Impaired Coronary Endothelial Vasorelaxation in a Preclinical Model of Peripheral Arterial Insufficiency.

The present study was designed to determine whether adult swine with peripheral artery insufficiency (PAI) would exhibit vascular dysfunction in vessels distinct from the affected distal limbs, the coronary conduit arteries. Moreover, we sought to evaluate the effect of exercise training on coronary vasomotor function in PAI. Eighteen female healthy young Yucatan miniature swine were randomly assigned to either occluded exercise trained (Occl-Ex, n=7), or occluded-sedentary (Occl-Sed, n=5), or non-occluded, non-exercised control (Non-Occl-Con, n=6) groups. Occl-Ex pigs were progressively trained by running on a treadmill (5days/week, 12 weeks). The left descending artery (LAD) and left circumflex (LCX) coronary arteries were harvested. Vasorelaxation to adenosine diphosphate (ADP), bradykinin (BK), and sodium nitro-prusside (SNP) were assessed in LAD's; while constrictor responses to phenylephrine (PE), angiotensin II (Ang II), and endothelin-1 (ET-1) were assessed in LCX's. Vasorelaxation to ADP was reduced in LADs from Occl-Sed and Occl-Ex pigs (P<0.001) as compared to Non-Occl-Con pigs; however, Occl-Ex pigs exhibited partial recovery (P<0.001) intermediate to the other two groups. BK induced relaxation was reduced in LADs from Occl-Ex and Occl-Sed pigs (P<0.001), compared to Non-Occl-Con, and exercise modestly increased responses to BK (P<0.05). In addition, SNP, PE, Ang II, and ET-1 responses were not significantly different among the groups. Our results indicate that 'simple' occlusion of the femoral arteries induces vascular dysfunction in conduit vessels distinct from the affected hindlimbs, as evident in blunted coronary vasorelaxation responses to ADP and BK. These findings imply that PAI, even in the absence of frank atherogenic vascular disease, contributes to vascular dysfunction in the coronary arteries that could exacerbate disease outcome in patients with peripheral artery disease. Further, regular daily physical activity partially recovered the deficit observed in the coronary arteries.

The effect of purinergic P2 receptor blockade on skeletal muscle exercise hyperemia in miniature swine.

PURPOSE: ATP could play an important role in skeletal muscle blood flow regulation by inducing vasodilation via purinergic P2 receptors. This study investigated the role of P2 receptors in exercise hyperemia in miniature swine. METHODS: We measured regional blood flow with radiolabeled-microsphere technique and systemic hemodynamics before and after arterial infusion of the P2 receptor antagonist reactive blue 2 during treadmill exercise (5.2 km/h, ~60 % VO2max) and arterial ATP infusion in female Yucatan miniature swine (~29 kg). RESULTS: Mean blood flow during exercise from the 16 sampled skeletal muscle tissues was 138 ± 18 mL/min/100 g (mean ± SEM), and it was reduced in 11 (~25 %) of the 16 sampled skeletal muscles after RB2 was infused. RB2 also lowered diaphragm blood flow and kidney blood flow, whereas lung tissue blood flow was increased (all P < 0.05). Infusion of RB2 increased arterial lactate concentration during exercise from 1.6 ± 0.5 to 3.4 ± 0.6 mmol/L and heart rate from 216 ± 12 to 230 ± 9 beats/min, whereas blood pressure was unaltered. Arterial ATP infusion caused a ~twofold increase in blood flow in 15 of the 16 sampled muscle tissues and this effect was abolished after RB2 infusion. CONCLUSIONS: These results indicate that P2 receptors play a role in regulating skeletal muscle blood flow during exercise in miniature swine.

Acute impact of intermittent pneumatic leg compression frequency on limb hemodynamics, vascular function, and skeletal muscle gene expression in humans.

The mechanisms by which intermittent pneumatic leg compression (IPC) treatment effectively treats symptoms associated with peripheral artery disease remain speculative. With the aim of gaining mechanistic insight into IPC treatment, the purpose of this study was to investigate the effect of IPC frequency on limb hemodynamics, vascular function, and skeletal muscle gene expression. In this two study investigation, healthy male subjects underwent an hour of either high-frequency (HF; 2-s inflation/3-s deflation) or low-frequency (LF; 4-s inflation/16-s deflation) IPC treatment of the foot and calf. In study 1 (n = 11; 23.5 ± 4.7 yr), subjects underwent both HF and LF treatment on separate days. Doppler/ultrasonography was used to measure popliteal artery diameter and blood velocity at baseline and during IPC treatment. Flow-mediated dilation (FMD) and peak reactive hyperemia blood flow (RHBF) were determined before and after IPC treatment. In study 2 (n = 19; 22.0 ± 4.6 yr), skeletal muscle biopsies were taken from the lateral gastrocnemius of the treated and control limb at baseline and at 30- and 150-min posttreatment. Quantitative PCR was used to assess mRNA concentrations of genes associated with inflammation and vascular remodeling. No treatment effect on vascular function was observed. Cuff deflation resulted in increased blood flow (BF) and shear rate (SR) in both treatments at the onset of treatment compared with baseline (P < 0.01). BF and SR significantly diminished by 45 min of HF treatment only (P < 0.01). Both treatments reduced BF and SR and elevated oscillatory shear index compared with baseline (P < 0.01) during cuff inflation. IPC decreased the mRNA expression of cysteine-rich protein 61 from baseline and controls (P <0 .01) and connective tissue growth factor from baseline (P < 0.05) in a frequency-dependent manner. In conclusion, a single session of IPC acutely impacts limb hemodynamics and skeletal muscle gene expression in a frequency-dependent manner but does not impact vascular function.

Intermittent pneumatic leg compressions enhance muscle performance and blood flow in a model of peripheral arterial insufficiency.

Despite the escalating prevalence in the aging population, few therapeutic options exist to treat patients with peripheral arterial disease. Application of intermittent pneumatic leg compressions (IPC) is regarded as a promising noninvasive approach to treat this condition, but the clinical efficacy, as well the mechanistic basis of action of this therapy, remain poorly defined. We tested the hypothesis that 2 wk of daily application of IPC enhances exercise tolerance by improving blood flow and promoting angiogenesis in skeletal muscle in a model of peripheral arterial insufficiency. Male Sprague-Dawley rats were subjected to bilateral ligation of the femoral artery and randomly allocated to treatment or sham groups. Animals were anesthetized daily and exposed to 1-h sessions of bilateral IPC or sham treatment for 14-16 consecutive days. A third group of nonligated rats was also studied. Marked increases in treadmill exercise tolerance (∼33%, P < 0.05) and improved muscle performance in situ (∼10%, P < 0.05) were observed in IPC-treated animals. Compared with sham-treated controls, blood flow measured with isotope-labeled microspheres during in situ contractions tended to be higher in IPC-treated animals in muscles composed of predominantly fast-twitch white fibers, such as the plantaris (∼93%, P = 0.02). Capillary contacts per fiber and citrate synthase activity were not significantly altered by IPC treatment. Collectively, these data indicate that IPC improves exercise tolerance in a model of peripheral arterial insufficiency in part by enhancing blood flow to collateral-dependent tissues.

Exercise training improves vasoreactivity in the knee artery.

Physical activity has been shown to enhance endothelial function of central and peripheral vascular beds. The primary purpose of the present study was to test the hypothesis that a short-term exercise training program would result in enhanced endothelium-dependent vasorelaxation of a major artery supplying blood flow to the knee joint, the middle genicular artery. Female Yucatan miniature swine were randomly assigned into exercise trained (n=7) or sedentary (n=7) groups. Exercise trained pigs underwent a daily exercise training program on treadmills for 7 days. In vitro assessment of vasorelaxation was determined in a dose response manner by administrating increasing doses of 3 different dilators; adenosine diphosphate, bradykinin, and sodium nitroprusside. The role of nitric oxide synthase and cyclooxygenase pathways in vasomotor responses was evaluated with specific inhibitors using nitro-L-arginine methyl ester and indomethacin incubation, respectively. The results of this investigation indicate that adenosine and bradykinin-induced endothelium-dependent vasorelaxation were significantly enhanced in middle genicular artery from exercise trained pigs (p<0.05). Endothelium-independent vasorelaxation was not altered with exercise training as determined by the response to sodium nitroprusside. The findings of the present investigation indicate that short-term exercise training enhances endothelial function of middle genicular artery through adaptations in the nitric oxide synthase and by non-nitric oxide synthase, non-cyclooxygenase pathways.

Impact of a single session of intermittent pneumatic leg compressions on skeletal muscle and isolated artery gene expression in rats.

Intermittent pneumatic leg compressions (IPC) have proven to be an effective noninvasive approach for treatment of patients with claudication, but the mechanisms underlying the clinical benefits remain elusive. In the present study, a rodent model of claudication produced by bilateral ligation of the femoral artery was used to investigate the acute impact of a single session of IPC (150 min) on hemodynamics, skeletal muscle (tibialis anterior), and isolated collateral artery (perforating artery) expression of a subset of genes associated with inflammation and vascular remodeling. In addition, the effect of compression frequency (15 vs. 3 compressions/min) on the expression of these factors was studied. In ligated animals, IPC evoked an increase of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant 1 (CXCL1) mRNA (P < 0.01) and immunostaining (P < 0.05), as well as a minor increase in VEGF immunostaining in the muscle endomysium 150 min postintervention. Further, collateral arteries from these animals showed an increased expression of MCP-1 (approximately twofold, P = 0.02). These effects were most evident in the group exposed to the high-frequency protocol (15 compressions/min). In contrast, IPC in sham-operated control animals evoked a modest initial upregulation of VEGF (P = 0.01), MCP-1 (P = 0.02), and CXCL1 (P = 0.03) mRNA in the muscle without concomitant changes in protein levels. No changes in gene expression were observed in arteries isolated from sham animals. In conclusion, IPC acutely up-regulates the expression of important factors involved in vascular remodeling in the compressed muscle and collateral arteries in a model of hindlimb ischemia. These effects appear to be dependent on the compression frequency, such that a high compression frequency (15 compressions/min) evokes more consistent and robust effects compared with the frequency commonly employed clinically to treat patients with claudication (3 compressions/min).

Impact of chronic intermittent external compressions on forearm blood flow capacity in humans.

During dynamic exercise, the vasculature embedded within skeletal muscle intermittently collapses due to increased intramuscular pressure (IMP). The aim of this study was to ascertain whether oscillations in IMP during muscle contractions independently contribute to exercise training-induced increases in blood flow capacity (BFC). Based on IMP measurements during handgrip exercise, we attempted to mimic the action of repeated vascular compressions by using external inflatable cuffs. Thus, 24 healthy young male subjects underwent a 4-week program (5 days/week, 1 h/day) of application of external compressions of the non-dominant forearm, while the dominant limb served as an internal control. To evaluate the impact of compression pressures of different magnitudes, subjects were randomly assigned to one of three groups: 50, 100 and 150 mmHg of external compression. Prior to the intervention and after 2 and 4 weeks of treatment, we measured peak forearm blood flow (PBF) (Doppler ultrasound) and calculated peak vascular conductance (PVC) following 10 min of forearm ischemia. In the 50 and 100 mmHg groups, application of intermittent compressions did not alter PBF in either control or intervention forearms. In the 150 mmHg group, there was a trend (P = 0.04) for greater increases in PBF from baseline after 4 weeks in the intervention forearm compared to the control forearm (delta PBF: 4.2 ± 2.5 vs. -2.1 ± 2.0 (ml(100 ml)(-1) min(-1)), in the intervention and control forearms, respectively), but the changes in PVC were not significant (P = 0.1). These findings suggest that repeated oscillations in IMP contribute minimally to exercise-induced increase in forearm BFC in healthy young humans.

Vascular endothelium-specific overexpression of human catalase in cloned pigs.

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

Effects of chronic nitric oxide synthase inhibition on endothelium-dependent and -independent relaxation in arteries that perfuse skeletal muscle of swine.

The purpose of this investigation was to test the hypothesis that chronic N(G)-nitro-l-arginine methyl ester (l-NAME) treatment produces differential effects on conduit artery and resistance arteriole relaxation responses to endothelium-dependent and -independent vasodilators in arteries that perfuse skeletal muscle of swine. To test this hypothesis, conduit skeletal muscle arteries and second-order skeletal muscle (2A) arterioles were harvested from 14 Yucatan swine that were chronically administered l-NAME and from 16 controls. In vitro assessments of vasorelaxation to increasing doses of acetylcholine (ACH), bradykinin (BK), and sodium nitroprusside (SNP) were performed in both conduit and 2A arterioles. l-NAME treatment produced a significant reduction in both BK and ACH relaxation responses in the conduit arteries. In contrast, the relaxation response and/or sensitivity to SNP were significantly greater in the intact, but not denuded, conduit arterial rings from chronically l-NAME-treated swine. There were no significant effects of chronic l-NAME treatment on vasodilation of skeletal muscle arterioles. These findings suggest (1) that unlike arterioles, skeletal muscle conduit arteries do not functionally compensate for a lack of NO through the upregulation of alternative vasodilator pathways; (2) that the greater relaxation response in conduit arteries of chronically l-NAME-treated swine to SNP can be explained by alterations to the endothelium.

Effects of posture on shear rates in human brachial and superficial femoral arteries.

Shear rate is significantly lower in the superficial femoral compared with the brachial artery in the supine posture. The relative shear rates in these arteries of subjects in the upright posture (seated and/or standing) are unknown. The purpose of this investigation was to test the hypothesis that upright posture (seated and/or standing) would produce greater shear rates in the superficial femoral compared with the brachial artery. To test this hypothesis, Doppler ultrasound was used to measure mean blood velocity (MBV) and diameter in the brachial and superficial femoral arteries of 21 healthy subjects after being in the supine, seated, and standing postures for 10 min. MBV was significantly higher in the brachial compared with the superficial femoral artery during upright postures. Superficial femoral artery diameter was significantly larger than brachial artery diameter. However, posture had no significant effect on either brachial or superficial femoral artery diameter. The calculated shear rate was significantly greater in the brachial (73 +/- 5, 91 +/- 11, and 97 +/- 13 s(-1)) compared with the superficial femoral (53 +/- 4, 39 +/- 77, and 44 +/- 5 s(-1)) artery in the supine, seated, and standing postures, respectively. Contrary to our hypothesis, our current findings indicate that mean shear rate is lower in the superficial femoral compared with the brachial artery in the supine, seated, and standing postures. These findings of lower shear rates in the superficial femoral artery may be one mechanism for the higher propensity for atherosclerosis in the arteries of the leg than of the arm.

Training-induced vascular adaptations to ischemic muscle.

Peripheral arterial insufficiency is a progressive degenerative disease associated with an increased morbidity and mortality. It decreases exercise tolerance and often presents with symptoms of intermittent claudication. Enhanced physical activity is one of the most effective means of improving the life of affected patients. While this occurs for a variety of reasons, vascular remodeling can be an important means for improved oxygen exchange and blood flow delivery. Relevant exercise-induced signals stimulate angiogenesis, within the active muscle (e.g. hypoxia), and arteriogenesis (enlargement of pre-existing vessels via increased shear stress) to increase oxygen exchange and blood flow capacity, respectively. Evidence from pre-clinical studies shows that the increase in collateral blood flow observed with exercise progresses over time of training, is accompanied by significant enlargement of isolated collateral vessels, and enhances the responses observed with angiogenic growth factors (e.g. VEGF, FGF-2). Thus, enhanced physical activity can be an effective means of enlarging the structure and function of the collateral circuit. Interestingly, disrupting normal NO production (via L-NAME) eliminates this increase in collateral blood flow induced by training, but does not disturb the increase in muscle capillarity within the active muscle. Similarly, inhibiting VEGF receptor kinase activity eliminates the increase in collateral-dependent blood flow, and lessens, but does not eliminate, angiogenesis within the calf muscle. These findings illustrate distinctions between the processes influencing angiogenesis and arteriogenesis. Further, sympathetic modulation of the collateral circuit does not eliminate the increase in collateral circuit conductance induced by exercise training. These findings indicate that structural enlargement of the collateral vessels is essential to realize the increase in collateral-dependent blood flow capacity caused by exercise training. This raises the potential that meaningful vascular remodeling can occur in patients with intermittent claudication who actively participate in exercise training.

Mechanisms for exercise training-induced increases in skeletal muscle blood flow capacity: differences with interval sprint training versus aerobic endurance training.

Skeletal muscle blood flow capacity (BFC) is increased by exercise training due to structural vascular remodeling (in the form of angiogenesis of capillaries and remodeling of the arterial tree within skeletal muscle) and/or altered control of vascular resistance. Changes in control can be central or the result of changes in reactivity of arteries and arterioles (due to changes in vascular smooth muscle and/or endothelium). The purpose of this review is to evaluate the relative importance of these mechanisms for increased BFC following interval sprint training (IST) and endurance exercise training (ET). Based on the results discussed herein we conclude that the importance of each of these mechanisms varies throughout muscle tissue due to interactions of muscle fiber-type composition and muscle fiber recruitment patterns during exercise. The distribution of vascular adaptive changes varies with mode of training. For example, IST has been shown to produce the greatest relative increase in contractile activity in fast-twitch, white, skeletal muscle (i.e. white gastrocnemius muscle (Gw) and Gw muscle exhibits the largest increase in oxidative capacity, capillary density, BFC, and changes in vascular cells with IST. In contrast, ET has been shown to produce the greatest relative increase in contractile activity in red gastrocnemius muscle (Gr), and Gr muscle exhibits the largest increase in oxidative capacity, capillary density, and BFC after ET training. Results demonstrate that the increases in BFC are not mediated solely by structural adaptation. Rather, changes in vascular control predominate in Gr and soleus muscle, while increases in arteriolar and capillary density predominate following IST in Gw. Finally, evidence indicates that ET and IST induce non-uniform changes in smooth muscle and endothelium throughout skeletal muscle arteriolar networks.

Production of endothelial nitric oxide synthase (eNOS) over-expressing piglets.

Vascular function, vascular structure, and homeostasis are thought to be regulated in part by nitric oxide (NO) released by endothelial cell nitric oxide synthase (eNOS), and NO released by eNOS plays an important role in modulating metabolism of skeletal and cardiac muscle in health and disease. The pig is an optimal model for human diseases because of the large number of important similarities between the genomic, metabolic and cardiovascular systems of pigs and humans. To gain a better understanding of cardiovascular regulation by eNOS we produced pigs carrying an endogenous eNOS gene driven by a Tie-2 promoter and tagged with a V5 His tag. Nuclear transfer was conducted to create these animals and the effects of two different oocyte activation treatments and two different culture systems were examined. Donor cells were electrically fused to the recipient oocytes. Electrical fusion/activation (1 mM calcium in mannitol: Treatment 1) and electrical fusion (0.1 mM calcium in mannitol)/chemical activation (200 microM Thimerosal for 10 min followed by 8 mM DTT for 30 min: Treatment 2) were used. Embryos were surgically transferred to the oviducts of gilts that exhibited estrus on the day of fusion or the day of transfer. Two cloned transgenic piglets were born from Treatment 1 and low oxygen, and another two from Treatment 2 and normal oxygen. PCR, RT-PCR, Western blotting and immunohistochemistry confirmed that the pigs were transgenic, made message, made the fusion protein and that the fusion protein localized to the endothelial cells of placental vasculature from the conceptuses as did the endogenous eNOS. Thus both activation conditions and culture systems are compatible with development to term. These pigs will serve as the founders for a colony of miniature pigs that will help to elucidate the function of eNOS in regulating muscle metabolism and the cardiorespiratory system.

Nonuniform effects of endurance exercise training on vasodilation in rat skeletal muscle.

Endurance exercise training (Ex) has been shown to increase maximal skeletal muscle blood flow. The purpose of this study was to test the hypothesis that increased endothelium-dependent vasodilation is associated with the Ex-induced increase in muscle blood flow. Furthermore, we hypothesized that enhanced endothelium-dependent dilation is confined to vessels in high-oxidative muscles that are recruited during Ex. To test these hypotheses, sedentary (Sed) and rats that underwent Ex (30 m/min x 10% grade, 60 min/day, 5 days/wk, 8-12 wk) were studied using three experimental approaches. Training effectiveness was evidenced by increased citrate synthase activity in soleus and vastus lateralis (red section) muscles (P < 0.05). Vasodilatory responses to the endothelium-dependent agent acetylcholine (ACh) in situ tended to be augmented by training in the red section of gastrocnemius muscle (RG; Sed: control, 0.69 +/- 0.12; ACh, 1.25 +/- 0.15; Ex: control, 0.86 +/- 0.17; ACh, 1.76 +/- 0.27 ml x min(-1) x 100 g(-1) x mmHg(-1); 0.05 < P < 0.10 for Ex vs. Sed during ACh). Responses to ACh in situ did not differ between Sed and Ex for either the soleus muscle or white section of gastrocnemius muscle (WG). Dilatory responses of second-order arterioles from the RG in vitro to flow (4-8 microl/min) and sodium nitroprusside (SNP; 10(-7) through 10(-4) M), but not ACh, were augmented in Ex (vs. Sed; P < 0.05). Dilatory responses to ACh, flow, and SNP of arterioles from soleus and WG muscles did not differ between Sed and Ex. Content of the endothelial isoform of nitric oxide synthase (eNOS) was increased in second-order, fourth-order, and fifth-order arterioles from the RG of Ex; eNOS content was similar between Sed and Ex in vessels from the soleus and WG muscles. These findings indicate that Ex induces endothelial adaptations in fast-twitch, oxidative, glycolytic skeletal muscle. These adaptations may contribute to enhanced skeletal muscle blood flow in endurance-trained individuals.

Alterations in PKC signaling underlie enhanced myogenic tone in exercise-trained porcine coronary resistance arteries.

The intracellular mechanisms underlying enhanced myogenic contraction (MC) in coronary resistance arteries (CRAs) from exercise-trained (EX) pigs have not been established. The purpose of this study was to test the hypothesis that exercise-induced alterations in protein kinase C (PKC) signaling underlie enhanced MC. Furthermore, we sought to determine whether modulation of intracellular Ca(2+) signaling by PKC underlies enhanced MC in EX animals. Male Yucatan miniature swine were treadmill trained (n = 7) at approximately 75% of maximal O(2) uptake for 16 wk (6 miles/h, 60 min) or remained sedentary (SED, n = 6). Diameter measurements in response to intraluminal pressure (60, 75, and 90 cmH(2)O) or 60 mM KCl were determined in single, cannulated CRAs ( approximately 100 microm ID) with and without the PKC inhibitor chelerythrine (CE, 1 microM). Confocal imaging of Ca(2+) signaling [myogenic Ca(2+) (Ca(m))] was also performed in CRAs of similar internal diameter after abluminal loading of the Ca(2+) indicator dye fluo 4 (1 microM, 37 degrees C, 30 min). We observed significantly greater MC in CRAs isolated from EX than from SED animals at 90 cmH(2)O, as well as greater reductions in MC after CE at all pressures studied. At intraluminal pressures of 75 and 90 cmH(2)O, CE produced greater decreases in Ca(m) in CRAs from EX than from SED animals (64% vs. 25%, P < 0.05). Inhibition of KCl constriction and Ca(m) by CE was also greater in EX animals (P < 0.05). Western blotting revealed significant increases in Ca(2+)-dependent PKC-alpha ( approximately 50%) but not Ca(2+)-independent PKC-epsilon levels in CRAs isolated from EX animals (P < 0.05). We also observed significant group differences in phosphorylated PKC-alpha levels. Finally, voltage-gated Ca(2+) current (VGCC) was effectively blocked by CE, bisindolylmaleimide, and staurosporine in isolated smooth muscle cells from CRAs, providing evidence for a mechanistic link between VGCCs and PKC in our experimental paradigm. These results suggest that enhanced MC in CRAs from EX animals involves PKC-dependent modulation of intracellular Ca(2+), including regulation of VGCCs.

Interval sprint training enhances endothelial function and eNOS content in some arteries that perfuse white gastrocnemius muscle.

The purpose of this study was to test the hypothesis that interval sprint training (IST) selectively increases endothelium-dependent dilation (EDD) and endothelial nitric oxide synthase and/or superoxide dismutase-1 protein content in arteries and/or arterioles that perfuse the white portion of rat gastrocnemius muscle (WG). Male Sprague-Dawley rats completed 10 wk of IST (n = 62) or remained sedentary (Sed) (n = 63). IST rats performed six 2.5-min exercise bouts, with 4.5 min of rest between bouts (60 m/min, 15% incline), 5 days/wk. EDD was assessed from acetylcholine (ACh)-induced increases in muscle blood flow measured in situ and by ACh-induced dilation of arteries and arterioles [first to third order (1A-3A)] that perfuse red gastrocnemius muscle (RG) and WG. Artery protein content was determined with immunoblot analysis. ACh-induced increases in blood flow were enhanced in WG of IST rats. eNOS content was increased in conduit arteries, gastrocnemius feed artery, and fourth-order arterioles from WG and fifth-order arterioles of RG but not in 2As from RG. EDD was examined in 2As and 3As from a subset of IST and Sed rats. Arterioles were canulated with micropipettes, and intraluminal pressure was set at 60 cmH2O. Results indicate that passive diameter (measured in 0 calcium PSS) of WG 2As was similar in IST and Sed, whereas diameter of WG 3As was greater in IST (96 +/- 8 microm) than Sed (73 +/- 4 microm). WG 2As and 3As of IST rats exhibited greater spontaneous tone, but sensitivity to stretch, phenylephrine, and sodium nitroprusside was similar to Sed arterioles. ACh-induced dilation was enhanced by IST in WG 2As but not in RG 2As or WG 3As. We conclude that IST induces vascular adaptations nonuniformly among arteries that perfuse WG muscle.

Arteriogenesis: role of nitric oxide.

Arteriogenesis is an important process for adapting the pre-existing circuit of vessels into functional collateral conduits for delivery of oxygen enriched blood to tissue distal to occlusion of a large, peripheral conduit artery. Recent evidence has shown that arteriogenesis is regulated by nitric oxide (NO), angiogenic factors and shear stress. NO significantly impacts vasomotor tone to enhance conductance of the newly recruited collateral arteries, and this effect is augmented by exercise training prior to arterial occlusion. NO-mediated increases in vascular conductance allows for greater collateral dependent blood flow to the tissue distal to occlusion. NO production is also critical to the efficacy of therapeutic arteriogenesis achieved by delivery of exogenous angiogenic growth factors (VEGF, FGF-2) or by exercise training. The critical role of NO in therapeutic arteriogenesis is independent of NO-mediated changes in vascular conductance and implies a central role in arteriogenic signaling events. Maintenance, or improvement, of NO production and signaling, such as with regular exercise, may improve endothelial cell function and thus may help preserve the arteriogenic potential of preexisting collateral networks.

Influence of coronary artery diameter on eNOS protein content.

The purpose of this study was to test the hypothesis that the content of endothelial nitric oxide synthase (eNOS) protein (eNOS protein/g total artery protein) increases with decreasing artery diameter in the coronary arterial tree. Content of eNOS protein was determined in porcine coronary arteries with immunoblot analysis. Arteries were isolated in six size categories from each heart: large arteries [301- to 2,500-microm internal diameter (ID)], small arteries (201- to 300-microm ID), resistance arteries (151- to 200-microm ID), large arterioles (101- to 150-microm ID), intermediate arterioles (51- to 100-microm ID), and small arterioles(<50-microm ID). To obtain sufficient protein for analysis from small- and intermediate-sized arterioles, five to seven arterioles 1-2 mm in length were pooled into one sample for each animal. Results establish that the number of smooth muscle cells per endothelial cell decreases from a number of 10 to 15 in large coronary arteries to 1 in the smallest arterioles. Immunohistochemistry revealed that eNOS is located only in endothelial cells in all sizes of coronary artery and in coronary capillaries. Contrary to our hypothesis, eNOS protein content did not increase with decreasing size of coronary artery. Indeed, the smallest coronary arterioles had less eNOS protein per gram of total protein than the large coronary arteries. These results indicate that eNOS protein content is greater in the endothelial cells of conduit arteries, resistance arteries, and large arterioles than in small coronary arterioles.

Differential adenosine sensitivity of diaphragm and skeletal muscle arterioles.

The hyperemic response in exercising skeletal muscle is dependent on muscle fiber-type composition and fiber recruitment patterns, but the vascular control mechanisms producing exercise hyperemia in skeletal muscle remain poorly understood. The purpose of this study was to test the hypothesis that arterioles from white, low-oxidative skeletal muscle are less responsive to adenosine-induced dilation than are arterioles from diaphragm (Dia) and red, high-oxidative skeletal muscle. Second-order arterioles (2As) were isolated from the white portion of gastrocnemius muscle (WG; low-oxidative, fast-twitch muscle tissue) and two types of high-oxidative skeletal muscle [Dia and red portion of gastrocnemius muscle (RG)] of rats. Results reveal that 2As from all three types of muscle dilated in response to the endothelium-dependent dilator acetylcholine (WG: 48 +/- 3%, Dia: 51 +/- 3%, RG: 74 +/- 3%). In contrast, adenosine dilated only 2As from WG (48 +/- 4%) and Dia (46 +/- 5%) but not those from RG (5 +/- 5%). Thus adenosine-induced dilator responses differed among 2As of these different types of muscle tissue. However, the results do not support our hypothesis because 2As from Dia and WG dilated in response to adenosine, whereas 2As from RG did not. We conclude that the adenosine responsiveness of 2As from rat skeletal muscle cannot be predicted only by the fiber-type composition or oxidative capacity of the skeletal muscle tissue wherein the arteriole lies.