INTRODUCTION

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THE HUMAN CARDIOVASCULAR SYSTEM is exquisitely adapted for maintaining arterial pressure and cerebral perfusion in the upright posture on Earth. However, when the force of gravity is removed during spaceflight, there is a cephalic fluid shift and an elimination of the head-to-foot hydrostatic pressure gradient (39, 41). The changes in the fluid volume and hydrostatic pressure distribution are thought to elicit adaptations within the cardiovascular system, which are subsequently rendered inappropriate on return to the Earth's gravitational environment (41). These microgravity-induced alterations of the cardiovascular system are primarily manifested as a diminished aerobic capacity (34, 41) and orthostatic intolerance (41). Although it is clear that there are several factors that contribute to the postflight orthostatic intolerance (41), one of the most prominent is orthostatic hypotension. The preponderance of recent evidence indicates that microgravity-induced orthostatic hypotension results from the body's inability to adequately elevate peripheral vascular resistance (2, 24).

To study these phenomena on Earth, the tail-suspended hindlimb-unloaded (HU) rat has been used to simulate the effects of microgravity. This model induces the cephalic fluid shift (14, 21, 30) and postural muscle unloading (26) that occur in microgravity. In addition, the HU animals manifest many of the cardiovascular adaptations that are characteristic of exposure to microgravity in humans, including a reduced aerobic capacity (12, 29), hypovolemia (25, 36), resting and exercise tachycardia (22), orthostatic hypotension (20), and a diminished capacity to elevate vascular resistance (22, 28, 45). Previous work with conduit arteries and skeletal muscle arterioles from HU rats indicates that at least part of the inability to elevate peripheral vascular resistance results from a blunting of myogenic autoregulation (5), a diminished responsiveness to vasoconstrictor stimuli (5, 9, 31), and a structural remodeling of some (7), but not all (43), vascular beds.

The inability to elevate peripheral vascular resistance has also been hypothesized to be the result of an enhanced vasodilatory responsiveness of the resistance vasculature. For example, an upregulation of vascular ₂-adrenergic receptors and the associated vasodilation could give rise to orthostatic hypotension. In support of this notion are studies that have reported an enhanced vascular -adrenergic receptor responsiveness after bed rest (3) and reported that the -receptor antagonist propranolol prevents presyncopal symptoms induced by tilt tests after bed rest (35). Therefore, the purpose of the present study was to test the hypothesis that ₂-adrenergic receptor-mediated vasodilation is enhanced in the resistance vasculature of hindlimb skeletal muscles from HU rats. Furthermore, we hypothesized that metabolite-induced (adenosine) and nitric oxide-mediated (sodium nitroprusside) vasodilation would also be augmented in skeletal muscle arterioles with simulated microgravity. To test these hypotheses, first-order arterioles were isolated from the soleus and superficial portion of gastrocnemius muscles to determine whether the effects of simulated microgravity on vasodilatory responsiveness vary according to muscle fiber composition.

	METHODS

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Animals. All procedures performed in this study were approved by the Texas A&M University Institutional Animal Care and Use Committee and conform to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, Bethesda, MD 20892].

Microvessel preparation and experimental design. With the use of a stereomicroscope, the feed arteries leading to the soleus and superficial portion of the gastrocnemius muscles were identified and isolated from each rat. At the point where the feed artery entered the muscle, the surrounding muscle fibers were carefully dissected away and a first-order (1A) arteriole was isolated. Isolated 1A arterioles were transferred to a Lucite vessel chamber containing PSS-albumin. One end of the arteriole was cannulated with a glass micropipette filled with filtered PSS-albumin solution and tied securely to the pipette with 11-0 nylon ophthalmic suture. The other end of each vessel was cannulated with a second micropipette and secured with suture.

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Data analysis. Student's t-tests were used to determine the significance of differences in the body mass, soleus muscle mass, the soleus muscle-to-body mass ratio, developed spontaneous tone, maximal vessel diameter, and maximal agonist-induced relaxation between control and HU groups. Vasodilatory responses were recorded as actual diameter (µm) and are presented relative to the possible change in diameter to normalize for differences between groups in starting diameter or maximal passive diameter. The percentage of maximal dilation was calculated according to the formula
where D is the measured diameter, D_i is the initial diameter recorded immediately before addition of the vasodilator agent, and D_m is the maximal diameter recorded. The vasodilator concentration-response curves were evaluated using repeated-measures ANOVA with one-within (agonist concentration) and one-between (experimental groups) factors. Planned contrasts were conducted at each molar concentration level to determine whether differences existed between groups. Vascular sensitivity (IC₅₀) was designated as the agonist concentration that produced 50% of the maximal vasodilator response. All IC₅₀ values were converted to log values for statistical comparison. Student's t-tests were used to determine whether differences in IC₅₀ values were significant between groups. All values are presented as means ± SE. A P < 0.05 was required for significance.

	RESULTS

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Body and soleus muscle mass. The body masses of control (427 ± 10 g) and HU (402 ± 7 g) rats were not different. Hindlimb unloading reduced soleus muscle mass from HU rats (109 ± 8 mg) relative to that of control animals (203 ± 9 mg). Similarly, the soleus-to-body mass ratio of HU rats (0.32 ± 0.02 mg/g) was lower than that of control animals (0.52 ± 0.03 mg/g). Soleus muscle atrophy, which is characteristic of reduced skeletal muscle weight-bearing activity, confirms the effectiveness of the hindlimb unloading intervention.

Vessel characteristics. The maximal intraluminal diameter of arterioles from the superficial portion of gastrocnemius muscle was similar between control and HU rats (Table 1). However, hindlimb unloading decreased the maximal diameter of soleus muscle arterioles (Table 1). Spontaneous tone developed before determination of the vasodilator concentration-response relationships was not different between soleus muscle arterioles from control and HU animals, but less spontaneous tone was developed in gastrocnemius muscle arterioles from HU rats (Table 1).

Vasodilator responses. Increases in the concentration of isoproterenol produced dose-dependent increases in vessel diameter of gastrocnemius and soleus (Fig. 1) muscle arterioles. Hindlimb unloading had no effect on these responses or on the maximal vasodilatory responses to isoproterenol (Table 1). Adenosine produced concentration-dependent increases in gastrocnemius and soleus muscle arteriolar diameters (Fig. 2). Vasodilatory responses induced by adenosine in gastrocnemius muscle arterioles from HU animals were less than those from control rats (Fig. 2; Table 1). Hindlimb unloading did not alter the vasodilatory response of soleus muscle arterioles along the range of adenosine concentrations (Fig. 2); however, the maximal vasodilation elicited by adenosine was lower in soleus muscle arterioles from HU rats (Table 1). Sodium nitroprusside induced dose-dependent increases in vessel diameter (Fig. 3). Hindlimb unloading reduced sodium nitroprusside-mediated vasodilation in both the gastrocnemius and soleus muscle arterioles (Fig. 3; Table 1). Hindlimb unloading did not alter the sensitivity of arterioles from either soleus or gastrocnemius muscles to any of the three vasodilators tested (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Dose-response relationship of superficial gastrocnemius muscle (A) and soleus muscle (B) first-order arterioles to isoproterenol. Values are means ± SE. For white gastrocnemius muscle arterioles, n = 10 and 9 rats in control and hindlimb unloaded (HU) groups, respectively. For soleus muscle arterioles, n = 9 and 10 rats in control and HU groups, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Dose-response relationship of superficial gastrocnemius muscle (A) and soleus muscle (B) first-order arterioles to adenosine. Values are means ± SE. For white gastrocnemius muscle arterioles, n = 10 and 9 rats in control and HU groups, respectively. For soleus muscle arterioles, n = 9 and 10 rats in control and HU groups, respectively. * Significant difference at P < 0.05. Repeated-measures ANOVA indicated that adenosine dose-response curves for soleus muscle arterioles were not different between groups. However, maximal dilation to adenosine was significantly lower in soleus muscle arterioles from HU rats (P < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Dose-response relationship of superficial gastrocnemius muscle (A) and soleus muscle (B) first-order arterioles to sodium nitroprusside. Values are means ± SE. For white gastrocnemius muscle arterioles, n = 10 and 9 rats in control and HU groups, respectively. For soleus muscle arterioles, n = 9 and 10 rats in control and HU groups, respectively. * Significant difference at P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Maximal vasodilatory response (A) and vascular sensitivity (IC50; B) to isoproterenol (Iso), adenosine (Ado), and sodium nitroprusside (SNP) in first-order (1A) arterioles from soleus and the superficial portion of gastrocnemius muscles of normal cage control rats. * Significant difference at P < 0.05.

	DISCUSSION

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The purpose of the present study was to test the hypothesis that vasodilation mediated through ₂-adrenergic receptor (isoproterenol), metabolite (adenosine), and nitric oxide (sodium nitroprusside) mechanisms is enhanced in the resistance vasculature of skeletal muscles with simulated microgravity. The data indicate that hindlimb unloading does not augment vasodilation of first-order arterioles from skeletal muscles composed primarily of slow-twitch (soleus muscle) or fast-twitch (superficial gastrocnemius muscle) fibers. On the contrary, hindlimb unloading diminished the maximal dilatory response elicited by adenosine and exogenous nitric oxide in arterioles from both the soleus and gastrocnemius muscles. These results suggest that orthostatic hypotension and the associated inability to adequately elevate peripheral vascular resistance is not the result of an enhanced vasodilatory responsiveness of the skeletal muscle resistance vasculature. However, the data do indicate that microgravity-induced reductions in aerobic capacity may be related to the diminished vasodilatory capacity of skeletal muscle arterioles.

Orthostatic intolerance after return to Earth from short-duration habitation in microgravity does not appear to be the result of a single factor. For example, orthostatic hypotension, one potential cause of orthostatic intolerance, may result from hypovolemia (c.f., Ref. 41) and inadequate elevations in stroke volume (c.f., Refs. 41, 42). However, there is convincing evidence from spaceflight and bed-rest studies that one of the primary factors in orthostatic hypotension is an inability to adequately elevate peripheral vascular resistance (2, 24, 40). This inability is not necessarily the result of a sympathetic neural dysfunction because it has been reported that both supine and standing plasma norepinephrine concentrations are actually elevated after flight (42). Thus there is reason to believe that the inability to adequately elevate peripheral resistance in humans is the result of an end-organ (vascular) deficit.

Cardiovascular alterations induced by microgravity have been simulated using the HU rat. HU rats manifest many of the cardiovascular adaptations that are characteristic of exposure to microgravity in humans, including orthostatic hypotension (20), hypovolemia (25, 36), and resting and exercise tachycardia (22). In addition, these animals possess a diminished capacity to elevate vascular resistance in fast-twitch skeletal muscle and visceral tissue (22, 28, 45). In these vascular beds, the inability to sufficiently elevate vascular resistance appears to result, at least in part, from deficits in myogenic autoregulation (5, 19) and vasoconstrictor responsiveness (5, 28). The mechanism of the reduced autoregulatory and vasoconstrictor responsiveness in visceral tissue is presently unknown but does not appear to be the result of structural alterations in the resistance vasculature (43). However, the diminished autoregulatory and vasoconstrictor responsiveness of the skeletal muscle microvasculature appears to result from smooth muscle atrophy and a corresponding thinning of the arteriolar wall (7). Results from the present study indicate that the inability of skeletal muscle resistance vessels to adequately elevate vascular resistance is not the result of an enhanced vasodilatory responsiveness. Similar vascular alterations to those of the HU rat have been described in humans after bed rest, i.e., diminished forearm vasoconstrictor and vasodilator responsiveness during a cold pressor test and reactive hyperemia, respectively (37). Thus these two models of microgravity produced similar vascular deficits in humans and animals; both models support the contention that microgravity induces a functional and, in some cases, a structural remodeling of the resistance vasculature.

Although alterations in the intrinsic vasodilatory properties of skeletal muscle arterioles do not appear to play a role in orthostatic hypotension, they may contribute to the hindlimb unloading-induced reduction in aerobic capacity (12, 29). Among the various potentially limiting factors of maximal oxygen consumption (O_2 max), the peripheral circulation is an important variable (32). Appropriate distribution of cardiac output among active and inactive tissues during exercise is paramount. Thus any microgravity-induced adaptation serving to diminish skeletal muscle perfusion or enhance visceral perfusion during exercise would lower O_2 max.

The vasodilators eliciting diminished responses in the skeletal muscle arterioles from HU rats were adenosine and sodium nitroprusside. Adenosine is a potent vasodilator that is formed in muscle cells with the hydrolysis of ATP during exercise (10). It readily diffuses from the intracellular compartment into the interstitium and binds with A₂ receptors on smooth muscle cells to induce relaxation via an adenylate cyclase mechanism. The reduction in maximal dilation induced by adenosine in isolated arterioles from soleus and gastrocnemius muscles of HU rats suggests that the resistance vasculature in muscles from these animals is less responsive to metabolite-mediated vasodilation, which is considered the primary mechanism for increasing skeletal muscle perfusion during exercise (10).

Sodium nitroprusside, an exogenous nitric oxide donor, similarly elicited a lower maximal dilation in arterioles from soleus and gastrocnemius muscles of HU rats. During exercise, local release of nitric oxide could originate from the vascular endothelium (33), hemoglobin (38), skeletal muscle cells (1), and nitroxidergic neurons (4). Previous investigators have utilized acetylcholine to determine the effects of hindlimb unloading on endothelium-dependent dilation to endogenous nitric oxide (6, 7, 17). These investigators reported reductions in acetylcholine-mediated vasodilation in the abdominal aorta (6) and skeletal muscle resistance arteries with hindlimb unloading (7, 17) but only in resistance arteries from the soleus muscle (7, 17). The diminished acetylcholine-induced dilation of soleus muscle arterioles appears to be associated with chronic reductions in blood flow and shear stress in HU rats (7) and is likely mediated through a reduced expression of endothelial cell nitric oxide synthase (ecNOS) (17).

Although there is general agreement in the literature concerning the effects of hindlimb unloading on acetylcholine-mediated vasodilation, the effects of unloading on vasodilation elicited through the exogenous nitric oxide donor sodium nitroprusside are less clear. For example, we have shown that hindlimb unloading increased maximal vasodilation induced by sodium nitroprusside in abdominal aortas preconstricted with 10⁴ M norepinephrine with no change in sensitivity (9). In contrast, abdominal aortas preconstricted with a lower dose of norepinephrine (10⁷ M) exhibited reduced sodium nitroprusside sensitivity with no change in maximal vasodilation (6). In the present study, we found that hindlimb unloading diminished maximal vasodilatory responses to sodium nitroprusside in gastrocnemius and soleus muscle arterioles (Fig. 3) with no change in sensitivity (IC₅₀, Table 1). Conversely, Jasperse et al. (17) reported an increased sensitivity to sodium nitroprusside with no change in maximal vasodilation in soleus muscle feed arteries from 2-wk-old HU rats. The reasons for such disparate results are presently unknown but may be related to the dynamic state of the vasculature, in particular that of soleus muscle, during the first several weeks of unloading. During the initial period of unloading (i.e., 10 min), soleus muscle blood flow and vascular shear stress are diminished (7, 22). Although soleus muscle blood flow remains low as long as the postural soleus muscle is unloaded (22), by 2 wk of unloading the soleus muscle resistance arteries have structurally remodeled so that the maximal diameter or luminal cross-sectional area of the vessels is smaller (present study and Refs. 5, 7, and 17) and shear stress has returned to control standing levels (7). Alterations in vascular wall shear stress have been shown to be a potent stimulus for adaptations in endothelium-mediated dilation and ecNOS expression (23, 27, 44). For example, it requires 4 wk for an exercise-induced hyperemia to enhance acetylcholine-elicited, nitric oxide-mediated vasodilation in rat aorta, which precisely corresponds to the time course of ecNOS protein upregulation (11). A similar adaptation appears to occur with hindlimb unloading, in which 2 wk of unloading produce a diminished endothelium-mediated dilation (7, 17) and ecNOS expression (17) that follow the initial decrease in wall shear stress. By 4 wk of unloading, endothelium-mediated dilation has again tracked the change in shear stress and returned to control standing levels (7). Thus the decrease and increase in endothelial cell release of nitric oxide may modulate the smooth muscle cell response to nitric oxide and, therefore, partially explain differences in vascular responses to sodium nitroprusside in HU rats. However, this does not appear to be the entire explanation, since sodium nitroprusside-induced relaxation was also diminished in arterioles from the gastrocnemius muscle of HU rats, a vascular bed that does not experience decreases in blood flow, calculated vascular shear stress, or endothelium-mediated dilation (7, 22).

The mechanism for the reduced sodium nitroprusside-mediated vasodilatory responsiveness could result from various factors, including decreases in cGMP production, increases in phosphodiesterase activity and subsequently the breakdown of cGMP, or decreases in the responsiveness of the smooth muscle cells to intracellular cGMP. The diminished dilatory responsiveness induced by adenosine is likely to involve alterations in the A₂ receptors on smooth muscle because alterations in the cAMP signal transduction mechanism would likely have affected the vasodilation induced by isoproterenol as well. Further experimentation will be necessary to determine which of these or other possibilities accounts for the hindlimb unloading-induced decreases in vasodilatory responsiveness of skeletal muscle arterioles.

Regardless of the mechanism, the collective findings from this study and others demonstrate that hindlimb unloading results in a diminished responsiveness of skeletal muscle arterioles to select vasodilators (present study and Refs. 7 and 17), a reduced vasodilatory capacity (i.e., maximal diameter) of arterioles from highly oxidative skeletal muscle (present study and Refs. 5, 7, and 17), and a diminished vasoconstrictor capacity in low oxidative muscle (5) and visceral tissue (19, 28). These vascular alterations appear to underlie the seemingly deleterious effects of simulated microgravity on the distribution of cardiac output during exercise, in which blood flow to highly oxidative muscle is lower and blood flow to visceral tissue and low oxidative muscle is greater (22, 45). Thus reductions in aerobic capacity may result from the imprecise matching of oxygen delivery to the oxidative capacity of muscle, a consequence of functional and structural vasculature alterations induced by simulated microgravity.

We examined arterioles from soleus (predominantly type I fibers) and the superficial portion of the gastrocnemius (predominantly type IIb fibers) muscles (8) because of the possibility that unloading would differentially affect the vasodilatory responses of arterioles from muscles composed of different fiber types. For example, several studies provide evidence that vascular responses and release of vasoactive substances differ in muscles of varying fiber composition. It has been shown that fast-twitch muscle shows greater vasodilation in response to epinephrine infusion, a ₂-adrenergic receptor-mediated dilation (15). In addition, slow-twitch muscles preferentially release adenosine during low-intensity exercise (18), and nitric oxide synthase inhibition preferentially affects the exercise hyperemia in highly oxidative muscle (16). Although hindlimb unloading produced similar alterations in the vasodilatory responses of arterioles from soleus and gastrocnemius muscles, intrinsic differences in the relaxation responses between soleus and gastrocnemius muscle arterioles from control animals were evident (Fig. 4). Similar to the findings of Hilton et al. (15), we found greater maximal vasodilation to the ₂-receptor agonist isoproterenol in gastrocnemius muscle arterioles and, additionally, greater maximal relaxation to adenosine. Gastrocnemius muscle arterioles were also more sensitive (i.e., lower IC₅₀) to isoproterenol, adenosine, and sodium nitroprusside than arterioles from soleus muscle. Furthermore, concentration-response data generated in a previous study (7) demonstrate that maximal responsiveness to acetylcholine is greater in isolated soleus muscle arterioles (soleus: 89 ± 5, gastrocnemius: 74 ± 10% relaxation). These data indicate that dilators acting directly on smooth muscle cells produce a more potent vasodilatory response in vessels from fast-twitch muscle and that dilators acting through the endothelium produce a more potent vasodilation in arterioles from slow-twitch muscle. We speculate that the greater vasodilatory responsiveness of arterioles from fast-twitch muscle to agents acting directly on the smooth muscle may result from the fact that these muscles are only recruited during high-intensity exercise (c.f., Ref. 8). Thus the resistance vasculature in this muscle type is seldom exposed to vasodilatory stimuli. Conversely, arterioles from tonically active slow-twitch muscles are chronically exposed to vasodilatory metabolites. Endothelium-dependent vasodilation, on the other hand, is closely linked to blood flow and maintaining vascular shear stress (11, 23, 27, 44). The high perfusion rate of slow-twitch muscle presumably enhances this mechanism of vasodilation, whereas the low blood flow rate to fast-twitch muscle provides little stimulus for endothelium-mediated vasodilation to be upregulated. These observations serve to emphasize the heterogeneity of microvascular blood flow control mechanisms and suggest that the mechanisms of vasodilation present in skeletal muscle arterioles are linked to the physical and chemical environment produced by or associated with the surrounding muscle fibers.

In summary, the purpose of this study was to test the hypothesis that vasodilation elicited through a ₂-adrenergic receptor-mediated pathway, a metabolite (adenosine), and a nitric oxide mechanism is enhanced by simulated microgravity in the skeletal muscle resistance vasculature. The results indicate that hindlimb unloading does not augment vasodilation mediated through any of these mechanisms in first-order arterioles from either a slow-twitch (soleus) or fast-twitch (superficial gastrocnemius) muscle. Rather, hindlimb unloading diminishes the maximal dilatory response elicited by adenosine and exogenous nitric oxide in arterioles from both these muscle types. These results suggest that orthostatic hypotension and the associated inability to adequately elevate peripheral vascular resistance are not the result of an enhanced vasodilatory responsiveness of the skeletal muscle resistance vasculature. Furthermore, the data indicate that microgravity-induced reductions in aerobic capacity may be related to the diminished vasodilatory responsiveness and capacity of skeletal muscle arterioles. Finally, the results demonstrate that vasodilatory responsiveness mediated through these various mechanisms differs in muscles composed of different fiber types, thus illustrating the heterogeneity of control mechanisms regulating skeletal muscle blood flow.