Lead and some other metals in the histochemical demonstration of rat liver glycogen phosphorylase activity

Summary Lead in a concentration of 0.25 mM was tried as a histochemical trapping agent for inorganic phosphate, liberated in the reversible reaction catalyzed by the liver glycogen phosphorylase.The reaction product, formed during the incubation and made visible with ammonium sulphide, was totally extracted with -amylase. Iodine staining after incubation was completely negative.The inhibitory effect on liver phosphorylase activity of several other metals was also studied histochemically. It was found that the inhibition generally increased with the molecular weight and concentration of the metals. It is concluded that Fe⁺⁺ could be useful as a trapping agent instead of lead.     

The effect of ischemia on glycogen phosphorylase activity in rat liver: a quantitative histochemical study

The effects of ischemia in vitro for 0-60 min at 37 degrees C on glycogen phosphorylase activity in rat liver have been studied under different feeding conditions. Glycogen phosphorylase activity was demonstrated with a recently developed quantitative histochemical method using a semipermeable membrane and the PAS-reaction. The cytophotometrically measured glycogen phosphorylase activity in livers from 24 h-fasted rats was approximately five times the activity in livers from normally fed rats. The activity in periportal areas was about 1.5 times higher than the activity in pericentral areas in livers from starved rats, but more or less evenly distributed in livers from fed rats. Enzyme activity in pericentral areas of livers from 24 h-fasted rats started to decrease after 20 min of ischemia. After 50-60 min of ischemia, the activity was decreased to approximately 25% of the control activity. Livers from normally fed rats showed unchanged activity in periportal and pericentral areas after 10-60 min of ischemia. It has been assumed that the activation of the enzyme was disturbed by ischemia, possibly as a consequence of plasma membrane damage.     

Histochemical demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary A new histochemical method for light microscopic demonstration of liver glycogen phosphorylase activity has been introduced in this study.The method demonstrates phosphorylase activity by precipitating phosphate ions, liberated in the reaction catalyzed by the enzyme, with Fe⁺⁺ present in the incubating medium. The precipitate is visualized as ferrous sulphide.The new glycogen, formed in the same reaction, can also be demonstrated in this method after staining with iodine.The lobular localization of the reaction products obtained according to this method in the liver, corresponds well to that obtained according to earlier methods for the demonstration of only new-formed glycogen.     

Diurnal variation in glycogen phosphorylase activity in rat liver. A quantitative histochemical study

The diurnal variations of the glycogen content and of glycogen phosphorylase activity in periportal and pericentral areas of rat liver parenchyma have been analyzed in periodic acid Schiff (PAS)-stained cryostat sections using quantitative microdensitometry. Glycogen content and phosphorylase activity were always higher in periportal areas than in pericentral areas throughout the daily cycle. The glycogen content was highest at the end of the active period during darkness and lowest at the end of the resting period. Phosphorylase activity appeared to be inversely correlated with the glycogen content in both areas. It is concluded that the glycogen content is regulated by phosphorylase activity, which may be due to local cAMP concentration.     

Electron microscopic demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary In this study a new electron microscopic method for the demonstration of liver glycogen phosphorylase activity has been presented.Prior to incubation the liver samples were shortly fixed in cold paraformaldehyde. Inorganic phosphate, liberated in the reaction catalyzed by the enzyme, were precipitated with iron (Fe⁺⁺) present in the incubating medium. Postfixation was performed in glutaraldehyde and osmium tetroxide.The ferrous phosphate precipitate was detected electron microscopically in unstained sections.The precipitate was mainly localized to endoplasmic membranes but also in glycogen particles. The method is imperfect in demonstrating phosphorylase activity bound to glycogen particles because of poor preservation of glycogen during treatment.     

Electron microscopic demonstration of glycogen and phosphorylase activity in rat hepatocytes

Synopsis The effect of fixation with a bicarbonate-buffered solution of paraformaldehyde and polyvinyl pyrrolidone (PVP) on the ultrastructural demonstration of glycogen and phosphorylase activity in rat hepatocytes has been studied. Phosphorylase was demonstrated by the precipitation of liberated phosphate ions with ferrous ions. 7.5% PVP was included in all steps in the procedure before post-fixation in osmium tetroxide.Glycogen particles were well preserved. Structures connecting membranes and glycogen particles were also evident. Phosphorylase activity was rapidly inhibited by the fixative; the fixation time was, therefore, kept very short. The final reaction product was localized on glycogen particles and on endoplasmic membranes in association with glycogen particles. The results support the view that endoplasmic membranes are involved in the metabolism of glycogen in hepatocytes.Paper presented at a symposium The changing directions of carbohydrate histochemistry at the Fifth International Congress of Cytochemistry and Histochemistry in Bucharest, Romania on 1 September 1976.     

The histochemical demonstration of aniline hydroxylase activity in rat liver

Synopsis A procedure is described for the histochemical demonstration of aniline hydroxylase activity in cryostat sections of rat liver. Tissue sections are incubated in a medium containing aniline; thep-aminophenol formed as a result of enzymatic action is coupledin situ with Fast Blue RR. The staining reaction is found to be confined to the cytoplasm of the hepatocytes. Confirmatory tests for true enzymatic staining reaction include the incubation of sections in medium from which aniline is omitted, and under conditions of enzyme inhibibition. A method for the quantitation of the histochemical staining reaction is also described.The histochemical reactions have been investigated on rat livers subjected to conditions eliciting microsomal enzyme stimulation and inhibition, bothin vitro andin vivo. A close correlation was found between the staining reactions observed and the results of the quantitative histochemical method and the biochemical estimations of aniline hydroxylase activity in liver microsomal fractions obtained by differential centrifugation.     

The effect of various aminoglycoside antibiotics on glycogen phosphorylase activity in liver and kidney medulla of rabbit

Glycogen phosphorylase activity in both liver and kidney medulla of rabbit was stimulated in the presence of caffeine by various aminoglycoside antibiotics in the following rank order: gentamicin greater than neomycin greater than amikacin = kanamycin greater than or equal tobramycin, while streptomycin did not affect the enzyme activity. In contrast, in the presence of AMP, the stimulatory action of antibiotics was not observed. Since in the gentamicin-treated rabbits stimulation of glycogen phosphorylase activity by about 30% in both liver and kidney medulla was accompanied by a decrease of liver glycogen content by about 60% it is likely that a decline in liver glycogen level following antibiotic treatment is due to an increased glycogen phosphorylase activity.     

Observations on the histochemically demonstrable liver glycogen phosphorylase activity and native glycogen under different nutritional conditions

Summary In this study a histochemical demonstration of glycogen phosphorylase activity and native glycogen in the livers from normally fed, overfed and starved rats was performed.It was found that the amount and localization of phosphorylase activity well corresponded to the amount and localization of the native glycogen. A change of the glycogen content in the liver also resulted in a change of the histochemically demonstrable liver glycogen phosphorylase activity.It is concluded that the presence of tissue bound glycogen and undissolved glycogenphosphorylase complexes are necessary for positive histochemical demonstration of liver glycogen phosphorylase activity.This work was supported by a grant from the Finnish Veterinary Medical Foundation.