Ca2+ dependency of ‘Ca2+‐independent’ exocytosis in SPOC1 airway goblet cells

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Abstract
SPOC1 airway goblet cells secrete mucin in response to P2Y2 receptor agonists and to secretagogues, phorbol 12-myristate 13-acetate (PMA) and ionomycin, which mobilize elements of the phospholipase C pathway, PKC and Ca2+, respectively. Previous studies demonstrated that mucin secretion from SLO-permeabilized, EGTA-buffered SPOC1 cells was stimulated by PMA at low Ca2+ levels (< 0.1 μm), consistent with the notion that regulated exocytosis may occur by Ca2+-independent pathways. We tested the alternative hypothesis that PMA-induced mucin secretion is, in fact, a Ca2+-dependent process under the conditions of low bulk Ca2+, one that is permitted in the typical SLO-permeabilized cell model by the slow binding kinetics of EGTA. Both IP3 and elevated bulk Ca2+ activated mucin secretion in SPOC1 cells buffered by EGTA, suggesting that IP3 generates a local Ca2+ gradient in the vicinity of the secretory granules to the degree necessary to trigger exocytosis. BAPTA, which binds Ca2+ approximately 100-fold faster than EGTA, diminished IP3-induced mucin release over a range of concentrations by ≥ 69%, yet maintained an essentially normal mucin secretory response to elevated bulk Ca2+ in permeabilized SPOC1 cells. BAPTA also diminished the mucin secretory response of permeabilized cells to PMA, relative to the EGTA-buffered control: at PMA below 30 nm, BAPTA abolished the secretory response, and at higher concentrations it was reduced significantly relative to the EGTA-buffered controls. PMA-induced secretion in EGTA was insensitive to heparin. These results suggest that Ca2+ is released locally during PMA-induced exocytosis, by an IP3-independent mechanism.