Experiments On Endothelial Cell Cultures

The cell experiments were done with immortalized murine microvascular brain endothelial bEND.3 cells which have a good expression of endothelial nitric oxide synthase and good reproducibility from generation to generation. These cells were cultivated to confluence in 75 cm2 flasks at 37°C under a controlled atmosphere containing 5% CO2 and 20% O2. At confluence, the bottom of the flask was covered by a cellular monolayer consisting of ca 7.5 ± 0.5 x 106 endothelial cells. Confluence and cell count are important parameters for NO synthesis [49] and are always verified by optical inspection via a stereomicroscope. The NO production is monitored via spin trapping of the NO radicals with iron-dithiocarbamate complexes and quantifying the yield of paramagnetic NO—Fe2+-DETC adducts with electron paramagnetic resonance spectroscopy.

Anoxia was imposed by replacing the growth medium containing 2.5 mM DETC, 10% fetal calf serum, 2 mM L-glutamine, 10 IU/ml penicillin and 100 mg/L streptomycin by the same argon-bubbled medium, then subsequently adding 10 ^M ferrous sulfate and flushing the flask with argon before closing it with an airtight top. After 20 min of NO trapping at 37°C, enzymatic activity was terminated by placing the flask on ice. The cellular fraction containing the Fe-DETC complexes was harvested by ultracentrifugation and snap-frozen in liquid nitrogen until EPR assay. Intracellular nitrite concentrations were determined by the colorimetric Griess assay of the lysated cellular fraction. Imposition of anoxia did not affect the viability of the cells (cell death as determined by trypan blue staining was below 0.1% for normoxic as well as anoxic cultures).

Upon NO trapping for 20 min in the controlled atmosphere with 5% CO2 and 20% O2, the cellular fraction from ca 7.5 ± 0.5 x 106 endothelial cells had acquired a yield of 110 ± 8 pmol paramagnetic MNIC (NO—Fe2+-DETC) as detected by EPR. This basal yield was obtained without any stimulus of the NO production from the cells. A typical EPR spectrum (Fig. 4A) showed a clear triplet hyperfine structure centered at g = 2.035 characteristic for MNIC. As expected for biological samples [53], a small contribution from paramagnetic Cu2+-DETC complexes was superposed. The most intense hyperfine line of this copper complex is visible at g = 2.01 in Fig. 4. Preincubation with Nm-nitro-L-arginine (NLA) diminished the MNIC yield in a dose-dependent manner (Fig. 4B).

Fig. 4. EPR spectra at 77 K from cellular fractions of ca 7.5 ± 0.5 x 106 endothelial cells after 20 min NO trapping at 37°C with iron-dithiocarbamate complexes.The arrow indicates one of the following. Curve a: 110 pmol MNIC formed under a controlled atmosphere containing 5% CO2 and 20% O2. Curve b: ca 25 pmol MNIC formed under the controlled atmosphere in the presence of 5 ^M NOS inhibitor NLA. Curve c: 160 pmol MNIC formed under anoxia. Curve d: ca 33 pmol MNIC formed under anoxia in the presence of 5 ^M NOS inhibitor NLA.

Fig. 4. EPR spectra at 77 K from cellular fractions of ca 7.5 ± 0.5 x 106 endothelial cells after 20 min NO trapping at 37°C with iron-dithiocarbamate complexes.The arrow indicates one of the following. Curve a: 110 pmol MNIC formed under a controlled atmosphere containing 5% CO2 and 20% O2. Curve b: ca 25 pmol MNIC formed under the controlled atmosphere in the presence of 5 ^M NOS inhibitor NLA. Curve c: 160 pmol MNIC formed under anoxia. Curve d: ca 33 pmol MNIC formed under anoxia in the presence of 5 ^M NOS inhibitor NLA.

These basal unstimulated yield of 110 ± 8 pmol compares favorably with a total MNIC yield of 400 ± 30 pmol as obtained when the cellular NOS production was stimulated by administration of 5 ^M Ca-ionophore A23187.

Interestingly, when anoxia was applied by argon, MNIC yields increased significantly over basal. In the absence of inhibitors, the anoxic yields from ca 7.5 ± 0.5 x 106 endothelial cells were ca 160 ± 10 pmol MNIC, these values being typically some 50% higher than basal in the presence of oxygen (Fig 4C). In the presence of specific NOS inhibitors like Nœ-nitro-L-arginine (NLA) and Nœ-nitro-L-arginine-methylester (l-NAME), the MNIC yield under anoxia was also diminished in a dose-dependent manner. The intensity of the EPR absorption from paramagnetic Cu2+-DETC complexes was not affected by anoxia. The yields of the trapping experiments are compiled in Table 2.

Preincubation with 10 mM of the heme-binding inhibitor imidazole diminished both oxic and anoxic yields to 80± 10 pmol. In contrast, preincubation for 20 min with the molybdenum-binding xanthine oxidase inhibitor oxypurinol did not affect the anoxic MNIC yield.

The kinetics of the anoxic NO release from the cells showed a linear increase with time up to ca 30 min after induction of anoxia, when the signal intensity saturated at an asymptotic value of ca 200 ± 20 pmol MNIC (cf Fig. 5).

The main and most prominent result from these cell culture experiments is the clear and large increase of MNIC yield upon introduction of anoxia. The anoxic NO production had a surprising duration of 30 min and its magnitude exceeded that of the conventional enzymatic pathway for NO production from arginine. In view of our earlier in vitro experiments [40] we attribute the anoxic NO production to the reduction of intracellular nitrite by

Table 2 Yields of MNIC adducts (in pmole) in the cellular fractions of a single 75 cm2 flask containing ca 7.5 x 106 endothelial cells. Trapping proceeded for 20 min at 37°C. The second row gives the preincubation time tinc (min) of the supplements. During preincubation the cells were kept at 37°C in an atmosphere with 5% CO2 and 20% O2. (With permission from Ref. [54])

Table 2 Yields of MNIC adducts (in pmole) in the cellular fractions of a single 75 cm2 flask containing ca 7.5 x 106 endothelial cells. Trapping proceeded for 20 min at 37°C. The second row gives the preincubation time tinc (min) of the supplements. During preincubation the cells were kept at 37°C in an atmosphere with 5% CO2 and 20% O2. (With permission from Ref. [54])

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