The effects of some non-steroidal anti-inflammatory agents on membrane-associated calcium in rabbit peritoneal neutrophils.

Several non-steroidal anti-inflammatory agents (NSAIAs) (flufenamate, flurbiprofen, indomethacin, phenylbutazone, piroxicam and salicylate), one anti-inflammatory steroid (hydrocortisone) and three compounds known to affect cellular calcium metabolism [nifedipine, calcium ionophore A23187, 8-(diethylamino)octyl 3,4,5,-trimethoxy benzoate hydrochloride (TMB-8)] were tested for their effects on membrane-associated calcium in rabbit peritoneal neutrophils using the fluorescent probe chlortetracycline (CTC). The NSAIAs reduced the level of fluorescence attained by incubating neutrophils with CTC, and caused an immediate reduction in the fluorescence of CTC-loaded neutrophils, both effects being concentration-related. Nifedipine, A23187 and TMB-8 also reduced the fluorescence of CTC-loaded cells. However, these measured reductions in fluorescence were due in whole or in part to a drug-induced drop in the autofluorescence of the neutrophils. After applying a correction factor which took account of this effect there was still an immediate concentration-related reduction in CTC-dependent fluorescence after the addition of all the NSAIAs. A23187 also caused a reduction in CTC fluorescence, but nifedipine, TMB-8 and hydrocortisone were inactive. Flufenamate, indomethacin and piroxicam reduced the drop in fluorescence brought about by N-formyl-methionyl-leucyl-phenylalanine (FMLP). These findings suggest that NSAIAs and A23187 displace membrane-associated calcium. Electron microscopical findings confirm that indomethacin and salicylate both affect membrane-associated calcium. Using the fluorescent probe Quin 2, an attempt was made to determine whether or not the displacement of membrane-associated calcium by NSAIAs was accompanied by changes in cytoplasmic calcium ion concentration. This was unsuccessful, however, since the effects of the NSAIAs on the fluorescence of Quin 2-loaded neutrophils was accounted for almost entirely by their effects on the autofluorescence of the cells.