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Long‐term carbachol treatment‐induced down‐regulation of muscarinic M 2 ‐receptors but not m 2 receptor mRNA in a human lung cell line
Author(s) -
Haddad E.B.,
Rousell J.,
Mak J.C.W.,
Barnes P.J.
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb16407.x
Subject(s) - carbachol , muscarinic acetylcholine receptor , forskolin , endocrinology , medicine , muscarinic acetylcholine receptor m1 , muscarinic acetylcholine receptor m2 , agonist , receptor , pirenzepine , biology , homologous desensitization , muscarinic acetylcholine receptor m3 , muscarinic antagonist , chemistry , stimulation
1 The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M 2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells). 2 Saturation studies performed with the non‐selective hydrophilic ([ 3 H]‐N‐methyl‐scopolamine, [ 3 H]‐NMS) and lipophilic ([ 3 H]‐quinuchdinyl benzilate, [ 3 H]‐QNB) muscarinic antagonists revealed a single class of high affinity binding sites. 3 Carbachol (1 mM) induced a rapid down‐regulation of [ 3 H]‐NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h. 4 The loss of [ 3 H]‐QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [ 3 H]‐NMS binding sites as a result of receptor sequestration. 5 Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin‐induced cyclic AMP accumulation was lost following 24 h carbachol stimulation. 6 The steady state level of muscarinic m 2 mRNA measured by Northern blot analysis was not affected by carbachol treatment over the time course investigated and half‐life studies with actinomycin D suggest that carbachol had no effect on the stability of m 2 mRNA. 7 The rate of transcription of m 2 muscarinic receptor gene as measured by nuclear RNA run‐on assay was unaltered by carbachol stimulation. 8 These results suggest that homologous sequestration, desensitization, and down‐regulation of M 2 muscarinic receptors in HEL 299 cells does not involve transcriptional or post‐transcriptional modifications of m 2 muscarinic receptor mRNAs.