Anoxic Cells Research Articles

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

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

The effect of extracellular calcium concentration and Ca-antagonist drugs on enzyme release and lactate production by anoxic heart cell cultures

Tissue cultures of neonate rat heart cells were subjected to anoxia for 16 h in the presence of 5 m m glucose and increasing concentrations of calcium in the incubation medium. When the calcium content of the culture medium was increased, release of intracellular enzyme was observed. Increased extracellular calcium concentration appeared to have no effect on normoxic tissue preparations. The time course of this anoxia-related change was examined. Increasing the calcium concentration of the medium caused an acceleration of the anoxia-induced rate of enzyme leakage when compared with anoxic cultures exposed to a low medium calicum concentration. The calcium-induced increase in anoxic enzyme release could be partially reversed by various concentrations of the calcium antagonist drugs verapamil and nifedipine. Verapamil and nifedipine both appear to reduce the extent to which an elevated medium calcium concentration is able to reduce myocyte ATP content under anoxic conditions. These drugs appear to be without effect on ATP content of anoxic cells exposed to a low calcium concentration on the medium. Calcium-induced increases in enzyme leakage cannot be modified by the provision of high medium glucose concentrations, and hypertonic solutions of mannitol exacerbate damage to myocytes under anoxic and hypercalcaemic conditions. These data suggest that high extracellular concentrations of calcium exacerbate damage to heart cells subjected to anoxia although the mechanism underlying this exacerbation is uncertain.

Protection of cultured renal tubular epithelial cells from anoxic cell swelling and cell death.

In order to study the relationship between cell swelling and cell death due to ischemia, we have developed an in vitro model by using primary cultures of renal tubular epithelial cells. With this model, we have studied two components of ischemia--namely, anoxia along with substrate deprivation. After 2 hr of anoxia in the absence of substrate, the cultured cells swelled and blebbed. Cells similarly treated in the presence of 8% polyethylene glycol, an oncotic agent, did not swell and bleb, and when cells were counted 18 hr later, similar numbers of cells were seen as in the untreated cultures. However, tubule cells exposed to anoxia without 8% polyethylene glycol had 50% fewer cells 18 hr later. Therefore, if cell swelling is prevented during 2 hr of anoxia, cell viability is improved.

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.

The electron affinity of some radiotherapeutic agents used in cancer therapy.

In order to evaluate whether chemotherapeutic compounds applied in cancer treatment might interact with radiation as anoxic cell sensitizers, the electron-affinic properties of DTIC, AIC, hydroxyurea, busulfan and cyclophosphamide were studied by pulse radiolysis. Reaction rates with hydrated electrons were determined for all these compounds. With the exception of DTIC, they all reacted much more slowly with electrons than do most electron-affinic sensitizers. One-electron reduction potentials were determined for DTIC, AIC and hydroxyurea. The values were all in the region for the onset of sensitization, with hydroxyurea as the most promising (E71 =- 0.0552V). For busulfan and cyclophosphamide no value could be determined, but these compounds are probably less electron-affinic than hydroxyurea. A possible application of chemotherapeutic agents as radiosensitizers is discussed.

The effect of hypoxic cell radiosensitizing drugs on cellular oxygen utilization

Various heterocyclic nitrocompounds ( nitroimidazoles, nitrofurans, nitropyrroles, nitrobenzenes) and thiol reactants (such as diamide and N- ethylmaleimide) that have been studied as anoxic cell radiosensitizers, alter cellular oxygen utilization. The effects are dependent upon the electron affinity and concentration of the drug, as well as on glucose and endogenous reducing substrates. Some of the drugs alter oxygen uptake by direct inhibition of electron transport, while others stimulate cellular oxygen utilization by forming oxygen-reactive nitro-intermediates. The production of these intermediates may be demonstrated with cyanide-inhibited cells or with purified microsomes. Ascorbate, in buffered solution, mimics cyanide-inhibited cells and purified microsomes by also producing oxygen-reactive nitro-intermediates. The importance of the drug effects on oxygen consumption is discussed with respect to radiation sensitization of multi-cellular systems in vitro and in vivo.

Release of alpha hydroxybutyrate from neonatal rat heart cell cultures exposed to anoxia and reoxygenation: comparison with impairment of structure and function of damaged cardiac cells.

Spontaneously beating monolayer cultures of neonatal rat heart cells first exposed to depletion of oxygen and metabolic substrates for 1 to 7 h (anoxia). Subsequently the cultures were resupplied with oxygen and substrates (reoxygenation). The release of alpha-hydroxybutyrate dehydrogenase (HBDH) from the cells, the extent of necrosis, and the changes in spontaneous contractile activity were measured. HBDH release was observed to start after 1 h of anoxia and to increase to 84% of intracellular HBDH activity after 7 h of anoxia. Reoxygenation of anoxic heart cells is associated with accelerated HBDH release (oxygen paradox). The activity of HBDH released by cardiac cells, the number of necrotic cells and the impairment of beating capacity of the cells depend on the duration of the anoxic period in a similar way. This study demonstrates that cardiac cell death can be assessed quantitatively and reliably by the measurement of the activity of HBDH released by the cells.

Hyperthermic Inactivation of Diploid Yeast and the Interaction of Damage Caused by Hyperthermia and Ionizing Radiation

Inactivation of diploid yeast by hyperthermia has been studied. DO and Dq decrease with temperature for euoxic and anoxic conditions. The Arrhenius plot shows a break at 52 degrees C; the inactivation energies above and below this temperature are 153 and 94kcal/mol respectively. Hyperthermia (20 min at 51 degrees C) also potentiates the lethal action of gamma rays in diploid yeast cells under both euoxic and anoxic conditions. The interaction between hyperthermic and radiation damage appears to be largely at the sublethal level. The euoxic cells, the hyperthermic potentiation decreases with increasing time between the application of hyperthermia and radiation, being completely lost after 24 hours. However, in the anoxic cells there was no decrease in the hyperthermic potentiation with increasing time interval. These results suggest that yeast cells are capable of repairing hyperthermic sublethal damage, but require oxygen to do so. Thus there is a similarity in the process of repair of sublethal damage caused by ionizing radiation on the one hand and hyperthermia on the other.

Alteration of hexose transport in avian erythrocytes by vinblastine and colchicine

Vinblastine and colchicine, compounds which effect the state of aggregation of microtubules, were investigated to determine if changes in the rate of sugar transport were produced by these compounds. Vinblastine accelerated 3- O-methylglucose entry into avian erythrocytes. At a concentration of 1.5 m m, transport was accelerated two-fold. The effect of vinblastine was not attributed to cell energy depletion or to increased entry by simple diffusion. Stimulation of transport did not require preincubation of the cells with vinblastine, and the effect was reversible. Colchicine (2 m m) inhibited 3- O-methylglucose entry in aerobic or anoxic intact red cells and in red cell ghosts. A change in the state of aggregation or activity of the microtubular system could represent a model for regulation of a membrane carrier. The present results would lend support to this model.

Enzyme release from anoxic rat heart cells in culture

Enzyme release from anoxic rat heart cells in culture

A relation between amino acid hydrophobicity and rate of uptake in Escherichia coli

Fumarate and ferricyanide increased the rate of amino acid uptake in anoxic cells of E. coli suspended in a glycerol medium. The stimulation correlated with the hydrophobicity of 16 amino acids transported by several carrier systems. Fumarate and ferricyanide increased membrane energization as measured by changes in transmembrane pH and electrical potential, or by quenching of fluorescence of 1-anilino-8-naphthalenesulfonate. The results suggest that a common, rate determining step among the amino acid transport systems is the transfer of the substrate from an aqueous to an apolar environment.

Survival curve of a human melanoma in nude mice.

Using cells from our tissue culture of human melanoma cell line Na 11, we transplanted 1 X 10)6) tumor cells sc into athymic nude mice. Tumors appeared after a latent period of 4-10 days; when they reached a mean volume of 100 mm3 we irradiated them with various doses of X-rays. Some tumors were irradiated while the mice were still alive; others were treated 10 minutes after the animals had been asphyxiated with nitrogen. All irradiation was done in the presence of oxygen. These tumors were excised, and cell suspensions were prepared; the cells formed colonies with a mean plating efficiency of 29%. In another series of experiments, we irradiated tumor cells in vitro 2 hours after excision, when most cells were fixed and presumably oxygenated. We then calculated survival curves for the tumor cells irradiated under these three conditions and found an average anoxic cell fraction of 85%, which was much higher than that reported in many other tumor systems. We explored several possible explanations for this phenomenon.

Affinity of intact Escherichia coli for hydrophobic membrane probes is a function of the physiological state of the cells.

The fluorescence parameters of several common membrane probes in the presence of whole E. coli have been examined. The probes included electrically neutral lipophilic molecules N-phenyl-1-naphthylamine, pyrene, and 1,6-diphenyl-1,3,5-hexatriene as well as the negatively charged molecule 8-anilino-1-naphthalene sulfonate. It is demonstrated in each case that certain fluorescence parameters are a function of the state of energization of the cells. All the probes appear to monitor structural changes in the E. coli envelope which accompany the energization and de-energization of the cells. tthe phenomenon is completely reversible as demonstrated by re-energizing anoxic cells by the addition of oxygen, or starved cells by the addition of substrate. All the results are qualitatively consistent with an increased binding of probe by de-energized cells and a subsequent expulsion of probe when the cells are re-energized. A pyrene substituted with a photosensitive group, 1-azidopyrene, has been synthesized. Photolysis in the presence of a suspension of energized E. coli reveals a relatively small amount of probe irreversibly bound to the cells. However, in the presence of cells that have been de-energized the amount of irreversibly bound probe is dramatically increased. This molecule should be useful for localizing the regions of the bacterial envelope that are involved in the structural changes being monitored in these experiments.

Cellular Response of a Rat Brain Tumour to a Therapeutic Neon Ion Beam

Clonogenic cell survival of in situ 9L brain tumors was measured in air-breathing rats and rats killed with nitrogen. Animals were irradiated with BEVALAC accelerated carbon ions in the plateau and spread-out Bragg peak regions of the specific ionization curve. The relative biological effectiveness (RBE) for carbon ions in the plateau was 1.2 ± 0.1 for oxic cells and 1.3 ± 0.1 for anoxic cells; in the Bragg peak the RBE was 1.6 ± 0.1 for oxic cells and 2.0 ± 0.1 for anoxic cells. The oxygen enhancement ratio (OER) in the plateau was 2.0 ± 0.1, not significantly different from the 2.1 ± 0.2 found for x rays. The OER in the Bragg peak was reduced to 1.7 ± 0.1. For cells having the same radiobiological sensitivity as in situ 9L cells, the neon ion beam may be therapeutically superior; however, this does not preclude selection of the carbon ion beam in many clinical situations.

The Effects of Nitrobenzene Derivatives on Oxygen Utilization and Radiation Response of an in Vitro Tumor Model

The effects of several nitrobenzene derivatives on the oxygen utilization and radiation response of multicell spheroids have been determined. In this system, the best radiosensitizer was nitrotoluene, previously reported to be a poor sensitizer of anoxic cells in monolayer cultures. The sensitization of the spheroid is interpreted as due to nitrotoluene's immediate inhibitory effect on cellular oxygen utilization resulting in a reoxygenation of the centrally located cells. The anoxic radiosensitizre dinitrobenzonitrile markedly stimulated oxygen utilization producing an immediate protection, presumably because the hypoxic fraction of the spheroid was increased. Some radiosensitization did occur with dinitrobenzonitrile at higher concentrations and after longer periods of exposure to the drug. The altered radiation response, caused by either nitrotoluene or dinitrobenzonitrile, agreed fairly well with the theoretical prediction of the radiation response as a function of oxygen utilization. A third anoxic s...

Biomedical use and dosimetry of pions of the sin 590 MeV isochrone cyclotrone. VII. Radiation therapy using negative pions (author's transl)

The scheme of radiation of Schumacher for high energy electrons is investigated. In it 3 high individual doses are given at first. In the second phase which follows immediately, weekly single doses of 500 rad are given until the tumor is completely destroyed. Reoxygenation and transformation of anoxic cells are important here for the number of surviving tumor cells but unfortunately still rather unknown. This uncertainty can be removed with the Pion therapy in which transformation does not take a large part. About 10 single doses of negative pions of 150-200 rad should be clearly superior to 5000 rad electron doses (D1 = 500 rad) and should not exceed the limits of tolerance with a weekly treatment. Finally the importance of contamination with electrons of the pionrays is examined. Exact measurement of this contamination is necessary.

The Postanoxic Regeneration of 5-Adenine Nucleotides in Rabbit Kidney Tissue during in Vitro Perfusion

The adenylate metabolism in rabbit kidney tissue was investigated, by isolated normothermic bloodless perfusion, during anoxic cell deterioration and postanoxic recovery in vitro. The amounts of hypoxanthine washed out during continuous anaerobic perfusion were shown to represent a definite loss of intracellular purine. No salvage of the endogenic liberated oxypurine was found to occur during postanoxic reoxygenation. The ability to restore the content of 5'-adenosine nucleotides failed after 60-120 min of anaerobic perfusion. Adenosine, adenine, inosine, hypoxanthine, and D-ribose added to the perfusate during reoxygenation were investigated with regard to their ability to stimulate postanoxic adenylate regeneration. Adenosine alone added in a concentration of more than 1 mmol/l showed a significant stimulation of the adenylate synthesis, with complete regeneration of the adenylate content after 180 min of preceding anoxia. In can be concluded that anoxia kidney deterioration is associated with a progressive loss of intracellular oxypurine, and a decreasing ability to regenerate adenylates after reoxygenation, which could be prevented by exogenic addition of adenosine. It appears reasonable to assume that the results obtained reflect the adenylate metabolism of a kidney graft during the postischaemic phase immediately after implantation.

Isolated rat hepatocytes as an experimental tool in the study of cell injury. Effect of anoxia

Rat hepatocytes were isolated by liver perfusion in the presence of collagenase and hyaluronidase and incubated in the absence or presence of oxygen. As a result of anoxia, there was a gradual increase in plasma membrane permeability, noted as an increase in succinate-stimulated oxygen uptake, a decrease in trypan blue exclusion frequency, a leakage of cytosolic lactate dehydrogenase activity and an increased proportion of swollen and disrupted cells. After anaerobic incubation for 30 minutes — but not for 60 minutes — there were signs of recovery from anoxic cell injury upon re-oxygenation. The changes in plasma membrane permeability properties in anoxia seemed to be preceded by a marked decrease in cellular ATP level; aerobic incubation of hepatocytes in the presence of an uncoupler of phosphorylation from respiration led to a similar decrease in cellular ATP concentration followed by similar disturbances in plasma membrane permeability properties. It is suggested that a disturbed plasma membrane function caused by a decreased energy level is of primary importance for the initiation of cell death in anoxia.

Ultrastructural and Physiological Changes in Piglet Oxyntic Cells During Histamine Stimulation and Metabolic Inhibition

Neonatal pig gastric mucosa was studied in order to correlate electrophysiological and secretory parameters with ultrastructural changes in membrane components of oxyntic cells. The non-stimulated tissue had a transmucosal resistance of about 130omega - cm2 while the oxyntic cells were characterized by numerous cytoplasmic tubulovesicles and short microvilli extending into patent glandular and canalicular lumina. Upon histamine-stimulation, the average rate of H+ secretion was 8.1 mueq - cm2 - hr-1 and the resistance decreased to 77omega - cm2. The changes were coupled with an immense elaboration of the oxyntic cell apical and canalicular surfaces with a concomitant decrease of tubulovesicles. Thus, the observed decrease in resistance was correlated to large increases in secretory membrane area. Anoxia inhibited H+ secretion while resistance increased to 211omega - cm2. Anoxic oxyntic cells were characterized by swollen mitochondria and occlusion of the lateral intercellular space and basal infoldings. Little change in the configuration of the secretory surfaces was noted, thereby suggesting that restriction of lateral and basal membranes might be responsible for the observed resistance increase. An electrical analogue of gastric mucosa is proposed on the basis of these morphological observations.

The potential of localized heating as a adjunct to radiation therapy.

Experimental studies have shown that (a) tumor cells may be more sensitive to heat than normal cells; (b) hyperthermia inactivates cellular repair mechanisms for radiation damage; and (c) heat may lower the OER for ionizing radiation (anoxic cells are at least as sensitive to hyperthermia as oxygenated cells). Localized hyperthemia produced by localized current fields in the range of 100 kHz-10 MHz by direct contact electrodes offers two major advantages: the eletrode configurations may be manipulated to obtain desired thermal dose distributions, and, since the mode of heating is essentially instantaneous, accurate temperature control can be maintained during treatment.