Characterization of a proposed oxytocin receptor in the uterus of the rat and sow.

[3H]Oxytocin, incubated with rat uterine segments, was bound specifically, and about equally, to the three particulate fractions obtained by homogenization and centrifugation at 1,000, 20,000, and 105,000 X g. There apparently was no binding in the cytosol fraction. [“H]Oxytocin, added directly to rat uterine particles sedimentmg between 1,000 and 20,000 x g, was bound with an apparent & of 1.8 X 10qv M; 0.18 pmole of oxytocin was bound per mg of protein. For the 20,000 x g particulate fraction of the uterus from a pregnant sow, the apparent Kd for oxytocin binding was 1.5 X 10mv M; 0.15 pmole of oxytocin was bound per mg of protein. The affinity of oxytocin for the 105,000 x g particles from sow uterus was about 3 times greater than for the 20,000 x g particles. The binding constants are of the same order as the concentration of oxytocin which elicits a half-maximal uterine response. The binding of [aH]oxytocin by sow uterine 20,000 x g particles was maximal by 40 min at 20’. Less binding occurred at 37 and 0” than at 20”. The competition for [sH]oxytocin binding sites by synthetic analogs of oxytocin was specific: oxytocin = [4-threonineloxytocin = [8-valineloxytocin > [&lysine]vasopressin >> [4-prolineloxytocin. Binding was optimal in the range of pH 7.4 to 7.8, was enhanced by Mn2+ and Co2+ > Mg2f > Zn2f, and was absent with 1 mu ethylenediaminetetraacetate. The concentrations of Mn2f giving maximal and half-maximal augmentation of binding were 5 and 0.4 mM, respectively. Ca2+, 5 mm, did not affect binding. A portion of the [aH]oxytocin appeared to be metabolized during the incubation with sow uterine particles. The uterine oxytocin receptor site is protein, at least in part, because binding was reduced by trypsin, -SH reagents such as N-ethylmaleimide, p-chloromercuribenzoate, and by thioglycollate. Phospholipases A and C inhibited binding; however, their inhibitory activities appeared to be separate from their catalytic activities. Neuraminidase, phospholipase D, butanedione, and sodium fluoride did not affect [aH]oxytocin binding. Binding was depressed by guanosine triphosphate, cytosine triphosphate, and uridine triphosphate.