Autoradiographic Studies of the Distribution of ³⁵Sulfate Label in Ferret Trachea: Effects of Stimulation

Abstract

Na₂ ³⁵SO₄ is among the common isotopic mucin precursors used to label airway secretory cells and monitor their active discharge in response to drugs. Previous work has established that ³⁵S is taken up by multiple cell types in the trachea, yet there is no direct evidence linking ³⁵S release with secretory activity by any specific cell type. In this study, we have used autoradiography to identify the sites of uptake and release of³⁵S in ferret trachea. Confirming work performed in other species, we found uptake sites include surface epithelium (ciliated and goblet cells), submucosal glands (serous and mucous cells) and cartilage. Extending these findings using a “pulse-chase” protocol, we found that ³⁵S turns over very rapidly in ciliated but not submucosal gland cells or cartilage. Specific grain density over epithelium declined from 0.12 ± 0.006 grains/μm² immediately after the pulse to 0.05 ± 0.004 grains/μm² at 4 h. In contrast, corresponding figures for the glands and cartilage showed no spontaneous loss of label during the same period. At 4 h, when the epithelium contained very little ³⁵S label, we exposed tracheal rings in vitro to neurotransmitter receptor agonists (bethanechol, phenylephrine, and isopro-terenol; all at 10⁵ M). Counts in the medium (determined by scintillation spectrometry) increased 2–3 times in response to each agonist. These increases were prevented by prein-cubating tracheal rings with appropriate antagonists. Autoradiography showed that stimulated glands contained many fewer silver grains than untreated or antagonist-blocked glands. In contrast, neither cartilage nor epithelium showed decreased labeling after stimulation.

These results indicate that (a) sulfated glycoconjugates turn over rapidly in the tracheal epithelium and may account for much sulfated material spontaneously released into organ culture medium and into the tracheal lumen, and (b) 4 h after Na₂ ³⁵SO₄ incubation, the major source of ³⁵S-labeled macromolecules released from ferret trachea by neural agonists is the submucosal glands.