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Articles in PresS. Am J Physiol Regul Integr Comp Physiol (March 2, 2006). doi:10.1152/ajpregu.00040.2006

Expression and function of potassium channels in the human placental vasculature. Mark Wareing, Xilian Bai, Fella Seghier, Claire M. Turner, Susan L. Greenwood, Philip N. Baker, Michael J. Taggart and Gregor K. Fyfe. Maternal and Fetal Health Research Centre, The University of Manchester, St. Mary’s Hospital, Hathersage Road, Manchester, M13 0JH, U.K.

Running title: Expression of placental potassium channels

Corresponding author: Dr Mark Wareing, Maternal and Fetal Health Research Centre, The University of Manchester, Division of Human Development, St. Mary’s Hospital, Hathersage Road, Manchester, M13 0JH, U.K. Tel: 0161-276-5474 Fax: 0161-276-6134 E-mail: [email protected]

Copyright © 2006 by the American Physiological Society.


In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of KV2.1, KV9.3, BKCa, KIR6.1 and TASK1 in chorionic plate arteries, veins and placental homogenate was assessed by RT-PCR and Western Blotting. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil and anandamide. Experiments were performed at 20%, 7% and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1 and TASK1channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1 and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.

Keywords: Placenta


Potassium channels




Introduction. Potassium (K) channels have an important role in the maintenance of smooth muscle tone via their effects on membrane potential and a variety of agonists can modify tone by alteration of K channel activity (8, 13). Several K channel subtypes have been identified in vascular smooth muscle cells (VSMCs) and altered function has been associated with cardiovascular disease (57).

In the human placenta, it has been proposed that Hypoxic FetoPlacental Vasoconstriction (HFPV) (47, 53) is a mechanism that could modulate blood flow by the diversion of blood from poorly to well oxygenated cotyledons. In perfused human placental cotyledon, reduced partial pressure of oxygen (pO2) triggered vasoconstriction yet, large diameter (>1mm) arterial / venous constriction was unaltered (12, 30, 33). HFPV may occur via modification of smooth muscle K channel activity (30).

In the lung, the effects of hypoxia (Hypoxic Pulmonary Vasoconstriction; HPV) on vessel contraction have been more thoroughly documented. HPV can be elicited in isolated pulmonary artery VSMCs without neuronal input (17) and is also observed in pulmonary veins (69); the endothelium is also thought to be a critical modulator of the process (2, 4, 26, 35). Recent studies of HPV suggest that K channels influence vascular tone directly and may also be involved in sensing the level of tissue oxygenation; they are therefore essential for the HPV response (4, 51, 67). These channels include members of the voltage-gated (KV), calcium-activated, two-pore domain and ATP-sensitive families. (4, 5, 10, 16, 19, 22, 23, 28, 51, 54, 56, 59).


Unlike the lung, there is little data on K channels in the fetoplacental vasculature. KV, KCa and KATP channel activity has been demonstrated electrophysiologically in VSMCs or endothelial cells from placental allantochorial vessels (24, 25). KV1.5, KV2.1 and BKCa have been demonstrated by RT-PCR, and variable expression of KV2.1, KV3.1b, KV1.5 and BKCa was documented in placental vessel homogenates (30). Most recently, calcitonin gene related peptide (CGRP)-induced glibenclamide-inhibitable vasodilatation of the fetoplacental vasculature has been demonstrated suggestive of a role for KATP channels (20). Thus, there is limited evidence on the role of K channels in the control of fetoplacental arterial tone and no previous published studies of fetoplacental venous tone.

Our hypothesis is that K channels have a role in the control of small vessel function in the human chorionic plate. We determined expression of mRNA (RT-PCR) and protein (Western Blotting) for KV2.1, KV9.3, TASK-1, BKCa and KIR6.1 in arteries, veins and placental villous homogenate. The rationale for choosing these channels was that they have been demonstrated previously in other tissues to directly (or indirectly in the case of KIR6.1) mediate altered vascular responsivity in relation to oxygenation. K channel function in arteries and veins was investigated pharmacologically. The influence of different levels of oxygen on K channel activity and vessel tone was also assessed (2%, umbilical artery (40), 7% (intervillous space (11)) and 21% (placental hyperoxia).


Materials and Methods. This work was performed with the approval of the ethics committee of Central Manchester and Manchester Children’s University Hospitals NHS Trust. Informed written consent was obtained for all tissue used in the study. The investigation conforms to the principles outlined in the Declaration of Helsinki (1).

Samples: Term (37-42 weeks gestation) placentas (N=95) were obtained post-delivery (vaginal or after elective Caesarean section) from women with otherwise uncomplicated pregnancies (no evidence of hypertension, intrauterine growth restriction or other medical disorders). Biopsies were taken within 20 mins of delivery and placed directly into ice-cold physiologic salt solution (PSS; in mM; 119NaCl, 25NaHCO3, 4.69KCl, 2.4MgSO4, 1.6CaCl2, 1.18KH2PO4, 6.05 glucose, 0.034 EDTA; pH 7.4).

RT-PCR: Umbilical arteries and vein were identified at the insertion of the umbilical cord in to the chorionic plate of the placenta. Chorionic plate small arteries and veins, which traverse the surface of the placenta, can be easily identified by tracing their origin from this insertion point prior to dissection using a stereomicroscope. Vessels were cut into short, 2-3 mm lengths, cleaned of blood placed into cryotubes prior to snap freezing in liquid N2. Placental tissue, comprising a section through the chorionic plate and villus tree, was also excised, rinsed in PSS and snap frozen.

After thawing on ice, total RNA was isolated from arterial, venous and whole placental tissue by homogenization in Trizol reagent (Invitrogen, Paisley, UK). RNA was reverse-transcribed using Moloney Murine Leukema Virus Reverse Transcriptase (RT) according to the manufacturer’s instructions (Invitrogen) in a Perkin-Elmer Cetus thermal cycler (Perkin


Elmer, Beaconsfield, UK). An initial RT-PCR, using I-actin primers, in which the RT was omitted, was performed to ensure that all RNA samples were not contaminated with genomic DNA. Subsequent RT-PCR, using standard techniques with a hot start, was employed using primer pairs previously described in other studies of human K channel expression, and optimised for use in our samples: KV2.1 5’-GCCTTCACCTCCATCCTCAACT-3’ (forward) with 5’ACTCATCGAGGCTCTGTAGCTCAG-3’

(reverse), annealing temperature (Ta) 64oC (56); KV9.3



(reverse), Ta 55oC (18); TASK-1 5’-CTCCTTCTACTTCGCCATCAT-3’ (forward) with 5’CATTCCACTTTTCTCCATGTAAGC-3’,

Ta 59oC (6). BLAST searches were performed to ensure

primers had no homology with any other known gene products. The number of cycles was 35 for each primer pair, with one cycle consisting of denaturation at 95oC for 60s, annealing at Ta for 60s and extension at 72oC for 60 sec; the exception to this was the extension times for KV2.1 and BKCa, which were 40 and 90s respectively. Appropriate positive (human brain RNA; Becton Dickinson Bioscience, Oxford, UK) and negative controls (water replacing template) were used at all times. I-actin was routinely amplified from all samples, confirming sample integrity and amplification capacity.

Western Blotting: Placental arteries, veins and whole placenta, different to those used for RNA extraction, were collected as described above. Samples were homogenized on ice in homogenisation buffer (in M): 0.01 HEPES, 0.001 EDTA, 0.25 sucrose (pH 7.4) with an antiprotease inhibitor cocktail (104 mM 4-(2-aminoethyl)benzenesulphonyl fluoride (AEBSF), 1.5 mM pepstatin A, 1.4 mM E-64, 3.6 mM bestatin, 2.1 mM leupeptin and 80 µM aprotinin;


Sigma-Aldrich, Poole, UK). Rat brain (animals killed by stunning followed by cervical dislocation according to UK Home Office guidelines) was used as a positive control for K+ channel protein expression. We routinely used the post nuclear supernatant, obtained after a spin at 4,000g for 10 min, for our blotting experiments. All sample protein concentrations were determined using a commercial protein assay kit (Bio-Rad, Hemel Hempstead, UK). Samples were stored at -80oC until used.

Protein from arteries, veins, placenta (50-100µg as indicated) and rat brain (50-70Mg) was mixed with a reducing loading buffer containing 1.25% I-mercaptoethanol (v/v), 2% SDS (w/v), 0.04% bromophenol blue (v/v) and 10% glycerol (v/v), in 0.05 mol l-1 Tris·HCl (pH 6.8) and heated at 95oC for 5 min. Proteins were then subsequently electrophorectically separated in 8-10% polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked for 1 hr using blocking buffer (1% dried milk powder (w/v) in 0.05% Tween 20 (v/v), Tris-buffered saline (TBS (in mol l-1): 0.015 Tris, 0.150 NaCl; pH 8.0).

Membranes were probed for 2h at room temperature with either anti-KV2.1 at 1:1000 (Upstate Biotech, Lake Placid, NY, USA), anti-BKCa (Alomone Labs, Jerusalem, Israel) at 1:500, antiKIR6.1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:500 or anti-TASK1 (Alomone Labs) at 1:100 in blocking buffer. KV2.1 rabbit polyclonal antibody was raised against residues 837-853 of rat KV2.1. BKCa rabbit polyclonal antibody was raised against residues 1184-1200 of mouse BKCa J-subunit. KIR6.1 rabbit polyclonal antibody was raised against residues 345-424 (C-terminus) of human KIR6.1. TASK1 rabbit polyclonal antibody was raised against the peptide (C-terminus) corresponding to residues 252-269 of the human TASK1 channel. Three 10min washes in TBS/0.05% Tween 20 were followed by incubation


with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody at a 1:2000 dilution (DAKO, Ely, UK) for 1h. After three 10min washes in TBS/0.05% Tween 20, membranes were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK). Appropriate negative controls without primary antibody or in the presence of competing peptide were also performed.

Myography: Chorionic plate small arteries (274±7µm; n=186) and veins (294±10µm; n=164) were cut into 2-3 mm lengths and mounted onto 40µm steel wires on a M610 wire myograph (Danish Myotech, Aarhus, Denmark), bathed in 6ml of PSS and warmed to 37oC. Vessels were normalised as described previously (62, 64) to 0.9 of L5.1kPa to mimic a physiological resting tension of approximately 25mmHg (39). Post-normalisation, vessels were equilibrated for 20min. Functional studies were performed in vessels normalised and equilibrated in 5% CO2 in air (termed 20% oxygen) to mimic placental hyperoxia, 5% CO2 in 5% oxygen (final dissolved oxygen content of 4.8-6.0%; termed 7% oxygen) to mimic intervillus space oxygenation or 5% CO2 in nitrogen (final dissolved oxygen content of 0.81.0%; termed 2% oxygen) to mimic placental hypoxia. Oxygenation was measured in the myograph chamber using a WPI oxygen meter (WPI Inc., USA; measurement accuracy +/1%). Following equilibration, concentration-response curves were constructed to the thromboxane mimetic U46619 (0.1-2000nM in 2min increments / 5min plateau (62, 64)). Placental vessel viability was assessed using 120mM KCl in PSS (equimolar substitution of KCl for NaCl). Vessels greater than 500µm in diameter were excluded from the study.

Role of K channels in the control of placental chorionic plate arterial and venous basal tone: The role of K channels in the control of placental vascular tone was assessed in unstimulated chorionic plate arteries and veins as follows:


Voltage-gated (KV) channels were inhibited with 4-aminopyridine (4-AP; 1mM)

Large conductance calcium activated K channels (BKCa) were inhibited with iberiotoxin (IBTX; 100nM) TWIK-related acid-sensitive K channels (TASK1) were inhibited with anandamide (AEA; 20µM)

ATP sensitive K channel (KATP) were opened with pinacidil (PIN; 50µM).

Basal tone was assessed pre- and 5min post-addition of the pharmacological agent.

Role of K channels in the control of placental chorionic plate arterial and venous constriction and relaxation: Following incubation of arteries and veins with K channel modulators for 5min, vessels were constricted with U46619 (0.1-2000nM). To assess the vasodilator effect of pinacidil, arteries were constricted with an EC80 dose of U46619. Once a stable constriction was achieved, relaxation was assessed with incremental doses of pinacidil (0.01-100µM). Time control vessels were performed in parallel (constricted with EC80 dose of U46619 only).

General chemicals: General chemicals and pharmacological agents were obtained from Sigma-Aldrich (Poole, Dorset, UK) or BDH (Poole, Dorset, UK). U46619 was obtained from Calbiochem (CN Biosciences (UK) Ltd., Nottingham, UK).

Statistical analysis: Vessel tension production was calculated as follows. To standardise for the length of the vessel segment, tension production in mN was divided by the length of the vessel segment in mm to give active wall tension RT (mN/mm). Active effective pressure (Pi in kPa), was calculated by dividing RT by the normalised internal radius (mm) of the vessel. An assessment of whether data was normally distributed was performed using the Kolomogorov-Smirnov normality test. Data for the effect of K channel inhibitors and openers


on basal tone were compared using Wilcoxon-signed rank (WSR) test. Relaxation was calculated as a percentage of the contraction achieved with EC80 dose of U46619. Concentration-response curves for contraction and relaxation were compared by repeated measures (RM-) ANOVA. Bonferroni post hoc test was used to assess statistical significance at individual concentrations of agonist. Data are expressed as mean ± standard error of the mean (SE) with n vessels from N placentas. P0.05; RMANOVA; data not shown). In arteries, IBTX did not affect the U46619 concentrationresponse relationship at 2% or 20% oxygenation (P>0.05; RM-ANOVA; Figure 4A,C). However, at 7% oxygenation maximal contraction with U46619 increased (P0.05; WSR test; Figure 4B) by IBTX.

ATP-sensitive (KATP) channels: 50µM pinacidil significantly decreased basal tone in unstimulated chorionic plate arteries and veins at 2% oxygenation (P0.05; WSR-test; data not shown). The effects of pinacidil were independent of oxygenation (P50µM (34). Thus a clarification of the role of TASK1 K channels in vascular tissues may await the development of more specific pharmacological tools.

IUGR In IUGR, umbilical artery Doppler waveforms indicate increased fetoplacental resistance compared to normal pregnancies (3, 38). Increased tone could be a consequence of aberrant KV channel function, as KV channel inhibition elicits increased tone in fetoplacental arteries and veins (Figure 3). However modified oxygenation which is also apparent in IUGR, did not alter 1) affects of 4-AP on basal tone 2) U46619-induced contraction or 3) vessel sensitivity to U46619. Hypersensitivity to U46619(46) and ET1 (48) has previously been demonstrated in IUGR. These effects may be via actions on KV or KATP channels (50, 65) but changes in oxygenation per se did not modify the actions of 4-AP or pinacidil on fetoplacental vessels. Conversely, KATP and KV channel function may be modified during ischaemia-reperfusion injury (31, 49) and by free radicals (21, 36) respectively, and perhaps these influences in addition to hypoxia are required to produce the IUGR phenotype.

Summary We demonstrated the presence of a number of K channels in chorionic plate arteries and veins using RT-PCR and Western blotting. Furthermore pharmacological manipulation K channels modified fetoplacental vascular function. In particular, administration of KATP channel openers may be a strategy to promote relaxation of the fetoplacental vasculature in


pathological states of inappropriately increased vascular tone. Further elucidation of the role for these channels in the control of fetoplacental vascular tone necessitates characterisation of vascular responses using pressure myography in the presence and absence of the endothelium.


Acknowlegements: Mark Wareing is supported by the British Heart Foundation. Support was also received from Central Manchester & Manchester University Hospitals NHS Trust (MW, GKF). The research was performed at the Tommy’s; the baby charity funded Maternal and Fetal Health Research Centre, Manchester.


References: 1.

World Medical Association Declaration of Helsinki. Recommendations guiding

physicians in biomedical research involving human subjects. Cardiovasc Res 35: 2-3, 1997. 2.

Aaronson PI, Robertson TP, and Ward JP. Endothelium-derived mediators and

hypoxic pulmonary vasoconstriction. Respir Physiolo Neurobiol 132: 107-120, 2002. 3.

Arbeille P. Fetal arterial Doppler-IUGR and hypoxia. Eur J Obstet Gynecol Reprod

Biol 75: 51-53, 1997. 4.

Archer SL, Souil E, Dinh-Xuan AT, Schremmer B, Mercier JC, El Yaagoubi A,

Nguyen-Huu L, Reeve HL, and Hampl V. Molecular identification of the role of voltagegated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J Clin Invest 101: 2319-2330, 1998. 5.

Archer SL, Wu XC, Thebaud B, Nsair A, Bonnet S, Tyrrell B, McMurtry MS,

Hashimoto K, Harry G, and Michelakis ED. Preferential expression and function of voltage-gated, O2-sensitive K+ channels in resistance pulmonary arteries explains regional heterogeneity in hypoxic pulmonary vasoconstriction: ionic diversity in smooth muscle cells. Circ Res 95: 308-318, 2004. 6.

Bai X, Greenwood SL, Glazier JD, Baker PN, Sibley CP, Taggart MJ, and Fyfe

GK. Localization of TASK and TREK, two-pore domain K+ channels, in human cytotrophoblast cells. J Soc Gynecol Investig 12: 77-83, 2005. 7.

Barry DM, Trimmer JS, Merlie JP, and Nerbonne JM. Differential expression of

voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ Res 77: 361-369, 1995. 8.

Beech DJ. Actions of neurotransmitters and other messengers on Ca2+ channels and

K+ channels in smooth muscle cells. Pharmacol Ther 73: 91-119, 1997.



Brakemeier S, Eichler I, Knorr A, Fassheber T, Kohler R, and Hoyer J.

Modulation of Ca2+-activated K+ channel in renal artery endothelium in situ by nitric oxide and reactive oxygen species. Kidney Int 64: 199-207, 2003. 10.

Buckler KJ. A novel oxygen-sensitive potassium current in rat carotid body type I

cells. J Physiol 498 ( Pt 3): 649-662, 1997. 11.

Burton GJ and Caniggia I. Hypoxia: implications for implantation to delivery-a

workshop report. Placenta 22 Suppl A: S63-65, 2001. 12.

Byrne BM, Howard RB, Morrow RJ, Whiteley KJ, and Adamson SL. Role of the

L-arginine nitric oxide pathway in hypoxic fetoplacental vasoconstriction. Placenta 18: 627634, 1997. 13.

Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H,

Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, and Rudy B. Molecular diversity of K+ channels. Ann N Y Acad Sci 868: 233-285, 1999. 14.

Cooper EJ, Wareing M, Greenwood SL, and Baker PN. Effects of oxygen tension

and normalization pressure on endothelin-induced constriction of human placental chorionic plate arteries. J Soc Gynecol Investig 12: 488-494, 2005. 15.

Cooper EJ, Wareing M, Greenwood SL, and Baker PN. Oxygen Tension and

Normalisation Pressure Modulate Nifedipine-sensitive Relaxation of Human Placental Chorionic Plate Arteries. Placenta, 2006. E Pub ahead of print. 16.

Cornfield DN, Reeve HL, Tolarova S, Weir EK, and Archer S. Oxygen causes

fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel. Proc Natl Acad Sci U S A 93: 8089-8094, 1996. 17.

Cornfield DN, Stevens T, McMurtry IF, Abman SH, and Rodman DM. Acute

hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells. Am J Physiol 265: L53-56, 1993.



Cui Y, Tran S, Tinker A, and Clapp LH. The molecular composition of K(ATP)

channels in human pulmonary artery smooth muscle cells and their modulation by growth. Am J Respir Cell Mol Biol 26: 135-143, 2002. 19.

Davies AR and Kozlowski RZ. Kv channel subunit expression in rat pulmonary

arteries. Lung 179: 147-161, 2001. 20.

Dong YL, Vegiraju S, Chauhan M, Gangula PR, Hankins GD, Goodrum L, and

Yallampalli C. Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone. Am J Physiol Heart Circ Physiol 286: H230-239, 2004. 21.

Duprat F, Guillemare E, Romey G, Fink M, Lesage F, Lazdunski M, and Honore

E. Susceptibility of cloned K+ channels to reactive oxygen species. Proc Natl Acad Sci U S A 92: 11796-11800, 1995. 22.

Duprat F, Lesage F, Fink M, Reyes R, Heurteaux C, and Lazdunski M. TASK, a

human background K+ channel to sense external pH variations near physiological pH. Embo J 16: 5464-5471, 1997. 23.

Fujita A and Kurachi Y. Molecular aspects of ATP-sensitive K+ channels in the

cardiovascular system and K+ channel openers. Pharmacol Ther 85: 39-53, 2000. 24.

Guiet-Bara A and Bara M. Evidence of K and Ca channels in endothelial cells of

human allantochorial placental vessels. Cell Mol Biol (Noisy-le-grand) 48 Online Pub: OL317-322, 2002. 25.

Guiet-Bara A, Ibrahim B, Leveteau J, and Bara M. Calcium channels, potassium

channels and membrane potential of smooth muscle cells of human allantochorial placental vessels. Bioelectrochem Bioenerg 48: 407-413, 1999. 26.

Gurney AM. Multiple sites of oxygen sensing and their contributions to hypoxic

pulmonary vasoconstriction. Respir Physiolo Neurobiol 132: 43-53, 2002.



Gurney AM, Osipenko ON, MacMillan D, and Kempsill FE. Potassium channels

underlying the resting potential of pulmonary artery smooth muscle cells. Clin Exp Pharmacol Physiol 29: 330-333, 2002. 28.

Gurney AM, Osipenko ON, MacMillan D, McFarlane KM, Tate RJ, and

Kempsill FE. Two-pore domain K channel, TASK-1, in pulmonary artery smooth muscle cells. Circ Res 93: 957-964, 2003. 29.

Gutterman DD, Miura H, and Liu Y. Redox modulation of vascular tone: focus of

potassium channel mechanisms of dilation. Arterioscler Thromb Vasc Biol 25: 671-678, 2005. 30.

Hampl V, Bibova J, Stranak Z, Wu X, Michelakis ED, Hashimoto K, and Archer

SL. Hypoxic fetoplacental vasoconstriction in humans is mediated by potassium channel inhibition. Am J Physiol Heart Circ Physiol 283: H2440-2449, 2002. 31.

Hiraoka M. Pathophysiological functions of ATP-sensitive K+ channels in

myocardial ischemia. Jpn Heart J 38: 297-315, 1997. 32.

Honore E, Barhanin J, Attali B, Lesage F, and Lazdunski M. External blockade of

the major cardiac delayed-rectifier K+ channel (Kv1.5) by polyunsaturated fatty acids. Proc Natl Acad Sci U S A 91: 1937-1941, 1994. 33.

Howard RB, Hosokawa T, and Maguire MH. Hypoxia-induced fetoplacental

vasoconstriction in perfused human placental cotyledons. Am J Obstet Gynecol 157: 12611266, 1987. 34.

Howlett AC and Mukhopadhyay S. Cellular signal transduction by anandamide and

2-arachidonoylglycerol. Chem Phys Lipids 108: 53-70, 2000. 35.

Hulme JT, Coppock EA, Felipe A, Martens JR, and Tamkun MM. Oxygen

sensitivity of cloned voltage-gated K(+) channels expressed in the pulmonary vasculature. Circ Res 85: 489-497, 1999.



Irvine JC, Favaloro JL, and Kemp-Harper BK. NO- activates soluble guanylate

cyclase and Kv channels to vasodilate resistance arteries. Hypertension 41: 1301-1307, 2003. 37.

Jackson WF, Konig A, Dambacher T, and Busse R. Prostacyclin-induced

vasodilation in rabbit heart is mediated by ATP-sensitive potassium channels. Am J Physiol 264: H238-243, 1993. 38.

Karsdorp VH, van Vugt JM, van Geijn HP, Kostense PJ, Arduini D, Montenegro

N, and Todros T. Clinical significance of absent or reversed end diastolic velocity waveforms in umbilical artery. Lancet 344: 1664-1668, 1994. 39.

Kleiner-Assaf A, Jaffa AJ, and Elad D. Hemodynamic model for analysis of

Doppler ultrasound indexes of umbilical blood flow. Am J Physiol 276: H2204-2214, 1999. 40.

Lackman F, Capewell V, Gagnon R, and Richardson B. Fetal umbilical cord

oxygen values and birth to placental weight ratio in relation to size at birth. Am J Obstet Gynecol 185: 674-682, 2001. 41.

Lacza Z, Snipes JA, Kis B, Szabo C, Grover G, and Busija DW. Investigation of

the subunit composition and the pharmacology of the mitochondrial ATP-dependent K+ channel in the brain. Brain Res 994: 27-36, 2003. 42.

Ling S, Sheng JZ, Braun JE, and Braun AP. Syntaxin 1A co-associates with native

rat brain and cloned large conductance, calcium-activated potassium channels in situ. J Physiol 553: 65-81, 2003. 43.

Lovren F and Triggle C. Nitric oxide and sodium nitroprusside-induced relaxation of

the human umbilical artery. Br J Pharmacol 131: 521-529, 2000. 44.

Matharoo-Ball B, Ashford ML, Arulkumaran S, and Khan RN. Down-regulation

of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition. Biol Reprod 68: 2135-2141, 2003.



Mills T, Wareing M, Bugg G, Greenwood SL, Sibley C, and Baker PN. Vascular

reactivity of chorionic plate small arteries is altered in IUGR. Placenta 25: A.21, 2004. 46.

Mills TA, Wareing M, Bugg GJ, Greenwood SL, and Baker PN. Chorionic plate

artery function and Doppler indices in normal pregnancy and intrauterine growth restriction. Eur J Clin Invest 35: 758-764, 2005. 47.

Myatt L. Control of vascular resistance in the human placenta. Placenta 13: 329-341,

1992. 48.

Neerhof MG, Silver RK, Caplan MS, and Thaete LG. Endothelin-1-induced

placental and fetal growth restriction in the rat. J Matern Fetal Med 6: 125-128, 1997. 49.

O'Rourke B. Pathophysiological and protective roles of mitochondrial ion channels. J

Physiol 529 Pt 1: 23-36, 2000. 50.

Park WS, Ko EA, Han J, Kim N, and Earm YE. Endothelin-1 acts via protein

kinase C to block KATP channels in rabbit coronary and pulmonary arterial smooth muscle cells. J Cardiovasc Pharmacol 45: 99-108, 2005. 51.

Patel AJ and Honore E. Molecular physiology of oxygen-sensitive potassium

channels. Eur Respir J 18: 221-227, 2001. 52.

Poling JS, Rogawski MA, Salem N, Jr., and Vicini S. Anandamide, an endogenous

cannabinoid, inhibits Shaker-related voltage-gated K+ channels. Neuropharmacology 35: 983991, 1996. 53.

Poston L, McCarthy AL, and Ritter JM. Control of vascular resistance in the

maternal and feto-placental arterial beds. Pharmacol Ther 65: 215-239, 1995. 54.

Quayle JM and Standen NB. KATP channels in vascular smooth muscle. Cardiovasc

Res 28: 797-804, 1994.



Sand AE, Andersson E, and Fried G. Effects of nitric oxide donors and inhibitors of

nitric oxide signalling on endothelin- and serotonin-induced contractions in human placental arteries. Acta Physiol Scand 174: 217-223, 2002. 56.

Shepard AR and Rae JL. Electrically silent potassium channel subunits from human

lens epithelium. Am J Physiol 277: C412-424, 1999. 57.

Sobey CG. Potassium channel function in vascular disease. Arterioscler Thromb Vasc

Biol 21: 28-38, 2001. 58.

Sobko A, Peretz A, and Attali B. Constitutive activation of delayed-rectifier

potassium channels by a src family tyrosine kinase in Schwann cells. Embo J 17: 4723-4734, 1998. 59.

Standen NB, Quayle JM, Davies NW, Brayden JE, Huang Y, and Nelson MT.

Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science 245: 177-180, 1989. 60.

Thengchaisri N and Kuo L. Hydrogen peroxide induces endothelium-dependent and

-independent coronary arteriolar dilation: role of cyclooxygenase and potassium channels. Am J Physiol Heart Circ Physiol 285: H2255-2263, 2003. 61.

Tokube K, Kiyosue T, and Arita M. Effects of hydroxyl radicals on KATP channels

in guinea-pig ventricular myocytes. Pflugers Arch 437: 155-157, 1998. 62.

Wareing M, Crocker IP, Warren AY, Taggart MJ, and Baker PN.

Characterization of small arteries isolated from the human placental chorionic plate. Placenta 23: 400-409, 2002. 63.

Wareing M, Greenwood SL, and Baker PN. Reactivity of human placental

chorionic plate vessels is modified by level of oxygenation: differences between arteries and veins. Placenta 27: 48-48, 2006.



Wareing M, Greenwood SL, Taggart MJ, and Baker PN. Vasoactive responses of

veins isolated from the human placental chorionic plate. Placenta 24: 790-796, 2003. 65.

Watanuki M, Horie M, Tsuchiya K, Obayashi K, and Sasayama S. Endothelin-1

inhibition of cardiac ATP-sensitive K+ channels via pertussis-toxin-sensitive G-proteins. Cardiovasc Res 33: 123-130, 1997. 66.

Yoshida H, Feig JE, Morrissey A, Ghiu IA, Artman M, and Coetzee WA. K ATP

channels of primary human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits. J Mol Cell Cardiol 37: 857-869, 2004. 67.

Yuan JX. Oxygen-sensitive K(+) channel(s): where and what? Am J Physiol Lung Cell

Mol Physiol 281: L1345-1349, 2001. 68.

Yuan XJ, Tod ML, Rubin LJ, and Blaustein MP. Contrasting effects of hypoxia on

tension in rat pulmonary and mesenteric arteries. Am J Physiol 259: H281-289, 1990. 69.

Zhao Y, Packer CS, and Rhoades RA. Pulmonary vein contracts in response to

hypoxia. Am J Physiol 265: L87-92, 1993.


Figure legends: Figure 1. K+ channel gene expression. Two representative examples of PCR products amplified from chorionic arterial (PA), venous (PV) samples and whole placenta (PL). PA1, PV1 and PL1, and PA2, PV2 and PL2 are matched samples from individual term placentas. – ve: dH20 negative control. +ve: human brain cDNA positive control, cDNA integrity confirmed with I-actin.

Figure 2. K+ channel protein expression. Membranes were probed with (A) anti-KV2.1 (1:1000), (B) anti-BKCa (1:500), (C) anti-KIR6.1 (1:500) or (D) anti-TASK1 (1:100). Primary antibody exposed to paired samples from a minimum of 3 term placentas. Exposure of primary antibody to its antigenic peptide or its omission resulted in signal ablation. Molecular weight standards (in kD) as indicated. RB-rat brain, PA- artery, PV-vein.

Figure 3. Functional responses to 4-AP. All data in 7% oxygenation. Effect of 4-AP on basal tone in arteries (A) and veins (B). Key: pre-4-AP (solid bar); 5 mins post-4-AP (1mM; hatched bar); All data mean +/- SE; P = Wilcoxon signed rank test. Effect of 4-AP on U46619-induced contraction in arteries (C) and veins (D). Key: P = Repeated measures ANOVA; * P

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