Regulation of Nrf2 and NF-κB during lead toxicity in bovine granulosa cells.

Lead (Pb), one of the pervasive and protracted environmental heavy metals, is believed to affect the female reproductive system in many species. The Nrf2 and NF-κB are the two key transcriptional factors regulating cellular redox status and response against stress and inflammation respectively, showing an interaction between each other. The aim of this study is to investigate the effect of Pb on bovine granulosa cells (GCs) and its association with the regulation of Nrf2 and NF-κB pathways. For this, bovine GCs were cultured in vitro and exposed to different doses of Pb for 2 h. Cellular response to Pb insult was investigated 24 h post treatment. Results showed that exposure of GCs to Pb-induced ROS accumulation and protein carbonylation. Additionally, GCs exhibited reduction in cell viability and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). This was accompanied by cell cycle arrest at G0/G1 phase. Moreover, Pb downregulated both Nrf2 and NF-κB and their downstream genes. Lead increased the expression of endoplasmic reticulum (ER) stress marker genes (GRP78 and CHOP) and the proapoptotic gene (caspase-3) while the antiapoptotic gene (BCL-2) was reduced. Our findings suggest that Pb-driven oxidative stress affected GCs proliferation, enhances ER stress, induces cell cycle arrest and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk. However, further functional analysis is required to explain different aspects of Nrf2 and NF-κB interactions under metal challenge.

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes.

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Randomized comparison of multipolar, duty-cycled, bipolar-unipolar radiofrequency versus conventional catheter ablation for treatment of common atrial flutter.

INTRODUCTION: Radiofrequency (RF) catheter ablation has been established as an effective and curative treatment for atrial flutter (AFL). Approved methods include a drag-and-drop method, as well as a point-by-point ablation technique. The aim of this study was to compare the acute efficacy and procedural efficiency of a multipolar linear ablation catheter with simultaneous energy delivery to multiple catheter electrodes against conventional RF for treatment of AFL. METHODS: Patients presenting to our department with symptomatic, typical AFL were enrolled consecutively and randomized to conventional RF ablation with an 8-mm tip catheter (ConvRF) or a duty-cycled, bipolar-unipolar RF generator delivering power to a hexapolar tip-versatile ablation catheter (T-VAC) group. For both groups, the procedural endpoint was bidirectional cavotricuspid isthmus block. RESULTS: Sixty patients were enrolled, 30 patients each assigned to ConvRF and T-VAC groups. Total procedure time (40.2 ± 15.8 min vs 60.5 ± 12.7 min), energy delivery time (8.5 ± 3.7 min vs 14.7 ± 5.2 min), radiation dose (14.5 ± 3.5 cGy/cm² vs 31.7 ± 12.1 cGy/cm²), and the minimum number of RF applications needed to achieve block (4.2 ± 2.4 vs 8.9 ± 7.2) were significantly lower in the T-VAC group. In 7 patients treated with the T-VAC catheter, bidirectional block was achieved with less than 3 RF applications, versus no patients with conventional RF energy delivery. CONCLUSION: The treatment of typical AFL using a hexapolar catheter with a multipolar, duty-cycled, bipolar-unipolar RF generator offers comparable effectiveness relative to conventional RF while providing improved procedural efficiency.

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration.

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10-100 µmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10-100 µM) on the membrane potential was measured with the fluorescence dye DiBAC4(3) and the effect on [Ca+2]i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10-100 µmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 µmol/L (35.1±1.8% as compared to control (p≤0.05; n=45)). As compared to the control, the addition of BbCI (100 µmol/L) caused a significant increase of systolic Ca2+ of 28.4±5.0% after 30 min incubation. HUVEC treatment with BbCI (100 µmol/L) showed a weak but significant decrease of the membrane potential of 9.5±0.9% as compared to control (p≤0.05; n=80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca2+ concentration and the membrane potential.

Oxidative and endoplasmic reticulum stress defense mechanisms of bovine granulosa cells exposed to heat stress.

In most mammalian species including cattle, heat stress has detrimental effects on ovarian function through disturbing estradiol production and viability of granulosa cells. However, effect of heat stress and underlying cellular defense mechanisms of bovine granulosa cells is not fully understood. Here, we aimed to investigate the effect of heat stress on granulosa cells function and the associated defense mechanism. For this an in vitro granulosa cell model was used to investigate the role of elevated temperature (41 °C) on granulosa cell functions at 24 h and 48 h exposure compared to the control cultured at 37 °C. The results showed that reactive oxygen species level was higher in cells under 41 °C at 24 h compared to control. In response to increased reactive oxygen species level, the expression of NRF2 and its antioxidant genes, CAT and PRDX1 were higher in bovine granulosa cells exposed to heat stress. Interestingly, heat stress markedly increased expression of endoplasmic reticulum stress marker genes; GRP78 and GRP94, in cultured bovine granulosa cells at 24 h, and higher protein accumulation of GRP78 accompanied by increased expression of apoptotic genes, BAX and CASPASE-3. Moreover, heat stress significantly decreased the bovine granulosa cells proliferation, which was supported by decreased in the expression of proliferation marker gene PCNA. All in all heat stress induce reactive oxygen species accumulation, apoptosis and reduced proliferation, which trigger the NRF2 mediated oxidative stress and endoplasmic reticulum stress response by bovine granulosa cells.

Histochemical assessment of early myocardial infarction using 2,3,5-triphenyltetrazolium chloride in blood-perfused porcine hearts.

INTRODUCTION: Although the chemical mechanism of the triphenyltetrazolium (TTC) reaction, for macroscopic detection of myocardial infarction, has been described previously, literature reports on correct tissue preparation and the use of this technique in intact large animals are lacking. METHODS: We investigated the special requirements for TTC staining in blood-perfused porcine hearts, validated the various handling steps and provided detailed information for precise and easy use of this histochemical method. The left anterior descending coronary artery was occluded for 45 min followed by 6 h of reperfusion in an open chest preparation using anesthetised domestic pigs. The hearts were excised and the organ-handling steps and TTC-staining procedure validated. RESULTS: The protocol includes (i) intracoronary saline perfusion, (ii) pressure-controlled determination of the non-ischemic region by Evans blue dye, (iii) a freeze-thaw cycle, (iv) a triphenyltetrazolium incubation period, and (v) a bleach cycle with 4% paraformaldehyde. The TTC-staining results were confirmed by histology of transitional regions of the infarct area, area-at-risk and non-risk-region. DISCUSSION: If some special features associated with blood-perfused porcine hearts are considered carefully, reliable results for subsequent infarct size calculations can be obtained and large potential errors excluded.

Calpain inhibition reduces infarct size and improves global hemodynamics and left ventricular contractility in a porcine myocardial ischemia/reperfusion model.

Calpains, a family of Ca2+-dependent cysteine proteases, are activated during myocardial ischemia and reperfusion. This study investigates the cardioprotective effects of calpain inhibition on infarct size and global hemodynamics in an ischemia/reperfusion model in pigs, using the calpain inhibitor A-705253. The left anterior descending coronary artery was occluded for 45 min and reperfused for 6 h. A bolus of 1.0 mg/kg A-705253 or distilled water was given intravenously 15 min prior to induction of ischemia and a constant plasma level of A-705253 was maintained by continuous infusion of 1.0 mg/kg A-705253 during reperfusion. Infarct size was assessed histochemically using triphenyltetrazolium chloride staining. Macromorphometric findings were verified by light microscopy on hematoxylin-eosin- and Tunel-stained serial sections. Global hemodynamics, including the first derivate of the left ventricular pressure (dP / dtmax), were measured continuously throughout the experiment. A-705253 reduced the infarct size by 35% compared to controls (P < 0.05). Hemodynamic alterations, including heart rate, aortic blood pressure, central venous pressure and left atrial pressure, were attenuated mainly during ischemia and the first 2 h during reperfusion by A-705253. Cardiac function improved, as determined by dP / dtmax, after 6 h of reperfusion (P < 0.003). Our results demonstrate that myocardial protection can be achieved by calpain inhibition, which decreases infarct size and improves left ventricular contractility and global hemodynamic function. Hence, the calpain-calpastatin system might play an important pathophysiological role in porcine myocardial ischemia and reperfusion damage and A-705253 could be a promising cardioprotective agent.

Mitochondrial dysfunction in sepsis: evidence from bacteraemic baboons and endotoxaemic rabbits.

Mitochondria, that provide most of the ATP needed for cell work, and that play numerous specific functions in biosyntheses and degradations, as well as contributing to Ca2+ signaling, also play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome, and by acute processes, are responsible for numerous diseases. The involvement of impaired mitochondria in the pathogenesis of sepsis is discussed. By means of the skinned fiber technique and high resolution respirometry, we have detected significantly reduced rates of mitochondrial respiration in heart and skeletal muscle of endotoxaemic rabbits. Mitochondria from heart were more affected than those from skeletal muscle. Decreased respiration rates were accompanied by reduced activities of complex I + III of the respiratory chain. Endotoxin-caused impairment was also detectable at the level of the Langendorff perfused heart, where the coronary vascular resistance was significantly increased. For an investigation of the influence of bacteraemia on the mitochondrial respiratory chain, baboons were made septic by infusion of high and low amounts of E. coli. For complex I + III and II + III, a clear dose-dependent decrease was detectable and in animals which died in septic shock, a further decrease of enzyme activities in comparison to the controls were found. These results are discussed in the light of current knowledge on the role of mitochondria in cell pathology in respect to sepsis. In conclusion, we present evidence that mitochondrial function is disturbed during sepsis. Besides ischaemic and poison-induced disturbances of mitochondrial function, sepsis is a further example of an acute disease where impaired mitochondria have to be taken into account.

Bradykinin and histamine generation with generalized enhancement of microvascular permeability in neonates, infants, and children undergoing cardiopulmonary bypass surgery.

OBJECTIVE: To investigate whether generation and liberation of bradykinin and histamine contribute to generalized edema formation in pediatric cardiopulmonary bypass surgery. DESIGN: Prospective observational study. SETTING: Pediatric heart surgery of a university hospital. PATIENTS: Forty-one neonates, infants, and children undergoing cardiopulmonary bypass to correct congenital cardiac anomalies. INTERVENTIONS: Plasma concentrations of bradykinin and histamine were determined before, during, and after cardiopulmonary bypass. Fluid balance was evaluated by control of fluid intake and output. MEASUREMENTS AND MAIN RESULTS: The susceptibility to generalized edema formation increased significantly (r = -.457; p <.005) with decreasing age. Approximately three times higher plasma concentrations of bradykinin (p <.001) were found at the onset of anesthesia and during the total observation period in patients with a fluid retention of >6% of body weight compared with patients with a lower retention rate. Plasma bradykinin reached significantly (p <.01) higher peak concentrations of 237.9 +/- 58.6 fmol/mL during cardiopulmonary bypass and of 227.5 +/- 90.7 fmol/mL during the early postoperative period in patients with severe edema formation in contrast to only 86.6 +/- 10.9 and 65.5 +/- 26.8 fmol/mL in patients with minor fluid retention. A tendency (p =.06) to slightly increasing histamine concentrations from 2.07 +/- 0.13 nmol/L at baseline to 3.32 +/- 1.41 nmol/L during 90 mins of cardiopulmonary bypass was only observed in patients with high fluid retention. CONCLUSIONS: Bradykinin seems to be essentially involved in the enhancement of microvascular permeability in pediatric cardiopulmonary bypass surgery, although a dominant causal role cannot be claimed by this study. Histamine, however, doesn't appear to play a major role and may only contribute as a cofactor. To what extent an increased expression of bradykinin-1 and bradykinin-2 receptors or a reduced potential of bradykinin-degrading enzymes is involved is the object of a further clinical study.

Calpain system and its involvement in myocardial ischemia and reperfusion injury.

Calpains are ubiquitous non-lysosomal Ca(2+)-dependent cysteine proteases also present in myocardial cytosol and mitochondria. Numerous experimental studies reveal an essential role of the calpain system in myocardial injury during ischemia, reperfusion and postischemic structural remodelling. The increasing Ca(2+)-content and Ca(2+)-overload in myocardial cytosol and mitochondria during ischemia and reperfusion causes an activation of calpains. Upon activation they are able to injure the contractile apparatus and impair the energy production by cleaving structural and functional proteins of myocytes and mitochondria. Besides their causal involvement in acute myocardial dysfunction they are also involved in structural remodelling after myocardial infarction by the generation and release of proapoptotic factors from mitochondria. Calpain inhibition can prevent or attenuate myocardial injury during ischemia, reperfusion, and in later stages of myocardial infarction.

Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema.

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 µM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

Omega-3 Fatty acids and vitamin d in cardiology.

Dietary modification and supplementation play an increasingly important role in the conservative treatment of cardiovascular disease. Current interest has focused on n-3 polyunsaturated fatty acids (PUFA) and vitamin D. Clinical trial results on this subject are contradictory in many aspects. Several studies indicate that n-3 PUFA consumption improves vascular and cardiac hemodynamics, triglycerides, and possibly endothelial function, autonomic control, inflammation, thrombosis, and arrhythmia. Experimental studies show effects on membrane structure and associated functions, ion channel properties, genetic regulation, and production of anti-inflammatory mediators. Clinical trials evaluating a possible reduction in cardiovascular disease by n-3 PUFA have shown different results. Supplementation of vitamin D is common regarding prevention and treatment of osteoporosis. But vitamin D also seems to have several effects on the cardiovascular system. Vitamin D deficiency appears to be related to an increase in parathyroid hormone levels and can predispose to essential hypertension and left ventricular hypertrophy, increased insulin resistance, and eventually to atherosclerosis and adverse cardiovascular events. Randomized prospective clinical trials are needed to determine whether vitamin D and omega-3 FA supplementation therapy should be recommended as a routine therapy for primary or secondary prevention of cardiovascular disease.

Transcatheter aortic valve implantation: our experience and review of the literature.

Transcatheter Aortic Valve Implantation [TAVI] is widespread worldwide as an alternative therapy procedure to the patients suffering from severe aortic valve stenosis. However, we shouldn't forget that the conventional surgical aortic valve replacement is still the gold standard therapy for severe aortic valve stenosis. For the patients who cannot be treated conventionally because of high risk comorbid diseases and older age, TAVI is an effective alternative therapy method. The indications should be limited, concerning the high mortality rate, 10% within 30 days of intervention. Long term efficacy data are still inadequate. Although the indications are restricted to older patients with a STS score >10 or log-Euro Score >20, age is not a definite indication for this treatment. The patients should be assessed by a heart team including a non-interventional cardiologist, interventional cardiologist, cardiac anesthesiologist and cardiac surgeon according to their general status, frailty and STS- Euro score. In other words, assessment and treatment of the patient by a heart team is the main factor besides the limited power of the scoring systems. The treatment should be applied to the patients with an aortic annulus diameter between 18-27 mm and a life expectancy of at least over 1 year. The currently ongoing investigations are focused on parameters like safety, efficiency and long term reliability of TAVI. The scientific and technical developments lead to new definitions and parameters regarding the treatment indications of severe aortic valve stenosis. In this review, we present the actual data about TAVI and also our own experiences.

Tako-tsubo cardiomyopathy in a 92-year old woman.

Tako-Tsubo cardiomyopathy (TTC) is an acute reversible cause of segmental myocardial dysfunction that is poorly understood and cannot be explained by the occlusion of a single coronary vessel. Its clinical presentation is similar to that of acute coronary syndrome and is often precipitated by a severe psychological or physical stress.

Quadripolar Left Ventricular Lead in a Patient with CRT-D Does Not Overcome Phrenic Nerve Stimulation.

Effective cardiac resynchronization therapy (CRT) requires an accurate atrio-biventricular pacing system. The innovative Quartet lead is a quadripolar, over-the-wire left ventricular lead with four electrodes and has recently been designed to provide more options and greater control in pacing vector selection. A lead with multiple pacing electrodes is a potential alternative to physical adjustment of the lead and may help to overcome high thresholds and phrenic nerve stimulation (PNS).

GRACE risk score as predictor of in-hospital mortality in patients with chest pain.

INTRODUCTION: Chest pain and chest discomfort are common problems in the acute care setting. Life-threatening causes of chest pain must be quickly differentiated from other less serious causes. There is a need to stratify risk rapidly in patients presenting to the emergency department (ED) with chest pain. This study evaluates the relationship between the GRACE risk score (GRS) and in-hospital mortality in patients presenting to the ED with chest pain of all causes. METHODS: We conducted a prospective study of a consecutive sample of 1,014 patients with chest pain and chest discomfort presenting to the medical ED of the University Clinic in Giessen, Germany. The GRS was calculated for each patient at admission. Additionally, the reason for admission into the hospital and the diagnosis on discharge or diagnosis of death were recorded. The relative risk between the risk groups was assessed, and the functional dependency between the GRS and observed in-hospital death was analyzed. RESULTS: A total of 94 patients died during the stay in the hospital, 83 patients with high risk, 9 with medium risk, and 2 with low risk. The risk of in-hospital death was 24.5% for high-risk patients, 2.6% for medium-risk patients, and 0.6% for patients with low risk. The correlation between the GRS and in-hospital mortality is strongly positive (p < 0.01). CONCLUSION: This study shows that the GRS accurately stratifies risk of intra-hospital mortality in patients presenting to the ED with chest pain and can guide patient triage and management.

Reduction of myocardial infarction by postischemic administration of the calpain inhibitor A-705253 in comparison to the Na(+)/H(+) exchange inhibitor Cariporide in isolated perfused rabbit hearts.

The calpain inhibitor A-705253 and the Na(+)/H(+)-exchange inhibitor Cariporide were studied in isolated perfused rabbit hearts subjected to 60 min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid solely or in combination at the beginning of reperfusion. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were performed. Myocardial infarct size and area at risk (transiently not perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 72.7+/-4.0% of the area at risk in untreated controls, but was significantly smaller in the presence of the inhibitors. The largest effect was seen with 10(-6) m A-705253, which reduced the infarcted area to 49.2+/-4.1% of the area at risk, corresponding to a reduction of 33.6%. Cariporide at 10(-6) m reduced the infarct size to the same extent. The combination of both inhibitors, however, did not further improve cardioprotection. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, heart rate, and in the release of lactate dehydrogenase and creatin kinase from heart muscle.

Reduction of myocardial infarction by calpain inhibitors A-705239 and A-705253 in isolated perfused rabbit hearts.

Two novel calpain inhibitors (A-705239 and A-705253) were studied in isolated perfused rabbit hearts subjected to 60-min occlusion of the ramus interventricularis of the left coronary artery (below the origin of the first diagonal branch), followed by 120 min of reperfusion. The inhibitors were added to the perfusion fluid in various final concentrations from the beginning of the experiments before the coronary artery was blocked. Hemodynamic monitoring and biochemical analysis of perfusion fluid from the coronary outflow were carried out. Myocardial infarct size and the area at risk (transiently non-perfused myocardium) were determined from left ventricular slices after a special staining procedure with Evans blue and 2,3,5-triphenyltetrazolium chloride. The infarcted area (dead myocardium) was 77.9+/-2.3% of the area at risk in untreated controls ( n =12). The infarct size was significantly reduced in the presence of both calpain inhibitors. The best effect was achieved with 10 -8 M A-705253 ( n =8), which reduced ( p <0.001) the infarcted area to 49.3+/-3.9% of the area at risk, corresponding to an infarct reduction of 61.8%. No statistical difference was observed between the experimental groups in coronary perfusion, left ventricular pressure, and in the release of lactate dehydrogenase and creatine kinase from heart muscle.

Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases.

During dermal injury and inflammation the serine proteases kallikreins cleave endogenous, multifunctional substrates (kininogens) to form bradykinin and kallidin. The actions of kinins are mediated by preferential binding to constitutively expressed kinin-B2 receptors or inducible kinin-B1 receptors. A feature of the kinin-B1 receptors is that they show low levels of expression, but are distinctly upregulated following tissue injury and inflammation. Because recent evidence suggested that kinin-B1 receptors may perform a protective role during inflammation, we investigated the specific occurrence of the kallikrein-kinin components in skin biopsies obtained from normal skin, patients undergoing surgery, basalioma, lichenificated atopic eczema, and psoriasis. The tissue was immunolabeled in order to determine the localisation of tissue pro-kallikrein, kallikrein, kininogen and kinin receptors. The kinin components were visualised in normal, diseased and traumatised skin, except that no labelling was observed for kininogen in normal skin. Of the five types of tissue examined, upregulation of kinin-B1 receptors was observed only in skin biopsies obtained following surgery. In essence, the expression of kinin-B1 receptors did not appear to be enhanced in the other biopsies. Within the multiple steps of the inflammatory cascade in wound healing, our results suggest an important regulatory role for kinin-B1 receptors during the first phase of inflammation following injury.

A novel water-soluble and cell-permeable calpain inhibitor protects myocardial and mitochondrial function in postischemic reperfusion.

The effects of the novel calpain inhibitor A-705239 were studied in isolated perfused rabbit hearts subjected to 45 min of global ischemia, followed by 60 min of reperfusion. During 15 min of perfusion the inhibitor accumulated in myocardial tissue up to 16 times the concentration in the perfusate. Almost complete recovery and survival of heart function (90%) was seen with an inhibitor concentration of 10(-8) M in the perfusion fluid when the compound was administered prior to ischemia. Left ventricular pressure amplitude and coronary flow showed significantly higher values during reperfusion in the presence of the inhibitor. A-705239 significantly reduced the release of creatine kinase, from 166+/-49 U/l in untreated hearts to 44+/-10 U/l, and diminished the release of lactate dehydrogenase from 118+/-20 U/l in untreated hearts to 63+/-4 U/l. Mitochondrial dysfunction following ischemia and reperfusion was markedly attenuated by the inhibitor. Thus, the state 3 respiration rate only decreased to 4.2 in contrast to 2.6 nmol O2/(min x mg s.w.) in untreated hearts, reflecting a reduced damage of oxidative phosphorylation. Furthermore, in the presence of the inhibitor the inner mitochondrial membranes became less permeable as indicated by a smaller leak respiration. The excellent properties of A-705239 should make this compound a valuable tool for further pharmacological studies.