Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA.
SPOC1 cells, which are a mucin-secreting model of rat airway goblet cells, possess a luminal P2Y2 purinoceptor through which UTP, ATP, and ATPgammaS stimulate secretion with EC50 values of approximately 3 microM. PMA elicits mucin secretion with high EC50 (75 nM) and saturation (300 nM) values. For the first time in airway mucin-secreting cells, the PKC isoforms expressed and activated by a secretagogue were determined using RT-PCR/restriction-enzyme mapping and Western blotting. Five isoforms were expressed: cPKCalpha, nPKCdelta and -eta, and aPKCzeta and -iota/lambda. PMA caused cPKCalpha and nPKCdelta to translocate to the membrane fraction of SPOC1 cells; only nPKCdelta so responded to ATPgammaS. Membrane-associated nPKCdelta and mucin secretion increased in parallel with ATPgammaS concentration and yielded EC50 values of 2-3 microM and maximum values of 100 microM. Effects of PMA to increase membrane-associated cPKCalpha and nPKCdelta saturated at 30 nM, whereas mucin secretion saturated at 300 nM, which suggests a significant PKC-independent effect of PMA on mucin secretion. A prime alternate phorbol ester-receptor candidate is the C1-domain protein MUNC13. RT-PCR revealed the expression of ubiquitous (ub)MUNC13-2 and its binding partner, DOC2-gamma. Hence, P2Y2 agonists activate nPKCdelta in SPOC1 cells. PMA activates cPKCalpha and nPKCdelta at high affinity and stimulates a lower affinity PKC-independent pathway that leads to mucin secretion.
Abdullah
Lubna H
LH
Cystic Fibrosis/Pulmonary Treatment and Research Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7248, USA.
Bundy
Jason T
JT
Ehre
Camille
C
Davis
C William
CW
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D020397:Goblet Cells / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D009077:Mucins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D011506:Proteins / Q000235:genetics; D058910:Purinergic P2 Receptor Agonists; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002273:Carcinogens; C490419:Doc2a protein, rat; D007527:Isoenzymes; D009077:Mucins; D009419:Nerve Tissue Proteins; D011506:Proteins; D058910:Purinergic P2 Receptor Agonists; D012333:RNA, Messenger; C485548:UNC13B protein, human; C117991:Unc13b protein, rat; C430713:Unc13d protein, rat; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate