Metabolic modulation of sympathetic vasoconstriction in exercising skeletal muscle

The tight coupling of oxygen supply and utilization in exercising skeletal muscle is the result of complex interactions between local mechanisms that control muscle blood flow and substrate utilization and systemic mechanisms that control cardiac output and arterial pressure. The role of the sympathetic nervous system in the integration of these responses, specifically the interaction between sympathetic activation and local vasodilator mechanisms in exercising muscle, has been an active area of research for many years yet remains incompletely understood. The functional consequence of sympathetic activation in exercising skeletal muscle has been the subject of considerable debate. Previous studies in animals and humans have suggested that sympathetic vasoconstricton in active muscle is (a) well maintained and serves to limit active hyperaemia, thereby preventing muscle blood flow from outstripping cardiac output in order to preserve blood pressure and vital organ perfusion or (b) greatly attenuated in order to optimize muscle perfusion, a concept that has been termed 'functional sympatholysis'. Studies performed over the past 70 years have provided conflicting evidence regarding the relative importance of sympathetic vasoconstriction vs. functional sympatholysis in exercising skeletal muscle. The focus of this review is mainly on recent studies in anaesthetized animal preparations and in conscious humans that have provided evidence for the metabolic modulation of sympathetic vasoconstriction in contracting skeletal muscle and have identified a number of key underlying mechanisms that extend the initial concept of sympatholysis.     

Zinc is both an intracellular and extracellular regulator of KATP channel function

Extracellular Zn²⁺ has been identified as an activator of pancreatic K_ATP channels. We further examined the action of Zn²⁺ on recombinant K_ATP channels formed with the inward rectifier K⁺ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn²⁺, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of K_ATP channels. External Zn²⁺, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn²⁺ dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 μ m , respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn²⁺ on the SUR subunit. Effects of internal Zn²⁺ were not due to Zn²⁺ leaking out, since they were unaffected by the presence of a Zn²⁺ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn²⁺. Therefore, Zn²⁺ is an endogenous K_ATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting K_ATP channels, and suggest a new role for Zn²⁺ as an intracellular signalling molecule.     

Mapping the architecture of the ATP-binding site of the KATP channel subunit Kir6.2

ATP-sensitive potassium (K_ATP) channels comprise Kir6.2 and SUR subunits. The site at which ATP binds to mediate K_ATP channel inhibition lies on Kir6.2, but the potency of block is enhanced by coexpression with SUR1. To assess the structure of the ATP-binding site on Kir6.2, we used a range of adenine nucleotides as molecular measuring sticks to map the internal dimensions of the binding site. We compared their efficacy on Kir6.2–SUR1, and on a truncated Kir6.2 (Kir6.2ΔC) that expresses in the absence of SUR. We show here that SUR1 modifies the ATP-binding pocket of Kir6.2, by increasing the width of the groove that binds the phosphate tail of ATP, without changing the length of the groove, and by enhancing interaction with the adenine ring.     

Pyridine nucleotide regulation of the KATP channel Kir6.2/SUR1 expressed in Xenopus oocytes

The pancreatic β-cell type of ATP-sensitive potassium (K_ATP) channel (Kir6.2/SUR1) is inhibited by intracellular ATP and ADP, which bind to the Kir6.2 subunit, and is activated by Mg-nucleotide interaction with the regulatory sulphonylurea receptor subunits (SUR1). The nicotinamide adenine dinucleotides NAD and NADP consist of an ADP molecule with a ribose group and a nicotinamide moiety attached to the terminal phosphate. Both these molecules block native K_ATP channels in pancreatic β-cells at concentrations above 500 μM, and activate them at lower concentrations. We therefore investigated whether NAD and NADP interact with both Kir6.2 and SUR1 subunits of the K_ATP channel by comparing the potency of these agents on recombinant Kir6.2ΔC and Kir6.2/SUR1 channels expressed in Xenopus oocytes. Our results show that, at physiological concentrations, NAD and NADP interact with the nucleotide inhibitory site of Kir6.2 to inhibit Kir6.2/SUR1 currents. They may therefore contribute to the resting level of channel inhibition in the intact cell. Importantly, our data also reveal that this interaction is dependent on the presence of SUR1, which may act by increasing the width of the nucleotide-binding pocket of Kir6.2.     

FAWSETS perfusion measurements in exercising skeletal muscle

Arterial spin labeling (ASL) techniques are now recognized as valid tools for providing accurate measurements of cerebral and cardiac perfusion. The labeling process used with most ASL techniques creates two problems, magnetization transfer (MT) effects and arterial transit time effects, that require compensation. The compensation process limits time resolution and hinders absolute quantification. MT effects are particularly problematic in skeletal muscle because they are large and change rapidly during exercise. The protocol presented here was developed specifically for quantification of perfusion in exercising skeletal muscle. The ASL technique that was implemented, FAWSETS, eliminates MT effects and arterial transit times. Localized, single-voxel perfusion measurements were acquired from rat hind limbs at rest, during ischemia and during three different levels of stimulated exercise. The results demonstrate sufficient sensitivity to determine the time constants for perfusion changes at onset of, and during recovery from, exercise and to distinguish the differences in the amplitude of the perfusion response to different levels of exercise. Additional measurements were conducted to demonstrate insensitivity to MT effects. The exercise protocol is easily adaptable to phosphorous magnetic resonance measurements, allowing the possibility to acquire local measurements of perfusion and metabolism from the same tissue in future experiments.