[(3)H]-
oxytocin was used to characterize the
oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-
oxytocin to USMC plasma membranes was dependent upon time, temperature and
membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1)
protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-
oxytocin binding showed that
oxytocin and
vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-
oxytocin in a concentration-dependent manner. The order of potencies for
peptide agonists and antagonists was:
oxytocin>[Asu(1,6)]-
oxytocin>AVP=
atosiban>d(CH(2))(5)
Tyr(Me)AVP>[Thr(4),Gly(7)]-
oxytocin>
dDAVP, and for nonpeptide antagonists was:
L-371257>
YM087>
SR 49059>
OPC-21268>
SR 121463A>
OPC-31260.
Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and
hyperplasia in USMC. The
oxytocin receptor antagonists,
atosiban and
L-371257, potently and concentration-dependently inhibited
oxytocin-induced [Ca(2+)](i) increase and
hyperplasia. In contrast, the V(1A) receptor selective antagonist,
SR 49059, and the V(2) receptor selective antagonist,
SR 121463A, did not potently inhibit
oxytocin-induced [Ca(2+)](i) increase and
hyperplasia. The potency order of antagonists in inhibiting
oxytocin-induced [Ca(2+)](i) increase and
hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-
oxytocin binding site found in human USMC is a functional
oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of
oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139