Inhibition of cromolyn-induced phosphorylation of a 78-kDa protein by phorbol esters in rat peritoneal mast cells

Disodium cromoglycate (cromolyn) is a well documented inhibitor of immunologically-induced histamine release from rat peritoneal mast cells and has been shown to stimulate the phosphorylation of a mast cell protein of apparent molecular mass 78,000 Da (78 kDa), an event which may be involved in terminating secretion. Here we aimed to determine the role of the ubiquitous enzyme, protein kinase C, in the phosphorylating activity of cromolyn by examining the effects of phorbol esters (activators of protein kinase C) on protein phosphorylation in [32P]orthophosphate loaded rat peritoneal mast cells. Protein kinase C-activating phorbol esters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA) and 4beta-phorbol 12,13-dibutyrate (PdBu) were found to potently inhibit cromolyn-induced phosphorylation when added to mast cells simultaneously with cromolyn (IC50 22 and 79 nM respectively). 4Alpha-phorbol 12,13-didecanoate (PdD), a phorbol ester which does not activate protein kinase C, had no effect on cromolyn-induced phosphorylation. Addition of TPA to mast cells previously exposed to cromolyn for 60 sec (i.e. when 78-kDa protein phosphorylation is maximal) also caused a very rapid dephosphorylation of the 78-kDa protein. Phosphorylation of the 78-kDa protein can also be induced by dibutyryl cyclic GMP and this action was similarly inhibited by TPA and PdBu. Cromolyn inhibited secretion induced by anti-IgE, but not by TPA, and thus inhibition of secretion by cromolyn is further correlated to its phosphorylation of the 78-kDa protein. The data suggest that the inhibitory action of cromolyn on mast cell secretion and phosphorylation of the 78-kDa protein are not mediated through a phorbol ester-sensitive protein kinase C, but more likely that such an enzyme could be involved in regulating dephosphorylation of the 78-kDa protein. Further explanations for this novel dephosphorylating activity of phorbol esters are discussed.