Potassium, contracting myocytes and rapid vasodilatation: peaking more than just our interest?

Following a single brief muscle contraction in humans, skeletal muscle blood flow rapidly increases in a monophasic pattern that reaches a peak response in ∼4–5 seconds. Further, vasodilatation via smooth muscle cell hyperpolarization appears to be an essential determinant for this phenomenon (Hamann et al. 2004). Although the existence of contraction-induced rapid vasodilatation is apparent, a clear explanation of the mechanism(s) underlying this response has been difficult to uncover. Due to the rapid nature of this response, original investigations focused upon potential neural mechanisms. Additionally, putative vasodilators known to assist in the regulation of blood flow during steady-state exercise have been evaluated under single contraction conditions. However, experiments evaluating the contribution of acetylcholine spillover, sympathetic dilator fibres, nitric oxide, and prostaglandins have demonstrated little to no involvement in contraction-induced rapid vasodilatation in humans. Given that these factors appear non-obligatory in the rapid dilatory response, investigations have continued to seek the elusive mechanism(s) of this vascular event. As previously mentioned, vascular smooth muscle cell hyperpolarization is obligatory for contraction-induced rapid vasodilatation (Hamann et al. 2004); however, its stimuli has yet to be determined. The close association between smooth muscle hyperpolarization and muscle activation suggests a common factor coupling these two events. The electromechanical coupling of muscle contraction is known to immediately increase potassium (K+) concentrations in the interstitial space and the plasma. Moreover, the K+ concentration produced by muscle contractions is similar to that known to result in vasodilatation (Mohrman & Sparks, 1974). The source of K+ is thought to originate from the ion transport associated with action potentials spreading along the sarcolemma of the muscle fibre (Fig. 1). One possibility is that during the propagation of an action potential throughout the muscle fibre, K+ is released from the contracting myocyte via voltage-gated K+ channels (KV) thus increasing extracellular K+ concentrations ([K+]o). Consequently, increasing [K+]o stimulates the opening of inward-rectifying K+ (KIR) channels and activation of Na+–K+ ATPases resulting in hyperpolarization and subsequent closing of voltage-gated calcium (Ca2+) channels on the smooth muscle cell, leading to vasodilatation. Therefore, it appears that K+ could contribute to contraction-induced rapid vasodilatation. Accordingly, the novel study by Armstrong et al. (2007) recently published in The Journal of Physiology sought to determine whether K+ is involved in producing the rapid vasodilatation in response to a single muscle contraction. Schematic demonstrating K+-induced rapid vasodilatation as a result of muscle contraction Motoneuron firing causes the release of acetylcholine at the neuromuscular junction resulting in depolarization of the skeletal myocyte membrane potential via the opening of ligand-gated Na+ channels. Na+ influx depolarizes the membrane potential above threshold, stimulating the opening of voltage-gated Na+ channels and further depolarization. This in turn stimulates voltage-gated K+ channels (K+v), resulting in K+ efflux and ensuing repolarization. These events occur throughout the sarcolemma and allow for propagation of action potentials down t-tubules ultimately resulting in skeletal muscle contraction. The efflux of K+ during the propagation of action potentials along the sarcolemma increases interstitial K+ concentrations. These K+ concentrations stimulate both Na+–K+ ATPase and inward-rectifying K+ channels (K+IR) located on the membrane of nearby vascular smooth muscle cells. The activation of these channels results in hyperpolarization and ensuing closure of voltage-gated Ca2+ channels. This leads to decreased [Ca2+]i, causing smooth muscle cell relaxation and decreased vascular tone. To demonstrate the blockade conditions from Armstrong et al. (2007), ‘X’ indicates the targets for channel inhibitors 3,4-diaminopyridine (DAP), barium chloride (BaCl2) and oubain, which inhibit K+v, K+IR and Na+-K+ ATPase activity, respectively. Through the use of an in situ hamster cremaster muscle preparation, these investigators measured the change in transverse arteriole diameter in response to a single, electrically stimulated muscle fibre contraction. Stimulation frequencies ranged from 4 to 80 Hz at 250 ms train durations. To test the involvement of K+ in contraction-induced rapid vasodilatation, single contractions of the cremaster muscle were performed before and after being superfused with 3,4-diaminopyridine (DAP; KV channel inhibitor), ouabain (Na+–K+ ATPases inhibitor), or barium chloride (BaCl2; KIR channel inhibitor) (Fig. 1) Following DAP, oubain or BaCl2 administration, peak vasodilatory responses at 4 s post-contraction were significantly blunted (about 66%, 60% and 65%, respectively) at most stimulation frequencies. Thus, these data indicate that K+ is a significant contributor to the peak rapid vasodilatory response following a single muscle contraction. During these experiments, baseline arterial diameter was noted to be significantly decreased during the BaCl2 administration. To evaluate whether this decrease in baseline diameter had an effect on vessel reactivity, arterioles were superfused with phenylephrine (PE), an α-adrenergic agonist, in an effort to match the baseline diameter to that observed during the BaCl2 condition. Thus, this PE group was constricted similarly to the BaCl2 group. Since the PE group was still able to dilate to the same extent as the control group, the investigators concluded that vessel reactivity was independent of vessel diameter. In a second set of experiments, an in vitro protocol was set forth to determine if the blunting of rapid vasodilatation was in fact due to the ability of K+ inhibition to modulate force generation of skeletal muscle rather than a direct effect upon smooth muscle hyperpolarization and subsequent relaxation. It is well known that contraction-induced rapid vasodilatation is proportional to force production (Kirby et al. 2007); thus a lower force generation would lead to an attenuated hyperaemic response. Within this protocol, BaCl2 and oubain exposure significantly blunted force production of the cremaster muscle during contraction at nearly all stimulation frequencies (∼50%), while DAP had a negligible effect on force production. Similarly, the vasodilatation at 4 s was inhibited by an average of ∼60% during BaCl2 and oubain application. Taking this reduction of force development into account, it is possible that KIR channel and Na+–K+ ATPases involvement in contraction-induced vasodilatation is overestimated. In contrast, the authors argued that decreased vasodilatation was independent of reduced force because a blunted vasodilatation was still observed despite the matched force production during DAP administration, which should limit the increase in extracellular K+ by inhibiting KV channels along the sarcolemma. However, interstitial K+ concentrations were not measured to confirm the efficacy of DAP to inhibit K+ efflux from skeletal myocytes. Nonetheless, the key finding of the present experiment was that K+ blockade significantly blunted the peak rapid vasodilatation at 4 s in response to a single muscle contraction, as primarily shown by KV channel inhibition via DAP. In a separate set of experiments to test the efficacy of K+ blockade, the controlled application of 15 mm K+ elicited a vasodilatation that was partially blunted under blockade conditions (∼50% to BaCl2 and ouabain). Therefore, the blunted but not abolished vasodilatation via KIR channel and Na+–K+ ATPases inhibition during single contractions may be explained by incomplete blockade. As such, if multiple K+ inhibitors were used together, this may have provided a more effective blockade, thus demonstrating a clearer role of K+ in contraction-induced rapid vasodilatation. It has been previously noted that the time to onset of vasodilatation in isolated rat arterioles bathed in K+ is at least 4 s (Wunsch et al. 2000), leading many to believe that K+ was too sluggish to account for rapid vasodilatation. However, the present study eloquently challenges this argument demonstrating that K+ administration via direct application to the vessel in situ can stimulate peak vasodilatation within 2 s. This suggests that K+ can stimulate vasodilatation in an adequate time frame as that observed following a muscle contraction, and further compliments the findings of reduced peak vasodilatation during K+ channel inhibition. The findings by Armstrong et al. (2007) are of particular interest in that, to date, no concrete explanation for the mechanism(s) of contraction-induced rapid vasodilatation exists. Specifically, these data, in addition to the previous findings from Hamann et al. (2004), largely suggest that elevated [K+]o from contracting myocytes can stimulate smooth muscle hyperpolarization and contribute to the peak contraction-induced rapid vasodilatory response. Unfortunately, the investigators did not show the dynamic vascular responses under any of the K+ inhibition conditions. With this information, these data could have provided tremendous insight into the dynamic regulation of the rapid vasodilatory response. For instance, does K+ solely contribute to the peak response at 4 s, or is it involved in the entire dynamic response: the initiation, the peak, and the sustained hyperaemia post contraction? As such, the present study demonstrated only a partial blunted peak vasodilatory response. Whether this is due to incomplete blockade or indicates the role for other factors is unclear. In this context, mechanical influences of muscle contraction have also been demonstrated to result in rapid vasodilatation (Kirby et al. 2007). However, the mechanisms underlying mechanically induced vasodilatation are largely unexplored. Thus, the integration between K+-induced vasodilatation, vascular smooth muscle hyperpolarization and mechanical influences would offer additional insight into this phenomenon. Further, whether K+ contributes to the sustained dilatation seen with increasing contraction intensity is also unknown. Corresponding measures of [K+]o and the total vasodilatory response during increasing single contraction intensities would assist in further defining the contribution of K+ to rapid vasodilatation. In summary, the recent study by Armstrong and colleagues provides the novel finding that K+ has a clear role in contraction-induced rapid vasodilatation, where inhibition of KIR channels, KV channels or Na+–K+ ATPases attenuate the magnitude of the peak rapid vasodilatory response at 4 s elicited via a single muscle contraction. Most importantly, through the use of a KV channel inhibitor which should limit the source of K+ from contracting myocytes, the peak rapid vasodilatory response was blunted independent of force production. However, whether the blunted vasodilatation represents only partial blockade efficacy or the existence of additional mechanisms cannot be determined. Nonetheless, an evaluation to what extent K+ contributes to the entire dynamic post-contraction hyperaemic response as well as whether other vasodilators play a role is necessary. As interest in contraction-induced rapid vasodilatation continues to ‘peak’, future investigations will certainly be required to gain further understanding since multiple factors inevitably provide the framework for this dynamic response. The authors would like to sincerely thank Dr Frank A. Dinenno and Dr Wyatt F. Voyles for their critical evaluation of this manuscript and support throughout this process.