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Ca 2 +-induced secretion by electropermeabilized human neutrophils . The roles of Ca 2 + , nucleotides and protein kinase C

Studies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Eiectropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomai constituents when exposed to micromolar levels of Ca 2 +. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3-1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2+-induced secretion in the presence of Mg 2÷. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADP~S ATPyS, and App[NH]p were effective. GTP-fS and GDP~S were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophiis which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca 2 + alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4 o C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorboi myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca 2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous AT]P. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca 2 + plus ATP in parallel with that induced by Ca 2 + plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electropermeabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the 'requirement' for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.