A Comparison of the Ability of Leu8- and Pro8-Oxytocin to Regulate Intracellular Ca2+ and Ca2+-Activated K+ Channels at Human and Marmoset Oxytocin Receptors

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the non-apeptide OT structure is highly conserved with leucine in the eighth position (Leu(8)-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro(8)-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (G(s)) or inhibition (G(i/o)) of adenylyl cyclase, stimulation of potassium channel currents (G(i)), and activation of phospholipase C (G(q)). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro(8)-OT was more efficacious than Leu(8)-OT in measures of G(q) activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro(8)-OT and Leu(8)-OT differed with respect to G(q) signaling. In both mOTR- and hOTR-expressing cells, Leu(8)-OT was more potent and modestly more efficacious than Pro(8)-OT in inducing hyperpolarization. In mOTR cells, Leu(8)-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for G(i/o) 10 activation; however, the Pro(8)-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a G(q) signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated channels.