Krebs-Henseleit Bicarbonate Research Articles

Recombinant Human Soluble Thrombomodulin (ART-123) Prevents Warm Ischemia-Reperfusion Injury in Liver Grafts from Non–Heart-Beating Donors

BackgroundLiver transplantation is an established treatment for end-stage liver disease. However, an ongoing problem worldwide concerning this treatment is the shortage of grafts. Although transplantation using grafts from non–heart-beating donors (NHBDs) is considered a promising solution, some researchers have reported that these liver grafts are associated with primary graft nonfunction and biliary complications. The purpose of this study was to establish a safe technique procuring liver grafts from marginal donors such as NHBDs. Materials and methodsMale Wistar rats were divided into three groups: (1) the heart-beating (HB) group, whose livers were retrieved from HB donors; (2) the non-HB (NHB) group, whose livers were retrieved from NHBDs that had experienced an apnea-induced agonal condition (for this group, livers were subjected to warm ischemia for 30 minutes after cardiac arrest); and (3) the recombinant human soluble thrombomodulin (ART-123) group, whose livers were retrieved in the same manner as the NHB group but pretreated with ART-123 (1 mg/kg) at the agonal stage. The livers were reperfused for 60 minutes with oxygenated Krebs-Henseleit bicarbonate buffer after cold preservation for 6 hours. ResultsBile production and portal flow volume in the ART-123 group were significantly higher than those in the NHB group. Alanine aminotransferase levels in the ART-123 group were significantly lower than those in the NHB group. Histological findings showed the narrowing of sinusoidal spaces and necroses in the NHB group were more severe than those in the ART-123 group. ConclusionsThese results suggest that thrombomodulin may improve the viability of liver grafts from NHBDs.

Protective effect of heme oxygenase-1 induction against hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion

AimsThe purpose of this study was to investigate the cytoprotective role of heme oxygenase-1 (HO-1) induction in hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion (I/R). Main methodsAnimals were fed an ethanol liquid diet or isocaloric control diet for 5weeks. Isolated perfused rat livers were preserved in Histidine–Tryptophan–Ketoglutarate at 4°C. After 24h of storage, livers were subjected to 120min of reperfusion with Krebs–Henseleit bicarbonate buffer at 37°C. Animals were pretreated with cobalt protoporphyrin (CoPP, 5mg/kg, i.p.) or zinc protoporphyrin (ZnPP, 25mg/kg, i.p.), HO-1 inducer and antagonist, respectively. Key findingsIn the model of ischemia/isolated perfusion, endogenous HO-1 was downregulated in the livers fed with ethanol diet (ED I/R). In ED I/R group, portal pressure and lactate dehydrogenase release were significantly increased, while bile output and hyaluronic acid clearance decreased compared to rats fed on control diet (CD I/R). Furthermore, hepatic glutathione content decreased and lipid peroxidation increased in the ED I/R group compared to the CD I/R group. These alterations were attenuated by upregulation of HO-1 with CoPP pretreatment. SignificanceOur results suggest that chronic ethanol consumption aggravates hepatic injury during cold I/R and it is likely due to downregulation of endogenous HO-1. Prior induction of HO-1 expression may provide a new strategy to protect livers against hepatic I/R injury or to increase the donor transplant pool through modulation of marginal alcoholic steatotic livers.

Multidrug donor preconditioning protects steatotic liver grafts against ischemia-reperfusion injury

Background Graft dysfunction of steatotic livers (SL) still remains a major challenge in liver transplantation. Different mechanisms are thought to be involved in the impaired tolerance of SL to ischemia-reperfusion injury. Thus, different pharmacologic strategies may need to be combined to effectively protect SL and to reduce graft dysfunction after transplantation. Therefore, we analyzed the effectiveness of a multidrug donor preconditioning (MDDP) procedure to protect SL from cold ischemia-reperfusion injury. Methods Liver steatosis was induced by a high-carbohydrate, fat-free diet. A total of 24 Sprague–Dawley rats were divided into 3 groups (n = 8 each), including a control group with nonsteatotic livers (Con), a vehicle-treated SL group (SL-Con), and a SL group undergoing MDDP (SL-MDDP), including pentoxyphylline, glycine, deferoxamine, N-acetylcysteine, erythropoietin, melatonin, and simvastatin. MDDP was applied before liver perfusion with 4°C histidine-tryptophan-ketoglutarate (HTK) solution and organ harvest. After 24 hours of cold storage in HTK, postischemic reperfusion was performed in an isolated liver reperfusion model using 37°C Krebs-Henseleit bicarbonate buffer. Results After 60 minutes of reperfusion, SL showed a significant reduction of bile flow as well as a marked increase of liver enzyme levels and apoptotic cell death compared with Con. This was associated with an increased malondialdehyde formation, interleukin-1 production, and leukocytic tissue infiltration. MDDP completely abolished the inflammatory response and was capable of significantly reducing parenchymal dysfunction and injury. Conclusions MDDP decreases SL injury after cold storage and reperfusion. The concept of MDDP as a simple and safe preoperative regime, thus may be of interest in clinical use, expanding the donor pool from marginal donors.

Decrease of gluconeogenesis by deramciclane is counteracted by cytochrome P450 inhibitors

The effect of deramciclane (EGIS-3886), a new anxiolytic drug, was investigated on carbohydrate metabolism in mouse isolated hepatocytes. Deramciclane is a substrate of hepatic cytochrome P450 enzymes. Its in-vivo administration has been shown to cause a dose-dependent liver glycogen depleting effect in dogs. Hepatocytes were prepared from male CFLP mice (fed or starved for 24 h) using the collagenase perfusion method. The cells were incubated in Krebs-Henseleit bicarbonate buffer at 37°C with constant bubbling of CO2/O2 (5:95, v/v). Glucose production of the cells was measured in deproteinized samples by a glucokinase-glucose 6-phosphate dehydrogenase method. Glycogen content of the cells was determined with the same method after hydrolysis. Glycogen breakdown in mouse isolated hepatocytes was not affected by deramciclane. Gluconeogenesis from alanine or pyruvate but not from fructose was inhibited by high concentrations (40 μM) of deramciclane. The inhibitory effect of deramciclane on glucose production (nmol min−1/106 cells) from alanine was inhibited by econazole (-0.97 ± 0.13 vs -2.21 ± 0.49, P < 0.05, s.e.m., n = 5) or by SKF 525A (-0.60 ± 0.21 vs -2.21 ± 0.49, P < 0.02). Addition of deramciclane did not affect the level of cAMP or glutathione in isolated hepatocytes. The study shows that oxidative biotransformation of deramciclane decreases glucose formation from three carbon unit precursors without any direct effect on glycogen metabolism. We concluded that glycogen depletion by long-term deramciclane administration may be mediated by decreased gluconeogenesis caused by increased NADPH consumption upon oxidative metabolism of deramciclane.

The pretreatment effects of pentoxifylline on aflatoxin B1-induced oxidative damage in perfused rat liver

This study was conducted to investigate the hepatoprotective and antioxidant effects of pentoxifylline (PTX) against aflatoxin B1 (AFB1) exposure in perfused rat livers by evaluating damage marker enzymes, antioxidant defense systems (glutathione, GSH) and lipid peroxidation (malondialdehyde, MDA). Sixteen rats were divided randomly into four experimental groups: control, PTX, AFB1 and AFB1 + PTX. Rats in the control group were infused with Krebs–Henseleit bicarbonate buffer. Rats in the AFB1-treated group received approximately 1 ppm and the PTX- treated group received 100 mg/kg intraperitoneally 24 h before surgery. Alanine aminotransferase and lactate dehydrogenase levels were increased by AFB1 and decreased by PTX. PTX also ameliorated the increased concentration of MDA caused by AFB1. PTX did not compensate for the decrease in GSH caused by AFB1. These results imply that PTX has an antioxidant effect by inhibiting free radicals, and prior treatment with PTX ameliorates the effects of AFB1-induced lipid peroxidation but does not compensate GSH depots.

Clenbuterol-Stimulated Glucose Uptake Activates both GS and GI Pathways through Β2-Adrenoceptor in Mouse Isolated Soleus Muscle

&beta;2-adrenoceptors activated by adrenaline can also couple to both Gs and Gi proteins. The former is associated with an increase in cAMP to illicit the effect of the catecholamine. In the later, &beta;2-AR induces PKA-catalysed phosphorylation of the receptor, which intends couples to Gi, at high concentration. We proposed that, clenbuterol which stimulates glucose uptake at low concentra-tion and inhibits it at high concentration might have identical signalling pathway as adrenaline. Mouse isolated muscles were pre-incubated in flasks containing 3 ml of Krebs-Henseleit Bicar-bonate buffer. After 120 min of pre-incubation, with the appropriate concentration of PTX the muscles were transferred to another incubation flask containing 3 ml of the same buffer and 0.3mCi 2-deoxy[1-14C]glucose containing varying concentrations of adrenaline, clenbuterol or 1nM insulin with or without PTX concentrations of 100ng/ml for adrenaline and clenbuterol and 0.1 or 1.0ng/ml for insulin, or 1M acetylcholine for 45 minutes. Adrenaline stimulated glu-cose uptake in isolated mouse soleus muscle at low concentration (10-10M) and inhibited it at high concentration (10-5M). The effect of the lower concentration was mediated through the &beta;2-AR coupling to the Gs protein and to the Gi protein at high concentration. Similarly, clenbuterol stimulated glucose uptake at lower concentration (10-11M) is mediated through the &beta;2-AR cou-pling to the Gs protein and to the Gi protein at the higher concentration (10-7M). These effects of high concentration of adrenaline and clenbuterol were supported by the fact that 100ng/ml PTX relieved their inhibitory effects. The higher concentration effect of clenbuterol was additionally supported by the fact that, 1M acetylcholine, relieved the inhibitory effect. Keywords: PTX, Pertusis toxin, G-proteins, Guanine nucleotide binding proteins, &beta;-AR, beta adrenoceptor, M2, muscarinic receptors

Multidrug donor preconditioning prevents cold liver preservation and reperfusion injury

Primary graft dysfunction still represents a major challenge in liver transplantation. We herein studied in an isolated rat liver perfusion model whether a multidrug donor preconditioning (MDDP) can not only reduce but also completely prevent cold ischemia-reperfusion injury. MDDP included curcumin, simvastatin, N-acetylcysteine, erythropoietin, pentoxyphylline, melatonin, glycine, and methylprednisolone. Postischemic reperfusion was performed after 24 h cold storage in histidine-tryptophan-ketoglutarate solution with 37°C Krebs Henseleit bicarbonate buffer. Cold hepatic ischemia-reperfusion resulted in a massive K(+) release, protein loss, and aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase elevation. This was associated with increased malondialdehyde formation, enhanced tumor necrosis factor-alpha and interleukin-6 production, pronounced leukocytic tissue infiltration, and apoptotic cell death. MDDP abolished the inflammation response and was capable of completely preventing the manifestation of parenchymal injury. Thus, MDDP potentiates the protective effects reported after single-drug donor preconditioning and may therefore be an interesting approach to improve the outcome in clinical liver transplantation.

The effects of ginseng total saponin, panaxadiol and panaxatriol on ischemia/reperfusion injury in isolated rat heart

The aim of the present study was to evaluate the protective effect of ginseng total saponin, panaxadiol and panaxatriol, which are the major components of Panax ginseng, against myocardial ischemia/reperfusion (I/R) injury in isolated rat hearts. Rats were orally administered once a day with total saponin (20mg/kg), panaxadiol (5mg/kg) and panaxatriol (5mg/kg) for consecutive 7days. On day 8, the hearts were isolated and perfused with Krebs-Henseleit bicarbonate buffer solution using Langendorff apparatus. After 30min of global ischemia, hearts were reperfused for 30min. Myocardial function, coronary flow and biochemical parameters, such as lactate dehydrogenase (LDH), creatine kinase (CK), adenosine triphosphate (ATP), malondialdehyde (MDA) and reduced glutathione (GSH) were measured. Total saponin and panaxatriol significantly improved I/R-induced myocardial dysfunction by increasing left ventricular development pressure, (−dP/dt)/(+dP/dt) and time to contracture. Moreover, the increases in the levels of LDH, CK and MDA and the decrease in the levels of GSH were attenuated by total saponin and panaxatriol. However, the ATP levels did not affected by total saponin, panaxadiol and panaxatriol pretreatment. Our findings suggest that pretreatment with ginseng total saponin, especially panaxatriol, ameliorates I/R-induced myocardial damage and this protection is caused by reducing oxidative stress.

Ischaemic Preconditioning and Postconditioning do not Affect Adenosine A1 and A2A Receptor Sensitivity

Ischaemic Preconditioning and Postconditioning do not Affect Adenosine A1 and A2A Receptor Sensitivity

Effect of anoxia and Polyscias filicifolia Bailey biomass tincture on the activity of tRNA and aminoacyl-tRNA synthetases in isolated pig heart

The aim of this study was to investigate effect of anoxia and Polyscias filicifolia Bailey biomass tincture on the activities of different tRNA and aminoacyl-tRNA synthetases in isolated pig heart. The isolated pig heart was perfused according to the modified method of Langendorf, using an artificial blood circulation apparatus. Anoxia 20 min in duration was performed by perfusion of isolated heart with Krebs-Henseleit bicarbonate buffer saturated with gas mixture (95% N(2) and 5% CO(2)). Control heart was perfused with the same buffer saturated with gas mixture (95% O(2) and 5% CO(2)). Effect of Polyscias filicifolia Bailey biomass tincture was evaluated by perfusion of isolated heart with a buffer containing tincture. Total tRNA and aminoacyl-tRNA synthetases were isolated from pig heart. Activities of tRNA and aminoacyl-tRNA synthetases were measured by the aminoacylation reaction using C(14)-amino acids. Anoxia 20 min in duration has caused a decrease in the acceptor activity of tRNA and increase in the activities of aminacyl-tRNA synthetases. Polyscias filicifolia Bailey tincture did not affect the acceptor activity of tRNA and activities aminacyl-tRNA synthetases. After 20-min anoxic perfusion with the buffer containing Polyscias filicifolia Bailey biomass tincture, the acceptor activities of tRNA increased to the control value and activities of aminacyl-tRNA synthetases reached the control value. The acceptor activity of tRNA from isolated pig heart decreased and activities of aminacyl-tRNA synthetases increased under anoxia. Perfusion with buffer containing tincture of Polyscias filicifolia Bailey biomass restored acceptor activities of tRNA and activities of aminacyl-tRNA synthetases.

Sivelestat reduces myocardial ischemia and reperfusion injury in rat hearts even when administered after onset of myocardial ischemia

Sivelestat, a neutrophil elastase inhibitor, has been shown to attenuate pulmonary injury during ischemia and reperfusion by improving microcirculation and may be effective as a cardioprotective agent. Isolated rat hearts were Langendorff-perfused (constant pressure, 75 mmHg) with oxygenated Krebs-Henseleit bicarbonate buffer (KHB). The optimal sivelestat concentration at 19 micromol/l was revealed because left ventricular developed pressure (LVDP) recovery in 19 micromol/l sivelestat was highest among 0.19, 1.9, 19, 190, and 1900 micromol/l sivelestat (26+/-10, 33+/-7, 56+/-5*, 35+/-2, and 15+/-5%, respectively; *P<0.01). In order to examine the optimal administration timing, sivelestat was administered at pre- and post-ischemic phases. LVDP recovery and troponin-T were observed in pre-, post-ischemic sivelestat groups and control. After 60 min-reperfusion, LVDP recoveries were 42+/-10*, 45+/-19*, and 14+/-5%, respectively (*P<0.01 compared to control), and troponin-T values were 4+/-1, 2+/-1**, and 8+/-2, respectively (**P<0.05 compared to control). Acetylcholine-induced increase in coronary flow was also investigated to examine the sivelestat's cardioprotective mechanism. Ischemia-reperfusion (I/R) impaired the acetylcholine-induced increase in coronary flow (maximal changes: sham, 125+/-11%; I/R, 98+/-3; P<0.01) and this impairment was attenuated by sivelestat-perfusion at reperfusion (maximal change: 112+/-7%; P<0.05 vs. I/R). Sivelestat attenuates coronary endothelial ischemia-reperfusion injury and improves myocardial protection even when administered at the reperfusion period. This suggests a role for sivelestat in the preservation of coronary endothelial function enhancing myocardial protection.

A Free Radical Scavenger, Edaravone, Prevents Ischemia-Reperfusion Injury in Liver Grafts From Non–Heart-Beating Donors

Background Due to the increase in liver transplantation, the donor shortage has become a serious problem, requiring marginal, non–heart-beating donors (NHBDs). The aims of this study were to evaluate the cytoprotective effect of edaravone, a free radical scavenger, on warm ischemia-reperfusion (I/R) injury of liver grafts from NHBDs. Methods Rat livers were harvested from heart-beating donors (HB group) or from NHBDs undergoing cardiac arrest for 30 minutes led by thoracotomy (NHB group), and reperfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after cold preservation for 6 hours. In another group (ED group), warm ischemic livers from NHBDs were reperfused with buffer containing edaravone (1 mg/L) after cold preservation. Results In the ED group, portal flow volume, bile production, and energy charge were significantly ameliorated. Lipid peroxidation, elevation of hepatic enzymes, and release of tumor necrosis factor-alpha and interleukin-1 beta were significantly alleviated, compared with the NHB group. Conclusions These results suggested that edaravone has suppressive effects on warm I/R injury in liver grafts from NHBDs.

Effects of Serine Protease Inhibitor and Prostaglandin I 2 on Liver Transplantation From Non–Heart-Beating Rat Donors

Objectives We sought to preserve the microcirculation as a keystone in liver transplantation from a non–heart-beating donor (NHBD). The purpose of this study was to investigate the cytoprotective effects of a serine protease inhibitor, nafamostat mesilate, and prostaglandin I 2 (PGI 2) on livers transplanted from NHBDs. Methods Male Wistar rats were used in five groups of nine rats each. In group 1, livers were retrieved from heart-beating donors (HB group); in group 2, livers were retrieved from NHBDs that had experienced agonal apnea (NHB group); in group 3, livers were retrieved in the same manner as in the NHBD group but were pretreated with nafamostat mesilate (NM), 0.2 mg/kg/h, (NM group); in group 4, livers were retrieved in the same manner as in the NHBD group but were pretreated with prostaglandin (PG) I 2, 33 ng/kg/h for 30 minutes (PG group); and in group 5, livers were retrieved in the same manner as in the NHBD group but were pretreated with NM plus PG, (NM+PG group). Livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissue were analyzed in one set of experiments. In another set of experiments, livers retrieved and after 1 hour of cold preservation were transplanted according to the Kamada method. Results In the NM+PG group, the values of interleukin-1β, tumor necrosis factor–α, and thromboxane B 2 were significantly lower than those in the NHB group. At histologic analysis, sinusoidal endothelial cells were well preserved in the NM+PG group. The number of survivors at 7 days after liver transplantation in the 5 groups were 9, 0, 1, 1, and 3, respectively. Conclusion The serine protease inhibitor, NM, and PGI 2 supported sinusoidal endothelial cells and preserved microcirculation.

Nitric oxide donor, NOC7, reveals biphasic effect on contractile force of isolated rat heart after global ischemia

Our purpose was to investigate whether the NO donor, 3-(2-hydroxy-1-methyl-2-nitroso-hydrazino)-N-methyl-1-propanamine (NOC7), restored cardiac function following global ischemia in an isolated rat heart model and whether intracellular messengers were involved in its effect. Isolated rat hearts (n = 36) were randomly divided into six groups. The sham control group was perfused with modified Krebs-Henseleit bicarbonate buffer (KHB) alone. The ischemic control group and the NOC7 groups were subjected to 35 min of global ischemia, followed by 30 min of reperfusion with KHB alone, or reperfusion with KHB including NOC7 at 0.2, 2, 20, or 200 microM, respectively. Left ventricular developed pressure (LVDP), the maximum and the minimal rate of rise in LVP (+/-dP/dt), and coronary flow were measured continuously. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels were measured in myocardium homogenate, using enzyme immunoassay (EIA) methods. NOC7 at 2 and 20 microM rescued myocardial performance (LVDP, 111.9 +/- 10.5% and 124.3 +/- 12.5% of baseline, respectively; P < 0.05 vs ischemic control) at 30 min after reperfusion. However, NOC7 at 200 microM reduced the LVDP to 55.3 +/- 6.0% of baseline. Coronary flows remain unchanged. The cAMP levels increased significantly from 0.83 +/- 0.44 pmol x mg(-1) protein in the ischemic control group to 1.79 +/- 0.39, 1.86 +/- 0.25, and 2.63 +/- 0.24 pmol x mg(-1) protein, in the groups with NOC7 at 2, 20, and 200 microM, respectively (P < 0.05). The cGMP level increased from 1.49 +/- 0.61 pmol x mg(-1) protein in the ischemic control group to 3.92 +/- 0.66 pmol x mg(-1) protein in the group with NOC7 at 200 microM alone (P < 0.05). NOC7 appeared to exert a biphasic effect on the contractile force of the isolated rat heart after 35-min global ischemia. The balance between intracellular cAMP and cGMP levels seemed to be involved in its mechanism.

Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion

Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.

The effects of alcohol binge on vessel reactivity

Alcohol (ALC) consumption is associated with alterations in blood pressure and changes in vascular function. Chronic and excessive ALC consumption increases the risk for hypertension and stroke, while moderate daily intake has been shown to reduce incidence of coronary artery disease leading to a reduction in the risk of myocardial infarction. The underlying mechanism has been suggested to be altered endothelial function, with moderate ALC consumption enhancing endothelial function, while chronic excessive consumption impairs function. Previous studies have shown that acute ALC intoxication (AAI) decreases blood pressure, enhances hypotension following hemorrhagic shock, and impairs pressor response to fluid resuscitation. This study examined the effects of AAI on blood vessel reactivity to phenylephrine (PE), acetylcholine (Ach), and nitroprusside (NP) ex‐vivo. Chronically instrumented, conscious male Sprague‐Dawley rats (300–350 g) received a primed continuous 15 hour intragastric ALC infusion (1.75g/kg + 300 mg/kg/hr). Thirty minutes after discontinuing ALC, animals were sacrificed for isolation of thoracic aorta and mesenteric arteries. Aortic and mesenteric ring segments (1–2 mm) were suspended in myograph baths containing Krebs‐Henseleit bicarbonate buffer, pH 7.4, gassed with 95% O2: 5% CO2. Arterial rings from AAI rats had decreased PE‐induced tension (aorta: 2.28 ± 0.09 vs. 2.6 ± 0.13 g; mesenteric artery: 1.70 ± 0.06 vs. 1.91 ± 0.10), greater (21%) Ach‐mediated relaxation and similar NP‐mediated relaxation. These results indicate that AAI favors vasodilatation and are consistent with enhanced endothelial dilator function. (Supported by DOD PR‐054196).

Role of Haptoglobin on the Uptake of Native and β-Chain [Trimesoyl-(Lys82)β-(Lys82)β] Cross-Linked Human Hemoglobins in Isolated Perfused Rat Livers

The role of haptoglobin in liver cell entry of acellular native hemoglobin, and cross-linked human hemoglobin, a potentially useful oxygen-carrier alternative in transfusion medicine, was examined in the recirculating, perfused rat liver preparation. Doses of tritiated native human or beta-chain [trimesoyl-(Lys82)beta-(Lys82)beta] cross-linked human hemoglobin were preincubated with haptoglobin-containing rat plasma or Krebs Henseleit bicarbonate buffer for 30 min and used for perfusion. Concentrations (dpm/ml) in reservoir, before and after separation of the hemoglobins and metabolites by gel filtration fast protein liquid chromatography column chromatography, were similar, showing mostly the presence of intact hemoglobin. Each hemoglobin species underwent a rapid distribution phase, followed by a protracted elimination phase. The radioactivity in bile at 3 h consisted of low molecular weight metabolites, and cumulative excretion was slightly higher when rat plasma was present: for native hemoglobin, 7.1 +/- 1.6% versus 9.2 +/- 2.1% dose; for cross-linked hemoglobin, 5.0 +/- 1.7% versus 7.2 +/- 0.8% dose. Data fit to a two-compartment model and physiologically based model revealed a significantly faster influx clearance (CL(influx)) over the metabolic intrinsic clearance (CL(int, met)). The ratios of CL(influx)/CL(int, met) were 125 and 535 for native hemoglobin in the absence and presence of rat haptoglobin, respectively, according to compartmental analyses; the ratios were 25 and 53, respectively, according to physiological modeling. The corresponding ratios for cross-linked hemoglobin in the absence and presence of rat haptoglobin were 55 and 81, respectively, and 24 and 70 for compartmental and physiological modeling. Although haptoglobin enhanced the hepatic internalization of the hemoglobins, the impact on the net clearance was lessened since degradation was the rate-limiting step.

Dramatic Accumulation of Triglycerides and Precipitation of Cardiac Hemodynamic Dysfunction during Brief Caloric Restriction in Transgenic Myocardium Expressing Human Calcium-independent Phospholipase A2γ

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Brief Ischemia-Reperfusion Performed After Prolonged Ischemia (Ischemic Postconditioning) Can Terminate Reperfusion Arrhythmias With no Reduction of Cardiac Function in Rats

Recently, it has been reported that ischemic postconditioning, a brief episode of ischemia-reperfusion performed after prolonged ischemia, can reduce ischemic myocardial injury. However, the effects of ischemic postconditioning on ischemia/reperfusion injury remain unclear. We investigated the effects of brief ischemia-reperfusion before (ischemic preconditioning) and after (ischemic postconditioning) prolonged ischemia on myocardial ischemia/reperfusion injury, especially reperfusion arrhythmias. Adult male Sprague-Dawley rats weighing about 400-500 g were used. The isolated heart was perfused using a working heart method (Krebs-Henseleit bicarbonate buffer). In the control group, after stabilization, diastolic global ischemia for 15 minutes was produced by a one-way ball valve with electrical pacing (330 bpm, 2.0 V). After ischemia, the heart was reperfused for 20 minutes. In the preconditioning and postconditioning groups, 5-minute global ischemia was produced before and after ischemia for 15 minutes with a 1 minute interval. An electrocardiogram was performed and left ventricular pressure (LVP, +dP/dt, -dP/dt) and CK activity in coronary effluent were measured during the protocol. Ischemic preconditioning did not affect the incidence or duration of reperfusion ventricular arrhythmias. Ischemic postconditioning could terminate reperfusion ventricular arrhythmias completely and reduced the duration of reperfusion ventricular arrhythmias significantly (P < 0.01). Furthermore, the recovery ratio of +dP/dt at 20 minutes after initial reperfusion was significantly higher in the postconditioning group than in the other groups. These results suggest that ischemic postconditioning can terminate reperfusion arrhythmias with no reduction of cardiac function, and may be useful for correcting stunned myocardium.

Generation of survival signal by differential interaction of p38MAPKα and p38MAPKβ with caveolin-1 and caveolin-3 in the adapted heart

Sphingomyelin breakdown product ceramide has recently been found to induce an adaptive response and reduce myocardial ischemia/reperfusion injury. Since activation of MAP kinases plays an essential role in myocardial adaptation to ischemic stress and since ceramide is involved in lipid raft formation where MAP kinases can be translocated in response to stress, we reasoned that preconditioning may potentiate the translocation of MAP kinases into the lipid raft. To test the hypothesis, rats were divided into five groups: (i) control, (ii) ischemia/reperfusion (I/R), (iii) I/R+C-2 ceramide, (iv) adapted and (v) adapted+desipramine, an inhibitor of ceramide formation. Isolated hearts were preperfused for 15 min with Krebs Henseleit bicarbonate (KHB) buffer in the absence or presence of 10 μM desipramine followed by adaptation induced by four cyclic episodes of 5 min ischemia and 10 min reperfusion. For myocardial adaptation to ischemia with ceramide, the hearts were perfused with 1 μM C-2 ceramide. All hearts were then subjected to 30 min ischemia and 2 h of reperfusion. As expected, both ischemic adaptation and ceramide adaptation made the heart resistant to I/R injury as evidenced by improved ventricular performance and reduced myocardial infarct size and cardiomyocyte apoptosis, which were significantly blocked with desipramine indicating the involvement of ceramide in ischemic adaptation. Ceramide also participated in the formation of lipid raft, and desipramine disrupted the raft formation. In the adapted hearts, there was an increased association of the proapoptotic p38MAPKα with caveolin-1 while there was a reduced association of anti-apoptotic p38MAPKβ with caveolin-3 indicating reduced amount of p38MAPKα and increased amount of p38MAPKβ were available to the adapted hearts thereby generating a survival signal. Desipramine decreased the association of P38MAPKα and C-2 ceramide increased the association of P38MAPKα with the lipid raft. The survival signal was further confirmed by increased phosphorylation of AKT and enhanced induction of expression of Bcl-2 during adaptation and its reversal with desipramine. The results indicated a unique ceramide signaling the ischemic and PC hearts involving lipid rafts, which generated a survival signal by differentially associating the p38MAPKα and p38MAPKβ with the caveolin-1 and caveoli-3, respectively.