INTRODUCTION

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IT IS WELL ESTABLISHED that the endothelium exerts a considerable local influence on microvascular tone and blood flow through the release of diffusible vasoactive factors such as nitric oxide (NO) and various cyclooxygenase products (15, 30). The release of these factors is governed by the immediate chemical environment of the endothelial cell (including the binding of various agents to membrane-bound receptors) and by physical forces acting on the vessel wall (2, 9, 12). In addition to a direct effect on vascular smooth muscle, endothelium-derived factors can indirectly influence microvascular tone by modulating the activity of other microvascular control mechanisms (23, 24, 26). Studies in rat cremaster muscle and canine epicardium have demonstrated that arteriolar constriction in response to exogenous norepinephrine (NE) is enhanced after inhibition of NO synthesis (19, 26). This laboratory (23, 24) reported has more recently reported that endothelium-derived NO normally limits arteriolar constriction during periods of increased sympathetic nerve activity in the rat intestine. This arteriolar NO release occurs despite a marked reduction in luminal shear stress, and it does not depend on the binding of neurally released NE to endothelial ₂-receptors (25).

The sympathetic constriction of resistance vessels is accompanied by a reduction in local blood flow (3, 4, 23) and a consequent fall in arteriolar wall O₂ levels (4). There is mounting evidence that a reduction in vascular wall PO₂ can promote vasodilation via the release of endothelium-derived NO (1, 10-12, 16, 21, 22, 27-29, 35). The aim of the current study was to investigate the functional significance of any relationship between arteriolar wall PO₂ and local NO activity in the intestine. More specifically, we tested the hypothesis that a reduction in arteriolar wall PO₂ serves as the stimulus for arteriolar NO production during periods of increased sympathetic nerve activity. If this hypothesis is correct, then minimizing the fall in arteriolar wall PO₂ should diminish the stimulus for NO release and lead to augmented sympathetic constriction. To test this prediction, arteriolar responses to sympathetic nerve stimulation were assessed under normal conditions and under a hyperoxic superfusate (to maintain arteriolar wall O₂ delivery) before and during inhibition of NO synthesis with N^G-monomethyl-L-arginine (L-NMMA). Arteriolar wall PO₂ was directly measured with O₂-sensitive microelectrodes to verify the effects of sympathetic nerve stimulation and increased superfusate O₂ content on arteriolar wall O₂ levels.

	METHODS

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All surgical and experimental procedures were approved by the West Virginia University Animal Care and Use Committee. Male Sprague-Dawley rats aged 8-9 wk (Harlan Sprague Dawley, Indianapolis, IN) were anesthetized with thiopental sodium (100 mg/kg ip) and placed on a heating mat to maintain a 37°C rectal temperature. To ensure adequate gas exchange, rats were intubated and ventilated with a rodent ventilator (Harvard Apparatus, South Natick, MA). Arterial pressure was measured directly with a Gould P23 ID pressure transducer (Cleveland, OH) connected to a cannula inserted into the right carotid artery.

The small intestine was prepared for microscopic observation as previously described (23). Briefly, a 14-cm loop of small intestine (ileum) was gently exteriorized through a midline abdominal incision. The loop was initially bathed in warm Normosol-R electrolyte solution (Abbott, Chicago, IL) and then continuously superfused with a physiological electrolyte solution (in mM: 119 NaCl, 25 NaHCO₃, 6 KCl, and 3.6 CaCl₂) that was warmed to 37°C and equilibrated with a gas mixture to either mimic normal in vivo conditions (5% O₂-5% CO₂-90% N₂) (5) or create a hyperoxic environment (20% O₂-5% CO₂-75% N₂). Isoproterenol (10 mg/l; Sigma, St. Louis, MO) and phenytoin (20 mg/l; Parke-Davis, Morris Plains, NJ) were added to the superfusate to suppress intestinal motility. At these concentrations, neither agent alters resting arteriolar tone in this vascular bed (7). After the ileum was exteriorized, two small incisions 6 cm apart were made by thermal cautery along the antimesenteric border, and chyme was flushed from the lumen through these incisions. The bowel was then secured over a transparent pedestal by four sutures tied to the antimesenteric border. Most of the preparation was covered with polyvinyl film with the superfusate flow directed beneath the film. With the normal (5% O₂) superfusate, this arrangement stabilizes solution PO₂ above the tissue at 40-50 mmHg (6).

After surgery, the rat was transferred to the stage of an Olympus BHTU intravital microscope (Hyde Park, NY) fitted with a CCD video camera (Dage MTI, Michigan City, IN). Video images were displayed on a Panasonic high-resolution video monitor and stored on videotape for off-line analysis. Observations were made with a ×10 eyepiece and Nikon ×10 water-immersion objective (final video magnification = ×730). Arteriolar inner diameters were measured with a video caliper (Microcirculation Research Institute, Texas A&M University) during videotape replay.

Arteriolar wall PO₂ was measured with Whalen-type O₂ microelectrodes (tip diameter = 2-3 µm, Diamond General, Ann Arbor, MI) that were calibrated immediately before and after each experiment. Data from electrodes exhibiting more than a 5% change in gain from pre- to postexperimental calibrations were discarded. For calibration, electrodes were placed in a tonometer (model 1251, Diamond General), and current output was recorded in superfusates equilibrated with 10% and 20% O₂ gas mixtures (PO₂= 71 and 142 mmHg, respectively). Zero-level PO₂ was determined by placing the electrode tip in an actively respiring yeast mixture as described by Whalen et al. (36).

For sympathetic nerve stimulation, a bipolar platinum electrode secured in a micromanipulator was used to stimulate the sympathetic postganglionic efferents in the sheath surrounding a mesenteric artery-vein pair upstream from the arteriole under study. The electrode and artery-vein pair were briefly raised above the superfusate, and the nerves were stimulated with square-wave pulses at supramaximal voltage (5-6 V) and a pulse duration of 10 ms. These stimulation parameters elicit frequency-dependent arteriolar constrictions that are abolished by the nonselective -receptor antagonist phentolamine (23) and the selective ₁-receptor antagonist prazosin (25), verifying that these responses are due to sympathetic nerve activation.

Experimental protocols. The first series of experiments was designed to define the relationship between arteriolar wall PO₂ and NO during periods of increased sympathetic nerve activity. During superfusion with either the normal (5% O₂) or hyperoxic (20% O₂) solution, a first-order arteriole was selected for study, and an O₂ microelectrode was positioned with the tip in light contact with the outer vessel wall. After a 1-min control period, the sympathetic nerves were stimulated for 1 min at 3, 8, or 16 Hz followed by a 3-min recovery period. This sequence was repeated two more times so that the arteriole was subjected to all three levels of increased sympathetic nerve activity, delivered in a random order. The superfusate was then changed (from normal to hyperoxic or from hyperoxic to normal), and the sequence of nerve stimulations was repeated. The nerve stimulation sequences under both the normal and hyperoxic superfusates were then repeated during continuous exposure of the vasculature to the NO synthase (NOS) inhibitor L-NMMA (1 × 10⁴ M superfusate concentration). Finally, adenosine was added to the superfusate (10³ M final concentration), and passive arteriolar diameter was measured.

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Data and statistical analysis. All data are expressed as means ± SE. Statistical analysis was carried out using commercially available software (SigmaStat, Jandel Scientific). ANOVA for repeated measures was used to compare responses to agonists or sympathetic nerve stimulation before and after a given treatment in the same animal, with post hoc analysis done via the Newman-Keuls multiple range procedure. Significance was assessed at P < 0.05 for all statistical tests.

	RESULTS

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Eleven Sprague-Dawley rats (274 ± 11 g body wt) were used in the first series of experiments to explore the relationship between arteriolar wall PO₂ and the influence of local NO on arteriolar sympathetic constriction. Under the normal superfusate, the first-order arterioles selected for study had a resting diameter of 63 ± 3 µm and a passive diameter of 88 ± 3 µm. Sympathetic nerve stimulation induced frequency-dependent constrictions, with arteriolar diameters being reduced to 55 ± 3, 48 ± 3, and 41 ± 3 µm during stimulation at 3, 8, and 16 Hz, respectively (Fig. 1). In the presence of 10⁴ M L-NMMA, resting arteriolar diameters were unchanged (60 ± 3 µm), but the responses to each level of sympathetic nerve stimulation were significantly enhanced. During L-NMMA exposure, stimulation at 3, 8, and 16 Hz reduced arteriolar diameters to 47 ± 2, 40 ± 3, and 35 ± 3 µm, respectively.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   First-order arteriole diameter responses to sympathetic nerve stimulation at 3 (A), 8 (B), and 16 (C) Hz under normal superfusate and normal superfusate + 104 M NG-monomethyl-L-arginine (L-NMMA). n = 11 vessels. *P < 0.05 vs. normal superfusate.

Resting arteriolar diameters under the hyperoxic superfusate were identical to those under the normal superfusate (63 ± 3 µm), but arteriolar responses to sympathetic nerve stimulation were significantly enhanced under these conditions (Fig. 2). Stimulation at 3, 8, and 16 Hz elicited steady-state arteriolar constrictions of 9 ± 2, 15 ± 1, and 21 ± 2 µm, respectively, from control under the normal superfusate versus 15 ± 2, 24 ± 3, and 33 ± 3 µm, respectively, from control under the hyperoxic superfusate. As under the normal superfusate, L-NMMA did not alter resting arteriolar diameters (62 ± 3 µm) under the hyperoxic superfusate. However, in contrast to the normal superfusate, L-NMMA no longer enhanced arteriolar responses to sympathetic nerve stimulation under the hyperoxic superfusate (Fig. 3). Stimulation at 3, 8, and 16 Hz reduced arteriolar diameters to 49 ± 2, 38 ± 2, and 30 ± 2 µm, respectively, in the absence of L-NMMA versus 49 ± 2, 40 ± 2, and 33 ± 2 µm, respectively, in the presence of L-NMMA.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Magnitude of arteriolar constriction (in µm) induced by sympathetic nerve stimulation at 3, 8, and 16 Hz under normal superfusate and 20% O2 superfusate. n = 11 vessels. *P < 0.05 vs. normal superfusate.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   First-order arteriole diameter responses to sympathetic nerve stimulation at 3 (A), 8 (B), and 16 (C) Hz under 20% O2 superfusate and 20% O2 superfusate + 104 M L-NMMA. n = 11 vessels.

Under the normal superfusate, resting arteriolar wall PO₂ averaged 69 ± 6 mmHg and tended to fall (but not significantly) during sympathetic nerve stimulation at 3 Hz. However, during nerve stimulation at 8 and 16 Hz, wall PO₂ rapidly fell to a new steady-state level that was dependent on stimulation frequency (Fig. 4). Exposure to the hyperoxic superfusate did not significantly change resting wall PO₂ (78 ± 6 mmHg), but the fall in wall PO₂ during sympathetic constriction was significantly attenuated under these conditions. Stimulation at 3, 8, and 16 Hz reduced arteriolar wall PO₂ to 59 ± 4, 50 ± 5, and 42 ± 5 mmHg, respectively, under the normal superfusate versus 70 ± 5, 62 ± 5, and 53 ± 5 mmHg, respectively, under the hyperoxic superfusate. The steady-state wall PO₂ reached during 3- and 8-Hz stimulation under the hyperoxic superfusate (70 ± 5 and 62 ± 5 mmHg) were not significantly different from resting wall PO₂ under the normal superfusate.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Arteriolar wall PO2 during sympathetic nerve stimulation at 3 (A), 8 (B), and 16 (C) Hz under normal superfusate and 20% O2 superfusate. n = 8 vessels. *P < 0.05 vs. normal superfusate.

The effectiveness of L-NMMA as a NOS inhibitor in the rat intestine was tested in the second series of experiments by evaluating arteriolar responses to A-23187 in five rats (259 ± 25 g) and arteriolar responses to A-23187 + L-NMMA in four rats (247 ± 26 g). Resting and passive arteriolar diameters averaged 73 ± 4 and 109 ± 9 µm, respectively, for the first group (n = 10 vessels) and 74 ± 3 and 94 ± 4 µm, respectively, for the second group (n = 10 vessels). Figure 5 shows arteriolar diameter responses to A-23187 in these two groups. In the first group, 1 × 10⁵ M and 2 × 10⁵ M A-23187 significantly increased arteriolar diameter to 96 ± 7 and 105 ± 8 µm, respectively (dilations of 29 ± 3 and 42 ± 5% from control). In the second group, L-NMMA completely abolished the vasoactive response to A-23187, with steady-state diameters in the presence of either concentration averaging 72 ± 5 µm or 4 ± 4% below control. This finding indicates that we were able to maximally inhibit NO synthesis with L-NMMA in this preparation.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   First-order arteriole diameters before and during application of A-23187 under normal superfusate and normal superfusate + 104 M L-NMMA. n = 10 vessels per group. *P < 0.05 vs. normal superfusate.

Eight rats (258 ± 18 g) were used in the third series of experiments to evaluate the effect of the hyperoxic superfusate on vascular smooth muscle responsiveness to NO. The arterioles studied in these experiments had resting and passive diameters of 62 ± 1 and 106 ± 3 µm, respectively (n = 8 vessels). The steady-state diameters reached during iontophoretic application of SNP at 5, 20, and 40 nA averaged 83 ± 3, 94 ± 3, and 103 ± 4 µm, respectively, under the normal superfusate and were not significantly different from those reached during SNP application under the hyperoxic superfusate (81 ± 6, 92 ± 5, and 102 ± 5 µm) (Fig. 6).

View larger version (42K): [in this window] [in a new window]   Fig. 6.   First-order arteriole diameter responses to iontophoretic application of sodium nitroprusside (SNP; 5, 20, and 40 nA ejection currents) under normal superfusate and 20% O2 superfusate. n = 8 vessels. *P < 0.05 vs. control diameter under same superfusate. P < 0.05 vs. diameter during 5 nA application.

Twenty rats (287 ± 7 g) were used in the fourth series of experiments to evaluate oxidative stress in the arteriolar wall under the normal or hyperoxic superfusates. The calculated light absorption values indicated that there was no difference in arteriolar wall formazan content (and therefore oxidant activity) between vessels exposed to TNBT under the normal superfusate (Absorption = 4.8 ± 0.9 units) and those exposed to TNBT under the hyperoxic superfusate (Absorption = 4.7 ± 0.5 units). After arterial infusion of HX + XO, arteriolar wall formazan content was significantly increased to the same level under both superfusates (Absorption = 7.9 ± 0.5 units under the normal superfusate and 7.9 ± 0.4 units under the hyperoxic superfusate), indicating elevated oxidant activity.

	DISCUSSION

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A reduction in O₂ availability can decrease arteriolar tone through a direct effect on the vessel wall and/or indirectly via changes in the metabolic state of nearby parenchymal cells (13, 16, 29). The mechanism(s) by which resistance vessels are directly sensitive to reduced O₂ can vary depending on the vascular bed. Harder et al. (17) reported that reduced O₂ levels in rat renal and cremaster muscle microvessels may lead to reduced formation of 20-hydroxyeicosatetraenoic acid, a constitutively-produced cytochrome P-450 metabolite that normally acts as a potent vasoconstrictor. In other cases, a fall in local PO₂ leads to increased release of one or more endothelium-derived vasodilator prostanoids. This has been demonstrated in vitro for resistance vessels in the rat heart (27), in arterioles isolated from the dog heart (22), and in the rat cremaster muscle (21). In contrast, a fall in PO₂ increases the release of endothelium-derived NO from resistance vessels in the coronary circulation of the pig (18) and guinea pig (28), in the diaphragm of the dog (35), and in the human forearm vasculature (1). There is also evidence of O₂-sensitive NO release in some vascular beds of the rat. Pries et al. (30) reported an inverse correlation between elevated microvascular O₂ levels and NO activity in the rat spinotrapezius muscle. The hypoxia-induced dilation of preconstricted rat aortic ring segments is also due to the release of endothelium-derived NO (16).

The results of our current study suggest that a reduction in arteriolar wall PO₂ may serve as a major stimulus for arteriolar NO production during sympathetic vasoconstriction in the rat intestine. Resting arteriolar wall PO₂ under the normal superfusate averaged 69 ± 6 mmHg, which is consistent with an earlier study in the rat intestine by Bohlen and Lash (8). They found that resting wall PO₂ averages 65-70 mmHg in first-order arterioles and as low as 50 mmHg in more distal arterioles, indicating a marked precapillary oxygen loss from these downstream vessels. Under the normal superfusate, sympathetic nerve stimulation produced frequency-dependent arteriolar constriction and a significant reduction in arteriolar wall PO₂ at stimulation frequencies of 8 and 16 Hz (Figs. 1 and 4). At all stimulation frequencies, the magnitude of sympathetic constriction was significantly increased in the presence of the NOS inhibitor L-NMMA (Fig. 1), which is consistent with a previous report by this laboratory in which this effect of L-NMMA was also completely reversed by excess L-arginine (23). Although mean arteriolar wall PO₂ was not significantly reduced during 3-Hz nerve stimulation under the normal superfusate (Fig. 4), wall PO₂ did fall by an average of 13 ± 4 mmHg in six of the eight vessels studied, and in those vessels L-NMMA significantly enhanced the sympathetic constriction. In the remaining two vessels where wall PO₂ did not fall under these conditions, L-NMMA had no effect on sympathetic constriction. These observations strongly suggest that endogenous NO plays an important role in limiting the magnitude of sympathetic arteriolar constriction when arteriolar O₂ levels fall in this vascular bed. We (24) have determined more recently that this NO is of endothelial origin, based on the loss of this NO influence after functional disruption of the endothelium.

In the current study, the high-O₂ superfusate that we used did not significantly increase resting arteriolar wall PO₂, but it did limit the fall in wall PO₂ that accompanies sympathetic nerve stimulation (Fig. 4). This is most likely because arteriolar wall O₂ levels are predominantly influenced by blood O₂ delivery during normal flow conditions, with extraluminal O₂ delivery from the superfusate only becoming an important contributor to wall oxygenation as blood flow falls (4). Under the O₂-enriched superfusate, arteriolar wall PO₂ remained at resting levels throughout the 3-Hz sympathetic nerve stimulation (Fig. 4). Under these conditions, the magnitude of the sympathetic constriction was increased and was no longer sensitive to L-NMMA (Figs. 2 and 3). Although arteriolar wall PO₂ still fell during the 8- and 16-Hz stimulations under the high-O₂ superfusate, sympathetic constriction was also increased and was no longer sensitive to L-NMMA. On the basis of our hypothesis, we might have expected these decreases in wall PO₂ to stimulate NO synthesis and thus make the arteriolar responses to sympathetic nerve stimulation sensitive to NOS inhibition. However, a closer inspection of the data reveals that, whereas wall PO₂ fell during the 8-Hz stimulation under increased superfusate O₂, the level to which it fell was not below the resting value for those vessels measured under the normal superfusate (Fig. 4). This suggests that a fall in wall O₂ levels may only promote NO release if PO₂ falls below some critical level. Taken together, the observations for sympathetic nerve stimulation at 3 and 8 Hz suggest a causal link between reduced arteriolar wall PO₂ and NO release during sympathetic constriction. However, the lack of an effect of L-NMMA on arteriolar responses to the 16-Hz nerve stimulation under the high-O₂ superfusate is inconsistent with this interpretation, because the level to which wall PO₂ falls under these conditions is similar to that measured during 8-Hz stimulation under the normal superfusate (where constriction was enhanced by L-NMMA). It may be that the dramatic (and possibly maximal) constriction of these vessels during this supraphysiological level of nerve stimulation in a high-O₂ environment was so overwhelming that locally released NO was simply unable to exert any modulating effect on the response.

In previous efforts to identify the stimulus for arteriolar NO release during sympathetic constriction in the rat intestine, this laboratory ruled out the possibility that neurally released NE was stimulating NO release by binding to endothelial ₂-receptors (25). Another possible stimulus that could act in concert with reduced arteriolar wall PO₂ is hemodynamic shear stress, which serves as an important trigger for continuous endothelial NO release during periods of normal and increased luminal flow (2, 9, 15). However, sympathetic nerve stimulation at 3, 8, and 16 Hz reduces average wall shear rate in first-order intestinal arterioles by 20%, 45%, and 73%, respectively (25), and Bohlen and Nase (9) reported a linear relationship between wall shear rate and periarteriolar [NO] for these vessels, with each 10% change in shear rate causing a 6% change in [NO]. If flow-dependent shear stress remained the predominant stimulus for endothelial NO release in these arterioles during sympathetic constriction, then Bohlen and Nase's calculations would predict a reduction in NO release of 12% during 3 Hz stimulation, 23% during 8 Hz stimulation, and 37% during 16 Hz stimulation. Because arteriolar NO release appears to be well maintained throughout sympathetic nerve stimulation (Fig. 1; Refs. 23 and 24), it appears unlikely that shear stress remains the primary stimulus for NO release under these conditions.

Instead of reflecting suppressed NO release, the enhanced sympathetic constriction and its resistance to L-NMMA under the hyperoxic superfusate could have been due to a reduction in arteriolar smooth muscle responsiveness to NO. However, our finding that arteriolar responses to the NO donor SNP were unchanged under the hyperoxic superfusate (Fig. 6) argues against this possibility. Alternatively, the apparent absence of arteriolar NO activity under the hyperoxic superfusate could have been due to accelerated NO breakdown by reactive oxygen species generated in the arteriolar wall (32). Although our direct measurements indicate that resting arteriolar wall PO₂ was not significantly increased during exposure to the hyperoxic superfusate, there was a clear trend toward higher resting wall PO₂ under these conditions (Fig. 4). We therefore used the TNBT reduction method to assess arteriolar oxidant activity in preparations exposed to either the normal or hyperoxic superfusate. This method has been previously used to demonstrate increased oxidative stress in arteriolar and venular walls of hypertensive Dahl rats (34) and normotensive rats fed a high salt diet (20). In the current study, exposure to the hyperoxic superfusate had no effect on arteriolar wall light absorption after TNBT exposure, arguing against increased oxidant activity in these vessels. Exposure to intravascular HX + XO changed arteriolar wall light absorption by the same amount under the normal superfusate and the hyperoxic superfusate (64-68%), indicating that the sensitivity of the TNBT reduction method is sufficient to detect increased oxidative stress under either superfusate.

The lack of an effect of L-NMMA on resting arteriolar tone in the intestine (Figs. 1 and 5) contrasts with findings in other vascular beds (2, 15, 26, 30) but is consistent with our previous findings in this preparation (24). In light of recent direct measurements verifying that NO is continuously released from these vessels in the resting state (9), local NOS inhibition would be expected to reduce arteriolar diameter. However, resting arteriolar tone is the result of the integration of numerous simultaneous vasoactive signals by the vascular smooth muscle, and the activity of local metabolic and myogenic control mechanisms may set vascular tone at some constant and optimal level for the tissue. These local regulatory mechanisms may be so highly developed in the intestine that the withdrawal of any single influence (such as NO in the presence of L-NMMA) is accompanied by a compensatory change in the activity of one or both of these systems, thereby preserving the level of tone. The lack of effect of increased superfusate O₂ on resting arteriolar tone (Fig. 6), which also contrasts with findings in some other vascular beds (3, 4, 13, 30), could simply reflect the fact that the level of O₂ in the superfusate was insufficient to significantly alter resting arteriolar wall PO₂ (Fig. 4). Therefore, a direct effect of oxygen on the vessel wall would be unlikely under these conditions.

Because molecular O₂ serves as a cosubstrate for all isoforms of NOS (14, 37), one might expect a fall in arteriolar wall PO₂ to limit arteriolar NO production instead of increasing it. This would undoubtedly be the case if the endothelial cell O₂ concentration reached rate-limiting levels, but during our experiments arteriolar wall PO₂ never fell to the level corresponding to the Michaelis-Menten constant of O₂ for either isolated endothelial NOS (eNOS, 6 mmHg) (31) or eNOS in intact endothelial cells (38 mmHg) (37). Therefore, we conclude that the reduction in arteriolar wall PO₂ during sympathetic constriction was probably not sufficient to limit endothelial NO synthesis in the current study. Our observations suggest that as arteriolar wall PO₂ falls, NO synthesis may in fact change in a biphasic manner. A fall in PO₂ from resting levels could initially stimulate NO synthesis, whereas a continued fall to lower levels would begin to suppress NO synthesis as O₂ becomes limited as a NOS cosubstrate. The mechanism by which reduced arteriolar wall PO₂ could lead to increased NO synthesis has not been clearly established. Some investigators have reported that a fall in PO₂ increases cytosolic Ca²⁺ levels in endothelial cells, which would increase both basal and stimulated NO production (12, 15). Alternatively, Bryan and Marshall (10, 11) suggested that a reduction in blood PO₂ can promote the release of adenosine from the endothelium of resistance vessels and that this adenosine acts in an autocrine fashion to hyperpolarize the endothelial cell membrane via activation of receptor-coupled ATP-sensitive potassium channels.

In summary, we have shown that if the fall in arteriolar wall PO₂ associated with neurogenic constriction is limited by increased superfusate O₂ delivery, then sympathetic neurogenic vasoconstriction is enhanced and is no longer sensitive to NOS inhibition. This effect cannot be explained by reduced vascular smooth muscle responsiveness to NO or by oxidative degradation of NO. These results suggest that during periods of increased sympathetic nerve activity, a flow-dependent fall in arteriolar wall PO₂ may serve as a stimulus for the release of endothelial NO, which in turn limits arteriolar neurogenic constriction.