Calcium-pH crosstalks in rat mast cells: modulation by transduction signals show non-essential role for calcium in alkaline-induced exocytosis

Alkalinization of cytosolic pH with ammonium chloride (NH4Cl) was reported to be a stimulus for mast cell degranulation. This paper studied the modulatory role of drugs that target protein kinase C (PKC), adenosine 3′,5′-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentrations of GF109203X (Gf) (50 nM) to inhibit calcium-dependent PKC isozymes. For calcium-independent isozymes, we used 500 nM Gf, and 10 μM rottlerin to specifically inhibit PKC δ, and chelerythrine as non-specific PKC inhibitor. Genistein (10 μM) and lavendustin A (1 μM) were used as unspecific TyrK inhibitors, and 10 nM wortmannin as a PI3K inhibitor. Chelerythrine and 50 nM Gf inhibit histamine release in the presence of external calcium. The inhibition caused by wortmannin was strictly internal calcium-dependent. cAMP-active drugs did not modify the response to NH4Cl. The effect of NH4Cl on histamine release was triggered by a transient elevation on cytosolic pH, which was simultaneous to an elevation on cytosolic calcium and followed by a probable Ca2+-H+ exchange after addition of external calcium. EGTA inhibit the response to suboptimal concentrations of NH4Cl, and BAPTA increased the effect of NH4Cl. There is a clear relationship between NH4Cl-mediated calcium release and histamine release, since those drugs that inhibit this release also inhibit NH4Cl-mediated histamine release; nevertheless, NH4Cl-mediated histamine release was possible in the absence of any calcium release, as shown with BAPTA. This data, in combination with the results with PKC inhibitors, suggest that calcium is not only unnecessary to trigger cell activation, but also that it may be a negative modulator of NH4Cl-mediated exocytosis.