Dissociation of the 47-Kilodalton Protein Phosphorylation From Degranulation and Superoxide Production in Neutrophils

The aim for the present studies is to examine the relationship between the phosphorylation of the 47-kDa protein and some neutrophil responses such as degranulation, the synergistic effect of PMA on calcium ionophore-induced degranulation, superoxide generation, and the priming of the oxidative burst produced by the chemotactic factor fMet-Leu-Phe and phorbol 12-myristate 13-acetate (PMA). Incubation of neutrophils with the protein kinase inhibitor 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7) inhibits the phosphorylation of the 47-kDa protein produced by PMA and fMet-Leu-Phe but does not affect lysozyme release induced by the same stimuli or fMet-Leu-Phe-induced N-acetyl-beta-glucosaminidase release. Furthermore, the synergistic effect of PMA on the A23187-induced degranulation is not inhibited by H-7. Also, pretreatment of the cells with H-7 inhibits superoxide production produced by PMA but not by fMet-Leu-Phe. The inhibitory effect of H-7 is more pronounced on the rate than on the extent of PMA-induced superoxide release. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, HA-1004, a less potent protein kinase inhibitor, has no inhibitory effect on superoxide generation produced by fMet-Leu-Phe or PMA. In the case of superoxide production, the addition of low concentrations of PMA to rabbit neutrophils primes these cells to the subsequent stimulation by fMet-Leu-Phe, dramatically increasing the effect. Conversely, low concentrations of fMet-Leu-Phe prime the cells to PMA stimulation. The stimulation by PMA of cells primed with fMet-Leu-Phe is inhibited by H-7. Moreover, the priming effect by PMA is also inhibited by H-7. This inhibition is less pronounced at a low concentration of PMA. On the other hand, in the case of human neutrophils, the priming effect of PMA is not inhibited by H-7. These results suggest several points. First, phosphorylation of the protein identified on two-dimensional gel electrophoresis as having a molecular weight of 47 kDa and PI of 4.9 is not necessary for degranulation produced by either PMA or fMet-Leu-Phe. Second, the phosphorylation of the 47-kDa protein is not necessary for superoxide generation, at least in the case of fMet-Leu-Phe and possibly for other stimuli. Third, in rabbit neutrophils, both the priming and stimulation of superoxide production with PMA are inhibited by H-7. In human neutrophils, the priming by PMA is not affected by H-7.(ABSTRACT TRUNCATED AT 250 WORDS)