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
- Received November 10, 1997.
- Accepted February 18, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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