Can in vitro techniques elucidate the mechanism of labour?

Abstract

51 CAN IN VITRO TECHNIQUES ELUCIDATE THE MECHANISM OF LABOUR? *S. Thornton , J. I. Gillespie, J. R. Greenwell, E. Davies *W. Dunlop Department of Physiological Sciences and *Department of Obstetrics, The Medical School, The University, Newcastle upon Tyne, UK. The factors controlling human labour remain elusive, due in part to our ignorance of the physiological mechanisms of uterine contraction. The underlying mechanism is important since its determination would allow a rational approach to the pharmacological manipulation of uterine activity. Within the myometrial cell, intracellular calcium ([Ca*+]i) is of fundamental importance in the initiation of contraction. In addition to controlling actin and myosin interaction it is the final mediator of numerous intracellular second messengers. Invasive physiological studies are inappropriate during human labour. We have developed an approach using isolated myometrial cells. These can retain the characteristics of the parent tissue.’ Cell culture has been combined with fluorescent indicator techniques in order to determine [Ca*+]i in single human myometrial cells. Myometrial biopsies were obtained, with informed consent, following hysterectomy or at caesarean section. Cells were separated by enzymatic digestion and cultured in 5% CO, and 95% air. Once confluent, cells were plated onto glass coverslips and studied after a further 3-4 days culture. Calcium was measured by loading cells with a calcium-sensitive fluorescent indicator dye (Fura-2) and the change in fluorescence ratio determined in single cells by microspectrofluorimetry. The extracellular environment was manipulated as required. Application of oxytocin (OT) or prostaglandin E, (PGE,) caused a transient increase in [Ca*+]i. In 4 of the 200 cells studied, spontaneous transient increases in [Ca’ ‘Ii occurred. This may represent spontaneous activity which occurs in the pregnant or nonpregnant uterus. Removal of extracellular calcium ([Ca*+]o) prevents calcium influx. In calcium-free medium, OT caused a small increase in [Ca’ ‘Ii but the response to PGE2 was completely prevented. This suggests that these agents have a different mechanism of action and intracellular stores of calcium may be released by OT but not PGE,. Spontaneous transient increases in [Ca* ‘Ii are prevented by removal of [Ca* +]o or application of 2mM Ni’ + (a calcium channel blocker) The OT antagonist (CAP 476) reduced the frequency of the transients. We conclude that physiological techniques can be used to determine the mechanism of action of agents which influence myometrial contractility: OT and PGE2 increase [Ca”+]i , but only OT releases intracellular stored calcium. Spontaneous increases in [Ca*+]i occur in some cells. These are caused by the influx of extracellular calcium and may by modified by the OT antagonist CAP 476.