The α1D-adrenergic receptor is expressed intracellularly and coupled to increases in intracellular calcium and reactive oxygen species in human aortic smooth muscle cells

BACKGROUND The cellular localization of the alpha1D-adrenergic receptor (alpha1D-AR) is controversial. Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the alpha1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled alpha1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity. RESULTS The adenovirally expressed alpha1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the alpha1D-AR was also demonstrated with immunocytochemistry using an alpha1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the alpha1A-alpha1B- and alpha1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other alpha1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the alpha1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the alpha1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The alpha1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the alpha1D-AR heterodimerizes with the beta2-AR and that desensitization of the beta2-AR results in alpha1D-AR desensitization. In the rat aorta, desensitization of the beta2-AR had no effect on contractile responses mediated by the alpha1D-AR. CONCLUSION Our results suggest that the dimerization of the alpha1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the alpha1D-AR.