Vasopressin receptors V1_aand V2 are not osmosensors

Abstract

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.

Evaluating the involvement of cerebral microvascular endothelial Na+/K+-ATPase and Na+-K+-2Cl–co-transporter in electrolyte fluxes in an in vitro blood–brain barrier model of dehydration

“…Biological activity of the vasopressin was therefore verified in V 1a R-expressing COS-7 cells in a parallel experimental series, which illustrated V 1a Rdependent down-regulation of AQP4 in V 1a R/AQP4expressing oocytes. 44 Ca 2þ imaging revealed lack of V 1a R activity in the primary cultures of bovine cerebral endothelium, which aligns well with low levels of V 1a R transcript in cerebral endothelium 12,25 and lack of V 1a R expression in capillary endothelial membrane as determined by immunohistochemistry and autoradiography. 26,52 Although we are unaware of the experimental explanation to the diversion from the report by O'Donnell et al 27 in which V 1a R-mediated NKCC1 activation was demonstrated, 27 a partial reason may be exposure of the co-culture to the glucocorticoid dexamethasone in order to induce BBB properties: 30 Dexamethasone exposure may abolish the vasopressin-dependent stimulation of NKCC1 activity.…”

“…(g) Ca 2+ response as a function of time determined in primary cultured endothelial monocultures loaded with Fura2-AM and treated with vasopressin (1 µM), ATP (100 µM), or control buffer. (h) Dose-dependent stimulation of IP 1 response by vasopressin in hV 1a R-transfected COS7 cells (figure adapted from Lykke et al., 44 n = 7). (i) Vasopressin-mediated stimulation of NKCC1 activity in hV 1a R/rNKCC1-expressing oocytes.…”

“…No vasopressin-dependent intracellular Ca 2þ response was observed, whereas addition of the positive control, ATP, to the extracellular medium produced a robust Ca 2þ response (Figure 4(g)). To ensure biological activity of the employed batch of vasopressin, we (in another project carried out simultaneously 44 ) tested the vasopressin-mediated inositol phosphate (IP) production in hV 1a R-expressing COS-7 cells. As illustrated in Figure 4(h), the V 1a R responded correctly to addition of vasopressin to the test medium with an EC 50 of 0.68 nM (figure adapted from Lykke et al 44 ).…”

“…To ensure biological activity of the employed batch of vasopressin, we (in another project carried out simultaneously 44 ) tested the vasopressin-mediated inositol phosphate (IP) production in hV 1a R-expressing COS-7 cells. As illustrated in Figure 4(h), the V 1a R responded correctly to addition of vasopressin to the test medium with an EC 50 of 0.68 nM (figure adapted from Lykke et al 44 ). These data indicate that the V 1a R is not functionally expressed in the cultured endothelial cells employed in these experiments.…”

Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias

“…Because mistrafficked mutants do not reach their biological site of action (so coupling cannot be assessed) and the use of pharmacoperones is a relatively new approach in drug discovery, there is little information regarding the characteristics of rescued mutants, such as whether they exhibit constitutive activity or second messenger bias. The WT V2R predominantly signals through coupling to Gs, however, it has been shown to couple to Gq by activating phospholipase C [ 15 , 16 ]. There are several V2R mutants that are retained in the ER, including the L83Q V2R mutant.…”