Vol. 53, Issue 6, 1034-1039, June 1998

Selective Inhibition of _1B-Adrenergic Receptor Expression and Function Using a Phosphorothioate Antisense Oligodeoxynucleotide

Pedro J. Gonzalez-Cabrera, Patrick L. Iversen,¹ Marvin F. Liu, Margaret A. Scofield, and William B. Jeffries

Creighton Nephrology Research Laboratory, Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178 (P.J.G.C., M.F.L., M.A.S., W.B.J.) and the Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68131 (P.L.I.)

To investigate _1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the _1B-adrenoceptor subtype in DDT₁ MF2 cells. We measured the cellular uptake of the AO and its effect on _1B-adrenoceptor mRNA expression, protein density, and coupling to phospholipase C. Cells treated with either a control oligodeoxynucleotide (CO) or medium alone served as control groups. Confocal microscopy demonstrated that DDT₁ MF2 cells internalized carboxyfluorescein-labeled (FAM) AO within 30 min. Analysis of cellular lysates showed that approximately 50% of the intracellular FAM-AO was present as an intact 18-mer for up to 48 hr. Incubation of cells with AO for 48 hr decreased _1B-adrenoceptor density ([³H]prazosin B_max) versus control groups by 12% (1 µM AO) and 72% (10 µM AO). In time course experiments, AO (10 µM) reduced _1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. _1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 µM AO versus controls. AO pretreatment (10 µM, 48 hr) reduced the maximum response to agonist-stimulated [³H]inositol phosphate accumulation. The maximal response of the full agonist norepinephrine was reduced by 30% after AO treatment, and by 73% for the partial agonist naphazoline. In contrast, AO did not affect histamine-stimulated total [³H]inositol phosphate accumulation. Thus, AO effectively reduced _1B-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit _1B-adrenoceptor function in vivo.