Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone.

The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3H-aldosterone-kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3H-aldosterone peak while the second 3H-aldosterone peak was eluted with Ve corresponding to Ve of free 3H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass.

Postnatal development of rat kidney plasma membrane Na-K-ATPase.

The postnatal development of the sodium transport mechanism in the rat kidney, as judged by the activity of renal plasma membrane Na-K-ATPase, was studied. Highly purified kidney plasma membranes as verified by electron microscopic and enzyme examinations, were used. Na-K-ATPase activity (mumol Pi/mg protein/h) increases exponentially from 16.9 +/- 1.94 found at birth to the mature level of 43.1 +/- 2.16 reached at the age of 41 days. This finding paralleled our results of SDS-polyacrylamide gel disc electrophoresis, showing a completely differentiated plasma membrane protein structure at birth, the maturation of which proceeds only as quantitative enrichment of some protein fractions in the further postnatal period.

In vitro inhibition of rat kidney plasma membrane Na-K-ATPase activity by triamterene.

In vitro experiments were performed to study the effect of 2,4,7-triamino-6-phenylpteridine (triamterene), a potassium sparing diuretic agent, upon the rat kidney plasma membrane Na-K-ATPase activity. Triamterene in the concentration range from 8X10(-13) mol/l to 8X10(-3) mol/l exerted a dose-dependent inhibitory effect of the rat kidney plasma membrane Na-K-Mg-ATPase and Na-K-ATPase activities--estimated IC50 values lay at about 8X10(-3) mol/l and 8X10(-7) mol/l, respectively. The diuretic did not influence the activity of Mg-ATPase, except at very high concentration. A Lineweaver-Burk analysis demonstrated that the inhibition of Na-K-ATPase by triamterene was non-competitive. The in vitro inhibitory effect, together with an in vivo effect observed before points to a possibility that the natriuresis caused by triamterene is due, at least partly, to a direct inhibition of active sodium transport across the kidney plasma membrane.

Binding (in vitro) of some antimitotic isatin derivatives to human serum albumin.

The binding of isatin and its mustard N-Mannich base, considerably biologically active compounds, to human serum albumin has been studied by equilibrium dialysis and ultrafiltration. The influences of ligand and macromolecule concentration, temperature and pH of the incubation medium have been demonstrated. The Scatchard plot of isatin binding to albumin shows a biphasic curve which indicates the presence of at least two different binding sites on albumin molecule. One site with a higher affinity, K1 = 2.25 X 10(3) M and n1= 25, and the other site with a lower affinity, i.e. higher capacity. In the cases of mustard Mannich base we could demonstrate the same type of curve, K1 = 2.20 X 10(5) M and n1 = 1.0, whereas another site has a lower affinity and greater number of binding sites.

Nephrotic changes in the rat induced by overdosage of triamterene.

Daily treatment of rats with 2,4,7-triamino-6-phenylpteridine (triamterene, Dyrenium) a potassium sparing diuretic, in daly doses of 1.5 mg, 3 mg and 4.5 mg/100 g body weight over the period of three weeks caused severe degenerative changes of renal cortical and medullary tubules resembling osmotic (sucrose) nephrosis. The undesirable side-effects were absent in the kidneys of intact rats receiving human therapeutic dose of triamterene (0.36 mg/100 g body weight) and even higher dose (1.5 mg/100 g body weight) but administered according to the prescription of treatment recommended by the manufacturer--daily over the first week and three times weekly after that. Rats .2 days after adrenalectomy administered triamterene in daily dose of 1.5 mg/100 g body weight, survived no more than 6--7 days of treatment. The results presented in this paper undoubtedly show that triamterene, although a mild diuretic agent, should be monitored in order to avoid undesirable side-effects. In addition care should be taken in patients with hypofunction of adrenal cortex.

The effect of triamterene upon the rat kidney plasma membrane Na-K-ATP-ase activity.

Triamterene (2,4,7-triamino-6-phenylpteridine) is a potassium-sparing diuretic whose mechanism of action is not clear. Experiments were performed to study the effect of triamterene upon the activity of Na-K-ATP-ase in the kidney plasma membranes. This enzyme, dissolved within the membrane bilayer, has been considered to be a biochemical vehicle for the active transport of sodium across the cell membrane. Intact and adrenalectomized rats were subjected to a five-day treatment with triamterene in a daily dose of 1.5 mg/100 g body weight. Triamterene was also administered to a group of intact, salt-loaded, rats. The activity of Na-K-ATP-ase in the kidney plasma membranes of intact and adrenalectomized rats treated with triamterene was decreased by 22.4% (p less than 0.05) and 37.2% (p less than 0.05), respectively. The activity of Na-K-ATP-ase in the renal plasma membranes of intact, salt-loaded, rats underwent greater decrease--63% (p less than 0.05). If the decreased activity of Na-K-ATP-ase in the kidney plasma membranes of rats treated with triamterene manifested the diuretic action of triamterene, results obtained in adrenalectomized rats would suggest that triamterene acts directly on the kidney, not via the adrenal glands.

Proline and glycine uptake by renal brushborder membrane vesicles.

Uptake of L-proline and glycine by rat renal brushborder membrane vesicles was seen to be osmotically sensitive, pH dependent,and occurred in the absence of proline and glycine metabolism. The uptake system for proline was Na+ gradient dependent, and exhibited a dual system for entry, Km1 = 0.067 mM and Km2 = 5.26 mM. The uptake of glycine was also Na+ gradient dependent, and exhibited a two Km system, Km1 = 0.22 mM and Km2 = 4.00 mM. Studies of proline and glycine interactions indicate a shared site which has a lower affinity and higher capacity for glycine than for proline. The high affinity glycine site and low affinity proline site do not appear to be shared.

Renal handling of [(14)C]mannose.

The uptake of D-[(14)C]mannose by adult rat kidney cortex slices has been examined. The process involved appeared to be saturable and oxygen dependent and to be inhibited by glucose, galactose, chi-methyl-D-glucoside, and phlorizin. Extensive metabolism of the sugar occurred. (14)CO(2) was produced to an extent comparable to that from D-glucose. Radioactive compounds accumulated in the tissue, approximately 50% of which were in the form of phosphorylated or other polar intermediates. The remaining neutral fraction consisted of mannose with a small amount of label in glucose and an unidentified compound which behaves like an alditol. Mannose concentration in the intracellular fluid was never greater than that of the medium.

The mechanism of liver splanchnomegaly produced by a transplantable pituitary mammotropic tumor in rats.

The mechanism of liver splanchnomegaly developed in rats bearing a transplantable pituitary mammotropic tumor which secretes large amounts of ACTH and prolactin has been studied. The results indicate that in the first phase of tumor growth liver enlargement could be due to hypertrophy, and later mainly to hyperplasia which overcomes hypertrophy. The adrenal glands were found to play an essential role in the process of liver splanchnomegaly because adrenalectomy prevented the disproportional growth of the liver. Evidence is presented showing that glucocorticoids are the dominant hormones responsible for the development of liver splanchnomegaly.

The role of the adrenal gland in the lipid accumulation process in the liver of rats bearing an acth and prolactin producing tumor.

The role of the adrenal glands in the development of fatty liver was investigated in rats bearing a transplantable pituitary mammotropic tumor which produces large quantities of ACTH and prolactin. The biochemical and histochemical and histochemical evidence obtained has demonstrated that the adrenal glands, particularly glucocorticoids, are essential for lipid accumulation in the liver of rats with tumor.

Inhibitory effect of prolactin on the development of fatty liver induced by ACTH in thrat.

The action and interaction of ACTH and prolactin in the development of fatty liver were investigated in intact rats treated with exogenous hormones. Administration of ACTH or of combinations of ACTH and GH to intact female rats was found to elicit significantly greater increase in liver total lipids content and concentration than administration of combinations of ACTH, or ACTH and GH, with prolactin. In addition, the results support the data reported by Bates et al. (6) that simultaneous application of GH, prolactin and ACTH reduces the effct of ACTH, upon adrenal gland weight.