Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing.

We tested whether mitochondria function as the O(2) sensor underlying hypoxic pulmonary vasoconstriction (HPV). In buffer-perfused rat lungs, rotenone, myxothiazol, and diphenyleneiodonium, which inhibit mitochondria in the proximal region of the electron transport chain (ETC), abolished HPV without attenuating the response to U46619. Cyanide and antimycin A inhibit electron transfer in the distal region of the ETC, but they did not abolish HPV. Cultured pulmonary artery (PA) myocytes contract in response to hypoxia or to U46619. The hypoxic response was abolished while the response to U46619 was maintained in mutant (rho(0)) PA myocytes lacking a mitochondrial ETC. To test whether reactive oxygen species (ROS) derived from mitochondria act as signaling agents in HPV, the antioxidants pyrrolidinedithiocarbamate and ebselen and the Cu,Zn superoxide dismutase inhibitor diethyldithiocarbamate were used. These abolished HPV without affecting contraction to U46619, suggesting that ROS act as second messengers. In cultured PA myocytes, oxidation of intracellular 2',7'-dichlorofluorescin diacetate (DCFH) dye increased under 2% O(2), indicating that myocytes increase their generation of H(2)O(2) during hypoxia. This was attenuated by myxothiazol, implicating mitochondria as the source of increased ROS during HPV. These results indicate that mitochondrial ATP is not required for HPV, that mitochondria function as O(2) sensors during hypoxia, and that ROS generated in the proximal region of the ETC act as second messengers in the response.

Thrombin increases fluid flux in isolated rat lungs by a hemodynamic and not a permeability mechanism.

Alpha-Thrombin increases endothelial protein permeability in vitro and induces weight gain in the isolated perfused lung. The objectives of this study were to determine whether thrombin increases endothelial permeability of the isolated perfused rat lung and whether a change in permeability or hemodynamics mediates the gain in lung weight. Endothelial protein permeability was assessed by regression analysis of 125I-labeled albumin clearance vs. fluid flux to determine the permeability-surface area product (PS) and the reflection coefficient (sigma). Thrombin (5 x 10(-8) or 5 x 10(-7) M) did not alter protein permeability from the control values of PS and sigma. Thrombin caused an overall increase in transvascular fluid flux, as depicted by a gain in lung weight. Pulmonary arterial and capillary pressures and arterial and venous resistances increased by 10 min after thrombin injection, and lung weight decreased due to arterial constriction. From 10 to 50 min, pressures and resistances decreased, but capillary pressure and venous resistance decreased to a lesser extent and, as a result, lung weight increased. Pretreatment with BQ-123, an endothelin-receptor antagonist, attenuated the sustained increases in pressures and resistances and the rate of lung weight gain. Indomethacin, a cyclooxygenase inhibitor, had no effect. These findings indicate that the increase in lung weight induced by thrombin results from an elevation of capillary pressure mediated, in part, by endothelin and is not due to an increase in endothelial protein permeability of the isolated perfused rat lung.

Oxidant-increased endothelial permeability: prevention with phosphodiesterase inhibition vs. cAMP production.

The present objective was to determine whether hydrogen peroxide (H(2)O(2)) increases transvascular albumin clearance and lung weight in an isolated rat lung and whether posttreatment with cAMP-enhancing agents can prevent these increases. Transvascular albumin clearance was assessed by (125)I-labeled albumin clearance ((125)I-albumin flux/perfusate concentration of (125)I-albumin) at a given fluid filtration. Nonlinear regression analysis of transvascular albumin clearance vs. fluid filtration yielded values for the permeability-surface area product (PS) and the reflection coefficient (sigma). H(2)O(2) decreased sigma from a control value of 0.93 to 0.38, did not change PS, and increased lung weight. Posttreatment with isoproterenol, a beta(2)-adrenergic-receptor agonist, reduced the H(2)O(2)-induced decrease in sigma to 0.65 and augmented the increase in lung weight. Posttreatment with CP-80633, a phosphodiesterase 4 inhibitor, further reduced the H(2)O(2)-induced decrease in sigma to 0.79 and blocked the rise in lung weight. In the presence of isoproterenol or CP-80633, H(2)O(2) increased PS. Therefore, H(2)O(2) increased the convective and diffusive clearances of albumin across an intact pulmonary vasculature. Furthermore, inhibition of cAMP metabolism more effectively attenuated the H(2)O(2)-induced increases in convective albumin clearance and lung weight as compared with stimulation of cAMP production.

Cytotoxicity induced by grape seed proanthocyanidins: role of nitric oxide.

Grape seed proanthocyanidin extract (GPSE) at high doses has been shown to exhibit cytotoxicity that is associated with increased apoptotic cell death. Nitric oxide (NO), being a regulator of apoptosis, can be increased in production by the administration of GSPE. In a chick cardiomyocyte study, we demonstrated that high-dose (500 microg/ml) GSPE produces a significantly high level of NO that contributes to increased apoptotic cell death detected by propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. It is also associated with the depletion of intracellular glutathione (GSH), probably due to increased consumption by NO with the formation of S-nitrosoglutathione. Co-treatment with L-NAME, a NO synthase inhibitor, results in reduction of NO and apoptotic cell death. The decline in reduced GSH/oxidized GSH (GSSG) ratio is also reversed. N-Acetylcysteine, a thiol compound that reacts directly with NO, can reduce the increased NO generation and reverse the decreased GSH/GSSG ratio, thereby attenuating the cytotoxicity induced by high-dose GSPE. Taken together, these results suggest that endogenous NO synthase (NOS) activation and excessive NO production play a key role in the pathogenesis of high-dose GSPE-induced cytotoxicity.