MODULATION OF SUPEROXIDE GENERATION IN IN-VIVO LIPOPOLYSACCHARIDE-PRIMED RAT ALVEOLAR MACROPHAGES BY ARACHIDONIC-ACID AND INHIBITORS OF PROTEIN-KINASE-C, PHOSPHOLIPASE A(2), PROTEIN SERINE-THREONINE PHOSPHATASE(S), PROTEIN-TYROSINE KINASE(S) AND PHOSPHATASE(S)

Ninety minutes after the i.v, injection of Escherichia coil lipopolysa ccharide (LPS) (1 mg/kg) into rats, phorbol 12-myristate 13-acetate (P MA)-stimulated superoxide anion (O-2(-)) secretion was enhanced in sus pensions of in vivo LPS-treated alveolar macrophages (AM Phi) when com pared with saline (SAL)-treated AM Phi. The purpose of this investigat ion was to dissect the in vitro mechanism of PMA-stimulated O-2(-) gen eration in both LPS and SAL-treated rat AM Phi, with a panel of inhibi tors of protein kinase C (PKC), protein serine-threonine phosphatase(s ) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), ph ospholipase A(2) (PLA(2)), cyclooxygenase (GO) and 5-lipoxygenase (5-L O). The following agents blocked PMA-stimulated O-2(-) generation in b oth LPS- and SAL-treated AM Phi (expressed as percentage of control): 1) PKC inhibitors: staurosporine: 100 nM, 7.0% (LPS) and 5.6% (SAL); s phingosine: 10 mu M, 21% (LPS) and 10.5% (SAL); 2) PTK inhibitor: geni stein: 100 mu M, 44% (LPS) and 31% (SAL); 3) PTP inhibitors: phenylars ine oxide, 10 mu M, 12.1% (LPS) and 18% (SAL); diamide, 1000 mu M, 10. 1% (LPS) and 10.5% (SAL); and 4) PLA(2) inhibitors: manoalide: 1 mu M, 29.3% (LPS) and 5.2% (SAL); scalaradial: 1 mu M, 7.7% (LPS) and 7.1% (SAL); and WAY 125,984: 10 CLM, 17.1 % (LPS) and 14.5% (SAL). In addit ion, it was observed that exogenously added arachidonic acid (AA)-stim ulated O-2(-) generation in a time- and dose-dependent manner in both LPS and SAL-treated AM Phi. The following inhibitors enhanced or did n ot affect PMA-stimulated O-2(-) generation in LPS- and SAL-treated AM Phi (expressed as percentage of of control): 1) PSP inhibitors: okadai c acid: 0.5 mu M, 117% (LPS) and 153% (SAL); calyculin A: 1 mu M, 112% (LPS) and 101% (SAL); 2) CO and 5-LO inhibitors: indomethacin: 10 mu M, 107% (LPS) and 90% (SAL); WY 50, 295: 1 mu M, 99% (LPS) and 103% (S AL); and 3) the PTP inhibitor orthovanadate upon prolonged preincubati on. in both in vivo LPS-or SAL-primed AM Phi, PMA-stimulated O-2- gene ration appears to be modulated by PKC, PLA(2), AA, PTK, PTP and PSP. N o modulatory role was evident for either CO or 5-LO metabolites. These findings might bear on the design of therapeutic approaches for the m odulation of O-2(-) release by AM Phi, in the early stages of sepsis a nd adult respiratory distress syndrome.