Effects Of ATX Research Articles

Astaxanthin Confers a Significant Attenuation of Hippocampal Neuronal Loss Induced by Severe Ischemia-Reperfusion Injury in Gerbils by Reducing Oxidative Stress.

Astaxanthin is a powerful biological antioxidant and is naturally generated in a great variety of living organisms. Some studies have demonstrated the neuroprotective effects of ATX against ischemic brain injury in experimental animals. However, it is still unknown whether astaxanthin displays neuroprotective effects against severe ischemic brain injury induced by longer (severe) transient ischemia in the forebrain. The purpose of this study was to evaluate the neuroprotective effects of astaxanthin and its antioxidant activity in the hippocampus of gerbils subjected to 15-min transient forebrain ischemia, which led to the massive loss (death) of pyramidal cells located in hippocampal cornu Ammonis 1-3 (CA1-3) subfields. Astaxanthin (100 mg/kg) was administered once daily for three days before the induction of transient ischemia. Treatment with astaxanthin significantly attenuated the ischemia-induced loss of pyramidal cells in CA1-3. In addition, treatment with astaxanthin significantly reduced ischemia-induced oxidative DNA damage and lipid peroxidation in CA1-3 pyramidal cells. Moreover, the expression of the antioxidant enzymes superoxide dismutase (SOD1 and SOD2) in CA1-3 pyramidal cells were gradually and significantly reduced after ischemia. However, in astaxanthin-treated gerbils, the expression of SOD1 and SOD2 was significantly high compared to in-vehicle-treated gerbils before and after ischemia induction. Collectively, these findings indicate that pretreatment with astaxanthin could attenuate severe ischemic brain injury induced by 15-min transient forebrain ischemia, which may be closely associated with the decrease in oxidative stress due to astaxanthin pretreatment.

The Effects of Drug Treatments for ADHD in Measures of Cognitive Performance.

Based on core symptoms of inattention and deficient impulse control, and the identification of effective pharmacotherapies such as amphetamine (AMP; Adderall®), methylphenidate (MPH; Ritalin®), and atomoxetine (ATX; Strattera®), ADHD is a clinical condition which provides opportunity for translational research. Neuropsychological tests such as the 5-Choice and Continuous Performance Tasks, which measure aspects of attention and impulse control in animals and humans, provide scope for both forward (animal to human) and reverse (human to animal) translation. Rodent studies support pro-attentive effects of AMP and MPH and effectiveness in controlling some forms of impulsive behavior. In contrast, any pro-attentive effects of ATX appear to be less consistent, the most reliable effects of ATX are recorded in tests of impulsivity. These differences may account for AMP and MPH being recognized as first-line treatments for ADHD with a higher efficacy relative to ATX. DSM-5 classifies three "presentations" of ADHD: predominantly inattentive type (ADHD-I), predominantly hyperactive/impulsive type (ADHD-HI), or combined (ADHD-C). Presently, it is unclear whether AMP, MPH, or ATX has differential levels of efficacy across these presentation types. Nonetheless, these studies encourage confidence for the forward translation of NCEs in efforts to identify newer pharmacotherapies for ADHD.

Atx regulates skeletal muscle regeneration via LPAR1 and promotes hypertrophy

Muscle differentiation is a multifaceted and tightly controlled process required for the formation of skeletal muscle fibers. Satellite cells are the direct cellular contributors to muscle repair in injuries or disorders. Here, we show that autotaxin (Atx) expression and activity is required for satellite cell differentiation. Conditional ablation of Atx or its pharmacological inhibition impairs muscle repair. Mechanistically, we identify LPAR1 as the key receptor in Atx-LPA signaling. Myogenic gene array and pathway analysis identified that Atx-LPA signaling activates ribosomal protein S6 kinase (S6K), an mTOR-dependent master regulator of muscle cell growth via LPAR1. Furthermore, Atx transgenic mice show muscle hypertrophic effects and accelerated regeneration. Intramuscular injections of Atx/LPA show muscle hypertrophy. In addition, the regulatory effects of Atx on differentiation are conserved in human myoblasts. This study identifies Atx as a critical master regulator in murine and human muscles, identifying a promising extracellular ligand in muscle formation, regeneration, and hypertrophy.

Atomoxetine Protects Against NMDA Receptor-mediated Hippocampal Neuronal Death Following Transient Global Cerebral Ischemia.

Atomoxetine has been widely used for the treatment of attention-deficit/ hyperactivity disorder. ATX has additional abilities such as antagonistic effects on the N-methyl-Daspartate receptors (NMDARs) and benefit effects in some animal models of neurological disorders. However, there were few studies regarding protective effects and related mechanisms of ATX against cerebral ischemic insults. The objective of this study is to investigate neuroprotection of ATX pretreatment and its mechanisms in the hippocampal cornu ammonis 1 (CA1) region following transient global cerebral ischemia in gerbils. Gerbils were subjected to transient global cerebral ischemia induced by the occlusion of common carotid arteries for 5 min. Thirty mg/kg of ATX was administrated intraperitoneally once daily for 3 days before ischemic surgery. To examine neuroprotective effects of ATX, we carried out neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. In addition, immunoreactivities of NMDAR1, NMDAR2A/B, B-cell lymphoma 2 (Bcl-2) and Bcl-2- associated X protein (Bax) are closely related with neuroexcitotoxicity. ATX pretreatment reduced ischemia-induced hyperactivity and protected CA1 pyramidal neurons from ischemia. Pretreatment with ATX significantly attenuated ischemia-induced increases of NMDAR1 and NMDAR2A/B immunoreactivities in the CA1 pyramidal neurons at early time following ischemia. In addition, significant ischemia-induced alterations of Bcl-2 and Bax immunoreactivities were not observed in the ATX-treated group following ischemia. These results show that pretreatment with ATX protected against ischemic neuronal via inhibition of ischemia-induced excitotoxicity at early time following transient global cerebral ischemia.

Clinical doses of atomoxetine significantly occupy both norepinephrine and serotonin transports: Implications on treatment of depression and ADHD

BackgroundAtomoxetine (ATX), a drug for treatment of depression and ADHD, has a high affinity for the norepinephrine transporter (NET); however, our previous study showed it had a blocking effect similar to fluoxetine on binding of [11C]DASB, a selective serotonin transporter (SERT) ligand. Whether the therapeutic effects of ATX are due to inhibition of either or both transporters is not known. Here we report our comparative PET imaging studies with [11C]MRB (a NET ligand) and [11C]AFM (a SERT ligand) to evaluate in vivo IC50 values of ATX in monkeys. MethodsRhesus monkeys were scanned up to four times with each tracer with up to four doses of ATX. ATX or saline (placebo) infusion began 2h before each PET scan, lasting until the end of the 2-h scan. The final infusion rates were 0.01–0.12mg/kg/h and 0.045–1.054mg/kg/h for the NET and SERT studies, respectively. ATX plasma levels and metabolite-corrected arterial input functions were measured. Distribution volumes (VT) and IC50 values were estimated. ResultsATX displayed dose-dependent occupancy on both NET and SERT, with a higher occupancy on NET: IC50 of 31±10 and 99±21ng/mL plasma for NET and SERT, respectively. At a clinically relevant dose (1.0–1.8mg/kg, approx. 300–600ng/mL plasma), ATX would occupy >90% of NET and >85% of SERT. This extrapolation assumes comparable free fraction of ATX in humans and non-human primates. ConclusionOur data suggests that ATX at clinically relevant doses greatly occupies both NET and SERT. Thus, therapeutic modes of ATX action for treatment of depression and ADHD may be more complex than selective blockade of NET.

Ranolazine Antagonizes The Effects Of Anemone Toxin-II On Intracellular Ca2+ Cycling In Whole Heart

The late sodium current (INa,L) is pathologically enhanced in several cardiac disease states, including ischemia, causing increased intracellular Na+ and Ca2+ loading and cellular dysfunction. Ranolazine (RAN) is a blocker of INa,L and this mechanism is thought to underlie its effectiveness at reversing many of the cellular effects of ischemia. The goal of this study was to determine if RAN antagonizes the effect of anemone toxin II (ATX-II), an INa,L enhancer known to increase Na+ influx, to alter intracellular Ca2+ cycling in individual myocytes of intact heart. Langendorff-perfused rat hearts were loaded with fluo-4AM (15μM) and placed in a chamber on the stage of a confocal microscope (contractions abolished with cytochalasin-D and blebbistatin). ATX-II (1nM) prolonged the early phase[j1] of basal Ca2+ transients (CaTs) in cells of hearts paced at a rate of 2 Hz[j2]. ATX-II slowed the rate of recovery of cellular CaTs (i.e., restitution) and promoted the development of CaT alternans at slower pacing rates. RAN (10μM) partially reversed the effects of ATX on restitution and alternans, shifting both to shorter intervals[j3] . In addition, pre-treatment with RAN reduced the effects of subsequent exposure to ATX-II on both restitution and alternans development and blunted the ATX-induced changes in basal CaTs. These effects are consistent with an action of RAN to block INa,L, reducing Na+ influx and resulting intracellular Ca2+ accumulation, and therefore suggest RAN treatment may reverse the effects of Ca2+ accumulation that occur in response to disease states in which INa,L is enhanced (such as ischemia). Consequently, RAN may also reduce the arrhythmias that might result from repolarization gradients established by Ca2+ alternans and the resulting action potential duration alternans.

Carba Analogs of Cyclic Phosphatidic Acid Are Selective Inhibitors of Autotaxin and Cancer Cell Invasion and Metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase-2) is an autocrine motility factor initially characterized from A2058 melanoma cell-conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA also activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect remains unresolved. Here we show that 3-carba analogs of cPA lack significant agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo.

Carba Analogs of Cyclic Phosphatidic Acid are Selective Inhibitors of Autotaxin and Cancer Cell Invasion and Metastasis

Autotaxin (ATX, nucleotide pyrophosphate/phosphodiesterase 2, NPP2) is an autocrine motility factor that was initially isolated from melanoma cell conditioned medium. ATX is known to contribute to cancer cell survival, growth, and invasion. Recently ATX was shown to be responsible for the lysophospholipase D activity that generates lysophosphatidic acid (LPA). LPA is also known to be an important mediator of cancer cell survival, growth and invasion. Production of LPA is sufficient to explain the effects of ATX on tumor cells. Cyclic phosphatidic acid (cPA) is a naturally occurring, stable analog of LPA in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Cellular responses to cPA generally oppose those of LPA despite activation of apparently overlapping receptor populations, suggesting that cPA activates cellular targets distinct from LPA receptors. cPA has previously been shown to inhibit tumor cell invasion in vitro and cancer cell metastasis in vivo. However, the mechanism governing this effect has remained unresolved. Here we show that carba analogs of cPA lack agonist activity at LPA receptors yet are potent inhibitors of ATX activity, LPA production, and A2058 melanoma cell invasion in vitro and B16F10 melanoma cell metastasis in vivo. This work was supported by USPHS & UT Cancer Institute Pilot Grant.

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant alpha-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, taufasth) and toxin-bound (slow component, tauslowh) channels. The relative amplitude of tauslowh allows an estimate of the IC50 values approximately 11 nM. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (tauh) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC50 = 51 nM). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses.

Effects of the Anemonia sulcata Toxin (ATX II) on Intracellular Sodium and Contractility in Rat and Guinea‐Pig Myocardium

The effects of the Anemonia sulcata toxin ATX II on action potentials and contractility of isolated papillary muscles and single myocytes from rat and guinea-pig hearts have been studied. ATX II prolonged the action potential in both rat and guinea-pig papillary muscle. Although it produced a positive inotropic effect in guinea-pig papillary muscle, it failed to do so in rat papillary muscle. However, in single rat and guinea-pig ventricular cells, it both prolonged the action potential and had a positive inotropic effect. We suggest that ATX II does not cause a positive inotropic effect in rat papillary muscle, because it induces Ca2+ overload. In single cells the positive inotropic effect was reduced by approximately 50% when the contractions were triggered by voltage clamp pulses of constant duration rather than by action potentials. This suggests that the inotropic effect of ATX II is in part the result of the prolongation of the action potential. The intracellular Na+ activity (a(i)Na) in single ventricular cells was measured with the Na(+)-sensitive fluorescent dye SBFI. After exposure of the cells to ATX II, a(i)Na was increased by a maximum of 1.9 +/- 0.3 and 2.2 +/- 0.3 mM in rat and guinea-pig cells, respectively. It is suggested that the positive inotropic effect of ATX II is also in part the result of the rise in a(i)Na.

Mucosal plexus and electrolyte transport across the rat colonic mucosa.

Histological and functional studies were performed on a preparation of rat colonic mucosa from which the myenteric and submucosal plexus were removed. This preparation, referred to as the mucosa preparation, was used to investigate the potential influence of the mucosal plexus on electrolyte transport. Two neuropharmacologically active agents were used: sea anemone toxin (ATX II) to stimulate the fibres of the mucosal plexus and tetrodotoxin (TTX) to block the fibres of the mucosal plexus. The morphology of the neuronal network of the mucosal plexus was visualized after the epithelium was removed and whole mount preparations of the lamina propria and circular muscle layer of muscularis mucosae were stained histochemically for acetylcholinesterase activity. Several levels of organization within the mucosal plexus were seen. Each crypt is encircled by a thin bundle of fibres near the top. These thin fibres connect with thicker bundles of fibres that encircle groups of two to five crypts in a broad band. These bundles of fibres are in turn connected to larger bundles of fibres which lie in a flat plane just below the crypts along the circular muscle layer of muscularis mucosae. In addition perikarya and ganglia were revealed within the mucosal plexus. The base-line net transport of Na+ and Cl- across the mucosa preparation was completely inhibited by ATX II (10(-6) M). This effect of ATX II on net Na+ and Cl- transport was accompanied with an increase in the short-circuit current (Isc), transmural conductance, and open-circuit potential difference across the mucosa preparation. The effect of ATX II on Isc was dose dependent with a half-maximal effective concentration at 5 X 10(-8) M-ATX II and a maximal effective concentration of 10(-7) M. ATX II was effective only when added to the serosal solution. Net Na+ and Cl- transport was restored by TTX (10(-6) M) to base-line values in ATX II-treated tissue. In addition the value of all three electrical parameters rapidly returned to the values measured before the addition of ATX II. TTX was effective in antagonizing the effects of ATX II only when added to the serosal solution. The results suggest that the regulation of electrolyte transport across the epithelium is at least one function of the mucosal plexus. Stimulation of the neurones within the mucosal plexus leads to the inhibition of electrolyte absorption.(ABSTRACT TRUNCATED AT 400 WORDS)

Class III Antiarrhythmic Action Linked with Positive Inotropy

Most antiarrhythmic drugs are more or less negatively inotropic. Positively inotropic properties, however, have been demonstrated for some class III antiarrhythmic drugs. To test the hypothesis that class III antiarrhythmic effect and positive inotropy may be linked, we used the sea-anemone polypeptide ATX II, which in isolated heart muscle preparations has been shown to specifically inhibit the inactivation of the sodium channel and thereby increase action potential duration and inotropy. We used 12 pentobarbital-anesthetized dogs. Atrial arrhythmias were induced by high-rate stimulation of the right atrium in 5 dogs. Cardiac electrophysiological effects were studied by His-bundle electrography, programmed electrical stimulation, and monophasic action potential (MAP) recordings in 7 autonomically blocked dogs. ATX II (1.0-5.0 micrograms/kg i.v.) converted the arrhythmias, and in the autonomically blocked dogs markedly increased atrial and ventricular refractoriness and ventricular MAP duration without influencing atrial or ventricular conduction velocities, heart rate, or AV-nodal refractoriness. ATX II induced a marked increase in left ventricular dP/dt max. The study indicates that ATX II has class III antiarrhythmic effect, and that the electrophysiological and positive inotropic effects of ATX II have a common mechanism.

Electrophysiological and neurobiochemical evidence for the blockade of a potassium channel by dendrotoxin

The effects of dendrotoxin (DTX), a toxic peptide from Dendroaspis angusticeps venom, were studied electrophysiologically on peripheral frog nerve fibres, and biochemically on large synaptosomes from rat brain. On nerve fibres, DTX reduced the amplitude and prolonged the duration of the action potential; even at 0.1 nmol/l DTX produced significant effects. Maximum block of potassium currents occurred at about 30 nmol/l. Turning on of the remaining current was slowed. Reversibility was incomplete. The reduction of potassium currents was between 31% and 85% at 85 nmol/l DTX (n = 8). The remainder appeared to be resistant to DTX. Sodium channels were not affected. On large synaptosomes DTX (above 1 nmol/l) produced a slight depolarization, indicated by an outward shift of the lipophilic cation tetraphenylphosphonium, and promoted the release of radioactivity after preloading with [3H] GABA. DTX had similar potency but lower efficacy in this respect than sea anemone toxin II (ATX II). In contrast to the effects of ATX II, those due to DTX were only partially inhibited by tetrodotoxin. The actions of 4-aminopyridine resembled those of DTX, but the latter was about 500 times more potent. The electrophysiological data provide direct evidence for blockade of a potassium channel by DTX. This action is sufficient to explain the biochemical observations, although additional effects on synaptosomes cannot be excluded.

Anemonia sulcatatoxin (ATX II) enhances spontaneous electrical activity and tension in chronically denervated rat diaphragm

In isolated strips of rat diaphragm denervated 9-21 days prior to experimentation, spontaneous action potentials were recorded extracellularly and twitch and resting tension were measured. The sea anemone toxin ATX II enhances the occurrence of spontaneous action potentials, increases resting tension and depresses twitch tension. These effects are essentially irreversible. In low sodium solution substituted with sucrose the effects of ATX II are attenuated, however, they fully develop upon return to normal sodium solution with a marked transient increase in the incidence of spontaneous action potentials and in resting tension. ATX II remains uneffective after pretreatment with tetrodotoxin. Reelevation of the extracellular sodium concentration after exposure to low sodium solution per se causes a marked increase in occurrence of fibrillation potentials, however the transient increase in resting tension was much smaller than in the presence of ATX II. Substitution of chloride with the impermeable anion methylsulphate enhances spontaneous activity and resting tension without an effect on twitch tension. Addition of ATX II elevates resting tension although the concomitant further increase in incidence of spontaneous action potentials is small. It is concluded that the increase in resting tension reflects a summation of the fibrillatory activity, but fibrillations become more effective when the preparations are exposed to ATX II. This finding points at the possible rôle of sodium ions in excitation contraction coupling of denervated skeletal muscle.

The effect of toxins fromAmeonia sulcata (Coelenterata) on neuromuscular transmission and nerve action potentials in the crayfish (Astacus leptodactylus)

1. The effects of three toxins (ATX I, II and III) from the sea anemoneAnemonia sulcata have been investigated on neuromuscular transmission and nerve action potentials in the crayfisch. 2. In the neuromuscular preparations the toxins produce a gradual increase in the amplitude of the excitatory junction potentials until they reach the threshold for the generation of electrically excitable membrane responses. This is followed by repetitive activity of the motor axons, first on single stimuli, later occurring spontaneously. 3. The effects of the three toxins differ only with regard to the effective concentrations: threshold concentrations amount to 5×10−9 M for ATX I and II, to 10−7 M for ATX III. 4. The toxins show no postsynaptic effects on the muscle fibre membrane and on their electrical responses upon direct stimulation. 5. All toxins prolong the action potentials of the giant axons in the abdominal nerve cord. The duration may reach more than one second. The rate of rise of the action potentials is not affected. In several cases, however, an increase of the amplitude of the action potential was observed. 6. The effects of ATX I and II are irreversible in contrast to those of ATX III. 7. It is suggested that the high potency of the toxins in the nanomolar range, their small size, the known composition and, in the case of ATX II, sequence of amino acids together with the specificity of action on the sodium channels, proved in different preparations, make these substances to important tools in the study of excitable membranes.

Persistence of Four Herbicides in Pond Water

This study, which was made in collaboration with the Army Corps of Engineers' Expanded Project for Aquatic Weed Control (Public Law 85‐500), reports the persistence of four herbicides in water and soils. The study considered only persistence as indicated by sampling and analytic techniques having a combined sensitivity of about 0.01 ppm. Fenac appeared to have the least potential for use in sources of potable water because of its long survival. The effects of heavy weed infestations on residue persistence were not evaluated. The analytic methods used for 3‐AT would not differentiate between 3‐AT and a principal metabolite (ATX). Although ATX is not phytotoxic, it is incorporated into the cellular protein of Esch. coli. This has not been demonstrated for mammals. Until the effects of ATX on mammalian biochemistry are determined, its presence in potable water is undesirable. As is the case with fenac, the survival of 3‐AT and ATX in water has to be evaluated in weed‐choked environments. Excluding toxicologic considerations, paraquat and diquat appear to have the greatest potential for use in sources of potable water supply. Both disappear rapidly in water and do not appear to be desorbed from bottom sediments. Because paraquat and diquat are strongly sorbed on certain clays, the introduction of such sorbents to the coagulation‐sedimentation phase of conventional water treatment might remove residues during the short period they remain in water.