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 myristate acetate-induced neutrophil oxygen radical release. Additionally, 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 radicals. Design: Prospective, randomized, laboratory study along with in vitro studies using flow cytometry and lucigenin-dependent chemiluminescence. Setting: Experimental laboratory. Subjects: Specific, pathogen-free guinea pigs and isolated human peripheral neutrophils. Interventions: Guinea pigs were randomized into three experimental groups: saline 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. Measurements and Main Results: We measured the wet/dry weight ratio as an index of pulmonary edema and we measured the concentration ratios of I-125-labeled albumin in lung tissue and in bronchoalveolar lavage fluid and compared the ratios with those values in plasma as indices of pulmonary permeability. We also studied the in vitro effect of H-7 on human neutrophil oxygen radical production, using flow cytometry and lucigenin-dependent chemiluminescence. By now cytometry, we measured oxygen radical production using the 2',7'-dichlorofluorescin and hydroethidine assays. The 2',7'-dichlorofluorescin assay mainly measures hydrogen peroxide, while the hydroethidine assay measures either superoxide anion alone or in combination with other oxygen intermediaries like hydrogen 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-dose group by electron microscopy. One hundred micromols of H-7 caused a small, significant decrease (23.3%, p <.05) in neutrophil oxygen radical production assessed by 2',7'-dichlorofluorescin. H-7 had no other effects on neutrophil oxygen radical production. H-7 did not stimulate neutrophil chemiluminescence; it decreased chemiluminescence. C onclusions: a) Protein kinase C inhibition with high-dose H-7 increased wet/dry weight and albumin in lung/plasma and bronchoalveolar lavage/plasma ratios in guinea pigs; b) the H-7 high-dose group demonstrated damaged pulmonary endothelium by electron microscopy; and c) since neutrophil oxygen radical production was not increased by H-7 as assessed by now cytometry and chemiluminescence, it appears that H-7-induced acute lung injury and endothelial damage are not mediated by increased neutrophil oxygen radical production.