Changes in functional and histological distributions of nitric oxide synthase caused by chronic hypoxia in rat small pulmonary arteries

Abstract

1. Chronic hypoxia (CH) increases lung tissue expression of all types of nitric oxide synthase (NOS) in the rat. However, it remains unknown whether CH-induced changes in functional and histological NOS distributions are correlated in rat small pulmonary arteries. 2. We measured the effects of NOS inhibitors on the internal diameters (ID) of muscular (MPA) and elastic (EPA) pulmonary arteries (100-700 micro m ID) using an X-ray television system on anaesthetized rats. We also conducted NOS immunohistochemical localization on the same vessels. 3. Nonselective NOS inhibitors induced ID reductions in almost all MPA of CH rats (mean reduction, 36+/-3%), as compared to approximately 60% of control rat MPA (mean, 10+/-2%). The inhibitors reduced the ID of almost all EPA with similar mean values (approximately 26%) in both CH and control rats. On the other hand, inducible NOS (iNOS)-selective inhibitors caused ID reductions in approximately 60% of CH rat MPA (mean, 15+/-3%), but did so in only approximately 20% of control rat MPA (mean, 2+/-2%). This inhibition caused only a small reduction (mean, approximately 4%) in both CH and control rat EPA. A neuronal NOS-selective inhibitor had no effect. 4. The percentage of endothelial NOS (eNOS)-positive vessels was approximately 96% in both MPA and EPA from CH rats, whereas it was 51 and 91% in control MPA and EPA, respectively. The percentage for iNOS was approximately 60% in both MPA and EPA from CH rats, but was only approximately 8% in both arteries from control rats. 5. The data indicate that in CH rats, both functional and histological upregulation of eNOS extensively occurs within MPA. iNOS protein increases sporadically among parallel-arranged branches in both MPA and EPA, but its vasodilatory effect is predominantly observed in MPA. Such NOS upregulation may serve to attenuate hypoxic vasoconstriction, which occurs primarily in MPA and inhibit the progress of pulmonary hypertension.

Figure 1

Typical angiograms of small pulmonary arteries obtained before (top panels) and after (bottom panels) nonselective NOS inhibitor (L-NMMA) injection in control (left panels) and chronically hypoxic (right panels) rats. Clear vasoconstrictions are chiefly observed in more distal side vessels ≲300 μm (arrowheads).

Figure 2

Mean values of NOS inhibitor-induced ID changes for muscular, transitional elastic, and classical elastic pulmonary arteries are shown in control (open column) and chronically hypoxic (solid column) rats. CH greatly enhanced ID reductions due to nonselective NOS inhibition (left) and iNOS-selective inhibition (right) primarily in muscular pulmonary arteries. *P<0.05, **P<0.01 vs control.

Figure 3

Relative frequency distributions of ID changes due to nonselective NOS inhibition (left) and iNOS-selective inhibition (right) are shown in control (broken line) and chronically hypoxic (solid line) rats. Dotted line indicates no ID change as defined in Methods section. Clear difference in distribution pattern between control and hypoxic rats is seen primarily in muscular pulmonary arteries (100–300 μm) in both types of NOS inhibition.

Figure 4

eNOS immunostaining of small muscular pulmonary arteries of control (a) and chronically hypoxic (b) rats. eNOS protein was detected in endothelium of hypoxic arteries (arrows), but not in control arteries. Scale bars=50 μm.

Figure 5

iNOS immunostaining of muscular pulmonary arteries of chronically hypoxic (a–c) and control (d) rats. (a) Low magnification view. iNOS-positive (arrow) and iNOS-negative (arrowhead) muscular pulmonary arteries are seen in the same section. (b, c) Higher magnification views of iNOS-positive and iNOS-negative arteries, respectively. (d) iNOS-negative muscular artery (arrowhead). Scale bars: (a) 500 μm, (b, c) 50 μm, (d) 100 μm.