Characterization of cholinergic muscarinic receptors in cow tracheal muscle membranes. Effect of maturation

Abstract

The parasympathetic nervous system is important in the control of basal airway muscle tone and caliber. We characterized muscarinic cholinergic receptors in isolated tracheal membranes from cows of three age groups (immature, less than 2 weeks; transition, 3-5 months; and mature, greater than 5 years) using l-[3H]quinuclidinyl benzilate (l-[3H]QNB) as the radioligand. There were significant decreases in the densities of l-[3H]QNB binding sites with maturation (Bmax: 2344 +/- 169 vs 1381 +/- 85 vs 1116 +/- 80 fmol/mg protein for tissues from immature, transition and mature cows respectively). No change in the dissociation constant was observed with maturation (Kd: 0.38 +/- 0.09 vs 0.55 +/- 0.06 vs 0.50 +/- 0.07 nM for tissues from immature, transition and mature animals respectively). The association and dissociation rate constants did not vary between tissues from immature and mature animals. The specific activity of the enzyme, acetylcholinesterase, was correlated with the density of l-[3H]QNB binding sites present in the tracheal homogenates; that is, with maturation, there were significant decreases in acetylcholinesterase activity [0.28 +/- 0.01 vs 0.16 +/- 0.02 vs 0.08 +/- 0.01 mol X l-1 X min-1 X (mg protein)-1 for tissues from immature, transition and mature animals respectively]. All competition binding studies using muscarinic antagonists exhibited single site binding and did not show any differences in drug affinities between the age groups. In contrast, multiple binding sites were observed with carbachol, methacholine and muscarine, and there were significant decreases in receptor affinities for the muscarinic agonists. No changes in the proportion of high and low affinity sites were found. These results indicate that with maturation there are alterations in the properties of muscarinic receptors in tracheal smooth muscle.