SELECTIVE-INHIBITION OF ALPHA(1B)-ADRENERGIC RECEPTOR EXPRESSION AND FUNCTION USING A PHOSPHOROTHIOATE ANTISENSE OLIGODEOXYNUCLEOTIDE

To investigate alpha(1B)-adrenoceptor function, we developed a phospho rothioate antisense oligodeoxynucleotide (AO) to inhibit the expressio n of the alpha(1B)-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on alpha(1B)-adrenocepto r mRNA expression, protein density, and coupling to phospholipase C. C ells treated with either a control oligodeoxynucleotide (CO) or medium alone served as control groups. Confocal microscopy demonstrated that DDT, MF2 cells internalized carboxyfluorescein-labeled (FAM) AO withi n 30 min. Analysis of cellular lysates showed that approximately 50% o f the intracellular FAM-AO was present as an intact 18-mer for up to 4 8 hr. Incubation of cells with AO for 48 hr decreased alpha(1B)-adreno ceptor density ([H-3]prazosin B-max) versus control groups by 12% (1 m u M AO) and 72% (10 mu M AO). In time course experiments, AO (10 mu M) reduced alpha(1B)-adrenoceptor density by 28, 64, and 68% versus cont rols after 24, 48, and 72 hr of exposure, respectively, alpha(1B)-Adre noceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 mu M AO versus controls. AO pretreatm ent (10 mu M, 48 hr) reduced the maximum response to agonist-stimulate d [H-3]inositol phosphate accumulation. The maximal response of the fu ll agonist norepinephrine was reduced by 30% after AO treatment, and b y 73% for the partial agonist naphazoline. In contrast, AO did not aff ect histamine-stimulated total [H-3]inositol phosphate accumulation. T hus, AO effectively reduced alpha(1B)-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit a lpha(1B)-adrenoceptor function in vivo.