Investigating the role of ion channels across the fetomaternal interface of the human placenta
Ali, Tayyba Yasmin (2012) Investigating the role of ion channels across the fetomaternal interface of the human placenta. PhD thesis, University of Nottingham.
Chorionic plate (CPA) and stem villous (SVA) arteries located at the fetal and maternal interface of the placenta respond to stimuli including hypoxia and acidic pH which can be the result of an intermittent blood supply. Unlike other vascular tissue the placenta lacks nervous control so any response to such stimuli will be autoregulated by ion channels. Members of the two pore domain potassium channel family (K2P) the Tandem of P domains in a weak inward rectifying (TWIK) related potassium channel (TREK-1) and the TWIK Related acid sensitive K+ channel (TASK-1/3) have been shown to respond to both intracellular and extracellular pH. The hypothesis that there is differential expression and modulation of these candidate ion channels in normal pregnancy was tested. Placentae (N) were collected with written informed consent from healthy patients undergoing elective Caesarean section at term (≥37 weeks). The functional responses of resistance sized arteries (≤500µm) (n) taken from the SVA and the CPA were characterised using wire myography. Vessels were pre-contracted with U46619 and the effect of extracellular pH was studied using 1M lactic acid to produce falls of 0.2 pH units over a range of pH 7.4-6.4. The effects of a variety of ion channel modulators along with tissue oxygenation (20%, 5% and 2% O2) were also investigated on the vascular response of CPA and SVA. Western blot analysis was performed on crude CPA and SVA tissue homogenates with separation by 12% SDS-PAGE to quantify expression of TASK-1/3 and TREK-1. The subcellular localisation of each ion channel was also examined with smooth muscle cells (SMC) cultured from the CPA and SVA by confocal immunofluorescence. CPA and SVA were equally positive for TASK 1/3 (N=31) and TREK 1 (N=40) at the protein level. SMC from CPA and SVA showed expression for TASK 1/3 (N=8) with an increased fluorescence stain around the peri nuclear region. TREK-1 (N=12) expression showed a linear organisation that closely overlapped with α actin IF stain. The acidic pH stimulation triggered a biphasic relaxation that was repeated with each subsequent pH insult. A change from pH 7.4-7.2 produced a 29 ± 3% (n=9) relaxation of CPA which increased to 61 ± 4% at the lowest pH of 6.4 in 20% pO2. Similarly, altering the pH of pre-constricted SVA caused a 21 ± 2% (n=6) fall at pH 7.2 with a maximum relaxation of 69 ± 2% at pH 6.4 (p<0.01). Lowering pO2 from 20% to 5% inhibited the relaxation response seen with CPA (45 ± 3%, n=8) and SVA (34 ± 3%, n=6) at pH 6.4. CPA were also treated with the TREK-1 blocker L-methionine (1mM) which increased the relaxation to 67 ± 7% (n=6 p<0.001) at pH 6.4. Similarly the TASK 1/3 blocker ZnCl2 (1mM) gave a maximum relaxation of 72 ± 5% (n=8 p<0.01) in 20% pO2. The TREK-1 opener riluzole demonstrated a potent relaxation with both CPA (75 ± 5%, n=6) and SVA (78 ± 5%, n=6) in 20% pO2. Our data has shown that tissue oxygenation and extracellular pH within the physiological range has an important role in controlling vasodilatation in the placenta. Protons are readily transported across the cell membrane and can activate a range of targets including the K2P channels. The relaxation by riluzole has not been previously reported and implicates a direct role for TREK-1 in controlling placental vessel function. However, when TREK-1 and TASK-3 channels were blocked, the response by CPA to lower pH was exaggerated, and reflects the complex pharmacology of pH on vascular function. This also suggests that K2P channel activity can be compensated for by other pH sensitive channels and work is currently underway to identify the role of other potential ion channels that may be involved in this pathway.
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