P2u 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 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.
Abdullah
L H
LH
Department of Medicine, University of North Carolina, Chapel Hill 27514, USA.
Davis
S W
SW
Burch
L
L
Yamauchi
M
M
Randell
S H
SH
Nettesheim
P
P
Davis
C W
CW
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D046508:Culture Techniques / Q000295:instrumentation / Q000379:methods; D017931:DNA Primers; D015870:Gene Expression; D005944:Glucosamine / Q000378:metabolism; D006001:Glycoconjugates / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009077:Mucins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D009092:Mucous Membrane / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D010477:Perfusion / Q000295:instrumentation / Q000379:methods; D016133:Polymerase Chain Reaction; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D014132:Trachea / Q000166:cytology; D014544:Uridine Triphosphate / Q000494:pharmacology*
D017931:DNA Primers; D006001:Glycoconjugates; D009077:Mucins; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D005944:Glucosamine; D014544:Uridine Triphosphate