Protein Expression And Function Of Calcium-Activated Potassium Channels In Human Endometrial Cells

BackgroudCa²⁺-activated K⁺(KCa) channels are a unique family of ion channels because they are capable of directly communicating calcium signals to changes in cell membrane potential required for cellular processes including but not limited to cellular proliferation and migration. Many cells also express Ca²⁺-activated K⁺(KCa) channels, which have the unique ability to translate changes in the level of the intracellular second messenger, Ca²⁺, to changes in membrane K⁺ conductance and, hence, resting membrane potential (Stocker, 2004; Swarthout and Walling, 2000). It is now possible to distinguish three families of KCa channels based on differences in their biophysical and pharmacological properties as well as genomic sequence. They are named large-conductance calcium-activated potassium channel (BKCa); Intermediate-conductance calcium-activated potassium channel (IKCa) and Small-conductance calcium-activated potassium channel (SKCa) with respect to their conductance. While all three classes of KCa channels share their regulation by intracellular Ca²⁺, they are otherwise rather distinct entities with different tissue distribution and presumed function. BKCa channels are known for their ability to regulate vascular tone of blood vessels (Sah and Davies, 2000; Sah and Louise Faber, 2002). SKCa channels are widely expressed in the nervous system where they are involved in regulating the firing frequency of many neurons (Bahia et al., 2005). IKCachannels on the other hand have been described in numerous cancer cells where they have been implicated in growth control.Embryo implantation is critical to embryogenesis and its development. Unfortunately, the mechanism of embryo implantation is unknown. Recently, the function of channel proteins in endometrium, including the relationship between channels and embryo implantation, development, has become a new hot spot in reproduction. The expression, disposition and function of many channels in endometrium have been studied,such as Cl , Na⁺,Ca²⁺ and Aquaporins. It is reported that CFTR(Cystic fibrosis transmembrane conductance regulator) has a critical role in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm, providing a link between defective CFTR and lower female fertility. It is firstly reported that there is a new aquaporin of aquaporin 2 expression in endometrium, as well as its distribution and regulation mechanism elucidated.In light of the well documented role that Ca2+ signaling plays in various cells, it is surprising that we know none about the expression and function of KCa channels in endometrial cells.The aim of the present study was to examine the protein expression of KCa in human uterine endometrium and their funtion of ovarian steroid hormone at the proliferative and Mid-secretory phase.Wetern blot and MTT were employed in the present study. Western blotting with antibodies specific for all three KCa channel types substantiated the expression of KCa channels in human uterine endometrium; The levels of endometrial KCa expression changed during the menstrual cycle. A significantly high level of BKCa was detected at the mid-secretory phase and IKCa, SKCa were detected at the proliferative phase. There may be an impotantant relationship between BKCa and embryo implantation. The use of selective pharmacological inhibitors shows inhibitation of endometrial cell growth by the IKCa channel specific inhibitor Tram-34. IKCa and SKCa may be involved in regultating endometrium proliferation.Materials and methods1. All women attended Women's hospital, School of Medicine, Zhejiang Universityfor IVF-ET treatment because of infertility due to tubule pathology. Endometrial samples were collected in the spontaneous cycle at 21st day (Mid-secretory phase) of the menstrual cycle(n=16); subjects undergoing elective surgey for leiomyoma uteri were recruited for proliferative samples(n=14). Western Blot was performed to identify KCa expression in endometrium. The results were semi-quantitified by Quantity One Software for Western blot.2. Four subjects undergoing elective surgey for leiomyoma uteri were recruited . Firstly, endometrial cells were primary cultured, then MTT was used to determine the viability of endometrial cells exposed to Tram-34, Apamin, E2. According to the OD results of human endometrial cells to 24 hours,48 hours, 72 hours, 96 hours exposure to them, a growth curve was drawed. The OD results of 96 hours were used for stastistical analysis.StastiticsAll datas were managed with SPSS13.0 for windows. One-way ANOVA, Tukey,T test were used for statistical analysis, two-tailed test with p<0.05 were considered significant.Results1. It was demonstrated that BKCa,IKCa,SKCa(SK3) were expressed in human endometrium at the proliferative and Mid-secretory phases.2. Semi-quantitative analysis result for BKCa, IKCa, SKCa(SK3) western-blotAfter normalizing each band of KCa with 6-actin from different samples, we found that the average level of BKCa expression in human endometrium at the Mid-secretory phase (1.24 ± 0.12) was significantly higher than that at the proliferative phase (0. 43 ± 0. 05) (p<0. 01) ; while IKCa expression in human endometrium at the proliferative phase (4.45 ± 0.70) was significantly higher than that at the Mid-secretory phase (0.95 ± 0.15) (p<0. 01), SKCa(SK3) expression in human endometrium at the proliferative phase (4. 80 ± 0. 76) was significantly higher than that at the Mid-secretory phase (0.98 ± 0.25)3. MTT was used to determine the viability of endometrial cells exposed to IKCa and SKCa(SK3) Special inhibitors, Tram-34 and ApaminAccording to the MTT OD results of human endometrial cells to 24 hours, 48 hours, 72 hours, 96 hours exposure to them,a growth assay was drawed .The OD results of 96 hours were used for stastistical analysis, control group (0.49±0.03); E₂ group (0.63±0.04); 1uM Tram-34 group (0.34±0.04); 1uM Apamin group (0.45 ± 0.03). Tram-34 group is significantly lower than the control group (p<0.05 ) and E₂ group (p<0.01) . Apamin group is significantly lower than E2 group (p<0.01) .There is no stastiscal difference between the other groups.Conclusions1. The expression of BKCa,IKCa,SKCa(SK3) in human endometrium during menstrual cycle were demonstrated in the present study, They are important ion channels in human endometrium.2. The average level of BKCa expression in human endometrium at the Mid-secretory phase was significantly higher than that at the proliferative phase ,which may regulate the production and delivery of factors at Mid-secretory phase.3. IKCa expression in human endometrium at the proliferative phase was significantly higher than that at the Mid-secretory phase, while Tram-34 inhibited endometrial cells growth. There may be an important relationship between endometrial cells growth and IKCa.4.SKCa (SK3) expression in human endometrium at the proliferative phase was significantly higher than that at the Mid-secretory phase. It may be involved in endometrial cells growth induced by E₂.