Effects Of Metabolic Inhibitors Research Articles

Effects of metabolic inhibition on conduction, Ca transients, and arrhythmia vulnerability in embryonic mouse hearts

Developing myocardium is more dependent on glycolysis than adult myocardium, yet the effects of selectively inhibiting glycolysis versus oxidative phosphorylation on embryonic heart function have not been well characterized. Accordingly, we investigated how selective metabolic inhibition affects membrane voltage and intracellular Ca (Ca(i)) transients in embryonic mouse hearts, including their susceptibility to arrhythmias. A total of 136 isolated embryonic mouse hearts were exposed to either 1) 2-deoxyglucose (2DG; 10 mM) or iodoacetate (IAA; 0.1 mM) with 10 mM pyruvate in place of glucose to selectively inhibit glycolysis or 2) the mitochondrial uncoupler protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 500 nM) with 10 mM glucose present to selectively inhibit oxidative phosphorylation. Using confocal imaging, we found that mitochondrial membrane potential monitored with tetramethylrhodamine methyl ester (200 nM) remained stable with 2DG or IAA but depolarized within 5 min after exposure to FCCP. IAA and FCCP decreased heart rate, inhibited Ca(i) transient amplitude, shortened action potential duration at 80% repolarization (APD(80)), and prolonged atrioventricular conduction time to similar extents. Although 2DG decreased heart rate and Ca(i) transient amplitude, it did not significantly affect APD(80) and AV conduction time. In addition, spontaneous arrhythmias occurred in 77 of 136 embryonic hearts (57%) after exposure to IAA (28/53) or FCCP (49/83). There were no significant differences in the types or incidence of arrhythmias induced by IAA and FCCP. These data support the idea that both glycolysis and oxidative phosphorylation play critical metabolic roles in regulating cardiac function in the embryonic mouse heart.

Effect of metabolic inhibitors on ATP and citrate content in PC3 prostate cancer cells

In normal prostate epithelial cells low m-aconitase activity decreases citrate oxidation leading to citrate accumulation. In prostate cancer cells m-aconitase activity is increased and citrate content is lower. The effect of inhibition of m-aconitase on ATP production by prostate cancer cells (PC3) is not known nor is the contribution of glycolysis versus respiration. ATP content of PC3 cells as affected by inhibition of m-aconitase (fluoroacetate (FA), zinc), inhibition of glycolysis (2DxG), or respiration (DNP, oligomycin) was determined. The ability to maintain ATP using glucose or glutamine as sole substrate was also determined. Intermediates including ATP, lactate, glucose, and glutamine were assayed in neutralized perchloric acid (PCA) cell extracts, virgin, and conditioned medium by enzymatic fluorometry. Data show that inhibition of m-aconitase, glycolysis, or respiration alone did not decrease ATP content. Inhibition of both glycolysis and respiration were required to decrease ATP content. PC3 cells were able to produce ATP with either glucose or glutamine as sole substrate. Though FA clearly inhibited m-aconitase there was no evidence that zinc had a similar effect. PC3 cells can support ATP production when m-aconitase is inhibited by using glycolysis or oxidation of substrate (e.g., glutamine) entering the TCA cycle distal to citrate.

Poly- β-hydroxybutyrate accumulation in Nostoc muscorum: Effects of metabolic inhibitors

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.

NS-398, a Cyclooxygenase-2-Specific Inhibitor, Delays Skeletal Muscle Healing by Decreasing Regeneration and Promoting Fibrosis

Nonsteroidal anti-inflammatory drugs are often prescribed after muscle injury. However, the effect of nonsteroidal anti-inflammatory drugs on muscle healing remains primarily controversial. To further examine the validity of using these drugs after muscle injury, we investigated the working mechanism of NS-398, a cyclooxygenase-2-specific inhibitor. In vitro experiments showed that NS-398 inhibited the proliferation and maturation of differentiated myogenic precursor cells, suggesting a detrimental effect on skeletal muscle healing. Using a mouse laceration model, we analyzed the in vivo effect of NS-398 on skeletal muscle healing at time points up to 4 weeks after injury. The in vivo results revealed delayed muscle regeneration at early time points after injury in the NS-398-treated mice. Compared to controls, lacerated muscles treated with NS-398 expressed higher levels of transforming growth factor-beta1, which corresponded with increased fibrosis. In addition, transforming growth factor-beta1 co-localized with myostatin, a negative regulator of skeletal muscle growth. We also found reduced neutrophil and macrophage infiltration in treated muscles, indicating that the delayed skeletal muscle healing observed after NS-398 treatment could be influenced by the anti-inflammatory effect of NS-398. Our findings suggest that the use of cyclooxygenase-2-specific inhibitors to treat skeletal muscle injuries warrants caution because they may interfere with muscle healing.

295. Peroxisome proliferator activated receptor-alpha is involved in H+-monocarboxylate transporter 2 and catalase protein expression in cultured preimplantation mouse embryos

Cleavage stage embryos consume pyruvate before switching to glucose as the major energy substrate for blastocyst formation. This switch is conditional, because freshly collected two-cell embryos form blastocysts without glucose by increasing pyruvate consumption. Zygotes cultured without glucose cannot adapt in this way and degenerate, but paradoxically demonstrate upregulation of the H+-monocarboxylate transporter protein, MCT2, in morulae. MCT2 is a high affinity transporter implicated in redox shuttling for peroxisomal beta-oxidation of fatty acids.3 Fatty acids may provide energy for embryos2 but peroxisomal beta-oxidation has not been explored in preimplantation development. Rat oocytes possess a primitive peroxisomal system.1 The possibility therefore exists that MCT2 may also be linked to fatty acid metabolism in embryos. Peroxisome proliferator activated receptor (PPAR)-alpha is a transcriptional regulator of fatty acid transport and beta-oxidation, and controls expression of catalase, a major peroxisomal enzyme. This investigation explores the role of PPAR-α in the glucose-driven control of MCT2 expression in mouse embryos. Zygotes (18 h post-hCG) were cultured in KSOM in the presence or absence of glucose, or KSOM with selective agonists of PPAR-α, fenofibrate and WY 14643. Expression of MCT2 and catalase was analysed by confocal laser scanning immunohistochemistry and western blot. Results confirm the presence of catalase throughout preimplantation development. With glucose, cytoplasmic immunoreactivity for catalase was punctate and diffuse, while MCT2 was localised to apical membranes of outer blastomeres in morulae. Without glucose, catalase and MCT2 expression were increased with notable localisation of catalase to nuclei. This response was reflected in morulae cultured in the presence of glucose and PPAR-α agonists. These data suggest that PPAR-α plays a role in controlling catalase and MCT2 expression in embryos, and that conditions in the absence of glucose are more conducive for PPAR-α activation. (1)Figueroa C, Kawada ME, Veliz LP, Hidalgo U, Barros C, Gonzalez S and Santos MJ (2000) Peroxisomal proteins in rat gametes. Cell Biochem Biophys 32, 259–268.(2)Hewitson LC, Martin KL and Leese HJ (1996) Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses. Mol Reprod Dev 43, 323–330.(3)McClelland GB, Khanna S, Gonzalez GF, Butz CE and Brooks GA (2003) Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system? Biochem Biophys Res Commun 304, 130–135.

Protection from the effects of metabolic inhibition and reperfusion in contracting isolated ventricular myocytes via protein kinase C activation

The protective effects of the PKC activator Phorbol 12-myristate 13-acetate (PMA) were investigated in electrically field stimulated (EFS) rat isolated ventricular myocytes following 7 min of metabolic inhibition induced by cyanide, iodoacetic acid and substrate removal, followed by reperfusion. PMA reduced reperfusion damage and increased functional recovery (response to EFS) following 10 min reperfusion from 20.0 ± 10.7% of control myocytes to 90.0 ± 7.2% following 5 min PMA pre-treatment (p<0.001). PMA significantly increased the time from the onset of MI before the myocytes failed to respond to EFS from 135 ± 19s in control cells to 200 ± 14s in PMA pre-treated cells (p<0.05). Additionally, there was an increase in the time to rigor with PMA pre-treated cells entering rigor 255 ± 17s after MI compared to 174 ±15s in control cell (p<0.05), indicating a delay in ATP depletion. During MI PMA pre-treated cells showed a significantly smaller increase in [Ca 2+]i compared to control myocytes. Following reperfusion the majority of PMA pre-treated myocytes recovered calcium transients in response to EFS and diastolic Ca 2+ levels not significantly different to those seen prior to metabolic inhibition. Activation of PKC is thought to involve translocation to the particulate fraction. Our results demonstrate the presence of PKC-α, β, γ, δ, ε, ι, λ/ζ in rat ventricular myocytes, all of which translocate to the membrane in response to PMA.

Effects of metabolic inhibition on phosphorylation levels of PKC isoforms in the guinea pig taenia caeci.

We investigated which isoform of protein kinase C (PKC) is responsible to metabolic inhibition in the guinea pig taenia caeci with respect to their phosphorylation levels. By Western blot analysis using isoform-specific antibodies, at least four isoforms of PKC, alpha, beta2, epsilon and zeta were identified in the taenia. Prolonged metabolic inhibition of hypoxia, hypoxia+glucose depletion, and addition of cyanide (all in the presence of high K+) for more than 60 min, but not glucose-depletion only, elicited dephosphorylation of PKCs, alpha, beta2 and epsilon, except zeta. Ca2+ depletion from the medium prevented the dephosphorylation of PKCs induced by hypoxia, and apparently inhibited the dephosphorylation induced by hypoxia+glucose depletion. Acute treatment with hypoxia for 10-30 min elicited a gradual dephosphorylation of PKCbeta2, but not of other tested PKC isoforms. Considering the ATP level under various metabolic conditions reported previously, PKCbeta2 is suggested to be primarily responsible to hypoxia, and its dephosphorylation is closely associated with the alteration of adenylate compounds in the cell. Re-oxygenation after prolonged hypoxia did not restore the phosphoryation level of any tested PKCs, suggesting that the dephosphorylation of PKCs is associated with the irreversible damage of the cell under hypoxia. Presumably, the dephosphorylaton of PKCs, particularly PKCbeta2, plays a role in the signal transduction pathway under metabolic inhibition of the taenia, as reported in proliferative and pathophysiological processes in many other cells.

The effects of metabolic inhibition on intracellular calcium and contractility of human myometrium

Objective Hypoxia occurs in the uterus during labour and may contribute to dysfunctional labours. We wanted to establish its effects on pregnant human myometrium and elucidate the mechanisms involved. Design Scientific study. Setting University Hospital and laboratories. Population or Sample Term pregnant women. Methods We measured contractions and intracellular [Ca2+] ([Ca2+]i), in biopsies from term pregnant women undergoing elective caesarean section, and used cyanide to block oxidative phosphorylation. Main outcome measures Changes in contractility and calcium. Results Although basal levels of [Ca2+]i and tone rose, spontaneous and agonist-induced Ca2+ transients and phasic contractions were rapidly reduced and abolished by cyanide. Neither stimulation of the uterus with oxytocin nor the Ca channel agonist, Bay K8644, prevented the changes produced by cyanide. The tonic force produced by depolarising the myometrium was also decreased by cyanide, but slowly recovered towards control levels, whereas [Ca2+]i was maintained throughout. Similar data were obtained when nitrogen, rather than cyanide, was applied to the depolarised uterus. Conclusions Impairment of oxidative phosphorylation is a potent depressor of phasic activity in human myometrium, irrespective of how it is produced, and our data suggest its effects lie at and beyond the surface membrane. Stimulation of the hypoxic uterus was not effective, which may explain the unpredictability of oxytocin application in some dysfunctional labours.

The effects of metabolic inhibition on intracellular calcium and contractility of human myometrium

Hypoxia occurs in the uterus during labour and may contribute to dysfunctional labours. We wanted to establish its effects on pregnant human myometrium and elucidate the mechanisms involved. Scientific study. University Hospital and laboratories. Term pregnant women. We measured contractions and intracellular [Ca(2+)] ([Ca(2+)](i)), in biopsies from term pregnant women undergoing elective caesarean section, and used cyanide to block oxidative phosphorylation. Changes in contractility and calcium. Although basal levels of [Ca(2+)](i) and tone rose, spontaneous and agonist-induced Ca(2+) transients and phasic contractions were rapidly reduced and abolished by cyanide. Neither stimulation of the uterus with oxytocin nor the Ca channel agonist, Bay K8644, prevented the changes produced by cyanide. The tonic force produced by depolarising the myometrium was also decreased by cyanide, but slowly recovered towards control levels, whereas [Ca(2+)](i) was maintained throughout. Similar data were obtained when nitrogen, rather than cyanide, was applied to the depolarised uterus. Impairment of oxidative phosphorylation is a potent depressor of phasic activity in human myometrium, irrespective of how it is produced, and our data suggest its effects lie at and beyond the surface membrane. Stimulation of the hypoxic uterus was not effective, which may explain the unpredictability of oxytocin application in some dysfunctional labours.

In vivo identification of aflatoxin-induced free radicals in rat bile.

Aflatoxin B1 (AFB1) is a potent hepatocarcinogen. We have recently detected [via electron spin resonance (ESR) spectroscopy] free radicals in vivo in rat bile following AFB1 metabolism using the spin trapping [alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN)] technique. The aim of the present study was to identify the trapped free radical intermediates from the in vivo hepatic metabolism of AFB1. Rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (1 g/kg i.p.), and bile was collected over a period of 1 h at 20 min intervals. On-line high performance liquid chromatography (HPLC) coupled to ESR was used to identify an arachidonic acid-derived radical adduct of 4-POBN in rat bile, and a methyl adduct of 4-POBN from the reaction of hydroxyl radicals with carbon-13-labeled dimethyl sulfoxide ((13)C-DMSO). The effect of metabolic inhibitors, such as desferoxamine mesylate (DFO), an iron chelator, 2-dimethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF) 525A, a cytochrome P-450 inhibitor, and gadolinium chloride (GdCl(3)), a Kupffer cell inactivator, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in radical formation as a result of DFO, SKF525A and GdCl(3) inhibition. Trapped 4-POBN radical adducts were also detected in rat bile following the in vivo metabolism of aflatoxin-M1, one of the hydroxylated metabolites of AFB1.

Differential Sensitivity of Inward Rectifier K+Channels to Metabolic Inhibitors

Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.

Effect of Metabolic Inhibitors on Red Light‐induced Resistance of Broad Bean (Vicia faba L.) against Botrytis cinerea

AbstractA possible relationship between the synthetic activities of broad bean leaves and red light‐induced resistance against Botrytis cinerea was examined. Both lesion formation and fungal development were suppressed under red light irradiation. In red light‐irradiated leaflets, restricted chocolate lesions were observed only on their inoculated adaxial surfaces, whereas black lesions extended to non‐inoculated abaxial surfaces in the dark. In microscopic studies, clear cell browning was recognized only in red light‐irradiated leaves. Treatment of leaves with an inhibitor of photosynthesis, 3‐(3,4‐dichlorophenyl)‐1,1‐dimethyl urea (DCMU) (25 μm), and an inhibitor of protein synthesis, cycloheximide (25 μg/ml), inhibited the induction of resistance by red light irradiation. Moreover, DCMU‐ and cycloheximide‐treated leaflets produced black necrotic lesions even under red light irradiation for 48 h, which extended to the non‐inoculated abaxial surface. DCMU did not affect fungal growth, spore production or spore germination of B. cinerea. By contrast, cycloheximide at 25 μg/ml suppressed mycelial growth, but not spore production, whereas cycloheximide at the same concentration completely inhibited spore germination. These results suggest that red light‐induced resistance possibly depends on host photosynthesis and protein synthesis.

Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML

The objective of this study was to determine the role of transport proteins in daunorubicin (Dnr) accumulation and efflux in leukemia cells from 36 patients with acute myeloid leukemia (AML). Mononuclear cells were isolated and incubated with 1 μM Dnr with/without addition of 3 μM cyclosporin A (CyA) or metabolic inhibitors (MI). Cellular Dnr concentration in leukemia blast cells was measured with flow cytometry. After washing and reincubation of the cells in drug-free medium, Dnr efflux was followed with/without addition of CyA or MI. Levels of mRNA expression for mdr1, multidrug resistance associated protein (mrp) and lung resistance protein (lrp) were determined with reverse transcriptase-polymerase chain reaction (RT-PCR). MI enhanced cellular Dnr accumulation to a higher extent than CyA whereas CyA reduced Dnr efflux more efficiently than MI ( P<0.001). There was a significant difference in Dnr accumulation between samples with low and high mdr1 mRNA levels but only in the presence of MI or CyA. Our results imply that other factors than P-glycoprotein (Pgp) are of major importance for in vitro Dnr accumulation in AML blasts and that the role of Pgp as a drug efflux pump is not conclusive.

In vivo detection of aflatoxin-induced lipid free radicals in rat bile

Aflatoxin B1 (AFB1), a potent hepatotoxin and hepatocarcinogen, is metabolized in the liver via cytochrome P-450 to an AFB1-8,9-epoxide intermediate. The formation of the AFB1-8,9-epoxide correlates with the pathological changes observed in numerous mammalian species. Oxidative damage has been postulated to play a major role in the mechanisms associated with AFB1-induced cytotoxicity and carcinogenecity in mammalian species. The aim of this study was to detect and identify free radical intermediates from the hepatic metabolism of AFB1 in vivo. Rat bile ducts were cannulated and rats were treated simultaneously with AFB1 (3 mg/kg i.p.) and the spin trapping agent 4-POBN (α-(4-pyridyl-1-oxide)- N- tert-butyl nitrone) (1 g/kg i.p.), and bile was collected over a period of 2 h at 20-min intervals. ESR spectroscopy was used to detect a carbon-centered radical adduct of 4-POBN in rat bile. The effect of metabolic inhibitors, such as deferoxamine mesylate (DFO), an iron chelator, and SKF 525A, a cytochrome P-450 inhibitor, on in vivo aflatoxin-induced free radical formation were also studied. It was found that there was a significant decrease in free radical formation by pre-treatment with both DFO and SKF 525A. This indicates that oxidation of AFB1 generates free radical species via CYP metabolism and an iron-mediated redox mechanism.

Glucose metabolism and pulsatile insulin release from isolated islets.

The effects of metabolic inhibition on insulin release and the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were studied in individually perifused pancreatic islets from ob/ob mice. The modest basal secretion in the presence of 3 mmol/l glucose was pulsatile with a frequency of approximately 0.2/min, although [Ca(2+)](i) was stable at approximately 100 nmol/l. Introduction of 11 mmol/l glucose resulted in large amplitude oscillations of [Ca(2+)](i) and almost 20-fold stimulation of average secretion manifested as increased amplitude of the insulin pulses without change in frequency. Inhibition of glycolysis with iodoacetamide or mitochondrial metabolism with dinitrophenol or antimycin A reduced glucose-stimulated secretion back to basal levels with maintained pulsatility. The [Ca(2+)](i) responses to the metabolic inhibitors were more complex, but in general there was an initial peak and eventually sustained elevation without oscillations. When introduced in the presence of 3 mmol/l glucose, the metabolic inhibitors tended to increase the amplitude of the insulin pulses, although the simultaneous elevation in [Ca(2+)](i) occurred without oscillations. The data indicate that pulsatile secretion is regulated by factors other than [Ca(2+)](i) under basal conditions and after metabolic inhibition. Although pulsatile secretion can be driven by oscillations in metabolism when [Ca(2+)](i) is stable, it was not possible from the present data to determine whether insulin pulses have a glycolytic or mitochondrial origin.

Differences in the Calcium-Handling Response of Isolated Rat and Guinea-Pig Cardiomyocytes to Metabolic Inhibition: Implications for Cell Damage

Species differences in response to hypoxic damage have been observed in studies using whole hearts. The aims of this study were to determine whether (i) species differences in response to simulated hypoxia could be detected at the level of the single myocyte, and (ii) there were any interspecies differences in the Ca2+ handling properties of the cells. Ventricular myocytes were isolated from hearts of adult rats and guinea-pigs and electrically stimulated on the stage of a fluorescence microscope. Cell length was measured using an edge-tracking device, and total intracellular [Ca2+] ([Ca2+]i) determined using indo-1. Cells were exposed to metabolic inhibition (MI) (2.5 mM NaCN and no glucose) to simulate hypoxia followed by washout of CN and re-addition of glucose ('reperfusion'). Following exposure to MI, rat cells underwent rigor contracture in 18.8+/-0.8 min (n = 80 cells), whereas the time was longer for guinea-pig cells (32.9+/-1.2 min, n = 83) (P<0.001). If cells were reperfused after 1-5 min in rigor, then rat cells showed improved morphological recovery compared with guinea-pig cells (P< 0.05); thereafter recovery decreased with increasing time spent in rigor, and was similar in both groups. In indo-1 loaded cells, [Ca2+]i was significantly increased in cells from both species at the end of MI; however, the actual increase was much higher in guinea-pig cells. Upon reperfusion, [Ca2+]i recovered fully in rat cells, but in guinea-pig cells there was no significant decrease. The restoration of [Ca2+]i to normal levels in rat cells following MI was associated with improved contractile recovery compared with guinea-pig cells. We conclude that rat cells are more resistant to effects of MI than are guinea-pig cells; this may be related to species differences in Ca2+ handling during and following exposure to MI.

Effects of metabolic inhibitors on ingestive behavior and physiology in preweanling rat pups

We have previously demonstrated that administration of 2-Mercaptoacetate (MA) stimulates independent intake after 1h in 12 and 15-day-old rat pups, but not younger pups. MA also produces decreases in β-HBA levels, consistent with the development of a role of altered fatty acid oxidation in modulating independent ingestion in rat pups by 12 days of age. The present experiments extended investigations of the role of changes in energy utilization in young rats by investigating the duration of the effects of altered fatty acid oxidation and the effects of combined blockade of fatty acid oxidation and glucose utilization. Pups were tested at 9, 12 or 15 days of age 3 or 6h following administration of a dose of 0, 11·4, 22·8, 45·6 or 91·2mg/kg MA. In pups aged 12 and 15 days, moderate doses of MA stimulated intake 3h, but not 6h, following administration. Administration of the highest dose of MA produced significant decreases in β-HBA levels in pups at all ages when tested after 3h, but not after 6h. In the second set of experiments, behavioral and physiological responses to administration of MA (0, 11·4 or 22·.8mg/kg) combined with 2-Deoxyglucose (2-DG: 0, 100 or 200mg/kg) were investigated in pups aged 6, 9, 12 or 15 days of age. The results demonstrated that while administration of 2-DG produced physiological responses, intake was not stimulated at any age by 2-DG alone or in combination with MA. In fact, in 12 and 15-day-old pups, administration of 2-DG blocked the stimulatory effects of administration of MA. Therefore, while altered utilization of glucose does not appear to be an effective stimulus for increased independent ingestion in pups at this age, altered fatty acid oxidation may be an early metabolic modulator of intake.

Effects of Low Oxygen Concentrations and Metabolic Inhibitors on Proteoglycan and Protein Synthesis Rates in the Intervertebral Disc

The intervertebral disc is the largest asvacular structure in the body; consequently, there are steep gradients in O2 concentration, with PO2 falling to as low as 1% O2 in the centre of the disc. We investigated the effect of O2 concentration on the rates of O2 consumption, lactate production, and sulphate and proline incorporation in bovine caudal discs. We also investigated the effects of metabolic inhibitors of energy production pathways on tracer incorporation. Samples from the outer annulus and nucleus pulposus were incubated for 24 hours in 1-21% O2. Rates were measured during the last 4 hours of incubation. As O2 concentration was reduced from 10 to 1% O2, O2 consumption rates fell by around 75% and lactate production rates almost doubled; the bovine discs thus showed a positive progressive Pasteur effect. Incorporation rates of [3H]proline and [35S]sulphate were lowest at 1% O2. In the nucleus, but not in the outer annulus, the rate of [35S]incorporation peaked at 5% O2, where it was 30% greater than at 21% O2 and 150% greater than at 1% O2. The competitive glycolysis inhibitor 2-deoxyglucose, the oxidative phosphorylation uncoupler 2,4-dinitrophenol, and the oxidative phosphorylation inhibitor sodium azide all markedly reduced sulphate incorporation. These results, together with previous measurements of CO2 production rates, suggest that a functionally significant fraction of the disc's energy is supplied by oxidative phosphorylation. However, low levels of PO2, 2,4-dinitrophenol, and sodium azide have been reported to reduce sulphate incorporation in articular cartilage, a tissue that derives its energy almost entirely from glycolysis.

Cardioprotection of preconditioning by metabolic inhibition in the rat ventricular myocyte. Involvement of kappa-opioid receptor.

To determine whether opioid receptors (ORs) are involved in the delayed cardioprotection of ischemic preconditioning (IP), the effect of severe metabolic inhibition (MI) with a glucose-free buffer that contained sodium cyanide and 2-deoxy-D-glucose on the viability of isolated rat ventricular myocytes was first determined 20 hours after preconditioning with a sublethal metabolic inhibition (MIP) with a glucose-free buffer that contained 2-deoxy-D-glucose and lactate for 30 minutes in the presence of OR antagonists. With the use of trypan blue exclusion as an index of cell viability, severe MI killed >60% of the cells and the value increased significantly after MIP. In the presence of 5x10(-6) mol/L nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, but not 5x10(-6) mol/L CTOP, a selective mu-OR antagonist, or 5x10(-6) mol/L naltrindole, a selective delta-OR antagonist, the cardioprotection of MIP was significantly attenuated. To verify the role of kappa-OR, we studied the effects of severe MI after pretreatment with the kappa-OR agonist U50,488H (UP) for 30 minutes. U50,488H at 3x10(-6) to 1x10(-4) mol/L increased cell viability concentration-dependently with an EC50 of 3.311x10(-6) mol/L. In the presence of 5x10(-6) nor-BNI, the cardioprotection of UP (3x10(-5) mol/L) was blocked. A time course study showed that UP-induced cardioprotection occurred in 2 windows: the first occurred approximately 1 hour later and the other occurred 16 to 20 hours later. Additional studies on cell contraction and intracellular Ca2+ ([Ca2+]i) revealed that both UP and MIP attenuated the inhibitory effects of severe MI on contractility and electrically induced [Ca2+]i transient in single ventricular myocytes. On blockade of protein kinase C, the delayed cardioprotections of UP and MIP were significantly attenuated. In conclusion, the results of the present study have provided evidence that kappa-OR mediates the cardioprotection of MIP, which may involve protein kinase C and [Ca2+]i.

O- and N-demethylation of Venlafaxine In Vitro by Human Liver Microsomes and by Microsomes from cDNA-Transfected Cells: Effect of Metabolic Inhibitors and SSRI Antidepressants

The biotransformation of venlafaxine (VF) into its two major metabolites, O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV) was studied in vitro with human liver microsomes and with microsomes containing individual human cytochromes from cDNA-transfected human lymphoblastoid cells. VF was coincubated with selective cytochrome P450 (CYP) inhibitors and several selective serotonin reuptake inhibitors (SSRIs) to assess their inhibitory effect on VF metabolism. Formation rates for ODV incubated with human microsomes were consistent with Michaelis-Menten kinetics for a single-enzyme mediated reaction with substrate inhibition. Mean parameters determined by non-linear regression were: Vmax = 0.36 nmol/min/mg protein, Km = 41 μM, and Ks 22901 μM (Ks represents a constant which reflects the degree of substrate inhibition). Quinidine (QUI) was a potent inhibitor of ODV formation with a Ki of 0.04 μM, and paroxetine (PX) was the most potent SSRI at inhibiting ODV formation with a mean Ki value of 0.17 μM. Studies using expressed cytochromes showed that ODV was formed by CYP2C9, −2C19, and −2D6. CYP2D6 was dominant with the lowest Km, 23.2 μM, and highest intrinsic clearance (Vmax/Km ratio). No unique model was applicable to the formation of NDV for all four livers tested. Parameters determined by applying a single-enzyme model were Vmax = 2.14 nmol/min/mg protein, and Km = 2504 μM. Ketoconazole was a potent inhibitor of NDV production, although its inhibitory activity was not as great as observed with pure 3A substrates. NDV formation was also reduced by 42% by a polyclonal rabbit antibody against rat liver CYP3A1. Studies using expressed cytochromes showed that NDV was formed by CYP2C9, −2C19, and −3A4. The highest intrinsic clearance was attributable to CYP2C19 and the lowest to CYP3A4. However the high in vivo abundance of 3A isoforms will magnify the importance of this cytochrome. Fluvoxamine (FX), at a concentration of 20 μM, decreased NDV production by 46% consistent with the capacity of FX to inhibit CYP3A, 2C9, and 2C19. These results are consistent with previous studies that show CYP2D6 and −3A4 play important roles in the formation of ODV and NDV, respectively. In addition we have shown that several other CYPs have important roles in the biotransformation of VF.