Identification and Characterization of Arginine Vasopressin Receptors in the Rat Testis*

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Abstract
Binding of neurohypophyseal peptides to interstitial cells of the rat testis was studied using [3H]AVP as the ligand. Interstitial cells were obtained from adult rat testis after collagenase dispersion and were incubated with [3H]AVP in the presence of absence of unlabeled AVP. Binding equilibrium was reached by 60 min at 4.degree. C, while incubation at higher temperatures (23.degree. and 37.degree. C) resulted in an apparent decrease in binding. Scatchard plot analysis of equilibrium binding data revealed the existence of 1 class of high affinity, low capacity binding sites (Kd = 1.0 .+-. 0.3 nM; maximal binding = 8.5 fmol/106 cells). The rate constants of association and dissociation were calculated to be 0.024 nM-1 and 0.009 min-1, respectively. Addition of naturally occurring neurohypophyseal hormones as well as their synthetic analogs inhibited [3H]AVp binding to testis cells, resulting in parallel displacement curves. The order of potencies for the native peptides was: AVP = lysine vasopressin = arginine vasotocin (IC50 5 .times. 10-10 M) > oxytocin = mesotocin (IC50, 4 .times. 10-7 M) > isotocin = glumitocin (IC50 > 10-6 M). Two potent vasopressor antagonists, d(CH2)5Tyr(Me)AVP ([1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine]AVP) and dPTyr(Me)AVP ([1-desaminopenicillamine-2-(O-methyl)tyrosine]AVP) competed for [3H]AVP binding with a higher affinity (IC50 .apprx. 10-11 M) than native AVP. A selective antidiuretic agonist, dDAVP (1-desamino-8-D-AVP), only competed weakly for receptor binding, while a specific oxytocic agonist, (Thr4,Gly7)oxytocin, did not affect AVP binding. The testis may contain the V1 receptor subtype. Studies on the intratesticular distribution of AVP receptors indicated minimal binding to cells derived from the seminiferous tubule, while most of the AVP-binding sites sediment with enriched fractions of Leydig cells after Metrizamide density gradient centrifugation. AVP-binding sites were also found in rat liver, kidney and anterior pituitary (10.7, 2.6 and 1.7 fmol/mg protein), whereas adrenal, cerebellum, prostate and hypothalamus were devoid of AVP-binding sites. The presence of high affinity, stereospecific receptors for AVP were demonstrated in the interstitial cell compartment of the rat testis. These V1 receptors may mediate the direct inhibitory action of neurohypophyseal hormones on testicular Leydig cell steroidogenesis.

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