GC-MS determination of nitrous anhydrase activity of bovine and human carbonic anhydrase II and IV.

The most widely recognized activity of the large family of the metalloenzyme carbonic anhydrases (CAs) is the diffusion-controlled hydration of CO2 to HCO3- and one proton, and the less rapid dehydration of HCO3- to CO2: CO2 + H2O ⇆ HCO3- + H+. CAs also catalyze the reaction of water with other electrophiles such as aromatic esters, sulfates and phosphates, thus contributing to lending to CAs esterase, sulfatase and phosphatase activity, respectively. Renal CAII and CAIV are involved in the reabsorption of nitrite, the autoxidation product of the signalling molecule nitric oxide (NO): 4 NO + O2 + 2 H2O → 4 ONO- + 4 H+. Bovine and human CAII and CAIV have been reported to exert nitrite reductase and nitrous anhydride activity: 2 NO2- + 2 H+ ⇆ [2 HONO] ⇆ N2O3 + H2O. In the presence of L-cysteine, NO may be formed. In the literature, these issues are controversial, mainly due to analytical shortcomings, i.e., the inability to detect authentic HONO and N2O3. Here, we present a gas chromatography-mass spectrometry (GC-MS) assay to unambiguously detect and quantify the nitrous anhydrase activity of CAs. The assay is based on the hydrolysis of N2O3 in H218O to form ON18O- and 18ON18O-. After pentafluorobenzyl bromide derivatization and electron capture negative-ion chemical ionization of the pentafluorobenzyl nitro derivatives, quantification is performed by selected-ion monitoring of the anions with mass-to-charge (m/z) ratios of 46 (ONO-), m/z 48 (ON18O- and 18ONO-), m/z 50 (18ON18O-) and m/z 47 (O15NO-, internal standard).

Skeletal Muscle Function in Young Patients With Cystic Fibrosis.

PURPOSE: Defects in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) cause CF. Absence of the CFTR may result in skeletal muscle dysfunction. Here, we tested skeletal muscle function in male adolescent patients with CF. METHODS: Ten CF and 10 control participants (age: 16.8 ± 0.6 years) performed 7 repetitive sets of maximum voluntary contractions (MVCs) and underwent an isometric fatigue test of the knee extensors. Electromyography (EMG) activity was recorded from the m. vastus lateralis (VL) and m. vastus medialis (VM). RESULTS: In CF, the MVC torque was lower and correlated with the predicted forced expiratory volume in one second (r = .73, p = .012, n = 10). The M-wave in the VL was shorter in CF than in controls (18.6 ± 0.5 vs. 20.3 ± 0.5 ms, p < .028). In the VM, both the M-wave (4.96 ± 0.61 vs. 7.97 ± 0.60 mV, p = .001) and the EMG (0.29 ± 0.04 vs. 0.47 ± 0.04 mV, p = .004) amplitudes were smaller in CF. CONCLUSION: The differences in the VL and VM EMG signals between the groups indicate that the lower MVC torque in CF did not result from the direct impact of a CFTR defect on the sarcolemmal excitability; the differences more likely resulted from the less developed musculature in the patients with CF.

Energy metabolism in intensively exercising calf muscle under a simulated orthostasis.

We conducted non-invasive methods to investigate the mechanisms how an orthostasis improves fatigue resistance in human calf muscle during intense exercise. Eleven healthy volunteers performed two series of ten intervals of maximum dynamic exercise (15 s) and recovery (45 s) at almost horizontal body position under both, control conditions (CON) and lower body negative pressure (LBNP, -40 mbar). As from the second work interval, LBNP significantly improved fatigue resistance shown as a lower reduction in work and in contraction velocity (P < 0.01). During each work interval, EMG showed a small increase in amplitude (P < 0.01) and a steep drop by 20% in median frequency (P < 0.01). Under LBNP, both EMG parameters completely recovered during subsequent rest, whereas under CON recovery was incomplete (P < 0.01). During the first work interval, consumption of phosphocreatine (PCr) was almost the same for both conditions. In periods of recovery under LBNP, resynthesis of PCr and inorganic phosphate were significantly faster. PCr reached 10 to 20% higher levels (P < 0.01). LBNP caused an initial increase in intracellular pH (0.08 U (P < 0.01)). The subsequent time courses of pH were similar for CON and LBNP. During work, pH steeply increased by about 0.3 U. During subsequent recovery, pH dropped to values between 6.3 and 6.5. LBNP caused significantly higher levels of total haemoglobin and oxy-haemoglobin (P < 0.05). A simulated orthostasis increased fatigue resistance during high intense interval exercise because of a faster PCr resynthesis and may be because of improvements in the maintenance of motoneuronal activity.

Human blood neutrophil responses to prolonged exercise with and without a thermal clamp.

The purpose of this study was to investigate the effects of prolonged exercise with and without a thermal clamp on neutrophil trafficking, bacterial-stimulated neutrophil degranulation, stress hormones, and cytokine responses. Thirteen healthy male volunteers (means +/- SE: age 21 +/- 1 yr; mass 74.9 +/- 2.1 kg; maximal oxygen uptake 58 +/- 1 ml x kg(-1) x min(-1)) completed four randomly assigned, 2-h water-immersion trials separated by 7 days. Trials were exercise-induced heating (EX-H: water temperature 36 degrees C), exercise with a thermal clamp (EX-C: 24 degrees C), passive heating (PA-H: 38.5 degrees C), and control (CON: 35 degrees C). EX-H and EX-C was comprised of 2 h of deep water running at 58% maximal oxygen uptake. Blood samples were collected at pre-, post-, and 1 h postimmersion. Core body temperature was unaltered on CON, clamped on EX-C (-0.02 degrees C), and rose by 2.23 degrees C and 2.31 degrees C on EX-H and PA-H, respectively. Exercising with a thermal clamp did not blunt the neutrophilia postexercise (EX-C postexercise: 9.6 +/- 1.1 and EX-H postexercise: 9.8 +/- 1.0 x 10(9)/liter). Neutrophil degranulation decreased (P < 0.01) similarly immediately after PA-H (-21%), EX-C, and EX-H (-28%). EX-C blunted the circulating norepinephrine, cortisol, granulocyte-colony stimulating factor, and IL-6 response (P < 0.01) but not the plasma epinephrine and serum growth hormone response. These results show a similar neutrophilia and decrease in neutrophil degranulation after prolonged exercise with and without a thermal clamp. As such, the rise in core body temperature does not appear to mediate neutrophil trafficking and degranulation responses to prolonged exercise. In addition, these results suggest a limited role for cortisol, granulocyte-colony stimulating factor, and IL-6 in the observed neutrophil responses to prolonged exercise.

The relationships between plasma potassium, muscle excitability and fatigue during voluntary exercise in humans.

The relationships between extracellular potassium elevation and EMG variables in relation to muscle fatigue were investigated during handgrip exercise in humans. Acid-base state, lactate, potassium ([K+](v)) and sodium in venous plasma, as well as variables of surface voluntary and evoked (M-wave) EMG were determined during repeated dynamic (DE) and static (SE) exercise (1 min exercise, 4 min rest). The different rises of [K+](v) were induced by randomly varied workloads. After 15 min of warming up, the M-wave area increased to 124.9 +/- 19.6% (P < 0.001) in comparison with the control value. Simultaneously, the [K+](v) decreased from 4.1 +/- 0.3 to 3.6 +/- 0.3 mmol l(-1) (P < 0.01). During both SE and DE, there were marked intensity-dependent signs of fatigue. The [K+](v) correlated with changes of the integrated EMG (r = 0.87, P < 0.001 for both DE and SE). Changes in the M-wave area during the exercise bouts correlated inversely with the [K+](v) (r = -0.73, P < 0.001). The M-wave area did not decrease below the control value at any intensity. The median frequency of the EMG decreased during exercise, depending on the exercise intensity (r = -0.73 for SE, r = -0.47 for DE, P < 0.001) with a maximal decrease to about 80% after SE with the maximal workload. The muscle action potential propagation velocity changed in the range of about +/-2%. For the first time, a negative relationship between venous potassium and M-wave area was shown during voluntary exercise. However, there was no evidence that the decrease in muscle performance was mainly caused by a decrease in sarcolemmal excitability resulting from a high extracellular [K+].