P-610: Intracellular angiotensin II expression alters the distribution of the AT1 receptor in VSMCs: Effects upon signal transduction and cellular proliferation

The objective of the study was to identify the functional outcome of intracellular versus extracellular angiotensin II:AT1 receptor interactions in vascular cells. Rat vascular smooth muscle cells (line A10) were transfected, independently and concurrently, with plasmids encoding fluorescent fusion proteins of rat angiotensin II (pECFP/AII) and the rat AT1a receptor (pAT1R/EYFP). The AII fusion protein possesses no secretory signal peptide and is maintained within these cells predominantly in the nucleus. AT1R/EYFP is absent from the nucleus when expressed exclusively or in untreated cells. AT1R/EYFP accumulates in the nucleus following exogenous AII treatment or when co-expressed with ECFP/AII. Furthermore, co-expression of ECFP/AII with AT1R/EYFP stimulates proliferation of VSMCs more than 40% (p < 0.001) compared to expression of either protein alone. Co-transfection of a control, pECFP/AIIC (which encodes a scrambled AII peptide fused to ECFP), with pAT1R/EYFP, has no growth effect. In view of the intracellular growth effects of ECFP/AII, we sought to elucidate the underlying signaling pathways. We found that extracellular AII treatment of AT1R/EYFP-expressing VSMCs activates cAMP response element-binding protein (CREB) as determined by one-hybrid assays. Co-expression of intracellular ECFP/AII with AT1R/EYFP similarly activates CREB. However, intracellular and extracellular AII activate CREB through different phosphorylation pathways. Exogenous AII treatment of AT1R/EYFP-expressing VSMCs activates p38MAPK and ERK1/2 phosphorylation as determined by western blot. The p38MAPK inhibitor, SB203580, and the ERK kinase inhibitor, PD98059, each partially inhibit exogenous AII-conferred CREB activation confirming that p38MAPK and ERK1/2 mediate CREB phosphorylation. In contrast, co-expression of ECFP/AII (intracellular AII) and AT1R/EYFP in VSMCs activates p38MAPK but not ERK1/2; inhibition of p38MAPK by SB203580 inhibits intracellular AII-induced CREB phosphorylation. Extracellular AII stimulates at least one pathway common to intracellular AII. This common pathway, in the case of exogenous AII, likely reflects intracellular signaling following internalization of receptor:ligand complex. Extracellular AII also stimulates a unique pathway, reflecting interaction with plasma membrane-associated AT1R.