alpha - and beta -phosphorylated amines and pyrrolidines, a new class of low toxic highly sensitive 31P NMR pH indicators. Modeling of pKa and chemical shift values as a function of substituents

Fourteen linear and cyclic alpha- and beta-aminophosphonates in which the P-atom is substituted by alkoxy groups have been synthesized and evaluated as (31)P NMR pH markers in Krebs-Henseleit buffer. pK(a) values varied with substitution in the range 1.3-9.1, giving potentially access to a wide range of pH. Temperature had a weak influence on pH and a dramatic increase in ionic strength slightly modified the pK(a) of the pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH).All compounds displayed a 4-fold better NMR sensitivity than inorganic phosphate or other commonly used phosphonates, as assessed by differences delta(b)-delta(a) between the chemical shifts of the protonated and the unprotonated forms. In isolated perfused rat hearts, a non-toxic concentration window of 1.5-15 mm was determined for three representative compounds. Using empirical linear relationships, the experimental values of pK(a), delta(a), and delta(b) have been correlated with the two-dimensional structure, i.e. the chemical nature of substituents bonded to the secondary amine and P-atom. The data suggest that DEPMPH and its cyclic and linear variants are ideal versatile (31)P NMR probes for the study of tenuous pH changes in biological processes.     

Effect of ischemia on NMR detection of phosphorylated metabolites in the intact rat heart

Phosphorus NMR spectroscopy is an important technique for the investigation of metabolism in tissues and intact organisms (including man). However, quantitation of the signals from an NMR experiment is difficult because it is not known from which regions of a cell metabolites are detected. It is generally believed that only metabolites free in the cytosol are observed. In this study a comparison of concentration measurements obtained by NMR and after freeze extraction was made in the normoxic and ischemic rat heart. The influence of ischemia was examined because of its potential effect on the level of phosphate metabolites in various compartments. The same fraction of ATP always appears visible to NMR, whereas inorganic phosphate is largely NMR invisible until after a period of ischemia and the phosphomonoesters are only partially observed early in ischemia.     

Sustained postischemic cardiodepression following magnesium-diltiazem cardioplegia

Magnesium-diltiazem cardioplegia was evaluated in the intact, perfused rat heart to determine whether the joint administration of these agents would adversely affect myocardial contractile and high-energy phosphate recovery following intermittent, normothermic global ischemic arrest. Sequential metabolic and functional analyses were performed on isolated perfused rat hearts during each phase of the experimental protocol: control (10 min), normoxic cardioplegia (10 min), intermittent global ischemic arrest (two 15-min periods separated by 2 min infusion of the normoxic cardioplegic perfusate), and normoxic postischemic control reperfusion (60 min). Four different cardioplegic solutions were evaluated: 30 mM KCl, 30 mM KCl with 2 mg diltiazem/liter, 20 mM MgCl2, and 20 mM MgCl2 with 2 mg diltiazem/liter. Myocardial phosphatic metabolite levels and intracellular pH were analyzed nondestructively in the intact hearts by phosphorus-31 NMR spectroscopy. Corresponding measurements of peak left intraventricular pressure, rate of peak pressure development (dP/dt), and contraction frequency were performed at the midpoint during each 5-min interval of 31P NMR signal averaging. Magnesium plus diltiazem-treated hearts were distinguished from all other groups by a marked delay in postischemic functional recovery consisting of a prolonged depression in contractility (34% of control, P less than 0.01) that persisted throughout the first 50 min of postischemic reperfusion. Diltiazem in combination with magnesium cardioplegia was detrimental to postischemic functional recovery, despite a rapid restoration of high-energy phosphate stores. The apparent adverse interactive effects of excess magnesium and diltiazem suggest that elective ischemic arrest with magnesium cardioplegia in combination with diltiazem may be contraindicated clinically. The mechanistic basis and drug specificity of this response require further clarification. The present findings appear to exclude ATP and PCr production, and structural causes as the basis for the observed aberrant functional recovery from global ischemia of magnesium plus diltiazem-arrested hearts.     

Rate of ATP synthesis in the perfused rat liver by 31P cryo-NMR

ATP concentrations in the perfused rat liver during normoxic perfusion, transient ischemia, and recovery from transient ischemia were measured using the modified 31P cryo-NMR method (Chance, B., Nakase, Y., Bond, M., Leigh, J. S., Jr., and McDonald, G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4925-4929). Transient ischemia was induced in the perfused livers of starved rats, and multiple freeze-trapped tissue samples were taken from each liver at short intervals (15-30 s) during ischemia or following reperfusion. The freeze-trapped tissue was pulverized together with an antifreezing agent and high energy metabolites were measured by 31P NMR at 243 K after thawing. By using the cryo-NMR technique, a biochemical time resolution of 2 s could be achieved. Absolute metabolite concentrations were calculated by comparing the peak areas with internal standards mixed into the samples. Good time resolution and reliable concentration measurements provided by the cryo-NMR method enable us to estimate the ATP synthesis rate in the perfused liver during reperfusion following transient ischemia. The rate of ATP synthesis in the normoxic perfusion was 1.95 mumol/min/g wet weight; the maximal ATP synthesis rate during the recovery phase from ischemia was 5.75 mumol/min/g wet weight.     

The 31P NMR visibility of ATP in perfused rat liver remains about 90%, unaffected by changes of metabolic state.

The 31P NMR visibility of ATP of the perfused rat liver was tested over a wide range of metabolic conditions, including normoxic and hypoxic perfusions, fructose loads, and various intervals of normothermic ischemia, for both ad libitum fed and 24-h fasted rats. The 31P NMR signal of ATP was compared to the concentration of ATP determined by enzymatic assays on liver biopsies performed at the end of NMR acquisition. In a first series of experiments, the NMR resonance of intracellular ATP was quantitated in absolute terms by applying the 1H NMR water signal as internal reference: during normoxic and hypoxic perfusions, a constant amount of ATP (0.43 +/- 0.19 mM, mean +/- SD), approximately 12% of the cellular ATP, is not detected by NMR. Nevertheless, there is a high correlation (slope = 0.96 +/- 0.09; r2 = 0.93) between the measurements of ATP by 31P NMR spectroscopy and by biochemical analysis. In a second series of experiments, there was a highly significant correlation between the NMR and analytical biochemical measurements of ATP for whole range of metabolic states, i.e., fructose loads (1.0-10 mM) and various intervals of normothermic ischemia (ranging from 2 to 12 min), indicating unchanged ATP visibility. Thus, as opposed to the studies of Murphy et al. [Murphy, E., et al. (1988) Biochemistry 27, 526-528], it is concluded that ATP at 37 degrees C remains almost entirely visible in the perfused rat liver, also during ischemia.     

Response of normal and reperfused livers to glucagon stimulation: NMR detection of blood flow and high-energy phosphates

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (Pi) to ATP ratio in normal livers as measured by ³¹P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the Pi/ATP ratio of 18%, 40%, and 40%, respectively. ¹⁹F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to deceases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.     

ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia

Interest is growing in the role of adenosine triphosphate (ATP) on P2 receptors during hypoxic/ischemic events in the brain. However, there is no direct evidence of an increase in extracellular ATP levels during cerebral ischemia in vivo. The aim of the present study was to evaluate ATP outflow from the rat striatum by the microdialysis technique associated with focal cerebral ischemia in vivo by intraluminal occlusion of the right middle cerebral artery (MCA). Between 1 and 4h after ischemia, rats showed a clear turning behavior contralateral to the ischemic side. Twenty-four hour after MCA occlusion, ischemic rats had definite neurological deficit and striatal and cortical damage. The ATP concentration (mean+/-S.E.M.) in the striatum of normoxic rats (n = 8) was 3.10+/-0.34 nM. During 220 min after MCA occlusion, the extracellular ATP levels significantly increased two-fold, being 5.90+/-0.61 nM (p < 0.01 versus normoxic level). ATP outflow showed a tendency to increase over time during the 220 min of ischemia. Since extracellular ATP is rapidly metabolized to adenosine, we also assessed ATP outflow in the presence of the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene-adenosine diphosphate (AOPCP, 1 mM) directly perfused into the striatum. The ATP concentration in normoxic rats (n = 8) was increased three-fold in the presence of the ecto-5'-nucleotidase inhibitor (9.57+/-0.26 nM). During 220 min of ischemia, extracellular ATP levels significantly increased 1.3-fold in AOPCP-treated rats (12.62+/-0.65 nM, p < 0.01 versus normoxic level). The present study confirms that ATP is continuously released in the brain and demonstrates for the first time that ATP outflow increases during ischemia in vivo. These results confirm that ATP may be an important mediator in brain ischemia.     

31P NMR spectroscopy, chemical analysis, and free Mg2+ of rabbit bladder and uterine smooth muscle

31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.     

Glycogen turnover and anaplerosis in preconditioned rat hearts

Using (13)C NMR, we tested the hypothesis that protection by preconditioning is associated with reduced glycogenolysis during ischemia. Preconditioned rat hearts showed improved postischemic function and reduced ischemic damage relative to ischemic controls after 30 min stop-flow ischemia and 30 min reperfusion (contractility: 30+/-10 vs. 2+/-2%; creatine kinase release: 41+/-4 vs. 83+/-15 U/g; both P<0.05). Preconditioning decreased preischemic [(13)C]glycogen by 24% (a 10% decrease in total glycogen), and delayed ischemic [(13)C]glycogen consumption by 5-10 min, reducing ischemic glycogenolysis without changing acidosis relative to controls. Upon reperfusion, glycogen synthesis resumed only after preconditioning. Glutamate (13)C-isotopomer analysis showed recovery of Krebs cycle activity with higher anaplerosis than before ischemia (23+/-4 vs. 11+/-3%, P<0.05), but in controls reperfusion failed to restore flux. Compared to control, preconditioning before 20 min ischemia increased contractility (86+/-10 vs. 29+/-14%, P<0.05) and restored preischemic anaplerosis (13+/-3 vs. 39+/-9%, P<0.05). Preconditioning is associated with reduced glycogenolysis early during ischemia. However, protection does not rely on major variations in intracellular pH, as proposed earlier. Our isotopomer data suggest that preconditioning accelerates metabolic and functional recovery during reperfusion by more efficient/active replenishment of the depleted Krebs cycle.     

Effect of low flow ischemia-reperfusion injury on liver function

The release of liver enzymes is typically used to assess tissue damage following ischemia-reperfusion. The present study was designed to determine the impact of ischemia-reperfusion on liver function and compare these findings with enzyme release. Isolated, perfused rat livers were subjected to low flow ischemia followed by reperfusion. Alterations in liver function were determined by comparing rates of oxygen consumption, gluconeogenesis, ureagenesis, and ketogenesis before and after ischemia. Lactate dehydrogenase (LDH) and purine nucleoside phosphorylase (PNP) activities in effluent perfusate were used as markers of parenchymal and endothelial cell injury, respectively. Trypan blue staining was used to localize necrosis. Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were measured in the perfusate as indicators of intracellular oxidative stress. LDH activity was increased 2-fold during reperfusion compared to livers kept normoxic for the same time period whereas PNP activity was elevated 5-fold under comparable conditions. Rates of oxygen consumption, gluconeogenesis, and ureagenesis were unchanged after ischemia, but ketogenesis was decreased 40% following 90 min ischemia. During reperfusion, the efflux rates of total glutathione and GSSG were unchanged from pre-ischemic values. Significant midzonal staining of hepatocyte nuclei was observed following ischemia-reperfusion, whereas normoxic livers had only scattered staining of individual cells. Reperfusion of ischemic liver caused release of hepatic enzymes and midzonal cell death, however, several major liver functions were unaffected under these experimental conditions. These data indicate that there were negligible changes in liver function in this model of ischemia and reperfusion despite substantial enzyme release from the liver and midzonal cell death.     

Magnetic resonance study of renal transplantation

In the last few years, we have focused our research effort on the magnetic resonance spectroscopic (NMR) studies of organ transplantation in the rat. P-31 NMR was employed to study changes in high-energy phosphates, intracellular pH in vivo of transplanted kidneys either during normal function, while undergoing the rejection process or subjected to other insults (e.g. ischemia, cyclosporine nephrotoxicity, urinary obstruction) which may also cause graft dysfunction. Nuclear magnetic resonance (NMR) parameters, specifically relative peak areas and intracellular pH, accurately distinguished among the different causes of graft dysfunction. Ureteral obstruction was clearly identified by elevations in the phosphodiester/urine phosphate peak. Ischemia and rejection were both associated with increases in inorganic phosphates and phosphomonesters and decreases in the beta-phosphate peak of adenosine triphosphate but were distinguishable from each other by differences in intracellular pH which was normal in rejected allografts (7.33 +/- 0.07, n = 3) and low in ischemic allografts (7.00 +/- 0.05, n = 3, p less than 0.05). Grafts insulted with cyclosporine toxicity were not distinguishable from normal allografts by any of the parameters studied. To determine the temporal relationship of NMR changes in allograft rejection, similar studies were performed serially in a group of rejecting (R) kidneys (n = 7) and compared with a control group of nonrejecting (NR) kidneys (n = 7). Major decrease in adenosine triphosphate (ATP) with increases in Pi and a marked increase in the Pi/ATP ratio were noted in the R allografts over time. The R allografts could be completely segregated from the NR allografts on the basis of the Pi/ATP ratio by day 7. These data suggest that 31P NMR spectroscopy may have potential clinical application in differentiating among the causes of graft failure of human renal allografts.     

Supersensitivity of P2X receptors in cerebrocortical cell cultures after in vitro ischemia

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.     

A metabolic index of ischemic injury for perfusion-recovery of cadaveric rat livers

With over 110,000 patients waiting for organ transplantation, the current crisis in organ transplantation is based on a lack of donors after brain-death (DBD). A very large alternative pool of donor organs that remain untapped are the donors after cardiac death (DCD), recovered after cardiac activity has ceased and therefore sustained some ischemic injury. Machine perfusion has been proposed as a novel modality of organ preservation and treatment to render such cadaveric organs, and in particular livers, transplantable. Two key issues that remain unaddressed are how to assess whether a DCD liver is damaged beyond repair, and whether machine perfusion has rendered an injured organ sufficiently viable for transplantation. In this work, we present a metabolic analysis of the transient responses of cadaveric rat livers during normothermic machine perfusion (NMP), and develop an index of ischemia that enables evaluation of the organ ischemic injury level. Further, we perform a discriminant analysis to construct a classification algorithm with >0.98 specificity to identify whether a given perfused liver is ischemic or fresh, in effect a precursor for an index of transplantability and a basis for the use of statistical process control measures for automated feedback control of treatment of ischemic injury in DCD livers. The analyses yield an index based on squared prediction error (SPE) as log(SPE) >1.35 indicating ischemia. The differences between metabolic functions of fresh and ischemic livers during perfusion are outlined and the metabolites that varied significantly for ischemic livers are identified as ornithine, arginine, albumin and tyrosine.     

NMR investigation of DNA conformational changes on base protonation: use of Cu2+ and pyrazole as probes

In order to evaluate models for the acid denaturation of DNA and to assess the potential importance of protonated bases in mutations and gene expression, an NMR investigation of DNA and nucleotides in the pH range 7-2 has been conducted. The changes in the imino proton spectral region are readily observed and quite dramatic on lowering pH. At pH 7.0, calf thymus DNA has imino proton signals for AT (13.6 ppm, 56% area) and GC (12.6 ppm, 44% area) base pairs but no peaks in the 10-12 ppm region. At pH 5 a broad peak(s) between 10 and 11 ppm was (were) observed, and it narrowed and shifted to 10.9 ppm at pH 3.2. The original GC area was lost by pH 3.2 while the AT area was reduced by 50%. Below pH 3 the remainder of the AT signal was lost, and the area of the 10.9 ppm peak increased. Over this pH range the aromatic proton signals of DNA sharpened, and the cytosine amino proton signals in DNA narrowed and shifted downfield. Addition of pyrazole in the pH 4-6 range caused broadening of the new resonance but had very little effect on the original signals. Addition of Cu2+ in the pH 4-6 range resulted in a large loss in area of the GC and the new upfield peak(s). However, at lower pH, the upfield peak was not totally broadened by Cu2+. At pH below 7, the broad 31P signal of calf thymus DNA shifted slightly downfield and sharpened.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis

The effectiveness of ischemic preconditioning (IP) against hepatic ischemia/reperfusion injury during human liver surgery is linked to decreased apoptotic cell death as well as preservation of the ATP content in liver tissue. Overproduction of Bcl-2 is reported in preconditioned organs. In human liver biopsies exhibiting steatosis and/or vascular injuries (mainly peliosis) induced by chemotherapy, we find that the expression of Bcl-2 in centrolobular and peliotic areas colocalizes with the autophagy protein Beclin 1 in IP livers. Increased expression of phosphorylated Bcl-2 in preconditioned livers is associated with a decreased immunoprecipitation of Beclin 1 and increased expression of LC3-II. The increased number of autophagic vacuoles seen by electron microscopy confirmed that IP could trigger autophagy in chemotherapy-injured livers, probably to reduce the pro-inflammatory necrotic cell death of hepatocytes or endothelial cells and to increase ATP levels. Indeed, necrosis is less frequent (p = 0.04) in IP livers than in the others although no change in apoptosis as assessed by TUNEL assay or caspase-3, -8 and -9 expressions is observed. In conclusion, Bcl-2 and Beclin 1 could be major targets in the regulation of cell death during ischemia/reperfusion injury modulating autophagy to switch on/off necrosis and/or apoptosis.     

Multinuclear NMR studies of intracellular cations in perfused hypertensive rat kidney.

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.     

Separate effects of ischemia and reperfusion on vascular permeability in ventilated ferret lungs.

In systemic organs, ischemia-reperfusion injury is thought to occur during reperfusion, when oxygen is reintroduced to hypoxic ischemic tissue. In contrast, the ventilated lung may be more susceptible to injury during ischemia, before reperfusion, because oxygen tension will be high during ischemia and decrease with reperfusion. To evaluate this possibility, we compared the effects of hyperoxic ischemia alone and hyperoxic ischemia with normoxic reperfusion on vascular permeability in isolated ferret lungs. Permeability was estimated by measurement of filtration coefficient (Kf) and osmotic reflection coefficient for albumin (sigma alb), using methods that did not require reperfusion to make these measurements. Kf and sigma alb in control lungs (n = 5), which were ventilated with 14% O2-5% CO2 after minimal (15 +/- 1 min) ischemia, averaged 0.033 +/- 0.004 g.min-1.mmHg-1.100 g-1 and 0.69 +/- 0.07, respectively. These values did not differ from those reported in normal in vivo lungs of other species. The effects of short (54 +/- 9 min, n = 10) and long (180 min, n = 7) ischemia were evaluated in lungs ventilated with 95% O2-5% CO2. Kf and sigma alb did not change after short ischemia (Kf = 0.051 +/- 0.006 g.min-1.mmHg-1.100 g-1, sigma alb = 0.69 +/- 0.07) but increased significantly after long ischemia (Kf = 0.233 +/- 0.049 g.min-1 x mmHg-1 x 100 g-1, sigma alb = 0.36 +/- 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of glutathione depletion during simulated ischemia-reperfusion of H9c2 cardiac myocytes

It has been reported that myocardial glutathione content is decreased during ischemia-reperfusion, but the mechanism of glutathione depletion has remained unclear. The present study tested whether osmotic stress is involved in the glutathione depletion during ischemia. Six hours of hypoxic acidosis with either high CO(2) tension or low HCO(3)(-) concentration, which simulates the ischemic condition, resulted in a significant decrease of glutathione content and the glutathione depletion was prevented by hyperosmolarity. High-CO(2) acidosis alone without hypoxia induced a similar degree of glutathione depletion. Intracellular pH was lowered by high-CO(2) acidosis to 6.41 ± 0.03 in 15 min. Meanwhile, the cell size gradually increased and reached ～110% in 10 min and the increased cell size was maintained for at least 30 min, which was also prevented by hyperosmolarity. Subsequent experiments observed the effects of simulated reperfusion on the glutathione content. Measured in 1 h after the hypoxic acidotic reperfusion, the glutathione content was further decreased compared to the level at the end of ischemia, which was not suppressed by increasing the osmolarity of reperfusion solution. The degree of glutathione depletion during hypoxic reperfusion with normal pH was similar to the hypoxic acidotic reperfusion group. On the other hand, normoxic reperfusion was not accompanied by further depletion of glutathione content. Based on these results, it was concluded that ischemia induces the glutathione depletion via osmotic stress, which results from intracellular acidification, and the glutathione content is further decreased during reperfusion through a mechanism other than oxygen toxicity.     

Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (K(ATP)) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (Fi(O(2)) = 0.21) or hypoxic (Fi(O(2)) = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts (n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 +/- 4%) than normoxic controls (43 +/- 4%). At 10 days of age, N(G)-nitro-L-arginine methyl ester (200 microM) and glibenclamide (3 microM) abolished the cardioprotective effects of chronic hypoxia (45 +/- 4% and 46 +/- 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 +/- 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 +/- 4%). L-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 +/- 4% and 39 +/- 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of K(ATP) channels.