Oxygen delivery and myocardial function in rabbit hearts perfused with cell-free hemoglobin.

Isolated rabbit hearts were perfused with Krebs-Henseleit buffer that contained 1.5 g/dl hemoglobin Ao [HbAo; PO2 at which half-saturation of hemoglobin occurs = 12 Torr], human hemoglobin cross-linked between alpha-chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha-Hb; PO2 at which half-saturation of hemoglobin occurs = 30 Torr), or fatty acid-free bovine serum albumin (BSA). Myocardial performance and oxygen uptake were determined at different aortic PO2's [arterial PO2 (PaO2)] by use of an isovolumic Langendorff preparation. Function and oxygen uptake were comparable among the three different groups of hearts at an average mean PaO2 of 557 Torr. As PaO2 decreased, myocardial function was preserved better in hearts perfused with hemoglobin than in hearts perfused with Krebs-Henseleit buffer alone or with BSA. Hearts perfused with either HbAo or alpha alpha-Hb exhibited similar 10% decreases in left ventricular developed pressure and rate of change in left ventricular developed pressure at PaO2 of 141 Torr compared with a 58% decrease with BSA. However, corresponding venous PO2's were lower with HbAo (20 Torr) than with alpha alpha-Hb (35 Torr), and oxygen uptake decreased by 36% with HbAo but remained constant with alpha alpha-Hb. These data suggest that although myocardial function can be sustained over a fairly broad range of hemoglobin oxygen affinities, tissue oxygen gradients and myocardial oxygen uptake are maintained better by cell-free hemoglobin with an oxygen affinity in the normal physiological range.     

Chromosome studies in newborn infants following exchange transfusion using countersexual blood]

The chromosomes of 6 (5 females, 1 male) newborns have been examined before and after exchange transfusion. The highest percentage of donor lymphocytes has been found immediately after the exchange transfusion (9%, range 5 to 16%). The percentage of donor lymphocytes had decreased to 4% after 24 hours and to 1% after 3 weeks. There was no hint of a clastogenic effect of phototherapy and exchange transfusion.     

尾加压素Ⅱ对正常及缺血-再灌注离体大鼠心脏的影响

在正常Langendorff灌流与缺血-再灌注(停灌20 min-复灌20 min)离体大鼠心脏模型,观察尾加压素Ⅱ(urotensin Ⅱ,UⅡ)对冠脉流量、心功能和心肌代谢的影响以及心肌UⅡ受体的功能,以探讨UⅡ的心脏效应。对正常心脏给予0.1、1和10 nmol/L UⅡ各5 min,然后换洗5 min,对停灌缺血-再灌注心脏在再灌注期给予1或10nmol/L UⅡ。监测心率、左室内压和左室内压升降的最大变化率等心功能指标,计算冠脉流量,测定冠脉流出液中总蛋白和肌红蛋白含量以及乳酸脱氢酶(LDH)活性。灌流结束后,测定心肌丙二醛(MDA)含量和质膜UⅡ结合位点(放射性配基结合法)。结果如下:(1)正常心脏灌流UⅡ后,冠脉流量和心功能呈浓度依赖下降,换洗后没有完全恢复。心肌蛋白、肌红蛋白和LDH漏出随UⅡ浓度的增加而增加,换洗后迅速减少。UⅡ组心肌MDA含量与对照组差异无显著性。(2)缺血-再灌注后,冠脉流量显著减少,心功能显著抑制,再灌注期心肌蛋白、肌红蛋白和LDH明显漏出;给予UⅡ后,上述变化增强,且高浓度组更强,与对照组差异有显著性(P<<0.01),再灌注后心肌MDA含量亦显著高于对照(P<0.01)。(3)缺血-再灌注心肌质膜UⅡ受体的B_(max)显著高于正常对照心肌(14.65±1.78vs20.53±1.98 fmol/mg pr,P<0.01),Kd值变化无统计学意义。上述结果表明,在正常     

Protein synthesis in perfused rat hearts after in vivo hyperthermia and in vitro cold ischemia

Isolated and perfused rat hearts can be maintained for up to 2.5 h with minimal synthesis of a stress protein with a relative mass (Mr) of 71 kilodaltons (SP71). Isolated hearts, subjected to 17 h of cold (4 degrees C) ischemia, upon perfusion (37 degrees C) synthesize a large amount of SP71. In the present study, the effect of in vivo hyperthermia on protein synthesis in isolated and perfused hearts was examined. Hearts were excised from rats subjected to a 15-min episode of hyperthermia (42 degrees C), either immediately (no recovery) or after 24 h of recovery. The excised hearts were perfused either immediately or after 17 h of cold ischemia. Hyperthermia (no recovery) increased [3H]leucine incorporation into SP71, while hyperthermia with a 24-h recovery did not increase incorporation into SP71 during perfusion (no ischemia). Hyperthermia (no recovery) increased the incorporation of [3H]leucine into SP71 seen after cold ischemia. Hyperthermia with a 24-h recovery decreased the incorporation of [3H]leucine into SP71 seen after cold ischemia. This reduction in synthesis of SP71 after 24-h recovery from hyperthermia could be caused by the accumulation of SP71 suppressing its own synthesis or a measure of protection (tolerance) induced by the hyperthermia.     

Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia

The beneficial effect of hemodilution on cerebral blood flow (CBF) during focal cerebral ischemia is mitigated by reduced arterial oxygen content (CaO2). In anesthetized cats subjected to permanent middle cerebral artery occlusion, the time course of regional CBF was evaluated after isovolemic exchange transfusion with either albumin or a tetrameric hemoglobin-based oxygen carrier. The transfusion started 30 min after arterial occlusion. We tested the hypothesis that bulk oxygen transport (CBF x CaO2) to ischemic tissue is increased by hemoglobin transfusion at a hematocrit of 18% compared with albumin-transfused cats at a hematocrit of 18% or control cats at a hematocrit of 30% and equivalent arterial pressure. In the nonischemic hemisphere, CBF increased selectively after albumin transfusion, and oxygen transport was similar among groups. In the ischemic cortex, albumin transfusion increased CBF, but oxygen transport was not increased above that of the control group. Hemoglobin transfusion increased both CBF and oxygen transport in the ischemic cortex above values in the control group, but the increase was delayed until 4 h of ischemia. Consequently, acute injury volume measured at 6 h of ischemia was not significantly attenuated. In contrast to the cortex, CBF in the ischemic caudate nucleus was not substantially increased by either albumin or hemoglobin transfusion. Therefore, in a large animal model of permanent focal ischemia in which transfusion starts 30 min after ischemia, tetrameric cross-linked hemoglobin transfusion can augment oxygen transport to the ischemic cortex, but the increase can be delayed and not necessarily provide protection. Moreover, an end-artery region such as the caudate nucleus is less likely to benefit from hemodilution.     

Experimental results using 3-bromopyruvate in mesothelioma: in vitro and in vivo studies

Over many years we have taken advantage of the special metabolism of cancer cells involving an increased consumption of glucose associated with lactic acid production even in the presence of oxygen, a phenomenon referred to as the “Warburg effect”, to counteract cancer cell growth. We have tested 3-bromopyruvate (3-BrPA), an inhibitor of pyruvate-associated reactions. Firstly, we tested this agent, in vitro, in two mesothelioma cell lines. Cellular response would appear to depend on the mode of administration (immediately or 24 h after seeding). Depending on the line, 3-BrPA induced a cytostatic or cytotoxic effect. This effect was accompanied by cell death induction even in cells highly refractory to cisplatin. Mitochondrial apoptotic death appeared to involve both lines; however, a different death pathway such as necrosis cannot be excluded. Interestingly, 3-BrPA leads to a diminution of the expression of the anti-apotptoic protein Mcl-1. We then tested 3-BrPA in vivo. Survival of nude mice bearing human mesothelioma was significantly prolonged (p < 0.0001). Toxicity and clinical studies should be performed to test 3- BrPA as local therapy for patients suffering from pleural or peritoneal mesothelioma. Association with cisplatin should be particularly considered.     

Bradykinin metabolism in rat hearts with left-ventricular hypertrophy following myocardial infarction

The aim of the present study was to assess the contribution of angiotensin I converting enzyme (ACE)and neutral endopeptidase (NEP) in the coronary degradation of bradykinin (BK) after left-ventricular hypertrophy following myocardial infarction (MI) in rats. Myocardial infarction was induced by left descendant coronary artery ligation, and the contribution of ACE and NEP in the degradation of exogenous BK after a single passage through the coronary bed was assessed at 2, 5, and 36 days post-MI. BK degradation rate (V(max)/Km) was found to be significantly lower in hearts at 36 days (3.30 +/- 0.28 min(-1)) compared with 2 days (4.39 +/- 0.32 min(-1)) for noninfarcted hearts, but this reduction was just above the statistical level of significance for post-MI hearts. In infarcted hearts, V(max)/Km was increased significantly 5 days post-MI (4.91 +/- 0.28 min(-1)) compared with the 2 and 36 day-groups (3.43 +/- 0.20 and 2.78 +/- 0.16 min(-1), respectively). The difference between noninfarcted and MI was significant only 2 days post-MI. Treatment with the vasopeptidase inhibitor, omapatrilat, showed that the relative contribution of ACE and NEP combined increased over time in infarcted hearts and became significantly higher 36 versus 2 days post-MI. Finally, the treatment with an ACE inhibitor (enalaprilat) and a NEP inhibitor (retrothiorphan) in the 36-day infarcted and noninfarcted hearts showed that the relative contribution of ACE in infarcted hearts was comparable with that of noninfarcted hearts, whereas the relative contribution of NEP was increased significantly in infarcted hearts. In conclusion, experimental MI in rats induces complex changes in the metabolism of exogenous BK. The changes resulted in an increased relative contribution of NEP 36 days after infarction.     

Hepcidin and hemojuvelin gene expression in rat liver damage: in vivo and in vitro studies

In this work, we used two rat models, partial hepatectomy (PH) and CCl(4) administration, to study the changes in iron pathways in response to hepatic damage. Liver injury induced changes in the hepatic gene expression of hepcidin, hemojuvelin (Hjv), several other proteins of iron metabolism, and several cytokines such as IL-1beta, IL-6, TNF-alpha, and IFN-gamma. Hepcidin gene expression was upregulated between 4 and 8 h with a maximum up to 16 h after surgery. However, Hjv gene expression was downregulated at the same time. An early upregulation of hepcidin (3 h) and downregulation of Hjv gene expression was found after CCl(4) administration. Transferrin receptor 1 and ferritin H gene expression was upregulated, whereas ferroportin 1 gene expression was downregulated. Hepatic IL-6 gene expression was upregulated early after PH and reached maximum 8 h after the PH. In CCl(4)-induced liver injury, IL-6, IL-1beta, TNF-alpha, and IFN-gamma upregulation were found at the maximum 12 h after the administration of the toxin. Treatment of isolated rat hepatocytes with IL-6 and, to a lesser extent, with IL-1beta but not with TNF-alpha or IFN-gamma dose dependently upregulated hepcidin and downregulated Hjv gene expression. In hepatic damage, changes of the hepatic gene expression of the main proteins involved in iron metabolism may be induced by locally synthesized mediators.     

Effects of taurine depletion on rat cardiac electrophysiology: in vivo and in vitro studies

Electrocardiograms were monitored in unanesthetized rats during treatment with drinking water containing guanidinoethyl sulfonate (GES), an inhibitor of taurine transport, which depleted cardiac taurine content. Treatment led to a selective prolongation of the QT interval which was highly correlated with the degree of taurine depletion (r2 = 0.92, p less than .001). Compared to controls, the duration of ventricular muscle action potentials was significantly increased in GES-treated rats, and this accounted for the prolongation of QT intervals. Oral taurine supplements reversed GES-induced cardiac taurine depletion and the associated increased duration of action potentials and QT intervals. In vitro superfusion with 0.2-10 mM GES or taurine had no effect on action potentials of control or GES-treated rats. These data indicate that intracellular taurine may play a role in regulating myocardial action potential duration, particularly during repolarization.     

In vivo and in vitro studies of hafnium-binding to rat serum transferrin

The binding of hafnium to rat serum transferrin was studied using the time differential perturbed angular correlation (TDPAC) technique. Hafnium is interesting as a toxic metal binding to transferrin because it behaves metabolically similarly to plutonium. The isotope 181Hf offers favorable access to the TDPAC-method. Samples were prepared in vivo by intravenous injection of Hf-NTA, Hf-citrate, and Hf-oxalate solutions, respectively, into Sprague-Dawley rats and in vitro by adding Hf-NTA solution to fresh rat serum. In both cases two specific electric quadrupole interactions were observed, which correspond to two well-defined binding configurations. They may be attributed to the N-terminal and the C-terminal binding site in the transferrin molecule. The 181Hf-distribution between these two binding states depends on pH, salt and hafnium concentrations, temperature, and incubation time. With a fast TDPAC-setup of four BaF2-detectors a time resolution of about 600 ps could be achieved. The specific binding configurations of 181Hf and the comparatively slow relaxation times lead to spectra of considerable accuracy.     

In vivo,ex vivo,and in vitro studies on apelin's effect on myocardial glucose uptake

Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2 mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100 nM) for 2 h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.     

The antitumor activity of liposomal aclarubicin in vitro and in vivo]

Study on antitumor activity of free and liposomal anthracycline antibiotic aclarubicin in vitro and in vivo showed that liposomal aclarubicin was characterised by activity against ascitic Ehrlich carcinoma comparable to that of free aclarubicin when used in a dose of 25 mg/kg. Liposomal antibiotic had a more pronounced antimetastatic action and showed no toxicity (in a dose of 30 mg/kg). Liposomal aclarubicin had a higher activating capacity with respect to the macrophage tumoricidal properties.     

In vitro and in vivo studies with adriamycin liposomes

Liposome entrapped adriamycin retains its full cytotoxic potential when tested under $\text{in}\text{vitro}$ conditions against murine leukemia L-1210 cells. $\text{In}\text{vivo}$ drug distribution studies indicate that, relative to the free drug, a lower proportion of adriamycin administered in the liposome form is delivered to the heart and kidneys at one and four hours after injection. When administered to normal mice in high doses, anionic adriamycin liposomes appear less harmful than equal doses of the free drug as judged by alterations in animal weight gain. In these studies, a noval double packing procedure has been used for the entrapment of adriamycin in phospholipid vesicles.     

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.     

Retinoid-sensitive steps in steroidogenesis in fetal and neonatal rat testes: in vitro and in vivo studies

Retinoic acid (RA) was recently shown to modify testosterone secretion of the fetal testis in vitro. We characterized this effect by culturing rat testes explanted at various ages, from Fetal Day 14.5 to Postnatal Day 3. In basal medium, RA inhibited, in a dose-dependent manner, both basal and acute LH-stimulated testosterone secretion by testes explanted on Fetal Days 14.5, 15.5, and 16.5. It had no effect on testes from older animals. The negative effect of RA did not result from a diminution in the number of Leydig cells but from a decrease in P450c17 mRNA levels and in LH-stimulated cAMP production. However, the RA-induced decrease in P450C17 mRNA levels was also observed with neonatal testes, suggesting that this enzymatic step is no longer rate limiting at this developmental stage. To study the physiological relevance of RA effects, we used fetuses and neonates issued from mothers fed a vitamin A-deficient (VAD) diet, resulting in a threefold decrease of plasma retinol concentration. On Fetal Day 18.5 and on Posnatal Day 3, testosterone secretion by the testis ex vivo was significantly increased in VAD animals. This shows that the endogenous retinol inhibits differentiation and/or function of fetal Leydig cells before Fetal Day 18.5 and is required for the normal regression of fetal Leydig cell function that occurs after Fetal Day 18.5. In conclusion, our results show that retinoids play a negative role on the steroidogenic activity during the differentiation of rat fetal Leydig cells.     

Tenascin gene expression in rat liver and in rat liver cells In vivo and in vitro studies

Tenascin is a major glycoprotein constituent of the extracellular matrix with a strong affinity to fibronectin; its distribution is believed to be temporarily and spatially limited. Tenascin gene expression is increased during wound healing processes. As repair mechanisms in chronic liver diseases resemble wound healing we studied tenascin gene expression in rat liver and in isolated rat liver cells. In normal rat liver a tenascin specific antiserum stains sinusoidal cells with fiber-like prolongations, which at the same time are desmin-positive (ITO-cells). In the CCl₄-acutely-damaged liver a strong tenascin staining is detected in cells located among the mononuclear cells of the inflammatory infiltrates in the areas of necrosis and in cells of the sinusoids. In CG⁴-chronically-damaged liver a strong tenascin staining is demonstrable in the connective tissue septa. In both cases, many of the tenascin-positive cells can be identified as desmin-positive by means of the double-staining fluorescence technique. The wall of larger vessels is always tensacin-negative. The staining pattern obtained with a fibronectin-specific antiserum is somewhat comparable with that of tenascin but the vessel wall was positive. Hepatocytes, Kupffer cells, ITO-cells and endothelial cells were isolated from rat liver and studied for their capacity to express the tenascin gene. Biosynthetically labeled tenascin was immunoprecipated from supernatants and cell lysates obtained from cultured ITO-cells and to a much lesser extent from intracellular lysates obtained from endothelial cells; its synthesis in ITO-cells increased during the time in culture. Tenascin was also identified immuno-cytochemically in increasing amount in ITO-cells in culture. We conclude that ITO-cells may play a major role in tenascin synthesis during liver fibrogenesis. Some of these results were presented at the Annual Meeting of the American Association Study of the Liver, Chicago, USA, 1990. G.R. holds a Hermann and Lilly Schilling professorship     

Peroxynitrite and myocardial contractility: in vivo versus in vitro effects

Generation of peroxynitrite (ONOO-) as a result of altered redox balance has been shown to affect cardiac function; however, inconsistencies in the data exist, particularly for myocardial contractility. The hypothesis that the cardiac impact of ONOO- formation depends on its site of generation, intravascular or intramyocardial, was examined. Cardiac contractility was assessed by pressure-volume analysis to delineate vascular versus cardiac changes on direct infusion of ONOO- into the right atria of conscious dogs both with normal cardiac function and in heart failure. Additionally, ONOO- was administered to isolated murine cardiomyocytes to mimic in situ cardiac generation. When infused in vivo, ONOO- had little impact on inotropy but led to systemic arterial dilation, likely as a result of rapid decomposition to NO2- and NO3-. In contrast, infused ONOO- was long lived enough to abolish beta-adrenergic (dobutamine)-stimulated contractility/relaxation, most likely through catecholamine oxidation to aminochrome. When administered to isolated murine cardiomyocytes, ONOO- induced a rapid reduction in sarcomere shortening and whole cell calcium transients, although neither decomposed ONOO- or NaNO2 had any effect. Thus, systemic generation of ONOO- is unlikely to have primary cardiac effects, but may modulate cardiac contractile reserve, via blunted beta-adrenergic stimulation, and vascular tone, as a result of generation of NO2- and NO3-. However, myocyte generation of ONOO- may impair contractile function by directly altering Ca2+ handling. These data demonstrate that the site of generation within the cardiovascular system largely dictates the ability of ONOO- to directly or indirectly modulate cardiac pump function.