Type of cell death and the role of acetylcholinesterase activity in neurotoxicity induced by paraoxon in cultured rat hippocampal neurons.

Organophosphate (Ops) neurotoxicity is attributed both to its well-known cholinergic and non-cholinergic effects. In the present study we compared enzymatic and morphologic changes in neurons exposed to paraoxon during one day and one week. The effect of exposure time is important in neurotoxicity of Ops. The longer the exposure time is the more damage is observed in neurons, although there are few investigations about the effect in the post-exposure period. Hippocampal cells were obtained from rat neonates and cultured in Neurobasal/B27. Paraoxon at 50 and 100 microM were added. Inverted microscope and electron microscope were used to study cell morphology and Neutral Red staining was used to measure viability. We also assayed caspase-3 and (acetylcholinesterase) AChE activity. Hoechst staining was utilized to determine the type of cell death. Culture medium was replaced after 24 h in one-day group, however, tests were all carried out at the end of the first week in both group. The results indicate that paraoxon reduced the viability in a dose-dependent manner. Our results do not confirm apoptosis in either group; it seems that the cell death in one-day exposure group was not AChE dependent. In conclusion, present data imply that the toxicity of paraoxon is both dose and duration dependent, which may even remain after the cessation of exposure.

Protein kinase C from bat brain: the enzyme from a hibernating mammal.

Protein kinase C (PKC) from brain of euthermic and hibernating bats (Myotis lucifugus) showed only one form as determined by hydroxylapatite chromatography, compared with three forms found in rat brain. Cross-reaction with antibodies to rabbit alpha, beta, and gamma isozymes showed that bat brain contained only PKC(gamma). During hibernation the activity of PKC in bat brain decreased to 63% of the euthermic value but the percentage that was membrane-associated did not change. Bat and rat brain PKC(gamma) were purified to homogeneity. Both enzymes phosphorylated all three of the substrates tested (FKKSFKL-NH2 peptide substrate, histone H1, protamine), the bat enzyme having significantly higher K(m) values than rat PKC for both peptide and histone. Both enzymes required phospholipids and Ca2+ for activation with rat brain PKC depending almost exclusively on phosphatidylserine. Bat PKC, however, made use of other phospholipids and showed relative activities of 100:81:33:42 for euthermic PKC and 100:91:45:35 for hibernator PKC with phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine (each at 50 microM), respectively. Activation of bat PKC by phosphatidylserine was temperature sensitive, being 3.5-fold at 4 degrees C (hibernating body temperature) compared with 14-18-fold at 33 degrees C (near euthermic body temperature). Arrhenius plots for bat brain PKC showed a sharp break below 10 degrees C; activation energies below this temperature were 11.5- and 5.2-fold greater than at higher temperatures for the enzyme from hibernating versus euthermic animals. By contrast, plots for the rat enzyme were linear over the range 0-42 degrees C. The data suggest that a sharp suppression of PKC activity by several mechanisms (reduced total activity, low temperature effects on activity and sensitivity to phospholipids) may be important to overall metabolic rate suppression during hibernation.

Liver protein kinase C isozymes: properties and enzyme role in a vertebrate facultative anaerobe.

Protein kinase C was purified to homogeneity from liver of the anoxia-tolerant turtle (Trachemys scripta elegans). Two isozymes were present and were identified as PKC alpha and PKC beta by hydroxylapatite chromatography and cross-reaction with specific antibodies to the mammalian isozymes. Kinetic characterization of the isozymes showed that both required phospholipids and Ca2+ for activation and both were inhibited by low concentrations of PKC inhibitors. The PKC alpha was activated more strongly by phosphatidylinositol and lysophosphatidylinositol compared with PKC beta. Treatment with trypsin did not activate turtle PKC isozymes, but generated inactive PKC beta, whereas PKC alpha was resistant to inactivation. Anoxia exposure of turtles in vivo, via submergence in N2-gassed water at 7 degrees C, altered the activity and subcellular distribution of PKC in liver. After 1 hr of anoxic exposure at 7 degrees C, the activity of membrane-bound PKC had increased by 2.4-fold and represented a translocation of 40% of PKC beta and more than 80% of PKC alpha from the cytosol to the membrane-associated fraction. With longer submergence, however, membrane-bound PKC activity was suppressed again. This two-phase response to anoxia by PKC suggests that an activation of PKC, through its translocation to the membrane, is important in mediating the initial metabolic responses to submergence, which include an activation of glycogenolysis during the hypoxia transition period. With sustained anoxia exposure, the subsequent reduction of PKC activity may be part of the overall mechanism of metabolic rate depression that allows endurance of prolonged anoxia.

Enzymatic control of glycogenolysis during anoxic submergence in the freshwater turtle Trachemys scripta.

Freshwater turtles Trachemys scripta elegans endure prolonged severe hypoxia, and even complete anoxia, while diving or hibernating underwater. Metabolic adaptations supporting survival include the activation of glycogenolysis and glucose output from liver, as well as strong metabolic rate depression. The present study analyzes the enzymes of both the phosphorolytic (glycogen phosphorylase, phosphorylase b kinase, cAMP-dependent protein kinase) and glucosidic (alpha-glucosidase) pathways of glycogenolysis in turtle organs. Turtles were subjected to 5 hr of submergence in N2-bubbled water at 7 degrees C and then activities of phosphorolytic and glucosidic enzymes were assayed in liver, heart, brain, and red and white skeletal muscle, and compared with aerobic controls. In vitro incubations also assessed protein kinase A control of phosphorolytic enzymes. A functional enzyme cascade system for the activation of glycogen phosphorylase was found in all organs, and both phosphorylase and phosphorylase kinase were stimulated by in vitro incubation with the catalytic subunit of cAMP-dependent protein kinase. Anoxic submergence led to significant increases in phosphorylase activities in liver and heart (phosphorylase a rose 2- and 2.5-fold, respectively) but phosphorylase kinase and protein kinase A activities in liver were reduced after 5 hr exposure. Both acidic (pH 4) and neutral (pH 7) forms of alpha-glucosidase were detected in all five organs with highest activities in liver. Activity of acid alpha-glucosidase, which degrades lysosomal glycogen, increased by 2-fold in liver during anoxic submergence. The data show that glycogen breakdown in turtle liver during anoxic submergence may result from coordinated activations of both the cytoplasmic phosphorolytic and the lysosomal glucosidic pathways of glycogenolysis.

cAMP-dependent protein kinase and anoxia survival in turtles: purification and properties of liver PKA.

The catalytic subunit of turtle (Trachemys scripta elegans) liver cyclic AMP-dependent protein kinase (PKAc) was purified to homogeneity with a final specific activity of 65,783 pmol phosphate transferred.min-1.mg protein-1. Subunit molecular weight was 42-43 kDa as determined by SDS-PAGE and Sephacryl S-300 chromatography. The isolectric point was pH 6.41 +/- 0.02. Turtle liver PKAc showed highest activity with kemptide as its substrate; activity with other artificial substrates, histone IIA and protamine, was only 21 and 11%, respectively, of the activity with kemptide. Km values were 83 +/- 6.5 microM for Mg.ATP and 11.7 +/- 0.5 microM for kemptide and enzyme activity was strongly reduced by inhibitors of mammalian PKA (H-89, PKA-1) but not by inhibitors of other protein kinases. The enzyme was also inhibited by salts, especially fluoride salts (I50 about 30 mM), and showed a sharp break in the Arrhenius plot (0-45 degrees C) with activation energy increasing by 4-fold from 27.9 +/- 1.85 to 115 +/- 2.5 kJ/mol for temperatures above versus below 15 degrees C. Temperature effects may be important in suppressing PKA function, and therefore PKA-mediated responses, in vivo to enhance anoxic survival time during winter hibernation under water. Analysis of the effects of in vivo anoxia exposure at 7 degrees C on PKA in turtle organs showed a rapid 2.3-fold increase in the amount of active enzyme in liver within 1 h of anoxic submergence accompanied by a 60% increase cAMP levels; with longer anoxia (5 or 20 h) the percentage of active PKA was suppressed to 2.1-3.7% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of anoxia on protein phosphatase in turtle organs: purification and properties of protein phosphatase type-1 from turtle liver.

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.

Control of glycogenolysis and effects of exercise on phosphorylase kinase and cAMP-dependent protein kinase in rainbow trout organs.

To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of alpha-glucosidases from rainbow trout liver.

Three forms of alpha-glucosidase were separable in trout liver by DE-52 chromatography, isoelectrofocusing, and gel filtration. Two forms showed acid pH optima, hydrolyzed glycogen, maltose, and 4-methylumbelliferyl alpha-glucoside, and were associated with the lysosomes. The third enzyme form was largely associated with microsomes and was present in the highest activity; it showed a neutral pH optimum and did not hydrolyze glycogen. Molecular weights were 181 +/- 2, 130 +/- 1.5, and 365 +/- 3 kDa for the acid types I and II and the neutral enzyme, respectively. Maximal activities and kinetic and physical properties of the three enzymes were compared in liver samples from control, resting fish versus fish that underwent exhaustive swimming exercise. The properties of liver acid alpha-glucosidase type I changed significantly in response to exercise; maximal activity increased by 80% and Km values for both glycogen and maltose dropped by 50% in exercised, versus control, fish. Under the same exercise condition, liver glycogen phosphorylase a activity also increased 4.4-fold. These changes in alpha-glucosidase type I are consistent with an activation of the enzyme, in parallel with phosphorylase activation, under physiological stress conditions that promote glycogenolysis and glucose export from liver. These results are, we believe, the first demonstration of the activation of the glucosidic route of glycogenolysis in response to a physiological stress and suggest that the glucosidic route has a significant role to play in complementing the phosphorolytic pathway in the metabolic response by liver to the fuel demands of working muscle.

Purification and molecular properties of glycogen phosphorylase b from trout white muscle.

Glycogen phosphorylase b (EC 2.4.1.1) was isolated from white skeletal muscle of rainbow trout (Oncorhynchus mykiss) and purified 214-fold to a final specific activity of 135 U/mg protein (assayed in the direction of glycogen breakdown at 21 degrees C) by using glycogen--concanavalin A, DEAE-Sephadex, and 3',5'-cAMP affinity chromatography. Purified phosphorylase b was a dimer with a native molecular weight of 193,000 and a subunit molecular weight of 87,000. Michaelis constants for glycogen, phosphate, and AMP were 128 microM, 31 mM and 142 microM, respectively, at pH 7.2; maximum activity of the enzyme was obtained at pH 7.5 and 25 degrees C. Glucose and ATP behaved as phosphorylase b inhibitors; glucose inhibition decreased at lower pH values. IMP did not affect the enzyme. The catalytic properties of trout phosphorylase b indicate that the enzyme would be virtually inactive at the physiological concentration of substrates and activators found in resting trout white muscle, but changes in cellular pH, ATP, Pi, and AMP levels during burst muscle work could allow phosphorylase b to augment phosphorylase a activity and make a substantial contribution to overall glycogenolysis in working trout white muscle.

Effects of an aqueous extract of Physalis alkekengi fruit on estrus cycle, reproduction and uterine creatine kinase BB-isozyme in rats.

Intraperitoneal injections of the aqueous extract of winter cherry fruits (Physalis alkekengi) to female rats produced 100% diestrus. The rats resumed their normal estrus cycle upon withdrawal of this extract. Although there was no significant decrease in the number of implantation sites, the number of pups born to rats decreased by 96% with extract administration. Treatment with this extract had no effect on body weight, uterus weight, plasma protein level or plasma total creatine kinase activity. However, the level of plasma progesterone was diminished by 44%. In addition, uterine creatine kinase BB-isozyme (an estrogen-induced protein) showed a time-dependent inhibition of activity from 55% to 82%.