Elwood Titus

THE ROLE OF THE RENIN-ANGIOTENSIN SYSTEM IN THE CONTROL OF ALDOSTERONE SECRETION

Within the last year, evidence (1-4) has accumulated to show that the kidney secretes a hormone which is a prime regulator of aldosterone secretion. The renal origin of an aldosteronestimulating hormone (ASH) has been demonstrated following acute blood loss (1-3), during chronic thoracic caval constriction (4), and during chronic Na depletion (4). Nephrectomizedhypophysectomized dogs failed to respond to acute hemorrhage with an increase in aldosterone secretion, and acute bilateral nephrectomy of hypophysectomized caval and hypophysectomized Nadepleted dogs resulted in a marked drop in aldosterone secretion. Furthermore, crude saline extracts of kidney produced a striking increase in aldosterone secretion (1-5). In malignant experimental renal hypertension, hyperaldosteronism was consistently present (6). These findings and the reports that renin preparations (6) and synthetic angiotensin II (6-8) increase the rate of aldosterone production suggest the possibility that ASH is renin. The present experiments were undertaken to determine the chemical nature of this ASH by fractionation of crude kidney extracts for aldosterone-stimulating and pressor activity. The renin content of kidneys from dogs with thoracic caval constriction and secondary hyperaldosteronism and from normal dogs has been compared. Since dogs with thoracic caval constriction and Na-depleted dogs do not have hypertension, the response in blood pressure to synthetic angiotensin

The uptake of labeled norepinephrine by isolated brain and other tissues of the cat

Summary Slices from brain and peripheral sympathetically innervated tissues of cats take up labeled norepinephrine to several times the concentration of this amine in the incubation media by a mechanism having the characteristics of active transport. The labeled norepinephrine remains in the cell in a site protected from metabolic degradation. Upon release from this site it is rapidly metabolized by monoamine oxidase in brain and o-methyl transferase in heart. Inhibition of metabolism by monoamine oxidase has no effect on uptake. Uptake is inhibited by reserpine and cardiac glycosides and may play a role in maintaining tissue stores of norepinephrine.

Uptake of tritium-labeled norepinephrine in brain and other tissues of cat in vitro.

Slices of cat cerebral cortex, heart, and spleen that have been incubated in media containing approximately 5 to 25 m�g of dl-norepinephrine-7H8 per milliliter contain levels of isotopic amine greater than those in the medium. The effects of norepinephrine concentration, reserpine, and ouabain on the uptake suggest that the amine enters cells both by diffusion and by a concentrating miechanism that is saturated at low levels of norepinephrine. The drugs inhibit the latter.

A comparison of microsomal (Na+ + K+)-ATPase with K+-acetylphosphatase.

Abstract Similar distributions of radioactivity were observed upon paper electrophoresis of pepsin-digested beef brain microsomes previously exposed to either [ 32 P]ATP or acetyl [ 32 P]phosphate in the presence of Na + and Mg 2+ . ATP and acetylphosphate were each competitive inhibitors of the other as substrate for a K + -requiring acetylphosphate (acylphosphate phosphohydrolase, EC 3.6.1.7) and for a (Na + + K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3). Despite these similarities, there were marked differences in the inhibitory effect of ouabain, oligomycin, and exposure to N -ethylmaleimide on the two enzymes. The data suggest that the K + -acetyl-phosphatase activity of the beef brain may represent a different entity than the ( Na + + K + )-ATPase.

Biosynthesis of cardiotonic sterols from cholesterol in the toad, Bufo marinus

Abstract 1. 1. The synthesis of the cardiotonic sterols, marinobufagin and marinobufotoxin, has been demonstrated in the toad, Bufo marinus . 2. 2. Approximately 2% of administered cholesterol-4-C 14 is converted to the sterol lactones in a 75-day period. No incorporation of acetate-C 14 was detected.

Pharmacologic Receptor Activity of Rabbit Aorta: Effect of Dithiothreitol and N-Ethylmaleimide

N-ethylmaleimide (NEM), an alkylating agent that covalently binds sulfhydryl groups, and dithiothreitol (DTT, Clelands reagent), which reduces disulfide bridges to sulfhydryl groups, affected drug-induced contractions of rabbit aorta. NEM depressed aortic responses to potassium chloride (KCl), norepinephrine, serotonin, histamine, and angiotensin II. This action was attributed to an interaction of NEM with sulfhydryl groups vital to either the membrane function or the contractile apparatus. DTT had minimal effects on responses to KCl, norepinephrine, and serotonin; it potentiated aortic contractions produced by histamine and abolished responses to angiotensin II. Enhancement of the histamine response by DTT was not accompanied by an effect on the histamine-metabolizing enzymes or by an alteration in the KB and pA2 values for pyrilamine. The results suggest that a disulfide bridge plays a prominent role in both histamine and angiotensin receptor activity of rabbit aorta. With regard to the histamine receptor system, two possibilities exist: (1) reduction of a disulfide bridge at some vital point increases receptor activity or (2) the histamine receptor system, normally in a reduced state, is oxidized in the artificial environment of the tissue bath and needs to be reduced to regain full activity.

The seperation of catecholamine storage vesicles from rat heart

Abstract The concept that acetylcholine is released from certain presynaptic nerve terminals in packets or quanta of uniform size led to the speculation that the transmitter might be stored in synaptic vesicles (1,2). The isolation of acetylcholine-containing pinched-off nerve endings (synaptosomes) from brain (3) and more recently the synaptic vesicles themselves (4,5) have stimulated investigations of the subcellular localization of biogenic amines in peripheral and central nervous tissue. In guinea pig brain the storage particles that contain norepinephrine (NE) and 5-hydroxytryptamine have been found to differ from those containing acetylcholine in that the first two have sedimentation characteristics more dense than the acetylcholine containing vesicles (6). Recent studies have shown that catecholamines in heart (7,8), brain (6,9) and splenic nerve (10) are stored in subcellular vesicles. Potter and Axelrod (7,11) reported that 30 minutes after the intravenous administration of DL-norepinephrine-7-H 3 to rats a sizable fraction of the labeled compound in several peripheral tissues appeared in a fraction associated with microsomes. These authors suggested that the radioactive NE is taken up from the circulation and fixed at the anatomical sites which bind endogenous NE. The morphological characterization of these sites was not possible with the Potter and Axelrod preparation. A modification of the density gradient centrifugation procedure of Potter and Axelrod (11) has made it possible to isolate NE containing particles of considerably greater homogeneity. Both endogenous NE and administered H 3 NE are localized in a particulate fraction which equilibrates in that part of the gradient which corresponds in density ot 0.45–0.5 M sucrose. Electronmicroscopic examination of this fraction has revealed numerous small vesicles approximately 500 A in diameter, some microsomal fragments but essentially no other subcellular organelles.

The effect of hydroxylamine and N-methylhydroxylamine on beef brain microsomal (Na+ + K+)-ATPase

Abstract Although both hydroxylamine and N -methylhydroxylamine inhibited beef brain microsomal (Na + + K + )-ATPase these reagents were found to be of only limited use in probing the mechanisms of this enzyme which is thought to involve a protein-bound acyl phosphate group. Results obtained with hydroxylamine were difficult to interpret owing to the potassium-like effects of traces of ammonia. Experiments with N -methylhydroxylamine did not permit differentiation between a mechanisms involving an acyl phosphate on the enzyme surface which was inaccessible to the reagent from one in which the acyl phosphate is not an intermediate. These findings were disappointing in view of the proven usefulness of hydroxylamine for the investigation of enzyme mechanisms involving a high energy acyl group.

Evidence for subclasses of SH groups in (Na+ + K+)-ATPase

Changes in the chemical reactivity of the sulfhydryl groups of (Na+ + K+)-dependent ATPase can be indicative of conformational changes induced by activating ions. Cyanylation of these groups by 5 mM 2-nitro-5-thiobenzoic acid caused a partial inhibition of enzymatic activity. Both this loss and the incorporation of radioactive cyanide from the 14C-labeled reagent were reduced by inclusion of 50 mM ATP and 150 mM Na+ in the incubation. When 10 mM Mg++ was added in addition, the inactivation was not different from that produced by cyanylation reagent alone, but the radioactive labeling of protein increased significantly. The data indicate that the sulfhydryl groups of this enzyme exist in two populations, one of which must be free if the enzyme is to function. The other, not essential for enzymatic activity, becomes accessible only when the Na+ and Mg++-dependent phosphorylation of the enzyme alters its conformation. Inactivation of the enzyme by freezing and thawing increases the incorporation of radioactivity but destroys the responsiveness of labeling to cations and ATP.

Characterization of pharmacological receptors

SummaryThe isolation of macromolecules that bind drugs and neurohumoral mediators at receptor sites will require analytical methods adequate to detect these substances in homogenates and crude mixtures. Although the binding of isotopically labeled agonists has been useful in a few instances, equilibrium binding studies of radioactive, lipophilic antagonists promises to be more useful with β-adrenergic receptors. Alkylation by radioactive reagents that seek out specific receptors or specific functional groups on protein have been used in attempts to label other receptors. Conformationally directed labeling of receptor and receptor-related proteins by the latter group of alkylating agents has been used with receptors for cardiac glycosides and may be applicable to other systems.