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Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202
Submitted 29 August 2002 ; accepted in final form 30 January 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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embolization; partial pressure of oxygen
The small intestine has one of the highest oxygen usages per gram of tissue
per minute, consuming 
20% of total body oxygen consumption at rest
(5). Mild oxygen depletion
compromises intestinal nutrient absorption
(6,
14) and gross oxygen depletion
results in severe tissue damage with major clinical morbidity and mortality.
Despite the great reliance of normal intestinal function on oxygen, tissue and
vascular PO2 during absorptive hyperemia are both
relatively unimportant mechanisms. First, the perivascular
PO2 actually is increased for all resistance arterioles
during nutrient absorption due to the large increase in blood flow
(2). Second, reduction in
PO2 in the villus during nutrient absorption explains
only about one-fourth of the increase in blood flow associated with absorptive
hyperemia (1). However, the
intestinal vasculature is exquisitely sensitive to oxygen deprivation and
responds with a large increase in perivascular NO concentration ([NO]), as we
(4) have reported in a study
that used NO-sensitive microelectrodes. In fact, the intestinal vasculature is
so sensitive to PO2 in the major resistance vessels that
we have found even minor problems in ventilation of anesthetized rats results
in elevated [NO] and an artificial exaggeration of the relative importance of
NO in what is thought to be "resting" vascular tone.
By utilizing NO-sensitive microelectrodes with sharpened tip diameters of 7–9 µm, we can use their spatial measurement properties to determine both the magnitude of changes in [NO] as PO2 is lowered and the relative changes in periarteriolar, perivenular, and general tissue [NO]. The spatial issue is of importance because the various sources of NO during a given perturbation could include arterioles, venules, and even tissue. Furthermore, most of the major resistance arterioles of the small intestine are paired with venules that are capable of producing NO. In fact, perivenular [NO] at rest and during the physiological stimulation associated with nutrient absorption (2) approach the arteriolar concentrations. These observations are particularly germane to oxygen depletion if venules release more NO during periods of oxygen deprivation. For example, venules have a much lower intravascular PO2 than do arterioles and the small intestine and skeletal muscle vasculatures experience a decrease in PO2 during increased metabolic activity at a time when intra-arteriolar PO2 is at or above resting PO2 levels (20). To reduce oxygen availability, we decreased the PO2 of the bathing media so that it became a sink for tissue oxygen and lowered the perivascular PO2. We established that the source of increased periarteriolar NO during oxygen deprivation was predominantly from the arterioles, although venules did exhibit robust NO formation at reduced perivascular PO2. In addition, we tested some of the major mechanisms thought to be of importance in increased NO formation during reduced oxygen availability. Our key findings are that increased NO formation was the dominant cause of vasodilation to reduced perivascular PO2 and that cyclooxygenase products, potassium channel activation, and adenosine were not responsible for the increased NO formation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Surgical preparation of small intestine. Adult male Sprague-Dawley rats (Harlan Sprague Dawley; Indianapolis, IN) were anesthetized with thiopental sodium (200 mg/kg; Abbott, Chicago, IL) injected subcutaneously at four locations over the thighs and lower back and placed on a heating mat to maintain a 37°C rectal temperature. One-fourth of the original dose was given intraperitoneally if supplemental anesthesia was needed. The trachea was intubated with a polyethylene (PE-240) catheter to ensure a patent airway and the left femoral artery was cannulated (PE-50) for the subsequent monitoring of blood pressure. The animal was given normal saline (0.5 ml · h–1 · 100 g body wt–1) to compensate for fluid loss by urine formation and mechanical ventilation. All animals were ventilated at a rate of 70 breaths/min, the typical ventilation frequency of conscious rats, and a tidal volume indicated from the Harvard Apparatus nomogram for small animals. In addition, the tidal volume was then adjusted up or down in small increments until the end-tidal CO2 tension was <40 mmHg (SC-219 CO2 monitor, Pyron; Menomonee, WI). With the fluid replacement and ventilatory support, the arterial pressure was virtually constant for 4–5 h after completion of surgery. Experiments were conducted only if mean arterial pressure was >90 mmHg and stable.
The small intestine was prepared for observation with a standard technique (3) that required a midline abdominal incision of 1.5–2 cm. The jejunal region of the bowel was located, and an 8- to 10-cm loop was exteriorized into a heated pool of saline and covered with plastic wrap (Saran Wrap, Dow; Indianapolis, IN). The bowel wall was slit along the antimesenteric border with a small thermal cautery and was intermittently wetted with saline to avoid drying of the tissue. With this technique, the nerves and vascular supply to the intestinal wall were left intact. The bowel contents were evacuated, and small threads were tied to the edges of the bowel incisions. The bowel was then draped with the mucosal surface downward over a translucent pedestal and held in place by the threads. A fluid chamber was lowered over the bowel and into the support device. A 4–5 ml/min flow of bicarbonate-buffered physiological solution was passed through the chamber after being heated to 37.5 ± 0.5°C. The support device was internally heated to 37.5 ± 0.5°C with heated circulating water. The physiological solution was equilibrated with 5% O2-5% CO2-90% N2, and the fluid lines were protected from equilibration with the atmosphere until the fluid entered a stainless steel heating and tissue support system.
After surgery, the animal was transferred to the stage of an intravital microscope (model BHMJ, Olympus; Hyde Park, NY) and observed with a closed-circuit television camera (model XC-77, Hamamatsu) coupled with a computerized digitizing and image analysis system (Image 1, Universal Imaging; West Chester, PA). Images were stored in digital format, and dimensions of the vessels were measured with the virtual caliper of the image analysis system. Linear dimensions were calibrated in the x- and y-dimensions with a stage micrometer marked in 10- and 100-µm units.
Perivascular PO2 measurements. Perivascular PO2 was measured with recessed-tip (15–20 µm) gold-plated microelectrodes (39). The electrode tip was sharpened to a diameter of 5–8 µm to facilitate penetration of intestinal tissue. The electrodes were calibrated and tested for a linear current-PO2 relationship in a precision tonomoter.
Perivascular NO measurements. The nanomolar [NO] was measured with
an adaptation of the polarographic technique for gold-plated, recessed-tip
glass microelectrodes, as developed by Buerk and colleagues
(7) and used in our past
studies (4,
6). The microelectrodes were
sharpened to an 8- to 10-µm outer diameter at the base of the sharpened
region. Each electrode had a tip recess of 10–20 µm beyond the
sharpened region, and the recess was coated with Nafion (Aldrich; Milwaukee,
WI). The Nafion coating decreased the random electrical noise of the
microelectrodes and essentially eliminated the interference of nitrate,
ascorbic acid, tyrosine, and norepinephrine at physiological concentrations
with measurements of NO. The microelectrodes were polarized at +0.8 V relative
to a carbon fiber reference electrode (World Precision Instruments; Sarasota,
FL), and the current generated was measured with an electrometer (model 610B,
Keithley; Cleveland, OH). A calibration curve in a gas tonometer at 37.5°C
was obtained on the morning of each experiment by measurement of the
microelectrode current at 0, 600, and 1,200 nM NO. The test saline
solution was cleared of oxygen and atmospheric NO by bubbling with pure
N2 to obtain a "0" nM [NO]. Commercial mixes of NO gas
in N2 were used to equilibrate the saline solutions with NO. The
600 and 1,200 nM NO concentrations are based on the parts per
million NO in N2 and solubility of NO in water at 37.5°C. Each
microelectrode was found to have a linear current-[NO] relationship and basal
currents at 0 nM [NO] were typically 5–10 pA. We used only
microelectrodes that generated >1 pA/1,000 nM NO. This sensitivity
translated to an 1-mV increase in output of the electrometer for each 4
nM elevation in [NO] or 80–120 mV above baseline for typical
periarteriolar [NO]. Uncertainties caused by tissue motion, electronic noise,
electrode drift characteristics, and very slow declines in microelectrode
sensitivity over time limit the resolution of the microelectrodes during in
vivo measurements to 10 nM NO. The slow electrical drift of the
microelectrode during experiments was compensated by the calculation of an
interpolated baseline over time during the individual measurements. Before and
immediately after each tissue measurement, the current generated by the
microelectrode 500 µm above the tissue surface was used as the 0 nM
reference. These data were used to calculate the rate of electronic drift and
an interpolated baseline (0 nM) for any given time. The typical duration of a
tissue or perivascular measurement was 10 min, and over such time frames,
worse case drift of the baseline would be 3% of the typical resting [NO]
on the surface of the arterioles before correction for drift. Separate
measurements for each step of a protocol were required to avoid risks of
intestinal motility on the microelectrode tip. The reference 0 nM equivalent
output voltage and the calibration output voltage-[NO] relationship were used
to calculate tissue [NO].
During perivascular measurements, the ideal micropipette penetration was
with the microelectrode shaft nearly parallel to the arteriole, with the
sharpened microelectrode tip appearing to touch the arteriolar wall. If the
microelectrode tip was pulled away from the vessel wall, [NO] decreased
dramatically and approached the tissue background [NO] of 100–150 nM at
100 µm from the vessel wall. Our goal for all measurements was to
achieve the highest possible [NO] for a given vessel, and we moved the
micropipette tip as required to maintain close contact of the vessel wall and
microelectrode tip.
CO2 embolization experiments. Localized embolization was used to functionally inactivate microvascular endothelial cells for several hours (26). A CO2-filled micropipette (4–6 µm outer tip diameter) was inserted with a micromanipulator into the lumen of a small feed artery immediately upstream from the first-order arteriole (1A) under observation. CO2 was ejected from the micropipette into the vessel lumen with the use of a pressure ejection system (Picospritzer II, General Valve; Fairfield, NJ). CO2 ejection consisted of two phases: an initial 0.05-s pulse delivered at 40–55 lb/in2 to verify that the pipette tip was inside the feed artery lumen, and a second 0.15-s pulse delivered at 40–55 lb/in2 to ensure complete filling of the downstream 1A. With the 0.15-s pulse there was a temporary cessation of blood flow in the 1A due to lodging of the CO2 embolus in the arteriolar lumen. After an average flow cessation of 25–40 s, the embolus dislodged and was carried away by blood flow with no visible thrombogenic effects. We did not observe bubbles in the paired venule as the arteriolar embolus broke down.
Experimental protocols. After a postsurgical period that typically lasted 45–60 min, a 1A or first-order venule (1V) was chosen for specific experimental protocols.
Effect of reduced oxygen availability on perivascular
PO2 and [NO]. The first series of experiments was
designed to determine the effect of reduced oxygen availability on
perivascular PO2. Whalen-type O2
microelectrodes were used to measure periarteriolar, perivenular, and
parenchymal PO2 during resting conditions and after
reduced oxygen availability. Reduced oxygen availability was achieved by
changing the superfusate solution percent oxygen from 5% to 0%. Our goal was
to reduce PO2 at the vessel wall 5–10 mmHg,
which was about the limit possible with a low O2-content saline
bath.
We evaluated the effect of reduced oxygen availability on perivascular [NO] and vascular diameter. NO-sensitive microelectrodes were used to measure periarteriolar and perivenular [NO] during resting conditions and after reduced oxygen availability. Periarteriolar and perivenular diameters were recorded simultaneously and the arteriolar or venular [NO] was measured.
Adenosine experiments. Adenosine ionizes in distilled water alkalized to pH 8 with NaHCO3. This allowed the iontophoretic release of adenosine while [NO] was measured essentially at the tip of the adenosine micropipette on the vessel wall. The iontophoretic current was increased in stages to cause minimal to maximal dilation as [NO] and vessel inner diameter were measured.
Effect of NO synthase inhibitor, cyclooxygenase inhibitor, and potassium channel blockade on arteriolar dilation during reduced oxygen availability. In three separate series of experiments, we determined the effect of a NO synthase inhibitor, cyclooxygenase inhibitor, and hyperpolarizing factor inhibitor on arteriolar dilation during reduced oxygen availability. In each case, the quality of the blockade was tested with the appropriate agonist, and these data are presented in RESULTS. In the first experiment, arteriolar diameter was measured at rest, during reduced oxygen availability, after addition of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10–4 M) into the bathing fluid, and during reduced oxygen availability + L-NAME. Direct measurement of [NO] served to confirm substantial suppression of NO production after L-NAME treatment.
In the second experiment, arteriolar diameter was measured at rest, during reduced oxygen availability, after addition of the cyclooxygenase inhibitor meclofenamate (3 x 10–5 M) into the bathing fluid, and during reduced oxygen availability + meclofenamate. The dilatory microvascular response to arachidonic acid was compared before and after meclofenamate application to verify the extent of cyclooxygenase inhibition (see RESULTS).
In the third experiment, arteriolar diameter was measured at rest, during reduced oxygen availability, after addition of the calcium-activated K+ channel blocker tetraethylammonium acetate (TEA; 10–3 M) into the bathing fluid, and during reduced oxygen availability + TEA. We blocked the calcium-activated potassium channels because they are generally considered the form of vascular smooth muscle potassium channels activated by endothelium-derived hyperpolarizing factors (11, 17, 38) To test for blockade, we compared the vascular dilation to NS-1619 (Sigma), a specific calcium-activated potassium channel agonist known to exist in the rat mesenteric vasculature (25), and 20 mM KCl before and during TEA application. Both agonists were substantially blocked by TEA (see RESULTS).
Effect of arteriolar embolization on periarteriolar [NO] and arteriolar and venular diameters during reduced oxygen availability. In these studies, we determined the effect of arteriolar embolization on periarteriolar [NO] during reduced oxygen availability. To accomplish this, NO-sensitive microelectrodes were used to measure periarteriolar NO concentration during resting conditions, during reduced oxygen availability, after arteriolar embolization, and during reduced oxygen availability + embolization.
Data and statistical analysis. All data are expressed as means ± SE, and statistical analysis was carried out with Sigma Stat software (Jandel Scientific; San Rafael, CA). Repeated measures procedures were used to compare responses before and after a given treatment in the same animal. To assess differences in vascular responses before versus after reduced oxygen availability, a comparison of mean values was made using ANOVA. For evaluation of differences in vascular responses to CO2 embolization before versus after reduced oxygen availability, comparisons were made using two-way ANOVA, with post hoc analysis via the Newman-Keuls multiple-range procedure.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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2–4 min.
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Under our conditions there was no significant change in tissue PO2. The site of tissue PO2 was the submucosal layer of tissue at the same depth in the intestinal wall as in the arterioles and venules we studied. The submucosal layer has access to oxygen from its own capillaries as well as those of the superficial muscle layers and the extensive capillary bed of the deeper glandular portions of the submucosa. Therefore, tissue PO2 in the submucosal layer was complexly influenced and did not represent the tissue environment between the superfusion fluid and the intestinal smooth muscle layers over the vessels observed.
Effect of reduced oxygen availability on perivascular [NO] and perivascular diameter. A total of five male Sprague-Dawley rats (305 ± 18 g) was used in these experiments. The mean resting diameters of 1As and 1Vs selected for study were 61 ± 3 and 105 ± 12 µm, respectively, and passive diameters were 87 ± 8 µm and 140 ± 12 µm, respectively. To determine the effect of reduced oxygen availability on perivascular [NO], NO-sensitive microelectrodes were used to measure arteriolar, venular and parenchymal NO. Figure 2 shows that resting periarteriolar and perivenular [NO] significantly increased from 397 ± 26 and 297 ± 34 nm during resting conditions to 695 ± 79 and 534 ± 66 nm, respectively, during reduced oxygen availability. Under these conditions, reduced oxygen availability caused significant arteriolar and venular dilation of 15 ± 3 and 13 ± 6%, respectively (Fig. 2B).
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Adenosine experiments: examples of the data obtained in three male Sprague-Dawley rats during application of adenosine. In each rat, as shown in Fig. 3, as the adenosine concentration was increased, the arteriole progressively dilated. The [NO] at the same point on the arteriolar wall either decreased or was relatively constant as the vessel dilated. Incomplete studies in other rats where we failed for technical reasons to reach maximum diameter also demonstrated relatively constant or mildly reduced [NO] as adenosine dilated the arterioles.
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Effect of NO inhibitor, cyclooxygenase inhibitor, and potassium channel inhibitor on arteriolar dilation during reduced oxygen availability. To determine whether NO, vasodilator cyclooxygenase products, or potassium channels activated are involved in the dilatory response to reduced oxygen availability, three separate protocols were performed. In the first series of experiments, a NO synthase inhibitor, L-NAME, was used to determine whether NO was involved in the dilatory response to reduced oxygen availability. To confirm the efficacy of 10–4 M L-NAME in the intestinal microvascular bed, a series of experiments utilizing ACh, an endothelium-dependent dilator, was first undertaken. In pilot studies, five male Sprague-Dawley rats (297 ± 15 g) were used to determine whether 10–4 M L-NAME provided acceptable suppression of NO-mediated dilation. Microiontophoretically applied ACh at 25, 50, 100, and 200 nA caused dose-dependent dilations that were attenuated by 64 ± 9%, 57 ± 5%, 45 ± 5%, and 39 ± 3%, respectively, in the presence of 10–4 M L-NAME (data not shown).
To determine the effect of L-NAME on dilatory responses to reduced oxygen availability, six male Sprague-Dawley rats (278 ± 18 g) were used. Application of 10–4 M L-NAME had no significant effect on the resting diameter for the overall data group, although about one-half of the arterioles did exhibit small reductions in resting diameter (pre-L-NAME diameters of 59 ± 3 µm vs. post-L-NAME diameters of 56 ± 4 µm). Figure 4A shows that NO inhibition with L-NAME significantly decreased arteriolar dilation to reduced oxygen availability in that the 14% dilation under natural conditions was reduced to 6% dilation in the presence of L-NAME.
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In the second series of experiments, a cyclooxygenase blocker (meclofenamate) was used to determine whether vasodilator cyclooxygenase products were involved in the dilatory response to reduced oxygen availability. Six Sprague-Dawley rats (246 ± 8 g) were used to test the efficacy of meclofenamate under the conditions of this study. Topical application of 10–2 M arachidonic acid evoked arteriolar dilation of 16 ± 4%. In the presence of 3 x 10–5 M meclofenamate, the dilation to arachidonic acid was completely abolished, confirming that meclofenamate was a potent inhibitor of cyclooxygenase activity in this vascular bed. Five more male Sprague-Dawley rats (328 ± 20 g) were used to assess the effect of cyclooxygenase inhibition on arteriolar dilation induced by reduced oxygen availability. Application of 3 x 10–5 M meclofenamate had no significant effect on the average resting diameter of the 1As selected for study (premeclofenamate diameters of 51 ± 4 µm vs. postmeclofenamate diameters of 50 ± 3 µm). Figure 4B shows that cyclooxygenase activity inhibition with meclofenamate had no effect on arteriolar dilation to reduced oxygen availability (12 ± 3% dilation under resting conditions vs. 11 ± 2% during superfusion with meclofenamate).
In the third series of experiments, we assumed that by blocking the vascular smooth muscle target of endothelium-derived hyperpolarizing factor(s), we might reveal the role of hyperpolarizing factor(s) in vasodilation associated with reduced perivascular PO2. We blocked potassium channels thought to respond to endothelium-derived hyperpolarizing factors (11, 17, 38) with TEA. We used 1 mM TEA because prior studies of arterioles have found this concentration to be effective (11, 17, 38).
A total of seven Sprague-Dawley rats (302 ± 10 g) was used to test
the efficacy of TEA under the conditions of this study. In none of the animals
did we find 1 mM TEA to have an effect on arteriolar diameter in the small
intestine (pre-TEA diameters of 64 ± 5 µm vs. post-TEA diameters of
62 ± 3 µm). In nine large arterioles of two rats, we compared
dilation to NS-1619, a specific agonist for calcium-activated potassium
channels, before and during TEA application. TEA reduced the dilation to
NS-1619 from 13.6 ± 3.5% before TEA to 3.2 ± 1.4% during TEA
application. In the next five animals using the same arteriole, which would
have its [NO] measured during reduced PO2, topical
application of 20 mM KCl evoked arteriolar dilation of 20 ± 3% in the
first five animals tested. The dilation to KCl was attenuated by 76 ±
4%; in the animals used to test NS-1619, the dilation to KCl was also
suppressed 75%. These results confirm that 1 mM TEA has specific
potassium channel blocking effects for intestinal arterioles and also has a
nonspecific potassium channel suppression to external 20 mM KCl.
A total of six male Sprague-Dawley rats (319 ± 8 g) was used to assess the possible effect of activated potassium channels as a contributor to arteriolar dilation induced by reduced oxygen availability. Figure 4C shows that inhibition of the effects of hyperpolarizing factors on potassium channels with TEA had no significant effect on arteriolar dilation to reduced oxygen availability (18 ± 3% dilation under resting conditions vs. 14 ± 3% during superfusion with TEA).
Effect of arteriolar embolization on periarteriolar [NO] and arteriolar dilation during reduced oxygen availability. To determine whether the arteriolar endothelium is the primary source of NO release during reduced oxygen availability, arteriolar embolization was used to functionally inactivate endothelial cells before and after reduced oxygen availability while NO was simultaneously measured with NO-sensitive microelectrodes. A total of four male Sprague-Dawley rats (290 ± 14 g) was used in these experiments. The mean resting diameter of the arterioles studies here was 57 ± 5 µm, with a mean passive diameter of 79 ± 6 µm measured at the end of the experimental period. CO2 embolization had a significant effect on resting diameter, causing arterioles to constrict by 17% (57 ± 5 µm during resting conditions vs. 46 ± 4 µm after embolization) and reduced basal NO release by 26% (501 ± 40 nm during preembolization conditions and 398 ± 36 nm after embolization). Figure 5 shows that CO2 embolization significantly attenuated the arteriolar NO response to reduced oxygen availability, with the [NO] nanomolar increase to reduced oxygen before embolization averaging 272 ± 12 nm, and that after embolization averaging 128 ± 17 nm.
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Vessel diameters were measured along with the [NO] measurements just described. The mean resting diameter of arterioles and venules studied here was 57 ± 5 and 121 ± 21 µm, respectively, with mean passive diameters of 79 ± 6 and 158 ± 16 µm measured at the end of the experimental period. The data in Fig. 6 show that CO2 embolization significantly attenuated the arteriolar and venular dilation to reduced oxygen availability. Before embolization, arterioles and venules dilated by 13 ± 1 and 12 ± 5%, respectively, to reduced oxygen availability, and after embolization, arteriolar and venular dilation under these conditions significantly decreased to 3 ± 1 and 4 ± 1%.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Effect of reduced oxygen availability on [NO] and vascular
diameter. In the strictest sense, hypoxia occurs when the oxygen
availability is decreased sufficiently to reduce oxygen consumption of a given
tissue. The test of this definition has been seldom met in studies of oxygen
depletion on microvessels. However, with this caveat in mind, oxygen
deprivation generally causes vasodilation in a variety of vascular beds and
species (19). It is also
documented in studies by other investigators that transient, "mild
hypoxia," or "reduced oxygen availability," causes dilation
of in vivo microvessels (22)
and isolated arterioles (12,
13,
18), although there is some
controversy in this area as to the exact degree of hypoxia needed to elicit a
dilatory response (10,
16,
31). In the current study,
reduced bathing fluid oxygen availability caused a decrease in periarteriolar
and perivenular PO2 of 15% and 8%, respectively
(Fig. 1). This drop in
perivascular PO2 was not considered to be
"hypoxic," but simply reflected reduced oxygen availability at the
vessel wall. Our goal was to use relatively small reductions in oxygen
availability that are far more likely to occur than hypoxic conditions. The
decrease in PO2 occurred within 45–60 s of
changing the bathing superfusate solution percent oxygen and was maintained
for 5 min for the conditions of this study. Our findings, as shown in Figs.
1,
2, and
6, demonstrated that both
arterioles and venules respond to reduced perivascular
PO2 with a localized increase in [NO] and simultaneous
vasodilation. The arteriolar and venular [NO] were locally determined by each
vessel because we measured the arteriolar and venular [NO] on vessel surfaces
facing the parenchymal tissue. These sites were at least 250 µm apart and
diffusion of NO between sites was highly unlikely. In effect, venules have
their own regulatory responses to PO2 and dilation is
associated with increased [NO]. It is important to point out that the
arteriolar [NO] at rest and during reduction in perivascular
PO2 was consistently higher than that measured for
paired venules. Therefore, these arterioles independently determined their own
NO responses. However, as shown in Fig.
2, venular perivascular PO2 did increase
during reductions in PO2 and at a distance that could
not be realistically influenced by the arteriolar [NO]. On the basis of these
observations, we propose that the PO2-NO production
relationships for the arterioles and venules studied occurred at different
ranges of PO2 but using similar mechanisms. The
regulatory issue seems reasonable on two fronts. First, venules must operate
at much lower PO2 values than even the smallest
arterioles and presumably adaptation to local circumstances has occurred.
Second, every perturbation that impaired increased [NO] and dilation by
arterioles in response to lowered PO2 caused a parallel
deficit for venular [NO] and dilation.
Depending on the microvascular bed and species studied, multiple vasoactive
factors have been implicated in eliciting vasodilation during reduced oxygen
availability. Among the most prominent include NO
(8,
9,
33,
34), hyperpolarizing factors
(13,
15), cyclooxygenase products
(12,
13,
23), and adenosine
(22,
30,
36). Recently, a myriad of in
vitro and in vivo studies (8,
9,
33,
34) have emphasized the key
role that NO plays in hypoxia-stimulated vasodilation. Our interest in tissue
oxygenation and NO formation was stimulated by Busse and colleagues
(8) and by Pohl and Busse's
(33) finding that hypoxia was
an important stimulus for NO-mediated vasodilation of isolated vessels. A
study by our laboratory (6)
demonstrated that when flow into a 1A was reduced by upstream occlusion of its
feed artery, there was significant oxygen desaturation and a concomitant
increase in [NO] by 27%. Our current findings are consistent with those
studies (33), showing that NO
is an important mediator of dilation in response to reduced oxygen levels.
Figure 4A shows that
local treatment with L-NAME attenuated dilation to reduced oxygen
availability by >60%.
Studies by and Pohl (32) strongly suggest that endothelial cells can act as "sensors" to oxygen levels. Thus there could be a sensing mechanism intimately related to endothelial cells and/or NO synthase that responds to reduced oxygen levels. To suppress endothelial regulation, we stunned them with intravascular CO2 embolization. The data in Fig. 5 demonstrate dramatic reductions in [NO] both at rest and in response to reduced oxygen levels postendothelial impairment with CO2 embolization. Nase and Boegehold (27) showed that localized gas embolization suppressed endothelium-dependent vasodilation to locally applied ACh without altering endothelium-independent dilation to sodium nitroprusside. A more recent study by our laboratory supported Nase and Boegehold's (6) finding for selective endothelial impairment by localized CO2 embolization. Embolization did not fully eliminate NO release at rest nor fully suppress responses to reduced perivascular PO2. Note in Fig. 5A that embolization decreased the resting [NO] but even more importantly, the response to lowered perivascular PO2 was reduced by about two-thirds, as is best appreciated in Fig. 5B. We have questioned where is the NO coming from after embolization at rest and during reduction of the PO2? There are three likely sources. First, the upstream portions of the arteries and large arteriole are fully intact and NO is likely being washed down in the blood. Second, the vessel portion that was embolized is stunned but still somewhat functional. Third, the nearby venular wall may be providing some of the arteriolar NO both at rest and during regional oxygen limitation. However, at the arteriolar site of measurement and embolization, NO responses to reduced PO2 were dramatically attenuated. We believe this localized suppression demonstrated that the primary site for NO release was from arteriolar endothelial cells when the tissue was intact. This is an important finding because both intestinal parenchymal cells and the venules can also produce NO in response to a variety of stimuli (28, 29). However, the data in Fig. 2A show that during reduced PO2 in the tissue bath, there was no significant increase in submucosal parenchymal cell [NO] and venular [NO] was less than that for arterioles.
In an attempt to determine whether there were secondary vasoactive factors involved in the increased [NO] response at reduced perivascular PO2, we evaluated whether potassium channels possibly linked to endothelium-derived hyperpolarizing factors, adenosine released from tissue, or dilator cyclooxygenase products played a role in vasodilation to reduced oxygen availability. The data in Fig. 4, B and C, indicated that blockade of cyclooxygenase products with meclofenamate and calcium-activated potassium channels sensitive to TEA had no effect on the dilatory response to reduced oxygen. With cyclooxygenase and calciumactivated potassium channel pharmacological blockades, resting diameter was not altered and the dilatory responses to arachidonic acid and 20 mM potassium chloride, respectively, were strongly suppressed. Direct application of ionized adenosine to the vessel wall and simultaneous measurement of NO at the site of application indicated that as expected the arterioles dilated but the [NO] did not increase (Fig. 3). Although we acknowledge that prior bioassay studies (12, 13) have demonstrated a role for hyperpolarizing factors, adenosine, and cyclooxygenase products in oxygen-related vasodilation, we believe the differences in past and current studies are related to the magnitude of oxygen deprivation. We used a small oxygen loss to simulate the small reductions in local vessel wall PO2 that occur quite frequently. In contrast, the majority of prior studies on this issue use much more severe reductions in oxygen availability. This raised a question that we cannot currently resolve as to whether regulation of NO formation linked to oxygen varies as a function of the degree of oxygen depletion. However, large reductions in tissue PO2 are relatively rare compared with small reductions in PO2 that accompany many forms of tissue and systemic responses.
Adenosine studies. Several laboratories (24, 35) have questioned whether large reductions in PO2 release adenosine to stimulate NO synthesis based on bioassay events after pharmacological suppression of endothelial NO synthase. We found adenosine applied to the vessel wall did not increase local [NO] (Fig. 3) as the arterioles dilate and therefore assumed adenosine was unlikely to cause vasodilation for mild reductions in PO2. Because we used a range of adenosine release that caused dilation from threshold dilation to near maximum, adenosine concentration per se did not seem to be the issue in causing or not causing NO release.
In conclusion, our study is consistent with the postulate that small perivascular reductions in oxygen availability stimulated endothelium-derived NO and caused concomitant arteriolar and venular dilation in the intact intestinal microcirculation. This influence represents a potentially important mechanism by which the local environment of a microvessel can modulate blood flow to meet small changes in oxygen requirements.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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