Pertussis toxin abolishes the inhibitory effects of prostaglandins E1, E2, I2 and F2 alpha on hormone-induced cAMP accumulation in cultured hepatocytes

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.     

Effect of prostaglandins on glycogenesis and glycogenolysis in primary cultures of rat hepatocytes--a role of prostaglandin D2 in the liver

16,16-Dimethylprostaglandin E2 (dimethylPGE2) increased the incorporation of glucose into glycogen in rat hepatocytes in primary culture and its stimulatory effect was blocked by pretreatment of the cells with pertussis toxin. In contrast, dimethylPGE2, prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha), but not prostaglandin D2 (PGD2), inhibited glucose incorporation in insulin-induced glycogenesis, and these inhibitory effects were not blocked by pretreatment with pertussis toxin. Prostaglandins and other stimuli (lipopolysaccharide, platelet-activating factor, phorbol ester and zymosan) did not increase the release of [14C]glucose from [14C]glycogen-labeled hepatocytes. On the other hand, under identical conditions except for the presence of glucagon, isoproterenol (beta-adrenergic response) or epinephrine (with propranolol, alpha 1-adrenergic response), dimethylPGE2 and PGE2 inhibited hormone-stimulated glycogenolysis but again PGD2 had no effect.     

Effect of prostaglandins and their analogues on hormone-stimulated glycogenolysis in primary cultures of rat hepatocytes

Hepatocytes were isolated by collagenase perfusion method from adult male rats, cultured and then prelabeled with [14C]glucose. The [14C]glycogen-labeled cells were used in experiments for effect of prostaglandins on hormone-stimulated glycogenolysis. Prostaglandin E1, prostaglandin E2 and 16,16-dimethylprostaglandin E2, but not prostaglandin D2 or prostaglandin F2 alpha, inhibited glycogenolysis stimulated by glucagon, epinephrine, isoproterenol (beta-adrenergic agonist) or epinephrine in the presence of propranolol (beta-antagonist) in primary cultured hepatocytes. The inhibitory effects on day 2 of cultures were approx. twice those on day 1. Dimethylprostaglandin E2 (10(-6)M) caused 60-70% inhibitions of the stimulations by these substances. In the case of the stimulation by glucagon, the inhibition further increased by 80-100% on day 3 of culture. Prostaglandin E1 and prostaglandin E2 caused less inhibition than dimethylprostaglandin E2 of all these stimulations. Dinorprostaglandin E1 (9 alpha,13-dihydroxy-7-ketodinorprost-11-enoic acid), which is a hepatocyte-metabolite of prostaglandin E1 and prostaglandin E2, and arachidonic acid did not have any inhibitory effects. These data indicate that the E series of prostaglandins may function as the regulation of hepatic glycogenolysis stimulated by epinephrine and glucagon, and that their rapid degradation system may contribute to the modulation of the action in liver.     

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.     

Inhibition of glucagon-stimulated cAMP accumulation and fatty acid oxidation by E-series prostaglandins in isolated rat hepatocytes

E-series prostaglandins have been shown to inhibit hepatic glucagon-stimulated glycogenolysis without inhibiting glycogenolysis stimulated by cAMP analogs. In the present studies, prostaglandin E2 and 16,16-dimethylprostaglandin E2 inhibited glucagon-stimulated cAMP accumulation in isolated rat hepatocytes by 25% and 46%, respectively, without affecting basal cAMP levels. Half-maximal inhibition of glucagon-stimulated cAMP accumulation occurred at approx. 10(-7) M 16,16-dimethylprostaglandin E2. 16,16-Dimethylprostaglandin E2 inhibited glucagon-stimulated palmitate oxidation in intact hepatocytes without affecting basal rates of palmitate oxidation. 16,16-Dimethylprostaglandin E2 had no effect on palmitate oxidation in a liver homogenate system. These studies demonstrate that prostaglandin E antagonizes the effects of glucagon on hepatic metabolism by inhibiting glucagon-stimulated cAMP accumulation.     

Possible coupling of prostaglandin E2 receptor with pertussis toxin-sensitive guanine nucleotide-binding protein in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin E2 (PGE2) stimulated the formation of inositol phosphates in a dose-dependent manner in the range between 10 nM and 10 microM. Pertussis toxin inhibited the effect of PGE2 dose-dependently in the range between 1 ng/ml and 1 micrograms/ml. In the cell membranes, pertussis toxin catalyzed ADP-ribosylation of a protein with an Mr of about 40,000. Pretreatment of membranes with 10 microM PGE2 in the presence of 2.5 mM MgCl2 and 100 microM GTP markedly attenuated this pertussis toxin-catalyzed ADP-ribosylation of the protein in a time-dependent manner. G12 was detected in these cells by immunoblotting with purified anti-G12 alpha antibodies. The results indicate the possible coupling of PGE2 signalling with pertussis toxin-sensitive GTP-binding protein, which is probably G12, in osteoblast-like cells.     

Structural specificity for prostaglandin effects on hepatocyte glycogenolysis.

Prostaglandins (PGs) are known to have effects on hepatic glucose metabolism. Some actions of PGs in intact liver systems may not involve PG effects directly at the level of the hepatocyte. To define the ability of structurally distinct prostaglandins to affect hepatocyte metabolism directly, the regulation of glycogenolysis was studied in hepatocytes isolated from male Sprague-Dawley rats. PGF and PGB2 inhibited glucagon-stimulated glycogenolysis in the hepatocyte system. Pinane thromboxane A2 (PTA2) and PGD2 had no effect on glucagon-stimulated glycogenolysis. Consistent with their inhibition of glucagon-stimulated glycogenolysis, PGF2 and PGF2 alpha inhibited glucagon-stimulated hepatocyte cyclic AMP accumulation. These actions of PGB2 and PGF2 alpha are identical with those previously reported for PGE2. Additionally, PGE2, PGF2 alpha and PGB2 inhibited glucagon-stimulated adenylate cyclase activity in purified hepatic plasma membranes. In contrast, PGF2 alpha, PGD2 and PTA2 were all without affect on basal rates of hepatocyte glycogenolysis or hepatocyte cyclic AMP content. PGE2 also inhibited glycogenolysis stimulated by the alpha-adrenergic agonist phenylephrine. Exogenous arachidonic acid was not able to reproduce the affects of PGE2 or PGF2 alpha on hepatocyte glycogenolysis, consistent with an extra-hepatocyte source of the prostaglandins in the intact liver. Thus PGE2 and PGF2 alpha act specifically to inhibit glucagon-stimulated adenylate cyclase activity. No prostaglandin tested was found to stimulate glycogenolysis. PGE2 and PGF2 alpha may represent intra-hepatic modulators of hepatocyte glucose metabolism.     

Pertussis toxin reverses prostaglandin E2- and somatostatin-induced inhibition of rat parietal cell H(+)-production.

In enzymatically dispersed enriched rat parietal cells we studied the effect of pertussis toxin on prostaglandin E2 (PGE2)- or somatostatin-induced inhibition of H(+)-production. Parietal cells were incubated in parallel in the absence (control cells) and presence of pertussis toxin (250 ng/ml; 4 h). [14C]Aminopyrine accumulation by both pertussis toxin-treated and control cells was used as an indirect measure of H(+)-production after stimulation with either histamine, forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (dbcAMP) alone and in the presence of PGE2 (10(-9)-10(-7) M) or somatostatin (10(-9)-10(-6) M). PGE2 inhibited histamine- and forskolin-stimulated [14C]aminopyrine accumulation but failed to alter the response to dbcAMP. Somatostatin was less effective and less potent than PGE2 in inhibiting stimulation by histamine or forskolin and reduced the response to dbcAMP. Pertussis toxin completely reversed inhibition by both PGE2 and somatostatin on histamine- and forskolin-stimulated H(+)-production but failed to affect inhibition by somatostatin of the response to dbcAMP. After incubation of crude control cell membranes with [32P]NAD+, pertussis toxin catalysed the incorporation of [32P]adenosine diphosphate (ADP)-ribose into a membrane protein of molecular weight of 41,000, the known molecular weight of the inhibitory subunit of adenylate cyclase (Gi alpha). Pertussis toxin treatment of parietal cells prior to the preparation of crude membranes almost completely prevented subsequent pertussis toxin-catalysed [32P]ADP ribosylation of the 41,000 molecular weight protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.     

Effects of oestrogen on adenylate cyclase system and glucose output in rat liver.

Effects of chronic oestrogen treatment on catecholamine- and glucagon-sensitive adenylate cyclase activity and glucose output in hepatocytes of castrated male rats were studied. In hepatocytes from male intact or castrated rats, the beta-adrenergic agonist isoprenaline did not stimulate adenylate cyclase activity and glycogenolysis, but glucagon markedly stimulated all these activities. Treatment of castrated animals with 17 beta-oestradiol for 7 days led to the appearance of beta-adrenergic-stimulated increases in both cyclic AMP generation and glucose output. The basal, glucagon- or fluoride-stimulated activities of adenylate cyclase of hepatic membranes prepared from oestrogen-treated rats were similar to those of control animals. Treatment with oestrogen did not influence the number or affinity of beta-adrenergic receptors. In hepatic plasma membranes from control rats, GTP failed to decrease the affinity of beta-adrenergic receptors for agonists, whereas the GTP-induced shift was apparently observed in those from oestrogen-treated animals. These results suggest that oestrogen is able to facilitate the coupling of hepatic beta-adrenergic receptors to the enzyme by increasing the effectiveness of receptor-guanine nucleotide regulation.     

Endothelin, a potent peptide agonist in the liver

Endothelin, a peptide mediator produced by vascular endothelial cells, caused sustained vasoconstriction of the portal vasculature in the perfused rat liver. The vasoactive effect of endothelin was accompanied by increased glycogenolysis and alterations in hepatic oxygen consumption. The endothelin-induced increase in the portal pressure was concentration-dependent with an EC50 of 1 nM. Endothelin-induced hepatic glycogenolysis was dose-dependent but exhibited a different EC50 than for the vasoconstrictive effects of endothelin. Hepatic vasoconstriction and glycogenolysis following endothelin infusion were inhibited when Ca2+ was removed from the perfusion medium. The endothelin-induced responses in the liver were not altered by prior infusion of phenylephrine (alpha-adrenergic agonist), isoproterenol (beta-adrenergic agonist), angiotensin II, glucagon, platelet-activating factor, or the platelet-activating factor antagonist, BN52021. However, repeated infusion of endothelin resulted in desensitization of the glycogenolytic response but was without a significant effect on hepatic vasoconstriction. Endothelin also stimulated metabolism of inositol phospholipids in isolated hepatocytes and Kupffer cells in primary culture. The present experiments demonstrate, for the first time, that endothelin is a very potent agonist in the liver eliciting both a sustained vasoconstriction of the hepatic vasculature and a significant increase in hepatic glucose output.     

Reciprocal expressions of alpha 1- and beta-adrenergic receptors, but constant expression of glucagon receptor by rat hepatocytes during development and primary culture

The stimulations of cyclic AMP formation and adenylate cyclase activity by glucagon and isoproterenol were both found to be highest in neonatal rat hepatocytes and to decrease during development. Adult hepatocytes still showed a considerable response to glucagon, but a negligible response to isoproterenol. The decrease in cyclic AMP formation during development can be explained in the case of the response to beta-adrenergic agonist as due to decrease of its receptor number, judging from binding of [125I]iodocyanopindolol to purified plasma membranes. But in the case of the glucagon response, the decrease in the response may be due to change of post-receptor components of the adenylate cyclase system, because the receptor number tended to increase during development, as shown by binding of [125I]iodoglucagon. Similarly, alpha 1-adrenergic receptors increased in number during development, but their IC50 value did not change, as measured by binding of [3H]prazosin to plasma membranes. Previous studies on primary cultures of adult rat hepatocytes showed that the beta-adrenergic response and its receptor number increased markedly during short-term culture (Nakamura, T., Tomomura, A., Noda, C., Shimoji, M., & Ichihara, A. (1983) J. Biol. Chem. 258, 9283-9289). However, in this work the amount of alpha 1-adrenergic receptor of adult rat hepatocytes was found to decrease by one third during 1-2 days culture. Therefore, changes of alpha 1- and beta-adrenergic receptors during development of rat liver and during primary culture of adult rat hepatocytes were reciprocal, although the directions of change in the two conditions were opposite. The additions of various hormones to primary cultures of adult rat hepatocytes did not affect the reciprocal changes of adrenergic receptors, suggesting that these hormones did not regulate the changes of the receptors.     

Studies on the lack of glucagon response to clycogenolysis and gluconeogensis and decrease in protein synthesis in isolated hepatocytes from hypophysectomized animals.

Effect of various concentrations of glucagon on gluconeogenesis and glycogenolysis was studied in isolated hepatocytes obtained from normal and hypophysectomized animals. Addition of glucagon (10^?10 to 10^?6M) stimulated glycogenolysis and gluconeogenesis by 2–3 fold in normal hepatocytes. However, this concentration of glucagon had only a slight effect in isolated hepatocytes obtained from hypophysectomized animals. This lack of glucagon response was not due to reduction in glycogen levels in isolated hepatocytes obtained from hypophysectomized animals. Studies on the incorporation of¹⁴C-alanine,¹⁴C-leucine and¹⁴C-valine showed a 3–5 fold decrease in the incorporation of these amino acids into protein in hypophysectomized animals compared to normal controls.     

Does lead exposure influence liver protein synthesis in rats?

Glycogenolysis was stimulated by catecholamines in in vitro cultures of hepatic tissue of Xenopus laevis. Dose response curves showed that adrenaline and isoprenaline were equally effective while noradrenaline and phenylephrine were progressively less effective in eliciting glycogen breakdown. Neither oxymetazoline nor methoxamine had any effect on glycogenolysis. Administration of adrenaline to cultures was followed within 1 min by a rise in tissue cyclic AMP concentration and within 2 min by an increase in phosphorylase a activity. Both these responses were blocked by propranolol but little affected by phenoxybenzamine. These findings suggest that catecholamines activate glycogenolysis via a beta-adrenergic receptor in X. laevis and that alpha-adrenergic receptors play no role in regulating hepatic glycogenolysis in this species.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

Correlation between cAMP, activation of cAMP-dependent protein kinase(s), and rate of glycogenolysis in isolated rat hepatocytes.

We have studied the correlation between cAMP-dependent protein kinase activation and rates of glycogenolysis in hepatocytes isolated from fed rats. With doses of 20 μM glucagon, the protein kinase was activated to a -cAMP/+cAMP ratio of 0.8 within 10 min and remained activated for up to 2 hours. A dose-response relationship between protein kinase activation and rates of glycogenolysis can be demonstrated to 0–20 μM glucagon. Glycogenolysis was stimulated greater than 2-fold after 2 hours of incubation with the higher doses of glucagon. Protein kinase activity ratios correlated well with the rates of glycogenolysis as the ratios varied from control levels of about 0.25 to the stimulated values of 0.5–0.6. However, as the ratios increased from 0.6 to 0.8, with higher doses of glucagon, there were no corresponding increases in the rates of glycogenolysis. These data may indicate (1) that activation of all of the protein kinase present in the liver cells is not necessary for maximal stimulation of glycogenolysis, or (2) that a specific protein kinase is involved in the intracellular control of glycogen breakdown in isolated rat hepatocytes.     

Glucagon desensitization of adenylate cyclase and stimulation of inositol phospholipid metabolism does not involve the inhibitory guanine nucleotide regulatory protein Gi, which is inactivated upon challenge of hepatocytes with glucagon.

Brief exposure of hepatocytes to glucagon, angiotensin or the protein kinase C activator TPA (12-O-tetradecanoylphorbol 13-acetate) caused the inactivation of the inhibitory guanine nucleotide regulatory protein Gi. Glucagon-mediated desensitization of glucagon-stimulated adenylate cyclase activity was seen in hepatocytes from both normal rats and those made diabetic with streptozotocin, where Gi is not functionally expressed. Normal glucagon desensitization was seen in hepatocytes from young animals, 6 weeks of age, which had amounts of Gi in their hepatocyte membranes which were some 45% of that seen in mature animals (3.4 pmol/mg of plasma-membrane protein). Streptozotocin-induced diabetes in young animals abolished the appearance of functional Gi in hepatocyte plasma membranes. Pertussis-toxin treatment of hepatocytes from both normal mature animals and those made diabetic, with streptozotocin, blocked the ability of glucagon or angiotensin or TPA to elicit desensitization of adenylate cyclase. The isolated B (binding)-subunit of pertussis toxin was ineffective in blocking desensitization. Neither induction of diabetes nor treatment of hepatocytes with pertussis toxin inhibited the ability of glucagon and angiotensin to stimulate the production of inositol phosphates in intact hepatocytes. Thus (i) Gi does not appear to play a role in the molecular mechanism of glucagon desensitization in hepatocytes, (ii) the G-protein concerned with receptor-stimulated inositol phospholipid metabolism in hepatocytes appears not to be a substrate for the action of pertussis toxin, (iii) in intact hepatocytes, treatment with glucagon and/or angiotensin can elicit the inactivation of the inhibitory G-protein Gi, and (iv) pertussis toxin blocks desensitization by a process which does not involve Gi.     

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.     

Signal transduction by tumor necrosis factor alpha is mediated through a guanine nucleotide-binding protein in osteoblast-like cell line, MC3T3-E1.

Transmembrane signalling mechanisms of tumor necrosis factor alpha (TNF alpha) were examined with special reference to the involvement of G-protein, in intact and permeabilized murine osteoblast-like cells. TNF alpha stimulated the release of 3H radioactivity from intact cells labeled with [3H]arachidonic acid within 10 min in a dose dependent manner and the production of lyso forms of phospholipids, an event presumably mediated through the activation of phospholipase A2. Production of cAMP and inositol 1,4,5-trisphosphate was not affected by TNF alpha. Pretreatment of the cells with pertussis toxin inhibited the liberation of [3H]arachidonate. GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) reduced the binding affinity of [125I]TNF alpha to beta-escin-permeabilized cells. The addition of TNF alpha together with an unhydrolyzable analog of GTP, GTP gamma S, to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid led to a release of the 3H radioactivity. The production of prostaglandin E2 (PGE2) was markedly stimulated by TNF alpha in a dose over 100 ng/ml, with a latent time of about 3 h, and the stimulation was abolished by pretreatment with pertussis toxin. The time and dose requirements for this process differed from those for the possible activation of phospholipase A2, thereby indicating that other process(es) in addition to the activation of phospholipase A2 may be responsible for the enhanced production of PGE2. The activity of cyclooxygenase (i.e. the combined activities of prostaglandin endoperoxide syntase and PGH2-PGE2 isomerase) was stimulated by TNF alpha with much the same time and dose requirements as for the production of PGE2, and the activation was found to be due to the increased amount of the enzyme, as assessed by a Western blot analysis with anti-cyclooxygenase antibody. This process was also sensitive to pertussis toxin. Therefore, receptors for TNF alpha in MC3T3-E1 cells apparently couple to G-protein sensitive to pertussis toxin and the coupling regulates the activations of phospholipase A2 and the de novo synthesis of cyclooxygenase.     

Neurotensin, bradykinin and somatostatin inhibit cAMP production in neuroblastoma N1E115 cells via both pertussis toxin sensitive and insensitive mechanisms

Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.