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Am J Physiol Heart Circ Physiol 291: H724-H731, 2006. First published March 31, 2006; doi:10.1152/ajpheart.01215.2005
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Leukocyte adherence inhibits adenosine-dependent venular control of arteriolar diameter and nitric oxide

Min-ho Kim and Norman R. Harris

Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana

Submitted 16 November 2005 ; accepted in final form 23 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Venular control of arteriolar perfusion has been the focus of several investigations in recent years. This study investigated 1) whether endogenous adenosine helps control venule-dependent arteriolar dilation and 2) whether venular leukocyte adherence limits this response via an oxidant-dependent mechanism in which nitric oxide (NO) levels are decreased. Intravital microscopy was used to assess changes in arteriolar diameters and NO levels in rat mesentery. The average resting diameter of arterioles (27.5 ± 1.0 µm) paired with venules with minimal leukocyte adherence (2.1 ± 0.3 per 100-µm length) was significantly larger than that of unpaired arterioles (24.5 ± 0.8 µm) and arterioles (23.3 ± 1.3 µm) paired with venules with higher leukocyte adherence (9.0 ± 0.5 per 100-µm length). Local superfusion of adenosine deaminase (ADA) induced significant decreases in diameter and perivascular NO concentration in arterioles closely paired to venules with minimal leukocyte adherence. However, ADA had little effect on arterioles closely paired to venules with high leukocyte adherence or on unpaired arterioles. To determine whether the attenuated response to ADA for the high-adherence group was oxidant dependent, the responses were also observed in arterioles treated with 10–4 M Tempol. In the high-adherence group, Tempol fully restored NO levels to those of the low-adherence group; however, the ADA-induced constriction remained attenuated, suggesting a possible role for an oxidant-independent vasoconstrictor released from the inflamed venules. These findings suggest that adenosine- and venule-dependent dilation of paired arterioles may be mediated, in part, by NO and inhibited by venular leukocyte adherence.

leukocyte adhesion; Tempol

RESTING ARTERIOLAR TONE can be determined by various endogenous vasoactive substances, the release of which depends on the metabolic state of the tissue, as well as on hemodynamic factors such as shear stress and pressure. In addition, when an arteriole is closely paired with a venule, vasoactive substances released from the venule also play an important role in regulating arteriolar diameter, depending on various stimulated conditions such as an increase in venular shear rate (26), an increase in muscle stimulation (16), and an exogenous venular infusion of vasoactive substances (5, 11). Nitric oxide (NO), prostaglandins, and adenosine have been implicated as mediators of venule-induced arteriolar dilation. Anatomically, various sizes of arterioles from large to small are paired with venules within a short diffusion distance in most tissues, and this close arrangement enables venular-arteriolar diffusion of vasoactive substances.

In this study, we hypothesized that endogenous adenosine induces venule-dependent dilation of closely paired arterioles in an NO-mediated mechanism, but with the response limited by the presence of adherent leukocytes in the venule. The rationale that led us to this hypothesis was obtained from several recent studies. Hammer et al. (7) observed a significantly larger resting diameter of paired than unpaired third-order arterioles in hamster cremaster muscle, consistent with the possibility that venular pairing mediates arteriolar diameter, even under resting conditions. In the mesenteric microcirculation, resting capillary flow velocities were positively correlated with the extent of close venular pairing of the feed arterioles (20). Moreover, acute NO synthase (NOS) inhibition completely reversed this correlation: NOS inhibition reduced capillary flow only when the feed arterioles were closely paired with venules. This result suggests that NO or other mediators dependent on NO are continuously released from venules and diffuse to paired arterioles to induce arteriolar dilation in resting conditions.

Although NO mediates venular-dependent arteriolar dilation during resting conditions, the extent to which NO diffuses from venule to arteriole is unknown and has been modeled recently by Kavdia and Popel (13). An alternative explanation is that another mediator diffuses from venule to arteriole in the NO-dependent dilatory mechanism. Cyclooxygenase metabolites play a major role in venule-mediated arteriolar dilation in functional hyperemia, but possibly not in resting conditions (7). Adenosine is a potent vasodilator in many vascular beds, and adenosine release is increased during hypoxia, ischemia, or metabolic stress, when O2 delivery is inadequate to meet the tissue demand (18, 32). Adenosine exogenously infused through venules has been shown to vasodilate paired arterioles (11). However, whether endogenous adenosine contributes to arteriolar vasodilatory function has not been determined.

In contrast to normal physiological conditions, the presence of inflammatory cells in venules may have a major influence on arteriolar constriction (8, 33). Zamboni et al. (33) found that arterioles in reperfused (previously ischemic) skeletal muscle, but only arterioles near inflamed venules (as assessed by the presence of adherent leukocytes), were constricted. Inhibition of venular leukocyte adherence attenuated arteriolar constriction in their model (34). Nellore and Harris (21) found a significant negative correlation between venular leukocyte adherence and NO concentration in the closely paired arteriole. If adenosine regulates resting arteriolar diameter in an NO-dependent mechanism, it is possible that oxidants released as a consequence of leukocyte-endothelial cell interactions may play a crucial role in arteriolar tone by decreasing NO bioavailability.

Therefore, the primary aim of this study was to determine whether adenosine mediates venule-dependent arteriolar dilation via an NO-mediated mechanism under normal physiological conditions. Another objective was to determine whether venular leukocyte adherence limits this response via an oxidant-dependent decrease in NO. As an experimental model, intravital microscopy of the rat mesenteric microcirculation was used to monitor changes in arteriolar diameters and NO levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Animal procedures were approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center. Male Wistar rats (250–300 g body wt) were housed two to three per cage in a controlled environment (12-h:12-h light-dark cycle) and given food and water ad libitum. The rats were anesthetized by an injection of thiobutabarbital (135 mg/kg ip; Inactin, Sigma T-133, St. Louis, MO). The right carotid artery was cannulated for systemic injection of saline during the experiment (~1 ml) and euthanasia solution at the end of the experiment. A segment of the small intestine was exteriorized through a midline abdominal incision, and the spontaneously breathing rat was placed on its right side on a Plexiglas board, so that a selected section of mesentery could be draped over a glass coverslip glued on a hole centered in the board. The exposed intestine, except for the selected mesenteric section under study, was covered with gauze soaked in bicarbonate-buffered saline (BBS, in mM: 131 NaCl, 4.7 KCl, 1.2 MgSO4, 20 NaHCO3, and 3.5 CaCl2). After the board was mounted onto the stage of an inverted microscope, the mesentery and intestine were kept moist with a 2 ml/min superfusion of BBS bubbled with 95% N2-5% CO2 and warmed to 37°C. Rectal temperature was maintained at ~37°C by positioning of an infrared heat lamp over the rat.

Video microscopy. The mesenteric microvessels were observed through a x40 objective (Nikon Plan Apo, 0.95 numerical aperature) using a 100-W halogen light source, and bright-field images were captured with a color camera (CCD-IRIS, Sony). The images were directed to a video-cassette recorder (SVO-9500 MD, Sony), and the taped images were used for playback analysis with an image grabber and image processor (Scion Image) for length and diameter measurements.

Local superfusion of adenosine deaminase. Local superfusion of adenosine deaminase (ADA, 0.42 U/ml; Sigma) was accomplished using a pressurized glass micropipette connected to a regulated pressure source. ADA metabolically inactivates adenosine by catalyzing the conversion of adenosine to inosine. ADA was applied directly to the tissue at an ejection pressure of 20 mmHg. Glass micropipettes were pulled with a pipette puller (model PUL-1, World Precision Instruments, Sarasota, FL) and blunted to a tip diameter of 10–15 µm. The micropipettes were filled with ADA mixed in BBS. Sulforhodamine (Sigma) was also included in the ADA solution to verify ADA flow from the micropipette with use of fluorescent images. For fluorescence microscopy, the preparation was illuminated with a 100-W mercury lamp, and sulforhodamine was briefly viewed in a rhodamine filter. The micropipette was positioned over selected mesenteric tissue between a paired arteriole and venule or tissue close to an unpaired arteriole (placed <10 µm from the arteriole) and then carefully lowered onto the tissue by means of a micromanipulator. After verification of fluorescent flow over the selected region of tissue, the fluorescence microscopy system was switched to bright-field microscopy, and the images were recorded continuously. Our preliminary data obtained from venule-paired arterioles demonstrated that the ADA concentration used in this study (0.42 U/ml) was sufficient to give a maximal response (Fig. 1).


Figure 1
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  		Fig. 1. Effects of increasing concentrations of adenosine deaminase (ADA) on changes in diameter of venule-paired arterioles.

 
Arteriolar NO measurement with use of a microelectrode. Periarteriolar NO concentration was monitored using an NO-selective microelectrode (model ISO-NO-3005, World Precision Instruments). In vitro calibration of the microelectrode was performed 30 min before each experiment. For calibration, the electrode was placed in a copper sulfate solution at 37°C, and a known concentration of S-nitroso-N-acetyl-DL-penicillamine, a stable NO-containing compound, was added. S-nitroso-N-acetyl-DL-penicillamine decomposes to NO and a disulfide by-product when dissolved in water in a reaction catalyzed by copper sulfate. The time course of current output was displayed on the instrument monitor. Figure 2A demonstrates a typical in vitro calibration, which shows linearity between the changes in output current and NO concentration. The sensor attached to the micromanipulator was placed over the arteriolar wall as close as possible to, and slightly pressed against, the vessel wall. The slight touch of the microelectrode on the arteriolar wall resulted in a transient initial signal, which returned to a stable value after 20–30 s. Before arteriolar NO measurements, the microelectrode was allowed to equilibrate for 30 min in the BBS superfusate bathing the mesentery.


Figure 2
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  		Fig. 2. A: sample calibration of microelectrode output current vs. nitric oxide (NO) concentration. B: time course of arteriolar microelectrode NO levels in the presence of 100 µM N{omega}-nitro-L-arginine methyl ester (L-NAME). Individual values obtained at 25 min from arterioles paired with high-leukocyte-adherence venules (PHL) and low-leukocyte-adherence venules (PLL) are shown at right.

 
To demonstrate the utility of the NO-selective microelectrode to measure NO concentrations in vivo, the electrode was placed on venule-paired arterioles, and the signal was monitored in response to NOS inhibition (with 100 µM N{omega}-nitro-L-arginine methyl ester; Sigma). Figure 2B shows the time course of arteriolar NO levels measured using the microelectrode, beginning with a 5-min baseline period with BBS superfusion. Infusion of N{omega}-nitro-L-arginine methyl ester for 20 min induced an ~55% decrease in arteriolar NO. However, this response was influenced dramatically by the number of adherent leukocytes in the closely paired venule, in which arterioles paired with high leukocyte adherence (PHL) demonstrated a small decrease in NO levels compared with arterioles with low leukocyte adherence (PLL) in the paired venule.

Venular leukocyte rolling and adherence. The number of rolling and adherent leukocytes was quantified during playback analysis of taped images. The number of rolling leukocytes was defined as the number of leukocytes that passed a selected cross section of venule per minute. Leukocyte adherence was defined as the number of leukocytes (per 100 µm length) that remained stationary on the venular wall for ≥30 s.

Experimental protocols. Unpaired arterioles and closely paired, parallel arterioles and venules (arteriovenular distance <20 µm) in the mesentery were selected. Experimental groups were divided as follows (Fig. 3) : 1) unpaired (UP) arterioles, 2) arterioles paired with venules with low leukocyte adherence (PLL, <5 adherent leukocytes per 100-µm length), and 3) arterioles paired with venules with high leukocyte adherence (PHL, ≥5 adherent leukocytes per 100-µm length). To determine whether venular pairing and venular leukocyte adhesion affect the resting diameters of paired arterioles, third-order UP and paired arterioles were selected, and resting diameters were measured. In each arteriole, an average diameter was obtained from three measurements along the vessel length.


Figure 3
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  		Fig. 3. Microscopic images of unpaired (UP) and paired arterioles. A: small UP arteriole. B: small PLL arteriole. C: small PHL arteriole. Leukocytes seen through the arteriole are in the surrounding interstitial space and are not adherent to the arteriolar endothelium. A, arteriole; V, venule.

 
To investigate the potential contribution of adenosine to arteriolar dilation and NO concentration, ADA was locally superfused over the selected tissue between paired arterioles and venules or, in separate experiments, near a UP arteriole. After 10 min of baseline measurement of arteriolar diameter and NO concentration, ADA was locally superfused for 15 min, and changes in arteriolar diameter and NO concentration were measured simultaneously.

To investigate the possible relation between arteriolar NO and oxidants induced by leukocyte-endothelial cell interactions, the free radical scavenger Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, 10–4 M; Sigma) was included in the BBS superfusate. After baseline measurement of arteriolar diameter and NO concentration for 10 min with superfusion of BBS, Tempol was superfused for an additional 15 min. The preparation was then superfused locally with ADA via micropipette, and the superfusion of Tempol was continued during the local superfusion of ADA. The experiments were performed in three groups of UP or paired (PHL and PLL) arterioles, and changes in arteriolar diameter and NO concentration were measured simultaneously throughout the experiment.

Statistics. GraphPad Instat (San Diego, CA) software was used for linear regression. Multiple data from individual sets of experiments were analyzed by repeated-measures ANOVA with Bonferroni’s corrections. Values are means ± SE. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In this study, to address the possible role of adenosine and leukocyte adhesion in venular control of resting arteriolar tone and NO bioavailability, arterioles were selected and categorized as venule paired (close parallel pairing with a venule) or UP. Venule-paired arterioles were divided further into two groups: PLL and PHL.

Influences of venular pairing and leukocyte adhesion on resting arteriolar diameter. The average resting diameter of the PLL group (27.5 ± 1.0 µm, total of 24 arterioles from 13 rats) was significantly larger than that of the UP (24.5 ± 0.8 µm, total of 29 arterioles from 15 rats) and PHL (23.3 ± 1.3 µm, total of 18 arterioles from 13 rats) groups (Fig. 4). The number of adherent leukocytes was significantly larger for the PHL (9.0 ± 0.5 per 100-µm length) than for the PLL (2.1 ± 0.3 per 100-µm length) group. No significant difference in resting diameter was observed between the UP and the PHL group. For closely paired arterioles and venules, a negative correlation was observed between resting arteriolar diameter and the number of venular adherent leukocytes (P = 0.028, r2 = 9.3%). However, no correlation was found between resting arteriolar diameter and the number of venular rolling leukocytes (P > 0.05). The mean arteriovenular distance (11.2 ± 1.4 and 9.5 ± 0.9 µm for PLL and PHL, respectively, P > 0.05) and diameter of the paired venule (43.7 ± 2.6 and 38.2 ± 1.4 µm for PLL and PHL, respectively, P > 0.05) were similar between the two groups of venule-paired arterioles.


Figure 4
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  		Fig. 4. Influence of venular pairing and leukocyte adherence on resting diameter of 3rd-order arterioles: resting arteriolar diameter in UP (n = 29) and paired (PHL and PLL, n = 42) arterioles. Error bars, SE. *P < 0.05 vs. unpaired. #P < 0.05 vs. PHL.

 
Effects of ADA on arteriolar diameter and NO. Figure 5 shows the changes in diameter and NO from the three groups of arterioles in response to local superfusion of ADA. Distribution of diameter changes among the groups is shown in Fig. 5A. A decrease in diameter of ≥0.4 µm (the limit of optical resolution) was observed in 67–77% of arterioles irrespective of group: 4 of 6 UP, 8 of 11 PLL, and 10 of 13 PHL arterioles. However, among these responsive arterioles, the level of constriction due to ADA was substantially greater (P < 0.05) in the PLL group (Fig. 5B), with the diameter decreasing by 18.3 ± 4.1% (compared with 3.8 ± 0.9% in the PHL group). In the arterioles of Fig. 5B, periarteriolar NO concentrations also were measured using an NO-selective microelectrode to investigate the possible role of NO in adenosine-induced arteriolar dilation (Fig. 5C). In baseline measurements, the level of arteriolar NO was significantly higher in the PLL group (521 ± 47 nM) than in the PHL and UP groups (408 ± 32 and 409 ± 23 nM, respectively). Local superfusion of ADA caused a significant decrease in arteriolar NO in the PLL group (17.3 ± 2.1%) and only small reductions in the PHL and UP groups (6.8 ± 1.2 and 2.2 ± 1.1%, respectively). The ADA-induced decrease of 18% in arteriolar diameter is similar to the 17% decrease in NO in the PLL group (Fig. 5D), suggesting that ADA-induced arteriolar constriction might involve a decrease in NO.


Figure 5
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  		Fig. 5. Changes in diameters and NO concentrations in response to ADA treatment for UP, PLL, and PHL arterioles. A: distribution of diameter changes. B–D: time course of diameter changes (B), time course of NO concentrations (C), and percent changes in arteriolar diameters and NO concentrations (D) after ADA treatment for arterioles that constricted in response to ADA. Error bars, SE. *P < 0.05 vs. UP. #P < 0.05 vs. PHL.

 
Effects of Tempol on changes in arteriolar diameter and NO. To determine whether superoxide might be responsible for the decreased resting diameter and NO bioavailability in PHL arterioles, changes in diameters and NO levels were measured after Tempol treatment (Fig. 6). As expected, Tempol treatment significantly increased arteriolar diameter (4.3 ± 0.1% increase) and NO level (29 ± 4.4%, increase from 395 to 509 nM). Tempol treatment also increased arteriolar diameter (2.4 ± 0.3%) and NO (11.0 ± 2.2%, increase from 470 to 524 nM) in the PLL group, but to a lesser degree than in the PHL group (P < 0.05, PHL vs. PLL). In UP arterioles, Tempol treatment did not affect arteriolar diameter (not significant compared with 0%) but resulted in a moderate increase in NO level (4.0 ± 0.8%, increase from 380 to 395 nM, significantly different from 0%). With the consideration that the resting NO level for the PHL group was ~20% lower than that of the PLL group (Fig. 5C), it appears that Tempol treatment fully restored the NO concentration of the PHL group to the level of the PLL group.


Figure 6
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  		Fig. 6. Effects of 10–4 M Tempol on diameters and NO concentrations for UP (n = 6), PLL (n = 7), and PHL (n = 7) arterioles. Error bars, SE. *P < 0.05 vs. UP. #P < 0.05 vs. PLL.

 
Effects of ADA on arteriolar diameter and NO in the presence of Tempol. To determine whether superoxide inhibition can restore adenosine-induced dilation of PHL arterioles, ADA was locally superfused in the presence of Tempol. The number of rolling and adherent leukocytes was not altered by Tempol and ADA treatment (Table 1). Among the arterioles that constricted in response to ADA, arteriolar NO in the PHL group was decreased significantly by 13.7 ± 0.9% after ADA treatment in the presence of Tempol, which is comparable to the 12.9 ± 1.5% decrease in the PLL group (Fig. 7). These results contrast significantly with the data presented in Fig. 5D, where ADA had a much smaller effect on arteriolar NO in the PHL group without Tempol. In the PHL group, treatment with ADA in the presence of Tempol also resulted in enhanced arteriolar constriction (8.0 ± 0.5%; Fig. 7) compared with ADA treatment only (3.8 ± 0.9%; Fig. 5D). However, the relative magnitude of the arteriolar constriction did not equal that of the PLL group: 15.0 ± 3.0 and 8.0 ± 0.5% decrease for the PLL and PHL groups, respectively (P < 0.05, PHL vs. PLL).


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  		Table 1. Number of rolling and adherent leukocytes in PLL and PHL groups before and after Tempol/ADA treatment

 

Figure 7
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  		Fig. 7. ADA-induced changes in diameters and NO concentrations in the presence of 10–4 M Tempol for UP (n = 3), PLL (n = 7), and PHL (n = 7) arterioles. Error bars, SE. *P < 0.05 vs. UP. #P < 0.05 vs. PHL.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Venular control of arteriolar perfusion has been the focus of several investigations in recent years. In two of the earliest studies, the vasoconstrictor norepinephrine (29) or the vasodilator adenosine (11) was injected into venules, and the response in arterioles closely paired with venules downstream of the injection site was observed. Whether endogenous adenosine performs the same vasodilatory function had not been established until our present study. Moreover, we demonstrate inhibition of adenosine-mediated venular dilation of arterioles when the venules are inflamed, as assessed by the accumulation of adherent leukocytes.

Resting arterial plasma adenosine concentrations are known to be lower than the threshold levels of adenosine required for arterial dilation (24). Similarly, at the downstream arteriolar level, there has been little evidence that adenosine plays a role in resting diameter. Sauls and Boegehold (28) reported no change in the diameter of first-order arterioles in rat intestine on application of ADA. Additionally, Proctor (25) found that ADA, when applied to third-order arterioles in hamster cremaster muscle, did not induce a significant change in diameter. Even though no statistical differences were reported in the latter study, resting arteriolar diameters were consistently ~10% smaller in the presence of ADA in the three experimental groups. Their arterioles were not segregated according to venular pairing; the resulting trend toward an ~10% smaller diameter is between our 1% and 18% ADA-induced constrictions in unpaired arterioles and arterioles paired with low-adherence venules, respectively.

In this study, the average distance between paired arterioles and venules was <15 µm, which is close enough to enable the diffusion of small mediators from venule to arteriole on the basis of the observation that adenosine molecules released from cannulated venules could dilate paired arterioles at a distance of >30 µm (11). These results support the hypothesis that venuloarteriolar diffusion results in increased arteriolar adenosine concentration above the threshold level for arteriolar dilation. This may explain our observation of larger resting arteriolar diameters for paired arterioles (with minimal leukocyte adhesion) than for UP arterioles. However, the origin of adenosine formation is not clear. Adenosine can be released from various types of cells. Recent studies suggest that adenosine can be formed intracellularly by vascular endothelium or blood cells during systemic hypoxia (18) and also by interstitial cells during muscle contractions (10). However, it is unlikely that venular endothelium is a source of adenosine during resting conditions, because resting arteriolar diameter was unaffected by disruption of the venular endothelium (27), indicating that mediators released directly from venular endothelium may not be involved in regulation of arteriolar diameter under resting conditions. Therefore, another possibility is that adenosine formed from extravascular cells diffuses into capillary beds, resulting in a higher venular adenosine concentration than in the feeding arterioles, with the concentration gradient providing a net interstitial flux of adenosine, in the direction of the arteriole, in a sufficient quantity to induce dilation. The countercurrent arrangement could facilitate the control system, whereby increased metabolism of the capillary bed would be sensed in the venular flow and a signal to increase blood flow and nutrient delivery would be delivered to the paired arteriole.

Adenosine is known to mediate vasodilation via endothelium-dependent and -independent mechanisms. Adenosine coupled to receptors on vascular smooth muscle triggers smooth muscle relaxation through cAMP-dependent (14) or cGMP-dependent (31) protein kinase pathways. On the other hand, adenosine also has been shown to mediate smooth muscle relaxation by endothelial NO synthesis in many vascular beds, such as the large coronary artery (1), coronary microvessels (9), intestinal microvessels (28), skeletal muscle (2), and hepatic microvessels (23). In this study, to investigate whether adenosine might mediate arteriolar dilation via an NO-dependent mechanism, we used an NO-selective microelectrode to measure periarteriolar NO concentration. The baseline NO concentration (~400 nM for UP arterioles) was almost identical to the value obtained from similar-sized arterioles of rat intestine [397 ± 26 nM (19)] measured using a recessed-tip glass microelectrode. Our results show decreases in arteriolar diameter induced by ADA in venule-paired vessels that are closely associated with concomitant decreases in perivascular NO concentrations (Fig. 5), supporting a role for NO in adenosine-mediated arteriolar dilation. These results are consistent with the possibility that adenosine coupled to receptors in the arteriolar wall might contribute to resting arteriolar dilation by triggering NO release. However, our experiments did not address the source of NO mediating the dilation. It is possible that adenosine triggers NO release via neuronal NOS in or near the arteriolar wall on the basis of a recent study of NO in the rat mesenteric microcirculation (12). Kashiwagi et al. (12) suggested that neuronal NOS could be a major source of NO in distal arterioles, with less contribution from endothelial NOS than in venules, capillaries, and more proximal arterioles.

In addition to the role of adenosine in maintaining resting dilation of venule-paired arterioles, another interesting finding of this study is that adenosine-dependent arteriolar dilation was affected by leukocyte adherence in paired venules. PHL arterioles had smaller resting diameters than PLL arterioles, and the arteriolar response of PHL arterioles to ADA was dramatically attenuated compared with that of PLL arterioles. One possible explanation is involvement of leukocyte-mediated scavenging of NO on the basis of the observation that ADA-induced arteriolar constriction was closely associated with a decrease in NO. This is similar to the arteriolar constriction observed in ischemia-reperfusion injury (34), in which venular adherent leukocytes contributed to the constriction of paired arterioles, with the response attenuated by injection of a monoclonal antibody against the adhesion molecule CD11/CD18.

The inflammatory response in postcapillary venules is associated with a significant increase in the recruitment of rolling, adherent, and emigrating leukocytes, resulting in an increased production of oxidants, such as the superoxide anion, which rapidly reacts with NO (6). The enhanced formation of oxidants attenuates NO bioavailability (17) and impairs endothelium-dependent vasodilation in various cardiovascular diseases such as hypercholesterolemia, hypertension, and diabetes (3). Our NO measurements support this hypothesis, with a significant 20% attenuation in resting perivascular NO in PHL arterioles compared with PLL arterioles. These data are also consistent with previous NO measurements in rat mesentery with cell-permeable diaminofluorescein-2-diacetate; decreased arteriolar and venular NO was observed with elevated leukocyte adhesion in the paired venule (21). Additionally, the superoxide dismutase mimetic Tempol significantly increased NO bioavailability in PHL arterioles, indicating that venular leukocytes might produce superoxide and, thereby, scavenge NO. Therefore, it is conceivable that arteriolar NO release triggered by adenosine might be inhibited by superoxide produced by the activation of adherent leukocytes in paired venules, resulting in arteriolar constriction (Fig. 8). However, it should be noted that superoxide dismutase activity also can trigger production of the vasodilator H2O2, which could contribute to arteriolar dilation.


Figure 8
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  		Fig. 8. Proposed roles of adenosine and adherent leukocytes in regulation of resting tone of venule-paired arterioles. With minimal leukocyte and platelet adhesion, diffusing adenosine (ADO) molecules may bind to receptors on the arteriolar wall. Binding of adenosine molecules to their receptors triggers release of NO and subsequent smooth muscle cell (SMC) relaxation. Inflamed venules with activated leukocytes or platelets contribute to SMC constriction by releasing vasoconstrictors or by producing oxidants that decrease NO bioavailability.

 
Another possible explanation for the attenuated response to ADA in PHL arterioles is an oxidant-independent involvement of other vasoconstrictors released from adherent platelets or leukocytes. Our data for resting arteriolar diameter in paired vessels indicate that PHL arterioles may be preconstricted by ~15% compared with PLL arterioles (Fig. 4). Diameters of UP (or more distantly paired) arterioles would be expected to be smaller than diameters of closely paired arterioles because of diffusion-limited dilation by venule-derived mediators. If the superoxide radical is entirely responsible for attenuation of adenosine-induced arteriolar dilation, it might be expected that Tempol would increase arteriolar diameter by ~15%. However, Tempol only partially increased arteriolar diameter by ~4% for the PHL group, and the arterioles were still constricted by ~11% compared with the PLL group. Additionally, the relative magnitude of arteriolar constriction in response to ADA was restored only partially to that of the PLL group, even though the NO level was fully restored by Tempol. These results suggest the involvement of an additional vasoconstrictor released from inflamed venules that may compete with the adenosine- and NO-dependent vasodilatory mechanism in regulating arteriolar tone. Adherent leukocytes release proteases (chymase and cathepsin G) that facilitate production of the vasoconstrictors endothelin-1 (ET-1) and angiotensin II. For example, cathepsin G on the neutrophil surface is able to convert not only angiotensinogen to angiotensin II but also angiotensin I to angiotensin II (22). Additionally, chymase and cathepsin G each cleaves the precursor Big ET-1 into ET-1 (15). Therefore, ET-1 and angiotensin II may be involved in a mechanism whereby leukocyte adherence contributes to venule-mediated arteriolar constriction. However, angiotensin II may contribute to arteriolar constriction via an NO-dependent mechanism, because angiotensin II can activate NADPH oxidase, which in turn results in superoxide production and NO inactivation (30).

We did not measure venular platelet adhesion in our present experiments, and we cannot rule out the possibility that platelet adhesion plays a significant role in venular-mediated arteriolar constriction. A recent study revealed that platelet adhesion within postcapillary venules was highly dependent on leukocyte adhesion, with ~45% of adherent leukocytes binding platelets in a model of ischemia-reperfusion (4). Activated platelets can release the potent vasoconstrictor thromboxane, which has been implicated in a recent study (8) in which platelet adherence in venules was associated with constriction of closely paired arterioles in a model of dextran sodium sulfate-induced inflammation.

Figure 8 shows the mechanism that has been proposed to explain the role of venular adenosine and adherent leukocytes in regulation of the resting diameter of closely paired arterioles. Under normal physiological conditions, there may be diffusional transport of adenosine molecules from venules to paired arterioles, which may contribute to arteriolar dilation partially via an NO-dependent mechanism. However, in pathophysiological conditions with inflamed venules, the paired venule may contribute to arteriolar constriction by providing oxidants and, thereby, decreasing NO bioavailability or providing various vasoconstrictors from activated platelets or leukocytes.

In summary, we found evidence that closely paired venules contribute to arteriolar dilation via an adenosine- and NO-dependent mechanism under normal resting conditions, whereas inflamed venules with adherent leukocytes contribute to arteriolar constriction by an oxidant-mediated inhibition of the adenosine pathway or release of additional vasoconstrictors produced at sites of leukocyte adhesion.


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 MATERIALS AND METHODS
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This work was funded by Juvenile Diabetes Research Foundation Grant 1-2003-159.


   ACKNOWLEDGMENTS
 
The authors thank Georgia Morgan First for proofreading and editing.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. R. Harris, Dept. of Molecular and Cellular Physiology, Louisiana State Univ., 1501 Kings Hwy., Shreveport, LA 71130 (e-mail: nharr6{at}lsuhsc.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Abebe W, Hussain T, Olanrewaju H, and Mustafa SJ. Role of nitric oxide in adenosine receptor-mediated relaxation of porcine coronary artery. Am J Physiol Heart Circ Physiol 269: H1672–H1678, 1995.[Abstract/Free Full Text]
  2. Bryan PT and Marshall JM. Cellular mechanisms by which adenosine induces vasodilation in rat skeletal muscle: significance for systemic hypoxia. J Physiol 514: 163–175, 1999.[Abstract/Free Full Text]
  3. Cai H and Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840–844, 2000.[Abstract/Free Full Text]
  4. Cooper D, Russell J, Chitman KD, Williams MC, Wolf RE, and Granger DN. Leukocyte dependence of platelet adhesion in postcapillary venules. Am J Physiol Heart Circ Physiol 286: H1895–H1900, 2004.[Abstract/Free Full Text]
  5. Falcone JC and Bohlen HG. EDRF from rat intestine and skeletal muscle venules causes dilation of arterioles. Am J Physiol Heart Circ Physiol 258: H1515–H1523, 1990.[Abstract/Free Full Text]
  6. Granger DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 6: 167–178, 1999.[CrossRef][Web of Science][Medline]
  7. Hammer LW, Ligon AL, and Hester RL. Differential inhibition of functional dilation of small arterioles by indomethacin and glibenclamide. Hypertension 37: 599–603, 2001.[Abstract/Free Full Text]
  8. Harris NR, Whatley JR, Carter PR, and Specian RD. Venular constriction of submucosal arterioles induced by dextran sodium sulfate. Inflamm Bowel Dis 11: 806–813, 2005.[CrossRef][Web of Science][Medline]
  9. Hein TW, Belardinelli L, and Kuo L. Adenosine A2A receptors mediate coronary microvascular dilation to adenosine: role of nitric oxide and ATP-sensitive potassium channels. J Pharmacol Exp Ther 291: 655–664, 1999.[Abstract/Free Full Text]
  10. Hellsten Y, Maclean D, Radegran G, Saltin B, and Bangsbo J. Adenosine concentrations in the interstitium of resting and contracting human skeletal muscle. Circulation 98: 6–8, 1998.[Abstract/Free Full Text]
  11. Hester RL. Venular-arteriolar diffusion of adenosine in hamster cremaster microcirculation. Am J Physiol Heart Circ Physiol 258: H1918–H1924, 1990.[Abstract/Free Full Text]
  12. Kashiwagi S, Kajimura M, Yoshimura Y, and Suematsu M. Nonendothelial source of nitric oxide in arterioles but not in venules: alternative source revealed in vivo by diaminofluorescein microfluorography. Circ Res 91: E55–E64, 2002.[Medline]
  13. Kavdia M and Popel AS. Venular endothelium-derived NO can affect paired arterioles: a computational model. Am J Physiol Heart Circ Physiol 290: H716–H723, 2006.[Abstract/Free Full Text]
  14. Kleppisch T and Nelson MT. Adenosine activates ATP-sensitive potassium channels in arterial myocytes via A2 receptors and cAMP-dependent protein kinase. Proc Natl Acad Sci USA 92: 12441–12445, 1995.[Abstract/Free Full Text]
  15. Maguire JJ, Kuc RE, and Davenport AP. Vasoconstrictor activity of novel endothelin peptide, ET-1(1–31), in human mammary and coronary arteries in vitro. Br J Pharmacol 134: 1360–1366, 2001.[CrossRef][Web of Science][Medline]
  16. McKay MK, Gardner AL, Boyd D, and Hester RL. Influence of venular prostaglandin release on arteriolar diameter during functional hyperemia. Hypertension 31: 213–217, 1998.[Abstract/Free Full Text]
  17. Minor RJ, Myers PR, Guerra RJ, Bates JN, and Harrison DG. Diet-induced atherosclerosis increases the release of nitrogen oxides from rabbit aorta. J Clin Invest 86: 2109–2116, 1990.[Web of Science][Medline]
  18. Mo FM and Ballard HJ. The effect of systemic hypoxia on interstitial and blood adenosine, AMP, ADP and ATP in dog skeletal muscle. J Physiol 536: 593–603, 2001.[Abstract/Free Full Text]
  19. Nase GP, Tuttle J, and Bohlen HG. Reduced perivascular PO2 increases nitric oxide release from endothelial cells. Am J Physiol Heart Circ Physiol 285: H507–H515, 2003.[Abstract/Free Full Text]
  20. Nellore K and Harris NR. L-Arginine and antineutrophil serum enable venular control of capillary perfusion in hypercholesterolemic rats. Microcirculation 9: 477–485, 2002.[CrossRef][Web of Science][Medline]
  21. Nellore K and Harris NR. Nitric oxide measurements in rat mesentery reveal disrupted venulo-arteriolar communication in diabetes. Microcirculation 11: 415–423, 2004.[Web of Science][Medline]
  22. Owen CA and Campbell EJ. Angiotensin II generation at the cell surface of activated neutrophils: novel cathepsin G-mediated catalytic activity that is resistant to inhibitors. J Immunol 160: 1436–1443, 1998.[Abstract/Free Full Text]
  23. Peralta C, Hotter G, Closa D, Prats N, Xaus C, Gelpi E, and Rosello-Catafau J. The protective role of adenosine in inducing nitric oxide synthesis in rat liver ischemia preconditioning is mediated by activation of adenosine A2 receptors. Hepatology 29: 126–132, 1999.[CrossRef][Web of Science][Medline]
  24. Phillis JW, O’Regan MH, and Perkins LM. Measurement of rat plasma adenosine levels during normoxia and hypoxia. Life Sci 51: PL-149–PL-152, 1992.[CrossRef][Web of Science][Medline]
  25. Proctor KG. Reduction of contraction-induced arteriolar vasodilation by adenosine deaminase or theophylline. Am J Physiol Heart Circ Physiol 247: H195–H205, 1984.[Abstract/Free Full Text]
  26. Saito Y, Eraslan A, and Hester RL. Importance of venular flow in control of arteriolar diameter in hamster cremaster muscle. Am J Physiol Heart Circ Physiol 265: H1294–H1300, 1993.[Abstract/Free Full Text]
  27. Saito Y, Eraslan A, Lockard V, and Hester RL. Role of venular endothelium in control of arteriolar diameter during functional hyperemia. Am J Physiol Heart Circ Physiol 267: H1227–H1231, 1994.[Abstract/Free Full Text]
  28. Sauls BA and Boegehold MA. Reduced PO2 and adenosine formation preserve arteriolar nitric oxide synthesis during sympathetic constriction in the rat intestine. J Vasc Res 38: 104–112, 2001.[CrossRef][Web of Science][Medline]
  29. Tigno XT, Ley K, Pries AR, and Gaehtgens P. Venulo-arteriolar communication and propagated response. A possible mechanism for local control of blood flow. Pflügers Arch 414: 450–456, 1989.[CrossRef][Web of Science][Medline]
  30. Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, and Munzel T. Increased NADH oxidase-mediated superoxide production in the early stage of atherosclerosis. Evidence for involvement of renin-angiotensin system. Circulation 99: 2027–2033, 1999.[Abstract/Free Full Text]
  31. West GA, Meno JR, Nguyen TK, Ngai AC, Simard JM, and Winn HR. cGMP-dependent and not cAMP-dependent kinase is required for adenosine-induced dilation of intracerebral arterioles. J Cardiovasc Pharmacol 41: 444–451, 2003.[CrossRef][Web of Science][Medline]
  32. Yada T, Richmond KN, Bibber RV, Kroll K, and Feigl EO. Role of adenosine in local metabolic coronary vasodilation. Am J Physiol Heart Circ Physiol 276: H1425–H1433, 1999.[Abstract/Free Full Text]
  33. Zamboni WA, Roth AC, Russel RC, Graham B, Suchy H, and Kucan JO. Morphologic analysis of the microcirculation during reperfusion of ischemic skeletal muscle and the effect of hyperbaric oxygen. Plast Reconstr Surg 91: 1110–1123, 1993.[Web of Science][Medline]
  34. Zamboni WA, Stephenson LL, Roth AC, Suchy H, and Russell RC. Ischemia-reperfusion injury in skeletal muscle: CD18-dependent neutrophil-endothelial adhesion and arteriolar constriction. Plast Reconstr Surg 99: 2002–2009, 1997.[Web of Science][Medline]



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