THG113.31, a specific PGF2alpha receptor antagonist, induces human myometrial relaxation and BKCa channel activation

Abstract

Background: PGF2alpha exerts a significant contractile effect on myometrium and is central to human labour. THG113.31, a specific non-competitive PGF2alpha receptor (FP) antagonist, exerts an inhibitory effect on myometrial contractility. The BKCa channel is ubiquitously encountered in human uterine tissue and plays a significant role in modulating myometrial cell membrane potential and excitability. The objective of this study was to investigate potential BKCa channel involvement in the response of human myometrium to THG113.31.

Methods: Single and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes, were investigated in the presence and absence of THG113.31. Functional studies investigated the effects of THG113.31 on isolated spontaneous myometrial contractions, in the presence and absence of the BKCa channel blocker, iberiotoxin.

Results: Single channel recordings identified the BKCa channel as a target of THG113.31. THG113.31 significantly increased the open state probability of these channels [control 0.023+/-0.006; 10 microM THG113.31 0.087+/-0.012 (P = 0.009); and 50 microM THG113.31 0.1356+/-0.018 (P = 0.001)]. In addition, THG113.31 increased whole-cell BKCa currents over a range of membrane potentials, and this effect was reversed by 100 nanoM IbTX. Isometric tension studies demonstrated that THG113.31 exerted a significant concentration-dependent relaxant effect on human myometrial tissue and pre-incubation of strips with IbTX abolished this effect on spontaneously occurring contractions.

Conclusion: These data suggests that activation of the BKCa channel may contribute, at least partially, to the uterorelaxant effect of THG113.31.

Figure 1

NS1619, a putative activator of BK_Ca channels, and IbTX, a specific BK_Ca channel blocker, serve to identify the BK_Ca channel in human uterine smooth muscle myocytes. Representative recordings from the same membrane patch (+40 mV) in the cell-attached configuration before and after addition of 20 μM NS1619 and subsequent addition of 100 nM IbTX. IbTX reversed NS1619-stimulated channel activity.

Figure 2

THG113.31 enhances BK_Ca channel activity in human myometrial smooth muscle cells. A, B & C Representative continuous recordings were recorded from the same cell attached patch (+40 mV) before (control) and 15 min after exposure to 10 & 50 μM THG113.31, respectively. Channel openings are upward deflections from the baseline (closed state) (dashed line).

Figure 3

THG113.31 opens BK_Ca channel activity in human myometrial smooth muscle cells in a concentration-dependent manner. A Activity plot of BK_Ca channel open probability in a single cell-attached patch before and 15 min after exposure to cumulative doses of 10 and 50 μM THG113.31. Vertical bars are channel activity during a 100-ms test pulse to +40 mV. Recording time under each condition was 10 s, as indicated on the time axis. Breaks in the time axis represent drug incubation periods when channel activity was not recorded. Period of drug exposure is indicated by horizontal lines above the histogram. B Each bar represents the average channel open probability obtained from cell attached patches (+40 mV) before and 15 min after additions of 10 and 50 μM THG113.31, respectively. *Significant increase in channel activity compared to control (n = 6; P < 0.005)

Figure 4

PGF_2α has no effect on BK_Ca channel activity. A Representative recordings from the same membrane patch (+40mV) in the cell-attached configuration after addition of 20 μM NS1619 and subsequent addition of 1 μM PGF_2α. PGF_2α did not reverse NS1619-stimulated channel activity. B Each bar represents the average channel open probability obtained from cell attached patches (n = 4; +40 mV) before and 15 min after each addition (n = 4). *Significant increase in channel activity compared to control. C & D THG113.31 activates the BK_Ca channel independently of the FP receptor. Representative recordings from the same membrane patch (+40mV) in the cell-attached configuration before and after addition of 1 μM PGF_2α and subsequent addition of 10 μM THG113.31. Each bar represents the average channel open probability obtained from cell attached patches (n = 3; +40 mV) before and 15 min after each addition (n = 3). *Significant increase in channel activity compared to control.

Figure 5

THG113.31 increases whole-cell currents in human uterine smooth muscle myocytes. A Perforated patch recordings (n = 3) from the same myocyte before and 15 min after exposure to 50 μM THG113.31, and then 5–10 min after cumulative addition of 100 nM IbTX. Tracings were taken from a range of potentials (-40 to +50 mV), with a holding potential of -60 mV. B The complete current (pA)-voltage (mV) relationship for steady-state outward current from the same cell as in A. Treatment conditions are the same as described for control, 50 μM THG113.31, and THG113.31 and 100 nM IbTX. C Average current-voltage relationship for uterine myocytes before and after 50 μM THG113.31, and then cumulative addition of 100 nM IbTX. Each point represents the mean number of cells ± SEM.

Figure 6

Effects of THG113.31 on spontaneous myometrial contractions. A Representative recordings of spontaneous contractions of pregnant human myometrium under control conditions and (B) the effects of cumulative additions of THG113.31 (1 nM, 10 nM, 100 nM, 1 μM and 10 μM) at 20 min intervals are shown. (C & D) The uterorelaxant effect was significantly attenuated by pre-incubation with the BK_Ca channel antagonist, IbTX

Figure 7

Dose response curves for THG113.31 and spontaneous myometrial contractions. Graphical representation of the effects of cumulatively increasing tissue bath concentrations of THG113.31 (1 nM, 10 nM, 100 nM, 1 μM and 10 μM) at 20 min intervals on spontaneous (shaded circle) contracting pregnant myometrium in the presence (shaded triangle) and absence (shaded square) of IbTX. A control trace showing uninterrupted spontaneous contractions in pregnant myometrium in the presence (open square) and absence (shaded circle) of IbTX, are shown for comparison. The symbols used represent the mean values (n = 6) within each group. Vertical error bars represent the standard error of the mean.