The annexins are a group of calcium and phospholipid binding proteins which may function as regulators of intracellular free Ca2+ or as mediators of Ca2+ action in the cell. Currently, the role of annexins in myometrium is unknown. We propose that annexins, under the control of steroid hormones, may regulate the contractile activity of the myometrium at parturition. Annexin II may regulate the formation of gap junctions which are essential for cell-cell coupling underlying the coordinate contraction of adjacent cells. Annexin VI may regulate the IP3-sensitive calcium release channel of the sarcoplasmic reticulum, leading to regulation of intracellular free Ca2+ and modulation of myosin light chain kinase activity. Our studies will include the following objectives: 1. Expression of annexins II and VI will be determined during normal gestation, in hormonally manipulated rats and in control and preterm human myometrial tissue. Intracellular localization of annexins II and VI will be performed, with selective co-localization by light and EM microscopy of annexin II with connexin 43 and of annexin VI with the IP3-sensitive calcium release channel. 2. The regulation of annexin II and VI expression by steroid hormones, using both hormonally manipulated rats and cultured myometrial cells will be determined. Annexins will be separated by subcellular fractionation. The nature and extent of covalent modifications in each subcellular fraction will be quantitated and evaluated to determine their role in regulation of annexin function. 3. The effect of suppression of connexin 43 or of annexin II using S- oligonucleotides antisense to their respective mRNAs will be examined. Concurrent measurements of gap junction formation and cell-cell coupling by electrophysiological methods will be made. The effect of agents which alter connexin and annexin activity (in particular, pp60arc tyrosine kinase) will be measured in conjunction with measurement of cell-cell coupling and subcellular localization of connexin 43. 4. The effect of annexin VI on the IP3-sensitive calcium release channel from the sarcoplasmic reticulum will be examined in lipid bilayer. These findings will be supported in vivo through the use of antisense against annexin VI to suppress intracellular concentration of annexin VI. Contractile properties and free calcium dynamics and ion channel activity will be examined following suppression of annexin VI in cultured myometrial cells. These studies will establish the role of annexins in two aspects of myometrial function which are essential to coordinated contractile activity. A more detailed understanding of the mediation and action of intracellular calcium will allow the development of novel approaches directed toward prevention of preterm labor.
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