Topic:Adenosine Triphosphate (ATP)
Myosin heavy chain-based fibre types in red cell hyper- and normovolaemic Standardbred trotters. An assumed link between red cell hypervolaemia, an excessive amount of training and impaired performance of hypervolaemic horses has led to a theory that the muscle fibres could be affected. Myosin heavy chain (MHC)-based fibre type composition in gluteus medius muscle of red blood cell normo- (NV) and hypervolaemic (HV) Standardbred trotters was evaluated using immunohistochemistry. Muscle biopsies were obtained from 13 NV and 16 HV horses. Serial transverse sections were cut and reacted with antibodies against different isoforms of the myosin heavy chains MHCI, MHCIIA and MHCIIX. Sections we...
Effects of prolonged training, overtraining and detraining on skeletal muscle metabolites and enzymes. Thirteen Standardbred horses trained intensively for 34 weeks and detrained for 12 weeks to investigate the effects of training, overtraining and detraining on muscle metabolites, buffering capacity and enzyme activities (CS, HAD and LDH). After a standardised exercise test to fatigue at 10 m/s (approximately 100% VO2max), there was significant depletion of [ATP], [PCr] and muscle [glycogen] and accumulation of muscle and plasma [lactate], [NH3] and elevated muscle temperature. After training, associated with increased run time to fatigue (148%), there was reduced depletion of muscle [glycogen...
Pro- and macroglycogenolysis in skeletal muscle during maximal treadmill exercise. The purpose was to investigate the degradation of proglycogen and macroglycogen in skeletal muscle during intense exercise. Ten Standardbred trotters performed a maximal treadmill exercise test comprising a warm-up period, an exercise period, starting at 7 m/s with increments of 1 m/s every 60 s until the onset of fatigue (mean +/- s.d. 246 +/- 32 s) and a walking recovery period. Muscle biopsies were taken at rest, immediately after exercise and 15 min postexercise. The exercise caused a marked anaerobic metabolism as shown by the decrease in both muscle ATP and creatine phosphate and increas...
Ribose supplementation in maximally exercising Thoroughbreds. A diverse group of studies, which are equine exclusive, indicate that ribose administered to myocardial and skeletal muscle tissue stimulates ATP production and recovery. This study investigated the effects of ribose supplementation on blood and muscle metabolites and performance in Thoroughbred geldings performing a maximal treadmill standardised exercise test (SET). In Experiment 1, 6 conditioned Thoroughbred geldings performed a baseline SET and horses were assigned to one of 2 experimental treatment groups, placebo or ribose, based on VO2max. The placebo treatment group received 0.07 g glu...
Effect of sodium bicarbonate administration on metabolic responses to maximal exercise. Administration of bicarbonate has been shown to cause metabolic alkalosis both in man and in horses and is, therefore, thought to increase the buffering capacity of the body and thereby delay the onset of fatigue. However, results regarding the influence of sodium bicarbonate loading on performance both in human athletes and in horses are conflicting. The aim of this study was, therefore, to investigate the metabolic response to a standardised treadmill exercise test to fatigue, in horses given bicarbonate (0.6 g/kg bwt), in comparison to horses given placebo (water). Five Standardbred trotter...
Effects of a P2Y(12) receptor antagonist on the response of equine platelets to ADP. Comparison with human platelets. Horses show susceptibility to platelet-related disorders. Equine platelets differ from human platelets in some of their responses, so information available about human platelets must be validated in the horse. Aggregation of platelets by ADP involves both P2Y(1) and P2Y(12) receptors on the platelet surface. We have compared the effect of the P2Y(12) antagonist, AR-C67085, on equine and human platelets in vitro using turbidimetric aggregometry to measure the rate and final extent of aggregation. Aggregation profiles, concentration-response curves and pA(2) values show that the rate of aggregat...
Effects of P2Y(1) and P2Y(12) receptor antagonists on ADP-induced shape change of equine platelets: comparison with human platelets. Platelet activation by adenosine 5' -diphosphate (ADP) is via both P2Y(1 )and P2Y(12) receptors and leads to shape change and aggregation. The effects on ADP-induced platelet shape change of two P2Y(1) antagonists, adenosine 3'-phosphate, 5'-phosphosulfate (A3P5PS) and 2-deoxy-N(6)-methyladenosine 3', 5'-diphosphate (MRS-2179) and a P2Y(12) antagonist 2-propylthio-D-beta,gamma-dichloromethylene-adenosine 5'-triphosphate (AR-C67085MX) were determined by turbidimetric aggregometry and scanning electron microscopy (SEM) on equine and human platelets. The platelet aggregation was inhibited during ...
ATPase and Adpase activities in synovial membrane of equine metacarpophalangeal joint. ATPase and ADPase activities capable of hydrolyzing nucleoside di- and triphosphates in the presence of Ca2+ are present in synovial membrane of metacarpophalangeal joint mainly associated to membrane fractions. These hydrolytic activities have been considered involved in the inflammatory process where ATP and ADP are inflammatory mediators while adenosine counteracts this effect. Both, subcellular localization and kinetic properties of these nucleotidase activities, suggest that could correspond to single enzyme called ATP-diphosphohydrolase or apyrase. The comparison of the activity on ATP-C...
Cytosolic ratio of malate dehyrogenase/lactate dehydrogenase activity in peripheral leukocytes of race horses with training. The activities of the enzymes involved in the malate-aspartate shuttle and m RNA expression of malate dehydrogenase (MDH), a crucial enzyme for the NADH shuttle that produces ATP in glucose metabolism in the peripheral leukocytes of horses, were measured to investigate the change in metabolic states with training. There were no significant differences in plasma glucose and immunoreactive insulin concentrations between race horses and riding horses, used as a comparable reference. The cytosolic and mitochondrial MDH activities in leukocytes of race horses were significantly higher than those of...
Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. Methods: Blood samples from 6 Thoroughbreds. Methods: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. Results: In samples incubated...
Horse lumbrical muscle: possible structural and functional reorganization in regressive muscle. An anatomical study of horse lumbrical muscle (Lm) was carried out by light and electron microscopy in combination with immunochemical and cytochemical methods. Paraffin sections were subjected to haematoxylin and eosin (H & E) and Masson's trichrome staining for morphometric analysis. Paraffin sections were also used for immunostaining by anti-PGP 9.5 for reaction with nerve-protein associated-structures, anti-heat-shock protein 70 (hsp 70) for detection of gene expression changes, anti-fast myosin for the determination of muscle fibre types, and for detection of apoptotic gene expression...
Changes in fibre type composition of gluteus medius and semitendinosus muscles of Dutch Warmblood foals and the effect of exercise during the first year postpartum. In order to obtain broader insights into the equine musculoskeletal system, we studied the fibre type composition of 2 locomotory muscles in biopsies from Dutch Warmblood foals taken at 3 different ages in the first year postpartum. The muscle fibre types were determined histochemically as well as immunohistochemically. ATPase-characterised IIB fibres appear to express either IId or type lIa plus IId myosin heavy chain (MHC). A high percentage of fibres classified as IIA with ATPase expressed both fast types of MHC. The type I classification by the 2 methods matched almost completely. There wa...
Effects of adenosine on bacterial lipopolysaccharide- and interleukin 1-induced nitric oxide release from equine articular chondrocytes. To determine whether adenosine influences the in vitro release of nitric oxide (NO) from differentiated primary equine articular chondrocytes. Methods: Articular cartilage harvested from the metacarpophalangeal and metatarsophalangeal joints of 11 horses (3 to 11 years old) without history or clinical signs of joint disease. Methods: Chondrocytes were isolated, plated at a high density (10(5) cells/well), and treated with adenosine, the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), bradykinin, or other agents that modify secondary messenger pathways alone or in combination ...
In vitro responses of equine colonic arterial and venous rings to adenosine triphosphate. To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. Methods: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. Methods: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine tri...
Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains. Combined methodologies of immunohistochemistry, histochemistry and photometric image analysis were applied: (1) to characterise control equine skeletal muscle fibres according to their myosin heavy chain (MyHC) composition and (2) to determine on a fibre-to-fibre basis the correlation between contractile [i.e. MyHC(s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [i.e. succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities, glycogen and phospholamban (PLB) contents], and morphological [i.e. cross-sect...
Calcium sensitivity of force production and myofibrillar ATPase activity in muscles from Thoroughbreds with recurrent exertional rhabdomyolysis. To determine whether the basis for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds lies in an alteration in the activation and regulation of the myofibrillar contractile apparatus by ionized calcium. Methods: 4 Thoroughbred mares with RER and 4 clinically normal (control) Thoroughbreds. Methods: Single chemically-skinned type-I (slow-twitch) and type-II (fast-twitch) muscle fibers were obtained from punch biopsy specimens, mounted to a force transducer, and the tensions that developed in response to a series of calcium concentrations were measured. In addition, myofibril preparation...
Alterations in systemic and local colonic hemodynamic variables associated with intravenous infusion of ATP-MgCl2 in healthy anesthetized horses. To characterize alterations in systemic and local colonic hemodynamic variables associated with IV infusion of ATP-MgCl2 in healthy anesthetized horses. Methods: 12 adult horses. Methods: Six horses were given ATP-MgCl2, IV, beginning at a rate of 0.1 mg of ATP/kg of body weight/min with incremental increases until a rate of 1.0 mg/kg/min was achieved. The remaining 6 horses were given an equivalent volume of saline (0.9% NaCl) solution over the same time period. Colonic and systemic hemodynamic variables and colonic plasma nitric oxide (NO) concentrations were determined before, during, and a...
Specificity and reversibility of the training effects on the concentration of Na+,K+-Atpase in foal skeletal muscle. The purpose of the present study was to determine whether training and detraining affect the Na+,K+-ATPase concentration in horse skeletal muscles, and whether these effects are specific for the muscles involved in the training programme. Twenty-four Dutch Warmblood foals age 7 days were assigned randomly to 3 groups: Box (box-rest without training), Training (box-rest with training: short-sprint) and Pasture (pasture without training). Exercise regimens were carried out for 5 months and were followed by 6 months of detraining. Five of the foals in each group were subjected to euthanasia at ag...
Quantitation of adenine nucleotides in equine colonic mucosal tissue using high performance liquid chromatography. The objectives were to use high performance liquid chromatography (HPLC) to validate an established method for adenine nucleotide separation in equine colonic mucosal tissue, to determine the inherent variability in the tissue and extraction method, and to determine the stability of ATP, ADP, and AMP in the tissue with time. Equine colonic mucosal tissue obtained from a single horse was immediately submersed in liquid nitrogen, and stored at -70 degrees C. Samples were lyophilized, extracted, and separated by HPLC. The limit of quantitation was 0.05 microg/mL. The coefficient of variation for ...
Prolonged food restriction and mild exercise in Shetland ponies: effects on weight gain, thyroid hormone concentrations and muscle Na(+),K(+)-ATPase. We determined the effects of food supply and low-intensity training on growth, serum thyroid hormone levels and the Na(+),K(+)-pump concentration in equine skeletal muscle. Twenty-two Shetland ponies were subjected to two different feeding regimes for 2(1/2) years (11 ponies per group): food restriction (body condition score kept at 2) or ad libitum fed (body condition score kept at 8). Five ponies in each group underwent low-intensity training. Gluteus medius muscle and serum samples were obtained in April 1998. Subsequently, all ponies were fed ad libitum and the training programme was stopp...
Effects of training on the concentration of Na+, K+-ATPase in foal muscle. We investigated the effect of training on the Na+, K+-ATPase concentration in foal skeletal muscle by measurement of [3H]ouabain binding. From the 7th day after birth, 12 foals were divided in 3 groups: a) staying in a box stall (Box); b) staying in a box stall with a training regimen of an increasing number of sprints per day (Training); and c) staying on pasture (Pasture). Euthanasia was performed after 5 months. In semitendinosus muscle, the concentration of [3H]ouabain binding sites (pmol/g wet wt) was 181 +/- 6 in the Box, 220 +/- 15 in the Training, and 197 +/- 8 in the Pasture group (al...
Actions and interactions of ADP, 5- HT, histamine and PAF on equine platelets. Platelets are thought to play a role in equine diseases such as acute laminitis and verminous arteritis and may be involved in allergic disease. Mediators implicated in the pathophysiology of these conditions activate platelets and responses may be enhanced by interactions between mediators. The present study compared platelet aggregation, thromboxane production and release of radiolabelled [(3)H]5- HT in response to 5- HT, histamine, ADP and PAF alone and in combination in vitro.PAF caused concentration-related aggregation, [(3)H]5- HT release and thromboxane production. In contrast, ADP caus...
Muscle adenine nucleotide degradation during submaximal treadmill exercise to fatigue. The aim was to investigate metabolic response in muscle during submaximal treadmill exercise to fatigue, with a special emphasis on adenine nucleotide degradation products such as inosine monophosphate (IMP) in muscle and hypoxanthine, xanthine and uric acid in plasma. Five Standardbred trotters performed treadmill exercise on 2 occasions, once at 7 m/s and once at 10 m/s. Venous blood samples were taken at rest, during exercise and at the end of exercise. Muscle biopsies were taken before and after exercise and muscle temperature was measured before and after exercise. Running time differed a...
Skeletal muscle Na(+)-K(+)-ATPase and K+ homeostasis during exercise: effects of short-term training. The objective of this study was to determine the effects of 10 consecutive days of moderate intensity training on 1) the concentration of middle gluteal muscle Na(+)-K(+)-ATPase as determined by vanadate-facilitated 3H[ouabain binding; and 2) plasma potassium regulation before, during and after exercise at 100% of the pre-training maximum rate of oxygen consumption (VO2max). Six mature, unfit Thoroughbred horses completed both incremental (for determination of VO2max) and high-intensity exercise protocols before (HI1) and after (HI2) training. There additional horses undertook no training or e...
Incremental treadmill exercise until onset of fatigue and its relationship to metabolic response and locomotion pattern. The aim was to study metabolic response and locomotion pattern in Standardbred trotters during incremental treadmill exercise performed by increasing speed by 1 m/s in 1 min steps (start 7 m/s) until the onset of fatigue. The test protocol included determination of oxygen uptake, heart rate (HR), stride length (SL) and stride frequency (SF). Venous blood samples were collected at rest, at the end of each exercise step and after 30 min of recovery. Muscle biopsies were taken at rest and post exercise and muscle temperature was measured after exercise. As horses fatigued at different speed steps...
Identification of calcium-activated potassium channels in cultured equine sweat gland epithelial cells. The patch-clamp recording technique was used to examine the properties of the K+ channels in cultured equine sweat gland epithelial cells. With symmetric K+ solutions (140 mM), a single population of K+ channels was identified with a slope conductance of 187 pS and a reversal potential of around 0 mV. The channel was selective for K+ over Na+. Channel activity was increased by membrane depolarization. A 10-fold increase in [Ca2+]i produced an approximate 60 mV negative shift in the open state probability (Popen)-voltage curve. Externally applied tetraethylammonium ions (TEA+) caused a rapid an...
Hemodynamic and metabolic alterations associated with intravenous infusion of a combination of adenosine triphosphate and magnesium chloride in conscious horses. To determine hemodynamic and metabolic effects of IV infusion of ATP-MgCl2 combination and maximal safe IV infusion rate in conscious horses. Methods: 6 adult female horses. Methods: All horses received an IV infusion of ATP-MgCl2 combination, beginning at a rate of 0.05 mg of ATP/kg of body weight/min, which was increased by 0.05 mg/kg/min increments at 10-minute intervals until a rate of 1.0 mg/kg/min was achieved. Data were collected prior to the start of the infusion, at the end of each infusion rate, and at 15-minute intervals for the next hour after discontinuation of the infusion. Measu...
Pyrimidine nucleotide-evoked inhibition of cyclic AMP accumulation in equine epithelial cells. Uridine triphosphate (UTP) evoked inhibition of adrenaline-evoked cAMP accumulation in cultured equine epithelial cells (EC50, 1.8 +/- 0.2 microM) and this effect was mimicked by 5-Br-UTP (EC50, 6.6 +/- 1.8 microM) and uridine diphosphate (UDP; EC50, 96 +/- 26 microM). This inhibitory action of UTP was abolished by pre-treating cells with pertussis toxin (10 ng ml-1, 24 h). UTP (EC50, 2.3 +/- 0.3 microM) and 5-Br-UTP (EC50, 29.4 +/- 9.4 microM) also increased intracellular free calcium ([Ca2+]i) whilst UDP did not; the two effects are thus differentially sensitive to these pyrimidine nucleotid...
CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa. Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, ...
Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants. The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of 'pure' I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling sit...