Pertussis toxin abolishes the inhibitory effects of prostaglandins E₁, E₂,I₂ and F_2α on hormone‐induced cAMP accumulation in cultured hepatocytes

Abstract

Several prostaglandins inhibit the cAMP response to glucagon and P-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 El (PGE1). Furthermore, whereas PGE1, PGE,, PGIz and PGF,, 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 PGEl and PGF,,. 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 PGEl was about 100-fold more potent as an inhibitor than PGFza. Pertussis toxin abolished the inhibitory effects of PGEl and PGF,, with almost identical time and dose requirements. The results obtained with PGEI, PGE2, PGI, and PGF,, suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.The formation of cAMP is subject to both positive and negative control. This regulation appears to be exerted through GTP-binding proteins (G proteins) that transduce stimulatory and inhibitory signals from the receptors to the catalytic moiety of the adenylate cyclase [l -51. In the case of inhibitory regulation, the understanding of the mechanisms involved has been facilitated by the observation [6] that receptor-mediated decreases in cAMP could be abolished by treating cells with pertussis toxin (islet-activating protein) [7, 81, which was subsequently found [9, 101 to act by ADPribosylating the CI subunit of an inhibitory G protein (Gi).Prostaglandins may both stimulate and inhibit the formation of CAMP [ll - cAMP accumulation by E prostaglandins is prevented by pertussis toxin in fat cells [32], mammary tumor cells [33] and kidney [34], suggesting a role of the Gi. It is not known whether different prostaglandins inhibit the cAMP system by a common mechanism. It is also unknown if the inhibitory and stimulatory effects of prostaglandins on the adenylate cyclase are mediated via the same receptors.In the liver, prostaglandins of the E series stimulate the adenylate cyclase only slightly [35 -371. However, many prostaglandins, including those of the E and F series, markedly inhibit the response to glucagon or epinephrine [19 -22, 38, 391. The purpose of the present study was to investigate the mechanisms behind the prostaglandin-induced attenuation of hormonal responsiveness and the role of the Gi protein in the regulation of cAMP in liver cells. We have used cultured hepatocytes, and examined the effect of pertussis toxin on cAMP responses to hormones and vari...

Glycogenolytic and antiglycogenolytic prostaglandin E2 actions in rat hepatocytes are mediated via different signalling pathways

“…2). This is in agreement with previously published data [32,341. By contrast, the glycogenolytic PGE, effect was rather enhanced.…”

“…2) indicating that the partial inactivation of a G, protein revealed the presence a G,-linked PGE, receptor signal chain normally obscured by the predominance of G, action. This is in agreement with a previous finding, that a small PGE,-mediated increase in cAMP formation was potentiated by initial treatment of hepatocytes with pertussis toxin [32].…”

“…perfusion and cell suspension with glucose-free media versus culture with glucosecontaining media have previously been used [37] to explain the lack of glycogenolytic PGE, effects in the latter studies with cultures . A slight PGE,-mediated increase in cAMP has previously been described in hepatocytes in the presence of the CAMP-phosphodiesterase inhibitor isobutyl methyl xanthine together with an inhibition of the glucagonmediated cAMP formation [32]. A potentiation of the glucagon-mediated glucose output at low (1 0-' M) concentrations of PGE, and an attenuation of the glucagon-mediated glucose output at high (10-'M) concentrations of PGE, in cell suspension in a glucose-free and lactate free medium has also been reported [28].…”

Inhibition of Interleukin-1β-Induced COX-2 and EP3 Gene Expression by Sodium Salicylate Enhances Pancreatic Islet β-Cell Function

“…In fat, PGE 2 decreases adenylate cyclase and cAMP generation and inhibits hormone-induced lipolysis (28 -32). In liver, PGE 2 also inhibits adenylate cyclase activity and decreases glucagon-stimulated glycogenolysis (33,34). In kidney, PGE 2 has been shown to decrease cAMP levels and vasopressin-induced water resorption by the collecting tubules (35)(36)(37).…”

Prostaglandin E receptor EP3γ isoform, with mostly full constitutive Gi activity and agonist‐dependent Gs activity

“…Among these subtypes, the EP3 receptor has been best characterized; it has been suggested to be involved in such PGE2 actions as contraction of the uterus [4], inhibition of gastric acid secretion [5], modulation of the neurotransmitter release [6], lipolysis in adipose tissue [7], and sodium and water reabsorption in the kidney tubules [8]. Various EP3 receptor-mediated actions are mediated through multiple signal transduction systems, and in addition, the doseresponse curve and potency of PGE2 vary with tissue, implying heterogeneity of EP3 receptors [9,10].…”