Human neutrophil secondary granule exocytosis is independent of protein kinase activation and is modified by calmodulin activity

Exocytosis of the secondary (2 degree) lysosomal granule is an important process in the activation of human neutrophils. Stored enzymes such as collagenase and gelatinase are released, and adhesion molecules from the granule membrane are inserted in the plasma membrane. This exocytosis is independent of azurophil granule release and respiratory burst activation. We investigated, using kinase and phosphatase inhibitors and activators of adenylate cyclase, common intracellular signalling mechanisms involved in exocytosis (vitamin B12 binding protein release) stimulated by different agonists. Exocytosis in response to tumour necrosis factor alpha (TNF alpha), phorbol myristate acetate (PMA) and the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was inhibited by the calmodulin antagonist N-(6-amino hexyl)-5-chloro-1-naphthalene sulphonamide (W7). Neither staurosporine, H7 nor genistein was inhibitory. In contrast, the same doses of W7 synergistically enhanced the exocytosis stimulated by the tyrosine phosphatase inhibitor sodium orthovanadate, while kinase inhibition by staurosporine or genistein dose-dependently inhibited the vanadate response. Furthermore, adenylate cyclase activation with prostaglandin E2 or dibutyryl cyclic AMP, inhibited exocytosis in response to TNF alpha and FMLP, while having no effect on the release induced by vanadate or PMA. Thus, 2 degree granule exocytosis stimulated by receptor-bound ligands is calmodulin-dependent, and is independent of protein kinase activity. In contrast, exocytosis in response to tyrosine phosphatase inhibition is antagonised by calmodulin, since the response to vanadate was enhanced synergistically by W7. Thus, depending on the initial stimulus, calmodulin may promote or inhibit 2 degree granule exocytosis by human PMN.