TRPV4 Stimulation Releases ATP via Pannexin Channels in Human Pulmonary Fibroblasts

We previously described several ionic conductances in human pulmonary fibroblasts, including one activated by two structurally distinct TRPV4 (transient receptor potential, vanilloid-type, subtype 4)-channel agonists: 4αPDD (4α-phorbol-12,13-didecanoate) and GSK1016790A. However, the TRPV4-activated current exhibited peculiar properties: it developed slowly over many minutes, exhibited reversal potentials that could vary by tens of millivolts even within a given cell, and was not easily reversed by subsequent addition of two distinct TRPV4-selective blockers (RN-1734 and HC-067047). In this study, we characterized that conductance more carefully. We found that 4αPDD stimulated a delayed release of ATP into the extracellular space, which was reduced by genetic silencing of pannexin expression, and that the 4αPDD-evoked current could be blocked by apyrase (which rapidly degrades ATP) or by the P2Y purinergic receptor/channel blocker pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and could be mimicked by exogenous addition of ATP. In addition, we found that the 4αPDD-evoked current was blocked by pretreatment with RN-1734 or HC-067047, by Gd³⁺ or La³⁺, or by two distinct blockers of pannexin channels (carbenoxolone and probenecid), but not by a blocker of connexin hemichannels (flufenamic acid). We also found expression of TRPV4- and pannexin-channel proteins. 4αPDD markedly increased calcium flashing in our cells. The latter was abrogated by the P2Y channel blocker PPADS, and the 4αPDD-evoked current was eliminated by loading the cytosol with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or by inhibiting Ca²⁺/calmodulin-sensitive kinase II using KN93. Altogether, we interpret these findings as suggesting that 4αPDD triggers the release of ATP via pannexin channels, which in turn acts in an autocrine and/or paracrine fashion to stimulate PPADS-sensitive purinergic receptors on human pulmonary fibroblasts.