Vittorio Tomasi

The role of lipids in the activity of adenylate cyclase of rat liver plasma membranes

Abstract The effect of mild lipid extraction and phospholipases on isolated rat liver plasma membrane was investigated. Extraction of membrane at 4 ° with a shaked mixture of petrol ether- n -butanol resulted in a dramatic reduction of the concentration of phosphatidylethanolamine (−97%) and phosphatidylserine (−99%), whereas phosphatidylinositol was reduced to about 30% of the initial value. Simultaneously the activity of adenylate cyclase as well as that of Mg 2+ -ATPase was strongly reduced −83 and −85%, respectively); another membrane enzyme, 5′-nucleotidase was slightly stimulated. The activity of solvent-treated adenylate cyclase was partially restored by addition of total membrane phospholipids or phosphatidylinositol, while phosphatidylserine was ineffective. Both phospholipase A and C reduced adenylate cyclase activity. However the effect of phospholipase C was prevented by serum albumin, while that of phospholipase A was not. This suggests that the effect of the latter enzyme may be related to alterations of phospholipids and (or) of membrane structure.

The role of phosphatidylserine in the hormonal control of adenylate cyclase of rat liver plasma membranes

The involvement of phospholipids in the responsiveness of adenylate cyclase of rat liver plasma membranes to hormones was studied, using a mild extraction procedure and phospholipase A and C treatment for the removal of membrane phospholipids. Mild extraction of membranes caused a reduction in the basal activity of adenylate cyclase which was also no longer sensitive to hormones and F−. Addition of phosphatidylserine almost completely restored the epinephrine responsiveness of the enzyme, but only partially restored the glucagon and F− sensitivity. Phosphatidylinositol, which restored the basal cyclase activity, had no effect on hormonal and F− sensitivity. Phosphatidylethanolamine and phosphatidylcholine had no effect. Treatment of membranes with phospholipase A and C abolished the hormonal and F− responsiveness of adenylate cyclase; phospholipase A also decreased its basal activity. The hormonal and F− responsiveness were partially reactivated with phosphatidylserine, but not with phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. These observations are consistent with the catalytic and regulatory subunits of plasma membrane adenylate cyclase both being lipoproteins, the former requiring phosphatidylinositol and the latter phosphatidylserine for proper functioning.

Modulation by cyclic AMP in vitro of liver plasma membrane (Na+—K+)-ATPase and protein kinases

Abstract The effect of cyclic AMP on (Na+—K+)-ATPase and protein kinase activities of isolated liver plasma membrane has been investigated. Theophylline and caffeine, several cyclic nucleotides (including cyclic AMP, dibutyryl cyclic AMP, cyclic GMP and cyclic UMP), and sodium butyrate reduced the activity of (Na+—K+)-ATPase. The most powerful inhibitor was dibutyryl cyclic AMP; however, its action is probably due to the combined effects of cyclic AMP and butyrate. The blocking agent propranolol prevented the action of both epinephrine and cyclic AMP. Liver plasma membranes contain protein kinase(s), which catalyze the transfer of terminal phosphate of ATP into protein of plasma membrane itself (endogenous phosphorylation) as well as into added proteins (exogenous phosphorylation). Endogenous phosphorylation was inhibited by cyclic AMP, dibutyryl cyclic AMP and cyclic GMP. On the other hand, exogenous phosphorylation was stimulated by cyclic AMP. The specific activity of the membrane enzyme catalyzing the latter reaction was found to be higher than that of the enzyme present in the 100 000 × g supernatant of rat liver. The parallel effect of cyclic AMP on (Na+—K+)-ATPase and on the phosphorylation of membrane protein, and the finding that the latter reaction involves the formation of acylphosphate bonds, may be indicative of a possible role of endogenous phosphorylation in the regulation of (Na+—K+)-ATPase activity.

Factors influencing the glucocorticoid-induced increase of ribonucleic acid polymerase activity of rat liver nuclei

Abstract Liver nuclei from adrenalectomized rats receiving cortisone have a nuclear RNA polymerase activity higher than controls. The induced and control polymerase activity present the same general characteristics. However, ammonium sulfate added in vitro enhances RNA polymerase activity of controls more than that of treated nuclei and obliterates the hormonal response. The stimulating effect of cortisone on RNA polymerase activity is prevented in vivo by puromycin and actinomycin D. Cortisone abolishes in vivo the toxic effects of histones; however, neither cortisone nor cortisol reverses in vitro the inhibitory action of these compounds on RNA polymerase activity. Irradiation with X-rays (600 r) results in a reduced response of RNA polymerase to cortisone. Liver RNA polymerase activity is very low at birth and reaches the adult values after about 15 days of extrauterine life. No response to cortisone or cortisol is observed in vivo before the 13th day of life, whereas other enzymic activities, such as tyrosine transaminase and alanine transaminase, respond much earlier. Therefore it cannot be stated at present that the increase of liver enzyme activities induced by glucocorticoids is mediated by RNA polymerase, at least in newborn rats. Administration of cortisone has no appreciable effect on the hydrolysis of pulse-labeled nuclear RNA, but it reduces the hydrolysis of microsomal RNA. The possibility is prospected that this effect together with the increased RNA polymerase activity may contribute to increase the amount of liver RNA, which is a typical effect of glucocorticoid treatment.

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE1 (prostaglandin E1). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF1α was less effective and neither PGE2 nor PGF2α had any effect. Dopamine and PGE, alone or in combination, did not modify low Km cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μM), which increased cyclic AMP as much as 10 μm‐dopamine, showed no synergistic effect when tested in the presence of PGE1 or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE1 Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE1 as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE1

Soluble and membrane-bound adenylate cyclase activity in Yoshida ascites hepatoma

Abstract In Yoshida hepatoma cells, adenylate cyclase was found to be present in the plasma membrane as well as in the supernatant fraction. Both activities were lower than that obtained with liver membranes. During tumor growth enzyme activity decreased in the membrane and rose in the supernatant. The response of soluble and membrane-bound adenylate cyclase to epinephrine, glucagon and fluoride was markedly different. The hormone concentrations required for maximal effect were higher for the tumor than for the liver enzyme. These findings suggest that tumor cells tend to escape hormonal control.

Cyclic AMP control by prostaglandin E1 in non-parenchymal liver cells

There is little doubt, despite some contradictory reports, that the liver is a main target of prostaglandin El (PGEI ). The PGEl -due increase of cyclic-AMP levels occurring when the drug is injected into rats [l] or when it is added to isolated rat liver hepatocytes [2] has been fully accounted for by a stimulatory effect on adenylate cyclase of particulate liver fractions [ 1 ] and isolated liver plasma membranes as well [3]. Using liver plasma membranes we have shown that a PGEl -sensitive cyclase is clearly distinct from glucagon and epinephrine-sensitive cyclases [3] ; in several tissues moreover PGEl action appears to be best explained by assuming an interaction with a specific receptor that does not share any property with polypeptide hormone or catecholamines receptors t4,51* In order to distinguish whether PGEl has to be considered an intercellular or an intracellular messenger it was important in our opinion to establish whether PGEl receptor is on the outer or on the inner layer of the plasma membrane. Experiments carried out in isolated hepatocytes point to an external rather than to an internal localization of PGEl receptor, thus favouring a role of PGEl as intercellular messenger [41. The results of experiments reported here, which indicate that PGEl dramatically increases cyclic-AMP levels in a discrete hepatocyte population not involving parenchymal cells, give a strong support to the role of

Prostaglandin E1-sensitive adenylate cyclase of rat liver plasma membranes

Abstract Adenylate cyclase of isolated rat liver plasma membranes is stimulated at least four-fold by prostaglandin E 1 (PGE 1 ) provided that EGTA and GTP are present in the assay system. The GTP-dependency is absolute, since in its absence the PGE 1 activation of adenylate cyclase is small and inconsistent. PGE 2 is much less effective while prostaglandins F 1α and F 2α are ineffective under all conditions tested. The stimulatory effect of PGE 1 is evident at concentrations around 0.5 uM, optimal stimulation requires 3 μM PGE 1 . Several lines of evidence suggest that PGE 1 interacts with plasma membrane binding sites separate from those specific for glucagon and epinephrine. First, the effect of combinations of PGE 1 with glucagon or epinephrine at optimal concentrations are additive, while not additive is the combination PGE 1 -NaF. Second, variations in the temperature of incubation between 25 and 45°C influence in a different way glucagon and PGE 1 -sensitive cyclase. Glucagon stimulation continuously increases with the increase of temperature while PGE 1 stimulation increases up to 30°C then it levels off and decreases at 45°C. Third, propranolol which nearly completely blocks epinephrine stimulation is much less effective on PGE 1 -sensitive cyclase. 0.1 mM phentolamine has little effect, if any, on hormonal sensitivity. Lubrol at very low doses (1 mg/100 ml) reduces the effect of epinephrine and glucagon, but increases that of PGE 1 .

Isolation and characterization of the plasma membrane from Yoshida hepatoma cells.

SummaryPlasma membranes isolated from Yoshida ascites hepatoma AH-130 by a modification of the method of T. K. Ray (Biochim. Biophys. Acta196: 1, 1970), were subfractionated into three fractions having densities (d) 1.12, 1.14 and 1.16 by discontinuous sucrose density-gradient. Membrane subfractions were characterized by electron-microscopy, by assay of marker enzymes and by lipid composition. All subfractions appeared to be essentially free from whole mitochondria, lysosomes and nuclei. Subfraction d 1.16 had, the highest 5′-nucleotidase, Mg++-ATPase and (Na++K+)-ATPase activities; cytochromec oxidase was undetectable in any fraction and glucose-6-phosphatase was measurable only in fraction d 1.14. Adenylate cyclase had the highest activity in fractions d 1.14 and 1.16. Cyclic AMP phosphodiesterase was nearly equally distributed in the fractions. Adenylate, cyclase, 5′-nucleotidase and Mg++-ATPase activities of tumor membrane were lower with respect to liver plasma membrane, while cyclic AMP phosphodiesterase and (Na++K+)-ATPase were found to have similar activities in the two membrane preparations. With respect to liver membrane, hepatoma membrane contained a higher amount of glycolipids and a higher amount of phospholipids accounted for mainly, by sphingomyelin, phosphatidylserine and phosphatidic acid. The possible significance of the decrease of adenylate activity in the hepatoma membrane is briefly discussed.

Synthesis and breakdown of rat liver RNA during the neonatal period

The incorporation of radioactivity from labeled pyrimidine precursors (6-14C-orotic acid and 2-14C-uridine) and from32P into liver RNA has been evaluated in fetuses and newborn rats. Very low values were recorded in the last days of intrauterine life and at birth. Soon after birth, there was a very sharp increase of the incorporation, and maximum values were attained at three days of life. Nuclear DNA-dependent RNA polymerase activity was also found to be very low in fetuses, but increased gradually after birth and reached the adult values only at about 25 days of of life. Ribonuclease activity of isolated nuclei and microsomal fractions were very high at birth and decreased in the first days of extrauterine life, attaining the lowest values in adult life. Similarly, autohydrolysis of endogenous nuclear or microsomal RNA was higher in young than in adult animals but did not undergo appreciable change during the first 3 weeks of life. Newborn liver nuclei and microsomes contained ribonuclease essentially in a free form, whereas in adult ones the latent form of enzyme was predominant. The significance of these findings in relation to the labeling of RNA, and with the changes in enzyme activities, liver size, and liver protein content in the perinatal period, is discussed.