Published online before print November 14, 2002, doi: 10.1161/01.RES.0000047529.26278.4D
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© 2002 American Heart Association, Inc.
UltraRapid Communication |
Nonendothelial Source of Nitric Oxide in Arterioles But Not in Venules
Alternative Source Revealed In Vivo by Diaminofluorescein Microfluorography
From the Departments of Biochemistry and Integrative Medical Biology and of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan.
Correspondence to Makoto Suematsu, MD, PhD, Professor and Chair, Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail msuem{at}sc.itc.keio.ac.jp
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Abstract |
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This study aimed to examine topographic distribution of microvascular NO generation in vivo. To this end, nitrosonium ion (NO+)–sensitive diaminofluorescein diacetate was superfused continuously on the rat mesentery and the fluorescence was visualized in the microvessels through laser confocal microfluorography. Two major sites exhibited a time-dependent elevation of the fluorescence: microvascular endothelia and mast cells. As judged by the fluorescence sensitivity to local application of different inhibitors of NO synthase (NOS), NO availability in arteriolar endothelium and mast cells appeared to be maintained mainly by NOS1, whereas that in venular endothelium greatly depends on NOS3. In venules, the magnitude of inhibitory responses elicited by the inhibitors was positively correlated with the density of leukocyte adhesion. NOS inhibitors significantly reduced, but did not eliminate, the NO+-associated fluorescence in arterioles, capillaries, and venules, suggesting alternative sources of NO in circulation for these microvessels. Immunohistochemistry for NOS isozymes revealed that NOS1 occurred not only in nerve fibers innervated to arterioles but also abundantly in mast cells. Laser flow cytometry of peritoneal cells in vitro revealed abundant expression of NOS1 in mast cells. Interestingly, NOS3 occurred in endothelia of capillaries and venules but not in those of distal arterioles with comparable diameters. These results suggest that the arterioles receive NO from nonendothelial origins involving NOS1 present in nerve terminals and mast cells, whereas venules depend on the endothelial NOS as a major source. Furthermore, nonenzymatic sources of NO from circulating reservoirs constitute a notable fraction throughout different classes of microvessels. The full text of this article is available at http://www.circresaha.org.
Key Words: diaminofluorescein • nitric oxide • neural NO synthase • mast cells • microcirculation
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Introduction |
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Bioavailability of nitric oxide (NO) in tissues largely depends on NO synthases, which are subdivided into three isoforms: neuronal (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3).1 NOS1 and NOS3 belong to constitutive enzymes that produce low levels of NO and require an increase in intracellular calcium to trigger their activities. NO released from the constitutive isozymes has been shown to play important roles for functional integrity of vascular systems under normal conditions. Maintenance of vascular patency to guarantee ample blood supply, prevention of unnecessary adhesion of platelets and leukocytes,1–3 stabilization of mast cells,4 regulation of vascular permeability,5 and modulation of vascular remodeling involving proliferation of vascular smooth muscle cells6 are such events executed by NO.
Until now, most NO available for vascular walls has been thought to derive from endothelial cells that express NOS3. This concept was also supported by observations in the NOS3 gene-targeted mice displaying an elevation of systemic blood pressure and baseline leukocyte rolling.6 On the other hand, another isozyme such as NOS1 has recently been suggested to serve as a major regulator of these vascular events mediated by NO in microvascular systems of varied organs.7–9 In addition, recent studies suggest generation of free NO from nonenzymatic reservoir in circulation including S-nitrosothiols and hemoglobin.10,11 The reservoir of this kind has been recognized as a major physiological source of NO in biological systems including vasculature.12 Sources of NO and their individual roles for regulation of vascular functions thus remain largely unknown. To address this question, in vivo distribution of NOS isozymes and its microtopographic correlation with actual bioavailability of NO in and around microvessels should be examined quantitatively. However, it has been technically difficult to determine the local availability of the gas in vivo.
We have attempted to overcome such a technical difficulty by applying laser confocal microfluorography using 4,5-diaminofluorescein diacetate (DAF-2DA). This compound is a membrane-permeable fluorescence precursor sensing intracellular NO generation.13 Once loaded in tissues, the reagent can enter cells, be hydrolyzed into DAF-2, and react specifically with nitrosonium ion (NO+), which derives from endogenously generated NO to yield the triazole form of DAF-2 (DAF-2T), the stable fluorogenic complex. The present data collected through this technique combined with immunohistochemistry of NOS isozymes have revealed heterogeneity in sources and bioavailability of NO between different hierarchy of microvessels such as arterioles, capillaries, and venules, shedding light on the necessity to modify the previous concept of NO-generating sites in and around vascular endothelium in vivo.
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Materials and Methods |
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The present study protocol was approved by the Animal Care Utilization Committee, Keio University School of Medicine, Tokyo, Japan. Male Wistar rats (250 to 280 g; CLEA Japan, Tokyo) were housed in a controlled environment and maintained on a standard pellet diet under standard pathogen-free conditions in accordance with NIH guidelines. All reagents used in the present study were purchased from Sigma, unless mentioned.
Intravital NO+ Imaging in Mesenteric Microcirculation With DAF-2
Rats were anesthetized with an intramuscular injection of pentobarbital sodium at 50 mg/kg, and the femoral artery and vein were cannulated with a polyethylene catheter (Atom, Tokyo). The abdomen was opened via a midline incision, and the ileocecal portion of the mesentery was mounted on a glass-bottom plastic stage as described elsewhere.14 The mesentery was superfused continuously with the Krebs-Henseleit buffer saturated with 95% N2/5% CO2 at 2.0 mL/min at 37°C. The mesenteric microcirculation was observed with transillumination through an inverted intravital microscope equipped with a 40x objective lens (Diaphot 300, Nikon/Sankei) to examine leukocyte adhesion in venules as described previously.14,15 The present microscopic system was also equipped with a silicon-intensified target camera (C2400-08, Hamamatsu Photonics) and a real-time laser confocal imaging system assisted by an 8-bit digital processor for determining gray level densities.16–18 Values for gain and offset of the camera as well as those for the laser power supply were digitally fixed throughout the experiments. As described previously, the system allowed us to assess alterations in microvascular diameters and the density of leukocytes adhered to microvessels and to semiquantitatively determine the fluorescence intensities at the area of interests.
To visualize microvascular distribution of NO in vivo, DAF-2 diacetate (DAF-2DA, Daiichi Pure Chemicals Co, Ltd) was superfused on the mesentery at varied concentrations in a range between 1.0 to 10 µmol/L during 20 minutes. As shown later in Results, we determined the optimal concentration of DAF-2DA that did not elicit venular leukocyte adhesion; the superfusion of the reagent at 10 µmol/L for 40 minutes caused a significant elevation of the density (8.3±2.1 versus 2.9±0.5 per 100-µm venular segment in the dye-free control; P<0.05), whereas that at 3 µmol/L did not elicit the changes (2.4±0.5 versus 3.1±0.5 per 100-µm venular segment). Thus, the maximum concentration of DAF-2DA was herein fixed at 3 µmol/L, unless mentioned. To capture microfluorographs of DAF-NO+ complex in the tissue, the mesentery was epi-illuminated at 488 nm by an argon laser power supply. The microscopic field containing an unbranched segment of the arteriole (15 to 40 µm in diameter) and its drainage venule (20 to 50 µm in diameter) was selected for observation. Some capillaries (4 to 6 µm in diameter) located occasionally in the observation field were also analyzed in different animals. In these experiments, only those that shared the same focusing plane as adjacent arterioles and/or venules were examined, and other capillaries that were dislocated from the focusing plane were discarded from analyses. The background fluorescence images were captured before the start of experiments. In the control group, the buffer containing 3 µmol/L DAF-2DA was saturated with 95% N2/5% CO2 to avoid autoxidation of the precursor and was superfused on mesentery for 40 minutes. The fluorescence images were captured with a short exposure time less than 0.5 seconds every 20 minutes. A series of such exposure time turned out to induce no notable photobleaching effects under the present experimental conditions (data not shown). As shown in Results, the focusing planes for arterioles and venules differed from those of mast cells in the interstitial space. We thus carefully obtained the optimal focusing planes for each component under transilluminated conditions before acquisition of the corresponding fluorescent images. In other groups, the mesentery was cosuperfused with varied concentrations of N
-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS, D-NAME as a negative control reagent for L-NAME, or 7-nitroindazole (7-NI), an inhibitor of NOS1.19
Data calibration of the fluorescence intensities was performed by determining gray levels of known concentrations of DAF-2 triazole (DAF-2T, Daiichi Pure Chemicals Co, Ltd), a stable compound yielded by the interaction between DAF-2 and NO+. As shown in Results, the relationship between 8-bit gray level intensities and the concentrations of DAF-2T was established. The gray levels in the areas of interests in captured microfluorographs were determined using a size-variable window (2x2 µm2) by the digital image processor (NIH Image 1.62/Power Macintosh 8100/100AV). Based on the aforementioned calibration line, the gray level data were converted into the corresponding concentrations of DAF-2T, being herein designated as apparent DAF-2T concentrations (DAF-2Tapp). Differences between the value measured at 40 minutes and that measured at 20 minutes were calculated in each site of measurements. As an index of local NO availability, the relative elevation of the apparent DAF-2T concentrations during the 20-minute observation period (%
DAF-2T) was defined as the following formula:
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As shown previously, mast cells located in the interstitial space of the mesentery have the ability to generate NO.4 To check the localization of these cells in the mesentery, 0.1% of toluidine blue solution was topically applied on the mesentery and washed out by a rinse with the dye-free buffer. Such a procedure was performed at the end of experiments when necessary, and this allowed us to examine distribution of these cells and its spatial correlation to those displaying positive DAF-2T fluorescence in vivo.
Fluorescence Immunohistochemistry of NOS Isozymes
Distribution of NOS isozymes in and around the mesenteric microvessels was examined immunohistochemically using monoclonal antibodies against NOS1, 2, and 3 (Transduction Laboratory, Inc) as described elsewhere.8,9 Briefly, the whole-mount mesenteric tissues were prepared by a fixation with 4% paraformaldehyde and followed by treatment with a primary antibody (Ab) for one of three isozymes, and its immunoreactivities were checked by a secondary anti–mouse IgG antibody labeled with FITC. The NOS immunoreactivities were finally visualized through laser confocal microscopy under epi-illumination at 488 nm according to our previous methods.15,20 Heterogeneity in distribution of NOS isozymes were also examined immunohistochemically using the antibodies in cerebral tissue slices. In these experiments, the NOS immunoreactivities were visualized using avidin-biotin complex/diaminobenzidine histochemistry as described previously.21
Flow Cytometrical Analyses of NOS Expression in Mast Cells In Vitro
Expression of each NOS isoform was examined in the mast cell–containing peritoneal lavage suspension by anti-NOS monoclonal antibodies.20 The peritoneal lavage collected from anesthetized rats contained approximately 5% of mast cells, whereas the rest of the cells were mainly resident macrophages.20 Two-color flow cytometrical analyses were conducted to determine the NOS expression in mast cells. The cells were fixed with 4% paraformaldehyde and saponin and treated with one of the monoclonal antibodies against NOS isoforms. The immunoreactivities of the NOS isozyme were visualized by the secondary anti–mouse IgG antibody labeled with FITC. The cells were further treated with the anti–mast cell antibody labeled with phycoerythrin (Amersham Biosciences Co). These cells served as samples for FACScan analyses (Becton-Dickinson, Inc).
Statistical Analyses
All data presented in the present study are expressed as mean±SE of experiments with given numbers. Differences in mean values were analyzed by one-way ANOVA with Fisher’s multiple comparison test. Values of P<0.05 were considered statistically significant.
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Results |
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DAF2-DA Senses Local NO Availability in Microvascular Endothelium and Mast Cells
Figure 1 illustrates a series of representative pictures showing time-dependent elevation of the DAF-2T fluorescence in the mesenteric microcirculation continuously superfused with 3 µmol/L DAF-2DA. By comparing them with the corresponding transmission image in Figure 1A, the NO+-sensitive probe turned out to become fluorescent in different cellular components in the tissue. As seen in a microfluorograph captured at 20 minutes after the start of the dye superfusion (Figure 1B), the fluorescence became evident first in endothelia of arterioles and venules, as judged by their continuous lining along the vascular walls. After the subsequent 20-minute superfusion, the intensities in the microvascular endothelium became further increased, while some cells in the interstitium also displayed notable fluorescence (Figure 1C). When the mesentery was finally superfused with 100 µmol/L of S-nitrosoglutathione, an NO donor, the fluorescence intensities in the microvascular endothelia as well as those in the interstitium were markedly elevated and new fluorescence activities emerged in the interstitial cells (Figure 1D). This observation showed that some cells that became newly fluorescent were loaded with the dye precursor but did not exhibit fluorescence because of a paucity of endogenous NO until it was supplied exogenously. When the focusing plane was adjusted onto the microvascular endothelium, fluorescent cells in the interstitium were often defocused because of the nature of the confocal microscopic system. We thus focused on the interstitial cells and attempted to identify their cell type separately. Asterisks in Figures 1E and 1F illustrated such cells in the interstitium that were strongly labeled with DAF-2T. Topical application of 0.1% toluidine blue solution on top of the mesentery revealed metachromasia of these cells (Figure 1G), indicating that a major population of the fluorescent cells in the interstitium involves mast cells. Furthermore, the ability of mast cells to become fluorescent seemed heterogeneous among the cells.
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Under the present experimental conditions, the intensities of DAF-associated fluorescence were calibrated with known concentrations of DAF-2T. As seen in Figure 1H, the 8-bit gray levels measured through a digital processor followed a sigmoid shaped curve as a function of logarithmic plots of DAF-2T concentrations in vitro. In a range between 10 nmol/L and 1 µmol/L, the plots exhibited clear linearity, and the apparent DAF-2T concentrations were calculated from the following formula in this range:
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where r2=0.995, P<0.05, and G indicates 8-bit gray levels.
Assuming that the fluorescence intensities of DAF-2T in solution are identical to those measured in tissues, the gray-level measurements were converted to the DAF-2Tapp values for determining %DAF-2T in the data presentation described later, unless mentioned.
Heterogeneous Sensitivity of DAF-2T Fluorescence to NOS Inhibitors
Figure 2 illustrates representative pictures showing effects of the superfusion of NOS inhibitors on intensities of DAF-2T fluorescence in response in the mesentery. As seen in Figure 2, top panels, and also shown in Figure 1, the basal 20-minute superfusion of the fluorescence precursor induced a notable elevation of DAF-2T fluorescence in endothelia of arterioles, capillaries, and venules, as well as in mast cells in the interstitium. When the mesentery was treated for 20 minutes with 100 µmol/L N-nitro-L-arginine methyl ester (L-NAME), further elevation of the fluorescence intensities in venules and mast cells was suppressed markedly. On the other hand, the same concentration of L-NAME did not suppress the fluorescence in either arterioles or capillaries. Effects of the application of 100 µmol/L 7-nitroindazole (7-NI), the inhibitor of NOS1, appeared to be different from those elicited by the L-NAME treatment. As seen in Figure 2, bottom panels, treatment with the NOS1 inhibitor markedly reduced the fluorescence in arteriolar endothelium and mast cells but decreased only modestly in venular endothelium. Because the DAF fluorescence intensities were not only determined by local NO availability but also influenced by the esterase activities in cells, we also examined if the NO+-insensitive esterase substrate such as 4-aminofluorescein (4-AF) diacetate could be hydrolyzed in microvascular endothelium and mast cells and generate fluorescence. As shown in Figure 2, bottom panels, endothelia of arterioles, capillaries, and venules, as well as mast cells exhibited comparable levels of the dye loading, and the following semiquantitative analysis confirmed this finding.
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Spatially different susceptibility of DAF-2T fluorescence between L-NAME and 7-NI treatments in vivo led us to semiquantitatively examine alterations in the NO+-dependent fluorescence. Figure 3 summarized quantitative evaluation of differences in the reduction of the fluorescence intensities (%DAF-2T) among the groups. Superfusion with 100 µmol/L L-NAME, but not with the same concentration of D-NAME, significantly reduced the DAF-2T fluorescence in venules and mast cells, but not in arterioles. When the concentration of L-NAME increased up to 1.0 mmol/L, the fluorescence in arterioles displayed a significant reduction. Under these circumstances, the intensities in venules and mast cells remained unchanged, indicating that 100 µmol/L is the concentration that sufficiently inhibit endogenous NO availability in these cellular components. These results suggest that NOS appears to serve as a source of NO in and around arterioles and venules. On the other hand, the NO availability in capillaries tended to decrease but without any statistical significance with 1.0 mmol/L L-NAME; further attention is given to this event in the Discussion. Data in the right corner of Figure 3 indicated that the erastase activities were almost comparable in different hierarchy of microvascular endothelia as well as in mast cells, displaying no statistical significance as judged from the fluorescence intensities calibrated with known concentrations of 4-AF. These results suggest that differences in the DAF-2Tapp values along varied cell types mainly result from those in local NO availability rather than those in amounts of the fluorescence precursor loaded in these cells.
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In agreement with representative microfluorographs in Figure 2, quantitative analyses of alterations in the DAF-2 fluorescence revealed that the superfusion of 7-NI caused different patterns of the fluorescence suppression. Namely, this NOS1 inhibitor at 100 µmol/L elicited a significant reduction of the DAF-2T fluorescence in arteriolar endothelium as well as in mast cells to the level comparable to the L-NAME–treated group. On the other hand, treatment with the same concentration of 7-NI did not fully suppress the DAF-2T fluorescence in endothelia of capillaries and venules. As mentioned in the Discussion, such a microtopographic pattern of the inhibition was obviously distinct from that observed in the L-NAME treatment. When the concentration of 7-NI was increased up to 1.0 mmol/L, the fluorescence in arteriolar endothelium and mast cells was unchanged, indicating that the concentration at 100 µmol/L is sufficient to abolish the fluorescence in these cellular components. On the other hand, 1.0 mmol/L of the inhibitor significantly attenuated the fluorescence in venules. Under these circumstances, the heterogeneous response of the fluorescence reduction between arterioles and venules disappeared as a consequence. This event could result from the nonspecific action of excess concentrations of 7-NI on NOS isozymes; however, the DAF fluorescence in capillaries exhibited only modest reduction without any significance on the application of 7-NI at this concentration.
In the groups treated with 100 µmol/L L-NAME or 7-NI, arterioles did not display any notable vasoconstriction (data not shown). When 1.0 mmol/L of L-NAME or 7-NI was superfused for 20 minutes, the diameter of arterioles became significantly reduced, being -15.4±3.2% or -13.1±3.8% versus the basal values (P<0.05), respectively, being statistically significant in the both groups.
Differential Expression of NOS Isozymes in the Rat Mesenteric Microcirculation
Heterogeneity in susceptibility of local NO+-associated fluorescence to different NOS inhibitors led us to hypothesize that NOS1 and NOS3 could occur in a distinct manner among different hierarchy of microvessels. To test this hypothesis, distribution of these isozymes was examined immunohistochemically using the whole-mount preparation of the mesentery. Figure 4A illustrates a representative laser-confocal picture of the mesentery stained with nonspecific mouse IgG; as seen, the basal nonspecific binding of the FITC-labeled IgG occurred in the intravascular space. Such immunoreactivities were not able to be eliminated by persistent rinsing procedures. When the anti-NOS3 monoclonal Ab was used as the primary antibody, arterioles did not exhibit any notable immunoreactivities on their endothelia, while only nonspecific binding of the fluorescence was detectable (Figure 4B). On the other hand, venules in the same preparations displayed marked immunoreactivities in their venular endothelia (Figure 4C). The immunoreactivities also occurred modestly in a capillary adjacent to the NOS3-negative arteriole (c and a in Figure 4D, respectively), being in good agreement with previously studies.22 These results suggest heterogeneity in the NOS3 expression among different hierarchy of microvessels.
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Observation that arteriolar endothelium can receive NO despite a paucity of in situ expression of NOS3 protein led us to further examine distribution of NOS1; as seen in Figure 4D, this isozyme occurred abundantly in interstitial cells as well as in nerve fibers that were innervated densely to an arteriole (Figure 4E) and its distal segment (Figure 4F). Furthermore, the density of nerve fibers was obviously smaller in venules (Figure 4G) than in arterioles. As judged by DAF-2T microfluorography, a major cellular component in the interstitium that exhibits the greatest bioavailability of NO appeared to be mast cells. We thus attempted to examine if the cells could actually express NOS1. As indicated by flow cytometric analyses of the isolated peritoneal cells collected from the peritoneal lavage (Figure 5), approximately 5% of the cells were positively stained with the anti–mast cell Ab. This result was in good agreement with previous studies showing percentage values of mast cells in the lavage.20 Under these circumstances, a majority of the anti–mast cell Ab–positive cells displayed considerable amounts of NOS1 expression, although histogram of the anti–mast cell Ab–positive cells displayed marked heterogeneity in the protein expression. By contrast, the anti–mast cell Ab–positive cells exhibited little expression of NOS2 or NOS3, if any. These results suggest that mast cells account for the major cell component expressing NOS1 in the interstitial cells.
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Different Sensitivity of Venular Leukocyte Adhesion Among NOS Inhibitors
Figure 6 illustrates differences in adhesive responses of leukocytes to different NOS inhibitors. Data were collected from the same rats prepared for DAF-2T microfluorography. As seen in the control, the superfusion of DAF-2DA at 3 µmol/L alone did not exhibit any notable increase in the density of venular leukocyte adhesion. On the other hand, the superfusion of 100 µmol/L L-NAME significantly increased the adhesion density in venules. These results were in good agreement with those reported previously by our and other studies examined in the absence of the fluorescence probe.2,3,23 L-NAME at 1.0 mmol/L caused further enhancement of the cell adhesion, although such an adhesive response appeared to involve effects of a significant vasoconstriction in the upstream arterioles as described above. On the other hand, the superfusion of 100 µmol/L 7-NI, the NOS1 inhibitor, elicited only modest but significant response of adhesion in venules. The adhesive response elicited by this concentration of 7-NI was significantly smaller than that elicited by 100 µmol/L L-NAME. When 7-NI was superfused at 1.0 mmol/L, the concentration causing a significant reduction of the DAF-2T fluorescence, the venular adhesion became further increased. We then examined the relationship between the reduction of local NO availability and the increase in venular leukocyte adhesion. In these analyses, data collected from 1.0 mmol/L L-NAME or 7-NI were discarded to eliminate effects of the reduction of local shear rates on the adhesive responses. As seen in Figure 6, bottom panel, there was a significant inverse correlation between the %DAF-2T values in venules and adhesive responses in situ, suggesting a critical role of venular NO availability in regulation of leukocyte adhesion.2,3,23
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Differential Expression of NOS Isozymes in the Proximal Arterioles in the Mesentery and Brain
A paucity of the NOS3 with relatively greater expression of NOS1 in the distal arterioles in the mesentery led us to examine if the proximal arterioles with greater diameters exhibited different expression patterns of these isozymes. Figure 7A illustrated a representative picture of a set of the proximal and distal arterioles in the mesenteric pedicle. As depicted by arrows, the proximal arteriole expressed notable NOS3 on its endothelial surface, whereas the expression in the small one was little, if any. When NOS1 was stained in the semiserial section, the immunoreactivities occurred not only on the proximal one but also in the wall of the distal arteriole (Figure 7B). Careful observation with high magnification revealed that the NOS1-positive nerve fibers traversing from the outside of the wall reached the inner side of these arterioles (arrowheads in Figure 7C). Collectively with microfluorographic observation that precapillary arterioles exhibited notable NOS1 with little NOS3 (Figure 4), these results suggested that NOS1 localization is evident over the entire hierarchy of arterioles in this organ. On the other hand, the expression of NOS3 is notable in the proximal arterioles, weakened with their diameters decreasing, and is restored in capillaries and venules.
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To address if such a concept is applicable to other organs, we attempted to examine distribution of the two NOS isozymes in the rat brain microcirculation. Figure 7D and 7E indicated a set of semiserial sections illustrating NOS3 and NOS1 localization in the brain slices, respectively. As seen, NOS3 immunoreactivities occurred in relatively large arterioles (pa and da), which were characterized by their thick smooth muscle layers and in capillaries (arrows) and venules (v) with few smooth muscle cells (Figure 7D). On the other hand, the NOS1 immunoreactivities appeared to occur not only in neurons of the cortex (arrowheads) but also in the arteriolar walls (arrowheads in Figure 7E). High-magnification micrograph revealed the NOS1 immunoreactivities not only inside but also outside the wall of this large arteriole (Figure 7F). Furthermore, scanning of deeper portions of the cortex allowed us to show the NOS1-positive distal arterioles (arrowheads in Figure 7G). Such dissociation and colocalization patterns of NOS1 and NOS3 in the brain appeared to be consistent with observations showing differential expression of these isozymes in the mesenteric microcirculation.
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Discussion |
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The present study is the first to reveal the microtopographic relationship between the enzymatic sources of NO and the actual bioavailability of the gas molecule in microvascular systems in vivo. The local availability of NO in arterioles and that in venules appears to depend on distinct enzymes such as NOS1 and NOS3, respectively. The results obtained in this study led us to recognize distinct mechanisms for delivery of NO among different hierarchy of microvessels and shed light on role of differential expression of NOS1 and NOS3 in this event. The fluoroprobe used in this study has been applied to detect NO generated from endothelial cells in culture or from isolated perfused vessels.24,25 However, in these studies, the fluorescence signals were neither calibrated nor determined quantitatively. There are several beneficial points of DAF-2 to detect NO in biological systems. Careful choices for the probe concentrations allowed us to visualize reliable NO-dependent signals without interfering with physiological behavior of leukocytes in venules. Such a feature seems to result from the property of the probe to capture only small amounts of NO+, which is secondarily generated from free NO; the percentage to trap NO is estimated to be approximately 10% versus total amounts of NO generated in vitro.13 Furthermore, a comparison with microfluorography by 4-AF, a NO+-insensitive analogue of DAF, serves as a negative control to confirm that the alterations in the DAF-2T fluorescence depend on these in local NO availability. This overcomes the weakness of the reagent as a probe to determine absolute concentrations of NO due to the absence of isosbestic points.
The immunohistochemistry for NOS1 and NOS3 in the rat mesentery and brain supports the following concept: the proximal arterioles express both isozymes, whereas the distal ones with smaller diameters express only NOS1 but little NOS3, if any. On the other hand, capillaries and venules seem to express NOS3. One of the important observations in the mesentery is that endothelia of the distal arterioles can receive NO abundantly despite the paucity of NOS expression in itself. NO available in these arterioles appears to be provided by NOS1 occurring in nerve fibers and mast cells adjacent to the microvascular walls, inasmuch as its inhibitor 7-NI suppressed the local DAF-2T fluorescence significantly. Many previous studies suggest that conducting arteries proximal to microvessels mainly use NO derived from its own endothelium-expressing NOS3 to maintain their patency to supply blood flow, whereas smaller arterioles could mainly use NO-independent mechanisms (eg, EDHF) for vasorelaxation to greater extents than those depending on NO.26,27 In this context, our immunohistochemical observation showing a paucity of NOS3 in the distal arteriolar endothelium is in good agreement with these functional studies ex vivo. Furthermore, the present observation was also confirmed by recent studies by Western blot analyses showing greater expression of the NOS3 protein in the mesenteric tissue containing venules than in that containing arterioles.28 However, the notion that the arterioles use NO generated from NOS1 expressed in nerve fibers and mast cells around them led us to reexamine if the aforementioned concept is applicable in vivo. Namely, most previous experiments suggesting lesser roles of NO in modulation of arteriolar tone were collected from the ex vivo perfused vessels undergoing the denervated, and connective tissue–free preparation, and could thus overlook roles for NOS1 around arterioles. Such an underestimation of the paracrine source of NO in arterioles is fully confirmed by the present observations showing their greater sensitivity to 7-NI than venules, and also supported by a recent study showing that NO-dependent component plays a greater role in arteriolar relaxation than NO-independent EDHF component in varied microvascular systems of rodents in vivo.29–31
Distinct from that in arterioles, NO bioavailability in venules seems to be supported through NOS3 in autocrine manner. When the mesentery was superfused with 7-NI at 100 µmol/L, a concentration leading to a significant NO reduction in arterioles, the local NO availability in venules exhibited only a modest reduction without any statistical significance. A heterogeneity in the NOS3 expression between arterioles and venules is obviously inconsistent with a notion that the enzyme can be induced in response to vascular wall shear stress in situ,32 and the mechanisms for such an event remain quite unknown. However, intense bioavailability of the endothelium-derived NO in venules could prevent unnecessary adhesion of platelets and/or leukocytes and reduce the risk of hemostasis in these low-shear microvessels. On the other hand, the same concentration of the NOS1 inhibitor significantly suppressed the NO availability in mast cells around venules. These results suggest that suppression of NOS1-derived NO in mast cells did not largely interfere with NO availability in the adjacent venules, and support a concept that the local availability in their endothelia is mainly maintained by their own NOS3 in an autocrine fashion. This notion was supported by the present observation that 7-NI induced only modest activation of venular leukocyte adhesion as compared with the same concentration of L-NAME, and also confirmed by a previous study using NOS3 knockout mice showing lesser adhesive impacts of the NOS1 inhibitor versus L-NAME.33 Although a role of NOS1 for mast cells to maintain NO in venules appears to be small under physiological circumstances, its role could contribute more greatly to local NO maintenance and modulation of leukocyte adhesion when NO derived from NOS3 in venular endothelium decreased under varied pathological conditions (eg, inflammation and ischemia/reperfusion).3,4,34 Such circumstances were well documented experimentally by previous studies showing that NOS3 knockout mice become highly susceptible to 7-NI to elicit venular leukocyte adhesion.33 Furthermore, it was previously demonstrated that tenuous balance between NO and superoxide anions could determine susceptibility of mast cells to degranulation stimuli, and the former serves as a stabilizer of the exocytosis in vitro and in vivo.4 Thus, perturbation of mast cell–derived NOS1 could alter the degranulation susceptibility and thereby modify local inflammatory responses via proinflammatory mediators released from the cells. Such a possibility should further be examined in different experimental models of disease conditions.
Finally, the present observation showing a paucity of the L-NAME–induced suppression of local NO availability in arterioles and capillaries raised an important question as to another source of NO besides NOS isozymes locally expressed in microcirculation. The lesser sensitivity of NO availability in the distal arterioles than that in venules to L-NAME is unlikely to result from different accessibility of the inhibitor between the two vascular categories, inasmuch as another inhibitor with comparably small molecular weight (eg, 7-NI) inhibited the arteriolar NO availability in the same preparation. Another important observation is that a considerable fraction of the DAF-2T fluorescence in the distal arterioles and venules was not abolished by sufficient amounts of NOS inhibitors; approximately 50% of the local DAF-2T signals could be derived from the nonenzymatic origins, assuming that 1 mmol/L of the NOS inhibitors is sufficient enough to eliminate the activities. These lines of evidence suggest the presence of nonenzymatic origins of NO in microcirculation. Among such sources, S-nitrosyl hemoglobin in circulating erythrocytes, which could release NO on a reduction of local oxygen pressure, is one to be taken into account.10,11,35 Such a possibility is supported by the fact that S-nitrosothiol serves as the major physiological source of NO+ that reacts with the fluoroprobe precursor used in the present study.12 Considering the recent observation that a drop in the intravascular oxygen tension occurs first in the distal arterioles rather than in capillaries,36 it is not unreasonable to speculate that such a source of NO could explain the presence of L-NAME– and 7-NI–insensitive fractions of local NO availability. We were unable to visually assess local NO in the proximal arterioles because the presence of thick adipose tissues limited DAF accessibility and interfered with reliable fluorescence imaging. Such technical difficulties prevented us from revealing the relationship between local NOS expression and actual NO bioavailability in the entire vascular system. Refinement and development of the present method are now underway in our laboratory to figure out roles of different sources of NO in regulation of the local gas availability and to understand their individual missions to maintain functional integrity of organ microcirculation.
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Acknowledgments |
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This study was supported by Health Science Research grants for Research on Advanced Medical Technology from the Ministry of Health and Welfare in Japan, by a grant for the 21st Century Center-of-Excellence Program from the Ministry of Education and Sciences Technology in Japan, by Grant-in-Aid for Creative Scientific Research (13GS0015), and by Japan Foundation of Cardiovascular Research.
Received September 19, 2002; revision received November 4, 2002; accepted November 5, 2002.
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