MUSCARINIC-INDUCED MUCIN SECRETION AND INTRACELLULAR SIGNALING BY HAMSTER TRACHEAL GOBLET CELLS

Hamster tracheal epithelial cell cultures were used to investigate mus carinic regulation of high-molecular-weight glycoconjugate (HMWG) secr etion by airway goblet cells. HMWG were radiolabeled with N-acetyl-D-[ 1-H-3] glucosamine, precipitated with trichloroacetic acid and phospho tungstic acid, and counted by liquid scintillation. Carbachol (100 mu M) increased HMWG secretion (166.6 +/- 18.7%, P < 0.001, n = 20), and this response was blocked by the muscarinic receptor antagonist atropi ne. Ca2+ may not be essential for carbachol response since 1) carbacho l-activated secretion was not inhibited by chelating extracellular Ca2 + with 1,2-bis(2-aminophenoxy)ethane-N, N,N', N'-tetraacetic acid (BAP TA) or by reducing both extracellular and intracellular Ca2+ with BAPT A-acetoxymethyl ester in low-Ca2+ medium; 2) the carbachol response wa s only partially blocked in low-Ca2+ medium; and 3) calcium ionophore did not stimulate HMWG secretion. However, carbachol-stimulated secret ion was abolished by pertussis toxin (PTX), indicating the involvement of a PTX-sensitive guanine nucleotide-binding regulatory protein (G p rotein), and by the protein kinase C (PKC) inhibitor chelerythrine chl oride. Furthermore, carbachol-stimulated secretion was not inhibited b y overnight incubation with phorbol 12-myristate 13-acetate. In conclu sion, carbachol-stimulated secretion of HMWG appears to be coupled to a PTX-sensitive G protein and requires the activation of a phorbol est er-insensitive PKC isoform.