Changes in mRNA expression induced by sustained noradrenaline stimulation are different for alpha1-adrenoceptor subtypes in HEK293 cells.
1. The aim of the present study was to investigate noradrenaline (NA)-induced regulation of alpha1-adrenoceptor (AR) mRNA expression in human embryonic kidney (HEK) 293 cells stably expressing cloned alpha1-AR subtypes with similar receptor densities. Stable transfection was performed by calcium phosphate precipitation. Receptor expression was detected by radioligand binding assay. The mRNA expression was measured by RNase protection assay. 2. alpha1-Adrenoceptor subtype mRNA respond in distinct ways following prolonged exposure to NA. The mRNA level of the alpha 1A-AR subtype was unchanged, the mRNA level of the alpha 1B-AR subtype was increased and the mRNA level of the alpha 1D-AR subtype declined time dependently. The protein kinase C (PKC) inhibitor calphostin C or RO 31-8220 abolished the NA-induced downregulation of alpha 1D-AR mRNA. Phorbol myristate acetate (PMA), a PKC activator, similarly repressed the effects of NA on alpha 1D-AR. However, calphostin C, RO 31-8220 or PMA had no effect on the induction of alpha 1B-AR mRNA by NA. The Ca2+-ATPase inhibitor thapsigargin or the calcium chelator 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM) had no effect on the repression of alpha 1D-AR mRNA, but did inhibit the induction of alpha 1B-AR mRNA by NA. Noradrenaline significantly decelerated the degradation of alpha 1B-AR mRNA, but had no effect on the degradation of alpha 1D-AR mRNA. 3. Thus, the mRNA expression of three alpha1-AR subtypes in HEK293 cells is differentially regulated through distinct signal transduction pathways under sustained NA stimulation. The upregulation of alpha 1B-AR mRNA is via the Ca2+ pathway, whereas the downregulation of alpha 1D-AR mRNA is via the PKC pathway.
Lei
Beilei
B
Institute of Vascular Medicine, Peking University Third Hospital, Beijing, People's Republic of China.
Zhang
Youyi
Y
Han
Chide
C
D000818:Animals; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D009638:Norepinephrine / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection / Q000379:methods
D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D009638:Norepinephrine