Dissociation of natriuresis and diuresis by oxytocin molecular forms in rats

Abstract

In the rat, oxytocin (OT) produces dose-dependent diuretic and natriuretic responses. Post-translational enzymatic conversion of the OT biosynthetic precursor forms both mature and C-terminally extended peptides. The plasma concentrations of these C-terminally extended peptides (OT-G; OT-GK and OT-GKR) are elevated in newborns and pregnant rats. Intravenous injection of OT-GKR to rats inhibits diuresis, whereas injection of amidated OT stimulates diuresis. Since OT and OT-GKR show different effects on the urine flow, we investigated whether OT-GKR modulates renal action by inhibition of the arginine-vasopressin (AVP) receptor V2 (V2R), the receptor involved in renal water reabsorption. Experiments were carried out in the 8-week-old Wistar rats receiving intravenous (iv) injections of vehicle, OT, OT-GKR or OT+OT-GKR combination. OT (10 μmol/kg) increased urine outflow by 40% (P<0.01) and sodium excretion by 47% (P<0.01). Treatment with OT-GKR (10 μmol/kg) decreased diuresis by 50% (P<0.001), decreased sodium excretion by 50% (P<0.05) and lowered potassium by 42% (P<0.05). OT antagonist (OTA) reduced diuresis and natriuresis exerted by OT, whereas the anti-diuretic effect of OT-GKR was unaffected by OTA. The treatment with V2R antagonist (V2A) in the presence and absence of OT induced diuresis, sodium and potassium outflow. V2A in the presence of OT-GKR only partially increased diuresis and natriuresis. Autoradiography and molecular docking analysis showed potent binding of OT-GKR to V2R. Finally, the release of cAMP from CHO cells overexpressing V2 receptor was induced by low concentration of AVP (EC50:4.2e-011), at higher concentrations of OT (EC50:3.2e-010) and by the highest concentrations of OT-GKR (EC50:1.1e-006). OT-GKR potentiated cAMP release when combined with AVP, but blocked cAMP release when combined with OT. These results suggest that OT-GKR by competing for the OT renal receptor (OTR) and binding to V2R in the kidney, induces anti-diuretic, anti-natriuretic, and anti-kaliuretic effects.

Fig 1. Dose-dependent effects of OT and OT-GKR on urine and electrolyte flow in rat kidney.

(A) Equimolar doses of oxytocin (OT) and oxytocin extended form (OT-GKR) were administered intravenously at different concentrations (1, 2, 4 and 10 μmol/kg) to assess urine, (B) sodium and (C) potassium outflow over a period of 5 hours. The effects of equimolar doses of OT and OT-GKR (10 μmol/kg) on (D) urine, (E) sodium and (F) potassium outflow. Both OT and OT-GKR were administered as a single treatment and in combination (OT+OT-GKR). Open circles represent OT and black squares represent OT-GKR. Results are expressed as relative values compared to the control (ctrl) saline injection. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s post-hoc test. *: p<0.05, **: p<0.01, ***: p<0.001 compared to the ctrl injection.

Fig 2. The effects of oxytocin (OT) and oxytocin extended form (OT-GKR) on diuresis, natriuresis and kaliuresis in the presence of an oxytocin receptor antagonist (OTA).

(A-A2) diuresis, (B-B2) natriuresis and (C-C2) kaliuresis. Measurement of urine and electrolyte outflow was performed following a normal volume injection (0.1 mL) and with blood volume expansion (5 mL, BVE). Statistical analysis was performed using a two-way ANOVA followed by a Bonferroni post-hoc test. *: p<0.05, **: p<0.01, ***: p<0.001 compared to the ctrl injection.

Fig 3. The effects of oxytocin (OT) and oxytocin extended form (OT-GKR) on diuresis, natriuresis and kaliuresis in the presence of a vasopressin receptor 2 antagonist (V2A).

(A-A2) diuresis, (B-B2), natriuresis and (C-C2) kaliuresis. Measurement of urine and electrolyte outflow was performed following a normal volume injection (0.1 mL) and with blood volume expansion (5 mL, BVE). Statistical analysis was performed using a two-way ANOVA.

Fig 4. Autoradiography kidney representations of AVP, OT-GKR and OT in competition with I¹²⁵AVP.

(A). Representative images of autoradiography registered by Phosphorimager. (B) The competitive binding efficiency of AVP, OT-GKR, OT, with I¹²⁵AVP. Statistical analysis: One-way ANOVA with a Dunnett’s post-test. *: p<0.05, **: p<0.01, ***: p<0.001 vs total binding displacement.

Fig 5. Schematic model of human vasopressin receptor (V₂R) binding with oxytocin (OT), oxytocin extended form (OT-GKR), and arginine vasopressin (AVP).

(A) The front upright view position (side view) of the receptor structure with OT. (B) The top intracellular view (i.e. rotation by 90^o out of plane) of OT-GKR inside the OTR binding site.(C) Conformational view of AVP, OT-GKR, and OT and (D) schematic model of human vasopressin V₂R binding with AVP, OT-GKR and OT. The amino acid residues in black circles have been proposed as OT docking sites, the red bars represent docking sites of OT-GKR and the green bars represent docking sites of AVP. Amino acid residues are identified by a 1-letter code in Table 1.

Fig 6. The effects of oxytocin (OT) and oxytocin extended form (OT-GKR) on intracellular calcium levels measured in Fura-2-loaded H9c2 cells.

(A). Percentage of calcium-responsive cells counted per microscopic field after treatment with equimolar concentrations (10⁻⁶ M) of OT and OT-GKR. (B) Kinetics of intracellular calcium levels normalized as the F340/F380 ratio in cells responding to OT-GKR and (C) in cells resistant to OT-GKR Data were obtained from 7 separate experiments. For (A), statistical analysis was performed using a Students t-test. Data are presented as mean ± SEM. ***: p<0.001, compared to OT-GKR-treated cells.

Fig 7. The effects of arginine vasopressin (AVP), oxytocin (OT), and oxytocin extended form (OT-GKR) on intracellular cAMP levels in AVP-R2 CHO-K1 cells.

(A) Release of cAMP from AVP-R2 CHO-K1 cells in response to increasing concentrations of AVP, OT, and OT-GKR. (B) Release of cAMP from AVP-R2 CHO-K1 cells treated with AVP as antagonist and in combination with OT-GKR used as agonist. OT-GKR was added in AVP-R2 CHO-K1 cells at the concentration of 10^-7M. (C) Release of cAMP from control CHO cells and AVP-R2 CHO-K1 cells containing the vasopressin receptor 2 (V₂R). Cells were stimulated with OT and OT-GKR in the presence of the V₂R antagonist (V2A, 10⁻⁵ M) or the oxytocin receptor antagonist (OTA, 10⁻⁶ M).