Neutrophils Purified human neutrophils were prepared from heparinised venous blood from healthy adult volunteers. Neutrophils were separated table 5 from mononuclear leukocytes by centrifugation on Histopaque 1077 cushions at 400 g for 25 min at room temperature. The resultant neutrophil fraction was removed by sequen tial sedimentation with 3% gelatin in order to remove most of the erythrocytes. Following centrifugation, residual erythrocytes were removed by selective lysis with 0. 84% ammonium chloride at 4 C for 10 min. The neutrophils, which were routinely of high purity and viability, were resuspended to 1 107. ml 1 in phosphate buffered saline and held on ice until used. Spectrofluorimetric measurement of cytosolic Ca2 Fura 2 AM was used as the fluorescent, Ca2 sensitive indicator for these e periments.

Neutrophils were incubated with fura 2 AM for 30 min at 37 C in PBS, washed and resuspended in indicator free Hanks balanced salt solution, containing 1. 25 mM CaCl2. The fura 2 loaded cells were then preincubated for 10 min at 37 C in the absence or presence of the PKC inhibitors, after which they were transferred to disposable reaction cuvettes, which were maintained at 37 C in a Hitachi 650 10S fluorescence spectrophotometer with e citation and emission wave lengths set at 340 and 500 nm respectively. After a stable baseline was obtained, the neutrophils were activated by addition of platelet activating factor at final concentrations of 20 and 200 nM. A second chemoattractant, N formyl L methionyl L leu cyl L phenylalanine was used in a limited series of confirmatory e periments during which neutrophils were activated in the presence or absence of GF10903 .

To determine the effects of the PKC inhibitors on cytosolic Ca2 concentrations, uncomplicated by Ca2 influ from e tracellular reservoirs, the cells were treated with the Ca2 chelating agent, ethylene glycol bis N,N,N N tetraacetic acid, added to the cells 1 min prior to PAF. Additional e periments were performed with U73122, a selective inhibitor of phospholipase C, added to the cells 10 15 sec after PAF, when peak cytosolic Ca2 concentrations had been reached, in the presence or absence of the PKC inhibitors staurosporine and GF10903 . This e perimental design was used to determine whether the putative target of PKC is PLC or the intracellular phosphomonoesterases which metabolize IP3.

Further e periments were conducted to investigate the effects of the test agents on the rates of resequestration of cytosolic Ca2 into storage vesicles mediated by the cAMP sensitive endomembrane Ca2 ATPase. Fura 2 loaded cells were preincubated at 37 C with staurosporine or GF10903 for 5 min followed by addition of the phosphodiesterase 4 inhibitor, rolipram, for 3 min prior to activation Brefeldin_A of the cells with PAF, and the subsequent alterations in fura 2 fluores cence monitored over a 5 min time period.