Neuronal nitric oxide synthase (NOS) regulates leukocyte-endothelial cell interactions in endothelial NOS deficient mice

Abstract

1. The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte - endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS(-/-) mice. 2. Baseline leukocyte rolling and adhesion revealed no differences between wild-type and eNOS(-/-) mice in either the cremasteric or intestinal microcirculations. 3. Superfusion with L-NAME (100 microM) caused a progressive and significant increase in leukocyte adhesion in both wild-type and eNOS(-/-) mice, without detecting differences between the two strains of mice. 4. Superfusion with 7-nitroindazole (100 microM), a selective inhibitor of nNOS, had no effect on leukocyte adhesion in wild-type animals. However, it increased leukocyte adhesion significantly in eNOS(-/-) mice, which was reversed by systemic L-arginine pre-administration. 5. Stimulation of the microvasculature with H(2)O(2) (100 microM) induced a transient elevation in leukocyte rolling in wild-type mice. Conversely, the effect persisted during the entire 60 min of experimental protocol in eNOS(-/-) mice either with or without 7-nitroindazole. 6. Semi-quantitative analysis by RT - PCR of the mRNA for nNOS levels in eNOS(-/-) and wild-type animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS(-/-) mice. 7. In conclusion, we have demonstrated that leukocyte-endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS(-/-) mice. Interestingly, unlike eNOS there was insufficient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.

Figure 1

Baseline leukocyte rolling flux (A) and leukocyte adhesion (B) in the mouse cremasteric postcapillary venules in wild-type and eNOS^−/− mice. The cremaster muscle was superfused with bicarbonate-buffered saline. Parameters were measured at 0, 30 and 60 min during buffer superfusion in wild-type (n=6) and eNOS^−/− animals (n=10). Results are presented as mean±s.e.mean. ^*P<0.05 or ^**P<0.01 relative to the respective control value (0 min).

Figure 2

Baseline leukocyte rolling flux (A) and leukocyte adhesion (B) in the venules of the mouse small intestine submucosa in wild-type and eNOS^−/− mice. After i.v. injection of FITC-dextran and rhodamine-6G, the small intestine submucosa was examined using epifluorescence microscopy. Parameters were measured at 0, 30 and 60 min in wild-type and eNOS^−/− mice (n=5 per group). Results are presented as mean±s.e.mean. ^*P<0.05 or ^**P<0.01 relative to the respective control value (0 min).

Figure 3

Effect of L-NAME on leukocyte rolling flux (A) and leukocyte adhesion (B) in the mouse cremasteric postcapillary venules in wild-type and eNOS^−/− mice. The cremaster muscle was superfused with bicarbonate-buffered saline. After the 30 min stabilization period, the baseline parameters (0 min) were determined. Then superfusion buffer was supplemented with L-NAME (100 μM). Parameters were measured 0, 15, 30 and 60 min after superfusion with L-NAME in wild-type and eNOS^−/− animals (n=5 in both groups). Results are presented as mean±s.e.mean. ^*P<0.05 or ^**P<0.01 relative to the respective control value (0 min).

Figure 4

Effect of 7-nitroindazole on leukocyte rolling flux (A) and leukocyte adhesion (B) in the mouse cremasteric postcapillary venules in wild-type and eNOS^−/− mice. Parameters were determined at 0, 15, 30 and 60 min after 7-nitroindazole (100 μM) superfusion in both wild-type and eNOS^−/− mice (n=4 in both groups). In one group of eNOS^−/− animals, L-arginine (1 mg kg⁻¹, i.v.) was administered 15 min prior 7-nitroindazole superfusion (n=4). Another group of eNOS^−/− mice was superfused with buffer for the same period of time as 7-nitroindazole (n=4). Results are presented as mean±s.e.mean. ^*P<0.05 or ^**P<0.01 relative to the respective control value (0 min). #P<0.01 relative to values detected in wild-type animals.

Figure 5

Effect of H₂O₂ on leukocyte rolling flux in the mouse cremasteric postcapillary venules in wild-type and eNOS^−/− mice. Representative example of alterations in leukocyte rolling flux after H₂O₂ superfusion in a wild-type animal (A). Representative example of alterations in leukocyte rolling flux after H₂O₂ superfusion in eNOS^−/− animal with or without 7-nitroindazole (B). After the 30 min stabilization period, the baseline values were determined (0 min). Parameters were measured 0, 15, 30, 45 and 60 min after superfusion with H₂O₂ (100 μM) in both wild-type and eNOS^−/− mice and H₂O₂ and 7-nitroindazole (100 μM) in eNOS^−/− mice (n=6 in all groups) (C). Results represents mean±s.e.mean of the numbers obtained after subtracting respective basal values. ^*P<0.05 relative wild-type animal value at each time point.

Figure 6

RT – PCR of nNOS and β Actin in brain and skeletal muscle tissue of wild-type animals and eNOS^−/− mice. The ratio nNOS/β Actin was 1.545 in wild-type mice and 3.154 in eNOS^−/− mice in brain tissue and 0.143 in wild-type mice and 0.621 eNOS^−/− mice in the skeletal muscle tissue. Results presented are representative of n=4 experiments.