Effect Of Angiotensin On Trpv4 Expression And Trpv4 Agonist Induced Calcium Transients In Hypothalamic Cell Line 4b

We have previously demonstrated that in bile duct ligated rats, an animal model of inappropriate vasopressin (AVP) release, TRPV4 protein expression and membrane trafficking is increased in AVP neurons and correlated with circulating angiotensin II (Ang II). Here, we used an in vitro approach with an immortalized AVP expressing cell line (4B cells) to investigate the possible regulation of TRPV4 by Ang II. We have characterized, for the first time, the presence of TRPV4 mRNA and protein 4B cells. After Ang II (1μM;1 hr) treatment, TRPV4 mRNA increased nearly 5‐fold (Control vs. Ang II; 1.12±0.28 vs. 4.98±0.65; p<0.001). Western Blot analysis showed significantly (p<0.001) increased TRPV4 levels in crude membrane fractions of Ang II treated cells compared to vehicle controls. Also, WB analysis revealed increased tyrosine phosphorylation of TRPV4 following Ang II treatment as compared to vehicle treated cells (p<0.001). In addition, calcium imaging experiments using Fura‐2AM dye demonstrated a significant increase in the magnitude of calcium transients elicited by theTRPV4 agonist, GSK1016790A (0.1nM, p<0.05) after 1hr incubation in Ang II. Thus, 4B cells may be an appropriate in vitro model to investigate the translational and posttranslational regulation of TRPV4 in hypothalamic neurons. R01 HL062569 & P01 HL88052