title: cANF causes endothelial cell hyperpolarization by activation of chloride channels.
abstract: OBJECTIVE
  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)).
  
  
  RESULTS
  Changes in EC E(m) was measured through non-invasive fluorescence imaging. EC were preincubated in the potentiometric dye, DiBAC(4)(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 Ca(2+). 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.
authors.affiliation: Vascular Research Laboratory, Providence VA Medical Center, Providence, RI 02908, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Simon
authors.forename: Gong Xin
authors.initials: GX
authors.lastname: Liu
authors.forename: Gideon
authors.initials: G
authors.lastname: Koren
authors.forename: Gaurav
authors.initials: G
authors.lastname: Choudhary
mesh_terms: D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D004594:Electrophysiology; D042783:Endothelial Cells / Q000187:drug effects*; D008564:Membrane Potentials / Q000187:drug effects*; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats
chemical_list: D018118:Chloride Channels; D010446:Peptide Fragments; C065367:atrial natriuretic factor (4-23)NH2, de-Gln(18)-de-Ser(19)-de-Gly(20,22)-de-Leu(21)-; D009320:Atrial Natriuretic Factor