Thomas Tobin

Rates of dissociation of enzyme-ouabain complexes and K0.5 values in (Na+ + K+) adenosine triphosphatase from different species

Abstract The half-lives of rabbit kidney and dog brain (Na+ + K+) adenosine triphosphatase (ATPase)-ouabain complexes were 10 and 89 min respectively. The ratios between these half-lives and those previously reported for guinea-pig kidney and rat brain enzyme-ouabain are more than sufficient to account for the differences in the K0.5 values for ouabain and those enzymes reported in the literature. The K0.5 values for the ouabain-enzyme interaction could be reduced up to 12-fold by pre-incubating the enzyme with Na+, Mg2+, ATP and ouabain prior to the addition of K+ to start the enzyme assay. These differences show that the K0.5 values are not obtained under equilibrium conditions and suggest that the true equilibrium position is intermediate between the measured K0.5 values. Ki values estimated from the dissociation rates of enzyme-ouabain fall between the measured K0.5 values. It appears that the differences in the dissociation rates alone are sufficient to account for the observed differences in the K0.5 values. The experiments also show a marked difference in glycoside sensitivity between rat brain and heart (Na+ + K+) ATPase.

Quabain: Temporal relationship between the inotropic effect and the in vitro binding to, and dissociation from, (Na++K+)-activated ATPase

SummaryThe time course of the inotropic response to ouabain in Langendorff preparations was compared with that of the in vitro ATP-dependent (3H)-ouabain binding to cardiac (Na++K+)-activated ATPase preparations, and subsequent dissociation, to determine the temporal relationship between the inotropic response and (Na++K+)-activated ATPase inhibition.Species differences were minimal either in the onset of inotropic response or the (3H)-ouabain binding. The rates of both loss of the inotropic response to ouabain during washout and the dissociation of the ouabain-enzyme complex, however, were rapid in guinea pig and rabbit (relatively ouabain-insensitive species) and slow in cat and dog (ouabain-sensitive species). The half-time of the loss of the inotropic response was similar to the half-time of the dissociation of the ouabain-enzyme complex in each species.Since ATP-dependent binding of cardiac glycosides has been related to enzyme inhibition, it was concluded that the time course of the inotropic response to ouabain parallels the time course of (Na++K+)-activated ATPase inhibition, and that the dissociation of ouabain from the enzyme may terminate the inotropic response.

A method for studying cutaneous pain perception and analgesia in horses

Pain perception and its alteration by analgesic drugs is difficult to measure in the horse. The latency to onset of flexion of a limb in response to a noxious thermal stimulus has been used as a nociceptive end point for analgesic studies in many species. While this method has been employed in the horse, it may be confounded by the spontaneous locomotor activity observed after administration of narcotic analgesics. Consequently, an alternative method of assaying narcotic analgesia that did not involve the equine locomotor apparatus was developed. This report describes the use of the heat-evoked skin-twitch reflex as a reproducible measure of pain threshold and its alteration by the narcotic analgesic fentanyl. This method is compared with the heat-evoked hoof-withdrawal reflex, and the apparatus necessary to elicit both reflexes in the horse is described. Fentanyl, administered at intravenous doses of 0.010, 0.005, and 0.0025 mg/kg, produced a dose-related prolongation of the skin-twitch reflex but failed to alter the latency to hoof withdrawal following noxious thermal stimulation. The skin-twitch reflex is therefore a more sensitive assay of narcotic analgesia in the horse than is the hoof-withdrawal reflex.

Dose-related effects of fentanyl on autonomic and behavioral responses in performance horses

The dose-related effects of intravenously administered fentanyl (0.010, 0.005, 0.0025 mg/kg) and saline were studied in mature performance horses using a rigorous experimental protocol. Fentanyl produced a dose-related prolongation of the skin twitch reflex latency but did not increase the hoof withdrawal reflex latency. Dose related increases in stepping frequency, cardiac and respiratory rats were observed following fentanyl, while changes in rectal temperature and pupil area were not. These data indicate that fentanyl, a prototypic mu-agonist, produces a syndrome characterized by analgesia, locomotor and sympathetic stimulation in the horse.

Clinical Use and Characteristics of the Corticosteroids

Corticosteroids possess potent anti-inflammatory activity and are commonly injected intra-articularly for local relief of inflammatory lesions in performance horses. However, the suppression of anabolic activity in the joint may lead to an increased rate of joint breakdown. Complications associated with intra-articular corticosteroid therapy include septic arthritis, which is usually due to inadvertent joint contamination at the time of corticosteroid injection, and steroid arthropathy, which is characterized by an accelerated rate of joint destruction and radiographic evidence of severe degenerative joint disease. Prognosis for both of these conditions is poor. Adverse effects of systemic corticosteroid therapy includes suppression of the hypothalamic-pituitary adrenal system, increased susceptibility to infection, and laminitis; however, moderate use of glucocorticoids does not permanently affect adrenal function.

Effects of monovalent cations on cardiac Na+, K+-ATPase activity and on contractile force.

SummaryThe relationship between Na+, K+-ATPase inhibition by monovalent cations and their inotropic effect was studied in guinea pig hearts. The activity of partially purified cardiac enzyme was assayed in the presence of 5.8 mM KCl and either 20 or 150 mM NaCl. Rb+ and Tl+ inhibited Na+, K+-ATPase activity, the magnitude of the inhibition by these cations being greater in the assay media containing lower Na+ concentrations. Tl+ produced a dose-dependent inhibition of Na+, K+-ATPase activity in the presence of 20 mM Na+ and 75 mM K+, a cationic condition similar to that of intracellular fluid. Other monovalent cations such as K+, Cs+, NH4+, Na+ or Li+ produced essentially no effect on the Na+, K+-ATPase activity or slightly stimulated it. In left atrial strips stimulated with field electrodes and bathed in Krebs-Henseleit solution (5.8 mM K+ and 145 mM Na+), addition of Cs+ failed to alter the isometric contractile force significantly. NH4+ and K+ caused a transient positive inotropic effect which was partially blocked by propranolol. The positive inotropic response to K+ was followed by a negative inotropic response. Rb+ produced a sustained, dose-dependent inotropic response reaching a plateau at 1–2 min, whereas Tl+ produced a dose-dependent positive inotropic effect which developed slowly over a 30-min period. The positive inotropic effects produced by Rb+ and Tl+ were insensitive to propranolol pretreatment. Concentrations of Tl+ and cardiac glycosides which produce similar inotropic effects appear to cause the same degree of Na+-pump inhibition. The onset of the positive inotropic response to Rb+ or Tl+ was not dependent on the number of contractions which is in contrast to the cardiac glycoside-induced inotropic response. Substitution of 20 mM LiCl for an equimolar amount of NaCl in Krebs-Henseleit solution produced a significantly greater inotropic response than that observed when sucrose was substituted for NaCl. It appears that, among monovalent cations, only sodium pump inhibitors produce a sustained positive inotropic response.

Cassaine: Mechanism of inhibition of Na++K+-ATPase and relationship of this inhibition to cardiotonic actions

The erythrophleum alkaloid cassaine shares many of the pharmacological actions of the cardiac glycosides but lacks the structural characteristics typical of cardiac glycosides. To further investigate the relationship between Na+ +K+ -ATPase inhibition and the cardiotonic actions of these drugs we investigated the interaction of cassaine with the Na+ +K+ -ATPase. Cassaine inhibited rat brain Na+ +K+ -ATPase with about one quarter of the apparent affinity of ouabain for this enzyme. This inhibition was non-competitive with respect to K+. Cassaine also inhibited this enzyme in the presence of Mg2+ and this inhibition was enhanced by Pi and antagonized by Na+. In the presence of Na+, Mg2+ and (gamma-32P)-ATP cassaine acted to stabilize the phosphorylated intermediate of Na+ +K+ -ATPase. Cassaine also acted to displace specifically bound (3H)-ouabain from this enzyme. These observations suggested that cassaine inhibited the Na+ +K+ -ATPase by interacting at the cardiotonic steroid binding sites of Na+ +K+ -ATPase. Consistent with this hypothesis, dog, guinea pig and rat heart Na+ +K+ -ATPase showed differing sensitivities to cassaine paralleling their differing sensitivities to ouabain. The principal difference between the interaction of cassaine and ouabain with Na+ +K+ -ATPase appeared to be the more rapid dissociation of cassaine from the cardiotonic steroid binding site(s) of Na+ +K+ -ATPase. In keeping with this the rates of offset of cassaine-induced inotropy in Langendorff perfused dog and guinea pig hearts were several times faster than those of ouabain-induced inotropy.

Nonsteroidal anti‐inflammatory agents and musculoskeletal injuries in Thoroughbred racehorses in Kentucky

Injuries sustained by horses during racing have been considered as an unavoidable part of horse racing. Many factors may be associated with the musculoskeletal injuries of Thoroughbred race horses. This study surveyed the amounts of nonsteroidal anti-inflammatory agents (NSAIDs) in injured horses biological system (plasma) at Kentucky racetracks from January 1, 1995 through December 31, 1996. During that period, there were 84 catastrophic cases (euthanized horses) and 126 noncatastrophic cases. Plasma concentrations of NSAIDs were determined by High Performance Liquid Chromatography in injured and control horses. The possible role of anti-inflammatory agents in musculoskeletal injuries of Thoroughbred race horses was investigated by comparing the apparent concentrations of NSAIDs in injured horses to concentrations in control horses. The plasma concentrations of phenylbutazone and flunixin were higher in injured horses than in control horses. Most injured and control horses did not have a detectable level of naproxen in their plasma samples. Further studies must be carried out to determine whether horses with higher plasma concentrations of NSAIDs have an altered risk of musculoskeletal injuries compared with other horses.

Pharmacokinetics of gabapentin in horses

*Department of Veterinary Biosciences, University of Illinois, College of Veterinary Medicine, Urbana, IL; Department of Biomedical Sciences, College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL; Livestock Disease and Diagnostic Center, College of Agriculture, University of Kentucky, Lexington, KY; Department of Large Animal Clinics, College of Veterinary Medicine, Nursing and Allied Health, Tuskegee University, Tuskegee, AL; 128C Maxwell Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA

Differential effects of phenylbutazone and local anesthetics on nociception in the equine

The effects of procaine, mepivacaine and phenylbutazone on pain perception in the equine were studied using two behavioral assays of nociception; the thermal evoked hoof withdrawal reflex and skin twitch reflex. Pain perception threshold was measured as the latency from onset of thermal stimuli to reflex withdrawal of the forelimb or contraction of the cutaneous musculature. Procaine 2% and mepivacaine 2% prolonged the hoof withdrawal reflex latency when administered locally by producing a block of the palmar and metacarpal nerves. Significant analgesia lasted 90 min and 210 min for procaine and mepivacaine, respectively. Phenylbutazone (7.3 mg/kg) failed to alter pain thresholds measured over a 36 h post-treatment period. However, pain thresholds rose over time with successive trials. These data suggest that in the equine (1) phenylbutazone does not alter normal cutaneous pain perception, and (2) successive presentation of painful stimuli increases nociceptive thresholds.