Affinity, potency and efficacy of [(D-Leu)2,Ile3,Thr4]-AVP, d[Cha4,Dab8]-AVP, and d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-AVP.

Affinity (Ki) and potency (EC50) data are indicated as mean ± SD (nM, n = 3); Ki values were calculated from IC50 values according to Cheng and Prusoff, assuming Kd values of 0.65 nM for OTR, 0.21 nM for V1aR and 0.14 nM for V1bR. Efficacy (Emax) data is normalized to the maximum IP1 formation by the control. #controls were OT at the OTR or AVP at the V1aR and V1bR; n.d., not determined.

The effect of AVP at the substimulatory glucose on β (A, B, F) and α (C, D, G) cells.

(A, C) Regions of interest (ROIs) were obtained with our analytical pipeline. Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 3 time periods where events per minute per ROI parameter was sampled for panels F and G. (bottom panel) Representative traces from ROIs indicated in A and C. (E) A scheme showing how the height, duration at half amplitude (halfwidth) and area under the curve (AUC) were measured for each Ca2+ event detected. (F, G) Events per minute per ROI were normally distributed, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD. The significant differences in AVP treatment at the substimulatory glucose were found in α cells only (*p ˂ 0.05).

The effect of AVP in the physiological stimulatory glucose range on β (A, B, E, G, H) and α (C, D, F) cells.

(A, C) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 3 time periods where events per minute per ROI, Halfwidth and AUC parameters were sampled for panels E-H. (bottom panel) Representative traces from ROIs indicated in A and C. (E, F) Forskolin significantly increased the number of Ca2+ oscillations per minute per ROI in β cells that were further stimulated by addition of AVP. A similar effect was observed also in α cells. (G, H) A non-significant but visible decrease in the events’ halfwidth and AUC values due to forskolin and AVP administration in β cells were noted. The parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD (*p ˂ 0.05, **p ˂ 0.01, **p ˂ 0.001).

The effect of physiological epinephrine concentration β (A, B, E) and α (C, D, F) cells.

(A, C) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 7 time periods where events per minute parameter was sampled for panels E and F. (bottom panel) Representative traces from ROIs indicated in A and C. (E, F) The analysis of events per minute per ROIs shows that inhibition by epinephrine of glucose-dependent activation is reversed with increasing vasopressin concentration (0.01, 0.1, 1, 10 and 100 nM) in α cells but not in β cells. The parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD (*p ˂ 0.05, **p ˂ 0.01, **p ˂ 0.001).

AVP modulates the activity of β (A, B, E-G, I, J) and α (C, D, G, H) cells in a concentration-dependent manner.

(A, C) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 7 time periods where events per minute per ROI and halfwidth parameters were sampled for panels F, H and J. (bottom panel) Representative traces from ROIs indicated in A and C. (E) Supraphysiological concentrations of AVP (above 10 nM) reduced insulin release from β cells below that measured at 8 mM glucose and forskolin alone. (F, H) Events per minute per ROI in β cells was gradually increased from 0.0001 nM to the physiological osmoregulatory range of AVP, between 0.01 and 0.1 nM, and then gradually decreased with further increases in AVP concentration. In the same islets the number of events per minute increased gradually in α cells also at higher AVP. (G) Pooling several islets revealed that bell-shaped distribution of the AUC could be found in both β and α cells, although the latter being shifted towards higher AVP concentration. (I) The AVP-dependent activation/inactivation curve for β cells was left-shifted at supraphysiological glucose concentration. (J) The halfwidth of events in β cells progressively declined with increased AVP concentration. All the parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD (*p ˂ 0.05, **p ˂ 0.01, **p ˂ 0.001).

Schematic structures and pharmacology of peptide probes [(D-Leu)2,Ile3,Thr4]-VP, d[Cha4,Dab8]-VP, and d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-VP.

(A) Schematic structures and amino acid compositions of oxytocin (OT), vasopressin (AVP), and their derivatives. (B) Radioligand displacement assay of [(D-Leu)2,Ile3,Thr4]-VP, d[Cha4,Dab8]-VP, and d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-VP. Concentration-dependent displacement of 3H-OT (0.65 nM) or 3H-AVP (0.21 nM for V1aR, 0.14 nM for V1bR) in HEK293 cell membranes expressing the human OTR, V1aR or V1bR by either test ligand or the respective control. Data is presented as specific binding by subtracting nonspecific from total binding normalized to the maximum binding of the radioligand in the absence of the peptides (n = 3). (C) Ligand induced formation of intracellular IP1 by 10 µM OT/AVP, [(D-Leu)2,Ile3,Thr4]-VP, d[Cha4,Dab8]-VP, and d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-VP demonstrating agonistic properties of [(D-Leu)2,Ile3,Thr4]-VP and d[Cha4,Dab8]-VP at V1bR, whereas in the sense of an antagonism no increased IP1 accumulation was observed at OTR and V1aR (n = 2). Activation data was normalized to maximum response generated by the control (OT for OTR, AVP for V1aR and V1bR). All data points are shown as mean ± SD. (D) Antagonistic properties of d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-VP at V1aR were assessed through concentration-dependent displacement of AVP according to the Schild method (n=3). (E) Concentration-response curves of AVP, [(D-Leu)2,Ile3,Thr4]-VP, and d[Cha4,Dab8]-VP were obtained for the V1bR in order to assess their potency (n = 3). Affinity constants (Ki), potency (EC50) and maximum efficacy (Emax) values for [(D-Leu)2,Ile3,Thr4]-VP, d[Cha4,Dab8]-VP, d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-VP, and the respective controls are listed in Table 1. Abbreviations: Cha = cyclohexylalanine; Dab = 2,4-diaminobutyric acid; d = desamino; X1 = D-Leu; X2 = d(CH2)5 (β-mercapto-β,β-cyclopentamethylenepropionic acid); YMe and Tyr(Me)= O-methylated tyrosine.

A specific V1b receptor agonist and V1a and oxytocin receptor antagonist, has a similar effect on β (A, B, E) and α (C, D, F) cells as AVP.

(A, C) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 2 time periods where events per minute per ROI and AUC parameters were sampled for panels E and F. (bottom panel) Representative traces from ROIs indicated in A and C. (E) There were no significant differences in the pooled AUC data for β cells treated with the [(D-Leu)2,Ile3,Thr4]-VP and forskolin or with forskolin only. Direct comparison of the AUC values before and after the ligand application shows a large heterogeneity of responses. (F) A significant increase in the events per minute per ROIs due to [(D-Leu)2,Ile3,Thr4]-VP administration in α cells was noted. (G) There was no significant effect on insulin release from slices after [(D-Leu)2,Ile3,Thr4]-VP exposure in comparison to stimulatory forskolin only. (H, I) The d[Cha4, Dab8]-VP (a specific V1b receptor agonist) and AVP produced similarly heterogeneous responses in β cells with non-significant changes in the AUC compared to forskolin only. All the parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD (*p ˂ 0.05, **p ˂ 0.01, **p ˂ 0.001).

A specific V1a receptor antagonist has no significant effect on β (A, B, E, F) and α (C, D) cells, but inhibits activity in smooth muscle cells

(G). (A, C) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. Inset labelled with green dashed line is expanded in panel G. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 3 time periods where events per minute per ROI and AUC parameters were sampled for panels E and F. (bottom panel) Representative traces from ROIs indicated in A and C. (E) There were no significant differences in the pooled AUC data for β cells treated with the d[(CH2)51, Tyr(Me)2, Dab5, Tyr9]-VP and forskolin or with forskolin only. Direct comparison of the AUC values before and after the antagonist application shows no difference in responses. (F) No significant differences in the events per minute per ROIs due to d[(CH2)51, Tyr(Me)2, Dab5, Tyr9]-VP administration in α cells were noted. (G) (left) Inset from C, showing location of two smooth muscle cells cells lining the blood vessel adjacent to the islet (right) Representative traces from ROIs on smooth muscle cells exposed to stimulatory glucose, forskolin, AVP, and d[(CH2)51, Tyr(Me)2, Dab5, Tyr9]-VP. AVP administration increased the frequency of Ca2+ oscillations in smooth muscle cells, but V1a receptor antagonist fully, but reversibly inhibited this activity. All the parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD.

Overview of the analysis of pharmacological modulation of V1b receptors in β cells in pancreatic tissue slices.

(A) The relative rates of Ca2+ oscillations obtained during the AVP concentration ramp experiment. The bell-shaped dependence curve rises at the lower pM range of AVP, followed by a relatively stable response around the peak of the curve, and then falls at higher concentrations (nM range) of AVP. (B, C, D) Normalized rates to the basal stimulatory conditions (8 mM glucose) in the context of single concentration static incubation with AVP, [(D-Leu)2, Ile3, Thr4]-VP and d[Cha4,Dab8]-VP are widely scattered. (E, F, G) Normalized rates to the basal stimulatory conditions (8 mM glucose) in the context of single concentration static incubation without AVP receptor agonists (control), with tolvaptan (V2 antagonist) and d[(CH2)51,Tyr(Me)2,Dab5,Tyr9]-VP (V1a antagonist) are only slightly scattered. (H) Different islets during their plateau phase stimulated by 8 mM glucose and forskolin can achieve different levels of IP3 receptor activation equivalent, contributing to the heterogeneity of further IP3R stimulation with AVP or specific V1b agonists.

The effect of physiological levels of glucose and forskolin on β (A, B, E, G, H) and α (C, D, F) cells.

(A, C) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B, D) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 3 time periods where events per minute per ROI parameter was sampled for panels E and F. (bottom panel) Representative traces from ROIs indicated in A and C. (E, F) Prolonged exposure to physiological glucose stimulation and low forskolin concentration resulted in a stable Ca2+ oscillations over almost an hour in β and α cells. The parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD.

Tolvaptan, a V2 receptor antagonist, has no significant effect on β cells.

(A) Indicated are the ROIs whose filtered traces correlate best with the average trace for the whole islet. (B) (top panel) Time distribution of all measured eventś halfwidths at their peak times. Stimulation protocol is indicated above. The color bar indicates the bin count in each time/halfwidth point. The dashed-line rectangles indicate the 3 time periods where events per minute per ROI parameter was sampled for the panel. (bottom panel) Representative traces from ROIs indicated in A (C) Tolvaptan had no significant effects on the number of events per minute per ROI. The parameters were distributed normally, we used one-way ANOVA and Tukey post-hoc test. The data are presented as mean +/-SD.

Overview of molecular structures, chromatograms, and mass spectra of synthesized compounds.

Amino acid residues that differ from native vasopressin were marked in different colors. Analytic RP-HPLC chromatograms were measured for product purity determination at 214 nm. Solvent A (ddH2O + 0.1% TFA) and B (ACN + 0.08% TFA) were used as eluents at 1 mL/min flow rate and a linear gradient of 5-65% B in 30 min on a Thermo Fisher Scientific Vanquish Horizon UHPLC system using a Kromasil Classic C18 column (4.6 x 150 mm, 300 Å, 5 µm). Mass spectra were measured for product identity verification through direct injection on a Thermo Scientific Dionex Ultimate 3000 system equipped with a Thermo Scientific MSQ Plus ESI-MS unit (positive ionization mode).

Name, sequence, purity, and mass identity of synthesized vasopressin analogs and vasopressin.

Amino acid residues that differ from native vasopressin were marked in different colors.