Surface Fluorometry Research Articles

Depressed calcium cycling contributes to lower ischemia tolerance in hearts of estrogen-deficient rats.

Estrogens enhance ischemia tolerance (IT) in the myocardium, the mechanism of which remains unclear. We investigated the effects of long-term estrogen deprivation on the intracellular calcium (Ca(2+)(i)) transient of the heart and its possible influence on IT. Hearts of ovariectomized (OVX) and sham-operated (control) adult female rats (some receiving estrogen therapy) were studied 10 weeks after surgical operation: control (n = 8), OVX (n = 10), sham-operated estrogen-substituted (n = 7), and ovariectomized estrogen-substituted (n = 9). In vivo heart function was assessed by echocardiography, whereas Ca(2+)(i) transients were recorded, concomitantly with left ventricular pressure and coronary flow, by Indo-1 surface fluorometry in isolated Langendorff-perfused hearts. Isolated hearts were subjected to a 30-minute global ischemia-30-minute reperfusion protocol. Left ventricular expression of myocardial sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), and Ser16-phosphorylated PLB was measured. Ovariectomy did not influence resting cardiac function in vivo or ex vivo. However, Ca(2+) removal was slower. During ischemia, Ca(2+)(i) elevation and ischemic contracture were more pronounced after ovariectomy. Postischemic restitution of inotropic function (developed pressure; +dP/dt(max)) and lusitropic function (-dP/dt(max)) and Ca(2+)(i) transient recovery (amplitude; ±dCa(2+)(i)/dt(max)) were decreased in OVX hearts. Sarcoendoplasmic reticulum Ca(2+)-ATPase expression was unaltered, whereas PLB and Ser16-phosphorylated PLB levels were higher after ovariectomy. All effects of ovariectomy were restored by estrogen therapy. Ovariectomy impairs myocardial Ca(2+) removal by increasing the expression of the SERCA2a inhibitor PLB. Defective Ca(2+) transport causes ischemic Ca(2+)(i) overload and insufficient postischemic recovery of Ca(2+)(i) transients, which entail depressed hemodynamic restitution. Protection of intact Ca(2+) cycling in the myocardium by estrogens plays a major role in enhancing IT.

Characterization of oxygen radical formation mechanism at early cardiac ischemia

Myocardial ischemia–reperfusion (I/R) causes severe cardiac damage. Although the primary function of oxymyoglobin (Mb) has been considered to be cellular O2 storage and supply, previous research has suggested that Mb is a potentially protective element against I/R injury. However, the mechanism of its protective action is still largely unknown. With a real-time fluorescent technique, we observed that at the onset of ischemia, there was a small burst of superoxide (O2•–) release, as visualized in an isolated rat heart. Thus, we hypothesize that the formation of O2•– correlates to Mb due to a decrease in oxygen tension in the myocardium. Measurement of O2•– production in a Langendorff apparatus was performed using surface fluorometry. An increase in fluorescence was observed during the onset of ischemia in hearts perfused with a solution of hydroethidine, a fluorescent dye sensitive to intracellular O2•–. The increase of fluorescence in the ischemic heart was abolished by a superoxide dismutase mimic, carbon monoxide, or by Mb-knockout gene technology. Furthermore, we identified that O2•– was not generated from the intracellular endothelium but from the myocytes, which are a rich source of Mb. These results suggest that during the onset of ischemia, Mb is responsible for generating O2•–. This novel mechanism may shed light on the protective role of Mb in I/R injury.

Early cardiac dysfunction is rescued by upregulation of SERCA2a pump activity in a rat model of metabolic syndrome

Various components of metabolic syndrome associate with cardiac intracellular calcium (Cai 2+) mishandling, a precipitating factor in the development of heart failure. We aimed to provide a thorough description of early stage Cai 2+-cycling alterations in the fructose-fed rat, an experimental model of the disorder, where insulin resistance, hypertension and dyslipidaemia act cooperatively on the heart. Rats were fed with fructose-rich chow. After 6 weeks, echocardiography was performed, which was followed by measurements of myocardial Cai 2+ transients recorded by Indo-1 surface fluorometry in isolated perfused hearts. Sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2a) activity was assessed by administration of its inhibitor cyclopiazonic acid (CPA). Mathematical model analysis of Cai 2+ transients was used to estimate kinetic properties of SR Ca(2+) transporters. Protein levels of key Ca(2+) handling proteins were also measured. Echocardiography showed signs of cardiac hypertrophy, but in vivo and ex vivo haemodynamic performance of fructose-fed rat hearts were unaltered. However, a decline in Ca(2+) sequestration capacity (-dCai 2+/dt and decay time of Cai 2+ transients) was observed. Model estimation showed decreased affinity for Ca(2+) (higher K(m) ) and elevated V(max) for SERCA2a. Diseased hearts were more vulnerable to CPA application. Fructose feeding caused elevation in SERCA2a and phosphorylated phospholamban (PLB) expression, while total PLB level remained unchanged. In early stage, metabolic syndrome primarily disturbs SERCA2a function in the heart, but consequential haemodynamic dysfunction is prevented by upregulation of SERCA2a protein level and phosphorylation pathways regulating PLB. However, this compensated state is very vulnerable to a further decline in SERCA2a function.

Hemodynamics and mitochondrial energy metabolism in right heart hypertrophy after acute hypoxic stress.

Excessive right heart hypertrophy was investigated under additional acute hypoxic stress to find out a possible contribution of mitochondrial dysfunction to sudden heart failure. Severe right heart hypertrophy in rats was induced by exposure to hypobaric pressure (46,663 Pa) for 4 weeks. Heart rate, isovolumic pressure and coronary flow were determined in the Langendorff mode of perfusion. After normoxia, the hearts were subdued to acute hypoxia/reoxygenation. Mitochondrial membrane potential was measured at the heart surface by fluorometry using 2-(dimethylaminostyryl)-l-ethylpyridinium iodide (DASPEI). At the end of each experiment mitochondria were isolated and ATP synthesis, ATPase, as well as creatine kinase activity were determined. Compared to normal hearts the heart rate is decreased in the hypertrophied group whereas right ventricular systolic and (end)diastolic pressure (adjusted to isovolumetric maxima) are increased. Coronary flow is decreased. Cytosolic creatine phosphate ATP levels and ATP/ADP ratios are significantly (p < 0.01) decreased. Furthermore, ATP synthesis and creatine kinase activities are diminished. At high ADP, respiration is loosely coupled or partially uncoupled. Acute hypoxia is particularly deleterious to hypertrophied hearts: Mitochondrial membrane potential as measured by heart surface fluorometry decreases extensively and is only very incompletely restored during reoxygenation. Rate-pressure product decreases precipitously and is restored during reoxygenation only to a very low extent. The results indicate an insufficient energy metabolism of mitochondria during acute hypoxia/reoxygenation which adds to the earlier described shifted isozyme pattern of myosin and decreased activities of myosin and sarcoreticular Ca2+ ATPase, leading to myocardial failure in right heart hypertrophy.

Acute effects of urocortin 2 on cardiac function and propensity for arrhythmias in an animal model of hypertension‐induced left ventricular hypertrophy and heart failure

To test acute effects of the corticotropin-releasing factor-related peptide urocortin 2 (Ucn2) on left ventricular (LV) function and the propensity for ventricular arrhythmias in the isolated heart of an animal model of hypertension-induced heart failure. Hearts from Dahl salt-sensitive rats with severe LV dysfunction were perfused according to Langendorff. Left ventricular developed pressure and the positive and negative derivatives of LV pressure were analysed before and after perfusion with Ucn2 (n = 15) or normal perfusion solution (control, n = 9). Intracellular calcium cycling parameters were assessed by surface fluorometry. Furthermore, monophasic action potential duration (MAPD) and ventricular fibrillation threshold (VFT) were determined, the latter by a train-of-pulses method at increasing voltage to scan the vulnerable period of repolarization. Urocortin 2 significantly affected intracellular calcium cycling and improved LV contractile function and relaxation. Compared with baseline values, Ucn2 significantly decreased MAPD at 30, 50, and 90% repolarization and significantly increased VFT compared with baseline values. No changes were observed in control experiments. Administration of Ucn2 rapidly improves LV function and increases VF threshold in failing, isolated rat hearts with increased propensity for ventricular arrhythmias. These observations suggest a potential use of Ucn2 as a safe and novel agent for the treatment of acute heart failure.

Hearts of surviving MLP-KO mice show transient changes of intracellular calcium handling

The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca(i)(2+)) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca(i)(2+) cycling, myocardial Ca(i)(2+)-transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with beta-agonist isoproterenol and the sarcoplasmic reticular Ca(2+)-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca(2+) handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. beta-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca(i)(2+)-level and a decrease in Ca(2+)-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca(i)(2+) handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.

Intracellular Ca2+ transients during fatigue in intact skeletal muscle: role of Ca2+ in low frequency fatigue

We previously reported a surface fluorometry method for measurement of intracellular Ca2+ in intact skeletal muscles. Here we employ this method to examine mechanisms of muscle fatigue. Mouse epitrochlearis (EPI) and diaphragm (DIA) muscles were mounted in vertical chambers, stretched to LO, and loaded with Rhod‐2 AM. Force and intracellular Ca2+ were recorded at several stimulation frequencies, and muscles were then contracted with periodic increases in train frequency (0.16, 0.2, 0.25, 0.33, 0.5, 1 train/sec). Fatigue was defined by a 50% reduction in force. EPI and DIA showed markedly different times to fatigue, as expected (~7.0 min for EPI vs. ~10.8 min for DIA). Ca2+ transients (peak ΔF) showed no initial decline, but later decreased by ~22% for EPI and ~27% for DIA. Baseline Rhod‐2 fluorescence increased in both muscles during fatigue (EPI ~77% of peak ΔF, DIA ~58% of peak ΔF), indicating mitochondrial Ca2+ accumulation. Following 15 min of recovery, max tetanic force recovered fully while forces at lower frequencies were reduced from baseline (low‐frequency fatigue). In contrast, intracellular Ca2+ transients recovered fully at both high and low stimulation frequencies in this preparation suggesting that the primary mechanism for low frequency fatigue is related to inhibition of the contractile elements and not to processes involved in Ca+2 release. NHLBI 53333.

Carbon monoxide inhibits myocardial oxygen free radical generation following the onset of ischemia

Reperfusion of the ischemic heart causes a burst of oxygen free radical (OFR) formation and secondary myocardial damage. However, questions remain on the mechanisms of OFR generation during ischemia. Recently, it has been reported that superoxide can derive from heme‐protein oxidation immediately following the onset of ischemia. Therefore, we tested the hypothesis that if OFR generation following ischemia involves heme‐protein oxidation then it should be inhibited by carbon monoxide (CO). Surface fluorometry was used to measure OFR generation during the onset of global ischemia in isolated rat heart. The fiber optic probe facing the left ventricle (LV) obtained the fluorescence signal from the heart. Hydroethidine (HE) was used as a sensitive probe to measure superoxide. When exposed to superoxide, HE is converted to ethidium (ET). Hearts were perfused with 44μM HE for 1.5 −5 min. We observed a rapid increase in ET signal with the onset of ischemia and that reached to a peak within 5 min. ET signal was completed blocked by a SOD mimic, MnPyP (250μM, P&lt;0.01). Importantly, CO (100μM) treatment significantly reduced ET signal to 30% of control (P&lt;0.01). Thus, there is a burst of OFR generation during the onset of myocardial ischemia in isolated rat heart. We report for the first time that CO inhibits OFR generation during myocardial ischemia, indicating that heme‐protein oxidation plays a crucial role in OFR generation.

O2delivery and redox state are determinants of compartment-specific reactive O2species in myocardial reperfusion

Reperfusion of the ischemic myocardium leads to a burst of reactive O(2) species (ROS), which is a primary determinant of postischemic myocardial dysfunction. We tested the hypothesis that early O(2) delivery and the cellular redox state modulate the initial myocardial ROS production at reperfusion. Isolated buffer-perfused rat hearts were loaded with the fluorophores dihydrofluorescein or Amplex red to detect intracellular and extracellular ROS formation using surface fluorometry at the left ventricular wall. Hearts were made globally ischemic for 20 min and then reperfused with either 95% or 20% O(2)-saturated perfusate. The same protocol was repeated in hearts loaded with dihydrofluorescein and perfused with either 20 or 5 mM glucose-buffered solution to determine relative changes in NADH and FAD. Myocardial O(2) delivery during the first 5 min of reperfusion was 84.7 +/- 4.2 ml O(2)/min with 20% O(2)-saturated buffer and 354.4 +/- 22.8 ml O(2)/min with 95% O(2) (n = 8/group, P < 0.001). The fluorescein signal (intracellular ROS) was significantly increased in hearts reperfused with 95% O(2) compared with 20% O(2). However, the resorufin signal (extracellular ROS) was significantly increased with 20% O(2) compared with 95% O(2) during reperfusion. Perfusion of hearts with 20 mM glucose reduced the (.)NADH during ischemia (P < 0.001) and the (.)ROS at reperfusion (P < 0.001) compared with 5.5 mM-perfused glucose hearts. In conclusion, initial O(2) delivery to the ischemic myocardium modulates a compartment-specific ROS response at reperfusion such that high O(2) delivery promotes intracellular ROS and low O(2) delivery promotes extracellular ROS. The redox state that develops during ischemia appears to be an important precursor for reperfusion ROS production.

Altered calcium handling is an early sign of streptozotocin-induced diabetic cardiomyopathy

The main objective of the present study was to determine alterations of calcium handling in the diabetic rat heart during the transition from adaptive to maladaptive phase of cardiomyopathy. By inhibiting the nuclear enzyme poly(ADP-ribose) polymerase (PARP), we also investigated the possible role of this enzyme in the sequence of pathological events. Six weeks after induction of type I diabetes by injection of streptozotocin in rats, the hearts were perfused according to Langendorff. Intracellular-free calcium (Ca(2+)(i)) levels were measured by surface fluorometry using Indo-1 AM. Cyclic changes in Ca(2+)(i) concentrations and hemodynamic parameters were measured simultaneously. The hearts were challenged by infusion of isoproterenol. Six weeks of diabetes resulted in reduced inotropy and lusitropy. The diabetic hearts (DM) expressed a significantly elevated end-diastolic Ca(2+)(i) level (control, 111-/+20 vs DM, 221-/+35 nM). The maximal transport capacity of SERCA2a and conductance of RyR2 were reduced. These changes were not accompanied by major alterations in the tissue content of SERCA2a, RyR2, phospholamban and Na(+)/Ca(2+) exchanger. In response to beta-adrenergic activation, SERCA2a transport capacity and RyR2 conductance were stunted in the DM hearts. Inhibition of PARP induced minor changes in the mechanical function and calcium handling of the DM hearts. In conclusion, the observed changes in contractility and in Ca(2+)(i) handling are most likely attributable to functional disturbances of SERCA2a and RyR2 in this transitional phase of diabetes. At this stage of diabetes, PARP does not appear to play a significant pathogenetic role in the alterations in contractile function and calcium handling.

Microvascular flow routes in muscle controlled by vasoconstrictors

Vasoconstrictors can either increase or decrease metabolism of the constant flow pump-perfused rat hindlimb. In addition, there is indirect evidence from vascular casts, surface fluorometry, dye entrapment studies, and fluorescent microsphere mapping of flow that this may be due to redistribution of flow between putatively nutritive and non-nutritive routes within muscle. In the present study, we used two methods in an attempt to identify perfused nutritive and non-nutritive vessels in muscle sections: (i) a combination of perfusion fixation with glutaraldehyde and post-perfusion Griffonia simplicifolia lectin and (ii) perfusion with rhodamine–dextran70 (lysine fixable) and post-fixation with formaldehyde. Perfusions involved vehicle only (control, a mix of nutritive and non-nutritive flow), 15 nM angiotensin II (AII) to increase, or 1 μM serotonin (5-HT) to decrease nutritive flow. Microscopic examination of muscle sections following AII showed an increase in perfused capillaries with fewer areas of under-perfusion, relative to control. In contrast, 5-HT caused a marked decrease in perfused capillaries relative to control and evidence that flow was carried by connective tissue vessels that on average were of greater diameter and were more sparsely distributed than capillaries. It is concluded that vasoconstrictors that alter hindlimb metabolism do so by intra-muscle redistribution between capillaries (nutritive) and non-nutritive (connective tissue) vessels within each muscle.

In vivo heat shock preconditioning mitigates calcium overload during ischaemia/reperfusion in the isolated, perfused rat heart

Heat shock (HS) pretreatment of the heart is effective in mitigating the deleterious effects of ischaemia/reperfusion. The main objective of this study was to determine whether the beneficial effect of HS is associated with the preservation of intracellular Ca2+ handling in the ischaemic/reperfused, isolated rat heart. Twenty-four hours after raising body core temperature to 42 degrees C for 15 min, rat hearts were perfused according to Langendorff and subjected to 30 min ischaemia followed by 20 min reperfusion. Cyclic changes of cytoplasmic calcium ion [Ca2+i] levels were measured by surface fluorometry using Indo-1 AM. Reperfused HS hearts showed improved recovery of contractile function compared with control hearts: end-diastolic pressure: 45+/-11 vs. 64+/-22 mmHg; developed pressure: 72+/-12 vs. 41+/-20 mmHg; maximum rate of pressure increase (+dP/dtmax): 1,513+/-305 vs. 938+/-500 mmHg/s; maximum rate of pressure decrease (-dP/dtmax): -1,354+/-304 vs. -806+/-403 mmHg/s. HS hearts displayed a significantly lower end-diastolic cytosolic [Ca2+] ([Ca2+]i) after reinstallation of flow. The dynamic parameters of the Ca2+i transients, i.e. the maximum rate of increase/decrease (+/-dCa2+i/dtmax) and amplitude, did not differ between reperfused control and HS hearts. The novel finding of this study is that improved performance of the HS-preconditioned heart after an ischaemic insult is associated with a reduced end-diastolic Ca2+i load, and most likely, preserved Ca2+ sensitivity of the myocardial contractile machinery.

Oxygen delivery modulates intracellular hydrogen peroxide generation during initial reperfusion of the isolated ischemic rat heart

Study objectives: Reactive oxygen species (ROS) generated during reperfusion of the ischemic heart play a major role in myocyte injury and may be dependent on changes in O₂ delivery (DO₂). We hypothesize that during low-flow reperfusion (eg, cardiopulmonary resuscitation after cardiac arrest), DO₂ modulates ROS formation and affects contractile recovery in the postischemic period. Methods: Isolated perfused rat hearts were loaded with 25 μM of dihydrofluorescein-diacetate (HFLUOR) and then subjected to 5 minutes of global ischemia. Controlled reperfusion consisted of 5 minutes of variable DO₂, ranging from 437.8 to 70.7 mL O₂/min before restoration of full baseline perfusion. Intracellular ROS was quantitated using surface fluorometry at the left ventricle to measure the oxidation of HFLUOR to fluorescein, reflecting H₂O₂ generation. Cellular NADH changes and contractile function were simultaneously measured. Results: At reperfusion, a burst of H₂O₂ was observed, which was significantly reduced by lowering DO₂ (354.4, 22.8 to 84.7; 4.2 mL O₂/min) during 5 minutes of controlled reperfusion (n=8/group, 0.47, 0.05 AU, versus 0.88; 0.14 AU, <i>P</i>=.035). However, this reduction did not correlate with improved functional recovery. Hearts reperfused with transient lower DO₂ before full reperfusion had a lower percentage of recovery of left ventricular–developed pressure (39.2%, 7.6% versus 73.5%; 6.3, <i>P</i><.01), and rate-pressure product (84.8%, 13.7 versus 27.4%; 4.9, <i>P</i><.01). Conclusion: Controlled reperfusion using an initial lower DO₂ reduces the intracellular ROS burst but does not improve recovery of postischemic contractile function.

The evaluation of brain CBF and mitochondrial function by a fiber optic tissue spectroscope in neurosurgical patients.

The brain of neurosurgical patients are exposed to various manipulations in the ICU or during surgery. Under such conditions brain O2 balance may become negative and as a result brain vitality and function will deteriorate. In order to evaluate brain vitality in real time it is important to measure more than one parameter. The multiparametric monitoring system used in our previous study to monitor comatose patients (Mayevsky et al., Brain Res. 740: 268-274, 1996) was changed into a "simplified" tissue spectroscope for real time monitoring of brain O2 balance. Mitochondrial function was evaluated by monitoring the NADH redox state by surface fluorometry. Microcirculatory blood flow was assessed by laser Doppler flowmetry. The combined optical probe was located on the surface of the brain during various neurosurgical procedures and the responses were recorded and presented in real time to the surgeon. A total of 32 patients were monitored during various procedures. The results could be summarized as follows: 1. Hypercapnia led to 3 different types of responses. In two patients the 'stealing' like event was recorded. In the other 7 patients the responses to high CO2 was not detectable. In the last group of 6 patients a clear CBF elevation was recorded with variable response of mitochondrial NADH. 2. Our monitoring device was able to evaluate the efficacy of the STA-MCA anastomosis during aneurysm surgery. 3. A significant correlation was recorded between CBF and NADH redox state during changes in blood pressure, papaverine injection, spontaneous drop in blood supply to the brain or during releasing of high ICP levels. We conclude that in order to evaluate the metabolic state of the brain during neurosurgical procedures it is necessary to monitor both CBF and mitochondrial NADH by using the tissue spectroscope.

New multiparametric monitoring approach for real-time evaluation of drug tissue interaction in vivo

Drug–tissue interactions can be evaluated at various levels of biological organization. The effects of different drugs are widely tested in various sliced tissues or intact organs under in vitro conditions. Very few techniques have been applied to monitor functional state of organs under in vivo conditions. In this overview, a new multiparametric monitoring approach (MPA) is presented that could be applied to various organs (brain, heart, kidney) in vivo exposed to compound effects. Tissue blood flow is evaluated by laser Doppler flowmetry, mitochondrial function by surface fluorometry reflectometry of NADH, and membrane integrity by extracellular level of K+, Ca2+, H+, or Na+. Other parameters, e.g., EEG, ECG, ICP, or temperature represent the functional state of the organ. The construction of the multiprobe assembly was adopted to various experimental as well as clinical situations to monitor various organs. Typical examples of the application of the MPA to test the effects of alcohol on the rat brain or antiischemic compounds in the gerbil brain are shown. The results collected using the MPA methodology allowed the differentiation between the various effects of a tested compound at the biochemical and physiological levels and provided suggestions as to a possible mode of action under in vivo conditions. Drug Dev. Res. 50:457–470, 2000. © 2000 Wiley-Liss, Inc.

Optical monitoring of NADH redox state and blood flow as indicators of brain energy balance.

Real-time evaluation of brain vitality in situ could be done by monitoring different parameters, which are complementary to each other. During the last 40 years the following four minimally invasive techniques were developed and applied to monitor the brain in situ: (1) Cerebral Blood Flow (CBF) using laser Doppler flowmetry (Stern et al., 1977; Dirnagl et al., 1989). (2) Hemoglobin oxygenation or saturation (HbO2) by dual wavelength reflectometry (Rampil et al., 1992) or spectral analysis (Frank et al., 1989). (3) Brain average oxygenation using oxygen electrodes (Mayevsky et al., 1980). (4) Mitochondrial redox state by monitoring of NADH fluorescence using surface fluorometry (Chance et al., 1962; Jobsis et al., 1971b; Mayevsky and Chance, 1982).

EFFECT OF ETHANOL ON THE METABOLISM OF TRICHLOROETHYLENE IN THE PERFUSED RAT LIVER

The effect of 2 m M ethanol, a concentration indicative of daily alcohol consumption, was investigated on trichloroethylene (TRI) metabolism in perfused Wistar rat liver. The study consisted of two parts: The first part studied TRI administration with or without ethanol. In the second study chloral hydrate (CH), an intermediate in TRI metabolism, was administered in the absence or presence of ethanol to phenobarbital (PB) treated or non-PB-treated rats. The concentrations of the metabolites, total trichloroethanol (TCE), and trichloroacetic acid (TCA) were measured by gas chromatography and intracellular reduced pyridine nucleotides by surface fluorometry. In the first study, ethanol infusion significantly increased the TCE/TCA ratio, TCE production rate, and percentage of reduced pyridine nucleotides, and decreased TCA production rate without an associated change in the sum of TCE and TCA formation rates. In the second study, ethanol infusion in the absence or presence of PB produced similar significant increases in the TCE/TCA ratio, TCE production rate, and percentage of reduced pyridine nucleotides, accompanied by a decrease in TCA formation. The observed shift in TRI metabolism in the presence of ethanol, from oxidation to TCA to reduction to TCE, suggests that alcohol exerts alterations in hepatic intracellular oxidation-reduction (redox) states.

ATP-independent membrane depolarization with ischemia in the oxygen-ventilated isolated rat lung.

We hypothesize that lung ischemic injury is related to cessation of flow leading to endothelial cell membrane depolarization and activation of oxidant-generating systems. Cell membrane potential was assessed in isolated, oxygen ventilated, Krebs-Ringer bicarbonate buffer-dextran-perfused rat lungs by lung surface fluorescence after infusion of bis-oxonol or 5,5',6,6'-tetrachloro-1, 1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), voltage-sensitive dyes. Surface fluorometry showed increased bis-oxonol fluorescence (34.7 +/- 3.3% above baseline) and decreased JC-1 fluorescence (24.5 +/- 4.5% below baseline) with ischemia, compatible with membrane depolarization. Fluorescence change was initiated within 1-2 min of the onset of ischemia and was rapidly reversible with reperfusion. Fluorescence changes varied with perfusion flow rate; maximal increase occurred with the transition from 1.8 ml/min to zero flow. Elevation of static intravascular pressure resulted in only a minor increase of bis-oxonol fluorescence. In situ subpleural fluorescence microscopy showed that endothelial cells are the major site of the increased bis-oxonol fluorescence signal with ischemia. These results indicate that endothelial cell membrane depolarization represents an early event with lung ischemia. Since the adenosine triphosphate content of lung was unchanged with ischemia in the O2-ventilated lungs, we postulate that membrane depolarization results from elimination of shear stress, possibly via inactivation of flow-sensitive K+-channels.

Detection of reactive oxygen produced by heat stress using a novel surface fluorometry technique: [formula omitted], Lawrence J. Berliner, Thomas L. Clanton. The Ohio State University, Dept. of Internal Medicine, Chemistry and Biophysics, Columbus, OH

Detection of reactive oxygen produced by heat stress using a novel surface fluorometry technique: [formula omitted], Lawrence J. Berliner, Thomas L. Clanton. The Ohio State University, Dept. of Internal Medicine, Chemistry and Biophysics, Columbus, OH

Intracellular generation of reactive oxygen species during nonhypoxic lung ischemia.

Surface fluorometry with 40 microM hydroethidine (HE) as a probe was used to detect oxidant generation in isolated, ventilated rat lungs during lung ischemia. Ethidium fluorescence due to HE oxidation was continuously monitored with 470 nm excitation and 610 nm emission. Fluorescence increased with ischemia in O2-ventilated lungs [0.98 +/- 0.08 arbitrary fluorescence units (AFU)/min vs. 0.58 +/- 0.07 with control perfusion]. HE oxidation during ischemia was prevented by N2 ventilation but was unaltered by preperfusion with superoxide dismutase. Ethidium fluorescence in homogenate prepared from lungs subjected to 1 h of nonhypoxic ischemia was increased (16.8 +/- 1.5 vs. 9.8 +/- 0.4 AFU/mg protein in control) but was unchanged in lungs that had been N2 ventilated. Microfluorographs of HE perfused and fixed lung sections demonstrated marked generalized increases in ethidium fluorescence with ischemia compared with control perfusion. Ischemia resulted in significant increases in tissue thiobarbituric acid reactive substance (176 +/- 13 vs. 44 +/- 3 pmol/mg protein for control) and in lung conjugated dienes (0.90 +/- 0.07 vs. 0.48 +/- 0.06 U/mg protein for control), indicating peroxidation of lung lipids. These results indicate that lung ischemia leads to intracellular oxidant generation that can be continuously monitored by surface fluorometry.