Acetylcholine-stimulated chloride flux in tracheal submucosal gland cells

The Cl content of isolated tracheal submucosal gland cells was studied using ³⁶Cl as a tracer. ³⁶Cl uptake reached a steady state within 10 min, yielding an estimate of intracellular Cl concentration of ~40 mM. Intracellular Cl fell rapidly when ouabain or furosemide was added, indicating that isolated tracheal submucosal gland cells concentrate Cl above its electrochemical equilibrium concentration. Acetylcholine (ACh) caused a Ca²⁺-dependent decline in cell Cl, with an effective concentration for a 50% response (EC₅₀) of 62 nM; this loss of cell Cl was blocked by atropine or pirenzepine. The EC₅₀ was 6 nM in cells when 95% of the acetylcholinesterase activity was abolished by diisopropylfluorophosphate (DFP) treatment. ACh continued to cause a decline in cell Cl even after a 7-day course of DFP treatment, which has been shown to abolish ACh-stimulated mucous glycoprotein secretion (23). After the 7-day course of DFP treatment, the EC₅₀ for ACh increased to 77 nM. Thus the Cl economy of the tracheal submucosal gland cell resembles that of cells in epithelia that secrete fluid; in addition, the transmitter-dependent loss of cell Cl is under long-term metabolic control of the cell.