Phosphatidic acid induces the release of β-glucuronidase but not lactoferrin from electropermeabilized human neutrophils

We studied the degranulation reaction of electropermeabilized human neutrophils induced by 1,2-didecanoyl-3-sn-phosphatidic acid (PA₁₀). PA₁₀ dose-dependently induced the release of β-glucuronidase, an enzyme of azurophil granules, but did not induce the release of lactoferrin, a protein of specific granules. The enzyme release by PA₁₀ absolutely required Ca²⁺, ATP, and Mg²⁺ and the concentrations for the half-maximal response were 2.5 μM, 60 μM, and 0.25 mM, respectively. Although Ca²⁺ alone at concentrations higher than 10 μM induced the release of both β-glucuronidase and lactoferrin, the extents of the release were far less than that of the β-glucuronidase release by PA₁₀. Phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol induced the release of lactoferrin alone at concentrations of Ca²⁺ below 0.5 μM while they induced the release of both β-glucuronidase and lactoferrin at higher Ca²⁺ concentrations, indicating that the degranulation induced by PA₁₀ is not mediated by diacylglycerol which might be formed from PA. The degranulation reactions induced by PA₁₀ and PMA were dose-dependently inhibited by staurosporine and calphostin C, protein kinase C inhibitors, although no direct activation of protein kinase C by PA₁₀ was observed. The extent of the β-glucuronidase release by PA₁₀ was not enhanced by the addition of PMA. Propranolol, which inhibits protein kinase C as well as phosphatidic acid phosphohydrolase, strongly inhibited the degranulation reactions induced by PA₁₀ and PMA. Ethanol, a metabolic modulator of phospholipase D, and cyclic AMP did not affect the degranulation reactions by PMA and PA₁₀. These observations suggest that selective enzyme release from azurophil granules of the permeabilized neutrophils might be induced through the activation of protein kinase C or some other protein kinase sensitive to the protein kinase C inhibitors and that phospholipase D may not be involved in the PMA-induced release.