Hormonal regulation of Cl transport in polar airway epithelia measured by a fluorescent indicator

Abstract

Cl transport mechanisms in polarized cultures of canine tracheal epithelium were examined using an Ussing-type chamber with independent mucosal and serosal perfusion. Cl activity was monitored continuously from fluorescence of entrapped 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). When added to the serosal (but not mucosal) solution, isoproterenol increased Cl fluxes across the apical membrane Cl more than fourfold. Apical Cl transport was sensitive to diphenylamine-2-carboxylate (DPC) but not to furosemide, whereas basolateral membrane Cl transport was sensitive to furosemide but not to DPC. Based on a mathematical model of Cl transport, we developed a sensitive protocol to measure hormone-sensitive Cl transport. In Cl-loaded cells in which basolateral Cl transport was partially inhibited by furosemide, mucosal Cl removal caused no Cl efflux before but rapid efflux (0.25 mM/s) after addition of isoproterenol or chlorophenylthio-cAMP. In the presence of indomethacin to block prostaglandin production, elevation of intracellular Ca by bradykinin or 4-bromo-A23187 did not cause Cl efflux, nor did Ca buffering with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid affect stimulation by the cAMP pathway. Phorbol 12-myristate 13-acetate increased Cl efflux submaximally (0.09 mM/s) but did not affect maximal stimulation by cAMP agonists. Methoxamine did not alter apical or basolateral membrane Cl transport.(ABSTRACT TRUNCATED AT 250 WORDS)