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Departments of Veterinary Biomedical Sciences and Physiology and The Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested the hypothesis that hindlimb unweighting (HLU) decreases endothelium-dependent vasodilation and expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) in arteries of skeletal muscle with reduced blood flow during HLU. Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 15) or control (n = 15) conditions for 14 days. ACh-induced dilation was assessed in muscle with reduced [soleus (Sol)] or unchanged [gastrocnemius (Gast)] blood flow during HLU. eNOS and SOD-1 expression were measured in feed arteries (FA) and in first-order (1A), second-order (2A), and third-order (3A) arterioles. Dilation to infusion of ACh in vivo was blunted in Sol but not Gast. In arteries of Sol muscle, HLU decreased eNOS mRNA and protein content. eNOS mRNA content was significantly less in Sol FA (35%), 1A arterioles (25%) and 2A arterioles (18%). eNOS protein content was less in Sol FA (64%) and 1A arterioles (65%) from HLU rats. In arteries of Gast, HLU did not decrease eNOS mRNA or protein. SOD-1 mRNA expression was less in Sol 2A arterioles (31%) and 3A arterioles (29%) of HLU rats. SOD-1 protein content was less in Sol FA (67%) but not arterioles. SOD-1 mRNA and protein content were not decreased in arteries from Gast. These data indicate that HLU decreases endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression primarily in arteries of Sol muscle where blood flow is reduced during HLU.
microgravity; physical inactivity; blood flow; acetylcholine; superoxide dismutase; microcirculation; endothelial nitric oxide synthase
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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HINDLIMB UNWEIGHTING (HLU) is a rodent model of physical inactivity that elicits a number of cardiovascular adaptations that occur during exposure to microgravity or bed rest (14). Included among these adaptations are reductions in maximal oxygen uptake (3, 18), endurance run time (28), cardiac output during exercise (27), and an impaired ability to increase skeletal muscle blood flow during exercise (18, 27). Previously, it has been proposed that blunted exercise hyperemia after HLU involves impaired endothelium-dependent vasodilation (2, 7). This hypothesis is supported by experimental findings indicating that in vitro vasodilator responses to flow or ACh are blunted in soleus (Sol) feed arteries (FA) and first-order (1A) arterioles after 14 days HLU (2, 7, 22).
One mechanism that may contribute to the detrimental effect of HLU on endothelium-dependent vasodilator responses is downregulation of endothelial nitric oxide synthase (eNOS), decreasing local production of nitric oxide (NO·). Indeed, eNOS expression is lower in Sol FA and 1A arterioles after HLU (7, 22). Because blood flow is chronically reduced in the Sol muscle during HLU (12), and eNOS expression is regulated by shear stress in blood vessels (15, 26), it is plausible that a signal for decreased eNOS expression in HLU rats is decreased blood flow and therefore shear stress. In addition, it is reasonable to predict that the detrimental effect of HLU on eNOS expression and endothelium-dependent vasodilator responses is localized to arteries and arterioles in skeletal muscle where blood flow is reduced during HLU. Consequently, the primary purpose of this study was to test the hypothesis that HLU selectively decreases in vivo endothelium-dependent vasodilator responses and eNOS expression in skeletal muscle arterial networks in which blood flow is chronically reduced during HLU.
The primary mechanism for NO· degradation in blood vessels is its
interaction with superoxide anion (O
Endothelium-dependent vasodilator responses were assessed in vivo by measuring the change in blood flow to the Sol and gastrocnemius (Gast) muscles after infusion of the endothelium-dependent vasodilator, ACh. Relative levels of eNOS and SOD-1 mRNA and protein expression were assessed in single FA and arterioles isolated from the Sol and Gast muscles. We focused on these two muscles because blood flow to Sol muscle is reduced during 14 days of HLU, whereas blood flow to the Gast is not decreased by HLU (12).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal Care
Before we initiated this study, approval was received from the Institutional Animal Care and Use Committee at the University of Missouri. Male Sprague-Dawley rats (300-350 g) were purchased from a commercial dealer (Harlan Sprague Dawley, Indianapolis, IN) and housed in the College of Veterinary Medicine's Animal Care Facility, which was maintained at 24°C with a 12:12-h light-dark cycle. Animals were provided food and water ad libitum. Rats were randomly assigned to HLU (n = 15) or cage control (Con; n = 15) conditions for 14 days. Rats were examined daily by the investigators and by veterinarians affiliated with the University of Missouri's College of Veterinary Medicine.HLU Procedures
HLU was accomplished via modification of the method described previously (13). In brief, rats were fitted with a small thoracic cast made from plaster of Paris. The rat's tail was cleaned with alcohol and allowed to dry, after which an adhesive strip with a metal clip was affixed to it. Rats were subsequently familiarized with HLU for 1-2 h/day for 3 consecutive days. At the end of the familiarization period, rats were exposed to 14 continuous days of HLU at a 45° head-down angle. A 14-day HLU protocol was utilized because previous studies revealed that blood flow to Sol muscle is reduced during 14 days of HLU, whereas blood flow to the Gast muscle is not decreased (12). In addition, it has been shown previously that 14 days of HLU is associated with decreased eNOS expression and attenuated endothelium-dependent vasodilator responses in Sol FA in vitro (7). The efficacy of the HLU protocol was assessed from measurements of muscle weight and muscle weight-to-body weight ratios.In Vivo Endothelium-Dependent Vasodilator Responses
At the end of the 14-day treatment period, endothelium-dependent vasodilator responses in the Sol and Gast were assessed in vivo with infusions of ACh (n = 9 rats/group). The procedures used were designed to allow two measures of blood flow (flow probe and radiolabeled microspheres) while increasing amounts of ACh were infused directly into the femoral artery via the epigastric artery (a branch of the femoral artery). This approach allowed for the assessment of local endothelium-dependent dilation in selected hindlimb muscles without producing systemic hypotension.Surgical procedures. Rats were anesthetized using an intraperitoneal injection of 100 mg/kg inactin (100 mg/ml; Sigma Chemical, St. Louis, MO). Inactin provided deep anesthesia while maintaining blood pressure >100 mmHg. A deep level of anesthesia was ensured before the start of data collection to avoid the need for supplemental doses of inactin (iv), because intravenous doses of inactin tended to decrease blood pressure to a lower steady state.
The anteriomedial portion of the left hindlimb was exposed, and the left femoral and epigastric arteries were exposed using blunt dissection. The distal portions of the epigastric artery and vein were tied off with suture, and a catheter (PE-10) was placed centrally in the epigastric artery. The catheter was secured to the muscle so the tip was flush with the lumen of the femoral artery. This allowed ACh to be infused directly into the femoral artery blood supply. A Transonic flow probe (0.5 V; Transonic Systems) was placed around the proximal portion of the left femoral artery. After placement of the probe, two or three drops of topical lidocaine (2%) were applied to the artery to relax any vasoconstriction induced by blunt dissection of the area. Lidocaine was applied directly onto the femoral artery to minimize potential downstream effects of lidocaine on Sol and Gast muscle blood flow. A catheter (PE-50) placed in the right femoral artery, advanced to the terminal aorta, was used for reference blood sample withdrawal. A catheter (PE-10) placed in the right femoral vein was used for administration of inactin and heparin. An additional catheter (PE-50) was inserted into the right carotid artery. A trachea tube was placed to ensure an open airway. All rats were heparinized with 300-400 units heparin administered intravenously before start of data collection. A low heparin dose was used in both groups to minimize possible confounding effects of heparin on skeletal muscle blood flow. Mean arterial pressure was measured from the carotid artery catheter by using a Statham pressure transducer. Heart rate was measured from the pulsatile pressure measurements. Heart rate, mean arterial pressure, and femoral artery blood flow were recorded on a Grass polygraph eight-channel recorder.Muscle blood flow.
Hindlimb blood flow responses to ACh were measured with the
labeled-microsphere method as described by Laughlin et al.
(10). In brief, radiolabeled microspheres
(85Sr, 46Sc, 113Sn), 15 µm in
diameter, were sonicated and vortexed for 60 s. Reference blood
withdrawal was initiated (0.68 ml/min) from the femoral artery 30 s before infusion of 0.1 ml of a microsphere solution (~10 µCi)
into the carotid artery catheter. After the infusion, the catheter was
flushed with 1.0 ml of saline and reference blood withdrawal was
continued for 30 s. Muscle blood flow measurements were made at
rest (pre-ACh) and two different rates of ACh infusion: 0.2-0.4
µg · kg
1 · min1 (low
dose) and 1.6-2.2
µg · kg1 · min1 (high
dose). The low-ACh dose was selected because it produced the first
consistent elevation in femoral artery blood flow as determined by the
Transonic flow probe. The high-ACh dose was defined as the dose that
doubled pre-ACh blood flow, or where three consecutive doses of ACh
caused no further increase in femoral artery blood flow. The isotope
used for a given dose of ACh was randomly selected. After the
low-ACh-dose sphere injection, ACh infusion rate was increased in 0.2 µg · kg1 · min1
increments for 2 min each until femoral artery blood flow appeared maximal, and the high-ACh-dose sphere was injected. Control saline infusions produced no significant change in femoral artery blood flow
(data not shown).
Isolation of Arterioles
At the end of the 14-day treatment period, a separate group of HLU (n = 6) and Con (n = 6) rats were anesthetized with pentobarbital sodium (100 mg/kg ip). The HLU rats were anesthetized while suspended to prevent their hindlimbs from bearing weight. The Sol and medial head of the Gast muscles were removed from the right leg and placed in iced (4°C) MOPS-buffered physiological saline solution containing (in 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, pH 7.4, and 1 g/100 ml bovine serum albumin. FA, 1A, second-order (2A) arterioles, and third-order (3A) arterioles were dissected from the Sol and Gast and placed individually in ribonuclease-free microcentrifuge tubes and frozen at
80°C for
subsequent use in measuring eNOS and SOD-1 mRNA expression. The
diameter and length of the unpressurized arteries were measured using
an Olympus dissecting microscope equipped with an eyepiece calibrated
to ±1 µm and recorded before freezing. An additional group of
arterioles (FA-3A) from the Sol and Gast was dissected in
physiological saline solution (without albumin), placed individually in
microcentrifuge tubes, and frozen at 80°C for use in measuring eNOS
and SOD-1 protein content. Arteries were classified using the
nomenclature system described previously by Wiedeman (24).
According to this ordering scheme, FA are defined as the last artery
before entering the epimycium. 1A arterioles arise from FA and
are the first arteries within the epimycium. 2A arterioles are branch
vessels arising from 1A arterioles, and 3A arterioles are branches of
2A arterioles. We further classified the fast-twitch Gast into its
Gastred and Gastwhite portions. Consequently,
mRNA and protein data for the Gast are presented for FA and 1A
arterioles that supply both the Gastred and
Gastwhite portions of the Gast and for 2A and 3A arterioles that are exclusive to the Gastred or Gastwhite.
Quantitation of eNOS and SOD-1 Expression
RT-PCR.
Relative differences in eNOS and SOD-1 mRNA expression in FA and
arterioles were assessed as described previously (25). In
brief, single FA or arterioles were homogenized by vortexing the sample
vigorously for 60 s. The sample was subsequently spun briefly in a
microcentrifuge, and the process was repeated four to five times until
the arteriole was completely digested. 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). Nine microliters of reverse-transcribed cDNA were used
in a PCR by using previously published primers and cycling conditions
for eNOS (25). A second nine-microliter aliquot of cDNA
was used in a PCR reaction by using primers specific for SOD-1. All
data were standardized by spiking the PCR reactions with 10 µCi of
[
32P]dCTP (3,000 Ci/mmol) and coamplifying eNOS or
SOD-1 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The
PCR-amplified products were electrophoresed on a 1.5% agarose gel, and
the eNOS, SOD-1, and GAPDH bands were excised, placed in separate
scintillation vials, and counted for 1 min in a liquid scintillation
counter as described previously (25). An eNOS-to-GAPDH and
SOD-1-to-GAPDH ratio was calculated for each vessel. The SOD-1 primers
were based on the rat SOD-1 sequence and were as follows: SOD-1 sense,
5'-CAT TCA CTT CGA GCA GAA GGC AAG TG-3'; and SOD-1 antisense, 5'-GTC
ATC TTG TTT CTC GTG GAC CAC C-3'. The GAPDH primers were based on the
rat sequence for GAPDH: sense, 5'-CAA GTT CAA TGG CAC AGT CAA GGC TG-3'; antisense, 5'-GTT GAA GTC ACA GGA GAC AAC CTG G-3'. GAPDH was
used as an internal standard because it is a constitutively expressed
enzyme whose mRNA level is not affected by flow in cultured endothelial
cells or isolated arterioles (4, 26). In addition, GAPDH
mRNA expression is not altered by 14 days of HLU in Sol FA or 1A
arterioles (7, 22). Consequently, expressing eNOS and
SOD-1 mRNA content relative to GAPDH allowed for relative comparisons
between arterioles isolated from Con and HLU rats.
Immunoblot analysis. Relative differences in eNOS and SOD-1 protein expression in FA and arterioles (1A-3A) from the Sol and Gast were assessed using immunoblot analysis as described by Jasperse et al. (7). Briefly, single FA or arterioles (matched for diameter and length) were solubilized in 20 µl Laemmli buffer (8) and boiled for 10 min. Protein samples were loaded onto 4-20% acrylamide gradient SDS gels (precast minigels, Bio-Rad), electrophoresed under reducing conditions, and transferred to polyvinylidene difluoride membrane (Hybond-ECL, Amersham). The membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris buffered saline-Tween (20 mmol/l Tris HCl, 137 mmol/l NaCl, and 0.1% Tween 20) and incubated overnight at room temperature with primary antibody against eNOS (1:1,600; catalog no. N30020, Transduction Laboratories) and GAPDH (1:10,000; catalog no. MAB374, Chemicon). Blots were subsequently incubated for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-mouse; catalog no. A8924, Sigma Chemical). Specific eNOS and GAPDH protein was detected by enhanced chemiluminescence (Amersham) and evaluated by densitometry using NIH Image software (National Institutes of Health, Bethesda, MD). All blots were reblocked for 1 h at room temperature and incubated overnight with a polyclonal antibody against SOD-1 (1:1,600; catalog no SOD-100, Stressgen). Blots were then incubated for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-rabbit; catalog no. A4914, Sigma Chemical). Data are expressed as relative densitometric units. GAPDH was used as an internal standard to control for small differences in protein loading.
Data Analysis
All values are means ± SE. Between-group differences in body mass, Sol mass, as well as eNOS and SOD-1 mRNA and protein expression were assessed using Student's t-tests for unpaired observations. Blood flow responses to ACh were analyzed by two-way ANOVA with repeated measures on one factor (ACh dose). When a significant F value was obtained, post hoc analyses were performed using Duncan's multiple-range test. Statistical significance was set at the P
0.05 probability level.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Efficacy of HLU Procedures
At the end of the 14-day treatment period, the body mass of HLU rats was slightly lower (5%) than Con rats (Table 1). In addition, Sol mass (37%) and Sol-to-body mass ratio (34%) were significantly lower in HLU rats, confirming the efficacy of HLU in inducing skeletal muscle atrophy.
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ACh-induced Vasodilation: Hemodynamics
Mean aortic pressures were in the range of 120-130 mmHg in both control and HLU rats. By experimental design, infusion of ACh produced minor decreases in blood pressure in HLU and Con rats. Heart rate was not altered in either group of rats (data not shown).ACh-induced Vasodilation: Regional Muscle Blood Flows
Blood flow responses to in vivo infusions of ACh, determined with the microsphere technique, are summarized in Fig. 1. Resting blood flow (measured before drug infusion) was significantly less in the Sol muscle of HLU rats than in Con rats (4.1 ± 0.7 vs. 13.0 ± 2.7 ml · min1 · 100 g1). In
contrast, resting blood flows were similar throughout the Gast muscle
of Con and HLU rats. ACh infusion significantly increased blood flow to
the Sol muscle of Con rats (Fig. 1). In contrast, ACh did not produce a
significant change in blood flow to the Sol in HLU rats. ACh infusion
did not significantly change blood flow to the Gastred,
Gastwhite, or Gastmixed portions in Con or HLU
rats.
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eNOS mRNA Expression
The effect of HLU on eNOS mRNA expression in FA and arterioles from the Sol muscle is summarized in Fig. 2. Semiquantitative PCR revealed that the eNOS-to-GAPDH ratio was significantly lower in Sol FA, 1A arterioles, and 2A arterioles (Fig. 2). eNOS mRNA content was not reduced by HLU in Sol 3A arterioles. eNOS mRNA expression was not decreased by HLU in FA or arterioles isolated from the Gast; however, the eNOS-to-GAPDH ratio was increased in 2A arterioles from Gastred and 3A arterioles from Gastwhite (Fig. 3).
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eNOS Protein Expression
Immunoblot analyses revealed that eNOS protein content was reduced in Sol FA and 1A arterioles (Fig. 2). In addition, these data indicate that eNOS protein expression was not significantly lower in Sol 2A or 3A arterioles. eNOS protein expression was not altered by HLU in FA or arterioles in the Gast (Fig. 3).SOD-1 mRNA Expression
Semiquantitative PCR revealed that the SOD-1-to-GAPDH ratio was significantly lower in Sol 2A arterioles and 3A arterioles of HLU vs. Con rats (Fig. 4). SOD-1 mRNA was not reduced by HLU in Sol FA or 1A arterioles. SOD-1 mRNA expression was not significantly reduced by HLU in FA or arterioles in the Gast (Fig. 5).
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SOD-1 Protein Expression
Immunoblot analyses revealed that SOD-1 protein content was reduced in Sol FA (Fig. 4). SOD-1 protein expression was not significantly lower in Sol 1A, 2A, or 3A arterioles. SOD-1 protein expression was not decreased by HLU in FA from the Gast (Fig. 5). In addition, HLU did not alter SOD-1 protein expression in 2A or 3A arterioles isolated from the Gastred or Gastwhite fiber portions but was decreased by HLU in 1A arterioles (Fig. 5).| |
DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The purpose of this study was to test the hypothesis that HLU decreases in vivo endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression throughout the arterial network in skeletal muscle where blood flow is reduced during HLU. The primary findings of this study are as follows. 1) Resting blood flow is decreased in the Sol muscle of HLU rats but not in Gast muscle. 2) ACh-induced vasodilation is blunted in Sol but not Gast muscle of HLU rats. 3) HLU decreases eNOS and SOD-1 mRNA expression in some branch orders of Sol arteries but not in Gast arteries. 4) HLU decreases eNOS and SOD-1 protein content in FA from Sol muscle but not FA from the Gast muscle. 5) HLU decreases eNOS protein expression in some arteriolar branch orders in Sol muscle but not Gast. These results are consistent with the hypothesis that decreased expression of eNOS and SOD-1 in Sol resistance arteries contributes to blunted endothelium-dependent vasodilator responses in the Sol muscle (Fig. 1) and isolated Sol arteries (7, 22), possibly through decreased production and increased degradation of NO·.
In rats, HLU has been shown to impair the ability to increase skeletal muscle blood flow during exercise (18, 27). Previous studies using isolated arteries and arterioles suggested the detrimental effect of HLU on exercise hyperemia may be due in part to blunted endothelium-dependent vasodilator responses (7, 22). Indeed, Jasperse et al. (7) reported that vasodilator responses to increases in intraluminal flow and ACh were blunted in isolated perfused Sol FA. In addition, previous studies indicated that endothelium-dependent dilation is decreased in isolated 1A arterioles from the Sol but not the Gast (2, 22). In the present study, endothelium-dependent vasodilator responses to ACh were assessed in vivo and the results indicate that endothelium-dependent dilation to ACh is blunted in the Sol but not in Gast. The finding that in vivo endothelium-dependent dilation is impaired in the Sol is in accord with previous studies showing blunted vasodilator responses in isolated cannulated arteries and arterioles (2, 7, 22). In addition, these data are consistent with the finding that HLU rats have an attenuated ability to increase blood flow to the Sol during treadmill exercise, whereas the ability to increase blood flow to the Gast is not altered by HLU (27).
In rats, HLU has been shown to decrease eNOS expression and endothelium-dependent vasodilator responses in Sol feed arteries and 1A arterioles (7, 22). The results of this study indicate the detrimental effects of HLU also occur in 2A arterioles but not Sol 3A arterioles. An important finding of this study was that HLU did not decrease eNOS expression in FA or arterioles isolated from the Gast.
The mechanisms by which HLU selectively decreases eNOS expression in vessels perfusing the Sol muscle are not clear. One potential mechanism for the HLU-induced decrease in eNOS expression is decreased shear stress. This speculation is based on experimental findings indicating that eNOS expression is regulated by shear stress in cultured endothelial cells, rat aorta, and porcine coronary arterioles (15-17, 19, 23, 26). It is plausible that chronically reduced blood flow to the Sol muscle during HLU contributes to a reduction in shear stress in arteries and arterioles perfusing this muscle. Indeed, McDonald et al. (12) demonstrated that Sol muscle blood flow was 70% lower during HLU than during weight-bearing conditions. Whether the reduction in Sol muscle blood flow in HLU rats reduces shear stress is not known. Estimates of shear stress calculated for arterioles isolated from Con and HLU rats suggest that shear stress in Sol arterioles may be reduced at the onset of HLU (2). Importantly, blood flow to the Gast is not reduced during 14 days of HLU (12), and eNOS expression was not reduced in arterioles perfusing this muscle. These data provide further support for the idea that the effects of HLU on eNOS expression in the Sol arterial network are mediated by reductions in blood flow and shear stress. However, further study is needed to determine whether shear stress is decreased by HLU in the Sol vasculature.
Decreased eNOS expression in arteries perfusing the Sol may provide one mechanism for blunted endothelium-dependent vasodilator responses in Sol muscle by impairing the ability of vascular endothelial cells to produce and release NO· (2, 7, 22). The finding that both eNOS expression and endothelium-dependent dilation were not altered in arteries isolated from the Gast in this study is in line with previously published data indicating that endothelium-dependent vasodilation is not altered by HLU in arterioles isolated from this muscle (2).
An additional mechanism that may contribute to blunted
endothelium-dependent vasodilator responses in Sol arteries and
arterioles in HLU rats is downregulation of SOD-1, increasing free
radical-mediated NO· degradation. The primary mechanism for NO·
degradation in blood vessels is its interaction with
O
It is conceivable that the blunted blood flow response to the endothelium-dependent vasodilator ACh in Sol muscle from HLU rats is due in part to HLU-induced vascular remodeling in this muscle. It has been reported previously that maximal passive diameter of Sol FA and 1A arterioles is smaller in HLU rats than Con rats (2, 7, 22). In addition, HLU-induced vascular remodeling does not occur in the Gast muscle (2).
Decreased eNOS expression may play an integral role in HLU-induced vascular remodeling that occurs in the Sol muscle (2, 7, 22). Langille and O'Donnell (9) reported previously that chronically reduced blood flow causes a reduction in arterial diameter of rabbit carotid arteries. In addition, experimental evidence indicates that endothelial-derived nitric oxide mediates this response because vascular remodeling associated with chronically reduced blood flow does not occur in mice with targeted disruption of the eNOS gene (20). Consequently, decreased eNOS expression in Sol FA and arterioles of HLU rats reported in this study may contribute to remodeling of the arteries to a smaller diameter and in turn to the blunted blood flow response to ACh.
It is important to consider possible differences in arteries and arterioles isolated from different areas of the Gast muscle. The data presented in Figs. 3 and 5 were obtained from the FA and arterioles of the medial head of the Gast muscle. The medial head of the Gast muscle of the rat is perfused by a single FA that branches from the popliteal artery. The arterioles from the Gastwhite muscle were branches from the only 1A arteriole in this muscle. The 2A and 3A arterioles in Gastwhite lead medially and superficially from the deep 1A arteriole. The 2A and 3A arterioles from the Gastred muscle were deep branches from the 1A arteriole, which lead toward the bone, next to the Sol and plantaris muscles. In contrast, blood flow data for Gastred and Gastwhite muscle, as presented in Fig. 1, are blood flow to the Gastwhite and Gastred portions of both the muscle's lateral and medial heads. We assumed that there are no differences between arterioles in the medial and lateral heads of the Gast muscle. We have not tested this assumption. This assumption indicates that Gast arterioles examined by Delp et al. (2) are similar to those we used in this study. Delp et al. indicate that the Gast 1A arterioles used in their studies were located in the Gastwhite portion of the muscle, suggesting that their arterioles came from the lateral head of the Gast muscle. The lateral head of the Gast, Sol, and plantaris muscles are perfused by FA that branch from the popliteal artery. The FA to the lateral Gast enters the superficial portion of the muscle forming the 1A arteriole.
In summary, the results of this study indicate that HLU decreases
endothelium-dependent vasodilation in Sol muscle where blood flow is
reduced during HLU. These data indicate one possible mechanism for
blunted endothelium-dependent vasodilator responses in the Sol muscle
of HLU rats is decreased eNOS expression leading to decreased
production of NO·. In addition these data are consistent with the
idea that a signal for decreased endothelium-dependent dilation and
eNOS expression is decreased blood flow (shear stress) because
vasodilator function and eNOS expression were not decreased in the Gast
muscle where blood flow is maintained during HLU. Decreased expression
of SOD-1 in sol FA may contribute to blunted endothelium-dependent
dilation by impairing the ability to scavenge O
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the expert technical assistance of Pam Thorne and Sarah Friskey.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-52490 and HL-36088 (to M. H. Laughlin), HL-55306 (to E. M. Hasser), HL-09739 (to C. R. Woodman), National Aeronautics and Space Administration Graduate Student Research Program Grant 098-13 (to W. G. Schrage), and American Heart Association Grant 9640177N (to E. M. Price).
Address for reprint requests and other correspondence: C. R. Woodman, Dept. of Veterinary Biomedical Sciences, W108 Veterinary Medicine, 1600 E. Rollins Rd., Univ. of Missouri, Columbia, MO 65211(E-mail: woodmanc{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 25 January 2001; accepted in final form 1 May 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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