Expression of stretch-activated two-pore potassium channels in human myometrium in pregnancy and labor

Abstract

Background: We tested the hypothesis that the stretch-activated, four-transmembrane domain, two pore potassium channels (K2P), TREK-1 and TRAAK are gestationally-regulated in human myometrium and contribute to uterine relaxation during pregnancy until labor.

Methodology: We determined the gene and protein expression of K2P channels in non-pregnant, pregnant term and preterm laboring myometrium. We employed both molecular biological and functional studies of K2P channels in myometrial samples taken from women undergoing cesarean delivery of a fetus.

Principal findings: TREK-1, but not TREK-2, channels are expressed in human myometrium and significantly up-regulated during pregnancy. Down-regulation of TREK-1 message was seen by Q-PCR in laboring tissues consistent with a role for TREK-1 in maintaining uterine quiescence prior to labor. The TRAAK channel was unregulated in the same women. Blockade of stretch-activated channels with a channel non-specific tarantula toxin (GsMTx-4) or the more specific TREK-1 antagonist L-methionine ethyl ester altered contractile frequency in a dose-dependent manner in pregnant myometrium. Arachidonic acid treatment lowered contractile tension an effect blocked by fluphenazine. Functional studies are consistent with a role for TREK-1 in uterine quiescence.

Conclusions: We provide evidence supporting a role for TREK-1 in contributing to uterine quiescence during gestation and hypothesize that dysregulation of this mechanism may underlie certain cases of spontaneous pre-term birth.

Figure 1. TRAAK K2P channel expression in human myometrium.

(A) PCR studies were carried out on both non-pregnant and pregnant tissues and human brain. (NTC, non template control). (B) Human brain served as a control and β-actin (500 bp) was used to adjust TRAAK gene expression (240 bp) relative to control gene expression. PCR transcripts at 240 base pairs were sequenced and matched known TRAAK sequence. (C) Expression of TRAAK in term and term laboring samples and in brain as a comparative control was determined by Q-PRC with the 18S ribosomal gene as an expression control.

Figure 2. TRAAK protein expression in human myometrium.

(A) Western blots were performed using a TRAAK-specific antibody (Santa Cruz) and stained using infrared fluorescence secondary from Licor Biosciences®. Individual lanes represent different patient samples and are shown to illustrate representative variation. GAPDH was employed as an expression control using simultaneous imaging. TRAAK expression is not increased by pregnancy compared to non-pregnant samples, nor was there any effect of labor (B). Representative Western blots are shown along with average data. Experiments were repeated three times on the same sample set and the results averaged for each sample. Data are Mean ± SEM compared by one-way analysis of variance.

Figure 3. TREK-1 K2P channel expression in myometrium.

(A) PCR studies were carried out on both non-pregnant and pregnant tissues and cells from term pregnancy myometrium and TREK-1 transcript confirmed by sequence analysis in each case. Individual lanes represent different patient samples and are shown to illustrate representative variation. (B) 18S ribosomal RNA was used to adjust TREK gene expression relative to control gene expression. (C) Expression of TREK in term and term laboring samples was determined by Q-PRC relative to the 18S ribosomal gene as an expression control (p<.001). Data are mean ± SEM; **  = p<0.01; ***  =  p<0.001 by unpaired T-test.

Figure 4. TREK-1 protein expression in myometrium.

(A) Western blots were performed using a TREK-1-specific antibody (Santa Cruz) and stained using infrared fluorescence secondary. GAPDH was employed as an expression control using simultaneous imaging. Individual lanes represent different patient samples and are shown to illustrate representative variation. TREK-1 expression is significantly increased by pregnancy compared to non-pregnant samples and, (B) remains unchanged during labor but expression is lower in preterm samples. Data are mean ± SEM, *  =  p<0.05; ***  =  p<0.001 by unpaired T-test.

Figure 5. GsMTx-4 increases OT-induced contractions.

Contractile bath experiments using the specific stretch-activated channel blocker, GsMTx-4 at doses of 0.9 to 1.8 µM, in the presence of OT were performed to observe the effect on non-laboring myometrium. OT-induced (1 µM) contractions were established, followed by a wash-out period. Tissue was then challenged with A) 0.9 µM GsMTx-4, causing increased duration of contractions, or (B) 1.8 µM GsMTx-4, causing attenuation of relaxation. Traces are representative of n = 4.

Figure 6. L-methionine ethyl ester increases frequency of contraction in a dose-dependent manner.

L-Mee increased contractile frequency in non-laboring myometrial strips in a concentration-dependent manner (30 µM to 300 mM). Iberiotoxin (100 nM) was present to block Ca²⁺-activated currents and tissues established spontaneous contractions before drug addition. (A) 30 µM L-Mee applied to the bath caused a slight increase in frequency of contraction, with recovery after washout. (B) 100 µM L-Mee caused a significant increase in frequency of contraction that was pronounced at 300 µM L-Mee with recovery after washout; 1 mM L-Mee caused a marked increase in frequency of contraction with modest, variable reduction in peak amplitude that remained after washout (not shown). (C) In a similar set of tissues, arachidonic acid (AA) significantly depressed contractions, an effect blocked by fluphenazine (FLU; 100 µM), while addition of fluphenazine alone had no significant effect. The effect of L-Mee to increase contractions was not different when added in the presence of 100 µM AA (not shown). Tissues returned to control tension after washout. Data are mean ± SEM, **  =  p<0.05, n = 7.