Relation of Chemical Attachment to Physiological Action of Vasopressin.

We have suggested in preliminary publications" 2 that the antidiuretic action of vasopressin in the mammalian kidney may depend on a sulfhydryl-disulfide interchange involving SH groups on the receptor and the S-S bridge of the hormone. Strong support for this concept is presented in the preceding paper' where it is shown that the vasopressin-induced increase in the permeability of a simple living membrane (the toad urinary bladder) depends on the availability of free and accessible SH groups on the membrane and where it is also shown that this action of vasopressin does not depend directly on the oxidative metabolism nor on the sodium transporting function of the membrane. The present work, employing tritium-labeled arginine vasopressin, provides direct evidence for this concept, for it has been found that there is a close correlation between the formation of hormone-receptor disulfide bridges and the physiological action of vasopressin on membrane permeability. Experimental Methods.-Highly purified arginine vasopressin was prepared from a partially purified Pitressin powder4 by ion exchange chromatography5 and then tritiated by exposure to H3 gas under electric discharge.6 Easily exchangeable tritium was removed by repeated solution and lyophilization from dilute acetic acid, following which the tritiated arginine vasopressin (H3AVP)l was separated from radiation degradation products by the same chromatographic procedures used originally to purify the natural hormone. This work is described in detail by Fong, Silver, Christman, and Schwartz.7 In addition to chromatographic and electrophoretic evidence for purity, rat antidiuretic assay and rat pressor assay showed biological activity of approximately 400 pressor units/mg, which is comparable to potencies that have been reported for highly purified preparations of natural arginine vasopressin.8'2 The specific radioactivity of the purified H3AVP was 426 Auc/mg. The procedure used to measure the hormonal effect on the permeability of the bladder is described in the preceding paper.3 In our hands Pitressin, Pitocin, Syntocinon, 13 arginine vasopressin, H3-arginine vasopressin, lysine vasopressin and arginine vasotocin'4 all showed activity of the same relative potencies as reported by Sawyer. 15 Exposure of bladders to tritiated arginine vasopressin (H3AVP) was carried out in the same manner as experiments with unlabeled hormone or by a modified procedure in which the bladders were filled with Ringer's solution diluted 1:5, and then tied as balloons as they were slipped off a tapered glass tube. These "balloons" were then placed in an aerated bath of full-strength Ringer's solution. Hormone and/or sulfhydryl-binding reagents were added to the latter solution to give final concentrations in the bath as follows: H3AVP 2.5 X 10-8 M (ca.