Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are usually identified with pathological states and mediators of cellular injury. However, over the last decade ROS and RNS have been identified in skeletal muscle under physiological conditions. Detection of ROS and RNS production by skeletal muscle cells is fundamental to the problem of differentiating between physiological and pathological levels. The goal of this paper is to review the techniques that have been used to detect ROS and RNS in skeletal muscle. Electron spin resonance, fluorescent assays, cyotchrome c reduction, chemiluminescence, hydroxylation of salicylate, and nitration of phenylalanine are some of the assay systems that have been used thus far. A large body of evidence now indicates that ROS and RNS are continually produced by many different skeletal muscle types studied in vivo, in situ, and in vitro. Under resting conditions, ROS and RNS are detectable in both intracellular and extracellular compartments. Production increases during both non-fatiguing and fatiguing muscle contractions. In the absence of disease, the individual molecular species detected in skeletal muscle include parent radicals for the ROS and RNS cascades: superoxide anions and nitric oxide. Both are generated at rates estimated to range from pmol-to-nmol/mg muscle/minute. Evidence indicates that hydrogen peroxide, hydroxyl radicals, and peroxynitrite are also present under physiological conditions. However, the molecular species that mediate specific biological effects remains largely undetermined, as do the sources of ROS and RNS within muscle fibers. Eventual delineation of the mechanisms whereby ROS and RNS regulate cellular function will hinge on our understanding of the production and distribution of ROS and RNS within skeletal muscle.

The rapid onset of vasodilatation within seconds of a single contraction suggests that the vasodilators involved may be products of skeletal muscle activation, such as potassium (K + ). To test the hypothesis that K + was in part responsible for the rapid dilatation produced by muscle contraction we stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of arterioles at a site of overlap with the stimulated muscle fibres before and after a single contraction stimulated over a range of stimulus frequencies (4, 10, 20, 30, 40, 60 and 80 Hz; 250 ms train duration). Muscle fibres were stimulated in the absence and presence of an inhibitor of a source of K + , the voltage dependent K + channel inhibitor 3,4-diaminopyridine (DAP, 3 × 10 −4 M) and inhibitors of the K + dilatory signal transduction pathway, either a Na + K + -ATPase inhibitor (ouabain; 10 −4 M) or an inward rectifying K + channel inhibitor (barium chloride, BaCl 2 ; 5 × 10 −5 M). We observed significant inhibitions of the rapid dilatation at all stimulus frequencies with each inhibitor. The dilatory event at 4 s was significantly inhibited at all stimulus frequencies by an average of 65.7 ± 3.6%, 58.8 ± 6.1% and 64.4 ± 2.1% in the presence DAP, ouabain and BaCl 2 , respectively. These levels of inhibition did not correlate with non-specific changes in force generation by skeletal muscle measured in vitro. Therefore, our data support that K + is involved in the rapid dilatation in response to a single muscle contraction across a wide range of stimulus frequencies.

Murrant, Coral L., and Ingrid H. Sarelius. Multiple dilator pathways in skeletal muscle contraction-induced arteriolar dilations. Am J Physiol Regulatory Integrative Comp Physiol 282: R969-R978, 2002; 10.1152/ajpregu.00405. 2001.-To determine whether nitric oxide (NO), adenosine (Ado) receptors, or ATP-sensitive potassium (KATP) channels play a role in arteriolar dilations induced by muscle contraction, we used a cremaster preparation in anesthetesized hamsters in which we stimulated four to five muscle fibers lying perpendicular to a transverse arteriole (maximal diameter ϳ35-65 m). The diameter of the arteriole at the site of overlap of the stimulated muscle fibers (the local site) and at a remote site ϳ1,000 m upstream (the upstream site) was measured before, during, and after muscle contraction. Two minutes of 4-Hz muscle stimulation (5-15 V, 0.4 ms) produced local and upstream dilations of 19 Ϯ 1 and 10 Ϯ 1 m, respectively. N -nitro-L-arginine (10 Ϫ4 M; NO synthase inhibitor), xanthine amine congener (XAC; 10 Ϫ6 M; Ado A1, A 2A, and A2B receptor antagonist), or glibenclamide (Glib; 10 Ϫ5 M; KATP channel inhibitor) superfused over the preparation attenuated the local dilation (by 29.7 Ϯ 12.7, 61.8 Ϯ 9.0, and 51.9 Ϯ 14.9%, respectively), but only XAC and Glib attenuated the upstream dilation (by 68.9 Ϯ 6.8 and 89.1 Ϯ 6.4%, respectively). Furthermore, only Glib, when applied to the upstream site directly, attenuated the upstream dilation (48.1 Ϯ 9.1%). Neither XAC nor Glib applied directly to the arteriole between the local and the upstream sites had an effect on the magnitude of the upstream dilation. We conclude that NO, Ado receptors, and KATP channels are involved in the local dilation initiated by contracting muscle and that both KATP channels and Ado receptor stimulation, but not NO, play a role in the manifestation of the dilation at the upstream site. microvasculature; adenosine; nitric oxide; adenosine 5Ј-triphosphate-sensitive potassium channels; metabolic control of blood flow IN SKELETAL MUSCLE, CHANGES in metabolic rate lead to closely related changes in blood flow (2,4,6,16,24,26,33). This very close coupling between muscle metabolism and tissue perfusion involves the coordination and integration of multiple mechanisms of blood flow control, including direct local dilation of the terminal ar-

To test the hypothesis that measurable changes in microvasculature dilation occur in response to a single short-duration tetanic contraction, we contracted three to five skeletal muscle fibres of the hamster cremaster muscle microvascular preparation (in situ) and evaluated the response of an arteriole overlapping the active muscle fibres. Arteriolar diameter (baseline diameter = 16.4 +/- 0.9 micro m, maximum diameter = 34.7 +/- 1.2 micro m) was measured before and after a single contraction resulting from a range of stimulus frequencies (4, 10, 20, 30, 40, 60, and 80 Hz) within a 250- or 500-ms train. Four and 10 Hz produced a significant dilation at 2.9 +/- 0.4 and 6.5 +/- 2.8 s, respectively, within a 250-ms train and 3.0 +/- 0.2 and 6.1 +/- 1.3 s, respectively, within a 500-ms train. Biphasic dilations were observed within a 250-ms train at 20 Hz (at 3.9 +/- 0.9 and 22.1 +/- 4.3 s), 30 Hz (at 2.7 +/- 0.3 and 17.5 +/- 2.9 s), and 40 Hz (at 3.8 +/- 0.4 and 23.2 +/- 2.6 s) and within a 500-ms train at 20 Hz (at 4.8 +/- 0.4 and 31.9 +/- 3.8 s) and 30 Hz (at 3.4 +/- 0.3 and 27.6 +/- 3.0 s). A single dilation was observed within a 250-ms train at 60 Hz (at 5.1 +/- 0.7 s) and 80 Hz (at 14.2 +/- 3.3 s) and within a 500-ms train at 40 Hz (at 9.9 +/- 3.2 s), 60 Hz (at 7.9 +/- 2.1 s), and 80 Hz (at 13.4 +/- 4.0 s). We have shown that a single contraction ranging from a single twitch (4 Hz, 250 ms) to fused tetanic contractions produces significant arteriolar dilations and that the pattern of dilation is dependent on the stimulus frequency and train duration.

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