Protects Rat Brain Research Articles

Electrical stimulation of cerebellar fastigial nucleus protects rat brain, in vitro, from staurosporine-induced apoptosis.

Electrical stimulation of the cerebellar fastigial nucleus (FN) elicits a prolonged ( approximately 10 days) and substantial (50-80%) protection against ischemic and excitotoxic injuries. The mechanism(s) of protection are unknown. We investigated whether FN stimulation directly protects brain cells against apoptotic cell death in an in vitro rat brain slice culture model. Rats were electrically stimulated in FN or, as control, the cerebellar dentate nucleus (DN). Coronal slices through the forebrain were explanted, exposed to staurosporine, harvested, and analyzed for caspase-3 activity by a fluorescence assay. FN, but not DN, stimulation significantly reduced staurosporine-induced caspase-3 activity by 39 +/- 7% at 3 h, 31 +/- 3% at 6 h and 26 +/- 4% at 10 h of incubation. Immunocytochemistry revealed FN-specific reductions in activated caspase-3 mainly in glial-like cells throughout the forebrain. FN stimulation also results in a 56.5% reduction in cytochrome c release upon staurosporine incubation. We conclude that neuroprotection elicited from FN stimulation can directly modify the sensitivity of brain cells to apoptotic stimuli and thereby suppress staurosporine induced apoptosis in adult rat brain slices. This model indicates that neuroprotection can be studied in vitro and provides new insight into the potential role of glial cells in ischemic protection of neurons induced by FN stimulation.

Permanent cerebral hypoperfusion: 'preconditioning-like' effects on rat energy metabolism towards acute systemic hypotension.

Chronic cerebrovascular disorders are often complicated by additional temporary ischaemic insults, resulting in substantial deterioration of brain energy metabolism. In the present study, chronic limitations of oxygen supply were induced in Wistar rats by 2 weeks of permanent bilateral common carotid artery occlusion (2-vo) to initiate a 'preconditioning-like' effect that protects rat brain energy metabolism against further acute systemic hypotension (15 min). Haemodynamic parameters, arterial blood gases and body temperature were monitored. Energy metabolites were determined in rat parietotemporal cerebral cortex: adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), phosphocreatine (PCr), and adenosine by the high-pressure liquid chromatography (HPLC) technique and lactate spectrophotometrically. After 2 weeks, permanent 2-vo led to a significant decrease in the concentrations of cortical tissue ATP and PCr, from 3.06+/-0.48 to 2. 09+/-0.28 and from 4.27+/-0.63 to 3.35+/-0.41 micromol/g, respectively. These changes were associated with a two-fold increase in AMP and adenosine. Acute systemic hypotension alone (non-preconditioning) reduced ATP and PCr drastically, to 0.97+/-0. 51 and 1.76+/-1.23 micromol/g. Tissue concentrations of lactate, AMP, and adenosine were markedly increased, three- to five-fold, in 'non-preconditioned' brain tissue. In contrast, after 2 weeks of 2-vo acute hypotension did not significantly alter the cortical energy state any further. The effects of preconditioning on tissue ATP and PCr were most pronounced at 5 min and 48 h after reperfusion. In conclusion, permanent 2-vo seems to activate compensatory mechanisms, which effectively protect the rat's cortical energy metabolism against an additional ischaemic attack ('preconditioning-like' effect).

The novel immunosuppressant SDZ-RAD protects rat brain slices from cyclosporine-induced reduction of high-energy phosphates.

1. SDZ-RAD, 40-O-(2-hydroxyethyl)-rapamycin, is a novel macrolide immunosuppressant. Because of its synergistic interaction, SDZ-RAD is under clinical investigation as immunosuppressant in combination with cyclosporine after organ transplantation. Neurotoxicity is a critical side-effect of cyclosporine. 2. We studied the effect of SDZ-RAD and its combination with cyclosporine on high-energy phosphates, phosphocreatine (PCr) and nucleoside triphosphates (NTP), in brain slices using 31P-magnetic resonance spectroscopy (MRS). 3. Cyclosporine significantly reduced high-energy phosphates after 2 h in a dose-dependent manner (100 micrograms l-1: 93 +/- 3% of control (NTP), 91 +/- 3% (PCr); 500 micrograms l-1: 84 +/- 2% (NTP), 73 +/- 2 (PCr); 5000 micrograms l-1: 68 +/- 3% (NTP), 55 +/- 5% (PCr); n = 6; P < 0.02). 4. In contrast, after perfusion for 2 h, SDZ-RAD (500 micrograms l-1 and 5000 micrograms l-1) significantly increased high-energy phosphate concentrations in the brain slices (P < 0.02). Even at the lowest concentration, SDZ-RAD protected brain energy metabolism against cyclosporine toxicity: 100 micrograms l-1 SDZ-RAD + 5000 micrograms l-1 cyclosporine: 86 +/- 3% (NTP), 83 +/- 7% (PCr), n = 3, P < 0.03 compared to cyclosporine alone. 5. As evaluated using an algorithm based on Loewe isobolograms, the effects of SDZ-RAD/cyclosporine combinations on brain energy reduction were antagonistic. Both drugs were found in mitochondria using h.p.l.c-MS analysis. 6. We conclude that cyclosporine inhibits mitochondrial high-energy phosphate metabolism, which can be antagonized by SDZ-RAD.

Clenbuterol induces growth factor mRNA, activates astrocytes, and protects rat brain tissue against ischemic damage

The induction of growth factor synthesis in brain tissue by β 2-adrenoceptor agonists, such as clenbuterol, is a promising approach to protect brain tissue from ischemic damage. Clenbuterol (0.01–0.5 mg/kg) reduced the cortical infarct volume in Long–Evans rats as measured 7 days after permanent occlusion of the middle cerebral artery. Dosages of clenbuterol higher than 1 mg/kg showed no cerebroprotective effect due to a decrease in blood pressure and an increase in plasma glucose level. The increase in the mRNA level of nerve growth factor (NGF), basic fibroblast growth factor (basic FGF), and transforming growth factor-β 1 (TGF-β 1) mRNA in cortical and hippocampal tissue occurred earlier after middle cerebral artery occlusion and was more pronounced in animals treated with clenbuterol than in controls. In addition, glial fibrillary acidic protein (GFAP) mRNA expression was enhanced in astrocytes 6 h after ischemia in clenbuterol-treated animals. The results suggest that growth factor synthesis is enhanced in activated astrocytes and that this could be the mechanism of clenbuterol-induced cerebroprotection after ischemia.

Neuroprotective effect of 5-HT1A receptor agonist, Bay×3702, demonstrated in vitro and in vivo

It has been shown recently that Bay×3702 ((−)-(R)-2-[4-[[(3,4-dihydro-2H-1-benzopyran-2-yl)methyl]amino]butyl]-1,2,-benzisothiazol-3(2H)-one 1,1-dioxide monohydrochloride), a highly potent and selective 5-HT1A receptor agonist, has a neuroprotective potency associated with its ability to inhibit ischemia-induced excessive release of glutamate. 5-HT1A receptors are highly expressed in brain areas, such as the hippocampus and the cerebral cortex, sensitive to neuronal damage induced by ischemic stroke or brain trauma. Therefore, we investigated whether Bay×3702 can rescue cultured hippocampal neurons subjected to excitotoxic damage. The hippocampal neurons exposed to 0.5 mM l-glutamate for 1 h had pronounced damage characteristic of neuronal necrosis as evaluated 18 h later by trypan blue staining and morphological criteria. However, treatment with Bay×3702 (0.001 to 1 μM) reduced the number of damaged neurons, and preserved cell morphology and integrity of the neuronal network. Bay×3702 was added immediately after the end of exposure to glutamate and was present until the evaluation of neuronal damage. Furthermore, the neuroprotective activity of Bay×3702 (0.1 μM) was abolished by WAY 100635 (N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl cyclo-hexanecarboxamide) (1 μM), a selective 5-HT1A receptor antagonist, indicating that the neurorescuing activity of Bay×3702 was mediated via stimulation of 5-HT1A receptors. Additionally, we attempted to find whether the drug could protect rat brain tissue from ischemic insult due to permanent occlusion of the middle cerebral artery in rats. Bay×3702 (12 and 40 μg/kg), infused within a period of 4 h, immediately after induction of ischemia greatly reduced cortical infarct volume (57 and 55% of controls, respectively) suggesting that this drug might be useful for the treatment of acute cerebral infarction.

Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury.

The role of tumor necrosis factor-alpha (TNF alpha) in brain injury is controversial. We studied the effect of anti-TNF-alpha antibody in a rat model of reversible middle cerebral artery occlusion. During focal ischemia and early reperfusion, TNF-alpha was rapidly and transiently released into circulation. Pretreatment with intravenous anti-TNF-alpha antibody reduced cortical (71%, P < 0.015) and subcortical (58%, P < 0.007) injury, enhanced the cerebral blood flow during reperfusion, and improved the neurologic outcome. This further supports the contention that TNF-alpha is a deleterious cytokine in stroke, whereas circulating antibody against TNF-alpha may protect brain from reperfusion injury.

Tirilazad does not protect rat brain from brachytherapy-induced injury

Acute and chronic brain injury are common sequelae of high-dose focused radiation, as in radiosurgery and brachytherapy. Development of protectors of radiation injury, which would work in brain but not in tumor, would help enhance the therapeutic ratio of focused-radiation therapy. Radiation protection by a clinically available 21-aminosteroid, Tirilazad, was studied in a rat brain brachytherapy model, both in tumor and non-tumor bearing animals. For the tumor model, 9L Glioma/SF line cells were implanted stereotactically into the right frontal lobe of F-344 rats and grew to a sphere of 5.0-mm diameter after 12 days. Animals received a standard brachytherapy dose of 80 Gy to a 5.5-mm radius volume administered by a high-activity removable iodine-125 seed. Radiation damage was evaluated 24 hours after removal of the seeds in all animals and again at 3 months in non-tumor-bearing animals, by T1-weighted gadolinium-enhanced and T2-weighted magnetic resonance imaging (MRI) on a 1.5-T unit. Treated animals received Tirilazad 5 mg/kg intravenously 15 minutes prior to implant, 1 hour after implant, every 6 hours for the duration of the implant, and for 24 hours after removal of the seed. Control animals were administered vehicle only. In both non-tumor-bearing and tumor-bearing rats, no difference in the volume of lesions on enhanced T1 or T2 MRI was seen between the Tirilazad-treated and control groups. In the non-tumor-bearing rats, volume of both T1 enhanced and T2 MRI lesions was significantly reduced at 3 months compared to the values at 24 hours. In the present model, Tirilazad failed to reduce the volume of radiation brain injury from brachytherapy as seen on MRI, studied acutely in tumor-bearing and non-tumor-bearing animals and also at 3 months in non-tumor-bearing rats.

Marchantinquinone, isolated from Reboulia hemisphaerica, as inhibitor of lipid peroxidation and as free radical scavenger

The antioxidant effect of marchantinquinone was studied using various models. Marchantinquinone inhibited Fe 2+-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC 50 15.3 ± 2.9 μM. Marchantinquinone was less effective than α-tocopherol and BHT in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH). The IC 0.200 were 37.5 ± 2.5, 9.2 ± 1.6 and 14.5 ± 2.5 μM, respectively. Marchantinquinone also scavenged peroxyl radical derived from 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) in aqueous system with a stoichiometric factor of 0.9 ± 0.1. Furthermore, it prevented conjugated-diene formation and apolipoprotein B (apo B) oxidation of LDL. However, marchantinquinone did not chelate Fe 2+ or possess Superoxide anion and hydroxyl radical scavenging activity. It also did not scavenge 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN)-derived peroxyl radical in hexane. These results indicate that marchantinquinone may be an effective antioxidant and can protect rat brain homogenate and LDL against oxidation.

Antioxidant properties of demethyldiisoeugenol

The antioxidant properties of demethyldiisoeugenol were investigated in this study using various models. Demethyldiisoeugenol inhibited Fe 2+-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC 50, 1.8 ± 0.1 m ̊ M . Demethyldiisoeugenol was more effective than α-tocopherol and BHT in reducing the stable free radical diphenyl-2-picrylhydrazyl (DPPH). It also scavenged superoxide anion generated by xanthine/xanthine oxidase and peroxyl radical (ROO .) derived from 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) in aqueous system with a stoichiometric factor of 1.3 ± 0.2. Furthermore, it prevented conjugated-diene formation and apolipoprotein B (apo B) oxidation of LDL. However, demethyldiisoeugenol did not scavenge 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN)-derived peroxyl radical in hexane. It also did not chelate Fe 2+, did not inhibit xanthine oxidase activity or possessed hydroxyl radical ( .OH) scavenging activity. Experimental results indicate that demethyldiisoeugenol is a potentially effective antioxidant and can protect rat brain homogenate and LDL against oxidation.

Circulation of red blood cells having high levels of 2,3-bisphosphoglycerate protects rat brain from ischemic metabolic changes during hemodilution.

We designed the present study to examine the effects of red blood cell oxygen-delivering capacity on ischemic brain metabolism during hemodilution with respect to red blood cell 2,3-bisphosphoglycerate content. A modification of red blood cell 2,3-bisphosphoglycerate content was achieved by an exchange transfusion of blood in which red blood cells were treated with either phospho(enol)pyruvate or inorganic phosphate in spontaneously hypertensive rats. Hematocrit values of circulating blood were varied from 30% to 20% during transfusion. Brain ischemia was produced in rats by bilateral carotid artery occlusion lasting 60 minutes. The concentrations of ATP and 2,3-bisphosphoglycerate in the blood and the ATP, phosphocreatine, and lactate concentrations in the brain were estimated by an enzymatic method. Red blood cell 2,3-bisphosphoglycerate concentration increased to 200% of the pretransfusion level after the transfusion in which red blood cells were treated with phospho(enol)pyruvate, whereas the concentration decreased to 80% after the transfusion in which red blood cells were treated with phosphate. Red blood cell ATP content did not differ significantly between the phospho(enol)pyruvate- and phosphate-treated groups after transfusion. When hematocrit was approximately 30%, the ischemic brain ATP and lactate contents did not differ between the nonischemic and ischemic groups. However, as hematocrit was reduced to less than 25% the ischemic brain ATP content remarkably decreased and the lactate content substantially increased in the 2,3-bisphosphoglycerate-subnormal red blood cell group. In contrast, the ischemic brain ATP and phosphocreatine contents in the 2,3-bisphosphoglycerate-enriched red blood cell group were preserved and as high as those in the nonischemic group under the same conditions. Cerebral ischemia was compensated with the increment of cerebral blood flow as a result of the reduction of hematocrit to optimal levels, but the extreme hemodilution induced insufficient oxygen supply to the brain tissue, resulting in a more marked impairment of brain metabolism despite an increase in cerebral blood flow. However, even in extreme hemodilution conditions the 2,3-bisphosphoglycerate-enriched red blood cells in circulating blood protected the brain from ischemic metabolic changes. These results suggest that the 2,3-bisphosphoglycerate-enriched red blood cells in the circulating blood may thus compensate for the insufficient oxygen supply in extremely anemic conditions by providing a sufficient supply of oxygen in the face of ischemic insult.

Antioxidative activity of 5,6,7,8-tetrahydrobiopterin and its inhibitory effect on paraquat-induced cell toxicity in cultured rat hepatocytes.

The in vitro antioxidative activity of 5,6,7,8-tetrahydrobiopterin (BPH4) was measured and the ability of BPH4 to prevent paraquat-induced cell damage was examined in cultured hepatocytes. The scavenging activity of BPH4 against superoxide anion radicals was assayed in two systems, i.e., xanthine/xanthine oxidase (X/XOD) and rat macrophage/phorbol myristate acetate (M psi/PMA) radical-generating systems. BPH4 showed an extremely strong superoxide anion radical-scavenging activity in both assay systems. Biopterin (BP) itself did not show any activity in the X/XOD system, but was effective in the M psi/PMA system. The antioxidative activities of BPH4 against both superoxide anion and hydroxyl radicals were confirmed by spin trapping-ESR spectrometry. BPH4 also protected rat brain homogenate against auto-oxidation. We further examined the effect of BPH4 on paraquat-induced cell toxicity in cultured rat hepatocytes. The paraquat-induced elevation of the release of lactate dehydrogenase (LDH), a marker enzyme for cytotoxicity from cultured hepatocytes was suppressed by BPH4 in a dose-dependent manner. The elevation of lipid peroxides simultaneously induced by paraquat was also inhibited by BPH4 in the same manner. These results suggest that BPH4 might be useful in the treatment of various diseases whose pathogenesis is active oxygen-related.

Neurotrophic factor mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism

Several conventional and novel growth factors (GFs) canprotect neurons against an array of insults including excitotoxins, hypoglycemia, hypoxia, free radicals and β-amyloid peptide. In tissue culture experiments basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and insulin-like growth factors (IGFs) protected neurons from several brain regions from metabolic/excitotoxic insults. In each case, the GF prevented the sustained elevation of [Ca2+]i that was causally involved in cell injury. Oxidative damage induced by iron, H2O2, 6-hydroxydopamine and MPP+ was attenuated by one or more GFs including bFGF, NGF, IGFs, and brain-derived neurotrophic factor (BDNF). Secreted forms of β-amyloid precursor protein (APPss) reduced [Ca2+]i and protected rat and human brain cells against excitotoxicity. The data indicate that: (1) neuroprotection is a major function of GFs in the brain; (2) the neuroprotective mechanism of GFs involves stabilization of [Ca2+]i and suppression of free radicals; (3) activation of GF signal transduction pathways may reduce brain damage in both acute (e.g. stroke and trauma) and chronic (e.g. Alzheimer's, Parkinson's and Huntington's diseases) neurodegenerative conditions.

On the blood-brain barrier to peptides: specific binding of atrial natriuretic peptide in vivo and in vitro

Using the intracarotid bolus injection technique, a saturable binding of [125I]atrial natriuretic peptide (ANP) was found in 8 blood-brain barrier (BBB)-protected rat brain regions as well as in the pineal gland, choroid plexus, neurointermediate and anterior lobes of the pituitary, i.e. structures lacking a BBB. The presence of specific ANP binding on the BBB, here shown for the first time by an in vivo approach, was evidenced concomitantly in vitro by incubation of isolated microvessels. A single-class high affinity binding without regional differences was obtained with Kd = 0.23 nM and Bmax = 120 fmol/mg protein. From that a density of 1,400 binding sites per endothelial cell was calculated, thought to be localized predominantly in the luminal membranes. In the in vivo study, the portion of the extracted peptide that, under the conditions used, may have crossed the BBB by passive diffusion amounted to less than 0.4% of the labeled ANP administered. ANP itself did not change the tightness of the BBB to the non-diffusible reference molecule [14C]inulin. In the BBB-free areas, ANP enhanced the inulin space by nearly 50%.

?-tocopherol protects rat brain ?-adrenoreceptors against damage by phospholipase A2

?-tocopherol protects rat brain ?-adrenoreceptors against damage by phospholipase A2

Oxygen protects rat brain from ischaemic injury

Interruption of the oxygen supply to the brain promotes functional and structural damage of the brain, such as oedema and swelling. The most prominent cellular reaction to this type of damage has been found to be trophic or proliferative changes in glial cells. We examined dynamic changes in glial cells adjacent to the infarcted tissue in response to oxygenation. Results showed that astrocyte proliferation was seen more at 3 than at 7 days after middle cerebral artery occlusion, while their numbers with and without oxygenation were almost the same. The proliferation of microglia, however, was seen more with than without oxygenation. However, microglia seem to be able to secrete an angiogenetic factor under hypoxic conditions, and are active in terms of superoxide dismutase (SOD). Cerebral blood flow is directly related to tissue oxygen content and its autoregulatory capacity has been found to be greater as the volume contracts. As a result, we suggest that the application of oxygen-treated microglia to ischaemic tissue could diminish tissue damage, and that the most effective oxygenation can be achieved by the withdrawal of blood and reduction of the intracranial pressure.

Studies on the binding of nucleotides by rat brain hexokinase

Various nucleoside di- and triphosphates have been compared with respect to their ability to protect rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activity against inactivation by chymotrypsin, glutaraldehyde, heat, and 5,5′-dithiobis(2-nitrobenzoic) acid. ATP could be distinguished from other nucleoside triphosphates in these comparisons, which may be related to the specificity with which ATP is utilized as a substrate. All nucleoside derivatives examined provided substantial protection against two or more of the above inactivating agents, indicating relatively nonspecific binding of nucleotides by brain hexokinase, consistent with a similar lack of specificity in the inhibition of this enzyme by nucleoside derivatives. The fluorescence of 2- p-toluidinylnaphthalene-6-sulfonate (TNS) and of tetraiodofluorescein (TIF) was enhanced by binding to brain hexokinase. TNS binding was not affected by the presence of various relevant metabolites (Glc, glucose 6-phosphate, ATP), nor did TNS inhibit the enzyme. In contrast, substantial (approximately 70%) decreases in the fluorescence of bound TIF resulted from the addition of various nucleoside derivatives, and TIF served as a competitive inhibitor of brain hexokinase. These observations are consistent with the view that TIF binds to a nucleotide binding site of the enzyme. The inability of nucleotides to totally displace TIF was taken to indicate the existence of an additional TIF binding site (or sites) discrete from the catalytic site, and probably identical to the site(s) at which TNS binds with no effect on catalytic activity. The effects of saturating levels of ATP and ADP were not additive indicating that both compounds were displacing TIF from the same site i.e., a common nucleotide binding site. Glc, mannose, and 2-deoxyglucose greatly enhanced the ability of nucleotides to displace TIF, while fructose, galactose, and N-acetylglucosamine did not, indicating the existence of interactions between hexose and nucleotide binding sites; the hexoses themselves were not effective at displacing TIF. The enhanced binding of nucleotides in the presence of the first three hexoses but not the latter three can be directly correlated with the relative ability of these hexoses to induce specific conformational changes in the enzyme. The hexoses themselves were not effective at displacing TIF. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate could also displace TIF, and as with the nucleotides, a maximum of approximately 70% decrease in fluorescence was observed and the effectiveness of glucose 6-phosphate was enhanced in the presence of Glc. Other hexose 6-phosphates tested were not effective at displacing TIF. The specificity with which hexose 6-phosphates displaced TIF could be correlated with their ability to induce specific conformational change in the enzyme. The results are discussed as they relate to the kinetic mechanism and allosteric regulation by nucleotides that have been proposed for this enzyme.

Ligand-induced conformational changes of rat brain hexokinase: Their role in determining the substrate specificity for hexoses and inhibitory effectiveness of hexose 6-phosphates

The effectiveness of Glc, mannose, 2-deoxyglucose, fructose, galactose, arabinose, and N-acetylglucosamine at protecting rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) from inactivation by chymotrypsin, glutaraldehyde, heat, and Ellman's reagent have been compared. The relative effectiveness at protecting against these inactivating agents decreases in the order Glc > mannose > 2-deoxyglucose > fructose, galactose, and arabinose, the last three providing no significant protection at all. The nonphosphorylatable substrate analog, N-acetylglucosamine, provides substantial protection against heat inactivation, but is ineffective against inactivation by the other agents. Similar inactivation studies were conducted using several hexose 6-phosphates. Glc-6- P and 1,5-anhydroglucitol-6- P provided substantial protection while 2-deoxyglucose-6- P, fructose-6- P, mannose-6- P, and galactose-6- P were all relatively ineffective at protecting hexokinase activity. The protective effect of these ligands is taken as an indication of ligand-induced conformational changes in brain hexokinase. The results are interpreted in terms of, and considered to support, a recently proposed model (J. E. Wilson, 1978, Arch. Biochem. Biophys. 185, 88–99) in which the suitability of a carbohydrate as a substrate depends directly on its ability to induce specific conformational changes prerequisite for subsequent catalytic events while the inhibitory effectiveness of a hexose 6-phosphate is likewise related to its ability to evoke appropriate conformational change in the enzyme. Synergistic interactions between hexose and hexose-6- P binding sites, first reported for Glc and Glc-6- P by Ellison et al. (1975 , J. Biol. Chem. 250, 1864–1871), have been confirmed. Thus, although fructose and galactose were themselves quite ineffective at providing protection against inactivation of hexokinase by chymotrypsin or glutaraldehyde, they greatly increased the protection afforded by suboptimal (with respect to degree of protection in the absence of added hexose) levels of Glc-6- P. Conversely, the 6-phosphates of fructose, galactose, mannose, and 2-deoxyglucose, which were themselves ineffective at protecting the enzyme activity, markedly enhanced the protection provided by suboptimal levels of Glc or mannose. Based on the relationship between this enhancement of Glc-dependent protection and the hexose-6- P concentration, the dissociation constants for the complexes of hexokinase with 2-deoxyglucose-6- P and mannose-6- P were estimated to be ⪖0.5 m m.

Metabolic interactions of glucose, lactate, and beta-hydroxybutyrate in rat brain slices.

Metabolic interactions of glucose, lactate, and beta-hydroxybutyrate in rat brain slices.