Abstract 5784: Altered Receptor Distribution and Expression Causing Vascular Myocytes Insensitivity to α1 Adrenergic Activation
Alpha1D-adrenergic receptor (α1D-AR) plays a critical role in controlling peripheral vascular resistance and blood pressure. However, unlike α1A- and α1B-AR subtypes that primarily locate at the plasma membrane and function efficiently, α1D-ARs are accumulated within intracellular compartments and poorly functioned in vitro. Using fluo4, BODIPY prazosin and subtype-specific antibodies to evaluate the native α1AR regulated Ca2+ signaling and their subcellular distribution in rat aortic and cardiac myocytes, we tested the hypothesis that α1D-ARs are altered during cell isolation or/and culturing procedures, causing the responding discordance between tissue and cells. In aortic rings and freshly isolated myocytes α1AR agonist phenylephrine (PE, 10−5 M) induced vasoconstriction, and intracellular Ca2+ increase and cell shortening, which could be abolished by a specific antagonist of α1D-AR BMY7378 (10−6 M), respectively. The cellular response to PE, however, was decreased to 55.2±4.9% in 1-day-and 0% in ≥2-day-cultured aortic myocytes (n= 40, and 238), but remained in all the cultured neonatal rat cardiomyocytes (n= 267). Similarly as the α1A-ARs in cultured cardiomyocytes, α1D-ARs distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, but mostly redistributed intracellularly in cultured aortic myocytes. Treating the culture medium with 2% charcoal/dextran (C/D) for 48 h caused restorations of the cell surface α1D-AR distribution in 34.67±4.1% (n=84) and partial Ca2+ signaling response to PE in 29.5±0.2% (n=81, p<0.05 vs. no C/D) cultured cells, suggesting an essential requirement for membrane expressed α1ARs for their efficient intracellular coupling. Additionally, the α1D-AR expression in the cultured aortic myocytes was reduced relative to the tissue. These results indicate that α1D-ARs can primarily express at the cell membrane in native system but occur internalized into the cytoplasm and declined in expression in culture condition, resulting in the intracellular uncoupling to α1adrenergic activation in vitro. Charcoal/dextran in culture medium partially prevents this receptor subtype redistribution.