Apoptosis is mediated by cytosolic phospholipase A2 during simulated ischaemia/reperfusion-induced injury in neonatal cardiac myocytes

It has become increasingly clear that apoptosis plays a major role in ischaemia/reperfusion (I/R)-induced cell death, but the molecular basis of this process remains to be elucidated. Therefore, the aim of this study was to investigate the role of cPLA(2) in MAPK phosphorylation and apoptosis in simulated ischaemia/reperfusion (SI/R)-induced injury in neonatal cardiomyocytes. Inhibition of cPLA(2) with AACOCF(3) significantly improved cell viability during SI/R (60.17+/-1.77 to 80.17+/-1.97%, p<0.05). The increase in cell viability was associated with a significant inhibition of p38 phosphorylation (135.3+/-4.47% to 87.94+/-10.71%, p<0.001) as well as with a significant decrease in caspase-3- (320.32+/-17.32% to 146.7+/-28.69%, p<0.01) and PARP-(263.9+/-8.15% to 154.7+/-2.24%, p<0.001) cleavage during SI/R. This study provides evidence for a role for cPLA(2) during SI/R-induced injury. It appears that p38 MAPK is a central role player in the signalling pathway involved.     

PARP-1 gene disruption in mice preferentially protects males from perinatal brain injury

Poly(ADP-ribose) polymerase-1 is over-activated in the adult brain in response to ischemia and contributes to neuronal death, but its role in perinatal brain injury remains uncertain. To address this issue, 7-day-old wild-type (wt) and PARP-1 gene deficient (parp+/- and parp-/-) Sv129/CD-1 hybrid mice were subjected to unilateral hypoxia-ischemia and histologic damage was assessed 10 days later by two evaluators. Poly(ADP-ribose) polymerase-1 knockout produced moderate but significant (p < 0.05) protection in the total group of animals, but analysis by sex revealed that males were strongly protected (p < 0.05) in contrast to females in which there was no significant effect. Separate experiments demonstrated that PARP-1 was activated over 1-24 h in both females and males after the insult in neonatal wt mice and rats using immnocytochemistry and western blotting for poly(ADP-ribose). Brain levels of NAD+ were also significantly reduced, but the decrease of NAD+ during the early post-hypoxia-ischemia (HI) phase was only seen in males. The results indicate that hypoxia-ischemia activates Poly(ADP-ribose) polymerase-1 in the neonatal brain and that the sex of the animal strongly influences its role in the pathogenesis of brain injury.     

Changes and Relationship of PAF and TNF in Rats with Myocardial Ischaemia and Reperfusion Injury

IN THIS STUDY IT IS REPORTED THAT: (1) the levels of blood platelet-activating factor and serum tumour necrosis factor significantly increased after coronary ligation and reperfusion, compared with sham-ligated controls, in an anaesthetized rat model; (2) compared with vehicle controls, pretreatment with the PAF antagonist BN 50739 (10 mg/kg, i.v.) produced significant decreases in infarct size (from 29.6 +/- 4.0% to 22.4 +/- 2.1%, p < 0.05 after 3 h ligation, and from 28.5 +/- 9.5% to 10.5 +/- 4.5%, p < 0.01 after 4 h reperfusion) and the level of serum TNF (from 10.4 +/- 7.7 U/ml to 3.9 +/- 4.8 U/ml, p < 0.05); and (3) a significan positive correlation was found between the level of blood PAF or serum TNF and infarct size. The present results indicate that PAF and TNF may be important mediators involved in myocardial ischaemia and reperfusion injury, and that PAF antagonists may exert a protective effect on ischaemic or reperfused myocardium by inhibiting the interaction of PAF and TNF.     

p38-MAPK and PKB/Akt,possible role players in red palm oil-induced protection of the isolated perfused rat heart?

It has been shown that dietary red palm oil (RPO) supplementation improves reperfusion function. However, no exact protective cellular mechanisms have been established. To determine a potential mechanism for functional improvement, we examined the regulation of both mitogen-activated protein kinases (MAPKs) and PKB/Akt in the presence and absence of dietary RPO supplementation in ischemia/reperfusion-induced injury. Wistar rats were fed a control diet or control diet plus 7 g RPO/kg diet for 6 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured before and after hearts were subjected to 25 min of total global ischemia. Hearts subjected to the same conditions were freeze clamped and used to characterize the degree of phosphorylation of extracellular signal-regulated kinase, p38, c-Jun NH(2)-terminal protein kinase (JNK) and PKB/Akt. Dietary RPO supplementation significantly improved aortic output recovery (72.1 +/- 3.2% vs. 54.0 +/- 3.2%, P < .05). This improved aortic output recovery was associated with significant increases in p38 and PKB/Akt phosphorylation during reperfusion when compared with control hearts. Furthermore, a significant decrease in JNK phosphorylation and attenuation of poly(ADP-ribose) polymerase cleavage occurred in the RPO-supplemented group during reperfusion. Our results suggest that dietary RPO supplementation caused differential phosphorylation of the MAPKs and PKB/Akt during ischemia/reperfusion-induced injury. These changes in phosphorylation were associated with improved functional recovery and reduced cleavage of an apoptotic marker, arguing that dietary RPO supplementation may confer protection via the MAPK and PKB/Akt signaling pathways during ischemia/reperfusion-induced injury.     

Caspase inhibition reduces cardiac myocyte dyshomeostasis and improves cardiac contractile function after major burn injury.

In the heart, thermal injury activates a group of intracellular cysteine proteases known as caspases, which have been suggested to contribute to myocyte inflammation and dyshomeostasis. In this study, Sprague-Dawley rats were given either a third-degree burn over 40% total body surface area plus conventional fluid resuscitation or sham burn injury. Experimental groups included 1) sham burn given vehicle, 400 microl DMSO; 2) sham burn given Q-VD-OPh (6 mg/kg), a highly specific and stable caspase inhibitor, 24 and 1 h prior to sham burn; 3) burn given vehicle, DMSO as above; 4) burn given Q-VD-OPh (6 mg/kg) 24 and 1 h prior to burn. Twenty-four hours postburn, hearts were harvested and studied with regard to myocardial intracellular sodium concentration, intracellular pH, ATP, and phosphocreatine (23Na/31P nuclear magnetic resonance); myocardial caspase-1, -3,and -8 expression; myocyte Na+ (fluorescent indicator, sodium-binding benzofurzan isophthalate); myocyte secretion of TNF-alpha, IL-1beta, IL-6, and IL-10; and myocardial performance (Langendorff). Burn injury treated with vehicle alone produced increased myocardial expression of caspase-1, -3, and -8, myocyte Na+ loading, cytokine secretion, and myocardial contractile depression; cellular pH, ATP, and phosphocreatine were stable. Q-VD-OPh treatment in burned rats attenuated myocardial caspase expression, prevented burn-related myocardial Na+ loading, attenuated myocyte cytokine responses, and improved myocardial contraction and relaxation. The present data suggest that signaling through myocardial caspases plays a pivotal role in burn-related myocyte sodium dyshomeostasis and myocyte inflammation, perhaps contributing to burn-related contractile dysfunction.     

The role of the neutrophil and formed elements of the blood in an in vitro model of reperfusion injury

Using The globally ischaemic isolated guinea-pig heart we conducted studies to assess the role of activated neutrophils (PMNs) and the role of the endothelium in reperfusion injury. Reperfusion injury was induced by a 20 min period of global ischaemia followed by a 30 min reperfusion with Krebs' buffer supplemented with f-Met-Leu-Phe (fMLP) and heparinized blood. Ischaemia alone or blood alone resulted in a complete recovery in contractile function measured by developed pressure, fMLP (500 muM) and blood, administered to normoxic hearts did not affect contractile function. The combination of 100 muM fMLP and blood beginning at reperfusion and continuing for 30 min decreased the recovery in contractile function (max. 33 +/- 6% reovery) while buffer and 100 pM fMLP resulted in a complete recovery in function. In hearts infused with buffer and neutropenic blood incubated with 100 muM fMLP a complete recovery in function was observed. Isolated peritoneal neutrophils, 7-70 x 10(5) PMN/ min, incubated with 100 muM fMLP and Krebs' solution decreased contractile function in a concentration-related manner (max. 44 +/- 11% recovery). Platelets, plasma or red blood cells alone incubated with fMLP did not decrease recovery in developed pressure. Platelets and PMN incubated with 100 muM fMLP did not, while red blood cells and PMN did, elicit a reduction in recovery in contractile function (34 +/- 4% recovery). A 20 min period of global ischaemia destroys the functional integrity of the endothelium (response to Ach). Pre-treatment of the heart with sufficient H(2)O(2) to functionally damage the endothelium, followed by infusion of Krebs' solution supplemented with blood and 100 muM fMLP also elicited a reduction in recovery of contractile function (42 +/- 15% recovery). In summary, partially activated neutrophils play a major role in reperfusion injury and there exists a cooperativity between the RBC and PMN in this model.     

Iloprost for the attenuation of ischaemia/reperfusion injury in a distant organ

The objective of this study was to investigate antioxidant and cytoprotective properties of iloprost in a distant organ after ischaemia reperfusion injury. Male Wistar rats were divided into two groups. After application of anesthaesia both hindlimbs were occluded. A 2-h reperfusion procedure was carried out after 60 min of ischemia. Study group (STU) rats (n=10) received 10 microg kg(-1) iloprost in 1 ml of saline from the tail vein 10 min before reperfusion. Control (CON) group rats (n=10) received an equal amount of saline. The rats were sacrificed by injection of a high dose of thiopentone sodium. Blood and tissue samples (right kidneys) were taken for analysis. Differences in malondialdehyde (MDA), myeloperoxidase (MPO), Na+-K+ ATPase and total antioxidant capacity (TAC) between the groups were analysed. MPO, MDA and TAC levels in the sera of CON and STU groups were 1.60+/-0.26 U l(-1), 11.42+/-5.23 nmol ml(-1), 8.30 x 10(-2)+/- 3.93 x 10(-2) nmol ml(-1) h(-1) and 1.07+/-0.11 U l(-1), 7.60+/-1.81 nmol ml(-1) and 0.15+/-3.23 x 10(-2) nmol ml(-1) h(-1) (p=0.0001, p=0.043 and p=0.0001 respectively). MPO, ATPase and MDA levels in kidneys for CON and STU groups were 1.24+/-0.58 U g(-1), 85.70+/-52.05 nmol mg(-1), 17.90+/-7.40 nmol ml(-1) and 0.78+/-0.31 U g(-1), 195.90+/-56.13 nmol mg(-1) and 10.10+/-0.99 nmol ml(-1) (p=0.046, p=0.0001 and p=0.009 respectively). When given prior to reperfusion, the positive effect of iloprost in the attenuation of distant organ reperfusion injury has been demonstrated.     

Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes

The effects of the pan-caspase inhibitor Q-VD-OPh on caspase activity, DNA fragmentation, PARP cleavage, 7A6 exposition, and cellular adhesivity to fibronectin were analyzed in detail in three different apoptotic systems involving two cell lines (JURL-MK1 and HL60) and two apoptosis inducers (imatinib mesylate and suberoylanilide hydroxamic acid). Q-VD-OPh fully inhibited caspase-3 and -7 activity at 0.05 μM concentration as indicated both by the measurement of the rate of Ac-DEVD-AFC cleavage and anti-caspase immunoblots. Caspase-8 was also inhibited at low Q-VD-OPh concentrations. On the other hand, significantly higher Q-VD-OPh dose (10 μM) was required to fully prevent the cleavage of PARP-1. DNA fragmentation and disruption of the cell membrane functionality (Trypan blue exclusion test) were both prevented at 2 μM Q-VD-OPh while 10 μM inhibitor was needed to inhibit the drug-induced loss of cellular adhesivity to fibronectin which was observed in JURL-MK1 cells. The exposition of the mitochondrial antigen 7A6 occurred independently of Q-VD-OPh addition and may serve to the detection of cumulative incidence of the cells which have initiated the apoptosis. Our results show that Q-VD-OPh efficiency in the inhibition of caspase-3 activity and DNA fragmentation in the whole-cell environment is about two orders of magnitude higher than that of z-VAD-fmk. This difference is not due to a slow permeability of the latter through the cytoplasmic membrane.     

Time Course of Peripheral Oxidative Stress as Consequence of Global Ischaemic Brain Injury in Rats

Free radicals play an important role in the pathogenesis of brain injury. This study evaluates the potential relationship between ischaemia/reperfusion (I/R)-induced brain injury, peripheral oxidative stress (lymphocyte DNA damage), plasma antioxidant potential and uric acid levels. We observed that 15 min of ischaemia were sufficient to significantly increase lymphocyte DNA damage that remained elevated at the end of early (3 h) reperfusion and at later (72 h) reperfusion time; this parameter was not significantly increased, when compared to preoperated levels. In parallel, antioxidant potential was elevated after 15 min of ischaemia, remained high at early (3 h) reperfusion and decreased again with longer (72 h) reperfusion. A close association between the plasma antioxidant status and the uric acid content has been confirmed by findings that changes in TRAP values positively correlate with uric acid concentration in rat plasma after ischaemic injury. Moreover, results of in vitro experiments with extra uric acid addition to control plasma have shown that uric acid contributes to a greater part of TRAP values. These results indicate a similar time course of brain I/R-associated oxidative stress and peripheral antioxidant defence status and/or oxidative stress in animal experiments.     

Baseline pulmonary physiologic values for the squirrel monkey (Saimiri sciureus).

Baseline pulmonary physiologic values were determined on 43 (421-910 g) male and 47 (425-604 g) female squirrel monkeys (Saimiri sciureus). Respiratory rate was found to be 55 +/- 1.9 (SE) breaths per minute for males and 58 +/- 1.7 breaths per minute for females. Tidal volume was 8.9 +/- 0.37 ml for males and 7.5 +/- 0.28 ml for females. Airway resistance for the male was 0.052 +/- 0.006 cm H2O/ml/second; while for the female it was 0.086 +/- 0.011 cm H2O/ml/second. Dynamic compliance was found to be 1.78 +/- 0.15 ml/cm H2O for males and 1.48 +/- 0.124 ml/cm H2O for females. An index of distribution of ventilation was 48 +/- 2.5 breaths for males and 42 +/- 1.7 breaths for females.     

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.     

Sex differences in endothelial STAT3 mediate sex differences in myocardial inflammation

Recent studies have shown that females have improved myocardial functional recovery, TNF receptor 1 (TNFR1) signaling resistance, and increased STAT3 phosphorylation following acute ischemia/reperfusion (I/R) compared with males. We hypothesized that 1) STAT3 deficiency in endothelial cells (EC) impairs myocardial functional recovery in both sexes, 2) EC STAT3 deficiency equalizes sex differences in functional recovery, and 3) knockout of EC STAT3 decreases activation of myocardial STAT3 and increases p38 MAPK activation following acute I/R. Isolated male and female mouse hearts from WT and EC STAT3 knockout (STAT3KO) were subjected to 20-min ischemia/60-min reperfusion, and +/- dP/dt were continuously recorded. Heart tissue was analyzed for the active forms of STAT3 and p38 MAPK as well as expression of caspase-8 (Western blot) following I/R. EC STATKO had significantly decreased myocardial functional recovery in both sexes (%recovered +dP/dt: male 51.6 +/- 3.1 vs. 32.1 +/- 13.1%, female 79.1 +/- 3.6 vs. 43.6 +/- 9.1%; -dP/dt: male 52.2 +/- 3.3 vs. 28.9 +/- 12%, female 75.2 +/- 4.1 vs. 38.6 +/- 10%). In addition, EC STAT3KO neutralized sex differences in myocardial function, which existed in WT mice. Interestingly, EC STAT3 deficiency decreased myocardial STAT3 activation but increased myocardial p38 MAPK activation in both sexes; however, this was seen to a greater degree in females. We conclude that EC STAT3 deficiency resulted in decreased recovery of myocardial function in both sexes and neutralized sex differences in myocardial functional recovery following I/R. This observation was associated with decreased activation of myocardial STAT3 and increased activation of p38 MAPK in EC STAT3KO heart after I/R.     

Effects of aging on responses to furosemide

Furosemide causes both diuretic and non-diuretic changes in renal function. We compared responses to intravenous furosemide 0.5 mg.kg-1 in 38 subjects (30 males, 8 females) aged 18 to 30 with those in 14 subjects (9 males, 5 females) aged 50 and over. There were no consistent differences attributable to gender. Older persons showed greater natriuresis (47 percent in males and 26 percent in females) but their increment in plasma renin activity was markedly reduced. The urinary excretion of thromboxane B2 was elevated in older subjects (58 +/- 10 vs. 30 +/- 4 ng/4 h, p less than 0.05 for males; 48 +/- 7 vs. 29 +/- 4 ng/4 hr, p less than 0.05 for females) while that of 6-keto prostaglandin F1 alpha was not different. While differences in the diuretic response to furosemide may be due to pharmacokinetic differences, the non-diuretic response differences may reflect age related changes in renal prostaglandin synthesis.     

Degradation of alpha-actin filaments in venous smooth muscle cells in response to mechanical stretch

Mechanical stretch has been shown to induce the degradation of alpha-actin filaments in smooth muscle cells (SMC) of experimental vein grafts. Here, we investigate the possible role of ERK1/2 and p38 MAPK in regulating this process using an ex vivo venous culture model that simulates an experimental vein graft. An exposure of a vein to arterial pressure induced a significant increase in the medial circumferential strain, which induced rapid alpha-actin filament disruption, followed by degradation. The percentage of SMC alpha-actin filament coverage was reduced significantly under arterial pressure (91 +/- 1%, 43 +/- 13%, 51 +/- 5%, 28 +/- 3%, and 19 +/- 5% at 1, 6, 12, 24, and 48 h, respectively), whereas it did not change significantly in specimens under venous pressure at theses times. The degradation of SMC alpha-actin filaments paralleled an increase in the relative activity of caspase 3 (3.0 +/- 0.7- and 1.7 +/- 0.4-fold increase relative to the control level at 6 and 12 h, respectively) and a decrease in SMC density (from the control level of 1,368 +/- 66 cells/mm(2) at time 0 to 1,205 +/- 90, 783 +/- 129, 845 +/- 61, 637 +/- 55, and 432 +/- 125 cells/mm(2) at 1, 6, 12, 24, and 48 h of exposure to arterial pressure, respectively). Treatment with a p38 MAPK inhibitor (SB-203580) significantly reduced the stretch-induced activation of caspase 3 at 6 h (from 3.0 +/- 0.7- to 2.2 +/- 0.3-fold) in conjunction with a significant rescue of alpha-actin filament degradation (from 43 +/- 13% to 69 +/- 15%) at the same time. Treatment with an inhibitor for the ERK1/2 activator (PD-98059), however, did not induce a significant change in the activity of caspase 3 or the percentage of SMC alpha-actin filament coverage. These results suggest that p38 MAPK and caspase 3 may mediate stretch-dependent degradation of alpha-actin filaments in vascular SMCs.     

Effects of male accessory sex gland secretions on early embryonic development in the golden hamster

The ventral prostates, dorsolateral prostates, coagulating glands, seminal vesicles and/or ampullary glands were bilaterally removed from adult male hamsters. Removal of these glands did not affect the fertilization rate and cleavage of the embryos at 48 h post coitum (p.c.). Air-dried preparations of the embryos showed a delay in cleavage at 72 h p.c. and a significant number of degenerated embryos was also found in females mated with males from which all the male accessory sex glands had been removed. A significant implantation loss was also observed at 122 h p.c. The results suggest that, in the golden hamster, removal of the male accessory sex gland causes a slower cleavage rate in embryonic development and a significant embryonic loss during pregnancy.     

Timing of microcirculatory injury from ischemia reperfusion

The low flow state that results from ischemia and reperfusion injury is a potentially reversible process that is important in numerous clinical situations. However, the point in time during the course of reperfusion where tissue injury becomes irreversible is unknown. This experiment evaluated the continuum of tissue damage in skeletal muscle after ischemic insult by quantifying the number of flowing capillaries and percentage muscle necrosis in a male Wistar rat skeletal muscle model. A gracilis muscle flap was raised on the vascular pedicle of 39 male Wistar rats and examined at 832x using intravital videomicroscopy. The numbers of flowing capillaries in five consecutive high-power fields were counted for baseline values. The flap was then subjected to 4 hours of global ischemia (except in sham animals, n = 7) by placing a microvascular clamp on the pedicle artery and vein. Upon reperfusion, flowing capillaries were counted in the same five high-power fields at intervals of 5, 15, 30, and 60 minutes, then at 2 to 8 (1-hour intervals), 24, and 48 hours. The gracilis muscle was then harvested at these intervals during reperfusion and assessed for viability. Compared with baseline, flowing capillaries from the ischemia and reperfusion group (mean +/- SEM) decreased significantly in the first 8 hours of reperfusion (7.7 +/- 0.2 to 3.2 +/- 0.3, p < 0.001) with minimal change noted from 8 to 48 hours. Percentage muscle necrosis increased progressively in ischemia and reperfusion preparations from 1 to 7 hours of reperfusion (16.5 +/- 2.6 percent to 38.9 +/- 1.2 percent, p < 0.001). No significant change in muscle necrosis in the ischemia and reperfusion group was noted between 7 and 48 hours. Sham preparations showed no change in the number of flowing capillaries through 3 hours of reperfusion, with a slight decrease at 24 hours. This rat gracilis microcirculation skeletal muscle model demonstrates a heterogeneous reperfusion injury. The decrease in flowing capillaries correlated with the increase in percentage necrosis and appeared to stabilize at the 7- to 8-hour interval. This finding may have important implications for the timing of interventions aimed at minimizing tissue damage from ischemia-reperfusion.     

Apoptosis-inducing factor is a major contributor to neuronal loss induced by neonatal cerebral hypoxia-ischemia

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)(Hq) mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX(Hq)) and female (X(Hq)X(Hq)) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH(2)-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.     

Sex-specific and exercise-acquired cardioprotection is abolished by sarcolemmal KATP channel blockade in the rat heart

The present study was conducted to determine whether the infarct sparing effect of short-term exercise is dependent on the operation of the myocardial sarcolemmal ATP-sensitive K(+) (K(ATP)) channel. Adult male and female Sprague-Dawley rats were exercised on a motorized treadmill for 5 days. Twenty-four hours following the training or sedentary period, hearts were isolated and exposed to 1 h of regional ischemia followed by 2 h of reperfusion on a modified Langendorf apparatus in the presence or absence of the sarcolemmal K(ATP) channel antagonist HMR-1098 (30 microM). Following the ischemia-reperfusion protocol, infarct size was determined as a percentage of the total ischemic zone at risk (ZAR). Short-term exercise reduced infarct size by 24% in males (32 +/- 2% of ZAR; P < 0.01) and by 18% in females (26 +/- 2% of ZAR; P < 0.05). Sarcolemmal K(ATP) channel blockade abolished the training-induced cardioprotection in both males and females, increasing infarct size to 43 +/- 3% and 52 +/- 4% of ZAR, respectively. In the absence of HMR-1098, infarct size was significantly lower in sedentary females than in males (33 +/- 4% vs. 42 +/- 2% of ZAR, respectively; P < 0.01). However, the presence of HMR-1098 abolished this sex difference, increasing infarct size by 58% in the sedentary females (P < 0.01) but having no effect on infarct size in sedentary males. This study demonstrates that the sex-specific and exercise-acquired resistance to myocardial ischemia-reperfusion injury is dependent on sarcolemmal K(ATP) activity during ischemia.     

The source of urinary epidermal growth factor in humans

To clarify the source of human urine EGF, we studied EGF renal clearance in 20 healthy, young adult subjects. Immunoreactive EGF was measured hourly in EDTA plasma, heparin plasma, serum and urine of 12 males and 8 females during a 3 h study period. Plasma and urine creatinine and creatinine clearance were measured and calculated hourly. Mean (and SEM) creatinine clearance was similar in males and females (118 +/- 12 vs 105 +/- 6 ml/min). EGF was not detectable in plasma, whereas relatively high levels were measured in serum (2.5 +/- 0.25 vs 1.5 +/- 0.18 ng/ml in males and females respectively p less than 0.05). Urine EGF excretion averaged 1641 +/- 233 ng/h in males and 1507 +/- 191 ng/h in females (p greater than 0.05). A significant correlation was observed between urine creatinine and urine EGF concentrations in both male (r = 0.98, p less than 0.01) and female (r = 0.94, p less than 0.01) subjects. EGF immunoreactivity in urine and serum eluted from G-75 sephadex columns similarly to recombinant 6000 Mr hEGF. Urine excretion of EGF approximated 1.5 micrograms/h or 25 ng/mg creatine. The high concentrations of EGF found in urine in the face of non-detectable levels of EGF in plasma favor the hypothesis that EGF in urine is derived from kidney synthesis and secretion. The significant positive correlation between urine creatinine and urine EGF suggests a functional correlation between glomerular filtration and the process of tubular EGF excretion.