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Manipulation of nitric oxide (NO) may enable control of progression and treatment of pulmonary hypertension (PH). Several approaches may modulate the NO-cGMP pathway in vivo. Here, we investigate the effectiveness of 3 modulatory sites: (i) the amount of l-arginine; (ii) the size of plasma NO stores that stimulate soluble guanylate cyclase; (iii) the conversion of cGMP into inactive 5′-GMP, with respect to hypoxia, to test the effectiveness of the treatments with respect to hypoxia-induced PH. Male rats (n = 80; 10/group) maintained in normoxic (21% O2) or hypoxic chambers (10% O2) for 14 days were subdivided in 4 sub-groups: placebo, l-arginine (20 mg/ml), the NO donor molsidomine (15 mg/kg in drinking water), and phoshodiesterase-5 inhibitor sildenafil (1.4 mg/kg in 0.3 ml saline, i.p.). Hypoxia depressed homeostasis and increased erythropoiesis, heart and right ventricle hypertrophy, myocardial fibrosis and apoptosis inducing pulmonary remodeling. Stimulating anyone of the 3 mechanisms that enhance the NO-cGMP pathway helped rescuing the functional and morphological changes in the cardiopulmonary system leading to improvement, sometimes normalization, of the pressures. None of the treatments affected the observed parameters in normoxia. Thus, the 3 modulatory sites are essentially similar in enhancing the NO-cGMP pathway, thereby attenuating the hypoxia-related effects that lead to pulmonary hypertension.

IntroductionNitric oxide (NO) is an essential signaling molecule modulating the endothelial adaptation during breath-hold diving (BH-diving). This study aimed to investigate changes in NO derivatives (NOx) and total antioxidant capacity (TAC), searching for correlations with different environmental and hyperbaric exposure.Materials and methodsBlood samples were obtained from 50 breath-hold divers (BH-divers) before, and 30 and 60 min after the end of training sessions performed both in a swimming pool or the sea. Samples were tested for NOx and TAC differences in different groups related to their hyperbaric exposure, experience, and additional genetic polymorphism.ResultsWe found statistically significant differences in NOx plasma concentration during the follow-up (decrease at T30 and increase at T60) compared with the pre-dive values. At T30, we found a significantly lower decrease of NOx in subjects with a higher diving experience, but no difference was detected between the swimming pool and Sea. No significant difference was found in TAC levels, as well as between NOx and TAC levels and the genetic variants.ConclusionThese data showed how NO consumption in BH-diving is significantly lower in the expert group, indicating a possible training-related adaptation process. Data confirm a significant NO use during BH-diving, compatible with the well-known BH-diving related circulatory adaptation suggesting that the reduction in NOx 30 min after diving can be ascribed to the lower NO availability in the first few minutes after the dives. Expert BH-divers suffered higher oxidative stress. A preliminary genetic investigation seems to indicate a less significant influence of genetic predisposition.

BackgroundSeveral mechanisms allow humans to resist the extreme conditions encountered during breath-hold diving. Available nitric oxide (NO) is one of the major contributors to such complex adaptations at depth and oxidative stress is one of the major collateral effects of diving. Due to technical difficulties, these biomarkers have not so far been studied in vivo while at depth. The aim of this study is to investigate nitrate and nitrite (NOx) concentration, total antioxidant capacity (TAC) and lipid peroxidation (TBARS) before, during, and after repetitive breath-hold dives in healthy volunteers.Materials and MethodsBlood plasma, obtained from 14 expert breath-hold divers, was tested for differences in NOx, TAC, and TBARS between pre-dive, bottom, surface, 30 and 60 min post-dive samples.ResultsWe observed a statistically significant increase of NOx plasma concentration in the “bottom blood draw” as compared to the pre-dive condition while we did not find any difference in the following samples We found a statistically significant decrease in TAC at the bottom but the value returned to normality immediately after reaching the surface. We did not find any statistically significant difference in TBARS.DiscussionThe increased plasma NOx values found at the bottom were not observed at surface and post dive sampling (T0, T30, T60), showing a very rapid return to the pre-dive values. Also TAC values returned to pre- diving levels immediately after the end of hyperbaric exposure, probably as a consequence of the activation of endogenous antioxidant defenses. TBARS did not show any difference during the protocol.

Oxygen administration is particularly relevant in patients undergoing surgery under general anesthesia and in those who suffer from acute or critical illness. Nevertheless, excess O2, or hyperoxia, is also known to be harmful. Toxicity arises from the enhanced formation of reactive oxygen species (ROS) that, exceeding the antioxidant defense, may generate oxidative stress. Oxidative stress markers are used to quantify ROS toxicity in clinical and non-clinical settings and represent a promising tool to assess the optimal FiO2 in anesthesia and critical care setting. Despite controversial, the guidelines for the regulation of FiO2 in such settings suggest the adoption of high perioperative oxygen levels. However, hyperoxia has also been shown to be an independent mortality risk factor in critically ill patients. In this literature review, we discuss the biochemical mechanisms behind oxidative stress and the available biomarkers for assessing the pro-oxidant vs antioxidant status. Then, we summarize recent knowledge on the hyperoxia-related consequences in the most common anesthesia and critical care settings, such as traumatic brain injury or cardiac arrest. To this purpose, we searched the PubMed database according to the following combination of key words: ("hyperoxia" OR "FiO2" OR "oxygen therapy") AND ("oxidative stress" OR "ROS" OR "RNS" OR "lipid peroxidation") AND ("anesthesia" OR "surgery" OR "intensive care"). We focused in the results from the past 20 years. Available evidence points toward a conservative monitoring and use of oxygen, unless there is solid proof of its efficacy.

Objective We previously demonstrated that the inhibition of phosphodiesterase type-5, an enzyme that degrades cGMP into inactive 5'-GMP, attenuates pulmonary remodeling and right ventricular (RV) hypertrophy (RVH) during exposure to chronic hypoxia (CH). The nitric oxide (NO) pathway is thought to play a major role in these changes. In this study, we investigate whether L-Arginine (L-ARG), a substrate of endothelial NO synthase (eNOS) and molsidomine (MOL), a NO donor, might alleviate the cardiovascular and pulmonary dysfunction led by CH. Methods Male rats (n=80; 10/group) were maintained in normoxic (21% O2) or hypoxic chambers (10% O2) for 14 days. Hypoxic rats were subdivided in four groups: untreated control, treated with L-ARG (45 mg/kg), eNOS inhibitor N-nitro-L-arginine methyl ester (L-NAME, 45 mg/kg) or MOL (15 mg/kg). Drugs were given daily in the drinking water. After sacrifice, we measured RV systolic pressure (RVSP), RV contractility (dP/dt), RVH, the lung/body weight ratio, the pulmonary vessels medial wall thickness, and the cardiac and pulmonary eNOS phosphorylation. Results Although CH increased RVSP, RV contractility and RVH, these increases were attenuated by L-ARG and MOL. Whereas L-ARG attenuated the RVH increase, MOL and L-NAME were ineffective. No treatment prevented the increase in lung/body weight ratio. Under all conditions, the lung tissue water content was unchanged, indicating no edema development. By contrast, CH rats developed a marked increase in medial wall thickness of small (0–100 mm) pulmonary arteries, while larger arteries were not affected. This increase was attenuated by L-ARG or MOL. Although CH decreased cardiac and pulmonary phosphorylated eNOS, L-ARG and MOL restored the normoxic level. Conclusions NO supplementation during CH attenuates RVSP, RVH and pulmonary remodeling, probably due to increased phosphorylation of eNOS. Despite normalizing RVSP, MOL does not influence RVH development.

Purpose In pulmonary hypertension (PH), hypoxia represents both an outcome and a cause of exacerbation. We addressed the question whether hypoxia adaptation might affect the mechanisms underlying PH alleviation through phosphodiesterase-5 (PDE5) inhibition. Methods Eight-week-old male Sprague-Dawley rats were divided into two groups depending on treatment (placebo or sildenafil, a drug inhibiting PDE5) and were exposed to hypoxia (10% O2) for 0 (t0, n = 9/10), 2 (t2, n = 5/5) or 4 (t4, n = 5/5) weeks. The rats were treated (0.3 mL i.p.) with either saline or sildenafil (1.4 mg/Kg per day). Results Two-week hypoxia changed the body weight (− 31% vs. − 27%, respectively, P = NS), blood hemoglobin (+ 25% vs. + 27%, P = NS) and nitrates+nitrites (+ 175% vs. + 261%, P = 0.007), right ventricle fibrosis (+ 814% vs. + 317%, P < 0.0001), right ventricle hypertrophy (+ 84% vs. + 49%, P = 0.007) and systolic pressure (+ 108% vs. + 41%, P = 0.001), pulmonary vessel density (+ 61% vs. + 46%, P = NS), and the frequency of small (< 50 µm wall thickness) vessels (+ 35% vs. + 13%, P = 0.0001). Most of these changes were maintained for 4-week hypoxia, except blood hemoglobin and right ventricle hypertrophy that continued increasing (+ 52% vs. + 42%, P = NS; and + 104% vs. + 83%, P = 0.04). To further assess these observations, small vessel frequency was found to be linearly related with the right ventricle-developed pressure independent of hypoxia duration. Conclusions Thus, although hypoxia adaptation is not yet accomplished after 4 weeks, PH alleviation by PDE5 inhibition might nevertheless provide an efficient strategy for the management of this disease.

Introduction: Nitric oxide (NO) plays an important role in the physiology and pathophysiology of diving, and the related endothelial dysfunction and oxidative stress roles have been extensively investigated. However, most available data have been obtained before and after the dive, whilst, as far as we know, no data is available about what happens during the water immersion phase of dive. The scope of this study is to investigate the Nitrate and Nitrite (NOX) concentration and the total plasma antioxidant capacity (TAC) before, during and after a single SCUBA dive in healthy scuba diving volunteers, as well as to look for evidence of a possible relationship with venous gas bubble formation. Materials and Methods: Plasma, obtained from blood of 15 expert SCUBA divers, 13 male and 2 female, was investigated for differences in NOX and TAC values in different dive times. Differences in NOX and TAC values in subjects previously known as "bubble resistant" (non-bubblers - NB) and "bubble prone" (Bubblers - B) were investigated. Results: We found a statistically significant increase of NOX plasma concentration in the "bottom blood draw" and in the "safety stop blood draw" as compared to the basal pre diving condition. We did not find any difference in NOX plasma concentration between the basal value and the post diving samples. We did not find any significant statistical difference in TAC in the bottom blood sample, while the safety-stop and the post-dive samples showed higher TAC values compared with the basal value. We did not find any difference in NOX and TAC mean values between non-bubblers and Bubblers. Discussion: Our protocol, by including underwater blood drawing, allowed to monitor plasma NOX changes occurred during diving activity, and not only by comparing pre and post diving values. It is particularly interesting to note that the increased NOX values found at the bottom and at the safety stop were not observed at post dive sampling (T0, T30, T60), showing a very rapid return to the pre-dive values. In this preliminary study we did not find any relationship between bubble formation and changes in NOX parameters and TAC response.

javax.xml.bind.JAXBElement@6f8948ff Hypoxia represents both an outcome of cardiopulmonary diseases and a trigger for severe pulmonary complications as pulmonary hypertension. Because nitric oxide (NO) is a critical mediator in the development of pulmonary hypertension, the modulators of its downstream function may become target of pharmacological interventions aimed at alleviating the impact of this condition. Here, we investigate the effects of an early administration of phosphodiesterase-5 inhibitor in rats where pulmonary artery hypertension was induced by chronic exposure to hypoxia. javax.xml.bind.JAXBElement@162dc677 Rats were divided into three groups: normoxic control, hypoxic with no treatments (2 weeks breathing an atmosphere containing 10% oxygen), and hypoxic treated with sildenafil (1.4 mg/Kg per day in 0.3 mL i.p.). After sacrifice, hearts and lungs were removed and harvested for analyses. javax.xml.bind.JAXBElement@1961a294 Sildenafil reduced hypoxia-induced right ventricle hypertrophy without effects in lung hypertrophy, and blunted the increase in right ventricle pressure without effects on left ventricle pressure. Furthermore, the NO-producing systems (i.e., the phosphorylation of the endothelial isoforms of NO synthase that was measured in both myocardial and lung tissues), and the blood NO stores (i.e., the plasma level of nitrates and nitrites) were up-regulated by sildenafil. We did not find significant effects of sildenafil on weight and hemoglobin concentration. Morphological analysis in lung biopsies revealed that 2-week hypoxia increased the frequency of small pulmonary vessels leaving large vessels unaffected. Finally, ultrastructural analysis showed that sildenafil down-regulated the hypoxia-induced increase in the thickness of the pulmonary basal lamina. javax.xml.bind.JAXBElement@7ae1b3f8 In this model of pulmonary hypertension, sildenafil contrasts the negative effects of hypoxia on pulmonary vascular and right ventricle remodeling. This action does not only encompass the canonical vasomodulatory effect, but involves several biochemical pathways. Although the human pathological model is certainly more complex than that described here (for example, the inflammatory issue), the potential role of phosphodiesterase-5 for long-term treatment, and perhaps prevention, of pulmonary hypertension is worthy of investigation.

The recruitment of bone marrow (BM)-derived progenitor cells to the lung is related to pulmonary remodelling and the pathogenesis of pulmonary hypertension (PH). Although sildenafil is a known target in PH treatment, the underlying molecular mechanism is still elusive. To test the hypothesis that the therapeutic effect of sildenafil is linked to the reduced recruitment of BM-derived progenitor cells, we induced pulmonary remodelling in rats by two-week exposure to chronic hypoxia (CH, 10% oxygen), a trigger of BM-derived progenitor cells. Rats were treated with either placebo (saline) or sildenafil (1.4 mg/kg/day ip) during CH. Control rats were kept in room air (21% oxygen) with no treatment. As expected, sildenafil attenuated the CH-induced increase in right ventricular systolic pressure and right ventricular hypertrophy. However, sildenafil suppressed the CH-induced increase in c-kit(+) cells in the adventitia of pulmonary arteries. Moreover, sildenafil reduced the number of c-kit(+) cells that colocalize with tyrosine kinase receptor 2 (VEGF-R2) and CD68 (a marker for macrophages), indicating a positive effect on moderating hypoxia-induced smooth muscle cell proliferation and inflammation without affecting the pulmonary levels of hypoxia-inducible factor (HIF)-1α. Furthermore, sildenafil depressed the number of CXCR4(+) cells. Collectively, these findings indicate that the improvement in pulmonary haemodynamic by sildenafil is linked to decreased recruitment of BM-derived c-kit(+) cells in the pulmonary tissue. The attenuation of the recruitment of BM-derived c-kit(+) cells by sildenafil may provide novel therapeutic insights into the control of pulmonary remodelling.

Background/Aims. The simultaneous supplementation of creatine and D-ribose has been shown to reduce apoptosis in vitro in non-irreversibly injured cultured ischemic cardiomyocytes through down-regulation of the signaling mechanisms governing adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (Akt). Here, we test the hypothesis that an analogous mechanism exists in vivo when the challenge is chronic exposure to hypoxia. Methods. Five week-old mice were exposed to an atmosphere containing 10% O2 for 10 days. Mice were gavaged daily with vehicle, creatine, D-ribose or creatine + D-ribose. After sacrifice, myocardial and pulmonary tissue were harvested for structural and biochemical analyses. Results. Hypoxia induced right ventricle hypertrophy and left ventricle apoptosis. Both phenotypes were slightly reduced by either creatine or D-ribose, whereas the simultaneous administration of creatine + D-ribose almost completely reversed the effects of hypoxia. Furthermore, creatine + D-ribose diminished the hypoxia-induced increases in the activity of AMPK, Akt and JNK, but not of ERK. Finally, the hypoxia-induced pulmonary overexpression of endothelin-1 mRNA was markedly reduced by creatine + D-ribose. Conclusion. The simultaneous administration of creatine + D-ribose confers additional cardiovascular protection with respect to that observed with either creatine or D-ribose. The mechanism stems from the AMPK and Akt signaling pathways. These findings may form the basis of a paradigm to re-energize non-irreversibly damaged cardiomyocytes, counteracting injury by triggering specific signaling pathways.

In patients undergoing exchange-transfusion with hemoglobin (Hb)-based oxygen (O2) carriers (HBOC), native Hb coexists with newly transfused Hb. The two Hb types share the same arterial and venous PO2, but their affinities for O2 vary. A simple spreadsheet model is described aiming at evaluating the contribution of each Hb type to the overall O2 transport characteristics as a function of the batch Hb concentration and O2 affinity in the HBOC solution, of the fraction of exchange-transfused blood/HBOC, and of the arterial PO2. This model helps to yield a quantitative estimate of how tissues with high or low O2 extraction respond to the changes cited above. The results show that the higher the exchange-transfusion ratio, the O2 transport to tissues becomes progressively impaired. However, this effect is more critical at low batch Hb concentration and high O2 affinity of the HBOC, especially for tissues/organs with high O2 extraction, whereas the arterial PO2 does not appear as critical.

OBJECTIVE Solid tumors contain underperfused regions where hypoxia-inducible factor-1α (HIF-1α) over-expression induces hypoxia adaptation and cell proliferation. We test the hypothesis that systemic hypoxia promotes prostate cancer growth in vivo and examine HIF-1α centrality in this effect. METHODS Male athymic mice were xenografted with 3 × 106 LNCaP cells per each flank and exposed for 28 days to either chronic hypoxia (CH, 10% O2) or CH with reoxygenation (CHReox, 3 times/week for 1 hr), with normoxia as control (n = 17, 9, and 20, respectively). At the end of the observation, mice were euthanized and tumors harvested for analyses. RESULTS The successful xenografts grew faster in CH and CHReox than in normoxia (first-order rate constants 0.15 ± 0.01, 0.18 ± 0.03, and 0.09 ± 0.01 day−1, P

Background/Aims. To alleviate ischemia-induced injury in the myocardium, a tissue that depends critically on energy-yielding processes, creatine may be used to enhance energy metabolism, whereas D- ribose may provide building blocks for ATP synthesis. We test the hypothesis that simultaneous supplementation of creatine+D-ribose protects non- irreversibly injured ischemic cardiomyocytes by reducing apoptosis. Results. When H9c2 cardiomyocytes were exposed to 24-h ischemia (1% O 2 with glucose deprivation), viability was severely compromised, but administration of 2.5 mM creatine + 5 mM D-ribose alleviated the fall in viability, whereas 2.5 mM creatine or 5 mM D-ribose did not. These findings correlated with up-regulation of protein kinase B (Akt) phosphorylation. Creatine+D-ribose also blunted adenosine monophosphate-activated protein kinase (AMPK) and down-regulated apoptosis by reducing caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Conclusions. Simulta- neous administration of creatine+D-ribose confers anti-ischemic protection that was absent when treating cardiomyocytes with either creatine or D-ribose. The involved mechanisms stem from the Akt and AMPK signaling pathways. These findings may form the basis of a paradigm whereby re-energization of non- irreversibly damaged cardiomyocytes is a critical step to counteract apoptosis.

Background and purpose: MP4 (Hemospan) is a Hb-based oxygen therapeutic agent, based on polyethylene-glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO-MP4 on myocardial infarct size following ischaemia and reperfusion in rats. Experimental approach: Kinetic measurements of CO-MP4 binding were used to evaluate the effects of PEG modification on Hb subunit structure/function and to calculate CO-MP4 equilibrium constants. CO transport by CO-MP4 was shown by ligand (O2/CO) partitioning between MP4 and red blood cell (RBC)-Hb. Pharmacological effects of CO-MP4 were studied on myocardial infarction in rats. Key results: CO binding kinetics show primary structural/functional effects on β chains in MP4, with α chains maintaining the ability to undergo tertiary conformational transition. CO confers long-term, room-temperature stability and is able to rapidly re-equilibrate between MP4 and RBCs. In a rat model of myocardial infarct, in contrast to oxy-MP4, CO-MP4 reduced infarct size when administered prior to the induction of ischaemia. Conclusions and implications: MP4 PEGylation chemistry modifies the individual function of Hb subunits, but results in an overall CO equilibrium constant similar to that for unmodified Hb. CO-MP4 is able to deliver CO to the circulation and reduces ischaemia/reperfusion injury in rats, providing the first evidence for this drug as a CO therapeutic agent. British Journal of Pharmacology (2008) 154, 1649–1661; doi:10.1038/bjp.2008.219; published online 9 June 2008

Long-Term Use of Sildenafil in the Therapeutic Management of Heart Failure Marco Guazzi, Michele Samaja, Ross Arena, Marco Vicenzi, Maurizio D. Guazzi We tested whether chronic phosphodiesterase 5 inhibition by sildenafil is beneficial in heart failure. A total of 46 heart failure patients were randomly assigned to placebo (23 patients) or sildenafil (23 patients), with 3- and 6-month assessments of cardiopulmonary exercise test performance, brachial artery flow-mediated maximal dilatation, and ergoreflex responses. Sildenafil and not placebo improved flow-mediated maximal dilatation, ergoreflex effect on ventilation, exercise ventilation to CO2production slope, and breathlessness, at both 3 and 6 months (p < 0.01). Findings suggest that benefits of sildenafil in heart failure are sustained and that an endothelium-mediated modulation of the muscle ergoreflex oversignaling may be at work. Objectives This study sought to test the functional exercise capacity and endothelial function in a cohort of chronic heart failure (CHF) patients treated with chronic type 5 phosphodiesterase (PDE5) inhibitor. Background In CHF, endothelial dysfunction is involved in muscle underperfusion, ergoreflex oversignaling, and exercise ventilation inefficiency. Inhibition of PDE5by improving endothelial dysfunction might be beneficial. Methods Stable CHF patients were randomly assigned to placebo (23 patients) or sildenafil at the dose of 50 mg twice per day (23 patients) in addition to their current drug treatment for 6 months, with assessments (at 3 and 6 months) of endothelial function by brachial artery flow-mediated dilatation (FMD), cardiopulmonary exercise testing, and ergoreflex response. Results In the sildenafil group only, at 3 and 6 months we observed reduction of systolic pulmonary artery pressure (from 33.7 to 25.2 mm Hg and 23.9 mm Hg), ergoreflex effect on ventilation (from 6.9 to 2.3 l·min−1and 1.9 l·min−1), ventilation to CO2production slope (VE/Vco2, from 35.5 to 32.1 and 29.8), and breathlessness (score) (from 23.6 to 16.6 and 17.2), and an increase of FMD (from 8.5% to 13.4% and 14.2%), peak Vo2(from 14.8 to 18.5 ml·min−1·kg−1and 18.7 ml·min−1·kg−1), and ratio of Vo2to work rate changes (from 7.7 to 9.3 and 10.1). All changes were significant at p < 0.01. In the sildenafil group, a significant correlation was found at 3 and 6 months between changes in FMD and those in ergoreflex. Changes in ergoreflex correlated with those in peak Vo2and VE/Vco2slope. No adverse effects were noted except for flushing in 3 patients. Conclusions In CHF, improvement in exercise ventilation and aerobic efficiency with sildenafil is sustained and is significantly related with an endothelium-mediated attenuation of exercising muscle oversignaling. Chronic sildenafil seems to be a remedy based on CHF pathophysiology and devoid of remarkable adverse effects.

Background. Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. Methods. Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 μg/kg ip) 15 min before LAD. Results. We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. Conclusion. We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.

The injury caused by myocardial reperfusion after ischemia can be contained by interventions aimed at reducing the inflammation and the oxidative stress that underlie exacerbation of tissue damage. Sphingolipids are a class of structural and signaling lipid molecules; among them, the inflammation mediator ceramide accumulates in the myocardium upon ischemia/reperfusion. Here, we show that, after transient coronary occlusion in mice, an increased de novo ceramide synthesis takes place at reperfusion in the ischemic area surrounding necrosis (area at risk). This correlates with the enhanced expression of the first and rate-limiting enzyme of the de novo pathway, serine palmitoyltransferase (SPT). The intraventricular administration at reperfusion of myriocin, an inhibitor of SPT, significantly protected the area at risk from damage, reducing the infarcted area by 40.9 % relative to controls not treated with the drug. In the area at risk, myriocin downregulated ceramide, reduced the content in other mediators of inflammation and reactive oxygen species, and activated the Nrf2–HO1 cytoprotective response. We conclude that an enhanced ceramide synthesis takes part in ischemia/reperfusion injury and that myriocin treatment can be proposed as a strategy for myocardial pharmacological postconditioning.

Although effective and safe, carotid endarterectomy (CEA) implies a reduced blood flow to the brain and likely an ischemia/reperfusion event. The high rate of uneventful outcomes associated with CEA suggests the activation of brain endogenous protection mechanisms aimed at limiting the possible ischemia/reperfusion damage. This study aims at assessing whether CEA triggers protective mechanisms such as brain release of erythropoietin and nitric oxide. CEA was performed in 12 patients; blood samples were withdrawn simultaneously from the surgically exposed ipsilateral jugular and leg veins before, during (2 and 40 min) and after clamp removal (2 min). Plasma antioxidant capacity, carbonylated proteins, erythropoietin, nitrates and nitrites (NOx) were determined. No changes in intraoperative EEG, peripheral and transcranial blood oxygen saturation were detectable, and no patients showed any neurologic sign after the intervention. Antioxidant capacity and protein carbonylation in plasma were unaffected. Differently, erythropoietin, VEGF, TNF-α and NOx increased during clamping in the jugular blood (2 and 40 min), while no changes were observed in the peripheral circulation. These results show that blood erythropoietin, VEGF, TNF-α, and NOx increased in the brain during uncomplicated CEA. This may represent an endogenous self-activated neuroprotective mechanism aimed at the prevention of ischemia/reperfusion damage.

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O 2 , hypoxia-inducible factor 1α (HIF-1α) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O 2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1α production by improved tissue O 2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1α expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1α in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O 2 for 10 days) with or without CEpo administered by daily s.c. injection (10 μg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1α and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1α expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.

The hypertensive effect observed with most cell-free haemoglobins has been proposed to result from NO scavenging. However, a newly developed PEG [poly(ethylene glycol)]-conjugated haemoglobin, MalPEG-Hb [maleimide-activated PEG-conjugated haemoglobin], is non-hypertensive with unique physicochemical properties: high O2 affinity, low co-operativity and large molecular radius. It is therefore of interest to compare the ligand-binding properties of MalPEG-Hb with unmodified cell-free HbA (stroma-free human haemoglobin). NO association rates for deoxy and oxyMalPEG-Hb and HbA were found to be identical. These results confirm the lack of correlation between hypertension and NO for a similar modified haemoglobin with high molecular radius and low p50 (pO2 at which haemoglobin is half-saturated with O2) [Rohlfs, Bruner, Chiu, Gonzales, Gonzales, Magde, Magde, Vandegriff and Winslow (1998) J. Biol. Chem. 273, 12128–12134]. The R-state O2 association kinetic constants were also the same for the two haemoglobins. However, even though the p50 of MalPEG-Hb is approx. half of that of HbA, the biphasic O2 dissociation rates measured at relatively high pO2 (150 Torr) were 2-fold higher, giving rise to a 2-fold lower R-state equilibrium association constant for MalPEG-Hb compared with HbA. Thus the O2 affinity of MalPEG-Hb is higher only at pO2 values lower than the intersection point of the O2 equilibrium curves for MalPEG-Hb and HbA. In summary, the present studies found similar rates of NO binding to HbA and MalPEG-Hb, eliminating the possibility that the lack of vasoactivity of MalPEG-Hb is simply the result of reduced molecular reactivity with NO. Alternatively, the unique O2-binding characteristics with low p50 and co-operativity suggest that the ‘R-state’ conformation of MalPEG-Hb is in a more T-state configuration and restricted from conformational change.

We measured time course and extent of xanthine dehydrogenase (XD) to xanthine oxidase (XO) conversion in ischemic human and rat intestine. To model normothermic no-flow ischemia, we incubated fresh biopsies for 0, 2, 4, 8 and 16h. At t = 0h, XO was less in humans than in rats (P0.0004), while XD was essentially the same (P = NS). After 16h incubation at 37 degrees C, there was no appreciable XD-to-XO conversion and no change in neither XO nor XD activity in human intestine. In contrast, the rat intestine had XO/(XO + XD) ratio doubled in the first 2h and then maintained that value until t = 16 h. In conclusion, no XO-to-XD conversion was appreciable after 16 h no-flow normothermic ischemia in human intestine; in contrast, XO activity in rats increased sharply after the onset of ischemia. An immunohistochemical labelling study shows that, whereas XO + XD expression in liver tissue is localised in both hepatocytes and endothelial cells, in the intestine that expression is mostly localised in epithelial cells. We conclude that XO may be considered as a major source of reactive oxygen species in rats but not in humans.

The method described here allows the quantitative simultaneous determination of adenosine 5′-triphosphate, adenosine 5′-diphosphate, adenosine 5′-monophosphate, adenosine, guanosine 5′-triphosphate, guanosine 5′-diphosphate, guanosine, inosine 5′-monophosphate, inosine, uric acid, xanthine, hypoxanthine and β-nicotinamide adenine dinucleotide by ion-pair high performance liquid chromatography. The chromatographic analysis requires 26 min per sample and allows the separation of the mentioned metabolites in a time as short as 16 min. Primary cultures of rat cerebellar granule cells were incubated in serum-free medium containing 25 mM KCl for 1.5–48 h and their acid extracts were injected onto column. Uric acid, inosine 5′-monophosphate, inosine, β-nicotinamide adenine dinucleotide, adenosine, adenosine 5′-monophosphate, guanosine 5′-diphosphate, adenosine 5′-diphosphate, guanosine 5′-triphosphate and adenosine 5′-triphosphate were identified and quantified, while hypoxanthine, xanthine and guanosine were below the detection limit. This method makes use of a single-step sample pre-treatment procedure which allows a greater than 91% recovery of the compounds of interest and provides the assay of the metabolites of interest in little amounts of cell extracts. Therefore, this method is suitable to evaluate the energetic state in a variety of cell types, both under normal and dismetabolic conditions, such as after the induction of apoptosis or necrosis.

Objective: The postoperative course of cyanotic children is generally more complicated than that of acyanotic children. A possible reason is reoxygenation injury at the beginning of cardiopulmonary bypass. In this study we tested the hypothesis that reoxygenation of chronically hypoxic hearts is worse than that of normoxic hearts. Methods: Two groups of rats (n = 9 each) were exposed to either room air (fraction of inspired oxygen, 0.21%) or chronic hypoxia (fraction of inspired oxygen, 0.10%) for 2 weeks. Hearts were then isolated and perfused for 30 minutes with hypoxic buffer (oxygen saturation, 10%), followed by 30 minutes of reoxygenation (oxygen saturation, 100%). Results: In hypoxic rats hematocrit values, hemoglobin concentrations, and red cells were higher (69% ± 6% vs 40% ± 6%, 219 ± 14 vs 124 ± 12 g/L, and 10.30 ± 0.6 vs 6.32 ± 0.5/μL/1000, respectively; P

It is still unclear if performance recovery in postischemic hearts is related to their tissue level of high-energy phosphates before reflow. To test the existence of this link, we monitored performance, metabolism and histological damage in isolated, crystalloid-perfused rat hearts during 20 min of low-flow ischemia (90% coronary flow reduction) and reflow. To prevent interference from different ischemia times and perfusing media compositions, the ischemic ATP level was varied by changing energy demand (electrical pacing at 330 min−1). Under full coronary flow conditions, work output, as well as ATP and phosphocreatine contents were the same in control, spontaneously contracting (n = 23) and paced (n = 21) hearts. During low-flow ischemia, the higher work output (p < 0.0001) in paced hearts decreased their tissue content of ATP, phosphocreatine and total adenylates and purines (p < 0.05), as opposed to maintained values in control hearts. During reflow, the recovery of mechanical performance and O2 uptake was 94 ± 5% and 110 ± 9% (p = NS vs. baseline) in controls, vs. 71 ± 5% and 74 ± 6% in paced hearts (p < 0.004 vs. baseline). The levels of ATP and total adenylates and purines remained constant in control, but were markedly depressed (p < 0.05 vs. baseline) in paced hearts. Phosphocreatine+creatine was the same in both groups. These data, together with the observed lack of creatine kinase leakage and of structural damage, indicate that myocardial recovery during reflow reflects the tissue level of ATP, phosphocreatine and total adenylates and purines during ischemia, regardless of physical cell damage.

The purpose of this study was to examine the bioenergetics and regulation of O2uptake (V˙o2) and force production in contracting muscle when blood flow was moderately reduced during a steady-state contractile period. Canine gastrocnemius muscle ( n = 5) was isolated, and 3-min stimulation periods of isometric, tetanic contractions were elicited sequentially at rates of 0.25, 0.33, and 0.5 contractions/s (Hz) immediately followed by a reduction of blood flow [ischemic (I) condition] to 46 ± 3% of the value obtained at 0.5 Hz with normal blood flow. TheV˙o2of the contracting muscle was significantly ( P < 0.05) reduced during the I condition [6.5 ± 0.8 (SE) ml ⋅ 100 g−1⋅ min−1] compared with the same stimulation frequency with normal flow (11.2 ± 1.5 ml ⋅ 100 g−1⋅ min−1), as was the tension-time index (79 ± 12 vs. 123 ± 22 N ⋅ g−1⋅ min−1, respectively). The ratio ofV˙o2to tension-time index remained constant throughout all contraction periods. Muscle phosphocreatine concentration, ATP concentration, and lactate efflux were not significantly different during the I condition compared with the 0.5-Hz condition with normal blood flow. However, at comparable rates of V˙o2and tension-time index, muscle phosphocreatine concentration and ATP concentration were significantly less during the I condition compared with normal-flow conditions. These results demonstrate that, in this highly oxidative muscle, the normal balance of O2supply to force output was maintained during moderate ischemia by downregulation of force production. In addition, 1) the minimal disruption in intracellular homeostasis after the initiation of ischemia was likely a result of steady-state metabolic conditions having already been activated, and 2) the difference in intracellular conditions at comparable rates ofV˙o2and tension-time index between the normal flow and I condition may have been due to altered intracellular O2tension.

Ear lobe blood pHa, PaCo2, PaO2, and O2 saturation (SaO2) were measured in healthy Caucasians and Sherpas at 3400 m (Namche Bazaar, Nepal, n = 4/5), 5050 m (Pyramid Laboratory, Lobuche, Nepal, n = 20/5) and 6450 m (Camp II of Mt Everest, n = 11/7). In the investigated altitude range, pHa increased progressively with altitude from 7.463 +/- 0.005 (mean +/- SE) to 7.496 +/- 0.006 in Caucasians whereas it remained essentially constant (7.45-7.46) in Sherpas. At all altitudes, PaCO2 was higher in Sherpas than in Caucasians (P < 0.02). By contrast, PaO2 and SaO2 were the same in Caucasians and Sherpas at all investigated altitudes. Moreover, in Caucasians sojourning for 3 weeks at 5050 m, PaCO2 kept decreasing whereas pHa, PaO2 and SaO2 remained constant. These data suggest that; (1) respiratory alkalosis was a common finding both in Caucasians and Sherpas; (2) at 6450 m. Sherpas were less alkalotic due to higher PaCO2 than Caucasians, possibly a consequence of a blunted ventilatory response; (3) at 6450 m, SaO2 and PaO2 were the same in Caucasians and Sherpas despite different PaCO2 values. The latter finding could be the consequence of one or more of the following adjustments in Sherpas: (1) an increased efficiency of alveolar O2 transfer, i.e. smaller alveolar-arterial O2 gradient; (2) a decreased (arterial-mixed venous) O2 difference possibly due to increased cardiac output; (3) a reduced increase of the [2,3-DPG]/[Hb] ratio; but not (4) an elevated gas exchange ratio (R). It is concluded that both physiological and biochemical variables contribute to optimize the O2 transport at altitude. Apparently a more efficient adaptation to hypoxia allows Sherpas to limit alkalosis through a lower ventilatory drive and to maintain SaO2 at the same PaO2 by decreasing the [2,3-DPG]/[Hb] ratio.

We investigated the role of carbohydrate antigen sialyl-Lewis a (sLea), an E-selectin ligand and epitope of tumor marker CA19.9, in the development of xenografts in nude mice. To this end, animals were inoculated with the human colon cancer cell line HCT-15, expressing no Lewis antigens, or with a clone expressing sLea (HCT-15-T5). The size of HCT-15-T5 xenografts appeared larger than those of HCT-15 and their average weight was over twice bigger. In both xenografts the mitotic index was found elevated, as determined by Ki-67 assay, and no apoptosis was detected in the tumor cells by both caspase 8 or TUNEL assays. Some apoptotic signals were instead detected in the vessels. Conversely, microvessel density, determined through CD-31 immunohistochemistry, was found 3.2-folds bigger in HCT-15-T5 xenografts (p

The aim of this work was to characterize the ion current that enters mouse metatarsal bones following damage to the cortex. We assessed both the spatial distribution of this current and its dependence on the presence of bicarbonate in the medium. We used a voltage-sensitive probe system vibrating in two dimensions and recorded the signal as function of the position of the probe with respect to the site of damage and of ion substitutions in the medium. When the cortex was damaged (50 microm cylindrical hole penetrating into the marrow cavity), we recorded a steady state net inward electrical current directed toward the site of damage. In nonbicarbonate media, the density of the current was maximal near the center of the hole and ranged from 6 to 18 microA/cm2. As the probe was moved off the center of the hole, measured current density decreased in a manner consistent with the hypothesis that the source of the inward current is localized to the hole. After changing bicarbonate concentration in the medium from 0 to 42 mM, the current density nearly doubled, then decayed back to its original level exponentially over 35 minutes. When the diaphysis of living bone was left intact the current density was close to background level either in the presence or absence of bicarbonate in the medium. Damaged dead bone did not drive any current higher than background level. We conclude that the vibrating probe technique is a powerful tool to characterize ion currents in injured bone, helping to understand the physiology of bone-plasma interface and the bone healing processes. The current density transiently doubled upon addition of bicarbonate, indicating that this ion may carry the electrical current in damaged bone, probably by pump-leak mechanisms operating at the bone-plasma interface.

The xanthine/xanthine oxidase reaction produces reproducible amounts of oxygen-derived free radicals that oxidize human oxyhemoglobin (Hb). We monitored the kinetics of the oxidation of stripped Hb (sHb), purified HbA0 and alpha-alpha cross-linked Hb (HbXL99 alpha) at [Hb] in the 5 to 150 microM (heme) range. For increasing [Hb], the oxidation halftime (t1/2) increased for all Hbs, but t1/2 was always less for HbXL99 alpha than for HbA0 and sHb. Such feature was attributed to the lower affinity for O2 of HbXL99 alpha and may represent a serious problem for use of this Hb as blood substitute.

UnlabelledAlthough chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R.Aim of the studyTo test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O₂) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout.ConclusionsSildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.

Training has been shown to induce cardioprotection. The mechanisms involved remain still poorly understood. Aims of the study were to examine the relevance of training intensity on myocardial protection against ischemia/reperfusion (I/R) injury, and to which extent the beneficial effects persist after training cessation in rats. Sprague-Dawley rats trained at either low (60% $$ {\dot{{V}}\text{O}}_{2\max } $$ ) or high (80% $$ {\dot{{V}}\text{O}}_{2\max } $$ ) intensity for 10 weeks. An additional group of highly trained rats was detrained for 4 weeks. Untrained rats served as controls. At the end of treatment, rats of all groups were split into two subgroups. In the former, rats underwent left anterior descending artery (LAD) ligature for 30 min, followed by 90-min reperfusion, with subsequent measurement of the infarct size. In the latter, biopsies were taken to measure heat-shock proteins (HSP) 70/72, vascular endothelial growth factor (VEGF) protein levels, and superoxide dismutase (SOD) activity. Training reduced infarct size proportionally to training intensity. With detraining, infarct size increased compared to highly trained rats, maintaining some cardioprotection with respect to controls. Cardioprotection was proportional to training intensity and related to HSP70/72 upregulation and Mn-SOD activity. The relationship with Mn-SOD was lost with detraining. VEGF protein expression was not affected by either training or detraining. Stress proteins and antioxidant defenses might be involved in the beneficial effects of long-term training as a function of training intensity, while HSP70 may be one of the factors accounting for the partial persistence of myocardial protection against I/R injury in detrained rats.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague–Dawley rats were maintained for two weeks under CH (10% O2) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 μmol/L LY-294002 (inhibitor of Akt), 10 μmol/L UO-126 (inhibitor of ERK1/2) or 10 μmol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate × developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.

Exposure to hypoxia triggers a variety of adverse effects in the brain that arise from metabolic stress and induce neuron apoptosis. Overexpression of the hypoxia-inducible factor-1α (HIF-1α) is believed to be a major candidate in orchestrating the cell’s defense against stress. To test the impact of HIF-1α on apoptosis during chronic hypoxia in vivo, we examined the protective effect of modulating the nitric oxide (NO)/cGMP pathway by sildenafil, a selective inhibitor of phosphodiesterase-5 (PDE-5). Male ICR/CD-1 mice were divided into 3 groups ( n = 6/group): normoxic (21% O2), hypoxic (9.5% O2), and hypoxic with sildenafil (1.4-mg/kg intraperitoneal injections daily). At the end of the 8-day treatment period, the mice were euthanized and cerebral cortex biopsies were harvested for analyses. We found that sildenafil: (1) did not significantly alter the hypoxia-induced weight loss and hemoglobin increase, but did augment plasma nitrates+nitrites and the tissue content of cGMP and phosphorylated (P) NO synthase III; (2) reversed the hypoxia-induced neuron apoptosis (terminal deoxynucleotidyl transferase positivity and double-staining immunofluorescence, P = 0.009), presumably through increased bcl-2/Bax ( P = 0.0005); and (3) did not affect HIF-1α, but rather blunted the hypoxia-induced increase in P-ERK1/2 ( P = 0.0002) and P-p38 ( P = 0.004). We conclude that upregulating the NO/cGMP pathway by PDE-5 inhibition during hypoxia reduces neuron apoptosis, regardless of HIF-1α, through an interaction involving ERK1/2 and p38.

The authors have investigated the relationships between the active calcium transport across the human red blood cell (RBC) membrane and the RBC aging processes in vivo and in vitro. For the study of this biological system, the authors have determined the active calcium uptake by inside-out membrane vesicles obtained from selected RBC populations. This model provided an optimal way to assess the biochemical and functional responses of the human cell to the oxidative stimulus triggered by the cellular aging processes. The activity of the calcium pump is indeed strictly correlated to the oxidative damage suffered by the RBC, being higher in the aged RBC. It appears that the main controller of the active calcium transport is the age-dependent protein inhibitor of the calcium pump.

Activation of the NO/cGMP pathway modulates smooth muscle cells relaxation and hence vasoconstriction, a major hindrance for the use of cell-free haemoglobin (Hb) as blood substitute, despite conjugation with 5-kDa maleimide poly(ethylene)-glycol (PEG) reduces vasoconstriction in vivo . We aimed at assessing how a recently developed PEGylated-Hb (Deoxy-PEGHb) and manipulation of the NO/cGMP pathway enable modulation of vasoconstriction in isolated rat hearts. Hearts were Langendorff-perfused with oxygenated Krebs–Henseleit (15 ml/min) while monitoring the coronary pressure (CPP) after injection (1 min) of 50 nM norepinephrine followed by a 1 μM Hb or Deoxy-PEGHb bolus, without altering the flow. Deoxy-PEGHb induced less vasoconstriction than Hb. Although the presence of PEG could contribute to vasoconstriction, Deoxy-PEGHb did not contain appreciable amounts of free PEG. Whereas reducing endothelial NO release by 0.2 mM l -NAME increased vasoconstriction, abolishing NO scavenging by Hb using its cyanomet derivative almost completely blunted it. Furthermore, maintaining intracellular cyclic GMP by inhibiting phosphodiesterase-5 with 0.02 mM sildenafil enabled control of Hb-induced vasoconstriction. We conclude that, although PEG-Hb represents a possible approach to limit Hb-induced vasoconstriction, manipulating the NO/cGMP pathway may provide a powerful way to circumvent this problem.

Phosphodiesterases (PDE) are a class of proteins whose most relevant biological activity concerns the modulation of intracellular levels of cyclic nucleotides, e.g., cGMP and cAMP. PDE isoenzyme 5 (PDE5) is specifically involved in cGMP inactivation in the smooth muscle cell. Chemical inhibition of PDE5 by sildenafil, tadalafil or vardenafil recently became a valid therapeutic option aimed at overexpressing the molecular pathway originated from nitric oxide and expressed via increased cell cGMP availability. Based on the optimal tolerability and proven efficacy in various human disorders, EMEA and FDA have approved PDE5 inhibition as an efficient therapy in some cardiovascular, pulmonary and vascular diseases. More specifically, PDE5 inhibition appears successful for the treatment of idiopathic arterial pulmonary hypertension. Furthermore, PDE5 inhibition resulted in important protective effects in the myocardium, i.e., antyhypertrophic and antiapoptic, as well as vascular functions, i.e., increased tolerance to ischemia/reperfusion injury and improved endothelial function, thereby implying a potential usefulness in the treatment of patients with heart failure and coronary artery disease. Evidence currently available for considering PDE5-inhibition an additional opportunity in the treatment of common cardiopulmonary disorders is here provided.

To improve current knowledge of the molecular mechanisms underlying exercise-induced cardioprotection in a rat model of mild exercise training, Sprague-Dawley rats were trained to run on a treadmill up to 55% of their maximal oxygen uptake for 1 h/day, 3 days/week, 14 weeks, with age-matched sedentary controls (n = 20/group). Rats were sacrificed 48 h after the last training session. Despite lack of cardiac hypertrophy, training decreased blood hemoglobin (7.94 +/- 0.21 mM vs. 8.78 +/- 0.23 mM, mean +/- SE, P = 0.01) and increased both plasma malondialdehyde (0.139 +/- 0.005 mM vs. 0.085 +/- 0.009 mM, P = 0.05) and the activity of Mn-superoxide dismutase (11.6 +/- 0.6 vs. 16.5 +/- 1.6 mU/microg, P = 0.01), whereas total superoxide dismutase activity was unaffected. When subjected to 30-min ischemia followed by 90-min reperfusion, hearts from trained rats (n = 5) displayed reduced infarct size as compared to controls (37.26 +/- 0.92% vs. 49.09 +/- 2.11% of risk area, P = 0.04). The biochemical analyses in the myocardium, which included gene expression profiles, real-time PCR, Western blot and determination of enzymatic activity, showed training-induced upregulation of the following mRNAs and/or proteins: growth-arrest and DNA-damage induced 153 (GADD153/CHOP), heme-oxygenase-1 (HO-1), cyclooxygenase-2 (Cox-2), heat-shock protein 70/72 (HSP70/72), whereas heat-shock protein 60 (HSP60) and glucose-regulated protein 75 (GRP75) were decreased. As a whole, these data indicate that mild exercise training activates a second window of myocardial protection against ischemia/reperfusion by upregulating a number of protective genes, thereby warranting further investigation in man.

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20–22 °C) and at 37 °C (P

In vivo exposure to chronic hypoxia is considered to be a cause of myocardial dysfunction, thereby representing a deleterious condition, but repeated aeration episodes may exert some cardioprotection. We investigated the possible role of ATP-sensitive potassium channels in these mechanisms. First, rats (n = 8/group) were exposed for 14 days to either chronic hypoxia (CH; 10% O2) or chronic hypoxia with one episode/day of 1-hr normoxic aeration (CH+A), with normoxia (N) as the control. Second, isolated hearts were Langendorff perfused under hypoxia (10% O2, 30 min) and reoxygenated (94% O2, 30 min) with or without 3 μM glibenclamide (nonselective K+ATP channel-blocker) or 100 μM diazoxide (selective mitochondrial K+ATP channel-opener). Blood gasses, hemoglobin concentration, and plasma malondialdehyde were similar in CH and CH+A and in both different from normoxic (P < 0.01), body weight gain and plasma nitrate/nitrite were higher in CH+A than CH (P < 0.01), whereas apoptosis (number of TUNEL-positive nuclei) was less in CH+A than CH (P < 0.05). During in vitro hypoxia, the efficiency (ratio of ATP production/pressure x rate product) was the same in all groups and diazoxide had no measurable effects on myocardial performance, whereas glibenclamide increased end-diastolic pressure more in N and CH than in CH+A hearts (P < 0.05). During reoxgenation, efficiency was markedly less in CH with respect to N and CH+A (P < 0.0001), and rate x pressure product remained lower in CH than N and CH+A hearts (P < 0.001), but glibenclamide or diazoxide abolished this difference. Glibenclamide, but not diazoxide, decreased vascular resistance in N and CH (P < 0.005 and < 0.001) without changes in CH+A. We hypothesize that cardioprotection in chronically hypoxic hearts derive from cell depolarization by sarcolemmal K+ATP blockade or from preservation of oxidative phosphorylation efficiency (ATP turnover/myocardial performance) by mitochondrial K+ATP opening. Therefore K+ATP channels are involved in the deleterious effects of chronic hypoxia and in the cardioprotection elicited when chronic hypoxia is interrupted with short normoxic aeration episodes.

The amplitude of coronary reactive hyperemia (CRH), elicited by 15 s of ischemia, is reduced in hearts subjected to 5 min of ischemic preconditioning (IP). F0F1ATP synthase activity and ATP concentration are also altered by IP. We hypothesized that F0F1ATP synthase is differently modulated by the inhibitor protein IF1during CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were obtained before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, after IP (Cprec), during CRHprec, and 4 min after CRHprec. F0F1ATP synthase activity, ATP concentration, and ATP-to-ADP ratio (ATP/ADP) were determined. Compared with CRHnp, IP blunted CRHprec. F0F1ATP synthase activity transiently increased during CRHnp, decreased 4 min after CRHnp, and returned to control 2 min later; it was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by IF1. During CRHnp, ATP concentration and ATP/ADP were reduced compared with Cnpand began to rise 6 min thereafter. During Cprec, both parameters were transiently reduced but increased during and after CRHprec. Hence, during CRHnp, F0F1ATP synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why a few seconds of occlusion do not induce IP. After IP, F0F1ATP synthase activity is blunted, and it is not affected by a subsequent 15 s of occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F0F1ATP synthase activity may be a feature of the preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of reduced ATP hydrolysis by cytoplasmic ATPases.

Transitions between rest and work, in either direction, and heavy exercise loads are characterized by changes of muscle pH depending on the buffer power and capacity of the tissues and on the metabolic processes involved. Among the latter, in chronological sequence: (1). aerobic glycolysis generates sizeable amounts of lactate and H(+) by way of the recently described, extremely fast (20-100 ms) "glycogen shunt" and of the excess of glycolytic pyruvate supply; (2). hydrolysis of phosphocreatine, tightly coupled with that of ATP in the Lohmann reaction, is known to consume protons, a process undergoing reversal during recovery; (3). anaerobic glycolysis sustaining ATP production in supramaximal exercise as well as in conditions of hypoxia and ischemia, is responsible for the accumulation of large amounts of lactic acid (up to 1 mol for the whole body). The handling of metabolic acids, i.e., acid-base regulation, occurs both in blood and in tissues, mainly in muscles which are the main producers and consumers of lactic acid. The role of both blood and muscle bicarbonate and non-bicarbonate buffers as well as that of lactate/H(+) cotransport mechanisms is analyzed in relation to acid-base homeostasis in the course of exercise. A section of the review deals with the analysis of the acid-base state of humans exposed to chronic hypoxia. Particular emphasis is put on anaerobic glycolysis. In this context, the so-called lactate paradox is revisited and interpreted on the basis of the most recent findings on exercise at altitude.

We tested the hypotheses that: (1) Ca 2 + handling and force production would be irreversibly altered in skeletal muscle during steady-state contractions when subjected to severe, prolonged hypoxia and subsequent reoxygenation; and (2) application of the cardio-protective drug trimetazidine would attenuate these alterations. Single, living skeletal muscle fibres from Xenopus laevis were injected with the Ca 2 + indicator fura 2, and incubated for 1 h prior to stimulation in 100 μM TMZ-Ringer solution (TMZ; n = 6) or standard Ringer solution (CON; n = 6). Force and relative free cytosolic Ca 2 + concentration ([Ca 2 + ] c ) were measured during continuous tetanic contractions produced every 5 s as fibres were sequentially perfused in the following manner: 3 min high extracellular P o 2 (159 mmHg), 15 min hypoxic perfusion (3-5 mmHg) then 3 min high P o 2 . Hypoxia caused a decrease in force and peak [Ca 2 + ] c in both the TMZ and CON fibres, with no significant (P < 0.05) difference between groups. However, basal [Ca 2 + ] c was significantly lower during hypoxia in the TMZ group vs. the CON group. While reoxygenation generated only modest recovery of relative force and peak [Ca 2 + ] c in both groups, basal [Ca 2 + ] c remained significantly less in the TMZ group. These results demonstrated that in contracting, single skeletal muscle fibres, TMZ prevented increases in basal [Ca 2 + ] c generated during a severe hypoxic insult and subsequent reoxygenation, yet failed to protect the cell from the deleterious effects of prolonged hypoxia followed by reoxygenation.

Altitude hypoxia is a major challenge to the blood O2 transport system, and adjustments of the blood-O2 affinity might contribute significantly to hypoxia adaptation. In principle, lowering the blood-O2 affinity is advantageous because it lowers the circulatory load required to assure adequate tissue oxygenation up to a threshold corresponding to about 5,000 m altitude, whereas at higher altitudes an increased blood-O2 affinity appears more advantageous. However, the rather contradictory experimental evidence raises the question whether other factors superimpose on the apparent changes of the blood-O2 affinity. The most important of these are as follows: (1) absolute temperature and temperature gradients within the body; (2) the intracapillary Bohr effect; (3) the red cell population heterogeneity in terms of O2 affinity; (4) control of altitude alkalosis; (5) the possible role of hemoglobin as a carrier of the vasodilator nitric oxide; (6) the effect of varied red cell transit times through the capillaries.

Lipids compete with glucose for utilization by the myocardium. Amino acids are an important energetic substrate in the heart but it is unknown whether they reduce glucose disposal. The molecular mechanisms by which lipids and amino acids impair insulin-mediated glucose disposal in the myocardium are unknown. We evaluated the effect of lipids and amino acids on the insulin stimulated glucose uptake in the isolated rat heart and explored the involved target proteins. The hearts were perfused with 16 mM glucose alone or with 6% lipid or 10% amino acid solutions at the rate of 15 ml/min. After 1 h of perfusion (basal period), insulin (240 nmol/l) was added and maintained for an additional hour. Both lipids and amino acids blocked the insulin effect on glucose uptake (P

Metabolic events during ischaemia are probably important in determining post-ischaemic myocardial recovery. The aim of this study was to assess the effects of the beta-blocker atenolol and the high energy demand in an ischaemia-reperfusion model free of neurohormonal and vascular factors. We exposed Langendorff-perfused isolated rat hearts to low-flow ischaemia (30 min) and reflow (20 min). Three groups of hearts were used: control hearts (n =11), hearts that were perfused with 2.5 micrograms l-1atenolol (n =9), and hearts electrically paced during ischaemia to distinguish the effect of heart rate from that of the drug (n =9). The hearts were freeze-clamped at the end of reflow to determine high-energy phosphates and their metabolites. During ischaemia, the pressure-rate product was 2.3+/-0.2, 5.2+/-1.1, and 3.3+/-0.3 mmHg 10(3)min in the control, atenolol and paced hearts, respectively. In addition, the ATP turnover rate, calculated from venous (lactate), oxygen uptake and flow, was higher in atenolol (11.2+/-1.7 micromol min-1) and paced (8.1+/-0.8 micromol min-1) hearts than in control (6.2+/-0.8 micromol min-1). At the end of reflow, the pressurexrate product recovered 75.1+/-6.4% of baseline in control vs 54.1+/-9.1 and 48.8+/-4.4% in atenolol and paced hearts (P0.05). In addition, the tissue content of ATP was higher in the control hearts (15.8+/-1. 0 micromol g(dw)(-1)) than in atenolol (10.5+/-2.6 micromol g(dw)(-1)) and paced (10.9+/-1.3 micromol g(dw)(-1)) hearts. Thus, by suppressing the protective effects of down-regulation, both atenolol and pacing apparently depress myocardial recovery in this model.

In this study, we examined the bioenergetic mechanisms underlying myocardial adaptation to O2-limited perfusion. Shortened O2 supply to contracting tissue results in nearly immediate metabolic and performance decline due to fast turnover rate of high-energy phosphates compared to their intracellular concentration.1 Thus, to maintain adequate ATP production, tissue is forced to divert from aerobic to anaerobic pathways: although less efficient than aerobic ones, glycolytic ATP production under hypoxic, high-flow conditions may account for up to half of total energy requirements.2 However, if low O2 supply is associated with reduced flow, the heart preferentially downregulates energy demand to meet supply.3 Whereas these processes were verified during sustained ischemia or hypoxia, it appears important to assess the mechanisms underlying acute regulation of performance. The main reason for this is the need to understand to a greater extent reperfusion injury and the generation of endogenous myocardial protection, both of which may be strictly linked to bioenergetic processes.

We investigated whether one or more factors control performance in O2-limited hearts. For this purpose, we measured the dynamics of myocardial adaptation to reduced O2 supply with a specially designed setup, analyzing early changes after reduction in either flow of the perfusion medium or its PO2. For 10 min, 38 isolated rat hearts underwent low-flow ischemia or hypoxemia, matched for O2 supply. Early during ischemia, developed pressure declined at a rate of 311 +/- 25 mmHg/s; lactate release increased and then leveled off to 3.4 +/- 0.7 mumol/min within 2 min. During hypoxemia, pressure dropped initially, as observed during ischemia. However, it then increased before slowly decreasing. Lactate release during hypoxemia peaked at 13.0 +/- 2.3 mumol/min after 2 min, leveling off to 3.5 +/- 1.3 mumol/min. Glycogen decreased by 52 and 81% in ischemic and hypoxemic hearts, respectively (P < 0.05). Reexposure to ischemia or hypoxemia induced comparable changes in both groups. We conclude that, at the beginning of ischemia, a single factor does limit myocardial performance. This variable, which remains undisturbed for 10 min, is presumably O2 availability. In contrast, approximately 20 s after induction of hypoxemia, glycolytic ATP production can partially override low O2 availability by providing most of the energy needed. During repeated restriction of O2 supply, O2 availability alone limits performance during both ischemia and hypoxemia.

We tested the hypothesis that residual oxygen supply during acute low-flow ischaemia or hypoxemia is a major regulator of myocardial performance, metabolism and recovery. Rat hearts were exposed for 20 min to either ischemia (coronary flow reduced to 10% of baseline), hypoxemia (oxygen content reduced to 10% baseline) or a “mixed” condition (combined ischaemia and hypoxemia). The oxygen supply (coronary flow × oxygen content) was matched in all groups ( n = 16 per group). Hypoxemic hearts had the highest performance (systolic and developed pressures, ±dP d t max and oxygen uptake) and content of IMP and AMP. Ischaemic hearts had the highest content of ATP, phosphocreatine, adenine nucleotides and purines. As flow and/or oxygenation were restored, post-ischemic hearts showed better functional and metabolic recovery than post-hypoxemic ones. “Mixed” hearts were more similar to hypoxemic ones during oxygen shortage but to ischemic ones during recovery. We conclude that as oxygenation is critically limiting, coronary flow is relatively more important than oxygen supply in determining myocardial function, metabolism and recovery, most likely secondary to changes in the metabolism of diffusible substances.