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Divisions of Critical Care and Pulmonary Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Ward, Michael E. Effect of inhibition of nitric oxide
synthesis on the diaphragmatic microvascular response to hypoxia. J. Appl. Physiol. 81(4):
1633-1641, 1996.
The purpose of this study was to determine the
effect of inhibition of nitric oxide (NO) release on the diaphragmatic
microvascular responses to hypoxia. In 
-chloralose-anesthetized
mongrel dogs, the microcirculation of the vascularly isolated ex vivo
left hemidiaphragm was studied by intravital microscopy. The diaphragm
was pump perfused with blood diverted from the femoral artery through a
series of membrane oxygenators. The responses to supramaximal
concentrations of sodium nitroprusside, moderate hypoxia (phrenic
venous PO2 27 Torr), and
severe hypoxia (phrenic venous PO2 15 Torr) were recorded before and after an infusion of
NG-nitro-L-arginine
(L-NNA; 6 × 10
4 M) into the phrenic
circulation for 20 min. Under control conditions, diaphragmatic blood
flow was 12.4 ± 1.1 ml · min1 · 100 g1. Diaphragmatic blood
flows recorded during moderate and severe hypoxia were 15.6 ± 1.2 and 24.3 ± 1.5 ml · min1 · 100 g1, respectively
(P < 0.05 for both compared with
control values). Treatment with
L-NNA reduced diaphragmatic
blood flow to 9.6 ± 0.8 ml · min1 · 100 g1 under control conditions
(P < 0.05) and caused arteriolar
vasoconstriction to a degree that was dependent on vessel size (i.e.,
larger vessels constricted more than smaller vessels).
L-NNA eliminated the increase in
blood flow during moderate hypoxia and inhibited arteriolar dilation by
an amount that was related to vessel size (i.e., dilation of larger
vessels was inhibited more than that of smaller vessels). Inhibition of
NO synthesis had no effect on the increase in diaphragmatic blood flow
(23.6 ± 1.9 ml · min1 · 100 g1;
P > 0.05 compared with that during
severe hypoxia before treatment with
L-NNA) or arteriolar diameters
during severe hypoxia. NO release plays a role in the diaphragmatic
vascular response to hypoxia, but this role is limited to dilation of
larger arterioles during hypoxia of moderate severity.
respiratory muscles; blood flow; arterioles; endothelium; endothelium-derived relaxing factor
INTRODUCTION FAILURE TO MAINTAIN SUBSTRATE SUPPLY to the diaphragm
in proportion to its metabolic need impairs the capacity to sustain spontaneous ventilation (2). Accordingly, the study of the mechanisms
by which diaphragm blood flow is regulated has been of high priority
among physicians seeking to develop treatment strategies for patients
with respiratory failure (2). Endothelial release of nitric oxide (NO)
has been found to be an important mechanism by which active hyperemia
(12), reactive hyperemia (32), and autoregulation of blood flow (31)
are mediated in this circulation. In each case, the vascular changes
serve the purpose of restoring the balance between
O2 availability and demand. A
central role for the
L-arginine-NO pathway in the
vascular adaptation to impaired diaphragmatic oxygenation therefore
appears to be emerging. The effect of inhibition of NO synthesis on the
diaphragmatic vascular response to hypoxia, however, has not been
specifically evaluated. Consequently, the role that hypoxia plays in
eliciting participation of the NO pathway in these vascular adjustments is unknown.
O2 tension is clearly an important
factor in regulating NO synthesis in in vitro systems (25). The
physiological importance of NO release in mediating vascular responses
in vivo, however, has been more difficult to establish. Dissimilar
effects of NO synthesis inhibition on a given vascular bed in different
species (19, 33) and among different vascular beds within the same species (16, 19) have been documented. Similarly, evidence has been
obtained supporting the position that modulation of NO release mediates
hypoxic vasodilation (24) or hypoxic vasoconstriction (20) or plays no
role at all in the hypoxic response (27) depending on the vasculature
under study. Because the results obtained in previous experiments
cannot be extrapolated to the diaphragm, further progress in this field
requires that the role of the NO pathway in this circulation be
specifically determined. The purpose of this study is to test the
hypothesis that inhibition of NO synthesis will alter the diaphragmatic
vascular adaptation to hypoxia and, by implication, that NO release is
an important mechanism by which local regulation of diaphragmatic
oxygenation is mediated.
METHODS Animal Preparation
-chloralose (60-80 mg/kg iv). Supplemental doses of
-chloralose were given as needed to eliminate jaw tone but maintain
knee reflexes. The animals were supine, intubated with cuffed
endotracheal tubes, and mechanically ventilated (initial tidal volume
15 ml/kg, frequency 15 breaths/min, adjusted to maintain arterial
PCO2 between 38 and 42 Torr).
Supplemental O2 was supplied
through the inspiratory line, and arterial
PO2 was maintained above 100 Torr. Positive end-expiratory pressure (5 cmH2O) was applied at the expiratory line. A catheter was placed in the aorta through the right
carotid artery to monitor arterial pressure, and another catheter was
placed in the right femoral vein to administer supplemental anesthesia.
Core body temperature was kept constant at ~37°C by a heating pad
placed under the animal.
Isolation of the Left Hemidiaphragm
The intercostal vessels and the left internal mammary artery were ligated, and the lower left ribs were removed. After the initial thoracotomy was made but before the ribs were removed, two silk sutures oriented perpendicular to the muscle fibers were placed 1 cm apart in the costal diaphragm. These were used to adjust the diaphragm to its approximate in situ length after removal from the animal (see below). Through an abdominal incision, the two halves of the diaphragm were divided. The stomach, liver, spleen, and left kidney were retracted to expose the left phrenic artery. The accessory branches from the aorta to the diaphragm were ligated. The phrenic artery was ligated proximally and cannulated by introducing a polyethylene catheter (PE-160, 1.14 mm ID, 1.57 mm OD, 3 cm long) into the distal portion. Phrenic arterial perfusion pressure (Pphr) was measured from a side port on this catheter. A second side port permitted infusion of drugs into the phrenic circulation with a syringe infusion pump (Harvard). The catheter was connected to an electromagnetic flow probe (1.91 mm ID; Carolina Medical Electronics) to measure phrenic arterial flow (
phr). The other side of the probe was connected to
a Y connector. One arm of the Y connector was attached to a catheter in
the left femoral artery from which the muscle could be autoperfused.
The other arm of the Y connector was secured to a perfusion pump (Cole Parmer) that took its inflow from a catheter in the right femoral artery. With the left femoral arterial catheter clamped, this pump was
used to perfuse the phrenic artery from the right femoral artery. All
animals were heparinized after catheterization of the phrenic artery.
The left hemidiaphragm was freed from the ribs and cartilages at its costal margin, and the crural diaphragm and the phrenic nerve were sectioned, freeing the muscle completely. The phrenic vein was cannulated directly, its effluent being collected and reinfused into the animal when appropriate. The entire left hemidiaphragm was then placed, abdominal surface up, in a Plexiglas chamber. The muscle was held flat by threads attached to its edges and to the walls of the chamber. The tension of the threads was adjusted such that the distance between the sutures placed in the costal diaphragm after the initial thoracotomy (see above) was restored to 1 cm. The temperature of the diaphragm was monitored with a flat unisurface tissue probe (SST1, Physitemp Instruments, Clifton, NJ) placed against the inferior surface of the muscle and maintained at 37°C with a heat exchanger (model P-7-14, Sci-Med, Minneapolis, MN) in line with the perfusion pump and a heat lamp when necessary. The muscle was sealed from the atmosphere by the bottom of the Plexiglas chamber below and above by an occlusive layer of Saran Wrap. Phrenic arterial and venous blood gases and O2 contents (CO-oximetry, Instrumentation Laboratory IL-482) were measured during steady state under each condition studied. At the end of the experiment, the animals were exsanguinated and the diaphragm was dissected free and weighed.
Microvascular Diameter Measurements
Diaphragmatic microvascular diameters were measured with a zoom (×0-6.3) stereomicroscope (Olympus SZ60) with ×2 objective mounted on a universal stand. The muscle was epi-illuminated by a fiber-optic ring light source (Olympus Highlight) focused on the surface of the muscle and transilluminated by a second dual-beam fiber-optic light source directed through the bottom of the Plexiglas chamber. The microscope was focused on the region of costal diaphragm adjacent to the central tendon. The arterioles in this region form a dense vascular arcade as they pass from the feed vessels traversing the central tendon into the body of the muscle. For each preparation, a field was selected that provided visualization of vessels with the widest possible range of diameters. The images were recorded with a charge-coupled device video camera (resolution 570 lines; Hitachi KPM1U) projected on a 15-in. monitor and recorded on SVHS video tape. Diameter measurements were made off-line by digitizing and importing the images into a computer with a frame grabber board (Data Translation DT3852a) and image-acquisition software (Data Translation Global Lab Image Version 4.21). This system was calibrated with a stage micrometer at the end of each experiment. After a stabilization period of ~15 min under each condition, during which steady state was established for transvascular resistance and arteriolar diameters, three images were collected over 30 s. As many vessels as could be clearly visualized (generally six to eight) within the field were measured at the same point in each image. Further magnification of the digitized image with the image-analysis software was used as needed to assist measurement of individual vessels. The reported diameters represent the means of the measurements from the three separately acquired images of each vessel. In no instance did these independent measurements differ by >0.5 µm, and the vast majority did not differ by >0.2 µm.Differentiation of arterioles from venules was accomplished by infusing saline into the phrenic artery and observing the order of blanching and reperfusion. Vessels identified as venules were excluded from the analysis.
Protocols
After a 30-min stabilization period and before any drugs were administered to the diaphragmatic circulation, recordings of arteriolar diameters,phr, and Pphr were made during
autoperfusion with normoxic normocapnic blood. Measurements made under
these condition were designated as baseline values. During the
remainder of the experiment, the flow rate of the perfusion pump was
adjusted manually to keep the steady-state Pphr the same as that
recorded under baseline conditions.
The diaphragm was then pump perfused with blood from the right femoral artery through the heat exchanger and a series of three membrane oxygenators (model 0400-2A, Canadian Cardiovascular Products, Pierrefonds, Quebec). The gas mixture delivered to the oxygenators was adjusted with a blender-and-regulator system that controlled flow from three separate tanks containing N2, CO2, and O2, respectively. With this system, any combination of these gases may be delivered, and the exact mixture may be adjusted to achieve the desired final phrenic venous blood gas values. Because the compliance of the oxygenators is large, all oscillations in flow resulting from the pump were effectively damped out. As a result, flow in the tubing directly upstream from the phrenic artery was nonpulsatile.
Group 1 (n = 7). In the group 1 animals, the effect of NG-nitro-L-arginine (L-NNA) on the diameters of diaphragmatic arterioles was evaluated. To determine whether linear heterogeneity in the effect of L-NNA exists, a reproducible index of vessel size that is not affected by treatment with L-NNA is required. The response to a supramaximal concentration of sodium nitroprusside (SNP) was, therefore, compared in these animals before and after L-NNA infusion. Control measurements were obtained during pump perfusion with the gas mixture delivered to the oxygenators adjusted to achieve phrenic arterial blood gas values approximating 100 Torr, 40 Torr, and 7.40 for PO2, PCO2, and pH, respectively. In random order, the following conditions were then presented: 1) infusion of a supramaximal concentration of SNP, 2) moderate hypoxia, and 3) severe hypoxia, as described in SNP, MODERATE HYPOXIA, and SEVERE HYPOXIA. After each presentation, gas tensions in the perfusing blood were returned to control values. Subsequent conditions were presented only after recovery of diaphragmatic blood flow and arteriolar diameters to their control values.
SNP. SNP (2 mg/ml) was infused into the phrenic artery at a rate of 1/100th of thephr. In preliminary studies, vasodilation was found to have reached 80% of maximum at a concentration of 2 µg/ml of SNP in this vascular bed. Maximal SNP-induced vasodilation was, therefore, taken as the diameter recorded during infusion of SNP
at a final concentration in the phrenic arterial blood of 20 µg/ml.
This concentration eliminated the dilation that normally follows
transient interruption of blood flow (reactive hyperemia) in all
animals. Further bolus injections of SNP did not result in additional
dilation of any of the vessels under observation. Because vasodilation
required a higher phr to maintain Pphr at the
baseline value, the rate of SNP infusion was adjusted so that the
concentration in the perfusing blood remained constant.
MODERATE HYPOXIA.
Moderate hypoxia was presented by adjusting the gas mixture to achieve
a target PO2 in the phrenic venous
blood (PvO2) of 25 Torr. Phrenic venous
PCO2 and pH were maintained constant
at their control levels through appropriate adjustments to the
CO2 and
N2 flow settings.
SEVERE HYPOXIA.
Severe hypoxia was produced by delivering a gas mixture containing 0%
O2 to the membrane oxygenators.
Phrenic venous PCO2 and pH were
maintained constant at their control levels through appropriate
adjustments to the CO2 and
N2 flow settings.
INHIBITION OF NO SYNTHESIS.
The diaphragm was perfused with normoxic normocapnic blood, and
diaphragmatic vascular resistance and arteriolar diameters were allowed
to return to their previous control values. A 6 × 102 M solution of
L-NNA was infused into the
phrenic circulation at a rate equal to 1/100th of the
phr (final concentration 6 × 104 M) for 20 min. This
concentration is the lowest that was found to completely reverse
vasodilation during an infusion of the endothelium-dependent vasodilator acetylcholine (ACh;
105 M) in this vascular bed
(30).
After completion of the L-NNA
infusion, measurements were repeated under control conditions and
during SNP infusion, moderate hypoxia, and severe hypoxia, presented in
random order as described in
SNP, MODERATE
HYPOXIA, and SEVERE
HYPOXIA.
Group 2 (n = 10). In this group, the
separate and combined effects of
L-NNA and indomethacin on
diaphragmatic blood flow during moderate hypoxia and during infusion of
prostaglandin E2
(PGE2) were assessed to
determine whether the effect of
L-NNA on the response to hypoxia
can be attributed to the inhibition of vasodilatory prostaglandins. The
isolated left hemidiaphragm was prepared as in the
group 1 animals. Five of the ten
animals in this group were pretreated with indomethacin (5 mg/kg iv).
Control measurements of Pphr and phr were obtained
during pump perfusion at the flow rate recorded during a prior period
of autoperfusion with target phrenic arterial blood gas values of 100 and 40 Torr for PO2 and
PCO2, respectively. The flow rate
required to maintain constant perfusion pressure was then determined
during moderate hypoxia (target PvO2 25 Torr) and during infusion of PGE2
(105 M). This concentration
of PGE2 produces 80% maximal
dilation in this circulation. Recordings were made at steady state
(i.e., after 15 min) under each condition.
L-NNA (final concentration 6 × 104 M) was then
infused into the phrenic circulation for 20 min, and measurements of
Pphr and phr were repeated under control conditions, during moderate hypoxia, and during infusion of
PGE2.
Data Analysis
The O2 consumption of the diaphragm was calculated as the product of the phrenic arteriovenous O2 content difference andphr.
The effect of L-NNA on arteriolar diameter was evaluated in two ways. The change in diameter during the application of each stimulus (SNP, moderate hypoxia, and severe hypoxia) was calculated as the diameter during application of the stimulus minus the corresponding control diameter. In addition, the difference in diameter before and after treatment with L-NNA was calculated as the diameter under each experimental condition (control, SNP, moderate hypoxia, and severe hypoxia) after treatment with L-NNA minus that recorded under the same condition before treatment with L-NNA. Both the change in diameter and the difference in diameter were normalized for vessel size by expressing them as a percentage of the maximum diameter during infusion of a supramaximal concentration of SNP. The maximum diameter was taken as the average of the diameters during SNP infusion before and after treatment with L-NNA. Linear regression was used to determine the contribution of vessel size to the variability in both the change in diameter and the difference in diameter after treatment with L-NNA compared with that before treatment with L-NNA under each condition.
Differences among mean values were tested for significance with analysis of variance corrected for repeated measures. If the analysis of variance revealed significant differences among the means (P < 0.05), the differences between individual means were tested post hoc with the Newman-Keuls procedure. Unless otherwise specified, values are reported as means ± SE.
RESULTS
Group 1
Pphr for the group 1 animals during autoperfusion with arterial blood averaged 125 ± 4 mmHg and was maintained at this value throughout the protocol, in keeping with the experimental design. During autoperfusion with arterial blood, the averagephr for the group
1 animals was 12.0 ± 1.2 ml · min1 · 100 g1. The average
O2 consumption of the
hemidiaphragm in the group 1 animals
under baseline conditions was 0.40 ± 0.07 ml · min1 · 100 g1 and did not differ
significantly from this value under any of the conditions studied.
Table 1 presents values for phrenic
arterial and venous blood gases under control and hypoxic conditions
before and after L-NNA infusion
in the group 1 animals.
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Figure 1 illustrates the diaphragmatic
blood flow before and after
L-NNA infusion during pump
perfusion at constant perfusion pressure in the group
1 animals under four conditions: control, SNP infusion,
moderate hypoxia, and severe hypoxia. Under control conditions, blood
flow was lower after L-NNA
infusion. Diaphragmatic blood flow during SNP infusion and during
severe hypoxia was unchanged after
L-NNA infusion. Before
L-NNA infusion, diaphragmatic
blood flow increased during moderate hypoxia. After
L-NNA infusion, blood flow
during moderate hypoxia did not differ from the corresponding control
value.
Effect of L-NNA on the arteriolar response to SNP. The effect of a supramaximal concentration of SNP was evaluated in the group 1 animals to establish the maximal arteriolar diameters for use as an index of vessel size. Because others (18) have suggested that the effect of NO donors (in submaximal concentrations) may be potentiated by inhibition of endogenous NO synthesis, the validity of this approach was tested by determining the effect of L-NNA on the response to SNP. The arteriolar diameters recorded during SNP infusion before and after treatment with L-NNA were highly correlated (r2 = 0.97), and the slope of this relationship approximates identity. A high correlation, however, may conceal lack of agreement because it is sensitive to range (i.e., the greater the range, the higher the correlation) and will not detect changes in scale (1). To overcome these shortcomings, the difference between the diameter recorded during SNP infusion after treatment with L-NNA and that recorded during SNP infusion before treatment with L-NNA is plotted as a function of the average of these two diameters in Fig. 2 (1). The mean difference between the measurements of arteriolar diameter obtained before and after treatment with L-NNA is not significantly different from zero. Because L-NNA does not affect the diameter during SNP infusion, the average maximum diameter was used as an index of vessel size and is referred to simply as the maximum diameter in the subsequent sections.
In Fig. 3, the change in diameter from control conditions (percent maximum) during SNP infusion before and after treatment with L-NNA is plotted against the maximum diameter for the group 1 animals. Both before and after L-NNA, SNP exerts a greater effect on smaller vessels than on larger vessels, resulting in an inverse relationship between the response to SNP and vessel size. The slope of this relationship is less steep (i.e., less negative) after L-NNA infusion (P < 0.05). Larger vessels exhibit a greater change in diameter in response to SNP after treatment with L-NNA. In smaller vessels, the response to SNP is similar to that before infusion of L-NNA. This finding may arise as a result of two effects. The diameter achieved during SNP infusion may be greater after L-NNA in larger vessels. Alternatively, treatment with L-NNA may have selectively reduced the diameter of larger vessels under control conditions. Because the maximum diameter of these vessels is the same after treatment with L-NNA (see above), the most likely explanation for this finding is that L-NNA reduced their control diameters.
Effect of L-NNA on control diameters. The effect of L-NNA on arteriolar diameter under control conditions in the group 1 animals is illustrated in Fig. 4. In Fig. 4, top, the control diameter of each vessel is plotted against its maximum diameter. Before L-NNA, the control diameter in smaller vessels is a smaller percentage of its maximum diameter than it is in larger vessels. After L-NNA, the control diameter of larger vessels is reduced, resulting in a lower slope for this relationship (P < 0.05).
In Fig. 4, bottom, the difference between the control diameter recorded after treatment with L-NNA and that before treatment with L-NNA is plotted against the maximum diameter. The effect of L-NNA on control diameter is related to vessel size and is greater in larger than in smaller arterioles. Effect of L-NNA on the arteriolar response to moderate hypoxia. The effect of L-NNA on the diaphragmatic arteriolar response to moderate hypoxia in the group 1 animals is illustrated in Fig. 5. In Fig. 5, top, the change in arteriolar diameter from control conditions during moderate hypoxia before and after infusion of L-NNA is plotted against the maximum diameter. Both before and after L-NNA, there is an inverse relationship between change in diameter and vessel size. Moderate hypoxia produces a greater response in smaller vessels than in larger vessels. In contrast to the effect on the response to SNP, however, larger vessels exhibit a smaller change in diameter during moderate hypoxia after treatment with L-NNA than before treatment. The response of smaller vessels to moderate hypoxia is relatively unchanged after L-NNA. As a result, the slope of this relationship during moderate hypoxia is steeper (more negative) after treatment with L-NNA. The effect of L-NNA on the response to moderate hypoxia is opposite of that on the response to SNP and represents the net influence of two effects. First, the control diameter of larger vessels is smaller (see Effect of L-NNA on control diameters) after treatment with L-NNA. This cannot be the only contributing factor, however, because, if it were, the result would have been similar to the effect of L-NNA on the response to SNP. L-NNA must, therefore, have exerted a second effect. Specifically, the diameter achieved by these vessels during moderate hypoxia must have been reduced.
In Fig. 5, bottom, the difference between the arteriolar diameter during moderate hypoxia after L-NNA and that before L-NNA is plotted against maximum diameter. This plot confirms that L-NNA reduces the diameter to which arterioles dilate during moderate hypoxia and that this effect is related to vessel size (i.e., L-NNA reduced the diameter of larger vessels to a greater extent than that of smaller vessels). Effect of L-NNA on the arteriolar response to severe hypoxia. Figure 6 illustrates the arteriolar response to severe hypoxia in the group 1 animals. In Fig. 6, top, the change in arteriolar diameter from the control value during severe hypoxia is plotted against maximum diameter. Both before and after treatment with L-NNA, smaller vessels exhibit a greater response to severe hypoxia than do larger vessels, and a negative correlation between the response to hypoxia and maximum vessel diameter exists. As during SNP infusion, and in contrast to the result obtained during moderate hypoxia, the slope of this relationship is less steep (i.e., less negative) after L-NNA than before L-NNA. In Fig. 6, bottom, the difference between arteriolar diameters during severe hypoxia before and after L-NNA is plotted against maximum diameter. Treatment with L-NNA does not affect the diameter to which diaphragmatic arterioles dilate during severe hypoxia. The effect of L-NNA on the change in diameter during severe hypoxia is, therefore, attributable to the selective reduction in the control diameter of larger vessels.
Group 2
During autoperfusion with arterial blood, Pphr for the group 2 animals was 126 ± 5 mmHg andphr averaged 13 ± 1.2 ml · min1 · 100 g1. Phrenic arterial
PO2,
PCO2, and pH under control conditions
were 104 ± 5 Torr, 42 ± 3 Torr, and 7.38 ± 0.02, respectively. During moderate hypoxia,
PvO2 was 25.5 ± 2 Torr, whereas
phrenic venous PCO2 and pH did not
differ from the control values. In animals pretreated with
indomethacin, diaphragmatic blood flow during moderate hypoxia was
similar to that recorded in the animals that were not treated with
indomethacin (16.5 ± 1.3 and 17.0 ± 1.2 ml · min1 · 100 g1, respectively).
L-NNA completely inhibited
vasodilation during moderate hypoxia in both indomethacin-treated and
untreated animals. L-NNA had no
effect on diaphragmatic blood flow during infusion of
PGE2 in either group (28.6 ± 2.0 vs. 27.8 ± 2.2 and 26.2 ± 2.2 vs. 27.4 ± 2.0 ml · min1 · 100 g1 in indomethacin-treated
and untreated animals, respectively).
DISCUSSION
The main findings of this study are that treatment of the diaphragmatic circulation with L-NNA 1) reduced diaphragmatic blood flow under control conditions and during moderate hypoxia, 2) had no effect on the diameter to which arterioles dilated during SNP infusion, 3) produced arteriolar vasoconstriction under control conditions that was related to vessel size, 4) inhibited the increase in diameter to which arterioles dilated during moderate hypoxia by an amount that was related to vessel size, and 5) had no effect on the diameter to which arterioles dilated during severe hypoxia.
Critique
The interpretation of the present findings depends on the specificity of L-NNA as an inhibitor of NO synthesis. In arterioles isolated from rat cremaster muscle, Koller et al. (15) found that L-NNA (104 to
103 M) inhibited the peak
arteriolar dilation to PGE2 and
proposed that L-NNA may inhibit
endothelial synthesis and/or the action of prostaglandins. In
the present study, L-NNA did not
affect the vascular response to
PGE2 and the change in
diaphragmatic blood flow during moderate hypoxia was not altered by
indomethacin. It is unlikely, therefore, that the effects of
L-NNA in the present study are
due to inhibition of prostaglandin-mediated dilation. Differences in
experimental conditions, particularly species and route of drug
administration, may explain this variation in results.
Although we have assumed that it is endothelial NO synthesis that is being inhibited by L-NNA, NO release by skeletal muscle has also been noted (3). In diaphragmatic myocytes, the neuronal isoform of NO synthase has been localized to the cell membrane (13, 14) and the endothelial isoform colocalizes with mitochondrial markers (14). It is possible, therefore, that inhibition of NO formation at these sites may have contributed to the observed effects.
Effect of L-NNA on the Response to SNP
L-NNA infusion altered neither the maximal diaphragmatic blood flow nor the maximum arteriolar diameters that could be attained by SNP infusion. Previous studies have demonstrated that inhibition of NO synthesis enhances sensitivity of smooth muscle to SNP and other nitrovasodilators (18). Increased soluble guanylate cyclase activity was proposed as the mechanism underlying this effect. As in the present study, however, inhibition of NO synthesis did not alter the maximum response to exogenous NO donors in these previous reports. The failure of L-NNA to alter the diameter of small arterioles under any of the conditions presented in the present study suggests that regulation of these vessels depends on mechanisms other than modulation of NO release. Because dilation of these vessels was elicited by SNP, this difference in the regulatory importance of the NO pathway among vessels of different sizes does not appear to be attributable to the impairment of guanylate cyclase activation or to events distal to this enzyme.Effect of L-NNA on Control Measurements
In the only previous study that has assessed the role of the NO pathway in the regulation of diaphragmatic arteriolar diameter, Boczkowski et al. (4) found that superfusion of the in vivo autoperfused rat diaphragm with L-NNA reduced the baseline diameter of second-order (40- to 45-µm) arterioles. Arterioles outside this range were not evaluated. ACh-induced dilation was not affected by superfusion with L-NNA in their preparation but was inhibited by combined treatment with L-NNA and indomethacin. Their findings contrast with those of the present study in which indomethacin altered neither the response to Ach nor the efficacy of L-NNA in reversing its effect. Interspecies variability, method of delivery of L-NNA and ACh, and the use of barbiturate rather than-chloralose anesthesia (8) in the rat
studies compared with the present canine experiments may account for
these discrepancies. The present findings extend those of Boczkowski et
al. (4) regarding the effect of inhibition of NO synthesis on
diaphragmatic arteriolar diameter under control conditions in that this
effect was demonstrated to be related to vessel size.
Studies in other preparations suggest that variation in the importance of the NO pathway among arterioles of different sizes may be a property common to a variety of tissues. Sun et al. (26) demonstrated that L-NNA reduces the baseline diameter in near-resistance arterioles (~100 µm) isolated from rat skeletal muscle. Terminal arteriolar diameters, in contrast, were found by Persson et al. (22) to be unaffected by NO inhibition. In cat skeletal muscle, Ekelund and Mellander (6) found that NG-monomethyl-L-arginine increased resistance preferentially in larger resistance arterioles (>25 µm). In the hamster cremaster muscle, Hester et al. (11) reported that NO inhibition with NG-nitro-L-arginine methyl ester decreased both resting diameter and functional dilation (active hyperemia) of first-order (65-µm) arterioles, resting diameter but not active dilation in second-order (45-µm) arterioles, and neither resting diameter nor active dilation in third-order (30-µm) arterioles. The present results extend these previous observations to include the canine diaphragm and, therefore, add support to the suggestion that a longitudinal gradient in the role of basal NO release may be a property of microcirculatory regulation in general (22).
Effect of L-NNA on the Response to Hypoxia
The role of the NO pathway in regulating the vascular response to hypoxia remains controversial. In support of a role for NO, relaxation of rabbit arterial rings during hypoxia is inhibited by NO scavengers (24). Similarly, Graser and Rubanyi (9) found that hypoxic dilation of the rat aorta is mediated by the release of NO and not by products of the cyclooxygenase pathway. In the guinea pig coronary circulation, the vasodilatory response to hypoxia is attenuated by L-arginine analogues (5, 21, 29) and release of guanosine 3
,5-cyclic monophosphate from the guinea pig heart increases during hypoxic perfusion (21). In other
preparations, however, participation of NO in the response to hypoxia
has been excluded. In arterioles isolated from rat cremaster muscle,
Messina et. al (17) found that inhibition of prostaglandin synthesis
completely suppresses dilation during superfusion with hypoxic buffer.
Similarly, Fredricks et al. (7) found that cyclooxygenase products are
the predominant mediators of hypoxic vasodilation in small arteries
(100-300 µm) from rat gracilis muscle and that inhibition of NO
synthesis had no effect. In dogs, Vallet et al. (27) reported that
systemic infusion of
NG-nitro-L-arginine
methyl ester had no effect on the vascular response of the hindlimb to
hypoxic perfusion. Factors that may have contributed to this
variability in results include differences in the species studied, the
blood vessel size and tissue of origin, and the severity of the hypoxic
stimulus.
It is because of the inability to generalize conclusions drawn from the current literature and because of the pivotal role that the NO pathway has been found to play in metabolic regulation of diaphragmatic blood flow (12) that the need for specific evaluation of the effect of the inhibition of NO synthesis on the response to hypoxia in this circulation was identified. The present results extend the known participation of NO release in hypoxic vascular responses to the canine diaphragmatic microcirculation. This study further adds to the existing knowledge concerning the role of NO in this setting by demonstrating that NO release is involved in the dilation of arterioles during moderate but not severe hypoxia and that the importance of this mechanism is in proportion to vessel size. The finding that linear heterogeneity exists in the role of the NO pathway in regulating the microvascular response to reductions in O2 availability complements the previous observation that a similar gradient exists in the contribution of this pathway to the response to increased metabolic activity (11).
The disparate effects of inhibition of NO synthesis at different levels of hypoxia suggest a change in the mechanisms regulating arteriolar smooth muscle tone as the imbalance between O2 availability and metabolic demand becomes more profound. In larger vessels, moderate reductions in luminal PO2 may serve as a stimulus for endothelial release of NO either directly through an endothelial O2-sensing mechanism (23) or because of downstream vasodilation with flow-induced NO-mediated recruitment of upstream arterioles. During severe hypoxia, L-NNA had no effect on diaphragmatic arteriolar diameters. Either dilation due to activation of other regulatory mechanisms was maximal and any further inhibitory influence of endothelium-derived NO was obscured or NO synthesis is limited by its dependence on O2 availability and does not normally mediate arteriolar dilation under such extreme conditions. Because the degree of dilation was not maximal (i.e., in comparison with the response to SNP), it is more likely that PO2 is a rate-limiting variable in NO production. This conclusion is compatible with observations in other preparations. For example, NO synthase activity in the bovine cerebellum is inhibited when environmental PO2 is reduced to 28 Torr (25). Similar conclusions have been drawn from studies in vascular tissues. Vedernikov and Hellstrand (28) found that ACh-induced relaxation of rabbit arterial rings is unaffected during exposure to 16% O2 but is eliminated at O2 concentrations below 4%. Similarly, Hashimoto et al. (10) have shown that hypoxia inhibits endothelium-dependent dilation of porcine coronary arteries in a biphasic manner. A rapid component is inhibited at PO2 values < 35 Torr, whereas a secondary slow component of the response persists at a PO2 of 25 Torr. Further studies are indicated to confirm this hypothesis and to determine the level of hypoxia at which O2 availability begins to limit the potential for participation of the NO pathway in the regulation of the diaphragmatic circulation.
ACKNOWLEDGEMENTS
The author thanks J. Petrella for expert technical assistance.
FOOTNOTES
Address for reprint requests: M. E. Ward, Rm. L3.05, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada (E-mail: mward{at}rvhmed.lan.mcgill.ca).
Received 12 September 1995; accepted in final form 6 June 1996.
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