Mitochondrial Calcium Accumulation Research Articles

Cardioprotection by nisoldipine: role of timing of administration.

Nisoldipine was administered at 10(-9) M, a dose lacking negative inotropism, to isolated and perfused rabbit hearts submitted to 60 min ischaemia (1 ml.min-1) followed by 30 min reperfusion. The drug was delivered either 30 min before ischaemia, at the onset and after 30 min of ischaemia and during reperfusion only. Cardiac protection was evaluated in terms of recovery of left ventricular pressure during reperfusion, release of creatine phosphokinase (CPK), mitochondrial function, tissue content of adenosine triphosphate (ATP) and creatine phosphate (CP), calcium homeostasis and the occurrence of oxidative stress, established measuring content and release of reduced and oxidized glutathione. The cytoprotective action of nisoldipine occurs in the absence of negative inotropism and is closely related to the time of administration. Optimal myocardial preservation is achieved when nisoldipine is given before or at the onset of ischaemia. Prophylactic administration of nisoldipine improved the recovery of the developed pressure from 15.9 +/- 1.0 (SE) mmHg to 47.8 +/- 1.9 mmHg, P < 0.01 and reduced the release of CPK from 830 +/- 29 to 229 +/- 27 mU.min-1 g-1 wet wt, P < 0.01. The accumulation of tissue and mitochondrial calcium was reduced from 58 +/- 11 and 49 +/- 9 to 14 +/- 6 and 10 +/- 4 mmol.kg-1 dry wt respectively, P < 0.01. This resulted in a significant (P < 0.01) preservation of all indices of mitochondrial function, allowing a higher recovery of ATP and CP after reperfusion (from 4.1 +/- 0.7 and 10.0 +/- 0.6 to 16.1 +/- 1.0 and 29.9 +/- 0.2 mumol.g-1 dry wt respectively, P < 0.001). Reperfusion-induced myocardial accumulation and release of oxidized glutathione were reduced from 0.493 +/- 0.07 nmol.mg-1 protein and 0.768 +/- 0.063 nmol.min-1 g-1 wet wt to 0.225 +/- 0.07 and 0.157 +/- 0.038 respectively, P < 0.01. Similar data were obtained when nisoldipine was given at the time of ischaemia, while administration 30 min after the onset of ischaemia showed only a trend towards protection. Nisoldipine lost its protective effect when given on reperfusion. A multifactorial analysis of the data suggest that the cardioprotective effect of nisoldipine is related to the maintenance of membrane integrity, possibly since nisoldipine is highly lipophilic.

Calcium-induced cytotoxicity in hepatocytes after exposure to extracellular ATP is dependent on inorganic phosphate. Effects on mitochondrial calcium.

In isolated mitochondria extensive uptake of Ca2+ in the presence of an "inducing agent," e.g. inorganic phosphate (Pi), causes permeabilization of the mitochondrial inner membrane and a collapse of the mitochondrial membrane potential. In this study we tested whether the effect of phosphate occurs in intact hepatocytes. Rat hepatocytes were incubated with ATP to induce a sustained increase in intracellular Ca2+ ([Ca2+]i), dissipation of the mitochondrial membrane potential, and cell death (Zoeteweij, J. P., van de Water, B., de Bont, H. J. G. M., Mulder, G. J., and Nagelkerke, J. F. (1992) Biochem. J. 288, 207-213). Omission of Pi from the incubation medium delayed the loss of viability. The nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) had similar effects on [Ca2+]i and viability, but now omission of extracellular Pi completely protected against cytotoxicity. Exposure to ATP or ATP gamma S induced a large cellular uptake of Pi. With the use of video-microscopy a significant increase in mitochondrial free calcium was observed before the onset of cell death. Accumulation of mitochondrial calcium was reduced when extracellular Pi was omitted. These results suggest that, after induction of high [Ca2+]i by ATP in hepatocytes, 1) mitochondria accumulate calcium which is associated with cell toxicity and 2) intracellular Pi increases which stimulates mitochondrial calcium uptake. These observations support a calcium-dependent mitochondrial dysfunction, induced by phosphate, as a valid model for ATP-induced cytotoxicity in hepatocytes.

Pathophysiology and treatment of focal cerebral ischemia

This article examines the pathophysiology of lesions caused by focal cerebral ischemia. Ischemia due to middle cerebral artery occlusion encompasses a densely ischemic focus and a less densely ischemic penumbral zone. Cells in the focus are usually doomed unless reperfusion is quickly instituted. In contrast, although the penumbra contains cells "at risk," these may remain viable for at least 4 to 8 hours. Cells in the penumbra may be salvaged by reperfusion or by drugs that prevent an extension of the infarction into the penumbral zone. Factors responsible for such an extension probably include acidosis, edema, K+/Ca++ transients, and inhibition of protein synthesis. Central to any discussion of the pathophysiology of ischemic lesions is energy depletion. This is because failure to maintain cellular adenosine triphosphate (ATP) levels leads to degradation of macromolecules of key importance to membrane and cytoskeletal integrity, to loss of ion homeostasis, involving cellular accumulation of Ca++, Na+, and Cl-, with osmotically obligated water, and to production of metabolic acids with a resulting decrease in intra- and extracellular pH. In all probability, loss of cellular calcium homeostasis plays an important role in the pathogenesis of ischemic cell damage. The resulting rise in the free cytosolic intracellular calcium concentration (Ca++) depends on both the loss of calcium pump function (due to ATP depletion), and the rise in membrane permeability to calcium. In ischemia, calcium influx occurs via multiple pathways. Some of the most important routes depend on activation of receptors by glutamate and associated excitatory amino acids released from depolarized presynaptic endings. However, ischemia also interfers with the intracellular sequestration and binding of calcium, thereby contributing to the rise in intracellular Ca++. A second key event in the ischemic tissue is activation of anaerobic glucolysis. The main reason for this activation is inhibition of mitochondrial metabolism by lack of oxygen; however, other factors probably contribute. For example, there is a complex interplay between loss of cellular calcium homeostasis and acidosis. On the one hand, a rise in intracellular Ca++ is apt to cause mitochondrial accumulation of calcium. This must interfere with ATP production and enhance anaerobic glucolysis. On the other hand, acidosis must interfere with calcium binding, thereby contributing to the rise in intracellular Ca++.

Cardioprotection by nisoldipine: Role of timing of administration

Nisoldipine was administered at 10−9M, a dose lacking negative inotropism, to isolated and perfused rabbit hearts submitted to 60 min ischaemia (1 ml.min−1) followed by 30 min reperfusion. The drug was delivered either 30 min before ischaemia, at the onset and after 30 min of ischaemia and during reperfusion only. Cardiac protection was evaluated in terms of recovery of left ventricular pressure during reperfusion, release of creatine phosphokinase (CPK), mitochondrial function, tissue content of adenosine triphosphate (ATP) and creatine phosphate (CP), calcium homeostasis and the occurrence of oxidative stress, established measuring content and release of reduced and oxidized glutathione. The cytoprotective action of nisoldipine occurs in the absence of negative inotropism and is closely related to the time of administration. Optimal myocardial preservation is achieved when nisoldipine is given before or at the onset of ischaemia. Prophylactic administration of nisoldipine improved the recovery of the developed pressure from 159±10 (SE) mmHg to 478±19 mmHg, P <0.01 and reduced the release of CPK from 830±29 to 229±27 mU. min−1 g−1 wet wt, P <0.01. The accumulation of tissue and mitochondrial calcium was reducedfrom 58±11 and49±9 to 14±6 and 10±4 mmol. kg−1 dry wt respectively, P <0.01. This resulted in a signficant ( P <0.01) preservation of all indices of mitochondrial function, allowing a higher recovery of ATP and CP after reperfusion (from 4.1±0.7 and 10.0±0.6 to 16.1±1.0 and 29.9±0.2 μmol.g−1 dry wt respectively, P <0.001). Reperfusion-induced myocardial accumulation and release of oxidized glutathione were reduced from 0.493±0.07 nmol.mg−1 protein and 0.768±0.063 nmol.min−1g−1 wet wt to 0.225±0.07 and 0.157±0.038 respectively, P <0.01. Similar data were obtained when nisoldipine was given at the time of ischaemia, while administration 30 min after the onset of ischaemia showed only a trend towards protection. Nisoldipine lost its protective effect when given on reperfusion. A multifactorial analysis of the data suggest that the cardioprotective effect of nisoldipine is related to the maintenance of membrane integrity, possibly since nisoldipine is highly lipophilic.

Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model.

The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated. Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation. Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains. Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin. In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically. Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion. The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity. We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally. Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S. Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells. Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium.

Postischaemic reperfusion injury in the isolated rat heart: effect of ruthenium red

The aim was to investigate the effect of attenuating mitochondrial calcium uptake with ruthenium red on myocardial function and the resultant necrosis following prolonged ischaemia and reperfusion in isolated rat hearts. Mitochondrial dysfunction, secondary to increased calcium uptake, has been implicated as an important mediator of reperfusion injury in the heart. To examine the role of mitochondrial calcium uptake in mediating ischaemic and reperfusion injury, isolated rat hearts were perfused with ruthenium red (n = 6), a polysaccharide dye which inhibits calcium uptake by mitochondria, and were compared to control perfused hearts (n = 7). After stabilisation, hearts were subjected to 60 min no flow ischaemia, immediately followed by 40 min reperfusion. Hearts were used from male Wistar rats weighing 300-350 g. Cardiac high energy phosphates (ATP, phosphocreatine, inorganic phosphate) and pH were continuously monitored during ischaemia and reperfusion using phosphorus magnetic resonance spectroscopy. Contractility (dP/dT), coronary flow, creatine kinase release, and the time to the onset of ischaemic contracture were also measured. No differences in metabolic abnormalities or time to peak contraction during ischaemia were found between groups, suggesting that ruthenium red does not alter the metabolic consequences of ischaemia. However, upon reperfusion, the following differences in the ruthenium red perfused hearts were observed when compared to control hearts (p less than 0.05): ATP and phosphocreatine recovery were more complete, myocardial contractility was greater, coronary flow was greater, and myocyte necrosis was attenuated. Combined with the known inhibitory effect of ruthenium red on mitochondrial calcium uptake, these data suggest that an important component of myocardial injury following ischaemia and reperfusion in the isolated rat heart is the result of mitochondrial calcium accumulation.

Effects of temperature on myocardial calcium homeostasis and mitochondrial function during ischemia and reperfusion

An isolated rabbit heart preparation was used to characterize the effects of hypothermia on the deterioration in mitochondrial respiratory function and on the calcium overload that occurs during ischemia and reperfusion. Hearts were perfused aerobically with an asanguineous solution for 120 minutes or made totally ischemic for 90 minutes at 37 degrees, 34 degrees, 28 degrees, 22 degrees C, respectively, and reperfused for 30 minutes at 37 degrees C. Mitochondrial function was assessed by measuring calcium content, yield, oxygen consumption, and adenosine triphosphate-producing capacities. In addition, the mechanical function of the hearts was measured together with tissue adenosine triphosphate, creatine phosphate, and calcium content. In a separate series of experiments, the effect of temperature on the initial rate of respiration-supported calcium accumulation of mitochondria from freshly excised, nonperfused rabbit hearts was determined. The hearts made ischemic at 37 degrees C were severely depleted of tissue adenosine triphosphate and creatine phosphate. Their mitochondria accumulated calcium and the oxidative phosphorylating activity was impaired. During reperfusion, tissue and mitochondrial calcium levels were substantially increased, state 3 of mitochondrial respiration was further impaired, and the adenosine triphosphate-generating capacities were severely reduced. Diastolic pressure increased and there was no recovery of developed pressure. Isolated mitochondrial function of hearts made ischemic at 28 degrees and 22 degrees C was protected. There was a less marked increase in tissue and mitochondrial calcium, and the initial rate and total production of adenosine triphosphate were maintained. In these hearts there was an almost complete recovery of mechanical performance at reperfusion, whereas the ischemia-induced depletion of tissue adenosine triphosphate and creatine phosphate was not significantly reduced by hypothermia. The hearts made ischemic at 34 degrees C were only partially protected. These data suggest that a decrease in temperature from 37 degrees to 22 degrees C during ischemia did not significantly prevent depletion of adenosine triphosphate at the end of ischemia but reduced tissue and mitochondrial calcium overload, maintaining mitochondrial function. Thus in our experiments the protective effect of hypothermia might be related to a direct reduction of tissue and mitochondrial calcium accumulation rather than to a slowing in rates of energy utilization. This possibility is supported by the finding that in freshly excised, nonperfused rabbit hearts, hypothermia significantly reduced the initial rate of mitochondrial calcium transport.

Inhibition by caltrin of calcium transport into spermatozoa, liver and heart mitochondria

The calcium-transport inhibitor, caltrin, isolated from bovine seminal fluid inhibits calcium accumulation by bovine epididymal spermatozoa, spermatozoal mitochondria, rat liver mitochondria and beef heart mitochondria. Respiration studies demonstrate a marked stimulation of oxygen consumption by caltrin in filipin-treated spermatozoa and rat liver mitochondria. A biphasic effect of caltrin on rat liver mitochondrial respiration was noted, with stimulation at low caltrin concentrations and inhibition as the concentration of caltrin is increased. The ability of caltrin to uncouple and/or inhibit respiration in filipin-treated spermatozoa and isolated liver mitochondria indicates that inhibition of mitochondrial calcium accumulation by caltrin results from one of these mechanisms. Only a marginal effect of caltrin on respiration of intact spermatozoa was observed; indicating that the plasma membrane is impermeable to this protein. The differential effect of caltrin on respiration of intact and permeabilized spermatozoa indicates that caltrin inhibition of Ca 2+ uptake into spermatozoa in vivo occurs at the level of the plasma membrane.

Changes in the mitochondrial calcium influx and efflux properties are responsible for the decline in sperm calcium during epididymal maturation

This study was undertaken to determine the role of calcium ion, a key regulator of the intensity and form of motility in mature demembranated sperm, in the development of motility during passage through the bovine epididymis. Cellular calcium levels in bovine caput and cauda epididymal spermatozoa were measured with three different techniques. 45Ca2+ uptake measurements revealed that net calcium uptake and Ca2(+)-Ca2+ exchange in caput spermatozoa were about 2 to 3 times higher than in caudal spermatozoa. Intracellular free calcium determination with the calcium fluorophore Fura 2 showed that the levels were 6 times higher in caput spermatozoa. The values for caput and caudal sperm were 875 +/- 55 nM (n = 15) and 155 +/- 6 nM (n = 24), respectively. Total cellular calcium levels quantitated by atomic absorption were 626 +/- 30 (n = 48) and 304 +/- 19 (n = 46) ng/10(8) sperm in caput and caudal epididymal sperm, respectively. At least one of the reasons for the high calcium content of caput epididymal sperm is the result of a higher rate and extent of mitochondrial calcium accumulation in caput compared to caudal sperm. Mitochondrial calcium uptake rates measured in digitonin permeabilized cells revealed uptake rates 2- to 3-fold higher in caput compared to caudal sperm. However, mitochondrial calcium efflux rates were identical in caput and caudal epididymal sperm. The efflux rates in both cell types were unaffected by external sodium levels but were found to be proportional to pH. Alkalinization or acidification of internal pH of intact sperm resulted in a corresponding lowering or elevation of cytoplasmic free calcium levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of pair-feeding and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on various biochemical parameters in female rats

Hypophagia is a common characteristics of the toxicity of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and may be responsible for some of the toxic manisfestations. Pair-feeding has been used in control animals to compensate for the hypophagia, but relatively few studies have assessed biochemical changes associated with pair-feeding versus weight loss induced by TCDD. Rats were treated with TCDD and killed 7 days post-treatment while pair-fed animals received an amount of diet equivalent to TCDD-treated partner animals. Ad libitum-fed rats were also used. No correlations were seen in altered calcium and iron homeostasis between pair-feeding and TCDD administration relative to ad libitium-fed animals. Pair-feeding resulted in greater alterations than TCDD administration in the subcellular distribution of iron in mitochondria, microsomes and cytosol. Pair-feeding also resulted in greater accumulation of calcium in mitochondria and microsomes in pair-fed as compared to TCDD-treated animals. Greater lipid peroxidation was observed in whole liver and nuclei nuclei of rats receiving TCDD relative to pair-fed animals. A significantly greater incidence of DNA single strand breaks occurred in hepatic nuclei of TCDD-treated animals as compared to pair-fed and ad libitum-fed animals. Significantly greater inhibition of hepatic glutathione peroxidase activity and thymic involution were observed in TCDD treated animals as compared to the pair-fed group. Although some similarities existed between TCDD-treated animals and pair-fed rats, the overall biochemical changes which were observed following TCDD administration cannot be attributed to weight loss associated with hypophagia.

Oxygen free radical-mediated heart injury in animal models and during bypass surgery in humans. Effects of alpha-tocopherol.

There is evidence that oxygen free radicals play a role in myocardial ischemic and reperfusion injury. We investigated the effect of ischemia and reperfusion on glutathione status. Reperfusion after prolonged ischemia (60 min) induced an important release of reduced (GSH) and oxidized (GSSG) glutathione, concomitant with an increase of tissue GSSG and no recovery of mechanical function, indicating that reperfusion results in oxidative stress. These alterations are associated with tissue and mitochondrial calcium accumulation, loss of mitochondrial function, and membrane damage. We also determined the arteriocoronary sinus difference for GSH and GSSG of 16 CAD patients undergoing coronary artery bypass. Patients were divided in two groups according to the length of clamping period: 25 +/- 2 min (group 1), and 55 +/- 6 min (group 2). In group 1, reperfusion resulted in a transient release of GSH, GSSG, CPK, and lactate, with return to preclamping values in 10 minutes. In group 2, reperfusion determined a sustained and pronounced release of GSH, GSSG, CPK, and lactate during declamping, suggesting the occurrence of an oxidative stress. Using an in vitro model, administration of alpha-tocopherol bound with albumin showed protection of mitochondrial function, improved recovery of contraction, and reduced oxidative stress during reperfusion.

Studies on prolonged acute regional ischemia: II. Implications of progression from dyskinesia to akinesia in the ischemic segment

This study analyzed the pattern of regional wall motion in 58 dogs undergoing 4 to 6 hours of left anterior descending coronary artery occlusion. Regional wall motion was measured by ultrasonic crystals and ischemic muscle either remained dyskinetic (-40% of control systolic shortening, n = 26) or progressed toward akinesia (less than 20% of control systolic shortening or greater than 50% reduction in passive lengthening, n = 32). Ten dogs underwent unmodified blood reperfusion. Regional blood flow (radioactive microspheres), histochemical damage (triphenyltetrazolium chloride staining), and mitochondrial function were determined. Hearts showing persistent dyskinesia had more collateral flow (12 versus 2 ml/100 gm/min, p less than 0.05), less histochemical damage (26% versus 63% area at risk/area of nonstaining, p less than 0.05), and better retention of mitochondrial oxidative phosphorylation capacity (adenosine triphosphate, 622 versus 444 nmol/mg protein/min, p less than 0.05), and tended toward mitochondrial calcium accumulation (48 versus 64 nmol/mg protein). Unmodified blood reperfusion after 4 hours of ischemia produced prompt akinesia (-2% +/- 3% systolic shortening) and was associated with increased edema (82% water content), caused the low-reflow phenomenon (19% control subendocardial flow, 13 ml/100 gm/min), and increased histochemical damage (69% triphenyltetrazolium chloride nonstaining, p less than 0.05). These findings suggest that persistent dyskinesia during early ischemia (first 6 hours) may reflect a relatively optimistic sign, as regression to akinesia occurs in muscle with less collateral flow, more impaired mitochondrial function, worsened calcium homeostasis, and more severe histochemical and ultrastructural damage. These observations imply that careful evaluation of ischemic wall motion may provide a valuable insight into potential muscle salvage.

Quantitation of bovine sperm cytoplasmic calcium with Quin-2 and Fura-2: Evidence that external calcium does not have direct access to the sperm cytoplasm

Internal calcium levels of sperm loaded with Quin-2 in the absence or presence of exogenous calcium were 63 ± 5 and 189 ± 19 nM, respectively. These values were similar to those determined by Fura-2. Surprisingly, however, dye loaded sperm depleted of internal calcium did not take up calcium from the medium into the cytoplasm upon re-addition of this ion. Uptake was rapid and maximal, however, if these cells were exposed to the calcium ionophores A23187 or ionomycin. Increasing [Quin-2] i progressively lowered [Ca 2+] i in spite of the presence of exogenous calcium during dye loading. This anomaly was not due to interference of the fluorophores with calcium uptake, since exogenous 45Ca 2+ was taken up at the same rate and to the same extent by control and fluorophore loaded sperm. This 45Ca 2+ uptake was mitochondrial and energy dependent. Also, inhibition of mitochondrial calcium accumulation during dye loading lowered [Ca 2+] i to values similar to those observed for calcium depleted sperm. These results suggest an extreme impermeability of the sperm plasma membrane to direct calcium entry into the cytoplasm while substantial amounts of calcium entry occurs into the sperm mitochondria.

Molecular mechanisms of gossypol action on sperm motility

1. 1. Gossypol acetic acid inhibits collective motility of ejaculated ram spermatoza. 2. 2. Oxygen consumption was stimulated at low gossypol concentrations and inhibited as the concentrations are increased. 3. 3. Gossypol inhibits respiration of permeabilized spermatozoa supported by durohydroquinome, which indicates a direct inhibition of mitochondrial electron transport chain. 4. 4. The rapid reduction of mitochondrial dependent motility, high uncoupling effect and almost complete inhibition of mitochondrial calcium accumulation, indicate that gossypol inhibits motility in a mechanism by which mitochondrial uncoupling is involved.

Altered calcium homeostasis and membrane integrity in myocardial cell injury.

Coronary occlusion leads to contractile arrest of ischemic myocardium and progressive metabolic derangements and ultrastructural alterations, which if of sufficient duration and severity, lead to irreversible myocardial injury. Based on a number of experimental observations, we and others have put forward the following general hypothesis regarding the pathogenesis of irreversible myocardial injury1,2. Myocardial ischemia causes oxygen deprivation which leads to depressed energy metabolism resulting in a reduced ATP level, increased lactate, and decreased pH. The metabolic changes are followed by discrete alterations in energy dependent membrane transport systems which lead to altered ionic composition of the cell, including an increase in cytosolic calcium. These membrane alterations may include altered ionic flux across the sarcolemma as well as release of calcium from mitochondria and sarcoplasmic reticulum. The elevated cytosolic calcium concentration has the potential to activate catabolic enzymes, including phospholipases. This may lead to acceleration phosphlipid degradation, increased membrane permeability, and further electrolyte derangements, including calcium accumulation. The excess cellular calcium load may also lead to ATP depletion by activation of calcium-dependent ATPases as well as by mitochondrial calcium accumulation which occurs at the expense of continued energy production. Thereafter, a vicious cycle of membrane injury and ATP depletion may ensue which terminates in irreversible cell injury. Observations pertinent to this hypothesis are reviewed in this chapter.KeywordsCoronary OcclusionTotal PhospholipidCalcium OverloadMembrane InjuryIrreversible InjuryThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

EXCITOTOXIC MECHANISMS IN HYPOGLYCAEMIC HIPPOCAMPAL INJURY

Light and electron microscopy were used to study the effect of hypoglycaemia on selectively vulnerable neurons of rat hippocampus with and without pharmacologic blockade of the N-methyl-D-aspartate (NMDA)-preferring receptor with 2-amino-7-phosphonoheptanoic acid (AP-7). In control hypoglycaemic hippocampi, dark cell change occurs predominantly in dentate granule cells. The topography and ultrastructural appearance of these changes is distinct from that produced by ischaemia or status epilepticus. In hypoglycaemia, mitochondrial calcium accumulation characteristic of ischaemia or status epilepticus is not seen. NMDA receptor blockade markedly attenuates the hypoglycaemic cell injury. Similar attenuation of ischaemic and epileptic brain damage by NMDA receptor blockade suggest that excessive neuronal excitation is a common mechanism of injury in each of the three conditions.

Programmed cell death. Cytochemical evidence for accumulation of calcium in mitochondria and its translocation into lysosomes: X-ray microanalysis in metamorphosing insect muscles.

The intersegmental muscles in the metamorphosing silkmoth Antheraea polyphemus were examined by two electron cytochemical procedures for demonstration of calcium compartmentation during the two-day period of degeneration after emergence. Muscle fibres were treated with either oxalate-pyroantimonate, or phosphate-pyroantimonate procedures. The elemental composition of the reaction product arising from the oxalate procedure was determined with electron probe X-ray microanalysis of unstained thin sections by energy dispersive spectrometry and wavelength dispersive spectrometry. The wavelength dispersive data revealed high peaks of calcium and antimony in the electron-dense precipitates. No reaction was obtained in muscles after treatment with the phosphate-pyroantimonate method. Shortly after the emergence of the moth, very few calcium deposits were found in the mitochondria, which also contained amorphous matrix densities. During the rapid lytic phase (17 and 30 h after ecdysis), the mitochondria, autophagic vacuoles sequestering mitochondria, and lysosomal dense bodies issuing from the latter were highly reactive in each muscle fibre. These results demonstrate that the collapse of tracheae (hypoxic conditions) is correlated with the calcium overload of mitochondria when the cell calcium homeostasis is apparently lost. Such calcium overload of the mitochondria appears to cause irreversible damage to these organelles which are then sequestered in autophagic vacuoles. This mitochondrial autophagic process leads to calcium translocation into a lysosomal compartment. We suggest that the calcium lysosomal stores may have a transient function of cell detoxification and stimulation of calcium-dependent degradative processes prior to the final muscle collapse.

Myocardial cAMP and calcium levels in the calcium paradox.

In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP.

Mitochondrial alterations in cisplatin-induced acute renal failure.

The mild reversible nonoliguric form of acute renal failure is perhaps the most common of many nephrotoxic side effects that occur secondary to cisplatin administration. The present studies were undertaken to gain insight into mitochondria alterations and morphological abnormalities underlying this form of renal failure. Following a single intraperitoneal injection of 5.5 mg/kg body wt of cisplatin changes in renal function, mitochondrial respiration, and calcium accumulation were measured serially over a 9-day period. Results indicate that reversible functional changes secondary to cisplatin are accompanied by changes in mitochondrial respiration and calcium accumulation. A decline in state 3 mitochondrial respiration precedes mitochondrial calcium accumulation. However, calcium accumulation begins to recover before mitochondrial respiration. At the peak of functional and biochemical injury morphological damage is extensive, and mitochondria are strikingly aberrant. The results demonstrate that changes in mitochondrial respiration and calcium accumulation occur secondary to cisplatin administration. Both of these effects may play a role in the renal cellular injury induced by cisplatin.

Neuronal alterations in hippocampus following severe hypoglycaemia: a light microscopic and ultrastructural study in the rat.

Because they induce similar neuropathological changes (ischaemic cell change with microvacuolization), it has been suggested that ischaemia, status epilepticus and hypoglycaemia produce cell death by similar mechanisms, especially those resulting from intracellular calcium accumulation. We have recently demonstrated microvacuolation of neurons, mitochondrial swelling (the electron microscopic correlate of microvacuolization) and massive mitochondrial calcium sequestration (using the pyroantimonate technique) following ischaemia or status epilepticus. We therefore studied the selectively vulnerable neurons of rat hippocampus by light and electron microscopy (including the pyroantimonate technique) following 30 and 60 min of EEG isoelectricity resulting from insulin hypoglycaemia. The neuropathology at the light and EM level is unique and different from that following status epilepticus or ischaemia. The most constant finding is dark cell change of the granule cells at the tip of the dentate gyrus. In contrast to status epilepticus and ischaemia, hippocampal pyramidal neurons are far less frequently involved. Microvacuoles are rarely seen and, when present, their ultrastructural correlate is swollen Golgi apparatus, not dilated mitochondria. No intracellular calcium accumulation is demonstrable with pyroantimonate technique. Thus the cellular alterations produced by hypoglycaemia differ in character and distribution from those produced by anoxia-ischaemia. Mitochondrial calcium accumulation is not prominent in cell death from hypoglycaemia. Whether calcium toxicity plays another, subtler role in hypoglycaemic brain injury is unknown.