Prolonged high fat/alcohol exposure increases TRPV4 and its functional responses in pancreatic stellate cells

The present study investigated transient receptor potential vanilloid type 4 (TRPV4) ion channels in pancreatic stellate cells (PSCs) isolated from rats with high-fat and alcohol diet (HFA)-induced chronic pancreatitis. TRPV4 is a calcium-permeable nonselective ion channel responsive to osmotic changes, alcohol metabolites arachidonic acid, anandamide, their derivatives, and injury-related lipid mediators. Male Lewis rats were fed HFA for 6-8 wk before isolation and primary culture of PSCs. Control PSCs were harvested from rats fed standard chow. Immunoreactivity for cytoskeletal protein activation product alpha-smooth muscle actin (alpha-SMA) and platelet-derived growth factor receptor-beta subunit (PDGFR-beta) characterized the cells as PSCs. TRPV4 expression increased in PSCs of HFA-fed rats and control cultures after alcohol treatment (50 mM). Cell responses to activation of inducible TRPV4 were assessed with live cell calcium imaging. Threefold increased and sustained intracellular calcium mobilization responses occurred in 70% of pancreatic stellate cells from HFA-fed rats in response to TRPV4 activators arachidonic acid, lipid second messenger, phorbol ester 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), and 50% hypoosmotic media compared with relatively unresponsive PSCs from control rats. Activation responses were attenuated by nonselective TRPV channel blocker ruthenium red. Tumor necrosis factor-alpha (TNF-alpha, 1 ng/ml, 16 h) increased responses to 4 alpha PDD in control PSCs. These findings implicate TRPV4-mediated calcium responses inducible after HFA exposure and inflammation in reactive responses of activated PSCs that impair pancreatic function, such as responsiveness to cytokines and the deposition of collagen fibrosis that precipitates ductal blockage and pain.