Naloxone reduces release of creatine kinase in the isolated ischemic rat heart

The effects of naloxone, propranolol, or both on the release of creatine kinase (CK) from the isolated ischemic rat heart were studied. Naloxone at concentrations of 1.1 and 3.6 mmole liter-1 in the perfusate at a rate of 1-2 ml min-1 reduced the release of CK from the isolated ischemic rat heart during myocardial ischemia in a dose-dependent manner. Propranolol at a concentration of 7 mumole liter-1 in the perfusate also reduced the release of CK. Addition of naloxone (1.1 mmole liter-1) to propranolol further reduced the release of CK. The effect of the joint administration of the two drugs seemed to be additive.     

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca²⁺ _i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca²⁺ _i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca²⁺ _i levels of the heart. The fluorescence ratio technique was applied to estimate Ca²⁺ _i. The disappearance of mechanical activity of the heart preceded that of the Ca²⁺ _i transient in the first 2 min of ischemia. The slope of upstroke of the Ca²⁺ _i transient, reflecting Ca²⁺ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca²⁺ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca²⁺ _i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca²⁺ _i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca²⁺ _i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca²⁺ _i handling and sustained depression of mechanical performance.     

Accumulation of specific ceramides in ischemic/reperfused rat heart; effect of ischemic preconditioning.

Ceramide signalling has been implicated in the mechanism of myocardial ischemia/reperfusion injury (IR). This study tested the hypothesis that ceramides containing a specific amino-linked acyl residue mediate the injury, and that ischemic preconditioning (IPC) affords myocardial protection because it prevents increased ceramide accumulation in IR myocardium. Perfused rat hearts were subjected either to the sham perfusion or to 30 min global ischemia, 30 min ischemia/30 min reperfusion (IR) or were preconditioned prior to the standard IR. The ventricles were harvested for biochemical assay that involved transmethylation of ceramide amino-linked acyl residues, and gas liquid chromatography measurement of acyl methyl esters. Fourteen ceramides containing myrystic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, arachidonic, eicosapentaenoic, behenic, docosapentaenoic, docosahexaenoic or nervonic acid were identified in the myocardium of rats. The total basal ceramide concentration in the myocardium was 135 nmol/g tissue, and it was increased by 14.1% and 48.4% in the ischemia and IR group, respectively. However, in fact, IR increased the accumulation of only 7 out of 14 ceramides identified in the heart (i.e., those containing palmitic, stearic, oleic, linoleic, and arachidonic acid), and the relative magnitude of these increases varied between the particular ceramides and was independent from their basal tissue concentration. IPC improved postischemic hemodynamic recovery and partially prevented the reperfusion-induced increases in these 7 ceramides, while the other ceramides were unaffected by IPC. These results support the role of the specific ceramide signalling in the mechanism of myocardial IR injury. We speculate that by preventing tissue accumulation of certain ceramides, IPC attenuates this signalling, that adds to the mechanism of myocardial protection afforded by IPC.     

Characterization of modes of release of amino acids in the ischemic/reperfused rat cerebral cortex

Brain extracellular levels of glutamate, aspartate, GABA and glycine increase rapidly following the onset of ischemia, remain at an elevated level during the ischemia, and then decline over 20-30 min following reperfusion. The elevated levels of the excitotoxic amino acids, glutamate and aspartate, are thought to contribute to ischemia-evoked neuronal injury and death. Calcium-evoked exocytotic release appears to account for the initial (1-2 min) efflux of neurotransmitter-type amino acids following the onset of ischemia, with non-vesicular release responsible for much of the subsequent efflux of these and other amino acids, including taurine and phosphoethanolamine. Extracellular Ca(2+)-independent release is mediated, in part by Na(+)-dependent amino acid transporters in the plasma membrane operating in a reversed mode, and by the opening of swelling-induced chloride channels, which allow the passage of amino acids down their concentration gradients. Experiments on cultured neurons and astrocytes have suggested that it is the astrocytes which make the primary contribution to this amino acid efflux. Inhibition of phospholipase A(2) attenuates ischemia-evoked release of both amino and free fatty acids from the rat cerebral cortex indicating that this group of enzymes is involved in amino acid efflux, and also accounting for the consistent ischemia-evoked release of phosphoethanolamine. It is, therefore, possible that disruption of membrane integrity by phospholipases plays a role in amino acid release. Recovery of amino acid levels to preischemic levels requires their uptake by high affinity Na(+)-dependent transporters, operating in their normal mode, following restoration of energy metabolism, cell resting potentials and ionic gradients.     

Effects of chronic caloric restriction on mitochondrial respiration in the ischemic reperfused rat heart

Dietary restriction increases life span and delays the development of age-related diseases in rodents. We have recently demonstrated that chronic dietary restriction is beneficial on recovery of heart function following ischemia. We studied whether the metabolic basis of this benefit is associated with alterations in mitochondrial respiration. Male Wistar rats were assigned to an ad libitum-fed (AL) group and a food restricted (FR) group, in which food intake was reduced to 55% of the amount consumed by the AL group. Following an 8-month period of restricted caloric intake, isolated working hearts perfused with glucose and high levels of fatty acids were subjected to global ischemia followed by reperfusion. At the end of reperfusion, total heart mitochondria was respiration was assessed in the presence of pyruvate, tricarboxylic acid intermediates, and palmitoylcarnitine. Recovery of heart function following ischemia was greater in FR hearts compared to AL hearts. Paralleling these changes in heart function was in increase in state 3 respiration with pyruvate. The respiratory control ratios in the presence of pyruvate and tricarboxylic acid intermediates were higher in FR hearts compared to AL hearts, indicating well-coupled mitochondria. Overall energy production, expressed as the ADP:O ratio and the oxidative phosphorylation rate, was also improved in FR hearts. Our results indicate that the beneficial effect of FR on recovery of heart function following ischemia is associated with changes in mitochondrial respiration.     

Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart

Brief, nonlethal episodes of ischemia in the mammalian heart provide cardioprotection against the detrimental effects of a longer duration ischemia. The manifestation of this preconditioning (PC) phenomenon is initiated by the enhanced phosphorylation state of signal transduction proteins. We reported previously that PC is decreased in the aged rat myocardium. Although the mechanism responsible for this loss is not understood, a reduction in the phosphorylation of critical proteins associated with PC may be postulated. Experiments were conducted to investigate whether PC in the aged heart can be restored with the inhibition of endogenous protein phosphatases thereby enhancing phosphorylation of signaling proteins. Levels of phosphatase activities were also assessed with adult heart aging. Hearts from young adult (3-4 mo.) and aged (21-22 mo.) Fischer-344 rats were perfused in the presence or absence of okadaic acid (OKA; 0.1 microM). Aged adult hearts were either not preconditioned or were preconditioned with two PC cycles (5 min ischemia/5 min reperfusion). Myocardial cellular death that developed with a subsequent ischemia was determined with triphenyltetrazolium. With PC, 55% of the aged heart after ischemia was no longer viable. OKA administered before or after ischemia reduced this ischemia-induced cellular death by 29%. Without PC, OKA reduced viability 18% only when present before and after the ischemic episode. OKA in the ischemic young heart during reperfusion reduced the loss of viability 31%. The Protein Phosphatase 2A (PP2A) activity was found to be up to 82% greater in ventricular myocardium of aged rats. In conclusion, aging-induced changes in protein dephosphorylation may be one mechanism reducing the manifestation of preconditioning in the aged heart.     

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of infarct size by orally administered des-aspartate-angiotensin I in the ischemic reperfused rat heart

Occlusion of the left main coronary artery for 45 min caused sizable infarct scaring of the left ventricular wall in the rat heart at 14 days post-reperfusion. Daily oral administration of des-aspartate-angiotensin I (DAA-I) for 14 days attenuated the area of the infarct scar and transmurality. The attenuation was dose-dependent and biphasic; maximum effective dose was 1524 nmol/kg, and doses higher than this were progressively inactive. The exact mechanism of the biphasic attenuation is not known, and receptor down-regulation by internalization, which has been implicated in a similar biphasic nature for the anticardiac hypertrophic action of DAA-I, could be a likely cause. Indomethacin (101 μmol/kg, i.p.), administered sequentially after the daily oral dose of DAA-I (1524 nmol/kg), completely inhibited the attenuation at 14 days post-reperfusion, indicating that prostaglandins may be involved in transducing the attenuation. The present findings support earlier indications that DAA-I exerts protective actions in cardiovascular pathologies in which angiotensin II is implicated. It is suggested that DAA-I exerts the cardioprotective action by acting on the same indomethacin-sensitive angiotensin AT₁ receptor. Although similar array of protective actions are also seen with another endogenous angiotensin, angiotensin-(1–7), the present findings demonstrate for the first time the ability of an endogenous angiotensin to reduce the infarct size of an ischemic-reperfusion injured rat heart.     

Malondialdehyde is a biochemical marker of peroxidative damage in the isolated reperfused rat heart

Concentration of MDA in isolated control, ischemic, and reperfused rat hearts was determined by using a new sensitive and reproducible HPLC method on the perchloric acid extract of the freeze-clamped tissues. By means of this HPLC assay for the direct measurements of MDA, concentrations of adenine nucleotide derivatives were also obtained in the same chromatographic run. Under the present experimental conditions, no detectable amount of MDA could be observed in control hearts while ischemic hearts showed 0.009moles/g d. w. of MDA (s. d. = 0.001), this value representing the sensitivity limit of the method employed. On the contrary, reperfused hearts showed 0.118moles/g d.w. of MDA (s.d. = 0.036), thereby indicating that this compound originates from an oxygen free radical-mediated breakdown of phospholipids and demonstrating the existence of quantifiable molecular damage occurring upon reperfusion. On the whole, our data demonstrate that MDA, if properly assayed, is a reliable index of peroxidative injury to biological systems. (Mol Cell Biochem116: 193–196, 1992)Abbreviations Ado Adenosine - d.w. dry weight - HPLC High-Performance Liquid Chromatography - Hyp Hypoxanthine - Ino Inosine - MDA Malondialdehyde - Xan Xanthine     

Detection of ferryl myoglobin in the isolated ischemic rat heart

Reflectance spectroscopy was utilized to monitor the oxidation states of myoglobin (Mb) in isolated, buffer-perfused rat hearts. Hearts were subjected to 30 min global, no-flow ischemia, followed by reperfusion under anoxic conditions. The addition of Na₂S to the buffer at reperfusion permitted the detection of ferryl myoglobin (MbIV) as its sulfmyoglobin derivative. The accumulation of MbIV was prevented by addition of ascorbic acid (1 mM), ergothioneine (2mM), or desferal (1mM) to the buffer prior to ischemia. Ascorbate and other agents have been previously shown to serve as one-electron reductants of MbIV. We propose that during the early phases of ischemia, deoxymyoglobin is oxidized to MbIV by residual H₂O₂. It also seems reasonable that the peroxidative activity of Mb(IV), during oxygenated reperfusion, might lead to cellular damage if this hypervalent form of Mb is not reduced.     

Alteration of 1,2-diacylglycerol content in ischemic and reperfused heart

Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN₃ were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase.A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05–0.8 µM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates.The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.Abbreviations Ap₅A P¹ - P⁵-di(adenosine-5) Pentaphosphate - ATP-DPH ATP Diphosphohydrolase - DCCD N,N Dicyclohexycarbodiimide - Fru-P₂ase Fructose 1,6-biphosphatase - SDS Sodium Dodecyl Sulfate - TLC Thin Layer Chromatography     

Ruthenium red-sensitive and Ruthenium Red-insensitive release of calcium by mitochondria isolated from rat liver and from rat heart

EGTA (ethanedioxybis(ethylamine)tetra-acetic acid) induced a release of Ca²⁺ from mitochondria isolated from both rat liver and rat heart that was inhibited by Ruthenium Red. The concentration of Ruthenium Red giving half-maximal inhibition was about 350 pmol/mg of protein, a value approximately 7 times greater than that giving half-maximal inhibition of the initial rate of Ca²⁺ transport. The EGTA-induced release of Ca²⁺ was temperature-dependent and was inhibited by the local anaesthetic, nupercaine.Pi, acetate, and tributyltin in the presence of Cl^?, inhibited the Ruthenium Red-sensitive Ca²⁺ release induced by EGTA, whereas these agents enhanced the Ruthenium Red-insensitive release of Ca²⁺ induced by acetoacetate in liver and heart mitochondria and by Na⁺ in heart mitochondria.     

Early preconditioning prevents the loss of endothelial nitric oxide synthase and enhances its activity in the ischemic/reperfused rat heart

Cardiac ischemia may be responsible for either the loss of endothelial nitric oxide synthase (eNOS) or changes in its activity, both conditions leading to coronary dysfunction. We investigated whether early ischemic preconditioning was able to preserve eNOS protein expression and function in the ischemic/reperfused myocardium. Langendorff-perfused rat hearts were subjected to 20 min global ischemia, followed by 30 min reperfusion (I/R). A second group of hearts was treated as I/R, but preconditioned with three cycles of 5 min-ischemia/5 min-reperfusion (IP). Cardiac contractility markedly decreased in I/R, consistently with the rise of creatine kinase (CK) activity in the coronary effluent, whilst ischemic preconditioning significantly improved all functional parameters and reduced the release of CK. Western blot analysis revealed that the amount of eNOS protein decreased by 54.2% in I/R with respect to control (p < 0.01). On the other hand, NOS activity was not significantly reduced in I/R, as well as cGMP tissue levels, suggesting that a parallel compensatory stimulation of this enzymatic activity occurred during ischemia/reperfusion. Ischemic preconditioning completely prevented the loss of eNOS. Moreover, both NOS activity and cGMP tissue level were significantly higher (p < 0.05) in IP (12.7 +/- 0.93 pmol/min/mg prot and 58.1 +/- 12.2 fmol/mg prot, respectively) than I/R (7.34 +/- 2.01 pmol/min/mg prot and 21.4 +/- 4.13 fmol/mg prot, respectively). This suggest that early ischemic preconditioning may be useful to accelerate the complete recovery of endothelial function by preserving the level of cardiac eNOS and stimulating the basal production of nitric oxide.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Evidence for alpha-1 adrenergic receptor regulation of atriopeptin release from the isolated rat heart

Atrial myocardium is the source of a recently described peptide hormone termed atriopeptin. Atriopeptin is thought to have a role in the regulation of systemic arterial pressure, fluid balance and plasma electrolyte homeostasis. Isolated rat hearts release atriopeptin into the coronary effluent, and we have found that this release is stimulated by the administration of norepinephrine, a compound with alpha and beta adrenergic properties. Infusion of the pure beta-receptor agonist, isoproterenol, failed to stimulate the release; however, the alpha-1 receptor agonist phenylephrine induced the release in a dose-dependent manner. The stimulation of atriopeptin release by norepinephrine and phenylephrine was inhibited by alpha-blockade with phentolamine. Administration of BHT-920, a selective alpha-2 agonist, had no effect on atriopeptin release. We conclude that atriopeptin secretion by the atrial myocyte is stimulated by activation of the alpha-1 adrenergic receptor. This finding suggests an involvement of the sympathetic nervous system in the physiologic regulation of the secretion of this hormone.     

Inhibition of ubiquinone synthesis in isolated rat heart under an ischemic condition

1. The biosynthesis of ubiquinone (UQ) in isolated rat heart under ischemic and hypoxic conditions was investigated. 2. Under ischemic perfusion, a greater amount of biosynthetic intermediates, 3-nonaprenyl and 3-decaprenyl-4-hydroxybenzoate (PPHBs) was accumulated and a smaller amount of UQ-9 and -10 was synthesized when compared with normal conditions. 3. The accumulation of PPHBs was observed without forming UQs during anaerobic perfusion. 4. Hydroxylation which is the following reaction of PPHBs for the biosynthesis of UQ in rat heart, was proceeded by the monooxygenase(s) depending upon the oxygen concentrations.     

Regulation of cardiomyocyte apoptosis in ischemic reperfused mouse heart by glutathione peroxidase

Apoptosis, a genetically controlled programmed cell death, has been found to play a role in ischemic reperfusion injury in several animal species including rats and rabbits. To examine whether this is also true for other animals, an isolated perfused mouse heart was subjected to 30 min of ischemia followed by 2 h of reperfusion. Experiments were terminated before ischemia (baseline), after ischemia, and at 30, 60, 90 and 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while DNA laddering was evaluated by subjecting the DNA obtained from the cardiomyocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The results of our study revealed that apoptotic cells appear only after 60 min of reperfusion as demonstrated by the intense fluorescence of the immunostained genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). Since our previous studies showed a role of glutathione peroxidase (GSHPx) in apoptotic cell death, we performed identical experiments using isolated hearts from GSHPx-l knockout mice and transgenic mice overexpressing GSHPx-l. GSHPx-l knockout mice showed evidence of apoptotic cell death even after 30 min of reperfusion. Significant number of apoptotic cells were found in the cardiomyocytes as compared to non-transgenic control animals. To the contrary, very few apoptotic cells were found in the hearts of the transgenic mice overexpressing GSHPx-l. Hearts of GSHPx-l knockout mice were more susceptible to ischemia/reperfusion injury while transgenic mice overexpressing GSHPx- 1 were less susceptible to ischemia reperfusion injury compared to non-transgenic control animals. The results of this study clearly demonstrate a role of GSHPx in ischemia/reperfusion-induced apoptosis in mouse heart.     

Somatostatin release from isolated perfused rat stomach.

Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and immunoreactively with synthetic somatostatin. Infusion of glucagon (10^?7 M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated by infusion of theophylline (10^?3 M) and dibutyryl cyclic AMP (10^?3 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.     

Volume-regulatory potassium release from isolated perfused rat kidney

The present study has been performed to test for cell volume regulatory potassium release from the isolated perfused rat kidney exposed to hypotonic perfusate and for its sensitivity to potassium channel blocker barium and calcium channel blocker verapamil. Replacement of 25 mmol/l NaCl with 50 mmol/l mannitol has little effect on effluent potassium activity, whereas subsequent omission of mannitol from the perfusate leads to a transient increase of effluent potassium activity, reflecting volume regulatory potassium release. Barium (1 mmol/l) leads to a marked transient decrease of effluent potassium activity, pointing to net cellular uptake of potassium. Verapamil (1 mumol/l) leads to a slight decrease of effluent potassium activity. Both barium and verapamil virtually abolish the rapid, transient increase of effluent potassium activity upon exposure to hypotonic perfusates. Thus, the substances either block or markedly retard volume regulatory potassium release. The apparent renal vascular resistance is transiently increased by exposure to hypotonic perfusates and by barium, but is reduced by verapamil. Cell volume regulation of isolated perfused mouse straight proximal tubules is retarded but not abolished by verapamil (0.1 mmol/l). In conclusion, cellular potassium release from rat kidney can be determined by continuous measurement of effluent potassium activity. The volume regulatory potassium release and cell volume regulation are impaired by both barium and verapamil. The persisting cell volume regulation could be due either to slow potassium release and/or some mechanism independent of potassium.