Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA

Abstract

SPOC1 cells, which are a mucin-secreting model of rat airway goblet cells, possess a luminal P2Y2 purinoceptor through which UTP, ATP, and ATPγS stimulate secretion with EC50 values of ∼3 μM. 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: cPKCα, nPKCδ and -η, and aPKCζ and -ι/λ. PMA caused cPKCα and nPKCδ to translocate to the membrane fraction of SPOC1 cells; only nPKCδ so responded to ATPγS. Membrane-associated nPKCδ and mucin secretion increased in parallel with ATPγS concentration and yielded EC50 values of 2–3 μM and maximum values of 100 μM. Effects of PMA to increase membrane-associated cPKCα and nPKCδ 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-γ. Hence, P2Y2 agonists activate nPKCδ in SPOC1 cells. PMA activates cPKCα and nPKCδ at high affinity and stimulates a lower affinity PKC-independent pathway that leads to mucin secretion.