Selective Inhibition of α1B-Adrenergic Receptor Expression and Function Using a Phosphorothioate Antisense Oligodeoxynucleotide
- Pedro J. Gonzalez-Cabrera1,
- Patrick L. Iversen1,2,
- Marvin F. Liu1,
- Margaret A. Scofield1 and
- William B. Jeffries1
- 1Creighton 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 2Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68131 (P.L.I.)
Abstract
To investigate α1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the α1B-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on α1B-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 α1B-adrenoceptor density ([3H]prazosinBmax) versus control groups by 12% (1 μm AO) and 72% (10 μm AO). In time course experiments, AO (10 μm) reduced α1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. α1B-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 α1B-adrenoceptor subtype expression and functionin vitro, suggesting a potential to selectively inhibit α1B-adrenoceptor function in vivo.
Footnotes
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Send reprint requests to: Dr. William B. Jeffries, Department of Pharmacology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178. E-mail: wbjeff{at}creighton.edu
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↵1 Current affiliation: Research and Development, AVI BioPharma Inc., Corvallis, OR 97333.
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This work was supported by grants from the American Heart Association, Nebraska Affiliate, and the Paul Teschan Research Fund of Dialysis Clinic, Inc., Nashville, TN.
- Abbreviations:
- AO
- antisense oligodeoxynucleotide
- CO
- control oligodeoxynucleotide
- FAM
- carboxyfluorescein
- DMEM
- Dulbecco’s modified Eagle’s medium
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- PBS
- phosphate-buffered saline
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received November 10, 1997.
- Accepted February 18, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
