Biomedical subjects

G Letko

Publications and source records attributed to G Letko.

At least 19 recordsLinked to original sources

PubMed2001

Randomized, placebo-controlled trial of lazaroid effects on severe acute pancreatitis in rats.

OBJECTIVES: To evaluate the therapeutic potential of lazaroids in severe necrotizing acute pancreatitis and to investigate the association between oxidative stress, protease activation, and local production of proinflammatory cytokines and the severity and lethality of the disease. BACKGROUND: Oxidative stress is a crucial factor in the pathophysiology of acute pancreatitis and its systemic complications. Treatment with antioxidants, however, failed to improve survival in most studies performed so far. Lazaroids are a novel class of antioxidants that potently protect pancreatic acinar cells against oxidant attack in vitro. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: Seventy-five anesthetized male Wistar rats (300-350 g). INTERVENTIONS: Severe acute pancreatitis was induced by retrograde injection of 3.5% taurocholate-sodium into the common bile-pancreatic duct. Interventions were performed to mimic the clinical situation, including continuous intravenous fluid substitution and administration of lazaroids in a therapeutic protocol. Therapy was started 1 hr after injection of the bile salt by using three different lazaroids, lactated Ringer's solution (placebo), and methylprednisolone as a corticosteroid control (n = 15 in each group). All the substances were given by continuous intravenous infusion throughout the 20-hr trial period. MEASUREMENTS AND MAIN RESULTS: Pancreatic homogenates and ascites were analyzed for indicators of oxidative stress, antioxidants, proteases, and proinflammatory cytokines. Pancreatic edema, morphologic pancreatitis severity, and pancreatic histopathology also were assessed. All three lazaroids and methylprednisolone diminished pancreatic tumor necrosis factor-alpha concentrations. Lethality was 33% in the placebo group. Neither the lazaroids nor methylprednisolone influenced survival. The local pancreatic and peritoneal concentrations of lipid peroxidation products, antioxidants, and proteases did not differ among the five groups. Nonsurviving rats, however, had a higher total protease activity in the pancreas and higher concentrations of trypsinogen activation peptide in ascites, as compared with surviving animals. There were no differences between survivors and nonsurvivors with regard to variables of oxidative stress and cytokines. CONCLUSIONS: Lazaroid application under clinically relevant conditions (i.e., after induction of fulminant acute pancreatitis) does not influence lethality or biochemical variables relevant to this disease. Protease activation rather than oxidative stress or local pancreatic cytokine production is an important determinant of disease severity and survival in acute pancreatitis. In experimental studies evaluating novel therapeutics, the same strict criteria should be applied as in the human setting.

Animals↗

PubMed1997

Similar morphological and intracellular biochemical changes in alcoholic acute pancreatitis and ischemic acute pancreatitis in rats.

Pancreatic hyperstimulation with simultaneous duct obstruction does not cause the typical features of acute pancreatitis, therefore the role of an additional challenge, such as either ethanol intoxication or short-term ischemia, was studied. Alcoholic pancreatitis was induced in 28 rats by acute ethanol intoxication (0.25 LD50) and an obstruction/hyperstimulation mechanism (clip of the biliopancreatic duct for 20 min and intravenous stimulation with 5 U of cholecystokinin and secretin each). Ischemic pancreatitis was performed by obstruction/hyperstimulation and subsequent pancreatic ischemia by clamping the supplying arteries for 40 min. The macro- and microscopic alterations were evaluated and graded by a scoring system. Additionally, the pancreas was removed in 50% of the animals and the pancreatic acini were prepared. From those acini the intracellular enzymes trypsinogen, kallikreinogen, amylase, lipase, glucuronidase, and acidic phosphatases were determined. While obstruction/hyperstimulation, 40 min of ischemia, or ethanol alone did not induce acute pancreatitis, a combination of obstruction/hyperstimulation with either ethanol or ischemia resulted in acute pancreatitis in 68 and 60% of treated rats, respectively. Similarly, both models were characterized by extrapancreatic fat necrosis and acinar necrosis at the periphery of the lobules. Almost all intracellular enzymes were elevated in both pancreatitis models compared to sham-operated controls. Both alcohol and ischemia were insults that sensitize the pancreas to develop acute pancreatitis after obstruction/hyperstimulation. Since the observed morphologic and enzymatic alterations in both models are very similar, alcohol and ischemia might have some common pathways by which they make the pancreas vulnerable to enzymatic attacks.

Acute Disease↗

PubMed1995

A method for in vivo assessment of reversible rat pancreatic ischemia using 31P NMR spectroscopy at 2.0 tesla.

A surgical method is described which allows in vivo assessment of reversible rat pancreatic ischemia using 31P NMR spectroscopy at 2.0 T. Phosphorous-31 NMR spectra acquired during the ischemic period show the expected increase in inorganic phosphate with a concomitant decrease in ATP levels and pH as compared to controls. Upon reperfusion, inorganic phosphate and ATP returned to control levels while pH recovered to a more alkaline value. This method provides a means of studying in vivo changes in high energy metabolite associated with acute pancreatitis (AP) and maintains the secretory ability of the gland so that different forms of AP, such as those arising from pancreatic juice edema, can be studied.

Adenosine Triphosphate↗

PubMed1994

[Mechanisms for cell damage in acute pancreatitis using isolated acinar cells].

Due to the complexity of interacting organ systems, in vivo the interpretation of results obtained from whole-animal experiments of acute pancreatitis remains difficult. To enlighten cause-and-effect-relationships, functional isolated parts of the pancreas are applied increasingly in research into the pathogenesis of the disease, therefore. By means of a collagenase digestion technique, intact acinar cells from normal as well as from pretreated rat pancreas could reproducibly be obtained in high yield. Animals were pretreated in situ by induction of either mesenteric ischemia/reperfusion, juice edema, or acute pancreatitis (AP). Pancreatic acinar cells isolated from these pretreated rats consumed oxygen at comparable rates under resting conditions throughout all experimental groups. A reduced stress capacity of cell respiration as well as a preterm decline in short-term culture was observed in the cells of the AP group, however. These results demonstrate that the induction of AP has found its reflection within the acinar cells themselves. Interestingly, these effects were not clouded by the isolation procedure. The manner of in-situ pretreatment of the respective animal proved to be decisive for later viability of isolated acinar cells in short-term culture. Uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (DNP) lead to an accelerated decline of the acinar cells in all groups. The intactness of cellular energy metabolism seems to play a crucial role in maintaining cellular integrity, therefore. In additional experiments in vitro, the intracellularly mediated cell damage by DNP was compared with one induced from the extracellular environment of isolated cells by antibodies directed against cell surface antigens plus complement.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1992

Influence of ethanol on survival of acinar cells isolated from rat pancreas.

The role of ethanol in precipitating acute pancreatitis has been studied in model experiments. The influence of ethanol on the survival of isolated pancreatic acinar cells (PAC) and a possible coaction with noxious agents (such as trypsin, chymotrypsin, temporary anoxia, and partial uncoupling of the oxidative phosphorylation by 2,4-dinitrophenol [DNP]) has been investigated. Isolated PACs withstood ethanol levels up to 180 mM without significant decrease of viability within 4 h incubation at 37 degrees C. A 90-min deprivation of oxygen was widely tolerated also. However, with a combination of both ethanol application and oxygen deprivation, a clearly forced cell damage was observed as was true with a combination of ethanol and chymotrypsin. The action of DNP or of extracellular trypsin on cell survival was not amplified by the addition of ethanol (180 mM). This study reveals two possible sites of action at which ethanol may contribute to the pathogenesis of acute pancreatitis.

2,4-Dinitrophenol↗

PubMed1992

Influence of trypsin-induced acute pancreatitis on survival and energy state of isolated acinar cells from rat pancreas.

To study the development of acute pancreatitis after intraductal trypsin instillation, at 4 hours, 1, 2, 4 and 6 days after this treatment and after instillation of physiologic saline viable acinar cells were isolated from rat pancreas. Gross anatomic and histologic findings were used to evaluate the time course of pathomorphologic changes. The isolated cells were incubated at 37 degrees C in Eagle's medium in a shaking water bath and the time course of their damage was studied. Additionally, by means of the active accumulation of the fluorophore rhodamine 6 G alterations of the mitochondrial membrane potential, an important parameter of the cellular energy metabolism was evaluated. The most severe histological damage was seen 1 and 2 days after trypsin instillation. At the same time yield and survivability of cells isolated, and their mitochondrial membrane potential reached a minimum. In the controls the time course of these parameters was very similar, but their decrease was less pronounced. Since a direct action of trypsin on acinar cells cannot be responsible for the findings presented a possible involvement of inflammatory cells and their products in the alteration of the cells and of their energy metabolism must be considered.

Acute Disease↗

PubMed1992

Influence of stepwise uncoupling and temporary anoxia on pancreatic enzyme secretion by isolated rat acini.

For special studies on pancreatic diseases a parameter is needed to record alterations of the cellular energy metabolism. In the in vitro model of isolated pancreatic acini, we investigated whether or not at standardized cholecystokinin stimulation the energy-consuming process of enzyme secretion can be used to monitor changes of the energy-supplying capacity. Rat pancreatic acini were isolated via collagenase digestion and characterized by basal and stimulated release of amylase and trypsin, oxygen uptake under resting and maximally uncoupled conditions and by their ability to accumulate actively rhodamine-6G, as a measure of the mitochondrial membrane potential. The stimulation of enzyme release did not find a measurable reflection in rhodamine-6G accumulation and in the respiratory rat. Stepwise uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (DNP) and temporary anoxia were used to simulate disturbances of the pancreatic energy metabolism in vitro. With increasing DNP concentration the enzyme release was significantly reduced. While after 30 min anoxia the enzyme release still exceeded that of unstimulated control, after 60 min anoxia there was no further response to hormonal stimulation. At standardized stimulation and after suitable calibration the enzyme release by acini may be used to monitor alterations of the pancreatic energy metabolism in vitro.

2,4-Dinitrophenol↗

PubMed1992

Effect of phospholipase A2 and ethanol on the survival of acinar cells isolated from the rat pancreas.

The study was undertaken to investigate the effect of phospholipase A2 (PLA2) on the viability of isolated pancreatic acinar cells (PACs) and its possible synergy with ethanol. The survival of PACs may be reduced by PLA2 in a dose-dependent manner. In the presence of 0.25 microgram PLA2.(10(6) cells)-1, all cells died within less than 4 h of incubation at 37 degrees C. A quantity of 180 mM ethanol, which alone was not lethal for PACs, increased the rate of cell death induced by PLA2 at a certain concentration of this enzyme. The present results imply that (1) PLA2 is able to damage PACs in the absence of additional substrates, and (2) at an appropriate activity of PLA2, ethanol may amplify the toxic effects of this lytic enzyme and force autodigestion of the pancreas.

Acute Disease↗

PubMed1991

Involvement of free radicals in the pathophysiology of chronic pancreatitis: potential of treatment with antioxidant and scavenger substances.

The present work reviews the evidence for an involvement of free radicals in the pathophysiology of chronic pancreatitis and the potential of treatment with antioxidant and scavenger substances. Preliminary results indicate that exposure of isolated pancreatic acinar cells to a reaction mixture containing hypoxanthine, xanthine oxidase, and chelated iron causes cell damage and death probably due to generation of superoxide anion and hydrogen peroxide. It still needs to be analyzed which scavengers and antioxidants are able to ameliorate the damage due to oxidant stress in cell models. Such knowledge from cellular studies might help to plan therapeutical trials to evaluate potentially effective antioxidants and scavengers in the experimental animal and in patients with pancreatitis. As yet there are no published studies about the role of free radicals in animal models of chronic pancreatitis. This fact is probably due to the shortcomings of the animal models available. Recent studies presented evidence that activation of oxygen-derived free radicals occurs in patients with chronic pancreatitis. There is also some evidence that the dietary intake of antioxidants may be reduced in patients with chronic pancreatitis. It was suggested that such reduction of antioxidant defenses in the face of an increased demand due to heightened induction of P450 activities may facilitate lipid peroxidation. However, as yet, there is no direct evidence that a reduction of dietary antioxidants with a simultaneous increase in P450 activity is the primary mechanism which initiates chronic pancreatitis without contribution of other factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Influence of anoxia, reoxygenation, and uncoupling on survival, respiration, and trypsin-inhibiting capacity of isolated pancreatic acinar cells.

In the pathogenesis of acute pancreatitis, the events and mechanisms increasing the digestibility of the pancreatic acinar cells are widely unknown. Therefore, the possible contribution of a disturbed energy supply (provoked by anoxia or partial uncoupling) to the induction of autodigestion was studied in experiments on acinar cells isolated from the pancreas. During incubation viability, respiration under normal and maximally stimulated conditions, and trypsin-inhibiting capacity (TIC) of these cells were determined. With increasing duration of anoxia, the portion of surviving cells was strongly diminished, and the number of cells with blebs and vesicularly transformed endoplasmic reticulum was increased. Although the endogenous respiration was not influenced up to 1.5 h of anoxia, 30 min of anoxia substantially decreased the capacity of oxidative energy production. The survival curves were characterized by a self-accelerating course of cell destruction. The alteration of the cellular energy metabolism found its reflection in the decreased TIC of the cells.

2,4-Dinitrophenol↗

PubMed1991

Development of acute pancreatitis in rats after single ethanol administration and induction of a pancreatic juice edema.

The effect of an acute gastral ethanol application and an additional induction of a pancreatic juice edema by an obstruction/hyperstimulation mechanism was studied in rats. Neither ethanol administration nor the combination of ductal obstruction and secretory stimulation resulted in an acute pancreatitis when applied independently. A combination of these factors, however, led to the development of AP in two-thirds of the rats treated in this manner. Its severity was evaluated by the degree of extrapancreatic fat necrosis, the increase in serum amylase and lipase activities, and by histologic investigations. It is assumed that a pancreatic juice edema, provoked by pancreatic secretion at temporary ductal obstruction, could be an important cofactor in formation of ethanol-induced acute pancreatitis.

Acute Disease↗

PubMed1991

Light and electron microscopic studies of lectin binding on the glycocalyx of rat pancreatic cells. I. Normal tissue and isolated cells.

Lectin binding of the glycocalyx of pancreatic tissue sections as well as of isolated pancreatic acini and acinar cells was studied of healthy wistar rats by light and electron microscopy. For light microscopy, we used FITC (WGA, RCAI, LCA) or peroxidase marked (WGA, RCAI, PNA, PHA, LCA, UEAI, LPA) as well as unmarked lectins (Con A, VAA I). Gold marked lectins were used for electron microscopy (WGA, RCAI, LCA, HPA, PNA, VAAI-B). Intact acinar cells in pancreatic tissue sections and isolated acini showed a strong binding of WGA, RACI, and HPA on the apical cell surface, whereas VAAI, UEAI, LCA, and Con A reacted strongly with the basolateral glycocalyx, but not with the apical surface. The 2 main domains of the glycocalyx of pancreatic cells showed their specific lectin binding so long as the junctional complexes between the cells are intact. The polarity of the cell surface of pancreatic acinar cells is discussed in regard to the possible function of the 2 domains.

Animals↗

PubMed1991

Studies on lipid peroxidation in pancreatic tissue. In vitro formation of thiobarbituric-acid-reactive substances (TBRS).

In pathogenic studies on acute pancreatitis the importance of a temporary ischemia on induction of autodigestion was demonstrated. Because of the involvement of oxygen-derived free radicals in the ischemia/reperfusion injury of other tissues we have investigated the influence of artificial oxidants, as FeCl3 and H2O2, on pancreatic tissue and isolated pancreatic acinar cells. Lipid peroxidation was determined as thiobarbituric-acid-reactive substances (TBRS). In these experiments the TBRS concentration was elevated within the first min of incubation with FeCl3. The exposure of pancreas homogenates and intact acinar cells to H2O2 had no remarkable effect on formation of TBRS. Under this condition the survival of cells was strongly reduced, while cells exposed to FeCl3 revealed a remarkably slower rate of cytolysis. The missing correlation between cell lysis and elevation of TBRS suggests that lipid peroxidation might not be essential process in pancreatic acinar cell damage.

Animals↗

PubMed1991

Transition of rat pancreatic juice edema into acute pancreatitis by single ethanol administration.

The present study was done to determine the influence of single oral, intravenous and intraperitoneal ethanol administration at pre-existing pancreatic juice edema on frequency and severity of acute pancreatitis (AP). The rats were divided into six control (isolated treatment) and five study groups (edema plus varied ethanol application). 24 h postoperatively quality and quantity of macroscopic and histopathologic changes were graded on an arbitrary scale. While in the controls AP was not induced in any rat, in the study groups extrapancreatic fat necrosis and histologic lesions of the pancreas were found in the majority of animals. The individual rats revealed a remarkable variation of the severity of changes. A detailed differentiation of pancreatic necroses revealed a dominance of intrapancreatic fat necrosis and acinar necrosis at the periphery of lobules. The findings observed were widely independent of the way of ethanol administration. The present approach offers a simple and reproducible animal model to study the influence of acute ethanol application on development of AP and hints on the involvement of disturbances of the pancreatic microcirculation and of acinar cell metabolism in the pathogenesis of ethanol-induced acute pancreatitis.

Acute Disease↗

PubMed1991

Influence of aprotinin and promazine on survival of isolated pancreatic acinar cells.

Aprotinin, a protease inhibitor, and promazine, an inhibitor of phospholipase A2, were tested for possible inhibition of pancreatic acinar cell (PAC) decline induced by uncoupling of oxidative phosphorylation with 2,4-dinitrophenol (DNP) or by temporary anoxia/reoxygenation. In incubates of acinar cells isolated from rat pancreas the presence of aprotinin did not influence the survival of cells treated with these noxae. This finding excludes that extracellulary acting trypsin, possibly released from damaged cells, contributes to further cell death. While promazine at concentrations of 15 to 20 nmol.(10(6) cells)-1 was well tolerated by untreated PAC, higher concentrations caused a clear reduction of cell viability. At optimum concentration promazine was without influence on DNP-treated cells, but it had a beneficial effect on survival and morphology of anoxia-treated PAC (p less than or equal to 0.05). Therefore, it can be assumed that after anoxia/reoxygenation the membrane phospholipase A2 becomes stimulated and causes phospholipid depletion with final death of the cells. It is suggested that such a mechanism may contribute to the initial cell damage in the pathogenesis of acute pancreatitis, too.

Acute Disease↗

PubMed1990

Decreased survival rate of pancreatic acinar cells isolated from rats with acute pancreatitis.

Viable acinar cells were isolated from normal rat pancreas as well as from pancreata pretreated in situ by a short-term ischemia without and with reperfusion, induction of a pancreatic juice edema, and acute pancreatitis (AP), respectively. The isolated cells were incubated at 37 degrees C in an oxygenated Krebs-Henseleit bicarbonate buffer lacking any nutrient. As an analog to in vivo acinar cell necrosis, the decline of the isolated cells was followed up in vitro. During the first 5-6 h of incubation, the percentage of damaged cells increased only slightly. A second phase of about 30 min followed, during which nearly all residual cells died. The mean half-life (t50) of the cells in all experimental groups ran to about 330 min, with the exception of the AP group (t50 = 132 min). The importance of an intact energy metabolism to prevent premature cell killing was underlined indirectly by the diminished t50 (about 120 min in all groups) of the cells exposed to excess 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation. The results suggest that experimental AP induced by a combination of biliary-pancreatic duct obstruction, stimulation of pancreatic secretion, and short-term pancreatic ischemia with subsequent reperfusion finds a reflection within the acinar cells themselves and that these effects are not clouded by the isolation procedure. Thus, the application of acinar cells isolated from pancreatic glands pretreated in situ may offer a new tool for pathophysiological research into AP at the cellular level.

2,4-Dinitrophenol↗

PubMed1990

Differences in time course of hepatocyte and pancreocyte damage after incubation with trypsin, chymotrypsin and 2.4-dinitrophenol.

The survival of isolated pancreatic acinar cells and of hepatocytes from rats was compared to mimic cell injury and death during the pathogenesis of acute pancreatitis in the presence of varying concentrations of trypsin, chymotrypsin and of the uncoupler 2.4-dinitrophenol (DNP). While hepatocytes were quickly damaged by trypsin they still excluded trypan blue though an excess amount of DNP was added. Pancreocytes revealed a reversed susceptibility to both noxae. The reduced sensitivity of pancreocytes to extracellular trypsin action could represent a special protectiveness against autodigestion and their increased susceptibility to an alteration of the cellular energy state stresses the importance of this factor to the survival of pancreatic tissue under normal and noxious conditions.

Animals↗

PubMed1990

Lactate and ornithine improve substrate hydrogen supply in isolated hepatocytes.

The influence of lactate and ornithine on the mitochondrial redox state as indicated by the 3-hydroxybutyrate/acetoacetate ratio was studied in isolated hepatocytes. The load on the hepatic energy metabolism when lactate and/or ornithine are additionally present can be higher than in the sole presence of octanoic acid. This was revealed by enhanced oxidative turnover rates and higher 3-hydroxybutyrate/acetoacetate ratios at identical rates of respiration. During gluconeogenesis from lactate, such an improved energy supply takes place through more intense oxidation of octanoic acid in the citric-acid cycle. In addition to octanoate oxidation, however, ornithine supplies substrate hydrogen through its catabolism to glutamate as was demonstrated by comparative studies in isolated rat liver mitochondria. The ambivalent character of lactate and ornithine with regard to loading and supplying the energy metabolism seems to substantially account for energetic homoeostasis of the liver.

3-Hydroxybutyric Acid↗