Inhibition of the α1D-adrenergic receptor gene by RNA interference (RNAi) in rat vascular smooth muscle cells and its effects on other adrenergic receptors

Sympathetic-induced vasoconstriction is mediated by various adrenergic receptor (AR) subtypes located on membranes of vascular smooth muscle cells (VSMC) located on the arterial wall, but is mostly attributed to activation of the α1D-AR. In order to study interaction and cross-talk among AR genes, we induced post-transcriptional silencing of the α1D-AR gene in cultured VSMC using the RNAi technique. A pSEC neo expression plasmid vector containing a small interfering RNA (siRNA) sequence selected to bind to the targeted mRNA of the α1D-AR gene was transfected into cultured VSMC from rat aorta. The RNA expression of all AR-subtype genes was assessed by Q-RT-PCR and the α1D and α2A-AR proteins quantified by Western blot. In siRNA-transfected cells, the α1D-AR protein levels decreased by 55%, 69% and 75% at 24 h, 48 h and 72 h, respectively (p < 0.03–0.01) with progressive increases in its gene expression by 50%–61% and concurrent increase in α2A-AR protein peaking at 48 h. Decreases were noted in expression of the α1A, α2A, and β3 AR genes. We conclude that post-transcriptional silencing of the α1D-AR gene leads to significant decrease in receptor protein despite reactive increase in gene expression. However, suppression of one AR leads to reactive changes in other subtypes, indicating that cross-talk among related genes, whose products have overlapping functions, may partly offset anticipated effects in vivo.