The objective of this proposal is to pursue studies on the regulation of membrane ion channels by growth factors and inflammatory mediators in the tunica muscularis mucosa (TMM). Our recent studies show that platelet-derived inflammatory factors, platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) enhance Ca2+ influx through voltage-dependent L-type Ca2+ channels in the TMM, the innermost smooth muscle layer of the G.I. tract. We propose to determine the mechanisms by which PDGF and LPA, acting via distinct signaling pathways, regulate specific ion channels and hence, Ca2+ influx which is an early essential step in cell proliferation and contraction. The first specific aim is to determine how PDGF and LPA enhance voltage-dependent Ca2+ currents. Preliminary studies show that PDGF activates a non-receptor tyrosine kinase pathway involving a complex formation of c-Src kinase and Focal Adhesion Kinase (FAK) in regulation of L-type of Ca2+ currents. Intracellular dialysis of antibodies to these signaling molecules attenuates basal Ca2+ currents and PDGF-Ca2+ channel coupling. Our studies also show that c-src kinase directly associates with the Ca2+ channel subunit of TMM. We will specifically study 1) biophysical properties of the modulation of the Ca2+ currents by PDGF and LPA, 2) the G protein subtypes that couple LPA activation of Ca2+ currents, and 3) assess the convergence of signaling pathways between LPA and PDGF.
The second aim i s to determine how intracellular Ca2+ release by PDGF and LPA affects other ion channels, which, in turn influence cell membrane potential and Ca2+ influx. Preliminary studies show that PDGF activates Ca2+ dependent Cl-, K+ and cation channels. We will investigate the role of c-src kinase and FAK in the modulation of these channels by PDGF and LPA. These studies will be carried out in single smooth muscle cells from the TMM using a) patch-clamp techniques, b) fluorescent dyes to measure intracellular Ca2+ and c) biochemical analyses to identify specific protein kinases. Elucidation of the cellular mechanisms of Ca2+ entry by growth factors and inflammatory mediator should advance our understanding of the regulation of TMM in health and disease.
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