Diagnosing overtraining in athletes using the two-bout exercise protocol.

OBJECTIVE: In this work, whether a two-bout exercise protocol can be used to make an objective, immediately available distinction between non-functional over reaching (NFO) and overtraining syndrome (OTS) was studied. DESIGN: Underperforming athletes who were diagnosed with the suspicion of NFO or OTS were included in the study. Recovery of the athletes was monitored by a sports physician to retrospectively distinguish NFO from OTS. SETTING: Sports medicine laboratory PARTICIPANTS: The protocol was started and completed by 10 underperforming athletes. NFO was retrospectively diagnosed in five athletes, and OTS was diagnosed in five athletes. INTERVENTIONS: A two-bout maximal exercise protocol was used to measure physical performance and stressinduced hormonal reactions. MAIN OUTCOME MEASUREMENTS: Exercise duration, heart rate and blood lactate concentration were measured at the end of both exercise tests. Venous concentrations cortisol, adrenocorticotrophic hormone (ACTH), prolactin and growth hormone were measured both before and after both exercise tests. RESULTS: Maximal blood lactate concentration was lower in OTS compared with NFO, while resting concentrations of cortisol, ACTH and prolactin concentrations were higher. However, sensitivity of these measures was low. The ACTH and prolactin reactions to the second exercise bout were much higher in NFO athletes compared with OTS and showed the highest sensitivity for making the distinction. CONCLUSIONS: NFO might be distinguished from OTS based on ACTH and prolactin reactions to a two-bout exercise protocol. This protocol could be a useful tool for diagnosing NFO and OTS; however, more data should be collected before this test can be used as the gold standard.

Hormonal responses during prolonged exercise are influenced by a selective DA/NA reuptake inhibitor.

OBJECTIVE: A decrease in dopamine activity is thought to lead to a reduction in motivation and arousal and therefore to the "central" component of fatigue. The purpose of the present study was to investigate the effects of a dopamine (DA) noradrenaline (NA) reuptake inhibitor, bupropion (Zyban), on exercise performance and on the hormonal response to exercise. METHODS: Eight healthy well trained male cyclists (Watt(max) 397+/-15 W) participated in the study. Subjects completed one maximal exercise test (to determine maximal power output Watt(max)), and two endurance performance tests (time trials) in a double blind randomised cross-over design. Subjects took either placebo capsules (lactose) or 2 x 300 mg bupropion (BUP). Blood samples were collected for adrenocorticotropin (ACTH), prolactin, cortisol, growth hormone, beta-endorphins, and catecholamines. RESULTS: Performance was not influenced by BUP (placebo: 89+/-1 min; BUP 2 x 300 mg: 89+/-0.7 min). All hormones increased during exercise in all trials. Cortisol plasma concentrations were significantly higher in the BUP trial at rest, at min 60, and at the end of exercise, while beta-endorphins were higher in the BUP trial at the end of exercise and during recovery, and ACTH at the end of exercise. CONCLUSION: From the present results, we can conclude that bupropion had a more marked central noradrenergic effect (compared to dopaminergic) on the hormonal response to exercise, but no effect on the outcome of performance.

Hormonal responses in athletes: the use of a two bout exercise protocol to detect subtle differences in (over)training status.

In overtrained athletes, several signs and symptoms have been associated with the imbalance between training and recovery. However, reliable diagnostic markers for distinguishing between well-trained, overreached (OR) and overtrained (OT) athletes are lacking. A hallmark feature of overtraining syndrome (OTS) is the inability to sustain intense exercise and recover for the next training or competition session. We therefore devised a test protocol utilizing two bouts of maximal work. With this test protocol we tried to establish a difference in hormonal responses between the training status of T and OR athletes. Seven well-trained cyclists participated in this study and were tested before and after a training camp. We also present the data of one OT motocross athlete who was clinically diagnosed as overtrained. All athletes performed two maximal exercise tests separated by 4 h. Blood was analyzed for cortisol, adrenocorticotrophic hormone (ACTH), growth hormone and prolactin (PRL). Performance decreased by 6% between the first and the second exercise test in the OR group and by 11% in the OT subject. Moreover, during the second exercise test there were more marked differences between the T and OR athletes; in particular, the OT subject did not show an increase in some of the hormonal responses. PRL increased only by 14% in the OT subject's second test and there was a 7% decrease in ACTH. The two exercise approach enables us to detect subtle performance decrements that will not be identified by one exercise trigger. The hormonal responses to the second exercise test were different between the T and OR athletes (the increase in the T group was higher than in the OR that was higher than in the OT). The results of the case presentation of an overtrained athlete provide evidence of an altered and dysfunctional hypothalamic-pituitary axis response to two bouts of maximal exercise. These findings can be used to develop markers for diagnosis of OTS and to begin to address the pathologic mechanism operative in the syndrome, as well as providing an outcome measure to evaluate possible therapeutic regimes.

Action of pinaverium bromide on calmodulin-regulated functions.

Pinaverium bromide at concentrations below 10(-5) M did not inhibit calmodulin-dependent enzymes such as phosphodiesterase and the Ca transport ATPase of the plasma membrane. At higher concentrations the compound interacted with the stimulation of those enzymes by calmodulin and also inhibited the calmodulin-independent activity. A similar inhibitory action was observed for the NaK ATPase. It is concluded that the inhibitory action of pinaverium bromide on smooth muscle concentration at concentrations below 10(-5) M was due to its interaction with the voltage-dependent Ca channels and not to its interference with the calmodulin-dependent activation of the contractile proteins.

Reconstitution of the purified calmodulin-dependent (Ca2+ + Mg2+)-ATPase from smooth muscle.

The purified calmodulin dependent (Ca2+ + Mg2+)-ATPase (CaMg ATPase) from porcine antral smooth muscle transports Ca2+ after reconstitution in lipid vesicles indicating that this enzyme is indeed a Ca2+-transport ATPase. For CaMg ATPase reconstituted in asolectin vesicles a good correlation was found between the time course of Ca2+ accumulation and the corresponding changes in CaMg ATPase activity. The ATPase activity was stimulated 8-fold by A23187, which further indicates a tight coupling between ATP hydrolysis and Ca2+ transport. Asolectin vesicles with incorporated enzyme accumulated Ca2+ with a ratio approaching one Ca2+ ion transported for each ATP hydrolyzed. For CaMg ATPase reconstituted in phosphatidylcholine vesicles on the other hand, Ca2+ transport and CaMg ATPase were poorly coupled as is shown by the approximately 3.5 fold stimulation by A23187. The activity of the CaMg ATPase when reconstituted in asolectin vesicles was stimulated 1.25 fold by calmodulin while in phosphatidylcholine a value of 4.25 was obtained. The CaMg ATPase activity of the enzyme reconstituted either in asolectin or phosphatidylcholine was, after its stimulation by A23187, still further stimulated by detergent by a factor of 5.

Tissue levels and purification by affinity chromatography of the calmodulin-stimulated Ca2+ -transport ATPase in pig antrum smooth muscle.

The Ca2+ -transport ATPase [Ca2+ + Mg2+)-ATPase) in a plasma membrane-rich fraction of porcine antrum (stomach) smooth muscle, is stimulated 2.9-times by calmodulin in the presence of 0.2 mg/ml saponin and reaches a value of 12.0 +/- 2.0 (4) mumol/100 mg protein (equivalent to 110 g wet tissue) per min at 37 degrees C and 10(-5) M [Ca2+]. Saponin was found to specifically potentiate the calmodulin-(Ca2+ + Mg2+)-ATPase interaction, even in the Triton X-100 solubilized enzyme. The conditions for purification of the (Ca2+ + Mg2+)-ATPase by affinity chromatography on a calmodulin-Sepharose 4B gel were optimized. The purified enzyme has a specific activity of 11.9 mumol/mg protein per min at 37 degrees C, 10(-5) M [Ca2+], 0.6 microM calmodulin, and shows a double polypeptide band at 140 and 150 kDa. The (Ca2+ + Mg2+)-ATPase can be incorporated in artificial liposomes that thereupon show an ATP-dependent Ca2+ uptake (Ca:ATP = 1.0). The magnitude of the calmodulin stimulation of the isolated enzyme depends on its phospholipid environment. When isolated in the presence of phosphatidylserine no calmodulin stimulation is observed. After reconstitution in phosphatidylcholine the calmodulin stimulation amounts to 4.05 +/- 0.63 (n = 12) times.

Antibodies to the calmodulin-binding Ca2+-transport ATPase from smooth muscle.

Antibodies were raised against a calmodulin-binding CaMg-ATPase (Ca2+-transport ATPase) from smooth muscle. The binding of these antibodies to a number of related Ca2+-transport ATPases was studied. Antibodies to the calmodulin-binding ATPase from porcine antrum (stomach) smooth muscle do not only bind to this CaMg-ATPase, but also to the corresponding enzyme in porcine erythrocytes. However, they do not bind to the CaMg-ATPase from sarcoplasmic reticulum of porcine skeletal muscle. The binding of these antibodies to the CaMg-ATPase of smooth muscle, does not inhibit the enzyme activity.

Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum.

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.

Demonstration of the phosphorylated intermediates of the Ca2+-transport ATPase in a microsomal fraction and in a (Ca2+ + Mg2+)-ATPase purified from smooth muscle by means of calmodulin affinity chromatography.

Ca2+ -dependent hydroxylamine-sensitive phosphorylated proteins can be demonstrated in a microsomal fraction of porcine antrum (stomach) smooth muscle and in a Ca2+ -transport ATPase ((Ca2+ + Mg2+)-ATPase) purified from this tissue by means of a calmodulin affinity technique. These phosphoproteins represent the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPases. In the (Ca2+ + Mg2+)-ATPase purified from smooth muscle the phosphorylated intermediate has an Mr of 130000 corresponding to the value found for erythrocyte (Ca2+ + Mg2+)-ATPase. In the smooth muscle microsomal fraction this 130 kDa phosphoprotein can also be seen, although its intensity is usually very low compared to a corresponding phosphorylation at Mr 100000. Including La3+ together with Ca2+ during phosphorylation of the microsomes increased selectively the steady state-level of the 130 kDa phosphoprotein over the value of the 100 kDa one. The 100 kDa Ca2+ -dependent phosphoprotein could either indicate the presence of a (Ca2+ + Mg2+)-ATPase of the same type of sarcoplasmic reticulum of skeletal muscle, or it could represent a proteolytic product of the 130 kDa phosphoprotein.

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

The effect of calmodulin on the active calcium-ion transport and (Ca2+ + Mg2+)-dependent ATPase in microsomal fractions of smooth muscle compared with that in erythrocytes and cardiac muscle.

The Ca2+ uptake and the (Ca2+ + Mg2+)-dependent ATPase of the porcine coronary-artery smooth-muscle microsomal fraction ('microsomes') are only slightly stimulated by calmodulin. The Ca2+ uptake after 2 min in the absence of oxalate, corrected for the ATP-independent binding, increased by a factor of 1.44, whereas the (Ca2+ + Mg2+)-dependent ATPase is stimulated 1.39 times. These findings contrast with the effect observed in human erythrocyte 'inside-out' microsomes. In these vesicles calmodulin increases the Ca2+ uptake after 20 min in an oxalate-free medium and the (Ca2+ + Mg2+)-dependent ATPase respectively by a factor of 3.82 and 6.18. The magnitude of the calmodulin stimulation of the Ca2+ transport in coronary-artery microsomes is similar to that observed in heart microsomes.