Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.
[(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
Tahara
A
A
Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan. tahara@yamanouchi.co.jp
Tsukada
J
J
Tomura
Y
Y
Wada
K i
Ki
Kusayama
T
T
Ishii
N
N
Yatsu
T
T
Uchida
W
W
Tanaka
A
A
D065092:Antidiuretic Hormone Receptor Antagonists; D001127:Arginine Vasopressin / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects; D005260:Female; D006801:Humans; D006965:Hyperplasia / Q000139:chemically induced / Q000473:pathology; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects*; D010121:Oxytocin / Q000031:analogs & derivatives / Q000494:pharmacology; D018045:Receptors, Oxytocin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000187:drug effects*; D017483:Receptors, Vasopressin / Q000819:agonists; D015290:Second Messenger Systems / Q000187:drug effects; D014599:Uterus / Q000166:cytology / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D065092:Antidiuretic Hormone Receptor Antagonists; D008024:Ligands; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin; D014662:Vasoconstrictor Agents; D001127:Arginine Vasopressin; D010121:Oxytocin; D002118:Calcium