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Lykke K
,
Assentoft M
,
Fenton RA
,
Rosenkilde MM
,
MacAulay N
.
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Herein, we investigated whether G protein-coupled signaling via the vasopressin receptors of the V1a and V2 subtypes (V1aR and V2R) could be obtained as a direct response to hyperosmolar challenges and/or whether hyperosmolar challenges could augment classical vasopressin-dependent V1aR signaling. The V1aR-dependent response was monitored indirectly via its effects on aquaporin 4 (AQP4) when heterologously expressed in Xenopus oocytes and V1aR and V2R function was directly monitored following heterologous expression in COS-7 cells. A tendency toward an osmotically induced, V1aR-mediated reduction in AQP4-dependent water permeability was observed, although osmotic challenges failed to mimic vasopressin-dependent V1aR-mediated internalization of AQP4. Direct monitoring of inositol phosphate (IP) production of V1aR-expressing COS-7 cells demonstrated an efficient vasopressin-dependent response that was, however, independent of hyperosmotic challenges. Similarly, the cAMP production by the V2R was unaffected by hyperosmotic challenges although, in contrast to the V1aR, the V2R displayed an ability to support alternative signaling (IP production) at higher concentration of vasopressin. V1aR and V2R respond directly to vasopressin exposure, but they do not have an ability to act as osmo- or volume sensors when exposed to an osmotic gradient in the absence or presence of vasopressin.
Figure 1. V1aR-dependent downregulation of AQP4. (A) Volume traces obtained from an uninjected oocyte (left panel) and an AQP4/V1aR-expressing oocyte challenged with an osmotic gradient of 50 mOsm mannitol for 30 sec. (B) Relative water permeability of oocytes expressing AQP4 (open circles; n = 5) or coexpressing AQP4/V1aR (filled circles, n = 20) exposed to 1 μmol/L vasopressin as marked by the black bar. (C) Relative water permeability of oocytes expressing AQP4 (open circles; n = 6) or coexpressing AQP4/V1aR (filled circles; n = 22) when exposed to repeated osmotic challenges. (D) Relative water permeability of oocytes coexpressing AQP4/mGluR1a and exposed to 500 μmol/L glutamate as indicated by the black bar (filled symbols, n = 8) or kept in control solution (open symbols, n = 8, not exposed to glutamate). The groups were compared with two-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. *P < 0.05.
Figure 2. V1aR-dependent internalization of AQP4. (A) Confocal laser scanning microscopy of oocytes expressing either AQP4 (left panel) or AQP4/V1aR (right panel) immune-labeled for AQP4. The upper panels are representative images of oocytes exposed to control solution without vasopressin for 80 min. The middle panels are representative images of oocytes kept in control solution for 20 min and then treated with 1 μmol/L vasopressin for 60 min. The lower panels are representative images of oocytes treated with a 50 mOsm hyperosmolar gradient for 30 sec every 10 min of an 80-min incubation period. (B) Oocyte plasma membrane fluorescence intensity normalized to that of the oocytes kept in control solution, n = 5 experiments with 3–6 oocytes per condition. The indicated groups were compared using one-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. *P < 0.05; ns, not significant.
Figure 3. Hyperosmolar effects on V1aR-dependent inositol phosphate (IP) production. (A) Vasopressin dose–response curve measured with IP production in COS-7 cells transfected with either vector alone or with V1aR, n = 7. (B) IP production of COS-7 cells transfected with either vector alone or with V1aR upon addition of vasopressin (1 μmol/L) or various hyperosmolar treatments as indicated on graph, n = 3–8. (C) Effect of hyperosmolar treatment (50 or 150 mOsm) on the vasopressin dose–response curve, n = 3–8. (D) cAMP production obtained in COS-7 cells transfected with either vector alone or with V1aR in response to vasopressin exposure (1 μmol/L) or various hyperosmolar treatments as indicated on graph, n = 4–6. Data obtained with V1aR-transfected cells were compared to those obtained with vector-transfected cells using two-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test and indicated with * (note: the difference between the vasopressin-induced cAMP production in vector- and V1aR-transfected cells was not statistically significant even when compared with Student’s t-test). Statistical significance of vasopressin-induced cAMP production in vector-transfected cells was determined with two-way ANOVA with a Dunnett’s multiple comparison test and indicated with #. #P < 0.05; ***P < 0.001; ns, not significant.
Figure 4. Hyperosmolar effects on V2R-dependent cAMP and inositol phosphate (IP) production. (A) Vasopressin dose–response curve measured with cAMP response of COS-7 cells transfected with either vector alone or with V2R, n = 5. (B) cAMP production of COS-7 cells transfected with either vector alone or with V2R upon addition of vasopressin (1 μmol/L) or various hyperosmolar treatments as indicated on graph, n = 4–6. (C) IP production (black and gray circles) and cAMP accumulation (dashed line, adapted from panel A) of COS-7 cells transfected with V2R upon vasopressin exposure, n = 4–7. Data obtained with V2R-transfected cells were compared to those obtained with vector-transfected cells using two-way ANOVA with Šídák’s multiple comparison post hoc test and indicated with *. Statistical significance of vasopressin-induced cAMP production in vector-transfected cells was determined with two-way ANOVA with a Dunnett’s multiple comparison test and indicated with #. #P < 0.05; ***P < 0.001; ns, not significant.
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