THE PROTEIN-KINASE-C INHIBITOR, H-7, INDUCES ACUTE LUNG INJURY IN GUINEA-PIGS

Objectives: To determine if the protein kinase C inhibitor, H-7, alone can cause acute lung injury. In cell studies, H-7 inhibited phorbol m yristate acetate-induced neutrophil oxygen radical release. Additional ly, one animal study demonstrated that H-7 inhibited phorbol myristate acetate-induced lung injury. There have been no studies on the effect of H-7 alone on lung function or on neutrophil release of oxygen radi cals. Design: Prospective, randomized, laboratory study along with in vitro studies using flow cytometry and lucigenin-dependent chemilumine scence. Setting: Experimental laboratory. Subjects: Specific, pathogen -free guinea pigs and isolated human peripheral neutrophils. Intervent ions: Guinea pigs were randomized into three experimental groups: sali ne control, H-7 low dose (2 mg/kg bolus + 0.2 mg/kg/hr), and H-7 high dose (6 mg/kg bolus + 0.5 mg/kg/hr). Human neutrophils were randomized into control and experimental groups. The effects of H-7 on pulmonary permeability in guinea pigs were examined over an 8-hr period. Measur ements and Main Results: We measured the wet/dry weight ratio as an in dex of pulmonary edema and we measured the concentration ratios of I-1 25-labeled albumin in lung tissue and in bronchoalveolar lavage fluid and compared the ratios with those values in plasma as indices of pulm onary permeability. We also studied the in vitro effect of H-7 on huma n neutrophil oxygen radical production, using flow cytometry and lucig enin-dependent chemiluminescence. By now cytometry, we measured oxygen radical production using the 2',7'-dichlorofluorescin and hydroethidi ne assays. The 2',7'-dichlorofluorescin assay mainly measures hydrogen peroxide, while the hydroethidine assay measures either superoxide an ion alone or in combination with other oxygen intermediaries like hydr ogen peroxide. Neutrophils (5 x 10(5)) were obtained by Ficoll-Hypaque gradient centrifugation and were incubated with H-7 (5, 25, 100 mu M) . In the H-7 high-dose group, wet/dry weight ratio, and I-125-labeled albumin ratios in lung/plasma, and bronchoalveolar lavage/plasma were significantly increased (p <.05 for each ratio). Pulmonary endothelial gap and subendothelial bleb formation were demonstrated in the high-d ose group by electron microscopy. One hundred micromols of H-7 caused a small, significant decrease (23.3%, p <.05) in neutrophil oxygen rad ical production assessed by 2',7'-dichlorofluorescin. H-7 had no other effects on neutrophil oxygen radical production. H-7 did not stimulat e neutrophil chemiluminescence; it decreased chemiluminescence. C oncl usions: a) Protein kinase C inhibition with high-dose H-7 increased we t/dry weight and albumin in lung/plasma and bronchoalveolar lavage/pla sma ratios in guinea pigs; b) the H-7 high-dose group demonstrated dam aged pulmonary endothelium by electron microscopy; and c) since neutro phil oxygen radical production was not increased by H-7 as assessed by now cytometry and chemiluminescence, it appears that H-7-induced acut e lung injury and endothelial damage are not mediated by increased neu trophil oxygen radical production.