Manganese stimulates calcium flux through the mitochondrial uniporter.

Mn2+ alters the balance between the simultaneous uptake and release of Ca2+ across the mitochondrial inner membrane toward a lower external level. Addition of as little as 0.5 microM Mn2+ to energised mitochondria from rat liver, rat heart or guinea-pig brain changed the level at which they buffered Ca2+ in the medium. That extramitochondrial Mn2+ was responsible was suggested by a partial decay in the shift in Ca2+ steady state at a rate similar to the rate at which Mn2+ was accumulated by the mitochondria. The alteration of transmembrane Ca2+ distribution by Mn2+ required that both Mg2+ and Pi be present, and was almost maximal at Mg2+ and Pi levels in the physiological range. Substitution of spermine or Ni2+ for Mg2+, or acetate for Pi, abolished the effect. In contrast to Sr2+, Mn2+ did not inhibit either EGTA- or Ruthenium red-induced release of Ca2+ from the mitochondria. However, when flux through the uniporter was rate-limiting, Mn2+ accelerated Ca2+ uptake. The stimulation showed hyperbolic kinetics, with an element of competition discernible in the Mn2+-Mg2+ interaction. Thus, extramitochondrial Mn2+ at levels occurring in vivo can alter the mitochondrial 'set-point' by stimulating Ca2+ influx through the uniporter.

Further evidence for biological effects resulting from ionizing radiation doses in the diagnostic X-ray range.

In this study we assessed cell response to ionizing radiation doses in the diagnostic X-ray range, using metabolic performance as the biological end point. HeLa cells were irradiated between 3.5 mGy (standard deviation (SD=2.5%) and 55.1 mGy (SD=0.9%) with 110 kV X-rays, and after 7 days metabolic capacity of cultures was measured spectrophotometrically with resazurin. Cells receiving 7.5 mGy or greater showed impaired proliferation. At 7.5 mGy this effect was prevented by nitric oxide synthase inhibitor N G-monomethyl-L-arginine but not by ascorbic acid or p38 MAPK inhibitor SB203580. We conclude that HeLa cell proliferation is adversely affected by doses within the range of some radiological examinations, through a mechanism involving nitric oxide.

Resazurin assay of radiation response in cultured cells.

We describe use of resazurin reduction for measurement of cell response to irradiation as a simple and non-destructive assay that complements the conventional colony forming assay and can readily be applied to both adherent and non-adherent cell cultures. The resazurin method yields data comparable with the colony forming assay as well as to assay of DNA synthesis (BrdU incorporation), giving an OER (oxygen enhancement ratio) of 2.5 at 60% isoeffect level versus 3.1 for the colony forming assay. Intraday and interday precisions for the resazurin assay were 4.1% and 5.2%, respectively.

Continuous measurements of cytoplasmic ATP in single cardiomyocytes during simulation of the "oxygen paradox".

OBJECTIVE: It is now possible to monitor cytoplasmic ATP in single cardiomyocytes and it has recently been shown that cardiomyocytes exposed for several minutes to metabolic inhibitors undergo an abrupt rigor mediated shortening which coincides with a sudden fall in cytoplasmic ATP, from approximately 150 mumol.litre-1 to a few micromolar or less. The objective of this work was to monitor cytoplasmic ATP during simulated reoxygenation of a poisoned cardiomyocyte. METHODS: Firefly luciferase was injected into a single cell and the light signal generated when luciferin was superfused was monitored. Calibration of the signal is complicated by a transient enhancement of the signal (possibly the result of complex luciferase kinetics), and by uncertainties about cytoplasmic pH. RESULTS: The data indicate that millimolar levels of cytoplasmic ATP are restored within 1-2 min of cyanide removal. CONCLUSIONS: Cytoplasmic free calcium is known to rise after poisoned cells undergo shortening, so it is conceivable that the restoration of cytoplasmic ATP in a cell in which free calcium is at micromolar levels may provide a plausible cellular mechanism for the "oxygen paradox". Reoxygenation induces large amplitude, but slow, oscillations in free calcium which, together with the millimolar levels of ATP indicated here, could provide the stimuli for generating the uncoordinated mechanical forces that are prevalent in the oxygen paradox.

Tri-iodothyronine increases intra-cellular calcium levels in single rat myocytes.

We have studied the effects of acute administration of triiodothyronine (T3) on cytosolic free calcium levels [Ca2+]i in single rat myocytes microinjected with aequorin. Ventricular myocytes were isolated by perfusing rat hearts with collagenase, and healthy, rod-shaped cells were injected to less than 1% of their volume with aequorin. The photons emitted from single cells were measured and a conversion to [Ca2+]i made on the basis of an in vitro calibration after the remaining aequorin had been discharged by cell lysis. Only cells that depolarized reversibly (showing elevated [Ca2+]i levels) when superfused with 80 mM KCl, and which gave a substantial signal on lysis with distilled water were used. The [Ca2+]i rose from a resting value of 150 +/- 56 nM (mean +/- SD, n = 14) by 127 +/- 47 nM on depolarization with 80 mM KCl. Application of T3 (1-100 nM) led to an increase (P less than 0.05) in [Ca2+]i (mean amplitude of 152 +/- 35 nM) before returning to baseline. The median duration of these events was 10 min (range = 1.4-34.4 min). The time to response was shorter when 100 nM T3 was applied (median and range; 6.8, 0-14 min) than when 1 nM T3 was used (16, 7.0-56.1 min) (P less than 0.05). To conclude, physiological concentrations of thyroid hormones caused rapid but transient stimulation of [Ca2+]i in single rat myocytes.

Failure of magnesium to protect isolated cardiomyocytes from effects of hypoxia or metabolic poisoning.

BACKGROUND: MgSO4 appears to reduce infarct size in animal models of myocardial infarction-reperfusion, but only if given before reperfusion. The mechanisms underlying this effect have not been established, nor has the discrepancy between these results and the lack of efficacy in the Fourth International Study of Infarct Survival (ISIS-4) been explained. HYPOTHESIS: The study was undertaken to examine the hypothesis that Mg2+ protects myocardium threatened by ischemia. METHODS: We studied the effects of extracellular magnesium [Mg2+](e) at 0.6 and 1.8 mmol/l on isolated myocytes exposed to 2.5 mmol/l NaCN + 5 mmol/l 2-deoxyglucose or to profound hypoxia. RESULTS: Nonpaced cells shortened at a mean of 8.8 min after onset of metabolic inhibition in [Mg2+](e) = 1.8 mmol/l, 9.6 min in [Mg2+](e) = 0.6 mmol/l (not significant). Paced cells shortened after 9.5 min in [Mg2+](e) = 1.8 mmol/l, 10.2 min at [Mg2+](e) = 0.6 mmol/l. On washout of inhibitors, 93% of cells underwent hypercontracture at [Mg2+](e) = 1.8 mmol/l, 94.8% at [Mg2+](e) = 0.6 mmol/l. During hypoxia, nonpaced cells in [Mg2+](e) = 1.8 mmol/l shortened after 67 +/- 11 min compared with 62.5 +/- 28 min at [Mg2+](e) = 0.6 mmol/l. Paced cells shortened after 25.8 +/- 12.9 min at [Mg2+](e) = 1.8 mmol/l and after 28.7 +/- 13.6 min at [Mg2+]e = 0.6 mmol/l. Although there was a trend toward longer survival at higher [Mg2+](e), the difference was small and not significant (p > 0.05, Student's paired t-test). CONCLUSION: We find no evidence to support the hypothesis that [Mg2+] protects myocardium threatened by ischemia. This is consistent with clinical data but contradicts data from animal experiments.

Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

Uptake, retention, and efflux of Ca2+ by mitochondrial preparations from skeletal muscle.

Functionally intact mitochondria, substantially free of contamination, were isolated from rabbit gastrocnemius muscle after protease digestion and their Ca2+-handling properties examined. When judged by their capacity to retain large Ca2+ loads and the magnitude of basal and Na+-stimulated Ca2+ effluxes, the most suitable isolation method was digestion of finely minced muscle in buffered isoosmotic KCl with low levels (0.4 mg/g) of trypsin or the bacterial protease nagarse, followed by differential centrifugation. Polytron disruption of skeletal muscle in both sucrose- and KCl-based media released mitochondria deficient in cytochrome c. Use of the divalent ion chelator EDTA rather than EGTA in the isolation medium sharply reduced Ca2+-dependent respiratory control and tolerance of the mitochondria to Ca2+ loads, probably by removing Mg2+ essential to membrane integrity. ADP-dependent respiratory control was not altered in mitochondria prepared in an EDTA-containing isolation medium. Purification of mitochondria on a Percoll density gradient did not improve their Ca2+-handling ability despite removal of minor contaminants. Mitochondria prepared by the protease method could accumulate micromole loads of Ca2+/mg while maintaining a low basal Ca2+ efflux. Addition of BSA to the assay medium slightly improved Ca2+ retention but was not essential either during isolation or assay. Ca2+-dependent state 3 respiration was maximal at pH 6.5-7.0 while respiratory control and Ca2+/O were optimal at pH 7.0-7.5. Neither Pi nor oxaloacetate induced Ca2+ release from loaded mitochondria when monitored for 30 min after ruthenium red addition. Na+-stimulated Ca2+ efflux had sigmoidal kinetics with a Hill coefficient of 3. Since skeletal muscle mitochondria can be isolated and assayed in simple media, functional deficiencies of mitochondria from diseased muscle are unlikely to be masked.

Modulation of rigor and myosin ATPase activity in rat cardiomyocytes.

Ischaemic myocardium undergoes calcium-independent contracture at millimolar tissue ATP, though in actomyosin solutions ATP must be reduced to micromolar before rigor complexes form. This contracture is associated with myosin ATPase activity that may contribute to tissue de-energization. Here we used isolated rat cardiomyocytes permeabilized with digitonin to analyse in parallel how rigor and myosin ATPase activity are modulated by metabolic conditions that develop during ischaemia. At pH 7.1 and 37 degrees C rigor and myosin ATPase showed co-ordinated bell-shaped dependence on ATP concentration over 3-1000 microM. Rigor, but not myosin ATPase, was inhibited by acidosis (pH 6.2), indicating reduced efficiency of cross-bridge cycling, while both parameters were stimulated by ADP (< or = 1 mM) and unaffected by inorganic phosphate (Pi, 30 mM), AMP, Mg2+, lactate or inhibition of adenylate kinase with diadenosine pentaphosphate. Combined acidosis and high ADP inhibited rigor, while Pi attenuated the enhancement of rigor by ADP. Thus, rigor complex formation activates myosin ATPase in the intact myofilament array, modulated by ADP, Pi and acidosis in the ranges that occur in ischaemia. There was no evidence that adenylate kinase might attenuate falling ATP/ADP ratio at the myofilaments. In combination these effects are sufficient to resolve the apparent discrepancy between ATP concentrations triggering rigor in actomyosin and onset of contracture in ischaemic myocardium. Since rigor contracture activates myosin ATPase it is likely to exacerbate ATP depletion and thereby limit vital cell functions. This positive feedback is consistent with the abrupt depletion of ATP observed in individual cardiomyocytes undergoing deenergization contracture.

Bioluminescent measurement in single cardiomyocytes of sudden cytosolic ATP depletion coincident with rigor.

The sequence of events that leads to irreversible injury of the ischaemic myocardium is poorly understood but it is axiomatic that lack of oxygen will impair regeneration of ATP. In the globally-ischaemic heart a contracture develops which is independent of raised cytoplasmic free Ca2+ and which has been attributed to activation of actomyosin by nucleotide-free actomyosin cross-bridges ('rigor complexes') which form at low ATP concentrations. Single, metabolically-poisoned or anoxic cardiomyocytes show comparable behaviour, shortening before a significant rise in cytoplasmic free Ca2+ occurs. To explain the close temporal relationship that exists between cell shortening and the onset of the free Ca2+ rise we have predicted that, during myocyte shortening, a precipitous fall in cytosolic ATP concentration occurs, the result of rigor-complexes activating myosin ATPase, which then perturbs ionic homeostasis. Here we show, by means of continuous measurements of cytosolic ATP using firefly luciferase microinjected into single, isolated cardiomyocytes, that cell shortening coincides with an abrupt fall in cytosolic ATP.

BDM drives protein dephosphorylation and inhibits adenine nucleotide exchange in cardiomyocytes.

Contractile dysfunction plays a key role in injury sustained by ischemic myocardium at reperfusion, whereas interventions that impede hypercontracture enhance recovery. In permeabilized adult rat cardiomyocytes, the negative inotrope 2,3-butanedione monoxime (BDM; 10-50 mM) inhibited rigor at low MgATP concentration but stimulated net ATP hydrolysis. Hydrolysis was attenuated by H-7, kaempferol, chelerythrine, and genistein. Evidently BDM opposed phosphorylation of both serine/threonine and tyrosine kinase target proteins, either directly or by enhancing protein phosphatase activity, in a futile cycle of ATP hydrolysis independent of cross-bridge cycling. Although 20 mM BDM did not affect the onset of rigor contracture in permeabilized cells at low MgATP, in intact cells exposed to the metabolic inhibitors cyanide and 2-deoxyglucose rigor onset was accelerated, indicating that BDM increases ATP depletion in quiescent cardiomyocytes. Conversely, in cells exposed to the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, BDM delayed the onset of contracture and hence ATP depletion, consistent with an inhibition of adenine nucleotide movement across the mitochondrial inner membrane. Such effects will limit the value of BDM as a cardioprotective agent at physiological temperature.

Cytosolic free Ca2+ in single rat heart cells during anoxia and reoxygenation.

Free Ca2+ in the cytosol ([Ca2+]i) of individual rat ventricle cells injected with aequorin was measured under anoxia. In glucose-free medium myocytes spontaneously shortened after about 60 min, although [Ca2+]i was still at or near resting levels. However, within minutes a net inward movement of Ca2+ across the sarcolemma developed and [Ca2+]i began to rise. Provided oxygen was readmitted before [Ca2+]i exceeded 2-3 microM, cells were able to restore [Ca2+]i to resting levels through caffeine-sensitive sequestration of Ca2+ in the sarcoplasmic reticulum. We suggest that Ca2+-independent shortening of anoxic cardiomyocytes reflects onset of rigor which triggers loss of [Ca2+]i homoeostasis.