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Departments of Veterinary Biomedical Sciences and Medical Physiology and 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) and the associated reduction
in soleus muscle blood flow causes decreased expression of endothelial
cell nitric oxide synthase (ecNOS) mRNA and protein and attenuated
endothelium-dependent vasodilator responses in rat soleus feed arteries
(SFA). Male Sprague-Dawley rats were exposed to HLU
(n = 12) or cage control (Con;
n = 12) conditions for 14 days. At the
end of this period, SFA were isolated, removed, and cannulated with two
glass micropipettes for examination of vasodilator responses or frozen
for analysis of ecNOS mRNA and protein expression. RT-PCR of RNA from
single SFA was used to measure ecNOS mRNA, and immunoblots on single
SFAs were used to measure ecNOS protein content. Results revealed that
both ecNOS mRNA and ecNOS protein expression were lower in SFA from HLU
rats. Dilation to increased intraluminal flow was attenuated in SFA from HLU rats (Con: 88 ± 8% vs. HLU: 53 ± 8%) as was maximal
vasodilation to acetylcholine
(10
9-104
M; Con: 88 ± 5% vs. HLU: 73 ± 5%). Sensitivity to the
endothelium-independent vasodilator sodium nitroprusside
(1010-104
M) was enhanced by HLU (EC50: Con:
4.46 × 107 M vs. HLU:
5.00 × 108 M).
Collectively, these data indicate that the chronic reduction in soleus
blood flow associated with the reduced physical activity during HLU
results in reduced expression of ecNOS mRNA and protein in SFA and
attenuated endothelium-dependent vasodilation.
microcirculation; acetylcholine; flow-induced dilation; sodium nitroprusside; microgravity
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN THIS STUDY WE EXAMINED the effect of hindlimb unweighting (HLU) on endothelial cell nitric oxide synthase (ecNOS) mRNA and protein expression and on vasodilator responses of rat soleus feed arteries. Our hypothesis was that reduced physical activity and the associated reduction in blood flow would decrease ecNOS mRNA and protein expression and endothelium-mediated vasodilator responses of soleus feed arteries.
HLU of rats is a model that has been widely used to simulate human bed rest or exposure to microgravity (25). HLU elicits adaptations that include a reduced maximal oxygen consumption (29), reduced blood volume (3), reduced exercising cardiac output (42), and altered distribution of blood flow during exercise (22, 42). The altered blood flow response is characterized by an impaired ability to redistribute blood flow from visceral regions to skeletal muscle as well as an impaired ability to shift blood flow from low-oxidative to high-oxidative regions among and within skeletal muscle (22, 42). One example of this altered distribution occurs in the soleus, a highly oxidative muscle in which blood flow is reduced both during unweighting (22) and after the unweighting period during standing (22) or during treadmill exercise (42). The mechanisms responsible for these changes in blood flow distribution are not completely understood but may involve alterations in metabolic demand (33), sympathetic nervous system activity (24, 39), vessel number (8, 9), vessel structure (4), and/or vascular control (6, 7). Indeed, Delp et al. (6, 7) have reported that HLU attenuated vasoconstrictor responses of rat aorta. However, the effect of HLU on vasodilator mechanisms in skeletal muscle resistance arteries has not been examined. Because HLU causes chronic reductions in blood flow to the soleus muscle (22, 42), and shear stress (related to blood flow) is known to modulate ecNOS gene expression (2, 28, 35, 41), it is reasonable to propose that ecNOS gene expression and endothelium-dependent vasodilator responses would be blunted by HLU.
Therefore, the purpose of this study was to test the hypothesis that HLU results in decreased ecNOS mRNA and protein expression and attenuated endothelium-dependent dilation in soleus feed arteries. This potential adaptation could contribute to the reduction in soleus muscle blood flow that has been observed in standing or running rats after HLU (22, 42).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Male Sprague-Dawley rats (wt = 250-275 g) were obtained (Sasco) and housed, one animal per cage, in a room with controlled temperature (24°C) and light (12:12-h light-dark cycle) conditions. Rats were provided food and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri.HLU
The HLU procedure has been described in detail previously (24). Rats were randomly assigned to HLU or control groups. HLU rats were acclimated to the unweighting procedure by unweighting the hindlimbs for 1-2 h/day for 3 consecutive days before the HLU procedure. The hindlimbs of HLU rats were then elevated with a harness attached to the proximal two-thirds of the tail. Rats were maintained in a suspension angle of 30-35°. A small thoracic cast made from plaster of Paris was applied to reduce lordosis and help prevent rats from reaching the tail apparatus. Rats were able to move freely about the cage by using their front limbs and were able to reach food and water without difficulty. The thoracic cast was also applied to control rats, but these rats were allowed to bear weight on all four limbs and to remain normally active during the unweighting period. The unweighting period lasted 14 days, on the basis of the previous work of McDonald et al. (22) and Woodman et al. (42) in which soleus muscle blood flow was compromised after HLU in hindlimb unweighted, weighted nonexercising, and weighted exercising rats. Furthermore, vasomotor responses in rat aorta are altered by this period of unweighting (7). Body weights were recorded before and after the unweighting period, and rats were closely monitored for adequate food and water intake, grooming behavior, and urination and defecation.Oxidative Enzyme Capacity
After dissection of the soleus feed arteries, the soleus muscle was removed and stored at
70°C until it was processed. Citrate synthase activity, a marker of oxidative enzyme capacity, was measured
spectrophotometrically by using whole muscle homogenate according to
the method of Srere (32).
Isolation of Feed Arteries
Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50.0 mg/kg) without allowing the hindlimbs to become weight bearing. An incision was made on the lateral surface of the lower leg, and the feed arteries to the medial portion of the soleus were carefully exposed as described in detail by Williams and Segal (38). The feed arteries were gently dissected free of the paired veins and connective tissue with a fine forceps, with care taken that the arteries not be touched with the forceps. The exposed tissue was kept moist with physiological saline solution (PSS) at all times during dissection. The isolated arteries were then cut on either end and frozen in ribonuclease-free microcentrifuge tubes at80°C
for subsequent quantitation of ecNOS mRNA or protein expression, or
they were transferred to a bath containing cold MOPS-buffered saline
for cannulation.
Quantitation of ecNOS mRNA and Protein
RT-PCR. Relative differences in ecNOS mRNA expression in soleus feed arteries were assessed by using a semiquantitative RT-PCR as described previously (40). In brief, arteries were homogenized in 50 µl of a LiCl lysis buffer (40). 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 by using previously published primers and cycling conditions for ecNOS (40). All data were standardized by coamplifying ecNOS with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and calculating an ecNOS-to-GAPDH ratio for each soleus feed artery. The GAPDH primers were based on the rat sequence for GAPDH and were as follows: GAPDH sense, 5'-CAT AGA CAA GAT GGT GAA GGT CGG-3'; and GAPDH antisense, 5'-GCC AAA GTT GTC ATG GAT GAC C-3'.
Immunoblot analysis. Frozen soleus feed arteries (n = 1/rat) were solubilized in 20 µl Laemmli buffer: 62.5 mM Tris, pH 6.8, 6 M urea, 160 mM 1,4-dithiothreitol, 2% SDS, and 0.001% bromophenol blue (15), boiled, and sonicated for 2 min. Cell lysates were subjected to SDS-PAGE under reducing conditions, and proteins were then transferred to polyvinylidene 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 mmol/l Tris · HCl, 137 mmol/l NaCl, and 0.1% Tween 20). The blots were incubated overnight at room temperature with primary antibody against ecNOS (1:1,600; Transduction Laboratories) followed by incubation for 1 h with secondary antibody (1:2,500; horseradish peroxidase-conjugated anti-mouse). Specific ecNOS protein was detected by enhanced chemiluminescence (ECL, Amersham) and evaluated by densitometry (National Institutes of Health Image). To allow for comparison among samples, all blots were reblocked for 1 h at room temperature and incubated overnight with a monoclonal antibody against GAPDH (1:1,600, Chemicon). ecNOS protein is expressed as arbitrary densitometric units after correction for differences in loading.
Determination of Vasodilator Responses
Preparation of arteries.
After isolation, feed arteries were transferred to a Lucite vessel
chamber containing cold (4°C) MOPS-buffered PSS (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 EDTA, and 3.0 MOPS, pH 7.4). Arteries
were cannulated on one end with a glass micropipette filled with
PSS-albumin (1 g/100 ml) solution. The artery was tied securely to the
pipette by using 11-0 opthalmic suture. The artery was flushed,
and the other end was cannulated and tied. The electrical resistances (LCR Bridge Circuit, model LCR-740, Leader Electronics) of pipettes were 150-250 k
. The resistances of pipettes were matched
(±0.5% during most experiments, but ±2.0% during some nonflow
experiments). After cannulation of arteries, the vessel bath was
transferred to the stage of an inverted microscope (Nikon Diaphot 200;
×20 or ×40 magnification; spatial resolution with either
magnification is <1 µm) coupled with a video camera (Javelin
Electronics, Los Angeles, CA), video monitor (Sony), video micrometer
(Microcirculation Research Institute, Texas A&M Univ.), and
Macintosh/MacLab data-acquisition system. Luminal diameter and pressure
were continuously monitored throughout the experiment and recorded on
the computer. The bath was gradually warmed and maintained at 37°C
for the duration of the experiment. Luminal pressure was set at 60 cmH2O initially and raised to 90 cmH2O after 30 min (1 mmHg = 1.36 cmH2O). This pressure was selected
because it is similar to the normal in vivo pressure measured in rat
soleus feed arteries by Williams and Segal (37). Micropipettes were
connected to independent reservoir systems, and pressure was measured
through sidearms connected to low-volume displacement pressure
transducers (Electromedics). Arteries were pressurized by elevating
both reservoirs to the same level. The bath solution was replaced every
15 min during the equilibration period.
Experimental protocols. The purpose of this portion of the study was to determine whether HLU (i.e., a period of chronic reduction in blood flow to the soleus muscle) would alter endothelium-mediated vasodilator responses of soleus feed arteries. Therefore, we selected an experimental protocol that utilized stimuli from three categories: a receptor-dependent, endothelium-dependent vasodilator (ACh); a receptor-independent, endothelium-dependent vasodilator (increased intraluminal flow); and an endothelium-independent vasodilator [sodium nitroprusside (SNP)]. SNP was utilized to determine whether hypothesized changes in the response to endothelium-dependent dilators resulted from changes in the endothelium or in the vascular smooth muscle. When more than one experimental intervention was performed in the same artery, the following guidelines were used to determine the order of interventions: responses to flow were examined first, followed by ACh, and last by SNP. Arteries were rinsed with fresh PSS numerous times between dose-response curves and allowed a 15- to 30-min recovery period before the next dose-response curve was initiated. In all cases, arteries reconstricted to within 10% of initial baseline diameters during the recovery period. At the end of each experiment, arteries were incubated for 1 h in calcium-free PSS containing 2.0 mM EDTA for determination of passive diameter.
The dose-response relationships to ACh (109-104
M) and SNP
(1010-104
M) were examined by cumulative addition of drug to the vessel bath.
Intraluminal flow through feed arteries was elicited by raising one
reservoir while lowering the opposite reservoir an equal distance. This
procedure has been shown to cause flow through the vessel lumen without
changes in midpoint intraluminal pressure (14). Pressure gradients of
2, 4, 6, 8, 10, 15, 20, 30, and 40 cmH2O were used to generate
increasing flow rates. Flows were calculated by using calibration
curves generated from measurements of flow made over the same pressure
gradient range with a ball flowmeter (Omega Engineering) in soleus feed
arteries and pipettes of similar size to those studied in the present
experiments (10, 11). The flowmeter was calibrated by using a perfusion
pump (model A99, Razel). The flow protocol was performed in soleus feed
arteries from 11 control and 12 HLU rats. Two feed arteries, one from a
control rat and one from a HLU rat, did not respond to the flow
protocol with any change in diameter. The endothelium of these arteries
appeared to be functional because they responded normally to ACh. These
two arteries were omitted from the data analysis for the flow-induced
dilation experiments.
Solutions and Drugs
Acetyl CoA used in the citrate synthase assay was obtained from Boehringer Mannheim. NaCl, KCl, and glucose were obtained from Fischer Scientific. Bovine serum albumin (>98% pure) was purchased from US Biochemical. All other reagents used in dose-response experiments were obtained from Sigma Chemical.Statistical Analysis
Three groups of rats were used in this study. A total of 12 control and 12 HLU rats were used in the vascular responses experiments, 6 control and 5 HLU for mRNA analysis, and 6 control and 6 HLU for determination of protein. When more than one feed artery from a rat was used in an experiment, data obtained from feed arteries of the same rat were averaged and counted as one observation. Thus n is equal to the number of rats for each experiment.Feed artery responses to various interventions were measured in actual
diameter (µm) and are presented relative to the possible change in
diameter to normalize for possible differences between groups in
beginning and maximal passive diameters. Percent possible dilation is
calculated as [(D DB)/(DP DB)] × 100, where D is the measured
diameter, DB is
the beginning diameter before the intervention was started, and
DP is the maximal
passive diameter measured after 1-h incubation in calcium-free PSS. Our
purpose was to determine whether HLU caused differences in the whole
dose-response curve, in the maximal response, or in the sensitivity as
reflected in the EC50.
Dose-response curves of the two groups were compared by using two-way
repeated-measures analysis of variance with one within comparison
(dose) and one between comparison (group), followed by Tukey's
multiple-comparison post hoc test. Student's unpaired t-tests were used when appropriate.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Characteristics of the rats, soleus muscles, and feed arteries are
shown in Table 1. The body weight of HLU
rats was slightly less than that of control rats, and the unweighting
period elicited a marked atrophy of the soleus muscle, with unweighted
soleus muscles weighing ~50% of the control muscles. Thus the soleus weight-to-body weight ratio was reduced 43% by HLU. Oxidative capacity
of the soleus muscle was also reduced by HLU. The number of soleus feed
arteries per soleus muscle was unchanged by HLU, as was the development
of spontaneous tone by feed arteries. However, the maximal passive
diameter of soleus feed arteries was lower in the HLU animals. A lower
maximal diameter with similar development of spontaneous tone meant
that arteries from HLU animals had lower baseline diameters but similar
relative diameters for the dose-response curves.
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ecNOS mRNA Expression
The effect of 14 days of HLU on the expression of ecNOS mRNA in soleus feed arteries is shown in Fig. 1. Semiquantitative PCR revealed that the ecNOS-to-GAPDH mRNA ratio was significantly lower in soleus feed arteries isolated from the HLU rats when compared with cage control rats.
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ecNOS Protein Expression
The effect of 14 days of HLU on the expression of ecNOS protein in soleus feed arteries is shown in Fig. 2. Immunoblot analysis indicated that ecNOS protein expression was significantly lower in soleus feed arteries of HLU rats than in arteries of control rats.
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Endothelium-Dependent Vasodilation
Repeated-measures ANOVA indicated that the response of feed arteries to increasing doses of ACh was not significantly different between groups (P = 0.08) (Fig. 3). However, when the maximal dilation of feed arteries to ACh was compared, these values were significantly lower in soleus feed arteries from HLU rats (control: 88 ± 5% vs. HLU: 73 ± 5%). In addition, the dilatory response to intraluminal flow was attenuated by HLU (Fig. 4). Similar to previous reports of soleus feed artery responses to flow (10, 11), feed arteries from both control and HLU rats dilated markedly to low levels of flow but did not dilate further to additional increases in flow. The dilation to low levels of flow was attenuated in arteries from HLU rats, and the plateau diameter during further increases in flow was thus lower in these arteries as well.
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Endothelium-Independent Vasodilation
The response to SNP was enhanced in feed arteries from HLU rats compared with arteries from casted control rats (Fig. 5). Although the dilation elicited by the greatest dose of SNP was similar in both groups, the EC50 of the response was less in arteries from HLU rats, indicating an increase in the sensitivity to SNP after HLU (control: 4.46 × 107 M vs. HLU: 5.00 × 108 M).
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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 hypotheses that ecNOS mRNA and protein expression are reduced and that endothelium-dependent dilation is impaired in soleus feed arteries isolated from HLU rats. We report that both ecNOS mRNA expression and ecNOS protein levels are lower in soleus feed arteries isolated from HLU rats. Furthermore, endothelial function is impaired, as evidenced by attenuated flow-induced dilation and a reduced maximal response to ACh in soleus feed arteries from HLU rats. These changes may contribute to the attenuated hyperemic response of the soleus muscle to exercise after HLU (22, 42).
In contrast to vasomotor responses to endothelium-dependent dilators, responses to the endothelium-independent dilator SNP were enhanced by HLU (Fig. 5). This indicated that the decreased response to the two endothelium-dependent stimuli in arteries from HLU rats was caused by a reduction in endothelial function rather than an attenuation of vascular smooth muscle function. The dilatory response to ACh in rat soleus feed arteries can be largely blocked by arginine analogs, indicating that the response is dependent primarily on endothelial release of nitric oxide (NO) (11). Thus the decreased maximal response to ACh taken together with enhanced responses to the NO donor SNP suggested that endothelial release of NO was attenuated by HLU. Collectively, these results indicate that flow- and ACh-stimulated release of NO from endothelial cells is attenuated by HLU and that enhanced vascular smooth muscle sensitivity to NO enables feed arteries to partially compensate for the attenuated endothelial function.
ecNOS mRNA, protein, and enzyme activity have previously been shown to be regulated by changes in flow or shear stress. Chronic increases in flow cause increases in all three variables in vivo in the rat aorta (27) and cause increased mRNA and protein levels in cultured bovine aortic cells (35). Cultured human umbilical vein endothelial cells exposed to shear stress showed increased ecNOS mRNA levels and increased promoter activation, indicating that ecNOS was transcriptionally controlled (43). Furthermore, the gene encoding human ecNOS appears to contain a shear stress response element in its 5' flanking region (23). In the present study, soleus feed arteries were chronically exposed to significantly reduced blood flow levels by HLU of the rats (22). After HLU, soleus feed arteries had reduced levels of both ecNOS mRNA and protein compared with feed arteries from control rats (Figs. 1 and 2). Although the relative rates of synthesis and degradation of ecNOS mRNA and ecNOS protein in soleus feed arteries are unknown, this evidence, when considered together with previous studies examining increases in flow, suggests that soleus feed artery ecNOS gene expression was transcriptionally downregulated. These data also indicate that basal levels of flow are needed for maintenance of normal ecNOS expression in soleus feed arteries.
Although endothelial production of NO appeared to be attenuated after HLU, feed artery sensitivity to SNP was enhanced (Fig. 5). Studies that have attempted to chronically increase blood flow by exercise training have generally reported unaltered (11, 20) or enhanced (20) responses to SNP in resistance vessels of skeletal muscle. In particular, feed arteries of the spinotrapezius muscle had enhanced responses to SNP after exercise training (20), but responses to SNP in feed arteries of the soleus muscle were unaltered by training (11). A previous study by Delp et al. (7) found no change in the sensitivity to SNP of aortic rings from HLU rats. However, in aortic rings with a maximal norepinephrine preconstriction, the maximal dilation to SNP was increased by HLU. These authors attributed these enhanced responses to increased sensitivity of the vascular smooth muscle to cGMP because dilation to 8-bromo-cGMP was also increased by HLU. The mechanism by which HLU increased sensitivity to SNP in soleus feed arteries in the present study is unknown.
Despite the enhanced smooth muscle sensitivity to NO, the response to increased intraluminal flow was attenuated in feed arteries from HLU rats (Fig. 4). This differed from the responses to ACh, in which the dose-response curves were not significantly different but only maximal ACh-induced dilation was reduced. These data suggest that some part of the flow-sensing and/or -signaling pathway and/or its coupling to ecNOS may be attenuated by HLU. Alternatively, NO may not be the major endothelial-derived relaxing factor mediating the response to flow. In arterioles of the rat cremaster, the cyclooxygenase inhibitor indomethacin did not affect the dilation to ACh but abolished flow-induced dilation (13). A similar situation may exist in soleus feed arteries in that flow-induced dilation may depend more on cyclooxygenase products than does ACh-induced dilation.
In addition to alterations in vasomotor function, soleus feed arteries also underwent structural adaptation to HLU as evidenced by the reduction in passive diameter (Table 1). McDonald et al. (22) reported that soleus blood flow was 72% lower during HLU compared with weight-bearing conditions. Reduction in blood flow for 2 wk by HLU in the present study resulted in a reduction in soleus feed artery passive diameter to 78% of that in control rats. To study the effect of reduced flow on the common carotid artery, Langille and O'Donnell (16) used rabbits in which the external carotid arteries were ligated for 2 wk. They reported that blood flow was reduced to 30% of control levels and that this treatment caused a reduction in arterial diameter to 79% of control levels. Thus our results agree very closely with theirs regarding the effect of chronically reduced flow on vessel structure. Furthermore, Chew and Segal (4) reported a reduction in passive diameter and an increase in wall thickness in the rat femoral artery after 10 wk of HLU. It is interesting that the vascular remodeling after chronic flow reduction appears to require an intact endothelium because when Langille and O'Donnell denuded arteries at the time of ligation the change in arterial diameter was abolished (16). Furthermore, this remodeling requires endothelium-derived NO because it is absent in ecNOS-knockout mice (30). Although technical limitations do not allow the measurement of feed artery diameters in conscious rats, the fact that the passive diameter of these arteries is reduced, the ability to develop spontaneous tone is not altered (Table 1), and endothelium-dependent dilator function is reduced indicates that feed artery diameter in conscious HLU rats may be reduced relative to control rats. The location of feed arteries proximal to the muscle is ideal for controlling total blood flow to individual muscles, and previous studies have demonstrated that feed arteries do in fact play an integral role in the control of skeletal muscle blood flow (17, 31, 38). Thus it is likely that alterations in both structure and function of soleus feed arteries contribute to the previously observed reduction in soleus blood flow of HLU rats (22, 42). Furthermore, a reduction in metabolic demand (33), a reduction in the capillary-to-fiber ratio of the soleus (8, 9), enhanced sympathetic nerve activity to the soleus (39), and attenuated baroreflex control of sympathetic nerve activity to hindlimb skeletal muscle (24) may all contribute to the altered distribution of blood flow elicited by HLU.
The results of this study also provide new information regarding the disparate effects of exercise training on vascular responsiveness in skeletal muscle resistance vessels (11). Although vasomotor responses of resistance vessels from some skeletal muscles are altered by exercise training (12, 18-20, 34, 36), training does not change vasomotor responses of soleus feed arteries (11). The explanation for this difference may lie in the fact that the soleus is a postural muscle with a relatively high blood flow rate even in nonexercising rats (1). This blood flow rate appears to be greater than the flow rate that causes maximal flow-induced dilation of soleus feed arteries (10). At least two factors suggest that acute (s) and long-term (h) responses to flow are closely related. First, increases in wall shear stress elicit both an immediate vasodilation (5) and a long-term increase in the expression of a number of proteins (5, 21), including ecNOS (27, 41). Second, some steps in the signaling cascade of both types of responses are shared. For example, both types of responses depend on integrin signaling and the activation of endothelial K+ channels (2, 26, 28). Thus available evidence supports the concept that both acute and longer-term endothelial cell responses to altered flow rates are elicited by similar ranges of shear stress and that, in soleus feed arteries, the signal for long-term adaptation to exercise is already saturated in nonexercising rats (10). The present data support this hypothesis by demonstrating that soleus feed arteries are capable of alterations in vasomotor control and that chronic reductions in flow result in attenuated endothelial function as well as reductions in ecNOS mRNA and protein expression.
In summary, endothelial cell expression of ecNOS mRNA and ecNOS protein was reduced by HLU. Furthermore, flow-induced dilation of rat soleus feed arteries is attenuated by HLU, and maximal dilation to ACh is also compromised. Thus both ecNOS gene expression and endothelium-mediated functional responses are decreased by HLU. In contrast, the vascular smooth muscle response to NO is enhanced by HLU. Although this enhancement does not normalize the response to endothelium-dependent agonists, it enables arteries to partially compensate for the attenuated release of endothelium-derived NO. Previously reported reductions in soleus blood flow (22, 42) after HLU may result in part from these adaptations.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the expert technical assistance of Pam Thorne, Tammy Strawn, 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-09739, HL-36088, and HL-55306.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. L. Jasperse, PO Box 10011, Dept. of Molecular Physiology and Biological Physics, Univ. of Virginia, Charlottesville, VA 22906.
Received 8 March 1999; accepted in final form 15 June 1999.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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