1 Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9040
2 Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York, New York 14642-8711
3 Cardiovascular Research Center, Massachusetts General Hospital, Boston, Massachusetts 02129
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ABSTRACT |
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endothelial nitric oxide synthase; neuronal nitric oxide synthase; arteriolar relaxation
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INTRODUCTION |
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For exercise to be sustained, the necessary increase in blood flow to contracting skeletal muscle can be achieved in part by a decrease in skeletal muscle vascular resistance (1). The exercise-induced hyperemic response of skeletal muscle vasculature or active/functional hyperemia is a well-established phenomenon; however, the precise mechanisms that regulate skeletal muscle perfusion during exercise remain incompletely understood. Factors implicated as vasoactive agents to mediate functional hyperemia include low oxygen tension (27), adenosine (28), potassium, pH, magnesium, and osmolarity. Although these factors all have vasodilatory effects in contracting skeletal muscle, their precise contributions to mediating the exercise-induced hyperemia are not well defined.
NO produced by eNOS and nNOS may also contribute to functional hyperemia (12, 16), because NO is formed in contracting skeletal muscle (2). After stimulation of cell surface receptors or mechanical shear stress in contracting muscle, the vascular endothelium produces NO as increased [Ca2+]i leads to activation of Ca2+/calmodulin-dependent eNOS (24). Additionally, fast-twitch skeletal muscle fibers contain nNOS colocalized to the sarcolemma with dystrophin (4, 5), but the contribution of nNOS-derived NO to functional hyperemia is less clear. We considered the possibility that NO produced from Ca2+/calmodulin-dependent nNOS in contracting fast-twitch skeletal muscle fibers could diffuse to adjacent vascular smooth muscle cells to elicit a localized relaxation response via the NO/cGMP-mediated pathway. To this end, we have previously shown that cGMP formation is increased in contracting extensor digitorum longus muscle (EDL) in vitro from C57Bl6 but not mdx mice (16). In Duchenne's muscular dystrophy (DMD) and in dystrophin-deficient mdx mice, both nNOS expression and activity are greatly reduced (5). These reductions may explain both the decrease in cGMP and the attenuated arteriolar dilation observed in contracting muscles from mdx mice compared with C57Bl6 mice (16). In addition, NO produced by nNOS in skeletal muscle antagonizes sympathetic -adrenergic vasoconstriction during muscle contraction in C57Bl6 but less so in mdx mice (32). This sympatholytic event appears to be fiber-type specific, because it occurs in contracting fast-twitch, glycolytic but not slow-twitch, oxidative muscles (31). Collectively, these findings (16, 31, 32) suggest that nNOS may play a specific functional role in the exercise-induced hyperemic response of contracting fast-twitch skeletal muscle fibers. However, in addition to the decrease in nNOS expression in mdx mice, the possibility that dystrophin-deficient muscles have coexisting derangements that interfere with the NO/cGMP signaling pathway could not be excluded.
In this report, we test the hypothesis that NO produced specifically by nNOS in contracting skeletal muscle stimulates the formation of cGMP and that this increase in cGMP is associated with a hyperemic response. We used fast-twitch (EDL and cremaster) muscles isolated from C57Bl6 mice as well as nNOS-deficient (nNOS-/-) and eNOS-deficient (eNOS-/-) mice to measure both cGMP formation and arteriolar relaxation in response to electrical stimulation. The use of nNOS-/- and eNOS-/- mice has the advantage of allowing assessment of individual contributions of nNOS or eNOS to cGMP formation and arteriolar responsiveness to skeletal muscle contraction. We also evaluated the potential signaling effects of NO on cGMP formation in slow-twitch soleus muscles, which have more eNOS and less nNOS compared with EDL muscles. We demonstrate that cGMP formation in contracting EDL muscles is nNOS dependent, but lack of eNOS also attenuates the response. We also show that the skeletal muscle vasodilatory response to contraction requires both nNOS and eNOS.
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METHODS |
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Isolation of muscles.
Mouse EDL and soleus muscles were isolated and mounted on Grass FTO3.C force transducers and bathed in physiological salt solution (PSS) containing (in mM) 120.5 NaCl, 4.8 KCl, 1.2 MgSO4, 1.5 CaCl2, 1.2 Na2PO4, 20.4 NaHCO3, 10.0 dextrose, and 1.0 pyruvate at 30°C. The buffer was continuously gassed with 95% O2-5% CO2, resulting in pH 7.6 at room temperature (16). Muscles were either electrically stimulated at 30 Hz for 15 s, incubated with 1 mM N-nitro-L-arginine (NLA) for 30 min or with 10 µM sodium nitroprusside (SNP) for 30 s, or subjected to specific combinations. After specific treatments, muscles were quick-frozen by tongs prechilled in liquid nitrogen and stored at -80°C.
Radioimmunoassay of cGMP in muscle extracts.
Briefly, trichloroacetic acid-extracted muscle samples were applied to individual alumina columns, which were then sequentially washed with 10 ml of water, 5 ml of 0.6 N HCl in 95% ethanol, 5 ml of 50% ethanol, and 2 ml of water. Alumina columns were then piggy-backed over Dowex 50W-X8 columns. Three milliliters of 200 mM ammonium formate was added to the alumina columns to elute nucleotides into the Dowex resin. The piggy-backed columns were placed over vials, and 1 ml of 200 mM ammonium formate was added. After the liquid was passed through the alumina resin, cGMP was further eluted from the Dowex 50W-X8 resin with 3 ml of water, and samples were lyophilized overnight. cGMP standards contained 5, 10, 20, 40, 50, 100, and 200 fmol of cGMP. Lyophilized samples and standards were acetylated by redissolving with 25 µl of 4% triethylamine and 25 µl of 2% acetic anhydride, mixed, and placed in an ice bath. 125I-cGMP in 90 mM sodium acetate buffer, pH 4.75, with 20,000 cpm in 50 µl, and 20 µl of the diluted anti-cGMP antibody in 0.1% -globulin were added to each tube and mixed. To estimate nonspecific binding, 50 µl of 0.1%
-globulin was added without anti-cGMP antibody. Samples were incubated overnight at 4°C. Heat-inactivated fetal bovine serum (50 µl) was added to each tube and immediately followed with 1 ml of 12% polyethylene glycol (PEG-8000) in 50 mM sodium acetate buffer, pH 6.2. Tubes were incubated for 1 h on ice and centrifuged (2,500 g, 30 min at 4°C). Immunoprecipitated complexes were washed with 1 ml of 12% PEG, then further incubated on ice for 20 min. After centrifugation, 125I radioactivity in immunoprecipitated complexes was measured in a gamma spectrometer.
Western blot analysis of nNOS and eNOS in muscle extracts.
Mouse skeletal muscles (EDL, soleus, or cremaster) were isolated from C57Bl6, nNOS-/-, and eNOS-/- mice, weighed, and homogenized in 20 vol of 50 mM Tris · HCl, pH 7.5, 1 mM EDTA, 0.1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml of pepstatin A, 2 µg/ml of leupeptin, 1 µg/ml of aprotinin, 1 mM N--p-tosyl-L-lysine chloromethyl ketone (TLCK), 2 µM tetrahydrobiopterin, and 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Protein determination of extracts was performed using a Bio-Rad DC Protein Assay kit. nNOS protein in muscle extracts was analyzed by electrophoresis of 20 µg of total protein from each muscle sample on a 6% SDS-polyacrylamide gel. eNOS protein was analyzed by electrophoresis of 60 µg of soluble protein (17,000 g supernatant fraction) from muscle extracts on a 6% SDS-polyacrylamide gel. Resolved proteins were transferred to nitrocellulose paper and subsequently probed for nNOS and eNOS. A positive nNOS marker prepared from mouse brain and an eNOS marker from mouse heart were also included. A polyclonal antibody raised against the NH2 terminus of rat brain nNOS was used (1:4,000) to detect nNOS, which includes µNOS, the alternatively spliced isoform specific to skeletal muscle (29), and a monoclonal eNOS antibody (Transduction Laboratories) was used (1:1,500) to detect eNOS. Horseradish peroxidase conjugated to goat anti-rabbit (nNOS) or anti-mouse (eNOS) IgG was used as secondary antibody. Immunoreactivity was detected by ECL chemiluminescence (Amersham). Relative nNOS and eNOS amounts were quantified by densitometric scanning of immunoblots. For relative comparisons, muscle samples were analyzed on the same immunoblot.
Vascular response measurements in mouse fast-twitch cremaster muscle.
Adult male mice were anesthetized with pentobarbital sodium (70 mg/kg ip), tracheotomized, maintained with supplemental anesthetic as needed via a jugular venous catheter, and kept at body temperature (37°C) by radiant heating. The right cremaster muscle was prepared and the microvasculature viewed as described elsewhere (16); like EDL, cremaster muscle consists predominantly of fast-twitch fibers (unpublished data). The prepared cremaster muscles were superfused with PSS containing (in mM) 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, 20 NaHCO3, 1.0 pyruvate, and 10.0 glucose. The buffer was equilibrated with 21% O2-5% CO2, balanced with N2, to give pH 7.4 at 37°C. Electrical field stimulation via silver foil electrodes was used to contract the muscle for 15 s at 30 Hz (0.2 ms duration, 13 V). Because flow-dependent mechanisms have been identified as possible contributors to local arteriolar responses, the muscles were clamped across their origins using a miniature arteriolar clamp to produce "no-flow" conditions comparable to those used in isolated EDL or soleus muscles. Following an equilibration period of 2030 min, baseline arteriolar diameter was measured, and the muscle was then contracted at 30 Hz for 15 s. Arteriolar diameter was measured within 3 s of the completion of the stimulation. The protocol was completed in C57Bl6, nNOS-/-, and eNOS-/- mice, either under control conditions or with 1 mM NLA added to the superfusate. Separate groups of animals were used for each experimental condition. One vessel was selected for observation in each preparation using predetermined criteria to select either a second or third branch distal to the main inflow vessel and to ensure that all were from the same anatomic location in the microvasculature. Arteriolar diameters were measured offline from videotapes using an electronic caliper calibrated against a videotaped stage micrometer. Maximum diameter of each vessel was taken as that produced by the addition of 10-4 M adenosine to the suffusate at the end of the experiment.
Statistics.
cGMP data were analyzed by a two-way ANOVA; simple effects were examined with a one-way ANOVA. Duncan's post hoc test was used to determine differences between means. Changes in arteriolar diameter were compared using a one-way ANOVA and Newman-Keuls post hoc test. For all analyses, differences were considered significant for P < 0.05. Values are means ± SE.
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RESULTS |
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cGMP formation in mouse fast-twitch EDL skeletal muscle.
Under resting conditions, EDL muscles from control mice contained 4.50 ± 0.93 fmol cGMP/mg wet wt tissue (n = 6). Compared with resting values, cGMP levels increased 166% with electrical stimulation (P < 0.05; Fig. 2). The NOS inhibitor NLA did not affect the resting level of cGMP but completely inhibited the increase due to electrical stimulation. Treatment of the muscles with 10 µM SNP significantly increased cGMP content to 167% of resting values (P < 0.05; Fig. 2).
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In EDL muscles isolated from eNOS-/- mice, resting levels of cGMP (2.64 ± 0.31 fmol cGMP/mg wet wt tissue, n = 19) were significantly lower than those from control or nNOS-/- mice (P < 0.05). Electrical stimulation did not result in increased cGMP formation in EDL from eNOS-/- mice. The mean absolute cGMP value from stimulated eNOS-/- EDL (3.92 ± 0.38; n = 8) was similar to resting values for C57Bl6 or nNOS-/- muscles. NLA did not significantly alter cGMP content in resting muscles but diminished cGMP content with electrical stimulation (P < 0.05; Fig. 2). The value of cGMP measured in EDL muscles from eNOS-/- mice treated with 10 µM SNP (4.35 ± 0.41; n = 4) was significantly less than the corresponding value from nNOS-/- mice (10.94 ± 1.39; n = 13, P < 0.05). This result raises the possibility that some aspect of the cGMP signaling pathway in EDL muscles from eNOS-/- mice may be compromised.
cGMP formation in mouse slow-twitch soleus muscle.
In resting slow-twitch soleus muscles from control mice, cGMP content was similar to that of EDL muscles (4.46 ± 0.50 fmol cGMP/mg wet wt tissue, n = 10). However, unlike the response of fast-twitch EDL muscles, cGMP formation was not increased in electrically stimulated soleus (Fig. 3). The absence of increased cGMP formation in response to stimulation correlates to the reduced nNOS protein content in these muscles (Fig. 1A). Thus, the contraction-dependent increase in cGMP formation appears to be specific for fast-twitch fibers.
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DISCUSSION |
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The significant increase in cGMP formation in electrically stimulated fast-twitch EDL muscles from control mice was totally abolished by the NOS inhibitor NLA, indicating a contraction-dependent activation of NOS (Ref. 16, and present study). In EDL muscles from nNOS-/- mice, cGMP content was not increased by electrical stimulation but was increased significantly by the NO donor SNP; therefore, the absence of cGMP formation with stimulation was due to the lack of nNOS and not an unresponsive sGC. These data support the hypothesis that nNOS activation during contraction is required for an increase in cGMP content.
To further discriminate the contribution of nNOS from that of eNOS in promoting cGMP production in contracting muscles, we employed eNOS-/- mice. Despite the nNOS contents comparable to control, cGMP formation was not significantly increased after stimulation. However, stimulation values were diminished by NLA treatment, indicating partial nNOS activity. Treatment with SNP produced a significant increase in cGMP content compared with the control value; however, the mean absolute cGMP responses of eNOS-/- EDL muscles following either stimulation or SNP treatment were lower compared with similar conditions for C57Bl6 EDL muscles. Moreover, the lower cGMP formation in SNP-treated eNOS-/- compared with nNOS-/- EDL muscles may indicate that the responsiveness of sGC to NO is compromised in fast-twitch muscles of eNOS-/- mice. Collectively, the cGMP data suggest that nNOS is activated during muscle contraction in vitro, but the responsiveness of sGC to NO may depend on the presence of eNOS.
cGMP formation in response to electrical stimulation is specific to fast-twitch and not slow-twitch muscles, because cGMP formation was not increased in stimulated soleus. Slow-twitch soleus muscles contain significantly less nNOS and more eNOS than either fast-twitch EDL or cremaster muscles. The amount of nNOS protein present in skeletal muscle fibers may be a critical determinant of its role in functional hyperemia. This apparent inverse relationship is demonstrated both in the differences between cGMP formation in stimulated EDL compared with soleus in vitro, as well as in the ability of electrically stimulated control vs. mdx hind limb to attenuate -adrenergic vasoconstriction (sympatholysis) during contraction in situ (32). For example, there is significant sympatholysis in mouse hind limb from control mice, which have fast-twitch muscles that demonstrate abundant nNOS protein, but attenuated sympatholysis from mdx and nNOS-/- mice (32), which have fast-twitch muscles that demonstrate a marked decrease in nNOS content and activity (5).
In agreement with the role of NO in sympatholysis (32), NO contributes a significant component to the dilation in stimulated isolated fast-twitch cremaster muscles. However, the dilation was not completely inhibited by NLA. This result suggests, not surprisingly, that there are both NO-dependent and NO-independent dilatory mechanisms, including metabolic mediators of functional hyperemia (27, 28). The arterioles studied in all three groups of mice were of similar size (maximal diameter), and all displayed considerable resting tone due to the presence of 21% oxygen in the superfusate. Estimated dilatory capacities in these vessels were consequently quite large, being ~27, 29, and 34 µm in control, nNOS-/-, and eNOS-/- mice, respectively. In stimulated cremaster muscles of control mice, the observed 2.5 times dilation from resting diameter was thus a dilatory response of ~45% of the maximal dilatory capacity. Although nNOS-/- mice also increased their diameter by a similar 2.3 times, this response constituted approximately only 22% of their dilatory capacity, while in the eNOS-/- group, the response was about 10% of their dilatory capacity. Because we used systematic criteria independent of size to select vessels for observation, the finding that maximal diameters were not different among the groups suggests that there was no significant compensatory change in microvascular anatomy at this level of the microvasculature in the nNOS-/- or eNOS-/- animals. Thus, we cannot attribute differences in arteriolar responses to effects of compensatory differences in the microvascular network.
In stimulated nNOS-/- cremaster muscles, arteriolar diameter increased to a value greater than the NLA-blocked preparation from control mice. This contrasts with the finding in EDL muscle that cGMP did not increase with stimulation in nNOS-/- mice. Assuming that the responsiveness of the cremaster arterioles is similar to the EDL, this prompts several questions: was there an increase in cGMP that we could not detect in our assay due to sensitivity limitations; and, did NO act via a cGMP-independent as well as dependent pathway? In stimulated EDL muscles from nNOS-/- mice, cGMP may have increased to a level which we could not detect, but because of the amplification inherent to signal cascades, this was enough to produce a modest dilation.
It is possible that eNOS in endothelial cells might be activated by the mechanical stress of contraction (e.g., vessel compression) indirectly to produce NO. Other potential activators of eNOS include acetylcholine and adenosine. For example, during stimulation acetylcholine might be released from nerve terminals (14) or from endothelial cells (25), and adenosine may be formed in the interstitium (9). However, cGMP formation was not increased in stimulated nNOS-/- EDL where only eNOS was present, and stimulation was not sufficient to increase cGMP in soleus muscles, despite the presence of significant eNOS protein. In regard to the soleus, we cannot, however, discount that there are distinct differences in the regulation of functional hyperemia relative to fast-twitch muscle.
In C57Bl6 and nNOS-/- mice, a contribution from NO is suggested by the observation that the stimulation-dependent arteriolar dilation was attenuated by NLA. This is in accord with studies on human forearm blood flow during exercise, which showed a significant attenuation of increased forearm blood flow with infusion of a NOS inhibitor during exercise (6). The moderate arteriolar dilation that occurred in response to stimulation in nNOS-/- mouse cremaster may be attributed to both NO-independent and cGMP-independent pathways. Metabolic effectors associated with muscle contraction could account for the NO-independent relaxation (e.g., potassium, pH). However, because dilation was partially attenuated in the stimulated nNOS-/- cremaster by NLA, it is possible that an NO-dependent but cGMP-independent pathway was also activated. A number of studies have reported cGMP-independent vasodilatory responses to NO in several vascular tissues (8, 20). Recently, it was shown that NO blocks the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by inhibiting the enzyme P4504A2. 20-HETE is a potent inhibitor of KCa channels (3), which leads to relaxation by hyperpolarization of smooth muscle cells and decreased [Ca2+]i; thus, inhibition of P4504A2 by NO can result in vasodilation independent of cGMP.
In stimulated eNOS-/- EDL, a modest increase in cGMP was blocked by NLA, yet there was a complete absence of effective dilation in stimulated eNOS-/- cremaster. This result suggests that there was a potential increase in NO with stimulation, but both NO-dependent and -independent relaxation pathways were compromised. nNOS and eNOS appear necessary for the complete contraction-induced NO-dependent arteriolar relaxation seen in control cremaster. This conclusion contrasts with reports indicating compensation by one isoform of NOS in the absence of the other isoform but supports the idea that compensation may depend on the location of the vessel in the circulatory system. For example, an nNOS-dependent mechanism appears to compensate for the absence of eNOS in mounting a vasodilatory response to acetylcholine in pial arterioles in eNOS-/- mice, because the vasodilation is blocked by 7-nitroindazole, an inhibitor of nNOS (21, 22). However, in a separate study with eNOS-/- mice, no nNOS-dependent compensatory relaxation mechanism was observed in carotid arteries treated with acetylcholine (7). In both cases, the sensitivity of carotid arteries and pial arterioles from eNOS-/- mice to SNP was increased (7, 21, 22), but this increased sensitivity was not observed in the eNOS-/- cremaster. Thus, compensatory mechanisms of nNOS or eNOS and related changes in sensitivity of sGC to NO may depend on the type and location of the vessel in the circulation. The attenuated cGMP formation with SNP in the eNOS-/- EDL identifies sGC as a possible candidate protein for decreased amount of activity in eNOS-/- fast-twitch skeletal muscles. Other possibilities in this cGMP signaling cascade should also be considered.
In summary, cGMP formation with muscle activation in vitro is due primarily to NO produced by nNOS and occurs in fast-twitch but not slow-twitch muscles. The cGMP response in fast-twitch muscle is attenuated in the absence of eNOS. In contracting isolated fast-twitch muscles, both nNOS and eNOS appear necessary for arteriolar dilation.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-06296 (to J. T. Stull) and HL-56574 (to I. H. Sarelius) and by a grant from the Muscular Dystrophy Association (to J. T. Stull).
Address for reprint requests and other correspondence: K. S. Lau, Dept. of Physiology, Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-9040 (E-mail: klau{at}mednet.swmed.edu).
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FOOTNOTES |
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REFERENCES |
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