ROLE OF ADENYL PURINES IN MYOMETRIAL FUNCTION

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This project proposes that adenyl purines, compounds known to cause contraction of uterus, participate in the regulation of human parturition. The specific aims of this research are to discover the origins, binding sites and molecular signaling events that subserve the contractile action of adenyl purines in uterine smooth muscle. The working hypothesis is that adenyl purines, both adenosine and ATP from one or more sources stimulate purinergic receptors in myometrial smooth muscle leading to uterine contraction. We propose that adenyl purines participate in, or initiate labor and as such, may provide a focal point for understanding preterm labor in humans. Studies will be conducted with tissues and freshly isolated cells from both pregnant guinea pigs and humans. The research plan will be separated into three areas of investigation. (1) We will determine the origins of adenyl purines in the uterus. HPLC and radiolabeling studies will investigate the role of putative purinergic nerves as well as the co-release of purines from adrenergic and cholinergic nerves in myometrium. Paracrine release of purines from the smooth muscle of myometrium itself, or from endometrium will also be tested. (2) The receptor targets for adenyl purines will be investigated with radioligand binding studies in cells and tissues and will determine the presence and distribution of purinergic receptors of the P1 and P2 subtype in circular and longitudinal smooth muscles. These methods will also be used to determine the nature of regulation of receptor affinity for agonist and the role of coupling proteins such as GTP-binding proteins in the molecular action of these receptors.(3) Pharmacological studies will characterize the actions of adenyl purines in pregnant uterus. Mechanical contraction of tissues, electrophysiological measurements of cellular ionic currents and biochemical measurements of receptor action will be made. Biochemical and electrophysiological studies will employ tissue segments and purified smooth muscle cells and will concentrate on the role of purines in the regulation of cellular Ca+2. Biochemical studies will include measurement of inositol phosphates, arachidonic acid release, cyclic nucleotide metabolism and the action of protein kinases. Cellular imaging using fluorescent pH dyes will be used to determine the regulation of phospholipase A2 through stimulation of N+/H+ exchange. Intracellular calcium fluctuations will be studied in cells and tissues using intracellular fluorescent dyes.