P-2U PURINOCEPTOR REGULATION OF MUCIN SECRETION IN SPOC1 CELLS, A GOBLET CELL-LINE FROM THE AIRWAYS

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in respon se to purinergic (P-2) agonists. High-molecular-mass glycoconjugates ( HMMGs) purified by CsCl-density-gradient centrifugation had a buoyant density of 1.45 g/ml, The purified HMMG material exhibited a single ma jor band with an apparent molecular mass of greater than 1000 kDa in S DS/polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin, [H-3]HMMG was resistant to proteoglycan-degradi ng enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMM G amino acid composition was enriched in Ser and Thr (sum 27%), Thus S POC1-cell HMMG possess the characteristics of mucin, Mucin secretion b y SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5'-[gamma-thio]triphosphate (100 mu M) 4-5-fold over a baseline of 4 ng/min, The three dose-effect rel ations were nearly identical (K-0.5 similar to 4 mu M). SPOC1 cells gr own on plastic and rat tracheal epithelial primary cells responded sim ilarly to ATP and/or UTP, SPOC1 cells failed to respond to other purin ergic agonists, either luminally or serosally, and consequently seem t o possess an apical membrane P-2u purinoceptor. SPOC1-cell total RNA w as probed for P-2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size wa s observed, which had a nucleotide base sequence identical with the ra t P-2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the con trol of a P-2u purinoceptor; they should prove useful in dissecting th e associated cellular regulatory pathways.