Nd acts via the cytochrome P450 (CYP) epoxygenasedependent formation of epoxyecosatrienoic acids (primarily 5′,6’EET) that straight activates the channel.26 In addition, nifedipine is in a position to increase the expression of CYP450, enhancing AA metabolization to 5′,6’EET and subsequently activating Trpv4.27 Hence, we hypothesized that pharmacological activation of Trpv4 could possibly restore the decreased [Ca2]i levels in cystic cells and thereby lower proliferation and cyst growth. In the present function, we found that cholangiocytes in the PCK rat (an animal model of ARPKD) and from patients with ARPKD and ADPKD overexpress Trpv4 and that its activation increases levels of [Ca2]i, suppressing cell proliferation and cyst growth in vitro, by a mechanism involving activation of Akt and inhibition on the BRaf/ERK1/2 signaling A-205804 Cytoskeleton pathway. In vivo, a certain Trpv4 activator, GSK1016790A, substantially decreases renal but not hepatic cystic regions.Gastroenterology. Author manuscript; accessible in PMC 2011 July 1.Gradilone et al.PageRESULTSTrpv4 is overexpressed in PCK rat cholangiocytesNIHPA Author Manuscript NIHPA Author Manuscript NIHPA Author ManuscriptAs shown in Figure 1A, major cultured PCK cholangiocytes overexpressedTrpv4 at mRNA levels by 8 occasions compared to standard cholangiocytes. Protein levels of Trpv4 had been also upregulated three times in freshly isolated PCK bile ducts, also as in cultured PCK rat cholangiocytes, PCKCCL (Figure 1B). Confocal microscopy confirmed the overexpression of Trpv4 in PCK rat liver (Figure 2A). When in standard ducts Trpv4 is mostly localized to cholangiocyte main cilia (as we reported),22 in PCK cholangiocytes, Trpv4 is predominantly expressed intracellularly (Figure 2A). Consistent with this observation, much more Trpv4 immunoreactivity was observed in cholangiocytes of human patients with ARPKD or ADPKD than in regular (Figure 2A). To additional analyze the site of Trpv4 expression, immunogoldelectron microscopy was performed. By this approach, and constant with confocal immunofluorescence microscopy and western blot, more immunogold particles were observed in cholangiocytes of PCK rats (862) in comparison to normal (18) (Figure 2B, C). Moreover, in normal rats, the particles had been predominantly localized to the apical domain, when in PCK rats; the majority of them had been intracellular (Figure 2B, C). To further explore Trpv4 expression, scanning immunogoldelectron microscopy was performed. By this method we detected, as previously reported,22 substantial Trpv4 expression on main cilium as well as on the apical membrane of typical bile ducts. In contrast, PCK bile ducts showed no Trpv4 staining on main cilia (Figure 2D). To be able to confirm the apparent Trpv4 mislocalization, Trpv4pEGFP was expressed in NRCs and PCKCCL. While NRCs showed a predominant ciliary localization of the Trpv4EGFP fusion protein, PCKCCL presented a far more diffuse, intracellular localization with no ciliary expression (Figure 2E). Trpv4 activators enhance intracellular calcium levels To test if Trpv4 activation induces a rise in [Ca2]i levels, PCK cholangiocytes had been incubated for 24 hrs together with the following activators: (i) 4PDD, (ii) 5′,6’EET, or (iii) combination of nifedipine and AA. Our information show that treatment with distinct concentrations of 4PDD increases [Ca2]i levels in a dosedependent manner (Figure 3A). The extra Trpv4 activators, 5′,6’EET and combination of nifedipine with AA (NifAA), elevated [Ca2]i also (Figure 3B). Quick ter.
