Muscarinic stimulation of submucosal glands in swine trachea

The properties of muscarinic acetylcholine receptors (mAChR) on tracheal explants and isolated submucosal gland cells were determined using [³H]quinuclidinyl benzilate ([³H]QNB) and N-[³H]methylscopolamine ([³H]NMS) as ligands. Analysis of competitive displacement of [³H]NMS binding by pirenzepine demonstrated the presence of M₁- (27 ± 2%) and M(2G)- (73 ± 2%) receptors on isolated tracheal submucosal gland cells (TSGC's) in control. Daily administration of diisopropylfluorophosphate (DFP) inhibited cholinesterase activity by >95%. After 7 days of DFP treatment, [³H]QNB binding to intact TSGC's decreased from 14.2 ± 0.6 to 6.3 ± 0.8 fmol/10⁶ cells; similarly, [³H]NMS binding fell from 8.1 ± 1.9 to 2.0 ± 0.8 fmol/10⁶ cells. The loss of mAChR's was predominantly of the M(2G) subtype with the relative proportion dropping to 33%. In addition, 90% of the receptors assumed the high-affinity state for carbachol displacement of [³H]NMS. Mucus secretion was quantitated by measuring the release of ³H-labeled mucus macromolecules from explants of tracheal submucosal glands and isolated cells. Acetylcholine (ACh), 2 x 10^-5 M, stimulated mucus secretion by 2.5 and 2.3 times the basal rate, respectively. Elimination of acetylcholinesterase (AChe) by DFP increased the ACh sensitivity by 18- and 5-fold. Tracheal explants or TSGC's obtained 2 h after an in vivo DFP treatment showed a 6- and 3-fold ACh stimulation. This ACh sensitivity decreased during the continued daily dosing with DFP such tht only a 1.3- and 1.1-fold ACh stimulation was apparent after 7 days of treatment. Thus tolerance to DFP was due to a decrease in mAChR density, particularly of the M(2G) subtype, and a shift to the high-affinity state for carbachol.