Active-Site Studies of Neurohypophyseal Hormones: Comparison of Oxytocin and Arginine-Vasopressin with Analogues Containing 4-d-Glutamine

Previous studies have indicated that oxytocin at the uterine smooth-muscle receptor and vasopressin at the mammalian antidiuretic receptor utilize different structural and conformational properties to produce their biological effects. In both cases, however, the Gln⁴ residue has been proposed to be of primary importance for receptor recognition (binding) but not critical for biological activity (transduction). On the basis of these considerations, it would be predicted that [D-Gln⁴] oxytocin and [D-Gln⁴, Arg⁸]vasopressin would be weak but full agonists at the uterine and antidiuretic receptors, respectively. We have synthesized and purified the two D-Gln⁴ analogues and examined their pharmacological activities in several assay systems for these hormones. In agreement with the predictions, [D-Gln⁴] oxytocin and [D-Gln⁴, Arg⁸] vasopressin have greatly reduced potency at the in vitro uterine and in vivo antidiuretic assay systems, respectively, and both appear to be full agonists in these assays. However, the effects are quantitatively different, with [D-Gln⁴]oxytocin possessing 3.3 ± 0.2 units/mg of uterotonic activity (1/₁₇₀) the potency of oxytocin) and [D-Gln⁴, Arg⁸]vasopressin possessing only 0.45 ± 0.01 unit/mg (¹/₁₁₀₀ the potency of arginine vasopressin) of antidiuretic activity. Based on carbon-13 nuclear magnetic resonance spectral data, both [D-Gln⁴]oxytocin and [d-Gln⁴, Arg⁸] vasopressin have very similar conformations to oxytocin and arginine-vasopressin, respectively. Interestingly [d-Gln⁴]oxytocin has much more reduced biological activities relative to the native hormone in the avian vasodepressor (∼0.04 unit/mg, ∼¹/₁₂₀₀₀ that of oxytocin) and milk-ejecting (0.09 ± 0.02 unit/mg, ∼¹/₄₅₀₀ that of oxytocin) assays and is a weak partial agonist in the pressor assay. [d-Gln⁴, Arg⁸] vasopressin is a weak full agonist in the pressor assay (0.260 ± 0.004 unit/mg, ¹/₁₉₀₀ that of arginine-vasopressin) and also has weak uterotonic (∼0.11 unit/mg) and avian vasodepressor (0.10 unit/mg) activities.