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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.

Abstract:

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

Human

Potassium channels

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artery

vein

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).

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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).

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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

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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

5’-CCATGATGTGAGTACCGACTCCTC-3’ (forward) with 5’-GAACTCCGACATGCTGTGAACG-3’ (reverse), Ta 55oC (56); BKCa J-subunit 5’-CAGACACTGACTGGCAGAGTCCTGG-3’ (forward) with 5’-GCATCGACCGTTTGTACCGGTCAGG-3’ (reverse), Ta 64oC (30); KIR6.1 5’TTGGCCAGAAAGAGTATCCCGGAG-3’

(forward) with 5’-CATTCCACTTTTCTCCATGTAAGC-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;

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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

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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:

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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

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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

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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.

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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.

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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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