Molecular Pharmacology, Vol 8, 230-240, Copyright © 1972 by the American Society for Pharmacology and Experimental Therapeutics

³H-Oxytocin Binding Sites in the Isolated Frog Skin Epithelium: Relation tO the Physiological Response

¹ Laboratoire de Physiologie Cellulaire, Collége de France, Paris 5^[unknown], France

The uptake of [³H]oxytocin by the isolated frog skin epithelial layer was studied under conditions that permitted simultaneous measurement of the biological response to the labeled hormone. Preliminary experiments showed the incorporation of a small amount of [³H]tyrosine into newly formed proteins since this incorporation can be blocked by first incubting the tissue with unlabeled tyrosine, puromycin, or cycloheximide. All further experiments were performed after the blockade of [³H]tyrosine incorporation. Measurement of [³H]oxytocin uptake as a function of the concentration of the labeled hormone in the medium showed the existence of two sets of binding sites. The sets differed in binding capacity and in affinity for oxytocin and its analogues. The sites with low binding capacity (1-2 pmoles/g) probably correspond to the receptors involved in the biological response (increase in active sodium transport), in accordance with the following criteria: (a) an apparent K value for binding (2.5 nM) identical with that determined from the dose-response relationship obtained for the same preparation; (b) a faster time course for binding than for the biological response; (c) [³H]oxytocin biniding in the presence of arginine-8-oxytocin and lysine-vasopressin similar to what might be expected from the relative biological potencies of these two analogues; and (d) parallel inhibition of the binding and biological response by O-methyltyrosine-carba-1-oxytocin, a competitive inhibitor of oxytocin on the frog skin epithelium. The second set of sites is characterized by a lower affinity (apparent K value, about 50 nM and a higher binding capacity (about 20 pmoles/g).

After the labeled hormone had been washed from the mediun, a significant amount of radioactivity was still present in the tissue, despite the complete reversal of the biological response. This fraction was released when dithiothreitol (10 mM) was added to the incubation medium during the rinsing period; it might correspond to covalent binding of the hormone to the structure through disulfide bonds and be unrelated to the biological effect. This interpretation is supported by the observation that O-methyltyrosine-carba-1-oxytocin, an oxytocin competitor lacking the disulfide bound, did not suppress this nonspecific binding of [³H]oxytocin.

This article has been cited by other articles:

				F.-l. Tan, S. J. Lolait, M. J. Brownstein, N. Saito, V. MacLeod, D. A. Baeyens, P. R. Mayeux, S. M. Jones, and L. E. Cornett Molecular Cloning and Functional Characterization of a Vasotocin Receptor Subtype That Is Expressed in the Shell Gland and Brain of the Domestic Chicken Biol Reprod, January 1, 2000; 62(1): 8 - 15. [Abstract] [Full Text]