Metabolic changes after injection of quinolinic acid or 6-hydroxydopamine in the rat striatum: a time-course study using cytochrome oxidase and glycogene phosphorylase a histochemistry.

Injection of excitotoxins, such as quinolinic acid (QA), into the striatum has been extensively used as an experimental model of Huntington's disease, while injection of 6-hydroxydopamine (6-OHDA) into the dopaminergic nigrostriatal pathway provides a well established model of Parkinson's disease. In the present study, we have examined the metabolic changes induced by an intrastriatal injection of QA or 6-OHDA using histochemical staining for the metabolic markers cytochrome oxidase (COx) and active glycogene phosphorylase (GPa). Intrastriatal injection of QA produced major changes in COx (decrease of staining) and GPa (increase of staining, except in the core of the lesion where the staining was virtually absent) histochemistry at the level of the striatum and of most of the other basal ganglia nuclei. Although attenuated over time, these changes persisted up to one year after the lesion. On the contrary, after the intrastriatal injection of 6-OHDA (which induces only a partial lesion of the nigrostriatal pathway), we did not observe any remarkable changes in COx or GPa staining. This study illustrates the discrepancies between the morphological changes and metabolic changes that are induced when using these experimental models of neurodegenerative disorders.

Physiological doses of oral casein affect hepatic glycogen metabolism in normal food-deprived rats.

In a previous study, administration of casein hydrolysate to food-deprived rats at a dose of 4 g/kg body wt resulted in an increase in portal plasma glucagon concentration. This was associated with an activation of phosphorylase a and a decrease in hepatic glycogen concentration. The present study was undertaken to determine whether similar results would be obtained with smaller doses. Doses of 1 and 2 g/kg body wt were administered to food-deprived rats. At a dose of 2 g/kg, portal plasma glucagon concentration was significantly elevated. This was associated with a slight increase in phosphorylase a activity (P < 0.05) and a 50% decrease in hepatic glycogen concentration (P < 0.01). At a dose of 1 g casein hydrolysate/kg body wt, changes in portal plasma glucagon concentration, phosphorylase a activity and hepatic glycogen concentration generally were not observed. Hepatic glucose, uridine diphosphoglucose, ATP and glucose-6-phosphate concentrations were unaffected by either dose of casein hydrolysate. The data indicate a dose-response relationship between casein hydrolysate administration and effects on glycogen metabolism in the liver. Protein-induced glycogenolysis is likely to occur when rats ingest a moderate amount of a pure protein meal.

Glycyl-histidyl-lysine interacts with the angiotensin II AT1 receptor.

Gly-His-Lys, a tripeptide isolated from human plasma that increases the growth rate of many cells, stimulated in dose-dependent fashion the activity of phosphorylase a in isolated rat hepatocytes. Such effect was associated to increases in both IP3 production and [Ca++]i. Interestingly, these effects of Gly-His-Lys were antagonized by losartan, a nonpeptide angiotensin II receptor antagonist (AT1 selective), which suggested that these receptors were involved in its effect. Binding competition experiments using the radioligand [125I][Sar1-Ile8]angiotensin II clearly indicated that Gly-His-Lys interacts with AT1 receptors. It was also observed that other histidine-containing tripeptides were also capable of interacting with these receptors.

Analysis of dissociation and unfolding of oligomeric proteins using a flat bed gel electrophoresis at high pressure.

A slab gel electrophoresis apparatus with the ability to operate over a pressure range of 10(-3) to 2 kbar is described. The system presented here is an improvement of a previous apparatus (A. A. Paladini, J. L. Silva, and G. Weber, Anal. Biochem. 161, 358-364, 1987). It consists of a flat bed gel, with a significantly enlarged buffer reservoir, which eliminates the requirement of high concentrations of running buffers, and at the same time allows shorter runs, leading to enhanced resolution and reproducibility. The application of the method to the dissociation of the tetramer glycogen phosphorylase a as a function of hydrostatic pressure is described. The flat geometry of the apparatus allows for the first time the analysis of the stability of oligomers and their constituent subunits to chemical denaturation by urea gradient electrophoresis gels at high pressure. Dimeric hexokinase shows a reversible cooperative unfolding transition with a midpoint at 3.8 M urea. In contrast, the monomers unfold at very low urea concentration (< 1.0 M). The observed differences in stability validates oligomerization as an important stabilizing element of the protein structure.

Metabolism of glycogen in submandibular glands of rats. Alteration by NaF.

Submandibular glands of rats injected with NaF solution (10 mg F-/kg body weight) were analysed for glycogen content and phosphorylase activity after various time intervals. In contrast to what has been reported for the liver, sodium fluoride caused increased glycogen content. Phosphorylase (a and total) activity was not affected, suggesting a different mechanism of action of F- in the submandibular gland. In vivo experiments demonstrated stimulation of glycogenesis.

Pitfalls in the histochemical demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres.

In the present communication, an investigation is described into the reliability of histochemical methods for the demonstration of alpha-glucan phosphorylase activity in glycogen-depleted skeletal muscle fibres. Human skeletal muscles with glycogen-depleted fibres from patients with diseases of the neuromuscular system and from subjects who had suffered from malignant hyperthermia were used for the study. The location of phosphorylase activity and glycogen was demonstrated with histochemical techniques. Biochemical techniques were used to assay the activity of phosphorylase and the content of glycogen. Biochemical determinations of phosphorylase activity did frequently not reveal significant differences between glycogen-depleted and non-depleted skeletal muscle fibres. In contrast, all histochemical methods investigated, showed little or no phosphorylase activity in the glycogen depleted fibres, indicating that none of the existing histochemical methods revealed reliable staining results in these fibres. Owing to the invalid staining results of the histochemical methods for glycogen-depleted muscle fibres, it is necessary that for metabolic studies a biochemical assay for phosphorylase activity is also to be performed.

Absence of insulinotropic glucagon-like peptide-I(7-37) receptors on isolated rat liver hepatocytes.

The effects of glucagon and the glucagon-like peptide GLP-1(7-37) were compared in rat liver hepatocytes. Glucagon elevated cAMP, elevated intracellular free calcium ([Ca2+]i), activated phosphorylase and stimulated gluconeogenesis, whereas GLP-1(7-37) was without effect on any of these parameters. GLP-1(7-37) did not block any of the actions of glucagon. The glucagon analog, des His1[Glu9] glucagon amide, was a partial agonist in liver, but also was an effective antagonist of glucagon actions in liver but not those of GLP-1(7-37) in islet B cells. It was concluded that in the rat, GLP-1(7-37) is a potent insulin secretagogue [1] but is without effect on liver.

Ovine fetal and maternal glycogen during fasting.

The present study was undertaken to assess the role of hepatic glycogen metabolism in fetal and maternal glucose homeostasis during a prolonged fast in the pregnant ewe. A control fed group of 13 ewes and 16 fetuses were compared to a 5-day-fasted group of 13 ewes and 17 fetuses, studied at 125 days gestation (term = 147 days). Tissue samples were obtained during pentobarbital anesthesia and frozen in liquid nitrogen. Protein, glycogen, active phosphorylase and total phosphorylase activity were determined. Fetal weight (3.61 vs. 2.86 kg) was decreased in the fasted group (p less than 0.001) while fetal hepatic glycogen was unchanged (59.8 vs. 52.4 mg/g tissue). Maternal liver glycogen decreased during fasting (38.2 vs. 4.0 mg/g tissue, p less than 0.001). Fetal active phosphorylase and total phosphorylase did not change between fed and fasted states (fed active phosphorylase 398 vs. fasted 441 and fed total phosphorylase 510 vs. fasted 574 mumol/h/g tissue). The maternal active phosphorylase and total phosphorylase decreased between fed and fasted (active phosphorylase 690 vs. 238 and total phosphorylase 981 vs. 599 mumol/h/g tissue, p less than 0.001). During fasting, the pregnant ewe depletes her hepatic glycogen stores, associated with a reduction in glycogen catabolizing enzyme activity. The fetus maintains a relatively large glycogen catabolizing enzyme activity, a relatively large glycogen reserve and substantial phosphorylase activity.

Detection of glycogen-debranching system in trophozoites of Entamoeba histolytica.

Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl-alpha-glucoside yielding successively 4-nitrophenyl-alpha-maltoside and 4-nitrophenyl-alpha-maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of Mr = 180,000 +/- 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.

Evidence in vivo for elevation of intracellular free Ca2+ in the liver after diquat, acetaminophen, and CCl4.

Several hepatotoxic agents with varied chemical mechanisms of toxicity (acetaminophen, diquat, and CCl4) depress membrane calcium pumps and/or enhance the permeability of membranes to calcium. To probe the relevance of these findings to maintenance of calcium homeostasis after toxins in vivo, we measured the activity of glycogen phosphorylase a, as an index of cytosolic free [Ca2+], in freeze-clamped liver samples obtained at several times after the toxin dose. Both acetaminophen and diquat caused significant increases of phosphorylase a activity, and activity remained elevated for several hours after the dose. Significantly, the administration prior to diquat of desferrioxamine, which offers protection against the liver necrosis and depression of microsomal Ca2+ accumulation observed after diquat alone (Tsokos-Kuhn et al., Mol Pharmacol 34: 209-214, 1988), decreased phosphorylase activation. Activation of phosphorylase was observed also after CCl4 administration, as previously reported by Long and Moore (Biochem Pharmacol 35: 4131-4137, 1986). We conclude that perturbations in liver membrane Ca2+ regulation observed after administration of these hepatotoxins in vivo correlate directly with phosphorylase a activity, thereby providing additional in vivo evidence for an alteration of Ca2+ homeostasis early in the development of the liver damage produced by these chemicals.

A dye-photosensitized reaction that generates stable protein-protein crosslinks.

Irradiation of fibrinogen with visible light for 30 s in the presence of 1-1000 microM fluorescein was found to crosslink fibrinogen both inter- and intramolecularly. Optimum crosslinking was achieved at dye concentrations of around 100 microM and the amount of crosslinking was shown to increase with pH. Crosslinking was inhibited in the presence of 50 microM tryptophan or tyrosine and enhanced in the presence of 5 mM histidine. Twice as much crosslinking was found to take place under anaerobic conditions. These observations are consistent with a dye-photosensitized reaction following the hydrogen abstraction pathway. The subunits of eukaryotic ribosomes and those of phosphorylase a were also crosslinked by the method described.

Inhibition of acetaminophen hepatotoxicity by chlorpromazine in fed and fasted mice.

Acetaminophen hepatotoxicity has been shown previously to be potentiated by fasting, and the mechanism of hepatotoxicity has been correlated with depletion of reduced glutathione and the resulting elevation of cytosolic calcium. Chlorpromazine inhibited the hepatotoxicity of acetaminophen in a dose-dependent manner in fed and fasted mice. A 6 mg/kg dose of chlorpromazine prevented the acetaminophen-promoted increase in SGPT levels and prevented hepatic necrosis. Chlorpromazine did not prevent the depletion of reduced glutathione by acetaminophen in fed or fasted mice, although it did decrease the extent of reduced glutathione depletion caused by acetaminophen in fed mice from 80% depletion to 67% depletion. We propose that chlorpromazine causes a negative sensitivity modulation to calcium in hepatocytes, as evidenced by chlorpromazine preventing the acetaminophen-stimulated rise in phosphorylase a activity. We also propose that fasting potentiates acetaminophen hepatotoxicity by causing a positive sensitivity modulation to calcium in hepatocytes via the actions of glucagon.

Cytotoxicity of N,N-bis(2-chloroethyl)-N-nitrosourea in hypoxic rat hepatocytes.

Incubation of rat hepatocytes with N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU, 10-100 microM) and 5% O2 caused a time-dependent loss of cell viability, whereas no cytotoxicity was observed when BCNU was incubated with hepatocytes and 95% O2. BCNU (50-100 microM) reduced intracellular glutathione concentrations by 40 and 80% in hepatocytes incubated in 95 and 5% O2, respectively. Intracellular glutathione disulfide concentrations were not altered by 95 or 5% O2 or by the presence of BCNU. The extracellular glutathione disulfide content of cells exposed to BCNU and 95% O2, but not to BCNU and 5% O2, exhibited a 150% increase. Incubation of hepatocytes with 100 microM BCNU and 5% O2 reduced the cellular energy charge from 0.85 to 0.58; no effect on energy charge was observed in hepatocytes incubated with BCNU and 95% O2. The decrease in energy charge was due to a decrease in cellular ATP content (66%) and increases in cellular ADP (180%) and AMP (50%) concentrations. The reduction in both cellular ATP and glutathione concentrations was paralleled by a rise in the activity of phosphorylase a, a sensitive indicator of cytosolic Ca2+ content. These findings indicate that hepatocytes incubated in 5% O2 are more vulnerable to BCNU-induced cytotoxicity than are hepatocytes incubated in 95% O2 and that this vulnerability is associated with the loss of both ATP and glutathione. This conclusion is supported by data showing (a) a similar hypoxia-dependent pattern of cytotoxicity in hepatocytes exposed to the BCNU degradation products 2-chloroethyl isocyanate, 2-chloroethanol, and 2-chloroethylamine and (b) little BCNU-induced cytotoxicity, no increase in phosphorylase a activity, and no loss of ATP with 5% O2 in the presence of adenosine (1 mM).

Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure. Studies in rat hepatocytes with phosphorylase a and quin2.

Carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca2+. Cytoplasmic Ca2+ concentrations were evaluated by two methods: indirectly by assaying the activity of glycogen phosphorylase a, and directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 +/- 2 to 130 +/- 12, 80 +/- 13, and 97 +/- 10 nmoles PO4(3-)/mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl4, DCE, or PE, cytosolic Ca2+ concentrations were elevated within 20 sec to 0.83 +/- 0.13, 0.59 +/- 0.06 and 0.99 +/- 0.14 microM Ca2+ respectively. Basal Ca2+ levels in these cells averaged 0.25 +/- 0.03 microM. Thus, CCl4 and PE apparently increased cytosolic Ca2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.

Determination of glycogen and enzymes of glycogen metabolism in human hair follicles.

The skin epithelium and its organelles use glycogen as well as glucose as source of energy. Therefore the characterisation of glycogen metabolism and the enzymes involved is important in the study of mechanisms regulating the normal or abnormal differentiation of skin organelles such as sebaceous glands and hair follicles. The present paper describes fluorimetric methods for the determination of glycogen and for the measurements of phosphorylase and phosphorylase kinase activity in one and the same lysate of minute tissue samples. The methods were tested for their suitability on freshly isolated human hair follicles and cultured hair follicle cells. The possible use of these techniques for studies on the pathophysiology of acne and hirsutism is discussed.

Injury to hepatocytes induced by a peptide toxin from the cyanobacterium Microcystis aeruginosa.

The freshwater, bloom forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin which causes death accompanied by liver necrosis. We show here that the time and dose-dependent blebbing of isolated hepatocytes is accompanied by the activation of phosphorylase a, with no changes in cyclic AMP levels, and by glutathione (acid-soluble thiols) depletion. These results suggest that the disruption of cytoskeletal structures is accompanied by disturbances in cellular calcium homeostasis and by decreased protection against oxidative damage to the cells.

Inhibition of liver endoplasmic reticulum calcium pump by CCl4 and release of a sequestered calcium pool.

One of the earliest effects observed in rat liver after CCl4 administration is inhibition of an ATP-dependent calcium pump found at the endoplasmic reticulum. This report confirms that the amount of calcium associated with the microsomal fraction is reduced after CCl4 administration and, for the first time, demonstrates time-, dose-, and metabolism-dependent relationships between inhibition of the liver microsomal calcium pump and the amount of calcium found in the microsomal fraction. Furthermore, release of calcium from the endoplasmic reticulum is shown to cause activation of a cytoplasmic enzyme that responds to increases of ionized calcium, glycogen phosphorylase. This suggests that the endoplasmic reticulum calcium pump sequesters an intracellular pool of calcium within the endoplasmic reticulum. This pool of calcium may be released into the cytoplasm as a consequence of inhibition of the calcium pump by CCl4.

Mechanism of stimulation of liver glycogen synthesis by fructose in alloxan diabetic rats.

In diabetic animals and humans, stimulation of liver glycogen synthesis has been reported after administration of a large parenteral fructose load. The effects of an oral fructose load have not been examined previously. In the diabetic state, glycogen synthase phosphatase activity is reduced, and synthase D (the inactive form) is a poor substrate for the phosphatase. Thus, activation of synthase to the synthase R and synthase I (R + I) (active) forms by fructose would not be expected. We have determined that oral fructose administration does stimulate glycogen synthesis and have examined the mechanism by which this is accomplished. In 24-h-fasted alloxan diabetic rats, basal liver glycogen was higher than in normal rats (8.3 +/- 1.8 vs. 3.0 +/- 0.5 mg/g wet wt). After fructose (4 g/kg) was given, the initial rate of glycogen synthesis was normal in diabetic rats, but total glycogen synthesis was reduced. By 240 min, liver glycogen increased to 18 +/- 4.0 mg/g wet wt in diabetic rats versus 30.5 +/- 1.5 mg/g wet wt in normal rats. Synthase R + I was low and did not increase significantly (0.063 +/- 0.006 to 0.064 +/- 0.010 U/g wet wt) after fructose administration to the diabetic animals. Phosphorylase a did not decrease significantly during the period of active glycogen synthesis. In the diabetic rats, glucose-6-phosphate increased by 84% (0.103 +/- 0.010 to 0.184 +/- 0.020 mumol/g wet wt) within 10 min and remained elevated above the control level. UDPglucose decreased from 0.336 +/- 0.013 to 0.271 +/- 0.011 mumol/g wet wt at 10 min and remained below the control level.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial relationship of histochemically demonstrable patches in the mouse superior colliculus.

Patches of high phosphorylase activity are found in the intermediate and dorsal deep grey layers of the mouse superior colliculus when either coronal or sagittal sections are cut. These patches indicate that the phosphorylase a activity is arranged in a continuous lattice composed of bands of high phosphorylase a activity with a width of 100-200 microns that surround pale islands of low activity. This lattice was demonstrated by cutting surface parallel sections through the partially flattened superior colliculus. An almost identical lattice is observed in sections incubated to demonstrate total phosphorylase or cytochrome oxidase (CYO) activity. This phosphorylase/CYO lattice extends over the entire area of the superior colliculus. A discontinuous staining pattern is also observed in the intermediate and deep grey layers of both sagittal and coronal sections incubated for acetylcholinesterase (AChE) activity. The staining is arranged in two discontinuous sheets of intense activity that are joined together by vertical streamers. In surface parallel sections the AChE activity is found to form a network pattern which extends over the entire extent of the superior colliculus but which becomes fainter at the anterior pole. The phosphorylase/CYO lattice is not in register with the AChE lattice and the two seem to be organized independently of each other despite occurring at the same depth.