SRC-KINASE MEDIATES ANGIOTENSIN II INDUCED POTENTIATION IN TRPV4 AGONIST EVOKED CALCIUM TRANSIENTS IN HYPOTHALAMIC IMMORTALIZED NEURONAL 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. Here, we used an in vitro approach with an immortalized AVP expressing neuroendocrine cell line (4B neurons) to investigate the possible regulation of TRPV4 by angiotensin II (Ang II). We characterized the presence of TRPV4 mRNA and protein in 4B cells. Ang II (100nM;1 hr) treatment significantly increased TRPV4 abundance (p. Purpose (a): ØInappropriate Vasopressin (AVP) release causes dilutional hyponatremia associated with heart and liver failure. Although the central molecular mechanisms that mediate inappropriate AVP release are not clear, plasma angiotensin II (Ang II) has been implicated as a factor in the pathogenesis of dilutional hyponatremia. Our previous studies using a rodent model of liver failure, have shown that increased TRPV4 expression in vasopressinergic neurons and elevated circulating AVP were blunted by normalization of the renin angiotensin system (RAS). Effects of circulating Ang II on neural networks may mediate cellular adaptations associated with changes in TRPV4 expression and/or sorting ØBased on our in vivo studies we speculate that modulation of transient receptor potential vanilloid (TRPV4) channels by means of changes in its membrane sorting could alter its gating, and thus contribute to changes in neural excitability that would be consistent with increased AVP release in rats with liver failure. To examine the effects of AngII treatment upon TRPV4 we utilized the rat hypothalamic AVP expressing neuronal cell line 4B. Methods (b): We used Western Blots to detect changes in TRPV4 protein in membrane fraction after drug treatments. In addition, we used calcium sensitive dye Fura 2-AM to detect changes in intracellular calcium after administration of a selective TRPV4 agonist - GSK 1016790A. Results (c): We characterized the presence of TRPV4 mRNA and protein in 4B cells. After Ang II (100nM;1 hr) treatment significantly increased TRPV4 levels in crude membrane fractions (p<0.001) and tyrosine phosphorylation of TRPV4 (p<0.001). Using calcium sensitive dye Fura-2AM, we noted that Ang II treated cells exhibited increased calcium transients in response to TRPV4 agonist, GSK1016790A (20nM, p<0.05). This increase was blocked by the Losartan (Ang II receptor antagonist) and Src-kinase inhibitor, PP2, but not by its analog PP3. Conclusions (d): Our data indicate that Ang II may facilitate TRPV4 trafficking and alter the phosphorylation status of TRPV4 through Src-kinases.