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Department of Health and Kinesiology, Texas A&M University, College Station, Texas
Submitted 27 May 2004 ; accepted in final form 18 November 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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nitric oxide; potassium channels; endothelin
A number of studies have shown that flow-induced vasodilation of skeletal muscle arterioles occurs through endothelial production of both NO and prostacyclin (PGI2) (11–13, 28); however, the contributions of these endothelial vasodilators to the flow-induced response vary among muscles of different location and fiber type (13, 25, 28). For example, flow-induced dilation of rat cremaster arterioles is unaffected by blockade of nitric oxide synthase (NOS), but the response to flow is eliminated by the cyclooxygenase (COX) inhibitor indomethacin (Indo) (13). Flow-induced vasodilation of isolated gracilis muscle arterioles is mediated by release of both endothelial NO and endothelial prostanoids (11, 12). In soleus muscle arterioles, endothelial NO contributes heavily to vasodilation induced by step increases in flow, but blockade of COX activity also inhibits the response, demonstrating a partial role for prostanoid vasodilators (25). In gastrocnemius muscle arterioles, inhibition of either COX or NOS reduces the vasodilator response to step increments in intraluminal flow (25). Furthermore, in arterioles from both soleus and gastrocnemius muscles, some flow-induced vasodilation persists when both NOS and COX are inhibited, indicating the contribution of yet another vasodilator pathway (25, 28). However, to date, no studies have examined the time dependence of vasodilator mechanisms activated by sustained shear stress in arterioles from locomotory skeletal muscle.
The primary goal of this study was to determine the time course of flow-induced vasodilation in locomotory skeletal muscle resistance arterioles and to identify differential mechanisms that mediate the initial and sustained phases of flow-induced dilation in these arterioles. Skeletal muscles of differing fiber type and oxidative capacity show different patterns of recruitment during exercise and are exposed to vastly different blood flow patterns at rest, at the onset of activation, and during sustained exercise (1, 2, 6, 18). Thus it is reasonable to expect that the time course of flow-induced vasodilation may differ among arterioles from muscles of different fiber types. Accordingly, we also sought to compare the time course of flow-induced vasodilation in first-order (1A) arterioles isolated from the soleus muscle, composed predominantly of slow-twitch fibers (6), with responses of arterioles from the superficial portion of the gastrocnemius muscle, composed predominantly of fast-twitch fibers (6).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Male Fischer 344 rats 4–6 mo of age (342 ± 3 g) were obtained from Harlan (Indianapolis, IN). The rats were housed in a temperature-controlled room (23 ± 1°C) with a 12:12-h light-dark cycle. Rat chow and water were provided ad libitum.
Isolation and cannulation of skeletal muscle arterioles. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). The soleus and gastrocnemius muscles were excised and placed in cold (4°C) physiological saline solution (PSS) containing (in mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS buffer, with 1 g/100 ml bovine serum albumin, pH 7.4 (PSS-albumin, PSSA).
Skeletal muscle arterioles were dissected and cannulated as previously described (26). Briefly, 1A arterioles were dissected out of both gastrocnemius and soleus muscles under a dissection microscope (Olympus SVH10) and transferred to a Lucite chamber for cannulation with micropipettes (45- to 60-µm diameter) of matched tip resistance. The microvessel chamber was transferred to the stage of an inverted microscope (Olympus IX70) equipped with a video caliper system (Panasonic BP310; Texas A&M Cardiovascular Research Institute) used to measure intraluminal diameter. Arterioles were pressurized to 70 cmH2O with two independent hydrostatic pressure reservoirs. Arterioles were perfused with and bathed in warm (37°C) PSSA and allowed to develop spontaneous tone. Only vessels that developed spontaneous tone were included in data analysis.
Time course of flow-induced vasodilation.
After equilibration at 70 cmH2O in the absence of flow (no pressure difference), vasodilator responses to shear stress were determined by altering the heights of the hydrostatic reservoirs in equal and opposite directions to generate a pressure difference without changing mean intraluminal pressure (17). All diameter measurements were determined in response to a pressure difference of 10 cmH2O, previously shown to elicit maximal dilation in 1A arterioles from both soleus and gastrocnemius muscles (25). Vessel diameter changes were monitored continuously for a 20-min period following initiation of intraluminal flow. Diameter measurements used in data analysis were extracted at time (t) = 0, 0.25, 0.50, 0.75, 1, 2, 3... 20 min. In a subset of vessels, the actual flow rate generated by a hydrostatic pressure difference of 10 cmH2O was computed from measurements of vessel radius and the velocity of red blood cells injected into the pipette tip. The vessels were cannulated on pipettes of similar inner tip diameters and resistance as those used in subsequent flow experiments. Volumetric flow (Q) was calculated from mean red blood cell velocity (Vrbc) and inner diameter (d) of these vessels according to the following equation (5, 17, 25):
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according to the following equation:
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is viscosity (0.8 cp), is calculated as described above, and r is vessel radius. Evaluation of endothelial mechanisms. Responses to intraluminal flow were evaluated after a 20-min incubation with one of the following: 1) control PSS or vehicle control (0.7% ethanol), 2) 1 x 10–5 M NG-nitro-L-arginine methyl ester (L-NAME), 3) 1 x 10–5 M Indo, 4) L-NAME + Indo, 5) 40 mM KCl, 6) L-NAME + the ETA receptor blocker BQ-123 (1 x 10–6 M) + the ETB receptor blocker BQ-788 (3 x 10–8 M) (21). Time control experiments were performed to establish that repeated exposures to flow did not alter vasodilator responses. Control flow responses did not differ from flow responses evaluated in the presence of ethanol vehicle. Responses in the presence of either Indo or L-NAME alone were not evaluated in the same arterioles. Responses in the presence of L-NAME + Indo were evaluated after completion of responses evaluated in the presence of L-NAME alone. Evaluation of repeated flow responses in the presence of L-NAME indicated that the effects of the inhibitor were similar with repeated exposure. A maximum of three 20-min exposures to flow was evaluated in any single arteriole. A minimum 20-min equilibration was allotted between successive exposures to flow to allow for reestablishment of spontaneous tone. At the end of each experiment the arteriole was washed twice in calcium-free PSS and treated with sodium nitroprusside (1 x 10–4 M) to achieve maximal dilation. The calcium-free PSS was similar to PSSA except that it contained 2 mM EDTA and CaCl2 was replaced with NaCl. The contribution of activation of Ca2+-activated K+ (KCa) channels to initial vasodilation was evaluated by exposure of arterioles to intraluminal flow for 2 min under control conditions and again after 20-min incubation with apamin (3 x 10–7 M) and charybdotoxin (1 x 10–7 M) (22).
Solutions and drugs. Stock solutions of L-NAME and BQ-123 were prepared in distilled water. Indo and BQ-788 stocks were prepared in 70% ethanol. Fresh dilutions of these stock solutions were prepared daily. All drugs were purchased from Sigma (St. Louis, MO).
Data analysis.
Vasodilator responses were recorded as actual diameters and subsequently expressed as a percentage of maximal dilation according to the following formula:
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RESULTS |
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44 ± 10%, >50% of the maximal dilation induced by flow. Correspondingly, in soleus muscle arterioles, the dilation to flow at t = 0.5 min was only 14 ± 9%, 25% of the maximal dilation to flow. By t = 2.0 min (gastrocnemius muscle) and t = 3.0 min (soleus muscle), arterioles had achieved a maximal response to flow, with no further significant dilation occurring during the remaining period of flow exposure. The magnitude of maximal dilation to flow was greater in gastrocnemius muscle arterioles (66 ± 6%) than in soleus muscle arterioles (38 ± 5%).
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Effects of endothelin blockade. Because shear stress has been shown to cause release of endothelin (14, 19, 27), we tested the effects of endothelin blockade on the flow-induced response in the presence of L-NAME. Blockade of ETA and ETB receptors in the presence of L-NAME reversed the constriction seen after t = 10 min when arterioles were treated with L-NAME alone (Fig. 7). In soleus muscle arterioles, endothelin receptor blockade resulted in responses to flow that were not different than those under control conditions at t = 20 min (Fig. 7A).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Flow-induced vasodilation may contribute to maintenance of skeletal muscle blood flow at rest and during conditions of increased activity. Flow-induced vasodilation of skeletal muscle arterioles has been shown to be an endothelium-dependent response to an increase in shear stress (4, 10, 12). Studies of isolated arterioles from various muscles have demonstrated that even very small increases in intraluminal flow produce robust vasodilation (4, 10, 12, 25, 28). An initial step increase in flow stimulates a strong vasodilator response, whereas subsequent increases in flow produce much smaller diameter changes (10, 12, 25, 28). In a study of Jasperse and Laughlin (10), maximal dilation to flow occurred at a flow rate of only 14 µl/min, a flow rate that exceeded estimated flow rates in soleus feed arteries of rats under physiological conditions of rest, walking, and running. These results suggested that flow-induced vasodilation is maximally activated under all of these conditions. Flow rates and shear stress have not been determined in 1A arterioles from locomotory skeletal muscle; thus we cannot determine whether the flow rate used in this study falls within the physiological range of flow for arterioles from either the soleus or the gastrocnemius muscle. However, the step increase in flow created by the onset of flow in this study is likely to be physiologically relevant, because flow through these arterioles is constantly adjusting in response to changes in muscular activity. Our results confirm that a small increment in flow generates a strong vasodilator response in 1A arterioles from soleus and gastrocnemius muscle but also indicate that the cellular mechanisms triggered by the onset of flow differ from those stimulated by steady-state flow. For example, only sustained exposure to flow triggers a constrictor response. Previous studies have not investigated the responses to a sustained increase in flow, a condition more relevant to the in vivo situation where arterioles are exposed to continuous flow. The current data suggest that flow modulates resistance in skeletal muscle through both constrictor and dilator mechanisms. The balance of these mechanisms depends not only on the magnitude of the flow rate to which the vessel is exposed but also on the time course of flow exposure. Thus different mechanisms triggered by flow may contribute to muscle blood flow responses at rest when blood flow is relatively constant or under physiological conditions of acutely elevated flow.
Our data also indicate that the relative contribution of the initial and sustained components of flow-induced vasodilation may differ among muscles of different fiber type. In addition to differences in muscle fiber composition and oxidative capacity (6), soleus and superficial gastrocnemius muscles vary in their recruitment order (1, 2, 6) and blood flow patterns at rest and during exercise (1, 18). Any of these differential characteristics could directly or indirectly influence the vasodilatory responses of the resistance vessels within the muscle. McCurdy et al. (20) reported that sensitivity to adenosine and sodium nitroprusside is greater in arterioles from the gastrocnemius muscle than in arterioles from the soleus muscle. Our data indicate that the initial response to flow was more rapid and pronounced in gastrocnemius muscle arterioles than in soleus muscle arterioles, confirming previous observations that the magnitude of dilation triggered by step increases in flow is greater in arterioles from the gastrocnemius muscle than in those from soleus muscle (25). Disparity in size and spontaneous tone development existed between arterioles from the soleus and gastrocnemius muscles, potentially contributing to the degree of difference in the flow-induced dilation observed here. Therefore, we also compared the magnitude of the initial flow-induced response in soleus and gastrocnemius muscle arterioles matched for size and spontaneous tone. In arterioles of similar size and spontaneous tone, the magnitude of flow-induced vasodilation remained significantly greater in arterioles from the gastrocnemius muscle (Fig. 2). The magnitude of exercise hyperemia in gastrocnemius muscle is far greater than that seen in soleus muscle (18). In contrast, soleus muscle receives a relatively high blood flow at rest compared with gastrocnemius muscle (18), and NOS inhibition preferentially affects the exercise hyperemia in highly oxidative muscle (7). Thus it seems likely that the more rapid and intense flow-induced vasodilation documented in gastrocnemius muscle may be related to the role of this muscle that is relatively quiescent at rest and recruited during more intense activity.
Butler et al. (4) recently reported that vasodilation in cremaster muscle arterioles elicited by changes in shear stress on a time scale of a few seconds was transient and highly rate sensitive: faster rates of changes in shear stimulated more potent dilation. In contrast, shear stress changes on a longer time scale resulted in a more modest but sustained vasodilation that was sensitive to the magnitude of shear stress. In cultured endothelial cells, a step increase in shear stimulates a rapid transient response that differs from the sustained response produced by prolonged exposure to shear stress (15, 16). A step increase in shear elicits a large and transient increase in NO, whereas exposure to a shear ramp results in more moderate production of NO (15). NO production in response to a step change in shear occurs through activation of a G protein, but ramp shear elicits NO production that is G protein independent (16). Similarly, our results indicate that flow-induced vasodilation in skeletal muscle arterioles is biphasic. A rapid dilation occurs in response to an initial increase in intraluminal flow, and a sustained vasodilator response persists over 20 min of exposure to flow; however, different endothelial pathways mediate these responses. Elevation of external [K+] inhibited the immediate dilation to flow but produced less inhibition of the sustained vasodilation than L-NAME treatment. Blockade of NOS with L-NAME also exhibited a biphasic effect on flow-induced dilation; however, in contrast to results in cultured endothelial cells, blockade of NOS abolished the vasodilation to sustained flow exposure but did not alter the response to the initial increase in flow. The difference between our results with NOS blockade and those of Kuchan and Frangos (15) may be due to influences present in intact arterioles that are lost in cells cultured under static conditions without the presence of smooth muscle or parenchymal cells.
The results of this study indicate that NO contributes to flow-induced vasodilation in arterioles from both soleus and gastrocnemius muscle arterioles in a time-dependent manner. In both soleus and gastrocnemius muscle arterioles, the initial vasodilator response to flow was not affected by treatment with L-NAME. We showed previously (25) that L-NAME treatment significantly reduces the vasodilation to step changes in flow in soleus but not in gastrocnemius muscle arterioles. The present data indicate that, although NO does not contribute to the immediate dilation to flow, a significant portion of the sustained response to flow is mediated by NO. The difference in the current findings and those previously reported may be due to the timing of the recordings of responses in previous work. In our previous study (25), diameter was recorded for up to 3 min and peak, steady-state diameter was reported after each step change in flow. Thus, between 2 and 3 min, diameter may have continued to increase through an L-NAME-sensitive mechanism. In this study, diameter continued to increase in soleus muscle arterioles between 2 and 3 min, and the vasodilation during this time period was reduced by treatment with L-NAME (Fig. 3A). Other investigators have reported varying contributions of NO to flow-induced vasodilation in arterioles from diverse skeletal muscles (11, 12, 28). In rat gracilis muscle arterioles, N
-nitro-L-arginine (L-NNA) reduced the response to step increases in luminal flow by 40% (11, 12). In soleus muscle, Schrage et al. (28) reported that L-NNA inhibited flow-induced vasodilation by 65–70% in 1A arterioles and abolished the response in second-order (2A) arterioles. Collectively, these data show that the role of NO in mediating flow-induced vasodilation varies with the location of arterioles, the muscle of origin, and the duration of flow exposure.
We found that treatment with Indo alone had no inhibitory effect on flow-induced vasodilation in either soleus or gastrocnemius muscle arterioles. The relative role of prostanoids in mediating flow-induced vasodilation has been shown to differ in arterioles from distinct muscles (12, 13, 25, 28). Our current findings are in agreement with the reports of Schrage et al. (28) showing that Indo had no effect on flow-induced vasodilation in 1A soleus muscle arterioles. In the current study, the only effect of Indo treatment appeared to be a slight enhancement of the initial response to flow in soleus muscle arterioles. In endothelial cultures, treatment with PGI2 and elevation of cAMP has been shown to inhibit endothelial cell calcium and inhibit the formation of NO (3). Thus it is possible that Indo treatment eliminates the negative feedback of PGI2 on NO production, leading to a greater initial vasodilation; however, our data coupled with the results of other studies indicate a relatively minor role for PGI2 in flow-induced vasodilation of arterioles from the soleus and gastrocnemius muscles.
The effect of fluid shear stress on endothelial release of endothelin has been studied in both in vitro and in vivo preparations (14, 19, 27). Kuchan and Frangos (14) showed that production of endothelin in cultured endothelial cells varied with the time and magnitude of shear stress exposure. Low levels of shear stress stimulated an increase in endothelin production, whereas longer exposure to higher levels of shear stress resulted in a diminution of endothelin production through a NO-cGMP pathway. Qiu and Tarbell (27) demonstrated in cultured endothelial cells grown in tubes that the balance between vasoconstrictor and vasodilator signals depends on whether the endothelium is exposed to steady shear, oscillatory shear, or oscillatory shear coupled with circumferential strain. In the iliac artery of the anesthetized dog, increases in pulsatile shear stress also resulted in release of endothelin and net vasoconstriction; blockade of endothelin receptors completely reversed the shear-induced constriction and resulted in a net vasodilation (19). In the present study, the net response to a sustained increase in flow was vasodilation under control conditions; however, application of L-NAME unmasked a significant vasoconstriction. Endothelin antagonists blocked this constriction, thus indicating that exposure of muscle arterioles to steady shear in the absence of NO results in endothelin production. Furthermore, our data are in agreement with the study of Kuchan and Frangos (14), in which they demonstrated shear stress-induced endothelin production that was countered by activation of an NO-cGMP pathway. Under control conditions, endothelin receptor antagonists did not potentiate flow-induced dilation in either soleus or gastrocnemius muscle arterioles (data not shown), indicating that under basal conditions flow-induced release of NO is sufficient to inhibit net endothelin production. Thus the predominant vasoconstriction recorded here in the presence of NOS blockade likely results from blockade of NO-mediated inhibition of endothelin production or increased endothelin production stimulated by greater shear stress in the absence of NO-mediated vasodilation. Collectively, these studies suggest that the net vasoactive response to increases in shear stress occurs as a result of integration of both vasodilator and vasoconstrictor effects. In isolated skeletal muscle arterioles, the primary mediator of the integrated response to sustained flow appears to be NO.
The inhibition of flow-induced vasodilation produced by treatment with 40 mM KCl suggests that exposure to flow activates a vasodilator pathway involving K+ channels in arterioles from both soleus and gastrocnemius muscles. Furthermore, the initial dilation to flow was significantly reduced by KCa channel blockade. Importantly, this inhibitory effect of KCa channel blockade was significantly greater in gastrocnemius muscle arterioles than in those from soleus muscle. These data suggest that KCa channel activation and subsequent smooth muscle hyperpolarization is an important mediator of initial flow-induced dilation in skeletal muscle arterioles. The greater contribution of these channels in gastrocnemius muscle arterioles may underlie the more rapid and pronounced dilation that occurs in these arterioles (compared with soleus muscle arterioles) at the onset of exposure to flow, i.e., flow exposure <2 min. Prevention of flow-induced vasodilation by membrane depolarization with 40 mM KCl and by KCa channel blockade suggests that endothelium-derived hyperpolarizing factor (EDHF) may participate in this response. EDHF has been identified as a significant contributor to flow-induced vasodilation in coronary arterioles and in gracilis muscle arterioles from mice lacking the endothelial NOS pathway (8, 24). We have not specifically demonstrated a role for EDHF in mediating either the immediate or sustained response to flow; however, our results do indicate that membrane hyperpolarization through activation of K+ channels participates in the response.
In conclusion, the results of this study indicate that the time course and magnitude of flow-induced vasodilation vary between arterioles from soleus and gastrocnemius muscles. Endothelial factors that contribute to flow-induced vasodilation differ with the time course of exposure to flow. NO formation contributes heavily to sustained flow-induced vasodilation. The initial vasodilatory response to flow involves K+ channel activation, and the role of NO in mediating the initial vasodilatory response may be regulated by PGI2 production. Sustained exposure to flow in the absence of NO triggers production of endothelin and subsequent constriction. The coordinated response to flow involves both vasoconstrictor and vasodilator mechanisms, and the contributions of these mechanisms vary with the time course of exposure to flow.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 min) in a subset of soleus and gastrocnemius muscle arterioles matched for size and degree of spontaneous tone. Spontaneous tone development was 48 ± 2% and 42 ± 3% in soleus (177 ± 1 µm) and gastrocnemius (174 ± 2 µm) muscle arterioles, respectively. Total dilation was significantly greater in gastrocnemius vs. soleus muscle arterioles (P < 0.05).
