Uterine Relaxing Factors: Molecular Aspects of Action

Colorado State University, Fort Collins CO

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Abstract

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the molecular mechanisms by which hormones influence uterine smooth muscle contraction and relaxation. This proposal focuses on defining cellular and subcellular mechanisms involved in crosstalk between major contractant and relaxant signaling pathways in the myometrium, in particular the role of scaffolding proteins and compartmentalization of signaling units in these mechanisms. Aim 1 will determine the mechanism responsible for the decline in myometrial plasma membrane-associated PKA prior to delivery in the pregnant rat and the relationship of this decline to the regulation of uterine contractile activity by cAMP. We will use a combination of muscle strip, cell culture and whole animal models to study basic mechanism and relate biochemical changes to effects on uterine contractile activity. Aim 2 will determine if similar changes in myometrial plasma membrane PKA occur in primate pregnancy in relation to labor. We will examine changes in tissue from both fundus and lower uterine segment in pregnant baboons at different stages in the transition from contractures to contractions and in myometrial tissues from women obtained at the time of cesarean section before or after the onset of labor. Mechanisms of regulation will be explored in vivo and in a pregnant human myometrial cell line. Aim 3 will determine the importance of the association of PKA and PP2B with AKAP79/150 in the control of myometrial PLCbeta3 phosphorylation. We will determine the phosphorylation status of PLCbeta3 in response to PKA activation in cells and tissue under conditions where we know Galpha(q) coupling is affected and will explore mechanisms by which PKA can influence activity and membrane association of itself, PP2B subunits and AKAP79/150 in cell culture and pregnant rat myometrial strip models. Aim 4 will determine how the constituents of OTR and cAMP regulatory pathways and K(Ca) channels are distributed between plasma membrane microdomains and assess whether distribution is affected by contractant or relaxant signals to influence signaling crosstalk. For these studies we will immunoblot, confocal microscopic immunolocalization and patch clamp methodology. Understanding the mechanisms for the changes in the influence of the cAMP pathway and signaling crosstalk balance will enhance understanding of myometrial activity during pregnancy and labor and may lead to design of improved methods for management of uterine contractile activity and the prevention of premature labor.