Inhibition of the alpha(1D)-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 alpha(1D)-AR. In order to study interaction and cross-talk among AR genes, we induced post-transcriptional silencing of the alpha(1D)-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 alpha(1D)-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 alpha(1D) and alpha(2A)-AR proteins quantified by Western blot. In siRNA-transfected cells, the alpha(1D)-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 alpha(2A)-AR protein peaking at 48 h. Decreases were noted in expression of the alpha(1A), alpha(2A), and beta(3) AR genes. We conclude that post-transcriptional silencing of the alpha(1D)-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.
Sun
Bei
B
Hypertension and Atherosclerosis Section of the Department of Medicine, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
Kintsurashvili
Ekaterina
E
Ona
Deborah
D
Ignjacev-Lazich
Ivana
I
Gavras
Irene
I
Gavras
Haralambos
H
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics* / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D022702:Receptors, Adrenergic, beta-3 / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
C547721:Adra1d protein, rat; C547770:Adra2a protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018340:Receptors, Adrenergic, alpha-1; D018341:Receptors, Adrenergic, alpha-2; D022702:Receptors, Adrenergic, beta-3