Role of Internal Ca2+ Stores in KCa Channel Feedback Control of Human Uterine Contractions

Abstract

Background Minute-to-minute regulation of intracellular calcium concentrations is essential to allow a laboring uterus to phasically contract and relax in a manner required for delivery. Cytosolic calcium levels increase through influx of calcium through L-type calcium channels and are amplified by SR calcium release through inositol 1, 4, 5-triphosphate receptor (IP3R) channels and calcium induced calcium release. A specific class of small conductance calcium-activated potassium (KCa2.3) channels has been strongly implicated in the negative feedback control of membrane potential and intracellular calcium levels. Uterine contractions can be suppressed by amplifying this calcium-dependent feedback system. CyPPA, a positive modulator of KCa2.3, reduces contraction amplitudes in human and rodent tissue. Previous studies of endothelial and neuronal cells suggest focused Ca2+ release from internal stores in close proximity to the plasma membrane, may play an important role in KCa channel control. Indeed, close proximity and interaction of endoplasmic reticulum Ca2+ release channels (e.g. IP3Rs) and KCa channels may regulate intracellular calcium in a variety of tissues. This study aims to assess whether internal stores contribute to KCa2.3 dependent feedback regulation of contraction in the human myometrium. Methods Myograph studies were performed on human uterine tissues collected from non-pregnant patients undergoing hysterectomy. Uterine strips were sutured end-to-end to form rings, mounted onto myograph pins, and allowed to develop spontaneous phasic contractions. Concentration-dependent suppression of contractions with the positive KCa2.3 modulator CyPPA (3 μM, 10 μM, 30 μM) was assessed following depletion of internal calcium stores (using the SERCA inhibitor cyclopiazonic acid, CPA) or following treatment with the vehicle DMSO (controls). Results Utilizing myographical recordings and LabChart software, contraction area under curve (AUC) was calculated and normalized to time. Addition of 3 μM CyPPA resulted in a control group mean AUC of 1.287 mN/min (± 0.417) and 2.261 mN/min (± 1.338) for the treatment group [n=5]. The mean AUC with 10 μM CyPPA was 1.129 mN/min (± 0.571) for the control group and 2.164 mN/min (± 1.387) for the treatment group. After 30 μM CyPPA the mean AUC for the control group was 0.734 mN/min (± 0.471) and 1.863 mN/min (± 1.364) for the treatment group. Our group found that CPA pretreated uterine tissue had a significantly higher AUC than the control group (p<0.5), after addition of 30 μM CyPPA. Further analysis demonstrated no significant difference with CyPPA concentrations of 3 and 10 μM, but did reveal a trend of reduced AUC in the control group compared to the treatment group. These findings suggest blocking SERCA and inhibiting SR reuptake of calcium may decrease CyPPAs ability to diminish contractions, especially at higher concentrations. Conclusions Phasic contractions of isolated human uterine strips vary considerably with respect to amplitude and frequency. Overall, the findings suggest internal calcium store release may play an important role in moderating uterine contractions and augmenting KCa2.3 channel suppression of uterine contractility. Extended studies will assess whether this mechanism can be exploited as an effective tocolytic strategy.