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

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

Functional coupling of beta(3)-adrenoceptors and large conductance calcium-activated potassium channels in human uterine myocytes

Journal articleContext: beta(3)-Adrenoreceptor modulation in human myometrium during pregnancy is linked functionally to myometrial inhibition. Maxi-K+ channels (BKCa) play a significant role in modulating cell membrane potential and excitability.Objective: This study was designed to investigate the potential involvement of BKCa channel function in the response of human myometrium to beta(3)-adrenoceptor activation.Design: Single and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes were obtained in the presence and absence of BRL37344, a specific beta(3)-adrenoreceptor agonist. The in vitro effects of BRL37344 on isolated myometrial contractions, in the presence and absence of the specific BKCa channel blocker, iberiotoxin ( IbTX), were investigated.Setting: The study was carried out at the Clinical Science Institute. Patients orOther Participants: Myometrial biopsies were obtained at elective cesarean delivery.Intervention: No intervention was applied.Main Outcome Measures: Open state probability of single channel recordings, whole cell currents, and myometrial contractile activity were measured.Results: Single-channel recordings identified the BKCa channel as a target of BRL37344. BRL37344 significantly increased the open state probability of this channel in a concentration-dependent manner ( control 0.031 +/- 0.004; 50 mu M BRL37344 0.073 +/- 0.005 (P < 0.001); and 100 mu M BRL37344 0.101 +/- 0.005 ( P < 0.001). This effect was completely blocked after preincubation of the cells with 1 mu M bupranolol, a nonspecific - adrenoreceptor blocker, or 100 nM SR59230a, a specific beta(3)- adrenoreceptor antagonist. In addition, BRL37344 increased whole- cell currents over a range of membrane potentials, and this effect was reversed by 100 nM IbTX. In vitro isometric tension studies demonstrated that BRL37344 exerted a significant concentrationdependent relaxant effect on human myometrial tissue (P < 0.05), and preincubation of these strips with IbTX attenuated this effect on both spontaneous and oxytocin- induced contractions (44.44 and 57.84% at 10(-5) M, respectively).Conclusions: These findings outline that activation of the BKCa channel may explain the potent uterorelaxant effect of beta(3)-adrenoreceptor agonists.Health Research Board (HRB) Ireland; HEA Ireland (Programme for Research in Third Level Institutions Cycle 1

Functional coupling of β3-adrenoceptors and large conductance calcium-activated potassium channels in human uterine myocytes

Context: beta(3)-Adrenoreceptor modulation in human myometrium during pregnancy is linked functionally to myometrial inhibition. Maxi-K(+) channels (BK(Ca)) play a significant role in modulating cell membrane potential and excitability. Objective: This study was designed to investigate the potential involvement of BKCa channel function in the response of human myometrium to beta(3)-adrenoceptor activation. Design: Single and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes were obtained in the presence and absence of BRL37344, a specific beta(3)-adrenoreceptor agonist. The in vitro effects of BRL37344 on isolated myometrial contractions, in the presence and absence of the specific BKCa channel blocker, iberiotoxin ( IbTX), were investigated. Setting: The study was carried out at the Clinical Science Institute. Patients or Other Participants: Myometrial biopsies were obtained at elective cesarean delivery. Intervention: No intervention was applied. Main Outcome Measures: Open state probability of single channel recordings, whole cell currents, and myometrial contractile activity were measured. Results: Single-channel recordings identified the BK(Ca) channel as a target of BRL37344. BRL37344 significantly increased the open state probability of this channel in a concentration-dependent manner ( control 0.031 +/- 0.004; 50 mu M BRL37344 0.073 +/- 0.005 (P < 0.001); and 100 mu M BRL37344 0.101 +/- 0.005 ( P < 0.001). This effect was completely blocked after preincubation of the cells with 1 mu M bupranolol, a nonspecific - adrenoreceptor blocker, or 100 nM SR59230a, a specific beta(3)- adrenoreceptor antagonist. In addition, BRL37344 increased whole- cell currents over a range of membrane potentials, and this effect was reversed by 100 nM IbTX. In vitro isometric tension studies demonstrated that BRL37344 exerted a significant concentrationdependent relaxant effect on human myometrial tissue (P < 0.05), and preincubation of these strips with IbTX attenuated this effect on both spontaneous and oxytocin- induced contractions (44.44 and 57.84% at 10(-5) M, respectively). Conclusions: These findings outline that activation of the BK(Ca) channel may explain the potent uterorelaxant effect of beta(3)-adrenoreceptor agonists

Human chorionic gonadotrophin relaxation of human pregnant myometrium and activation of the bkcachannel

The uterorelaxant effect of human chorionic gonadotropin (hCG) is regarded as an important mediator in maintenance of uterine quiescence during pregnancy with clinical potential for tocolysis, the mechanisms of which are unknown. The large conductance calcium-activated K(+) channel (BK(Ca)) 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 the involvement of BK(Ca) channel function in the response of human myometrial cells to hCG. Single electrophysiological BK(Ca) channel recordings from freshly dispersed myocytes were obtained in the presence and absence of increasing hCG concentrations. Isometric tension studies, investigating the effects of hCG on isolated myometrial contractions, in the presence and absence of the BK(Ca) channel blocker, iberiotoxin, were performed. The hCG significantly increased the open-state probability of these channels in a concentration-dependent manner [control 0.036 +/- 0.01; 1 IU/ml hCG 0.065 +/- 0.014 (P +/- 0.262); 10 IU/ml hCG 0.111 +/- 0.009 (P = 0.001); and 100 IU/ml hCG 0.098 +/- 0.004 (P = 0.007)]. In vitro functional studies demonstrated that hCG exerted a significant concentration-dependent relaxant effect on human myometrial tissue. This effect was significantly attenuated by preincubation with iberiotoxin (P < 0.05). These findings outline that activation of BK(Ca) channel activity may explain the potent uterorelaxant effect of hCG

Loci associated with ischaemic stroke and its subtypes (SiGN): a genome-wide association study

BACKGROUND: The discovery of disease-associated loci through genome-wide association studies (GWAS) is the leading genetic approach to the identification of novel biological pathways underlying diseases in humans. Until recently, GWAS in ischaemic stroke have been limited by small sample sizes and have yielded few loci associated with ischaemic stroke. We did a large-scale GWAS to identify additional susceptibility genes for stroke and its subtypes. METHODS: To identify genetic loci associated with ischaemic stroke, we did a two-stage GWAS. In the first stage, we included 16 851 cases with state-of-the-art phenotyping data and 32 473 stroke-free controls. Cases were aged 16 to 104 years, recruited between 1989 and 2012, and subtypes of ischaemic stroke were recorded by centrally trained and certified investigators who used the web-based protocol, Causative Classification of Stroke (CCS). We constructed case-control strata by identifying samples that were genotyped on nearly identical arrays and were of similar genetic ancestral background. We cleaned and imputed data by use of dense imputation reference panels generated from whole-genome sequence data. We did genome-wide testing to identify stroke-associated loci within each stratum for each available phenotype, and we combined summary-level results using inverse variance-weighted fixed-effects meta-analysis. In the second stage, we did in-silico lookups of 1372 single nucleotide polymorphisms identified from the first stage GWAS in 20 941 cases and 364 736 unique stroke-free controls. The ischaemic stroke subtypes of these cases had previously been established with the Trial of Org 10 172 in Acute Stroke Treatment (TOAST) classification system, in accordance with local standards. Results from the two stages were then jointly analysed in a final meta-analysis. FINDINGS: We identified a novel locus (G allele at rs12122341) at 1p13.2 near TSPAN2 that was associated with large artery atherosclerosis-related stroke (first stage odds ratio [OR] 1·21, 95% CI 1·13-1·30, p=4·50 × 10-8; joint OR 1·19, 1·12-1·26, p=1·30 × 10-9). Our results also supported robust associations with ischaemic stroke for four other loci that have been reported in previous studies, including PITX2 (first stage OR 1·39, 1·29-1·49, p=3·26 × 10-19; joint OR 1·37, 1·30-1·45, p=2·79 × 10-32) and ZFHX3 (first stage OR 1·19, 1·11-1·27, p=2·93 × 10-7; joint OR 1·17, 1·11-1·23, p=2·29 × 10-10) for cardioembolic stroke, and HDAC9 (first stage OR 1·29, 1·18-1·42, p=3·50 × 10-8; joint OR 1·24, 1·15-1·33, p=4·52 × 10-9) for large artery atherosclerosis stroke. The 12q24 locus near ALDH2, which has previously been associated with all ischaemic stroke but not with any specific subtype, exceeded genome-wide significance in the meta-analysis of small artery stroke (first stage OR 1·20, 1·12-1·28, p=6·82 × 10-8; joint OR 1·17, 1·11-1·23, p=2·92 × 10-9). Other loci associated with stroke in previous studies, including NINJ2, were not confirmed. INTERPRETATION: Our results suggest that all ischaemic stroke-related loci previously implicated by GWAS are subtype specific. We identified a novel gene associated with large artery atherosclerosis stroke susceptibility. Follow-up studies will be necessary to establish whether the locus near TSPAN2 can be a target for a novel therapeutic approach to stroke prevention. In view of the subtype-specificity of the associations detected, the rich phenotyping data available in the Stroke Genetics Network (SiGN) are likely to be crucial for further genetic discoveries related to ischaemic stroke