Mechanism of C-type natriuretic peptide-induced endothelial cell hyperpolarization

• Published in: American journal of physiology. Lung cellular and molecular physiology 2009-02, Vol.296 (2), p.L248-L256
• Main Authors: Simon, Aaron, Harrington, Elizabeth O, Liu, Gong Xin, Koren, Gideon, Choudhary, Gaurav
• Format: Article
• Language: English
• Subjects: Animals; Cells; Cells, Cultured; Cyclic GMP - analogs & derivatives; Cyclic GMP - pharmacology; Cyclic GMP-Dependent Protein Kinases - antagonists & inhibitors; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Enzyme Inhibitors - pharmacology; Fluorescence; Guanylate Cyclase - genetics; Guanylate Cyclase - metabolism; Immunoblotting; Ions; Membrane Potentials - drug effects; Membranes; Natriuretic Agents - pharmacology; Natriuretic Peptide, C-Type - pharmacology; NG-Nitroarginine Methyl Ester - pharmacology; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - genetics; Nitric Oxide Synthase Type III - metabolism; Peptides; Potassium; Potassium Channels, Calcium-Activated - antagonists & inhibitors; Potassium Channels, Calcium-Activated - metabolism; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; Pulmonary Artery - drug effects; Pulmonary Artery - metabolism; Rats; Receptors, Atrial Natriuretic Factor - antagonists & inhibitors; Receptors, Atrial Natriuretic Factor - genetics; Receptors, Atrial Natriuretic Factor - metabolism; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Small Interfering - pharmacology; Soluble Guanylyl Cyclase
• ISSN: 1040-0605; 1522-1504
• DOI: 10.1152/ajplung.90303.2008

1 Vascular Research Laboratory, Providence VA Medical Center, and 2 Department of Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island Submitted 7 May 2008 ; accepted in final form 25 November 2008 C-type natriuretic peptide (CNP) has a demonstrated hyperpolarizing effect on vascular smooth muscle cells. However, its autocrine function, including its electrophysiological effect on endothelial cells, is not known. Here, we report the effect of CNP on the membrane potential ( E m ) of pulmonary microvascular endothelial cells and describe its target receptors, second messengers, and ion channels. We measured changes in E m using fluorescence imaging and perforated patch-clamping techniques. In imaging experiments, samples were preincubated in the potentiometric dye DiBAC 4 (3), and subsequently exposed to CNP in the presence of selective inhibitors of ion channels or second messengers. CNP exposure induced a dose-dependent decrease in fluorescence, indicating that CNP induces endothelial cell hyperpolarization. CNP-induced hyperpolarization was inhibited by the K + channel blockers, tetraethylammonium or iberiotoxin, the nonspecific cation channel blocker, La 3+ , or by depletion or repletion of extracellular Ca 2+ or K + , respectively. CNP-induced hyperpolarization was also blocked by pharmacological inhibition of PKG or by small interfering RNA (siRNA)-mediated knockdown of natriuretic peptide receptor-B (NPR-B). CNP-induced hyperpolarization was mimicked by the PKG agonist, 8-bromo-cGMP, and attenuated by both the endothelial nitric oxide synthase (eNOS) inhibitor, N -nitro- L -arginine methyl ester ( L -NAME), and the soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3- a ]quinoxalin-1-one. Presence of iberiotoxin-sensitive, CNP-induced outward current was confirmed by perforated patch-clamping experiments. We conclude that CNP hyperpolarizes pulmonary microvascular endothelial cells by activating large-conductance calcium-activated potassium channels mediated by the activation of NPR-B, PKG, eNOS, and sGC. large-conductance calcium-activated potassium channels; ion channel Address for reprint requests and other correspondence: G. Choudhary, Providence VA Medical Center, 830 Chalkstone Ave., Providence, RI 02908 (e-mail: gaurav_choudhary{at}brown.edu )