Abstract

To investigate α_1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the α_1B-adrenoceptor subtype in DDT₁ MF2 cells. We measured the cellular uptake of the AO and its effect on α_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 DDT₁MF2 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 α_1B-adrenoceptor density ([³H]prazosinB_max) versus control groups by 12% (1 μm AO) and 72% (10 μm AO). In time course experiments, AO (10 μm) reduced α_1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. α_1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 μmAO 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 α_1B-adrenoceptor subtype expression and functionin vitro, suggesting a potential to selectively inhibit α_1B-adrenoceptor function in vivo.