Central dopaminergic D1 agonists and treatment of Parkinson disease

A glucose analogue labelled with iodine-123 in position 6 has been synthesized: [123I]-6-deoxy-6-iodo-D-glucose (6DIG). The aim of this study was to examine its biological behaviour in order to assess whether it could be used to evaluate glucose transport with SPECT. To establish whether 6DIG enters the cells using the glucose transporter, four biological models have been used: human erythrocytes in suspension, neonatal rat cardiomyocytes in culture, isolated perfused rat hearts, and biodistribution in mice. 6DIG competed with D-glucose to enter the cells and its entry was increased by insulin and inhibited in the presence of cytochalasin B. The biological behaviour of 6DIG was similar to that of 3-O-methyl-D-glucose. 6DIG is a tracer of glucose transport which is very promising for clinical studies.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.     

Subcellular calcium transport in failing hearts due to calcium deficiency and overload

Mitochondrial and heavy microsomal fractions were isolated from rat hearts perfused for different intervals with Ca2+-free medium, as well as from hearts reperfused with control medium after perfusion with Ca2+-free medium. Contractile failure due to intracellular calcium deficiency produced by perfusing the isolated rat hearts with Ca2+-free medium resulted in a marked decline of calcium binding and uptake activities of the mitochondrial fraction without any effect on the microsomal fraction. On the other hand, inability of the rat hearts to recover their contractile force due to intracellular calcium overload produced by reperfusion for 10 min with control medium after 5-20 min of perfusion with Ca2+-free medium was associated with decreased microsomal calcium-binding and uptake activities and increased mitochondrial calcium-binding and uptake activities. When the hearts perfused with Ca2+-free medium in the presence of low sodium (35 mM) for 5 min were reperfused with control medium, the contractile force recovered completely, and appreciable augmentation in mitochondrial calcium transport or depression in microsomal calcium transport as seen in conditions of intracellular calcium overload did not occur. These results suggest dramatic alterations in calcium-transporting properties of mitochondria and sarcoplasmic reticulum in hearts failing due to intracellular calcium deficiency and calcium overload, respectively.     

Effect of soleus unweighting on stimulation of insulin-independent glucose transport activity

Unweighting of the rat soleus by tail-cast suspension results in increased insulin action on stimulation of glucose transport, which can be explained, at least in part, by increased insulin binding and enhanced glucose transporter protein levels. Glucose transport is also activated by an insulin-independent mechanism stimulated by in vitro muscle contractions or hypoxia. Therefore, the purpose of this study was to determine if soleus unweighting leads to an enhanced response of the insulin-independent pathway for stimulation of glucose transport. The hindlimbs of juvenile male Wistar rats were suspended by a tail-cast system for 3 or 6 days. Glucose transport activity in isolated soleus strips (approximately 18 mg) was then assessed by using 2-deoxy-[1,2-3H]glucose (2-DG) uptake. Insulin (2 mU/ml) had a progressively enhanced effect on 2-DG uptake after 3 and 6 days of unweighting (+44 and +72% vs. control, respectively; both P < 0.001). At these same times, there was no difference between groups for activation of 2-DG uptake by maximally effective treatments with contractions (10 tetanuses), hypoxia (60 min), or caffeine (5 mM). These results indicate that the enhanced capacity for stimulation of glucose transport after soleus unweighting is restricted to the insulin pathway, with no apparent enhancement of the insulin-independent pathway.     

Conclusive evidence for distinct transporters for 5-hydroxytryptamine and noradrenaline in pulmonary endothelial cells of the rat

The aims of this study were to obtain conclusive evidence about the roles of a 5-hydroxytryptamine [5-HT] transporter and uptake1 in the dissipation of 5-HT in the lungs of the rat and to compare the properties of the 5-HT transporter in rat lungs with that in other tissues, including brain and platelets. In the first part of the study, the IC50 values of a range of selective inhibitors and substrates of the 5-HT transporter or uptake1 were determined for inhibition of uptake of 5-HT or noradrenaline in intact perfused lungs of rats. Monoamine oxidase was inhibited and, in experiments with noradrenaline, catechol-O-methyltransferase was also inhibited. Initial rates of uptake of 5-HT or noradrenaline were measured in lungs perfused with 2 nmol/l 3H-5-HT or 3H-noradrenaline for 2 min, in the absence or presence of at least three concentrations of paroxetine, citalopram, fluoxetine, 7-methyltryptamine, tryptamine, nisoxetine, imipramine, 5-HT, desipramine, (+)-oxaprotiline, cocaine or tyramine. The results showed that pharmacologically distinct transporters are involved in the uptake of 5-HT and noradrenaline in rat lungs, since there was no significant correlation between the IC50 values for inhibition of 5-HT and noradrenaline uptake in the lungs. However, there were significant correlations between the IC50 values for (a) inhibition of 5-HT uptake in rat lungs and of uptake by the 5-HT transporter in rat brain and (b) inhibition of noradrenaline uptake in rat lungs and of uptake1 in rat phaeochromocytoma PC-12 cells. The results support the conclusion that 5-HT uptake in rat lungs occurs, at least predominantly, by a 5-HT transporter which is very similar to or the same as that in other tissues, such as the brain, and provide further evidence for transport of noradrenaline by uptake1. Further experiments were carried out to determine whether there is any transport of 5-HT by uptake1 or of noradrenaline by the 5-HT transporter in rat lungs. Lungs were perfused with 2 nmol/l 3H-5-HT or 3H-noradrenaline for 2 min in the absence or presence of 1 mumol/l citalopram, desipramine, or citalopram and desipramine. The results showed that there was no evidence of any transport of 5-HT in the lungs by uptake1 or of noradrenaline by the 5-HT transporter, in that desipramine had no effect on 5-HT uptake (in the absence or presence of citalopram) and citalopram had no effect on noradrenaline uptake (in the absence or presence of desipramine). The final series of experiments was carried out to determine whether, at high concentrations of the amine, there is any interaction of 5-HT with uptake1 or of noradrenaline with the 5-HT transporter. Noradrenaline, at a concentration of 10 mumol/l, did not affect 5-HT uptake in lungs perfused with 2 nmol/l 3H-5-HT for 2 min (uptake1 inhibited), but 50 mumol/l 5-HT inhibited noradrenaline uptake by 56% in lungs perfused with 2 nmol/l 3H-noradrenaline for 2 min (5-HT transporter inhibited). These and the above results show that the 5-HT transporter appears to be exclusively responsible for 5-HT uptake in rat lungs, despite the possible interaction of 5-HT at high concentrations with the uptake1 transporter in the cells. On the other hand, noradrenaline is transported exclusively by uptake1 in the lungs, and there is no evidence that it interacts with the 5-HT transporter, even at high concentrations.     

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.     

SPECT imaging of dopamine transporters in human brain with iodine-123-fluoroalkyl analogs of beta-CIT

Iodine-123-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane (beta-CIT) is a useful SPECT tracer for imaging the dopamine transporter. Its slow kinetics, however, necessitate imaging on the day after the injection. Two N-omega-fluoroalkyl analogs of beta-CIT, the fluoropropyl and fluoroethyl compounds (beta-CIT-FP and beta-CIT-FE, respectively), characterized by faster kinetics in baboons, were tested in humans as potential tracers for the dopamine transporter. Four healthy volunteers were injected with [123I]-beta-CIT-FP and another four were injected with [123I]beta-CIT-FE. SPECT data were acquired for 1149 +/- 590 min and 240 +/- 30 min, respectively. Both tracers demonstrated high brain uptake (6.37% +/- 0.37% and 7.8% +/- 1.5% of the injected dose, respectively). Activity concentrated with time in the striatal area, reaching a peak within 30 min, with little or no washout for [123I]beta-CIT-FP and a faster washout for [123I]beta-CIT-FE (14.7% +/- 6.9%). Occipital and midbrain activity showed similar patterns, displaying a peak within 15 min and rapid washout, followed by stable levels at approximately 100 min for both tracers. The ratio of peak specific striatal-to-peak specific midbrain activity was 9.1 +/- 1.8 for [123I]beta-CIT-FP and 7.7 +/- 0.7 for [123I]beta-CIT-FE, showing high in vivo selectivity for the dopamine transporter. These preliminary results suggest that both compounds could be used as SPECT (labeled with 123I) or PET (labeled with 18F) radiotracers to image the dopamine transporters in the living human brain.     

Deficient transport of dehydroascorbic acid in the glucose transporter protein syndrome

The glucose transporter protein syndrome (GTPS) is caused by defective transport of glucose across the blood-brain barrier via the glucose transporter GLUT1, resulting in hypoglycorrhachia, infantile seizures, and developmental delay. Recent reports indicated that GLUT1 is a multifunctional transporter. We investigated the transport of vitamin C in its oxidized form (dehydroascorbic acid) via GLUT1 into erythrocytes of 2 patients with GTPS. In both patients, uptake of oxidized vitamin C was 61% of the mothers' values. Our findings are consistent with recent observations that vitamin C is transported in its oxidized form via GLUT1. We speculate that impaired transport of this substrate and perhaps other substrates in GTPS might contribute to the pathophysiology of this condition.     

The role of providers in implementation of the National Kidney Foundation-Dialysis Outcomes Quality Initiative: Fresenius Medical Care North America perspective

Cadmium (Cd) is reported to produce cardiotoxicity at doses and exposure conditions that cause no effect in kidney or liver. The purpose of the present investigation was to examine the cytotoxicity of Cd to neonatal rat cardiomyocytes in primary culture and to elucidate the transport characteristics of Cd in these cells at a nontoxic concentration. Cd concentrations of 0.1 microM and higher that are well tolerated by hepatocytes and renal cortical epithelial cells were toxic to the cardiomyocyte. The plot of initial uptake rate of Cd at various concentrations was nonlinear suggesting that, in addition to simple diffusion, other processes may also be involved. These processes required metabolic energy as pretreatment with dinitrophenol or sodium fluoride inhibited 58 and 59% of the Cd uptake, respectively. The uptake of Cd was also affected by the incubation temperature and lowering the temperature from 37 to 4 degreesC reduced Cd uptake over 30 min by 61%. Cd uptake required interaction with membrane sulfhydryl groups; pretreatment with p-chloromercuribenzenesulfonic acid or mercuric chloride reduced Cd uptake by 46 and 58%, respectively. Cd utilized the transport pathways for calcium (Ca), zinc (Zn), and copper (Cu). Coincubation with 1.26 mM Ca competitively inhibited Cd uptake by 77%. In the presence of Ca, 30 microM Zn or Cu further inhibited Cd accumulation competitively by as much as 63 and 32%, respectively. Cd could enter the cardiomyocytes through Ca channels and Ca channel blocker, verapamil, inhibited up to 76% of Cd uptake. From the above results it can be concluded that Cd is highly toxic to the cardiomyocytes. A majority of Cd enters these cells through transport processes that exist for Ca, Zn, and Cu. The transport processes utilized by Cd are temperature sensitive and dependent on metabolic energy. Furthermore, these involve membrane sulfhydryl groups and include Ca channels.     

Sensing by intrahepatic muscarinic nerves of a portal-arterial glucose concentration gradient as a signal for insulin-dependent glucose uptake in the perfused rat liver

In vivo, insulin increases net hepatic glucose uptake efficiently only in the presence of a portal-arterial glucose gradient. In isolated perfused rat livers supplied with a glucose gradient (portal 10 mM/arterial 5 mM) insulin-induced glucose uptake was blocked by atropine; in livers not supplied with the gradient (portal = arterial 5 mM) insulin-dependent glucose uptake was elicited by acetylcholine. Apparently, the gradient was sensed and transformed into a metabolic signal by intrahepatic nerves, releasing acetylcholine to muscarinic receptors.     

Persistent myocardial ischemia increases GLUT1 glucose transporter expression in both ischemic and non-ischemic heart regions

Persistently ischemic myocardium exhibits increased glucose uptake which may contribute to the preservation of myocardial function and viability. Little is known about the specific molecular events which are responsible for this increase in uptake. Therefore, we investigated whether myocardial ischemia induces the gene expression of the major cardiac facilitative glucose transporters, GLUT4 and GLUT1. We determined the expression of myocardial glucose transporter mRNAs and polypeptides after 6 h of regional ischemia in a dog model by semi-quantitative Northern blotting and immunoblotting. GLUT1 but not GLUT4 expression was significantly increased in both ischemic and non-ischemic regions from the experimental hearts when compared to surgical control and normal hearts. GLUT1 mRNA expression was increased 3.4-fold and GLUT1 polypeptide expression was increased 1.7-fold in ischemic hearts when compared to normal or surgical-control hearts. There were no significant regional differences in GLUT1 expression in either normal or ischemic hearts. However, there was a tendency for GLUT1 mRNA expression to be highest in the non-ischemic regions from the 6-h ischemia hearts. These findings suggest that myocardial ischemia induces a factor or factors which stimulate GLUT1 expression in non-ischemic as well as ischemic myocardial regions. Increased GLUT1 expression may play a role in augmenting glucose uptake during ischemia.     

Acceleration of glycolysis in erythrocytes by the antidiabetic agent M16209

The effects of M16209 (1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin), an antidiabetic agent and aldose reductase inhibitor, on glycolysis were studied in rat and human erythrocytes in vitro. M16209 increased lactate production from glucose when incubated with rat and human erythrocytes, and also increased glucose consumption in rat erythrocytes. The rates of production of lactate in rat erythrocytes treated with M16209 at 10, 25 and 50 microM were 113, 118 and 123%, respectively, of those in vehicle treated cells. Sorbinil (aldose reductase inhibitor), tolbutamide (sulfonylurea), and buformine (biguanide) did not increase lactate production in rat erythrocytes when tested at 50 microM. On the other hand, M16209 did not affect lactate production from D-glyceraldehyde in rat erythrocytes. At 100 microM the agent decreased both glucose-6-phosphate and fructose-6-phosphate in rat erythrocytes, and increased fructose-1,6-bisphosphate; at 10 microM it also increased 6-phosphofructokinase activity in rat hemolysates. These findings suggest that M16209 accelerates glycolysis in erythrocytes via activation of 6-phosphofructokinase.     

Primary active transport of peptidic endothelin antagonists by rat hepatic canalicular membrane

The biliary excretion mechanism of three derivatives of BQ-123, an anionic cyclopentapeptide, was examined using isolated canalicular membrane vesicles (CMVs) from Sprague-Dawley rats. The uptake by CMV of BQ-485, a linear peptide, BQ-518, a cyclic peptide, and compound A, a cyclic peptide with a cationic moiety, was stimulated by ATP. An "overshoot" phenomenon and saturation were observed for the ATP-dependent uptake of these three peptides. The Michaelis-Menten constants (Km) for the uptake of BQ-485 and BQ-518 were comparable to the inhibition constants (Ki) for their inhibitory effects on ATP-dependent [3H]BQ-123 uptake. The uptake of BQ-485 showed the highest value and was inhibited by BQ-123 with a Ki that was comparable to the Km for BQ-123 uptake. The ATP-dependent uptake of BQ-123, BQ-485, and BQ-518 was much lower in CMVs from Eisai hyperbilirubinemic rats, a strain having a hereditary defect of the canalicular multispecific organic anion transporter (cMOAT). These results suggest that both BQ-485 and BQ-518 principally share the cMOAT transporter with BQ-123. Compound A almost completely inhibited BQ-123 uptake, although its ATP-dependent uptake was much lower than that of the other three peptides. The ATP-dependent uptake of compound A was not very different in Sprague-Dawley rats and Eisai hyperbilirubinemic rats and was not inhibited by S-(2, 4-dinitrophenyl)-glutathione, a typical substrate for cMOAT. Thus, although compound A inhibits cMOAT-mediated transport, its own transport by cMOAT is minimal and mediated by another transporter. This low degree of primary active transport by cMOAT may be the principal reason for its relatively longer residence in the circulation.     

Amyloid beta-peptide impairs glucose transport in hippocampal and cortical neurons: involvement of membrane lipid peroxidation

A deficit in glucose uptake and a deposition of amyloid beta-peptide (A beta) each occur in vulnerable brain regions in Alzheimer's disease (AD). It is not known whether mechanistic links exist between A beta deposition and impaired glucose transport. We now report that A beta impairs glucose transport in cultured rat hippocampal and cortical neurons by a mechanism involving membrane lipid peroxidation. A beta impaired 3H-deoxy-glucose transport in a concentration-dependent manner and with a time course preceding neurodegeneration. The decrease in glucose transport was followed by a decrease in cellular ATP levels. Impairment of glucose transport, ATP depletion, and cell death were each prevented in cultures pretreated with antioxidants. Exposure to FeSO4, an established inducer of lipid peroxidation, also impaired glucose transport. Immunoprecipitation and Western blot analyses showed that exposure of cultures to A beta induced conjugation of 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, to the neuronal glucose transport protein GLUT3. HNE induced a concentration-dependent impairment of glucose transport and subsequent ATP depletion. Impaired glucose transport was not caused by a decreased energy demand in the neurons, because ouabain, which inhibits Na+/K(+)-ATPase activity and thereby reduces neuronal ATP hydrolysis rate, had little or no effect on glucose transport. Collectively, the data demonstrate that lipid peroxidation mediates A beta-induced impairment of glucose transport in neurons and suggest that this action of A beta may contribute to decreased glucose uptake and neuronal degeneration in AD.     

Angiotensin I modulates Ca-transport systems in the rat heart through angiotensin II

The objective of this study was to determine the effect of angiotensin I (Ang I) treatment in vivo on two major Ca-transport systems-the L-type voltage dependent calcium channel (L-VDCC) and the Na/Ca exchanger in rat heart. For our experiments we used four groups of rats, treated differently with saline, Ang I, the ACE inhibitor enalapril and/or combination of both for 6 days, every 24 h. We observed an increase in the activity, and also in mRNA expression of the Na/Ca exchanger, after repeated administration of Ang I in vivo. The maximal binding capacity of Ca-antagonist PN 200-110, which binds to the alpha 1 subunit of the L-VDCC was elevated from 0.8-1.85 pg/mg protein. mRNA expression of the voltage-dependent calcium channels of L-type system was also upregulated by Ang I administration, but not when enalapril was applied simultaneously with Ang I. These results demonstrate that in vivo application of the Ang I significantly modulates not only the activity, but also expression of the Na/Ca exchanger and the L-VDCC in rat hearts through angiotensin II (Ang II). Since in the in vitro experiments on the isolated cardiomyocytes, Ang II (100 nM) increased the calcium uptake after depolarization, and the AT1 receptor agonist losartan prevented this increase, we assume that this regulation might involve the AT1 receptors.     

Control of glucose utilization in working perfused rat heart

Metabolic control analyses of glucose utilization were performed for four groups of working rat hearts perfused with Krebs-Henseleit buffer containing 10 mM glucose only, or with the addition of 4 mM D-beta-hydroxybutyrate/1 mM acetoacetate, 100 nM insulin (0.05 unit/ml), or both. Net glycogen breakdown occurred in the glucose group only and was converted to net glycogen synthesis in the presence of all additions. The flux of [2-3H]glucose through P-glucoisomerase (EC 5.3.1.9) was reduced with ketones, elevated with insulin, and unchanged with the combination. Net glycolytic flux was reduced in the presence of ketones and the combination. The flux control coefficients were determined for the portion of the pathway involving glucose transport to the branches of glycogen synthesis and glycolysis. Major control was divided between the glucose transporter and hexokinase (EC 2.7.1.1) in the glucose group. The distribution of the control was slightly shifted to hexokinase with ketones, and control at the glucose transport step was abolished in the presence of insulin. Analysis of the pathway from 3-P-glycerate to pyruvate determined that the major control was shared by enolase (EC 4.2.1.1) and pyruvate kinase (EC 2.7.1.40) in the glucose group. Addition of ketones, insulin, or the combination shifted the control to P-glycerate mutase (EC 5.4.2.1) and pyruvate kinase. These results illustrate that the control of the metabolic flux in glucose metabolism of rat heart is not exerted by a single enzyme but variably distributed among enzymes depending upon substrate availability, hormonal stimulation, or other changes of conditions.     

Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase

The overall disposition and hepatobiliary transport of BQ-123, an anionic cyclopentapeptide, and three analogs were examined in rats in vivo. Total body clearance (CLtotal) and biliary excretion clearance (CLbile, p) exhibited 4- to 8-fold differences between the compounds, with those for BQ-485 and compound A having the highest and lowest values, respectively. The CLbile, p values of BQ-485, BQ-123, and BQ-518 were almost equal to the CLtotal, suggesting that hepatobiliary transport is the major elimination pathway for these compounds. Hepatic uptake clearance (CLuptake, vivo) and biliary excretion clearance (CLbile, h/fT), which was defined for the hepatic unbound concentration, were separately determined to examine the hepatic uptake and excretion processes, respectively. Both the CLuptake, vivo and CLbile, h/fT of BQ-485 were higher than those of BQ-123, whereas the corresponding values for BQ-518 were similar to those for BQ-123. The CLuptake, vivo and CLbile, h/fT of compound A were, respectively, approximately two thirds and one half those of BQ-123, suggesting that the lower CLbile, p value is due to the low efficiency of both the uptake and excretion processes. The CLuptake, vivo of these four peptides in vivo was similar to the extrapolated values based on the carrier-mediated transport activity previously assessed in vitro in isolated rat hepatocytes. The primary active transport previously assessed in an in vitro study in canalicular membrane vesicles was also highest for BQ-485 and lowest for compound A, similar to CLbile, h/fT in vivo. Thus, the transporters on both the sinusoidal and canalicular membranes determine the efficiency of the peptide overall elimination from the circulation.     

mRNA expression of glycolytic enzymes and glucose transporter proteins in ischemic myocardium with and without reperfusion

It is known that ischemia commonly increases exogenous glucose utilization by accelerating glucose uptake and flux rates through the Embden-Meyerhof pathway. Constitutive enzymes regulate the rate of glycolysis and in turn are regulated by product inhibition and allosteric controls. The purpose of this report was to test whether mRNA abundance for select glycolytic enzymes, and glucose transport proteins, is also modified. Six intact working pig hearts with coronary flow controlled by extracorporeal perfusion were compared at the following conditions: (1) aerobic control perfusion; (2) ischemia affected by a 60% decrease in left anterior descending (LAD) coronary perfusion: (3) ischemia again affected by a 60% decrease in LAD flow followed by a 40-min interval of aerobic reflow; (4) an intermittent ischemia and reflow protocol including four cycles of similar LAD flow reductions (5 min per cycle) interspersed with 15-20 min of aerobic reperfusion; (5) a 4-day model designed to produce myocardial chronic hibernation: and (6) mild ischemia induced by a 40% decrease in LAD flow for 85 min to produce certain adaptations compatible with short-term hibernation. In each heart, mRNA abundance was measured from LAD and circumflex (LCF) perfused myocardium for hexokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and the two glucose transporter isomers, GLUT 4 and GLUT 1. mRNA data from LAD myocardium in intervention hearts were normalized to those from LAD tissue in the control heart (LADc) and with LCF values in the same intervention hearts. Signal variance around unity in the LAD tissue, with respect to that of the LCF myocardium, in the control heart compared closely (44 and 41% in two separate runs, respectively). GLUT 1/GLUT 4 ratios in the LAD and LCF beds of this heart also agreed closely. LAD/LADc ratios were increased for hexokinase (1.69), phosphofructokinase (3.69), and glyceraldehyde-3-phosphate dehydrogenase (2.29) in the ischemia heart and for phosphofructokinase (3.90), glyceraldehyde-3-phosphate dehydrogenase (2.20), GLUT 4 (1.55) and GLUT 1 (2.20) in the ischemia/reflow heart. There was no evidence of excess signal in the intermittent ischemia/reflow, chronic hibernation, or mild ischemia hearts. Altered signal from LCF myocardium was also suggested. These data indicate that mRNA abundance for select glycolytic enzymes and transporter proteins is increased in ischemic myocardium with or without reperfusion and offers a possible mechanism for increased protein activity in settings of diminished regional coronary flow.     

Relation between enzyme release and irreversible cell injury of the heart under the influence of cytoskeleton modulating agents

The effects of agents modulating the cytoskeleton, taxol (microtubuli stabilizing), vinblastine (microtubuli destabilizing) and cytochalasin D (actin destabilizing) (10(-6) M each) on enzyme and ATP release as well as on irreversible cell injury were investigated in isolated perfused hypoxic and reoxygenated rat hearts. Enzyme (creatine kinase (CK)) and ATP concentration were assayed in the interstitial transudate and venous effluent. Irreversible cell injury was determined from trypan blue uptake and nuclear staining (NS) of cardiomyocytes in histologic sections. ATP release from nonneuronal cells was only detectable in the interstitial transudate and was not significantly altered by the agents. In controls total CK release (about 4% of total CK) exceeded the percentage of irreversibly injured cells by a factor of 8. Taxol and cytochalasin D abolished the hypoxia/reoxygenation induced interstitial CK release and reduced total CK release to a highly significant extent. The percentage of irreversible injured cells was even more diminished by these agents resulting in a ratio of CK/NS of 40. The effect of cytochalasin D apparently is the consequence of decreased contractile performance as shown by analogous depression by butonedione monoxine (BDM), whereas contractile activity was not altered by taxol. Vinblastine had no influence on CK release but increased the number of irreversibly injured cells significantly. In conclusion, cytoskeletal elements apparently participate in the hypoxia/reoxygenation induced process of release of cytosolic enzymes (CK) and irreversible injury in a different way and extent. Taxol exhibits a cytoprotective effect in isolated perfused rat hearts as evaluated by the extent of enzyme release and irreversible cell injury.