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Biomedical subjects

C E Ganote

Publications and source records attributed to C E Ganote.

At least 19 recordsLinked to original sources
PubMed1992

Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on metabolically inhibited and ischaemic isolated myocytes.

Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 microM okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nM and a nearly full effect during metabolic inhibition at 1 microM concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

Adenosine Triphosphate↗
PubMed1992

Flow cytometric analysis of isolated adult cardiomyocytes: vinculin and tubulin fluorescence during metabolic inhibition and ischemia.

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

Animals↗
PubMed1991

Effects of 2,3-butanedione monoxime (BDM) on contracture and injury of isolated rat myocytes following metabolic inhibition and ischemia.

The relationship between myocardial cell contracture and injury during total metabolic inhibition (amylobarbital and iodoacetic acid) and ischemia was examined, using 5-50 mM butanedione monoxime (BDM) as an inhibitor of contracture. BDM had no apparent effect on control myocytes during 180 min incubations, but inhibited contracture following anoxia or ischemia in a dose-dependent fashion, as directly quantitated by length/width ratios. Cellular ATP levels decreased at a similar rate in the absence or presence of BDM, following metabolic inhibition. BDM-mediated inhibition of contracture was associated with accelerated cell injury, as defined by: the uptake of an extracellular marker (trypan blue) by the cardiomyocytes, by direct analysis of myoglobin released into the supernatant and by ultrastructural demonstration of defects in sarcolemmal membrane integrity. Calcium was not required for BDM's enhancement of injury, in that cells incubated in calcium free-EGTA buffer showed a similar BDM-mediated acceleration of injury. In the presence or absence of calcium, enhancement of injury was more marked in cells osmotically stressed with a brief incubation in hypotonic buffer, than in cells resuspended in isotonic media. It is concluded that BDM enhances development of osmotic fragility of inhibited or ischemic cardiomyocytes and that contracture is not a necessary contributing factor to myocardial cell death.

Adenosine Triphosphate↗
PubMed1991

Effects of the phospholipase inhibitor mepacrine on injury in ischemic and metabolically inhibited adult isolated myocytes.

The phospholipase inhibitor mepacrine has been shown to delay cell death of metabolically inhibited cultured cardiomyocytes. The present study was initiated to determine if mepacrine also delays cell death and development of osmotic fragility of both metabolically inhibited and ischemic adult rat cardiomyocytes. Isolated myocyte suspensions were incubated with 3 mmol/l (millimolar) iodoacetic acid and 6 mmol/l amytal (inhibited) or were pelleted into a slurry and layered with oil (ischemic) in the presence and absence of 10 or 50 mumol/l (micromolar) mepacrine. Rates of contracture, cell viability as determined by trypan blue permeability, cell viability after osmotic swelling in 170 mOsm media (osmotic fragility), and cell morphology were monitored. Mepacrine had no effects on rates of contracture, but was found to significantly delay cell death during isotonic incubations of both metabolically inhibited and ischemic cells. In contrast, mepacrine had no effect on the development of osmotic fragility. Incubation of metabolically inhibited myocytes in calcium-free media did not delay contracture or cell injury, but did attenuate the protective effects of mepacrine. This study confirms previous reports that mepacrine protects cells from injury, extends the observations of protection to ischemic isolated adult myocytes, but shows that development of osmotic fragility is not inhibited by mepacrine.

Animals↗
PubMed1978

Pathologic changes induced by an euthanasia agent.

Dogs and cats killed by intravenous injection of either 0.3 ml/kg body weight T-61 or 100 mg/kg body weight pentoarbital and necropsied at less than 5 minutes or at 15 minutes after injection did not have gross or microscopic pathological changes. However, dogs and cats killed with T-61 at a dose of 1.0--1.5 ml/kg body weight and necropsied at 15 minutes after injection had significant gross and microscopic pathological lesions. Grossly, the lungs were severely edematous, did not collapse, and were deep red. Microscopically, the lungs had severe pulmonary edema and endothelial necrosis. Endothelial swelling of glomerular tuft vessels was also present. These lung and kidney lesions are classified as an euthanasia artefact.

Amides↗
PubMed1977

Cellular swelling and irreversible myocardial injury. Effects of polyethylene glycol and mannitol in perfused rat hearts.

Irreversible injury was produced in Langendorf-perfused rat hearts by 60 minutes of hypoxic, substrate-free perfusion at 37 C. Upon reoxygenation, hearts suddenly released large amounts of creatine phosphokinase (CPK) and over 60% of cells contained contraction bands and appeared irreversibly injured by light and electron microscopic criteria. Ten percent polyethylene glycol (PEG) or mannitol (420 mOsmol/liter) prevented or reduced swelling of rat heart slices incubated in vitro in the cold or under anoxic conditions. Both PEG and mannitol inhibited oxygen-induced CPK release after 60 minutes of hypoxia. Cells from protected hearts contained contraction bands but remained structurally intact. The results of this study provide evidence that cell swelling may play an important role in the pathogenesis of oxygen-induced enzyme release and irreversible myocardial cell injury.

Animals↗
PubMed1976

Mitochondrial structure and function in acute myocardial ischemic injury.

Changes in both the structure and function of mitochondria occur in the dog heart as a consequence of severe ischemia produced by acute coronary occlusion. Brief periods of severe ischemia (reversible injury) produced no significant change in mitochondrial ultrastructure and no defects in pyruvate or succinate metabolism. However, periods of ischemia of 40-60 minutes' duration (irreversible injury) produced striking structural changes including swelling, an increase in matrix space, disorganization of cristae, and the appearance of amorphous matrix densities. After 60 minutes of severe ischemia, one or more amorphous densities were present in each mitochondrial profile. These osmiophilic structures contained lipid but have not been characterized further. Their presence was typical of the irreversible state. Mitochondria of irreversibly injured cells were fragile, and consequently were more difficult to isolate than mitochondria of control tissue. Furthermore, after isolation from tissue injured by 60 minutes of ischemia, they showed markedly defective function.

Animals↗
PubMed1976

Oxygen-induced enzyme release after irreversible myocardial injury. Effects of cyanide in perfused rat hearts.

The effects of 5 mM potassium cyanide (KCN) on creatine phosphokinase (CPK) release and cellular morphology were studied. Rat hearts were perfused with substrate-deficient media gassed with O2 or N2 (O2 medium, N2 medium) at 37 C, and effluent was collected for creatine phosphokinase analysis. Tissue fixation was with glutaraldehyde for light and electron microscopy. Experiments included the following: a) continuous perfusion with O2- or N2-medium in the presence of KCN; b) 45 or 60 minutes of perfusion with N2-medium followed by O2-medium for 15 or 180 minutes, respectively; c) 45 minutes of perfusion with N2-medium with KCN added 15 minutes before reoxygenation with O2-medium plus KCN; (4) 60 minutes of N2-medium plus KCN followed by O2-medium plus KCN for 180 minutes; d) as a control for irreversible injury, 21 minutes of perfusion with calcium-free O2-medium followed by 2.5 mM calcium-O2-medium ("calcium paradox"). The following results were seen: a) Initial CPK release occurred about 30 minutes later from hearts perfused with O2-medium plus KCN than from hearts perfused with N2-medium plus KCN. b) Upon reoxygenation after either 45 or 60 minutes of anoxia, hearts had a sudden peak of oxygen-induced CPK release. Most irreversibly injured cells were massively swollen and had sarcolemmal defects and contraction bands. Reversibly injured cells in the same hearts resembled normal myocardium. A previously unrecognized third population of cells is described. These cells were characterized by contraction bands but were not swollen, had intact sarcolemma, and contained both normal and damaged mitochondria with intramatrical calcium accumulation granules. It could not be determined if these cells were reversibly injured or in an early stage of irreversible injury. c) KCN added 15 minutes before reoxygenation of hearts after 45 minutes of anoxia inhibited the sudden peak of oxygen-induced CPK release but not a slow sustained release. Small to moderate numbers of cells in these hearts contained contraction bands. d) After 60 minutes, KCN completely inhibited both oxygen-induced CPK release and contraction band formation. e) Addition of calcium to calcium-free hearts caused both massive CPK release and contraction band formation. It is concluded that: the beginning of CPK release from oxygenated KCN-inhibited hearts requires about 30 minutes longer than from anoxic hearts; KCN can inhibit both oxygen-induced CPK release and contraction bands in irreversibly injured rat myocardial cells; sudden contracture of myocardial cells as occurs in the calcium paradox can result in massive CPK release; contraction bands occur in nonswollen cells, hence contraction bands can occur independently of massive cell swelling or membrane rupture. It is postulated that there may be two stages of irreversible myocardial injury; a) loss of control of contraction and b) progressive loss of mitochondrial and membrane integrity.

Animals↗
PubMed1975

Distribution of coronary arterial flow in acute myocardial ischemia.

Thioflavin S (TS), a fluorescent dye, was used to visualize the distribution of coronary flow within the area of ischemia produced by circumflex artery occlusions. In the ischemic region, TS failed to penetrate the subendocardium and was seen in the subepicardium. Even though collateral flow was noted in the subepicardium, studies with methylene blue showed that it was inadequate to prevent the development of ischemia. The proportion of the posterior papillary muscle and subjacent myocardium showing TS nonfluorescence was similar after 15 and 60 minutes of ischemia and correlated with maximum lead II ST segment elevation and the percent of grossly injured myocardium found at 60 minutes postocclusion. The results suggest that flow to ischemic myocardium is reduced to the greatest extent in the subendocardium, ie, the site where irreversible injury first appears.

Acute Disease↗