Abstract
The molecular and pharmacological characteristics of muscarinic receptor subtypes in the rat parotid acinar cell line, PAR-C5, were determined and compared with native rat parotid glands to evaluate the PAR-C5 cell line as a model to study receptor-mediated secretion. Reverse transcription-polymerase chain reaction (RT-PCR) identified mRNAs for M3, M4, and M5receptor subtypes in both PAR-C5 cells and parotid glands. Specific [N-methyl-3H]scopolamine binding in PAR-C5 and parotid membranes was to a single class of sites with meanK D values of 0.38 and 0.64 nM, respectively. Binding affinities (K I values) of muscarinic receptor subtype-selective drugs were obtained in side-by-side experiments comparing PAR-C5 cells with parotid glands. Nonlinear regression analysis indicated that competition binding curves for drugs in PAR-C5 cells and parotid glands fit best to a one-site binding model. K I values (nM) in PAR-C5 cells and parotid glands, respectively, for atropine (1.0, 2.1), darifenacin (1.2, 2.0), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (2.9, 2.4), tripitramine (220, 180), pirenzepine (320, 720), and methoctramine (1400, 1700) were consistent with their known affinities at the M3 receptor subtype. Affinities (K B values) of muscarinic receptor subtype-selective drugs for blocking methacholine-stimulated Ca2+ mobilization were determined to show which subtype mediates Ca2+-dependent secretion in Fura-2-loaded PAR-C5 cells. K B values (nM) for atropine (0.44), 4-DAMP (0.38), pirenzepine (140), and methoctramine (320) for blocking Ca2+ responses correlated well with their known affinities at the M3 receptor (r 2 = 0.99). These results show that at the level of mRNA, receptor protein and function, PAR-C5 cells and parotid glands are similar, establishing PAR-C5 cells as an important model for muscarinic receptor-mediated secretion.
Footnotes
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Send reprint requests to: Dr. Charles S. Bockman, Department of Pharmacology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE 68178. E-mail: cbockman{at}creighton.edu
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This study was supported by National Institute of Dental and Craniofacial Research Grant DE-12530 to C.S.B.
- Abbreviations:
- RT-PCR
- reverse transcription-polymerase chain reaction
- 4-DAMP
- 4-diphenylacetoxy-N-methylpiperidine methiodide
- S
- sense
- AS
- antisense
- bp
- base pair
- HBK
- HEPES-buffered Krebs' solution
- Received December 5, 2000.
- Accepted January 24, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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