Molecular and pharmacological characterization of muscarinic receptor subtypes in a rat parotid gland cell line: Comparison with native parotid gland

The molecular and pharmacological characteristics of muscarinic receptor su btypes in the rat parotid acinar cell line, PAR-C5, were determined and com pared with native rat parotid glands to evaluate the PAR-C5 cell line as a model to study receptor-mediated secretion. Reverse transcription-polymeras e chain reaction (RT-PCR) identified mRNAs for M-3, M-4, and M-5 receptor s ubtypes in both PAR-C5 cells and parotid glands. Specific [N-methyl-H-3] sc opolamine binding in PAR-C5 and parotid membranes was to a single class of sites with mean K-D values of 0.38 and 0.64 nM, respectively. Binding affin ities (K-I values) of muscarinic receptor subtype-selective drugs were obta ined in side-by-side experiments comparing PAR-C5 cells with parotid glands . Nonlinear regression analysis indicated that competition binding curves f or 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, fo r atropine (1.0, 2.1), darifenacin (1.2, 2.0), 4-diphenylacetoxy-N-methylpi peridine methiodide (4-DAMP) (2.9, 2.4), tripitramine (220, 180), pirenzepi ne (320, 720), and methoctramine (1400, 1700) were consistent with their kn own affinities at the M-3 receptor subtype. Affinities (K-B values) of musc arinic receptor subtype-selective drugs for blocking methacholine-stimulate d Ca2+ mobilization were determined to show which subtype mediates Ca2+-dep endent secretion in Fura-2-loaded PAR-C5 cells. K-B values (nM) for atropin e (0.44), 4-DAMP (0.38), pirenzepine (140), and methoctramine (320) for blo cking Ca2+ responses correlated well with their known affinities at the M-3 receptor (r(2) = 0.99). These results show that at the level of mRNA, rece ptor protein and function, PAR-C5 cells and parotid glands are similar, est ablishing PAR-C5 cells as an important model for muscarinic receptor-mediat ed secretion.