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LETTERS |
O2 kinetics
-nitro-L-arginine methyl ester (L-NAME) on muscle oxygen uptake 
kinetics in the pump-perfused canine hind-limb model, and concluded that ‘the inhibition of mitochondrial respiration by nitric oxide (NO) does not limit the kinetics of adjustment of oxidative metabolism at exercise onset’. This conclusion contradicts the results of previous studies which have demonstrated a significant speeding of 
kinetics with L-NAME administration in horses and humans (Kindig et al. 2001, 2002; Jones et al. 2003, 2004; Wilkerson et al. 2004). We contend that limitations inherent in the experimental model utilized by Grassi et al. (2005) might at least partly explain the differences in the results obtained. Methodology
In their study, Grassi et al. (2005) utilized a pump-perfused canine hind-limb preparation, while those studies in which a speeding of 
kinetics with L-NAME was reported utilized whole-body conscious exercise. The canine hind-limb model, while exceptionally useful for examining a wide variety of muscular responses during contractions (as arterial inflow and contraction intensity can be rigorously controlled), might not be the ideal methodology for investigating 
kinetics. In addition, experimental order cannot be randomized (the control condition was always first) and it is therefore impossible to know whether or not some residual effect from the initial contraction period might have affected the preparation and, thus, the results. Indeed, it is worth noting that initial blood pressure was very high in the L-NAME condition (see Table 1), to a level wherein preparations can become oedematous and vascular conductance can fall precipitously (Poole et al. 2004). As the matching of oxygen delivery to oxygen utilization 
is a critical determinant of the driving pressure for O2 into the myocyte and thus diffusing capacity (McDonough et al. 2005), it is worth noting that if the 
ratio is negatively impacted for any of a variety of reasons (increased heterogeneity of flow, compromised muscle/tissue integrity, structural and/or mechanical impediments to flow leading to oedema, etc.) then O2 extraction will be blunted, the achievable 
reduced and 
kinetics slowed. Furthermore, Laughlin & Schrage (1999) noted that cannulation of the venous circulation can alter vascular mechanics, particularly the interaction between contractions and venous vascular responsivity. Thus, it remains a possibility that the combination of maximal vasodilatation, pump-perfusion at high-arterial inflow and cannulation of the venous outflow altered vascular mechanics in such a way as to negatively impact 
matching and thus 
kinetics in these studies.
As 
must be determined via Fick's equation in the canine hind-limb model, using the preset arterial inflow and direct arterial and venous sampling, the fidelity of the 
measurements depends upon the timing of the arterial and venous sampling. As the arterial samples were only taken at rest, immediately prior to and following the contractions period, and the venous samples were taken at rest, every 5–7 s during contractions (first 75 s) and every 30–45 s thereafter, the arterial and venous samples were not precisely matched. This appears to have been a particular problem at the immediate onset of contractions (see Fig. 2).
Another important issue is that the canine gastrocnemius complex contains only highly oxidative muscle (Parsons et al. 1985); however, the influence of NOS inhibition on 
kinetics appears to be greatest at higher exercise intensities where type II muscle fibres are likely to be recruited (Wilkerson et al. 2004), in keeping with the reportedly greater NOS activity in type II fibres (Kobzik et al. 1994). The hind-limb musculature of the dog is very oxidative and, unlike the muscle of almost all other species, may not respond to training (cf. Parsons et al. 1985 and Hepple et al. 2000 with Bebout et al. 1993). As NO-mediated inhibition of mitochondrial oxidative metabolism is inversely dependent upon intracellular PO2 (Brown, 2000), it is plausible that NOS inhibition would have a smaller effect upon the canine hind-limb, particularly as the muscle was maximally vasodilated (which elevates PO2) and canine hind-limb muscle is already ‘finely tuned’ to a high level of extraction and performance (the fundamental time constant for both conditions was 
10 s, extraordinarily fast; see Fig. 4).
Data analyses
The ability to accurately measure 
kinetics is determined principally by the number of data points collected across the rest-to-contractions transition, the fidelity of those data points, and the confidence that those data points represent a ‘normal sample’. The study of Grassi et al. (2005) involved a relatively small muscle mass, a low sampling frequency, and a single transition from rest to contractions, all of which would reduce confidence in the parameters estimated from the model fits and possibly mask any ‘true differences’ that might have existed between the conditions (Lamarra et al. 1987). In addition, although Grassi et al. (2005) state that a 
‘slow component’ was evident in two animals, confidence in using a higher-order model would be limited since only three to five data points existed beyond 75 s of exercise. Certainly, several of the individual model fits displayed in Fig. 2 of Grassi et al. (2005) could be considered questionable.
Biopsy data were obtained from superficial muscle at three time points, rest, 1 min into contractions and 15 s prior to end-contractions. In a preparation that is not inherently stable, it is impossible to determine the impact of muscle excision upon 
kinetic parameter estimation. Notwithstanding the possibility that superficial muscle samples might not necessarily reflect the overall muscle energetic state, it is noteworthy that the estimated substrate-level phosphorylation over the entire 4 min period was 38% lower in the L-NAME compared to the control condition. Although inter-animal variability in response and a small sample size (n
= 6) precluded this difference reaching statistical significance, these data, along with the reduced muscle fatigue (see Fig. 1), certainly suggest that the muscle ‘O2 deficit’ was reduced with L-NAME (Kindig et al. 2001, 2002; Jones et al. 2003), further questioning the conclusions drawn from the analysis of the 
data.
Data interpretation
We were surprised that Grassi et al. (2005) described the previously reported changes induced by L-NAME administration as ‘small’ when the average speeding of the phase II 
kinetics in five studies from our laboratories was 41% (range: 15–78%) (Kindig et al. 2001, 2002; Jones et al. 2003, 2004; Wilkerson et al. 2004). It seems very unlikely that differences of this magnitude could be explained simply by a ‘distortion’ caused by possible drug-induced alterations in cardiac output (see Barstow et al. 1990).
One of the main stimuli for NO release is increased shear stress (Sanders et al. 2000), which would be negligible in the pump-perfused hind-limb model. However, Grassi et al. (2005) did not consider that their methods might have inhibited NO release in both the control and L-NAME conditions, potentially invalidating any comparison. It is also known that L-NAME has somewhat peculiar effects in dogs, including elevating the resting and exercising ‘steady-state’ metabolic rate (Shen et al. 1994; see Fig. 3) yet these potentially confounding effects were not discussed.
In conclusion, while we applaud Grassi et al. for presenting novel data regarding the effects of NOS inhibition upon 
kinetics, we would stress that their results and conclusions might only be applicable to the non-physiological (unchanged 
and maximal vasodilatation throughout; synchronous tetanic contractions), highly oxidative, cannulated, hind-limb canine muscle preparation. The bold conclusion that NO does not limit 
kinetics is therefore not warranted in other models and species.
Department of Internal MedicineUniversity of Texas SouthwesternMedical Center, Dallas, TX 75390, USAEmail: paul.mcdonough{at}utsouthwestern.edu
School of Sport and Health SciencesUniversity of Exeter, Exeter EX1 2LU, UK
Departments of Kinesiology, Anatomyand Physiology, Kansas State UniversityManhattan, KS 66506-5802, USA
References
Barstow
TJ, Lamarra
N
&
Whipp
BJ (1990). Modulation of muscle and pulmonary O2 uptakes by circulatory dynamics during exercise. J Appl Physiol
68, 979–989.
Bebout
DE, Hogan
MC, Hempleman
SC
&
Wagner
PD (1993). Effects of training and immobilization on 
and Do2 in dog gastrocnemius muscle in situ. J Appl Physiol
74, 1697–1703.
Brown GC (2000). Nitric oxide as a competitive inhibitor of oxygen consumption in the mitochondrial respiratory chain. Acta Physiol Scand 168, 667–674.[CrossRef][Medline]
Grassi
B, Hogan
MC, Kelley
KM, Howlett
RA
&
Gladden
LB (2005). Effects of nitric oxide synthase inhibition by L-NAME on oxygen uptake kinetics in isolated canine muscle in situ. J Physiol
568, 1021–1033.
Hepple
RT, Hogan
MC, Stary
C, Bebout
DE, Mathieu-Costello
O
&
Wagner
PD (2000). Structural basis of muscle O2 diffusing capacity: evidence from muscle function in situ. J Appl Physiol
88, 560–566.
Jones
AM, Wilkerson
DP, Koppo
K, Wilmshurst
S
&
Campbell
IT (2003). Inhibition of nitric oxide synthase by L-NAME speeds phase II pulmonary 
kinetics in the transition to moderate-intensity exercise in man. J Physiol
552, 265–272.
Jones
AM, Wilkerson
DP, Wilmshurst
S
&
Campbell
IT (2004). Influence of L-NAME on pulmonary O2 uptake kinetics during heavy-intensity cycle exercise. J Appl Physiol
96, 1033–1038.
Kindig
CA, McDonough
P, Erickson
HH
&
Poole
DC (2001). Effect of L-NAME on oxygen uptake kinetics during heavy-intensity exercise in the horse. J Appl Physiol
91, 891–896.
Kindig CA, McDonough P, Erickson HH & Poole DC (2002). Nitric oxide synthase inhibition speeds oxygen uptake kinetics in horses during moderate domain running. Respir Physiol Neurobiol 132, 169–178.[CrossRef][Medline]
Kobzik L, Reid MB, Bredt DS & Stamler JS (1994). Nitric oxide in skeletal muscle. Nature 372, 546–548.[CrossRef][Medline]
Lamarra
N, Whipp
BJ, Ward
SA
&
Wasserman
K (1987). Effect of interbreath fluctuations on characterizing exercise gas exchange kinetics. J Appl Physiol
62, 2003–2012.
Laughlin MH & Schrage WG (1999). Effects of muscle contraction on skeletal muscle blood flow: when is there a muscle pump? Med Sci Sports Exerc 31, 1027–1035.
McDonough
P, Behnke
BJ, Padilla
DJ, Musch
TI
&
Poole
DC (2005). Control of microvascular oxygen pressures in rat muscles comprised of different fibre types. J Physiol
563, 903–913.
Parsons
D, Musch
TI, Moore
RL, Haidet
GC
&
Ordway
GA (1985). Dynamic exercise in foxhounds II. Analysis of skeletal muscle. J Appl Physiol
59, 190–197.
Poole DC, Behnke BJ, McDonough P, McAllister RM & Wilson DF (2004). Measurement of muscle microvascular oxygen pressures: Compartmentalization of phosphorescent probe. Microcirculation 11, 317–326.[CrossRef][Medline]
Sanders
DB, Kelley
T
&
Larson
D (2000). The role of nitric oxide synthase/nitric oxide in vascular smooth muscle control. Perfusion
15, 97–104.
Shen
W, Xu
X, Ochoa
M, Zhao
G, Wolin
MS
&
Hintze
TH (1994). Role of nitric oxide in the regulation of oxygen consumption in conscious dogs. Circ Res
75, 1086–1095.
Wilkerson
DP, Campbell
IT
&
Jones
AM (2004). Influence of nitric oxide synthase inhibition on pulmonary O2 uptake kinetics during supra-maximal exercise in humans. J Physiol
561, 623–635.
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