The skeletal muscle microcirculation: if this is the hippodrome for the chariots of vasoactivity, who is the charioteer?

The publication of the hot topic review article by Poole et al. (2013) in Experimental Physiology, which nicely assimilated our growing understanding of skeletal muscle microcirculatory control, has highlighted the current interest in this area. The skeletal muscle capillary bed possesses a large surface area and thin diffusional barrier that facilitate the exchange of O2, CO2, substrates and metabolites between the blood and the myocytes. During the transition from rest to exercise, greater metabolic demand requires increased O2 delivery to the active muscle. In such conditions, intravital microscopy of the skeletal muscle microcirculation, also coming from the work of Drs Poole, Musch and co-workers, reveals a pattern of red blood cell movement that could be described as chaotic, perhaps even analogous to race day in the hippodrome. However, this is most certainly not random movement, but rather a finely regulated distribution of O2-carrying ‘chariots’ that appear to be remarkably well linked to O2 demand. Not surprisingly, much of the signalling for this increased O2 delivery is likely to originate from within the microcirculation itself. Although the exact mechanisms responsible for the regulation of skeletal muscle blood flow are still poorly understood, it is becoming apparent that several bioactive molecules probably have crucial roles in this process. One such vasoactive signalling molecule is extracellular ATP Kirby et al. (2013). ATP is present in venous plasma draining skeletal muscle at rest, and increases in conditions of hypoxia and skeletal muscle contraction when blood flow is elevated. Furthermore, intra-arterial infusion of ATP into both the brachial and the common femoral arteries has been demonstrated to elicit an increase in blood flow similar to that during exercise. This somewhat circumstantial evidence supports the role of ATP in effecting functional hyperaemia. Previously, four potential sites of extracellular ATP production have been implicated as possible sources of this ATP-induced vasodilatation, as follows: the contracting skeletal muscle; co-release with noradrenaline during sympathetic neuron activation; endothelial cells in response to shear stress; and red blood cells in response to mechanical deformation and the unloading of O2 from haemoglobin. ATP must then bind to purinergic P2Y receptors located on the luminal surface of endothelial cells to cause vasodilatation. The exact mechanism by which ATP-mediated P2Y receptor stimulation causes vasodilatation is not completely understood, but it appears to act, at least in part, by increasing nitric oxide and prostaglandin synthesis (Mortensen et al. 2009) and by stimulating hyperpolarization by means of inwardly rectifying potassium channels (Crecelius et al. 2012). Additionally, ATP has been demonstrated to override sympathetically mediated vasoconstriction (Rosenmeier et al. 2004). Regardless of the exact mechanism, intravascular ATP, which appears to increase during exercise, results in vasodilatation. Published in the May issue of the Experimental Physiology, Kirby et al. (2013) attempted to determine which of these four sources (sympathetic neurons, skeletal muscle, endothelial cells or erythrocytes) was most likely to be responsible for the increased intraluminal ATP, and therefore ATP-induced vasodilatation, during exercise. Venous plasma was collected from the forearm muscle and assayed for ATP concentration. As previously demonstrated, venous plasma ATP increased during rhythmic hand-grip exercise at 15% of the subjects’ maximal voluntary contraction. Employing a rather elegant integrative physiology study design to test the hypothesis that co-released ATP from sympathetic neurons may diffuse into the vessel lumen causing vasodilatation, the authors used lower-body negative pressure to stimulate sympathetic nerve activity during both rest and exercise. However, venous plasma ATP did not change during sympathetic nervous system activation in either condition, indicating that sympathetic neurons do not contribute significantly to intravascular ATP. Finally, using a potentially confounding yet creative approach, the investigators inflated a blood pressure cuff on the brachium to eliminate both the supply of erythrocytes and endothelial shear stress from the forearm. Despite continued muscle contractions and the hypoxic environment induced by the cuff, both of which would be expected to augment ATP release, venous plasma ATP remained unchanged, suggesting that the skeletal myocyte is not a significant source of the extracellular ATP. One of the potentially confounding issues with this approach, elevated sympathetic nerve activity due to cuff-induced ischaemia, worked in favour of the authors, providing further evidence against sympathetic nerve activity-induced ATP release. The authors concluded that elevations in venous plasma ATP rely on intact perfusion of the contracting muscle, pointing to endothelial cells and, more likely, erythrocytes as the primary source of intravascular ATP that helps to facilitate exercise-induced hyperaemia. As red blood cells traverse the vascular system from the O2-rich environment of the lungs to the relatively hypoxic skeletal muscle microcirculation, due to a change in iron's electron spin state, haemoglobin undergoes a conformational change from relaxed (R) to tense (T). Interestingly, this R-to-T transition not only explains the efficient regional capture and release of O2, but may also be linked to the regulation of blood flow. Specifically, although in typical conditions haemoglobin O2 saturation and are predictably related, by divorcing these two parameters Gonzalez-Alonso et al. (2001) demonstrated that the haemoglobin conformational change was more important in regulating skeletal muscle blood flow than . Indeed, it is becoming more apparent that the R-to-T transition of haemoglobin has ramifications, beyond the binding and unbinding of O2 and the discharging of CO2, with the carriage and delivery of a third gas with exceptional vasoactive properties, NO (McMahon et al. 2002). Thus, NO, riding upon the erythrocyte chariots of bioactivity, is preferentially released in areas where O2 is at low concentration, facilitating vasodilatation and resulting in hyperaemia. However, it appears that ATP may be an equally proficient charioteer, also being released by the conformational change in haemoglobin from the R to T state, facilitating the matching of O2 supply to O2 demand (González-Alonso et al. 2002). As our knowledge of these and, perhaps, other charioteers of vasoactivity develops, it will be interesting to determine their individual and potentially collective role in the diminished hyperaemic response in pathologies such as diabetes, hypertension and atherosclerosis, as well as healthy ageing.