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Am J Physiol Heart Circ Physiol 292: H2737-H2744, 2007. First published January 12, 2007; doi:10.1152/ajpheart.00548.2006
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Impaired NO-mediated vasodilation with increased superoxide but robust EDHF function in right ventricular arterial microvessels of pulmonary hypertensive rats

Masahito Kajiya,1,2 Masanori Hirota,1 Yousuke Inai,1 Takahiko Kiyooka,1 Taro Morimoto,1 Tatsuo Iwasaki,1 Kousuke Endo,3 Satoshi Mohri,1 Juichiro Shimizu,1 Toyotaka Yada,4 Yasuo Ogasawara,4 Keiji Naruse,1 Tohru Ohe,2 and Fumihiko Kajiya1,4

Departments of 1Cardiovascular Physiology and 2Cardiovascular Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; and Departments of 3Physiology and 4Medical Engineering and Systems Cardiology, Kawasaki Medical School, Kurashiki, Japan

Submitted 29 May 2006 ; accepted in final form 5 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary hypertension (PH) causes right ventricular (RV) hypertrophy and, according to the extent of pressure overload, eventual heart failure. We tested the hypothesis that the mechanical stress in PH-RV impairs the vasoreactivity of the RV coronary microvessels of different sizes with increased superoxide levels. Five-week-old male Sprague-Dawley rats were injected with monocrotaline (n = 126) to induce PH or with saline as controls (n = 114). After 3 wk, coronary arterioles (diameter = 30–100 µm) and small arteries (diameter = 100–200 µm) in the RV were visualized using intravital videomicroscopy. We evaluated ACh-induced vasodilation alone, in the presence of N{omega}-nitro-L-arginine methyl ester (L-NAME), in the presence of tetraethylammonium (TEA) or catalase with or without L-NAME, and in the presence of SOD. The degree of suppression in vasodilation by L-NAME and TEA was used as indexes of the contributions of endothelial nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), respectively. In PH rats, ACh-induced vasodilation was significantly attenuated in both arterioles and small aretries, especially in arterioles. This decreased vasodilation was largely attributable to reduced NO-mediated vasoreactivity, whereas the EDHF-mediated vasodilation was relatively robust. The suppressive effect on arteriolar vasodilation by catalase was similar to TEA in both groups. Superoxide, as measured by lucigenin chemiluminescence, was significantly elevated in the RV tissues in PH. SOD significantly ameliorated the impairment of ACh-induced vasodilation in PH. Robust EDHF function will play a protective role in preserving coronary microvascular homeostasis in the event of NO dysfunction with increased superoxide levels.

acetylcholine; N{omega}-nitro-L-arginine methyl ester; tetraethylanmonium; catalase; superoxide dimutase; endothelium-derived hyperpolarizing factor

PULMONARY HYPERTENSION (PH) causes pressure overload and hypertrophy, finally leading to failure, in the right ventricle (RV) of the heart. Although the right coronary perfusion pressure remains relatively unchanged, the high pressure and hypertrophy of the RV affect the coronary hemodynamics (8, 13), and patients with PH and RV hypertrophy might have symptoms indistinguishable from those of angina pectoris (4, 30). In chronic PH, coronary flow regulation is the only mechanism that maintains the oxygen supply to the RV, since no further increase in the absorption of oxygen is possible (31). The endothelium-dependent vasodilation of coronary small arteries (SAs, diameter = 100–200 µm) isolated from the RV has been shown to be lower in rats with chronic PH (33), whereas in acute PH, endogenous nitric oxide (NO) regulates right coronary blood flow (37). However, the contributions of NO and endothelium-derived hyperpolarizing factor (EDHF) to the vasodilation of arterial microvessels of different sizes, i.e., SAs (diameter = 100–200 µm) and arterioles (diameter < 100 µm), in the RV in chronic PH have not been clarified. Moreover, the coronary microvascular tone in hypertrophied RV is greatly affected by the surrounding myocardial and interstitial tissues. Therefore, using direct in vivo visualization is desirable when evaluating RV coronary microvascular vasoreactivity in PH. An increase in the oxidative stress in the RV myocardium has been suggested to be a key factor in RV dysfunction in PH (1, 2, 29).

We hypothesized that the vasodilation of RV coronary arterial microvessels of different sizes, induced by NO and EDHF in PH, might be differently affected under conditions of increased superoxide. To test this hypothesis, we observed arterial microvessels (diameter = 30–200 µm) in the RV in vivo in control and monocrotaline (MCT)-treated PH rats using intravital videomicroscopy and evaluated the endothelium-dependent vascular responses to ACh in the absence of prostacyclin under following conditions: ACh alone, in the presence of an NO or an EDHF inhibitor, in the presence of both NO and EDHF inhibitors, and in the presence of superoxide dismutase (SOD). Endothelium-independent vasodilation induced by sodium nitroprusside (SNP) was also examined.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Five-week-old male Sprague-Dawley rats (130–160 g, Japan SLC) were injected subcutaneously with 60 mg/kg MCT to induce PH (n = 126) or with saline (n = 114) as controls.

This study conformed to the Guidelines on Animal Experiments of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan, and to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH), and protocols were approved by the Animal Care and Use Committee of Okayama University, Japan.

Surgical procedures. Three weeks after treatment with MCT or saline, the rats in both groups were anesthetized with pentobarbital sodium (50 mg/kg ip). After tracheotomy, the rats were mechanically ventilated with air supplemented by 90% oxygen, at a rate sufficient to maintain physiological parameters within the normal range (pH = 7.35–7.45; PCO2 = 25–40 mmHg; and PO2 > 80 mmHg). The right internal carotid artery was cannulated and used for continuous monitoring of arterial pressure, whereas the left femoral vein was cannulated and used for the administration of drugs. To monitor pressure in the RV, a fluid-filled catheter was introduced through the right atrium. ECGs were recorded using standard limb leads. The heart was exposed by a median sternotomy, and the descending aorta was encircled with an elastic suture so that the coronary perfusion pressure could be adjusted by aortic snaring. When the physiological parameters of the rat deviated from the normal range, the experiment was discontinued.

Intravital videomicroscopy. To visualize the subepicardial arterial microvessels in the RV, we used our own intravital needle-probe videomicroscope. The spatial resolution of this system in moving images is 2.5 µm at a x200 magnification, with a maximum depth of field of about 250 µm, as previously described (35). In brief, our videomicroscope system (VMS-70A; Nihon Kohden, Tokyo, Japan) consists of a needle probe, a camera body containing a charge-coupled device camera, a lens with a light guide, a camera controller, a light source, a monitor, and a digital videocassette recorder. The images on the charge-coupled device were converted into color video signals at 30 frames/s and recorded onto digital videotape.

Stabilization of the visual field for microscopy. To stabilize the visual field for observation of the subepicardial microvessels while the RV was beating, we supported the rat heart with a strip of soft elastic bandage, anchored at its four edges, to maintain a constant in situ position (9). The supporting bandage also acted as a pressure absorber to minimize compression by the probe. The tip of the probe was gently placed on the surface of the free wall of the RV where it had minimal hemodynamic effects.

Observation of RV coronary arterial microvessels. To identify the subepicardial arterial microvessels (diameter = 30–200 µm) in the RV, indocyanine green was introduced by retrograde injection through the catheter in the ascending aorta, and the microvessels were observed with the needle probe. After dye injection, arterial microvessels immediately turned green, whereas venous microvessels only became green after a considerable delay. The time sequence of vascular images was transferred to a Macintosh computer (Power Mac G4; Apple Computer; Cupertino, CA) with simultaneous recordings of arterial blood pressure (BP), RV pressure, and ECG. After conversion into grayscale images, the internal diameters of the microvessels at the end of diastole were measured using software with a freeze-frame modality (NIH image 1.63; NIH, Bethesda, MD). The edges of the microvessels were determined automatically or with a manual border-tracing procedure using a density profile (35).

Measuring vasodilation and the vasodilatory contribution of NO and EDHF. To evaluate the contribution of NO and EDHF alone, all experiments were performed in rats treated with indomethacin (10 mg/kg iv) to block cyclooxygenase, based on an earlier study (26). This had little effect on the magnitude of vasodilation in our pilot study. All drugs were obtained from Sigma Chemical.

First, the endothelium-dependent vasodilation induced by ACh (2 µg·kg–1·min–1 iv) was examined under three conditions: ACh alone, in the presence of the NO inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) (36), and in the presence of L-NAME plus tetraethylammonium (TEA; 200 µg·kg–1·min–1 iv), which inhibits vascular calcium-activated potassium (KCa) channels. More than one EDHF candidate exists, i.e., epoxyeicosatrienoic acids, potassium, gap junctions, and hydrogen peroxide, and the contribution of each of them to endothelium-dependent vasodilation might vary depending on the species tested and the vessels used. However, in general, the hyperpolarizing mechanism of EDHF is considered to be mediated by KCa channels on vascular smooth muscle. Whereas TEA at high doses blocks a number of potassium channels, TEA at low doses is fairly specific for the KCa channels (20).

The difference between the percent vasodilation induced by ACh alone and in the presence of L-NAME was used as the relative index of the contribution of NO. Similarly, the difference between the percent vasodilation induced in the presence of L-NAME and in the presence of L-NAME + TEA was used as an index of the contribution of EDHF.

We also evaluated the effect of TEA alone on ACh-induced vasodilation in arterioles in both control and PH rats to clarify the effect of the EDHF inhibitor alone. In addition, we evaluated the possible inhibition of ACh-induced vasodilation in arterioles by catalase, since hydrogen peroxide is a well-recognized candidate for EDHF (24, 36). The vasodilation was evaluated in the presence of catalase (15,000 U·kg–1·min–1 iv) and in the presence of L-NAME + catalase.

To evaluate the possible effect of increased superoxide production of the RV on endothelium-dependent vasodilation in PH, ACh-induced vasodilation was also evaluated after SOD injection (25,000 U/kg iv).

In addition, to evaluate endothelium-independent vasodilation, SNP (1 µg·kg–1·min–1 iv) was continuously administered. In these experiments, we administered ACh and SNP by intravenous infusion, since intracoronary administration is technically difficult in in vivo rat models. Systemic infusion of ACh (2 µg·kg–1·min–1 iv) and SNP (1 µg·kg–1·min–1 iv) in rats has been reported to increase coronary blood flow by 46% and 42%, respectively, without affecting the heart rate (HR) (5). In our pilot study, there was no significant difference in ACh-induced vasodilation using doses of 2 and 4 µg·kg–1·min–1. However, the lower dose did not affect the hemodynamics, whereas the higher dose significantly reduced both HR and BP. There was no significant difference in SNP-induced vasodilation at doses of 1 and 2 µg·kg–1·min–1. We therefore decided to use 2 µg·kg–1·min–1 ACh and 1 µg·kg–1·min–1 SNP. The dose of L-NAME needed to completely inhibit NO, and the dose of TEA needed to completely inhibit EDHF were also determined in our pilot study, i.e., we tested 5, 10, and 20 mg/kg L-NAME and 100, 200, and 600 µg·kg–1·min–1 TEA.

Evaluation of RV hypertrophy. At the end of the experiments, hypertrophy of the RV was assessed in two ways: from the heart-to-body-weight ratio and from the ratio of the weight of the RV free wall to the weight of the left ventricle (LV) free wall plus the interventricular septum.

Measurement of superoxide production in RV. Superoxide production in RV tissues from control rats (n = 7), control rats after SOD treatment (25,000 U/kg iv; n = 7), rats with PH (n = 7), and rats with PH after SOD treatment (n = 7) was determined by lucigenin-derived chemiluminescence (LDCL), as described previously, with some modifications (3, 15, 34). Samples (5–10 mg) of the RV free wall were cut, immediately minced, and incubated in 5 ml of air-equilibrated Krebs-Henseleit buffer solution (pH 7.4) for 5 min at 37°C. The samples were placed in tubes containing 5 µM lucigenin in a final volume of 2 ml Krebs-Henseleit buffer, and the superoxide-induced LDCL of the samples was measured with a luminescence reader (Lumicounter 2500, Microtec, Chiba, Japan) for 5 min. Superoxide levels were reported as relative light units after subtracting background luminescence and were normalized to dry tissue weight (in mg).

Histology. Coronary arterial microvessels (30–200 µm in diameter) in the RV of PH and control rats were stained with hematoxylin and eosin and examined by light microscopy.

Statistical analysis. All results are expressed as means ± SE. The relationship between the percent dilation and the vascular diameter of the microvessels was evaluated by correlation analysis. The percent dilation for microvessels of different diameters was analyzed by one-way ANOVA, followed by Scheffé's post hoc test. The Student's t -test was used for both paired and unpaired comparisons. The criterion for statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
RV pressure overload and hypertrophy in MCT-treated rats. Comparing the physiological profiles of MCT-treated and control rats (Table 1) showed a great increase in the systolic pressure in the RV of MCT-treated rats, whereas there was no significant difference in the mean BP in the two groups. The heart-to-body-weight ratios and the weight ratios of the RV free wall to the LV free wall plus interventricular septum were significantly larger in MCT-treated rats than in control animals (P < 0.001), demonstrating the successful induction of PH.


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  		Table 1. Physiological profiles of the two experimental groups

 
Hemodynamics. As summarized in Table 2, BP at baseline significantly increased in the presence of L-NAME and L-NAME + TEA, compared with ACh alone, in both groups (P < 0.01). In contrast, there was no significant difference in the BP of rats treated with L-NAME and L-NAME + TEA. In all groups, the HR and BP were constant and comparable before and after the administration of the vasodilators. TEA alone and catalase alone did not affect the hemodynamics, whereas L-NAME + catalase significantly increased BP at baseline (P < 0.01; data not shown).


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  		Table 2. Hemodynamics

 
In vivo RV coronary microvascular images. Figure 1 shows representative color images of the subepicardial arterial and venous microvessels in the RV of a control rat, which were obtained by intravital needle-probe videomicroscopy. The coronary venous microvessels lay in the superficial layer of the myocardium in the RV, and the coronary arterial microvessels were embedded at a deeper level. However, we were able to visualize both types of microvessel, since the focal depth of our videomicroscope was 250 µm. There were no apparent morphological differences in the images of the coronary arterial microvessels (diameter = 30–200 µm) in the control and MCT-treated rats.


Figure 1
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  		Fig. 1. Color images of the coronary microvasculature in a control rat. A: venous microvessels were located in the superficial layer of the myocardium, and arterial microvessels were embedded at a deeper level. B: arterial microvessels were the first vessels stained after retrograde injection of indocyanine green into the coronary artery from the ascending aorta. Scale bars represent 100 µm.

 
Endothelium-dependent vasodilation by ACh in arterial microvessels and the vasodilatory contribution of NO and EDHF. The scatter plots in Fig. 2 show the relationship between the diameter of microvessels at baseline and the degree of vasodilation induced by ACh. In control animals, the degree of vasodilation was inversely correlated with vessel size. L-NAME depressed vasodilation by a similar percentage (parallel downward shift) in microvessels of all sizes. The addition of TEA further reduced, or even abolished, vasodilation. In rats with PH, ACh-induced vasodilation markedly reduced, and the effect of L-NAME was also much lower compared with that in the controls. However, the addition of TEA dramatically reduced vasodilation, as in the control animals.


Figure 2
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  		Fig. 2. ACh-induced vasodilation in microvessels of different diameters in control rats and those with pulmonary hypertension (PH). A: in control rats, the degree of vasodilation was inversely associated with the vessel diameter. N{omega}-nitro-L-arginine methyl ester (L-NAME) depressed vasodilation to a similar extent in microvessels of all diameters, and, additionally, tetraethylammonium (TEA) further reduced or abolished the residual vasodilation. B: in PH, ACh-induced vasodilation was much lower than in the controls. The effect of L-NAME was almost negligible, whereas TEA had a significant effect. The numbers of vessels were 32 in 22 rats for ACh alone in control, 32 in 21 rats for L-NAME in control, 27 in 25 rats for L-NAME + TEA in control, 32 in 27 rats for ACh alone in PH, 33 in 30 rats for L-NAME in PH, and 18 in 15 rats for L-NAME + TEA in PH. NS, not significant.

 
To clarify the effects of NO and EDHF in vessels of different sizes in the control and MCT-treated rats, the vessels were divided into two categories: arterioles (diameter < 100 µm) and SAs (diameter = 100–200 µm). Figure 3 shows the magnitude of ACh-induced vasodilation for these two groups. In rats with PH, the magnitude of ACh-induced vasodilation was significantly lower than that in the controls for both arterioles (5.2 ± 1.5% vs. 10.8 ± 3.5% in controls; P < 0.001) and SAs (3.8 ± 1.0% vs. 6.0 ± 1.9% in controls; P < 0.001). In both arterioles and SAs, ACh-induced vasodilation was significantly suppressed by L-NAME (P < 0.05) and was further reduced by the addition of TEA (P < 0.001). However, the extent of suppression varied for vessels of different sizes and with different inhibitors. Inhibition of vasodilation by L-NAME in PH was markedly attenuated in both arterioles (1.0% vs. 4.0% in controls) and SAs (1.0% vs. 3.6% in controls), indicating that the contribution of NO to vasodilation is much lower in PH in both arterioles and SAs. The inhibition of vasodilation by the addition of TEA in PH was lower than in control rats in arterioles (3.1% vs. 5.4% in controls), whereas the percent inhibitions were similar in SAs (2.0% in PH vs. 1.8% in controls). Notably, the relative contribution of EDHF to vasodilation in PH increased in both arterioles (59% vs. 50% in controls) and SAs (54% vs. 29% in controls), indicating the robust vasodilatory properties of EDHF in PH.


Figure 3
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  		Fig. 3. Comparison of ACh-induced vasodilation in arterioles (<100 µm diameter; A) and small arteries (SAs, diameter = 100–200 µm; B) in control rats and rats with PH. In PH rats, ACh-induced vasodilation was significantly lower than in the controls in both arterioles and SAs (P < 0.001 for both). ACh-induced vasodilation of arterioles and SAs was significantly suppressed by L-NAME and was further reduced by the addition of TEA in both control and PH rats. However, in rats with PH, the effect of L-NAME was markedly attenuated in both arterioles and SAs compared with its effect in the controls. The effect of TEA on the suppression of vasodilation was reduced in PH compared with the control rats for arterioles, but the effects were similar for the SAs.

 
Figure 4A shows the effect of TEA alone and catalase alone on ACh-induced vasodilation in arterioles. TEA suppressed the vasodilation significantly in both controls and PH (by 5.3% and 3.0%, respectively; both P < 0.001). Catalase alone also decreased the vasodilation by 5.0% in controls and 3.2% in PH (both P < 0.001), comparable with the results of TEA. Figure 4B shows the comparison of the suppression in vasodilation by TEA and catalase in the presence of L-NAME. Catalase attenuated the vasodilation by 5.2% in controls and by 3.1% in PH (both P < 0.001). The degree of suppression by catalase was similar to that by TEA in the presence of L-NAME. It is noticeable that the effects of both agents were almost the same irrespective of the absence or presence of L-NAME.


Figure 4
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  		Fig. 4. Individual effects of TEA and catalase on ACh-induced vasodilation of arterioles in the absence (A) and presence (B) of L-NAME. Both TEA and catalase alone almost equally suppressed ACh-induced vasodilation in both control and PH rats (P < 0.001). In the presence of L-NAME, the two agents also attenuated the vasodilation in both groups (P < 0.001). The degree of suppression by both agents was almost similar to each other in both control and PH rats. The numbers of vessels were 16 in 12 rats for ACh alone in control, 17 in 14 rats for ACh alone in PH, 8 in 5 rats for TEA in control, 8 in 6 rats for TEA in PH, 8 in 5 rats for catalase in control, 8 in 5 rats for catalase in PH, 16 in 12 rats for L-NAME in control, 18 in 16 rats for L-NAME in PH, 16 in 15 rats for L-NAME + TEA in control, 10 in 9 rats for L-NAME + TEA in PH, 8 in 5 rats for L-NAME + catalase in control, and 8 in 5 rats for L-NAME + catalase in PH.

 
Endothelium-dependent vasodilation following SOD administration. Figure 5 shows the effect of SOD on the magnitude of ACh-induced vasodilation in arterioles and SAs. In control rats, SOD had no effect on ACh-induced vasodilation in either arterioles or SAs. In rats with PH, SOD significantly ameliorated the impaired vasodilation induced by ACh, improving it by 45% (P < 0.001) in arterioles and by 38% (P < 0.01) in SAs. The degree of vasodilation after SOD was almost compatible with controls in SAs (not significant between control after SOD and PH after SOD), but the amelioration was still incomplete in arterioles (P < 0.01 between the two).


Figure 5
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  		Fig. 5. ACh-induced vasodilation after SOD administration. In both arterioles (A) and SAs (B), SOD had no effect on ACh-induced vasodilation in control rats but significantly ameliorated the decrease in ACh-induced vasodilation in rats with PH. The numbers of vessels were 32 in 22 rats for control, 16 in 14 rats for control after SOD, 32 in 27 rats for PH, and 26 in 23 rats for PH after SOD.

 
Superoxide production in RV. Figure 6 shows the results of directly measuring superoxide production by LDCL in RV tissue in the presence and absence of SOD. Superoxide production was significantly higher in tissue from rats with PH compared with control rats (290 ± 52 vs. 158 ± 25 counts·min–1·mg–1 in controls; P < 0.001). Administering SOD significantly attenuated the superoxide production, reducing it by 28% (P < 0.01) in control rats and by 49% (P < 0.001) in rats with PH. Superoxide levels in PH rats treated with SOD were comparable with the normal levels seen in control rats.


Figure 6
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  		Fig. 6. Superoxide production in the right ventricle measured by lucigenin-derived chemiluminescence. Superoxide production was significantly higher in rats with PH compared with controls. SOD treatment significantly attenuated superoxide production in both groups, with a greater reduction in rats with PH, resulting in superoxide levels almost comparable with those in the control rats. cpm, Counts/min.

 
Endothelium-independent vasodilation by SNP in arterial microvessels. The scatter plot in Fig. 7A shows the relationship between the diameter of microvessels and the percent dilation induced by SNP in both control and PH rats. In the control animals, the vasodilation caused by SNP was larger in smaller vessels and decreased linearly with the vessel size. In PH rats, the vasodilation induced by SNP was lower, and, interestingly, this effect was most noticeable in smaller arterioles.


Figure 7
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  		Fig. 7. Comparison of sodium nitroprusside (SNP)-induced vasodilation in controls and rats with PH. A: scatter plot showing the association of vessel diameter and SNP-induced vasodilation. In control rats, SNP-induced vasodilation was inversely correlated with vessel diameter. B: comparison of vasodilation induced by SNP in arterioles (<100 µm) and SAs (100–200 µm). Vasodilation in rats with PH was significantly reduced in arterioles (**P < 0.01) compared with that in controls but was preserved in SAs. The numbers of vessels were 20 in 17 rats for control and 20 in 15 rats for PH.

 
When the effects of SNP on arterioles and SAs were analyzed separately (Fig. 7B), the vasodilation seen in PH was significantly lower for arterioles (5.3 ± 1.3% vs. 7.3 ± 1.9% in controls; P < 0.01) but was not significantly different for SAs (4.2 ± 1.2% vs. 4.6 ± 0.8% in controls).

Histology. Histologically, the RV coronary arterial microvessels in rats with PH were similar to those in the controls, although myocardial cell hypertrophy was clearly seen in the RV samples from PH rats. Similarly, intravital microscopy showed no morphological differences in the RV coronary arterial microvessels in rats with PH.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings from this study were as follows. First, RV coronary microvascular endothelium-dependent vasodilation in response to ACh was impaired in rats with PH in both arterioles and SAs. Second, NO-mediated vasodilation in these rats was markedly decreased in both arterioles and SAs, whereas the vasodilatory effect of EDHF was relatively robust. Third, SOD ameliorated the impaired endothelium-dependent vasodilation in PH, indicating a role for increased superoxide. Fourth, SNP-induced endothelium-independent vasodilation in PH was impaired in smaller arterioles but preserved in SAs. To the best of our knowledge, these are the first direct in vivo observations of the RV coronary microcirculation in PH rats.

We used a MCT-induced PH rat model in this study. MCT, which is a pyrrolizidine alkaloid, is converted in the liver to MCT pyrrole and causes remodeling of pulmonary arteries and, subsequently, PH (11, 18). MCT pyrrole is mainly retained within the pulmonary arteries; its direct effect on arteries in other organs, such as the coronary artery, is considered to be minimal (32). Using this model of PH, we confirmed by light microscopy that the RV coronary arterial microvessels showed no significant morphological differences compared with controls. Therefore, we believe that this model can be used to study coronary microcirculation in RV hypertrophy resulting from pressure overload without other complications.

When using needle-probe videomicroscopy to visualize coronary microvessels in the RV of rats, great care must be taken in manipulating the lens probe. However, we were concerned that direct external compression by the lens tip might have affected the behavior of the microvasculature. To examine any such effect of the lens tip, we measured the pressure it exerted at the end of diastole using a miniature pressure gauge (model P-7; Konigsberg Instruments, Pasadena, CA). The lens-tip pressure was kept to a minimum (<2 mmHg), and the RV and LV pressures did not change. In dogs, we also confirmed that the pressure due to the lens probe was close to zero near the end of diastole (16). Thus we were confident that we could visualize coronary microvessels in situ in the rat RV with minimal interference from the microscopy system.

Although we did not measure epicardial coronary arterial flow directly, we evaluated capillary red blood cell flow velocity in the RV in vivo using a high-resolution intravital videomicroscope in an additional experiment (14), since this is a good index of coronary flow velocity. Infusion of ACh (2 µg·kg–1·min–1 iv) increased red blood cell flow velocity in both control rats and rats with PH without any change in vessel diameter, and the increment was much smaller in PH (13 ± 4%, n = 15, vs. 31 ± 18% in controls, n = 17; P < 0.01), roughly corresponding to the flow increment estimated from diameter increases of arterial microvessels according to Poiseuille's law.

In this study, we focused on the nonselective KCa channel inhibitor TEA, since TEA at low doses is fairly specific for the KCa channels. Node et al. (27) reported that TEA caused similar effects on coronary blood flow during hypoperfusion as did iberiotoxin and charybdotoxin, both of which are highly specific blockers of large-conductance KCa channels. TEA is a well-established tool to evaluate KCa-mediated EDHF in in vivo studies (12, 36), since TEA is much safer to use in living bodies compared with the toxins iberiotoxin and charybdotoxin. Therefore, we evaluated TEA-related EDHF-mediated vasodilation. Catalase, in which several KCa channels are involved, significantly suppressed ACh-induced vasodilation in arterioles both in control and PH rats, suggesting that hydrogen peroxide exerts an important vasodilator effects in both controls and PH. The suppression in vasodilation by catalase was almost comparable with TEA, indicating that TEA at the dose in our study blocked KCa channels almost specifically.

Endothelium-dependent vasodilation in the RV of PH rats decreased in both arterioles and SAs. In control rats, NO was active in both arterioles and SAs with a remarkable contribution to SAs, whereas EDHF was the predominant mediator in arterioles. The relative balance between NO and EDHF in control animals is consistent with earlier reports (17, 26). NO-mediated vasodilation in PH was markedly lower in both arterioles and SA, indicating that NO dysfunction is a pivotal factor in endothelial dysfunction. In contrast, EDHF-mediated vasodilation in PH was reduced in arterioles but unchanged in SAs, and the relative contribution of EDHF to vasodilation was increased in both arterioles and SAs. This robust and sustained vasodilatory action of EDHF in PH is consistent with the suggestion that EDHF might be a crucial compensatory or reserve mechanism for maintaining organ blood flow in situations of NO dysfunction (7, 23, 25). In a preliminary study, we demonstrated that the RV coronary capillary flow response to ACh in spontaneously hypertensive rats was preserved in the absence of NO by robust EDHF activity (10). In PH, EDHF might also function to maintain RV coronary capillary flow in NO dysfunction. NO itself may have direct activity on KCa channels. However, this effect may be little, if any, in our experimental model since the results of L-NAME and TEA in different orders were similar to each other.

Superoxide production in RV myocardial tissue was increased in PH rats compared with in controls. SOD administration reduced this and ameliorated the impairment in ACh-induced vasodilation in PH. These results indicated that increased superoxide production of RV in PH was a pivotal cause of endothelial dysfunction, especially NO dysfunction. On the other hand, it is possible that increased superoxide production in PH-RV also contributes to the robust EDHF function via the increment of the production of hydrogen peroxide, and SOD administration may further increase its production, causing an enhancement of vascular relaxation. In this connection, it was reported that superoxide itself has little effect on the function of large-conductance KCa channels in vascular smooth muscle cells (6).

A decrease in NO bioavailability could result from either decreased production or increased inactivation. Park et al. (28) reported that endothelial NO synthase gene expression increased in the RV in PH rats. Increased endothelial NO synthase production suggested that increased superoxide scavenged NO. There are several possible sources of superoxide, including NADPH oxidase (21) and xanthine oxidase (19). Elevated expression of genes of the renin-angiotensin system in the RV has been reported in PH rats (28), and this might augment NADPH oxidase activity. De Jong et al. (1) reported enhanced expression and activity of xanthine oxidase in the RV in PH rats, and any of these sources could increase superoxide production in PH-RV. An increase in SOD activity in the RV has been reported in PH rats (29). Although the upregulation of SOD might act to regulate increased superoxide production of the RV in PH, it might not be sufficient to entirely suppress the augmented superoxide levels.

SNP-induced endothelium-independent vasodilation in PH rats was impaired in arterioles but preserved in SAs. Sun and Ku (33) showed that the SNP-induced vasodilation of SAs isolated from the RV was unchanged in PH rats, indicating no impairment in vascular smooth muscle cell function. The mechanical stress arising from the overload of RV pressure and hypertrophy might impair SNP-induced vasodilation in arterioles, despite the preservation of smooth muscle cell function. Arterioles may be more sensitive to the perivascular mechanical stress compared with SAs, since lower intravascular pressure in arterioles (than in SAs) results in smaller transluminal pressure and, hence, limits the increase in vascular cross-sectional area during vasodilation. The similar mechanism may cause incomplete amelioration of the arteriolar vasodilation by SOD in PH. Ito et al. (13) reported an increase in perivascular collagen in arterioles of <60 µm diameter, but not in larger arterial microvessels, in the RV of the 12-wk pulmonary artery-banding rat model. Increases in the collagen matrix can cause an increase in the passive stiffness of vessels at higher pressure (22). Although we did not observe such histological changes in our model, which might have been due to its relatively short duration, there might be changes in the micromechanical properties and stress around smaller arterioles.

Clinically, RV dysfunction is an important prognostic factor in PH. Although the most important treatment for PH is the reduction of pulmonary arterial resistance, pharmacological interventions to preserve coronary microvascular vasoreactivity in the hypertrophied RV myocardium could be a useful therapy for maintaining RV function, and antioxidants might be useful in this context.

In conclusion, NO-mediated vasodilation of coronary arterial microvessels decreased markedly with increased superoxide production of the RV in PH, whereas EDHF-mediated vasodilation was robust. EDHF might play a protective role against NO dysfunction in preserving the homeostasis of coronary microcirculation in PH. Endothelium-independent vasodilation of RV coronary arterial microvessels in PH was reduced in arterioles but preserved in SAs, probably due to changes in extravascular micromechanical properties and stress in RV pressure-overloaded hypertrophy.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was partly supported by Scientific Research Grants 17390230, 17390231, and 13854030 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by Cardiovascular Disease Research Grants 14A-1 from the Ministry of Health, Labor and Welfare, Japan.


   ACKNOWLEDGMENTS
 
We thank the Engineering Work Center of the Faculty of Engineering (Okayama University) and the Central Research Laboratory of Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences for technical assistance. We thank the encyclopedic pathological knowledge of Prof. Chikao Yutani.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Kajiya, Dept. of Cardiovascular Physiology, Okayama Univ. Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 5-1 Shikata-cho, 2-chome, Okayama, 700-8558, Japan (e-mail: mkajiya{at}md.okayama-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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