Selective inhibition of alpha1B-adrenergic receptor expression and function using a phosphorothioate antisense oligodeoxynucleotide.
To investigate alpha1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the alpha1B-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on alpha1B-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 alpha1B-adrenoceptor density ([3H]prazosin Bmax) versus control groups by 12% (1 microM AO) and 72% (10 microM AO). In time course experiments, AO (10 microM) reduced alpha1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. alpha1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 microM AO versus controls. AO pretreatment (10 microM, 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 alpha1B-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit alpha1B-adrenoceptor function in vivo.
Gonzalez-Cabrera
P J
PJ
Creighton Nephrology Research Laboratory, Department of Pharmacology, Creighton University School of Medicine, Omaha, Nebraska 68178, USA.
Iversen
P L
PL
Liu
M F
MF
Scofield
M A
MA
Jeffries
W B
WB
D058668:Adrenergic alpha-1 Receptor Antagonists; D006868:Hydrolysis; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology*
D058668:Adrenergic alpha-1 Receptor Antagonists; D016376:Oligonucleotides, Antisense; D010716:Phosphatidylinositols; D018340:Receptors, Adrenergic, alpha-1; D013873:Thionucleotides