title: Mechanism of C-type natriuretic peptide-induced endothelial cell hyperpolarization.
abstract: 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(omega)-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.
authors.affiliation: Providence VA Medical Center, Providence, RI 02908, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Simon
authors.forename: Elizabeth O
authors.initials: EO
authors.lastname: Harrington
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; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008564:Membrane Potentials / Q000187:drug effects*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D045283:Natriuretic Agents / Q000494:pharmacology*; D020098:Natriuretic Peptide, C-Type / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011651:Pulmonary Artery / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071756:Soluble Guanylyl Cyclase; D000099242:Atrial Natriuretic Factor Receptor B
chemical_list: D006152:Cyclic GMP; D017869:Cyclic GMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D006162:Guanylate Cyclase; D019331:NG-Nitroarginine Methyl Ester; D045283:Natriuretic Agents; D020098:Natriuretic Peptide, C-Type; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D024681:Potassium Channels, Calcium-Activated; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017461:Receptors, Atrial Natriuretic Factor; D018160:Receptors, Cytoplasmic and Nuclear; D000071756:Soluble Guanylyl Cyclase; D000099242:Atrial Natriuretic Factor Receptor B; C016276:8-bromocyclic GMP; C496316:Nos3 protein, rat