名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成3年3月8日 葛谷雅文氏の博士論文として提出された

Addition of lysophosphatidic acid (LPA) to rat aorta-derived endothelial cells significantly induced preproendothelin-1 (preproET-1) mRNA expression. PreproET-1 mRNA levels reached a plateau within 1 h after the addition of 0.5 μM LPA and declined after 2 h. The induction was superinduced by cycloheximide and was blocked by actinomycin D. Suramin, an LPA receptor antagonist, abolished the induction of preproET-1 mRNA by LPA. Protein kinase C inhibitors, H7 and bisindolylmaleimide, were able to block the induction. Transient transfection experiment revealed that the elevated preproET-1 mRNA was a result of the activation of ET-1 gene activity. Electrophoretic mobility shift assay revealed that LPA stimulated the binding of AP-1. The secreted level of ET-1 was elevated 2.3-fold after 12 h of stimulation with LPA. Our results suggest that the upregulation of preproET-1 by LPA may serve to augment and prolong the vasoconstriction action of LPA.

Endothelial cells separate the bloodstream from the underlying tissue and play a crucial role in vascular homeostasis. They also form an important barrier for vascular drug delivery. This thesis contains preliminary studies targeted at understanding the mechanisms of binding and transport across endothelial cells cultured in vitro. Specifically, the first study investigates how the recombinant source of Factor IX (FIX), a blood coagulant protein used in the treatment of Hemophilia B, impacts surface ligand binding (FIX to its specific receptors) to bovine aortic endothelial cells (BAECs). Competitive binding experiments between 125I-FIX and FIX were undertaken to quantify the interaction of recombinant and transgenic FIX with BAECs and human collagen IV and determine if there was a measurable difference in binding affinity. Results indicate limited specific binding of 125I-FIX to BAECs and no binding to human collagen IV. Concrete conclusions were not drawn from this data due to technical issues during the experimental process. The second study investigates insulin-like growth factor-I (IGF-I) transport across both BAEC and MAC-T cells, a mammary epithelial cell line, cultured on tissue culture inserts. IGF-I is a circulatory growth factor implicated in the regulation of cell division and tissue proliferation. Competitive binding experiments between 125I-IGF-I and unlabeled protein (IGF-I, Y60L-IGF-I, a mutant of IGF-I, and IGF Binding Protein-3 (IGFBP-3)) were undertaken to quantify the binding and transport of IGF-I under various experimental conditions. Results confirmed earlier work from the Williams' laboratory indicating that 125I-IGF-I transport was enhanced by incubation with its non-receptor-binding analog, Y60L-IGF-I, but cell surface associated 125I-IGF-I was decreased by its presence. Other studies were undertaken but conclusive results could not be drawn. / Master of Science

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