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Am J Physiol Heart Circ Physiol 275: H776-H782, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 3, H776-H782, September 1998

Depression of endothelial and smooth muscle cell oxygen consumption by endotoxin

Roberto Motterlini1, Heinz Kerger3, Colin J. Green1, Robert M. Winslow2, and Marcos Intaglietta3

1 Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow HA1 3UJ, United Kingdom; and Departments of 2 Medicine and 3 Bioengineering, University of California, San Diego, La Jolla, California 92093

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

An optical method based on the oxygen-dependent quenching of a phosphorescent probe (palladium-porphyrin) was used to investigate the effect of bacterial endotoxin [lipopolysaccharide (LPS)] on oxygen consumption (VO2) by vascular cells. Endothelial (EC) and smooth muscle (SMC) cells from pig aorta were suspended in culture medium in the presence of palladium-porphyrin and transferred to glass capillary tubes that were sealed to create a hypoxic environment. Measured PO2 changed as a function of time in a highly predictable fashion when cell suspensions were exposed to agents or treatment known to affect cellular metabolism. Both EC and SMC showed a significant decrease in VO2 as cell density increased, and SMC VO2 was significantly higher than EC (1.94 ± 0.09 vs. 1.0 ± 0.15 nmol · min-1 · 106 cells-1). Exposure to LPS (1 µg/ml) caused a decrease in VO2 of 46% and 15% for EC and SMC, respectively. Pretreatment of cells with N-acetyl-L-cysteine, a substrate for glutathione synthesis with antioxidant properties, restored VO2 to normal values after exposure to LPS. These data suggest that endotoxin impairs VO2 in cells derived from the vascular wall and indicate the importance of EC and SMC respiration in maintaining vascular homeostasis under conditions of sepsis.

lipopolysaccharides; vascular wall; oxygen tension; N-acetyl-L-cysteine; oxidative stress

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SEVERE ENDOTOXEMIC SEPSIS is characterized by inadequate tissue oxygenation and altered oxygen distribution, leading to reduced oxygen extraction in different organs. The pathogenesis of impaired oxygen extraction in septic states is related to a cascade of responses including capillary blockage by activated neutrophils and platelets, release of cytokines and vasoactive substances, and the consequent damage to the vascular tissue (8, 13, 33). Vascular endothelial cell injury and increased permeability caused by oxygen free radicals appear to be major determinants in organ dysfunction mediated by endotoxin.

Previous reports have shown that cultured endothelial cells exposed to bacterial endotoxin [lipopolysaccharide (LPS)] cause changes in endothelial barrier function (11) and that glutathione precursors reduce the susceptibility of endothelium to free radical-induced damage by potentiating intracellular antioxidant defense systems (19). The endothelium-derived relaxing factor (EDRF), which has been characterized as nitric oxide (NO) or an S-nitrosothiol derivative (21, 23), also plays a critical role in maintaining both blood flow and tissue oxygenation. There are both clinical and experimental findings (7, 13, 15) that support the concept that endotoxin-mediated sepsis induces depletion of EDRF and glutathione, and a recent report (27) suggested a possible physiological significance of EDRF-NO in the control of tissue respiration.

The evaluation of cellular oxygen uptake and utilization is a fundamental parameter to assess the potential metabolic capabilities of tissues and organs undergoing endotoxin-induced sepsis. The measurement of intra- and extravascular oxygen tension (PO2) by means of polarographic oxygen electrodes is a conventional procedure for the invasive assessment of tissue oxygen consumption. An optical method based on the oxygen-dependent quenching of phosphorescent palladium-porphyrin complexes has been recently developed for the noninvasive measurement of tissue PO2 in vivo (26, 31). We have adapted this technology to the measurement of PO2 in vitro using glass capillary tubes loaded with cells of the vascular tissue. The vascular wall is a site of synthesis and conversion of a variety of substances, including NO and prostaglandins (23, 34), but very limited data are available on the oxygen utilization of its constituent cells. This information is important to establish the significance of alterations in oxygen consumption partition between vascular endothelial and smooth muscle cells under pathophysiological conditions such as ischemia-reperfusion and septic shock.

At present it is not known whether endotoxins lead to an impairment of oxygen consumption in the vascular wall. Thus the aim of the present study was to investigate the effect of endotoxin on oxygen consumption of vascular cell suspensions in conditions of limited oxygen availability. Cells sealed in glass capillary tubes containing palladium-porphyrin were kept at room temperature, and PO2 in the suspending medium was monitored over time. The phosphorescence technique samples oxygen more uniformly than the electrode method because oxygen measurements by the phosphorescence method are uniform throughout the medium, whereas the electrode method automatically introduces an oxygen gradient at the location of the measurement. We conducted a series of experiments in which cells were exposed to treatments that affect cellular metabolism and oxygen utilization to validate the method used for the PO2 measurement. We report that the phosphorescence decay technique is a reliable method for determining changes in PO2 of endothelial and smooth muscle cells in suspension after exposure to LPS. The beneficial effect of N-acetyl-L-cysteine, a precursor of glutathione with potent antioxidant characteristics, on the preservation of oxygen consumption by LPS-treated vascular cells was also tested.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents. Palladium-meso-tetra(4-carboxyphenyl)porphyrin was supplied by Porphyrin Products (Logan, UT) and bound to albumin using a method previously described (31). Endothelial cell growth medium (EGM) was obtained from Clonetics (San Diego, CA), and fetal bovine serum (FBS) was from HyClone Laboratories (Logan, UT). Bacterial endotoxin (LPS; Escherichia coli O128:B12), N-acetyl-L-cysteine (NAC), and all other reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified. Endotoxin was prepared in sterile PBS at concentrations of 0.5 mg/ml and stored at -20°C until use.

Cell culture and experimental design. Vascular endothelial (EC) and smooth muscle (SMC) cells of the pig thoracic aorta (cell lines AG08472 and AG08473, respectively) were purchased from Coriell Cell Repositories (Camden, NJ). Cells were cultured in 75-cm2 T flasks (Corning) and grown in EGM supplemented with 10% (vol/vol) FBS and antibiotics. Confluent cells were subcultured in a 1:3 ratio by brief trypsinization using 0.05% trypsin containing 0.5 mM EDTA. Cells between passages 5 and 8 were used in all studies within 24 h after growth to confluence. Harvested cells were centrifuged for 5 min at 2,000 g and washed two times with PBS. The pellet was resuspended in EGM containing 10% FBS, and cell viability was assessed by the trypan blue exclusion method (>95% all times). The number of cells in suspension was determined with the use of a hemacytometer, and aliquots of 1 × 106 cells/ml (final concentration) were prepared. Cell suspensions were then incubated with endotoxin (LPS) and/or other agents in aerobic conditions for 30 min. The following groups were studied: EC and SMC without treatment (control); EC (or SMC) + LPS (1 µg/ml); EC (or SMC) + LPS + NAC (0.5 mM); and EC (or SMC) + NAC (0.5 mM). After the addition of 10 µl of palladium-porphyrin solution (0.15 mg/ml), cell suspensions were drawn into glass capillary tubes (1.6 × 100 mm), which were then sealed at both ends, avoiding the entrapment of any air bubbles. A small inert metal bar was placed inside the capillary tube to mix the suspension before PO2 measurements. PO2 was determined at intervals of 30 min, and cell suspensions were kept at room temperature in the dark, because palladium-porphyrin is a light-sensitive compound.

Determination of PO2 in cell suspensions. PO2 values were determined by the previously described (16, 17, 31) method of oxygen-dependent quenching of phosphorescence emitted by albumin-bound metalloporphyrin complexes after pulsed-light excitation. This technique has been used extensively for the noninvasive assessment of intravascular and tissue PO2 in vivo. PO2 measurements in vitro on various types of cells have also been reported (24, 25), but no data are available regarding the use of this technique for the evaluation of PO2, oxygen uptake, and utilization of vascular EC and SMC (see Advantages with use of phosphorescence method and validation of technique). The relationship between phosphorescence lifetime (tau ) and oxygen tension is given by the Stern-Volmer equation: tau 0/tau  = 1 + kq · tau 0 · PO2, where tau 0 and tau  are the phosphorescence lifetimes in the absence of molecular oxygen and at a given PO2, respectively, and kq is the quenching constant, with both factors being pH and temperature dependent. Palladium-porphyrin bound to serum albumin and diluted in saline (0.9% NaCl, Elkins-Sinn, Cherry Hill, NJ) to a final concentration of 0.15 mg/ml was used as a phosphorescent dye. An aliquot of this solution was added to each cell suspension before measurement of PO2. The final concentration of porphyrin in the capillary tubes was 1.5 µg/ml.

Phosphorescence was excited by light pulses (30 Hz) generated by a 45-W xenon strobe arc (EG&G ElectroOptics, Salem, MA) while PO2 measuring sites in the capillary tubes containing cells were microscopically vignetted by an adjustable slit in such a fashion that a segment of the capillary (0.5 mm of tube length) was fully illuminated by the pulsed-light excitation. The lifetime of the strobe arc was 4 µs, and the shape of the beam of pulsed light in the excitation region was a circle of ~150 µm in diameter. Two measurements in different areas of the glass capillary were taken at each time point, and the average of the values was used. Filters of 420 and 630 nm were used for porphyrin excitation and phosphorescence emission. Phosphorescence signals were captured by a photomultiplier (EMI, 9855B, Knott Elektronik, Munich, Germany). One hundred twenty-eight decay curves were averaged, visualized, and saved using a digital oscilloscope (Hitachi Oscilloscope V-1065, 100 MHz, Hitachi Denshi, Japan). Decay time constants were determined by computer, fitting the averaged decay curves to a single exponential using the Stern-Volmer equation and predetermined parameters tau 0 (600 µs) and kq (325 mmHg-1 · s-1) evaluated for room temperature (22°C) and pH 7.4. The pH of the suspending medium, measured with a blood gas analyzer at the end of the incubation period, did not significantly change from the initial pH value.

Oxygen consumption rates (VO2) were obtained by measuring the PO2 in sealed capillary tubes over time and finding the slope of the resulting linear plot. The following equation was used: VO2 = s · alpha , where s is the slope of the PO2 curve (in mmHg/s) and alpha  is the solubility of oxygen in water (1.59 nmol/mmHg at 22°C, 2.64 nmol/mmHg at 4°C). VO2 was expressed as nanomoles of oxygen per minute per 106 cells.

Advantages with use of phosphorescence method and validation of technique. The phosphorescence method uses oxygen throughout the medium in proportion to the absorbed excitation light, providing a more direct measurement of PO2 in the medium. In fact, the only oxygen gradients within the medium are those due to the respiration of the cells, because no additional gradients are created by the measuring technique within the volume being analyzed. Oxygen gradients occur at the boundary between the illuminated region defined by the vignetting slit and the remainder of the nonexcited fluid; however, these have a minimal effect on the measurement because the oxygen consumption by the porphyrin during the excitation period (90 flashes) lowered PO2 in the excited region by 0.4 mmHg. In contrast, an oxygen electrode creates a concentration gradient from the cell-containing medium to the oxygen-consuming electrode. This gradient causes a diffusion flux of oxygen that generates electric current, which is measured and related to the bulk PO2. As a consequence, the relationship between the measured current and the actual PO2 in the medium is complex and for exact measurements requires calibration of the electrode. This difference in the principle of operation has additional implications, because in the electrode method there is a diffusion boundary layer between the electrode surface and the medium that may be affected by stirring or motion of the medium. Perturbations in the diffusion boundary layer from conditions existing at the time of calibration affect the current that develops and therefore the PO2 values. In our implementation of the phosphorescence technique, the only diffusion boundary layer present is that between the oxygen-consuming cells and the bulk of the medium. These oxygen concentration gradients can be calculated for a single cell and, for the measured rates of decay of PO2 in our cell suspensions, are in the order of 0.2 mmHg between the cell surface and the bulk of the medium.

A series of control experiments was carried out to validate the use of phosphorescence decay for the assessment of PO2 in vitro. Initial studies with albumin-bound palladium-porphyrin revealed that, in the absence of cells, PO2 decreased significantly over time (PO2 = 65 mmHg at 2-h time point) in capillary tubes containing the phosphorescence dye alone at concentrations >0.15 mg/ml (measurements taken every 5 min for 2 h). This indicates that a direct interaction between palladium-porphyrin and molecular oxygen occurs when the dye is repeatedly excited by light and that photoactivated oxygen consumption takes place at relatively high concentrations of porphyrin. This effect was virtually eliminated by using a lower concentration of albumin-bound palladium-porphyrin (1.5 µg/ml), reducing the number of flashes to 90 per measurement (30 flashes/s for 3 s), and extending the interval between measurements to 30 min over a 5-h interval.

To ensure that the results obtained were not due to uncontrolled parameters, glass capillary tubes were loaded with dead cells and PO2 was determined over time. Nonviable cells were obtained from confluent EC growing in a 75-cm2 flask and previously incubated in PBS for 96 h under hypoxic conditions (95% N2-5% CO2). Floating cells were collected at the end of the incubation period, and cell viability was assessed by trypan blue exclusion before cells were transferred to the glass capillary.

An additional experiment was performed by maintaining capillary tubes containing EC at 4°C, because a decreased temperature diminishes cellular metabolism and oxygen consumption. Cells were collected by trypsinization, resuspended in EGM, and placed in a refrigerator at 4°C for 30 min. After the addition of palladium-porphyrin, cells were rapidly transferred to a glass capillary tube and sealed at both ends, and PO2 was measured. Samples were immediately returned to 4°C until the following PO2 measurement. Control cells were also stimulated by the redox cycler menadione (final concentration 10 µM), which uncouples mitochondrial oxidative phosphorylation, thus increasing VO2 and resulting in a faster decrease in PO2 over time.

Finally, a series of experiments was conducted to evaluate changes in VO2 in relation to cell density. Aortic EC and vascular SMC were suspended in glass capillaries at various concentrations (3 × 105-4 × 106 cells/ml of culture medium), and PO2 was determined over time. In these experiments, cells were used at passage 5 and harvested from subculture after 4 days, because it has been reported that the cellular metabolism of cultures may change depending on age (6).

Data analysis. All values are expressed as means ± SD of four to five independent experiments; error bars are not visible in Figs. 1-6 when SD < 2%. Because data distribution conformed to a binomial normal distribution, ANOVA and Student's t-test were used for statistical analysis. Differences between groups were considered to be significant at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The time course of PO2 of aortic EC in suspension incubated under various conditions is shown in Fig. 1. Capillary tubes loaded with EC suspended in culture medium at room temperature (22°C) and sealed at both ends showed a steady (linear slope) decrease in PO2 over time. VO2 in this group was 1.00 ± 0.15 nmol · min-1 · 106 cells-1, and the time at which PO2 is 50% of its initial value (t50) was 1.71 h (Table 1). The drop in PO2 was considerably attenuated when cells were stored at 4°C, as indicated by a significant shift of the linear plot to the right; VO2 decreased to 0.61 ± 0.32 nmol · min-1 · 106 cells-1, and t50 increased to 4.47 h (P < 0.05). Only minor changes in PO2 over time were detected in glass capillaries containing dead EC obtained by prolonged incubation in a hypoxic environment (cell viability <5%, VO2 = 0.01 ± 0.0 nmol · min-1 · 106 cells-1, t50 = 62.5 h). Incubation of EC with menadione, a redox cycler that uncouples oxidative phosphorylation, increased VO2 to 1.41 ± 0.05 nmol · min-1 · 106 cells-1 and decreased t50 to 0.93 h (P < 0.05; Table 1).


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  		Fig. 1.   Time dependence of PO2 in endothelial cell (EC) suspensions exposed to various agents and treatments. Cells (1 × 106 cells/ml) were transferred to glass capillary tubes, and PO2 was measured by a phosphorescence decay technique as reported in MATERIALS AND METHODS. Values at each time point are expressed as means ± SD of 4-5 independent experiments. MD, menadione (10 µM).

                              
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  		Table 1.   O2 consumption rate and t50 in cell suspensions treated with various agents

The effect of cell density on PO2 and VO2 by EC and SMC is shown in Figs. 2 and 3, respectively. VO2 decreased as cell number increased in both types of cells. VO2 of EC decreased from 1.12 ± 0.12 to 0.60 ± 0.02 nmol · min-1 · 106 cells-1 as cell density increased from 5 × 105 to 4 × 106 cells/ml. Similarly, increasing the concentration of SMC from 3 × 105 to 2.4 × 106 cells/ml caused VO2 to decrease from 4.22 ± 0.10 to 1.44 ± 0.09 nmol · min-1 · 106 cells-1.


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  		Fig. 2.   Time course of PO2 in EC suspensions at various cell densities (, 4 × 106; open circle , 2 × 106; bullet , 1 × 106; and , 5 × 105 cells/ml). EC were suspended in complete culture medium, sealed in glass capillary tubes, and kept at room temperature for determination of PO2 at different time points. Inset: correlation between endothelial oxygen consumption rate (VO2) and cell density. VO2 was calculated from slope of linear plot between PO2 and time as described in text. All values are means ± SD of 4-5 independent experiments.


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  		Fig. 3.   Time course of PO2 in vascular smooth muscle cell (SMC) suspensions at various cell densities (, 2.4 × 106; open circle , 1.2 × 106; bullet , 6 × 105; and , 3 × 105 cells/ml). Preparation of cell suspensions and PO2 determination at different time points were performed as described for experiments with EC. Inset: correlation between VO2 in suspended SMC and cell density. All values are means ± SD of 4-5 independent experiments.

As shown in Fig. 4, EC treated with LPS (1 µg/ml) had a significantly reduced VO2 (0.54 ± 0.29 vs. 1.00 ± 0.15 nmol · min-1 · 106 cells-1, P < 0.05), and t50 was 3.86 h. This effect was completely reversed by the presence of NAC (0.5 mM), a precursor of glutathione and scavenger of oxygen free radicals. Incubation of EC with LPS in the presence of NAC resulted in a VO2 of 1.07 ± 0.02 nmol · min-1 · 106 cells-1 and a t50 of 1.44 h (compared with 1.71 h in the control group). Incubation of EC with NAC showed a VO2 not significantly different from that of EC alone (data not shown).


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  		Fig. 4.   Effect of endotoxin [lipopolysaccharide (LPS)] on changes in PO2 of EC suspensions. EC (1 × 106 cells/ml) suspended in culture medium were incubated for 30 min in presence of 1 µg/ml LPS or LPS + 0.5 mM N-acetyl-L-cysteine (NAC) and subsequently transferred to glass capillary tubes for PO2 determination. Control group is represented by untreated EC. Values are means ± SD of 4 experiments.

Similar results were obtained from glass capillary tubes loaded with vascular SMC, although in this case we observed a faster decrease in PO2 compared with that in EC. At similar cell densities (106 cells/ml), SMC consumed more oxygen than EC (1.94 ± 0.09 vs. 1.00 ± 0.15 nmol · min-1 · 106 cells-1; Fig. 5), and treatment with LPS caused VO2 to decrease. VO2 of SMC exposed to LPS decreased from 2.64 ± 0.09 to 2.25 ± 0.14 nmol · min-1 · 106 cells-1 (P < 0.05, Fig. 6); however, LPS-treated SMC incubated in the presence of NAC showed a 15% increase in VO2 (3.11 ± 0.12 nmol · min-1 · 106 cells-1) relative to control (P < 0.05). Incubation of SMC with NAC showed a VO2 not significantly different from that of SMC alone (data not shown).


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  		Fig. 5.   Time dependence of PO2 in suspensions of EC and SMC. EC (1 × 106 cells) and SMC (1.2 × 106 cells/ml) were sealed in glass capillary tubes, and PO2 was determined at various time points. Inset: comparison between VO2 in EC and SMC suspended in glass capillaries at similar cell densities. Values are means ± SD of 4 experiments. * P < 0.01 vs. SMC.


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  		Fig. 6.   Effect of endotoxin (LPS) on changes in PO2 of SMC suspensions. SMC (5 × 105 cells/ml) suspended in culture medium were incubated for 30 min in presence of 1 µg/ml LPS or LPS + 0.5 mM NAC and subsequently transferred to glass capillary tubes for PO2 determination. Control group is represented by untreated SMC. Values are means ± SD of 4 experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of this in vitro study show that, under conditions of reduced oxygen availability, endotoxin impairs oxygen consumption of vascular cells suspended in culture medium. Endothelial and smooth muscle cells transferred and sealed in glass capillary tubes consumed the solubilized oxygen available in the suspending medium at a relatively fast rate, as indicated by a gradual and time-dependent decrease in PO2. Exposure to endotoxin produced a depression in oxygen consumption by both types of cells, and N-acetyl-L-cysteine, a substrate for glutathione synthesis with antioxidant properties, completely restored the cellular capability for oxygen utilization. These data suggest that decreased oxygen utilization in vascular tissues may be responsible for the alteration of vascular functions mediated by endotoxin and indicate the importance of endothelial and smooth muscle respiration in maintaining vascular homeostasis under conditions of sepsis.

The oxygen-dependent quenching of phosphorescence from the palladium-porphyrin probe was used to determine PO2 changes in cell suspensions. This extremely sensitive optical method has been developed for measuring oxygen dependence of oxidative phosphorylation in isolated rat liver mitochondria (32, 37) and for the noninvasive monitoring of PO2 in normal tissues (26, 31) and in tumors in vivo (30, 36). The present study uses the same principle to investigate the effect of endotoxin on oxygen uptake by cells isolated from vascular tissue. To our knowledge, this is the first attempt to use this optical method to determine changes in PO2 and oxygen consumption of viable vascular cells (endothelial and smooth muscle) exposed to various agents and treatments.

The phosphorescence decay method appears to be as reliable as other methods commonly used for in vitro PO2 determination (e.g., Clark electrode) because all measurements were consistent with the metabolic demand associated with the treatment to which cells were subjected. In a separate study, phosphorescence decay oxygen measurements were compared with simultaneous measurements made in tissue using recessed gold cathode microelectrodes with tip diameters of ~5 µm (unpublished observations). This comparison was made in avascular tissue areas of the hamster subcutaneous connective tissue, in which it was found that the optical phosphorescence and the electrode techniques agree with a maximum divergence of 3% between measurements over the tissue PO2 range of 5-40 mmHg.

Our results for endothelial cell suspensions at a 106 cell/ml concentration show that oxygen is consumed at the rate of 1.0 nmol · min-1 · 106 cells-1. This result is comparable to the rate of 1.45 nmol · min-1 · 106 cells-1 obtained by Kjellstrom et al. (18) using a microrespirometric technique. These values are lower than 87.5 nmol · min-1 · 106 cells-1, obtained by Bruttig and Joyner (6) with a manometric technique, and higher than 0.13 nmol · min-1 · 106 cells-1, found by James et al. (14) using electron paramagnetic resonance (EPR) spectroscopy, where the disparity in results may be due to differences in experimental conditions. It should be noted that cellular metabolism can substantially vary depending on the conditions of incubation; the presence or absence of serum, growth factors, and hormones; and the type of cell line being used.

Cell suspensions exposed to hypothermic conditions (4°C), which should diminish metabolic activity, resulted in a much slower decrease in PO2 over time and, therefore, a significantly reduced oxygen consumption rate. Reduction of cell viability (<5%) by prolonged exposure to hypoxia left PO2 virtually unchanged because nonviable cells utilized a very small amount of oxygen in the closed system. Stimulation of viable vascular cells with low concentrations (10 µM) of the redox cycler menadione uncoupled oxidative phosphorylation and cells consumed oxygen at a faster rate. Menadione, which can be toxic and mediates cellular injury at concentrations >100 µM, has been shown to cause inhibition of prostaglandin synthesis at sublethal doses (<20 µM) in cultured porcine endothelial cells (3). This effect was accompanied by a severalfold increase in oxygen consumption that was suggested to be mediated by hydrogen peroxide and semiquinone formation in mitochondrial membranes. Our results indicate that, by affecting cellular metabolism, oxygen consumption falls in a highly predictable fashion, and measurements of PO2 can be precisely taken when palladium-porphyrin is added at very low concentrations to a cell suspension.

Our data show that cellular oxygen utilization in vitro is inversely related to the density of both cell types, because oxygen consumption decreases as cell density increases. These findings are in agreement with a previous report that cellular oxygen consumption, determined manometrically in flasks containing cultured vascular cells, decreased as cell concentration increased (6). This phenomenon may be due to the conservation of substrate supply and regulation of metabolic demand and may represent a generalized characteristic of cells in culture. The phenomenon becomes, perhaps, more apparent when cells in suspension and homogeneously distributed in a closed system utilize the available oxygen in conditions of progressive hypoxia, as in the case of our experiments. Under these conditions, the phosphorescence decay method shows that, in the resting state, smooth muscle cells consume more oxygen than aortic endothelial cells. These results are in agreement with the findings of Bruttig and Joyner (6) indicating that oxygen consumption by both segments and cultured cells of arterial smooth muscle was significantly higher than in cells and tissues of endothelial origin.

In these experiments we show for the first time that endotoxin impairs oxygen utilization by cells derived from the vascular wall. Endotoxin, the LPS component of the outer membrane of Gram-negative organisms, has been reported to mediate vascular endothelial cell injury (22), and it has been implicated in the pathogenesis of bacterial sepsis and endotoxic shock (10, 29). Alteration of microvascular functions due to oxygen free radical generation and consequent damage to vascular cells can significantly contribute to decrease the oxygen extraction capabilities of tissues and limit oxygen availability to the cells. Although increased oxygen demand and reduction in tissue oxygen extraction have been demonstrated in whole animals and in various different organs after endotoxin treatment (5, 35), direct evidence for diminished endothelial oxygen consumption is as yet undocumented. Using an in vitro system, we demonstrated that LPS decreased oxygen utilization by aortic endothelial cells and, to a lesser extent, smooth muscle cells. Our findings are partially in agreement with a previous study in which EPR spectroscopy was used for the in vitro evaluation of cellular oxygen uptake in various cell types (14). In that report it was shown that LPS inhibits mitochondria-associated respiration of nonvascular cells in a dose- and time-dependent manner, but no effect on endothelial oxygen consumption rate was observed on treatment with LPS.

Preincubation of endotoxin with N-acetyl-L-cysteine in both endothelial and smooth muscle cell suspensions resulted in complete preservation of cellular oxygen utilization. N-acetyl-L-cysteine is a nontoxic drug that enters cells readily and can replenish intracellular glutathione, a tripeptide that serves as the major cellular defense against oxidative stress (4, 12). A protective effect of N-acetyl-L-cysteine can also be attributed to its intrinsic antioxidant properties, given that, as a sulfhydryl donor, it may contribute to scavenging endotoxin-mediated free radical formation (1). In septic conditions, administration of N-acetyl-L-cysteine has been shown to improve oxygen extraction capabilities as well as myocardial function (28, 38), effects that have been associated with the attenuation of cytokines and tumor necrosis factor toxicity (2).

Increasing evidence suggests that N-acetyl-L-cysteine affects microcirculatory blood flow and tissue oxygenation by inducing vasodilatation (28, 38). These in vivo effects of N-acetyl-L-cysteine appear to be related to its interaction with NO and the subsequent formation of the more stable S-nitrosothiol, whose properties closely resemble those of EDRF (21). The present finding showing that oxygen extraction is restored in vitro by N-acetyl-L-cysteine in LPS-treated cells is indicative of a local beneficial action of this drug in sustaining oxygen utilization by cellular components. Although the protective mechanism of N-acetyl-L-cysteine in vitro is unknown, it is possible that reduced thiol groups play a role in maintaining the integrity of mitochondria against oxidative stress, thus preserving their respiratory functions (9).

One of the mechanisms of endotoxin-mediated inhibition of respiratory function in vascular cells could be related to the stimulation of NO production from inducible NO synthase (iNOS). Smooth muscle and endothelial cells express iNOS when appropriately stimulated, and exposure to LPS can result in a higher NO generation (20). An in vitro study (27) indicated that skeletal muscle mitochondrial oxygen consumption is significantly inhibited by stimulators of endogenous NO production from the vascular endothelium and by exogenous NO released by the spontaneous decomposition of S-nitroso-N-acetylpenicillamine (SNAP) (27). Accordingly, we observed that exposure of smooth muscle cells to SNAP (0.5 mM) resulted in a slower decrease in PO2 over time, with consequent impairment of oxygen utilization (data not shown). Whether endogenous NO stimulation by LPS elicits a similar response is questionable because of the multifactorial effect of endotoxin on vascular functions. However, it should be noted that the time required for the stimulation of inducible proteins (iNOS) is 2-6 h, which does not account for the results found in our experimental conditions. Because a much slower decrease in PO2 had already occurred during the first 2 h of incubation with endotoxin, it is unlikely that depression in oxygen consumption by iNOS-derived NO is the major cause for the respiratory inhibition observed in vascular cell suspensions. Therefore, the protective effect of N-acetyl-L-cysteine would be attributed more to its scavenging and antioxidant properties rather than to direct binding of NO to the sulfhydryl groups of this drug.

In conclusion, using an in vitro phosphorescence decay technique, we have demonstrated that LPS mediates alteration of oxygen consumption in vascular endothelial and smooth muscle cells. Preservation of cellular oxygen consumption by N-acetyl-L-cysteine, an antioxidant drug experimentally used for the treatment of endotoxemic states, is consistent with the improved oxygen extraction capabilities of tissues observed in septic shock. These data emphasize the importance of vascular cell respiration during sepsis and indicate that, in conditions of limited oxygen supply, decreased utilization of oxygen can be attenuated by preserving vascular integrity and function.

    ACKNOWLEDGEMENTS

The authors are grateful to Darren Whittemore for valuable help in carrying out culture of endothelial and smooth muscle cells.

    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-48018 (R. M. Winslow) and a grant from the National Heart Research Fund, Leeds, United Kingdom (R. Motterlini).

Address for reprint requests: R. Motterlini, Vascular Biology Unit, Dept. of Surgical Research, Northwick Park Inst. for Medical Research, Watford Rd., Harrow, UK HA1 3UJ.

Received 2 December 1997; accepted in final form 12 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(3):H776-H782
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