Selective Inhibition of a1B-Adrenergic Receptor Expression and Function Using a Phosphorothioate Antisense Oligodeoxynucleotide

To investigate a1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the a1B-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on a1B-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 a1B-adrenoceptor density ([ H]prazosin Bmax) versus control groups by 12% (1 mM AO) and 72% (10 mM AO). In time course experiments, AO (10 mM) reduced a1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. a1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 mM AO versus controls. AO pretreatment (10 mM, 48 hr) reduced the maximum response to agonist-stimulated [H]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 histaminestimulated total [H]inositol phosphate accumulation. Thus, AO effectively reduced a1B-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit a1B-adrenoceptor function in vivo. a1-Adrenergic receptors are a subfamily of G protein-coupled receptors that mediate the actions of catecholamines. Based on cloning and pharmacological data, it is known that a1-adrenergic receptors can be classified into three subtypes (a1A-, a1B-, and a1D-adrenergic receptors). We (Scofield et al., 1995) and others (Perez et al., 1994; Price et al., 1994) have shown that the genes for each of the subtypes are expressed in discrete, tissue-specific patterns. Each of the a1-adrenergic receptor subtypes has been shown to mediate distinct physiological functions. For example, the a1B-subtype mediates activation of glycogenolysis in rat liver (Garcia-Sainz and Macias-Silva, 1995). The a1A-subtype is involved in the contraction of human prostate smooth muscle (Forray et al., 1994), and contraction of rat aorta seems to be mediated at least in part by the a1D-subtype (Buckner et al., 1995; Piascik et al., 1995). Despite these examples, a major challenge to the determination of the function of each of the a1-adrenergic receptor subtypes is the paucity of available pharmacological tools to distinguish among them. Competitive antagonists such as 5-methyl-urapidil can be used experimentally to distinguish the a1A-adrenergic receptor subtype from the other two subtypes (Gross et al., 1988). Unfortunately, antagonists with good selectivity for the a1Band a1D-adrenergic receptors are currently lacking. Some studies have reported that the alkylating agent chloroethylclonidine can distinguish between the a1Aand a1B-adrenergic receptor subtypes (Minneman et al., 1988); however, chloroethylclonidine irreversibly alkylates both the a1Band the a1D-adrenergic receptors nonselectively (Hirasawa et al., 1997; Xiao and Jeffries,