Reversal of inhibitory pathways in neutrophils by protein kinase antagonists: a rational approach to the restoration of depressed cell function?

Neutrophil functions are sensitive to both stimulatory and inhibitory pathways. For example, the endogenous hormones histamine, prostaglandin E1, adenosine, and catecholamine were found to inhibit the oxidant responses of human neutrophils by formyl peptide to 6.2, 16.8, 11.4, and 15.4%, respectively, of the initial response. The inhibition of cell function is mimicked by dibutyryl cAMP and forskolin, consistent with a pathway involving cAMP and an A kinase. Because of likely roles of kinases in both stimulatory and inhibitory pathways, we evaluated the potential for regulating either pathway by kinase inhibitors. Preincubation of intact neutrophils with membrane-permeable but nonspecific protein kinase antagonists blocked the isoproterenol-mediated inhibition of superoxide generation. The isoquinoline sulfonamides H-7, H-8, and H-9 at 100 microM reversed inhibition to 60.1, 66.6, and 70.9%, respectively, of the response of control cells. H-9 also antagonized the inhibition of superoxide production induced by other agents that regulate intracellular cAMP (prostaglandin E1, histamine, adenosine, forskolin, and dibutyryl cAMP). A synthetic peptide used as a specific but impermeable protein kinase A antagonist restored superoxide production inhibited by isoproterenol and cAMP up to 70% in electroporated cells. A small number of proteins are targets of cAMP-dependent phosphorylation in electroporated cells, and phosphorylation is inhibited in the presence of the peptide inhibitor. Taken together, these data show that a peptide inhibitor and isoquinoline sulfonamides reverse the inhibition of the respiratory burst in neutrophils evoked by the inhibitory pathways. Drugs that reverse the effect of endogenous inhibitors of neutrophil activation (by restoring cell function) have important therapeutic implications in restoring cell functions in patients whose cell functions are depressed under physiological conditions.