cANF causes endothelial cell hyperpolarization by activation of chloride channels

Objectives: Natriuretic peptides bind with natriuretic peptide receptor (NPR)-C, which can alter cellular function through its interaction with the G_i protein complex. NPR-C has been found to mediate the activation of K⁺ channels and non-selective cation channels in vascular smooth muscle and cardiac fibroblast cells, respectively. However, the electrophysiological effect of NPR-C activation on endothelial cells (EC) has not been previously examined. In this study we sought to elucidate the effect of cANF(4-23), a selective NPR-C ligand, on EC membrane potential (E_m). Methods/results: Changes in EC E_m was measured through non-invasive fluorescence imaging. EC were preincubated in the potentiometric dye, DiBAC₄(3) and subsequently exposed to cANF(4-23), in the presence of selective inhibitors of ion-channels or second messengers. NPR-C expression in rat lung microvascular endothelial cells was assessed by RT-PCR. cANF(4-23) induced a sustained decrease in EC cellular fluorescence, indicating endothelial cell hyperpolarization. The cANF-induced hyperpolarization could not be attenuated by TEA, barium, ouabain or by the reduction of extracellular Ca²⁺. Further, the cANF-induced hyperpolarization was insensitive to inhibition of G_i and protein kinase G (PKG), downstream messengers of NPRs. However, the Cl^- channel inhibitors, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, niflumic acid, and hypertonic saline attenuated the cANF-induced hyperpolarization. Perforated patch clamp recordings confirmed the cANF-induced current was carried by Cl^- and could be inhibited by niflumic acid. RT-PCR confirmed expression of NPR-C in vascular smooth muscle cells but not in EC. Conclusions: cANF causes hyperpolarization that is most likely mediated via activation of Cl^- channels by a PKG and G_i independent mechanism.