ryanodine and Reperfusion-Injury

Ischemia-reperfusion injury is the major complication of abdominal aortic surgery, and it mainly affects the lower extremities and remote organs. In the present study, the electrophysiological alterations in diaphragm that underlie the post-operative respiratory dysfunction were investigated. Wistar Albino rats were randomly divided into two groups: SHAM (only laparotomy was performed) and IR (abdominal aorta was clamped for 30min and reperfused for 2h). Following the operational period diaphragm muscles were isolated and electrophysiological experiments were carried out in-vitro. 3nM Ryanodine application, Na

Topics: 4-Aminopyridine; Action Potentials; Analysis of Variance; Animals; Biophysics; Diaphragm; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Agonists; Ischemia; Male; Muscle Contraction; N-Methylaspartate; Potassium Channel Blockers; Rats; Rats, Wistar; Reperfusion Injury; Ryanodine; Time Factors

This study further investigates the mechanisms responsible for the effects of acute and severe hypoxia, and subsequent reoxygenation, on the contractility of isolated rat mesenteric arteries. In noradrenaline (NA)-contracted arteries, hypoxia caused a relaxation to near baseline levels. Reoxygenation resulted in an immediate transient contraction before tension returned more slowly to prehypoxia levels. Similar responses to hypoxia were observed in tissues precontracted by addition of KCl (60 mM) or U46619 (10 microM); however, the transient contraction upon reoxygenation was absent (KCl) or reduced (U46619). Responses to hypoxia were independent of changes in intracellular calcium ([Ca2+]i), while those to reoxygenation were accompanied by corresponding changes in [Ca2+]i and were completely abolished by ryanodine. In NA-contracted tissues, all responses were unaffected by endothelial removal or by inhibitors of nitric oxide synthase and cyclooxygenase. The K+ channel blockers triethylamine (TEA), glibenclamide, and 4-aminopyridine (4-AP) had no effect on the responses to hypoxia. The transient contractile response to reoxygenation was, however, significantly reduced in the presence of 4-AP. The response to reoxygenation, but not that to hypoxia, was inhibited by the antioxidant dithiothreitol (DTT) and the NAD(P)H-oxidase inhibitor diphenyliodonium (DPI). These data suggest that hypoxic vasodilation occurs independently of reductions in [Ca2+]i. Alternatively, transient contractions on reoxygenation are dependent upon the generation of reactive oxygen species and the release of stored Ca2+.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 4-Aminopyridine; Adenosine Triphosphate; Animals; Anti-Arrhythmia Agents; Calcium; Endothelium, Vascular; Ethylamines; Glyburide; Hypoxia; Male; Mesenteric Arteries; Muscle Contraction; Norepinephrine; Oxygen; Potassium; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Wistar; Reperfusion Injury; Ryanodine

Elevated levels of free fatty acids (FFA) have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the effects of immunosuppressants agents, calcineurin inhibitors and blockade of endoplasmic reticulum (ER) calcium channels on free fatty acid formation and efflux in the ischemic/reperfused (I/R) rat brain. Changes in the extracellular levels of arachidonic, docosahexaenoic, linoleic, myristic, oleic and palmitic acids in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical cup technique. A 20-min period of ischemia elicited large increases in the efflux of all six FFAs, which were sustained during the 40 min of reperfusion. Cyclosporin A (CsA) and trifluoperazine, which reportedly inhibit the I/R elicited opening of a mitochondrial permeability transition (MPT) pore, were very effective in suppressing ischemia/reperfusion evoked release of all six FFAs. FK506, an immunosuppressant which does not directly affect the MPT, but is a calcineurin inhibitor, also suppressed the I/R-evoked efflux of FFAs, but less effectively than CsA. Rapamycin, a derivative of FK506 which does not inhibit calcineurin, did not suppress I/R-evoked FFA efflux. Gossypol, a structurally unrelated inhibitor of calcineurin, was also effective, significantly reducing the efflux of docosahexaenoic, arachidonic and oleic acids. As previous experiments had implicated elevated Ca(2+) levels in the activation of phospholipases with FFA formation, agents affecting endoplasmic reticulum stores were also evaluated. Dantrolene, which blocks the ryanodine receptor (RyR) channel of the ER, significantly inhibited I/R-evoked release of docosahexaenoic, arachidonic, linoleic and oleic acids. Ryanodine, which can either accentuate or block Ca(2+) release, significantly enhanced ischemia/reperfusion-elicited efflux of linoleic acid, with non-significant increases in the efflux of myristic, arachidonic, palmitic and oleic acids. Xestospongin C, an inhibitor of the inositol triphosphate (IP(3)R) channel, failed to affect I/R-evoked FFA efflux. Thapsigargin, an inhibitor of the Ca(2+)-ATPase ER uptake pump, elicited significant elevations in the efflux of myristic, arachidonic and linoleic acids, in the absence of ischemia. Collectively, the data suggest an involvement of both ER and mitochondrial Ca(2+) stores in the chain of events which lead to PLA(2) activation and

Topics: Animals; Calcineurin; Calcineurin Inhibitors; Calcium Channels; Cerebral Cortex; Cyclosporine; Dantrolene; Endoplasmic Reticulum; Enzyme Inhibitors; Fatty Acids, Nonesterified; Gossypol; Immunosuppressive Agents; Macrocyclic Compounds; Male; Oxazoles; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ryanodine; Sirolimus; Tacrolimus; Thapsigargin; Trifluoperazine

Junctional SR membrane vesicles have been isolated from chronically failing human hearts explanted at transplant operations. Vesicles have been incorporated into artificial planar phospholipid bilayers and the activity of single calcium-release channels investigated under voltage-clamp conditions. The properties of these channels are similar to those previously reported from normal animal tissue and do not provide evidence that the function of individual calcium-release channels is altered in the failing heart. Using radio-labelled ryanodine binding as a specific marker for the calcium-release channel, we demonstrate that, in the sheep heart, ischaemia results in the degradation of the calcium-release channel. The activation of proteases and oxidant stress in the ischaemic and re-perfused post-ischaemic myocardium are likely mediators of cell injury. Using the protease trypsin and the photosensitisation of rose bengal to generate the reactive oxygen species (ROS) singlet oxygen and superoxide radicals we demonstrate a direct effect on the calcium-release channel in vitro. Exposure of junctional SR vesicles to trypsin or oxidant stress resulted in the progressive loss of specific ryanodine binding and the degradation of high molecular weight proteins identified by polyacrylamide gel electrophoresis. The activity of single channels was also modified during exposure to proteolysis or oxidant stress; an initial increase in channel opening was observed followed by irreversible loss of channel function. Degradation of specific proteins, such as the calcium-release channel, may contribute to contractile dysfunction in the ischaemic and reperfused post-ischaemic myocardium.

Topics: Adult; Aged; Animals; Calcium Channels; Coronary Disease; Disease Models, Animal; Endopeptidases; Enzyme Activation; Heart Failure; Humans; Middle Aged; Reperfusion Injury; Ryanodine; Sarcoplasmic Reticulum; Sheep; Superoxides

The effect of normothermic ischemia and ischemia/reperfusion on the function of cardiac sarcoplasmic reticulum (CSR) was investigated using a modified Langendorff perfusion of isolated rat hearts. The function of the CSR was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates. The contribution of the ryanodine-sensitive portion of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The Ca2+ uptake rate of the CSR decreased progressively with increasing duration of ischemia, but this depression was much less when uptake was assayed in the presence of ryanodine. The depression in CSR Ca2+ uptake preceded ischemic contracture. Ryanodine and ruthenium red stimulated uptake almost equally in control hearts, but ruthenium red was much less effective than ryanodine after ischemia. This difference could not be overcome by increasing the ruthenium red concentration. These results confirm the suggestion that the Ca2+ release channel is inappropriately opened after ischemia. The CSR uptake rates were almost completely restored at 15 minutes of reperfusion after 5 and 10 minutes of ischemia but were only partially restored after 15 minutes of ischemia. At reperfusion, mechanical function (end-diastolic pressure and peak systolic developed pressure) was markedly depressed after only 15 minutes of ischemia. The degree of "stunning" correlated well with the depression of CSR function in individual hearts. The decreased Ca2+ uptake of the CSR was not due to a buildup of ADP in the homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; In Vitro Techniques; Male; Myocardium; Rats; Rats, Inbred Strains; Reperfusion Injury; Ryanodine; Sarcoplasmic Reticulum; Stimulation, Chemical

Restoration of blood flow to the ischemic myocardium prevents continuing cell necrosis, but reperfusion may cause irreversible damage to potentially salvable tissue, possibly through the generation of toxic reactive oxygen species. Intracellular calcium overload, secondary to membrane lipid peroxidation, has been proposed as a general pathogenic mechanism. However, using the photosensitisation of rose bengal to generate singlet oxygen and superoxide radicals, we demonstrate a direct effect of reactive oxygen species on the cardiac sarcoplasmic reticulum calcium-release channel. Exposure of heavy sarcoplasmic reticulum vesicles to reactive oxygen species in vitro resulted in the progressive loss of specific [3H]ryanodine binding and the degradation of high molecular weight proteins identified by polyacrylamide gel electrophoresis. The gating of single channels incorporated into artificial planar phospholipid bilayers was modified during the exposure to reactive oxygen species: an initial increase in open probability being followed by irreversible loss of channel function. Degradation by reactive oxygen species of specific proteins, such as the calcium-release channel, may contribute to in vivo reperfusion injury.

Topics: Animals; Calcium Channels; Free Radicals; Heart; In Vitro Techniques; Oxygen; Reperfusion Injury; Rose Bengal; Ryanodine; Sarcoplasmic Reticulum; Sheep; Superoxides