ryanodine and Necrosis

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Calcium; Cyclosporine; Fluoresceins; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Rats; Rats, Wistar; Reactive Oxygen Species; Ruthenium Compounds; Ryanodine; Thapsigargin; Tiopronin

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.

Topics: Animals; Apoptosis; bcl-X Protein; Blotting, Western; Caffeine; Calcium; Calcium Channels; Cell Survival; Chelating Agents; CHO Cells; Chromatin; Cricetinae; Cytosol; DNA, Complementary; Dose-Response Relationship, Drug; Egtazic Acid; Endoplasmic Reticulum; Microscopy, Confocal; Muscle, Skeletal; Necrosis; Phosphodiesterase Inhibitors; Plasmids; Proto-Oncogene Proteins c-bcl-2; Rabbits; Ryanodine; Ryanodine Receptor Calcium Release Channel; Time Factors; Transfection

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Apoptosis; Benzodiazepines; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cell Survival; Cells, Cultured; Cerebellum; Cysteine Proteinase Inhibitors; Dizocilpine Maleate; Electric Conductivity; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Ion Channel Gating; Kainic Acid; Necrosis; Neurons; Nifedipine; Oligopeptides; Phosphodiesterase Inhibitors; Rats; Ryanodine; Sodium; Sucrose; Thapsigargin

As a result of oxidative stress to membrane lipid matrix, the peroxidation of polyunsaturated fatty acids induced the transient formation of lipid hydroperoxides (ROOH). The aim of this study was to evaluate the damaging effects of ROOH on the cardiac cell and the link between the alterations observed and intracellular calcium overload.. Necrosis of cultured rat cardiac cells was determined by measuring the release of lactate dehydrogenase (LDH). In guinea-pig papillary muscles, action potential (AP) and isometric tension were recorded with standard microelectrodes and a transducer, respectively. The reactive oxygen species (ROS) scavenging properties of tested compounds were determined using a cell-free model of lipid photoperoxidation.. 15(S)-HpETE (15(S)-hydroperoxyeicosatetraenoic acid), an arachidonic acid hydroperoxide, induced a concentration-dependent loss of cardiomyocytes membrane integrity. The release of LDH induced by 15(s)-HpETE (30 microM) was prevented by a ROS scavenger, BW755C (10 microM), but not by a sarcolemmal calcium channel blocker, Amlodipine (10 microM), or a calcium overload protective agent, R56865 (10 microM). Cardiomyocytes necrosis induced by calcium paradox was prevented by Amlodipine (10 microM) and R56865 (10 microM), but not by BW755C (10 microM). Superfusion of papillary muscles with 15(S)-HpETE (20 microM) induced a membrane depolarization and a marked reduction in the AP amplitude and duration. Concomitantly, a transient positive inotropic effect and a progressive rise in diastolic tension were observed. These alterations were maximal after 15 min and associated with delayed after-depolarizations (DADs) and after-contractions. Every alteration was inhibited by BW755C (10 microM) and R56865 (30 microM), but not by Amlodipine (1 microM). Ryanodine (1 microM), a blocker of sarcoplasmic reticulum calcium channel, only prevented the appearance of DADs and after-contractions. Only BW755C exhibited ROS scavenging properties.. ROOH induced enzyme leakage and electromechanical alterations in cardiac cells. These effects of ROOH implicated oxidative mechanisms and resulted in an intracellular calcium overload.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Action Potentials; Amlodipine; Animals; Benzothiazoles; Calcium; Calcium Channel Blockers; Cells, Cultured; Dose-Response Relationship, Drug; Free Radical Scavengers; Free Radicals; Guinea Pigs; L-Lactate Dehydrogenase; Leukotrienes; Lipid Peroxides; Membrane Potentials; Myocardium; Necrosis; Piperidines; Rats; Rats, Wistar; Respiratory Burst; Ryanodine; Thiazoles; Vasoconstrictor Agents