Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E₁ or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue.In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Effect of high level section of the spinal cord on the cyclic AMP system in rat liver

The effect of high level section of the spinal cord upon the hepatic cyclic AMP system was investigated in the rat. We report that transection of the spinal cord dramatically decreases the basal level of cyclic AMP from 0.88 nmol/g liver to 0.36 nmol/g at 1 h and to 0.20 nmol/g at 4 h. This was not due to increased activity of cyclic AMP phosphodiesterase or to decreased activity of basal adenylate cyclase. The sensitivity of adenylate cyclase to its usual effectors in vitro was not impaired. It is proposed that the lowering of liver cyclic AMP below its basal level after high level section of the spinal cord is due to decreased levels of hepatic catecholamines and/or plasma glucagon.     

Effect of high level section of the spinal cord on the cyclic AMP system in rat liver.

The effect of high level section of the spinal cord upon the hepatic cyclic AMP system was investigated in the rat. We report that transection of the spinal cord dramatically decreases the basal level of cyclic AMP from 0.88 nmol/g liver to 0.36 nmol/g at 1 h and to 0.20 nmol/g at 4 h. This was not due to increased activity of cyclic AMP phosphodiesterase or to decreased activity of basal adenylate cyclase. The sensitivity of adenylate cyclase to its usual effectors in vitro was not impaired. It is proposed that the lowering of liver cyclic AMP below its basal level after high level section of the spinal cord is due to decreased levels of hepatic catecholamines and/or plasma glucagon.     

Lack of correlation between hormonal effects on cyclic AMP and glycogenolysis in rat liver.

Portions of liver were obtained by biopsy from rats infused with various concentrations of glucagon or epinephrine and analyzed for cyclic AMP, glycogen, phosphorylase activity, and glycogen synthetase I activity. The response of tissue cyclic AMP to glucagon or epinephrine was far less sensitive than other metabolic parameters; at certain lower doses of glucagon or epinephrine, glycogen decomposed without a simultaneous increase in the hepatic level of cyclic AMP. It is probable that hormonal activation of adenylate cyclase results in an increase of cyclic AMP only in its small “active” pool without detectable changes in its much larger inactive or bound pool. Though the active cyclic AMP is expected to be released into the circulation or to be labeled with [³H]adenine in preference to the inactive nucleotide, neither the increase of cyclic AMP in the vena cava in vivo nor the incorporation of [³H]adenine into tissue cyclic AMP in liver slices in vitro exhibited more sensitivity to glucagon than the hepatic level of cyclic AMP as a whole. Thus, it remains to be settled whether cyclic AMP is compartmentalized in the cell or plays no essential role in the stimulation of hepatic glycogenolysis induced by small doses of hormones.     

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.     

Prostaglandin deficiency promotes sensitization of adenylyl cyclase

Inhibition of prostaglandin synthesis by the drug indomethacin suppresses the synthesis of the cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), and leads to a metabolic state comparable to type II diabetes. It was of interest whether prostaglandin-deficiency likewise causes sensitization of adenylyl cyclase, as this has been reported for the diabetic state. In liver plasma membranes of indomethacin-treated male rats, basal and forskolin-stimulated cyclic AMP synthesis remained unchanged when compared to untreated control rats. In control rats, stimulation of cyclic AMP synthesis by fluoride (2.2-fold) or glucagon (3.5-fold) was much lower than stimulation by forskolin (6.6-fold). In contrast, in indomethacin-treated rats, stimulation of cAMP synthesis by fluoride (4.6-fold) or glucagon (5.2-fold) nearly matched the stimulation by forskolin (6.4-fold). The level of alpha1-adrenergic receptors was slightly reduced, from 450 to 320 fmol/mg protein, by the indomethacin treatment. Independent of the treatment by indomethacin, stimulation of cyclic AMP synthesis by adrenaline failed, in agreement with the low density of adrenergic beta-receptors. In conclusion, PGE deficiency sensitizes adenylyl cyclase in rat liver for G protein-coupled receptors (glucagon) and also for fluoride.     

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.     

Activation of renal adenylate cyclase by forskolin: assessment of enzymatic activity in animal models of the secondary hyperparathyroid state

The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptor-mediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.     

Prostaglandin E1 effects on cyclic AMP and glycogen metabolism in rat liver.

Prostaglandins E₁ or E₂ (PGE₁, PGE₂)¹ stimulated adenylate cyclase(s) from particulate fractions of whole liver homogenates 5- to 6-fold, but caused only slight (1.5- to 2-fold) stimulation of the enzyme from homogeneous hepatocytes. In contrast, glucagon stimulated enzyme from hepatocytes 12- to 15-fold and enzyme from whole liver 8- to 10-fold. Accordingly, most of the total prostaglandin-sensitive adenylate cyclase in cell suspensions was recovered in fractions containing non-parenchymal cells, and most of the total glucagon-sensitive activity was recovered with hepatocytes. PGE₁ did not change adenosine-3′,5′-monophosphate (cyclic AMP) concentrations, or alter cyclic AMP increases caused by glucagon in hepatocytes. Glucagon consistently increased hepatocyte cyclic AMP concentrations and stimulated glycogenolysis by 35 to 40%. PGE₁ did not affect basal or glucagon-stimulated glycogenolysis in the intact cells.     

Elevation of Intracellular Cyclic AMP and Stimulation of Adenylate Cyclase Activity by Vasoactive Intestinal Peptide and Glucaeon in the Retinal Pigment Epithelium

Both vasoactive intestinal peptide (VIP) and glucagon rapidly elevated cyclic AMP levels in embryonic chick retinal pigment epithelium (RPE), in culture as well as in freshly dissected tissue. In cultured cells, the half-maximal activities of VIP and glucagon were 5 X 10(-8) M and 3 X 10(-8) M, respectively. After 3 min of reaction, VIP elevated intracellular cyclic AMP by 100-fold; elevation with glucagon was up to 10-fold. Both neuropeptides stimulated adenylate cyclase activity in RPE membranes. Glucagon showed a half-maximal activity of 1 X 10(-8) M. VIP remained more effective than glucagon in stimulating adenylate cyclase activity, but the dose-response curve was shifted to a higher concentration range when compared to that of the VIP-elevated intracellular cyclic AMP.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values.

Treatment of intact hepatocytes with glucagon led to the rapid desensitization of adenylate cyclase, which reached a maximum around 5 min after application of glucagon, after which resensitization ensued. Complete resensitization occurred some 20 min after the addition of glucagon. In hepatocytes which had been preincubated with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), glucagon elicited a stable desensitized state where resensitization failed to occur even 20 min after exposure of hepatocytes to glucagon. Treatment with IBMX alone did not elicit desensitization. The action of IBMX in stabilizing the glucagon-mediated desensitized state was mimicked by the non-methylxanthine cyclic AMP phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxylbenzyl)-2-imidazolidinone]. IBMX inhibited the resensitization process in a dose-dependent fashion with an EC50 (concn. giving 50% of maximal effect) of 26 +/- 5 microM, which was similar to the EC50 value of 22 +/- 6 microM observed for the ability of IBMX to augment the glucagon-stimulated rise in intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with IBMX did not alter the ability of either angiotensin or the glucagon analogue TH-glucagon, ligands which did not increase intracellular cyclic AMP concentrations, to cause the rapid desensitization and subsequent resensitization of adenylate cyclase. It is suggested that, although desensitization of glucagon-stimulated adenylate cyclase is elicited by a cyclic AMP-independent process, the resensitization of adenylate cyclase can be inhibited by a process which is dependent on elevated cyclic AMP concentrations. This action can be detected by attenuating the degradation of cyclic AMP by using inhibitors of cyclic AMP phosphodiesterase.     

Adenosine 3',5'-monophosphate formation by preparations of rat liver soluble guanylate cyclase activated with nitric oxide, nitrosyl ferroheme, S-nitrosothiols, and other nitroso compounds

Cyclic AMP formation from ATP was stimulated by unpurified and partially purified soluble hepatic guanylate cyclase in the presence of nitric oxide (NO) or compounds containing a nitroso moiety such as nitroprusside, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), nitrosyl ferroheme, and S-nitrosothiols. Cyclic AMP formation was undetectable in the absence of NO or nitroso compounds and was not stimulated by fluoride or glucagon, indicating the absence of adenylate cyclase activity. The nitroso compounds failed to activate, whereas fluoride or glucagon activated, adenylate cyclase in washed rat liver membrane fractions. Cyclic GMP formation from GTP was markedly stimulated by the soluble hepatic fraction in the presence of NO or nitroso compounds. Cyclic AMP formation by partially purified guanylate cyclase was competitively inhibited by GTP and cyclic GMP formation is well-known to be competitively inhibited by ATP. Therefore, it appears that activated guanylate cyclase, rather than adenylate cyclase, was responsible for the formation of cyclic AMP from ATP. Formation of cyclic AMP of cyclic GMP was enhanced by thiols, inhibited by hemoproteins and oxidants, and required the addition of either Mg2+ or Mn2+. Further, several nitrosyl ferroheme compounds and S-nitrosothiols stimulated the formation of both cyclic AMP and cyclic GMP by the soluble hepatic fraction. These observations support the view that soluble guanylate cyclase is capable, under certain well-defined conditions, of catalyzing the conversion of ATP to cyclic AMP.     

Uncoupling of the Glucagon Receptor-Adenylate Cyclase System by Glucagon in Cloned Differentiated Rat Hepatocytes

Abstract

The ability of glucagon to induce a state of desens it ization to glucagon responsiveness has been examined in a cloned line of normal, differentiated, diploid rat hepatocytes (RL-PR-C). These cells, which respond to glucagon with increased production of cyclic AMP, become refractory to further stimulation of cyclic AMP synthesis following a 4 hour exposure period of the cells to the hormone. Refractoriness to glucagon was demonstrated over a wide range of hormone concentrations (10^?12 to 10^?6 M). In desensitized cells that were subsequently washed free of the hormone, recovery from refractoriness was complete by 20 hours. The mechanism underlying this desensitization does not appear to involve decreased receptor numbers, increased efflux of cyclic AMP from the cells, increased degradation of cyclic AMP by phosphodtesterase, or an alteration in the catalytic unit of the adenylate cyclase enzyme system. By elimination, the diminished cellular cyclic AMP responsiveness to glucagon in normal RL-PR-C hepatocytes may involve a reversible uncoupling of glucagon receptors from adenylate cyclase. In addition, late passage, spontaneously transformed RL-PR-C hepatocytes were found to exist in a state in which glucagon receptors are permanently uncoupled from adenylate cyclase.     

Islet-activating protein blocks glucagon desensitization in intact hepatocytes.

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5'-[beta-thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Comparison of the effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism.

The effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism are compared. Both peptides stimulated glycogenolysis as a result of an increase in phosphorylase a activity and enhanced glucose synthesis from [2-14C]pyruvate, although neither had any significant effect on pyruvate kinase activity. Although the magnitudes of the effects of both peptides on glycogenolysis were comparable and unaffected by the presence of insulin. [Leu]enkephalin proved to be more efficacious in enhancing gluconeogenesis, the response being comparable with that to glucagon. Both effectors decreased the intracellular concentration of cyclic AMP in hepatocytes when incubated under control conditions and after addition of sub-optimal concentrations of glucagon. This was correlated with the ability of the two peptides to inhibit both basal and hormone-stimulated adenylate cyclase activity in purified liver plasma membranes.     

Hormone responsiveness of plasma membrane-bound enzymes in normal and regenerating rat liver.

The hormonal responsiveness of plasma membrane-bound enzymes (Na-+-K-+)-ATPase and adenylate cyclase has been investigated in normal and regenerating rat liver. (Na-+-K-+)-ATPase basal activity is not affected by surgery and only slightly affected by partial hepatectomy; its response to epinephrine and cyclic AMP is decreased only 15 h after hepatectomy. Adenylate cyclase activity of plasma membranes from untreated animals is stimulated by parathyroid hormone and thyroxine; partial hepatectomy increased basal activity as well as the stimulation exerted by the aforementioned hormones, when glucagon and epinephrine sensitivity is essentially unaltered.     

Enzymatic,biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTPγS. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5′-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.