Skeletal muscle contractions cause a large increase in blood flow to the active skeletal muscle. Despite a long history of research, the exact mechanism that matches the level of blood flow to the metabolic demand of the skeletal muscle is not fully understood (10,13). The contraction-induced release of vasoactive skeletal muscle metabolites has been put forward as a potential link between both events (“metabolic theory”) (6). Relevant metabolites released during exercise are, among others, inorganic phosphate (7), magnesium (26), hydrogen (3), adenosine (24), lactate (20), and potassium (K+) (12). In a previous study, we have shown that raising the K+ concentration with only 1 mM induces a strong vasodilatation through an endothelium-independent mechanism (2). Moreover, the cumulative release of vasoactive metabolites causes a pronounced increase in local osmolarity (“hyperosmolarity,” HO), which as such is an independent, vasoregulatory factor in skeletal muscle arterioles of both man (16) and animals (17,27).
Although most metabolites released during exercise might have a potential role in the exercise hyperemia, none of them can on its own account for the whole exercise-induced hyperemia (6,10). In the early 1970s, Skinner and Costin (28) demonstrated a synergism between several vasoactive metabolic factors, such as hypoxia, K+, and HO, in perfused dog isolated gracilis muscles. It was suggested that the presence of one factor significantly increases the sensitivity of the skeletal muscle arterioles for another metabolic factor. In the present study, we considered the possibility that exercise hyperemia results from the combined action of two (or more) mechanisms, that is, the rise in K+ concentration and HO. The aim of the present study is to identify the influence of a hyperosmotic environment on K+-induced relaxations in small isolated rat gluteal arteries. As we found a sensitizing effect of HO on the K+-induced relaxations, we investigated the potential involvement of the endothelium, KATP channels, Na+/K+ ATPase, KIR channels, and volume regulated anion channels (VRAC).
MATERIALS AND METHODS
This study was approved by the local ethics committee for animal experimentation of the Faculty of Medicine and Health Science (Ghent University) and was in adherence with the ACSM animal care standards. Experiments were performed on gluteal skeletal muscle arteries from adult female Wistar rats (180–285 g), previously killed by stunning and cervical dislocation.
Small gluteal arteries (ranging between 101 and 322 μm in diameter) were dissected and stored in ice-cold Krebs-Ringer bicarbonate (KRB) solution. Surrounding connective tissue and neighboring veins were carefully removed from the specimen. Segments of the artery (length ± 2 mm) were put into an organ bath containing 10 mL KRB solution and mounted in a myograph for isometric tension recording. Two stainless steel wires (40 μm in diameter) were guided through the lumen of the segments: one wire was connected to a force-displacement transducer, the other to a micrometer. Thereafter, the vessels equilibrated in oxygenated (95% O2 and 5% CO2) and heated (37°C) KRB solution (pH 7.4) for approximately 30 min. Next, they were stretched to an optimal lumen diameter, which corresponds to 90% of the diameter when passive transmural pressure is 100 mm Hg (21). Vessels were then contracted twice by adding 120 mM K+ and 10−5 M norepinephrine (NOR).
Removal of the endothelium.
An L-shaped micropipette was positioned at the proximal side of the segment. Gas containing 95% O2–5% CO2 was bubbled through the vessel lumen for 2 min. Thereafter, segments were allowed to equilibrate for 30 min. Removal of the endothelium was confirmed by the absence of a relaxing response to acetylcholine (ACh).
The experiments were performed using a KRB solution with following composition (mM): NaCl 135, KCl 5, NaHCO3 20, glucose 10, CaCl2 2.5, MgSO4 1.3, KH2PO4 1.2, and EDTA 0.026. Modified KRB solution containing 120 mM K+ (K120) was prepared by equimolar replacement of NaCl with KCl. HO was obtained by adding 30 mM (S30) or 60 mM of sucrose (S60), mannitol (M60) or urea (U60) to the KRB solution. Norepinephrine bitartrate, acetylcholine chloride, glibenclamide (Glib), 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), mannitol, indomethacin (Indo), Nω-nitro-L-arginine (L-NA), and ouabain were purchased from SIGMA (St. Louis, MO). Barium chloride (Ba2+), sucrose, sodium nitroprusside (SNP), and potassium chloride were obtained from MERCK (Darmstadt, Germany). Urea was purchased from J. T. Baker chemical (Phillipsburg, U.S.). All solutions were water based, except for NPPB and glibenclamide, which were dissolved in DMSO.
Control and experimental protocols were always performed on the same blood vessels to obtain paired observations. In all experiments, the vessels were precontracted by adding 10−6 M NOR, which elicits approximately 80% of the maximum contraction. ACh concentration-response curves (from 10−9 to 10−5 M) were made by cumulatively adding higher concentrations to the organ bath. K+ concentration-response curves were made by adding only one concentration of K+ (1 mM (K1), 2 mM (K2), or 3 mM (K3)) to the precontracted artery. The maximum relaxation was used for calculations. After 5 min, the solution was removed and replaced by fresh KRB solution. Thereafter, the preparations were precontracted again and a higher concentration of K+ was added. In some experiments, preparations were incubated for a period of 10 min (10−5 M NPPB, 3 × 10−5 M Ba2+, 10−4 M L-NA and 5 × 10−5 M ouabain), 15 min (S30, S60, M60 or U60), or 20 min (10−5 M glibenclamide and 10−5 M indomethacin) before precontraction and subsequent addition of K+. All concentrations are expressed as final molar concentrations in the organ bath.
All values shown are mean ± standard error of mean (SEM). Relaxations are expressed as a percentage change (relaxation) in tone developed by NOR. A two-way repeated measures analysis (protocol × concentration) was used to evaluate statistical significance in the K+ experiments. Follow-up univariate tests were used to assess the statistical significance between corresponding control and experimental group for a particular concentration of K+. Statistical analysis was performed using SPSS (version 11.0). Statistical significance was defined as P < 0.05; N indicates the number of arteries tested.
Characteristics of the arteries.
The gluteal arteries used in this study had a mean intraluminal diameter of 245 ± 6 μm (N = 68). Precontraction of the arteries with NOR (10−6 M) evoked a mean sustained increase in isometric force of 10.92 ± 0.47 mN (N = 68). The cumulative addition of increasing concentrations of ACh significantly (P < 0.001) relaxed all blood vessels (mean maximum relaxation of 72.28 ± 2.16% at 10−6 M; N = 68). Arteries failing to relax at least 50% in response to ACh were excluded from this study because of potential damage to the endothelium.
Potassium-induced relaxations in control and HO conditions.
Application of 1, 2, and 3 mM K+ evoked significant (P < 0.001, N = 41) concentration-dependent vasodilatations in all gluteal arteries tested (K1: 31.04 ± 3.54%; K2: 56.16 ± 3.74%; K3: 76.75 ± 3.56%). To incubate the blood vessels in a moderate or a high HO environment, respectively, 30 mM (S30) or 60 mM (S60) sucrose was added to the KRB solution. The application of sucrose did not alter the baseline tension (0.04 ± 0.31 mN; NS; N = 18) nor the contraction force in response to NOR (“Control”: 9.84 ± 0.56 mN; “S60”: 10.64 ± 0.70 mN; NS; N = 18). In the moderate HO condition, the K+-induced relaxations were significantly increased (F = 28.18, P < 0.005, N = 6) compared with control conditions (“Control”: K1: 28.55 ± 10.12%; K2: 59.74 ± 6.53%; K3: 83.51 ± 4.34%; “S30”: K1: 56.70 ± 6.80%, P < 0.01; K2: 74.41 ± 7.86%, NS; K3: 90.88 ± 3.15%, NS). This effect was more pronounced in high HO conditions in which relaxations to K1, K2, and K3 were all significantly increased (F = 251.05, P < 0.001, N = 6) in the presence of a high concentration of sucrose (“Control”: K1: 18.22 ± 6.38%; K2: 66.50 ± 8.55%; K3: 52.07 ± 10.40%; “S60”: K1: 62.97 ± 9.51%, P < 0.05; K2: 92.38 ± 2.17%, P < 0.05; K3: 93.56 ± 3.70%, P < 0.05) (Fig. 1A). In addition, the sensitizing effect of HO is relatively specific since neither the ACh-induced EDHF-response (“Indo–L-NA”: 10−9 M: 9.27 ± 2.65%; 10−8 M: 26.45 ± 1.51%; 10−7 M: 52.17 ± 6.04%; 10−6 M: 45.42 ± 15.25%; 10−5 M: 41.24 ± 13.92%; “Indo–L-NA–S60”: 10−9 M: 9.59 ± 2.48%; 10−8 M: 28.57 ± 11.37%; 10−7 M: 46.03 ± 10.65%; 10−6 M: 51.24 ± 14.69%; 10−5 M: 56.34 ± 13.78%; F = 0.405, NS, N = 4, Fig. 1B), nor the relaxation induced by the NO–donor SNP (“Control”: 10−9 M: 9.60 ± 3.11%; 10−8 M: 35.03 ± 7.95%; 10−7 M: 72.70 ± 6.27%; 10−6 M: 86.40 ± 4.43%; 10−5 M: 95.18 ± 0.82%; “S60”: 10−9 M: 1.88 ± 0.62%; 10−8 M: 20.25 ± 4.02%; 10−7 M: 68.20 ± 3.96%; 10−6 M: 82.60 ± 3.25%; 10−5 M: 90.40 ± 5.26%; F = 1.86, NS, N = 4, Fig. 1C) were significantly increased on HO preincubation.
The sensitizing effect of the HO environment on K+-induced relaxations was largely reversible upon washout (30 min) (F = 0.10, NS, N = 4) (“Control-pre”: K1: 23.00 ± 12.94%; K2: 66.63 ± 9.63%; K3: 79.21 ± 6.03%; “S60”: K1: 72.98 ± 10.88%; K2: 95.05 ± 3.16%; K3: 97.23 ± 2.15%; “Control-post”: K1: 33.56 ± 8.37%; K2: 59.34 ± 15.13%; K3: 48.96 ± 21.25%).
In some experiments, 60 mM mannitol was used instead of sucrose. Similar to what was seen in the presence of sucrose, the K+-induced relaxations were also significantly increased (F = 15.44, P < 0.01, N = 6) in the presence of mannitol (“Control”: K1: 36.92 ± 8.50%; K2: 57.33 ± 8.60%; K3: 84.75 ± 5.07%; “Mannitol”: K1: 55.45 ± 7.08%, P < 0.05; K2: 79.21 ± 9.56%, P < 0.05; K3: 89.89 ± 4.98%, NS) (Fig. 2A).
In another set of experiments, 60 mM urea was used instead of sucrose. The presence of urea did not significantly alter the K+-induced relaxations (F = 0.318, NS, N = 6) (“Control”: K1: 30.86 ± 10.02%; K2: 60.43 ± 11.21%; K3: 60.32 ± 9.52%; “Urea”: K1: 27.27 ± 10.59%; K2: 53.30 ± 13.97%; K3: 77.65 ± 11.29%) (Fig. 2B).
K+-induced relaxations in HO conditions: underlying mechanisms.
In some experiments, the vascular endothelium was removed. Endothelium denudation (ED) was established by the presence of a largely reduced ACh-induced relaxation (4.39 ± 1.49% at 10−5 M; N = 7). Despite the absence of the endothelium, the K+-induced relaxations continued to be significantly increased (F = 24.48, P < 0.005, N = 7) in the presence of S60 compared with control conditions (“ED”: K1: 22.33 ± 7.43%; K2: 34.21 ± 5.65%; K3: 81.75 ± 5.47%; “ED-S60”: K1: 54.32 ± 13.67%, P < 0.05; K2: 64.72 ± 10.26%, P < 0.01; K3: 93.31 ± 5.82%, NS) (Fig. 3).
In some experiments, the ATP sensitive K+ channel (KATP channel) inhibitor glibenclamide (10 μM) was added to the organ bath (Fig. 4A). Glibenclamide did not significantly influence (F = 3.00, NS, N = 6) the K+-induced relaxations in isoosmotic control conditions (“Control”: K1: 38.21 ± 4.02%; K2: 65.43 ± 5.32%; K3: 85.44 ± 2.08%; “Glib”: K1: 58.90 ± 6.46%; K2: 69.27 ± 4.39%; K3: 82.75 ± 2.60%). In the presence of glibenclamide, the addition of S60 still evoked an increase in K+-induced relaxations (F = 98.39, P < 0.001, N = 6) (“Glib”: K1: 51.47 ± 8.78%; K2: 66.08 ± 10.81%; K3: 91.97 ± 2.58%; “Glib-S60”: 71.41 ± 9.70%, P < 0.05; K2: 83.88 ± 8.89%, P < 0.01; K3: 97.60 ± 1.63%, P < 0.05).
In two other sets of experiments, the Na+- K+ pump inhibitor ouabain (50 μM) or the Kir-channel inhibitor Ba2+ (30 μM) were added to the organ bath. As demonstrated in Figure 4B, ouabain had no inhibitory effect on the enhancement of the K+-induced relaxation by HO (F = 15.50, P < 0.05, N = 6) (“Ouabain”: K1: 10.87 ± 1.74%; K2: 37.52 ± 8.54%; K3: 60.95 ± 10.53%; “Ouabain-S60”: K1: 41.53 ± 8.67%, P < 0.05; K2: 73.95 ± 3.26%, P < 0.01; K3: 72.24 ± 5.05%, NS). In contrast, the application of Ba2+ totally abolished the sensitizing effect of HO in K1, K2, and K3 conditions (F = 1.02; NS; N = 6) (“Ba2+”: K1: 14.55 ± 6.10%; K2: 14.08 ± 3.46%; K3: 36.93 ± 5.87%; “Ba2+-S60”: K1: 18.84 ± 7.18%; K2: 25.91 ± 10.93%; K3: 40.22 ± 5.70%) (Fig. 4C).
K+-induced relaxations and the volume regulated anion channels (VRAC).
In these experiments the VRAC inhibitor NPPB was applied to the organ bath. In some arteries (6 of 20 blood vessels tested), this resulted in an abolishment of the precontraction force induced by NOR. In the other arteries, the contraction force was only slightly attenuated (“Control”: 12.15 ± 0.86 mN; “NPPB”: 10.34 ± 1.20 mN; NS). As seen in Figure 5, the K+-induced relaxations were significantly increased (F = 6.01, P < 0.05, N = 8) in the presence of NPPB compared with control conditions (“Control”: K1: 19.81 ± 5.82%; K2: 33.39 ± 8.22%; K3: 67.34 ± 11.18%; “NPPB”: K1: 54.54 ± 10.62%, P < 0.05; K2: 74.13 ± 8.57%, P < 0.005; K3: 67.21 ± 10.60%, NS).
The effect of NPPB on the K+-induced relaxations was largely reversible upon washout (30 min) (F = 0.57, NS, N = 6) (“Control-pre”: K1: 15.52 ± 4.16%; K2: 29.20 ± 12.99%; K3: 69.42 ± 15.54%; “NPPB”: K1: 55.49 ± 12.46%; K2: 70.67 ± 13.13%; K3: 71.84 ± 12.29%; “Control-post”: K1: 25.05 ± 11.79%; K2: 16.09 ± 2.06%; K3: 43.17 ± 18.09%).
In the presence of Ba2+, the sensitizing effect of NPPB was abolished (F = 0.458, NS, N = 6) (“Ba2+”: K1: 10.90 ± 2.63%; K2: 31.38 ± 4.39%; K3: 56.34 ± 4.89%; “Ba2+-NPPB”: K1: 15.45 ± 4.13%; K2: 27.69 ± 4.06%; K3: 61.11 ± 5.91%) (Fig. 6).
Several regulatory mechanisms of skeletal muscle perfusion have been proposed to participate in the exercise-induced hyperemic response, including neural, endothelial, myogenic, and metabolic mechanisms (4,10,13). The “metabolic theory” claims that active skeletal muscle fibers release vasorelaxing metabolites, such as K+, adenosine, and lactate, which increase the blood flow in adjacent arterioles (6). K+ ions are released from active skeletal muscle fibers and accumulate in the interstitial fluid from about 5 mM up to 9.5 mM when the intensity of exercise increases (11). In a previous study, we reported that a raise in K+ concentration of only 1 mM already induces a potent relaxation in small skeletal muscle arteries (2). This observation is confirmed in the present study: increasing the K+ concentration with 1, 2, or 3 mM resulted in a strong vasorelaxation of 30.50%, 55.43%, and 79.49%, respectively, supporting that K+ might be a potent mediator of the exercise hyperemia. However, the extent to which the increase in K+ concentration contributes to the exercise hyperemia is thought to be limited (30) and variable in the course of exercise (25% at the onset, about 65% during steady state) (12). Therefore, K+ alone cannot fully account for the exercise hyperemia.
It has also been suggested that the exercise-induced increase in osmolarity plays a role in exercise hyperemia (18). The accumulation of several metabolites during exercise rapidly increases osmolarity in the venous blood up to more than 40 mosmol·L−1 (15,16,19,25), implying that osmolarity in the skeletal muscle tissue itself might raise to even higher levels (16). Lundvall et al. (16) investigated the role of HO in the hyperemic response to forearm contractions in men and found a strong relationship between the increase in venous osmolarity and the concomitant raise in blood flow. Furthermore, Massett et al. (17) exposed isolated rat skeletal muscle arterioles to increasing concentrations of sucrose and mannitol and demonstrated significant, concentration-dependent vasodilations. However, HO cannot be fully responsible for the exercise hyperemia since increasing the osmolarity (by infusing sucrose, glucose, etc.) up to the level attained during exercise does not cause an increase in blood flow equivalent to the value observed during exercise hyperemia in both electrically stimulated gracilis dog (18) and calf cat skeletal muscle (25).
Because none of the vasorelaxing mechanisms alone accounts for the whole exercise hyperemia (6,10), we considered the possibility that the exercise hyperemia results from the combined action of two (or more) mechanisms, that is, the rise in K+ concentration and HO. Skinner and Costin (27,28) have demonstrated that a change in concentration of K+ or in osmolarity alters the vasoactive properties of the other simultaneously present metabolic factor, in other words that one mechanism sensitizes the blood vessel for the other. In the present study, we found a clear interaction between K+ and HO: the presence of 60 mM sucrose significantly sensitized the gluteal blood vessels to small increases in K+. Remarkably, the sensitizing effect was already present at lower concentrations of sucrose (30 mM), suggesting a physiological role of HO in the regulation of the exercise hyperemia. These results may clarify the role of different metabolites in the exercise hyperemia. Skeletal muscle contraction causes an accumulation of K+ in the interstitial space and, simultaneously, an increase in tissue osmolarity. Even though the changes in K+ concentration are modest during moderate exercise, in interplay with a simultaneous increase in osmolarity, it may importantly contribute to the magnitude of the vasodilation.
The sensitizing effect is not sucrose-specific because increasing osmolarity with mannitol evoked a similar increase of the K+-induced relaxations. Additionally, the lack of a sensitizing effect in the presence of urea, which freely diffuses through the cell membrane, suggests that the influence of sucrose and mannitol can be attributed to hypertonicity, rather than hyperosmolarity as such.
Hypertonic solutions induce cell shrinkage, which might cause a deformation of some membrane structures (e.g., ion channels, ion transport systems) that might become more or less active (8,14). Massett et al. (17) investigated the vasorelaxing effect of HO on rat skeletal muscle arterioles and concluded that an increase in osmolarity acts directly on endothelial KATP channels causing a vasodilation. Also, other studies on cerebral and coronary blood vessels indicate that HO directly influences the endothelium (1,9). Therefore, in the present study, the endothelial layer was removed to find out whether endothelial cells are involved in the sensitizing effect of HO on K+-induced relaxations. Removal of the endothelium has no effect on the sensitizing effect of HO, suggesting that the increase in osmolarity exerts its effect directly on the smooth muscle cells. Further, we investigated the role of the KATP channels in the sensitizing effect of HO. In the blood vessels used in this study, the presence of glibenclamide did not inhibit the sensitizing effect of HO, suggesting no role for the KATP channels.
It is well known that an increase in osmolarity blocks VRAC, and more specifically, the volume regulated chloride channels (23,29) that are present on vascular smooth muscle cells (31). Therefore, we investigated whether a pharmacological inhibition of those channels with NPPB could mimic the sensitization of HO on the K+-induced relaxations. In the presence of NPPB, the K+-induced relaxations were significantly larger. This suggests that a blockade of the VRAC might be involved in the sensitization of K+-induced relaxations by HO. Doughty et al. (5) reported a similar sensitization in mesenteric arteries: both the application of sucrose and NPPB evoked stronger relaxations compared with the control conditions. However, it should be noted that NPPB has nonspecific inhibitory effects on other ion channels such as Ca2+ sensitive K+ channels and L-type Ca2+ channels. It is not clear, however, what effect hyperosmolarity has on these channels.
Raising the extracellular K+ concentration causes a vasodilation in skeletal muscle arterioles via two independent mechanisms, both located on the smooth muscle cells in skeletal muscle arterioles (2). The addition of small amounts of K+ evokes an activation of the Na+–K+ pumps and stimulates the inward rectifying K+ channels (Kir-channels), thereby causing a smooth muscle hyperpolarization and relaxation. To elucidate the role of these mechanisms in the sensitizing effect of HO on the K+-induced relaxations, we repeated the experiments in the presence of ouabain, a Na+- K+ ATPase inhibitor, and Ba2+, a Kir-channel blocker. In the presence of ouabain, a significant sensitization was still observed, suggesting no role for the Na+-K+ pump in the sensitization. Remarkably, Ba2+ abolished the stimulating effect of HO on the K+-induced relaxations, indicating a significant role for the Kir-channels.
As already mentioned, the similarity of the sensitizing influence of NPPB and HO suggests that a blockade of the VRAC might be involved. To further validate this hypothesis, we investigated whether inhibition of the Kir-channels also influences the NPPB-induced sensitization of K+-induced relaxations. Indeed, we found that Ba2+ significantly abolished this effect.
From all these results, we hypothesize that HO inhibits the VRAC on the smooth muscle cells, decreasing the outward Cl− current and thus inducing a limited hyperpolarization. This hyperpolarization sensitizes the Kir-channels, which are known to be more sensitive in the presence of more negative membrane potentials (22). As a result, activation of these channels with K+ therefore evokes a larger relaxation in HO conditions than in isoosmotic conditions.
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