The endogenous production of hydrogen sulphide in intrauterine tissues

Background: Hydrogen sulphide is a gas signalling molecule which is produced endogenously from L-cysteine via the enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). The possible role of hydrogen sulphide in reproduction has not yet been fully investigated. It has been previously demonstrated that hydrogen sulphide relaxes uterine smooth muscle in vitro. The aim of the present study was to investigate the endogenous production of hydrogen sulphide in rat and human intrauterine tissues in vitro. Methods: The production of hydrogen sulphide in rat and human intrauterine tissues was measured in vitro using a standard technique. The expression of CBS and CSE was also investigated in rat and human intrauterine tissues via Western blotting. Furthermore, the effects of nitric oxide (NO) and low oxygen conditions on the production rates of hydrogen sulphide were investigated. Results: The order of hydrogen sulphide production rates (mean +/- SD, n = 4) for rat tissues were: liver (777 +/- 163 nM/min/g) > uterus (168 +/- 100 nM/min/g) > fetal membranes (22.3 +/- 15.0 nM/min/g) > placenta (11.1 +/- 4.7 nM/min/g), compared to human placenta (200 +/- 102 nM/ min/g). NO significantly increased hydrogen sulphide production in rat fetal membranes (P < 0.05). Under low oxygen conditions the production of hydrogen sulphide was significantly elevated in human placenta, rat liver, uterus and fetal membranes (P < 0.05). Western blotting (n = 4) detected the expression of CBS and CSE in all rat intrauterine tissues, and in human placenta, myometrium, amnion and chorion. Conclusion: Rat and human intrauterine tissues produce hydrogen sulphide in vitro possibly via CBS and CSE enzymes. NO increased the production of hydrogen sulphide in rat fetal membranes. The augmentation of hydrogen sulphide production in human intrauterine tissues in a low oxygen environment could have a role in pathophysiology of pregnancy

I_to reduction in cardiomyocytes isolated from the left atrial poisterior wall (LAPW).

<p>(a) Example voltage-sensitive, Ca²⁺ independent, macroscopic K⁺ currents evoked in cardiomyocytes isolated from the left atrial appendage (LAA, left) and LAPW (right). Voltage protocol is shown inset. (b-d) LAA and LAPW I/V relationships for the peak outward K⁺ current, I_to and steady state K⁺ current. Data presented as mean ± SEM. * denotes P<0.05 LAA (N = 16 cells) v LAPW (N = 12 cells), two way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis.</p

I_KACh is depleted in left atrial posterior wall (LAPW) cardiomyocytes.

<p>(a) Current traces demonstrating isolation of BaCl₂ sensitive (I_K1) and CCh induced (I_KACh) currents in a single left atrial cardiomyocyte. Voltage protocol is shown inset. (b & c) Comparison of LAA and LAPW I/V relationships for I_K1 and I_KACh. The dashed lines indicate mean best fit I_K1 and I_KACh I/V curves with liquid junction potential correction, for both LAA and LAPW. Data presented as mean ± SEM. * denotes P<0.05 LAA (N = 25 cells) v LAPW (N = 17 cells), two way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis.</p

Action potential (AP) prolongation and heterogeneity in the left atrial posterior wall (LAPW).

<p>(a) Examples of left atrial (LA) isochronal action potential duration (APD) distribution maps at 30 and 70% repolarisation. (b) A raw fluorescence image of an LA loaded with Di-4-ANEPPS, along with the 9 region grid used for quantitative regional analysis. (c) Example optical action potentials (OAPs) recorded from the 9 different LA regions during 10Hz pacing. The green dotted line indicates APD70. (d) Box and whisker plot of APD70 values measured in each LA region. * denotes P<0.05 vs regions 7,8,9 inclusive, + P<0.05 vs region 7 only, one way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis, N = 18 LA. Inset: Heat map depicting mean APD70 values of the 9 LA regions of the LA. (e) Example isochronal APD70 distribution maps of the same LA at 10 and 1Hz (same scale). (f) Mean APD70 at 10 and 1Hz for the left atrial appendage (LAA) and left atrial posterior wall (LAPW). * denotes P<0.05 LAA v LAPW, one way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis, N = 5 LA. (g) LA gradients at 10 and 1Hz. * denotes P<0.05 LAA v LAPW, paired t-test, N = 5 LA.</p

Ion channel expression differences between the left atrial posterior wall (LAPW) and left atrial appendage (LAA).

<p>(a-c) Comparisons of K⁺, Na⁺ and background/leak channel gene expression, between the LAPW and LAA, measured using Taqman Low Density Array (TDLA). Control sample was the LAA. ** and *** denote P<0.01 and P<0.001 respectively, LAA v LAPW, paired t-test, N = 9 LA.</p

Action potential differences between cardiomyocytes in the left atrial posterior wall (LAPW) and left atrial appendage (LAA).

<p>(a) Example intracellular recording trace demonstrating the stimulation protocol used to achieve sufficient action potential rate adaptation. (b) Example transmembrane action potentials (TAPs) taken from the LAA and LAPW of the same left atrium. TAPs are aligned at the resting membrane potential (RMP). The green vertical line indicates action potential duration at 90% repolarisation (APD90). (c-f) Box and whisker plots and individual values comparing the RMP, APD50-90, action potential amplitude (APA) and dV/dt (Vmax), of the LAA and LAPW, at 10Hz pacing frequency. **, *** denotes P<0.01 and P<0.001, LAA v LAPW, one way repeated measures Analysis of Variance (ANOVA) with Bonferroni post hoc analysis, or paired t-test; N = 20 LA.</p