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J Appl Physiol 89: 1483-1490, 2000;
8750-7587/00 $5.00
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Vol. 89, Issue 4, 1483-1490, October 2000

Hindlimb unweighting alters endothelium-dependent vasodilation and ecNOS expression in soleus arterioles

William G. Schrage, Christopher R. Woodman, and M. Harold Laughlin

Departments of Physiology and Veterinary Biomedical Sciences and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to test the hypothesis that endothelium-dependent dilation is impaired in soleus resistance arteries from hindlimb-unweighted (HLU) rats. Male Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 14) or weight-bearing control (Con, n = 14) conditions for 14 days. After the 14-day treatment period, soleus first-order (1A) arterioles were isolated and cannulated with micropipettes to assess vasodilator responses to an endothelium-dependent dilator, ACh (10-9-10-4 M), and an endothelium-independent dilator, sodium nitroprusside (SNP, 10-9-10-4 M). Arterioles from HLU rats were smaller than Con arterioles (maximal passive diameter = 140 ± 4 and 121 ± 4 µm in Con and HLU, respectively) but developed similar spontaneous myogenic tone (43 ± 3 and 45 ± 3% in Con and HLU, respectively). Arteries from Con and HLU rats dilated in response to increasing doses of ACh, but dilation was impaired in arterioles from HLU rats (P = 0.03), as was maximal dilation to ACh (85 ± 4 and 65 ± 4% possible dilation in Con and HLU, respectively). Inhibition of nitric oxide (NO) synthase (NOS) with Nomega -nitro-L-arginine (300 µM) reduced ACh dilation by ~40% in arterioles from Con rats and eliminated dilation in arterioles from HLU rats. The cyclooxygenase inhibitor indomethacin (50 µM) did not significantly alter dilation to ACh in either group. Treatment with Nomega -nitro-L-arginine + indomethacin eliminated all ACh dilation in Con and HLU rats. Dilation to sodium nitroprusside was not different between groups (P = 0.98). To determine whether HLU decreased expression of endothelial cell NOS (ecNOS), mRNA and protein levels were measured in single arterioles with RT-PCR and immunoblot analysis. The ecNOS mRNA and protein expression was significantly lower in arterioles from HLU rats than in Con arterioles (20 and 65%, respectively). Collectively, these data indicate that HLU impairs ACh dilation in soleus 1A arterioles, in part because of alterations in the NO pathway.

skeletal muscle; microcirculation; acetylcholine; microgravity; physical inactivity; indomethacin; Nomega -nitro-L-arginine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXERCISE CAPACITY IS REDUCED in astronauts after spaceflight and in rats exposed to 14 days of hindlimb unweighting (HLU) (3, 11, 16, 26). Humans and rats exhibit classic signs of cardiovascular deconditioning brought about by the physical inactivity associated with a non-weight-bearing environment. The deconditioning is characterized by reductions in maximal oxygen consumption (3, 11, 16, 26), endurance capacity (16), and blood volume (5) and an impaired ability to augment cardiac output during exercise (11, 25). In addition, HLU rats exhibit altered blood flow distribution during standing and exercise, such that blood is not diverted from the gastrointestinal tract to the more active muscle (12, 25). Specifically, the soleus muscle, a muscle predominantly active in normal posture and running, has reduced resting blood flow (12) and lower exercise hyperemia after 14 days of HLU (25). Decreased soleus blood flow after HLU might be due to impaired dilator pathways. To evaluate mechanisms controlling soleus blood flow after prolonged inactivity, we used the HLU rat model based on that of Morey (14), which mimics the cephalic fluid shifts, removal of weight bearing from the soleus, and reduction in exercise capacity experienced by astronauts.

Two weeks of HLU have been reported to result in decreased dilation of rat aorta in response to ACh (2). Because resistance arteries control blood flow within and between muscles, alterations in vasodilator pathways in resistance arteries by HLU might contribute to lower soleus blood flow at rest and during exercise in these animals. Recently, Jasperse et al. (8) reported that soleus feed arteries isolated from HLU rats exhibit impaired endothelium-dependent dilation and that endothelial cell nitric oxide (NO) synthase (ecNOS) mRNA and protein content are diminished in feed arteries from HLU rats. The lower ecNOS mRNA and protein levels suggest one mechanism for impaired endothelium-dependent dilation in soleus feed arteries, since it is known that approximately one-half of the ACh dilation in soleus feed arteries from normal rats is mediated by ecNOS (7). These authors concluded that HLU was associated with impaired NO-mediated dilation caused in part by a downregulation of ecNOS expression (8).

With these data in mind, the purpose of this study was to determine whether endothelial dysfunction induced by HLU also occurs in soleus arterioles. In addition, we sought to determine which vasodilator mechanisms mediate ACh dilation in soleus arterioles [NO, prostacyclin (PGI2), or endothelium-derived hyperpolarizing factor (EDHF)] and whether the relative contribution of these dilators is altered by HLU. Therefore, the following experiments were designed to test the hypothesis that arterioles from the soleus of HLU rats exhibit impaired vasodilator responses, due in part to alterations in the NO pathway. We tested this hypothesis by examining vasodilator responses of soleus first-order (1A) arterioles to an endothelium-dependent dilator (ACh) and an endothelium-independent dilator [sodium nitroprusside (SNP)]. In addition, we tested the hypothesis that ecNOS expression is lower in arterioles from HLU rats.

Limited data are available concerning vascular control in arteries from such muscles as the soleus, which is active during posture and locomotion. Therefore, it is important to understand the mechanisms regulating arteriole caliber and whether vascular control is altered by HLU. The results of this study may elucidate mechanisms of vascular control in resistance arteries after HLU and may help explain why soleus blood flow is decreased in HLU rats and ultimately whether altered control of blood flow contributes to reduced exercise capacity on return to a normal weight-bearing environment.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Male Sprague-Dawley rats (Harlan; 275-300 g) were housed two per cage in a room with constant temperature (24°C) and a 12:12-h light-dark cycle. Food and water were provided ad libitum. The Institutional Animal Care and Use Committee of the University of Missouri approved all experimental protocols.

HLU Procedures

Rats were randomly assigned to HLU (n = 14) or control (Con, n = 14) groups for 14 days, as described previously (8, 13). The 14-day protocol was used, because previous studies indicate that 14 days of HLU are associated with chronic reductions in soleus muscle blood flow during unweighting, an impaired ability to increase blood flow during exercise after HLU (12, 16, 25), and reduced exercise capacity (3, 26). Rats were housed individually in Plexiglas cages and suspended in a manner that prevented the hindlimbs from touching any surface while allowing free, 360° movement about the cage on the forelimbs. The height of the tail was raised high enough to prevent weight bearing of the hindlimbs and to elicit cephalic fluid shifts, which resulted in a suspension angle of 40-45°. A cast was placed around the thorax to prevent the rat from interfering with the suspension apparatus. Con rats wore a thoracic cast for 14 days but experienced normal weight-bearing activity during the 14-day treatment period. Con rats were housed individually in standard metabolic cages for the 14-day period. All rats had unrestricted access to food and water throughout the study and were monitored daily by researchers and regularly by University of Missouri veterinarians to ensure their health and well-being.

Isolation and Cannulation of Soleus Arterioles

After the 14-day treatment period, rats were anesthetized with pentobarbital sodium (100 mg/kg), and the left soleus muscle was removed, weighed, and stored at -70°C. Citrate synthase activity of the soleus muscle was measured to assess muscle oxidative enzyme capacity according to the method of Srere (17). The right soleus was removed, weighed, and placed in ice-cold (4°C) physiological saline solution (PSS) + albumin (PSSA). PSS contained (mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EGTA, and 25.0 MOPS and 10 g/l albumin (pH 7.4). After muscle dissection revealed the arteriolar network, 1A arterioles (~800-1,500 µm long) were carefully dissected free of connective tissue, removed from the muscle, and transferred to a 2-ml vessel chamber containing cold PSS. The 1A arterioles were defined as the first arteries within the epimycium that were supplied by the feed arteries (21).

Under a microscope, one end of the 1A arteriole was tied with 11-0 suture to a glass micropipette (37-47 µm ID), and blood was flushed out of the arteriole with PSSA. The free end of the arteriole was then tied to a size-matched micropipette. Under an inverted microscope (Nikon Diaphot 200; ×40 magnification), the arteriole image was displayed. Internal arteriole diameter was measured with a video micrometer (Microcirculation Research Institute, Texas A & M University) calibrated to <1 µm. Data were acquired and stored with the Macintosh/MacLab data acquisition system. Pressure with no flow was applied to the vessel lumen by raising to the same level two reservoirs connected to the micropipettes. We selected an intraluminal pressure of 80 cmH2O on the basis of reports of intravascular pressure in similar arterioles of other skeletal muscles, according to Dodd and Johnson (4) and Fenton and Zweifach (6), and the fact that soleus feed artery pressure in vivo has been reported to be 90 cmH2O (22).

Each arteriole was set at its in situ length, pressurized, and warmed in the bath to 37°C and checked for leaks over a 5-min period. Pressure and diameter were continuously recorded by the Macintosh MacLab system and stored on a hard disk. The arteriole was washed several times with warm PSS (37°C) over a 1-h equilibration period, during which time the pressure was incrementally increased from 30 to 80 cmH2O in the first 30 min. Arterioles were exposed to 80 mM KCl to test for responsiveness. If arterioles did not constrict to KCl by 20% of initial diameter, the arteriole was considered nonfunctional and discarded. After KCl was replaced with PSS, all arterioles developed >= 20% spontaneous tone.

Experiments to Assess Vasodilator Function

Dose-response relationships to ACh were designed to determine whether ACh-induced dilation is different between Con and HLU rats. In addition, we assessed the relative contribution of known mediators of ACh dilation (NO, PGI2, and EDHF) and whether these dilators contribute differently in arteries from Con and HLU rats. Therefore, responses were studied first to ACh in all rats and then to ACh with inhibitors of the vasodilator pathways. The experimental protocol (Fig. 1) included four dose-response curves for each arteriole: 1) ACh alone, 2) ACh + Nomega -nitro-L-arginine (L-NNA), 3) ACh + L-NNA + indomethacin (Indo), and 4) SNP. The order of inhibitors was reversed in one-half of the rats in each group (Fig. 1).


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  		Fig. 1.   Protocol for vasodilator experiments. During the initial 1-h period, the arteriole was warmed to 37°C and pressurized to 80 cmH2O. The arteriole was then challenged with 80 mM KCl (KCl). Four dose-response relationships were examined after the arteriole exhibited stable spontaneous tone for >= 10 min. ACh was applied cumulatively to the bath (10-9-10-4 M in log increments). Next, the arteriole was incubated with 300 µM Nomega -nitro-L-arginine (L-NNA) for 20 min. After the arteriole exhibited stable tone, a second ACh dose-response relationship was examined in the presence of L-NNA. A third dose-response curve to ACh was examined in the presence of L-NNA + 50 µM indomethacin (Indo). In a second group of rats, Indo was given first, and then L-NNA + Indo was administered. In our hands, ACh responses are repeatable for >= 3 successive curves. To assess effects of hindlimb unweighting (HLU) on vascular smooth muscle function, responsiveness to sodium nitroprusside (SNP, 10-9-10-4 M) was examined after the ACh protocols. Maximal arteriole diameter (passive diameter) was determined by replacing physiological saline solution (PSS) with Ca2+-free PSS (2 mM EDTA) for >= 30 min (Zero Ca++).

ecNOS Expression

The arterioles used for RT-PCR were dissected in cold PSSA, as described above. Length and diameter were recorded, and each arteriole was removed and placed into individual RNase-free tubes and frozen (-70°C) until use. Arterioles used for immunoblots were acquired in an identical manner, except the soleus was placed in ice-cold PSS without albumin, since 1% albumin interferes with the small amounts of protein in single arterioles.

RT-PCR. Relative differences in ecNOS mRNA expression in soleus 1A arterioles were assessed using a semiquantitative RT-PCR, as described previously (8, 23). Briefly, single arterioles were homogenized in 50 µl of LiCl lysis buffer. Poly(A)+ RNA was isolated from the crude lysate with paramagnetic oligo(dT) polystyrene beads (Dynal), and first-strand cDNA synthesis was performed in a 20-µl volume (Superscript Preamplification System, GIBCO-BRL Life Technologies). Eight microliters of the reverse-transcribed cDNA were used in a PCR reaction with use of previously published primers and cycling conditions for ecNOS (23). All data were standardized by coamplifying ecNOS with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculating an ecNOS-to-GAPDH ratio for each soleus arteriole. The GAPDH primers were based on the rat sequence for GAPDH and were as follows: 5'-CAA-GTT-CAA-TGG-CAC-AGT-CAA-GGC-TG-3' (sense) and 5'-GTT-GAA-GTC-ACA-GGA-GAC-AAC-CTG-G-3' (antisense).

Immunoblots. Relative differences in ecNOS protein expression in soleus 1A arterioles were assessed using immunoblot analysis, as described previously (8). Briefly, arterioles (1/rat in 6 Con and 6 HLU rats) were dissolved in 20 µl of Laemmli buffer (10) containing 62.5 mM Tris (pH 6.8), 6 M urea, 160 mM dithiothreitol, 2% SDS, and 0.001% bromphenol blue; boiled; and sonicated for 2 min. Cell lysates were subjected to SDS-PAGE under reducing conditions, and proteins were transferred to polyvinylidine difluoride membrane (Hybond-ECL, Amersham). The membrane was blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline-Tween (20 mM Tris · HCl, 137 mM NaCl, and 0.1% Tween 20). The blots were incubated overnight at room temperature with primary antibody against ecNOS (1:1,600; Transduction Laboratories) and GAPDH (1:10,000; Chemicon). Blots were subsequently incubated for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-mouse). Specific ecNOS and GAPDH proteins were detected by enhanced chemiluminescence (Amersham) and evaluated by densitometry (NIH Image). GAPDH was used as an internal standard to control for small differences in loading, as described previously (8). Data are expressed as relative densitometric units.

Solutions and Drugs

Reagents were obtained from Sigma Chemical (St. Louis, MO). ACh and SNP were dissolved in water and PSS, respectively. L-NNA was dissolved in hydrochloric acid, which did not alter the pH of PSS. Indo was dissolved in ethanol [0.6% (vol/vol) final concentration]. In control experiments, ethanol did not alter steady-state diameter.

Statistical Analysis

Dose-response data were originally expressed as absolute diameter, relative (to passive) diameter, and percentage of possible change in diameter to account for any differences in beginning diameter, myogenic tone, and maximal diameter. Relative diameter data (relative to maximal passive diameter, which was defined as 1.0) are presented here, because maximal diameters were different between groups. Percent possible dilation was used to identify the ACh (or SNP) dose producing half-maximal response (EC50) and to determine whether maximal dilation was different between groups. EC50 values were determined using linear regression (Basica IC50) and expressed as logarithms. Student's unpaired t-tests were used to compare the Con with the HLU group. Percent possible dilation was calculated as [(Ddose - DB)/(DP - DB)] × 100, where Ddose is measured diameter for a given dose of ACh (or SNP), DB is baseline diameter before ACh, and DP is maximal passive diameter.

All dose-response curves were analyzed by two-way ANOVA, with repeated measures on one factor (dose of ACh or SNP). Post hoc analyses were performed using Fisher's protected least significant difference, Duncan's new multiple range, Student-Newman-Keuls, and Tukey's compromise tests. In all cases, the post hoc tests gave the same results. Comparisons of maximal percent possible dilation to ACh or SNP, EC50, passive diameter, percent myogenic tone, body mass, soleus mass, soleus mass-to-body mass ratio, and citrate synthase activity were analyzed by unpaired Student's t-test. Level of significance for all analyses was P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Efficacy of HLU Procedures

At the end of the 14-day treatment period, there was no difference in body mass of HLU and Con rats (Table 1). Soleus mass and the soleus mass-to-body mass ratio were 35 and 34% lower, respectively, in HLU rats (Table 1). In addition, citrate synthase activity was 23% lower in soleus muscles from HLU rats. Collectively, these data confirm the efficacy of the HLU protocol in inducing muscle atrophy and reduced oxidative capacity in response to HLU.

                              
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  		Table 1.   Characteristics of Con and HLU rats and Soleus 1A arterioles

Arteriole Characteristics

Arterioles were obtained from the middle of the soleus muscle. Measurement of passive vessel diameter revealed that HLU 1A arterioles were 16% smaller than Con arterioles (140 ± 4- vs. 121 ± 4-µm maximal passive diameter at 80 cmH2O intraluminal pressure; Table 1). Both groups of arterioles exhibited spontaneous tone (43 and 45% in Con and HLU, respectively) before the first ACh curve. Thus arterioles from HLU rats exhibited smaller stable diameter in the presence of spontaneous tone (79 ± 7 vs. 66 ± 4 µm). Both groups responded similarly to 80 mM KCl (not shown), indicating that vascular smooth muscle vasoconstrictor responsiveness signaled by voltage-gated Ca2+ channels was not altered by HLU.

Dilation to ACh

Soleus 1A arterioles from Con (n = 14) and HLU (n = 14) rats exhibited a dose-dependent dilation to ACh (Fig. 2). ANOVA indicated that the dilation response to ACh for HLU rats was different from that for Con rats (P = 0.03), suggesting a blunted response. In addition, the maximal response to ACh was significantly lower in HLU rats (85 ± 4 vs. 65 ± 4% possible dilation). The sensitivity to ACh, reflected in the concentration of ACh that produced half-maximal response (EC50), was similar between groups (1.6 ± 1.9 × 10-7 and 2.6 ± 2.8 × 10-7 M in Con and HLU, respectively, P = 0.30).


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  		Fig. 2.   Comparison of ACh dilation in control (Con) and HLU rats. Values are means ± SE for 14 Con and 14 HLU rats. Whether expressed as absolute diameter (A) or relative to maximal passive diameter (B), arterioles from HLU rats dilated less to ACh than did arterioles from Con rats (ANOVA P = 0.03). * Significant (P < 0.05) difference between Con and HLU for a specific ACh dose.

In arterioles from two Con and two HLU rats, the endothelium was destroyed (denuded) to verify that ACh dilation is an endothelium-dependent process. Before denudation, arterioles were exposed to KCl (Fig. 1). After KCl was washed out and arterioles attained stable diameter for 10 min, they were challenged with 10-4 M ACh, which produced 60-90% possible dilation. Subsequently, 3 ml of air were passed through the lumen to denude the arterioles. After denudation, arterioles were recannulated and pressurized, checked for leaks, and exposed to KCl. Denuded arterioles displayed no leaks and slightly decreased myogenic tone but an unaltered response to 80 mM KCl. After myogenic tone was stable for 5 min, denuded arterioles were challenged with 10-4 M ACh. ACh dilation was abolished in denuded arterioles. SNP response curves performed in three of the four arterioles indicated that vascular smooth muscle responsiveness to NO was not altered by denudation (70-95% possible dilation). Collectively, these data indicate that ACh dilation is an endothelium-dependent response.

Endothelium-Independent Dilation

Dilation to SNP produced 78 ± 6 and 80 ± 6% possible dilation in arterioles from Con (n = 11) and HLU (n = 10) rats, respectively (Fig. 3). Neither the maximal response nor the sensitivity to SNP was altered by HLU (5.5 ± 3.2 × 10-7 and 1.7 × 10-7 ± 5.6 × 10-8 M in Con and HLU, respectively, P = 0.30), and ANOVA indicated that the curves were not different (P = 0.98).


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  		Fig. 3.   Comparison of SNP dilation in Con and HLU rats. Values are means ± SE for 11 Con and 10 HLU rats. ANOVA indicated no significant difference in the dilator response to SNP (P = 0.98) whether expressed as absolute diameter (A) or relative to maximal passive diameter (B). Data are a subset of the 14 rats used for Fig. 2.

Basal Release of Endothelium-Dependent Dilators

In arterioles from Con rats, L-NNA treatment alone or in combination with Indo tended to decrease resting diameter slightly; however, neither treatment significantly altered baseline diameter (Fig. 4A). Treatment with Indo, L-NNA, or L-NNA + Indo did not alter baseline diameter in HLU 1A arterioles, whereas Indo tended to increase the diameter of arterioles from Con rats (not significant; Fig. 4B). Thus, although these results are consistent with a small basal release of NO in Con arterioles, statistical analysis indicates that there is no significant effect of Indo or L-NNA on basal, spontaneous tone in these arterioles.


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  		Fig. 4.   Effect of treatment with L-NNA or L-NNA + Indo on arteriolar diameter. Values are means ± SE for 6-8 rats/treatment. ANOVA indicated that stable diameter was not significantly altered by L-NNA or Indo. A: diameter was not altered by L-NNA treatment in arterioles from Con (CC) (n = 7, P > 0.65) or HLU (n = 6, P > 0.80) rats. B: diameter was not altered by Indo in arterioles from Con (n = 7, P > 0.30) or HLU (n = 8, P > 0.95) rats. No Tx, no treatment.

Relative Roles of NO and PGI2 in ACh Dilation

Treatment with 300 µM L-NNA blocked ~40% of the ACh dilation in Con rats (Fig. 5A) and abolished ACh dilation in arterioles from HLU rats (Fig. 5B). L-NNA + Indo abolished ACh dilation in Con rats. L-NNA + Indo did not alter the ACh + L-NNA response in HLU rats. Treatment with L-NNA + Indo also abolished the between-group differences. When the order of inhibitors was reversed, 50 µM Indo did not significantly alter ACh dilation in Con or HLU arterioles (Fig. 5, C and D), and L-NNA + Indo abolished dilation to ACh in Con and HLU rats.


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  		Fig. 5.   Effects of treatment with L-NNA, Indo, or L-NNA + Indo on the vasodilator response to ACh. Values are means ± SE for 6-8 rats/treatment. A: in Con rats (n = 7), L-NNA inhibited ~40% of ACh dilation, whereas L-NNA + Indo eliminated ACh dilation. B: in HLU rats (n = 6), L-NNA abolished ACh-dilation and L-NNA + Indo did not change this effect. C: in Con rats (n = 7), ACh dilation was not significantly altered by Indo, but L-NNA + Indo eliminated ACh dilation. D: in HLU rats (n = 8), Indo alone did not alter ACh dilation, whereas L-NNA + Indo abolished ACh dilation.

Regardless of the order in which inhibitors were given, ACh dilation was less in arterioles from HLU than from Con rats (Figs. 6A and 7A). A primary difference between Con and HLU rats was that L-NNA abolished all ACh dilation in HLU rats (Fig. 6B). L-NNA + Indo abolished ACh dilation and eliminated group differences (Fig. 6C). Interestingly, Indo treatment did not significantly alter the response to ACh in Con or HLU rats (Fig. 5, C and D), but Indo abolished differences between Con and HLU rats to ACh (Fig. 7B). L-NNA + Indo abolished ACh dilation and eliminated the difference between Con and HLU rats (Figs. 6C and 7C).


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  		Fig. 6.   Effects of L-NNA and L-NNA + Indo on ACh dilation. Values are means ± SE from 7 Con and 6 HLU rats. A: ACh dilation is impaired in arterioles from HLU rats. B: L-NNA treatment completely abolished ACh dilation in HLU rats and reduced dilation by only ~40% in Con rats. C: L-NNA + Indo abolished ACh dilation in Con and HLU rats and eliminated the group difference. * Significant (P < 0.05) difference between HLU and Con at that ACh dose.



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  		Fig. 7.   Effects of Indo or L-NNA + Indo on ACh dilation. Values are means ± SE from 7 Con and 8 HLU rats. A: ACh dilation is impaired in arterioles from HLU rats. B: Indo did not significantly affect ACh dilation but did abolish the group difference. C: similar to L-NNA + Indo in Fig. 6C, L-NNA + Indo treatment abolished dilation in arterioles from Con and HLU rats. * Significant (P < 0.05) difference between HLU and Con at that ACh dose.

ecNOS Expression

The ecNOS-to-GAPDH mRNA ratio was 20% lower in arterioles from HLU rats than in Con soleus 1A arterioles (Fig. 8A). In similar fashion, protein levels from HLU rats were 65% lower than those from Con rats (Fig. 8B).


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  		Fig. 8.   Effect of HLU on endothelial cell nitric oxide synthase (ecNOS) mRNA and protein expression. Values are means ± SE for arterioles from 6 Con and 6 HLU rats. A: arterioles from HLU rats displayed 20% lower ecNOS mRNA levels than Con rats. B: arterioles from HLU rats contained 65% less protein than arterioles from Con rats. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C, Con; H, HLU. * Significantly different from Con (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to test the hypothesis that HLU results in decreased endothelium-dependent vasodilation in soleus resistance arteries. The key findings of this study are as follows: 1) arterioles from HLU rats dilate less in response to ACh; 2) the relative contribution of NO and PGI2 mediating ACh dilation is altered by HLU; 3) ecNOS mRNA and protein expression is decreased in soleus arterioles from HLU rats; and 4) HLU appears to alter the interaction between NOS and cyclooxygenase (COX) dilator pathways. On the basis of these findings, we conclude that HLU impairs endothelium-dependent dilation in soleus 1A arterioles, in part as a result of decreased ecNOS expression and altered contribution of NOS mediating ACh dilation.

Soleus muscles from HLU rats were 35% smaller in absolute and relative terms (Table 1). Oxidative capacity was also diminished in HLU soleus muscle, as indicated by a 23% reduction in citrate synthase activity. These changes are similar to data from previous studies using HLU rats (1, 8, 26) and confirm that our HLU model elicits classical responses to HLU in rats that also exhibit impaired exercise blood flow responses (12, 16, 25). The findings that soleus 1A arterioles from HLU rats were smaller than Con arterioles and exhibited similar spontaneous tone are in agreement with previously published data indicating that feed arteries and second-order arterioles remodel during HLU (1, 8). It is not known whether other arterioles remodel in soleus muscle or whether the remodeling is signaled by the low soleus blood flow during 14 days of HLU. However, arteriole passive diameter in the white portion of the gastrocnemius, a muscle that does not exhibit a reduction in blood flow during HLU, is not altered by 2 wk of HLU (1).

Soleus 1A arterioles from HLU rats dilated less to ACh, suggesting that endothelium-dependent dilator mechanisms are impaired after HLU (Fig. 2). Impaired dilation to ACh has also been reported in soleus feed arteries from HLU rats (8). Thus the first two branches of resistance arteries controlling blood flow to the soleus muscle exhibit impaired endothelium-dependent vasodilation after HLU. Interestingly, the decrement in functional dilation in HLU rats was more pronounced in 1A arterioles than in feed arteries (8). Because 1A arterioles are the first arteries supplying blood to the soleus, it is possible that limitations in vasodilator response induced by HLU contribute to impaired vasodilation and, therefore, lower soleus blood flow during standing (12) and light exercise (25).

Altered ACh-induced dilation could be the result of changes in endothelium and/or vascular smooth muscle. Our results indicate that the response of vascular smooth muscle to the NO donor SNP was not different between Con and HLU 1A arterioles. These results suggest that impaired ACh dilation in 1A arterioles from HLU rats is mediated by alterations in the endothelium and not vascular smooth muscle. Jasperse et al. (8) reported previously that soleus feed arteries from HLU rats exhibited enhanced sensitivity to SNP. These investigators proposed that feed arteries partially compensated for lower NO production with increased responsiveness of vascular smooth muscle to NO. Although our EC50 data suggest a trend toward greater sensitivity of HLU arterioles, ANOVA indicated that the SNP responses were similar (P = 0.98) between groups, and the t-test on the EC50 values indicated no between-group differences (P = 0.3). Our interpretation of these results (Fig. 3) is that the impaired ACh-induced dilation of soleus 1A arterioles from HLU rats is primarily the result of adaptations in endothelium and not responsiveness of vascular smooth muscle to NO.

The endothelial cell lining of arterial walls has received much attention for its role in vascular regulation. Endothelium-dependent dilation appears to be mediated via NO, PGI2, and EDHF. Therefore, we sought to determine the relative contribution of these dilators in soleus arterioles and to determine whether the relative importance of these pathways was altered by HLU. To this end, we used inhibitors of NOS (L-NNA) or COX (Indo) and combined inhibition of NOS and COX pathways (L-NNA + Indo).

Previous studies have shown that ACh dilation in resistance arteries from Con rats appears similar across different skeletal muscle (8, 18). Our results indicate no significant basal release of NO or PGI2 from soleus arterioles of Con rats (Fig. 4). Also, Indo treatment had no significant effect on ACh-induced dilation in soleus arterioles from Con rats (Fig. 5C). In contrast, treatment with L-NNA decreased ACh-induced dilation by 40-50% (Fig. 5A). Arterioles did not dilate significantly after treatment with L-NNA + Indo. These results indicate that ACh-induced dilation of soleus 1A arterioles is not mediated by release of EDHF, since treatment with L-NNA + Indo abolished dilation. Results further indicate that release of NO from NOS is responsible for much of the ACh-induced dilation. The fact that treatment with L-NNA + Indo abolished dilation suggests that PGI2 release from the COX pathway contributes to ACh-induced dilation in arterioles from Con rats. However, it is not clear why treatment with Indo alone had no significant effect (Fig. 5). It is possible that blocking COX with Indo indirectly enhances the inhibitory effects of L-NNA by decreasing endothelial cell Ca2+ levels, which would diminish the NO production via Ca2+-dependent activation of ecNOS. Alternatively, PGI2 production may increase during inhibition of NOS and decrease endothelial cell cGMP, counteracting the direct smooth muscle effects of attenuated NO production during ACh stimulation (15). Finally, endothelium removal demonstrates that ACh-induced dilation of soleus 1A arterioles is an endothelium-dependent process. In general, ACh-induced dilation in arterioles from the soleus muscle of Con rats is similar to results obtained from arteries of other skeletal muscles (8, 18).

HLU appeared to produce some interesting changes in the contribution of NO and PGI2 to ACh-induced dilation of soleus 1A arterioles. Similar to arterioles from soleus muscles of Con rats, arterioles from HLU soleus muscles show no evidence of basal release of NO or PGI2 (Fig. 4). Also, similar to Con results, Indo did not have a significant effect on ACh-induced dilation in arterioles from HLU soleus muscle (Fig. 5D). Perhaps the most important difference between Con and HLU soleus arterioles was seen after treatment with L-NNA (Figs. 5, A and B, and 6B). Arterioles from HLU rats treated with L-NNA did not dilate significantly in response to ACh (Fig. 5B), whereas L-NNA treatment only blocked 40-50% of ACh-induced dilation in Con arterioles. Consistent with the notion that ACh-induced dilation of soleus 1A arterioles from HLU rats is mediated almost entirely by NO release is the observation that arterioles exhibited similar responses after treatment with only L-NNA and after treatment with L-NNA + Indo (Fig. 5).

The finding of lower ecNOS mRNA and protein expression in arterioles from HLU rats (Fig. 8) is consistent with the idea that impaired dilation to ACh in soleus arterioles from HLU rats is due to alterations in the NO pathway. Similar reductions in ecNOS expression were seen in soleus feed arteries from HLU rats (8). Lower ecNOS expression may result in decreased NO production, leading to decreased responsiveness to ACh. The finding that blockade of the NO pathway in arterioles from HLU rats abolished ACh dilation but only blunted ACh dilation in Con arterioles is surprising (Fig. 6B). One possible explanation for these results is that arterioles from HLU rats also have decreased PGI2-mediated dilation. Thus increased PGI2 production may have compensated for NOS inhibition in arterioles from Con but not HLU rats (15). If COX expression was decreased by HLU, then we would expect that arterioles from HLU rats would not be able to compensate for NOS inhibition with L-NNA. Our results do not indicate whether COX expression and/or part of the COX signaling pathway is changed by HLU. However, our results clearly indicate that the relative contribution of NO and PGI2 in ACh dilation is altered by HLU.

It is possible that reduced ecNOS expression in arterioles from HLU rats involves chronic reductions in blood flow. McDonald et al. (12) reported that soleus blood flow is decreased during 14 days of HLU. In addition, it has been shown previously that ecNOS protein and mRNA expression is regulated by flow and shear stress in cultured endothelial cells (19) and in blood vessels (24). ecNOS expression has been shown to be decreased in soleus feed arteries of HLU rats (8), which experience a chronic reduction in soleus blood flow during unweighting (12). Consistent with the idea that blood flow can influence ecNOS expression, humans and rats with heart failure (and presumably less physical activity and, therefore, less muscle blood flow) exhibit impaired dilator responses to ACh infusions in the forearm and gracilis arteries, respectively (9, 20). These results (8, 9, 19, 20, 24) support the notion that one signal for lower ecNOS expression in soleus 1A arterioles could be reduced blood flow and, therefore, reduced shear stress during HLU.

In conclusion, our results reveal that HLU leads to decreased ecNOS expression and impaired ACh dilation in soleus 1A arterioles. It is possible that impaired endothelium-dependent dilation after HLU contributes to the blunted exercise hyperemia response and diminished exercise capacity after HLU.


    ACKNOWLEDGEMENTS

We are grateful for the expert technical assistance of Pam Thorne, Tammy Strawn, Denise Holiman, and Sarah Friskey.


    FOOTNOTES

This work was supported by National Institutes of Health Grant HL-36088 (M. H. Laughlin) and Individual National Research Service Award HL-09739 (C. R. Woodman) and National Aeronautics and Space Administration Graduate Student Research Program Grant 098-13 (W. G. Schrage).

Address for reprint requests and other correspondence: M. H. Laughlin, E102 Veterinary Biomedical Sciences, University of Missouri, Columbia, MO 65211 (E-mail: LaughlinM{at}missouri.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.

Received 21 March 2000; accepted in final form 25 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 89(4):1483-1490
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