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Background-Nitric oxide (NO) production is increased in postischemic myocardium, and NO can control mitochondrial oxygen consumption in vitro. Therefore, we investigated the role of endothelial NO synthase (eNOS)-derived NO on in vivo regulation of oxygen consumption in the postischemic heart. Methods and Results-Mice were subjected to 30 minutes of coronary ligation followed by 60 minutes of reperfusion.Myocardial oxygen tension (PO 2 ) was monitored by electron paramagnetic resonance oximetry. In wild-type, N-nitro-L-arginine methyl ester (L-NAME)-treated (with 1 mg/mL in drinking water), and eNOS knockout (eNOS Ϫ/Ϫ ) mice, no difference was observed among baseline myocardial PO 2 values (8.6Ϯ0.7, 10.0Ϯ1.2, and 10.1Ϯ1.2 mm Hg, respectively) or those measured at 30 minutes of ischemia (1.4Ϯ0.6, 2.3Ϯ0.9, and 3.1Ϯ1.4 mm Hg, respectively). After reperfusion, myocardial PO 2 increased markedly (PϽ0.001 versus baseline in each group) but was much lower in L-NAME-treated and eNOS Ϫ/Ϫ mice (17.4Ϯ1.6 and 20.4Ϯ1.9 mm Hg) than in wild-type mice (46.5Ϯ1.7 mm Hg; PϽ0.001). A transient peak of myocardial PO 2 was observed at early reperfusion in wild-type mice. No reactive hyperemia was observed during early reperfusion. Endothelial NO decreased the rate-pressure product (PϽ0.05), upregulated cytochrome c oxidase (CcO) mRNA expression (PϽ0.01) with no change in CcO activity, and inhibited NADH dehydrogenase (NADH-DH) activity (PϽ0.01) without alteration of NADH-DH mRNA expression. Peroxynitrite-mediated tyrosine nitration was higher in hearts from wild-type mice than in eNOS Ϫ/Ϫ or L-NAME-treated hearts. Conclusions-eNOS-derived

We tested correlations between lipophilicity parameters and the partitioning of sensitizers into membranes. For this purpose we investigated 17 porphyrins and two chlorins having various chemical structures. Some of these compounds possess an amphiphilic structure (including hematoporphyrin, deuteroporphyrin, mesoporphyrin, chlorin e6 and more). The others are very symmetrical sensitizers [meso-tetra(N-methyl-4-pyridyl)porphyrin, tetra-benzoporphyrin, coproporphyrin I dihydrochloride (CP), meso-tetra(4-carboxyphenyl)porphyrin (TCP) and meso-tetra(m-hydroxyphenyl)chlorin]. Our investigation also included two series of hematoporphyrins and protoporphyrins with varying lengths of alkylcarboxylate side groups. The partitioning of these compounds between the bulk aqueous phase and liposomes was studied by fluorescence methods, and a liposome-binding constant, Kb, was obtained. It was found that CP and TCP do not incorporate into the lipid phase at pH 7.3. An n-octanol-water partition coefficient (log P) and a distribution coefficient (log D) were predicted with a modeling software. The values of log D were also obtained experimentally. We found that for the studied molecules, the predicted log D correlated well with the measured values. The values of log D as well as log P, in turn, did not correlate nicely, for the whole group of studied compounds, with the binding constants to liposomes. However, in the case of porphyrins that share a similar structure, the Kb showed good linear correlation with both log P and log D. For the series of hematoporphyrins and protoporphyrins with different lengths of alkylcarboxyl groups, it was shown that prolongation of this group caused an increase in the lipophilicity and the liposome-binding constant. This effect is more pronounced for the proto- than for the hematoporphyrin series. The results highlight the possible use, as well as limitations, of lipophilicity parameters for the prediction of membrane binding.

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A new technique is described for simultaneous determination of intra- and extracellular oxygen concentrations [partial pressure of O(2) (pO(2))] in bovine lung microvascular endothelial cells (BLMVECs) using electron paramagnetic resonance (EPR) oximetry. The measurements were performed in BLMVEC suspensions of a 20- micro l volume containing 4,000 cells. The extracellular pO(2) was measured using a trityl EPR probe [triarylmethyl (TAM), 10 micro M], a tricarboxylate anion radical, that stays exclusively in the extracellular space. The intracellular oxygen was measured using a pre-internalized particulate spin probe, lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-naphthalocyanine (LiNc-BuO). Because there is a wide discrepancy in the reported values of cellular oxygenation by and large due to differences in the methods employed, we utilized the dual EPR probe technique to measure the oxygen gradient that apparently exists across the cell membrane. The intra- and extracellular pO(2) values were 139 +/- 2.5 and 157 +/- 3.6 mm Hg, respectively, for cells exposed to room air. A fairly smaller gradient of oxygen was observed in cells exposed to 7.5% oxygen (pO(2) = 57 mm Hg). In summary, this study confirms the feasibility of simultaneous and accurate measurements of intra- and extracellular pO(2) using LiNc-BuO and TAM EPR oximetry probes.