Abstract
We recently reported that α2A-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 μM) in BE(2)-C cells that express both α2- and β2-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by β2-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the α2A-versus α2B-ARs have been reported, the present study examines the effects of simultaneous activation of α2B- and β2-ARs on α2B-AR number and signaling. We studied NG108 cells that naturally express α2B-ARs, and BN17 cells, NG108 cells transfected to express the human β2-AR. In NG108 cells, α2B-AR desensitization and down-regulation require treatment with 20 μM EPI or NE; GRK expression was not changed. In BN17 cells expressing β2-ARs, the threshold EPI concentration for α2B-AR desensitization and down-regulation was reduced to 0.3 μM; 10 μM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The β-AR antagonist alprenolol (1 μM) and GRK3 antisense (but not sense) DNA blocked 0.3 μM EPI- and 10 μM NE-induced desensitization and down-regulation of the α2B-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of α2B- and β2-ARs results in a 67-fold decrease in the threshold concentration of EPI required for α2B-AR down-regulation. This lower threshold for down-regulation is associated with α2B- and β2-AR dependent up-regulation of GRK3 expression.
Footnotes
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The research presented in this publication was supported in part by a grant to D.C.E. from the American Heart Association, Texas Affiliate, and a grant to K.M.S. from the Texas Higher Education Coordinating Board Advanced Research Program (00 36 52-011402001).
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doi:10.1124/jpet.104.069674.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; EPI, epinephrine; NE, norepinephrine; PGE1, prostaglandin E1; ODN, oligodeoxynucleotide; AR, adrenoceptor; ISO, isoproterenol; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline/Tween 20; Alp, alprenolol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; [3H]RX821002, (1,4-[6,7)(n)-3H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride.
- Received April 15, 2004.
- Accepted June 10, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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