Reversal of changes of lipid peroxide, xanthine oxidase and superoxide dismutase by cardio-protective drugs in isoproterenol induced myocardial necrosis in rats

In myocardial necrosis produced by isoproterenol (beta-adrenergic agonist) marked increase in creatine phosphokinase, phospholipase and significant decrease in cardiac glycogen and phospholipid levels were observed. The enhanced levels of lipid peroxides, xanthine oxidase activity and lowering of superoxide dismutase may lead to excessive formation of free radicals resulting in cardiac cell damage. Nifedipine--a calcium antagonist, Propranolol--a beta-blocker and guggulsterone a lipid lowering agent showed marked reversal of these metabolic changes related to ischemia induced by isoproterenol.     

Histochemistry of the glycogen body of the turkey spinal cord

Summary Enzyme histochemical studies of the glycogen body of the turkey showed very little activity of suocinic dehydrogenase and cytochrome oxidase in the glycogen body cells, and marked activity of lactic dehydrogenase, NAD-diaphorase and the hexosemonophosphate shunt enzymes. Gradients of histochemical staining intensity for lactic and succinic dehydrogenase in the glycogen body and spinal cord were confirmed by biochemical assays of homogenates of these tissues. It was concluded that glycogen body metabolism is predominantly glycolytic. Alkaline phosphatase activity was weak; acid phosphatase activity was moderate. There was no acetyl cholinesterase or nonspecific cholinesterase activity in the glycogen body.This investigation was supported by U. S. Public Health Grant B 3250.Submitted in partial fulfillment of Masters' degree requirements.     

An enzyme histochemical study of isoproterenol-induced myocardial necroses in rats

Summary The effect of isoproterenol on myocardial metabolism in rats was studied using qualitative and quantitative histochemical techniques. The activity and location of 20 enzymes that play a role in the aerobic and anaerobic pathways of energy metabolism were qualitatively examined. The activity and location of some hydrolytic enzymes and the glycogen content were also qualitatively studied. For the quantitative study the activity of 10 enzymes was measured.The isoproterenol injections induced necrosis with inflammatory infiltrates. The muscle fibres in the necrotic regions were characterized by defective aerobic energy metabolism and increased glycolytic capacity. There was a depletion of the glycogen reserves in the necrotic fibres. These fibres showed a markedly increased activity of enzymes belonging to the oxidative branch of the pentose phosphate pathway. The implication of this increase for the metabolism of the myocardial cells is discussed. The activity of acid phosphatase in the pathological muscle fibres was strongly increased. The inflammatory cells in the necrotic areas were characterized by preponderantly anaerobic metabolism.Dedicated to Prof. H. G. Goslar in honour of his 70th birthday.     

Ultrastructure of isoproterenol-induced myocardial damage in chick embryos

Isoproterenol is known to cause severe myocardial lesions when given in toxic doses to adult homoiotherms. Previous studies on chick embryos revealed myocardial damage with scattered necroses in the outer layer of ventricular myocardium. The present ultrastructural study, performed on embryos 6 to 20 days old, has shown various types of cellular lesions; mainly cellular oedema, mitochondrial swelling, necroses of isolated cardiac muscle cells, fatty degeneration, accumulation of glycogen, and signs of increased proteosynthesis in the surviving muscle cells. Morphological features of the lesions differed from those which are known to be induced by isoproterenol in adult animals and seemed to depend on the stage of embryonic development.     

Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.     

Histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow (Capricornis crispus, Artiodactyla, Mammalia) with special reference to glandular structures

The histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow ( Capricornis crispus ) was studied by light microscopic histochemical methods, particularly lectin histochemistry. The eccrine glands present exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-D-galactose and N-acetyl-neuraminic acid) especially in the cells of the secretory acini, the free surface of the collecting duct cells also showed distinct positive reactions with most of the histochemical methods. The thick epidermis of the nasolabial skin contained smaller amounts of glycoproteins. The results obtained are discussed with regard to possible functions of the glandular secretions. This substance mixture may particularly improve water retention on the skin surface, and protect against physical damage as well as microbial contamination. There seem to be no basic differences of muzzle functions between wild and domesticated bovine species.     

Physiological adaptation in relation to hyperosmotic stress in the epidermis of a fresh-water teleost Barbus sophor (Cypriniformes, Cyprinidae): a histochemical study.

The changes in distribution of mucopolysaccharides, glycogen and protein bound sulphydryl groups of cysteine in the various cellular components of the epidermis of Barbus sophor along with its structural alterations as a result of hyperosmotic stress, have been described using histochemical techniques. The hyperosmotic saline induces a cyclic secretion of acid mucopolysaccharides by the mucous cells. Simultaneously the polygonal cells also show a marked disturbance in the processes of mucogenesis and keratinization, indicating an inverse relationship between the degree of keratinization and the amount of mucus secreted by epidermis. The role of glycogen in the polygonal cells has been discussed in relation to disturbed mucogenesis. The appearance of intercellular spaces in basal layer and middle layer has been correlated with the passage for movement of increased amounts of nutrients through the skin.     

Achieving elongated lesions employing cardiac cryoablation: a preclinical evaluation study

Cardiac cryoablation applied for treating cardiac arrhythmias has shown promising results after intervention, particularly for the creation of elongated lesions. A model for simulating and assessing cryoablation interventions was developed, evaluated and validated with animal experiments. We employed two simulations of different freezing outlet settings for a loop shaped cryocatheter, applying Pennes heat equation for cardiac tissue. Our experiments demonstrated that an equidistantly spaced freezing outlet distribution of 5mm led to an improved formation of lesions, i.e., elongated lesions were observed throughout the transmural cardiac volume and on the epicardial structure. A complete transmural frozen lesion was not achieved with a freezing outlet distance of 10mm. These simulation results could be experimentally verified by morphological and histological examinations. Using our simulation model we were able to optimize the intervention procedure by predicting and assessing the freezing process. This should further increase the success rate of cardiac cryoablation in clinical interventions.     

实验性肝癌糖原和癌基因N-ras表达的研究

通过应用原位杂交和组织化学技术,对二乙基亚硝胺诱发的大鼠肝细胞肝癌中糖原和癌基因Ｎ－ｒａｓ表达的研究,发现从诱癌早期到晚期,肝细胞内的糖原由储积而逐渐丧失。Ｎ－ｒａｓ在诱癌的第１～２周即出现阳性表达,随诱癌过程的延长,阳性表达的细胞数和范围逐渐增加,至诱癌晚期甚至在癌结节内均转为阴性。对肝组织连续切片中糖原和Ｎ－ｒａｓ表达的对比观察发现,糖原ＰＡＳ反应与Ｎ－ｒａｓ反应同步,糖原ＰＡＳ反应具有与Ｎ－ｒａｓ一致的异质性,其阳性与阴性病变分布与Ｎ－ｒａｓ表达重叠。提示Ｎ－ｒａｓ基因表达可能在肝癌的启动过程中发挥重要作用,并且可能涉及对糖原基因的调控。     

Unusual histochemical pattern in preneoplastic hepatic foci characterized by hyperactivity of several enzymes

In a stop-experiment using the hepatocarcinogen N-nitrosomorpholine, as well as glycogenotic and related lesions, hepatocellular foci with a different histochemical pattern were identified. The outstanding features of these hepatic foci, which may progress to hepatocellular adenoma, were increased activities of mitochondrial glycerol-3-phosphate dehydrogenase (mG3PD), glycogen synthase, pyruvate kinase and glucose-6-phosphatase detected by enzyme histochemistry. Since no decrease in activity of any of the enzymes examined were seen in these foci, compared with normal liver, the term enzymatically hyperactive focus (EHF) is proposed for this type of lesion. Only at the stage of overtly nodular growth did these lesions exhibit some of the characteristic changes seen in nodules developing from glycogenotic foci, namely elevated activities of glucose-6-phosphate dehydrogenase, gamma-glutamyl transferase and glutathione-S-transferase P as well as decreased activities of adenosine-triphosphatase, glucose-6-phosphatase and adenylate cyclase. Some of these enzymes have been used widely in morphometric studies as markers for preneoplastic and neoplastic lesions. The inability to detect early EHF may lead to an underestimation of preneoplastic liver lesions in quantitative studies. Although there are apparent differences in the histochemical patterns of glycogen storing foci and early EHF, these differences tend to disappear during progression to overtly neoplastic lesions. In studies comparing the phenotypic alterations in different types of preneoplastic hepatic lesions, the recognition of EHF may contribute to the distinction of obligatory from facultative phenomena during transformation.     

Intranuclear synthesized and native glycogen particles in human gastric cancer: Ultrastructure and histochemistry

Summary Ultrastructural and histochemical studies on human gastric cancer cells disclosed the presence of native and synthesized glycogen particles. The glycogen particles were investigated in the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system and non-incubated native glycogen in human gastric adenocarcinoma tubulare.It was observed that focal synthesis localized in the intracytoplasmic matrix and intranucleus. Intranuclear synthesized glycogen appeared as a rosette form ranging from 1100 to 1300 Å in diameter and free particles ranging from 325 to 900 Å in diameter. The synthesis of glycogen appeared in the nucleus as well as in the cytoplasm of the human gastric cancer cells, and the synthesized glycogen was observed as a group of particles. Newly formed glycogen particles appeared occasionally in the interchromatin area as a large macromolecular structure of rosette form.Native glycogen appeared as a free-particle (250–333 Å, medium=300 Å) and aggregated rosette from (694–1050 Å, medium=917 Å) in the autophagosome of gastric cancer cells. There was not, however, a native glycogen particle in the nuclei of gastric cancer cells.Under certain conditions the nuclei of gastric cancer cells can acquire the capacity to synthesize glycogen.     

Isoproterenol-induced responses in the regional polyamine metabolism of the rat heart

Rats were treated with a single dose of isoproterenol (25 mg/kg s.c.) and the levels of polyamines determined in various parts (right ventricle, basis, medial part and apex of the left ventricle) of the heart 24, 48 and 72 hours after the injection. The isoproterenol treatment produced marked alterations in the concentrations of cardiac polyamines. The most apparent changes were seen in the apex and medial part of the left ventricle where spermidine concentration exhibited a biphasic response with peaks at 24 and 72 hours. In the basis of the heart the spermidine concentration was significantly elevated only at 24 hours. In the right ventricle the spermidine level was significantly higher than control at 72 hours. Spermidine/spermine ratio was augmented in all cardiac tissues examined over the 72-hour period. Results appear to show that the isoproterenol-induced alterations in cardiac polyamine metabolism were not uniformly distributed in the various regions of the heart.     

Glycogen phosphorylase hyperactive foci of altered hepatocytes in aged rats

In untreated 12- to 24-month-old rats, the enzyme histochemical pattern of 45 focal hepatic lesions was investigated in serial sections. In addition to previously characterized glycogen storage foci, a new type of enzymatically altered hepatic focus was found. The outstanding feature of this was an increased glycogen phosphorylase activity. The frequent appearance of glycogen phosphorylase hyperactive foci simultaneously exhibiting excessive glycogen storage suggests a close relationship to the well known glycogen storage foci representing an early stage in the sequence of cellular changes which lead to hepatic tumors.     

Qualitative and quantitative histochemical changes in the caecum and liver of turkeys infected with Histomonas meleagridis.

Changes in the amount and distribution of acid and alkaline phosphatase, non-specific esterase, glycogen, lipid and acid mucopolysaccharide in the caecal wall and liver of turkey poults infected with Histomonas meleagridis have been studied histochemically. A microdensitometer was used to measure changes in activity and distribution of acid phosphatase and non-specific esterase in the caecal mucosa. During the course of the infection there is a marked reduction in activity and distribution of acid phosphatase and non-specific esterase but little change in the amounts and distribution of alkaline phosphatase, glycogen, lipid and acid mucopolysaccharide in the wall of the main part of the caecum. Similar, but smaller, changes occurred in the wall of the neck region of the caecum. In the liver most changes occurred in the immediate vicinity of the parasites. Initially, there was a reduction in the amount of glycogen in the parasitic lesions but later in the infection there was a marked loss of glycogen in all regions of the liver. Changes in the caecum are apparently brought about by the parasite prior to and after invasion of the caecal tissues; changes in the liver occur after it has been invaded.     

Resurgence of glycogen synthesis and storage capacity in cultured hepatoma cells.

This paper describes a new cultured hepatoma cell line referred as ZHC cells, derived from the ascitic Zajdela rat hepatoma. Since 1963, the dedifferenciated in vivo transplanted ascitic cells were characterized by the absence of glycogen as in generally the case in all fast growing hepatic tumors. In 1974, we succeeded in adapting these tumor cell to in vitro defined growth conditions, where we observed the progressive recovery of the ability to synthesize and to store large amounts of glycogen, as shown by histochemical, ultrastructural and biochemical studies. It can now be considered as an established cell line in which the reverted phenotype has been stable for 3 years.     

Liver glycogen metabolism during short-term insulin-induced hypoglycemia in fed rats

The activities of glycogen phosphorylase and synthase during infusions of glucagon, isoproterenol, or cyanide in isolated liver of fed rats submitted to short-term insulin-induced hypoglycemia (IIH) was investigated. A condition of hyperinsulinemia/hypoglycemia was obtained with an intraperitoneal injection of regular insulin (1.0 U kg(-1)). The control group received ip saline. The experiments were carried out 60 min after insulin (IIH group) or saline (COG group) injection. The rats were anesthetized and after laparotomy, blood was collected from the vena cava for glucose and insulin measurements. The liver was then infused with glucagon (1 nM), isoproterenol (2 microM), or cyanide (0.5 mM) during 20 min and a sample of the organ was collected for determination of the activities of glycogen phosphorylase and synthase 5 min after starting and 10 min after stopping the infusions. The infusions of cyanide, glucagons, and isoproterenol did not change the activities of glycogen synthase and glycogen phosphorylase. However, glycogen catabolism was decreased during the infusions of glucagon and isoproterenol in IIH rats, being more intense with isoproterenol (p < 0.05), than glucagon. It was concluded that short-term IIH promoted changes in the liver responsiveness of glycogen degradation induced by glucagon and isoproterenol without a change in the activities of glycogen phosphorylase and synthase.     

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP – kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase – MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

Zero-stress state of intra- and extraparenchymal airways from human, pig, rabbit, and sheep lung.

Alterations in airway wall anatomic properties and the consequential effects on airway narrowing have been assessed by use of computational models. In these models, it is generally assumed that at zero transmural pressure the airway wall exists in a zero-stress state. Many studies have shown that this is often not the case, as evidenced by a nonzero opening angle. In this study, we measured the opening angle of airway rings at zero transmural pressure to test this assumption. The airway tree was dissected from human, pig, sheep, and rabbit lungs. Airways were excised from the tree, and the opening angle was measured. There were obvious species and regional differences in opening angle. Rabbit airways from both extraparenchymal and intraparenchymal sites exhibited marked opening angles (7-82 degrees). Extraparenchymal airways from sheep had large opening angles (up to 50 degrees), but ovine intraparenchymal airways had small opening angles. Measurable opening angles were rarely observed in human and porcine airways of any size. The assumption of a stable zero-stress state at zero transmural pressure is therefore valid for human and porcine, but not rabbit and sheep, airways.     

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.