An inhibitory and beneficial effect of chlorpromazine and promethazine (C + P) on hyperglycolysis through HIF-1α regulation in ischemic stroke.

BACKGROUND: After ischemic stroke, the increased catabolism of glucose (hyperglycolysis) results in the production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). A depressive or hibernation-like effect of C + P on brain activity was reported to induce neuroprotection. The current study assesses the effect of C + P on hyperglycolysis and NOX activation. METHODS: Adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C + P with or without temperature control, or phloretin [glucose transporter (GLUT)-1 inhibitor], or cytochalasin B (GLUT-3 inhibitor). We detected brain ROS, apoptotic cell death, and ATP levels along with HIF-1α expression. Cerebral hyperglycolysis was measured by glucose, protein expression of GLUT-1/3, and phosphofructokinase-1 (PFK-1), as well as lactate and lactate dehydrogenase (LDH) at 6 and 24 h of reperfusion. The enzymatic activity of NOX and protein expression of its subunits (gp91phox) were detected. Neural SHSY5Y cells were placed under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with C + P treatment. Cell viability and protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured. A HIF-1α overexpression vector was transfected into the cells, and then protein levels of HIF-1α, GLUT-1/3, PFK-1, and LDH were quantitated. In sham-operated rats and control cells, the protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured at 6 and 24 h after C + P administration. RESULTS: C + P reduced the protein elevations after stroke in HIF-1α, glycolytic enzymes, as well as in ROS, cell death, glucose and lactate, but raised ATP levels in the brain. In ischemic rats exposed to GLUT-1/3 inhibitors, ROS, cell death, glucose, and lactate were all decreased, as well as GLUT-1, GLUT-3, LDH, and PFK-1 protein levels. C + P decreased ischemia-induced NOX activation by reducing the enzymatic activity and protein expression of the NOX subunit gp91phox, as was observed in the presence of GLUT-1/3 inhibitors. These markers were significantly decreased following C + P administration with the induced hypothermia, while C + P administration with temperature control at 37 °C induced lesser protection after ischemia stroke. In the OGD/reoxygenation model, C + P treatment increased cell viability and diminished protein levels of HIF-1α, GLUT-1, GLUT-3, PFK-1, LDH, and gp91phox. However, in OGD with HIF-1α overexpression, C + P was unable to effectively reduce the upregulated GLUT-1, GLUT-3, and LDH. In normal conditions, C + P reduced HIF-1α and the levels of key glycolytic enzymes depending on its pharmacological effect. CONCLUSION: C + P, partially depending on hypothermia, attenuates hyperglycolysis and NOX activation through HIF-1α regulation.

Effect of lithium salts on lactate dehydrogenase, adenylate kinase, and 1-phosphofructokinase activities.

Inhibitions of 30 nM rabbit muscle 1-phosphofructokinase (PFK-1) by lithium, potassium, and sodium salts showed inhibition or not depending upon the anion present. Generally, potassium salts were more potent inhibitors than sodium salts; the extent of inhibition by lithium salts also varied with the anion. Li(2)CO(3) was a relatively potent inhibitor of PFK-1 but LiCl and lithium acetate were not. Our results suggest that extents of inhibition by monovalent salts were due to both cations and anions, and the latter needs to be considered before inhibition can be credited to the cation. An explanation for monovalent salt inhibitions is proffered involving interactions of both cations and anions at negative and positive sites of PFK-1 that affect enzyme activity. Our studies suggest that lithium cations per se are not inhibitors: the inhibitors are the lithium salts, and we suggest that in vitro studies involving the effects of monovalent salts on enzymes should involve more than one anion.

Calmodulin upregulates skeletal muscle 6-phosphofructo-1-kinase reversing the inhibitory effects of allosteric modulators.

6-phosphofructo-1-kinase (PFK) is a calmodulin (CaM)-binding protein that plays a key role on the regulation of glycolysis. Each PFK monomer binds two CaM molecules inducing the dissociation of the active tetrameric conformation of the enzyme into dimers, thus inhibiting it. Recently, we have reported that the binding of one CaM per PFK monomer promotes the dimerization of the enzyme although maintaining its full catalytic activity. The present work aims to understand the regulatory role of these active PFK dimers induced by CaM. We show that the inhibition of PFK activity by ATP (>1 mM) is abolished in the presence of CaM. CaM decreases the affinity of PFK for its substrates, fructose-6-phophate and ATP. Moreover, CaM activates PFK in the presence of citrate and lactate, two inhibitory metabolites that induce the dimerization of PFK tetramers, as well as potentiate the stimulatory action of ADP and fructose-2,6-bisphosphate. Under all the conditions tested CaM induces the formation of PFK dimers, supporting that these CaM-bound dimers are active and less susceptible to inhibition by allosteric ligands. In the end, we suggest that CaM binding to PFK, which is stimulated by Ca(2+), represents an important way to increase the glycolytic pathway in cells.

Fructose 1,6-diphosphate administration attenuates post-ischemic ventricular dysfunction.

BACKGROUND: Cardiomyocyte energy production during ischemia depends upon anaerobic glycolysis inefficiently yielding two ATP per glucose. Substrate augmentation with fructose 1,6-diphosphate (FDP) bypasses the ATP consuming steps of glucokinase and phosphofructokinase thus yielding four ATP per FDP. This study evaluated the impact of FDP administration on myocardial function after acute ischemia. METHODS: Male Wistar rats, 250-300 g, underwent 30 min occlusion of the left anterior descending coronary artery followed by 30 min reperfusion. Immediately prior to both ischemia and reperfusion, animals received an intravenous bolus of FDP or saline control. After 30 min reperfusion, myocardial function was evaluated with a left ventricular intracavitary pressure/volume conductance microcatheter. For bioenergetics studies, myocardium was isolated at 5 min of ischemia and assayed for ATP levels. RESULTS: Compared to controls (n=8), FDP animals (n=8) demonstrated significantly improved maximal left ventricular pressure (100.5+/-5.4 mmHg versus 69.1+/-1.9 mmHg; p<0.0005), dP/dt (5296+/-531 mmHg/s versus 2940+/-175 mmHg/s; p<0.0028), ejection fraction (29.1+/-1.7% versus 20.4+/-1.4%; p<0.0017), and preload adjusted maximal power (59.3+/-5.0 mW/microL(2) versus 44.4+/-4.6 mW/microL(2); p<0.0477). Additionally, significantly enhanced ATP levels were observed in FDP animals (n=5) compared to controls (n=5) (535+/-156 nmol/g ischemic tissue versus 160+/-9.0 nmol/g ischemic tissue; p<0.0369). CONCLUSIONS: The administration of the glycolytic intermediate, FDP, by intravenous injection, resulted in significantly improved myocardial function after ischemia and improved bioenergetics during ischemia.

Clotrimazole decreases human breast cancer cells viability through alterations in cytoskeleton-associated glycolytic enzymes.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.

Environmental cadium exposure and metabolic responses of the Nile tilapia, Oreochromis niloticus.

The contamination of water by metal compounds is a worldwide environmental problem. This study was undertaken to evaluate the impact of short-term cadmium exposure on metabolic patterns of the freshwater fish Oreochromis niloticus. The fish were exposed to 320, 640, 1,280 and 2,560 microg/l sublethal concentrations of Cd++ (CdCl2) in water for 7 days. The specific activities of the enzymes phosphofructo kinase (PFK-E.C.2.7.1.11.), lactate dehydrogenase (LDH-E.C.1.1.1.27.) and creatine kinase (CKE.C.2.7.3.2.) were decreased in white muscle after cadmium treatments, indicating decreases in the capacity of glycolysis in this tissue. Cadmium exposure induced increased glucose concentration in white muscle of fish. On the other hand, cadmium exposure at sublethal concentrations increased phosphofructo kinase and LDH in red muscle of fish. Cadmium significantly decreased total protein concentrations in liver and white muscle regardless of tissue glycogen levels. The data suggest that cadmium acts as a stressor, leading to metabolic alterations similar to those observed in starvation.

Effects of the Na(+)/H(+) exchanger monensin on intracellular pH in astroglia.

Stimulation of astroglial glucose utilization by the Na(+)/H(+) exchanger monensin is only partially blocked by ouabain. The present studies show that monensin also raises intracellular pH in astroglia. Because increased pH stimulates phosphofructokinase activity, the ouabain-insensitive portion of the stimulation of cerebral glucose utilization (CMR(glc)) appears to be due to stimulation of glycolysis by intracellular alkalinization.

Growth factor-mediated signal transduction and redox balance in isolated digestive gland cells from Mytilus galloprovincialis Lam.

In mammalian cells, a growing body of evidence indicates a relationship between cellular redox balance and tyrosine kinase-mediated cell signalling. The phosphorylative cascade activated by extracellular signals is inhibited by reducing conditions and stimulated by oxidative stress, in particular at the level of mitogen activated protein kinase (MAPK) activation. The mussel Mytilus typically shows variations in antioxidant defence systems and decreases in glutathione content in response to both natural and contaminant environmental stressors. In isolated mussel digestive gland cells, both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) have been recently demonstrated to activate tyrosine kinase receptors leading to multiple responses; among these, stimulation of the key glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). The present study investigates the possible relationship between the tyrosine kinase-mediated metabolic effects of growth factors and cellular redox balance in mussel cells. The results demonstrate that the effects of growth factors on glycolytic enzymes were abolished by cell pretreatment with the antioxidant N-acetyl-cysteine (NAC). On the other hand, in cells where the glutathione content and synthesis were lowered either in vitro (by cell pretreatment with buthionine sulfoximine (BSO)), or in vivo (by mussel exposure to Cu(2+)) the metabolic effects of growth factors were unaffected. Moreover, the results show that, in both control and glutathione-depleted cells, growth factors can also regulate the level of glutathione apparently by modulating, via phosphorylative mechanisms involving MAPK activation, the activity of gamma-glutamylcysteine synthetase (GCS), the rate limiting enzyme in GSH biosynthesis. Overall, this study extends the hypothesis that cell signalling is intimately related to redox balance in marine invertebrate cells.

Effects of Ca2+ on erythrocyte membrane skeleton-bound phosphofructokinase, ATP levels, and hemolysis.

Erythrocyte Ca2+ overload is known to occur in several different disease states, and to affect the erythrocyte membrane deformability. We show here that an increase in intracellular Ca2+ concentration in erythrocytes, induced by ionomycin, caused a reduction in ATP levels. Concomitant to the fall in ATP, a marked activation of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme in glycolysis, in the membrane skeleton fraction occurred. The increase in the membrane skeleton-bound PFK activity was most probably mediated by Ca2+, as direct addition of Ca2+ to the membrane skeleton fraction from the erythrocyte induced an enhancement of the bound PFK activity. Time-response curves revealed that erythrocyte hemolysis did not occur during the first 30 min of incubation with ionomycin, when the membrane skeleton-bound PFK was activated. Longer incubation time resulted in solubilization of the membrane skeleton-bound PFK and a concomitant hemolysis of the erythrocytes. These results suggest that the Ca2+-induced activation of membrane skeleton-bound PFK, and thereby glycolysis, the sole source of energy in erythrocytes, may be a defense mechanism to surmount the damage induced by high Ca2+ levels.

Ribose 1,5-bisphosphate regulates rat kidney cortex phosphofructokinase.

Phosphofructokinase (EC 2.7.1.11) is a major enzyme of the glycolytic pathway, catalyzing the conversion of fructose 6-phosphate to fructose 1,6-bisphosphate. In this study, we demonstrated the effect of ribose 1,5-bisphosphate on phosphofructokinase purified from rat kidney cortex. Ribose 1,5-bisphosphate relieved the phosphofructokinase from ATP inhibition and increased the affinity for fructose 6-phosphate at nanomolar concentrations. These activating effects of ribose 1,5-bisphosphate were enhanced in the presence of AMP. Ribose 1,5-bisphosphate reduced the inhibition of the phosphofructokinase induced by citrate. These results suggest that ribose 1,5-bisphosphate is an activator of rat kidney cortex phosphofructokinase and synergistically regulates the enzyme activity with AMP.

Shared biochemical properties of glucotoxicity and lipotoxicity in islets decrease citrate synthase activity and increase phosphofructokinase activity.

Diabetic states are characterized by a raised serum/islet level of triglycerides and a lowered EC50 (concentration at half-maximal stimulation) for glucose-induced insulin secretion. Culturing islets with long-chain fatty acids (FAs) replicates the basal insulin hypersecretion. In a previous study, we showed that the mechanism involved deinhibition of hexokinase by a 60% decrease in glucose-6-phosphate (G-6-P). The key event was proposed to be an increased phosphofructokinase (PFK) Vmax secondary to an upregulatory effect of the FA metabolite, long-chain acyl-coenzyme A (LC-CoA). We now show another contributory factor, a lowered content of the PFK inhibitor citrate. Citrate synthase Vmax and citrate levels were lowered 45% in rat islets cultured with 250 micromol/l oleate for 24 h. Both effects were reversed by triacsin C, an inhibitor of fatty acyl-CoA synthetase, the enzyme that generates LC-CoA. Culturing islets with high doses of glucose (16.7 mmol/l) for 48 h should also raise cytosolic LC-CoA. As predicted, citrate synthase Vmax was lowered and PFK Vmax was increased, both in a triacsin C-reversible fashion. These results show shared selected functional and biochemical properties in beta-cells of so-called glucotoxicity and lipotoxicity.

Fru 2,6-P2 and citrate: intracellular distribution in citrus tissues and effect on grapefruit leaf PFP.

Grapefruit leaf PFP was studied for its activation by fructose 2,6-bisphosphate (Fru 2,6-P2) in the forward and reverse reactions. In the glycolytic reaction, a Ka of 4.0 +/- 0.12 nM was obtained. This constant is affected by the presence of increasing concentrations of citrate (1, 5 and 20 nM) with a Ka(citrate) of 4.5 +/- 0.09, 6.9 +/- 0.05 and 38.2 +/- 1.4 respectively. The inhibition mode of citrate is competitive with Fru 2,6-P2, but non-linear in relation of increasing concentrations of the inhibitor. The intracellular distribution and concentration of the key regulatory metabolite Fru 2,6-P2 was further investigated in citrus leaves and juice cells. Fru 2,6-P2 was only found in the cytosol of juice cells. Fru 2,6-P2 was detected under both conditions with higher concentrations found under aerobiosis.

Failure of a two-state model to describe the influence of phospho(enol)pyruvate on phosphofructokinase from Escherichia coli.

A linked-function analysis is presented of the influence of the inhibitor phospho(enol)pyruvate (PEP) on the binding of fructose 6-phosphate (Fru-6-P) and MgATP to phosphofructokinase (PFK) from Escherichia coli. The results of this analysis indicate that the previously described inhibition of Fru-6-P binding by MgATP [Johnson, J. L., & Reinhart, G. D. (1992) Biochemistry 31, 11510-11518] is almost completely independent of the inhibition by PEP. Moreover, with or without the presence of MgATP, the inhibition by PEP does not conform to the behavior expected if PEP and Fru-6-P bind exclusively to different enzyme forms since the formation of a ternary complex with both PEP and Fru-6-P bound is clearly evident at high concentrations of Fru-6-P and PEP. van't Hoff analyses of the coupling interactions between PEP and Fru-6-P in the presence and absence of MgATP indicate that these couplings are driven by enthalpy. However, the influence that PEP has on MgATP binding is small and changes from being activating to being inhibitory at temperatures above 40 degrees C, revealing the importance of a compensating entropy component to the coupling interactions. The four functionally defined enzyme forms that contribute to the coupling between Fru-6-P and PEP were evaluated structurally using the fluorescence properties of the single intrinsic tryptophan as a probe. The steady-state and dynamic fluorescence emission and polarization properties of the tryptophan, as well as its susceptibility to I- quenching, indicate that the flexibility of PFK in the vicinity of the tryptophan is perturbed by the binding of ligands. The properties of free PFK do not lie between those established by the binding of Fru-6-P and PEP individually, indicating that it is structurally distinct. The properties of the ternary complex lie between those of the singly-ligated forms. Though an equilibrium mixture of two conformations of ternary complex cannot therefore be formally ruled out, no evidence obtained to date requires the presumption of this mechanistic complication.

Analysis of phosphofructokinase subunits and isozymes in ascites tumor cells and its original tissue, murine mammary gland.

Phosphofructokinase (PFK) subunits and isozymes have been examined in ascites tumor cells and murine mammary gland, the tissue from where this tumor originated. Ascites tumor was found to contain predominantly the C-type subunit, whereas the L-type subunit was more abundant in mammary gland. An altered M-type subunit of lower electrophoretic mobility was found in both cell types and no M4 homotetramers were detected in either of them. Characteristic regulatory properties of ascites tumor PFK, i.e. insensitivity to fructose-1,6-P2 activation and inhibition by P-enolpyruvate, were also observed in the mammary gland isozyme. The nature of these properties and the contribution of the distinct subunit types to fructose-1,6-P2 activation are discussed.

Actividad de la 6-fosfofructo-2-quinasa y contenido de fructosa-2.6-bifosfato en glándulas parotidas de ratas despues del tratamiento con isoproterenol / Activity of 6-phosphofructo-2-quinase (PFK-2) and contents of fructose-2.6-biphosphate in parotid glands of the rats after treatment of isoproterenol

La sialadenosis en animales de laboratorio puede ser provocada por la administración de un agonista betadrenérgico conocido como isoproterenol. Su efecto produce hipertrofia e hiperplasia de las glándulas salivales, especialmente de la glándula parótida. La 6-fosfofructo-2-quinasa (PKF-2) es una enzima bifuncional que cataliza tanto la síntesis como la degradación de la fructosa-2,6-difosfato. este metabolito es un potente activador de la 6-fosfofructo-1-quinasa (PKF-1) enzima clave en el proceso glucolítico. En este trabajo determinamos la actividad de la PKF-2 y el contenido de fructosa-2,6-bifosfato en glándulas parótidas de ratas, al ser estimuladas con varias dosis de isoproterenol

Stimulation of antioxidative enzymes by paraquat in cultured Vero cells.

Different cellular and biochemical cytotoxicity indicators have been assessed to evaluate the damages caused in Vero monkey kidney fibroblasts after 24 h exposure to paraquat (PQ), a widely used bipyridyl herbicide highly toxic through the active oxygen species that it generates by redox cycling. Cell viability, estimated by the relative neutral red uptake (EC50 = 0.5 mM), was more sensitive to PQ than cell proliferation, measured by total protein content (EC50 = 5 mM). Cell growth was more extensively inhibited in the presence of fetal bovine serum than in its absence. PQ exposure was paralleled with higher intracellular specific activities of lactate dehydrogenase and phosphofructokinase, directly assayed in the 96-wells culture plates, whereas those of succinate dehydrogenase raised only 1.35-fold and hexosaminidase was almost unaltered by PQ. The intracellular specific activities of several antioxidative enzymes were also directly determined in the microtiter plates. At the highest PQ concentration used (10 mM) glutathione reductase activity increased 4-fold, while superoxide dismutase and glucose-6-P dehydrogenase activities increased 2- and 1.8-fold compared to untreated control cells. An 1.9-fold raise in glutathione-S-transferase activity was also observed in exposed cells. The results show the action in Vero cells of a complex regulatory defensive network against PQ-induced damages.

A pH-dependent allosteric transition in Ascaris suum phosphofructokinase distinct from that observed with fructose 2,6-bisphosphate.

Ascaris suum phosphofructokinase exhibits dramatic shifts in its circular dichroic spectra in the pH range 6 to 8. These shifts are quite distinct from those induced by the activators AMP and fructose 2,6-bisphosphate. Concomitant with these pH-induced spectral shifts, the enzyme also displays changes in its allosteric behavior. Inorganic ions such as K+, NH+4, SO4(2-), and PO4(3-) also cause CD-spectral shifts similar to those produced by a change in pH. Based on the evidence derived from gel filtration and sedimentation equilibrium studies, the observed CD-spectral shifts are interpreted as due to conformational changes in the enzyme tetramer rather than due to a change in its aggregation state. Further, since the pK value of 6.4 obtained from pH dependence of increase in ellipticity at 210 nm agrees very well with the pK value of 6.8 for the loss of ATP inhibition due to modification of a histidine residue (G. S. J. Rao, B. A. Wariso, P. F. Cook, and B. G. Harris (1987) J. Biol. Chem. 262, 14068-14073), it is concluded that a single histidine residue in the ATP-inhibitory site acts as a trigger for the structural changes accompanying ATP inhibition of the enzyme. This view is strongly supported by the observation that the enzyme desensitized to ATP inhibition by chemical modification of a histidine residue in the ATP-inhibitory site shows absolutely no change in its CD spectrum in the pH range 6 to 8. This study demonstrates that the mechanism of activation of phosphofructokinase at higher pH and by inorganic ions involves conformational transitions that are quite distinct from those induced by AMP and fructose 2,6-bisphosphate. A scheme is presented that incorporates all of the different states of the enzyme dependent upon effectors and pH.

Urea and salt effects on enzymes from estivating and non-estivating amphibians.

The effects of urea, cations (K+,NH4,Na+,Cs+,Li+), and trimethylamines on the maximal activities and kinetic properties of pyruvate kinase (PK) and phosphofructokinase (PFK) from skeletal muscle were analyzed in two anuran amphibians, an estivating species, the spadefoot toad Scaphiopus couchii, and a semi-aquatic species, the leopard frog Rana pipiens. Urea, which accumulates naturally to levels of 200-300 mM during estivation in toads, had only minor effects on the Vmax, kinetic constants and pH curves of PK from either species and no effects on PFK Vmax or kinetic constants. Trimethylamine oxide neither affected enzyme activity directly or changed enzyme response to urea. By contrast, high KCl (200 mM) lowered the Vmax of toad PFK and of PK from both species and altered the Km values for both substrates of frog PFK. Other cations were even more inhibitory; for example, the Vmax of PK from either species was reduced by more than 80% by the addition of 200 mM NH4Cl, NaCl, CsCi, or LiCl. High KCl also significantly changed the Km values for substrates of toad lactate dehydrogenase and strongly reduced the Vmax of glutamate dehydrogenase and NAD-dependent isocitrate dehydrogenase in both species whereas 300 mM urea had relatively little effect on these enzymes. The perturbing effect of urea on enzymes and the counteracting effect of trimethylamines that has been reported for elasmobranch fishes (that maintain high concentrations of both solutes naturally) does not appear to apply to amphibian enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of maternal nicotine exposure on neonatal lung carbohydrate metabolism.

The influence of maternal nicotine exposure (1 mg/kg body mass/day) during pregnancy and lactation on energy metabolism of lung tissue of neonatal rats were investigated. The glucose turnover of the lung tissue of the neonatal rats exposed to nicotine via the placenta and mother's milk was 86.4% higher than that of the controls. Glycolysis was however suppressed by 22.7% (P < 0.01). The adenine nucleotide pool (ATP+ADP+AMP) was 32.8% higher for the lungs of the 3 week old neonates exposed to nicotine than that of the control rat lung. After 4 weeks of nicotine withdrawal glycolysis of those animals exposed to nicotine were still inhibited to the same extent than during exposure. The adenine nucleotide pool was 69.95% higher than that of the controls. It is proposed that the inhibition of glycolysis was due to the high ATP/ADP ratio of the lungs of the nicotine exposed rats.