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Am J Physiol Regul Integr Comp Physiol. Jun 2009; 296(6): R1771–R1782.
Published online Apr 22, 2009. doi:  10.1152/ajpregu.00096.2009
PMCID: PMC2692785

Integration of skeletal muscle resistance arteriolar reactivity for perfusion responses in the metabolic syndrome

Abstract

Previous study suggests that with evolution of the metabolic syndrome, patterns of arteriolar reactivity are profoundly altered and may constrain functional hyperemia. This study investigated interactions between parameters of vascular reactivity at two levels of resistance arterioles in obese Zucker rats (OZR), translating these observations into perfusion regulation for in situ skeletal muscle. Dilation of isolated and in situ resistance arterioles from OZR to acetylcholine, arachidonic acid (AA), and hypoxia (isolated arterioles only) were blunted vs. lean Zucker rats (LZR), although dilation to adenosine was intact. Increased adrenergic tone (phenylephrine) or intralumenal pressure (ILP) impaired dilation in both strains (OZR>LZR). Treatment of OZR arterioles with Tempol (superoxide dismutase mimetic) or SQ-29548 (prostaglandin H2/thromboxane A2 receptor antagonist) improved dilator reactivity under control conditions and with increased ILP, but had minimal effect with increased adrenergic tone. Arteriolar dilation to adenosine was well maintained in both strains under all conditions. For in situ cremasteric arterioles, muscle contraction-induced elevations in metabolic demand elicited arteriolar dilations and hyperemic responses that were blunted in OZR vs. LZR, although distal parallel arterioles were characterized by heterogeneous dilator and perfusion responses. α-Adrenoreceptor blockade improved outcomes at rest but had minimal effect with elevated metabolic demand. Treatment with Tempol or SQ-29548 had minimal impact at rest, but lessened distal arteriolar perfusion heterogeneity with increased metabolic demand. In blood-perfused gastrocnemius of OZR, perfusion was constrained primarily by adrenergic tone, while myogenic activation and endothelium-dependent dilation did not appear to contribute significantly to ischemia. These results of this novel, integrated approach suggest that adrenergic tone and metabolic dilation are robust determinants of bulk perfusion to skeletal muscle of OZR, while endothelial dysfunction may more strongly regulate perfusion distribution homogeneity via the impact of oxidant stress and AA metabolism.

Keywords: peripheral vascular disease, microcirculation, regional blood flow, obesity

epidemiological studies have clearly demonstrated that the prevalence of obesity (9), impaired glycemic control (21), dyslipidemia (4), and hypertension (30) is continuing to increase in Western society and in most developed economies worldwide (24). While these systemic pathologies have been well documented to increase risk for the progressive evolution of peripheral vascular disease, these conditions are frequently present as comorbidities, leading to the genesis of a combined pathological state termed the “metabolic syndrome.” Given the impact of peripheral vascular disease on mortality and quality of life (19), psychosocial health (7), and direct (health care) and indirect (lost productivity) economic costs to society (19), considerable emphasis has been placed on the study of peripheral vascular disease in animal models of the metabolic syndrome. As one of the critical hallmarks of peripheral vascular disease in afflicted individuals is an evolving ischemia within skeletal muscle, an integrated understanding of the regulation of arteriolar tone and resistance across levels of metabolic demand is critical.

The obese Zucker rat (OZR; fa/fa) is an animal model of the metabolic syndrome that, due to its characteristic dysfunctional leptin receptor gene, experiences a chronic hyperphagia and rapidly becomes obese compared with its control strain, the lean Zucker rat (LZR; 5). With continued hyperphagia, OZR become progressively insulin-resistant, dyslipidemic (hypercholesterolemia and severe hypertriglyceridemia) and moderately hypertensive (5, 18). As such, the OZR represents a complete model of the metabolic syndrome and, owing to the origin in a chronic excess caloric intake, an appropriate and relevant model for the human condition.

Previous study from our laboratory and by others has demonstrated that multiple relevant pathways of both dilator and constrictor reactivity are altered in OZR with development of the metabolic syndrome, and this can contribute to an impaired ability to effectively match skeletal muscle perfusion with metabolic demand (10, 11, 34, 35, 36). Among these impacted pathways are those of endothelium-dependent origin, such as wall shear rate (3, 15), reduced Po2 (16, 17), and an array of pharmacological challenges, including acetylcholine (15) and arachidonic acid (17, 3537). From the perspective of constrictor reactivity, previous studies suggest that adrenergic constriction (10, 20) or signaling mechanisms contributing to adrenergic constriction (26, 32) may be enhanced in arterioles of OZR, as well as myogenic (i.e., pressure-induced) activation (13) and responses to thromboxane A2 (17, 35, 36). However, despite the presence of this growing body of knowledge, how these myriad stimuli interact to determine vessel dimension, and ultimately tissue perfusion, remains completely unknown. The purposes of the present study were to determine the integration of multiple relevant pathways for dilator and constrictor reactivity in the establishment of arteriolar tone (in vitro) and in situ vascular resistance/functional hyperemia in both cremaster and gastrocnemius muscles as a means for determining the most complete picture possible of how development of the metabolic syndrome can negatively impact skeletal muscle perfusion in OZR. Additionally, the role of elevated oxidant stress and alterations to arachidonic acid metabolism were factored into these analyses.

MATERIALS AND METHODS

Animals.

Male lean and obese Zucker rats (Harlan) fed standard chow and drinking water ad libitum were housed in the animal care facility at either the West Virginia University Health Sciences Center or the Medical College of Wisconsin, and all protocols received prior IACUC approval. At ~17 wk of age, rats were anesthetized with injections of pentobarbital sodium (50 mg/kg ip) and received tracheal intubation to facilitate maintenance of a patent airway. In all rats, a carotid artery and an external jugular vein were cannulated for determination of arterial pressure and for infusion of supplemental anesthetic and pharmacological agents, as necessary. Blood samples were drawn from the venous cannula for determination of glucose and insulin concentrations (Linco) as well as cholesterol/triglyceride levels (Waco) and nitrotyrosine (Oxis).

Preparation of isolated skeletal muscle resistance arterioles.

In all rats, the intramuscular continuation of the gracilis artery was removed and cannulated (14), and the vessel was equilibrated in a physiological salt solution at 80% of the animal's mean arterial pressure (22). Arterioles were divided into one of four major groups, those where vascular reactivity was evaluated in response to: 1) increasing concentrations of acetylcholine (10−10 M–10−6 M), 2) increasing concentrations of arachidonic acid (10−10–10−6 M), 3) reductions in superfusate/perfusate oxygen concentration (21, 15, 10, 5, or 0% O2; balance N2), and 4) increasing concentrations of adenosine (10−10 M–10−6 M). For initial experiments, arteriolar responses to the dilator stimuli were conducted under control conditions and during imposed conditions: 1) increased phenylephrine concentration (10−10 M–10−7 M to simulate increasing adrenergic tone) and 2) altered intralumenal pressure (from −20 mmHg to +40 mmHg from equilibration pressure, in 10 mmHg increments to simulate myogenic activation). Subsequently, arterioles were treated with the superoxide dismutase mimetic Tempol (10−4 M) or the antagonist for the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor SQ-29548 (10−5 M) to assess the contribution of vascular oxidant stress and alterations to arachidonic acid metabolism. At the conclusion of all experiments involving isolated arterioles, arteriolar wall mechanics were assessed under Ca2+-free conditions as described previously (2, 12).

Preparation of in situ blood-perfused hindlimb.

The left hindlimb of each animal was isolated in situ (11, 12), and the sciatic nerve was tied into a stimulating electrode. A microcirculation flow probe (Transonic 0.5/0.7 PS) was placed on the femoral artery to monitor muscle perfusion, and heparin (1500 IU/kg) was infused via the jugular vein to prevent blood coagulation. In individual experiments, rats received intravenous infusion of the α12 adrenoreceptor antagonist phentolamine (10 mg/kg), Tempol (50 mg/kg), or SQ-29548 (10 mg/kg). Effectiveness of these interventions was assessed by monitoring the changes in arterial pressure in response to intravenous infusion of phenylephrine (10 μg/kg), methacholine (10 μg/kg), or U-46619 (10 μg/kg), respectively.

Upon completion of the surgical preparation, the gastrocnemius muscle was allowed 30 min of self-perfused rest and was then stimulated via the sciatic nerve to perform bouts of isometric twitch contractions (1 or 3 Hz, 0.4 ms duration, 5V) lasting for 3 min, with continuous monitoring of arterial pressure and femoral artery blood flow. At the conclusion of these procedures, the muscle was removed, cleared of nonmuscular tissue, and its mass was determined for subsequent perfusion normalization.

Preparation of in situ cremaster muscle.

In a separate series of experiments, an in situ cremaster muscle from each animal was prepared for study using intravital microscopy as described previously (15). Following a 30-min period of equilibration after the surgical/experimental preparation, a third-order arteriole (~40 μm diameter) was selected for investigation. Arterioles were selected based on the following criteria: 1) distance from any site of incision, 2) presence of significant vascular tone (assessed by brisk dilator response to challenge with 10−3 M adenosine), 3) clearly discernible walls, 4) a rapid and stable level of erythrocyte perfusion, and 5) the presence of two clearly defined daughter branches (fourth order; ~20 μm diameter) that also met criteria 1–4. Please see Fig. 1 for a schematic representation of these experiments.

Fig. 1.
Schematic of in situ cremasteric arteriolar preparation used for assessing parent and daughter arteriolar mechanical and hemodynamic/perfusion responses to pharmacological challenge and muscle contraction. Open arrows represent determination of parent ...

The mechanical (on-screen videomicrometer) and perfusion (optical Doppler velocimetry) responses of both the “parent” (third order) and “daughter” (fourth order) arterioles were assessed in response to topical application of increasing concentrations of acetylcholine (10−10–10−6 M), arachidonic acid (10−10–10−6 M), and adenosine (10−10–10−6 M). Additionally, the cremaster muscle was stimulated to contract for 3 min at both 1- and 3-Hz contractions by using electrodes placed on opposite sides of the muscle (Grass SD9). Stimulation parameters followed those established previously for transilluminated, striated muscle preparations (34, 36), and assessments of both mechanical and perfusion responses were determined immediately following cessation of the electrical stimulation. All procedures were performed under control conditions in LZR and OZR, and following treatment of the in situ cremaster muscle with Tempol (10−3 M), SQ-29548 (10−4 M), and/or phentolamine (10−5 M) within the superfusate solution.

Data and statistical analyses.

Mechanical responses of isolated and in situ arterioles following pharmacological challenge were fit with a semilogarithmic regression equation y = a0 + b1 log(x), by using least squares analysis. In this equation, y represents arteriolar diameter, a0 is an intercept term, x is the nontransformed agonist concentration, and b1 represents the slope of the relationship between agonist concentration and arteriolar diameter. For responses to hypoxia, this reduces to a linear regression equation y = a0 + b1 (x).

Subsequent presentation of isolated arteriolar mechanical responses will utilize “sensitivity,” which represents the slope coefficient from the regression analyses. As such, each data point in Figs. 25 represents the slope of the stimulus-response relationship (to acetylcholine, hypoxia, arachidonic acid, or adenosine) for that specific set of independent variables (phenylephrine concentration or intralumenal pressure). For integration of individual stimulus-response slope coefficients into a surface relationship, data are interpolated using a running average technique that determines mean values between adjacent data points within the surface as a data smoothing procedure.

Fig. 2.
A: dilator sensitivity of arterioles from lean Zucker rats (LZR; upper surfaces) and obese Zucker rats (OZR; lower surfaces) to acetylcholine with increasing adrenergic and myogenic tone. Sensitivity is defined as the slope coefficient describing arteriolar ...
Fig. 5.
A: dilator sensitivity of arterioles from LZR (upper surfaces) and OZR (lower surfaces) to adenosine with increasing adrenergic and myogenic tone. Sensitivity is defined as the slope coefficient describing arteriolar dilation to increasing concentrations ...

Determination of arteriolar perfusion for in situ cremaster muscle of LZR and OZR was calculated as: Q = (V·1.6−1)·(πr2)·(0.001). In this equation, Q represents arteriolar perfusion (nl·s−1), V represents red cell velocity (mm·s−1), and r represents arteriolar radius (1).

All data are presented as means ± SE. However, for the three-dimensional presentations of isolated arteriolar reactivity and for some of the data describing in situ cremasteric arteriolar responses, error terms are omitted for increased clarity and ease of interpretation. Statistically significant differences in measured and calculated parameters were determined using analysis of variance. In all cases, the Student-Newman-Keuls post hoc test was used when appropriate, and P < 0.05 was taken to reflect statistical significance.

RESULTS

Table 1 presents basic characteristics of animal groups in the present study. At ~17 wk, OZR were heavier than LZR and exhibited moderate hypertension. Plasma insulin levels were elevated in OZR, as were both indices of dyslipidemia (total cholesterol and triglycerides). Furthermore, plasma nitrotyrosine was also increased in OZR vs. LZR, indicative of a chronic increase in vascular oxidant stress.

Table 1.
Characteristics of animal and arteriolar groups for the present study

Isolated resistance arteriolar reactivity.

Fig. 2 presents the relationship describing arteriolar sensitivity to acetylcholine across levels of adrenergic and myogenic tone. As presented in Fig. 2, A and B, arteriolar sensitivity to acetylcholine in LZR was increased over that in OZR at comparable levels of adrenergic or myogenic tone. Arteriolar sensitivity to acetylcholine in OZR was nearly abolished with increased adrenergic or myogenic tone; an effect that was also present in LZR, although to a much lesser extent. Following treatment with Tempol, the impact of elevated intralumenal pressure on arteriolar sensitivity to acetylcholine in OZR was abolished, while the effect of adrenergic tone on dilator reactivity was largely intact, as a strong adrenergic-based reduction to acetylcholine sensitivity remained (Fig. 2, C and D).

Figure 3 presents arteriolar sensitivity to arachidonic acid in LZR and OZR with altered adrenergic tone and intralumenal pressure. Under control conditions, arterioles of LZR demonstrated a greater sensitivity to arachidonic acid, regardless of phenylephrine concentration or myogenic tone (Fig. 3, A and B). When arterioles from OZR were treated with Tempol (Fig. 3, C and D), sensitivity to arachidonic acid was improved across nearly all combinations of pressure and adrenergic tone, although this improvement was biased toward blunting the impact of myogenic activation rather than adrenergic tone. Interestingly, the impact of treating arterioles of OZR with SQ-29548 resulted in an even stronger improvement in sensitivity to arachidonic acid, as negative impact of both myogenic activation and even modest increases in adrenergic tone on dilator reactivity were attenuated (Fig. 3, E and F).

Fig. 3.
A: dilator sensitivity of arterioles from LZR (upper surfaces) and OZR (lower surfaces) to arachidonic acid with increasing adrenergic and myogenic tone. Sensitivity is defined as the slope coefficient describing arteriolar dilation to increasing concentrations ...

Arteriolar sensitivity from LZR and OZR in response to reduced Po2 with changes in myogenic and adrenergic tone under control conditions and in response to pharmacological treatment with Tempol or SQ-29548 is summarized in Fig. 4. Under control conditions (Fig. 4, A and B), arteriolar sensitivity to hypoxia was reduced in OZR vs. LZR at nearly all levels of adrenergic or myogenic tone. Treatment with Tempol (Fig. 4, C and D) improved sensitivity to hypoxia in OZR, and throughout much of the range of myogenic activation and lower range of adrenergic tone, restored it to levels determined in LZR. Similarly, treatment with SQ-29548 exhibited a directionally consistent, although less robust, improvement to arteriolar sensitivity to hypoxia (Fig. 4, E and F). As with sensitivity to acetylcholine and arachidonic acid, these improvements were biased toward ameliorating the effects of myogenic tone, as the impact of increased adrenergic tone on impairing arteriolar sensitivity to reduced Po2 was unaffected.

Fig. 4.
A: dilator sensitivity of arterioles from LZR (upper surfaces) and OZR (lower surfaces) to hypoxia with increasing adrenergic and myogenic tone. Sensitivity is defined as the slope coefficient describing arteriolar dilation to reduced oxygen tension at ...

Figure 5 presents arteriolar sensitivity to adenosine for both LZR and OZR across all employed conditions of altered adrenergic and myogenic tone. While changes to arteriolar sensitivity were present under these conditions, sensitivity to adenosine remained high throughout and demonstrated little difference between LZR and OZR, as evidenced by the fact that the surfaces for LZR and OZR are nearly superimposable.

In situ cremaster muscle arteriolar reactivity and perfusion.

The reactivity of in situ cremasteric parent arterioles in response to application of increasing concentrations of acetylcholine (Fig. 5A), arachidonic acid (Fig. 5B), or adenosine (Fig. 5C) are summarized in Fig. 6. While arteriolar dilation to both acetylcholine and arachidonic acid were reduced in OZR vs. LZR, treatment with Tempol improved dilation to both agonists, while application of SQ-29548 increased responses to arachidonic acid only, as evidenced by an increase in the slope coefficient for that relationship. Application of phentolamine normalized arteriolar diameter under control conditions but had no impact on reactivity to increasing concentrations of acetylcholine or arachidonic acid. Arteriolar dilation to adenosine was robust in both strains (Fig. 6C) and, with the exception of the shift in resting diameter following adrenoreceptor blockade, dilator responses to adenosine were not impacted by application of phentolamine, Tempol, or SQ-29548.

Fig. 6.
Dilator responses of in situ cremaster muscle parent arterioles from OZR and LZR in response to increasing concentrations of acetylcholine (A), arachidonic acid (B), and adenosine (C). Data (means ± SE) are presented for arteriolar responses under ...

The mechanical and hemodynamic responses of in situ parent arterioles following muscle contraction are presented in Fig. 7. At rest, and with both 1- and 3-Hz contractions, parent arteriolar dilation was decreased in OZR vs. LZR (Fig. 7A), and application of either Tempol or SQ-29548 had minimal impact on these differences, except at 3-Hz contraction, wherein the Tempol-induced reduction in vascular oxidant stress resulted in an enhanced functional dilation in OZR. While phentolamine normalized arteriolar diameter under resting conditions, this improvement was less discernible with increased metabolic demand. However, combined adrenoreceptor and PGH2/TxA2 receptor blockade, with antioxidant treatment resulted in an effect wherein functional dilation was normalized at low metabolic demand (the impact of the phentolamine) and at both 1- and 3-Hz contractions (the impact of SQ-29548 and Tempol). Arteriolar erythrocyte velocity demonstrated a mild increase in all groups with elevated metabolic demand, although these relationships did not exhibit significant differences between groups (Fig. 7B). Combining these data, Fig. 7C presents parent arteriolar perfusion with cremaster muscle contraction. Similar to dilator responses, arteriolar hyperemia was reduced in OZR vs. LZR, and treatment with Tempol or SQ-29548 did not impact this difference at rest or with 1-Hz contraction, but improved perfusion at 3-Hz contraction. In contrast, treatment with phentolamine demonstrated a reverse relationship, improving perfusion at low metabolic demand, but less so at 3-Hz contraction. Combined treatment with the three agents profoundly improved arteriolar perfusion at rest and with both levels of increased metabolic demand.

Fig. 7.
Data describing the responses of in situ cremaster muscle parent arterioles under rest conditions and in response to increasing metabolic demand (muscle contraction). Data (means ± SE) are presented for arteriolar diameter (A), erythrocyte velocity ...

The examination of both parent and arising daughter arterioles within the cremaster muscle allows for an estimation of perfusion heterogeneity at bifurcations, and potentially tissue perfusion heterogeneity, within the skeletal muscle microcirculation. Table 2 presents initial conditions within in situ cremaster muscle daughter arterioles originating from the parent. From each animal, the dimension and hemodynamic properties within both daughter arterioles was compiled into this table. As such, while trends toward differences are present for arteriolar dimension and perfusion, the magnitude of the error terms are such that these differences failed to reach statistical significance.

Table 2.
Initial conditions within in situ cremasteric daughter arterioles under the experimental conditions of the present study

Figure 8 presents data describing the responses of cremasteric daughter arterioles of LZR and OZR with elevated metabolic demand. While the patterns of vasodilation and hyperemia mirror those presented for parent arterioles in Fig. 7, these data are organized to provide insight into the relative responses between the two daughter arterioles arising from the single parent in response to muscle contraction (please see Fig. 1 for schematic representation). As shown in Fig. 8A, the difference in diameter between daughter arterioles is greater in OZR than in LZR at rest, and while this divergence is unaffected by treatment with Tempol or SQ-29548, it is eliminated by adrenoreceptor blockade. With elevated metabolic demand, this disparity in daughter arteriolar diameter becomes more severe between LZR and OZR, although it becomes increasingly correctable via treatment of endothelial dysfunction (Tempol, SQ-29548), and resistant to amelioration via adrenoreceptor blockade. However, at all levels of metabolic demand, combined treatment with Tempol, SQ-29548, and phentolamine dramatically improved the disparity between daughter arteriolar diameters in OZR. The disparity in daughter arteriolar erythrocyte velocity under resting conditions and with elevated metabolic demand between LZR and OZR is summarized in Fig. 8B. In OZR, the difference in RBC velocity between daughter arterioles is elevated vs. that in LZR, at each level of metabolic demand. Treatment of cremaster muscle with Tempol, SQ-29548, or phentolamine (or all three) attenuated this heterogeneity in erythrocyte velocity between LZR and OZR. Combining these data, Fig. 8C presents daughter arteriolar perfusion heterogeneity between OZR and LZR at rest and with muscle contraction. For arterioles in OZR, the heterogeneity between daughter arteriolar perfusion is both greater than in LZR and remarkably stable with elevated metabolic demand. Treatment with phentolamine reduced this heterogeneity at rest, but was much less effective with increased metabolic demand. Conversely, treatment of cremaster muscle with Tempol or SQ-29548 also reduced the disparity between LZR and OZR, and became increasingly effective with increased metabolic demand. In all cases, simultaneous treatment with all three agents normalized daughter arteriolar perfusion homogeneity. Treatment of the in situ cremaster muscle of LZR with Tempol, SQ-29548, and/or phentolamine did not result in a consistent, statistically significant alteration in perfusion outcomes (data not shown).

Fig. 8.
Data describing the responses of in situ cremaster muscle daughter arterioles under rest conditions and in response to increasing metabolic demand (muscle contraction). Data (means ± SE) are presented as the heterogeneity between daughter arteriolar ...

Contributions of arteriolar reactivity to functional hyperemia.

Table 3 presents the effectiveness of intravenous treatment of animals with Tempol, SQ-29548, and phentolamine on reducing oxidant stress, antagonizing PGH2/TxA2 receptors, and blocking adrenergic receptors, respectively. Intravenous infusion of methacholine had a minimal depressor response in OZR under normal conditions; treatment with Tempol improved this response to a level that was not different from that in LZR. Treatment of OZR with SQ-29548 almost completely abolished pressor responses to U-46619. Finally, treatment of OZR with phentolamine severely attenuated pressor responses to an intravenous infusion of phenylephrine.

Table 3.
Treatment effectiveness of intravenous infusion of Tempol, SQ-29548, and phentolamine in OZR

Figure 9 presents data describing bulk skeletal muscle perfusion in OZR under resting conditions (Fig. 9A) and in response to low (1 Hz, Fig. 9B) and moderate (3 Hz, Fig. 9C) elevations in metabolic demand. Gastrocnemius muscle mass did not differ significantly between Zucker rat strains at this age, averaging 1.24 ± 0.19 g in LZR and 1.10 ± 0.11 g in OZR. While significant functional hyperemia was evident in both strains, skeletal muscle blood flow in OZR was reduced below that in LZR at each level of metabolic demand. Infusion of Tempol or SQ-29548, or both was largely without effect on skeletal muscle perfusion in OZR at any metabolic intensity. In contrast, blockade of α12 adrenergic receptors with phentolamine improved blood flow responses to skeletal muscle of OZR across this range of metabolic demand to an extent that they were not significantly different from that in untreated LZR. Treatment of LZR with Tempol, SQ-29548, and/or phentolamine did not result in a consistent, statistically significant alteration in perfusion outcomes (data not shown).

Fig. 9.
Blood flow to in situ gastrocnemius muscle of LZR and OZR at rest (A) and in response to 1- (B) and 3-Hz (C) contraction. Data (means ± SE) are presented for responses under control conditions and following treatment of the animal with intravenous ...

DISCUSSION

With evolution of metabolic syndrome, a central negative microvascular outcome within skeletal muscle is an inability of the vasculature to deliver and distribute blood in a manner that satisfies metabolic demand. As this limitation could result from lesions to signaling patterns underlying dilation, competition against dilation by constrictor responses, or factors independent of reactivity (e.g., structural changes to vessel walls which limit expansion despite relaxation or a reduction in microvessel density), an integrated understanding of how different processes of vascular reactivity interact and impact perfusion is critical. The purpose of the present study was to determine how major pathways of skeletal muscle resistance arteriolar reactivity in OZR interact to affect net vascular tone within the proximal microcirculation as well as determining distal arteriolar mechanical and perfusion responses to imposed challenges. Additionally, we assessed the contributing roles of elevated vascular oxidant stress and altered arachidonic acid metabolism to these processes. Finally, these results from isolated and in situ arterioles were translated into blood-perfused skeletal muscle, where their contributions to restrained functional hyperemia in OZR were evaluated.

The primary observation for integrated reactivity of isolated arterioles in OZR is that adrenergic and myogenic tone can both have a profound impact on dilator responses to acetylcholine, arachidonic acid, and hypoxia. In all cases, elevated intralumenal pressure or phenylephrine concentration blunted dilator responses to these three stimuli, and this effect was increased in arterioles from OZR vs. LZR. In contrast, dilator responses to adenosine were largely unaffected by adrenergic or myogenic tone in either strain, with the only consistent effect being a reduced initial diameter as a result of the increased tone, rather than an impact on the slope of the adenosine concentration-response relationship. Similarly, these results suggest that adrenergic reactivity may be the dominant constrictor influence in OZR that, in addition to establishing initial arteriolar diameter, is resistant to modulation from dilator influences. Clearly, this can have major implications for not only bulk perfusion to tissue, but also (and potentially more importantly) for perfusion distribution within tissue.

In isolated resistance arterioles, blockade of the PGH2/TxA2 receptor profoundly improved the dilator reactivity of arterioles from OZR in response to both elevated arachidonic acid concentration and reduced Po2. Furthermore, the ability of myogenic tone to restrain dilation was attenuated following treatment of arterioles with SQ-29548, suggesting that the impact of pressure-induced constriction on endothelium-dependent dilation in OZR may have been predominantly mediated via an increased activation of PGH2/TxA2 receptors. Based on our recent study (17) demonstrating an increased arteriolar TxA2 generation with hypoxia (a response normally dependent on production of PGI2; 14, 22, 23), this beneficial effect of SQ-29548 may reflect the combination of preventing both arachidonic acid- or hypoxia-induced generation/release of TxA2 and activation of PGH2/TxA2 receptors with myogenic activation alone in arterioles of OZR.

Decreased oxidant stress with Tempol treatment, while profoundly effecting patterns of reactivity between acetylcholine, arachidonic acid, or hypoxia with increased adrenergic or myogenic tone in both strains, revealed some intriguing issues. For acetylcholine-, arachidonic acid-, and hypoxia-induced dilation, Tempol improved arteriolar sensitivity in OZR with no adrenergic tone but had a negligible impact on responses with increased phenylephrine levels. In contrast, normalizing oxidant stress improved dilator responses with elevated myogenic tone, suggesting that alterations to both arachidonic acid metabolism and elevated vascular oxidant tone contribute to dilator impairments with increased myogenic tone. While the extent to which these represent interdependent effects cannot be confidently determined from the present study, previous evidence suggests a key role for oxidant stress in altering arachidonic acid metabolism, resulting in increased TxA2 production (17, 27, 38, 39). The impact of Tempol on integrated resistance arteriolar reactivity in OZR may be interpreted as the result of two distinct outcomes of the normalization of vascular oxidant stress. First, improvements to oxidant stress should correct elements of altered arachidonic acid metabolism and PGH2/TxA2 receptor activation that are reflective of acute alterations to increased oxidant stress. Second, previous studies have suggested that a minority contributor to hypoxic dilation in LZR is endothelial production/release of NO (16). While this effect is not always identifiable as statistically significant, it is frequently present. As such, we speculate that any contribution of vascular NO production to hypoxic dilation will be enhanced by the reduction in oxidant tone and resulting decrease in scavenging of NO by superoxide radicals.

From the perspective of vascular tone regulation and the establishment of resistance arteriolar diameter, results from interrogation of in situ cremaster muscle of LZR and OZR are consistent with those discussed above, as third-order arterioles exhibited directionally consistent impairments to acetylcholine- and arachidonic acid-induced dilation compared with responses in proximal resistance arterioles. Additionally, treatment of the cremaster muscle with Tempol or SQ-29548 improved dilator reactivity to these agonists, while adrenoreceptor blockade restored resting diameter, with minimal impact on agonist-induced responses. Finally, responses to adenosine were largely intact in third-order arterioles of both LZR and OZR. However, use of this preparation under conditions of muscle contraction provides critical insight into microvascular hyperemia associated with increased metabolic demand. While third-order arteriolar dilation and hyperemia were blunted in OZR vs. LZR with increasing metabolic demand, the ameliorative impact of adrenoreceptor blockade or improved endothelial function were spatially distinct. Treatment with phentolamine improved outcomes at the lowest metabolic demand and had a decreasing benefit with increasing muscle contraction frequency, while improvements to endothelial function with Tempol and SQ-29548 exhibited the reciprocal relationship. While application of these pharmacological treatments impacts the distal cremasteric microcirculation rather than proximal resistance arterioles, these observations suggest that constraints on functional hyperemia within skeletal muscle in OZR may be reflective of a multivariate process that is dependent on metabolic intensity, where adrenergic constraint contributes to ischemia at rest, and endothelial dysfunction becomes more significant with increased metabolic demand.

This concept of endothelial function may be more apparent during the examination of daughter arteriolar responses to cremaster muscle contraction. As presented in Fig. 8, daughter arteriolar diameter/dilator and hemodynamic/perfusion responses to muscle contraction were more heterogeneous in OZR vs. LZR, both at rest and with elevated metabolic demand. While adrenoreceptor blockade reduced heterogeneity in these responses at rest, this effect was minimized with muscle contraction, providing additional support for the concept that adrenergic tone can represent a constraining influence on arteriolar dilation/perfusion in OZR, although perhaps less effectively in more distal levels of the skeletal muscle microcirculation. In contrast, improving endothelial function in OZR with Tempol or SQ-29548 demonstrated an increasing effectiveness in reducing daughter arteriolar perfusion heterogeneity as metabolic demand increased. Taken together, these results suggest that effective endothelial function may be critical for the ability to match local perfusion distribution with skeletal muscle metabolic demand at higher levels of resolution.

On the basis of results presented in Fig. 9, it is apparent that for OZR manifesting the metabolic syndrome, not only is bulk blood flow to skeletal muscle reduced vs. that in LZR at rest and with mild elevations in metabolic demand, but this reduction may largely be a function of an increased adrenergic tone, likely at more proximal resistance arterioles (which would not have been accessible to intervention in the cremaster muscle preparation). However, infusion of Tempol or SQ-29548, or both, had no discernible impact on either resting perfusion or during functional hyperemia in the gastrocnemius muscle of OZR. As these interventions appeared to be effective (Table 3) when taken in context with the results in isolated vessels and in situ cremasteric arterioles, the implication may be that neither impairments to endothelial function in the metabolic syndrome nor interventions that can ameliorate this dysfunction significantly impact skeletal muscle perfusion at rest or with mild elevations in metabolic demand. It is important to note that these interpretations contrast with recent work in Hester's group, which suggest that adrenergic tone does not represent a constraint on skeletal muscle perfusion in OZR (25) and that the limiting influence may reside within arachidonic acid metabolism and activation of the PGH2/TxA2 receptor (3436). While the source for the discrepancy between the current results and the previous studies are not clear at this time, Hester's group employed OZR at a younger age and utilized a different muscle preparation (spinotrapezius).

Taken together, and with results from studies cited above, these data suggest a conceptual framework for interrogating impaired skeletal muscle perfusion in the metabolic syndrome. First, the major contributors to ischemia in OZR appear to be dependent on metabolic demand. At lower levels of metabolic demand, an adrenergic constraint on bulk perfusion may dominate. However, while there is an increased severity of adrenergic restraint on perfusion in skeletal muscle of OZR, this becomes progressively alleviated through metabolic sympatholysis (although this relationship is right-shifted, requiring a greater degree of metabolism to elicit a comparable suppression of adrenergic tone). With further elevations in metabolic demand, previous results suggest that hyperemia becomes increasingly constrained by arteriolar wall remodeling (12, 31) and a progressive microvascular rarefaction (12). What has not been demonstrated in OZR is a critical role for the endothelium in contributing to reducing bulk perfusion to skeletal muscle with increased metabolic demand. While this lack of a demonstrated role for the endothelium in bulk hyperemia at the organ level of resolution could reflect a compensated state wherein acute interventions cannot overcome a chronic disease condition, the present results neither support nor refute this concept. However, these results do suggest a critical importance for the vascular endothelium in maintaining perfusion distribution at higher levels of resolution, potentially through the impact of communicated responses (6, 8, 29), sensing of arteriolar wall shear stresses (28, 29, 33), or arachidonic acid metabolism (17, 35, 36). Clearly, this role for the endothelium would be in addition to its established involvement in other outcomes, including contributions to angiogenesis/angiostasis, antithrombotic activity, and leukocyte adhesion, among others. The ability to construct and integrate these roles for the vascular endothelium into a holistic understanding of function in health, and dysfunction in disease, continues to represent a critical area for future investigation.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-64668 and American Heart Association Grants SDG-0330194N and EIA-0740129N.

Acknowledgments

The authors thank Milinda E. James for expert technical assistance, as well as the support provided through the Analytical Biochemistry and Statistical Modeling Facilities in the Center for Cardiovascular and Respiratory Sciences at the West Virginia University Health Sciences Center for the performance of this study. The authors also express their sincere thanks to Dr. Julian H. Lombard from the Medical College of Wisconsin for his support, encouragement, and helpful suggestions during the performance of this study and to Dr. Lombard and Dr. Robert L. Hester from the University of Mississippi Medical Center for critical evaluation of this manuscript.

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