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 nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu⁸-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro⁸-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, Pro⁸-OT was more efficacious than Leu⁸-OT in measures of G_q activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro⁸-OT and Leu⁸-OT differed with respect to G_q signaling. In both mOTR- and hOTR-expressing cells, Leu⁸-OT was more potent and modestly more efficacious than Pro⁸-OT in inducing hyperpolarization. In mOTR cells, Leu⁸-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for G_i/o activation; however, the Pro⁸-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 K⁺ channels.