Min Of Perfusion Research Articles

Energy Metabolism of Rat Skeletal Muscle Modulated by the Rate of Perfusion Flow

In order to advance our understanding of the phenomenon of flow-induced increases in the metabolism of the relaxed muscle, the metabolic rate of the isolated rat gracilis muscle was investigated at 28 degrees C in vitro. The muscle was perfused with cell-free Krebs-Henseleit bicarbonate buffer containing 5% bovine serum albumin and 5 mM glucose, saturated with a gas mixture of 95% O2 and 5% CO2 and simultaneously superfused with a medium saturated with with a low O2 gas mixture (1% O2, 5% CO2 and 94% N2). Two different perfusion flow rates (0.054 and 0.100 ml min-1) have been used. Their influence on oxygen consumption and lactate production has been measured. After a 100 min perfusion period, the muscle was freeze-clamped and analysed for ATP, phosphocreatine, creatine, lactate, pyruvate, inorganic phosphate and glycogen content. The energy state of the cell and the proportions of glycolytic and mitochondrial fluxes of ATP synthesis were evaluated. During perfusion at the low flow rate of 0.054 ml min-1, the oxygen uptake was 45 +/- 9 nmol min-1 (g wet wt)-1, accompanied by a dominance of anaerobic glycolytic synthesis of ATP over mitochondrial ATP synthesis, even though the total delivery of oxygen to muscle was three times higher than oxygen consumption. Increasing the perfusion flow rate to 0.100 ml min-1 increased the oxygen uptake to 120 +/- 6 nmol min-1 (g wet wt)-1, thus leading to a prevalence of mitochondrial ATP synthesis over glycolytic ATP synthesis. The inner stores of glycogen served as the main substrate of energy metabolism and the role of exogenous substrates in the flow-stimulated increase of oxygen uptake was negligible. The increase in perfusion rate also enhanced the energy state of the muscle fibres, which was expressed either as the creatine charge or as the value of the change of Gibbs free energy of ATP hydrolysis. Data indicate that the change of perfusion flow rate per se, apart from oxygen and exogenous substrate supply, elicits changes in the regulation of energy metabolism within non-contracting skeletal muscle under open microcirculation.

Role of Ischemic Preconditioning on Ischemia-Reperfusion Injury of the Lung

Ischemia-reperfusion injury of the lung frequently occurs after cardiopulmonary bypass, after pulmonary thromboembolectomy, and especially during lung transplantation. The protective effects of preconditioning on the heart, liver, bones, and various other organs have been previously evaluated. In this comparative study, we used isolated guinea pig lungs to show the effects of preconditioning on lung ischemia. The lungs (n = 10 in each group) were mounted on a modified Langendorff perfusion apparatus and perfused by Krebs-Henseleit solution for 30 min. We applied an ischemic preconditioning (5 min ischemia + 5 min perfusion, two times) in the experimental group. After 3 h of normothermic ischemia, the lungs were reperfused for 30 min. Pulmonary artery pressures and malondialdehyde (MDA) and glutathione (GSH) levels of the tissue and the perfusate were measured before and after the ischemic period and also at the end of reperfusion. Electron microscopic evaluation was done on randomly selected lungs of three animals in each group at the end of the experiment. Both MDA and GSH levels of tissue and perfusate decreased in the experimental group after reperfusion, although the reduction in GSH levels did not reach statistical significance. The increase in pulmonary artery pressure was lower in the preconditioning group after reperfusion. Our data showed that ischemic preconditioning of the lung may have a protective effect in ischemic-reperfusion injury.

Preconditioning prevents the negative inotropic action of phenylephrine in rat isolated stunned papillary muscle

The aim of the present study was to establish a model of ischemic preconditioning of rat isolated papillary muscle and to investigate its effect on the simulated ischemia-induced disturbances in contractility and responsiveness to isoproterenol and phenylephrine. Experiments were performed in rat left ventricle papillary muscle. The following parameters were measured: force of contraction (Fc), velocity of contraction (+dF/dt), velocity of relaxation (−dF/dt), time to peak contraction (ttp), and relaxation time at the level of 10% of total amplitude (tt 10). After 60 min of simulated ischemia induced by the perfusion of isolated tissues with no-substrate solution aerated by 95% N 2/5% CO 2, all of the measured parameters were markedly decreased. There was not complete recovery of Fc, +dF/dt and −dF/dt after 60 min of reperfusion. Positive inotropic action of isoproterenol does not differ before and after simulated ischemia. In contrast, phenylephrine induces a positive inotropic action in non-treated, but a significant negative one in simulated ischaemia/reperfusion treated preparations. The latter effect of phenylephrine was reversed by chloroethylclonidine (CEC), a selective blocker of α 1b-adrenoceptor, but not by WB-4101, a selective blocker of α 1a-adrenoceptor. Ischemic preconditioning of rat isolated cardiac tissue induced by the 5 min perfusion with no-substrate solution, aerated by 95% N 2/5% CO 2, in the presence of fast electrical pacing (BCL shortened from 2000 ms to 700 ms) and 10 min reperfusion, significantly improves a recovery of the contractility and prevents phenylephrine negative inotropic action.

Separation of carrier mediated and vesicular release of GABA from rat brain slices

In this study the temperature dependence of [ 3H]GABA release from brain slices evoked by electrical field stimulation and the Na +/K +ATPase inhibitor ouabain was investigated. [ 3H]GABA has been taken up and released from hippocampal slices at rest and in response to electrical field stimulation (20 V, 10 Hz, 3 msec, 180 pulses) at 37°C. When the bath temperature was cooled to 7°C, during the sample collection period, the tissue uptake and the resting outflow of [ 3H]GABA were not significantly changed. In contrast, the stimulation-induced tritium outflow increased both in absolute amount (Bq/g) and in fractional release and the S 2/S 1 ratio was also higher at 7°C. Perfusion of the slices with tetrodotoxin (TTX, 1 μM) inhibited stimulation-induced [ 3H]GABA efflux indicating that exocytotic release of vesicular origin is maintained under these conditions. 15 min perfusion with ouabain (10–20 μM) induced massive tritium release both in hippocampal and in striatal slices. However, the fraction of [ 3H]GABA outflow evoked by ouabain was much higher in the hippocampus than in the striatum. Sequential lowering the bath temperature from 37°C to 17°C completely abolished ouabain-induced [ 3H]GABA release in both brain regions, indicating that it is a temperature-dependent, carrier-mediated process. When the same experiments were repeated under Ca 2+ free conditions, cooling the bath temperature to 17°C, although substantially decreased the release but failed to completely abolish the tritium outflow evoked by ouabain, a significant part was maintained. Our results show that vesicular (field stimulation-evoked) and carrier-mediated (ouabain-induced) release of GABA is differentially affected by low temperature: while vesicular release is unaffected, carrier-mediated release is abolished at low bath temperature. Therefore, lowering the temperature offers a reliable tool to separate these two kinds of release and makes possible to study exclusively the pure neuronal release of GABA of vesicular origin.

Mild therapeutic hypothermia for postischemic vasoconstriction in the perfused rat liver.

Mild hypothermia, a promising therapy being evaluated for various clinical situations, may suppress the formation of reactive oxygen species during reperfusion and may ameliorate microcirculatory perfusion failure (the "no-reflow phenomenon"). Isolated rat livers underwent 30 min of perfusion, 2.5 h of ischemia, and 3 h of reperfusion. The temperature was maintained at 34 degrees C (mild hypothermia, n = 5) or 38 degrees C (normothermia, n = 6) for all three periods by perfusion of a modified Krebs Henseleit solution, air surface cooling, or both. A third group of livers was normothermic before and during ischemia and mildly hypothermic during reperfusion (reperfusion hypothermia, n = 6). Control livers had 3 h of perfusion at normothermia. Chemiluminescence (a measure of the generation of reactive oxygen species) and hepatic vascular resistance were monitored simultaneously to evaluate the effect of temperature on the formation of reactive oxygen species and the development of no reflow. Also measured were thiobarbituric acid reactive species and lactate dehydrogenase, as indicators of oxidative stress and cell injury. Mild hypothermia decreased formation of reactive oxygen species and postischemic increases in vascular resistance. Reperfusion hypothermia also decreased postischemic increases in vascular resistance, but not as effectively as did mild hypothermia. Levels of thiobarbituric acid reactive species were lower for reperfusion hypothermia than for mild hypothermia at only 0 and 30 min of reperfusion. Lactate dehydrogenase was significant only at 0 min of reperfusion for the normothermic group. Oxygen consumption did not change. The prevention of hepatic vascular injury by suppression of oxidative stress may be an important protective mechanism of mild hypothermia.

ATP-sensitive potassium channel activation mimics the protective effect of ischaemic preconditioning in the rat isolated working heart after prolonged hypothermic storage.

1. Ischaemic preconditioning (IP) can significantly reduce the extent of infarct size, contractile dysfunction and necrosis in hearts from a number of animal species. Activation of ATP-sensitive potassium channels has been implicated in this process. The aims of the present study were to determine the extent to which IP preserves haemodynamic function in the rat isolated working heart model after prolonged hypothermic storage and to examine the involvement of activation of potassium channels in this process. 2. Hearts from Wistar rats were perfused on a Langendorff apparatus. After stabilization in working mode, baseline measurements of heart rate, aortic flow, coronary flow and cardiac output were performed. Hearts were randomized to one of six treatment groups: (i) untreated control; (ii) IP; (iii) 3 min perfusion with 200 mumol/L pinacidil; (iv) pinacidil vehicle; (v) 3 min perfusion with 10 mumol/L glibenclamide before IP; and (vi) 3 min perfusion with glibenclamide then pinacidil. Hearts were stored in an extracellular-based preservation solution for 6 or 12 h at 2-3 degrees C, remounted on the perfusion apparatus, stabilized as before and then haemodynamic measurements were repeated, after which time heart water contents were determined. 3. Recovery of haemodynamic function was markedly enhanced in the IP and pinacidil-treated groups compared with untreated and vehicle controls. These beneficial effects were completely blocked by glibenclamide. These results suggest that strategies for activating potassium channels in donor hearts may protect organs during hypothermic storage prior to transplantation.

Propofol Decreases Cell Volume and Intracellular Calcium, and Rearranges Actin Filaments in Astroglial Cells

Background : Regulation of cell volume is one of the major physiologic functions in living cells. Intracellular calcium ion and cytoskeletal component of cell membrane play pivotal roles in cell volume regulation. The significance of astroglial cell swelling is in its relation to delayed and permanent neuronal death. The aim of the current study is therefore to elucidate the effect of propofol on the cell volume, [Ca2+]i and actin filaments in astroglial cells. Methods : Astroglial cell line U1242MG astrocytoma cells were used in vitro. To investigate the alterations of cell volume and [Ca2+]i by propofol, flow cytometry system was used. Actin filaments were determined by tetramethylrhodamine isothiocyanate (TRITC)-phalloidin staining. Results : Treatment with propofol 10μg/ml for 15 min or 2 h perfusion reduced significantly both cell volume and [Ca2+]i. Cell volume was reduced 3∼4% in either duration of perfusion with propofol. Decreases in [Ca2+]i level in the presence of propofol was duration-dependent. Fifteen min perfusion with propofol caused 5% decrease in [Ca2+]i, but 2 h perfusion decreased [Ca2+]i further to 10%. The changes of actin filaments after propofol treatment for 30 min were apparently observed under fluorescent microscope. Conclusions : Changes of cell volume, [Ca2+]i and actin filaments are occurred by propofol in astroglial cells. Rearrangement of actin filaments may be associated with volume changes by propofol in astroglial cells. (Korean J Anesthesiol 1999; 36: 863∼868)

Evaluation of serum uric acid changes in different forms of hepatic vascular inflow occlusion in human liver surgeries

Uric acid values in serum have been analyzed as one of the markers to predict cellular damage due to ischemia reperfusion injury in the field of organ transplantation. The present study was conducted to confirm that uric acid values in serum could be an efficient marker of ischemic injury of liver parenchyma following hepatic vascular occlusion in human liver surgery. The changes in serum uric acid values were analyzed at fixed intervals during different liver surgeries. Significant increases in serum uric acid values were observed in patients who received the Pringle's maneuver in which hepatic vascular inflow was manipulated with a repetition of 15 min occlusion and 5 min perfusion, whereas almost no changes in uric acid values were found in both groups of patients who received the hemilobal occlusion of the Glisson's triad in which the right or left vessels were manipulated with a repetition of 30 min occlusion and 5 min perfusion and the “control method” in which the hepatic vessels of the lesion side were previously cut before liver resection. Uric acid values in serum increased in patients of Pringle's maneuver compared to those of the hemilobal occlusion of the Glisson's triad and the control method though these procedures were used in larger hepatectomies rather than Pringle's maneuver. The results indicated that serum uric acid values do not always reflect the severity of ischemia of the liver parenchyma but reflect intestinal congestion because marked intestinal congestion was observed in patients of Pringle's maneuver but not in patients of the hemilobal occlusion of the Glisson's triad and the control method. The evaluation of the severity of the ischemic injury of the liver should be done with caution when uric acid is used as a marker in human liver surgery.

The chronic effect of vascular endothelial growth factor on individually perfused frog mesenteric microvessels.

1. Hydraulic conductivity (Lp) of the wall of perfused microvessels has previously been shown to be chronically increased 24 h after a 10 min perfusion with vascular endothelial growth factor (VEGF). In order to investigate this further, Lp and the effective oncotic pressure difference (f3DeltaPi) acting across the vessel walls was measured before exposure to VEGF and 24 h later after the mesentery had been replaced in the abdominal cavity. 2. Acute 10 min perfusion with VEGF did not chronically change f3DeltaPi despite chronically increasing Lp 6.8 +/- 1.2-fold. This suggests that pathways formed 24 h after perfusion with VEGF which increase hydraulic conductivity of the capillary walls have the same reflection coefficient as those present before VEGF. 3. Acute 10 min perfusion with VEGF significantly increased the diameter of vessels after 24 h by 48 +/- 13%. To determine whether this was due to changes in the compliance of the vessel wall, the distensibility of microvessels was measured before and 24 h after perfusion with VEGF. The distensibility was increased 45 +/- 15% by VEGF but this was not great enough to account for the increase in diameter. 4. The chronic increase in Lp could be attenuated by inhibition of nitric oxide synthase with L-NAME. In addition, the chronic increase in permeability was correlated with the acute response to VEGF (r = 0.71, P < 0.01) suggesting that the acute and chronic changes may be related. 5. These results show that VEGF chronically increases Lp without affecting the oncotic reflection coefficient. This may be due to reduced pore path length, or increased small pore numbers, which are properties of fenestrated capillaries. They also show that VEGF increases microvascular distensibility and diameter.

Consequences of the inhibition of the sarcoplasmic reticulum calcium ATPase on cardiac function and coronary flow in rabbit isolated perfused heart: role of calcium and nitric oxide.

The effects of thapsigargin (Tg) and cyclopiazonic acid (CPA), two selective blockers of the sarcoplasmic reticulum Ca2+-ATPase were studied in rabbit isolated perfused hearts. Tg and CPA were infused into the hearts for 60 min followed by 60 min of wash-out. Left-ventricular developed pressure (LVDP), left-ventricular end diastolic pressure (LVEDP) and the relaxation time constant,tau, were assessed with a fluid-filled LV intraventricular balloon. Both Tg and CPA induced a concentration-dependent reduction in LVDP and dose-dependently altered diastolic function parameters LVEDP and tau. After 60 min of perfusion, both Tg (0.01, 0.1 and 1.0 microM) and CPA (0.1, 1.0 and 10.0 microM) decreased LVDP from 98+/-1 mmHg in control to 83+/-4; 81+/-5 and 55+/-7 mmHg and to 91+/-3, 80+/-5 and 65+/-4 mmHg, respectively. LVEDP increased from 5+/-1 mmHg in controls to 6+/-0.2, 10+/-1 and 29+/-4 mmHg and to 7+/-0.2, 9+/-1 and 11+/- mmHg; while tau elevated from 28+/-1 ms to 32+/-1, 38+/-4 and 99+/-18 ms and to 34+/-1, 38+/-2 and 48+/-4 ms in Tg (0.01, 0.1 and 1.0 microM) and CPA (0.1, 1.0 and 10.0 microM), respectively. The effects of Tg were more pronounced than those of CPA and were modulated by extracellular Ca2+. With 1 mm Ca2+, both agents Tg (0.03 microM) and CPA (0.1 microM) produced a vasodilatation (81.7+/-2. 6 and 89.1+/-3.1% of pre-drug values, respectively). Pretreatment of the hearts with L-NMMA, a specific inhibitor of nitric oxide production, completely abolished the relaxing effect of Tg and CPA as well as the production of cGMP. These data show that the two SR-Ca2+ ATPase inhibitors, Tg and CPA, are negatively inotropic and lusitropic agents and that both Tg and CPA induce a vasodilatation mediated by a NO-dependent mechanism.

Upper T Mini-Sternotomy for Aortic Valve Operations

New minimally invasive approaches for cardiac surgical procedures are constantly being developed in the hope of decreasing patient morbidity and enhancing the postoperative recovery. This report reviews the use of an upper T mini-sternotomy approach to aortic valve surgery. Nine consecutive nonselected patients (5 men, 4 women, mean age, 66 years) underwent isolated aortic valve replacement with the use of this approach. Two patients had isolated aortic valve stenosis, three had isolated aortic valve incompetence, and four patients had mixed aortic valve disease. In all cases, an excellent view of the aortic valve was obtained, aortic valve replacement with a bileaflet mechanical prostheses was performed, and no intraoperative difficulties were encountered. Mean aortic cross-clamp time was 83 min and mean cardiopulmonary bypass perfusion time was 97 min. All patients were extubated in the operating room at the end of the surgical procedure, and there were no postoperative complications. All patients were discharged home on postoperative day 3, and there were no late complications. Through an upper T mini-sternotomy, aortic valve surgery can be performed in the conventional manner using standard surgical instruments with no alteration in cardiopulmonary bypass and myocardial protection routines. With this method, postoperative pain is reduced and patient recovery is expeditious.

Astrocytes are more vulnerable than neurons to cellular Ca 2+ overload induced by a mitochondrial toxin, 3-nitropropionic acid

The differential effects of 3-nitropropionic acid on cultured neurons and astrocytes (of cortical and striatal origin) were examined by studying intracellular Ca 2+ changes using imaging techniques with fura-2. The neurons and astrocytes whose intracellular Ca 2+ concentration was recorded were identified later by immunocytochemical staining for microtubule-associated protein 2 and glial fibrillary acidic protein, respectively. 3-Nitropropionic acid (1.7 mM) irreversibly increased intracellular Ca 2+ in astrocytes (27%) and, to a significantly smaller extent, in neurons (10%). The latency to onset of the intracellular Ca 2+ increase was longer in neurons (45 min) than in astrocytes (29 min). Thus, a differential susceptibility of astrocytes and neurons was observed. The 3-nitropropionic acid-induced astrocytic and neuronal Ca 2+ accumulations were both due to influx of Ca 2+, as the increases were absent in Ca 2+-free medium. An inhibitor of the Na +–Ca 2+ exchanger (2′,4′-dichlorobenzamil), greatly reduced the intracellular Ca 2+ increase in astrocytes, but not in neurons. This indicates that the intracellular Ca 2+ increase in astrocytes is primarily mediated by a reverse operation of the Na +–Ca 2+ exchange system, whereas in neurons it is mediated by a different mechanism. In addition, we noted that astrocytic cell death occurred in 9% of cells at 60 min or more after the start of a 40 min perfusion with 3-nitropropionic acid, while only 4% of neurons died. In astrocytes, cell death was preceded by blebbing of the cell membrane, and by a sustained increase in intracellular Ca 2+ followed by an abrupt further elevation occurring just before cellular collapse. The present results indicate that astrocytes are more vulnerable than neurons to 3-nitropropionic acid-induced cellular Ca 2+ overload and toxicity, and hence support the hypothesis that, in part, 3-nitropropionic acid-induced neurotoxicity could be secondary to astrocytic cell death caused by Ca 2+ overload.

Association between the low threshold calcium spike and activation of NMDA receptors in guinea-pig substantia nigra pars compacta neurons.

The aim of this study was to examine the interaction between N-methyl-D-aspartate (NMDA) receptor activation and the low threshold calcium spike (LTS) of phasically firing neurons in the rostral part of the substantia nigra pars compacta (SNpc) in mid-brain slices. Bath perfusion of 10 microM NMDA gradually increased the LTS area and the effect reached a maximum after 6 min of perfusion. This enhancement of the LTS by NMDA was blocked both by a competitive and non-competitive NMDA receptor antagonist, 50 microM D-AP5 and 10 microM MK801, respectively, demonstrating that this effect of NMDA was mediated through NMDA receptors. Prolonged exposure to increasing concentrations of NMDA (0.1-100 microM) progressively decreased the LTS area. The higher doses led to an irreversible marked depolarization and decrease of the membrane resistance. These results suggest that the LTS of SNpc neurons can trigger a NMDA receptor-dependent response which may have physiological and pathological roles.

Early alterations in intracellular and alveolar surfactant of the rat lung in response to endotoxin.

The aim of this study was to characterize early ultrastructural, biochemical, and functional alterations of the pulmonary surfactant system induced by Salmonella minnesota lipopolysaccharide (LPS) in rat lungs. Experimental groups were: (1) control in vitro, 150 min perfusion; (2) LPS in vitro, 150 min perfusion, infusion of 50 microg/ml LPS after 40 min; (3) control ex vivo, 10 min perfusion; (4) LPS ex vivo, lungs perfused for 10 min from rats treated for 110 min with 20 mg/kg LPS intraperitoneally. Morphometry of type II pneumocytes showed that LPS increased stored surfactant. Lamellar bodies were increased in size, but decreased in numerical density, suggesting that giant lamellar bodies observed in LPS-treated lungs may result from fusion of normal bodies. Structural analysis of alveolar surfactant composition showed that LPS elicited an increase in lamellar body-like and multilamellar forms. Bronchoalveolar lavage (BAL) material from LPS-treated lungs was decreased in phospholipids. BAL bubble surfactometer analysis showed a reduction in hysteresis area caused by LPS. We conclude that LPS leads to alterations of intracellular and alveolar surfactant within 2 h: fusion of lamellar bodies, reduction in surfactant secretion, and changes in alveolar surfactant transformation, composition, and function, which may contribute to the development of respiratory distress.

An ELISA technique for quantitation of human xenoantibodies binding to pig cells: application in patients with pig kidneys extracorporeally connected to the circulation.

A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Gal(alpha)1-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70-90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody binding to xenogenic target cells than conventional titration techniques.

Heterogeneity and underlying mechanism for inotropic action of endothelin-1 in rat ventricular myocytes.

1. To clarify the mechanisms underlying the positive inotropic action of endothelin-1 (ET-1), we investigated the effect of ET-1 on twitch cell shortening and the Ca2+ transient in rat isolated ventricular myocytes loaded with a fluorescent Ca2+ indicator indo-1. 2. There was a cell-to-cell heterogeneity in response to ET-1. ET-1 (100 nM) increased twitch cell shortening in only 6 of 14 cells (44%) and the increase in twitch cell shortening was always accompanied by an increase in the amplitude of the Ca2+ transient. 3. The ET(A)- and ET(B)-receptors antagonist TAK-044 (100 nM) almost reversed both the ET-1-induced increases in twitch cell shortening and in the Ca2+ transient. In the ET-1 non-responding cells, the amplitude of the Ca2+ transient never increased. 4. Intracellular pH slightly increased (approximately 0.08 unit) after 30 min perfusion of ET-1 in rat ventricular myocytes. However, ET-1 did not change the myofilament responsiveness to Ca2+, which was assessed by (1) the relationship between the Ca2+ transient amplitude and twitch cell shortening, and by (2) the Ca2+ transient-cell shortening phase plane diagram during negative staircase. 5. We concluded that there was a cell-to-cell heterogeneity in the positive inotropic effect of ET-1, and that the ET-receptor-mediated positive inotropic effect was mainly due to an increase in the Ca2+ transient amplitude rather than to an increase in myofilament responsiveness to Ca2+.

Relative Importance of Adenosine A1and A3Receptors in Mediating Physiological or Pharmacological Protection from Ischemic Myocardial Injury in the Rabbit Heart

Although ischemic preconditioning (IP) in several species can be pharmacologically mimicked by selective adenosine A1 or A3 receptor agonists, it is currently unclear which receptor subtype (A1 and/or A3) is physiologically involved in mediating IP. To investigate this question, we determined (a) the affinity of adenosine for rabbit adenosine A1 and A3 receptors, and (b) the effects of selective rabbit A1 receptor blockade on IP and adenosine-mediated cardioprotection in a rabbit Langendorff model of myocardial ischemia-reperfusion injury. Adenosine was 19-fold selective for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to recombinant rabbit A1 v rabbit A3 receptors (A1 Ki: 28 nm; A3 Ki 532 nm). Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion, and infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). Ischemic preconditioning (5 min global ischemia and 10 min reperfusion) or adenosine (20 micro M, 5 min) perfusion reduced infarct size (IA/AAR) to 17+/-3 and 14+/-2%, respectively (controls: 59+/-2%). Ischemic preconditioning and adenosine-mediated cardioprotection were completely blocked (57+/-2 and 61+/-4% IA/AAR, respectively) in the presence of a rabbit A1-selective concentration (50 nm) of the antagonist BWA1433 (rabbit A1 Ki: 3 nm; A3 Ki; 746 n m). Thus, whereas recent studies have demonstrated that selective A1 or A3 receptor agonists can both pharmacologically mimic IP, the results of the present study suggest that the adenosine-mediated component of IP in the isolated rabbit heart is preferentially mediated by adenosine A1 receptors, potentially due to adenosine's selectivity for this receptor subtype.

EXTRACELLULAR DOPAMINE AND GABA CONCENTRATIONS IN THE NIGRASTRIATAL SYSTEM AND THALAMUS OF RATS UNDER PROPOFOL ANESTHESIA

S502 Many reports have suggested that enhancement of the inhibition (GABA (A-mediated)) or depression of excitatory (glutamate-mediated) neurotransmission, or both, may account for the dominant depressant effects of anesthetics on the central nervous system. Recently, several studies have implied that propofol may interact with dopamine receptors [1,2]. However, there is no evidence that propofol alters the release of dopamine and GABA. Therefore, the goal of this study is to examine the effects of propofol on the concentrations of dopamine and GABA in the nigrostriatal system and thalamus by using in vivo microdialysis. Materials and methods: Sprague-Dawley female rats weighing 200-250 g (n=36) were randomly assigned to two groups: group C (n=18) were free moving rats serving as controls and the microdialysis probe was implanted in the striatum (S) (n=6), the substantia nigra (SNR) (n=6) or ventrolateral thalamus (VL)(n=6). Group Propofol (P) rats (n=18) were anesthetized with propofol at a 50 mg/kg/hour infusion rate and the probes were in the same brain parts as described above (n=6 each). Microdialysis probes (CMA PC12, length 1mm for SN or VL, and 2mm for S) were stereotactically implanted in the left S, SNR or VL of each rat. Group C rats were prepared for implantation of microdialysis probe guide cannula under isoflurane anesthesia on day 1. On day 2, probes were inserted into each part described above. After 2 hours recovery, the five dialysates were sampled every 10 min (perfusion rate 1.0 [micro sign]l/min). In group P, 2 hours' administration of propofol after probe implantation was followed by five consecutive samplings. Dialysates were analyzed by HPLC. Each concentration was corrected according to the recovery rate of each probe. Data were analyzed by Mann-Whitney tests and are presented as means +/- SD. P<0.05 was considered statistically significant. Results: Physiological parameters (blood pressure, body and brain temperatures, etc.) did not differ significantly between the two groups. In group C, baseline concentrations of dopamine were 16.4 +/- 1.4nM (S), 3.1 +/- 0.2nM (SNR) and 8.0 +/- 0.3nM (VL) and GABA concentrations were 215.2 +/- 9.3nM (S), 80.0 +/- 3.3nM (SNR) and 45.9 +/- 3.4nM (VL), respectively. In group P, propofol decreased the dopamine by 21% (p<0.005) in VL. However, it increased the dopamine by 233% (p<0.005) in SNR. Propofol increased GABA by 199% (p<0.005) and 229% (p<0.005) in SNR and VL, respectively. There were no significant dopamine or GABA concentration changes in S. Discussion: A large dose of propofol stimulated dopaminergic fibers or inhibited uptake of dopamine in the nigrostriatal system. Propofol also increased GABA concentrations in SNR and VL. The increase in the dopamine concentration in SNR is suspected to be one of the mechanisms by which propofol induces involuntary movement. These results may explain why propofol has not only hypnotic effect, also has an anesthetic property.

Effect of selenium on the uptake of methyl mercury across perfused gills of rainbow trout Oncorhynchus mykiss

The uptake of methyl mercury was measured across the perfused gills of rainbow trout Oncorhynchus mykiss. The effect of selenium, either in the blood (perfusion medium), or in the water was investigated. Methyl mercury was effectively taken up from the water across the gills into the perfusate. The uptake rate reached a stable level after 30 min perfusion. When the gills were placed in mercury free water after exposure to mercury in the water for 1 h, they continued to liberate significant amounts of accumulated mercury into the perfusate. Exposure to selenite (SeIV) or selenate (VI) (0.075–0.75 μM) in the external medium did not affect the uptake of methyl mercury across the gills or the liberation of the metal from the gills. Internal selenite or selenate (7.5 μM) augmented the uptake of methyl mercury across the gills and internal selenite also increased the amounts of liberated methyl mercury from the gills in the unload period. Internal selenium, increased the mercury accumulation in the gills, whereas, external selenium did not alter the mercury accumulation in the gills. Uptake of selenium from the water across the gills occurred very slowly.

A Porcine Model for Investigation of Hyperthermic Isolated Liver Perfusion

Our study was aimed at developing a reliable method of hyperthermic isolated liver perfusion (HILP) in pigs and at assessing its local and systemic side effects. HILP was performed via the hepatic artery and portal vein in 15 animals. The perfusate consisted of blood (200 ml), oxypolygelatine (500 ml), Ringer's solution (1000 ml), and trapped intrahepatic blood. HILP was carried out for 45 min at a mean perfusate inflow temperature of 41.2°C. The mean portal flow and pressure were adjusted to 500 ml/min and 20-25 mm Hg; the mean arterial flow and pressure were 130 ml/min and 40-60 mm Hg, respectively. After 20 min of perfusion the mean temperature in the right and the left liver lobe were 40.8°C and 40.3°C and remained almost constant over the whole perfusion period. Liver enzymes (alanine aminotransferase and aspartate aminotransferase) and serum lactate levels showed slight increases after perfusion but normalized within 1 week. Histology of liver parenchyma showed only mild pathological changes, which were also reversible within 7 days. The presented method of HILP is a safe and reproducible technique for isolated hyperthermic liver perfusion. Based on this animal model, experimental HILP with different chemotherapeutic agents can be investigated in order to assess hepatic and systemic toxicity of this therapy.