Paolo Luly

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.

Insulin effect in vitro on human erythrocyte plasma membrane

The effect of porcine insulin has been tested in vitro on human erythrocyte plasma membrane (Na+−K+) and Mg2+-ATPase activities as well as on membrane fluidity. The results indicate that the hormonal treatment significantly inhibits (Na+−K+)-ATPase activity, and at the same time decreases membrane fluidity.

Effect of free fatty acids and cholesterol in vitro on liver plasma membrane-bound enzymes.

The effect of cholesterol and fatty acid treatment in vitro was tested on rat liver plasma membrane-bound enzymes and lipid fluidity. The observed alterations of membrane fluidity affect both (Na+−K+)-ATPase and Mg2+-ATPase activities but not 5′-nucleotidase; basal adenylate cyclase as well as its hormonal sensitivity were differentially affected by changes of membrane microenvironment.

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.

Insulin binding and internalization in rat hepatocytes during prenatal and postnatal life.

Insulin binding to isolated rat hepatocytes was studied during prenatal and postnatal life. Results show that in hepatocytes isolated from prenatal, postnatal and adult rat there is a constant increase in the number of insulin binding sites per cell, whereas the affinity of plasma membrane receptors for the hormonal ligand remains unaltered from prenatal to adult hepatocytes. Autoradiographic studies indicate a greater internalization of hormone during prenatal life and, taking into account the increase of cell size, suggest an unchanged surface density of receptor sites before and after birth.

Stimulation of (Na+K+)-ATPase of rat liver plasma membrane by amino acids

Abstract The effect of twelve l -amino acids on the activity of liver plasma membrane (Na+ue5f8K+)-ATPase has been tested. Histidine and arginine significantly enhanced the activity. The activtion by histidine showed saturation kinetics with an apparent Ka of about 8 mM, and was evident over a wide range of Na+ concentrations. The same amino acid did not significantly affect the Mg2+-dependent ATPase activity.

Insulin-sensitive adenosine 3′,5′-cyclic monophosphate phosphodiesterase of hepatocyte plasma membrane

Adenosine 3,5-cyclic monophosphate phosphodiesterase (EC 3.1.4.17) has been investigated in rat liver as to its insulin sensitivity. Hormone action has been assayed in vitro on a liver homogenate purified by DEAE-cellulose column chromatography, on isolated hepatocytes, on isolated plasma membranes. The DEAE-cellulose chromatography purified homogenate showed no sensitivity to insulin, whereas isolated hepatocytes incubated in presence of insulin showed increased phosphodiesterase activity in a plasma membrane-containing fraction. The plasma membrane-bound enzyme, which shows both high and low affinity components, was significantly stimulated after hormonal treatment; this effect being dependent on a V increase of the low Km form.

Effect of colchicine on rat liver plasma membrane.

Colchicine effect has been tested on rat liver plasma membrane-bound enzymes after in vitro or in vivo treatment. It appears that the in vitro treatment does not affect 5-nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase, whereas adenylate cyclase is sensitive to both in vitro and in vivo treatment, the latter condition being also effective for 5-nucleotidase.

Effect of chlorpropamide and phenformin on rat liver: the effect on plasma membrane-bound enzymes and cyclic AMP content of hepatocytes in vitro.

Chlorpropamide and phenformin inhibited (Na+ - K+)-ATPase and stimulated a high affinity cyclic AMP-phosphodiesterase of isolated liver plasma membrane when tested in vitro. In addition, the two drugs decreased the intracellular cyclic AMP content of isolated hepatocytes without being effective on plasma membrane-bound adenylate cyclase. The results suggest that the plasma membrane plays an important role in the mechanism of action of the two hypoglycemic drugs, but do not exclude the presence of intracellular targets.

A study of the hormonal control in vitro of Na+-K+ activated ATPase of isolated liver plasma membrane

Abstract The activity of rat liver plasma membrane-bound Na+-K+ ATPase was modulated in vitro by epinephrine, glucagon and insulin through an action probably mediated by cyclic AMP. Epinephrine, glucagon and cyclic AMP inhibited the activity of this enzyme; insulin did not influence the baseline activity but prevented partially the effect of the former hormones. Rat liver plasma membranes contain protein kinase activity. In the absence of exogenous substrate, phosphorylation of membrane constituents occurred very rapidly and was inhibited by cyclic AMP, both in the presence and absence of Ca++ ions. When an exogenous substrate (protamine) was added to the system, phosphorylation was linear with time, at least up to 15 min; the reaction was stimulated by cyclic AMP, and the effect of this compound was abolished when Ca++ was added to the medium. Hydroxylamine removed about 35% of labeled phosphate incorporated—both in the presence and absence of cyclic AMP—in membrane preparations incubated in the absence of protamine, while it had no effect in its presence. These results suggest that, as a consequence of endogenous phosphorylation, part of labeled phosphate was incorporated in membranes through an acyl linkage. As Na+-K+ ATPase catalyzes two main reaction steps, the first being represented by a phosphate group transfer from ATP to a free carboxylic radical belonging to enzyme protein, these results very probably indicate that the marked effect exerted by cyclic AMP on ATPase activity may be ascribed to the inhibition of the formation of such a linkage, due to the cyclic nucleotide itself. The second step of ATPase reaction was investigated by model reactions. The hydrolysis of p-nitrophenyl phosphate was not modified by cyclic AMP while that of acetyl phosphate was inhibited. As in the latter case, the reaction concerns the hydrolysis of an acyl phosphate group, it seems probable that also the second step of ATPase reaction is inhibited by cyclic AMP.