P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways

Abstract

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in response to purinergic (P2) 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 major band with an apparent molecular mass of greater than 1000 kDa in SDS/ polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin. [3H]HMMG was resistant to proteoglycan-degrading enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMMG amino acid composition was enriched in Ser and Thr (sum 27%). Thus SPOC1-cell HMMG possess the characteristics of mucin. Mucin secretion by SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5'-[gamma-thio]triphosphate (100 microM) 4-5-fold over a baseline of 4 ng/min. The three dose-effect relations were nearly identical (K0.5 approximately 4 microM). SPOC1 cells grown on plastic and rat tracheal epithelial primary cells responded similarly to ATP and/or UTP. SPOC1 cells failed to respond to other purinergic agonists, either luminally or serosally, and consequently seem to possess an apical membrane P2u purinoceptor. SPOC1-cell total RNA was probed for P2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size was observed, which had a nucleotide base sequence identical with the rat P2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the control of a P2u purinoceptor; they should prove useful in dissecting the associated cellular regulatory pathways.

Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

“…MUC5AC and MUC5B are the major gel-forming mucins in both bovine cervical mucus and human respiratory mucus (43)(44)(45). To produce large volumes of purified MUC5AC/5B mucins, 50 ml of fresh bovine cervical mucus was solubilized in 6 M guanidinium chloride, and mucins were purified by double isopycnic density gradient centrifugation (46). Fractions were collected, weighed for density, and assessed for DNA (A 260 ), protein (A 270 ), and sialic acid.…”

MARCKS Protein Is a Key Molecule Regulating Mucin Secretion by Human Airway Epithelial Cells in Vitro

“…1 (3)(4)(5)(6) or cGMP-dependent protein kinase (PKG) (7) in the secretory process. However, coordinated interactions or "cross-talk" between these two protein kinases in regulation of mucin secretion have not been demonstrated, nor have signaling events downstream of protein kinase activation that ultimately lead to the exocytotic release of mucin granules been elucidated.…”

nPKCε, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

“…Standard curves were generated from purified SPOC1 mucins applied to each plate, and the results were expressed as nanograms mucin released per culture. Importantly, in Western blots analyses of SPOC1 cell extracts, soybean agglutinin stains only high-molecular-weight glycoconjugates (3).…”

“…Two such mediators, the nucleotides ATP and UTP, appear to be major agonists in goblet cell mucin secretion acting via P2Y 2 purinoceptors (P2Y 2 -Rs) (15,17,31,62). P2Y 2 -R activation by agonist elicits a coupling with PLC via G q , production of the primary intracellular messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP 3 ) from phosphatidylinositol-4,5-bisphosphate (PIP 2 ), followed by activation of protein kinase C (PKC) and mobilization of Ca 2ϩ (62). Pharmacological studies in goblet cells have shown that mucin secretion can be stimulated via intracellular messenger pathways by the DAG mimic phorbol-1,2-myristate-13-acetate (PMA) (15,17,30,31), by IP 3 (52), and by the Ca 2ϩ -mobilizing agents ionomycin and thapsigargin (15,30,51).…”