Tannin inhibits the cAMP-β-adrenergic receptor pathway in bovine tracheal epithelium

Abstract

Tannin, isolated from cotton bracts, inhibits chloride secretion in airway epithelium. In bovine tracheal epithelial cells, tannin (25 μg/ml) blunted isoproterenol (Iso)-stimulated adenosine 3′,5′-cyclic monophosphate (cAMP) accumulation. Inhibition was time and dose dependent, with 52 ± 5% (mean ± SE, n = 6) inhibition at 60 min and 82 ± 9% ( n = 3) inhibition at 8 h. Inhibition was reversible starting at 4 h. Low-molecular-mass tannin (1,000–5,000 Da) had no effect on Iso-stimulated cAMP accumulation, whereas N-acetylcysteine, which interacts with cysteine residues, blocked the effects of tannin on Iso-stimulated cAMP accumulation. Tannin exposure (25 μg/ml for 30 min) had no effect on the dissociation constant ( K d) for [3H]dihydroalprenolol (DHA) (0.41 ± 0.03 nM, n = 3) but decreased maximal binding from 252 ± 32 to 162 ± 36 fmol/mg protein. Using single-point analysis and [3H]CGP-12177, we determined that tannin (25 μg/ml for 4 h) decreased surface β-adrenergic receptor density from 26.4 ± 4.3 ( n = 12) to 11.9 ± 3.0 fmol/mg protein and that the decrease was dose dependent. Agonist binding affinity by Iso displacement of DHA demonstrated a two-site model ( K d values = 27 ± 9 and 2,700 ± 600 nM) and a ratio of high- to low-affinity receptors of 1:1. Tannin (25 μg/ml) steepened the curve and shifted it to the right, as did Gpp(NH)p. Gpp(NH)p had no further effect on the shape or position of the displacement curve in the presence of tannin. In contrast, when polymer length was decreased by oxidation, tannin had no effect on the DHA displacement curve. These data demonstrate that tannin reversibly desensitizes bovine tracheal epithelial cells to Iso, decreases β-adrenergic receptor density, and uncouples the receptor from its stimulatory G protein. These data also suggest that the polymer length of tannin and its interaction with cysteine residues are important for these effects. These studies provide additional evidence for the role of tannin in the occupational lung disease byssinosis.