Address for reprint requests and other correspondence: F. A. Dinenno, Colorado State University, 220 Moby-B Complex, Fort Collins, CO 80523-1582 (e-mail: ude.etatSoloC@onneniD.knarF).
Received 2018 Feb 22; Revised 2018 Apr 30; Accepted 2018 May 1. Copyright © 2018 the American Physiological SocietyThe increase in interstitial potassium (K + ) during muscle contractions is thought to be a vasodilatory signal that contributes to exercise hyperemia. To determine the role of extracellular K + in exercise hyperemia, we perfused skeletal muscle with K + before contractions, such that the effect of any endogenously-released K + would be minimized. We tested the hypothesis that local, intra-arterial infusion of potassium chloride (KCl) at rest would impair vasodilation in response to subsequent rhythmic handgrip exercise in humans. In 11 young adults, we determined forearm blood flow (FBF) (Doppler ultrasound) and forearm vascular conductance (FVC) (FBF/mean arterial pressure) during 4 min of rhythmic handgrip exercise at 10% of maximal voluntary contraction during 1) control conditions, 2) infusion of KCl before the initiation of exercise, and 3) infusion of sodium nitroprusside (SNP) as a control vasodilator. Infusion of KCl or SNP elevated resting FVC similarly before the onset of exercise (control: 39 ± 6 vs. KCl: 81 ± 12 and SNP: 82 ± 13 ml·min −1 ·100 mmHg −1 ; both P < 0.05 vs. control). Infusion of KCl at rest diminished the hyperemic (ΔFBF) and vasodilatory (ΔFVC) response to subsequent exercise by 22 ± 5% and 30 ± 5%, respectively (both P < 0.05 vs. control), whereas SNP did not affect the change in FBF (P = 0.74 vs. control) or FVC (P = 0.61 vs. control) from rest to steady-state exercise. These findings implicate the K + ion as an essential vasodilator substance contributing to exercise hyperemia in humans.
NEW & NOTEWORTHY Our findings support a significant and obligatory role for potassium signaling in the local vasodilatory and hyperemic response to exercise in humans.
Keywords: blood flow, exercise hyperemia, potassium, vasodilationThe potassium ion (K + ) has long been recognized as a vasoactive substance that could serve to match blood flow and oxygen delivery to local metabolic demand in contracting skeletal muscle (16, 32, 65). Plasma and interstitial [K + ] increase during muscle contractions in amounts commensurate with contractile activity, and the rise in [K + ] parallels the hyperemic response to exercise (25, 32, 35, 36, 38, 42, 48, 64). At concentrations in the range observed during exercise, extracellular K + hyperpolarizes the vasculature and elicits vasodilation by stimulating inwardly-rectifying K + (KIR) channels and the Na + /K + -ATPase (7, 9, 34, 64, 71, 73). Thus, during muscle contraction, an increase in extracellular [K + ] and subsequent activation of hyperpolarizing pathways is thought to be a functional signal contributing to exercise hyperemia.
At the onset of exercise, there is an immediate vasodilation that serves to increase muscle blood flow in a contraction intensity-dependent manner, which is followed by a second, slower phase that continues until steady-state hyperemia is achieved (43, 60, 75). In this context, it is generally thought that the factors that initiate vasodilation may differ from those that sustain it during steady-state exercise (24, 61). Initial studies in the canine hindlimb by Mohrman and Sparks (48) demonstrated that the time course and magnitude of K + release in response to a brief tetanus are sufficient to contribute to rapid vasodilation, and more recently, Armstrong and colleagues (3) demonstrated in the hamster cremaster muscle that pharmacological inhibition of voltage-dependent K + channels to block K + release from skeletal muscle reduces the vasodilation evoked by a single contraction. Consistent with these observations, we recently demonstrated that inhibition of the downstream pathways involved in K + -mediated vasodilation (KIR channels and the Na + /K + -ATPase) attenuates the hyperemic response following a single muscle contraction (~50%), as well as the vasodilatory response from exercise onset to steady-state hyperemia (~30%) during rhythmic exercise in the human forearm (10, 15).
Collectively, these observations support a role of K + in exercise hyperemia, yet whether K + directly mediates vasodilation in contracting human muscle is unclear. The conclusions drawn from studies blocking downstream pathways of K + -mediated vasodilation are limited due to the fact that KIR channels and the Na + /K + -ATPase can be activated by a variety of mechanical and chemical factors, particularly during exercise. In this regard, KIR channels are sensitive to fluid shear stress (1, 51) as well as hyperpolarization that arises from sources other than elevated extracellular [K + ] (11, 22, 66, 68). For instance, vasoactive substances that initiate Ca 2+ signaling in the endothelium, such as circulating ATP and metabolites of cytochrome P450 epoxygenase, can stimulate calcium-activated K + (KCa) channels to elicit hyperpolarization and activate KIR channels independent of extracellular [K + ] (9, 22, 26, 66–68). Thus, there is no direct evidence that K + -mediated vasodilation contributes to exercise hyperemia in humans, and the relative importance of this mechanism remains unclear.
In the present study, we adopted an approach similar to that of Hester et al. (31) to further investigate the role of K + in exercise hyperemia in humans. In theory, the vasodilatory response to changes in extracellular [K + ] during muscle contraction could be reduced by artificially elevating the concentration of K + perfusing the skeletal muscle vasculature before the onset of muscle contractions thus limiting further recruitment of this pathway and blunting the dilatory response to exercise. Therefore, we tested the hypothesis that local, intra-arterial infusion of potassium chloride (KCl) to elevate extracellular [K + ] would impair the vasodilation observed in response to rhythmic handgrip exercise in humans.
After providing written, informed consent, a total of 17 young, healthy adults (7 women and 10 men) participated in the present study with approval from the Colorado State University Institutional Review Board. Participants were sedentary to moderately active, normotensive, nonsmokers, and not taking any medication aside from oral contraceptives (two participants). All studies were performed in accordance with the Declaration of Helsinki.
Whole body dual X-ray absorptiometry was used to determine body composition and forearm volume (FAV) of the experimental forearm for normalization of drug doses (9, 12).
Following local anesthesia (2% lidocaine), a 20-gauge, 7.6 cm catheter was placed in the brachial artery of the nondominant forearm under aseptic conditions for local administration of study drugs via a 3-port connector. The catheter was continuously flushed at 3 ml/h with heparinized saline and connected to a pressure transducer for measurement of arterial pressure waveforms and determination of mean arterial pressure (MAP) (13, 37). A three-lead electrocardiogram (Cardiocap/5, Datex-Ohmeda, Louisville, CO) was used to determine heart rate (HR).
Prior to the experiment, maximal voluntary contraction (MVC) of the experimental forearm was determined as the average of 3 squeezes of a handgrip dynamometer (Stoelting, Chicago, IL) that were within 3% of each other (MVC mean: 36 ± 3 kg, range: 24–54 kg). Participants performed dynamic forearm contractions using a handgrip pulley system to lift a weight corresponding to 10% MVC. The weight was lifted 4–5 cm over the pulley with a duty cycle of 1 s contraction to 2 s relaxation (20 contractions/min) using visual and auditory feedback to ensure correct timing. This mild exercise intensity, which corresponds to ~30% of maximal forearm work rate (54), was chosen to facilitate comparison with previous work from our laboratory (15). Rhythmic handgrip exercise at 10% MVC does not elicit significant changes in cardiac output or reflex activation of the sympathetic nervous system (53, 57); thus, our experimental model isolates the effects of skeletal muscle contractions on local vascular control mechanisms.
All vasoactive drugs were infused via the brachial artery catheter to elicit a localized effect within the forearm vasculature. Saline was utilized as a control infusate. To determine the contribution of extracellular [K + ] to vasodilation and exercise hyperemia, KCl (Hospira, Lake Forest, IL) was infused to elevate extracellular [K + ] before the onset of muscle contractions. Because of concerns regarding subject safety and comfort, the dose of KCl was kept constant across subjects at 0.20 mmol/min, which is equal to the largest dose of KCl given by our laboratory previously without subject discomfort (9, 29).
The increase in blood flow that occurs as a result of KCl infusion necessitates comparison with a “high flow control” condition, as changes in resting blood flow per se may alter the response to exercise. Thus, sodium nitroprusside (SNP) was infused as a control vasodilator to match the K + -induced vasodilation observed at rest. The nitric oxide donor SNP was chosen because inhibition of nitric oxide synthesis does not impact rapid vasodilation or the rest to steady-state blood flow response to exercise (6, 14, 56). SNP was initially infused at 0.20 μg/dl FAV/min, and the infusion rate was intentionally adjusted to match the level of hyperemia observed during KCl infusion (final dose: 0.16 ± 0.04 μg·dl FAV −1 ·min −1 ).
In a separate control experiment to confirm that the dose of KCl infused in the present study does not impair general vascular responsiveness, the endothelium-dependent vasodilator acetylcholine (ACh) (Miochol-E; Novartis, Basel, Switzerland) was infused at 2 μg·dl FAV −1 ·min −1 under control conditions and during infusion of KCl (0.20 mmol/min).
Participants arrived at the laboratory in the morning following an overnight fast and 24 h abstention from exercise, alcohol, and caffeine. Participants lay supine with the experimental arm abducted to 90° and elevated slightly above heart level on a tilt-adjustable table throughout the study visit. All experiments were performed in a cool, temperature-controlled laboratory (20–22°C) with a fan directed toward the experimental arm to minimize the contribution of skin blood flow to overall forearm hemodynamics.
The primary experimental protocol is illustrated in Fig. 1 . In 11 young subjects (5 women and 6 men; age: 21 ± 1 yr; height: 172 ± 2 cm; weight: 70.8 ± 3.1 kg; body mass index: 23.8 ± 0.8; body fat: 26.1 ± 2.3%; FAV: 917 ± 58 ml; means ± SE), we sought to determine the effect of KCl infusion at rest on subsequent hemodynamic responses to muscle contractions. Thus, rhythmic handgrip exercise was performed during 1) control conditions, 2) intra-arterial infusion of KCl to elicit local increases in extracellular [K + ] before the initiation of contractions, and 3) intra-arterial infusion of SNP as a “high flow control” to match KCl-induced vasodilation before muscle contractions. The order of drug conditions was pseudorandomized and counterbalanced across participants, and each trial was separated by 15 min rest. During the loading period of drug infusions at rest, participants performed three single dynamic forearm contractions to facilitate delivery of the infusion to the vasculature of the muscle fibers recruited for this type of contraction (11, 15). Baseline hemodynamics were allowed to return to steady-state (≥ 3 min rest) during continued drug infusion before participants began rhythmic handgrip exercise at 10% MVC for 4 min to evaluate onset and steady-state exercise hyperemia and vasodilation.
Experimental protocol. After arterial catheterization and instrumentation, the hemodynamic response to rhythmic handgrip (RHG) exercise was assessed during infusion of saline, KCl, or sodium nitroprusside (SNP) in separate trials. Three single dynamic contractions were performed to facilitate drug delivery during the loading period of drug infusions. Following single contractions, drug infusions were continued, and hemodynamics were allowed to return to steady state (≥ 3 min rest) before measurements were made at baseline, during the onset of RHG contractions, and throughout 4 min of exercise. The order of drugs was counter-balanced across subjects, and each trial was separated by 15 min rest. MVC, maximal voluntary contraction.
Changes in membrane excitability resulting from elevated extracellular [K + ] may alter vascular responsiveness to other stimuli; thus, in a separate control experiment, the vasodilatory response to ACh was observed at rest under control conditions (saline) and during infusion of KCl to confirm that the low dose of KCl used in this study does not impair vascular responsiveness to a nonexercise vasodilatory stimulus. In six young adults (2 women and 4 men), saline or KCl was infused for 3 min to reach steady-state, then ACh was infused for an additional 3 min and the magnitude of the vasodilatory response was quantified. The order of trials was counterbalanced across subjects.
Data were collected at 250 Hz and stored on a computer for offline analysis with data acquisition and signal-processing software (WinDaq; DATAQ Instruments, Akron, OH). Baseline and steady-state FBF, MAP, FVC, and HR reflect 30 s averages of the resting time period immediately preceding the onset of muscle contractions or ACh infusion and the end of 4 min of rhythmic handgrip exercise or 3 min of ACh infusion. Because of differences in baseline hemodynamics between drug conditions, hyperemic and vasodilatory responses for each contraction trial were expressed as the absolute change in FBF and FVC from baseline. For subjects in whom we were able to accurately obtain data throughout the onset of exercise (n = 8), FBF and MAP were analyzed in 3-s bins corresponding to each contraction/relaxation (1:2 s) cycle through the first 3 min (15). In the event that the MBV signal quality obtained during a 3-s contraction cycle was altered due to operator error or excessive movement, a mathematical average of MBV from the preceding and subsequent bins was used.
All data are presented as means ± SE. In all protocols, hemodynamic variables were assessed using two-way (drug condition × time point) repeated-measures ANOVA. When a significant F value was observed, Tukey’s post hoc testing was performed to compare drug conditions within each time point. One-way repeated-measures ANOVA for drug condition was used to assess changes from baseline, and two-way (drug condition × sex) repeated-measures ANOVA was used to assess sex differences. In the event of nonnormal distributions, data were log transformed before analysis. Significance was set a priori at P < 0.05.
Forearm hemodynamics were similar at rest before initiating infusion of saline, SNP, or KCl (data not shown), and systemic hemodynamics remained largely unchanged throughout all trials and conditions ( Table 1 ). As intended, infusion of KCl or SNP elevated resting FBF and FVC similarly before the onset of muscle contractions ( Table 1 and Fig. 3 ). Small increases in baseline HR (4 beats/min) and MAP (3–4 mmHg) were observed during infusion of KCl compared with saline control and SNP, although these did not reach statistical significance.
Forearm and systemic hemodynamics
| Condition | FBF, ml/min | MAP, mmHg | FVC, ml·min −1 ·100 mmHg −1 | HR, beats/min |
|---|---|---|---|---|
| Resting baseline (n = 11) | ||||
| Control | 35 ± 6 | 90 ± 2 | 39 ± 6 | 58 ± 4 |
| KCl | 76 ± 11 † | 94 ± 3 | 81 ± 12 † | 62 ± 4 |
| SNP | 74 ± 12 † | 91 ± 2 | 82 ± 13 † | 58 ± 4 |
| Steady-state 10% MVC RHG (n = 11) | ||||
| Control | 167 ± 22 | 92 ± 2 | 179 ± 22 | 64 ± 4 ‡ |
| KCl | 180 ± 24 * | 99 ± 3 † * | 181 ± 23 * | 67 ± 4 ‡ |
| SNP | 204 ± 28 † | 93 ± 3 | 215 ± 26 † | 63 ± 4 ‡ |
Values are means ± SE. FBF, forearm blood flow; FVC, forearm vascular conductance; HR, heart rate; MAP, mean arterial pressure; MVC, maximal voluntary contraction; RHG, rhythmic handgrip.
* P < 0.05 vs. SNP at same time point, † P < 0.05 vs. control at same time point, ‡ P < 0.05 vs. baseline within condition.
Steady-state exercise hyperemia and vasodilation. Infusion of sodium nitroprusside (SNP) or KCl elevated forearm blood flow (FBF) (A) and forearm vascular conductance (FVC) (B) similarly at rest. KCl attenuated the subsequent hyperemic (ΔFBF) (C) and vasodilatory (ΔFVC) (D) responses to rhythmic handgrip exercise, whereas SNP did not (n = 11, 5 women and 6 men). *P < 0.05 KCl vs. SNP, †P < 0.05 KCl vs. control, ‡P < 0.05 SNP vs. control.
In all trials, a small (~5 beats/min) increase in HR was observed during rhythmic handgrip exercise ( Table 1 ). MAP during exercise was higher in the KCl trial compared with control and SNP ( Table 1 ), although the change in MAP from rest to exercise was similar across trials (control: 2 ± 1, KCl: 5 ± 2, SNP: 2 ± 2 mmHg; P = 0.29).
In control conditions, FBF and FVC increased rapidly from rest at the onset of muscle contractions ( Fig. 2 ). Elevation of resting blood flow with SNP augmented the change in FBF and FVC during the initial 30 s of rhythmic contractions but did not otherwise affect the response to exercise ( Figs. 2 and and3). 3 ). In contrast, infusion of KCl at rest diminished the immediate hyperemic (ΔFBF) ( Fig. 2C ) and vasodilatory (ΔFVC) ( Fig. 2D ) response to subsequent handgrip exercise within 15 and 30 s of exercise onset (compared with SNP and control, respectively), and the decrement continued throughout 4 min of contractions ( Fig. 3, C and D ). Steady-state FBF ( Fig. 3A ) and FVC ( Fig. 3B ) were significantly reduced in the KCl trial compared with SNP (P < 0.05) and were not different versus control. The change in FBF (Fig. 3C ) and FVC ( Fig. 3D ) from rest to steady-state exercise was similar during SNP and control conditions but was significantly reduced during KCl by 22 ± 5% and 30 ± 5%, respectively. Across drug trials, there was no effect of sex on the change in FBF or FVC from rest to steady-state exercise (main effect of sex: ΔFBF, P = 0.17; ΔFVC, P = 0.19), and importantly, there was no sex by drug interaction (ΔFBF, P = 0.82; ΔFVC, P = 0.74); however, these results should be interpreted with caution considering the limited statistical power to detect sex differences with this sample size.
Dynamic hyperemic and vasodilatory responses to repeated muscle contractions. Change in forearm blood flow (ΔFBF) and forearm vascular conductance (ΔFVC) from baseline during 10% maximal voluntary contraction (MVC) rhythmic handgrip exercise is shown in A and B. Data represent 3 s bins corresponding to each contraction-to-relaxation cycle during exercise. Statistical analyses were performed on 15 s averages (five 3 s bins), which are shown in C and D. Infusion of KCl before the onset of muscle contractions significantly reduced exercise hyperemia and vasodilation compared with control conditions or infusion of sodium nitroprusside (SNP) (n = 8, 4 women and 4 men). *P < 0.05 KCl vs. SNP, †P < 0.05 KCl vs. control, ‡P < 0.05 SNP vs. control.
In a separate control experiment, infusion of KCl increased resting FVC from 31 ± 3 to 57 ± 6 ml·min −1 ·100 mmHg −1 . KCl did not reduce steady-state vasodilation to subsequent infusion of ACh ( Fig. 4 ; ΔFVC; P = 0.25 vs. control), indicating that the vascular response to a nonexercise vasodilatory stimulus was not impaired.
Vasodilatory response to acetylcholine (ACh). Infusion of KCl did not reduce the subsequent change in forearm vascular conductance (ΔFVC) in response to ACh (P = 0.25 vs. control; n = 6, 2 women and 4 men).
The purpose of the present study was to investigate the role of K + -mediated vasodilation in exercise hyperemia in humans. The premise of our experimental approach was to perfuse the muscle with K + at rest before muscle contractions such that the effect of any endogenously-released K + on exercise hyperemia would be reduced. Utilizing this approach, we demonstrate a clear contribution of endogenous K + to the initial vasodilation at the onset of muscle contractions as well as the sustained, steady-state exercise hyperemia for the first time in humans.
K + released from skeletal muscle fibers upon repolarization is uniquely suited to initiate vasodilation in parallel with skeletal muscle activation in a rapid, feedforward manner. Initial attempts to investigate the role of K + in exercise hyperemia employed hemodialysis or dietary K + restriction to induce K + depletion in dogs, which attenuates both K + release and the hyperemic response to electrically stimulated contractions (2, 28, 39). However, because hypokalemia interferes with development of muscle tension, results from these studies cannot distinguish the effects of K + release from altered metabolic demand during sustained exercise (28). More recent data from the hamster cremaster preparation indicate that K + released from skeletal muscle initiates local vasodilation via KIR channels and the Na + /K + -ATPase in response to an electrically-stimulated contraction (3). In line with these observations, we have demonstrated that inhibition of K + -mediated hyperpolarization through combined blockade of KIR channels and the Na + /K + -ATPase reduces the total vasodilatory response to a single forearm contraction by 55%–75% in humans (10). Moreover, we have shown that activation of KIR channels accounts for ~50% of the increase in FBF at the onset of rhythmic handgrip exercise and ~30% of steady-state exercise hyperemia in humans (15). However, given that KIR channels and the Na + /K + -ATPase can be activated by multiple vasodilatory stimuli, the exact stimulus for activation of these pathways in humans remained speculative.
In the present study, when KCl was infused before contractions to reduce the effects of endogenously-released K + , the initial vasodilatory response to exercise was significantly attenuated, and this persisted throughout the full 4 min of rhythmic contractions. In contrast, when SNP was infused as a control vasodilator, the initial hyperemic and dilatory response was augmented, but this normalized by 45 s and was not different from control (saline) conditions. The effect of KCl on the hyperemic and vasodilator response from rest to steady-state exercise was a reduction of ~25% and 30%, respectively, which is similar in magnitude to what we observed in a previous study upon inhibition of KIR channels and the Na + /K + -ATPase (15). Collectively, these data strongly implicate the K + as a requisite vasodilator substance contributing to exercise hyperemia in humans.
It is important to highlight the magnitude of the effect of KCl infusion in the present study, as the skeletal muscle blood flow response to contractions is minimally impacted by inhibition of many vasodilatory pathways (15, 33, 56). Furthermore, infusion of other dilator substances to elevate resting blood flow does not affect the rest-to-exercise blood flow response. Indeed, the changes in FBF and FVC during exercise were maintained at control levels when SNP was infused in the present study. Similarly, prolonged infusion of either ATP or adenosine, two putative factors thought to contribute to exercise hyperemia, has no effect on the vasodilatory response to superimposed exercise bouts (52, 59). Thus, the diminished vascular response to contractions during KCl infusion is specific to K + and does not represent a generalized effect of altering baseline blood flow.
Although the results from the present investigation during steady-state exercise are consistent with our previous blockade experiments, infusion of KCl in the present study resulted in a lesser decrement in the initial vasodilatory response to contractions compared with combined inhibition of KIR channels and the Na + /K + -ATPase in our laboratory (15). This was most evident during the initial 15 s of exercise when KCl had a modest impact on ΔFVC (~15% reduction relative to SNP) compared with a ~60% reduction in vasodilation with inhibition of KIR channels and the Na + /K + -ATPase (15). By 30 s, the discrepancy was greatly diminished (KCl effect: ~30% compared with a ~40% reduction with inhibition of KIR channels and the Na + /K + -ATPase), and by 60 s, both interventions reduced vasodilation by ~25%. The smaller reduction at the onset of contractions in the present study may reflect differences in baseline vascular tone before exercise onset (vasodilation in the present study vs. vasoconstriction in our previous study), or an inability to fully activate K + -mediated signaling pathways at rest in our experimental design. Regarding the latter, the site of action of exogenous K + administered intra-arterially is unclear, and it is possible that K + did not reach the exact sites within the muscle interstitium, myoendothelial extracellular space, or cellular microdomains (21, 41, 55) that are normally activated by endogenous K + release. Further, because extracellular K + is vasodilatory up to a concentration of 15–20 mM, a higher dose of KCl would be required to completely prevent the ability of endogenous K + to elicit vasodilation. Additionally, because a variety of stimuli can initiate hyperpolarization through KIR channels and the Na + /K + -ATPase, we speculate that other factors capable of activating these pathways may be involved in vasodilation at the onset of exercise. In line with this, Sinkler and Segal (61) recently demonstrated that pharmacological inhibition of small- and intermediate-conductance KCa channels reduces rapid vasodilation to a brief, electrically-stimulated contraction of the mouse gluteus maximus, whereas inhibition of KCa channels did not impact the steady-state response to rhythmic contractions. Thus, substances which initiate hyperpolarization via KCa channels may contribute to KIR channel activation, particularly at the onset of contractions.
The increase in interstitial [K + ] during exercise is largely attributed to K + efflux from skeletal muscle, which occurs primarily via voltage-gated K + channels and skeletal muscle KIR channels during action potential repolarization (3, 17). Increased extracellular [K + ] alters the voltage-dependence of KIR channels, and the ensuing K + efflux produces hyperpolarization that closes voltage-gated Ca 2+ channels and leads to vascular smooth muscle relaxation (7, 19, 40, 66). At the same time, elevated [K + ] stimulates the electrogenic Na + /K + -ATPase to hyperpolarize vascular smooth muscle and elicit vasodilation (7, 19, 22, 30, 44, 71). In humans, inhibition of KIR channels alone (34) or in combination with the Na + /K + -ATPase (9) abolishes vasodilation to exogenous KCl.
In addition to efflux from skeletal muscle, K + is released as an endothelium-derived hyperpolarizing factor (21). Endothelial cell Ca 2+ signaling in response to shear stress or agonists such as ATP activates small- and intermediate-conductance KCa channels (1, 41, 70) and KIR channels on the endothelium (22, 68). Ultimately, K + efflux from endothelial cells and direct transmission of endothelium-derived hyperpolarization to adjacent smooth muscle cells activate KIR channels and the Na + /K + -ATPase on vascular smooth muscle (21–23). Furthermore, endothelial KIR currents have recently been shown to amplify vasodilatory signals arising from endothelium-dependent agonists by boosting hyperpolarization (66, 68).
Endothelial cell hyperpolarization initiated within the arteriolar network of contracting skeletal muscle spreads to adjacent cells through gap junctions and is conducted along the endothelium to facilitate vasodilation of upstream vessels (58). This ascending vasodilation is functionally important as it couples downstream signaling events with relaxation of feed arteries to increase tissue perfusion; indeed, in the absence of feed artery vasodilation, muscle blood flow is restricted despite robust vasodilation of downstream resistance vessels (58). In isolated arterioles, physiologically relevant doses of KCl stimulate local dilation at the site of administration and initiate a conducted response (18); thus, we speculate that increases in [K + ] may be involved in the ascending vasodilatory response to skeletal muscle contractions. However, because measurements at the brachial artery reflect bulk blood flow whereas direct measurement of microvessel diameter is required to examine propagated vasodilation, it is not currently possible to investigate this in humans.
An important consideration with our approach is whether the concentration of K + was sufficient to obviate the effects of endogenously-released K + . In a subset of four subjects, resting venous [K + ] increased from 3.8 ± 1 to 5.2 ± 0.5 mM when KCl was infused intra-arterially, which is comparable to the rise in venous [K + ] observed during moderate-intensity forearm or knee extensor exercise (36, 63, 73). However, as we did not measure interstitial [K + ], it is unclear whether our mode of K + administration (intra-arterial KCl) altered its concentration in the muscle interstitium. During exercise, high interstitial [K + ] results in a gradient between interstitial and plasma [K + ] that only partially equilibrates (32, 35), whereas infusion of K + reverses this gradient (34). Thus, while our dose of KCl was sufficient to observe significant decrements in the vasodilatory response to exercise, it is likely that our results underestimate the contribution of endogenous K + to contraction-induced dilation.
Moderate elevations in extracellular [K + ] (up to a concentration of 15–20 mM) cause membrane hyperpolarization and vasodilation, whereas higher doses result in depolarization and vasoconstriction (7, 16). Such changes in membrane excitability could impair vascular responsiveness in general; indeed, it has been demonstrated that nonphysiological concentrations of K + used to depolarize membrane potential can reduce vasodilation to other stimuli, including muscle contractions (27, 45, 74). However, our control data clearly indicate that the low dose of KCl used in this study does not reduce the vasodilatory response to ACh, and further, the concentration of K + draining the forearm in the present study (see above) was much lower than concentrations used to depolarize membrane potential in these previous studies. Thus, given that vasodilatory sensitivity to ACh is not impaired during KCl infusion, the effect of KCl on vasodilation and exercise hyperemia in the present study does not reflect a generalized reduction in vascular responsiveness resulting from altered membrane excitability.
A reduction in skeletal muscle excitability secondary to a decline in the sarcolemmal [K + ] gradient has been proposed as an important contributor to muscle fatigue (62). Force production is slightly reduced (~10%) in mouse skeletal muscle when extracellular [K + ] is increased to 8 mM, and sharp decrements in force production occur at concentrations of 10–12 mM (8). Although interstitial [K + ] has been reported to reach 10–12 mM in skeletal muscle during exhaustive, high-intensity exercise at workloads exceeding ~80% of maximal effort (49, 50, 69, 72), smaller increments are observed during mild-to-moderate workloads such that interstitial [K + ] reaches ~5–7 mM at intensities ranging from ~15% to 60% of maximal work rate (25, 35, 42, 46, 49). Considering the moderate increase in plasma [K + ] and the mild contraction intensity employed in the present protocols, we feel it is unlikely that skeletal muscle force production was altered by KCl infusion. Moreover, all subjects successfully performed handgrip contractions throughout the study. Therefore, if a decline in muscle excitability did occur, the compensatory increase in muscle fiber recruitment necessary to complete contractions would suggest that our results underestimate the role of K + .
The present investigation was limited to one mild intensity of rhythmic exercise. A single brief muscle contraction elicits an immediate hyperemic response that is largely independent of changes in tissue metabolism, thereby allowing investigation of feedforward mechanisms which contribute to contraction-induced hyperemia (47). We have previously demonstrated that inhibition of K + -mediated signaling through combined blockade of KIR channels and the Na + -K + -ATPase reduces the total vasodilatory response to a single forearm contraction by 55%–75% in humans (10). Although we were unable to utilize the single contraction model in the present study because of safety concerns with extending the infusion time and total dose of KCl, future studies utilizing this approach would provide valuable insight regarding the role of K + in contraction-induced rapid vasodilation. Additionally, because K + is released from skeletal muscle in parallel with muscle fiber recruitment, we speculate that the contribution of K + to exercise hyperemia may be greater at higher exercise intensities. However, safety concerns with prolonged administration of KCl preclude repeated trials to test this hypothesis using multiple exercise intensities within the same subject. Moreover, given the limitations of a low dose of KCl, the current experimental approach would likely further underestimate the role of K + with increasing exercise intensities. Future studies should address whether downstream signaling pathways are activated to a greater extent with increasing exercise intensity and muscle fiber recruitment.
Although the mechanisms underlying the augmented initial response to exercise during the high-flow control condition (SNP) compared with control conditions remain speculative, this observation highlights the importance of employing a control vasodilator to account for changes in blood flow and shear stress at baseline. However, the augmented hyperemic and dilatory response to exercise in the SNP trial quickly normalized to control levels with repeated contractions.
Vascular hyperpolarization is an important mechanism where vasodilatory stimuli originating within tissue beds can be conducted upstream to larger feed arteries to coordinate vasodilation across the vascular network (4, 20). Data from experimental animals suggest that with advanced age, chronic activation of K + channels increases the loss of electrical current from endothelial cells and impairs electrical conduction along the endothelium, which may lead to impaired blood flow control in older adults (5). Although this age-associated increase in endothelial KCa channel activity is thought to be due to oxidative stress-mediated increases in hydrogen peroxide (5) and potentially elevated sympathetic nerve activity (4), infusion of KCl at rest in the present study may mimic the increased current leak and changes in membrane potential observed in the aging endothelium. Thus, we speculate that our results may have implications regarding the mechanisms underlying age-associated impairments in exercise hyperemia.
Muscle contractions evoke a rapid increase in blood flow to meet the elevated metabolic demand of active skeletal muscle. An increase in interstitial K + ions and subsequent activation of hyperpolarizing pathways has been proposed to play an important role in exercise hyperemia; yet inferences regarding the role of K + in humans have previously been limited to observational studies and those employing pharmacological blockade of downstream targets. Here, we demonstrate that artificially elevating the concentration of K + perfusing the vasculature to minimize the effects of endogenously-released K + blunts the increase in blood flow in response to subsequent muscle contractions. The collective observations support a significant and obligatory role for K + signaling in the hyperemic response to exercise in humans.
This research was supported by NIH award HL-119337 (to F. A. Dinenno and M. J. Joyner).
No conflicts of interest, financial or otherwise, are declared by the authors.
C.M.H. and F.A.D. conceived and designed research; J.D.T., C.M.H., G.J.L., J.C.R., and F.A.D. performed experiments; J.D.T. and C.M.H. analyzed data; J.D.T., C.M.H., G.J.L., J.C.R., M.J.J., and F.A.D. interpreted results of experiments; J.D.T. prepared figures; J.D.T. drafted manuscript; J.D.T., C.M.H., and F.A.D. edited and revised manuscript; J.D.T., C.M.H., G.J.L., J.C.R., M.J.J., and F.A.D. approved final version of manuscript.
We thank the subjects who volunteered to participate and Matt Racine for contributions to this project.
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