The α(1D)-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 α(1D)-adrenergic receptor (α(1D)-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 α(1D)-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled α(1D)-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 α(1D)-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the α(1D)-AR was also demonstrated with immunocytochemistry using an α(1D)-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the α(1A)-α(1B)- and α(1D)-ARs in these aortic smooth muscle cells. Therefore, the presence of the other α(1)-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the α(1D)-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the α(1D)-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The α(1D)-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the α(1D)-AR heterodimerizes with the β(2)-AR and that desensitization of the β(2)-AR results in α(1D)-AR desensitization. In the rat aorta, desensitization of the β(2)-AR had no effect on contractile responses mediated by the α(1D)-AR. CONCLUSION: Our results suggest that the dimerization of the α(1D)-AR with other ARs does not alter the cellular expression or functional response characteristics of the α(1D)-AR.