Carbon monoxide exposure in rat heart: evidence for two modes of toxicity

Rat hearts were perfused for 30 min with buffer equilibrated with CO. Mean data for hearts exposed to 0.01% CO show a 15% decrease in heart rate (HR) during exposure followed by recovery, with a further 17% decrease post exposure. Examination of time courses from individual perfusions shows that in 10 hearts exposed to 0.01% CO HR responded in different ways: no response (5 hearts); decrease during exposure followed by recovery (3 hearts); and decrease post exposure (2 hearts). There was a strong association between CO-induced HR decrease and release of creatine kinase into the perfusate, both of which were not prevented by the antioxidants, ascorbate, and Trolox C. Perfusate flow rate declined post exposure (4.9% and 8.9% with 0.01% and 0.05% CO, respectively) and this was prevented by antioxidants. CO may have two, independent, cardiotoxic effects; these may be mediated by CO-induced elevation of oxidant production, H2O2 in one case and peroxynitrite in the other.     

Dobutamine responsiveness,PET mismatch,and lack of necrosis in low-flow ischemia: is this hibernation in the isolated rat heart?

The clinical hallmarks of hibernating myocardium include hypocontractility while retaining an inotropic reserve (using dobutamine echocardiography), having normal or increased [18F]fluoro-2-deoxyglucose-6-phosphate (18FDG6P) accumulation associated with decreased coronary flow [flow-metabolism mismatch by positron emission tomography (PET)], and recovering completely postrevascularization. In this study, we investigated an isolated rat heart model of hibernation using experimental equivalents of these clinical techniques. Rat hearts (n = 5 hearts/group) were perfused with Krebs-Henseleit buffer for 40 min at 100% flow and 3 h at 10% flow and reperfused at 100% flow for 30 min (paced at 300 beats/min throughout). Left ventricular developed pressure fell to 30 +/- 8% during 10% flow and recovered to 90 +/- 7% after reperfusion. In an additional group, this recovery of function was found to be preserved over 2 h of reperfusion. Electron microscopic examination of hearts fixed at the end of the hibernation period demonstrated a lack of ischemic injury and an accumulation of glycogen granules, a phenomenon observed clinically. In a further group, hearts were challenged with dobutamine during the low-flow period. Hearts demonstrated an inotropic reserve at the expense of increased lactate leakage, with no appreciable creatine kinase release. PET studies used the same basic protocol in both dual- and globally perfused hearts (with 250MBq 18FDG in Krebs buffer +/- 0.4 mmol/l oleate). PET data showed flow-metabolism "mismatch;" whether regional or global, 18FDG6P accumulation in ischemic tissue was the same as (glucose only) or significantly higher than (glucose + oleate) control tissue (0.023 +/- 0.002 vs. 0.011 +/- 0.002 normalized counts. s-1x g-1x min-1, P < 0.05) despite receiving 10% of the flow. This isolated rat heart model of acute hibernation exhibits many of the same characteristics demonstrated clinically in hibernating myocardium.     

Effect of glycerol level in Tris-basek extender and equilibration period on quality of frozen goat semen

Twenty ejaculates, 4 from each of 5 native goats, were collected using an artificial vagina, and the effects of glycerol level (4, 6.4 and 9 %) and the equilibration period (1, 3 and 5 h) were studied by split-sample technique. The extender used was Tris egg yolk citric acid fructose glycerol extender. The semen was frozen in 0.5-ml French straws by exposure for 10 min to liquid nitrogen vapor, 5 cm above the liquid nitrogen level. After 14 h of storage in liquid nitrogen, the straws were thawed in water at 37 degrees C for 12 - 15 sec. The percentage of progressively motile sperm (PPM) and the percentage of damaged acrosomes (PDA) were studied after equilibration and after thawing. The mean PPM after thawing was found to be 64.0 +/- 0.90, 66.92 +/- 0.54 and 63.65 +/- 1.07 when semen was frozen with 4, 6.4 and 9 % glycerol and 61.48 +/- 0.81, 65.05 +/- 0.78 and 68.03 +/- 0.87 in 1-, 3- and 5-h equilibrated semen, respectively. The mean PDA after thawing was 7.12 +/- 0.88, 8.23 +/- 0.76 and 10.58 +/- 0.84 when semen was frozen with 4, 6.4 and 9 % glycerol and 7.0 +/- 0.74, 9.0 +/- 0.95 and 9.93 +/- 0.81 in 1-, 3- and 5-h equilibrated semen, respectively. Both PPM and PDA differed significantly (P<0.01) between glycerol levels, between equilibration periods and between stages (after equilibration and after thawing). The PPM also differed significantly due to equilibration period x stage interaction (P<0.01) and glycerol level x stage interaction (P<0.05). The PDA did not differ significantly due to interactions. When the differences between pairs of means were tested by least significant difference, it was found that after equilibration PPM was not significantly affected by either glycerol level or equilibration period, while after thawing, it was significantly higher (P<0.05) for 6.4 % glycerol and 5-h equilibrated semen than for 4 or 9 % glycerol and 1- or 3-h equilibrated semen, respectively. The PDA was lower with 4 % glycerol and 1-h equilibrated semen.     

Exposure of neonatal rats to carbon monoxide alters cardiac adaptation to aortic constriction.

We tested the hypothesis that a 32-day exposure of newborn rats to 500 ppm carbon monoxide (CO) would alter the adaptive response of the heart to aortic constriction in adulthood. At 110 days of age aortic constriction or sham operations were performed, and hearts were studied 28 days later. Aortic constriction increased left ventricular (LV) mass by 40% over the control value of 611 +/- 27 mg; this adaptive response was not altered by CO exposure. Aortic constriction and CO exposure increased right ventricular (RV) mass by 10 and 11%, respectively, over the control value of 185 +/- 10 mg. The effects of both experimental procedures on RV mass were additive (23%). Peak LV pressure development (dP/dtmax) in vitro increased 29% after aortic constriction in the nonexposed rats. CO exposure blunted the increase in peak LV systolic pressure due to aortic constriction. Maximum positive and negative dP/dtmax decreased by 19% after aortic constriction and were unaffected by CO exposure. The percentage of alpha-myosin heavy chain (MHC) in the ventricles was 94 +/- 2% in the control group and was decreased to 81 +/- 3% by aortic constriction. In contrast, the percentage of alpha-MHC was 87 +/- 2% for CO-exposed rats and was not significantly altered after aortic constriction. In vitro coronary flow was increased 18% in hearts of adult rats exposed to CO as neonates. Exposure of neonatal rats to CO induced chronic adaptations in the myocardium, some of which became evident in adulthood only when hearts were challenged by aortic constriction.     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.     

Contribution of malonyl-CoA decarboxylase to the high fatty acid oxidation rates seen in the diabetic heart

Myocardial glucose oxidation is markedly reduced in the uncontrolled diabetic. We determined whether this was due to direct biochemical changes in the heart or whether this was due to altered circulating levels of insulin and substrates that can be seen in the diabetic. Isolated working hearts from control or diabetic rats (streptozotocin, 55 mg/kg iv administered 6 wk before study) were aerobically perfused with either 5 mM [(14)C]glucose and 0.4 mM [(3)H]palmitate (low-fat/low-glucose buffer) or 20 mM [(14)C]glucose and 1.2 mM [(3)H]palmitate (high-fat/high-glucose buffer) +/-100 microU/ml insulin. The presence of insulin increased glucose oxidation in control hearts perfused with low-fat/low-glucose buffer from 553 +/- 85 to 1,150 +/- 147 nmol x g dry wt(-1) x min(-1) (P < 0. 05). If control hearts were perfused with high-fat/high-glucose buffer, palmitate oxidation was significantly increased by 112% (P < 0.05), but glucose oxidation decreased to 55% of values seen in the low-fat/low-glucose group (P < 0.05). In diabetic hearts, glucose oxidation was very low in hearts perfused with low-fat/low-glucose buffer (9 +/- 1 nmol x g dry wt(-1) x min(-1)) and was not altered by insulin or high-fat/high-glucose buffer. These results suggest that neither circulating levels of substrates nor insulin was responsible for the reduced glucose oxidation in diabetic hearts. To determine if subcellular changes in the control of fatty acid oxidation contribute to these changes, we measured the activity of three enzymes involved in the control of fatty acid oxidation; AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and malonyl-CoA decarboxylase (MCD). Although AMPK and ACC activity in control and diabetic hearts was not different, MCD activity and expression in all diabetic rat heart perfusion groups were significantly higher than that seen in corresponding control hearts. These results suggest that an increased MCD activity contributes to the high fatty acid oxidation rates and reduced glucose oxidation rates seen in diabetic rat hearts.     

Inhibition of p38 MAPK alpha/beta reduces ischemic injury and does not block protective effects of preconditioning

We examined the effect of inhibition of p38 mitogen-activated protein kinase (MAPK) alpha/beta during ischemia and preconditioning by using the inhibitor SB-202190. Isolated rat hearts were perfused with Krebs-Henseleit buffer, while left ventricular developed pressure (LVDP) and (31)P nuclear magnetic resonance spectra were acquired continuously. After 20 min of ischemia and 25 min of reperfusion, recovery of LVDP in untreated hearts was 32 +/- 4%, whereas hearts treated with SB-202190 5 min before ischemia recovered 59 +/- 7% of their pretreatment LVDP. Preconditioning improved functional recovery to 65 +/- 5%, which was unaffected by SB-202190 treatment, added either throughout the preconditioning protocol (56 +/- 5% recovery) or during the final reperfusion period of preconditioning (71 +/- 11% recovery). Necrosis was assessed after 40 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC) staining and creatine kinase release. The untreated group had 54 +/- 8% necrotic myocardium, whereas the SB-202190-treated group had 32 +/- 7% and the preconditioned group had 21 +/- 4% necrotic tissue by TTC staining.     

缺血预处理合并低温及晶体停搏液对兔未成熟心肌的保护作用

本文拟探讨缺血预处理(ischemic preconditioning,IP)合并低温及晶体停搏液对幼兔的离体心脏是否具有心肌保护作用.采用Langendorff离体心脏灌注模型,灌注液为Krebs-Henseleit液(K-H液).取3～4周龄幼兔心脏,在第一部分实验中分为Con、IP1、IP2、IP3组(n=6),分别给予0、1、2、3次IP,其后各组心脏均在20℃低温下停灌2 h,37℃常温下再灌注30 min.在第二部分实验中分为SConI、SCon2、SCon3、SIPl、SIP2、SIP3组(n=8),其中SIPl、SIP2、SIP3组给予2次IP后灌注St.Tho-mas Ⅱ晶体停搏液(CCS)使心脏停搏,然后分别使心脏在32℃、25℃、20℃下停灌30、90和120min,其后各组均在37℃再灌注30 min.SConl,SCon2,SCon3三组则不给予IP,继续灌注20min后灌注CCS使心脏停搏,然后分别在32℃、25℃、20℃下停灌30、90和120 min,其后各组均在37℃再灌注30 min.以Maclab/4 s生理实验系统记录平衡末、缺血前、再灌注后1、3、5、10、20、30 min时心率(HR)、左心室发展压(LVDP)以及左心室内压上升及下降最大速率(±dp/dtmax),测定再灌注末心肌组织中ATP和丙二醛(MDA)的含量,以及超氧化物歧化酶(SOD)的活性.在20℃低温停灌且停灌期间不给予CCS时,再灌注末IP2组LVDP×HR、+dp/dtmax和-dp/dtmax的恢复率分别为96%±21%、101%±19%和84%±15%,显著高于Con组和IP3组(P＜0.01,P＜0.05);心肌组织的ATP含量亦高于Con组(P＜0.01).在不同低温停灌且停灌期间给予CCS时,再灌注末SIP1、SIP2组的LVDP×HR、+dp/dtmax分别恢复到87%±14%、99%±26%(P＜0.05,vs SConl group)和87%±16%、102%±20%(P＜0.05,vs SCon2 group);心肌组织的ATP含量均分别显著高于SCon1组和SCon2组(P＜0.05),心肌组织MDA含量亦分别低于SCon1组和SCon2组(P＜0.05).上述结果提示,IP对在20℃低温停灌的兔未成熟心脏具有一定的心肌保护作用,2次IP的保护效应优于1次或3次IP.在停灌期间应用CCS,IP的心肌保护作用随停灌期间低温程度的升高而减弱.     

Response of rat coronary circulation to carbon monoxide and nitrogen hypoxia

The effects of nitrogen (N2) or carbon monoxide (CO) hypoxia on coronary flow were assessed in the isolated nonworking rat heart perfused via the aorta with oxygenated (95% O2-5% CO2) Kreb's Henseleit solution. After 30 min, the hearts were challenged with solutions containing either CO (10% CO-85% O2-5% CO2) or N2 (10% N2-85% O2-5% CO2) for 2 min (Challenge I). After recovery in oxygenated solution, the hearts were challenged with the alternate test solution (Challenge II). There were no significant differences in heart rate or pulse pressure between the hearts challenged with CO or N2. Coronary flow was significantly higher in the hearts challenged with CO regardless of the challenge sequence. Coronary flows (ml X min-1 X g dry wt) in the CO- and N2-treated hearts, respectively, were 61.5 +/- 4.5 and 52.9 +/- 2.3 after Challenge I, and 64.3 +/- 2.6 and 56.4 +/- 3.0 after Challenge II. Because PO2 and oxygen content were the same in both test solutions, the results suggest that CO has a direct effect on coronary artery vascular smooth muscle.     

The influence of lactate,pyruvate and glucose as exogenous substrates on free radical defense mechanisms in isolated rat hearts during ischaemia and reperfusion

Previous studies have shown that exogenous lactate impairs mechanical function of reperfused ischaemic hearts, while pyruvate improves post-ischaemic recovery. The aim of this study was to investigate whether the diverging influence of exogenous lactate and pyruvate on functional recovery can be explained by an effect of the exogenous substrates on endogenous protecting mechanisms against oxygen-derived free radicals. Isolated working rat hearts were perfused by a Krebs-Henseleit bicarbonate buffer containing glucose (5 mM) as basal substrate and either lactate (5 mM) or pyruvate (5 mM) as cosubstrate. In hearts perfused with glucose as sole substrate the activity of glutathione reductase was decreased by 32% during 30 min of ischaemia (p<0.10 versus control value), while the activity of superoxide dismutase and catalase was reduced by 27 and 35%, respectively, during 5 min of reperfusion (p<0.10 versus control value). The GSH level in the glucose group was reduced by 29% following 30 min of ischaemia and 35 min of reperfusion (p<0.10). In lactate- and pyruvateperfused hearts there were no significant decreases of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activity during 30 min of ischaemia, 5 min of reperfusion or 35 min of reperfusion. In pyruvate-perfused hearts the glutathione peroxidase activity was even increased by 43% during 30 min of ischaemia (p<0.05). Glutathione levels (reduced and oxidized) did not markedly change in the lactate and pyruvate groups. Thus, the endogenous defense mechanism against oxygen-derived free radicals is compromised at the onset of reperfusion when glucose as sole substrate is present, while addition of lactate or pyruvate prevents reduction of the endogenous capacity to scavenge oxygen-derived free radicals. The equivocal relationship between endogenous scavenging enzyme activity and haemodynamic recovery indicates that involvement of the endogenous antioxidants, if any, in functional recovery of the post-ischaemic heart is complex. Pyruvate may exert protective effects on mechanical function after mild ischaemia by functioning as exogenous scavenger in itself, as pyruvate is able to react with hydrogen peroxide.     

Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo- 1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo- 1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai.     

The effect of a low molecular weight inhibitor of lipid peroxidation on ultrastructural alterations to ischemia-reperfusion in the isolated rat heart

The effects of H290/51, a novel indenoindole derivative inhibitor of lipid peroxidation, on ultrastructural changes during cardiac ischemia-reperfusion injury were investigated. Langendorff-perfused rat hearts were exposed to 30 minutes of global ischemia followed by 20 minutes of reperfusion: Group A: Control hearts with standard buffer perfusion with vehicle added. Group B: H290/51 (10(-6) mol/l) added to buffer throughout stabilisation and reperfusion. In an additional Group C, where hearts were given H290/51, but not subjected to ischemia, the ultrastructure was preserved till the end of reperfusion. Absolute volumes and calculated volume fractions (Vv) of tissue and subcellular components were assessed with quantitative stereologic morphometry. After ischemia the increase in volume of extracellular interstitium was inhibited by H290/51 (247 +/- 80 vs. 159 +/- 50 microl, mean +/- SD, groups A and B, respectively, p<0.05). The Vv (interstitium/myocard) was higher in control hearts (0.318 +/- 0.062 vs. 0.206 +/- 0.067, p<0.05). Vv (cell edema/myocyte) was higher in the control group (0.144 +/- 0.07 vs. 0.083 +/- 0.033, p<0.05). Vv (myocyte/myocard) was higher in group B after ischemia than in the control group (0.622 +/- 0.071 vs. 0.707 +/- 0.052, p<0.05). The decreased Vv (capillary/myocard) after ischemia was inhibited by H290/51. After reperfusion there was no difference between groups. Treatment with H290/51 reduced edema and ensured better preserved sarcolemmal membrane structure during ischemia. The effect was no longer present after reperfusion.     

Effects of hypercapnia on brain pHi and phosphate metabolite regulation by 31P-NMR

The ability of brain cells to regulate intracellular pH (pHi) and several phosphate metabolites was evaluated during 1 h of hypercapnia (inspiratory CO2 fraction of 0.10 and 0.05) in anesthetized rats by 31P high-field (145.6 MHz) nuclear magnetic resonance spectroscopy. Body temperature was maintained at 37 +/- 0.5 degrees C. Fully relaxed spectra were obtained for controls and 30-50 min after CO2 loading and CO2 withdrawal. Spectra were taken serially every 2.5 min after gas mixtures were changed. Brain pHi decreased 0.10 +/- 0.02 units [7.06 +/- 0.01 (SE)] to 6.96 +/- 0.01 (P less than 0.001) after 30-50 min of 10% CO2 breathing, and arterial pH decreased 0.24 +/- 0.01 units. Brain pHi decreased by 0.045 +/- 0.01 units (7.05 +/- 0.01 to 7.01 +/- 0.01, P less than 0.05) during 5% CO2 breathing. Brain pHi returned to control values after 30-50 min of CO2 washout in both groups. In three of six animals breathing 10% CO2, there was an undershoot in brain pHi by 0.07-0.09 units between 2.5 and 20 min of hypercapnia. Three animals exhibited an overshoot in pHi by 0.06-0.11 units between 7.5 and 17.5 min during CO2 washout. Phosphocreatine-to-Pi and Pi-to-beta-ATP ratios changed during hypercapnia and returned to base line after withdrawal of CO2. The findings of a smaller brain pHi change than arterial pH change and undershoots and overshoots in pHi support the view that pHi regulation involves active processes such as transmembrane ion transport.     

Pressure-distension relationship in arteries and arterioles in response to 5 wk of horizontal bedrest

We hypothesized that exposure to prolonged recumbency (bedrest), and thus reductions of intravascular pressure gradients, increases pressure distension in arteries/arterioles in the legs. Ten subjects underwent 5 wk of horizontal bedrest. Pressure distension was investigated in arteries and arterioles before and after the bedrest, with the subject seated or supine in a hyperbaric chamber with either one arm or a lower leg protruding through a hole in the chamber door. Increased pressure in the vessels of the arm/leg was accomplished by increasing chamber pressure. Vessel diameter and flow were measured in the brachial and posterior tibial arteries using Doppler ultrasonography. Electrical tissue impedance was measured in the test limb. Bedrest increased (P < 0.01) pressure distension threefold in the tibial artery (from 8 +/- 7% to 24 +/- 11%) and by a third (P < 0.05) in the brachial artery (from 15 +/- 9% to 20 +/- 10%). The pressure-induced increase in tibial artery flow was more pronounced (P < 0.01) after (50 +/- 39 ml/min) than before (13 +/- 23 ml/min) bedrest, whereas the brachial artery flow response was unaffected by bedrest. The pressure-induced decrease in tissue impedance in the leg was more pronounced (P < 0.01) after (16 +/- 7%) than before (10 +/- 6%) bedrest, whereas bedrest did not affect the impedance response in the arm. Thus, withdrawal of the hydrostatic pressure gradients that act along the blood vessels in erect posture markedly increases pressure distension in dependent arteries and arterioles.     

Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.     

Reversibility of acute alcohol cardiac depression: 31P NMR in hamsters

Isolated hamster hearts were perfused with 2% ethanol for 30 min and then reequilibrated with control medium. One group of hamsters was pretreated with verapamil. Another group received diltiazem. Myocardial verapamil levels were 9.5 +/- 0.7 mg/g dry wt; diltiazem levels were 22 +/- 7 mg/g dry wt. Energy metabolites were assessed by using 31P NMR standardized with high-pressure liquid chromatography of freeze-clamped tissue. Intracellular calcium was measured by atomic absorption spectrophotometry, marking the extracellular space with K(CoEDTA). After 30 min of perfusion, untreated hamster hearts showed a 74% decrease in developed pressure, a marked increase in end-diastolic pressure, a decrease of ATP from 9.8 to 8.8 mmol, and an increase of Pi from 6.7 to 9.8 mmol, but no change of phosphocreatine (PCr) or intracellular pH (pHi). Verapamil pretreatment partially prevented cardiac depression during alcohol perfusion. Whereas diltiazem had no protective effect. After reequilibration, developed pressure and oxygen consumption significantly exceeded control values. ATP decreased to 8 mmol; pHi, PCr, and Pi showed no significant change. Verapamil-pretreated hearts showed better performance than untreated hearts without change in PCr and Pi, whereas ATP dropped slightly to 8.7 mmol. Thus, functional cardiac depression resulting from acute alcohol exposure is reversible. Increased intracellular calcium levels during alcohol exposure normalized after the removal of alcohol. There was no major change in high-energy phosphates during alcohol exposure or after the removal of alcohol. Verapamil protects the heart from functional depression during alcohol exposure without affecting energy resources.     

Role of S-nitrosothiol transport in the cardioprotective effects of S-nitrosocysteine in rat hearts

The objective of this study was to determine if prior exposure of rat hearts to S-nitrosocysteine (CysNO) was able to provide protection against reperfusion injury. We probed NO release using the extracellular NO scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid transport system L (L-AT), a known transporter of CysNO, using the L-AT competitor, L-leucine (L-Leu). Isolated (9- to 12-week-old Wistar male) rat hearts (six to eight per group) were perfused with CysNO (10 microM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of ischemia. Cardiac function was assessed before, during, and after treatment and during 120 min of reperfusion after ischemia. Functional recovery (rate-pressure product) was significantly improved in the CysNO group compared to hearts in the CysNO+L-Leu group and the control group (p<0.05). Necrosis, measured by triphenyltetrazolium chloride staining, was significantly reduced in CysNO hearts (p<0.05) and this improvement was reversed by L-Leu. The NO scavenger oxyHb (20 microM) was perfused either concomitant with CysNO or just before ischemia. In neither case did oxyHb affect the cardioprotection afforded by CysNO. OxyHb alone, given in either time window, did not alter the course of ischemia-reperfusion injury. When nitrite was used in place of CysNO, no protective effects were observed. Perfusion with CysNO increased tissue S-nitrosothiol (RSNO) levels from an unmeasurable background to a value of about 15.7+/-4.1 pmol RSNO/mg protein, as measured by triiodide-based chemiluminescence in the presence and absence of mercury(II) chloride. In the presence of L-Leu, this value dropped to 0.4+/-0.3 pmol RSNO/mg protein. This study demonstrates that exposure to CysNO before ischemia increases tissue S-nitrosothiol levels, improves postischemic contractile dysfunction, and attenuates necrosis. The mechanism of cardioprotection requires the uptake of CysNO via the L-AT and does not seem to involve NO release either during CysNO exposure or during ischemia. This suggests that the protective effects of CysNO are mediated through the posttranslational modification of cellular proteins through an NO-independent transnitrosation mechanism.     

Preserved postischemic heart function in sucrose-fed type 2 diabetic OLETF rats

Cardiovascular disease is one of the most important causes of morbidity and mortality in diabetes mellitus, but there has been controversy over functional impairment of diabetic hearts and their tolerance to ischemia. We studied ischemic heart function in type 2 diabetic rats with different degrees of hyperglycemia and its relationship with cardiac norepinephrine release. Otsuka Long-Evans Tokushima Fatty rats (OLETF) and age-matched Long-Evans Tokushima Otsuka normal rats (LETO) were used. One group of OLETF rats was given 30% sucrose in drinking water (OLETF-S). Hearts were isolated and perfused in a working heart preparation and subjected to 30 min ischemia followed by 40 min reperfusion at age of 12 months. Hemodynamics and coronary norepinephrine overflow were examined. Fasting plasma glucose in OLETF increased markedly at 12 months and sucrose administration exacerbated hyperglycemia in diabetic rats (LETO 6.6 +/- 0.5, OLETF 8.3 +/- 0.7, OLETF-S 15.0 +/- 1.7 mmol/L, P < 0.01). Basic cardiac output in OLETF was decreased as compared with LETO and OLETF-S (LETO 29.4 +/- 2.5, OLETF 24.0 +/- 2.4, OLETF-S 27.0 +/- 0.9 ml/min/g, P < 0.05) and remained very low after ischemia, while in OLETF-S it was well preserved (OLETF 4.2 +/- 2.1, OLETF-S 13.7 +/- 2.6 ml/min/g, P < 0.01). Correspondently, cardiac norepinephrine released during ischemia and reperfusion was lower in OLETF-S (OLETF 2.3 +/- 1.0, OLETF-S 0.7 +/- 0.1 pmol/ml, P < 0.01). Thus, OLETF hearts were more vulnerable to ischemia but sucrose feeding rendered their hearts resistant to ischemia. Less norepinephrine release may play a role in preventing postischemic functional deterioration in sucrose-fed diabetic hearts.     

Effects of broad band electromagnetic fields on HSP70 expression and ischemia-reperfusion in rat hearts

Although exposure to broad band (0.2-20 MHz) electromagnetic fields (EMF) is part of the treatment of several diseases, little is known as to their effects on myocardial protein expression and resistance to ischemia-reperfusion (I/R). We exposed Sprague-Dawley rats to either high (H, 10 min/day at 200 V/m, 36.1 microT) or low (L, 2 min/day at 30 V/m, 11.4 microT) intensity broad band EMF for 15 days. At the end of the treatment, myocardial HSP70 was 32 +/- 8% (mean +/- SEM) higher in L (P = 0.01) than in control (C), whereas in H it remained the same as in C. Electron microscopy revealed sporadic ruptures of mitochondrial cristae in H hearts, with no differences in other parameters. Malondialdehyde was increased in treated hearts (P < 0.05), but especially in H (P = 0.008). To assess the protective role of HSP70 during I/R, hearts were Langendorff-perfused with Krebs-Henseleit. After I/R, C hearts displayed depressed rate. pressure (-13 +/- 7%) and increased end-diastolic (+9.2 +/- 2.8 mmHg) and perfusion pressures (+30 +/- 10 mmHg). In H and L, rate. pressure recovery was similar to C (-2 +/- 21% and -12 +/- 16%, respectively, P = NS). In contrast, both end-diastolic and perfusion pressures were higher in L than in H (30.8 +/- 5.4 vs 18.2 +/- 3.5, P = 0.01, and 54 +/- 8 vs 21 +/- 8 mmHg, P = 0.01, respectively) indicating diastolic derangement in L. In conclusion, the effects of broad band EMF on HSP70 appear to be biphasic, and HSP70 overexpression might not be directly related to improved protection against I/R.