Expression and function of potassium channels in the human placental vasculature.

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, which are 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 voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. 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 TASK1 channels 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.

Reactivity of human placental chorionic plate vessels from pregnancies complicated by intrauterine growth restriction (IUGR).

A successful pregnancy is dependent on liberal placental perfusion via the maternal and fetal circulations. Doppler waveform analyses of umbilical arteries suggest increased resistance to flow in the fetoplacental circulation of pregnancies complicated by intrauterine growth restriction (IUGR). Neither the site nor the mediators responsible for this altered vascular reactivity are known, to date. In placentas in normal pregnancy, reduced oxygenation promotes contraction of the in vitro-perfused placental cotyledon and modulates agonist-induced contraction of chorionic plate arteries and veins. Placental oxygenation has also been suggested to be reduced in IUGR. We tested the hypothesis that oxygen tension could directly modify placental chorionic plate vessel vasoreactivity in IUGR. Small arteries and veins from the chorionic plate were dissected from biopsies from placentas of pregnancies complicated by IUGR and were studied using parallel wire myography. Vasoconstriction at 20%, 7%, and 2% oxygen was assessed utilizing the thromboxane mimetic U46619. Experiments were also performed in the presence of 4-aminopyridine (4AP), a blocker of voltage-gated potassium channels. Increased oxygenation reduced venous vasoconstriction but did not modify arterial vasoconstriction. 4AP increased basal tone in arteries and veins. We suggest that venoconstriction in response to hypoxia may provide a mechanism for increased fetoplacental vascular resistance associated with IUGR.

TASK channel expression in human placenta and cytotrophoblast cells.

OBJECTIVE: The multinucleate syncytiotrophoblast is the transporting epithelium of the human placental villus, formed throughout pregnancy by fusion and differentiation of underlying mononucleate cytotrophoblast cells. Similar to other epithelia, K+ channels will impact on syncytiotrophoblast transport properties during its development and differentiation. Therefore we investigated expression and activity of the two-pore domain K+ channels TASK1 and 2 in relation to gestation and differentiation, using villous tissue from first and third trimester and cultured cytotrophoblast cells at mononucleate and multinucleate stages of culture. METHODS: Quantitative real-time polymerase chain reaction (PCR), immunofluorescence, and 86Rb+ (K) efflux were used to investigate TASK channel expression and function. RESULTS: TASK2 mRNA expression was higher in first trimester than term (10 to 13 vs 38 to 40 weeks, P < .05). Other K+ alpha-subunit mRNAs, including TASK1, remained unaltered but the regulatory BKCa beta-subunit, like TASK2, was higher in first trimester than term (P < .001). Immunofluorescence showed that TASK2 had an intracellular localization within the trophoblast of first trimester villi but was less abundant and restricted to stem villi at term. TASK2 also showed intracellular localization in mononucleate cytotrophoblast cells in culture and expression was lost with multinucleation. By contrast, TASK1 was localised, independently of cell nucleation, to cytotrophoblast cell plasma membranes. 86Rb+ (K) efflux was measured from multinucleated cytotrophoblast cells. Both basal and pH 8.0-stimulated efflux was inhibited by the TASK1 antagonist anandamide (n = 5 for both conditions; P < .01 and P < .001, respectively). CONCLUSION: TASK1 and 2 are expressed in placental trophoblast cells and TASK1 activity may have a role in regulating syncytiotrophoblast homeostasis and/or solute transport functions.

Expression of TASK and TREK, two-pore domain K+ channels, in human myometrium.

Two-pore domain K+ channels are an emerging family of K+ channels that may contribute to setting membrane potential in both electrically excitable and non-excitable cells and, as such, influence cellular function. The human uteroplacental unit contains both excitable (e.g. myometrial) and non-excitable cells, whose function depends upon the activity of K+ channels. We have therefore investigated the expression of two members of this family, TWIK (two-pore domain weak inward rectifying K+ channel)-related acid-sensitive K+ channel (TASK) and TWIK-related K+ channel (TREK) in human myometrium. Using RT-PCR the mRNA expression of TASK and TREK isoforms was examined in myometrial tissue from pregnant women. mRNAs encoding TASK1, 4 and 5 and TREK1 were detected whereas weak or no signals were observed for TASK2, TASK3 and TREK2. Western blotting for TASK1 gave two bands of approximately 44 and 65 kDa, whereas TREK1 gave bands of approximately 59 and 90 kDa in myometrium from pregnant women. TASK1 and TREK1 immunofluorescence was prominent in intracellular and plasmalemmal locations within myometrial cells. Therefore, we conclude that the human myometrium is a site of expression for the two-pore domain K+ channel proteins TASK1 and TREK1.

Localization of TASK and TREK, two-pore domain K+ channels, in human cytotrophoblast cells.

OBJECTIVE: Two-pore domain K+ channels (K2P), an emerging K+ channel subfamily, contribute to setting membrane potential in both electrically excitable and nonexcitable cells and, as such, influence cellular function. The multinucleate syncytiotrophoblast of human placenta, formed from the fusion of mononucleate cytotrophoblast cells, is a transporting epithelium whose function likely depends on the activity of K+ channels. We have therefore investigated the gene expression of two members of this family, TASK and TREK, in cultured human cytotrophoblast cells, and have also investigated protein expression in cytotrophoblast cells and placenta. METHODS: We used reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemistry to investigate the gene and protein expression of TASK and TREK isoforms in both isolated cytotrophoblast cells and term placental tissue. RESULTS: In cytotrophoblast cells, mRNAs encoding TASK1, 2, 4, 5, and TREK1 were detected, whereas weak or no signals were observed for TASK3 and TREK2. Western blotting for TASK1 in cytotrophoblast cells gave two bands of approximately 78 and 150 kd; TREK1 gave bands of approximately 90 and 130 kd. TASK1 immunofluorescence in placenta colocalized with cytokeratin-7, a trophoblast-specific marker. TREK1 predominantly stained cells around the villous perimeter and this staining was colocalized with propidium iodide nuclear staining. CONCLUSION: Human cytotrophoblast cells from term placenta are a site of expression for various K2P genes, two of which, namely, TASK1 and TREK1, are transcribed into protein.