okadaic-acid and Hypoxia

Alzheimer's disease (AD) is the most common cause of progressive decline of memory function in aged humans. To study about a disease mechanism and progression, animal models for the specific disease are needed. For AD, although highly valid animal models exist, none of the existing models recapitulates all aspects of human AD. The pathogenic mechanisms involved in AD are diverse and thus it is difficult to recapitulate human AD in model organisms. Intracerebroventricular (ICV) injection of okadaic acid (OKA), a protein phosphatase 2A (PP2A) inhibitor, in rats causes neurotoxicity associated with neurofibrillary degeneration. However, this model lacks amyloid pathology as observed in AD. We aimed at combining two different treatments and hence producing a better animal model of AD which may mimic most of the neuropathological, neurobehavioral, and neurochemical changes observed in AD. For this, OKA (200 ng) was microinjected bilaterally into the hippocampus of male Wistar rats followed by exposure of same rats to hypoxic conditions (10%) for 3 days. The result of which, the combination model exhibited tau hyperphosphorylation along with Aβ upregulation as evident by western blotting and immunohistochemistry. The observed changes were accompanied with dysfunction of neurotransmitter system, i.e., decreased acetylcholine activity and expression. This combinatorial model also exhibited cognitive deficiency which was assessed by Morris water maze and avoidance tests along with enhanced oxidative stress which is thought to be a major player in AD pathogenesis. Taken together, we established an easily reproducible and reliable rat model for sporadic dementia of Alzheimer's type in rats which allows effective testing of new therapeutic strategies.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Avoidance Learning; Cognitive Dysfunction; Disease Models, Animal; Hippocampus; Hypoxia; Male; Maze Learning; Microinjections; Neurons; Okadaic Acid; Oxidative Stress; Rats, Wistar; Stereotaxic Techniques

Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined.

Topics: Animals; Hypoxia; Long-Term Potentiation; Male; Okadaic Acid; Phosphoprotein Phosphatases; Phrenic Nerve; Rats; Rats, Sprague-Dawley

Abnormal phosphorylation of microtubule-associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37 degrees C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)-1, PP-2A, glycogen synthase kinase 3 (GSK-3), extracellular signal-regulated protein kinase (ERK) and C-jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF-1), Ser422 (R145), Ser199/Ser202 (Tau-1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP-2A and the level of dephosphorylated PP-2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP-2A inhibitor, okadaic acid (OA, 0.75 microm), completely prevented tau from acute anoxia-induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK-3 and PP-1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP-2A may play a key role in tau dephosphorylation induced by acute anoxia.

Topics: Acute Disease; Animals; Brain; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hypoglycemia; Hypoxia; In Vitro Techniques; JNK Mitogen-Activated Protein Kinases; Male; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Rats; Rats, Wistar; tau Proteins

Excitotoxic cell death (ECD) is characteristic of mammalian brain following min of anoxia, but is not observed in the western painted turtle following days to months without oxygen. A key event in ECD is a massive increase in intracellular Ca(2+) by over-stimulation of N-methyl-d-aspartate receptors (NMDARs). The turtle's anoxia tolerance may involve the prevention of ECD by attenuating NMDAR-induced Ca(2+) influx. The goal of this study was to determine if protein phosphatases (PPs) and intracellular calcium mediate reductions in turtle cortical neuron whole-cell NMDAR currents during anoxia, thereby preventing ECD. Whole-cell NMDAR currents did not change during 80 min of normoxia, but decreased 56% during 40 min of anoxia. Okadaic acid and calyculin A, inhibitors of serine/threonine PP1 and PP2A, potentiated NMDAR currents during normoxia and prevented anoxia-mediated attenuation of NMDAR currents. Decreases in NMDAR activity during anoxia were also abolished by inclusion of the Ca(2+) chelator -- BAPTA and the calmodulin inhibitor -- calmidazolium. However, cypermethrin, an inhibitor of the Ca(2+)/calmodulin-dependent PP2B (calcineurin), abolished the anoxic decrease in NMDAR activity at 20, but not 40 min suggesting that this phosphatase might play an early role in attenuating NMDAR activity during anoxia. Our results show that PPs, Ca(2+) and calmodulin play an important role in decreasing NMDAR activity during anoxia in the turtle cortex. We offer a novel mechanism describing this attenuation in which PP1 and 2A dephosphorylate the NMDAR (NR1 subunit) followed by calmodulin binding, a subsequent dissociation of alpha-actinin-2 from the NR1 subunit, and a decrease in NMDAR activity.

Topics: Animals; Calcium; Calmodulin; Cerebral Cortex; Egtazic Acid; Female; Hypoxia; Imidazoles; Marine Toxins; Okadaic Acid; Oxazoles; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Protein Phosphatase 1; Pyrethrins; Receptors, N-Methyl-D-Aspartate; Turtles

We hypothesized that increased myofibrillar type 1 protein phosphatase (PP1) catalytic activity contributes to impaired aortic smooth muscle contraction after hypoxia. Our results show that inhibition of PP1 activity with microcystin-LR (50 nmol/l) or okadaic acid (100 nmol/l) increased phenylephrine- and KCl-induced contraction to a greater extent in aortic rings from rats exposed to hypoxia (10% O(2)) for 48 h than in rings from normoxic animals. PP1 inhibition also restored the level of phosphorylation of the 20-kDa myosin light chain (LC(20)) during maximal phenylephrine-induced contraction to that observed in the normoxic control group. Myofibrillar PP1 activity was greater in aortas from rats exposed to hypoxia than in normoxic rats (P < 0.05). Levels of the protein myosin phosphatase-targeting subunit 1 (MYPT1) that mediates myofibrillar localization of PP1 activity were increased in aortas from hypoxic rats (193 +/- 28% of the normoxic control value, P < 0.05) and in human aortic smooth muscle cells after hypoxic (1% O(2)) incubation (182 +/- 18% of the normoxic control value, P < 0.05). Aortic levels of myosin light chain kinase were similar in normoxic and hypoxic groups. In conclusion, after hypoxia, increased MYPT1 protein and myofibrillar PP1 activity impair aortic vasoreactivity through enhanced dephosphorylation of LC(20).

Topics: Animals; Aorta; Blotting, Western; Cells, Cultured; Enzyme Inhibitors; Hypoxia; Male; Marine Toxins; Microcystins; Muscle Contraction; Muscle, Smooth, Vascular; Myofibrils; Myosin Light Chains; Okadaic Acid; Oxygen; Peptides, Cyclic; Phenylephrine; Phosphoprotein Phosphatases; Phosphorylation; Potassium Chloride; Protein Phosphatase 1; Rats; Rats, Sprague-Dawley

To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 micromol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 micromol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na(+)/Ca(2+) overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism.

Topics: Animals; Cantharidin; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Indoles; Ischemic Preconditioning; Male; Maleimides; Muscle Fibers, Skeletal; Myocardial Reperfusion Injury; Myocardium; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Phosphatase 2; Rats; Rats, Wistar; Ventricular Pressure

Atrial natriuretic peptide (ANP) and its analog, atriopeptin III (APIII), inhibit carotid body chemoreceptor nerve activity evoked by hypoxia. In the present study, we have examined the hypothesis that the inhibitory effects of ANP and APIII are mediated by cyclic GMP and protein kinase G (PKG) via the phosphorylation and/or dephosphorylation of K(+) and Ca(2+) channel proteins that are involved in regulating the response of carotid body chemosensory type I cells to low-O(2) stimuli. In freshly dissociated rabbit type I cells, we examined the effects of a PKG inhibitor, KT-5823, and an inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), on K(+) and Ca(2+) currents. We also investigated the effects of these specific inhibitors on intracellular Ca(2+) concentration and carotid sinus nerve (CSN) activity under normoxic and hypoxic conditions. Voltage-dependent K(+) currents were depressed by hypoxia, and this effect was significantly reduced by 100 nM APIII. The effect of APIII on this current was reversed in the presence of either 1 microM KT-5823 or 100 nM OA. Likewise, these drugs retarded the depression of voltage-gated Ca(2+) currents induced by APIII. Furthermore, APIII depressed hypoxia-evoked elevations of intracellular Ca(2+), an effect that was also reversed by OA and KT-5823. Finally, CSN activity evoked by hypoxia was decreased in the presence of 100 nM APIII, and was partially restored when APIII was presented along with 100 nM OA. These results suggest that ANP initiates a cascade of events involving PKG and PP2A, which culminates in the dephosphorylation of K(+) and Ca(2+) channel proteins in the chemosensory type I cells.

Topics: Alkaloids; Animals; Atrial Natriuretic Factor; Calcium; Carbazoles; Carotid Body; Carotid Sinus; Cells, Cultured; Cyclic GMP-Dependent Protein Kinases; Drug Synergism; Electrophysiology; Enzyme Inhibitors; Hypoxia; Indoles; Nervous System; Okadaic Acid; Patch-Clamp Techniques; Peptide Fragments; Phosphoprotein Phosphatases; Potassium; Protein Kinase Inhibitors; Protein Phosphatase 2; Rabbits; Reference Values

Hypoxia induces bronchodilation in vivo and in vitro, but the mechanisms are still unclear. To evaluate whether an extra- or intracellular free Ca(2+) ion is involved in the mechanisms of hypoxic relaxation, we simultaneously measured cytosolic Ca(2+)levels and tensions in both intact and denuded guinea-pig tracheal strips precontracted with histamine (100 microM), and assessed the effect of hypoxia on guinea-pig tracheal rings precontracted with okadaic acid (10 microM) and calyculin-A (0.1 approximately 10 microM) under an extracellular Ca(2+)-free state. The exposure of tracheal rings to hypoxia induced an immediate decrease of tracheal tension without decrease in intracellular free Ca(2+)levels. In the presence of okadaic acid but not calyculin-A, hypoxic air exposure caused significant transient reductions in tracheal tone. Further, thapsigargin (5 microM or 10 microM) did not affect hypoxic bronchodilation, suggesting that the release of intracellular Ca(2+) does not take a role in hypoxic bronchodilation. Hypoxic dilation decreased ATP content in epithelium-intact rings but not epithelium-denuded rings, indicating a relationship between hypoxic dilation and change of adenine nucleotide in epithelium-intact rings. Our findings indicate that the epithelium dependent mechanisms of hypoxic relaxation of guinea pig tracheal rings preconstricted with histamine may not be related to the mobilization of extra and intra-cellular Ca(2+).

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Enzyme Inhibitors; Guinea Pigs; Hypoxia; Male; Marine Toxins; Muscle Relaxation; Muscle, Smooth; Okadaic Acid; Oxazoles; Oxygen; Thapsigargin; Trachea

Glucose transport in skeletal muscle can be mediated by two separate pathways, one stimulated by insulin and the other by muscle contraction. High-fat feeding impairs glucose transport in muscle, but the mechanism remains unclear. FVB mice (3 weeks old) were fed a high-fat diet (55% fat, 24% carbohydrate, 21% protein) or standard chow for 3-4 weeks or 8 weeks. Insulin-stimulated glucose transport, assessed with either 2-deoxyglucose or 3-O-methylglucose was decreased 35-45% (P < 0.001) in isolated soleus muscle, regardless of diet duration. Similarly, glucose transport stimulated by okadaic acid, a serine/threonine phosphatase inhibitor, was also 45% lower with high-fat feeding, but the glucose transport response to hypoxia or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) (which are stimulators of the "contraction pathway") was intact. Hexokinase I, II, and total activity were normal in soleus muscle from high-fat-fed mice. GLUT4 expression in soleus muscle from the high-fat-fed mice was also normal, but the insulin-stimulated cell surface recruitment of GLUT4 assessed by exofacial photolabeling with [3H]-ATB bis-mannose was reduced by 50% (P < 0.001). Insulin-receptor substrate 1 (IRS-1) associated phosphatidylinositol (PI) 3-kinase activity stimulated by insulin was also reduced by 36% (P < 0.001), and expression of p85 and p110b subunits of PI 3-kinase was normal. In conclusion, high-fat feeding selectively impairs insulin-stimulated, but not contraction-pathway-mediated, glucose transport by reducing GLUT4 translocation to the plasma membrane. This appears to result from an acquired defect in insulin activation of PI 3-kinase. Since effects of okadaic acid on glucose transport are independent of PI 3-kinase, a second signaling defect may also be induced.

Topics: Animals; Biological Transport; Blood Glucose; Cell Compartmentation; Dietary Fats; Enzyme Inhibitors; Female; Glucose Transporter Type 4; Hexokinase; Hypoxia; Insulin; Insulin Resistance; Male; Mice; Monosaccharide Transport Proteins; Muscle Proteins; Muscle, Skeletal; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Signal Transduction; Sulfonamides

Protein phosphatase type 1 (PP-1) was analyzed in organs of the red-eared slider turtle, Trachemys scripta elegans, a species capable of long-term anoxia survival. During anoxic submergence at 7 degrees C, PP-1 activity in liver rapidly decreased to 63% of the control value within the first hour and remained suppressed over the subsequent 20 h of anoxia. PP-1 activity was also suppressed in red skeletal muscle during anoxia and dropped transiently (after 1 h) in brain but did not change in heart or white muscle. PP-1 was purified from turtle liver using polyethylene glycol fractionation and chromatography on DEAE-cellulose, blue dextran, Sephacryl S-200, and ADP-agarose. A 3000-fold purification was achieved with a final specific activity of 3156 nmol released min-1 mg protein-1 using 32P-labeled phosphorylase a as the substrate. Turtle liver PP-1 was a monomer of molecular mass 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or 38 +/- 2 kDa by Sephacryl S-200 gel filtration. The enzyme was inhibited by okadaic acid (Ki 12.6 +/- 1.4 nM) and AMP (Ki 23 +/- 2 microM) as well as by ADP, ATP, and IMP. Regulation of liver PP-1 appears to be an integral part of anoxia-induced changes in liver glycogenolysis and metabolic rate suppression.

Topics: Adenosine Monophosphate; Animals; Brain; Dose-Response Relationship, Drug; Ethers, Cyclic; Heart; Hypoxia; Liver; Muscle, Skeletal; Myocardium; Okadaic Acid; Phosphoprotein Phosphatases; Tissue Distribution; Turtles

Gamma-Aminobirtyric acid (GABA) is one of the major neurotransmitters in the mammalian central nervous system (CNS). The activation of post-synaptic GABAA receptor-chloride channel complex is thought to underlie inhibitory postsynaptic potentials ubiquitously in various CNS regions. GABAA receptors are modulated by convulsant, hypnotic-anticonvulsant, anxiolytic and anxiogenic agents and endogenous agents such as nurosteroids and intracellular calcium, ATP, and cyclic AMP. The function of GABAA receptor in CNS neuron is also affected by some pathophysiological processes, e.g., anoxia. For example, it is currently believed that delayed neuronal death after brain ischemia results from excessive cell excitability and/or loss of inhibition. In the present study, we investigated how the GABA-gated chloride current is affected by anoxic conditions. All experiments were carried out on neurons freshly dissociated from rat CNS by the use of both conventional and nystatin perforated patch recording configurations. The GABA response showed a considerable rundown with time in anoxic condition. The rundown was prevented by adding either ouabain or SPAI-I (Na+-K+ ATPase inhibitor-I), suggesting that the experimental anoxia reduced GABA response by decreasing intracellular ATP synthesis. This result was also confirmed by finding that the direct decrease of intracellular ATP concentration using a conventional whole-cell patch recording mode inhibited the GABA-gated chloride response in mammalian CNS neurons.

Topics: Adenosine Triphosphate; Animals; Brain; Electric Conductivity; Ethers, Cyclic; gamma-Aminobutyric Acid; Hypoxia; Kinetics; Magnesium; Neurons; Okadaic Acid; Ouabain; Rats; Rats, Wistar; Sodium; Sodium-Potassium-Exchanging ATPase

Anoxia-induced depletion of cellular ATP may affect the degree of protein phosphorylation due to kinase inhibition. In this study, protein phosphorylation was measured in rabbit kidney proximal tubules under normoxic or anoxic conditions in a medium containing 32P. During the first 20 min of normoxia, phosphate incorporation was linear, averaging 17 +/- 5 pmol.mg protein-1.min-1 and was 70% inhibited by the protein kinase C inhibitor chelerythrine chloride. Phosphorylation measurements initiated simultaneously with anoxic conditions (95% N2-5% CO2) significantly reduced the initial rate to 58% of control, saturating after 15 min, and reaching 28 +/- 5% of the normoxic value after 60 min of incubation. The phosphatase inhibitor calyculin A did not affect the initial rate of phosphate incorporation by anoxic tubules but increased phosphate incorporation at 60 min to 43 +/- 4% of normoxia. Addition of 32P after 15 min of anoxia abolished phosphate incorporation, demonstrating that kinase activity was completely inhibited. Cellular phosphate uptake was measured and found not to be rate limiting for phosphorylation. Chelerythrine chloride increased lactate dehydrogenase (LDH) release during normoxia, and calyculin A decreased anoxia-induced LDH release, suggesting that protein phosphorylation events may control plasma membrane permeability.

Topics: Alkaloids; Animals; Benzophenanthridines; Ethers, Cyclic; Hypoxia; In Vitro Techniques; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Marine Toxins; Okadaic Acid; Oxazoles; Phenanthridines; Phosphates; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinase C; Proteins; Rabbits; Reference Values