Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease

OBJECTIVES: Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. METHODS: Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37 degrees C followed by reperfusion (n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31-nuclear magnetic resonance spectroscopy. RESULTS: In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 +/- 2 vs 70 +/- 4 mm Hg, p < 0.05), lower diastolic pressures (7 +/- 1 vs 12 +/- 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. CONCLUSIONS: We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion.     

Mirror, mirror on the wall, who''s the thinnest one of all? Effects of self-awareness on consumption of full-fat, reduced-fat, and no-fat products

The aim of this study was to determine whether adenosine receptor blockade before ischemia would enhance the degree of stunning and induce a sustained decrease in glucose uptake after reperfusion. METHODS: Stunning was induced in 14 anesthetized swine by partially occluding the left anterior descending artery (LAD) for 20 min (> 80% flow reduction). Seven animals were pretreated with the nonspecific adenosine receptor blocker 8-phenyltheophylline (8-PT; 5 mg/kg), which decreased reactive hyperemia by an average of 38%. Myocardial glucose uptake was assessed 1 hr following reperfusion with PET and the glucose analog 18F-fluorodeoxyglucose (FDG). RESULTS: Before ischemia, systolic shortening in the LAD region was 15% +/- 6% in the control group and 16% +/- 4% in the 8-PT group and in both groups was reduced to - 1% +/- 2% during ischemia. After reperfusion, systolic shortening was 7% +/- 3% in the control group and 2% +/- 3% in the 8-PT group (p < 0.05). Myocardial oxygen consumption before ischemia was 4.58 +/- 3.03 micromol/min/g in the control group and 4.44 +/- 1.83 micromol/min/g in the 8-PT group (ns) and neither were different after reperfusion. In the postischemic LAD region, myocardial glucose uptake was 0.18 +/- 0.15 micromol/min/g in the control group and was similar to that of the 8-PT group (0.17 +/- 0.08 micromol/min/g; ns). CONCLUSION: The nonspecific adenosine blocker 8-PT enhanced the degree of stunning when given before ischemia but did not induce a sustained effect on myocardial glucose uptake after reperfusion.     

Is a high glycogen content beneficial or detrimental to the ischemic rat heart? A controversy resolved

A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effect of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose. 31P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na+-H+ exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemia pH in these hearts, leading to increased Na+-H+ exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.     

Use of immunoblot assay to define serum antibody patterns associated with Helicobacter pylori infection and with H. pylori-related ulcers

OBJECTIVE: We evaluated the effect of pretreatment with nitric oxide precursor before ischemia on recovery with reperfusion in rat hearts. METHODS: Isolated rat hearts were perfused with Krebs-Henseleit buffer without (C group) or with 3 mmol/L L-arginine (A group) before 30 minutes of ischemia. The left ventricular function, including heart rate, developed pressure, maximal dp/dt, and coronary flow, were measured before pretreatment and after 10 and 30 minutes of reperfusion. Cyclic guanosine monophosphate (by radioimmunoassay), calcium (by absorption spectrophotometry), and inositol 1,4,5-triphosphate synthesized from tritiated myo-inositol (by ion-exchange chromatography preceding counting) were measured at the same times and immediately after ischemia. RESULTS: Recovery of ventricular function was significantly greater in the A group than in the C group. Pretreatment increased postischemic cyclic guanosine monophosphate content compared with the preischemic level (from 1.06 +/- 0.12 to 1.94 +/- 0.09 pmol/mg protein, p < 0.05). No change in cyclic guanosine monophosphate was evident in the C group. In the C group, inositol triphosphate content increased after 10 minutes of reperfusion beyond the preischemic level (from 0.53 +/- 0.023 to 1.15 +/- 0.045 cpm x 10(-3)/gm, p < 0.05) as did calcium at 30 minutes (from 4.12 +/- 0.164 to 6.86 +/- 0.544 mmol/gm dry weight). In the A group, both of these increases were significantly attenuated. CONCLUSION: These data suggest that L-arginine pretreatment may reduce calcium overload by increasing cyclic guanosine monophosphate production, which in turn downregulates inositol triphosphate synthesis during reperfusion.     

Post-ischemic stimulation of 2-deoxyglucose uptake in rat myocardium: role of translocation of Glut-4

Myocardial ischemia elicits translocation of the insulin-sensitive glucose transporter GLUT-4 from intracellular membrane stores to the sarcolemma. Because glucose metabolism is of crucial importance for post-ischemic recovery of the heart, myocardial uptake of [3H]-labeled 2-deoxyglucose and subcellular localization of GLUT-4 were determined during reperfusion in isolated rat hearts perfused with medium containing 0.4 mm palmitate and 8 mm glucose. Hearts were subjected to 20 min of no-flow ischemia, followed by reperfusion for up to 60 min. Subcellular localization of GLUT-4 was determined by cell fractionation followed by immunoblotting. After 15 and 60 min of reperfusion uptake of 2-deoxyglucose was significantly higher (91+/-9 and 96+/-8 nmol/min/g wet weight, respectively) as compared to control values (65+/-1 nmol/min/g wet weight). Ischemia elicited translocation of GLUT-4 to the sarcolemma, which persisted after 15 min of reperfusion. However, after 60 min of reperfusion the subcellular distribution of GLUT-4 was similar to control hearts. In conclusion, reversal of ischemia-induced translocation of GLUT-4 to the sarcolemma is rather slow, possibly facilitating glucose uptake early during reperfusion. However, myocardial uptake and phosphorylation of 2-deoxyglucose remains enhanced late during reperfusion, when pre-ischemic distribution of GLUT-4 is almost completely restored, indicating that additional mechanisms are likely to be involved in post-ischemic stimulation of glucose uptake.     

Effects of intermittent reperfusion on brain pHi, rCBF, and NADH during rabbit focal cerebral ischemia

BACKGROUND AND PURPOSE: The use of intermittent reperfusion versus straight occlusion during neurovascular procedures is controversial. This experiment studied the effects of intermittent reperfusion and single occlusion on intracellular brain pH (pHi), regional cerebral or cortical blood flow, and nicotinamide adenine dinucleotide (NADH) fluorescence during temporary focal ischemia. METHODS: Twenty fasted rabbits under 1.0% halothane anesthesia were divided into four groups: (1) nonischemic controls, (2) 60 minutes of uninterrupted focal ischemia, (3) 2 x 30-minute periods of focal ischemia separated by a 5-minute reperfusion, and (4) 4 x 15-minute periods of focal ischemia separated by three 5-minute reperfusion periods. Focal ischemia was produced by occlusion of both the middle cerebral and ipsilateral anterior cerebral arteries. After the final occlusion, there was a 3-hour reperfusion period in all groups. Regional cerebral and cortical blood flow, brain pHi, and NADH fluorescence were measured with in vivo panoramic fluorescence imaging. RESULTS: During occlusion, regional cerebral and cortical blood flows and NADH fluorescence values were not different among the groups. Brain pHi was significantly lower in the 4 x 15-minute group compared with the 1 x 60-minute group (6.57 +/- 0.02 versus 6.73 +/- 0.06; P < .03) but not significant when compared with the 2 x 30-minute group. During the short reperfusion periods, all parameters returned to normal except for NADH fluorescence levels, which remained elevated. During the postischemic final reperfusion period, there was a mild brain alkalosis of approximately 7.1 in all groups. There were no significant differences in NADH fluorescence among groups during the final reperfusion. Regional cerebral and cortical blood flow returned to near normal values in all groups. CONCLUSIONS: This study demonstrates that intermittent reperfusion during temporary focal ischemia has different effects on the intracytoplasmic and the intramitochondrial compartments: worsening of brain cytoplasmic pHi but no significant differences in the oxidation/reduction level of mitochondrial NADH.     

The relationship between the adenine nucleotide metabolism and the conversion of the xanthine oxidase enzyme system in ischemia-reperfusion of the rat small intestine

The time course of the energy metabolism after reperfusion, the relationship between the conversion of xanthine dehydrogenase to xanthine oxidase (D-to-O conversion) during ischemia, and the changes of the energy metabolism after reperfusion were studied using an ischemia-reperfusion model in the small intestine of the rat. The rat jejunum underwent an occlusion of the superior mesenteric artery and vein for either 30 minutes (group 1, n = 6) or 90 minutes (group 2, n = 6) with collateral interruption, and then it was reperfused. The contents of the adenine nucleotides in the small intestine of the rat were measured by high-performance liquid chromatography (HPLC) before ischemia, and 30, 60, and 90 minutes of ischemia, as well as 30, 60, 120, and 180 minutes after reperfusion. The recovery level of adenosine triphosphate (ATP) in group 1 (6.05 +/- 0.80 mumol/g dry weight) 30 minutes after reperfusion was significantly higher than that in group 2 (2.28 +/- 1.12 mumol/g dry weight) (P < .001). In addition, the ATP content after reperfusion in group 2 did not change from 30 to 180 minutes after reperfusion. The D-to-O conversion during ischemia in group 1 was not significantly greater than that before ischemia; however, that of group 2 did increase significantly during ischemia (P < .005). These results suggest that the tissue damage from ischemia-reperfusion injury after reperfusion under 90 minutes' ischemia is accomplished within the first 30 minutes after reperfusion. Therefore, the ATP level at 30 minutes after reperfusion may be useful for the evaluation of intestinal viability. Thus, the conversion of the xanthine oxidase enzyme system might play an important role in the expression of ischemia-reperfusion injury.     

Aminosulfonic acid buffer preserves myocardium during prolonged ischemia

Prevention of myocardial acidosis during global ischemia in operative cardiopreservation was explored in two series of dogs where acid-base control was the only variable. A specifically designed aminosulfonic acid buffer composition, 3:1 molar equivalents NaMOPS to HEPES, 0.2 mol/L, was compared with NaHCO3 (pH 8). Dissolved in standard cardioplegic solution it was given every 30 minutes by coronary infusion at 20 degrees C during 3 hours of global ischemia. Glass electrode intramyocardial pH, adenosine triphosphate (ATP) level, left ventricular contractility (Dp/Dt) and compliance (-Dp/Dt), and other cardiovascular parameters were measured frequently throughout ischemia and for 75 minutes thereafter. In the buffer group (n = 6) myocardial pH remained above entry levels throughout the study period, adenosine triphosphate level remained normal during ischemia, and Dp/Dt and -Dp/Dt at 75 minutes of reperfusion were above entry levels. In the NaHCO3 group (n = 6) pH declined and remained depressed throughout ischemia, adenosine triphosphate level fell steadily and significantly throughout the experiment, and Dp/Dt and -Dp/Dt never regained entry levels. The difference in each parameter between the two groups was statistically significant (p < 0.05). We conclude that control of myocardial acid-base equilibrium alone during global ischemia will preserve myocardial function and minimize reperfusion injury.     

Role of preischemic glycogen depletion in the improvement of postischemic metabolic and contractile recovery of ischemia-preconditioned rat hearts

BACKGROUND: Ischemic preconditioning (IPC) attenuates acidosis during prolonged ischemia and improves contractile and metabolic parameters during subsequent reperfusion. Glycogen depletion induced by IPC is proposed as a potential mechanism. METHODS AND RESULTS: We studied the influence of manipulations of preischemic glycogen levels (Pre-G, micromol glucose/g wet wt) on contractile and metabolic (via 31P-nuclear magnetic resonance) parameters during 30 minutes of ischemia and recovery in four groups of isovolumic rat hearts: First, control (Con, n=18, mean Pre-G, 21.5+/-0.8); second, after two 5-minute IPC periods (IPC, n=12, Pre-G, 11.3+/-0.7); third, a control group in which Pre-G was depleted by glucose-free, acetate perfusion (Con-LowG, n=9, Pre-G, 7.9+/-1.2); and fourth, an IPC group in which Pre-G was raised by glucose and lactate perfusion such that Pre-G was similar to Con (IPC-HiG, n=11, Pre-G, 20+/-1.4). Manipulation of Pre-G significantly altered the pH fall during 30 minutes of ischemia (Con, 5.76+/-.03, Con-LowG, 6.26+/-.07; IPC-HiG, 5.91+/-.02, IPC, 6.05+/-.09). IPC-HiG hearts had significantly worse metabolic recovery (PCr, 70+/-7 versus 91+/-3% initial; IPC-HiG versus IPC, P<.05) and contractile recovery (end-diastolic pressure, 52+/-5 versus 29+/-5 mm Hg, P<.05) than IPC hearts but better recovery than Con (%PCr, 56+/-6% and end-diastolic pressure, 72+/-6 mm Hg). An ischemic rise in intracellular magnesium occurred and was atttenuated in preconditioned hearts. CONCLUSIONS: Pre-G levels before ischemia influence but are not the sole determinants of the extent of acidosis during prolonged ischemia and of metabolic and contractile recovery during reperfusion in control and preconditioned hearts.     

Increase in incidence of insulin-dependent diabetes mellitus among children in Finland

It has been proposed that NO may function as an endogenous cardioprotectant. We have investigated whether modulation of NO levels (detected in coronary effluent by chemiluminescence) by a blocker of its synthesis, by supplementation of its precursor, and by administration of an NO donor can influence reperfusion arrhythmias in the isolated rat heart. Rat hearts were perfused with modified Krebs' solution and subjected to 5, 35, or 60 minutes of left regional ischemia followed by 10 minutes of reperfusion. NG-Nitro-L-arginine methyl ester (L-NAME), which blocks NO synthase, increased the incidence of reperfusion-induced ventricular fibrillation (VF) from 5% in the control condition to 35% after 60 minutes of ischemia (n = 20, P < .05). The profibrillatory effect of L-NAME was prevented in hearts coperfused with 1 or 10 mmol/L L-arginine (an NO precursor) but persisted in hearts coperfused with D-arginine (1 mmol/L). L-NAME did not increase VF susceptibility in hearts reperfused after 5 or 35 minutes of ischemia. L-NAME caused sinus bradycardia (264 +/- 10 versus 309 +/- 5 bpm in control groups, P < .05) and reduced coronary flow before ischemia (6.2 +/- 0.6 versus 9.2 +/- 0.6 mL.min-1.g-1 tissue in controls, P < .05). L-NAME reduced coronary effluent NO levels after 60 minutes of ischemia; during the first minute of reperfusion, values were reduced from 1457 +/- 422 to 812 +/- 228 pmol.min-1.g-1 (P < .05). This effect was prevented by coperfusion with L-arginine (10,344 +/- 1730 pmol.min-1.g-1, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

PET 2-fluoro-2-deoxyglucose uptake in rat prostate adenocarcinoma during chemotherapy with gemcitabine

This study was performed to investigate the effect of the new chemotherapeutic agent gemcitabine on glucose transport and metabolism in prostate carcinoma in vitro and in vivo. METHODS: After transplantation of rat prostate adenocarcinoma cells, dynamic PET measurements with fluorine-18-labeled 2-fluoro-2-deoxy-D-glucose (18FDG) were performed in 15 animals before and 1 day after therapy with 90 mg/kg of body weight (n = 8) and 180 mg/kg of body weight (n = 7) gemcitabine. In the second examination, the animals received a simultaneous injection of 18FDG and [3H]thymidine. Quantitative evaluation of the PET data was done using the standardized uptake value (SUV) as well as a three-compartment pharmacokinetic model. Furthermore, the incorporation of [3H]thymidine into the DNA was determined. In vitro measurements of the FDG, 3-O-methylglucose and thymidine uptake were performed immediately and 4 hr after a 24-hr incubation period with different doses of gemcitabine. RESULTS: FDG-SUV and the metabolic rate of FD 3 utilization did not change significantly after therapy. However, the values for the transport rate constants K1 and K2 increased significantly. The incorporation of thymidine into the DNA of treated tumors showed an 80% decline as compared with a control group. In the cell culture experiments, a dose-dependent increase of FDG (up to 178%) and 3-O-methylglucose uptake (up to 305%) was demonstrated. The thymidine uptake showed a 96% decline in the nucleic acid fraction and an increase of up to 337% in the cytoplasmic fraction. CONCLUSION: The more global measures of FDG metabolism as SUV and metabolic rate of FDG utilization were unchanged after therapy, while DNA synthesis and cell viability declined. However, in vitro and in vivo evidence of an enhancement of glucose transport is presented, indicating that quantification by modelling may be superior for the evaluation of metabolic effects during chemotherapy.     

Incremental iron overload during reperfusion progressively augments oxidative injury

OBJECTIVE: To determine if a relationship exists between the extent of iron-catalyzed injury and the degree of tissue iron overload during reperfusion. METHODS: To selectively increase tissue iron only during early reperfusion, isolated, buffer perfused rabbit hearts were exposed to 20 microM Fe(2+)-100 microM ADP during the last 3 minutes of ischemia and the initial 4 minutes of reperfusion. Control groups were exposed to ADP and iron-ADP regimens that did not increase intracellular iron. All the hearts received 30 minutes of normothermic global ischemia and 30 minutes of reperfusion. Heart function was monitored continuously throughout each experiment. Tissue iron and biochemical markers were analyzed at the end of experiments. RESULTS: Hemodynamic recovery was decreased and tissue lipid peroxide levels were increased in the 20 microM Fe(2+)-100 microM ADP group compared to controls. The recoveries of developed pressure and positive/negative dP/dT at 30 minutes of reperfusion were negatively correlated with tissue iron levels, while cytosol and membrane lipid peroxide levels correlated positively with the iron levels during reperfusion. CONCLUSION: The extent of oxidative injury during reperfusion was directly related to the tissue iron burden present during reperfusion. Increased lipid peroxidation was the principal chemical marker of iron-catalyzed injury.     

Enhancement of postischemic myocardial stunning by calcium overload in hearts of diabetic rats

The effects of Ca2+ concentration on postischemic myocardial stunning were studied in isolated working hearts of rats with streptozotocin-induced diabetes and of age-matched control rats. During reperfusion after 10 min of ischemia, hearts from control rats showed complete recovery of cardiac function of Ca2+ concentrations of 1.25, 1.88, and 2.50 mmol/L, while the recovery of diabetic rats was decreased only at a Ca2+ concentration of 2.50 mmol/L. Although myocardial Na+ and Ca2+ concentrations were comparable between control and diabetic rats, only diabetic rats showed increases in myocardial concentration of Na+ during ischemia and Ca2+ during reperfusion at a Ca2+ concentration of 2.50 mmol/L. Results suggest that diabetic rat hearts are vulnerable to postischemic stunning via an overload of calcium.     

Pitfalls in oncologic diagnosis with FDG PET imaging: physiologic and benign variants

A rapidly emerging clinical application of positron emission tomography (PET) is the detection and staging of cancer with the glucose analogue tracer 2-[fluorine-18]fluoro-2-deoxy-D-glucose (FDG). Proper interpretation of FDG PET images requires knowledge of the normal physiologic distribution of the tracer, frequently encountered physiologic variants, and benign pathologic causes of FDG uptake that can be confused with a malignant neoplasm. One hour after intravenous administration, high FDG activity is present in the brain, the myocardium, and--due to the excretory route--the urinary tract. Elsewhere, tracer activity is typically low, a fact that allows sensitive demonstration of tracer accumulation in many malignant neoplasms. Interpretive pitfalls commonly encountered on FDG PET images of the body obtained 1 hour after tracer administration can be mistaken for cancer. Such pitfalls include variable physiologic FDG uptake in the digestive tract, thyroid gland, skeletal muscle, myocardium, bone marrow, and genitourinary tract and benign pathologic FDG uptake in healing bone, lymph nodes, joints, sites of infection, and cases of regional response to infection and aseptic inflammatory response. In many instances, these physiologic variants and benign pathologic causes of FDG uptake can be specifically recognized and properly categorized; in other instances, such as the lymph node response to inflammation or infection, focal FDG uptake is nonspecific.     

Contribution of glycogen and exogenous glucose to glucose metabolism during ischemia in the hypertrophied rat heart

Although hypertrophied hearts have increased rates of glycolysis under aerobic conditions, it is controversial as to whether glucose metabolism during ischemia is altered in the hypertrophied heart. Because endogenous glycogen stores are a key source of glucose during ischemia, we developed a protocol to label the glycogen pool in hearts with either [3H]glucose or [14C]glucose, allowing for direct measurement of both glycogen and exogenous glucose metabolism during ischemia. Cardiac hypertrophy was produced in rats by banding the abdominal aorta for an 8-week period. Isolated hearts from aortic-banded and sham-operated rats were initially perfused under substrate-free conditions to decrease glycogen content to 40% of the initial pool size. Resynthesis and radiolabeling of the glycogen pool with [3H]glucose or [14C]glucose were accomplished in working hearts by perfusion for a 60-minute period with 11 mmol/L [3H]glucose or [14C]glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate, and 100 mumol/mL insulin. Although glycolytic rates during the aerobic perfusion were significantly greater in hypertrophied hearts compared with control hearts, glycolytic rates from exogenous glucose were not different during low-flow ischemia. The contribution of glucose from glycogen was also not different in hypertrophied hearts compared with control hearts during ischemia (1314 +/- 665 versus 776 +/- 310 nmol.min-1.g dry wt-1, respectively). Glucose oxidation rates decreased during ischemia but were not different between the two groups. However, in both hypertrophied and control hearts, the ratio of glucose oxidation to glycolysis was greater for glucose originating from glycogen than from exogenous glucose. Our data demonstrate that glycogen is a significant source of glucose during low-flow ischemia, but the data do not differ between hypertrophied and control hearts.     

Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion

Involvement of ceramide signaling in the initiation of apoptosis induction in myocardial cells by in vitro and in vivo ischemia and reperfusion was analyzed. Synthetic cell permeable C2-ceramide induced apoptotic death of rat neonatal cardiomyocytes in vitro. In vitro ischemia (oxygen/serum/glucose deprivation) led to a progressive accumulation of ceramide in cardiomyocytes. After 16 hours of simulated in vitro reperfusion (readdition of oxygen, serum and glucose), the level of ceramide in surviving cells was found to have returned to baseline, whereas, levels in nonadherent dead cells remained high. In the rat heart left coronary artery occlusion model, ischemia with the subsequent reperfusion, but not ischemia alone, induced apoptosis in myocardial cells as demonstrated by DNA electrophoresis and measurement of soluble chromatin degradation products. The content of ceramide in ischemic area was elevated to 155% baseline levels at 30 minutes, and to 330% after 210 minutes of ischemia. Ischemia (30 minutes) followed by reperfusion (180 minutes) increased the ceramide level to 250% in the ischemic area. The combination of results obtained in both in vitro and animal models demonstrate for the first time that ceramide signaling can be involved in ischemia/reperfusion death of myocardial cells.     

Nitric oxide scavenging by hemoglobin or nitric oxide synthase inhibition by N-nitro-L-arginine induces cortical spreading ischemia when K+ is increased in the subarachnoid space

We investigated the combined effect of increased brain topical K+ concentration and reduction of the nitric oxide (NO.) level caused by nitric oxide scavenging or nitric oxide synthase (NOS) inhibition on regional cerebral blood flow and subarachnoid direct current (DC) potential. Using thiopental-anesthetized male Wistar rats with a closed cranial window preparation, brain topical superfusion of a combination of the NO. scavenger hemoglobin (Hb; 2 mmol/L) and increased K+ concentration in the artificial cerebrospinal fluid ([K+]ACSF) at 35 mmol/L led to sudden spontaneous transient ischemic events with a decrease of CBF to 14+/-7% (n=4) compared with the baseline (100%). The ischemic events lasted for 53+/-17 minutes and were associated with a negative subarachnoid DC shift of -7.3+/-0.6 mV of 49+/-12 minutes' duration. The combination of the NOS inhibitor N-nitro-L-arginine (L-NA, 1 mmol/L) with [K+]ACSF at 35 mmol/L caused similar spontaneous transient ischemic events in 13 rats. When cortical spreading depression was induced by KCl at a 5-mm distance, a typical cortical spreading hyperemia (CSH) and negative DC shift were measured at the closed cranial window during brain topical superfusion with either physiologic artificial CSF (n=5), or artificial CSF containing increased [K+]ACSF at 20 mmol/L (n=4), [K+]ACSF at 3 mmol/L combined with L-NA (n=10), [K+]ACSF at 10 mmol/L combined with L-NA (five of six animals) or [K+]ACSF at 3 mmol/L combined with Hb (three of four animals). Cortical spreading depression induced longlasting transient ischemia instead of CSH, when brain was superfused with either [K+]ACSF at 20 mmol/L combined with Hb (CBF decrease to 20+/-20% duration 25+/-21 minutes, n=4), or [K+]ACSF at 20 mmol/L combined with L-NA (n=19). Transient ischemia induced by NOS inhibition and [K],ACSF at 20 mmol/L propagated at a speed of 3.4+/-0.6 mm/min, indicating cortical spreading ischemia (CSI). Although CSH did not change oxygen free radical production, as measured on-line by in vivo lucigenin-enhanced chemiluminescence, CSI resulted in the typical radical production pattern of ischemia and reperfusion suggestive of brain damage (n=4). Nimodipine (2 microg/kg body weight/min intravenously) transformed CSI back to CSH (n=4). Vehicle had no effect on CSI (n=4). Our data suggest that the combination of decreased NO. levels and increased subarachnoid K+ levels induces spreading depression with acute ischemic CBF response. Thus, a disturbed coupling of metabolism and CBF can cause ischemia. We speculate that CSI may be related to delayed ischemic deficits after subarachnoid hemorrhage, a clinical condition in which the release of Hb and K+ from erythrocytes creates a microenvironment similar to the one investigated here.     

Synergistic upregulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappa B

BACKGROUND: OG-VI is a solution composed of 30 mmol/l inosine, 30 mmol/l sodium 5'-guanylate, 30 mmol/l cytidine, 22.5 mmol/l uridine and 7.5 mmol/l thymidine; it limits myocardial stunning in dogs. We examined whether adenosine A1 receptors were involved in the mechanism of action of OG-VI. METHODS: Dogs anesthetized with pentobarbital were subjected to 20 min of left anterior descending coronary artery ligation followed by 30 min of reperfusion. Saline, OG-VI in several doses, adenosine or inosine was infused at 0.1 ml/kg/min, starting 30 min before the ischemia. In some experiments, 1 or 3 mg/kg 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, was injected intravenously 15 min before the start of the OG-VI infusion. The percentage myocardial segment shortening (%SS) was measured by sonomicrometry. The tissue concentration of ATP was measured in the 30-min-reperfused hearts. RESULTS: In the saline group, %SS that had been decreased by ischemia returned toward pre-ischemic values after reperfusion, although the metabolic recovery was incomplete, with a low concentration of ATP. The %SS was almost completely restored by 12 and 1.2 mumol/kg/min OG-VI, but 0.4 mumol/kg/min was less effective. Administration of adenosine or inosine did not modify the changes in %SS during ischemia/reperfusion. Pretreatment with DPCPX worsened the recovery of %SS during reperfusion after ischemia in both the saline and the OG-VI groups. Infusion of DPCPX (3 mg/kg) with saline caused the animals to die shortly after the onset of ischemia. However, the enhancement of %SS recovery during OG-VI reperfusion was observed in the presence of DPCPX. CONCLUSION: OG-VI improves the recovery of %SS during reperfusion after brief ischemia in a dose-dependent manner. This effect is not brought about by stimulation of adenosine A1 receptors.     

Fasting does not impair insulin-stimulated glucose uptake but alters intracellular glucose metabolism in conscious rats

Effects of 24-h and 48-h fasting on maximal insulin-stimulated whole-body and muscle glucose uptake, glycogen synthesis, and glycolysis were studied in conscious rats by combining the glucose clamp technique with tracer methods. Fasting decreased body weight and basal plasma glucose, plasma insulin, hepatic glucose output, and glucose clearance (P < 0.05 for all). However, maximal insulin-stimulated whole-body glucose uptake, normalized to body weight, was almost identical in fed, 24-h fasted, and 48-h fasted rats (191 +/- 8, 185 +/- 14, and 182 +/- 5 mumol.kg-1.min-1, respectively; P > 0.7). Similarly, rates of insulin-stimulated glucose uptake by four different skeletal muscles, estimated by the 2-deoxyglucose injection technique, were not different among the three groups. In contrast to glucose uptake, insulin-stimulated whole-body glycolysis was decreased significantly after fasting (36% after 48 h fasting; P < 0.05), whereas insulin-stimulated whole-body glycogen synthesis was increased (44% after 48 h fasting; P < 0.05). In fed rats, glycolysis was the major pathway for glucose metabolism during hyperinsulinemia, accounting for 60 +/- 5% of glucose uptake. This fraction was decreased significantly by fasting (P < 0.01), so that after a 48-h fast, glycolysis accounted for only 40 +/- 3% of insulin-stimulated glucose uptake and glycogen synthesis became predominant pathway, accounting for 60 +/- 3% of whole-body glucose utilization. Whole-body patterns of glucose metabolism during hyperinsulinemia were paralleled by glucose metabolism in individual muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the 21-aminosteroid U-74389G on ischemia and reperfusion injury of the ascending colon in horses

Sixteen horses were allotted at random to 3 groups: vehicle only; low dosage (vehicle and 3 mg of U-74389G/kg of body weight); high dosage (vehicle and 10 mg of U-74389G/kg). These solutions were given prior to reperfusion. The ascending colon was subjected to 2 hours of ischemia followed by 2 hours of reperfusion. Before, during, and after ischemia, full-thickness colonic tissue biopsy specimens were obtained for measurement of malondealdehyde (MDA) concentration and myeloperoxidase activity and for morphologic evaluation. Although increases were not significant, MDA concentration and myeloperoxidase activity increased during ischemia and reperfusion. Administration of U-74389G did not have significant effects on MDA concentration and myeloperoxidase activity. However, the lower dosage tended (P = 0.08) to reduce myeloperoxidase activity at 30 and 60 minutes of reperfusion. In horses of the vehicle-only group, ischemia induced a decrease in mucosal surface area that was continued into the reperfusion period (P < or = 0.05). Administration of U-74389G at both dosages (3 and 10 mg/kg) prevented the reperfusion-induced reduction in mucosal surface area, which was significant at 60 minutes (high dosage; P = 0.05) and 90 minutes (low and high dosages; P = 0.02). After initial reduction in horses of all groups, mucosal volume increased for the initial 60 minutes of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)