0031-3998/08/6302-0176 PEDIATRIC RESEARCH Vol. 63, No. 2, 2008 Copyright © 2008 International Pediatric Research Foundation, Inc. Printed in U.S.A. Developmental Absence of the O2 Sensitivity of L-Type Calcium Channels in Preterm Ductus Arteriosus Smooth Muscle Cells Impairs O2 Constriction Contributing to Patent Ductus Arteriosus BERNARD THE´ BAUD, XI-CHEN WU, HIDEMI KAJIMOTO, SANDRA BONNET, KYOKO HASHIMOTO, EVANGELOS D. MICHELAKIS, AND STEPHEN L. ARCHER Department of Pediatrics [B.T.], Division of Neonatology, Vascular Biology Group [X.-C.W., H.K., K.H., E.D.M.], University of Alberta, Edmonton, T6G 2J3, AB, Canada; Department of Medicine [S.L.A.], Section of Cardiology, University of Chicago, Chicago, Illinois 60637 ABSTRACT: Patent ductus arteriosus (PDA) complicates the hos- Failure of DA closure after birth complicates the hospital pital course of premature infants. Impaired oxygen (O2)-induced course of preterm infants. PDA is associated with an increased vasoconstriction in preterm ductus arteriosus (DA) contributes to incidence of chronic lung disease, intraventricular hemor- PDA and results, in part, from decreased function/expression of rhage, and necrotizing enterocolitis (4). Both medical and O -sensitive, voltage-gated potassium channels (Kv) in DA smooth 2 surgical interventions to close the DA, although usually ef- muscle cells (DASMCs). This paradigm suggests that activation of fective, are associated with additional morbidity (5). the voltage-sensitive L-type calcium channels (CaL), which increases 2ϩ The crucial role of endothelium-derived relaxing and con- cytosolic calcium ([Ca ]i), is a passive consequence of membrane depolarization. However, effective Kv gene transfer only partially stricting factors in regulating DA tone is well established (6). matures O2 responsiveness in preterm DA. Thus, we hypothesized However, O2 constricts the DA in the absence of endothelium that CaL are directly O2 sensitive and that immaturity of CaL function (7), suggesting that the core of the O2-sensing mechanism is in preterm DA contributes to impaired O2 constriction. We show that intrinsic to the DA smooth muscle cell (DASMC). Potassium ϩ preterm rabbit DA rings have reduced O2- and 4-aminopyridine (Kv (K ) channels in the vascular smooth muscle cells (SMCs) blocker)–induced constriction. Preterm rabbit DASMCs have re- regulate vascular tone through modulation of the membrane 2ϩ ϩ duced O2-induced whole-cell calcium current (ICa) and [Ca ]i. BAY 2ϩ potential (EM) (8). Closure of K channels leads to vasocon- K8644, a CaL activator, increased O2 constriction, ICa, and [Ca ]i in striction by depolarizing EM. Depolarization opens voltage- preterm DASMCs to levels seen at term but had no effect on human 2ϩ ␥ gated L-type calcium (Ca ) channels (CaL), thereby increas- and rabbit term DA. Preterm rabbit DAs have decreased and 2ϩ ␣ ing influx of extracellular Ca . In term rabbit (9) and human increased subunit protein expression. We conclude that the CaL in (10) DAs, O -induced constriction is initiated by the inhibition term rabbit and human DASMCs is directly O2 sensitive. Functional 2 immaturity of Ca O sensitivity contributes to impaired O constric- of O2 and 4-aminopyridine (4-AP)–sensitive voltage-gated, L 2 2 ϩ tion in premature DA and can be reversed by BAY K8644. (Pediatr K channels (Kv), including Kv1.5 and Kv2.1. Preterm rabbit Res 63: 176–181, 2008) DAs have reduced O2 constriction due, in part, to decreased function and expression of O2-sensitive Kv (11). Kv1.5 or n the hypoxic environment in utero, the ductus arteriosus Kv2.1 gene transfer partially (50%) “rescues” the develop- I(DA), a vital fetal artery that connects the pulmonary artery mental deficiency, conferring O2 responsiveness to preterm to the aorta, is widely patent and shunts more than half of the rabbit DAs and human DAs (11). However, O2 responsiveness right heart’s cardiac output away from the nonventilated lung is not completely restored, suggesting additional mechanisms into the umbilicoplacental circulation, where gas exchange contribute to O2-induced constriction in the DASMCs. In resistance pulmonary artery SMC, another cell from the spe- takes place (1). Within minutes of birth, the increased PO2 constricts the DA (2) and simultaneously dilates the pulmo- cialized O2-sensing system (12), the CaL have intrinsic O2 nary circulation (3). The response of the term DA to oxygen sensitivity in addition to their response to Kv inhibition– (O2) rarely fails; however, in humans, approximately 50% of induced depolarization of EM (13). We suggest that DASMC preterm DAs do not close, despite adequate oxygenation (4). CaL are more active participants in DA constriction to O2 than was previously recognized, responding not just to membrane depolarization but directly to increased PO2. We tested the Received March 19, 2007; accepted September 14, 2007. Correspondence: Stephen L. Archer, M.D., Department of Medicine, Section of hypothesis that the CaL are intrinsically O2 sensitive in rabbit Cardiology, University of Chicago, MC 6080, 5841 S. Maryland Avenue, Chicago, IL and human DASMCs. We also tested the hypothesis that 60637; e-mail: [email protected] Dr. Archer is supported by NIH-RO1-HL071115. Drs. Michelakis, The´baud, and Archer are supported by a Canada Research Chair (CRC), the Canada Foundation for 2؉ Abbreviations: 4-AP, 4-aminopyridine; [Ca ]i, cytosolic calcium; CaL, Innovation, the Alberta Heart and Stroke Foundation, the Canadian Institutes for Health 2ϩ L-type Ca channels; DA, ductus arteriosus; EM, membrane potential; ICa, Research (CIHR), and the Alberta Cardiovascular and Stroke Research Centre ϩ (ABACUS). Drs. Michelakis and The´baud are supported by the Alberta Heritage whole-cell calcium current; Kv, voltage-gated K channels; SMC, smooth Foundation for Medical Research. Dr. The´baud is supported by the Stollery Foundation. muscle cell 176 2ϩ O2-SENSITIVE Ca CHANNELS IN THE DUCTUS 177 reduced expression and/or function of these channels contrib- stage of an inverted microscope and perfused with a warmed normoxic utes to impaired O constriction in preterm rabbit DA. Hanks’ solution (32–33°C). Background fluorescence was recorded from each 2 dish of cells and subtracted before calculation of the 340- to 380-nm ratio. Emission was measured at 510 nm. Duration of each measurement was METHODS 1200 s. Immunoblotting. DAs were flash frozen in liquid N2 and homogenized in All procedures were approved by the Animal Welfare and the Human buffer containing an antiprotease cocktail (Sigma Chemical Co.) and run on ␣ ␥ studies committees of the University of Alberta. All investigators had access 7.5%–10% gels. Specific antibodies against CaL subunits 1c and 2 were to the data and take responsibility for its integrity. purchased from US Biologic (Cedarlane Laboratories Ltd., Hornby, Ontario) Rabbit DAs. New Zealand White rabbits (n ϭ 50) were delivered by and Sigma Chemical Co.-Aldrich, respectively. Expression was quantified cesarean section at gestational d 26 (preterm) or 30 (term), as previously using densitometry and expressed as the percentage of the loaded protein described (11,14). The endothelium-intact DA was used within 5 min of density, measured using the Ponceau stain. ϭ Ϯ ϭ Ϯ Ϯ harvest and was maintained hypoxic (pH 7.40 0.08, PO2 31 1mm Statistics. Values are expressed as means SEM. All sample sizes are Hg) until PO2 was intentionally increased. listed in the figures. Intergroup comparisons were performed with a t test or Rabbit DASMCs. DASMCs were obtained by enzymatic digestion as factorial repeated-measures analysis of variance, as appropriate. Fisher’s described (11,14). probable least significant differences test was used for post hoc comparisons. Human DASMCs. Human DAs were obtained from hypoplastic left heart A p value Ͻ0.05 was considered statistically significant. term infants during the Norwood procedure, and the SMCs were isolated by enzymatic digestion, as described (11,14). Tension measurements in isolated preterm and term rabbit DA rings. RESULTS Isolated rabbit DAs were placed in an organ bath and equilibrated in hypoxic ϭ Ϯ Krebs solution (PO2 31 1 mm Hg to mimic in utero conditions), at the Decreased O -induced constriction in preterm rabbit DA experimentally derived optimal resting tension values of 400 mg (preterm) 2 and 800 mg (term), as previously described (11,14). To investigate the is restored by a CaL opener. In both term and preterm DAs, contribution of maturational differences in CaL function to diminished O2 O2-induced constriction increased in proportion to PO2 (Fig. constriction, the response of preterm and term DA rings to increased PO was 2 1A, B). O2-induced constriction was significantly weaker in compared in the presence and absence of the CaL opener BAY K8644 Ϫ6 Ϫ6 ϩ preterm DAs (Fig. 1A, B). Pretreatment with the Ca opener (10 M) or the CaL inhibitor nifedipine (10 M). The responsiveness to K L channel inhibitors Kv inhibitor 4-AP and iberiotoxin (IBTX), a highly specific BAY K8644 enhanced O -induced constriction in preterm ϩ 2 inhibitor of large conductance calcium-sensitive K channels (BKCa) was DAs to a magnitude similar to that achieved in term DAs (Fig. also compared. 2ϩ 1A, B). In contrast, BAY K8644 had no effect on O -induced Whole cell patch clamp. The effect of PO2 on whole-cell Ca current (ICa) 2 and EM were measured in freshly dispersed preterm and term rabbit DASMCs constriction in term DAs. BAY K8644 had no effect on and human DASMCs, using voltage and current clamp protocols, as previ- 2ϩ phenylephrine-induced constriction in preterm and term DAs ously described (11,15) and detailed in the online supplement. Ca channel 2ϩ current recordings were obtained using the whole-cell configuration. Barium (data nor shown). The Ca channel inhibitor nifedipine (20 mM) was used as a charge carrier because its favorable conduction by the blunted O2 constriction in both preterm and term DAs (by CaL increases current amplitude.
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