吡那地尔缺血后处理对心脏再灌注损伤细胞凋亡的作用

目的:研究缺血/再灌注(I/R)对大鼠心肌细胞凋亡蛋白表达的影响以及ATP敏感钾通道(KATP)开放剂（吡那地尔）缺血后处理(IPO)的作用。方法: 24只Wistar雄性大鼠,随机分为对照组、I/R组、吡那地尔IPO组及吡那地尔+格列本脲IPO组,每组6只动物(n=6)。在大鼠I/R过程中给予吡那地尔IPO后,对心肌细胞中Bax、Bcl-2和纤维连接蛋白(FN)凋亡蛋白的早期变化以及表达采用免疫组化染色法和图像分析技术进行定量分析。结果: 心肌I/R后,心肌细胞中Bax、FN蛋白的表达上调、Bcl-2蛋白的表达下调;吡那地尔IPO组Bax、FN蛋白的表达下调、Bcl-2蛋白的表达上调,各组平均吸光度(A)值有显著性差异(P<0.05)。结论: 吡那地尔本身具有抗细胞凋亡的作用,以其进行IPO具有减轻I/R损伤的作用。     

Effects of selective cyclooxygenase isoform inhibition on systemic prostacyclin synthesis and on platelet function at rest and after exercise in healthy volunteers

To test the hypothesis that selective inhibition of cyclooxygenase (COX)-2 would result in exercise-induced platelet activation by causing a shift in the endogenous thromboxane (TX)/prostacyclin balance, a double blind, randomized study comparing aspirin (300?mg/d) with rofecoxib (25?mg/d) (cross-over design, 14 days washout between treatments) in n?=?10 trained healthy volunteers was carried out. Physical exercise resulted only in a minor platelet activation, as reflected by the expression of basal or ADP-stimulated platelet activation markers or basal plasma concentrations of TXB₂. Aspirin significantly reduced TXB₂ in plasma while rofecoxib significantly increased TXB₂ in urine. Although no increase in systemic prostacyclin concentration was observed, there was a significant exercise-related increase in both platelet cAMP and cGMP without any drug-related effects. It is concluded that, in trained healthy volunteers, selective inhibition of COX-1 (aspirin) or COX-2 (rofecoxib) does not affect systemic prostacyclin synthesis after physical exercise. However, our data do not exclude the possibility that in subjects at risk for atherothrombotic complications (e.g. patients with advanced atherosclerotic disease) COX-2 inhibitors may result in platelet activation by inhibiting endothelial prostacyclin formation.     

Increased prostacyclin release from perfused hearts of acutely diabetic rats

Summary The release of prostacyclin and PGE₂ from the isolated perfused hearts of acutely diabetic (streptozotocin 100 mg/kg) rats was studied and compared with hearts from control animals. Prostacyclin and PGE₂ were measured by a differential bioassay technique. No basal release of either prostaglandin was detected. However, after addition of arachidonic acid, a dose dependent release of prostacyclin and PGE₂ was noted. Prostacyclin was identified as the major prostaglandin. Release of prostacyclin and PGE₂ from acutely diabetic rat hearts was increased 2–3 times compared to control hearts. No release of prostaglandin endoperoxides was observed in either group of hearts.     

CB2 cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion

Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB₂ cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB₂ selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27% ± 1.91) as compared to vehicle-treated mice (31.77% ± 2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-α, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-α induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils.     

Effect of prostacyclin infusion during low-flow ischaemia in the isolated perfused rat heart

Summary Although prostacyclin (PGI₂) has been shown to exert a protective effect on ischaemic hearts its precise mode of action remains obscure. Possible explanations include protection of the high energy phosphate stores (ATP and CP), maintenance of homeostasis with respect to Ca²⁺, and an antiaggregatory effect. The following experiments were undertaken to investigate these possibilities, using isolated, spontaneously beating rat hearts perfused with Krebs-Henseleit solution. Ischaemia was induced at 37°C for 30 min by reducing the flow rate from 10.0 to 0.1 ml/min, and was followed by reperfusion. PGI₂ was given as a constant infusion (20 ng/ml). The hearts were frozen and assayed for ATP and CP, or digested in HNO₃ and assayed for Ca²⁺. Peak developed tension was recorded throughout. The results show that PGI₂ slowed the rate of decline of developed tension during low flow perfusion, and hastened the recovery of contractions on reperfusion. These effects could not be accounted for in terms of an improved supply of ATP or CP, or an altered tissue Ca²⁺. The protective effect of PGI₂ on isolated, buffer-perfused hearts may be a reflection of a generalized, but underfined, mechanism of cell preservation which has also been observed in other systems.     

Antiarrhythmic effect of ischemic preconditioning during low-flow ischemia

Abstract. Short episodes of ischemia (ischemic preconditioning) protect the heart against ventricular arrhythmias during zero-flow ischemia and reperfusion. However, in clinics, many episodes of ischemia present a residual flow (low-flow ischemia). Here we examined whether ischemic preconditioning protects against ventricular arrhythmias during and after a low-flow ischemia and, if so, by what mechanism(s).Isolated rat hearts were subjected to 60 min of low-flow ischemia (12% residual coronary flow) followed by 60 min of reperfusion. Ischemic preconditioning was induced by two cycles of 5 min of zero-flow ischemia followed by 5 and 15 min of reperfusion, respectively. Arrhythmias were evaluated as numbers of ventricular premature beats (VPBs) as well as incidences of ventricular tachycardia (VT) and ventricular .brillation (VF) during low-flow ischemia and reperfusion. Ischemic preconditioning significantly reduced the number of VPBs and the incidence of VT and of VF during low-flow ischemia. This antiarrhythmic effect of preconditioning was abolished by HOE 140 (100 nM), a bradykinin B₂ receptor blocker. Similar to preconditioning, exogenous bradykinin (10 nM) reduced the number of VPBs and the incidence of VT and of VF during low-flow ischemia. Furthermore, the antiarrhythmic effects of both ischemic preconditioning and bradykinin were abolished by glibenclamide (1 µM), a non-specific blocker of ATP-sensitive K⁺ (K_ATP) channels. Finally, the antiarrhythmic effects of both ischemic preconditioning and bradykinin were abolished by HMR 1098 (10 µM), a sarcolemmal K_ATP channel blocker but not by 5-hydroxydecanoate (100 µM), a mitochondrial K_ATP channel blocker. In conclusion, ischemic preconditioning protects against ventricular arrhythmias induced by low-flow ischemia, and this protection involves activation of bradykinin B₂ receptors and subsequent opening of sarcolemmal but not of mitochondrial K_ATP channels.     

The critical role of intracellular zinc in adenosine A2 receptor activation induced cardioprotection against reperfusion injury

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A₂ receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A₁/A₂ receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30 min ischemia followed by 2 h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl₂ given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl₂ inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA.     

Ischemia-reperfusion arrhythmias and lipids: Effect of human high- and low-density lipoproteins on reperfusion arrhythmias

The effect of high- and low-density lipoproteins separated from human serum on the postischemic reperfusion arrhythmias was investigated. The hearts were perfused by working heart mode with Krebs Henseleit bicarbonate buffer containing arachidonic acid (1 μg/ml) for 5 minutes. Whole heart ischemia was induced by the use of a one-way ball valve, and hearts were perfused for 15 minutes followed by 20 minutes of reperfusion. Physiologic concentrations of high- and low-density lipoproteins were constantly infused through the atrial route during ischemic perfusion. Coronary effluent was collected via pulmonary artery cannulation for subsequent radioimmunoassay of thromboxane B₂ and 6-keto-prostaglandin F_1α, the major stable metabolites of thromboxane A₂ and prostacyclin, respectively. The incidence of ventricular arrhythmias during reperfusion was 6/6 (100%), 1/6 (17%), and 6/6 (100%) in control, high-density lipoprotein and low-density lipoprotein infusion groups, respectively. There was no significant difference in coronary flow among the three groups throughout the perfusion. Both thromboxane B₂ and 6-keto-prostaglandin F_1α increased significantly during ischemia compared with preischemic values in all groups of hearts. However, the ratio of these two parameters varied in control and low-density lipoprotein infusion groups during ischemia, while there was no significant change in the high-density lipoprotein infusion group. These results provide the possibility that arachidonate metabolites may be involved in the regulation of ischemia-reperfusion arrhythmias and that high-density lipoprotein that was infused during ischemia markedly inhibits the incidence of ischemia-reperfusion-induced ventricular arrhythmias, due in part at least, to stabilizing the arachidonate metabolites during ischemic perfusion.     

JAK2/STAT3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Numerous data indicate that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IRI. Melatonin has potent activity against IRI and may regulate JAK2/STAT3 signaling. This study investigated the protective effect of melatonin pretreatment on myocardial IRI and elucidated its potential mechanism. Perfused isolated rat hearts and cultured neonatal rat cardiomyocytes were exposed to melatonin in the absence or presence of the JAK2/STAT3 inhibitor AG490 or JAK2 siRNA and then subjected to IR. Melatonin conferred a cardio‐protective effect, as shown by improved postischemic cardiac function, decreased infarct size, reduced apoptotic index, diminished lactate dehydrogenase release, up‐regulation of the anti‐apoptotic protein Bcl2, and down‐regulation of the pro‐apoptotic protein Bax. AG490 or JAK2 siRNA blocked melatonin‐mediated cardio‐protection by inhibiting JAK2/STAT3 signaling. Melatonin exposure also resulted in a well‐preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase (SOD) activity, and decreased formation of mitochondrial hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), which indicates that the IR‐induced mitochondrial oxidative damage was significantly attenuated. However, this melatonin‐induced effect on mitochondrial function was reversed by AG490 or JAK2 siRNA treatment. In summary, our results demonstrate that melatonin pretreatment can attenuate IRI by reducing IR‐induced mitochondrial oxidative damage via the activation of the JAK2/STAT3 signaling pathway.     

Bradykinin B2 Receptor Antagonist FR173657 Ameliorates Small Bowel Ischemia–Reperfusion Injury in Dogs

Bradykinin mediates acute inflammation by increasing microvascular permeability, vasodilation, leukocyte migration and accumulation, and the production of arachidonic acid via phospholipase A₂ activation. Arachidonic acid metabolites, or eicosanoids, are potent modulators of biological functions, particularly inflammation. Bradykinin exerts its inflammatory effects via the bradykinin B₂ receptor. The aim of this study was to evaluate the effect of a bradykinin B₂ receptor antagonist, FR173657 (FR), on intestinal ischemia-reperfusion (I/R) injury. Twenty-eight mongrel dogs were divided into four groups (n = 7 per group). Group I underwent I/R alone, Group II underwent I/R and received FR treatment, Group III was sham operated, and Group IV was sham operated and received FR treatment. The FR treatment consisted of FR continuously from 30 min prior to ischemia to 2 hr after reperfusion. In the I/R procedure, the superior mesenteric artery (SMA) and vein were clamped for 2 hr and then released to permit reperfusion for 12 hr. The intramucosal pH (pH_i), SMA blood flow, and mucosal tissue blood flow were measured during the reperfusion period. The serum thromboxane B₂ and 6-keto-prostaglandin F₁ levels were determined, and tissue samples were examined histologically. Results showed that tissue blood flow, pH_i, and SMA blood flow after reperfusion were maintained in Group II in comparison with Group I. Histopathological examination showed less severe mucosal damage after reperfusion in Group II than in Group I. The serum thromboxane B₂ and 6-keto-prostagland in F₁ levels were significantly lower in Group II than in Group I (P < 0.05). We conclude that FR treatment appears to have clear protective effects on small bowel I/R injury by inhibiting the release of eicosanoids.     

Contractile activity and prostacyclin generation in isolated coronary arteries from diabetic dogs

Summary The present study was aimed at determining the generation of prostacyclin (PGI₂)-like-material in coronary arteries from normal and diabetic (pancreatectomized) dogs as well as the contractile responses to prostacyclin of preparations from normal, diabetic and insulin-treated diabetic animals. PGI₂ produced a dose-dependent relaxation of coronary arteries from normal dogs. In contrast, those from diabetic animals were not relaxed; indeed, at low concentrations PGI₂ failed to evoke any effect but at higher ones it induced a distinct contraction. In arteries from diabetic animals treated with insulin, PGI₂ induced a biphasic contractile effect, which lay between that of normal controls and untreated diabetics. In addition the basal generation of PGI₂-like-material by coronary arteries was significantly higher in the diabetic (141±0.2 pg/mg, mean±SEM) than in normal dogs (59±0.2 pg/mg). The present experiments demonstrate that the generation of PGI₂-like-substance is significantly increased in coronary arteries from diabetic dogs, but the same vessels are unable to respond to added authentic PGI₂ with relaxation; on the contrary they react with a distinct positive contractile response.     

丹酚酸B对大鼠心肌缺血再灌注损伤内皮素及TXA2/PGI2系统的影响

目的　探讨丹酚酸 B(Sal B)对大鼠心肌缺血再灌注损伤 (MIRI)内皮素释放 (ET)及血栓素 /前列环素 (TXA2 / PGI2 )系统的影响。方法　大鼠心肌缺血 30 min后再灌注 1 2 0 min造成大鼠心肌缺血再灌注损伤模型 ,放免法测定给药前后血浆中 ET、TXB2 及 6- Keto- PGF1α的含量 ,观察 Sal B对 MIRI心肌的保护作用。结果　 Sal B可以减少 ET及 TXB2 的释放 ,提高 PGI2 的含量。结论　 Sal B可以减轻 MIRI,可能通过减少 ET的释放 ,改善 TXA2 / PGI2 系统的平衡状态 ,减轻心肌细胞的损伤。     

Nitroflurbiprofen, a nitric oxide-releasing cyclooxygenase inhibitor, improves cirrhotic portal hypertension in rats

BACKGROUND & AIMS: We studied whether administration of nitroflurbiprofen (HCT-1026), a cyclooxygenase inhibitor with nitric oxide (NO)-donating properties, modulates the increased intrahepatic vascular tone in portal hypertensive cirrhotic rats. METHODS: In vivo hemodynamic measurements (n = 8/condition) and evaluation of the increased intrahepatic resistance by in situ perfusion (n = 5/condition) were performed in rats with thioacetamide-induced cirrhosis that received either nitroflurbiprofen (45 mg/kg), flurbiprofen (30 mg/kg, equimolar concentration to nitroflurbiprofen), or vehicle by intraperitoneal injection 24 hours and 1 hour prior to the measurements. Additionally, we evaluated the effect of acute administration of both drugs (250 micromol/L) on the intrahepatic vascular tone in the in situ perfused cirrhotic rat liver (endothelial dysfunction and hyperresponsiveness to methoxamine) and on hepatic stellate cell contraction in vitro. Typical systemic adverse effects of nonsteroidal anti-inflammatory drugs, such as gastrointestinal ulceration, renal insufficiency, and hepatotoxicity, were actively explored. RESULTS: In vivo, nitroflurbiprofen and flurbiprofen equally decreased portal pressure (8 +/- 0.8 and 8.4 +/- 0.1 mm Hg, respectively, vs 11.8 +/- 0.6 mm Hg) and reduced the total intrahepatic vascular resistance. Systemic hypotension was not aggravated in the different treatment groups (P = .291). In the perfused cirrhotic liver, both drugs improved endothelial dysfunction and hyperresponsiveness. This was associated with a decreased hepatic thromboxane A(2)-production and an increased intrahepatic nitrate/nitrite level. In vitro, nitroflurbiprofen, more than flurbiprofen, decreased hepatic stellate cells contraction. Flurbiprofen-treated rats showed severe gastrointestinal ulcerations (bleeding in 3/8 rats) and nefrotoxicity, which was not observed in nitroflurbiprofen-treated cirrhotic rats. CONCLUSIONS: Treatment with nitroflurbiprofen, an NO-releasing cyclooxygenase inhibitor, improves portal hypertension without major adverse effects in thioacetamide-induced cirrhotic rats by attenuating intrahepatic vascular resistance, endothelial dysfunction, and hepatic hyperreactivity to vasoconstrictors.     

Lack of thromboxane A2 involvement in the arrhythmias occurring during acute myocardial ischemia in dogs

Summary Coronary artery occlusion (CAO) followed by reperfusion of the ischemic myocardium has been associated with the onset of ventricular arrhythmias. It has been suggested that platelet aggregates in the ischemic area may release thromboxane A₂ (TxA₂) which may then be responsible for the arrhythmias that occur during reperfusion. To study this possibility, the effect of TxA₂ synthetase inhibition on arrhythmias was examined in anesthetized dogs during occlusion and for 60 minutes following release. Imidazole (30 mg/kg) was infused intravenously for 10 minutes, followed by continuous infusion of 100 mg/kg/hr for 125 minutes. The left anterior descending coronary artery was occluded, 5 minutes after the initial dose, for 60 minutes. Three minutes after release of CAO, TxB₂ concentrations were significantly higher in the arterial blood of vehicle-treated animals (2.06±0.53 pmoles/ml) than in either CAO + imidazole (0.66±0.16 pmoles/ml) or sham-CAO animals receiving imidazole (0.66±0.09 pmoles/ml). However, CAO dogs whether receiving imidazole or 0.9% NaCl generated a significantly greater number of ectopic beats during and after occlusion than sham-CAO animals. Therefore, release of TxA₂ does not appear to be a major causative factor in the generation of reperfusion arrhythmias in dogs following coronary artery occlusion.Predoctoral Fellow of the Ischemia-Shock Research Center of Jefferson Medical College.     

NHE-1 and NBC during pseudo-ischemia/reperfusion in rabbit ventricular myocytes

Despite many studies into the pathophysiology of cardiac ischemia-reperfusion injury, a number of key details are as yet undisclosed. These include the timing and magnitude of the changes in both Na(+)/H(+) exchange (NHE-1) and Na(+) -- HCO(3)(-) -cotransport (NBC) transport rates. We fluorimetrically measured H(i)(+) fluxes (J(NHE-1) and J(NBC)) and Na(i)(+) fluxes in single contracting rabbit ventricular myocytes subjected to metabolic inhibition, pseudo-ischemia (i.e. metabolic inhibition and extracellular acidosis of 6.4), and pseudo-reperfusion. Metabolic inhibition and pseudo-ischemia inhibited NHE-1 by 43 +/- 3.1% and 91 +/- 3.6%, and NBC by 66 +/- 5.4% and 100%, respectively. Inhibition was due to both an acidic shift of the pH(i) at which NHE-1 and NBC become quiescent (set-point pH(i)) and a reduction of the steepness of the pH(i) -- H(i)(+) flux profiles. NHE-1 and NBC did not contribute to Na(i)(+) loading during metabolic inhibition (Na(i)(+) 18 +/- 1.7 mM) or pseudo-ischemia (Na(i)(+) 21 +/- 1.7 mM), because pH(i) acidified less than set-point pH(i)'s. Upon pseudo-reperfusion NBC recovered to 54 +/- 7.3% but NHE-1 to 193 +/- 11% of aerobic control flux, and set-point pH(i)'s returned to near neutral values. Metabolic inhibition and reperfusion caused an acid load of 18 +/- 3.2 mM H(+) 94% of which were extruded by the hyperactive NHE-1. At pseudo-reperfusion Na(i)(+) rose sharply to 31 +/- 5.8 mM within 1.5 min and that coincided with hypercontracture. Cariporide not only prevented the Na(i)(+) transient, but also inhibited pH(i) recovery and prevented hypercontracture. Our results are consistent with the view that NHE-1 is active during metabolic inhibition if, like in whole hearts, pH(i) is driven more acidic than NHE-1 set-point pH(i). Furthermore, either an acidic pH(i) or absence of additional Na(i)(+) loading during reperfusion, or both, limit ischemia-reperfusion injury.     

Induction of cyclooxygenase protein and stimulation of prostaglandin E2 release by epidermal growth factor in cultured guinea pig gastric mucous cells

The present study was undertaken to investigate whether epidermal growth factor (EGF) could stimulate prostaglandin E₂ release, and if so, by what mechanism EGF would exert such an effect in gastric mucosal cells. In cultured guinea pig gastric mucous cells, EGF dosedependently stimulated prostaglandin E₂ release, with maximal stimulation observed at 10 ng/ml. EGF stimulated an increase in cyclooxygenase activity, which was reduced by protein synthesis inhibitor, actinomycin D, and cycloheximide. EGF also stimulated the enzyme protein synthesis estimated by Western blot analysis, whereas EGF did not stimulate phospholipase A₂ activity. These results suggest that such an effect of EGF onde novo synthesis of cyclooxygenase protein and prostaglandin E₂ release may be involved at least in part in the mechanism of EGF-induced local regulation of gastric mucosal integrity.     

Inflammation and platelet activation in peripheral arterial occlusive disease

OBJECTIVES

Epidemiological evidence indicates that inflammation accompanies the progression of atherosclerosis. The aim of the present cross-sectional study was to define relationships between platelet activation and inflammation in patients with mild to severe (stages II to IV) peripheral arterial occlusive disease (PAOD) and matched controls. The effect of chronic administration of low-dose acetylsalicylic acid was investigated.

METHODS

Subjects were studied on a single occasion. C-reactive protein (CRP) and two indexes of in vivo platelet activation were measured – the urinary excretion of 11-dehydrothromboxane (TX) B₂ by immunoassay and circulating platelet-monocyte aggregates (PMAs) by flow cytometry.

RESULTS

Plasma PMAs and urinary 11-dehydro-TXB₂ were significantly increased in PAOD patients compared with controls (P<0.01 for all). A positive correlation between 11-dehydro-TXB₂ and CRP was found in the study population (r_s=0.63, P<0.001). Using logistic regression analysis, CRP was the only independent correlate of 11-dehydro-TXB₂ (β_CRP=11.9, P<0.01), whereas only the presence of PAOD was an independent predictor of high PMA levels (β_PAOD=13.7, P=0.001). Chronic administration of acetylsalicylic acid reduced 11-dehydro-TXB₂, but not PMA and CRP.

CONCLUSIONS

There is evidence that platelet activation in patients with PAOD is related to the vascular disease and is dependent on the severity of inflammation.     

Targeted disruption of PDE3B,but not PDE3A,protects murine heart from ischemia/reperfusion injury

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B^−/− heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B^−/− mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B^−/− mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca²⁺-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B^−/− heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3–enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.The two cyclic nucleotide phosphodiesterase type 3 (PDE3) subfamilies PDE3A and PDE3B are products of separate but homologous genes. PDE3 isoforms hydrolyze both cAMP and cGMP with high affinity (K_m <1 μM) in a mutually competitive manner and are important regulators of cyclic nucleotide signaling pathways and responses in cardiomyocytes and vascular smooth muscle (1). PDE3A and PDE3B exhibit different patterns of expression. PDE3A is more abundant in platelets, airway and vascular smooth muscle, and cardiovascular tissues, whereas PDE3B is relatively more highly expressed in tissues that are important in regulating energy metabolism, including liver, pancreatic β cells, brown adipose tissue (BAT), and white adipose tissue (WAT) (2). Little is known about their differential localization and functions when PDE3A and PDE3B are present in the same cell. To gain further insight into specific PDE3A and PDE3B functions in physiological contexts, we have generated and studied PDE3A^−/− and PDE3B^−/− mice (3, 4).PDE3 inhibitors, e.g., milrinone, are thought to enhance myocardial inotropic responses via cAMP/PKA regulation of Ca²⁺ cycling in the sarcoplasmic reticulum (SR) (1, 5). The PDE3 inhibitor cilostazol (6–9) and the PDE5 inhibitor sildenafil (10, 11) have been reported to protect hearts against ischemia/reperfusion (I/R) injury in various species. Fukasawa et al. (8) have suggested that cilostazol exerts its cardioprotective effect by activating mitochondrial Ca²⁺-activated K⁺ (mitoK_Ca) channels, whose opening protects hearts against infarction (12). Furthermore, studies have shown that the opening of mitoK_Ca channels is potentiated by cAMP-dependent PKA signaling (13), whereas PKC potentiates mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channel activation (14). Kukreja and his associates have suggested that the cardioprotective effects of sildenafil are mediated by activation of both mitoK_ATP (10) and mitoK_Ca channels (11).Ischemic preconditioning (PreC), a process in which brief intermittent episodes of ischemia and reperfusion protect the heart from subsequent prolonged ischemic injury (15), initiates a number of cardioprotective signaling pathways at the plasma membrane, which are transduced to mitochondria (16). According to the “signalosome” hypothesis, cardioprotective [e.g., G protein-coupled receptor (GPCR)-induced or ouabain-induced] signals are delivered to mitochondria by specialized caveolae-derived vesicular structures, signalosomes, which contain a wide variety of receptors (e.g., GPCRs) and signaling molecules (e.g., Akt, Src, eNOS, and PKCε) that are assembled in lipid rafts and caveolae (17). In recent years, the role of lipid rafts and caveolae in cardiovascular signaling has attracted much attention (18), and adenylyl cyclases and PDEs have emerged as key players in shaping and organizing intracellular signaling microdomains (19–21).Accumulating evidence implicates the mitochondrial permeability transition (MPT) pore as a key effector of cardioprotection against I/R injury, and reperfusion-induced elevation of reactive oxygen species (ROS) can trigger the opening of the MPT pore, resulting in ischemic injury, apoptosis, and cell death (16). A wide range of cardioprotective signaling pathways converge on glycogen synthase kinase-3β (GSK-3β), and its inhibition directly and/or indirectly regulates MPT pore-regulatory factors (e.g., cyclophilin D and voltage-dependent anion channels) and antiapoptotic Bcl-2 family members (22). Physical association between mitochondria and the endoplasmic reticulum (ER) [via mitochondria-associated ER membranes (MAMs)] (23) or the SR (24) also may reduce reperfusion-induced mitochondrial Ca²⁺ overload and consequent oxidative stress and thus block MPT pore opening (25).In this study, we report that, 24 h after in vivo coronary artery ligation, I/R or, in a Langendorff cardiac I/R model system, infarct size is reduced in PDE3B^−/− heart, but not in PDE3A^−/− heart, compared with WT heart. This protective effect is most likely caused by reduced production of ROS and reduced Ca²⁺-induced MPT pore opening in PDE3B^−/− mitochondria. The mechanism(s) for cardioprotection in PDE3B^−/− mice may be related to cAMP/PKA-induced opening of mitoK_Ca channels and assembly of ischemia-induced caveolin-3–enriched fraction (ICEF) signalosomes in which various cardioprotective molecules accumulate, resulting in functional cardiac preconditioning. Our results also suggest that the increased physical interaction between mitochondria and transverse tubules (T-tubules) (indirectly via the SR at dyads or directly) in PDE3B^−/− heart may be involved in ICEF/signalosome delivery of cardioprotective molecules to mitochondria, leading to reduced ROS generation and increased resistance to Ca²⁺-induced MPT pore opening in PDE3B^−/− mitochondria. Although PDE3A is more highly expressed than PDE3B in cardiovascular tissues, our findings of cardioprotection against I/R injury in PDE3B^−/− mice but not in PDE3A^−/− mice and the different subcellular locations of PDE3A and PDE3B in cardiomyocytes [PDE3A colocalizes with sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) on SR membranes, and PDE3B localizes with caveolin-3 in T-tubules along Z-lines] may reflect an important example of individual PDEs at distinct subcellular sites regulating the compartmentalization of specific cAMP/PKA-signaling pathways (19, 21). In this case, PDE3B, located in regions where cardiomyocyte mitochondria, T-tubules, and SR may be in close proximity, may regulate stress responses and/or the assembly of ICEF signalosomes or other specific cardioprotective pathways.     

Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini

BACKGROUND & AIMS: The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are the principal Ca2+-release channels in cells and are believed to serve distinct roles in cytosolic Ca2+ (Ca(i)2+) signaling. This study investigated whether these receptors instead can release Ca2+ in a coordinated fashion. METHODS: Apical and basolateral Ca(i)2+ signals were monitored in rat pancreatic acinar cells by time-lapse confocal microscopy. Caged forms of second messengers were microinjected into individual cells and then photoreleased in a controlled fashion by either UV or 2-photon flash photolysis. RESULTS: InsP3 increased Ca(i)2+ primarily in the apical region of pancreatic acinar cells, whereas the RyR agonist cyclic adenosine diphosphate ribose (cADPR) increased Ca(i)2+ primarily in the basolateral region. Apical-to-basal Ca(i)2+ waves were induced by acetylcholine and initiation of these waves was blocked by the InsP3R inhibitor heparin, whereas propagation into the basolateral region was inhibited by the cADPR inhibitor 8-amino-cADPR. To examine integration of apical and basolateral Ca(i)2+ signals, Ca2+ was selectively released either apically or basolaterally using 2-photon flash photolysis. Ca(i)2+ increases were transient and localized in unstimulated cells. More complex Ca(i)2+ signaling patterns, including polarized Ca(i)2+ waves, were observed when Ca2+ was photoreleased in cells stimulated with subthreshold concentrations of acetylcholine. CONCLUSIONS: Polarized Ca(i)2+ waves are induced in acinar cells by serial activation of apical InsP3Rs and then basolateral RyRs, and subcellular release of Ca2+ coordinates the actions of these 2 types of Ca2+ channels. This subcellular integration of Ca2+-release channels shows a new level of complexity in the formation of Ca(i)2+ waves.