The role of adenylate cyclase in ischemic preconditioning in the rat heart: a cytochemical study

Using catalytic cytochemistry the AC activity was studied during ischemic preconditioning (IP) (5 min occlusion of LAD and 10 min reperfusion) followed by 30 min regional ischemia in isolated Langendorff-perfused rat heart. In controls the specific precipitate of AC reaction was found on the sarcolemma (SL) and the junctional sarcoplasmic reticulum (JSR) of cardiomyocytes. After prolonged ischemia the reaction product was absent, whereas IP followed by prolonged ischemia protected the AC activity on SL and JSR. IP-induced enhancement of AC activity in this model was accompanied by significant reduction of ischemia/reperfusion fibrillation. The results suggest involvement of AC system in mechanisms of IP.     

Evidence for extracellular deamination of adenosine in the rat heart

• 1.1. In rat heart perfused with adenosine (10⁻⁶M), dilazep (10⁻⁴M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused.
• 2.2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular.
• 3.3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%.
• 4.4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries.
• 5.5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.

     

Morphine treatment enhances extracellular ATP enzymolysis and adenosine generation in rat astrocytes

Recent studies have shown that astrocytes play important roles in ATP degradation and adenosine (a well known analgesic molecule) generation, which are closely related to pain signaling pathway. The aim of this study was to investigate whether morphine, a well known analgesic drug, could affect the speeds of ATP enzymolysis and adenosine generation in rat astrocytes. Intracellular calcium concentration ([Ca(2+)](i)) of astrocyte was measured by flow cytometry, and the time points that morphine exerted notable effects were determined for subsequent experiments. Cultured astrocytes were pre-incubated with morphine (1 μmol/L) and then were incubated with substrates, ATP and AMP, for 30 min. The speeds of ATP enzymolysis and adenosine generation were measured by high performance liquid chromatography (HPLC). The results showed that both 1.5 and 48 h of morphine pre-incubation induced maximal ATP enzymolysis speed in astrocytes among all the time points, and there was no statistical difference of ATP enzymolysis speed between morphine treatments for 1.5 and 48 h. As to adenosine, morphine pre-incubation for 1.5 h statistically increased adenosine generation, which was degraded from AMP, in cultured astrocytes compared with control group. However, no difference of adenosine generation was observed after 48 h of morphine pre-incubation. These results indicate that treatment of morphine in vitro dynamically changes the concentrations of ATP and adenosine in extracellular milieu of astrocytic cells. In addition, astrocyte can be regarded as at least one of the target cells of morphine to induce changes of ATP and adenosine levels in central nervous system.     

MitoK ATP -dependent changes in mitochondrial volume and in complex II activity during ischemic and pharmacological preconditioning of langendorff-perfused rat heart

It has been proposed that activation of the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) is part of signaling pathways triggering the cardioprotection afforded by ischemic preconditioning of the heart. This work was to analyze the mitochondrial function profile of Langendorff-perfused rat hearts during the different phases of various ischemia-reperfusion protocols. Specifically, skinned fibers of ischemic preconditioned hearts exhibit a decline in the succinate-supported respiration and complex II activity during ischemia, followed by a recovery during reperfusion. Meanwhile, the apparent affinity of respiration for ADP (which reflects the matrix volume expansion) is increased during preconditioning stimulus and, to a larger extent, during prolonged ischemia. This evolution pattern is mimicked by diazoxide and abolished by 5-hydroxydecanoate. It is concluded that opening the mitoK_ATP channel mediates the preservation of mitochondrial structure-function via a mitochondrial matrix shrinkage and a reversible inactivation of complex II during prolonged ischemic insult.     

ATP as a source of interstitial adenosine in the rat heart.

The contribution of neuronal ATP to interstitial adenosine levels was investigated in isolated perfused rat hearts. Ventricular surface transudates, representing interstitial fluid, were analyzed for norepinephrine, ATP, and adenosine. Exocytotic release of norepinephrine was induced by electrical stimulation of cardiac efferents emanating from the stellate ganglion. Ganglion stimulation increased contractility, interstitial norepinephrine, ATP, and adenosine. Interstitial adenosine was 11- to 27-fold higher than interstitial ATP, suggesting that the released ATP is unlikely the only source of adenosine. In the presence of AOPCP (alpha,beta-methyleneadenosine 5'-diphosphate), an ecto-5'-nucleotidase inhibitor, the ganglion-stimulated increase in interstitial ATP and adenosine reached levels similar to those in the absence of AOPCP, also suggesting that adenosine does not derive from extracellular ATP. The perfusate Ca2+ was raised from 1 to 4 mM to determine the importance of the enhanced contractile function on the levels of norepinephrine, ATP, and adenosine. The results were increases in contractility and interstitial norepinephrine, ATP, and adenosine, which were not suppressed with atenolol, indicating a norepinephrine-independent release of ATP and adenosine. Reserpine treatment and administration of guanethidine depleted the catecholamine stores and diminished the catecholamine release, respectively. However, neither agent altered Ca2+-induced increases in ATP and adenosine. It is concluded that the amount of neuronal-derived ATP is low and most likely does not contribute significantly to interstitial levels of adenosine. Furthermore, elevations in interstitial norepinephrine, ATP, and adenosine are associated with neuronal-independent increases in contractile function.     

Evidence for calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart.

Previous studies have suggested that calcitonin gene-related peptide (CGRP) may play an important role in the mediation of ischemic preconditioning. In the present study, we examined the release of CGRP during ischemic preconditioning and the effect of preconditioning frequency on this effect in the isolated rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant cardiac dysfunction and an increase in the release of creatine kinase (CK) during reperfusion. Preconditioning with one, two or three cycles of 5-min ischemia and 5-min reperfusion caused a marked improvement of cardiac function and a decrease in the release of CK, and there was no difference in the degree of improvement among groups. The protective effects of ischemic preconditioning were abolished by the CGRP receptor antagonist CGRP(8-37). A single preconditioning cycle induced a significant increase in the release of CGRP in the coronary effluent. In the hearts treated with two or three preconditioning cycles, the level of CGRP was highest in the first cycle, and was gradually decreased with increasing number of cycles of preconditioning. These results suggest that the protective effects of ischemic preconditioning are mediated by endogenous CGRP in the isolated rat heart.     

Role of mitochondrial and sarcolemmal K(ATP) channels in ischemic preconditioning of the canine heart

We tested whether mitochondrial or sarcolemmal ATP-sensitive K(+) (K(ATP)) channels play a key role in ischemic preconditioning (IP) in canine hearts. In open-chest beagle dogs, the left anterior descending artery was occluded four times for 5 min each with 5-min intervals of reperfusion (IP), occluded for 90 min, and reperfused for 6 h. IP as well as cromakalim and nicorandil (nonspecific K(ATP) channel openers) markedly limited infarct size (6.3 +/- 1.2, 8.9 +/- 1.9, and 7.2 +/- 1.6%, respectively) compared with the control group (40.9 +/- 4.1%). A selective mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate, partially blunted the limitation of infarct size in the animals subjected to IP and those treated with cromakalim and nicorandil (21.6 +/- 3.8, 25.1 +/- 4.6, and 19.8 +/- 5.2%, respectively). A nonspecific K(ATP) channel blocker, glibenclamide, completely abolished the effect of IP (38.5 +/- 6.2%). Intracoronary or intravenous administration of a mitochondria-selective K(ATP) channel opener, diazoxide, at >100 micromol/l could only partially decrease infarct size (19.5 +/- 4.3 and 20.1 +/- 4.4%, respectively). In conclusion, mitochondrial and sarcolemmal K(ATP) channels independently play an important role in the limitation of infarct size by IP in the canine heart.     

Differential role of sarcolemmal and mitochondrial K(ATP) channels in adenosine-enhanced ischemic preconditioning

Adenosine-enhanced ischemic preconditioning (APC) extends the protection afforded by ischemic preconditioning (IPC) by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. The purpose of this study was to determine whether APC is modulated by ATP-sensitive potassium (K(ATP)) channels and to determine whether this modulation occurs before ischemia or during reperfusion. The role of K(ATP) channels before ischemia (I), during reperfusion (R), or during ischemia and reperfusion (IR) was investigated using the nonspecific K(ATP) blocker glibenclamide (Glb), the mitochondrial (mito) K(ATP) channel blocker 5-hydroxydecanoate (5-HD), and the sarcolemmal (sarc) K(ATP) channel blocker HMR-1883 (HMR). Infarct size was significantly increased (P < 0.05) in APC hearts with Glb-I, Glb-R, and 5-HD-I treatment and partially with 5-HD-R. Glb-I and Glb-R treatment significantly decreased APC functional recovery (P < 0.05 vs. APC), whereas 5-HD-I and 5-HD-R had no effect on APC functional recovery. HMR-IR significantly decreased postischemic functional recovery (P < 0.05 vs. APC) but had no effect on infarct size. These data indicate that APC infarct size reduction is modulated by mitoK(ATP) channels primarily during ischemia and suggest that functional recovery is modulated by sarcK(ATP) channels during ischemia and reperfusion.     

血红素氧合酶-1在离体大鼠心肌缺血预适应中的作用

目的:分别观察给予HO-1诱导剂和抑制剂对心肌相对缺血再灌注损伤和缺血预适应的影响,探讨HO-1在缺血预适应中的作用.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC)、HO-1诱导剂 缺血/再灌损伤组(HM)、HO-1抑制剂 缺血预适应组(ZP).心肌缺血/再灌损伤采用相对缺血/再灌损伤模型,缺血预适应则为相对缺血5min恢复5min,反复2次.测定心功能、MDA及HO-1活性变化.结果:HM组HO-1活性升高,心功能恢复率均显著高于IR组(P＜0.01),MDA含量显著低于IR组(P＜0.05).ZP组活性降低,心功能恢复率显著低于PC组(P＜0.05),MDA含量显著高于PC组(P＜0 05).结论:HO-1是缺血预适应释放的内源性活性物质之一.     

Calcitonin gene-related peptide-mediated ischemic preconditioning in the rat heart: influence of age

In the present study, we examined whether age-related reduction of ischemic preconditioning is related to calcitonin gene-related peptide (CGRP) release in the rat heart. Thirty minutes of global ischemia and 40 min of reperfusion caused a significant decrease of cardiac function and a marked increase of creatine kinase (CK) release at 2, 6 and 20 months of age. Ischemic preconditioning and pretreatment with CGRP for 5 min significantly improved cardiac function and reduced CK release during reperfusion at 2 and 6 months of age but not at 20 months of age. The content of CGRP in the coronary effluent during ischemic preconditioning was significantly increased in the first cycle at 2, 6 months of age but not at 20 months of age. These results suggest that the protection afforded by ischemic preconditioning is decreased in aging hearts, and the age-related change may be related to reduction of the release and effect of CGRP in the rat heart.     

Influence of fasting on the effects of ischemic preconditioning in the ischemic-reperfused rat heart

The effects of fasting and ischemic preconditioning (IP) on heart function of Langendorff-perfused rat hearts exposed to 25 min global ischemia plus 30 min reperfusion (RP), were correlated with lactate release and tissue-levels of long-chain acyl carnitine (LCCa) and CoA (LCCoA). IP was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. Creatine kinase leakage was measured to assess the extent of cardiac injury. Fasting reduced the ischemic-induced contracture, improved RP recovery of mechanical function, reduced lactate release and increased the end-ischemia LCCoA and LCCa levels. Both in the fed and the fasted rat hearts IP delayed the pacemaker depression, reduced the amplitude of ischemic contracture and improved the RP recovery of contraction. However, IP reduced creatine kinase and lactate release only in the fed rat hearts. IP had no effects on tissue LCCa and LCCoA in both groups. These data suggest that: 1) beneficial effects of fasting may be ascribed, at least in part, to a reduced lactate production which may attenuate ischemic myocyte acidification and to the accumulation of fatty acyl esters which would favour citric acid cycle replenishment during RP. 2) beneficial effects of IP could be in part explained by the reduction of lactate production in the fed group although data obtained with the fasted rat heart indicate that another mechanisms must also be involved in the effects of IP. 3) accumulation of LCCoA and LCCa is not involved in the noxious effects of ischemia as well as in the protection effected by IP.     

血红素氧合酶-1 mRNA在离体大鼠心肌缺血预处理中的变化

目的:观察血红素氧合酶-1(HO-1)mRNA在缺血预适应中的变化.方法:实验动物随机分为对照组(CN)、缺血/再灌损伤组(I/R)、缺血预适应 缺血/再灌损伤组(PC).结果:PC组的心功能恢复率高于I/R组(P<0.05),MDA含量低于I/R组(P<0.05),HO-1 mRNA又高于I/R组(P<0.05).结论:HO-1mRNA表达上调与缺血预适应保护缺血/再灌注损伤心肌有关.     

Suppression of ischemic and reperfusion ventricular arrhythmias by inhalational anesthetic-induced preconditioning in the rat heart

Inhalational anesthetic-induced preconditioning (APC) has been shown to reduce infarct size and attenuate contractile dysfunction caused by myocardial ischemia. Only a few studies have reported the effects of APC on arrhythmias during myocardial ischemia-reperfusion injury, focusing exclusively on reperfusion. Accordingly, the aim of the present study was to examine the influence of APC on ventricular arrhythmias evoked by regional no-flow ischemia. APC was induced in adult male Wistar rats by 12-min exposures to two different concentrations (0.5 and 1.0 MAC) of isoflurane followed by 30-min wash-out periods. Ventricular arrhythmias were assessed in the isolated perfused hearts during a 45-min regional ischemia and a subsequent 15-min reperfusion. Myocardial infarct size was determined after an additional 45 min of reperfusion. The incidence, severity and duration of ventricular arrhythmias during ischemia were markedly reduced by APC. The higher concentration of isoflurane had a larger effect on the incidence of ventricular fibrillation than the lower concentration. The incidence of ventricular tachycardia and reversible ventricular fibrillation during reperfusion was also significantly reduced by APC; the same was true for myocardial infarct size. In conclusion, we have shown that preconditioning with isoflurane confers profound protection against myocardial ischemia- and reperfusion-induced arrhythmias and lethal myocardial injury.     

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.     

Essential role of EGFR in cardioprotection and signaling responses to A1 adenosine receptors and ischemic preconditioning

Transactivation of epidermal growth factor receptor (EGFR) may contribute to specific protective responses (e.g. mediated by δ-opioid, bradykinin, or muscarinic receptors). No studies have assessed EGFR involvement in cardioprotection mediated by adenosine receptors (ARs), and the role of EGFR in ischemic preconditioning (IPC) is unclear. We tested EGFR, matrix metalloproteinase (MMP), and heparin-binding EGF (HB-EGF) dependencies of functional protection via A(1)AR agonism or IPC. Pretreatment of mouse hearts with 100 nM of A(1)AR agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) or IPC (3 × 1.5-min ischemia/2-min reperfusion) substantially improved recovery from 25-min ischemia, reducing left ventricular diastolic dysfunction up to 50% and nearly doubling pressure development and positive change in pressure over time (+dP/dt). Benefit with both CCPA and IPC was eliminated by inhibitors of EGFR tyrosine kinase (0.3 μM AG1478), MMP (0.3 μM GM6001), or HB-EGF ligand (0.3 ng/ml CRM197), none of which independently altered postischemic outcome. Phosphorylation of myocardial EGFR, Erk1/2, and Akt increased two- to threefold during A(1)AR agonism, with responses blocked by AG1478, GM6001, and CRM197. Studies in HL-1 myocytes confirm A(1)AR-dependent Erk1/2 phosphorylation is negated by AG1478 or GM6001, and reduced with CRM197 (as was Akt activation). These data collectively reveal that A(1)AR- and IPC-mediated functional protection is entirely EGFR and MMP dependent, potentially involving the HB-EGF ligand. Myocardial survival kinase activation (Erk1/2, Akt) by A(1)AR agonism is similarly MMP/HB-EGF/EGFR dependent. Thus MMP-mediated EGFR activation appears essential to cardiac protection and signaling via A(1)ARs and preconditioning.     

内源性阿片物质参与大鼠缺血预处理的心肌保护作用

实验以离体灌流的ＳＤ大鼠心脏为模型,用ｋ特异性拮抗剂的ＭＲ２２６６研究ｋ阿片受体的阻断与缺血预处理的关系,用放射免疫分析法研究ＩＰ及长时间缺血对心肌强啡肽Ａ１－１３浓度的影响,探索Ｋ阿片物质在ＩＰ过程中的作用和地位。     

胞外ATP在呼吸道中的作用

ATP不但是各种细胞的能量来源,而且更是一种自分泌或旁分泌的胞外信使,参与细胞一系列的生物学效应。ATP从呼吸道上皮细胞中释放,在调节呼吸道表面液体量的平衡、黏膜纤毛清除能力和呼吸道防御功能方面起重要作用,并参与呼吸道疾病及炎症的发生。本文对ATP从呼吸道上皮释放的途径,ATP调节呼吸道上皮离子转运的机制,ATP对呼吸道平滑肌的双重调节作用,以及ATP参与呼吸道疾病和炎症的发生机制等方面予以综述。