Functional determination of oxytocin affinity in near-term pregnant rat myometrium: effect of chronic hypoxia

Abstract

We designed the present study to determine: (1) if phenoxybenzamine can be used as an irreversible blocker for oxytocin receptors, and as such to determine oxytocin affinity, (2) if prolonged hypoxic exposure alters oxytocin receptor coupling efficacy of oxytocin receptors to post-receptor mediated mechanisms in the rat myometrium. Rats were exposed to room air (control), or to continuous hypoxia (10.5% O 2) from day 19 through day 21 (2-day exposure). On day 21, one uterine horn was removed and used for in vitro study of myometrial contractile responses to oxytocin, while the other was used for oxytocin receptor analysis. In normoxic tissues, phenoxybenzamine (20 μM) decreased the maximum contractile response ( E MAX) to oxytocin (155±17 vs. 66±19 g s/cm 2) and oxytocin binding sites ( B MAX: 253±35 vs. 114.9±21.3 fmol/mg protein). A similar degree of reduction in E MAX and B MAX were observed in hypoxic tissues. The oxytocin dissociation constant ( K A) in the normoxic rat was 2.8±0.7 nM, which was not different from the chronic hypoxic rat (3.3±0.9 nM). Analysis of receptor occupancy–response curves indicated no oxytocin receptor reserve in both normoxic and hypoxic myometrium. However, for a given fraction of the total oxytocin receptors occupied, hypoxic tissue elicited a lower contractile response to oxytocin. We conclude that: (1) phenoxybenzamine is a useful tool to functionally study oxytocin receptor kinetics, (2) prolonged hypoxic exposure does not affect the oxytocin affinity, (3) no spare receptors for oxytocin are present in the rat myometrium, and (4) prolonged exposure to hypoxia decreases oxytocin receptor–effector coupling efficiency in rat myometrium.