Anders Kalsen

β2‐Adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise‐induced reductions in Na+–K+‐ATPase Vmax in trained men

From animal models, it is well established that β2‐adrenergic stimulation increases contractile force, rates of Ca2+ release and uptake from the sarcoplasmic reticulum, and Na+–K+‐ATPase activity of skeletal muscles. However, these effects are unexplored in humans. Here we report that β2‐adrenergic stimulation with the high dose selective β2‐adrenoceptor agonist terbutaline elicits positive inotropic and lusitropic effects on non‐fatigued m. quadriceps that are associated with enhanced rates of Ca2+ release and uptake from the sarcoplasmic reticulum in trained men. However, we also observed that the positive inotropic and lusitropic effects of β2‐adrenergic stimulation on m. quadriceps were blunted when muscle fatigue developed. Furthermore, we show that β2‐adrenergic stimulation counteracts exercise‐induced reductions in Na+–K+‐ATPase Vmax (maximum rate of activity) and elevates glycolytic activity during high intensity exercise. These findings are important for our understanding of the role of β2‐adreceptor activation in regulation of ion handling and contractile properties of non‐fatigued and fatigued skeletal muscles in humans.

Combined inhalation of beta2 -agonists improves swim ergometer sprint performance but not high-intensity swim performance.

There is a high prevalence of asthma and airway hyperresponsiveness (AHR) in elite athletes, which leads to a major use of beta2‐agonists. In a randomized double‐blinded crossover study, we investigated the effects of combined inhalation of beta2‐agonists (salbutamol, formoterol, and salmeterol), in permitted doses within the World Anti‐Doping Agency 2013 prohibited list, in elite swimmers with (AHR, n = 13) or without (non‐AHR, n = 17) AHR. Maximal voluntary isometric contraction of m. quadriceps (MVC), sprint performance on a swim ergometer and performance in an exhaustive swim test at 110% of VO2max were determined. Venous plasma interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) were measured post‐exercise. No improvement was observed in the exhaustive swim test, but swim ergometer sprint time was improved (P < 0.05) in both groups from 57 ± 1.7 to 56 ± 1.8 s in AHR and 58.3 ± 1 to 57.4 ± 1 s in non‐AHR. MVC and post‐exercise plasma IL‐6 increased (P < 0.05) with beta2‐agonists in both groups, whereas IL‐8 only increased in AHR. In summary, inhalation of beta2‐agonists, in permitted doses, did not improve swim performance in elite swimmers. However, swim ergometer sprint performance and MVC were increased, which should be considered when making future anti‐doping regulations.

Mechanisms underlying enhancements in muscle force and power output during maximal cycle ergometer exercise induced by chronic β2-adrenergic stimulation in men

The study was a randomized placebo-controlled trial investigating mechanisms by which chronic β2-adrenergic stimulation enhances muscle force and power output during maximal cycle ergometer exercise in young men. Eighteen trained men were assigned to an experimental group [oral terbutaline 5 mg/30 kg body weight (bw) twice daily (TER); n = 9] or a control group [placebo (PLA); n = 9] for a 4-wk intervention. No changes were observed with the intervention in PLA. Isometric muscle force of the quadriceps increased (P ≤ 0.01) by 97 ± 29 N (means ± SE) with the intervention in TER compared with PLA. Peak and mean power output during 30 s of maximal cycling increased (P ≤ 0.01) by 32 ± 8 and 25 ± 9 W, respectively, with the intervention in TER compared with PLA. Maximal oxygen consumption (V̇o2max) and time to fatigue during incremental cycling did not change with the intervention. Lean body mass increased by 1.95 ± 0.8 kg (P ≤ 0.05) with the intervention in TER compared with PLA. Change in single fiber cross-sectional area of myosin heavy chain (MHC) I (1,205 ± 558 μm(2); P ≤ 0.01) and MHC II fibers (1,277 ± 595 μm(2); P ≤ 0.05) of the vastus lateralis muscle was higher for TER than PLA with the intervention, whereas no changes were observed in MHC isoform distribution. Expression of muscle proteins involved in growth, ion handling, lactate production, and clearance increased (P ≤ 0.05) with the intervention in TER compared with PLA, with no change in oxidative enzymes. Our observations suggest that muscle hypertrophy is the primary mechanism underlying enhancements in muscle force and peak power during maximal cycling induced by chronic β2-adrenergic stimulation in humans.

The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis.

The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti-Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO2max and a 20-km time-trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0-24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol Cmax was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·∙h) than EX (16.9±2.9 ng/mL·∙h)(P<0.01) and EXD (16.1±3.2 ng/mL·∙h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright

Urine concentrations of oral salbutamol in samples collected after intense exercise in endurance athletes.

Our objective was to investigate urine concentrations of 8 mg oral salbutamol in samples collected after intense exercise in endurance athletes. Nine male endurance athletes with a VO2max of 70.2 ± 5.9 mL/min/kg (mean ± SD) took part in the study. Two hours after administration of 8 mg oral salbutamol, subjects performed submaximal exercise for 15 min followed by two, 2-min exercise bouts at an intensity corresponding to 110% of VO2max and a bout to exhaustion at same intensity. Urine samples were collected 4, 8, and 12 h following administration of salbutamol. Samples were analyzed by the Norwegian World Anti-doping Agency (WADA) laboratory. Adjustment of urine concentrations of salbutamol to a urine specific gravity (USG) of 1.020 g/mL was compared with no adjustment according to WADAs technical documents. We observed greater (P = 0.01) urine concentrations of salbutamol 4 h after administration when samples were adjusted to a USG of 1.020 g/mL compared with no adjustment (3089 ± 911 vs. 1918 ± 1081 ng/mL). With the current urine decision limit of 1200 ng/mL for salbutamol on WADAs 2013 list of prohibited substances, fewer false negative urine samples were observed when adjusted to a USG of 1.020 g/mL compared with no adjustment. In conclusion, adjustment of urine samples to a USG of 1.020 g/mL decreases risk of false negative doping tests after administration of oral salbutamol. Adjusting urine samples for USG might be useful when evaluating urine concentrations of salbutamol in doping cases.

Inhaled Beta2-Agonist Increases Power Output and Glycolysis during Sprinting in Men.

PURPOSE The aim of the present study was to investigate the effects of the beta2-agonist terbutaline (TER) on power output and muscle metabolism during maximal sprint cycling. METHODS In a randomized double-blind cross-over design, nine moderately trained men (VO2max = 4.6 ± 0.2 L · min(-1)) conducted a 10-s cycle sprint after inhalation of either 15 mg of TER or placebo (PLA). A muscle biopsy sample was collected before and <10 s after the sprint and was analyzed for metabolites. RESULTS The mean power and peak power during the sprint were 8.3% ± 1.1% and 7.8% ± 2.5% higher (P < 0.05) with TER than with PLA, respectively. Moreover, the net rates of glycogenolysis (6.5 ± 0.8 vs 3.1 ± 0.7 mmol glucosyl units · kg dry weight(-1) · s(-1)) and glycolysis (2.4 ± 0.2 vs 1.6 ± 0.2 mmol glucosyl units · kg dry weight(-1) · s(-1)) were higher (P < 0.05) with TER than with PLA. After the sprint, adenosine triphosphate (ATP) was reduced with PLA (P < 0.05) but not with TER. During the sprint, there was no difference in the breakdown of phosphocreatine (PCr) between treatments. Estimated anaerobic ATP utilization was 9.2% ± 4.0% higher (P < 0.05) with TER than with PLA. After the sprint, ATP in Type II fibers was lowered (P < 0.05) by 25.7% ± 7.3% with PLA but was not reduced with TER. Before the sprint, PCr in Type II fibers was 24.5% ± 7.2% lower (P < 0.05) with TER than with PLA. With PLA, breakdown of PCr was 50.2% ± 24.8% higher (P < 0.05) in Type II fibers (vs Type I fibers), whereas no difference was observed between fiber types with TER. CONCLUSION The present study shows that a TER-induced increase in power output is associated with increased rates of glycogenolysis and glycolysis in skeletal muscles. Furthermore, as TER counteracts a reduction in ATP in Type II fibers, TER may postpone fatigue development in these fibers.

Urine Concentrations of Inhaled Salmeterol and its Metabolite a-Hydroxysalmeterol in Asthmatic and Non-Asthmatic Subjects

Salmeterol is a long-acting beta2-agonist, which is on the WADA prohibited list, but can be used by athletes in therapeutic doses by inhalation. The prohibited list, however, contains no urinary threshold for salmeterol, which gives athletes the opportunity to inhale unlimited doses of salmeterol. In doping controls, metabolites may be used as markers for misuse of substances. No studies have determined urine concentrations of α-hydroxysalmeterol, the metabolite of salmeterol. Furthermore, the metabolism and excretion of salmeterol may vary between asthmatics and nonasthmatics. We determined the serum and urinary concentrations of salmeterol and its metabolite α-hydroxysalmeterol after inhalation of 100 μg salmeterol in ten asthmatics and ten non-asthmatics. Blood samples were collected at baseline and ½, 1, 2, 3, 4 and 6 hours after the administration of salmeterol. Urine samples were collected at baseline and 4, 8 and 12 hours after administration. The urinary concentration of salmeterol following enzymatic hydrolysis of the glucuronide fraction was 0.38 ± 0.26 ngmL-1 in asthmatics and 0.38 ± 0.22 ngmL-1 in non-asthmatics, 4 hours after inhalation. The highest median serum concentration (Cmax) was 0.07 ± 0.03 ngmL-1 in asthmatics after 30 minutes (Tmax) and 0.06 ± 0.03 ngmL-1 in non-asthmatics. No statistical differences were found in Cmax or Tmax of salmeterol between asthmatics or non-asthmatics in neither the serum nor urine samples. The highest median urinary concentration of α-hydroxysalmeterol following enzymatic hydrolysis of the glucuronide fraction was 2.86 ± 1.75 ngmL-1 in asthmatics, 4 hours after inhalation of salmeterol. In non-asthmatics, it was 2.73 ± 2.08 ngmL-1. The asthmatics had a significantly (p<0.05) higher concentration after 12 hours compared with non-asthmatics. In doping control, α-hydroxysalmeterol may be a more suitable marker for excessive use of inhaled salmeterol, due to its higher concentration in urine.

Effect of formoterol, a long-acting β2-adrenergic agonist, on muscle strength and power output, metabolism and fatigue during maximal sprinting in men

The aim was to investigate the effect of the long-acting β2-adrenergic agonist formoterol on muscle strength and power output, muscle metabolism, and phosphorylation of CaMKII Thr(287) and FXYD1 during maximal sprinting. In a double-blind crossover study, 13 males [V̇o2 max: 45.0 ± 0.2 (means ± SE) ml·min(-1)·kg(-1)] performed a 30-s cycle ergometer sprint after inhalation of either 54 μg of formoterol (FOR) or placebo (PLA). Before and after the sprint, muscle biopsies were collected from vastus lateralis and maximal voluntary contraction (MVC), and contractile properties of quadriceps were measured. Oxygen uptake was measured during the sprint. During the sprint, peak power, mean power, and end power were 4.6 ± 0.8, 3.9 ± 1.1, and 9.5 ± 3.2% higher (P < 0.05) in FOR than in PLA, respectively. Net rates of glycogenolysis and glycolysis were 45.7 ± 21.0 and 28.5 ± 13.4% higher (P < 0.05) in FOR than in PLA, respectively, and the decrease in ATP content was lower (P < 0.05) in FOR than in PLA (3.7 ± 1.5 vs. 8.0 ± 1.6 mmol/kg dry weight). There was no difference in breakdown of phosphocreatine and oxygen uptake between treatments. Before and after the sprint, MVC and peak twitch force were higher (P < 0.05) in FOR than in PLA. No differences were observed in phosphorylation of CaMKII Thr(287) and FXYD1 between treatments before the sprint, whereas phosphorylation of CaMKII Thr(287) and FXYD1 was greater (P < 0.05) in FOR than in PLA after the sprint. In conclusion, formoterol-induced enhancement in power output during maximal sprinting is associated with increased rates of glycogenolysis and glycolysis that may counteract development of fatigue.

Intra-individual variability in the urine concentrations of inhaled salmeterol in male subjects with reference to doping analysis – impact of urine specific gravity correction

Since 2010, the World Anti-Doping Agency (WADA) has introduced urinary thresholds for some beta2-agonists. In doping analysis urine samples of beta2-agonists are not corrected for the Urine Specific Gravity (USG) by the WADA laboratories. Several studies have observed high differences in the urine concentrations of beta2-agonists when correction for USG is compared with no correction, as well as high inter-individual variability between subjects. However, no studies have measured the intra-individual variability after inhalation of the long-acting beta2-agonist salmeterol. As such, the purpose of this study was to measure the intra-individual variability in the urine concentrations of salmeterol and its metabolite α-hydroxysalmeterol. Furthermore, to highlight the variability between corrected and uncorrected urine samples for USG. Urine samples from 20 subjects were analyzed for USG, urine excretion and urine concentrations of salmeterol and α-hydroxysalmeterol. Seven of the subjects underwent a second visit with the same procedures. At each visit 100 μg salmeterol was administered by inhalation. Urine samples were collected before administration of the drug (T0) and 4 (T4), 8 (T8) and 12 (T12) hours after administration. The mean relative differences in the urine concentrations of salmeterol and α-hydroxysalmeterol between USG corrected and uncorrected samples were 43 ± 44, 27 ± 42 and 56 ± 87% at T4, T8 and T12, respectively. The intra-individual variability in the urine excretion of salmeterol and α-hydroxysalmeterol during visits one and two were 12.6 and 21.8%, respectively. The intra-individual variability of salmeterol and α-hydroxysalmeterol in the urine concentrations were significantly higher when uncorrected for USG with 43.0 and 43.7% versus 20.4% (p<0.01) and 28.0% (p<0.05), respectively. Correction for USG reduces inter-individual and intra-individual variability in urine concentrations of salmeterol and α-hydroxysalmeterol.

Beta2‐adrenoceptor agonist salbutamol increases protein turnover rates and alters signalling in skeletal muscle after resistance exercise in young men

Animal models have shown that beta2‐adrenoceptor stimulation increases protein synthesis and attenuates breakdown processes in skeletal muscle. Thus, the beta2‐adrenoceptor is a potential target in the treatment of disuse‐, disease‐ and age‐related muscle atrophy. In the present study, we show that a few days of oral treatment with the commonly prescribed beta2‐adrenoceptor agonist, salbutamol, increased skeletal muscle protein synthesis and breakdown during the first 5 h after resistance exercise in young men. Salbutamol also counteracted a negative net protein balance in skeletal muscle after resistance exercise. Changes in protein turnover rates induced by salbutamol were associated with protein kinase A‐signalling, activation of Akt2 and modulation of mRNA levels of growth‐regulating proteins in skeletal muscle. These findings indicate that protein turnover rates can be augmented by beta2‐adrenoceptor agonist treatment during recovery from resistance exercise in humans.