Characterization of Endothelial Cell Internalization of AP4A

We will determine whether Ap4A is internalized by binding to the low affinity binding site on the bovine aortic endothelial cell surface by characterizing the effects of P2 agonists, P2 antagonists and various guanosine dinucleotides on Ap4A internalization. The P2 agonists, P2 antagonists and guanosine dinucleotides only interact with the low affinity binding site. The mechanism of Ap4A internalization will be accomplished by determining whether protease activation and/or macromolecular biosynthesis are required for internalization. We will also determine whether Ap4A is internalized by an endocytic or transporter system. We will determine whether Ap4A binding to endoplasmic reticulum induces the release of calcium from calcium release channels. This will be accomplished by characterizing the binding of Ap4A to endoplasmic reticulum and also by determining whether Ap4A induces calcium release from endoplasmic reticulum receptors. To determine whether the internalization of Ap4A induces the synthesis of nitric oxide we will determine whether the internalization of Ap4A induces nitric oxide synthesis in the presence and absence of inhibitors of nitric oxide formation.