title: Functional characterization of voltage-dependent Ca(2+) channels in mouse pulmonary arterial smooth muscle cells: divergent effect of ROS.
abstract: Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca(2+) channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca(2+) or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K(+)-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca(2+) currents were -2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of -70 mV). Window currents were between -40 and +10 mV with a peak at -15.4 mV. Nifedipine inhibited currents with an IC(50) of 0.023 μM, and 1 μM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K(+)-mediated increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K(+) but not in aorta and MA. In contrast, H(2)O(2) inhibited high K(+)-mediated increase in [Ca(2+)](cyt) and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of Ca(V)1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.
authors: Department of Medicine, Section of Pulmonary, Critical Care, Sleep and Allergy Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors: Eun A
authors: EA
authors: Ko
authors: Jun
authors: J
authors: Wan
authors: Aya
authors: A
authors: Yamamura
authors: Adriana M
authors: AM
authors: Zimnicka
authors: Hisao
authors: H
authors: Yamamura
authors: Hae Young
authors: HY
authors: Yoo
authors: Haiyang
authors: H
authors: Tang
authors: Kimberly A
authors: KA
authors: Smith
authors: Premanand C
authors: PC
authors: Sundivakkam
authors: Amy
authors: A
authors: Zeifman
authors: Ramon J
authors: RJ
authors: Ayon
authors: Ayako
authors: A
authors: Makino
authors: Jason X-J
authors: JX
authors: Yuan
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D051379:Mice; D009119:Muscle Contraction / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011651:Pulmonary Artery / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015220:Calcium Channels; D017382:Reactive Oxygen Species
keywords: pulmonary arterial smooth muscle; reactive oxygen species; vascular contraction