The classic view of angiotensin II (All), the primary biologically active component through which the renin angiotensin system mediates hemodynamic and electrolyte effects, is that it functions as a blood-borne hormone. In the last decade, it has become increasingly clear that All can be generated in tissues, act as a local hormone, and confer autocrine and/or paracrine effects. Our published studies show that expression of intracellular All stimulates cell growth and that the growth stimulation is accompanied by cAMP response element-binding protein (CREB) phosphorylation and PDGF A-chain upregulation. We now propose to extend these studies to generate mice transgenic for two different expression cassettes directing intracellular AII production. In addition, we will cross the transgenic mice with null-Aogen mice to determine whether intracellular expression of Aogen (angiotensinogen) and subsequent intracellular generation of All can compensate for an absence of extracellular All and, thus, reverse the kidney and blood pressure phenotypes of the knockout mice. These studies will apply a cDNA construct which we have designed to encode a form of Aogen, devoid of the signal sequence (which directs secretion), but possessing an intact AI/AII-encoding region. This modified Aogen is growth stimulatory for H4-II-E-C3 rat hepatoma cells. These studies will likewise apply an intracellular fluorescent fusion protein of All that we have shown to stimulate growth and CREB phosphorylation in cells expressing AT1 receptor fusion proteins. Both exogenous All and intracellular All induce AT 1R trafficking to cell nuclei; the receptor trafficking pathway for each will be established. We will identify and compare kinase pathways responsible for CREB phosphorylation following intracellular versus extracellular All stimulation. Collectively, these studies will define the relative roles and relative importance of intracellular and extracellular All, and the potential for internalization or intracellular generation of All in vivo in unmanipulated systems.
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