Ashley Allshire

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.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.

[8] Calcium ion transport in mitochondria

Publisher Summary This chapter discusses the calcium ion transport in mitochondria. Mitochondria are organelles capable of accumulating and releasing calcium ion (Ca 2+ ) in the cell. Calcium channels and pumps in the plasma membrane determine the overall cellular Ca 2+ content. Mammalian mitochondria accumulate Ca 2+ mainly by an electrogenic mechanism that drives Ca 2+ into the matrix at the expense of the membrane potential generated by the respiration or hydrolysis of Adenosine triphosphate (ATP) while efflux of Ca 2+ occurs mainly by other electroneutral mechanisms. Under physiological conditions, the distribution of Ca 2+ between cytosol and mitochondria is, thus, influenced by the kinetic properties of the influx and efflux mechanisms. Increase in permeability is correlated with the transition of an increasing proportion of the mitochondrial population from an aggregated to an orthodox configuration.

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.

Causes and effects of changes in cytosolic free calcium in the hypoxic myocardial cell

We review cytosolic free calcium (Cai) measurements in hypoxic single cardiomyocytes, isolated heart tissue and intact myocardium. In single cells the seminal event may be a shortening which is (largely) Cai-independent and probably corresponds to hypoxic contracture of intact tissue. This shortening is soon followed by a Cai rise and net Ca2+ ingress across the sarcolemma which is sensitive to the Na+ electrochemical gradient, suggesting that Na-Ca exchange occurs, and that a Nai imbalance precedes the Cai rise. Reoxygen-ation of single cells triggers spontaneous mechanical activity (and oscillation of Ca2+ between cytosol and sarcoplasmic reticulum) analogous to reoxygenation arrhythmias in intact myocardium, and provided that Cai has not risen above several micromolar it is returned to resting levels. We interpret the Cai-independent shortening as a rigor which activates the myosin ATPase and thereby accelerates ATP depletion so that ATPase-linked ion pumps in the cell membranes become limited thermodynamically. An ensuing Nai rise leads through depressed Na-Ca exchange to a Cai rise. Such a model highlights rigor-compex mediated activation of myosin S1-ATPase as the fundamental target for interventions to ameliorate ischemic damage to the myocardium.

Tipping the balance towards tolerance: the basis for therapeutic immune modulation by gold?

Gold salts have long been used in the treatment of rheumatoid arthritis. However, the basis for their therapeutic immune-modulating properties has never been satisfactorily explained. Furthermore, treatments are often marred by the development of adverse immune reactions such as hypersensitivity and even exacerbation of autoimmunity. We would like to propose a hypothesis to explain the basis for both the beneficial and adverse immune-modulating effects of gold in the treatment of rheumatoid arthritis. If accepted, this hypothesis will allow for the development of safer and more effective treatments with gold salts. The principle underlying this hypothesis also has broader implications for how immune hypersensitivity and tolerance are perceived.

Acute reperfusion injury of myocardium

Summary In ischemic-reperfused myocardium severe cellular injury may develop rapidly upon re-oxygenation (‘oxygen paradox’). Several causes of this acute form of reperfusion injury have now been identified. During oxygen depletion cardiomyocytes accumulate Ca2+ in the cytosol. When re-energized upon re-oxygenation high cytosolic Ca2+ causes uncontrolled contractile activation, leading to hypercontracture of the myofibrils. Protection against hypercontracture can be achieved by various means, for example, by prolongation of ischemic intracellular acidosis or by attenuation of the oscillatory elevations of cytosolic Ca2+ in the early phase of re-oxygenation. Structural damage provoked by hypercontracture is favored by increased cytoskeletal fragility of the cardiomyocytes. In reperfused myocardium cardiomyocytes are also challenged by osmotic stress. Oxidative injury reduces sarcolemmal stability upon re-oxygenation and thereby reduces the tolerance of cardiomyocytes to osmotic stress. All these causes contributing to acute reperfusion injury are rooted within the myocardial cells. They are independent of exogenous, blood-borne factors that can additionally modify the reperfusion situation.