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Divisions of Pulmonary and Critical Care Medicine, Royal Victoria Hospital and Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The in vitro responses to ACh, flow, and
hypoxia were studied in arterioles isolated from the diaphragms of
rats. The endothelium was removed in some vessels by low-pressure air
perfusion. In endothelium-intact arterioles, pressurized to 70 mmHg in
the absence of luminal flow, ACh
(10
5 M) elicited dilation
(from 103 ± 10 to 156 ± 13 µm). The response to ACh was
eliminated by endothelial ablation and by the nitric oxide synthase
antagonists NG-nitro-L-arginine
(L-NNA;
105 M) and
NG-nitro-L-arginine methyl ester
(L-NAME,
105 M) but not by
indomethacin (105 M).
Increases in luminal flow (5-35 µl/min in 5 µl/min steps) at
constant distending pressure (70 mmHg) elicited dilation (from 98 ± 8 to 159 ± 12 µm) in endothelium-intact arterioles. The response to flow was partially inhibited by
L-NNA,
L-NAME, and indomethacin and
eliminated by endothelial ablation and by concurrent treatment with
L-NAME and indomethacin. The
response to hypoxia was determined by reducing the periarteriolar
PO2 from 100 to 25-30 Torr by
changing the composition of the gas used to bubble the superfusing
solution. Hypoxia elicited dilation (from 110 ± 9 to 165 ± 12 µm) in endothelium-intact arterioles but not in arterioles from which
the endothelium had been removed. Hypoxic vasodilation was eliminated
by treatment with indomethacin and was not affected by
L-NAME or
L-NNA. In rat diaphragmatic
arterioles, the response to ACh is dependent on endothelial nitric
oxide release, whereas the response to hypoxia is mediated by
endothelium-derived prostaglandins. Flow-dilation requires that both
nitric oxide and cyclooxygenase pathways be intact.
respiratory muscles; blood flow; autoregulation; vascular smooth muscle
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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TO GENERATE THE RESPIRATORY pressure swings necessary to sustain spontaneous ventilation, substrate and oxygen delivery to the diaphragm must be maintained in proportion to its metabolic demand. Many of the vascular responses that preserve this balance are now recognized to be mediated by mechanisms localized to the endothelium. Of these, the vasodilator responses to flow and hypoxia are of particular relevance. Flow dilation is required to maximize perfusion and to prevent unopposed myogenic vasoconstriction from overriding metabolic regulatory mechanisms (21). Hypoxic vasodilation permits diaphragmatic oxygenation to be maintained during periods when systemic oxygen delivery is declining (35).
In most preparations, the mechanisms that mediate endothelium-dependent vasoregulation include modulation of the release of nitric oxide (NO), vasodilator prostaglandins, or both (16). In rat arterioles isolated from nondiaphragmatic skeletal muscle, inhibition of NO synthesis has no effect on hypoxic vasodilation, whereas, on the contrary, cyclooxygenase inhibitors block the hypoxic response (24). In arterioles from rat hindlimb and cremaster muscles, flow dilation is also consistently reported to involve vasodilator prostanoid release, whereas the role of NO varies between these circulations (16, 17). To the extent that endothelium-dependent responses in the phrenic circulation resemble those in other muscles, these results suggest that inhibition of NO release will have little effect on the response to hypoxia in diaphragmatic arterioles but may decrease the response to flow. In contrast, inhibiting prostaglandin release should block hypoxic vasodilation and eliminate or significantly attenuate flow dilation. Such extrapolation is risky, however, because the effects of inhibitors of nitric oxide synthase (NOS) and cyclooxygenase on basal diameter and on the response to the endothelium-dependent vasodilator ACh in diaphragmatic microvessels have been reported to differ from their effects on arterioles in peripheral skeletal muscles (5). Therefore, the mechanisms that mediate the responses to physiological stimuli in diaphragmatic arterioles may also differ and must be specifically determined. Accordingly, the present study was carried out to evaluate the role of the endothelium in flow- and hypoxia-induced vasorelaxation in diaphragmatic arterioles and to use available pharmacological probes to determine the relative contribution of NO and prostaglandin release in mediating these responses.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Arteriole Isolation
Studies were performed on diaphragmatic arterioles from male Sprague-Dawley rats (200-250 g). One arteriole from each rat was used. The diaphragms were removed immediately after decapitation of animals and placed in cold (0-4°C) oxygenated, bicarbonate-buffered, physiological salt solution [PSS; (in mM) 119 NaCl, 4.7 KCl, 3.5 MgSO4, 24.9 NaHCO3, 3.7 CaCl2, 1.18 KH2PO4, and 5 glucose, pH 7.4, as well as 400 µM EDTA]. The muscle was pinned to the bottom of the silicone-lined base of a dissecting dish. A first-order arteriole, identified as the first major arteriole penetrating the muscle, was selected from the costal part of the diaphragm. A segment 1 mm in length was cleared from the adhering tissue and transferred to a Plexiglas vessel chamber (Living Systems, Burlington, VT) containing PSS. Inflow and outflow micropipettes were matched for resistance to flow, and the system was arranged to have mirror symmetry, with the axis of symmetry located at the middle of the arteriolar segment. This resulted in equal resistances of the two sides of the system (from pressure transducer to the tip of the pipette). The proximal end of the arteriole was mounted to the inflow cannula and secured with 12-0 suture. The perfusion pressure was then increased to 20 mmHg with a pressure-servo micropump system (Living Systems) taking its inflow from a reservoir of PSS. After the arteriole was cleared of clotted blood, its distal end was mounted to the outflow cannulas. Both the inflow and outflow cannulas were connected to microflow pumps (Living Systems). The inflow pump was a calibrated constant-flow pump, whereas the outflow pump was regulated by a servo system to maintain a constant downstream pressure. This allowed flow to be established by changing proximal and distal pressures by an equal amount but in opposite directions, such that the average of the upstream and downstream pressures (midpoint luminal pressure) remained constant (19). In preliminary studies we measured midpoint intraluminal pressure by micropuncture by using a servo-null pressure transducer (micropressure system model 900, WPI) over a pressure range of 0 to 100 mmHg at each of the flows used in the present study. As others have reported previously (19), we found no difference between the average of upstream and downstream pressures and the directly measured midpoint pressure at any of the flow rates studied. The arteriole was set to its in situ length by using an eyepiece micrometer. The outflow cannula was closed, and the transmural pressure (i.e., intraluminal pressure relative to atmospheric pressure) was slowly increased to 70 mmHg by adjusting the downstream pressure target for the servomechanism regulating the outflow pump. This pressure was chosen because previous micropuncture studies have documented intraluminal pressures in this range in rat skeletal muscle arterioles of this size in vivo (20). The pressure-servo system was then placed in manual mode, whereby a stable pressure value indicated that there was no leak in the system. Vessels in which a leak was detected were discarded.The apparatus was transferred to an inverted microscope (Nikon TMS-F,
×20 objective). Measurements of internal diameter were made by
using a high-resolution charge-coupled device video camera (Hitachi
KPC503) and a video caliper (Living Systems) calibrated by using a
stage micrometer. The vessel was continuously superfused with PSS
flowing through the chamber at a rate of 6 ml/min. The chamber was
warmed to 37°C by using a heat exchanger in line with the
superfusion pump over 60 min and maintained at this temperature throughout the experimental protocol. Chamber temperature and pH were
monitored continuously by using a probe (Oakton series 35616, Singapore), and samples of the superfusing buffer were periodically
drawn from the chamber for gas analysis (model 995, AVL Instruments,
Graz, Austria). A Plexiglas cover excluded ambient air from the
chamber. During the 1-h stabilization period, the reservoir containing
the superfusate and the vessel chamber itself were bubbled with gas,
the composition of which was adjusted, using separate tanks and
regulators for O2,
CO2, and
N2, to achieve PO2 and
PCO2 values in the vessel chamber of 100 and 40 Torr, respectively. Under these conditions, the vessels gradually developed spontaneous tone independently of vasoconstrictor agents. Endothelium-dependent and -independent dilation were evaluated in all vessels by determining the ability of ACh
(105 M, Sigma Chemical) and
sodium
1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO; 104 M, Research
Biochemicals International), respectively, to inhibit intrinsic tone.
On completion of each experiment, the internal diameter was recorded
with the vessels in the passive state at a distending pressure of 70 mmHg. The passive state was achieved by bathing the arterioles in
calcium-free PSS containing EGTA (4 mM) and adenosine
(104 M).
Endothelial Removal
In vessels in which the endothelium was to be removed, the responses to ACh and DEA/NO were evaluated after the initial equilibration period as above. The intraluminal pressure was reduced to 20 mmHg, the stopcock on the outflow cannula was opened, and the arterioles were perfused with 2 ml air. The arterioles were then perfused with PSS for 10-15 min at 20 mmHg to flush the separated endothelial layer from the vessel lumen and out of the cannula system. The outflow cannula was closed, the intraluminal pressure was restored to 70 mmHg, and the dilatory responses to ACh and DEA/NO were once again determined. In previous histological studies (23), elimination of vasodilation in response to ACh with an intact vasodilator response to NO donors after this procedure has been shown to be associated with ablation of the endothelial cell layer and, in the present study, was taken as evidence of successful endothelial removal.Experimental Protocols
Effect of L-arginine analogs on the responses to ACh, DEA/NO, and prostaglandin E2 (PGE2). In initial experiments, the effect of the NOS antagonists NG-nitro-L-arginine (L-NNA) and NG-nitro-L-arginine methyl ester (L-NAME) on the dilatory response to ACh, DEA/NO, and PGE2 were evaluated to determine whether differences in responsiveness to flow or hypoxia after treatment with these agents could be attributed to nonspecific effects.
Studies were carried out in arterioles in which the endothelium was intact, in the absence of luminal perfusion. At the end of the equilibration period, a baseline diameter at 70 mmHg intraluminal pressure was recorded. The responses to ACh (105 M), DEA/NO
(104 M), and
PGE2
(109 M) were then evaluated
by adding these agents to the superfusate. The internal diameter was
recorded after the arterioles had reached steady state. The order of
exposure to ACh, DEA/NO, and PGE2
was randomized, and vessels were allowed to return to their baseline diameters before exposure to the next agent. Either
L-NAME
(105 M,
n = 7) or
L-NNA
(105 M,
n = 7) was then added to the
superfusate. The baseline diameter was recorded, and the responses to
ACh, DEA/NO, and PGE2 were determined again in the presence of the antagonist.
Effects of indomethacin on the responses to ACh and DEA/NO. Previously, elimination of the dilatory response of rat diaphragmatic arterioles to ACh in vivo was found to require concurrent inhibition of NOS and cyclooxygenase (5). Experiments were, therefore, included to determine whether the dilatory response to ACh is altered in these vessels by treatment with indomethacin and whether or not inhibition of cyclooxygenase affects the ability to suppress this response by treatment with L-NAME.
Studies were carried out in arterioles in which the endothelium was intact, at 70 mmHg intraluminal pressure, in the absence of luminal perfusion. Reproducibility of ACh- and DEA/NO-induced vasodilation was tested in five arterioles. After the initial stabilization period, the responses to ACh (105 M)
and DEA/NO (104 M) were
assessed in random order, and the vessels were allowed to return to
their baseline diameters before addition of the second agent. This
procedure was repeated twice, for a total of three measurements of the
response to each agent. In a separate group of arterioles
(n = 7), the responses to ACh
(105 M) and DEA/NO
(104 M) were evaluated, and
then indomethacin (105 M)
was added to the superfusate. The baseline diameter was recorded, and
the vasodilator responses to ACh and DEA/NO were again determined. L-NAME
(105 M) was then added to
the superfusate. After the baseline diameter in the presence of both
antagonists was recorded, the responses to ACh and DEA/NO were reassessed.
Response to flow.
Flow-diameter relationships were obtained in seven arterioles in which
the endothelium was left intact and in seven arterioles in which the
endothelium had been removed. Perfusate flow was increased from 0 to 35 µl/min in 5 µl/min steps, whereas the intraluminal pressure at 70 mmHg was maintained. Diameter was recorded after the vessel had reached
steady state, 10 min after each change in flow. In separate groups of
endothelium-intact arterioles (n = 7/group), indomethacin
(105 M),
L-NAME
(105 M), or
L-NNA
(105 M), or else both
L-NAME and indomethacin
(105 M for both,
n = 7) were added to the superfusion
and perfusion solutions. After a further 15-min equilibration period,
the response to increasing flow over the range from 0 to 35 µl/min at
a distending pressure of 70 mmHg was determined.
Response to hypoxia. After the baseline diameter in the absence of flow was recorded, at an intraluminal pressure of 70 mmHg and a chamber PO2 of 100 Torr, arterioles were exposed to hypoxia by bubbling the superfusate and the vessel chamber with a gas mixture containing no oxygen (chamber PO2 = 25-30 Torr). The flow rate of CO2 was adjusted to maintain PCO2 in the vessel chamber at 40 Torr. Internal diameter was recorded after the arteriole had reached steady state ~20 min after the composition of the gas used to bubble the superfusate was changed. The PO2 in the chamber was then returned to 100 Torr, and vessel diameter was allowed to return to its previous baseline value. Reproducibility of the hypoxic response was tested in five arterioles in which the endothelium was left intact. Three hypoxic exposures were undertaken. Diameter was allowed to return to its previous baseline value at a chamber PO2 of 100 Torr between each hypoxic epoch.
In seven arterioles the baseline diameter at a PO2 of 100 Torr was recorded. The gas mixture bubbling the perfusate and the vessel chamber was switched to 0% oxygen. Steady-state diameter under hypoxic conditions was recorded, the PO2 in the vessel chamber was restored to 100 Torr, and the diameter was allowed to return to its previous baseline value. The endothelium was then removed as described above. The baseline diameter at a PO2 of 100 Torr was recorded, and the arterioles were exposed to hypoxia a second time. The chamber PO2 was restored to 100 Torr, and the vessels were allowed to return to their previous baseline diameters. L-NAME (105 M) and indomethacin
(105 M) were then added to
the superfusate, and the response to hypoxia was again recorded in
deendothelialized arterioles in the presence of both antagonists.
In 21 arterioles in which the endothelium was left intact, the baseline
diameter at a PO2 of 100 Torr was
recorded, the gas bubbling the superfusate and vessel chamber was
switched to 0% O2, and the
response to hypoxia was determined. The vessel chamber
PO2 was then restored to 100 Torr,
and the diameter was allowed to return to its previous baseline value.
Either indomethacin (105 M,
n = 7),
L-NAME
(105 M,
n = 7), or
L-NNA
(105 M,
n = 7) was added to the superfusate,
and the baseline diameter in the presence of the antagonist at a
chamber PO2 of 100 Torr was recorded.
The gas mixture used to bubble the reservoir and the vessel chamber was
again switched to 0% O2, and the
response to hypoxia was recorded. In the vessels treated with
indomethacin, L-NAME
(105 M) was then added to
the superfusate. The baseline diameter was measured, and the response
to hypoxia was determined in the presence of both antagonists.
Data Analysis
Comparisons of multiple means were performed by ANOVA corrected for repeated measures when appropriate. If the ANOVA revealed significant overall differences, variations among individual means were evaluated post hoc by using the Student-Newman-Keuls procedure. Results are expressed as the means ± SE for n number of arterioles, with P < 0.05 representing significance.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The internal diameters of the arterioles used in this study averaged
103 ± 10 µm at the end of the initial equilibration period. This
value is less than the diameter recorded under passive conditions at an
intraluminal pressure of 70 mmHg (179 ± 12 µm), indicating that
by the end of the equilibration period the arterioles had developed
spontaneous tone. The average diameters during relaxation with ACh (156 ± 13 µm) and DEA/NO (172 ± 11 µm) were also significantly greater than at the end of the equilibration period. In arterioles from
which the endothelium was to be removed, ACh elicited an average change
in diameter equal to 49 ± 8% of the baseline diameter before the
procedure and 
6.4 ± 4% after endothelial ablation. The
average diameter during treatment with DEA/NO did not differ from that
recorded under passive conditions.
Effects of L-NAME and L-NNA on the Responses to ACh, DEA/NO, and PGE2
The effects of treatment with L-NAME and L-NNA on baseline diameter and on the responses to ACh, DEA/NO, and PGE2 are presented in Fig. 1, top. Both L-NAME and L-NNA decreased baseline diameter and completely blocked ACh-induced dilation without significantly affecting the dilatory response to DEA/NO or PGE2.
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Effects of Indomethacin on the Responses to ACh and DEA/NO
After stabilization at a chamber PO2 of 100 Torr and a distending pressure of 70 mmHg, the diameter of the five endothelialized arterioles in which reproducibility of ACh-induced dilation was assessed averaged 95 ± 11 µm. During the first, second, and third exposures to ACh, these arterioles dilated by 44 ± 5, 43 ± 7, and 46 ± 6% of baseline diameter, respectively. During all exposures to DEA/NO, tone was eliminated and the diameters did not differ from those recorded under passive conditions. Diameters of the seven arterioles in which the responses to ACh and DEA/NO were tested before and after treatment with indomethacin and during treatment with the combination of indomethacin and L-NAME are presented in Fig. 1, bottom. Indomethacin did not alter the response to ACh or DEA/NO. Simultaneous treatment with indomethacin and L-NAME decreased baseline diameter and completely blocked ACh-induced dilation without affecting the dilatory response to DEA/NO. The effects of concurrent treatment with indomethacin and L-NAME on baseline diameter and on the response to ACh did not differ from those in arterioles treated with L-NAME alone (Fig. 1, top).Response to Flow
In Fig. 2, the relationships between flow and diameter in endothelialized and deendothelialized arterioles are compared. In endothelialized vessels, flow elicited dilation, whereas in deendothelialized arterioles increasing flow had no effect on diameter. Figure 3 illustrates the effect of treatment with L-NAME, L-NNA, indomethacin, and both indomethacin and L-NAME on the response to increasing flow in arterioles in which the endothelium was left intact. Lines representing the flow-diameter relationships recorded in endothelialized and deendothelialized arterioles (data presented in Fig. 2) are included in Fig. 3 for reference. The response to flow was attenuated (difference from untreated endothelium-intact arterioles, P < 0.05) but not eliminated in vessels treated with either L-arginine analogs or with indomethacin. Inhibition of both NO and prostaglandin release by concurrent treatment with L-NAME and indomethacin eliminated the flow response and mimicked the effect of endothelial ablation (P > 0.05 for difference from endothelium-denuded arterioles).
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Response to Hypoxia
After stabilization at a chamber PO2 of 100 Torr and a distending pressure of 70 mmHg, the diameter of the five endothelialized arterioles in which reproducibility of hypoxic dilation was assessed averaged 102 ± 13 µm. During the first, second, and third hypoxic exposures, these arterioles dilated by 61 ± 5, 62 ± 7, and 64 ± 5% of baseline diameter, respectively.In Fig. 4,
top, the results of the studies done
on arterioles in which the endothelium was removed before the second
hypoxic exposure are presented. No significant response to hypoxia was observed after removal of the endothelium. Concurrent treatment with
L-NAME and indomethacin had no
effect on the diameter under either normoxic or hypoxic conditions
after endothelial ablation. Figure 4,
middle, illustrates the effects of
L-NAME and
L-NNA on baseline diameter and
the dilatory response to hypoxia in arterioles in which the endothelium
was left intact. Both L-arginine
analogs decreased the baseline diameter under normoxic conditions, but neither agent significantly altered the response to hypoxia. In Fig. 4,
bottom, the effects of treatment with
indomethacin alone and of concurrent treatment with indomethacin and
L-NAME in endothelium-intact arterioles are illustrated. Treatment with indomethacin abolished hypoxic vasodilation. Treatment with
L-NAME in the presence of indomethacin reduced the baseline diameter but had no additional effect
on the diameter during exposure to hypoxia.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, the effects of inhibiting NO and prostaglandin release on the endothelium-dependent dilatory responses to ACh, flow, and hypoxia were compared. The results show that in isolated diaphragmatic arterioles 1) vasodilation in response to ACh is abolished by inhibition of NO release with L-arginine analogs and is not affected by inhibiting cyclooxygenase with indomethacin, 2) flow dilation is endothelium dependent and mediated by corelease of NO and prostaglandins, and 3) hypoxic vasorelaxation is mediated by endothelium-derived vasodilator prostaglandins and is not affected by inhibition of NO release.
The effects of inhibition of cyclooxygenase and NOS have been studied previously in the rat diaphragmatic microcirculation (5). In that study, Boczkowski et al. (5) determined the effects of topical application of L-NNA and the cyclooxygenase inhibitor mefanamic acid on the diaphragmatic microvasculature by in vivo microscopy. L-NNA and, to a lesser extent mefanamic acid, reduced the baseline diameter of second-order arterioles (baseline luminal diameters of 40-45 µm), and the combined effect of these agents was greater than the sum of their individual effects. Neither L-NNA nor mefanamic acid inhibited ACh-induced dilation; however, this response was completely eliminated by the simultaneous application of both antagonists. These findings were unexpected because neither synergistic interaction between the effects of inhibitors of NOS and cyclooxygenase on basal diameter nor failure of L-NNA to inhibit the response to ACh is observed in arterioles from peripheral skeletal muscles. The discrepancy was attributable to heterogeneity in the mechanisms by which endothelium-dependent responses are mediated among vascular beds (5). In the present study, arterioles were isolated from neurohumoral influences, regional hemodynamic differences related to the geometry of the microvascular network, and the effects of metabolic mediators arising from the surrounding striated muscle. Under these conditions, baseline diaphragmatic arteriolar diameter did not change significantly after treatment with indomethacin. L-Arginine analogs reduced basal diameter; however, indomethacin did not accentuate this effect. Furthermore, both L-NAME and L-NNA eliminated the dilatory response to ACh even in the absence of concurrent inhibition of cyclooxygenase. These results resemble, qualitatively, those observed in arterioles from other rat skeletal muscle (15). The differences between our present results and those obtained in vivo indicate that extrinsic regulatory influences are important determinants of basal tone and the response to ACh in the intact diaphragmatic circulation. Therefore, the role of the endothelium in the responses to physiological stimuli, as determined in the present study, may also differ from that observed in vivo, where the effects of hypoxia and flow interact with those of other stimuli. Nonetheless, understanding these interactions requires that the contributing elements initially be separated, and our study provides valuable information concerning the mechanisms intrinsic to the arteriolar wall that participate in these responses.
The mechanisms by which flow elicits vasodilation remain a topic of controversy. In in vitro studies in rabbit ear arterial network (12), canine femoral artery segments (30), piglet cerebral artery segments (31), and porcine coronary arterioles (18), flow dilation has been abolished by inhibitors of NOS, suggesting exclusive NO-related mediation of the response. In arterioles from the rat cremaster muscle, in contrast, indomethacin was found to eliminate flow dilation (17), and it was concluded that, in this vascular bed, the response was entirely dependent on prostaglandin release. Both of these results contrast with subsequent findings (16) in arterioles from rat gracilis muscle, in which the flow response was attenuated by both L-NNA and indomethacin and eliminated when these antagonists were given concurrently. Regulation of arteriolar tone by flow is even more complex in other vascular beds. In rat basilar artery (10) and feline femoral artery (22), inhibitors of cyclooxygenase and NOS do not entirely eliminate flow dilation, and involvement of as yet unknown, nonprostanoid, non-NO-related endothelium-derived mediators has been proposed. In studies in isolated rabbit ear and cerebral artery segments (4, 11), furthermore, endothelial ablation did not abolish flow relaxation, indicating that mechanisms localized to the arteriolar smooth muscle may also participate and that the release of endothelium-derived mediators is not, necessarily, obligatory. This lack of consistency highlights the importance of interpreting these data in the context of the experimental conditions under which they were obtained. The results of the present study demonstrate that, in arterioles from the rat diaphragmatic circulation pressurized in vitro to 70 mmHg, flow dilation is endothelium dependent and that both NO and prostaglandin release must be intact for the full response to occur. Among the vascular beds previously studied, therefore, the mechanisms that mediate flow dilation in diaphragmatic arterioles most resemble those identified in arterioles from limb muscle (16).
In isolated skeletal muscle arterioles, reductions in ambient PO2 were found to elicit dilation, which was entirely eliminated by removal of the endothelium (23). The present findings demonstrate a similar predominance of endothelium-dependent mechanisms in hypoxic dilation in diaphragmatic arterioles. Previously, studies in rabbit aortic and femoral artery segments (28) have shown that the NO scavengers hemoglobin and dithiothreitol inhibit endothelium-dependent hypoxic relaxation. In addition, in guinea pig heart (7, 27) and canine diaphragm (34) perfused at constant pressure, L-arginine analogs inhibit the increase in flow that accompanies reductions in oxygenation. These results suggest that NO is responsible, at least in part, for hypoxic vasodilation. Others (33) have challenged this conclusion on the basis of the lack of specificity of NO scavengers. Furthermore, in organs perfused at constant pressure, the increase in flow that accompanies hypoxia will, itself, stimulate endothelial NO release (16). L-Arginine analogs could, therefore, impair dilation in these preparations through inhibition of flow-mediated relaxation rather than by blockage of hypoxia-induced NO synthesis. In the present study, which was carried out at zero flow, neither L-NNA nor L-NAME altered the response to hypoxia. This finding argues strongly against a primary role for stimulation of NO synthesis in hypoxic dilation in rat diaphragmatic arterioles.
Controversy also exists concerning the participation of vasodilator prostaglandins in hypoxic vasorelaxation. There is evidence to support a role for involvement of the cyclooxygenase pathway in coronary vasodilation in response to hypoxia in rabbit (26) and rat (1, 3) heart as well as in isolated large (8, 29, 32)- and small (9, 23)-vessel segments. Conversely, indomethacin infusion did not alter the magnitude of hypoxic coronary dilation in the isolated canine (13) or guinea pig (6, 7) heart. The studies that may be most directly compared with the present experiments are those of Fredricks et al. (9) in small arteries from rat gracilis muscle and of Messina et al. (23, 25) that were carried out in arterioles isolated from the rat cremaster muscle. Indomethacin was found to inhibit dilation of isolated gracilis muscle arteries (9) and to completely eliminate the hypoxic response in cremaster muscle arterioles (23). In the present study, treatment with indomethacin inhibited the response and mimicked the effect of endothelial ablation. In agreement with the conclusions of previous studies in arterioles from other rat skeletal muscles, therefore, we found that the hypoxic response is mediated by endothelial prostaglandin release. The present results extend those of the previous studies and enhance their significance by demonstrating the contribution of this mechanism to hypoxic vasodilation in diaphragmatic arterioles.
Diaphragm function is insensitive to changes in oxygen supply over a broad range (2). This is because of potent vascular responses that defend diaphragmatic oxygenation during periods of reduced systemic oxygen delivery. Mediators released by the endothelium have been found to play key roles in several of these responses, including active hyperemia (14), reactive hyperemia (37), and autoregulation of diaphragm blood flow (36). In each case, changes in flow and periarteriolar PO2 serve as important regulatory stimuli. Our present results indicate that both of these variables exert their influence on diaphragmatic arteriolar tone through modulation of endothelium-derived relaxing factor release. The endothelium, therefore, plays a central role in regulating the balance between diaphragm oxygen supply and demand. Endothelial dysfunction occurs in such diverse conditions as hypercholesterolemia, diabetes mellitus, cigarette smoking, and heart failure and after prolonged episodes of hypoxia. In patients with these disorders, impairment of endothelium-dependent vasoregulation will handicap the responses that defend diaphragmatic substrate supply and contribute to the development of respiratory insufficiency.
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ACKNOWLEDGEMENTS |
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The author thanks Stephen Nuara for technical expertise.
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FOOTNOTES |
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This study was funded by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Canada. M. E. Ward is a scholar of the Medical Research Council of Canada.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. E. Ward, The Meakins-Christie Laboratories, 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2 (E-mail: mward{at}meakins.lan.mcgill.ca).
Received 15 July 1998; accepted in final form 21 January 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Brown, I. P.,
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