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The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is the master regulator of genes known to be involved in antioxidant, and anti-inflammatory processes, metabolic regulation, and other cellular functions. Here, we also hypothesize a core role for it in endogenous neuroprotection, i.e., the natural adaptive mechanisms protecting the brain from ischemia–reperfusion (I/R) episode. An example of endogenous neuroprotection is ischemia-resistance of the hippocampal regions comprising the CA2, CA3, CA4 and dentate gyrus subfields (here abbreviated to CA2-4,DG) which can be contrasted with the ischemia-vulnerable CA1 region. In the work detailed here, we used a gerbil model of transient cerebral ischemia to examined Nrf2 activation in CA1 and CA2-4,DG, in a control group, and post I/R episode. Data obtained indicate enhanced Nrf2 activity in CA2-4,DG as compared with CA1 in the control, with this difference seen to persist even after I/R. While I/R does indeed cause further activation of Nrf2 in CA2-4,DG, it is associated with slight and transient activation in CA1. Sub-regional differences in Nrf2 activity correlate with immunoreactivity of Keap1 (an Nrf2 suppressor) and Nrf2 target proteins, including heme oxygenase 1, the catalytic and modulatory sub-units of glutamate-cysteine ligase, and glutathione peroxidase 1. Pharmacological Nrf2 activation by sulforaphane results in protection of CA1 after I/R episode. Our results therefore suggest that high Nrf2 activity in CA2-4,DG may guarantee resistance of this region to I/R, potentially explaining the differential sensitivities of the hippocampal regions.

Ischemic stroke is the leading cause of death and long-term disability worldwide, and, while considerable progress has been made in understanding its pathophysiology, the lack of effective treatments remains a major concern. In that context, receiving more and more consideration as a promising therapeutic method is the activation of natural adaptive mechanisms (endogenous neuroprotection) - an approach that seeks to enhance and/or stimulate the endogenous processes of plasticity and protection of the neuronal system that trigger the brain's intrinsic capacity for self-defence. Ischemic preconditioning is a classic example of endogenous neuroprotection, being the process by which one or more brief, non-damaging episodes of ischemia-reperfusion (I/R) induce tissue resistance to subsequent prolonged, damaging ischemia. Another less-known example is resistance to an I/R episode mounted by the hippocampal region consisting of CA2, CA3, CA4 and the dentate gyrus (here abbreviated to CA2-4, DG). This can be contrasted with the ischemia-vulnerable CA1 region. There is not yet a good understanding of these different sensitivities of the hippocampal regions, and hence of the endogenous neuroprotection characteristic of CA2-4, DG. However, this region is widely reported to have properties distinct from CA1, and capable of generating resistance to an I/R episode. These include activation of neurotrophic and neuroprotective factors, greater activation of anti-excitotoxic and anti-oxidant mechanisms, increased plasticity potential, a greater energy reserve and improved mitochondrial function. This review seeks to summarize properties of CA2-4, DG in the context of endogenous neuroprotection, and then to assess the potential utility of these properties to therapeutic approaches. In so doing, it appears to represent the first such addressing of the issue of ischemia resistance attributable to CA2-4, DG.

Diabetes-induced vasculopathies are linked to inflammation mediated by mutually inhibitory nuclear factor-kappaB (NF-κB) and nuclear factor erythroid 2-related factor 2 (Nrf2). NF-κB is activated by superoxide (O

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.

One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism involving activation (nuclear translocation) of the transcription factor nuclear factor-Nuclear factor-κB (Nf-κB-p65). Up-regulation of y(+)LAT2 transporter is coupled with increased inducible nitric oxide synthase (iNOS) expression, which leads to increase nitric oxide (NO) synthesis and protein nitration.

The function of the sino-atrial node (SAN), the pacemaker of the heart, is known to decline with age, resulting in pacemaker disease in the elderly. The aim of the study was to investigate the effects of ageing on the SAN by characterizing electrophysiological changes and determining whether changes in gene expression are involved. In young and old rats, SAN function was characterized in the anaesthetized animal, isolated heart and isolated right atrium using ECG and action potential recordings; gene expression was characterized using quantitative PCR. The SAN function declined with age as follows: the intrinsic heart rate declined by 18 ± 3%; the corrected SAN recovery time increased by 43 ± 13%; and the SAN action potential duration increased by 11 ± 3% (at 75% repolarization). Gene expression in the SAN changed considerably with age, e.g. there was an age-dependent decrease in the Ca(2+) clock gene, RYR2, and changes in many ion channels (e.g. increases in Na(v)1.5, Na(v)β1 and Ca(v)1.2 and decreases in K(v)1.5 and HCN1). In conclusion, with age, there are changes in the expression of ion channel and Ca(2+) clock genes in the SAN, and the changes may provide a partial explanation for the age-dependent decline in pacemaker function.

Background— Heart failure (HF) causes a decline in the function of the pacemaker of the heart—the sinoatrial node (SAN). The aim of the study was to investigate HF-induced changes in the expression of the ion channels and related proteins underlying the pacemaker activity of the SAN. Methods and Results— HF was induced in rats by the ligation of the proximal left coronary artery. HF animals showed an increase in the left ventricular (LV) diastolic pressure (317%) and a decrease in the LV systolic pressure (19%) compared with sham-operated animals. They also showed SAN dysfunction wherein the intrinsic heart rate was reduced (16%) and the corrected SAN recovery time was increased (56%). Quantitative polymerase chain reaction was used to measure gene expression. Of the 91 genes studied during HF, 58% changed in the SAN, although only 1% changed in the atrial muscle. For example, there was an increase in the expression of ERG, K v LQT1, K ir 2.4, TASK1, TWIK1, TWIK2, calsequestrin 2, and the A1 adenosine receptor in the SAN that could explain the slowing of the intrinsic heart rate. In addition, there was an increase in Na + -H + exchanger, and this could be the stimulus for the remodeling of the SAN. Conclusions— SAN dysfunction is associated with HF and is the result of an extensive remodeling of ion channels; gap junction channels; Ca 2+ -, Na + -, and H + -handling proteins; and receptors in the SAN.

Ammonia neurotoxicity is associated with overactivation of N-methyl-D-aspartate (NMDA) receptors leading to enhanced nitric oxide and cyclic GMP synthesis and to accumulation of reactive oxygen and nitrogen species. Ammonia is detoxified in the brain via synthesis of glutamine, which if accumulated in excess contributes to astrocytic swelling, mitochondrial dysfunction and cerebral edema. This study was aimed at testing the hypothesis that the activity of the NMDA/NO/cGMP pathway is controlled by the ammonia-induced production of Gln in the brain. Ammonium chloride (final concentration 5 mM), infused for 40 min to the rat striatum via a microdialysis probe, caused a significant increase in Gln (by 40%), NO oxidation products (nitrite+nitrate=NOx) (by 35%) and cGMP (by 50%) concentration in the microdialysate. A Gln synthetase inhibitor, methionine sulfoximine (MSO, 5 mM), added directly to the microdialysate, completely prevented ammonia-mediated production of Gln, and paradoxically, it increased ammonia-mediated production of NOx and cGMP by 230% and 250%, respectively. Of note, MSO given alone significantly reduced basal Gln concentration in the rat striatum, had no effect on the basal NOx concentration, and attenuated basal concentration of cGMP in the microdialysate by 50%. The results of the present study suggest that Gln, at physiological concentrations, may ameliorate excessive activation of the NO-cGMP pathway by neurotoxic concentrations of ammonia. However, in view of potential direct interference of MSO with the pathway, exogenously added Gln and less toxic modulators of Gln content and/or transport will have to be employed in further studies on the underlying mechanisms.

OBJECTIVES: We tested the hypothesis that low-density lipoprotein-cholesterol induces angiotensin II type 1 receptor upregulation that, in turn, accounts for enhanced oxidative stress, and the subsequent endothelial dysfunction in patients with coronary artery disease. METHODS: Brachial artery flow-mediated vasodilation, serum 8-iso-prostaglandin F2alpha (8-isoprostane), and angiotensin II type 1 receptor density on platelets were measured in 19 patients with coronary artery disease, at entry and after 12 weeks of simvastatin therapy, 40 mg/day. RESULTS: At entry there was a significant linear correlation between: angiotensin II type 1 receptor density and plasma low-density lipoprotein-cholesterol; plasma 8-isoprostane and angiotensin II type 1 receptor density; and flow-mediated vasodilation and 8-isoprostane. Simvastatin therapy reduced low-density lipoprotein-cholesterol, downregulated angiotensin II type 1 receptor, decreased 8-isoprostane, and improved flow-mediated vasodilation. The slopes of the presimvastatin and the postsimvastatin angiotensin II type 1 receptor/low-density lipoprotein relationships did not significantly differ, indicating that simvastatin caused a downregulation of angiotensin II type 1 receptor that could be predicted by the low-density lipoprotein reduction. In addition, simvastatin-mediated changes in 8-isoprostane could be predicted by angiotensin II type 1 receptor downregulation, and flow-mediated vasodilation improvement by changes in 8-isoprostane. A significant correlation existed between simvastatin-mediated changes in 8-isoprostane and angiotensin II type 1 receptor. CONCLUSION: The results of this study are consistent with the hypothesis that in coronary artery disease, the impairment of endothelial function is strongly associated with oxidative stress, oxidative stress with cellular angiotensin II type 1 receptor density, and the angiotensin II type 1 receptor density with low-density lipoprotein-cholesterol, suggesting cause-effect relationships between these variables. In support for this notion, these baseline associations were not significantly disturbed by low-density lipoprotein-lowering therapy with simvastatin.

Objectives: A hypothesis was tested that a reaction product between superoxide (O2−) and nitric oxide (NO) mediates post-ischemic coronary endothelial dysfunction that ischemic preconditioning (IPC) protects the endothelium by preventing post-ischemic cardiac O2− and/or NO formation, and that the opening of the mitochondrial ATP-dependent potassium channel (mKATP) plays a role in the mechanism of IPC. Methods: Langendorff-perfused guinea-pig hearts were subjected either to 30 min global ischemia/30 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mKATP opener, diazoxide (0.5 μM). Coronary flow responses to acetylcholine (ACh) and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of O2− and NO, and functional recoveries were followed during reperfusion. Results: IR impaired the ACh response by approximately 60% and augmented cardiac O2− and NO outflow. Superoxide dismutase (150 U/ml) and NO synthase inhibitor, l-NMMA (100 μM) inhibited the burst of O2− and NO, respectively, and afforded partial preservation of the ACh response in IR hearts. NO scavenger, oxyhemoglobin (25 μM), afforded similar endothelial protection. IPC and diazoxide preconditioning attenuated post-ischemic burst of O2−, but not of NO, and afforded a complete endothelial protection. Diazoxide given after 30-min ischemia increased the O2− burst and was not protective. The effects of IPC and diazoxide preconditioning were not affected by HMR-1098 (25 μM), a selective blocker of plasmalemmal KATP, and were abolished by glibenclamide (0.6 μM) and 5-hydroxydecanoate (100 μM), a nonselective and selective mKATP blocker, respectively. 5-Hydroxydecanoate produced similar effects, whether it was given as a continuous treatment or was washed out prior to IR. Conclusion: The results suggest that in guinea-pig heart: (i) a reaction product between O2− and NO mediates the post-ischemic endothelial dysfunction; (ii) the mKATP opening serves as a trigger of the IPC and diazoxide protection; and (iii) the mKATP opening protects the endothelium in the mechanism that involves the attenuation of the O2− burst at reperfusion.

Inhibition of Na(+)/H(+) (NHE) and Na(+)/Ca(2+) (NCE) exchangers prevents myocardial ischemia/reperfusion injury by preventing cardiomyocyte Ca(2+) overload. We hypothesized that it may influence ischemic/reperfused myocardium also indirectly by preventing endothelial Ca(2+) accumulation, and thereby by attenuating reperfusion-induced formation of nitric oxide (NO) and/or oxygen free radicals. Langendorff-perfused rat hearts were subjected to 30-min ischemia and 30-min reperfusion. Myocardial outflow of NO (nitrite+nitrate) and hydroxyl radical (*OH, salicylate method), and functional recoveries were followed during reperfusion. In all groups, there was a transient rise in NO and *OH outflow upon reperfusion. An inhibitor of NHE, cariporide (10 microM) [(4-Isopropyl-3-methylsulfonyl-benzoyl)-quanidine methanesulfonate], and an inhibitor of the reverse mode of NCE, KB-R7943 (5 microM) (2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate), decreased NO and *OH formation, reduced contracture, and improved the recovery of mechanical function during reperfusion, compared to the untreated hearts. The formation of NO was reduced by 40% by 100 microM N(G)-methyl-L-arginine acetate salt (L-NMMA, NO synthase inhibitor), and not affected by 50 microM L-NMMA. *OH formation, contracture, and the functional recoveries were affected neither by 50 nor by 100 microM L-NMMA. Also, the effects of cariporide and KB-R7943 were unaffected by 100 microM L-NMMA. This study shows for the first time that the inhibition of NHE and NCE attenuates post-ischemic myocardial formation of NO and *OH, suggesting that prevention of Ca(2+) overload is cardioprotective via these mechanisms. The results indicate, however, that NO synthase pathway did not interfere with the protection afforded by NHE or NCE in our model.

The superoxide‐forming NADPH oxidase homologues, Nox1, Nox2, and Nox5, seem to mediate the pro‐atherosclerotic vascular phenotype. The hydrogen peroxide‐forming Nox4 afforded vascular protection, likely via NF‐E2‐related factor‐2 (Nrf2) activation and/or Nox2 downregulation in transgenic mice. We hypothesized that oxidative stress in the intact vasculature involves, aside from the upregulation of the superoxide‐forming Noxs, the downregulation of the Nox4/Nrf2 pathway. Guinea‐pigs and rats were studied either in winter or in summer, and the streptozotocin diabetic rats in winter. Plasma nitrite, and superoxide production by isolated hearts were measured, while frozen tissues served in biochemical analyses. Summer in both species and diabetes in rats downregulated myocardial Nox4 while reciprocally upregulating Nox2 and Nox5 in guinea‐pigs, and Nox2 in rats. Simultaneously, myocardial Nrf2 activity and the expression of the Nrf2‐directed heme oxygenase‐1 and endothelial NO synthase were reduced while activity of the nuclear factor κB (NF‐κB) and the expression of NF‐κB‐directed inducible NO synthase and the vascular cell adhesion molecule‐1 were increased. Cardiac superoxide production was increased while plasma nitrite was decreased reciprocally. Analogous disregulation of Noxs, Nrf2, and NF‐κB, occurred in diabetic rat kidneys. Given the diversity of the experimental settings and the uniform pattern of the responses, we speculate that: (1) chronic vascular oxidative stress is a nonspecific (model‐, species‐, organ‐independent) response involving the induction of Nox2 (and Nox5 in guinea‐pigs) and the NF‐κB pathway, and the repression of Nox4 and the Nrf2 pathway; and (2) the systems Nox2‐NF‐κB and Nox4‐Nrf2 regulate each other negatively.

Over 20 years ago, the Sadowski group separated two mouse lines, one with high (HA) and the other with low (LA) sensitivity to swim stress-induced analgesia (SSIA). Recently, we proposed that increased leakage of the blood-brain barrier (BBB) in the HA line created the difference in the response to SSIA. To search for further evidence for this hypothesis, differences in the levels of the BBB proteins occludin and claudin-5 were analysed. In addition, we sought to evaluate practical differences in BBB permeability by examining the antinociceptive levels in HA and LA mouse lines after IV administration of peptides that have limited access to the CNS. Western blot was used to analyse the differences between occludin and claudin-5. To evaluate the functional differences between the BBB of HA and LA mice, the antinociception levels of endomorphin I, biphalin and AA2016 (peptides with limited BBB permeabilities) in the tail flick test were examined. The expression levels of occludin and claudin-5 in the HA mouse line were lower than in the LA and control mice. Central antinociception of the opioid peptides were significantly higher in the HA line than in the LA and control lines. Our data support the hypothesis that BBB leakage is responsible for the differences between the HA and LA mouse lines. Although SSIA confirmed BBB differences between both lines, it is not limited to the opioid system and could be a useful model for studying the role of the BBB in molecular communications between the periphery and CNS.

The role of adenosine and ATP-sensitive potassium channels (K ATP ) in the mechanism of ischemic preconditioning (IPC)-induced protection against the post-ischemic endothelial dysfunction was studied. Langendorff-perfused guinea-pig hearts were subjected either to 40 min of global ischemia and 40 min reperfusion or were preconditioned prior to the ischemia/reperfusion with three cycles of either 5 min ischemia/5 min reperfusion (IPC) or 5 min infusion/5 min wash-out of adenosine, adenosine A 1 receptor agonist, N 6 -cyclohexyladenosine (CHA) or K ATP opener, pinacidil. The magnitude of coronary flow reduction caused by NO-synthase inhibitor, N ω -nitro- l -arginine methyl ester ( l -NAME), served as an index of a basal endothelium-dependent vasodilator tone. Coronary overflows produced by a bolus of acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of agonist-induced endothelium-dependent and endothelium-independent vascular function, respectively. The coronary flow, LVDP, ACh response and l -NAME response were reduced by 8, 32, 41 and 54%, respectively, while SNP response was not changed in the hearts subjected to ischemia/reperfusion. ACh response was fully restored, l -NAME response was partially restored, and SNP response was not affected in the hearts subjected to IPC. The post-ischemic recoveries of coronary flow and LVDP were not improved by IPC. The protective effect of IPC on the ACh response was mimicked by adenosine, CHA, and pinacidil. The protective effect of IPC, CHA and pinacidil was abolished by K ATP antagonist, glibenclamide. The IPC protection was affected neither by a non-specific adenosine antagonist, 8-p-sulfophenyltheophylline, nor by a specific adenosine A 1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Our data indicate that: (1) IPC affords endothelial protection in the mechanism that involves activation of K ATP , but not adenosine A 1 receptors; (2) exogenous adenosine and A 1 receptor agonist afford the protection, which might be of a potential clinical significance; (3) the endothelial dysfunction is not involved in the mechanism of myocardial stunning in guinea-pig hearts.

The function of the sino-atrial node (SAN), the pacemaker of the heart, is known to decline with age, resulting in pacemaker disease in the elderly. The aim of the study was to investigate the effects of ageing on the SAN by characterizing electrophysiological changes and determining whether changes in gene expression are involved. In young and old rats, SAN function was characterized in the anaesthetized animal, isolated heart and isolated right atrium using ECG and action potential recordings; gene expression was characterized using quantitative PCR. The SAN function declined with age as follows: the intrinsic heart rate declined by 18 ± 3%; the corrected SAN recovery time increased by 43 ± 13%; and the SAN action potential duration increased by 11 ± 3% (at 75% repolarization). Gene expression in the SAN changed considerably with age, e.g. there was an age-dependent decrease in the Ca(2+) clock gene, RYR2, and changes in many ion channels (e.g. increases in Na(v)1.5, Na(v)β1 and Ca(v)1.2 and decreases in K(v)1.5 and HCN1). In conclusion, with age, there are changes in the expression of ion channel and Ca(2+) clock genes in the SAN, and the changes may provide a partial explanation for the age-dependent decline in pacemaker function.

The effects of perfusate calcium reduction, allopurinol and dimethylthiourea on reperfusion-induced arrhythmias and purine wash-out in isolated rabbit and rat hearts were compared. The overall incidence of reperfusion-induced ventricular tachycardia (VT) was 88% and 94% and that of ventricular fibrillation (VF) was 44% and 88% in the control rabbit and rat hearts, respectively. VF was reduced to 10% and 0% in rat and rabbit hearts subjected to perfusate calcium reduction (0.4 mM for 1 min before ischemia and for 1 min before and throughout reperfusion), respectively. In allopurinol, 1 mM, perfused rat hearts the overall incidence of VF was not changed and only the incidence of a sustained VF (that lasting for at least 10 min) was reduced. VT and VF were prevented in allopurinol-perfused rabbit hearts. Dimethylthiourea, 10 mM, reduced the incidence of VF in rat hearts to 16% and did not significantly affect VT and VF in rabbit hearts. In untreated rat hearts, the major purine compounds washed out upon reperfusion were inosine, hypoxanthine, xanthine and urate. Allopurinol augmented the wash-out of adenosine and abolished that of xanthine and urate. In untreated rabbit hearts, the major purine washed out were inosine, adenosine and hypoxanthine. Allopurinol did not cause further increase in adenosine wash-out in rabbit hearts. We speculate that: (1) calcium mediated arrhythmogenic mechanism is operating both in reperfused rat and rabbit heart; (2) free radical mediated mechanism is of an importance only in rat heart; (3) neither a decreased free radical production secondary to xanthine oxidase inhibition nor the augmentation of adenosine wash-out is a likely explanation for the antiarrhythmic effect of allopurinol in reperfused hearts; and (4) high level of myocardial adenosine accumulation during ischemia, probably secondary to low xanthine oxidase activity, may play a role of a natural defence mechanism in ischemic/reperfused rabbit heart.

Seasonality in endothelial dysfunction and oxidative stress was noted in humans and rats, suggesting it is a common phenomenon of a potential clinical relevance. We aimed at studying (i) seasonal variations in cardiac superoxide (O(2)(-)) production in rodents and in 8-isoprostane urinary excretion in humans, (ii) the mechanism of cardiac O(2)(-) overproduction occurring in late spring/summer months in rodents, (iii) whether this seasonal O(2)(-)-overproduction is associated with a pro-inflammatory endothelial activation, and (iv) how the summer-associated changes compare to those caused by diabetes, a classical cardiovascular risk factor. Langendorff-perfused guinea-pig and rat hearts generated ~100% more O(2)(-), and human subjects excreted 65% more 8-isoprostane in the summer vs. other seasons. Inhibitors of NADPH oxidase, xanthine oxidase, and NO synthase inhibited the seasonal O(2)(-)-overproduction. In the summer vs. other seasons, cardiac NADPH oxidase and xanthine oxidase activity, and protein expression were increased, the endothelial NO synthase and superoxide dismutases were downregulated, and, in guinea-pig hearts, adhesion molecules upregulation and the endothelial glycocalyx destruction associated these changes. In guinea-pig hearts, the summer and a streptozotocin-induced diabetes mediated similar changes, yet, more severe endothelial activation associated the diabetes. These findings suggest that the seasonal oxidative stress is a common phenomenon, associated, at least in guinea-pigs, with the endothelial activation. Nonetheless, its biological meaning (regulatory vs. deleterious) remains unclear. Upregulated NADPH oxidase and xanthine oxidase and uncoupled NO synthase are the sources of the seasonal O(2)(-)-overproduction.

Currently, a high carbohydrate/low fat diet is recommended for patients with heart failure and/or hypertension; however, the potentially important role that the composition of dietary fat and carbohydrate might play in the development of LVH and heart failure has not been well characterized. Recent studies demonstrate that cardiomyocyte hypertrophy can also be triggered by activation of insulin signalling pathways, altered adipokine levels or the activity of peroxisome proliferator-activated receptors (PPARs), suggesting that metabolic alterations play a role in the pathophysiology of LVH and heart failure. Hypertensive patients with high plasma insulin or metabolic syndrome have a greater occurrence of LVH, which could be due to insulin activation of the serine-threonine kinase Akt and its downstream targets in the heart, resulting in cellular hypertrophy. PPARs also activate cardiac gene expression and growth, and are stimulated by fatty acids and consumption of a high fat diet. Dietary intake of fats and carbohydrate, the resultant effects of plasma insulin, adipokine, and lipid concentrations, may affect cardiomyocyte size and function, particularly following cardiac injury or with chronic hypertension. This review discusses potential mechanisms by which dietary carbohydrates and fats can affect cardiac growth, metabolism and function, particularly in the context of pressure overload LVH.

Objective: A proposal that injury in ischaemic/reperfused rat heart is critically dependent on the availability of free iron rather than on the efficiency of O2 and H2O2 production was examined. Methods: Isolated working rat hearts from 152 male Wistar rats (200-250 g weight), subjected to 20-40 min of global ischaemia and reperfused for 30 min, were perfused with 10 μmol·litre−1 Fe [III] or Fe [11] and/or 0.6 mmol·litre−1 desferrioxamine, 10 mmol·litre−1 dimethylthiourea, and 1 mmol·litre−1 allopurinol. Curves relating the recoveries of haemodynamic functions and the reperfusion lactate dehydrogenase release to the duration of the preceding ischaemic period were constructed. Morphological examination was also performed. Results: In the untreated hearts, the duration of ischaemia resulting in 50% loss of cardiac output was 29 min. This time was decreased to 24 min and 20 min by Fe[III] and Fe[II], respectively, and was increased to 36 min and 37 min by desferrioxamine and dimethylthiourea, respectively. Desferrioxamine prevented the effect of Fe [III] but not that of Fe[II], whereas dimethylthiourea prevented the effect of Fe [II]. Neither the effect of Fe [III] nor that of Fe [II] was prevented by allopurinol which, however, proved to be beneficial in the untreated hearts. Conclusions: The beneficial effect of desferrioxamine and dimethylthiourea suggest that it is intensification of the Fenton reaction by iron which accounts for iron induced aggravation of the reperfusion injury. Thus we speculate that the availability of free iron, rather than O2 and H2O2, is a limiting factor in the development of injury in an ishaemic/reperfused rat heart. What remains unclear is why allopurinol is unable to prevent iron induced changes.

Oxidative stress may promote chronic inflammation and contribute to accelerated atherogenesis in patients with coronary artery disease (CAD). Sulodexide, a glycosaminoglycan consisting primarily of heparin, has been shown to affect oxidative stress in experimental settings. The purpose of this pilot study was to determine the effect of sulodexide administration on oxidative stress, inflammation and plasma lipids in patients with proven stable CAD.Fifty-six optimally treated male CAD patients (pts), mean age 57+/-6 yrs, were randomized to either 8 weeks of sulodexide treatment (SUL, n=28), or to a control group (n=28). At baseline and at the end of the study, all pts underwent full clinical and standard laboratory plasma level assessment of lipids, markers of inflammation, and 8-isoprostane, as a sensitive index of oxidative stress.At entry the 2 groups did not differ significantly in terms of age, coronary risk factors, clinical status and concomitant medication. SUL treatment appeared to be safe and caused a significant decrease in the level of plasma 8-isoprostane (77.4 vs 44.5 pg/ml, p0.0001) compared with controls (75.7 vs 68.3 pg/ml, p=NS). In contrast, neither LDL cholesterol (2.71 vs 2.72 mmol/l) and triglycerides (1.38 vs 1.43 mmol/l), nor markers of inflammation - fibrinogen (3.7 vs 3.6 g/l), C-reactive protein (0.14 vs 0.13 mg/l), leukocyte count (6.33 vs 6.32x10(9)/l) - were affected by SUL treatment.Sulodexide administration resulted in significant reduction in oxidative stress in stable CAD patients, and neither the changes in cholesterol metabolism nor in systemic inflammation underlay this effect.

In failing hearts, coronary flow is normal, but the coronary flow reserve (CFR) is reduced, so demand-induced ischemia (DII) may occur in response to greater demand for O(2). The objectives of this study were: (i) to verify that dobutamine stimulation produces DII in isolated rat hearts having, like failing hearts, increased left ventricular end-diastolic pressure (LVEDP) and hence reduced CFR and (ii) to study the effects of stimulation of glucose oxidation and of inhibition of fatty acid oxidation in this new model of DII. Isolated rat hearts perfused with 11 mM glucose and 0.6 mM palmitate (or no palmitate) were studied. Stepwise increments in the volume of a balloon placed in LV resulted in reciprocal impairment of CFR, supporting the role of the extravascular compressive forces in determining CFR. CFR was 1.82+/-0.1 and 1.32+/-0.1 (p0.05) in the hearts with LVEDP set to 5 mmHg (controls) and 40 mmHg (expanded), respectively. In controls, dobutamine increased coronary flow, myocardial oxygen consumption (MVO(2)), LVDP, mechanical efficiency, and the rates of palmitate and glucose oxidation, however, the effluent lactate concentration remained unchanged. In the expanded hearts vs. controls, dobutamine-induced increases in coronary flow and MVO(2) were reduced by approximately 50%, the increases in LVDP, efficiency, and rates of glucose and fatty acid oxidation were completely prevented, and lactate production greatly increased with dobutamine, indicating DII. Pyruvate dehydrogenase activator, dichloroacetate (DCA 1 mM) and a putative inhibitor of fatty acid beta-oxidation, trimetazidine (5 microM), both increased the rate of glucose oxidation and attenuated myocardial lactate production during DII, however they did not improve myocardial function during DII. Likewise, palmitate-free perfusion had no beneficial effect during DII although it attenuated lactate production. In the hearts subjected to palmitate-free perfusion plus DCA, lactate overproduction during DII was completely abolished, however, the deterioration of LVDP and mechanical efficiency was only partially prevented. Thus, greater demand for O(2) induces DII in the expanded hearts with reduced CFR. Lactate overproduction secondary to an imbalance between glycolysis and glucose oxidation is not a primary factor adversely affecting cardiac mechanical function during DII. Interventions shifting this balance toward glucose oxidation are not beneficial in the setting of DII in our model although they are known to effectively mitigate contractile dysfunction in the post-ischemic myocardium.

It is known that adenosine 5'-triphosphate (ATP) is a cotransmitter in the heart. Additionally, ATP is released from ischemic and hypoxic myocytes. Therefore, cardiac-derived sources of ATP have the potential to modify cardiac function. ATP activates P2X(1-7) and P2Y(1-14) receptors; however, the presence of P2X and P2Y receptor subtypes in strategic cardiac locations such as the sinoatrial node has not been determined. An understanding of P2X and P2Y receptor localization would facilitate investigation of purine receptor function in the heart. Therefore, we used quantitative PCR and in situ hybridization to measure the expression of mRNA of all known purine receptors in rat left ventricle, right atrium and sinoatrial node (SAN), and human right atrium and SAN. Expression of mRNA for all the cloned P2 receptors was observed in the ventricles, atria, and SAN of the rat. However, their abundance varied in different regions of the heart. P2X(5) was the most abundant of the P2X receptors in all three regions of the rat heart. In rat left ventricle, P2Y(1), P2Y(2), and P2Y(14) mRNA levels were highest for P2Y receptors, while in right atrium and SAN, P2Y(2) and P2Y(14) levels were highest, respectively. We extended these studies to investigate P2X(4) receptor mRNA in heart from rats with coronary artery ligation-induced heart failure. P2X(4) receptor mRNA was upregulated by 93% in SAN (P0.05), while a trend towards an increase was also observed in the right atrium and left ventricle (not significant). Thus, P2X(4)-mediated effects might be modulated in heart failure. mRNA for P2X(4-7) and P2Y(1,2,4,6,12-14), but not P2X(2,3) and P2Y(11), was detected in human right atrium and SAN. In addition, mRNA for P2X(1) was detected in human SAN but not human right atrium. In human right atrium and SAN, P2X(4) and P2X(7) mRNA was the highest for P2X receptors. P2Y(1) and P2Y(2) mRNA were the most abundant for P2Y receptors in the right atrium, while P2Y(1), P2Y(2), and P2Y(14) were the most abundant P2Y receptor subtypes in human SAN. This study shows a widespread distribution of P2 receptor mRNA in rat heart tissues but a more restricted presence and distribution of P2 receptor mRNA in human atrium and SAN. This study provides further direction for the elucidation of P2 receptor modulation of heart rate and contractility.

Study objective — The aim was to evaluate the regulation of contractility by autonomic stimulation in the necrotic stage of cardiomyopathy in male Syrian hamsters. Design — The electrical and contractile activity of isolated intracellularly stimulated ventricular myocytes has been recorded and dose-response curves to [Ca2+]o, and to β adrenergic, α adrenergic, and muscarinic agonists and antagonists were examined. Experimental material — Ventricular cardiomyocytes were isolated by enzymatic dissociation of hearts from 90 to 120 day old cardiomyopathic hamsters CHF 147 and from age matched non-myopathic controls: CHF 148 or golden hamsters. Measurements and main results — The membrane potential was recorded by suction (patch) electrodes. The contractile activity was recorded by a video system as the shortenings of the myocytes. The contractile response (EC50) to β adrenergic stimulation (isoprenaline) showed a bimodal distribution: 60% of the myopathic myocytes responded like the controls, while in the remaining 40% the sensitivity was significantly decreased. The electrical activity and β adrenergic receptor density were not different from the controls. The α adrenergic stimulation (by phenylephrine) was enhanced, while response to the muscarinic agonist carbachol (in the presence of isoprenaline) was attenuated in the myopathic cells. Sensitivity to [Ca2+]o was unchanged. Conclusions — Profound changes occur in the contractile response of myocytes from cardiomyopathic hamster to stimulation by mediators of the autonomic nervous system, which at this necrotic stage are not related to any significant changes in basal contractile response to [Ca2+]o, to the electrical activity, or to the number of β adrenergic receptors.

Brain l -glutamine (Gln) accumulation and increased activity of the NO/cGMP pathway are immediate consequences of acute exposure to ammonia. This study tested whether excess Gln may influence NO and/or cGMP synthesis. Intrastriatal administration of the glutaminase inhibitor 6-diazo-5-oxo- l -norleucine or the system A-specific Gln uptake inhibitor methylaminoisobutyrate increased microdialysate Gln concentration and reduced basal and ammonia-induced NO and cGMP accumulation. Gln applied in vivo ( via microdialysis) or in vitro (to rat brain cortical slices) reduced NO and cGMP accumulation in the presence and/or absence of ammonia, but not cGMP synthesis induced by the NO donor sodium nitroprusside. Attenuation of cGMP synthesis by Gln was prevented by administration of l -arginine (Arg). The l -arginine co-substrates of y + LAT2 transport system, l -leucine and cyclo -leucine, mimicked the effect of exogenous Gln, suggesting that Gln limits Arg supply for NO synthesis by interfering with y+LAT2-mediated Arg uptake across the cell membrane.

Study objective – It has been proposed that oxygen free radicals trigger reperfusion arrhythmias. The mechanism of these arrhythmias is not clear. Thus, the effect of H2O2 on cellular action potentials was examined. Design – Trabecular muscles were superfused either with H2O2 alone or with H2O2 in combination with iron ions or an iron chelating agent or various scavengers of oxygen free radicals. The effect of reduction of the superfusate calcium from 1.8 to 0.2 mmol·litre−1 on H2O2 induced changes was also studied. Experimental material – Thin trabecular muscles isolated from the hearts of guinea pigs (200-300 g) of either sex were used. Measurements and main results – H2O2 (0.6 mmol·litre−1) caused a reproducible sequence of changes consisting of an initial increase in plateau height and in action potential duration, followed after 12-14 min by rapid action potential shortening accompanied by resting membrane depolarisation, reduction in action potential amplitude and dV/dtmax, and by occasional appearance of late afterdepolarisations, leading finally to loss of excitability. This sequence of changes was: (1) accelerated by higher concentrations of H2O2, FeCl3 (0.1 mmol·litre−1), and FeCl2 (0.1 mmol·litre−1); (2) prevented by dimethylthiourea (10 mmol·litre−1) and desferrioxamine (2 mmol·litre−1); (3) not influenced by superoxide dismutase (150 units·ml−1), mannitol (5-50 mmol·litre−1) or PBN (50 μmol·litre−1); and (4) not prevented by a reduction of the superfusate calcium. Conclusions – The electrophysiological alterations induced by H2O2 are caused by a hydroxyl radical formed intracellularly in the iron catalysed Fenton reaction.

The postsynaptic density (PSD) is a dynamic multi-protein complex attached to the postsynaptic membrane composed of several hundred proteins such as receptors and channels, scaffolding and adaptor proteins, cell-adhesion proteins, cytoskeletal proteins, G-proteins and their modulators and signaling molecules including kinases and phosphtases. This review focuses on the prominent PSD scaffolds proteins such as members of the MAGUK (membrane-associated guanylyl kinase), Shank (SH3 domain and ankyrin repeat-containing protein) and Homer families. These molecules interact simultaneously with different kinds of receptors and modulate their function by linking the receptors to downstream signaling events. For example PSD 95, a main member of MAGUK family, interacts directly with carboxyl termini of NMDA receptor subunits and clusters them to the postsynaptic membrane. In addition, PSD 95 is involved in binding and organizing proteins connected with NMDAR signaling. Based on the modular character and ability to form multiproteins interactions, MAGUK, Shank and Homer are perfectly suited to act as a major scaffold in postsynaptic density.

SDF-1, a chemokine secreted by injured tissues, may be instrumental in chemoattracting CXCR4(+) stem cells (SCs) for repair of infarcted myocardium. We hypothesize that the myocardial SDF-1 expression determines also the engraftment and beneficial effects of SCs transplanted into the infarcted heart. Myocardial infarction (MI) was induced in rats by coronary artery ligation. The animals were either sacrificed at 2, 7, 16, 21 or 28 days after MI or were re-operated at 2, 7 or 14 days after MI to receive SCs transplantation, and were sacrificed 14 days later. SCs transplantation consisted of 3 x 15 microl injections of SCs isolated from foetal rat liver (FLSCs) into the myocardium bordering the infarction zone (5 x 10(6) cells/heart, labelled with PKH2 Green Fluorescent Cell Linker, approximately 20% CXCR4(+)). In the MI border zone, SDF-1 and CXCR4 immunostaining was transiently increased after MI, picking at 2 days and down regulating to the sham level by 21 days after MI. Simultaneously, an increased incorporation of CXCR4(+) and CD133(+) cells into capillaries was evident. AMD1300, a blocker of CXCR4, prevented the post-MI expression of CXCR4. In the MI border zone, the cardiomyocyte cross-sectional diameter increased and capillary/cardiomyocyte ratio decreased systematically during the 28 post-MI days, while an interstitial collagen accumulation demonstrated transient increase. FLSCs did not survive in the non-infarcted hearts. In infarcted hearts, FLSCs survived best when they were injected at 2 days after MI. The survival was negligible again when the injection was performed at 14 days after MI. FLSCs transplanted at 2 days after MI caused a further rise in SDF-1, CXCR4, and CD133 expression, compared with the untreated infarcted hearts. Only FLSCs transplanted at 2 days, but not later, attenuated cardiomyocyte hypertrophy and increased capillary/cardiomyocyte ratio in the MI border zone. These results suggest that myocardial signalling for homing of the endogenous and the exogenous SCs is transiently activated early after MI, that SDF-1 is instrumental in this process, and that there is only a narrow time-window after MI when SCs transplantation results in their efficient myocardial engraftment and beneficial anti-remodelling effect.

The hypothesis was tested that endothelin-1 (ET-1)-induced superoxide (O(2)(-)) generation mediates post-ischemic coronary endothelial injury, that ischemic preconditioning (IPC) affords endothelial protection by preventing post-ischemic ET-1, and thus O(2)(-), generation, and that opening of the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the mechanism of IPC. Furthermore, the study was aimed at identifying the source of O(2)(-) mediating the endothelial injury. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min washout of mK(ATP) opener diazoxide (0.5 mM). Coronary flow responses to acetylcholine (ACh) served as a measure of endothelium-dependent vascular function. Myocardial outflow of ET-1 and O(2)(-) and functional recoveries were followed during reperfusion. NADPH oxidase and xanthine oxidase (XO) activities were measured in cardiac homogenates. IR augmented ET-1 and O(2)(-) outflow and impaired ACh response. All these effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. Superoxide dismutase and tezosentan (a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. IR augmented also the activity of NADPH oxidase and XO. Apocynin treatment, that resulted in NADPH oxidase inhibition, prevented XO activation and O(2)(-) generation in IR hearts. The inhibition of XO, either by allopurinol or feeding the animals with tungsten-enriched chow, prevented post-ischemic O(2)(-) generation, although these interventions had no effect on the NADPH activity. In addition, the post-ischemic activation of NADPH oxidase and XO, and O(2)(-) generation were prevented by IPC, tezosentan, thenoyltrifluoroacetone (mitochondrial complex II inhibitor), and tempol (cell-membrane permeable O(2)(-) scavenger). In guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates post-ischemic endothelial dysfunction; (ii) IPC and diazoxide afford endothelial protection by attenuating the ET-1, and thus O(2)(-) generation, and the mK(ATP) opening triggers the protection; (iii) the NADPH oxidase maintains the activity of XO, and the XO-derived O(2)(-) mediates the endothelial injury, and (iv) ET-1 and O(2)(-) (probably of mitochondrial origin) are upstream activators of the NADPH oxidase-XO cascade, and IPC prevents the cascade activation and the endothelial dysfunction by preventing the ET-1 generation.

Evidence indicates that ischemia/reperfusion (IR) results in endothelial dysfunction and neutrophil adhesion in the post-ischemic myocardium and that ischemic preconditioning (IPC), superoxide dismutase (SOD), and anti-endothelin-1 (ET-1) interventions prevent these effects. We tested the hypothesis that ET-1-induced superoxide (O(2)(-)) generation mediates endothelial injury and neutrophil accumulation in the IR heart, that IPC protects the endothelium and prevents the adhesion by attenuating post-ischemic ET-1, and thus O(2)(-), generation, and that the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the IPC-induced protection. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mK(ATP) opener diazoxide (0.5 microM). Neutrophils were infused to the hearts at 15-25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and nitroprusside (SNP) served as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of ET-1 and O(2)(-), P-selectin expression, neutrophil adhesion and functional recoveries were followed during reperfusion. IR augmented ET-1 and O(2)(-) outflow, P-selectin expression, and neutrophil adhesion, and impaired ACh response. These effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. SOD (150 U/ml) and tezosentan (5 nM, a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. The preventive effect of IPC, SOD and tezosentan on P-selectin expression preceded their effect on neutrophil adhesion. These data suggest that in guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates the post-ischemic endothelial dysfunction, P-selectin expression and neutrophil adhesion; (ii) IPC and diazoxide afford protection by attenuating the ET-1, and thus O(2)(-) generation; (iii) the mK(ATP) opening triggers the IPC protection; (iv) endothelial injury promotes post-ischemic neutrophil adhesion, but not vice versa.

The study was aimed at testing the hypothesis that a toxic product of the reaction between superoxide (O(2)(-)) and nitric oxide (NO) mediates, not only endothelial dysfunction, but also endothelium-glycocalyx disruption, and increased neutrophil (PMN) accumulation in the heart subjected to ischemia/reperfusion (IR) injury. Accordingly, we studied if scavengers of either O(2)(-) or NO, or a compound that was reported to attenuate cardiac production of peroxynitrite, would prevent endothelial injury and subsequent PNM adhesion in IR heart. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/35 min reperfusion, and infusion of PMN between 15 and 25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and -independent vascular function, respectively. PMN adhesion and endothelium glycocalyx ultrastructure were assessed in histological preparations. IR impaired the ACh, but not SNP, response by approximately 60%, caused endothelium-glycocalyx disruption, and approximately nine-fold increase in PMN adhesion. These alterations were prevented by superoxide dismutase (150 U/ml), NO synthase inhibitor, L-NAME (10 microM), NO scavenger, oxyhemoglobin (25 microM), and NO donor, SNAP (1 microM), and were not affected by catalase (600 u/ml). The glycocalyx-protective effect of these interventions preceded their effect on PMN adhesion. The data imply that PMN adhesion in IR guinea-pig heart is a process secondary to functional and/or structural changes in coronary endothelium, and that a toxic product of the reaction between superoxide and NO mediates these endothelial changes.

Cardiac ischemia/reperfusion leads to coronary endothelial dysfunction, mediated by superoxide anion (O2-), but not hydroxyl radical (*OH). Ischemic preconditioning and mitochondrial ATP-dependent potassium channel opener (diazoxide) protect endothelium in the mechanism involving attenuation of O2- burst at reperfusion. We hypothesize that the endothelial protection involves upregulation of myocardial anty-O2- defense. Langendorff-perfused guinea-pig hearts were subjected to global ischemia/reperfusion (IR) or were preconditioned prior to IR with three cycles of ischemia/reperfusion (IPC) or infusion/washout of 0.5 microM diazoxide. Coronary flow responses to acetylcholine were measures of endothelium-dependent vascular function. Myocardial outflow of O2- and of *OH during reperfusion and myocardial activities of superoxide dismutase (SOD) and catalase were measured. IR impaired acetylcholine response and augmented cardiac O2- and *OH outflow. IPC, diazoxide, and SOD (150 IU/ml) attenuated O2- outflow, increased *OH outflow and protected endothelium. There were no differences in Cu/Zn-SOD, Mn-SOD and catalase activities between sham-perfused and IR hearts and only catalase activity was increased in the IPC hearts. We speculate that: (i) IPC and diazoxide endothelial protection involves activation of some SOD-like anti-O2- mechanism resulting in attenuation of O2- burst and increase in *OH burst, (ii) improved SOD activity might have not been detected because it was confined to a small, although functionally important, enzyme fraction, like that bound to the endothelial glycocalyx.

Evidence indicates that nitric oxide (NO) suppresses myocardial oxygen consumption (MVO(2)) and regulates myocardial substrate oxidation, however data from in vivo and isolated heart preparations are conflicting. In addition, cardiac endothelin (ET-1) release has been shown to increase with inhibition of NO synthase (NOS), however the effects of ET-1 on myocardial energetics is not clear. We employed the isolated rat heart model to assess the role of NO and ET-1 on myocardial function and metabolism. Oxidation of glucose and FFA was measured using [U-(14)C]glucose and [9,10-(3)H]palmitate. NOS inhibition with N(G)-methyl-L-arginine acetate salt (L-NMMA, 50 microM), resulted in an increase in MVO(2) at a given rate of myocardial external workload, and no change in myocardial glucose or FFA oxidation. ET-1 (25 pM), which caused coronary vasoconstriction similar to that produced by L-NMMA, also increased MVO(2) without an effect on cardiac workload, or substrate oxidation, suggesting a role for ET-I in the regulation of myocardial energetics. We assessed also the effect of ET(A)/ET(B) receptor blockade (tezosentan; 5 nM) on MVO(2) and glucose and FFA oxidation and observed no effect, suggesting that basal ET-1 production does not play a role in regulating MVO2 or substrate selection. In conclusion, inhibition of NOS or the addition of ET-1 resulted in an increase in MVO2, but did not affect glucose or FFA oxidation.

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

Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (*OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating *OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce *OH. The addition of Fe2+ and Cu+ (0-20 microM) to KH resulted in a concentration-dependent increase in *OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 microM) did not result in *OH formation, these ions mediated significant *OH production in the presence of a number of reducing agents. The *OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 microM, 5 microM or 10 microM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in *OH formation. For each Fe2+ concentration tested, the *OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one.(i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting *OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the *OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate *OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.

This study addressed the hypothesis that endothelin promotes neutrophil accumulation in ischemic/reperfused myocardium, not only via its direct effect on neutrophils, but also because it mediates post-ischemic endothelial injury. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/35 min reperfusion, and infusion of neutrophils between 15 and 25 min of reperfusion. The infusion of the endothelin ET(A)/ET(B) receptor antagonist, tezosentan, the endothelin ET(A) receptor antagonist, BQ 123 [cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-], and superoxide dismutase was terminated at reperfusion, 5 min before the start of the neutrophil infusion, to avoid the contact of the drugs with neutrophils. Coronary flow responses to acetylcholine and nitroprusside were used as measures of endothelium-dependent and -independent vascular function, respectively. Neutrophil adhesion and endothelium glycocalyx ultrastructure were assessed in histological preparations. Ischemia/reperfusion resulted in a 54%-impaired acetylcholine response, endothelium glycocalyx disruption, and enhanced neutrophil adhesion (21.6% of microvessels contained neutrophils vs. 2.6% in sham group), the latter prevented by a selectin blocker, sulfatide, 20 microg/ml. These alterations were completely prevented by 0.5 and 5 nM, but not 0.05 nM, tezosentan, and were greatly attenuated by BQ 123, 1 and 10 nM. The glycocalyx-protective effect of these interventions preceded their effect on neutrophil adhesion. Superoxide dismutase, 150 IU/ml, reported before by us to protect post-ischemic endothelium glycocalyx, here prevented the post-ischemic endothelial dysfunction and neutrophil adhesion. The data imply that neutrophil adhesion in ischemic/reperfused guinea-pig heart is a selectin-dependent process, secondary to mostly endothelin ET(A) receptor- and free radical-mediated functional and/or structural changes in the coronary endothelium. Thus, endothelin ET(A)/ET(B) as well as ET(A) receptor antagonists may be useful in attenuation of the inflammatory response in ischemic/reperfused heart. The antagonists may be effective because of their direct effect on neutrophils, as demonstrated by others, and because they provide endothelial protection, as demonstrated here.

The effect of nitric oxide (NO) synthase inhibition on apoptosis of cardiomyocytes during ischemia/reperfusion was investigated. Isolated perfused guinea-pig hearts were subjected to 35 min ischemia (I) followed by 30 min reperfusion (IR) in the presence or absence of NO synthase inhibitors, L-NAME or L-NMMA or a superoxide scavenger, SOD. Apoptosis was assessed by immunohistochemistry (TUNEL assay, Bax protein staining), by spectrophotometric measurement of cytochrome oxidase activity (COX), and by ultrastructural analysis. Inhibition of NOS significantly increased apoptosis with activation of Bax protein and decrease of COX. SOD infusion had a protective effect on these apoptotic markers. The results suggest that endogenous NO synthesis during I/R protects the heart against apoptotic cell death.

Transient ischemia has been shown to impair endothelium-dependent, but not endothelium-independent, coronary vasodilation, indicating selective endothelial dysfunction. Here a hypothesis was tested that agonist mediated activation of protein kinase C (PKC) and the related overproduction of the oxidative species contribute to the mechanism of the endothelial dysfunction. Perfused guinea-pig hearts were subjected either to 30 min global ischemia/30 min reperfusion or to 30 min aerobic perfusion with a PKC activator, phorbol ester (1 n M, PMA). Coronary flow responses to a bolus of acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Salicylate hydroxylation was used as the assay for the myocardial hydroxyl radical (.OH) formation. Both ischemia/reperfusion and PMA impaired the ACh response and augmented the myocardial.OH production. The effect of ischemia/reperfusion on the ACh response: (i) was fully prevented by a PKC inhibitor, chelerythrine (2microM) and a mixed endothelin blocker, bosentan (20microM); (ii) was partially prevented by an endothelin converting-enzyme inhibitor, phosphoramidon (40microM), and superoxide dismutase (150-500 U/ml, SOD) and (iii) was affected neither by catalase (600 U/ml) nor by losartan (20microM) and captopril (250microM), nor by prazosin (10microM). SOD, but not bosentan, partially prevented the effect of PMA on the ACh response. None of the interventions studied affected the SNP response. The reperfusion-induced.OH release was attenuated by chelerythrine and bosentan, was not affected by prazosin and was increased by SOD. These results implicate the following sequence of events in the mechanism of the post-ischemic endothelial dysfunction: ischemia/reperfusion, endothelin-induced PKC activation, increased production of superoxide and/or some of its toxic metabolite, damage to the endothelium and endothelial dysfunction. The results argue against the contribution of angiotensin II, adrenergicalpha(1)-receptors and kinins in the mechanism of the post-ischemic endothelial dysfunction in guinea-pig hearts.

It has been demonstrated that ischemic preconditioning (IPC) affords protection against the post-ischemic endothelial dysfunction. Here, a hypothesis was tested that IPC, by protecting the endothelium, prevents also the adherence of granulocytes (PMNs) in the post-ischemic heart. Langendorff-perfused guinea-pig hearts were subjected to 30 min ischemia/30 min reperfusion (IR) and peritoneal PMNs were infused between 15 and 25 min of the reperfusion. Acetylcholine (ACh)-induced coronary vasodilatation and nitrite outflow were used to measure endothelial function and coronary flow response to sodium nitroprusside (SNP) served as a measure of endothelium-independent vascular function. The endothelial adherence of PMNs to the coronary microvessels was assessed in histological preparation of the myocardium. In the hearts subjected to IR, ACh-induced vasodilatation and nitrite outflow were reduced by 55% and 69%, respectively, SNP response remained unaltered, and 22% of microvessels were occupied by PMNs, as compared to 2% in the sheam perfused hearts. These alterations were attenuated by IPC (3 x 5 min ischemia). A selectin blocker, sulfatide, prevented IR-induced PMNs adherence and did not affect the responses to ACh and SNP. These data demonstrate that IR leads to the endothelial dysfunction and to the selectin-mediated PMNs adhesion in the isolated guinea-pig and that IPC attenuates both alterations. We speculate that the pro-adhesive effect of IR is secondary to the endothelial injury and that the anti-PMNs action represents a novel cardioprotective mechanism of IPC.

The effect of ischemic preconditioning and superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME.(1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts.

Antiarrhythmic effects and intracellular electrophysiological properties of a new antiarrhythmic compound (-)trans-4-[2-hydroxy-3(N-isopropylamino)-propoxyimino]-cis-car ane (9) were studied in several models of arrhythmia and in isolated guinea-pig myocardial preparations. Compound 9 prevented the aconitine-induced arrhythmia, reversed the ouabin-induced arrhythmia depressed the maximum rate depolarization (Vmax), and shortened the action potential duration (ADP) and the effective refractory period (ERP). The data indicate that compound 9 is an effective antiarrhythmic presumably with a class 1 B mechanism of action.

Evidence indicates that ATP-sensitive potassium channels (KATP) participate in the metabolic regulation of coronary flow and that this regulation is attenuated when endothelial production of nitric oxide (NO) is blocked. A hypothesis tested in this study was that, in hearts with the impaired NO-pathway, either with an inhibitor or as a result of ischemia/reperfusion, a coronary vasodilator response to KATP stimulation is impaired as well. In Langendorff perfused rat hearts, a blocker of NO synthesis (N omega-nitro-L-arginine, L-NOARG, 10 microM) and KATP inhibitor (glibenclamide, 0.6 microM) reduced the basal coronary flow by 44% and 29%, respectively. Glibenclamide caused a further 25% drop in the flow in L-NOARG perfused hearts. To determine the responsiveness of coronary resistance vessels to KATP stimulation and NO, dose-response curves (DRC) for KATP opener, pinacidil-, and NO-donor, 3-morpholino-syndomine-hydrochloride (SIN-1)-induced increase in coronary flow were constructed, respectively. The pinacidil DRC was shifted to the right by glibenclamide and L-NOARG and to the left by SIN-1 and adenosine. The L-NOARG-induced effect was reversed by L-arginine. The SIN-1 DRC was shifted to the right by glibenclamide and not affected by L-NOARG. Another NO synthesis blocker, L-NG-monomethylarginine (L-NMMA, 50 microM), caused a 43% drop in coronary flow in the untreated hearts and only 24% drop in the hearts subjected to 20 min global ischemia and 40 min reperfusion. The pinacidil DRC obtained at reperfusion showed a 2.3-fold rightward shift as compared to the DRC obtained before ischemia/reperfusion. Similar displacement of the pinacidil DRC was observed also in L-NMMA perfused hearts and in L-NMMA-perfused hearts which were subjected to ischemia/reperfusion. These results indicate that in the isolated rat heart: (1) NO and KATP, acting simultaneously, participate in the setting of the vasodilator component of the basal coronary flow; (2) The responsiveness of coronary microcirculation to KATP stimulation is attenuated when endothelial NO-pathway is impaired either pharmacologically or by ischemia/reperfusion.

The effect of ischemic preconditioning and Superoxide dismutase (SOD) on endothelial glycocalyx and endothelium-dependent vasodilation in the postischemic isolated guinea-pig hearts was examined. Seven groups of hearts were used: group 1 underwent sham aerobic perfusion; group 2 was subjected to 40 min global ischemia without reperfusion; group 3, 40 min ischemia followed by 40 min reperfusion; group 4 was preconditioned with three cycles of 5 min global ischemia followed by 5 min of reperfusion (IPC), prior to 40 min ischemia; group 5 was subjected to IPC prior to standard ischemia/ reperfusion; group 6 underwent standard ischemia/reperfusion and SOD infusion (150 U/ml) was begun 5 min before 40 min ischemia and continued during the initial 5 min of the reperfusion period; group 7 was subjected to 80 min aerobic perfusion with NO-synthase inhibitor, L-NAME, to produce a model of endothelial dysfunction independent from the ischemia/reperfusion. Coronary flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were used as measures of endothelium-dependent and endothelium-independent vascular function, respectively. Reduction in coronary flow caused by NO-synthase inhibitor, L-NAME, served as a measure of a basal endothelium-dependent vasodilator tone. After completion of each experimental protocol, the hearts were stained with ruthenium red or lanthanum chloride for electron microscopy evaluation of the endothelial glycocalyx. While ischemia led only to a slightly flocculent appearance of the glycocalyx, in ischemia/reperfused hearts the glycocalyx was disrupted, suggesting that it is the reperfusion injury which leads to the glycocalyx injury. Moreover, the coronary flow responses to ACh and L-NAME were impaired, while the responses to SNP were unchanged in the ischemia/reperfused hearts. The disruption of the glycocalyx and the deterioration of ACh and L-NAME responses was prevented by IPC. In addition, the alterations in the glycocalyx and the impairment of ACh responses were prevented by SOD. The glycocalyx appeared to be not changed in the hearts subjected to 80 min aerobic perfusion with L-NAME. In conclusion: (1) the impairment of the endothelium-dependent coronary vasodilation is paralleled by the endothelial glycocalyx disruption in the postischemic guinea-pig hearts; (2) both these changes are prevented by SOD, suggesting the role of free radicals in the mechanism of their development; (3) both changes are prevented by IPC. We hypothesize, therefore, that alterations in the glycocalyx contribute to the mechanism of the endothelial dysfunction in the postischemic hearts. (Mol Cell Biochem 186: 87-97, 1998)