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Nitric oxide modulates oxygen consumption by arteriolar walls in rat skeletal muscle

Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan

Submitted 28 April 2005 ; accepted in final form 15 July 2005

	ABSTRACT

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To study the role of nitric oxide (NO) in regulating oxygen consumption by vessel walls, the oxygen consumption rate of arteriolar walls in rat cremaster muscle was measured in vivo during flow-induced vasodilation and after inhibiting NO synthesis. The oxygen consumption rate of arteriolar walls was calculated based on the intra- and perivascular PO₂ values measured by phosphorescence quenching laser microscopy. The perivascular PO₂ value of the arterioles during vasodilation was significantly higher than under control conditions, although the intravascular PO₂ values under both conditions were approximately the same. Inhibition of NO synthesis, on the other hand, caused a significant increase in arterial blood pressure and a significant decrease in arteriolar diameter. Inhibition of NO synthesis also caused a significant decrease in both the intra- and perivascular PO₂ values of the arterioles. Inhibition of NO synthesis increased the oxygen consumption rate of the vessel walls by 42%, whereas enhancement of flow-induced NO release decreased it by 34%. These results suggest that NO plays an important role not only as a regulator of peripheral vascular tone but also as a modulator of tissue oxygenation by reducing oxygen consumption by vessel walls. In addition, enhancement of NO release during exercise may facilitate efficient oxygen supply to the surrounding high metabolic tissue.

flow-induced vasodilation; N-nitro-L-arginine methyl ester; oxygen transport; vascular tone; vascular endothelial cells

In vivo studies have also demonstrated that NO modulates tissue metabolism (3, 5, 17, 18, 28, 30). Shen et al. (30) reported that inhibition of NO synthesis caused an increase in oxygen consumption in conscious dogs, and they concluded that basal constitutive NO release by capillary endothelium regulates peripheral tissue oxygenation. Crystal et al. (7), however, found that whole body oxygen consumption in anesthetized dogs was unaffected by inhibition of NO synthesis and, after administration of NO donor, proposed that basally released endogenous NO has a tonic systemic vasodilator effect but no effect on oxygen consumption. In addition, Chang et al. (3) suggested that the major action of inhibition of NO synthesis on oxygen consumption was through the alteration of vascular tone. Thus the potential physiological relevance of NO-mediated control of tissue metabolism in vivo remains to be established.

Duling and Berne (9) were first to observe that PO₂ drops in arterioles, and they suggested that a significant amount of oxygen diffuses from the arteriolar network. Subsequent studies using a phosphorescence quenching method have consistently supported this finding, but PO₂ decreases in arterioles are too great to be explained by diffusion alone (15, 32, 40). The possibility of a large amount of oxygen being consumed by arteriolar walls has been raised. The oxygen consumption rates of the walls of functional arterioles estimated on the basis of intra- and perivascular PO₂ values (39) were two orders of magnitude higher than previously reported for endothelial cells and smooth muscle cells in suspension and isolated blood vessel segments in vitro (19, 20). Oxygen consumption by arteriolar walls also depends on vascular tone. The vascular smooth muscle contractions significantly increase vessel wall oxygen consumption (11), whereas vascular smooth muscle relaxation decreases the PO₂ gradient across the arteriolar wall, suggesting the decrease in vessel wall oxygen consumption (14). Our most recent study (33) also demonstrated the effects of reducing vascular tone on oxygen consumption by vessel walls in skeletal muscle. The results suggested that oxygen consumption by arteriolar walls depends on the workload of the vascular smooth muscle.

Because NO induces vasodilation by reducing the vascular tone, we hypothesized that endothelium-derived NO modulates arteriolar wall oxygen consumption. To test this hypothesis, we carried out the present study to determine the vessel wall oxygen consumption rates of arterioles in skeletal muscle in vivo when increased NO release by vascular endothelial cells was induced by an increase in blood flow and when NO production was blocked with N-nitro-L-arginine methyl ester (L-NAME).

	MATERIALS AND METHODS

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Animal preparation. Experiments were performed on nine adult male Wistar rats weighing 150–200 g. All animal procedures were approved by the University of Tokyo Animal Care and Use Committee. Animals were anesthetized with urethane (1 g/kg) intramuscularly, and a tracheotomy was performed to facilitate spontaneous breathing. A carotid artery was cannulated to measure arterial blood pressure and arterial blood gases, and a jugular vein was cannulated to facilitate injection of the intravascular phosphorescence probes and administration of drugs. The cremaster muscle was spread out in a special bath chamber with an optical port for transillumination, and the surface of the muscle was suffused with a 37°C Krebs solution with 5% CO₂ in 95% N₂, adjusted to pH 7.3–7.4. After a 20-min equilibration period, suffusion was stopped, and polyvinylidene chloride film was placed on the muscle to prevent dehydration and hyperoxia. All animals whose mean arterial pressure fell to <60 mmHg during the experiment were excluded.

Experimental protocols. Intra- and perivascular PO₂ measurements of first-order (1A) arterioles were made 300–500 µm past the branch from the central cremasteric artery. The intravascular PO₂ measurements were made at the center of the vessel 20 min after injection of a Pd-porphyrin solution (25 mg/kg) into the cannulated jugular vein over a period of 2 min. They were immediately followed by perivascular PO₂ measurements 20 µm from the internal surface of the same arterioles to avoid uncertainty arising from the inability to precisely locate the outer surface of the vessel wall. This arrangement would have little influence on measured perivascular PO₂ values, because a fall in PO₂ in the surrounding tissue has a significantly shallower decay compared with that within the vessel wall. After making the PO₂ measurements under control conditions, we made intra- and perivascular PO₂ measurements at the same sites during flow-induced dilation. The dilation was induced by the parallel arteriolar bifurcation occlusion method (22), in which mechanical occlusion of one branch causes an increase in blood flow in the unoccluded branch. The occluding needle (tip diameter 150 µm) was positioned over the central cremasteric artery, a minimum of 500 µm distal to the bifurcation. The PO₂ measurements were started 20 s after occlusion, and both the intra- and perivascular PO₂ measurements were performed within a period of 40 s. After a 10-min rest period, the nonselective NO synthase (NOS) inhibitor L-NAME was infused into the cannulated jugular vein in a dose of 21 mg/kg over a period of 1 min, and 5 min later, the intra- and perivascular PO₂ measurements were made at the same sites. To assess the reproducibility of measurements, we repeated all intra- and perivascular PO₂ measurements after moving the intra- and perivascular sampling site 10 µm downstream along the vessel, and PO₂ values were obtained by averaging these data. The total duration of the experiment was <1 h after exposure of the cremaster muscle.

Hemodynamic changes. The changes in the arteriolar internal diameter under control conditions, during flow-induced dilation, and after L-NAME administration were analyzed in video images off-line. Changes in arterial blood pressure after L-NAME administration were also monitored. In some experiments, to check whether arteriolar flow velocity increased during occlusion, relative changes in arteriolar flow velocity were monitored using a laser-Doppler flowmeter (FLO-C1EL; Omega Wave, Tokyo, Japan).

Phosphorescence quenching laser microscope. Observations of the microcirculation and measurements of intra- and perivascular PO₂ were made with a modified Nikon microscope and the oxygen-dependent quenching of phosphorescence decay technique described previously (31). Pd-meso-tetra(4-carboxyphenyl)porphyrin (Pd-porphyrin; Porphyrin Products, Logan, UT) bound to bovine serum albumin was used as the phosphorescent probe for oxygen-dependent quenching. The phosphorescent probe was excited by epi-illumination with an N₂/dye pulse laser (LN120C; Laser Photonics, Lake Mary, FL) with a 535-nm line at 20 Hz through the objective lens (CF Plan x20/0.40 EPI ELWD; Nikon, Tokyo, Japan). The average optical power and pulse width of the laser were 1.2 mW and 300 ps, respectively. The surface of the epi-illuminated tissue was 10 µm in diameter. The excitation pulse energy is estimated to be 60 µJ/pulse at the outlet of the laser. However, the energy is reduced by the time it travels to the tissue surface because the laser beam is induced by fiber optics and is irradiated through an objective lens; thus it appears to have relatively little impact on the measurement. The phosphorescent emissions were captured by a photomultiplier (C6700; Hamamatsu Photonics, Hamamatsu, Japan) through a long-pass filter at 610 nm and a pinhole-like aperture with a diameter of 20 µm. The phosphorescence signals were then converted to 10-bit digital signals at intervals of 3 µs. A total of 10 pulses were used to obtain a mean phosphorescence decay curve, and PO₂ values were determined according to the rectangular model of PO₂ distribution (12, 13), expressed as

(1)

where I(t) is light intensity at time t, I₀ is initial light intensity at t = 0, ₀ and are the phosphorescence lifetimes in the absence of oxygen and in the area being measured, respectively, k_q is the quenching constant, and is the half-width of the rectangular distribution. The decay curve was fit to Eq. 1 by using the least-squares approximation, and 10 curves were averaged to determine the PO₂ value. The length of time used for fitting the decay curve was 150 µs. The PO₂ value was finally obtained from the average of two PO₂ values measured at the same site. All data with a correlation coefficient r < 0.900 between the measured and theoretical curves were excluded.

Determination of the oxygen consumption rate of arteriolar walls. The oxygen consumption rate of the arteriolar wall was determined using a modified Krogh capillary-tissue model for an arteriolar wall having cylindrical geometry, as previously described (33). Briefly, assuming that the arteriole is cylindrical and has an outer radius and inner radius of R_o and R_i, respectively, the oxygen consumption rate per unit tissue volume per unit of time in its wall (QO₂) was expressed as

(2)

where PO_2peri and PO_2in represent the PO₂ values of the outer surface of the arteriolar wall and within the arteriole, respectively. _t and D_t represent oxygen solubility and oxygen diffusivity, respectively, in the arteriolar wall, for which values of 3.0 x 10^–5 ml·g^–1·mmHg^–1 and 1.5 x 10^–5 cm²/s, respectively, were used. Therefore, the oxygen consumption rate of the arteriolar wall was determined by utilizing the measured intra- and perivascular PO₂ values of the arteriole. Because of the uncertainty of the location of the outer boundary of the vessel wall, the outer radius was assumed to be 10% larger than the inner radius (39), which was measured on video-recorded images.

Data analysis. Values are means ± SD. Data were analyzed using a one-way ANOVA test. Differences between groups were determined using a t-test with the Bonferroni correction. Differences with a P value <0.05 were considered statistically significant.

	RESULTS

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Systemic arterial PO₂, PCO₂, and pH were measured with a blood analysis system (Series 2000; Diametrics Medical, St. Paul, MN) in samples from the carotid arteries after the microvascular PO₂ measurements were performed. Arterial PO₂ averaged 89.7 ± 6.0 mmHg, and arterial PCO₂ and pH averaged 48.8 ± 8.7 mmHg and 7.33 ± 0.05, respectively. The internal diameter of the 1A arterioles under control conditions, during occlusion, and after administration of the nonselective NOS inhibitor L-NAME was 94 ± 12, 102 ± 9, and 83 ± 11 µm, respectively. These values were used to calculate the oxygen consumption rates of arteriolar walls under each of the experimental conditions.

Changes in hemodynamics during parallel occlusion. Measurements of blood flow velocity were made in four of the nine rats, in the same arterioles as the PO₂ measurements were made. The maximum values of flow velocity and internal diameter during the occlusion period (60 s) were recorded as the values of velocity and diameter during occlusion. The individual change in internal diameter of arterioles for each preparation of nine rats and mean percent changes in blood flow velocity before and during occlusion are shown in Fig. 1. The parallel occlusion increased internal diameter 13% and increased flow velocity 39% relative to the values before occlusion. This clearly demonstrates that the occlusion caused an increase in flow velocity in the unoccluded arteriole and induced flow-dependent vasodilation. The flow velocity and diameter values during occlusion were both significantly higher than before occlusion.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Individual change in internal diameter of arterioles for each preparation of 9 rats (top) and mean percent changes in blood flow velocity (bottom) before and during parallel occlusion. Flow-induced vasodilation was performed using the parallel arteriolar bifurcation occlusion method (22), which, by mechanically occluding one branch, causes an increase in blood flow in the unoccluded branch. The parallel occlusion increased internal diameter 13% and increased flow velocity 39% relative to the values before occlusion. Velocity values are means ± SD; n = 4 rats. *P < 0.05, significantly different from control group.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Microvascular hemodynamic changes during inhibition of nitric oxide (NO) synthesis by N-nitro-L-arginine methyl ester (L-NAME). Individual change in internal diameter of arterioles for each preparation of 9 rats (top) and the change in mean arterial blood pressure (bottom) are shown before and after administration of L-NAME. The peak mean blood pressure and minimum internal diameter during the 5 min after administration of L-NAME were recorded as the values during NO synthesis inhibition. Administration of L-NAME decreased internal diameter 15% and increased mean arterial blood pressure 77% relative to the control group. Blood pressure values are means ± SD; n = 9 rats. Significantly different from control group **(P < 0.01).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Individual values of intra- and perivascular PO2 of arterioles before (control) and during parallel occlusion-induced vasodilation (occluded) and after administration of L-NAME for each preparation of 9 rats. Administration of L-NAME decreased both the intra- and perivascular PO2 values of the arterioles, possibly as a result of the decrease in blood perfusion induced by L-NAME.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Average intra- and perivascular PO2 values of arterioles before and during parallel occlusion-induced vasodilation and after administration of L-NAME, calculated from the data shown in Fig. 3 for each preparation of 9 rats. The intravascular PO2 values of the arterioles under all conditions were significantly lower than the systemic arterial PO2 value (89.7 ± 6.0 mmHg). Values are means ± SD; n = 9 rats. *P < 0.05; **P < 0.01, significantly different from control group.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Individual values of the oxygen consumption rates in the walls of arterioles before and during parallel occlusion-induced vasodilation and after administration of L-NAME, calculated from the intra- and perivascular PO2 data shown in Fig. 3 for each preparation of 9 rats (top). Average values of oxygen consumption rates calculated from the above data also are shown (bottom). The increase in NO release induced by blood flow decreased oxygen consumption by 34%, whereas NO synthase inhibition by L-NAME increased arteriolar wall oxygen consumption by 42%. Values are means ± SD; n = 9 rats. **P < 0.01, significantly different from control group.

	DISCUSSION

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The recent development of the phosphorescence quenching technique (41, 42) has demonstrated significant drops in the oxygen levels of arterioles (16, 31, 37, 39), suggesting that arterioles play an important role as an oxygen supplier to the surrounding tissue and that the arteriolar wall consumes a large amount of oxygen (15, 36, 39, 40). The oxygen consumption rate of arteriolar walls estimated by intra- and perivascular PO₂ values under control conditions in the present study was 1.7 x 10^–2 ml·s^–1·g^–1. It was slightly lower than the 2.8 x 10^–2 ml·s^–1·g^–1 reported for cat pial arterioles (10) and the 6.5 x 10^–2 ml·s^–1·g^–1 reported for rat mesenteric arterioles (39). These differences might have occurred, in part, because of technical problems with the phosphorescence quenching technique for perivascular PO₂ measurements, because the perivascular measurement site is a relatively contained stationary fluid whose PO₂ value tends to be lowered by the measurement process itself. On the other hand, many of the data obtained from endothelial cells and smooth muscle cells suspensions or vessel segments in vitro (19, 20) have been 100–1,000 times lower than in vivo, which have been on the order of 10^–5 ml·s^–1·g^–1. The fact that the suspensions and segments are not subject to functional in vivo conditions may explain the low oxygen consumption. Our previous study (33) demonstrated that the oxygen consumption rate is highest in the arteriolar wall upstream and sequentially decreases in a downstream direction under resting conditions. During vasodilation induced by papaverine, on the other hand, the oxygen consumption rates at all sites decrease to similar levels, suggesting that the high oxygen consumption rate of arteriolar walls under resting conditions is likely dependent on the workload of vascular smooth muscle.

It is well known that endothelial NO regulates peripheral vascular tone and controls capillary perfusion. Our finding that inhibition of NO synthesis increases the oxygen consumption rate by vessel walls and that enhancement of flow-induced NO release decreases it suggests that NO plays an important role not only as a regulator of peripheral vascular tone but also, by reducing oxygen consumption in vessel walls, as a modulator of tissue oxygen consumption. Increasing NO release by microvascular endothelium, for example, by exercise, may facilitate efficient oxygen supply to the surrounding tissue, whereas decreases in NO production in aged animals and elderly humans may become an additional risk factor for ischemia. Our results emphasize the physiological importance of NO as a modulator of peripheral tissue oxygenation.

We evaluated oxygen consumption by arteriolar walls during NO-induced vasodilation and during vasoconstriction induced by inhibition of NO production. Recent microvascular studies have reported that oxygen consumption by arteriolar walls depends on the level of vascular tone (40). Friesenecker et al. (11) demonstrated the contribution of vascular smooth muscle contraction to oxygen consumption by microvessel walls during arginine vasopressin-induced vasoconstriction. Their findings that intra- and perivascular PO₂ levels decrease and the PO₂ gradient across the vessel wall increases during vasoconstriction were compatible with our findings during vasoconstriction induced by inhibition of NO synthesis and suggest that a large amount of oxygen is consumed by vessel walls. The PO₂ values obtained under control and vasoconstricted conditions were approximately the same, except that their perivascular PO₂ value during vasoconstriction was 40% lower than in our study. Because oxygen consumption by arteriolar walls depends on the magnitude of vascular smooth muscle contraction, the 51% decrease in arteriolar diameter by vasopressin and 15% decrease by inhibition of NO synthesis may explain the lower perivascular PO₂ value in their study. In our previous study (33), oxygen consumption by the arteriolar wall during vasodilation was reported. Papaverine-induced vasodilation increased the arteriolar diameter 17% and decreased the oxygen consumption rate 55% of the arteriolar wall. The flow-induced vasodilation in the present study, on the other hand, increased diameter 13% and decreased oxygen consumption by 34%. The variable ratio of oxygen consumption rate to diameter (QO₂/D) is 3.2 during papaverine-induced vasodilation and 2.6 during NO-induced vasodilation. These results strongly support a direct correlation between arteriolar wall oxygen consumption and vascular tone. Furthermore, it is considered that NO decreases vessel wall oxygen consumption by decreasing the workload of the vascular smooth muscle rather than by direct modulation of oxygen uptake by NO inhibition of mitochondrial cytochrome c oxidase.

Many studies have shown that NO synthesis has an inhibitory effect on oxygen consumption in vivo (3, 5, 7, 17, 30). Shen et al. (30) demonstrated that inhibition of NO synthesis resulted in both vasoconstriction and an increase in oxygen consumption in conscious dogs but that vasoconstriction was not the main reason for the increase in oxygen consumption, because administration of methoxamine caused an increase in vascular resistance without increasing oxygen consumption. Chang et al. (3), on the other hand, suggested that the major action of inhibition of NO synthesis on oxygen consumption was dependent on the level of metabolic demands. They showed that resting oxygen consumption by the diaphragm is unaffected by inhibition of NO synthesis, despite the significant decrease in oxygen consumption during contraction. In addition, the opposite result has been reported in that inhibition of endothelial function in hindlimb has been associated with a reduction in limb oxygen consumption simply due to vasoconstriction (25). These discrepancies might be caused, in part, by the differences of vascular tone level during experiments. Thus further investigation in the context of vascular tone and oxygen consumption may be needed.

Fewer studies have analyzed the relationship between NO and oxygen consumption by investigating the effects of flow-induced NO release than inhibition of NO synthesis. In the present study the parallel arteriolar bifurcation occlusion method (22) was used to induce flow-dependent vasodilation. Intravascular pressure of unoccluded arteriole may rise during occlusion, but the effect of the increase in pressure on vasodilation is small. Most of the vasodilation was induced by the increase in flow-dependent NO release, because arteriolar diameter increased only 2.5% during occlusion by L-NAME or L-NAME plus indomethacin (data not shown), whereas it increased 13% during occlusion. The 2.5% increase in diameter may have been caused by intravascular pressure changes. In our previous study (33), intra- and perivascular PO₂ values of the same arterioles were measured during papaverine-induced vasodilation. A similar PO₂ response during vasodilation induced by varapamil was reported by Hangai-Hoger et al. (14). Both the intra- and perivascular PO₂ values were significantly higher during papaverine- or varapamil-induced vasodilation than the control values, whereas only perivascular PO₂ increased during NO-dependent vasodilation. The higher intravascular PO₂ levels during papaverine- and varapamil-induced vasodilation could largely be explained by an increase in blood perfusion. The results of the present study showing an increase in perivascular PO₂ levels but no change in intravascular PO₂ levels clearly demonstrate that the increase in NO release by vascular endothelium further improved tissue oxygenation by decreasing oxygen consumption by vessel walls.

Hemodynamic responses to inhibition of NO release by vascular endothelium have been reported. The inhibition of NO synthesis by L-NAME in the present study raised mean arterial pressure by 75%, i.e., from 78 to 137 mmHg, a rate of increase that is much higher than the increase of 26%, i.e., from 94 to 118 mmHg, reported by Chen and Hu (4). The mean arterial pressure value of 78 mmHg in our study appears to be relatively low. These differences may be attributable to the age difference between rats, which were 6 wk old in our study and 22–24 wk old in their study. It is a well-known fact that NO production by vascular endothelium decreases with age. In addition, the effects of inhibition of NO synthesis on arteriolar dimension and intravascular and tissue PO₂ levels in 2A arterioles (diameter 48 µm) in a hamster cheek pouch preparation were recently reported (2). Inhibition of NO synthesis by N^G-monomethyl-L-arginine decreased arteriolar diameter by 10% and decreased intravascular PO₂ and tissue PO₂ by 14 and 50%, respectively. These responses consistently support our findings that the vasoconstriction induced by inhibition of NO synthesis increases arteriolar oxygen consumption and results in tissue hypoxia.

In conclusion, NO plays an important role not only as a regulator of peripheral vascular tone but also as a modulator of peripheral tissue oxygenation. The increase in NO release by vascular endothelium facilitates efficient oxygen supply to the surrounding tissue, reducing oxygen consumption by the vessel walls as well as by increasing blood flow. Conversely, decreases in NO production in elderly humans or hypertension may be an additional risk factor for ischemia in peripheral tissue, in addition to being a risk factor for angiopathy.

	GRANTS

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This study was supported by a Grant-in-Aid for Scientific Research (15300156, 17300143) from Japan Society for the Promotion of Science.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Shibata, Dept. of Biomedical Engineering, Graduate School of Medicine, Univ. of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan (e-mail: shibatam{at}m.u-tokyo.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.

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