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

Abstract

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 (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 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 Ca 21 -dependent activation of K 1 channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca 21 -activated K 1 channels.

Leu8 and Pro8 oxytocin agonism differs across human, macaque, and marmoset vasopressin 1a receptors

“…While OT acted as a partial agonist at human AVPR1a, OXT (both Leu 8 -OXT and Pro 8 -OXT) instead acted as a superagonist at marmoset AVPR1a. This is especially important from an evolutionary context because the Callitrichid clade has evolved widespread Pro 8 -OXT expression of the OXT ligand, and the Pro 8 ligand produces stronger behavioral effects 44,45 , potency and efficacy effects at marmoset OXTR 24,25 , and efficacy effects at marmoset AVPR1a (this study). It is unclear whether the higher agonism and coupling efficiency of OXT at marmoset AVPR1a is an important or conserved mechanism underlying the potential coevolution between OXTR and AVPR1a variability with socially monogamous phenotypes in primates 15,46 .…”

“…These pharmacological findings also partially align with previous work examining effects of Leu 8 -OXT and Pro 8 -OXT signaling at OXTR. Pro 8 -OXT exhibited modestly higher potencies than Leu 8 -OXT at primate OTRs 25 ; Pro 8 -OXT produced more efficacious Ca 2+ responses at marmoset OXTR but not at human OXTR 24 ; and Pro 8 -OXT produced lower recruitment of β-arrestin and less receptor desensitization and internalization at both human OXTR and AVPR1a, where only human receptors were tested 23 . Perhaps the most compelling finding from this study was that OXT exhibits differential agonism at human and marmoset AVPR1a.…”

“…Similar to OXT signaling at OXTR, Pro 8 -OXT was more efficacious than Leu 8 -OXT at marmoset but not human AVPR1a. Previous pharmacological studies of marmoset OXTR did not explicitly test if different OXT ligands were partial agonists at marmoset and human OXTR 24 , and that study did not make direct comparisons of OXT agonism to AVP agonism at OXTR. However, AVP appears to be a full agonist relative to both Leu 8 -OXT and Pro 8 -OXT at primate OXTR based on data reported across human, marmoset, macaque, and titi monkey OXTR 25 .…”

“…Pharmacological evidence also supports the notion that Pro 8 -OXT is more efficacious and potent than Leu 8 -OXT at primate OXTR. Pro 8 -OXT binds with higher affinity at primate OXTR (in both Pro 8 -OXT and Leu 8 -OXT expressing primates) and produces greater calcium signaling responses compared with Leu 8 -OXT 24,25 , but others have reported minimal signaling differences 23 . These data strongly suggest these evolutionary changes in the OXT ligand lead to important functional consequences presumably through OXT activation of OXTR.…”

Oxytocin Receptor Signaling in Vascular Function and Stroke

“…Whereas the suspected G i /G o pathway is less well-defined, it has been demonstrated that G iβγ signaling leads to p38 MAPK activation and aids cells in adaptive processes to physiological stressors through transcriptional activators and direct effects on cell stabilizing proteins (Hoare et al, 1999). Potential hyperpolarizing effects through G i could occur through interactions with Ca 2+dependent K + channels, as proposed based on recent studies using alternative peptides and chelators (Pierce et al, 2019). Hence, in addition to the well-established role for OXT as a contraction influencing hormone, it appears to be involved in cellular differentiation, migration, proliferation, responses to stressors, dilation, and inflammation.…”