A preliminary study of an alternative method for evaluating skin sensitizing potential of chemicals.

BACKGROUND: In order to comply with the European legislation concerning the risk assessment of skin sensitizers, considerable progress has been made in developing alternative methods, such as human cell line activation test (h-CLAT). H-CLAT is based on cytometric measurement of fluorescence emitted by anti-CD54 and anti-CD86 antibodies in THP-1 cells. Following this method, a range of substances have been analyzed; the emitted fluorescence, generally at low intensity, has caused problems concerning the interpretation of results. AIM: Find an alternative parameter to h-CLAT for evaluating the sensitizing potential of chemicals. MATERIALS AND METHODS: Cells have been analyzed with flow cytometry after treatment with sensitizing compounds administered at non-cytotoxic concentrations. RESULTS: Sensitizers were able to inducealterations in cell morphology to a more 'condensed' one allowing the identification of cells under microscope as a 'sensitized' subpopulation. These variations cause similar modifications in 'scattering' parameters, making cells easily monitorable by flow cytometry. No changes have been observed in cells treated with non-sensitizers or in untreated cells. CONCLUSION: This method based on the analysis of forward scatter and side scatter parameters, can be used as an alternative method for identifying sensitization potential of chemical compounds.

CONTEXTE: Pour se conformer à la législation européenne en matière d'évaluation des risques des sensibilisants cutanés, beaucoup des progrès ont été fait dans le développement de méthodes alternatives comme le test d'activation de la lignée cellulaire humaine (H-CLAT). H-CLAT se base sur la mesure cytométrique de la fluorescence émise par les anticorps anti-CD54 et anti-CD86 dans les cellules THP-1. Suivant cette méthode, une gamme de substances a été analysée; la fluorescence émise, généralement de faible intensité, a posé des problèmes d'interprétation des résultats. OBJECTIF: Trouver un paramètre alternatif à h-CLAT pour évaluer le potentiel sensibilisant des produits chimiques. MATÉRIAUX ET MÉTHODES: Les cellules ont été analysées par cytométrie en flux après le traitement avec des composés sensibilisants administrés à des concentrations non cytotoxiques. RÉSULTATS: Les sensibilisateurs ont pu induire des altérations de la morphologie cellulaire en une morphologie plus condensée, permettant l'identification des cellules au microscope comme sous-population sensibilisée. Ces variations entraînent des modifications similaires des paramètres de "scattering", rendant les cellules facilement contrôlables par cytométrie en flux. Aucun changement a été observé dans les cellules traitées avec des agents non sensibilisants ni dans les cellules non traitées. CONCLUSION: Cette méthode basée sur l'analyse des paramètres de "forward" et "side scatter" peut être utilisée comme méthode alternative pour identifier le potentiel de sensibilisation des composés chimiques.

Response of colonic motility to dopaminergic stimulation is subverted in rats with nigrostriatal lesion: relevance to gastrointestinal dysfunctions in Parkinson's disease.

BACKGROUND: Constipation is extremely common in patients with Parkinson's disease (PD) and has been described in PD animal models. In this study, we investigated whether a PD-like degeneration of dopaminergic neurons of the substantia nigra can influence peristalsis in colonic segments of rats by impacting on enteric dopaminergic transmission. METHODS: Male, Sprague-Dawley rats received a unilateral injection of neurotoxin 6-hydroxydopamine (6-OHDA), or saline, into the medial-forebrain-bundle. Peristaltic activity was recorded in isolated colonic segments, in baseline conditions and following exposure to combinations of D2 receptor (DRD2) agonist sumanirole and antagonist L-741626. Dopamine levels and DRD2 expression were assessed in the ileum and colon of animals. We also investigated the involvement of the dorsal motor nucleus of the vagus (DMV) - a potential relay station between central dopaminergic denervation and gastrointestinal (GI) dysfunction - by analyzing cytochrome c oxidase activity and FosB/DeltaFosB expression in DMV neurons. KEY RESULTS: We observed profound alterations in the response of colonic segments of 6-OHDA lesioned animals to DRD2 stimulation. In fact, the inhibition of colonic peristalsis elicited by sumanirole in control rats was absent in 6-OHDA-lesioned animals. These animals also showed reduced DRD2 expression in the colon, along with elevation of dopamine levels. No significant changes were detected within the DMV. CONCLUSIONS & INFERENCES: Our results demonstrate that selective lesion of the nigrostriatal dopaminergic pathway subverts the physiological response of the colon to dopaminergic stimulation, opening new perspectives in the comprehension and treatment of GI dysfunctions associated with PD.

Colonic mucosal mediators from patients with irritable bowel syndrome excite enteric cholinergic motor neurons.

BACKGROUND: Mediators released in the mucosal milieu have been suggested to be involved in visceral hypersensitivity and abdominal pain in patients with irritable bowel syndrome (IBS). However, their impact on myenteric neurons remains unsettled. METHODS: Mucosal biopsies were obtained from the descending colon of patients with IBS and controls. Mucosal mast cells were identified immunohistochemically. The impact of spontaneously released mucosal mediators on guinea pig electrically stimulated longitudinal muscle myenteric plexus (LMMP) preparations was assessed in vitro by means of selective receptor antagonists and inhibitors. KEY RESULTS: Patients with IBS showed an increased mast cell count compared with controls. Application of mucosal mediators of IBS to LMMPs potentiated cholinergic twitch contractions, an effect directly correlated with mast cell counts. Enhanced contractions were inhibited by 50.3% with the prostaglandin D2 antagonist BW A868C, by 31.3% and 39% with the TRPV1 antagonists capsazepine and HC-030031, respectively, and by 60.5% with purinergic P2X antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Conversely, the serotonin1-4, histamine1-3, tachykinin1-3 receptor blockade, and serine protease inhibition had no significant effect. CONCLUSIONS & INFERENCES: Colonic mucosal mediators from patients with IBS excite myenteric cholinergic motor neurons. These effects were correlated with mast cell counts and mediated by activation of prostanoid receptors, TRPV1, and P2X receptors. These results support the role of mucosal inflammatory mediators and mast cell activation in altered motor function of IBS.

Mitochondrial alterations, oxidative stress and neuroinflammation in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder characterized by the progressive deterioration of neuronal networks. The primary cause and sequence of its progression are only partially understood but abnormalities in folding and accumulation of insoluble proteins such as beta-amyloid and Tau-protein are both associated with the pathogenesis of AD. Mitochondria play a crucial role in cell survival and death, and changes in mitochondrial structure and/or function are related to many human diseases. Increasing evidence suggests that compromised mitochondrial function contributes to the aging process and thus may increase the risk of AD. Dysfunctional mitochondria contribute to reactive oxygen species which can lead to extensive macromolecule oxidative damage and the progression of amyloid pathology. Oxidative stress and amyloid toxicity leave neurons chemically vulnerable. The mitochondrial toxicity induced by beta-amyloid is still not clear but may include numerous mechanisms, such as the increased permeability of mitochondrial membranes, the disruption of calcium homeostasis, the alteration of oxidative phosphorylation with a consequent overproduction of reactive oxygen species. Other mechanisms have been associated with the pathophysiology of AD. Inflammatory changes are observed in AD brain overall, particularly at the amyloid deposits, which are rich in activated microglia. Once stimulated, the microglia release a wide variety of pro-inflammatory mediators including cytokines, complement components and free radicals, all of which potentially contribute to further neuronal dysfunction and eventually death. Clinically, novel approaches to visualize early neuroinflammation in the human brain are needed to improve the monitoring and control of therapeutic strategies that target inflammatory and other pathological mechanisms. Similarly, there is growing interest in developing agents that modulate mitochondrial function.

Effects of esomeprazole on glutathione levels and mitochondrial oxidative phosphorylation in the gastric mucosa of rats treated with indomethacin.

Proton pump inhibitors exert their preventive and healing effects on gastropathy induced by nonsteroidal anti-inflammatory drug (NSAIDs) by a dual action: the antisecretory and the antioxidant effect. The latter was investigated by using esomeprazole against indomethacin-induced gastric mucosa lesions in rats and assessed by a histomorphometric analysis. Treatment by intragastric gavage were 1% methocel as vehicle; esomeprazole 10, 30, or 60 micromol/kg; indomethacin 100 micromol/kg; and esomeprazole 10, 30, or 60 micromol/kg plus indomethacin 100 micromol/kg. The evaluation of glutathione (GSH) levels and respiratory chain complex activities [nicotinamide adenine dinucleotide, reduced (NADH)-ubiquinone oxidoreductase, succinate dehydrogenase, cytochrome C reductase, cytochrome oxidase] was performed in the isolated gastric mucosa. Esomeprazole (10-60 micromol/kg) dose dependently reversed, up to complete recovery, the inhibitory effect of indomethacin on GSH levels (approximately 60% inhibition) and mitochondrial enzyme activities (inhibition ranging from 60% to 75%). Indomethacin-induced mucosal injuries were reduced by esomeprazole. Thus, in addition to inhibiting acid secretion, the gastroprotective effect of esomeprazole can be ascribed to a reduction in gastric oxidative injury.

Energy expenditure and nutritional adequacy of rehabilitation paraplegics with asymptomatic bacteriuria and pressure sores.

OBJECTIVE: To measure resting energy expenditure (REE) in a group of people with postacute paraplegia, quantify the impact of asymptomatic bacteriuria and pressure sore(s) on patients' metabolic rate, and estimate the adequacy of patients' nutritional intakes to preserve patients' protein levels. MATERIAL AND METHODS: Ten males with post-acute paraplegia aged 42.1+/-18.7 years. We evaluated: height, body mass index (BMI), resting energy expenditure (REE), total daily calorie requirement (E), 24-h urine creatinine excretion (Cru), creatinine index (CI), and nitrogen balance (NB). RESULTS: Subjects with paraplegia showed high erythrocyte sedimentation rates. As a group, they had normal resting calorie consumption when REE was normalized for unit of urine creatinine (REE/Cru), it was higher in patients than in controls. Six of the 10 patients had a low calorie intake: of these only three had a negative nitrogen balance. CONCLUSION: In conclusion, the resting energy expenditure of the subjects with significant bacteriuria and pressure sore(s) of 23.7 kcal/kg/day suggests that a large portion of patients may have an inadequate calorie protein intake to preserve their nutritional status. The clinical significance of this study is that 28.5 kcal/kg/day may be the lower calorie threshold to meet the metabolic demands of people with apyretic paraplegia with bacteriuria and pressure sore(s).

Neuromuscular junction disassembly and muscle fatigue in mice lacking neurotrophin-4.

Neurotrophin-4 (NT-4) is produced by slow muscle fibers in an activity-dependent manner and promotes growth and remodeling of adult motorneuron innervation. However, both muscle fibers and motor neurons express NT-4 receptors, suggesting bidirectional NT-4 signaling at the neuromuscular junction. Mice lacking NT-4 displayed enlarged and fragmented neuromuscular junctions with disassembled postsynaptic acetylcholine receptor (AChR) clusters, reduced AChR binding, and acetylcholinesterase activity. Electromyographic responses, posttetanic potentiation, and action potential amplitude were also significantly reduced in muscle fibers from NT-4 knock-out mice. Slow-twitch soleus muscles from these mice fatigued twice as rapidly as those from wild-type mice during repeated tetanic stimulation. Thus, muscle-derived NT-4 is required for maintenance of postsynaptic AChR regions, normal muscular electrophysiological responses, and resistance to muscle fatigue. This neurotrophin may therefore be a key component of an activity-dependent feedback mechanism regulating maintenance of neuromuscular connections and muscular performance.

Effects of the beta(2)-agonist clenbuterol on respiratory and limb muscles of weaning rats.

The aim of this study was to analyze the effects of chronic administration of the beta(2)-agonist clenbuterol (1.5 mg x kg(-1) x day(-1) for 4 wk in the drinking water) on respiratory (diaphragm and parasternal intercostal) and hindlimb (tibialis and soleus) muscles in young rats during postnatal development (21 to 49 postnatal days). The treatment resulted in very little stimulation of muscle growth. Significant slow-to-fast transitions in the expression of myosin heavy chain isoforms and significant increases in the myofibrillar ATPase activity were found in the diaphragm and soleus, whereas tibialis anterior and intercostal muscles did not show any significant fiber-type alteration. Decrease of oxidative enzyme activities and increase of glycolytic enzyme activities were also observed. It is concluded that whereas the growth stimulation is age dependent and only detectable in adult rats, the fiber-type transformation is also present in weaning rats and particularly evident in the soleus and diaphragm. The fiber-type transformation caused by clenbuterol might lead to an enhancement of contractile performance and also to a reduced resistance to fatigue.

The effects of aging on enzyme activities and metabolite concentrations in skeletal muscle from sedentary male and female subjects.

Aging affects the metabolic capacity of skeletal muscle, in particular the glycolytic and respiratory capacities. The purpose of this study was to quantify biochemical alterations due to aging in muscular metabolic capacity in human skeletal muscles in sedentary subjects. The activities of various marker enzymes and metabolites related to glycolysis, Krebs' cycle and the electron transfer chain and high energy phosphate compounds were measured in muscle biopsies from the rectus abdominis, vastus lateralis, and gluteus maximus muscles of 76 sedentary subjects (32 males and 44 females) between 15 and 91 yr. No significant differences between males and females were found, but changes related to age were: a decrease in hexokinase and lactate dehydrogenase activities in the rectus abdominis; a decrease in citrate synthase activity and citrate in the vastus lateralis; an increase in pyruvate kinase activity and a decrease in ATP and creatine phosphate concentrations in the gluteus maximus. These data suggest that distinct muscles may respond differently to aging regardless of sex in sedentary subjects.

Peripheral plasma amino acid abnormalities in rehabilitation patients with severe brain injury.

OBJECTIVE: Acute severe brain injury causes an increased mobilization of amino acids from tissue. The plasma amino acid profile of patients undergoing rehabilitation after brain injury is unknown. This study was aimed at delineating the plasma amino acid profile of rehabilitation patients with brain injury. DESIGN: Peripheral plasma aminogram, lactate, pyruvate, glycerol, ketone body, and carnitine concentrations were determined in 11 patients with brain injury (34.6+/-15 years old, 60+/-16.8 days after injury) and in 8 controls. Resting energy expenditure and nitrogen balance were also determined. RESULTS: (1) All essential amino acids and about 50% of nonessential amino acids were significantly lower in brain injury patients than in controls (p < .05). (2) Plasma amino acids were lower irrespective of either energy and protein intake or nitrogen balance. (3) Total carnitine concentration and esterified/free carnitine ratio were higher in brain injury patients than in controls (p < .05). CONCLUSIONS: Rehabilitation patients with brain injury may have an important reduction of their plasma aminogram. Muscle tissue depletion and the persistence of a hypercatabolic state caused by subclinical infections, pressure sores, and immobility may contribute to this reduction.

Despite good compliance, very low fat diet alone does not achieve recommended cholesterol goals in outpatients with coronary heart disease.

AIM: A low-saturated, low-cholesterol diet is important in the treatment of hypercholesterolaemia in patients with coronary heart disease. The aim of this study was to investigate the efficacy of a very low fat diet to achieve a targeted serum low density lipoprotein (LDL) cholesterol level (3.37mmol x l-1 were investigated 12-14 weeks after an acute coronary event. After overnight fasting each patient had (a) his resting energy expenditure measured (indirect calorimetry using standard protocol) and (b) venous blood sampled from a forearm vein to determine lipid profile. All the patients were randomly allocated to four groups of treatment: Group A on a very low fat diet (resting energy expenditure-fat diet, where fat intake was

Metabolite concentrations in skeletal muscle of different aged rats submitted to hypoxia and pharmacological treatment with nicergoline.

The energy metabolism of the gastrocnemius and soleus muscles in young-adult, mature, and senescent rats was evaluated after 72 h of continuous exposure to normobaric hypoxia or normoxia. The effects of treatment with the alpha-adrenergic antagonist nicergoline were also investigated. In the gastrocnemius muscle we evaluated the concentrations of some significative metabolites involved in anaerobic glycolysis and the Krebs' cycle, free amino acids related to the Krebs' cycle, ammonia, some energy mediators, and the energy store creatine phosphate. In the soleus muscle a selection of these was evaluated. In both muscles aging was similarly characterized by a decrease in muscular creatine phosphate concentration, while the energy mediators and the energy charge potential remained unchanged. Singly, some gastrocnemius muscle metabolites showed linear changes in their concentrations with aging, while for the soleus muscle the only linear change regarded glucose-6-phosphate. Continuous normobaric hypoxia induced greater changes at the age of 4 and 24 months than at 12 months. Chronic treatment with nicergoline modified the influence of hypoxic conditions on muscle metabolites concentrations only in some cases, regardless of the age of the animals. Further investigations are necessary before any firm conclusions can be drawn about the pharmacological activity of nicergoline on hypoxia in aged rats.

Effect of L-carnitine on myocardial metabolism: results of a balanced, placebo-controlled, double-blind study in patients undergoing open heart surgery.

The effects of L-carnitine on cardiac performance after open heart surgery were evaluated in a balanced, placebo-controlled, double-blind study in 38 patients. Preoperative haemodynamic status was good in all of them. Seventeen subjects underwent mitral valve replacement and 19 patients coronary artery bypass grafting. Five grams L-carnitine were given intravenously over 2 h, twice daily for 5 consecutive days; moreover, 10 g L-carnitine in 1500 ml cardioplegia were administered through the aortic root after aortic cross-clamping. Surgery was always planned on treatment day 3. The post-ischaemic functional recovery of the heart was assessed by clinical parameters, as well as by biochemical and ultrastructure evaluations on biopsy specimens. No differences were found between the control and the treatment group with respect to all clinical parameters of cardiac performance after cardiopulmonary bypass. At anaesthesia induction, serum carnitine was significantly increased in treated patients, but carnitine concentrations in the right atrial biopsy obtained just before aortic declamping were similar in the two groups. In patients with mitral valve replacement, L-carnitine therapy was associated with significantly higher concentrations of pyruvate, ATP and creatine phosphate in papillary muscle. Glycogen levels were also higher in the treated group, but the difference was not statistically significant. Myocardial ultrastructure on septal biopsies, obtained within 5 min from weaning from extracorporeal circulation, showed better preservation scores for all considered parameters (nucleus, sarcoplasmic reticulum, mitochondria and cellular oedema) in the treated subjects, although the difference reached statistical significance only for nuclei. When biochemical and ultrastructural data are considered, these findings suggest that L-carnitine improves myocardial metabolism. However, it cannot be concluded that L-carnitine provides an advantageous support therapy for well-compensated patients requiring cardiac surgery. In contrast, the positive effects of L-carnitine on cardiac recovery after bypass might become clinically relevant in the surgical setting for haemodynamically compromised patients, in which further investigations are required.

Altered muscle energy metabolism in post-absorptive patients with chronic renal failure.

Skeletal muscle biopsies were performed on 12 healthy sedentary subjects and on 22 non-dyalized chronic renal failure patients (CRF) on a free diet and after overnight fasting. Parathormone, glucagon and insulin were determined at the same time of biopsies. CRF patients showed significantly low ATP and creatine phosphate levels. Regarding enzyme activities, a high hexokinase Vmax was found, while the pyruvate kinase activity was lower than in the control group. For the tricarboxylic acid cycle, citrate synthase, succinate dehydrogenase and malate dehydrogenase activities were higher; total NADH cytochrome c reductase activity was also high, while cytochrome oxidase activity was slightly lower. Both alanine aminotransferase and aspartate aminotransferase activities were considerably high in comparison with the control group. In conclusion, our study revealed a hypermetabolic TCA cycle, but impaired oxidative phosphorylation, which partly explained the reduced ATP concentration. Excessive protein intake and hormonal derangements may play a role in these metabolic changes.

Biochemical analysis of muscle biopsy in overnight fasting patients with severe chronic heart failure.

To better characterize the role of skeletal muscle in chronic heart failure we studied energetic charge, metabolites and enzyme activity in the energy production pathway. We selected 15 males with severe chronic heart failure (NYHA class III, stable clinical conditions and in normal nutritional status) and seven controls. Controls and patients were submitted to biopsy of the vastus lateralis muscle in resting and fasting conditions. Hormone profiles were also evaluated. Our results showed near normal ATP, ADP and AMP concentrations, but there were substantially more reductions in glycogen (46 +/- 5 vs 77 +/- 6 mumoles glycosidic units.g-1 fresh tissue) and creatine phosphate (5 +/- 1 vs 13 +/- 1 mumoles.g-1 fresh tissue) in patients than in controls. We also found a reduction in glycolytic activity (pyruvate kinase 1009 +/- 79 vs 1625 +/- 26 nmoles. min-1.mg protein-1), despite normal tricarboxylic acid cycle velocity, an increase in alanine amino-transferase (964 +/- 79 vs 425 +/- 34 nmoles. min-1.mg protein-1) and in aspartate aminotransferase (515 +/- 44 vs 291 +/- 56 nmoles.min-1.mg protein-1). An increase was also observed in total NADH cytochrome c reductase (128 +/- 14 vs 68 +/- 5 nmoles.min-1.mg protein-1), while cytochrome oxidase activity was normal. The cortisol/insulin ratio was slightly elevated (77 +/- 4 vs 32 +/- 12). In conclusion, normonutritive patients with severe heart failure show an imbalance in the energy production/utilization ratio. The impairment is probably due both to a decrease in production and an increase in consumption of energy owing to greater cellular workload and/or a hypercatabolic state.

Pyruvate dehydrogenase deficiency in a child responsive to thiamine treatment.

We report the clinical features in a 4-year-old child who was investigated for a suspected metabolic disorder but was subsequently diagnosed as having a pyruvate dehydrogenase deficiency. A muscle biopsy was performed and the data obtained suggested thiamine treatment which resulted in a regression of the clinical findings and a return to normal values of blood lactic and pyruvic acids. The interruption of thiamine supplementation after 1 year of treatment led to a prompt recurrence of the previous clinical and biochemical symptoms.

Instrumental and metabolic evaluation of patients affected by peripheral arterial occlusive disease (PAOD) following surgical revascularization surgery.

Experiments were performed on eight subjects affected by peripheral arterial occlusive disease (PAOD) of the lower limbs. Each patient was submitted to Ecodoppler, angiography and the "Treadmill test". Two bioptic muscle of these patients. A sample was used for the spectrophotometric and spectrophotofluorimetric determinations of: glycogen, pyruvate, lactate, citrate, alpha-ketoglutarate, malate, aspartate, glutamate, AMP, ADP, ATP and creatine phosphate (CP). The other bioptic sample was used to determine the following enzyme activities: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, malate dehydrogenase, total NADH cytochrome c reductase, cytochrome oxidase, aspartate aminotransferase and alanine aminotransferase. Patients showed an increase in lactate dehydrogenase, total NADH cytochrome c reductase and succinate dehydrogenase activities, a decrease in glycogen, ATP and CP concentrations. Telethermographic data showed patient muscle thermic emission quantitatively different from control group. The telethermographic test can be used as an additional diagnostic tool to determine and monitor the efficiency of a muscle undergoing metabolic failure.

Effects of hypoxia on enzyme activities in skeletal muscle of rats of different ages. An attempt at pharmacological treatment.

The activities of enzymes related to energy metabolism in the gastrocnemius and soleus muscles in young-adult (4 months), mature (12 months) and senescent (24 months) rats were compared after 72 h of continuous exposure to normobaric hypoxia or normoxia after alpha-adrenergic antagonist nicergoline or saline solution had been given intraperitoneally for 30 consecutive days. The maximum rates (Vmax) of the following enzyme activities in the crude extract and/or the mitochondrial fraction of each muscle specimen were evaluated: (1) for the anaerobic glycolytic pathway: hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase; (2) for the tricarboxylic acid cycle; citrate synthase and malate dehydrogenase; (3) for the electron transfer chain; cytochrome oxidase; and (4) for the NAD+/NADH redox state: total NADH cytochrome c reductase. The significant differences between the enzyme activities at different ages or under different experimental conditions in the two tissue preparations of the two muscles were determined by ANOVA. MCA and ETA were used to evaluate the net effects of the experimental conditions. Ageing did not seem to affect the soleus and gastrocnemius muscles in the same way. Changes were seen only in the glycolytic pathway enzymes in the crude extract from the gastrocnemius muscle. In the soleus muscle changes in enzyme activities as a function of ageing were also found in the mitochondrial fraction. We also found that hypoxia caused greater changes in 12-month-old rats than in those of other ages (especially in the enzyme activities of the gastrocnemius muscle). Finally out data show that only in certain cases was the pharmacological treatment able to modify the influence of hypoxic conditions on the levels of enzyme activities, regardless of the age of animals.

Biochemical evaluations in skeletal muscles of primates with MPTP Parkinson-like syndrome.

The toxic effects of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in primates can be exploited for investigating the physiopathology of Parkinson's disease which may also cause functional alterations of skeletal muscles, whose biochemical modifications have been studied very little. Some enzyme activities related to energy transduction in skeletal muscles were evaluated (gastrocnemius, soleus and biceps) from MPTP-treated monkeys. Systemically administered MPTP altered the enzyme activities related to: (i) the anaerobic glycolytic pathway (decrease in hexokinase and phosphofructokinase activities; increase in lactate dehydrogenase activity); (ii) the tricarboxylic acid cycle (decrease in malate dehydrogenase activity); (iii) the electron transfer chain (decrease in cytochrome oxidase activity related to complex IV). No alteration in mitochondrial Complex I was observed. Treatment with an ergot alkaloid derivative (dihydroergocryptine) modified some alterations in the muscle enzyme activities and reduced the rigidity and some autonomic dysfunction.

Modifications by chronic intermittent hypoxia and drug treatment on skeletal muscle metabolism.

The energy metabolism was evaluated in gastrocnemius muscle from 3-month-old rats subjected to either mild or severe 4-week intermittent normobaric hypoxia. Furthermore, 4-week treatment with CNS-acting drugs, namely, alpha-adrenergic (delta-yohimbine), vasodilator (papaverine, pinacidil), or oxygen-increasing (almitrine) agents was performed. The muscular concentration of the following metabolites was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactate-to-pyruvate ratio; citrate, alpha-ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creatine phosphate. Furthermore the Vmax of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The adaptation to chronic intermittent normobaric mild or severe hypoxia induced alterations of the components in the anaerobic glycolytic pathway [as supported by the increased activity of lactate dehydrogenase and/or hexokinase, resulting in the decreased glycolytic substrate concentration consistent with the increased lactate production and lactate-to-pyruvate ratio] and in the mitochondrial mechanism [as supported by the decreased activity of malate dehydrogenase and/or citrate synthase resulting in the decreased concentration of some key components in the tricarboxylic acid cycle]. The effect of the concomitant pharmacological treatment suggests that the action of CNS-acting drugs could be also related to their direct influence on the muscular biochemical mechanisms linked to energy transduction.