascorbate-dependent system in a membranous suspension demonstrated an opposite effect, i. e. fast inhibition of Na, K-ATPase. Possible reasons for the different effects of lipid peroxidation in vivo under stress and on Na, K-ATPase activity in vitro are discussed. It is concluded that activation of Na K-ATPase is a mechanism which is responsible for acceleration of reflex conditioning and for the maintenance of the conditioned reflexes in stress-exposed animals.     

Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells

The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The beta-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of beta-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 degrees C was not associated with a change in anisotropy.     

Isolation of plasma membranes from bovine corpus luteum possessing adenylate cyclase, 125I-hCG binding and NaK-ATPase activities

Plasma membranes from bovine corpora lutea have been purified by sucrose density gradient centrifugation. The purified membranes, in addition to binding ¹²⁵I-hCG, also possess hCG-stimulated adenylate cyclase and NaK-ATPase. The relative purification of ¹²⁵I-hCG binding, adenylate cyclase and NaK-ATPase on the basis of the specific activities in the whole homogenate were 7.8, 6.4 and 2.6, respectively. The presence of both the hormone sensitive adenylate cyclase and ¹²⁵I-hCG binding activities suggest that these plasma membranes might possess the ‘receptor’ for gonadotropin.     

Isolation, purification and characterization of regulatory properties of adenylate cyclase from rabbit heart]

Rabbit heart membranes possessing the adenylate cyclase activity were isolated and purified by extraction with high ionic strength solutions and centrifugation in the sucrose density gradient. It was shown that the membranes are characterized by a high percentage of cholesterol (molar ratio cholesterol/phospholipids is 0.24) and an increased activity of Na, K-ATPase, which suggests the localization of adenylate cyclase in the sarcolemma. During centrifugation in the sucrose density gradient the activities of andenylate cyclase and Na,K-ATPase are not separated. Treatment of heart sarcolemma with a 0.3% solution of lubrol WX results in 10--20% solubilization of adenylate cyclase. Purification of the enzyme in the membrane fraction is accompanied by a decrease in the activity of phosphodiesterase; however, about 2% of the heart diesterase total activity cannot be removed from the sarcolemma even after its treatment with 0.3% lubrol WX. Epinephrine and NaF activate adenylate cyclase without changing the pH dependence of the enzyme. The alpha-adrenergic antagonist phentolamine has no effect on the adenylate cyclase activation by catecholamines, glucagon and histamine; the beta-adrenergic antagonist alprenolol competitively inhibits the effects of isoproterenol, epinephrine and norepinephrine, having no effect on the enzyme activation by glucagon and histamine. There is no competition between epinephrine, glucagon and histamine for the binding site of the hormone; however, there may occur a competition between the hormone receptors for the binding to the enzyme. A combined action of several hormones on the membranes results in the averaging of their individual activating effects. When the hormones were added one after another, the extent of adenylate cyclase activation corresponded to that induced by the first hormone; the activation was insensitive to the effect of the second hormone added. It is assumed that the outer membrane of myocardium cells contains a adenylate cyclase and three types of receptors, each being capable to interact with the same form of enzyme. The activity of adenylate cyclase is determined by the type of the receptor, to which it is bound and by the amount of the enzyme-receptor complex.     

The distribution of ATPase activities in purified transverse tubular membranes

Vesiculated fragments of transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes were purified from heterogeneous microsomal membrane fractions of chicken breast muscle by a modification of an iterative calcium-oxalate loading technique. The distribution of ATPase activities were determined for the TT and SR and were compared to enriched fractions of sarcolemma (SL) membranes. The TT membranes were characterized by high rates of magnesium-stimulated ATPase (Mg-ATPase) and 5′-nucleotidase activities but were virtually devoid of calcium-stimulated, magnesium-dependent ATPase (Ca,Mg-ATPase) activity. Moderate levels of a latent sodium and potassium-stimulated ATPase (Na,K-ATPase) were observed for TT membranes when unmasked with valinomycin and monensin. In contrast to the behavior of TT membranes, highly purified SR membranes displayed an active Ca,Mg-ATPase but negligible Na,K-ATPase, Mg-ATPase, and 5′-nucleotidase activities. High levels of Na,K-ATPase and 5′-nucleotidase activities were observed for SL membranes; however, the SL displayed no appreciable Ca,Mg-ATPase and Mg-ATPase activities. The lack of significant Mg-ATPase activity in the SR and SL fractions suggested that the Mg-ATPase was uniquely associated with the TT membranes. The TT Mg-ATPase was further characterized by its pH and temperature dependences, and its sensitivity to pharmacologic agents. The Mg-ATPase of the TT was insensitive to inhibition by sodium azide and oligomycin in concentrations shown to exert maximum inhibition on the F₁ ATPase of submitochondrial particles. The Mg-ATPase was also resistant to the effects of ouabain and orthovanadate in concentrations which abolished the Na,K-ATPase and Ca,Mg-ATPase activities of the SL and SR, respectively. The Mg-ATPase displayed temperature and pH optima (25 °C, pH 7.3) which were distinguishable from the Ca,Mg-ATPase (45 °, pH 7.0) of highly purified SR fractions but which were very similar to the temperature and pH dependencies of the mixed microsomal fractions (MMF) from which the TT membranes were derived. Similarities in the pH and temperature dependencies of the TT and MMF Mg-ATPases plus the absence of appreciable Mg-ATPase activity in highly purified SR membranes suggests that the “basic” Mg-ATPase often seen in crude SR fractions may originate from TT membrane contamination. The resistance of the TT Mg-ATPase to inhibition by the pharmacologic agents tested plus its unique temperature and pH dependences indicate that this ATPase is distinguishable from other ATPases and may, therefore, be of value as a specific biochemical marker for TT membranes.     

Role of Extracellular Adenosine in Ethanol-Induced Desensitization of Cyclic AMP Production

Abstract: The decrease in receptor-stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol-induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol-induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4-day exposure to ethanol decreased basal as well as 2-chloroadenosine- and forskolin-stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2-day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2-chloroadenosine- and forskolin-stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum-free defined media. The ethanol-induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24-h exposure of PC12 cells to 2-chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin-stimulated cyclic AMP levels were increased. Similar results were obtained after a 4-day exposure of PC12 cells to 2-chloroadenosine or 5'- N -ethylcarboxamido-adenosine. The present results indicate that the ethanol-induced decrease in receptor-stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.     

Differential Effect of w3 PUFA Supplementations on Na,K-ATPase and Mg-ATPase Activities: Possible Role of the Membrane w6/w3 Ratio

Several functional properties of Na,K-ATPase are strongly dependent on membrane fatty acid composition, but the underlying mechanism is still not well defined. We have studied the effects of two types of supplementations enriched in the w3 polyunsaturated fatty acids on the Na,K-ATPase and Mg-ATPase activities in sciatic nerve (SN) and red blood cells (RBC). Eight groups of rats, controls and diabetics, received a standard diet, supplemented or not with 30 or 60 mg/kg/day of docosahexaenoic acid (DHA) or with soybean for eight weeks. Diabetes induced significant decrease of Na,K-ATPase activity in SN (-23%) and RBC (-25%), without affecting Mg-ATPase activity. In RBC, soybean and DHA supplementations caused significant increases in Na,K-ATPase activity (in various range, +13% to +145%) in all groups, and in Mg-ATPase activity in control soybean (+65%), control and diabetic DHA high dose (+39%, +53%) and diabetic DHA low dose (+131%) groups. In SN, the soybean caused a significant decrease in Na,K-ATPase activity (-26%) and still more in the diabetic group (-53%). The DHA diet induced a slight decrease in activity in control groups, whilst during diabetes, at high dose, we noted an aggravation of this decrease (-36%). Mg-ATPase activity was not modified by supplementations except for the low dose of DHA where the activity was slightly decreased in the control group (-16%). The supplementations induced multiple tissue-specific modifications in the membrane fatty acid composition of RBC and of SN homogenates. Several specific correlations have been found between variations in fatty acids amounts and Na,K-ATPase activity in these tissues but only in RBC for Mg-ATPase activity. Indeed, we observed that the variations in Na,K-ATPase activity are positively and significantly correlated with changes in the omega6/omega3 ratio in SN as well as in RBC. These data clearly show, for the first time, that the diet could modulate the Na,K-ATPase activity via the omega6/omega3 ratio in the membranes. A similar correlation was observed with Mg-ATPase activity in RBC, suggesting also a dietary regulation of the enzyme; but for the SN, this activity might be regulated by a different omega6/omega3 ratio or by another pathway.     

Phospholipids and ATPase activities in the developing chick brain.

During the embryogenesis of chick brain an increase in the specific activities of Na,K- and Mg-ATPase takes place. We determined the quantities of the phospholipids phosphatidylserine, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and sphingomyelin in the developing chicken brain by quantitative thin-layer chromatography and found changes in their composition during the first 18 days after incubation. The increase in the Na,K-ATPase activity was proportional to the increase in phosphatidylserine and phosphatidylcholine; the increase in the Mg-ATPase activity was approximately proportional to the increase in phosphatidylethanolamine.     

Conditioned media from cultured cystic fibrosis fibroblasts inhibits Na/K ATPase activity

Conditioned culture media taken from fibroblast cell lines derived from skin biopsies of control or of patients with Cystic Fibrosis (CF) were incubated with membranes of rat submandibular glands. The Na/K - ATPase activity of these membranes was inhibited when treated with CF-media, including both ouabain sensitive and insensitive activities. However, the membrane associated Mg-ATPase, Ca-ATPase, and both basal and hormone-stimulated adenylate cyclase activities were relatively unaffected. Thus, a factor or factors produced by CF-fibroblasts was shown to be active in a cell-free system derived from an exocrine gland.     

The influence of changes in the phospholipid pattern of intact fibroblasts on the activities of four membrane-bound enzymes.

Human skin fibroblasts, grown to confluency in the presence of 32P for random labelling of the phospholipids, showed upon 24 h incubation in the presence of either 8 mM L-serine or 4 mM ethanolamine an increased content of phosphatidylserine (150% of control cells) or phosphatidylethanolamine (116% of control cells), respectively. Concomitantly the phosphatidylcholine correspondingly decreased. Upon cell harvesting and gentle enzyme preparation the base-treated cells demonstrated a significantly higher unstimulated, fluoride- and thyrotropin-stimulated activity of adenylate cyclase. The activities of total ATPase, ouabain-sensitive ATPase, 5'-nucleotidase and gamma-glutamyltransferase remained unaltered. When subjecting enzyme preparations from fibroblasts to ultrasonication the activity of adenylate cyclase decreased progressively with energy applied, whereas the activities of the other enzymes were unaltered ((K+ + Na+)-ATPase, 5'-nucleotidase) or even increased (Mg2+-ATPase, gamma-glutamyltransferase). The results have a bearing upon the regulatory function of the phospholipid microenvironment of membrane-bound enzymes.     

Adrenergic receptors and adenylate cyclase activity in hepatocytes of the streptozotocin-diabetic rat.

Effects of short and long exposure to the diabetic state induced by an injection of streptozotocin to young female rats on glucagon- and catecholamine-sensitive adenylate cyclase activity and adrenergic receptors of hepatic membranes have been studied. The short period of exposure to the diabetic state exhibited an increase in the sensitivity of the enzyme to isoproterenol without changes in the affinity and the number of beta-adrenergic receptors. The increased response of adenylate cyclase activity to isoproterenol was accompanied with a greater GTP-induced lowering of the affinity to the beta-adrenergic agonist in diabetic membranes than in the controls. The chronic diabetic state produced a decrease in the adenylate cyclase activity to hormonal or non-hormonal stimuli with a fall in the number of alpha- and beta-adrenergic receptors. These results suggest that the observed effects of the diabetic state on hormonally sensitive adenylate cyclase activities and their receptor binding sites of the hepatic membranes would vary depending on the duration and/or severity of the diabetic state experimentally induced.     

Ethanol and guanine nucleotide binding proteins: a selective interaction

Guanine nucleotide binding proteins (G proteins) play key roles in signal transduction, including the coupling of hormone and neurotransmitter receptors to adenylate cyclase, ion channels, and polyphosphoinositide metabolism. One member of this family of proteins, Gs, appears to represent a specific site of action of ethanol in the central nervous system. Ethanol is often perceived as a nonspecific drug, and its anesthetic effects may in fact arise from relatively nonspecific interactions with cell membrane lipids. However, recent investigations point to a selective effect of low concentrations of ethanol to promote the activation of Gs, and thus to enhance adenylate cyclase activity. Ethanol seems to have little or no effect on the function of other identified G proteins. After chronic ingestion of ethanol by animals, or chronic exposure of cells in culture to ethanol, the sensitivity of adenylate cyclase to stimulation by guanine nucleotides and agonists that act via Gs is decreased. The mechanism of this change may involve qualitative and/or quantitative alterations in Gs, and seems to vary in different cell types. Studies of human platelets and lymphocytes also reveal differences in adenylate cyclase activity between alcoholics and control subjects. The differences are consistent with involvement of Gs, and do not appear to reverse upon cessation of alcohol exposure. The results suggest that the platelet and/or lymphocyte adenylate cyclase system may provide a biochemical marker of genetic predisposition to alcoholism.     

Ethanol Does Not Modify Opiate-Mediated Inhibition of Striatal Adenylate Cyclase

Ethanol increases the activity of "basal," guanine nucleotide- and dopamine-stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D-Ala2,D-Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor-coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.     

Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Effects of NaCl concentration on adenylate cyclase regulation by prostaglandins and guanine nucleotides

Na+ has been implicated as a requirement for the inhibition of adenylate cyclase by hormones and neurotransmitters. This study examines effects of salt concentration on neuroblastoma plasma membranes that occur in the absence of an inhibitory hormone. The adenylate cyclase response to stimulatory agonists (GTP plus PGE1 (3), PGI2 or PGE2) was influenced by NaCl. As the [NaCl] increased to 150 mM, an increase in maximal activity and a decrease in apparent affinity was observed. At concentrations above 150 mM, NaCl decreased prostaglandin affinity and progressively decreased maximal activation. The GTP requirement was not altered by 30 or 150 mM NaCl in the presence of PGE1 or PGI2. The rate of Gpp(NH)p stimulated activity increased as the [NaCl] was increased in the assay. This increased rate was conserved when membranes activated in the presence of Gpp(NH)p and NaCl were reassayed in the absence of guanine nucleotide or salt. The salt evoked rate increase was proportionally greater at submaximal MgCl2 concentrations. The concentration requirement for Mg2+ was reduced by salt for adenylate cyclase in the presence of GTP or Gpp(NH)p. However, the enzyme stimulated by hormone exhibited a Mg2+ requirement that was low in the absence of salt and could not be further reduced by increased [NaCl]. Alternative monovalent cations (150 mM Li+, K+, Cs+, but not choline or tetramethylammonium) and anions (SO4=) substituted for NaCl. The observed effects were reversible upon washing the membranes and neither ouabain nor tetrodotoxin altered the response. These effects may result from a conformational alteration of a protein particularly sensitive to neutral salts in the assay.     

Inhibition of gonadotropin-stimulated adenylate cyclase by dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA)

Protease inhibitors are known to suppress basal, fluoride-, and hormone-stimulated adenylate cyclase activities. The thrombin inhibitor, dansyl-arginyl-(4'-ethyl)piperidine amide (DAPA), also specifically inhibits the binding of gonadotropins to their receptors. Our studies were undertaken to find a concentration of DAPA that would specifically inhibit gonadotropin-stimulated adenylate cyclase without significantly altering basal, fluoride-, isoproterenol-, or prostaglandin E1-stimulated cyclase. Basal adenylate cyclase activity was not inhibited by DAPA in either human chorionic gonadotropin (hCG)- or follicle-stimulating hormone (FSH)-responsive rat ovarian plasma membranes. Human chorionic gonadotropin-stimulated cyclase was completely inhibited by DAPA at a concentration of 2.96 mM; the ID50 was 1.32 mM. Follicle-stimulating hormone-stimulated cyclase was completely inhibited by a DAPA concentration of 4.44 mM, and the ID50 was 1.75 mM. Dansyl-arginyl-(4'-ethyl)piperidine amide (2.96 mM) inhibited isoproterenol-, prostaglandin E1-, and fluoride-stimulated cyclase in hCG-responsive membranes by 11%, 28%, and 35%, respectively. Dansyl-arginyl-(4'-ethyl)piperidine amide (4.44 mM) inhibited fluoride- and prostaglandin-stimulated cyclase in FSH-responsive membranes by 10% and 11%, respectively. The data show that appropriate concentrations of DAPA can antagonize gonadotropin-stimulated adenylate cyclase while only minimally affecting fluoride- and other receptor-activated cyclase activities.     

Altered Pattern of Na,K-ATPase Activity and mRNA During Chronic Alcohol Consumption by Juvenile and Adolescent Rats

The effect of chronic ethanol consumption on cerebral cortical activity of Na,K-ATPase was determined in Long-Evans (LE) rats fed an ethanol-containing diet beginning at different stages of development. Na,K-ATPase activity was operationally resolved into α1 and α2/3 isozyme activities. There was no significant difference in Na,K-ATPase activities before and after alcohol consumption in the preparations from adult rats. However, for rats beginning alcohol consumption as adolescents, the α2/3 activity was significantly elevated following chronic alcohol consumption. Both LE and Sprague–Dawley rats showed this same selective increase in cortical α2/3 activity when rats began alcohol consumption as juveniles. The shift in cortical α2/3 activity was not observed in cerebellum or subcortical forebrain and was reversible when rats were fed ethanol throughout the normal adolescent period and then withdrawn and tested 2 weeks later (during the adult period). Levels of isoform-specific mRNA were determined in preparations of cerebral cortices of rats showing elevated α2/3 isozyme activities. In these preparations, isoform specific α2 and α3 mRNA was significantly elevated. There was no effect of ethanol feeding on cortical α1 mRNA. These findings indicate that the longer term effects of ethanol on the developing brain include elevated Na,K-ATPase activity and a mechanism that is pre-translational and isoform specific.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Protein kinase C stimulates adenylate cyclase activity in prolactin-secreting rat adenoma (GH4C1) pituicytes by inactivating the inhibitory GTP-binding protein Gi

The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and thyroliberin exerted additive stimulatory effects on prolactin release and synthesis in rat adenoma GH4C1 pituicytes in culture. Both TPA and thyroliberin activated the adenylate cyclase in broken cell membranes. When combined, the secretagogues displayed additive effects. TPA did not alter the time course (time lag) of adenylate cyclase activation by hormones, guanosine 5'-[beta,gamma-imino]triphosphate or forskolin, nor did it affect the enzyme's apparent affinity (basal, 7.2 mM; thyroliberin-enhanced, 2.2 mM) for free Mg2+. The TPA-mediated adenylate cyclase activation was entirely dependent on exogenously added guanosine triphosphate. ED50 (dose yielding half-maximal activation) was 60 microM. Access to free Ca2+ was necessary to express TPA activation of the enzyme, however, the presence of calmodulin was not mandatory. TPA-stimulated adenylate cyclase activity was abolished by the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, by the protein kinase C inhibitor polymyxin B and by pertussis toxin, while thyroliberin-sensitive adenylate cyclase remained unaffected. Experimental conditions known to translocate protein kinase C to the plasma membrane and without inducing adenylate cyclase desensitization, increased both basal and thyroliberin-stimulated enzyme activities, while absolute TPA-enhanced adenylate cyclase was maintained. Association of extracted GTP-binding inhibitory protein, Gi, from S49 cyc- murine lymphoma cells with GH4C1 cell membranes yielded a reduction of basal and hormone-stimulated adenylate cyclase activities, while net inhibition of the cyclase of somatostatin was dramatically enhanced. However, TPA restored completely basal and hormone-elicited adenylate cyclase activities in the Gi-enriched membranes. Finally, TPA completely abolished the somatostatin-induced inhibition of adenylate cyclase in both hybrid and non-hybrid membranes. These data suggest that, in GH4C1 cells, protein kinase C stimulation by phorbol esters completely inactivates the n alpha i subunit of the inhibitory GTP-binding protein, leaving the n beta subunit functionally intact. It can also be inferred that thyroliberin conveys its main effect on the adenylate cyclase through activation of the stimulatory GTP-binding protein, Gs.