CRH And Endogenous H₂S Differentially Modulate Human Myometrial Contractility Before And During Labour

Premature birth remains a major cause of infant morbidity and is a serious health and financial burden to society. The pre-excitement of uterine smooth muscle will lead to preterm labor. And the etiology for premature labor is not clear. While in human the events that trigger parturition are not well defined, prior to the onset of parturition, a series of coordinate events prepare the myometrium to respond to contractant signals and to be less responsive to relaxant signals.Placental CRH production increases exponentially as pregnancy progresses toward labor, and it has been proposed that CRH might act as a placental clock regulating the length of human gestation. The precise biological role of placental CRH during pregnancy and labor remains unknown, but the human myometrium appears to be a major target of their actions. In vitro studies in our laboratory suggest that, CRH acts on CRH-R1 to inhibit spontaneous contractions in term myometrium from women who were not undergoing labour, but not those who were undergoing labour. But the mechanisms of CRH different modulation on human myometrial contractility before and during labour is unknown.Hydrogen sulfide (H₂S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H₂S produced locally in the control of uterine contractility during labour is unknown.The aims of the present study were to explore effects of CRH and endogenous H₂S on contractility of human term myometrium, and compare these effects in labouring and nonlabouring myometrium. Further, study the mechanisms of CRH and endogenous H₂S different modulation in human myometrium contractility before and during labor.Results:1. CRH (10-10-10-7 mol/L) enhance oxytocin-induced contractions and produced dose-dependent increase in [Ca²⁺]_i in myometrium from women who were undergoing labour, but not those who were not undergoing labour.2. The effects, CRH increase in [Ca²⁺]_i in myometrium cells from women underwent labour, were reversed by CRH receptor type 1 (CRHR1) antagonist antalarmin, but not by CRH receptor type 2 (CRHR2) antagonist astressin 2b. And CRHR1 small interfering RNA (siRNA) abrogate the effect of [Ca²⁺]_i increase in laboring myometrium cell stimulated by CRH.For the nonlabouring myometrium, CRH did not induce [Ca²⁺]_i transient in myometium cells. Antalarmin(10-9-10-6 mol/L) dose-dependently increased [Ca²⁺]_i in the cells, whereas astressin 2b did not. The effects of antalarmin could be blocked by exogenous CRH, suggesting that endogenous CRH has tonic inhibitory effects on [Ca²⁺]_i . And CRHR1 siRNA abrogate the effect of [Ca²⁺]_i increase in nonlaboring myometrium cell stimulated by antalarmin. CRHR1 exert the contrasting roles of maintaining myometrial relaxation before labor onset and stimulating contractility during labor.3. The possible signaling pathways involved in the CRH regulation of [Ca²⁺]_i were also investigated.For the labouring myometrium, CRH (10-9-10-6 mol/L) induced active Gαi expression and dose-dependently decreased cAMP production in the cells. Blocking phospholipase C (PLC) activity with U73122 and application of IP₃ receptor antagonist blocked CRH-evoked [Ca²⁺]_i transient in laboring myometrium cells. CRH (10-10-10-7 mol/L) dose-dependently induced phosphorylated PLC-?3 expression and increased in level of IP₃ in labouring myometrium cells. These effects were partly block by Gαi antibody and totally block by GP-2A, the inhibitor of Gαq.For nonlabouring myometrium, CRH (10-10-10-7 mol/L) induced active Gαs expression and antalarmin (10-9-10-6 mol/L) dose-dependently decreased cAMP production in the cells. Forskolin, the adenylate cyclase activator, reversed antalarmin-induced [Ca²⁺]_i transient. SQ 22536, an adenylate cyclase inhibitor, induced [Ca²⁺]_i transient. Our results suggest that CRH act on CRHR1 to induce [Ca²⁺]_i transient in labouring myometrium, but inhibit [Ca²⁺]_i in nonlabouring myometrium. Before labour onset, CRHR1 may coupled with Gs protein and CRH enhances AC-PKA signaling pathway to decrease [Ca²⁺]_i in myometrium cells. During labour, CRHR1 may couple with Gq protein and CRH activates PLC/IP₃ signaling pathway to increase [Ca²⁺]_i in myometrium cells.4. Cumulative administration of L-cysteine (10-7-10-2mol/L), a precursor of H₂S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10-3mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.Conclusion:Our results suggest that CRH act on CRHR1 to induce [Ca²⁺]_i transient in labouring myometrium, but inhibit [Ca²⁺]_i transient in nonlabouring myometrium. Before labour onset, CRHR1 may coupled with Gαs protein and CRH stimulates AC-PKA signaling pathway to decrease [Ca²⁺]_i in myometrium cells. During labour, CRHR1 may couple with Gαq protein and Gαi protein, and CRH activates PLC/IP₃ signaling pathway to induced [Ca²⁺]_i transient in myometrium cells, thereby facilitating contraction.H₂S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H₂S synthetic enzymes is down-regulated during labour, suggesting that H₂S is one of factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.