1.Regulation Of BK_Ca Channel Expression In Human Pregnant Myometrial Cells Mediated By CRH 2.Expression Of Synthetases Responsible For H₂S Production And K_ATP Channel In Human Labor And Nonlabor Myometrium

Numerous physiological signals which trigger uterine myometrial contractility at term, orchestrate changes in uterine excitability via ion-channel modulation. Large conductance calcium-activated potassium channel (BKCa), which expression level is highest among potassium ion-channels of uterine, plays an important role in maintaining uterine myometrial quiescent state. Corticotropin-Releasing Hormone (CRH) takes a vital part in the progression of pregnancy and initiation of labor in most mammalian species including human. During pregnancy, CRH and its related peptides secreted by placenta can modulate dilatation condition of uterine via different kinds of CRH receptors. However, the molecular targets and mechanism of its regulation are uncovered. In the first part of present study, we conducted experiments to determine the modulation of CRH on BKCa via different receptors in myometrial cells.Studies over recent years demonstrate that gas molecules such as NO and CO can relax urine myometrium via GC-cGMP pathway. As the third gas molecule, H2S comes under observation that it causes activation of K+channels leading to membrane hyperpolarization and relaxation of vascular smooth muscle cells. While ATP sensitive potassium channel also contribute to maintain the urine quiescent status in the progression of pregnancy. In the second part, we conducted experiments to observe expression level of H2S synthetases, and the H2S production rate in nonlabor myometrium and labor myometrium. Meanwhile the expression level of KATP subunits on different status myometrial tissue is also under observation.Main results:1. Regulation of BKCa expression mediated by CRH via different receptors1) Both CRHR1 and CRHR2 expressed in cultured myometrial cells in vitro showed by immunofluroscence respectively.2) RIA measured the endogenous CRH level which is higher in cultured labor myometrial cells than nonlabor myometrial cells.3) Antalarmin, the specific CRHR1 antagonist, down-regulated the mRNA and protein expression ofαsubunit andβsubunit in nonlabor myometrial cells, while up-regulated their expression in labor myometrial cells. Specific CRHR2 antagonist astressin 2B upregulated the mRNA and protein expression of a subunit andβsubunit in nonlabor myometrial cells, down-regulates expression of a subunit, while has no effects on (3 subunit expression.4) Exogenous CRH induced the mRNA and protein expression of a subunit andβsubunit in nonlabor myometrial cells which can be blocked by antalarmin and astressin. The mRNA and protein expression of a subunit andβsubunit was down-regulated by exogenous CRH in labor myometrial cells which can also be blocked by antalarmin and astressin. These results suggest that effect mediated by CRH in myometrial cells mainly via CRHR1.5) Primary nonlabor myometrial cells infected with CRHR1 siRNA or CRHR2 siRNA respectively, the results confirmed that CRH via CRHR1 up-regulated expression of BKca in nonlabor myometrial cells, and CRHR2 mediated the opposite effects. 2. Expression of synthetases responsible for H2S production and ATP-sensitive potassium channels in human labor and nonlabor myometrium.1) Both H2S synthetases, CBS and CSE, express in myometrium showed by immunochemistry.2) The mRNA and protein expression of CBS were significantly down-regulated in term in labor (TL) biopsies compared to those of term not in labor (TNL) samples, while the mRNA expression of CSE downgraded as well, but the protein level remained constant. And expression of CBS is higher than CSE which may promote that CBS plays leading role to produce H2S in the mechanism of relax myometrium.3) To define the real-time kinetics of H2S production by myometrial cells, we used miniaturized H2S micro-respiration sensor to measure its production. Results showed that higher level of H2S production in nonlabor myometrial cells than labor cells. It suggests that H2S involves in maintain of uterus in pregnancy progress.4) Four KATP channel subunits, namely Kir6.1, Kir6.2, SUR1, SUR2B, were identified in pregnant myometrium and localized to myometrial smooth muscle.5) The mRNA and protein expression of Kir6.1 and Kir6.2 were significantly down-regulated in term in labor (TL) biopsies compared to those of term not in labor (TNL) samples. The protein, but not mRNA expression of SUR1 was significantly increased in the TL group compared with TNL group. No significant difference in SUR2B expression at either the mRNA or protein level between TNL and TL groups was observed. ConclusionIn conclusion, these results suggest that the expression of BKCa, one of the most important ion channels in human myometrium, is under a dynamic control in human pregnancy and parturition. CRH can regulation the expression of BKca via its different receptors by means of paracrine and atuocrine in human myometrium, which plays a role in maintaining pregnancy and initiation of parturition. And the down-regulation of the expression of H2S synthetases as well as KATP channel core subunits Kir6.1 and Kir6.2 in myometrium following labor may contribute, in part, to enhance uterine contractility during parturition.