Release Of Cellular Enzymes Research Articles

Albumin-derived perfluorocarbon-based artificial oxygen carriers: A physico-chemical characterization and first in vivo evaluation of biocompatibility

Until today, artificial oxygen carriers have not been reached satisfactory quality for routine clinical treatments. To bridge this gap, we designed albumin-derived perfluorocarbon-based nanoparticles as novel artificial oxygen carriers and evaluated their physico-chemical and pharmacological performance.Our albumin-derived perfluorocarbon-based nanoparticles (capsules), composed of an albumin shell and a perfluorodecalin core, were synthesized using ultrasonics. Their subsequent analysis by physico-chemical methods such as scanning electron-, laser scanning- and dark field microscopy as well as dynamic light scattering revealed spherically-shaped, nano-sized particles, that were colloidally stable when dispersed in 5% human serum albumin solution. Furthermore, they provided a remarkable maximum oxygen capacity, determined with a respirometer, reflecting a higher oxygen transport capacity than the competitor Perftoran®. Intravenous administration to healthy rats was well tolerated. Undesirable effects on either mean arterial blood pressure, hepatic microcirculation (determined by in vivo microscopy) or any deposit of capsules in organs, except the spleen, were not observed. Some minor, dose-dependent effects on tissue damage (release of cellular enzymes, alterations of spleen’s micro-architecture) were detected.As our promising albumin-derived perfluorocarbon-based nanoparticles fulfilled decisive physico-chemical demands of an artificial oxygen carrier while lacking severe side-effects after in vivo administration they should be advanced to functionally focused in vivo testing conditions.

The role of calcium and nitric oxide during liver enzyme release induced by increased physical forces as evidenced in partially hepatectomized rats

Although increased plasma enzyme activities could be diagnostic for tissue damage, the mechanisms controlling cellular enzyme release remain poorly understood. We found a selective and drastic elevation of serum enzyme activities accompanying rat liver regeneration after partial hepatectomy (PH), apparently controlled by a mechanism dependent on flow-bearing physical forces. In fact, this study assesses a putative role of calcium mobilization and nitric oxide (NO) production underlying rat liver enzyme release. The role of increased shear stress (by enhancing viscosity during perfusion) and the participation of cell calcium and NO were tested in isolated livers subjected to increasing flow rate. After PH, there was a drastic elevation of serum activities for liver enzyme markers, clearly predominating those of mitochondrial localization. Liver enzyme release largely depended on extracellular calcium entry, probably mediated by stretch-sensitive calcium channels, as well as by increasing NO production. However, these effects were differentially observed when comparing liver enzymes from cytoplasmic or mitochondrial compartments. Moreover, a possible role for cell-mediated mechanotransduction in liver enzyme release was suggested by increasing shear stress (high viscosity), which also selectively affected the release of the enzymes tested. Therefore, we show, for the first time, that flow-induced shear stress can control the amount of hepatic enzymes released into the bloodstream, which is largely regulated through modifications in cell calcium mobilization and production of liver NO, events markedly elevated in the proliferating rat liver.

The improved everted gut sac: a simple method to study intestinal P-glycoprotein

P-glycoprotein (P-gp) is an energy-dependent transporter protein located in the apical membrane of intestinal mucosal cells. It is believed that it may limit the bioavailability of many orally administered drugs, by transporting them back into the intestinal lumen following their absorption by the enterocytes. To demonstrate the activity of the intestinal P-gp in vitro, we have used the `improved' rat everted gut sac system, where good tissue viability and metabolic activity is maintained by incubating the everted sacs in tissue culture medium. Digoxin was used as the test drug. After 90 min incubation, its transport across the intestinal mucosa was enhanced 2.5-fold by the P-gp inhibitor verapamil, and 5-fold by the inhibitor quinidine. In the presence of the three drugs, the sacs showed uniform kinetics and good viability, as assessed by the active transport of glucose and lack of release of cellular enzymes. The improved everted gut sac system has potential as a simple and efficient tool to evaluate the role of P-gp in the intestinal absorption of drugs, and to screen for putative P-gp inhibitors that may improve the bioavailability of drugs susceptible to transport by P-gp.

Improved viability and metabolic behavior of hepatocytes after liver storage in the presence of a succinic acid ester.

Selected esters of succinic acid were recently proposed as novel nutrients to support ATP generation in cells endangered by an imbalance between the formation and breakdown of this adenine nucleotide. In the present study, a new ester, glycerol-1,2,3-trimethylsuccinate, was examined for its potential beneficial effect in the procedures preceding liver transplantation. The viability and metabolic behavior of hepatocytes were examined after perfusion and storage of rat livers for 20 hr at 4 degrees C with a Belzer UW-CSS solution in the absence or presence or 2 mM glycerol-1,2,3-trimethylsuccinate. Although it failed to affect significantly the release of cellular enzymes during storage and the protein or glycogen content of the liver, and was unable to prevent the storage-induced decrease in both biosynthetic activity and D-[U-14C]glucose incorporation into glycogen in isolated hepatocytes, the ester restored to a close-to-normal value the viability of the hepatocytes and opposed the starvation-like effects of liver storage upon both the conversion of D-[U-14C]glucose to 14CO2 and radioactive amino acids and the de novo generation of 14C-labeled D-glucose from [2-14C]pyruvate. Because succinic acid esters are efficiently metabolized in several cell types, the present results suggest that such esters may have a wide field of application in transplantation procedures.

Cytotoxicity of radiocontrast agents on polarized renal epithelial cell monolayers

Radiocontrast-induced nephropathy is a clinically important complication of coronary angiography. The cellular mechanisms of radiocontrast-induced renal dysfunction are not clear. Since tubular transport functions depend on the polarity of renal epithelial cells, we investigated the effects of radiocontrast agents on polarized tubular cells in vitro. We studied the effects of iso-iodine concentrations (37 and 74 mg iodine/ml) of an ionic (diatrizoate) and a non-ionic (iopamidol) monomeric radiocontrast agent and of hyperosmolal mannitol control solutions on filter-grown renal epithelial cell (MDCK, LLCPK) monolayers in vitro. The cytotoxicity was assayed by measurement of cell viability, transepithelial resistance, inulin permeability and (polarized) cellular enzyme release. The polarized MDCK cell phenotype was assessed by transmission electron microscopy and indirect immunofluorescence microscopy using monoclonal antibodies against specific apical (gp135) and basal (gp60, uvomorulin) MDCK surface markers. The radiocontrast agents reduced cell viability to a greater extent than hyperosmolal mannitol solutions in both cell lines; diatrizoate was more toxic than iopamidol. LLCPK cells were more susceptible to radiocontrast cytotoxicity than MDCK cells. This cytotoxicity was associated with an alteration of MDCK cell polarity as assessed by the redistribution of surface marker proteins. Diatrizoate is more toxic than iopamidol, which is partly related to its higher osmolality. The cytotoxicity of radiocontrast agents induces a redistribution of polarized membrane proteins which could contribute to the pathophysiology of radiocontrast-induced nephropathy.

Time course of cellular enzyme release in dog heart injury.

The transport time of enzyme from heart to plasma was studied in two experimental models. First, the enzyme alanine aminotransferase was slowly infused into the left ventricular wall in open-chest dogs. The half-life for the washout of alanine aminotransferase activity into plasma was 20 +/- 4 minutes (mean +/- SEM, n = 8) and was not different in ischemic and normally perfused tissue. From measurements of arteriovenous differences in alanine aminotransferase activity and left ventricular blood flow, it was concluded that 77 +/- 14% of total enzyme washout from ischemic tissue occurred by direct entry into the bloodstream. The corresponding value for the vascular permeability-surface area product was 264 +/- 55 ml.kg-1.hr-1. For a second model, we studied myocardial enzyme release into plasma after abrupt heart injury induced by 10 minutes of calcium-free coronary perfusion followed by reintroduction of calcium (calcium-paradox mechanism). The half-life for the release into plasma was 1.9 +/- 0.2 hours (mean +/- SEM, n = 6) and was again not influenced by sustained ischemia. Slower washout, as observed for this second model, is consistent with increased interstitial protein space and corresponds to a permeability--surface area product between 135 and 285 ml.kg-1.hr-1. These results were used to calculate the time course of cellular enzyme leakage from the rate of enzyme release into plasma in various forms of heart injury. Significant shifts between the time curves of evolving cellular injury and enzyme release into plasma are observed after 2 hours of ischemia followed by coronary reperfusion, but not after permanent ischemia.

Chromatin structure, DNA synthesis and transcription through the lifespan of human embryonic lung fibroblasts

The presence in terminal embryonic fibroblasts of small molecular weight (MW) DNA independent of bulk DNA could be ascertained by three different techniques performed in parallel. This alteration was not artifactually induced, either by high pH and the detergent used or by the release of cellular enzymes. An increased thermolability of old chromatin was also observed. Cells with altered chromatin synthesized DNA and RNA according to a pattern similar to young type nuclei. Long-term treatment with hydrocortisone significantly increased the cell yield but did not prevent, in the late passages, the occurrence of old-type chromatin; the nucleolar filamentous masses, however, maintained a ‘young’ pattern. Short-term treatment induced only a moderate reversion in the appearance of chromatin lesions. Direct evidence was obtained of increased gene expression in the presence of hydrocortisone.

PREDICTION OF PNEUMOCONIOSIS RISK BY BIOASSAYS OF PARTICULATE FROM OCCUPATIONAL EXPOSURES

A set of short-term animal bioassays was evaluated for its ability to predict occupational risk of pneumoconiosis. The method uses dust samples from granite and talc industries that have been previously studied epidemiologically. A sampler has been developed to collect respirable dust, comparable to that obtained from personal samplers worn by exposed workers, in quantities suitable for bioassays (> 100 mg). The collected dust was mechanically removed from the membrane filter and ultrasonically resuspended in normal saline for intratracheal instillation in hamsters. The dust size distribution was determined by scanning electron microscopy. Addition of lung surfactant to the saline solution apparently changed the size distribution. X-ray diffraction was used to characterize and quantitate crystalline silica. Standardized respirable dusts (aluminium oxide and quartz) were also used. Pulmonary bioassays in hamsters were used to estimate the inflammatory response, oedema, release of cellular enzymes and alteration of in situ phagocytosis after exposure to instilled dusts. The inflammatory response was estimated by measuring changes in the number and type of cells present in multiple lung lavages. Oedema was quantitated by measurements of serum albumin in the lung lavage fluid. Alterations in in situ phagocytosis were assayed by the ‘lambda’ technique, which estimates the fraction of instilled radioactive gold colloid ingested by pulmonary phagocytic cells. The enzymes measured included lactate dehydrogenase, β- N -acetylglucosaminidase, peroxidase and elastase.

Characterization of the human complement (c3b) receptor with a fluid phase C3b dimer.

The interaction of C3b receptor with C3b, the major cleavage product of C3, elicits important biologic functions, such as enhanced phagocytosis and release of cellular enzymes. We determined the binding kinetics and binding isotherm of C3b-receptor interaction by using human cells and fluid phase C3b generated by trypsin cleavage of purified native C3. 125I labeled C3b was separated into 2 molecular species, a dimer and a monomer by column chromatography. We found that dimeric C3b bound to human erythrocyte C3b receptors with an affinity that was more than 25 times that of the monomer. 125I dimeric C3b did not bind to sheep red blood cells, which lack the C3b receptor, nor to trypsinized or 2-mercaptoethanol treated normal human red blood cells, 2 methods for abrogating the immune adherence activity. Binding of 125I fluid phase C3b dimer to the C3b receptor was specific, saturable (about 90 ng of C3b dimer bound maximally per 1 X 10(9) red blood cells), reversible (in the presence of a 100-fold molar excess of unlabeled ligand), and of moderate affinity (Kd about 9.53 nM). The equilibrium binding constants were similar with the various cells tested. Binding was characterized by rapid on and off rates and did not exhibit ligand cooperativity. This specific interaction reached a steady state within 10 to 15 min at 0 degrees C; 50% of specifically bound ligand dissociated from its binding site on human red blood cells in approximately 1 min at 0 degrees C. The density of C3b receptors on human red blood cells, polymorphonuclear leukocytes, monocytes, and B lymphocyte-enriched preparations was 360, 20,000, 30,000, and 21,000 receptors/cell, respectively.

The effects of incubation conditions on enzyme release from anoxic heart cell cultures

Cultured myocytes from neonate rats were subjected to varying concentrations of glucose and serum under normoxic and anoxic conditions. Cellular metabolism was measured in terms of glucose utilization and lactate production. Leakage of cytoplasmic lactate dehydrogenase (LDH) into the medium was measured to give an indication of cell injury. The effect of variations in temperature on these parameters was evaluated. Myocytes cultured under anoxic conditions for 16 h utilized more glucose than normoxic cells with a concomitant increase in lactate production. When the initial glucose concentration in the medium was below 3 m m, anoxic myocytes released large amounts of LDH. Also, at low initial glucose concentrations there was a surge of anoxic LDH release between 12 and 15 h of incubation. The addition of exogeneous lactate to anoxic cultures had no influence on enzyme leakage. However, reduced enzyme leakage was observed when medium lactate was increased under normoxic conditions. An increased concentration of serum in the medium stimulated glucose metabolism in both normoxic and anoxic myocytes. Despite this enhancement of glycolysis, anoxic cells released significant amounts of LDH into the medium. LDH release by anoxic myocytes is minimal in medium containing 1 to 2% v/v serum. Maximum glucose metabolism and lactate production at 5 m m glucose occurred between 37 to 40°C. The relation between LDH release and temperature was not marked. Significant release of LDH did occur in anoxic cultures incubated between 37°C and 40°C when compared with normoxic cultures at the same temperature. At 4°C, in the absence of glucose metabolism, LDH release occurred from both normoxic and anoxic cells. These data demonstrate that significant cellular enzyme release occurs during anoxia: (a) below a threshold glucose concentration and (b) in proportion to the serum content of the medium.

Biological effects of chrysotile after SO 2 sorption : II. Effects on alveolar macrophages and red blood cells

An experimental study has been carried out using different biological and biochemical in vivo and in vitro tests for assessing the toxicity of natural UICC (A) chrysotile and SO 2-sorbed UICC (A) chrysotile and for detecting a possible synergistic effect. For in vivo studies, rabbits received an intratracheal injection of chrysotile fibers suspended in physiological saline (PS). The control group received only PS; all animals were sacrificed after 68 hr. The alveolar free cells were harvested by pulmonary lavage. The results have shown that chrysotile induces a decrease in the free cell population but there was no significant difference in the number of viable cells or in the nature of cells harvested between the two chrysotile groups. Enzymatic activities of the alveolar macrophages from animals injected with SO 2-sorbed chrysotile showed a significant increase of the enzymes LDH and acid phosphatases. The LDH increase could be related to the affinity of the enzyme regarding some chemical forms of SO 2. In vitro studies using alveolar macrophages harvested by pulmonary lavage have shown no differences between the two chrysotile groups when cell viability and enzyme release were studied. The toxic effect was due to chrysotile fibers (decrease of 38% in cell viability and increase in cellular enzyme release when compared with control). When rabbit red blood cells were used, both natural and SO 2 chrysotile fibers showed the same hemolytic activity. The failure to detect high differences between the two chrysotile groups may be related to the chemical form of sorbed SO 2.

Mechanisms of cellular enzyme release. II. Inhibition of sarcolemmal enzymes by myopathy-inducing agents.

A defective membrane mechanism has been suggested [Arch. Neurol. 33, 315 (1976)] for the pathogenesis of Duchenne muscular dystrophy. The characteristic clinical and biological findings, including leakage of cellular enzymes into the serum in the disease, have been duplicated by the imipramine/serotonin rat myopathy model. Sarcolemma was prepared from quadriceps femoris muscles of control and myopathy-affected animals. The activities of sarcolemmal adenosinetriphosphatase and acetylcholinesterase were inhibited in vitro by imipramine and serotonin. The inhibition by imipramine of these sarcolemma-bound enzyme systems decreased the Vmax and increased the Km. This mixed type of inhibition is consistent with an imipramine-induced interference at these enzyme sites and a disruption of lipid-protein interrelations. We hypothesize that such conformational membrane changes might contribute to the leakage of macromolecules such as enzymes from the cell interior.

Mechanisms of cellular enzyme release. I. Alteration in membrane fluidity and permeability.

Above-normal plasma enzyme activities resulting from increased release of intracellular macromolecules are an important diagnostic feature of Duchenne muscular dystrophy. These and other biochemical and histological characteristics of this disease are faithfully duplicated when imipramine and serotonin are administered to the rat. Imipramine, but not serotonin alone, causes release of enzymes from rat diaphragms and human lymphocytes and release of hemoglobin from erythrocytes in vitro. Ouabain causes neither. Imipramine-induced enzyme release is decreased by adding ATP to the specimen in vitro or in hypertonic solution. Imipramine inhibits the capping phenomenon (an aggregation of antigen/antibody complexes of the membrane) of human B lymphocytes labeled with fluorescein-conjugated antihuman immunoglobulins. Serotonin alone has no such effect, but, administered together with imipramine, it potentiates the inhibition of capping by imipramine.

Cell killing by simian virus 40: variation in the pattern of lysosomal enzyme release, cellular enzyme release, and cell death during productive infection of normal and simian virus 40-transformed simian cell lines

Simian virus 40 (SV40) growth on rhesus kidney cells and on the T-22 line of SV40-transformed green monkey kidney (GMK) cells is largely limited by the low plating efficiency of SV40 on these cells. In addition, a fraction of the rhesus kidney and T-22 cells are resistant to infection by SV40. Nevertheless, 72-h viral yields per infected rhesus kidney and T-22 cell are nearly equivalent to that obtained on normal GMK cells and are independent of the multiplicity of infection. Despite the production of high viral yields, infected rhesus kidney and T-22 cells are killed slowly by SV40. Monolayers of these cells are also refractory to plaque formation by SV40. SV40 induces the release of lysosomal N-acetyl-beta-glucosaminidase into the cytoplasmic fractions of rhesus kidney and T-22 cells to an extent equal to that observed during infection of rapidly killed normal GMK cells. In contrast, damage to the plasma membrane, as indicated by the release of the cellular enzymes lactic dehydrogenase and glutamic oxaloacetic transaminase into the overlay media, occurred to a much greater extent in the normal GMK cells than in the rhesus kidney or T-22 cells. Neither a lysosomal hydrolase mechanism nor viral release appear to be responsible for this phenomenon. The different rates and extents of the SV40 cytocidal process on these cells do not result from the differences in the viral plating efficiency on them.

Release of enzymes from the liver.

DIFFERENT lesions of an organ lead to distinct enzyme patterns in the serum. Our knowledge about the mechanisms affecting the release of cellular enzymes from damaged tissues, however, is poor.