Krebs-Henseleit Bicarbonate Buffer Research Articles

Effect of esaxerenone on ischemia and reperfusion injury in rat hearts.

In myocardial infarction, the addition of mineralocorticoid receptor blockers (MRBs) to standard therapies, such as angiotensin-converting enzyme inhibitors or beta-blockers, reportedly reduces mortality and cardiac events. We investigated whether the non-steroidal MRB esaxerenone has cardioprotective effects and its protective mechanisms. Isolated rat hearts were Langendorff-perfused (constant pressure, 80 mmHg) with oxygenated Krebs-Henseleit bicarbonate buffer and reperfused for 60 min; afterwards, recovery of function (left ventricular pressure, measured with an intraventricular balloon) and myocardial injury were measured. In a preliminary study, we determined the optimal concentration of esaxerenone required for myocardial protection. Next, esaxerenone was administered in the pre- and post-ischemic phases to determine the optimal timing of administration. In addition, we assessed coronary flow response to acetylcholine (ACh) with and without esaxerenone. We examined whether esaxerenone-induced cardioprotection was prevented by targeting putative components in the preconditioning manner (the mitochondrial ATP-sensitive potassium (KATP) channel). Myocardial protection by esaxerenone was observed when esaxerenone was administered before ischemia but not after ischemia. The coronary flow response to ACh was significantly better in the esaxerenone group than in the control group. The cardioprotective effect of esaxerenone was eliminated by the mitochondrial KATP channel blocker, 5-hydroxy decanoate. This study confirmed the myocardial protective effect of the pre-ischemic administration of esaxerenone. Esaxerenone may contribute to coronary endothelial protection and exert pharmacological preconditioning via the mitochondrial KATP channel.

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model.

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of developing pigs.

Enterocytes of young pigs are known to use glutamine, glutamate, and glucose as major metabolic fuels. However, little is known about the roles of aspartate, alanine, and fatty acids as energy sources for these cells. Therefore, this study simultaneously determined the oxidation of the amino acids and glucose as well as short- and long-chainfatty acids in enterocytes of developing pigs. Jejunal enterocytes were isolated from 0-, 7-, 14- and 21-day-old piglets, and incubated at 37°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: D-[U-14C]glucose, 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM L-[U-14C]palmitate, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate by enterocytes from all age groups of piglets increased (P < 0.05) gradually with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, and glucose by enterocytes from 0- to 21-day-old pigs and of alanine from newborn pigs were much greater (P < 0.05) than those for the same concentrations of palmitate, propionate, and butyrate. Compared with 0-day-old pigs, the rates of oxidation of glutamate, aspartate, glutamine, alanine, and glucose by enterocytes from 21-day-old pigs decreased (P < 0.05) markedly, without changes in palmitate oxidation. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes of pigs was limited during their postnatal growth. At each postnatal age, the oxidation of glutamate, glutamine, aspartate, and glucose produced much more ATP than alanine, propionate, butyrate and palmitate. The degradation of glutamate was initiated primarily by glutamate-pyruvate and glutamate-oxaloacetatetransaminases. Our results indicated that amino acids (glutamate plus glutamine plus aspartate) are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of post-hatching developing chickens.

This study determined the oxidation of amino acids, glucose and fatty acid in enterocytes of developing chickens. Jejunal enterocytes were isolated from 0-, 7-, 21-, and 42-d-old broiler chickens, and incubated at 40°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM [U-14C]palmitate, D-[U-14C]glucose, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. 14CO2 produced from each 14C-labeled substrate was collected for determination of radioactivity. Among all the substrates studied, glutamate had the greatest rate of oxidation in enterocytes from 0- to 42-d-old chickens. Glutamate transaminases, rather than glutamate dehydrogenase, may be primarily responsible for initiating glutamate degradation. Rates of amino acid and fatty acid oxidation by cells increased (P < 0.05) with increasing their extracellular concentrations from 0.5 to 5mM. Rates of glutamate and glucose oxidation in enterocytes decreased (P < 0.05) with increasing age, and rates of glutamine, aspartate, propionate, and butyrate oxidation were lower (P < 0.05) in 42-d-old chickens than in 0-d-old chickens. By contrast, oxidation of palmitate at 2mM increased (P < 0.05) by 118% in cells from 42-d-old chickens, compared with 0-d-old chickens. Compared with glutamate, oxidation of glutamine, aspartate, alanine, propionate, butyrate, and palmitate was limited in cells from all age groups of chickens. Collectively, these results indicate that glutamate is the major metabolic fuel in enterocytes of 0- to 42-d-old chickens.

Oxidation of energy substrates in tissues of largemouth bass (Micropterus salmoides).

This study tested the hypothesis that amino acids are oxidized at higher rates than glucose and palmitate for ATP production in tissues of largemouth bass (LMB, a carnivorous fish). Slices (10 to 50 mg) of liver, proximal intestine, kidney, and skeletal muscle isolated from LMB were incubated at 26°C for 2h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5mM D-glucose) containing either D-[U-14C]glucose, 2mM L-alanine plus L-[U-14C]alanine, 2mM L-aspartate plus L-[U-14C]aspartate, 2mM L-glutamate plus L-[U-14C]glutamate, 2mM L-glutamine plus L-[U-14C]glutamine, 2mM L-leucine plus L-[U-14C]leucine, or 2mM palmitate plus [U-14C]palmitate. In parallel experiments, tissues were incubated with a [U-14C]-labeled tracer and a mixture of unlabeled substrates [alanine, aspartate, glutamate, glutamine, leucine, and palmitate (2mM each) plus 5mM glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In separate experiments, O2 consumption by each tissue was measured in the presence of individual or a mixture of substrates. The activities of key metabolic enzymes were also measured. Results indicated that the liver and skeletal muscle had a limited ability to oxidize glucose and palmitate to CO2 for ATP production in the presence of individual or a mixture of substrates due to low activities of carnitine palmitoyltransferase-I, hexokinase and pyruvate dehydrogenase. In the presence of individual substrates, each amino acid was actively oxidized by all the tissues. In the presence of a mixture of substrates, glutamine and glutamate were the major metabolic fuels in the proximal intestine and kidney, as glutamine for the liver and aspartate for skeletal muscle. All the tissues had high activities of glutaminase, glutamate dehydrogenase, and transaminases. At the same extracellular concentration of amino acids (2 mM) in a mixture of energy substrates, glutamine was the major metabolic fuel for the liver of the LMB, glutamine and glutamate for the proximal intestine and kidneys, and aspartate for the skeletal muscle. Glutamine plus glutamate plus aspartate generated 60-70% of ATP in LMB tissues.

220-LB: Glucagon Promotes Hepatic Autophagy by AMPK-Mediated mTORC1 Inhibition

Glucagon plays an important role in hepatic glucose metabolism as well as protein/amino acid metabolism but detailed knowledge about glucagon’s role in hepatic autophagy is limited. We hypothesized that glucagon regulate hepatic protein metabolism partly through activation of AMP-kinase (AMPK) that in turn lead to inhibition of mammalian target of rapamycin complex1 (mTORC1) and activation of Uncoordinated-51 like autophagy activating kinase-1 (ULK1) resulting in increased autophagy. In order to test this hypothesis, we infused chronically catheterized awake mice with [13C5]-glutamine [0.225 mg/(kg-min)] for 120 min after which a second line was connected infusing somatostatin [4 µg/(kg-min)] and insulin [0.1 mU/(kg-min)], with (n=7) or without glucagon [10 ng/(kg-min)] (n=6) for 90 min. This raised plasma glucagon concentration ~18-fold compared to control. Glucagon infusion increased phosphorylation of hepatic: AMPKThr172, RaptorSer792, ULK1Ser555 (P=0.03, P=0.03, and P=0.06, respectively, compared to control), and resulted in hepatic Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II accumulation (P=0.02), a marker of autophagy, assessed by immunoblotting. Glucagon treatment also increased the 13C enrichment (m+3) in liver glucose (P&amp;lt;0.0001), indicating increased conversion of [13C5]-glutamine to glucose as well as increased plasma urea concentrations (P=0.04). Consistent with these in vivo results, parallel studies performed in liver slices obtained from overnight fasted male rats (n=6) incubated in Krebs-Henseleit bicarbonate buffer supplemented with [3-13C]-alanine (1 mM), glucagon (250 µM) treatment increased: glucose production (P=0.01), urea production (P=0.0005), and intracellular levels of leucine (P=0.045). In conclusion, our data suggest that glucagon promotes hepatic proteolysis and ureagenesis by AMPK-mediated suppression of mTOC1 activity, leading to increased hepatic autophagy. Disclosure K.D. Galsgaard: None. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp &amp; Dohme Corp. J.J. Holst: Advisory Panel; Self; AstraZeneca, Merck Sharp &amp; Dohme Corp., Novo Nordisk A/S, Zealand Pharma A/S. Other Relationship; Spouse/Partner; Antag Therapeutics. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, LLC, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Consultant; Self; Novo Nordisk A/S. Consultant; Spouse/Partner; Novo Nordisk A/S. Other Relationship; Self; Gilead Sciences, Inc., iMetabolic Biopharma Corporation. Other Relationship; Spouse/Partner; iMetabolic Biopharma Corporation. Other Relationship; Self; Maze Therapeutics. K. Petersen: Advisory Panel; Spouse/Partner; Merck Sharp &amp; Dohme Corp. Research Support; Self; Merck Sharp &amp; Dohme Corp., National Institute of Diabetes and Digestive and Kidney Diseases. Research Support; Spouse/Partner; National Institute of Diabetes and Digestive and Kidney Diseases. A. Nasiri: Employee; Spouse/Partner; Medtronic. G. Cline: None. X. Zhang: None. J. Lee: None. B.T. Hubbard: None.

253 Glutamate and glutamine are the major metabolic fuels in enterocytes of suckling piglets

Abstract Glutamate and glutamine are known to be important energy substrates in pig enterocytes, and aspartate has also been reported to be extensively catabolized by the rat small intestine. However, little is known about the relative role of amino acids, glucose and fatty acids in ATP production by enterocytes. In the present study, enterocytes isolated from 0-, 7-, 14- and 21-day-old piglets were used to determine the rates of oxidation of amino acids, fatty acids and glucose. Enterocytes were incubated at 37oC for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and one of the following: 0.5–5 mM L-[U-14C]glutamate, 0.5–5 mM L-[U-14C]glutamine, 0.5–5 mM L-[U-14C]aspartate, 0.5–5 mM L-[U-14C]alanine, 0.5–2 mM L-[U-14C]palmitate, D-[U-14C]glucose, 0.5–5 mM [U-14C]propionate, or 0.5–5 mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate in enterocytes from all age groups of piglets increased (P &amp;lt; 0.01) progressively with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, glucose by enterocytes from 0- to 21-day-old pigs were much greater (P &amp;lt; 0.01) than those for the same concentrations of alanine, palmitate, propionate, and butyrate. In cells from all age groups of piglets, rates of oxidation of, and ATP production from 5 mM glutamate or 5 mM glutamine were greater (P &amp;lt; 0.01) than those from 5 mM glucose and aspartate. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes was limited. At each postnatal age, the oxidation of glutamate and glutamine produced more ATP than any other substrates. Our results indicated that glutamate and glutamine are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs. Because of limited uptake of arterial glutamate by enterocytes, dietary glutamate is essential to the integrity and function of the pig small intestine.

251 Oxidation of energy substrates in tissues of Largemouth bass (Micropterus salmoides)

Abstract Because of growing interest in producing Largemouth bass (HMB) as a source of high-quality protein for human consumption worldwide, it is imperative to understand the metabolism of nutrients (including amino acids and carbohydrate) in this aquatic animal. The present study tested the hypothesis that amino acids are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for tissues of LMB fed a 45%-crude protein diet. The liver, intestine, kidney, and skeletal muscle were isolated from juvenile LMB and incubated at 26 °C (the body temperature of LMB) for 2 h in 1 ml of oxygenated Krebs–Henseleit bicarbonate buffer (pH 7.4) containing a mixture of nutrients (2 mM glutamate, 2 mM glutamine, 2 mM aspartate, 2 mM alanine, 2 mM leucine, 5 mM glucose, and 2 mM palmitate). The rate of oxidation of each energy substrate was determined by using [U-14C]-labeled glutamate, glutamine, aspartate, alanine, leucine, glucose, or palmitate and collecting 14CO2 from each tracer. Results indicated that aspartate, glutamate and glutamine were extensively oxidized in all the four tissues and contributed to 67% of total ATP production. Glutamate contributed to more ATP than glutamine in the intestine, whereas similar amounts of ATP were produced from glutamate and glutamine in the liver, kidneys and skeletal muscle. In all the four tissues, rates of oxidation of alanine, leucine, palmitate and glucose were low and each of those nutrients contributed to &amp;lt; 10% of total ATP production. Together, the oxidation of aspartate, glutamate, glutamine, alanine plus leucine provided 82–85% of total ATP for the liver, intestine, kidney, and skeletal muscle. We conclude that amino acids, rather than glucose and long-chain fatty acids, are the primary energy substrates in the major tissues of Largemouth bass.

Ex Vivo Method to Simultaneously Evaluate Glucose Utilization, Uptake, and Production in Rat Liver.

A novel ex vivo method to simultaneously evaluate hepatic glucose utilization, uptake, and production was developed in rats. The right lateral lobe of the liver was perfused with Krebs-Henseleit bicarbonate buffer containing 5 mmol/L uniformly labeled 13C-glucose ([U-13C]-glucose). The whole glucose concentration in the perfusate was measured by colorimetric assay, and the concentrations of [U-12C]-glucose (natural isotope) or [U-13C]-glucose were estimated on the basis of the abundance ratio of [U-12C]-glucose or [U-13C]-glucose, which were measured by GC-MS. The difference in whole glucose and [U-13C]-glucose concentrations between the baseline and effluent perfusate represents hepatic glucose utilization and glucose uptake, respectively. The [U-12C]-glucose concentration in the effluent perfusate corresponds to hepatic glucose production. With this method, we clarified the precise mechanism that underlies the hepatic impairment of diabetic animals and pharmacological effects of anti-diabetic agents. Thus, this method is useful for the pathophysiological and pharmacological research of type 2 diabetes.

Cytoprotective Effects of Mesenchymal Stem Cells During Liver Transplantation from Donors After Cardiac Death in Rats

BackgroundLiver transplantation from donors after cardiac death (DCD) might increase the pool of available organs. Recently, some investigators reported the potential use of mesenchymal stem cells (MSCs) to improve the outcome of liver transplantation from DCD. The aim of this study was to evaluate the cytoprotective effects and safety of MSC transplantation on liver grafts from DCD. MethodsRats were divided into 4 groups (n = 5) as follows: 1. the heart-beating group, in which liver grafts were retrieved from heart-beating donors; 2. the DCD group, in which liver grafts were retrieved from DCD that had experienced apnea-induced agonal conditions; 3. the MSC-1 group, and 4. the MSC-2 group, in which liver grafts were retrieved as with the DCD group, but were infused MSCs (2.0 × 105 or 1.0 × 106, respectively). The retrieved livers were perfused with oxygenated Krebs-Henseleit bicarbonate buffer (37°C) through the portal vein for 2 hours after 6 hours of cold preservation. Perfusate, bile, and liver tissues were then investigated. ResultsBile production in the MSC-2 group was significantly improved compared with that in the DCD group. Based on histologic findings, narrowing of the sinusoidal space in the both MSC groups was improved compared with that in the DCD group. ConclusionsMSCs could protect the function of liver grafts from warm ischemia-reperfusion injury and improve the viability of DCD liver grafts. In addition, we found that the infusion of 1.0 × 106 MSCs does not obstruct the hepatic sinusoids of grafts from DCD.

Donor Treatment With a Hypoxia-Inducible Factor-1 Agonist Prevents Donation After Cardiac Death Liver Graft Injury in a Rat Isolated Perfusion Model.

The protective role of hypoxia-inducible factor-1 (HIF-1) against liver ischemia-reperfusion injury has been well proved. However its role in liver donation and preservation from donation after cardiac death (DCD) is still unknown. The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver condition. Male SD rats (6 animals per group) were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592 (Selleck, 6 mg/kg of body weight) or its vehicle (dimethylsulfoxide). Six hours later, cardiac arrest was induced by bilateral pneumothorax. Rat livers were retrieved 30 min after cardiac arrest, and subsequently cold stored in University of Wisconsin solution for 24 h. They were reperfused for 60 min with Krebs-Henseleit bicarbonate buffer in an isolated perfused liver model, after which the perfusate and liver tissues were investigated. Pretreatment with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate, decreased perfusate liver enzyme release, histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model. The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased PDK1 production. Pretreatment with FG-4592 in DCD donors resulted in activation of the HIF-1 pathway and subsequently protected liver grafts from warm ischemia and cold-stored injury. These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention to prevent injury to DCD allografts.

Amino acids are major energy substrates for tissues of hybrid striped bass and zebrafish.

Fish generally have much higher requirements for dietary protein than mammals, and this long-standing puzzle remains unsolved. The present study was conducted with zebrafish (omnivores) and hybrid striped bass (HSB, carnivores) to test the hypothesis that AAs are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for their tissues. Liver, proximal intestine, kidney, and skeletal muscle isolated from zebrafish and HSB were incubated at 28.5°C (zebrafish) or 26°C (HSB) for 2h in oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4, with 5mM D-glucose) containing 2mM L-[U-14C]glutamine, L-[U-14C]glutamate, L-[U-14C]leucine, or L-[U-14C]palmitate, or a trace amount of D-[U-14C]glucose. In parallel experiments, tissues were incubated with a tracer and a mixture of unlabeled substrates [glutamine, glutamate, leucine, and palmitate (2mM each) plus 5mM D-glucose]. 14CO2 was collected to calculate the rates of substrate oxidation. In the presence of glucose or a mixture of substrates, the rates of oxidation of glutamate and ATP production from this AA by the proximal intestine, liver, and kidney of HSB were much higher than those for glucose and palmitate. This was also true for glutamate in the skeletal muscle and glutamine in the liver of both species, glutamine in the HSB kidney, and leucine in the zebrafish muscle, in the presence of a mixture of substrates. We conclude that glutamate plus glutamine plus leucine contribute to ~80% of ATP production in the liver, proximal intestine, kidney, and skeletal muscle of zebrafish and HSB. Our findings provide the first direct evidence that the major tissues of fish use AAs (mainly glutamate and glutamine) as primary energy sources instead of carbohydrates or lipids.

Resveratrol Prevents Warm Ischemia–Reperfusion Injury in Liver Grafts From Non–Heart-Beating Donor Rats

BackgroundSuccessful liver transplantation from non–heart-beating donors (NHBDs) might enlarge donor source. Some studies have reported that resveratrol (RES), an activator of sirtuins, has cytoprotective effects on ischemia-reperfusion (I/R) injury. The purpose of this study was to investigate the effects of RES on warm I/R injury in rats. MethodsMale Wister rats were divided into 5 groups: (1) the heart-beating (HB) group, whose livers were retrieved from HB donors; (2) the NHB group, whose livers were retrieved under apnea-induced NHB conditions; (3) the ethanol group, retrieved in the same manner as the NHB group with ethanol (10 μL) as a solvent; (4) the RES-1 group, retrieved in the same manner as the NHB group and pretreated with RES (0.4 mg/kg, dissolved in 10 μL ethanol); and (5) the RES-2 group, retrieved in the same manner as the NHB group and pretreated with RES (2 mg/kg, dissolved in 10 μL ethanol). The resected livers were perfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after 6 hours of cold preservation, after which the perfusate and liver tissues were investigated. ResultsThe bile production, portal vein flow volume, tumor necrosis factor-α level, and adenosine triphosphate level in the RES-2 group were significantly improved compared with in the NHB group. Histology revealed numerous well-preserved sinusoidal endothelial cells in the RES-2 group. ConclusionsRES might reduce warm I/R injury and improve the viability of liver grafts from NHBDs. We considered that this method may represent a promising approach for clinical liver transplantation from NHBDs.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings.

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Journal-related Activities and Other Special Activities at the 2012 American Society of Anesthesiologists Annual Meeting

Journal-related Activities and Other Special Activities at the 2012 American Society of Anesthesiologists Annual Meeting

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.

The use of a perfluorochemical emulsion as a vascular perfusate in drug absorption.

In-vitro simultaneous luminal and vascular perfusion using the perfluorochemical emulsion, FC-43 emulsion, as a vascular perfusate, was examined for drug absorption in rat jejunum. The intestinal membrane in this system was found to retain its normal barrier functions for drug transport, as evident from the following: (i) stable absorption clearance of tritiated water and salicylic acid at steady-state, (ii) agreement of this clearance with that by in-situ single-pass luminal perfusion, (iii) active transport of D-glucose and its inhibition by phloridzin and (iv) normal glutamate pyruvate transaminase activity in the intestinal mucosa. FC-43 emulsion gave a more normal absorption site blood flow than the usual vascular perfusate containing erythrocytes and albumin in Krebs-Henseleit bicarbonate buffer solution. Consequently, this emulsion was useful for examining the contribution of blood flow resistance toward drug absorption. The rate-limiting steps of absorption of tritiated water, antipyrine and salicylic acid were examined by perfusion using FC-43 emulsion. The absorption of tritiated water was almost completely blood flow-limited and its absorption clearance may possibly be an approximated absorption site blood flow. The contribution of blood flow resistance to total resistance for antipyrine absorption exceeded that for salicylic acid absorption.

Protein kinase inhibitors attenuate cardiac swelling-induced amino acid release in the rat.

Rat Langendorff heart preparations have been used to study the efflux of cardiac amino acids into coronary artery perfusates during brief (5-min) periods of exposure to hyposmotic stress (70 mM NaCl). Coronary flow rates, heart rates and intra-aortic pressures were recorded. Amino acid levels were measured by high-performance liquid chromatography. Hyposmotic stress caused marked percentage increases in taurine, glutamate and aspartate levels in the coronary perfusate, with smaller increases in phosphoethanolamine, glycine and alanine and non-significant increases in serine and glutamine. Amino acid levels declined during reperfusion with isosmotic Krebs-Henseleit bicarbonate buffer. Inhibition of protein kinase C with chelerythrine chloride (5 microM) depressed the osmotically-induced release of aspartate, glutamate, taurine and glycine. The protein tyrosine kinase inhibitor, genistein, reduced the anisosmotic efflux of aspartate, glutamate, taurine and phosphoethanolamine. Lavendustin A, another inhibitor of tyrosine kinase, depressed the osmotically evoked release of aspartate, glutamate and taurine. These studies demonstrate the involvement of protein kinase C and tyrosine kinases in the efflux of amino acids from the osmotically challenged rat heart and imply that these enzymes are involved in the mechanisms responsible for volume regulation by cardiac cells.