Contrasting effects of a convulsant (CHEB) and an anticonvulsant barbiturate (phenobarbitone) on amino acid release from rat brain slices

The effects of a convulsant barbiturate, 5(2-cyclohexylidine-ethyl)-5-ethyl barbituric acid (CHEB), and phenobarbitone (PhB) on the release of exogenous D-aspartate and GABA from slices of rat cerebral cortex were investigated. While PhB inhibited potassium-evoked release of D-aspartate more so than that of GABA, CHEB potently inhibited potassium-evoked GABA release and stimulated evoked D-aspartate release, in a concentration-dependent manner. These actions are consistent with the observed in vivo convulsant and anticonvulsant properties of these barbiturates. CHEB, but not PhB also elevated spontaneous efflux of both amino acids. The actions of these barbiturates were further studied in calcium- and sodium-free media, and in the presence of tetrodotoxin and ruthenium red, agents known to alter ion flux across neuronal membranes. The results obtained indicate that different ionic mechanisms may be involved in the release of excitatory and inhibitory amino acid transmitters.     

The human blood-brain barrier glucose transporter (GLUT1) is a glucose transporter of gray matter astrocytes

Human and monkey brain sections were examined by immunohistochemical light and electron microscopy to determine the distribution of GLUT1, a glucose transporter isoform associated with erythrocytes and endothelial cells of the human blood-brain barrier. Protein immunoblotting of fractionated human brain membranes was performed to determine the distribution of molecular forms of the transporter. GLUT1 staining was abundant in erythrocytes and cerebral endothelium of gray and white matter but was also present diffusely in gray matter neuropil when viewed by light microscopy. Immunoelectron microscopy confirmed the gray matter and vascular localization of GLUT1, with specific GLUT1 staining seen in erythrocytes, gray and white matter endothelial cells, astrocyte foot processes surrounding gray matter blood vessels, and in astrocyte processes adjacent to synaptic contacts. No astrocytic staining was identified in white matter. Astrocyte GLUT1 staining was identified only in mature gray matter regions; undifferentiated regions of preterm (22–23 weeks gestation) cortex had GLUT1 staining only in blood vessels and erythrocytes, as did germinal matrix. Immunoblots of adult human frontal cortex revealed that two forms of GLUT1 (45 and 52 kDa) were present in unfractionated brain homogenates. Immunoblots of vessel-depleted frontal lobe revealed only the 45 kDa form in gray matter fractions, and depleted in membranes prepared from white matter regions. We conclude that the GLUT1 isoform of glucose transporter is present both in endothelium of the blood-brain barrier and in astrocytes surrounding gray matter blood vessels and synapses. Furthermore, the form present in astrocytes is likely to have a lower molecular weight than the form found in cerebral endothelium. The GLUT1 transporter may play an important role not only in astrocyte metabolism, but also in astrocyte-associated pathways supporting neuronal energy metabolism. © 1995 Wiley-Liss, Inc.     

Effect of tacrine on intracellular calcium in cholinergic SN56 neuronal cells

We have found earlier that the depolarization-induced release of acetylcholine from the brain could be inhibited by tacrine (tetrahydroaminoacridine) but the mechanism of this action of tacrine was not clarified (S. Tu?ek, V. Dole?al, J. Neurochem. 56 (1991) 1216). We have now investigated whether tacrine has an effect on the changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) induced by depolarization. Experiments were performed on the cholinergic SN56 neuronal cell line with Fura-2 fluorescence technique of calcium imaging. The depolarization by 71 mmol/l K⁺ evoked minimum increases of [Ca²⁺]_i up to day 5 in culture. Then the response gradually increased and reached a plateau after 7 days in culture. A similar time course was observed for acetylcholinesterase activity. The effect of K⁺ ions was concentration-dependent and the concentration of 71 mmol/l K⁺ evoked maximum [Ca²⁺]_i responses. The increases of [Ca²⁺]_i did not occur in the absence of extracellular calcium. They were mediated by high voltage-activated calcium channels of the L-type and the N-type. Nifedipine (2 μmol/l; L-type calcium channel blocker) and ω-conotoxin GVIA (100 nmol/l; N-type calcium channel blocker) diminished the response to 71 mmol/l K⁺ by 53% and 39%, respectively, and their effects were additive (decrease to 8% of controls). Non-selective inorganic blocker of voltage-activated calcium channels LaCl₃ (0.1 mmol/l) decreased the response by 83%. Tacrine attenuated the [Ca²⁺]_i response in a concentration-dependent manner. At a concentration of 10 μmol/l it inhibited the [Ca²⁺]_i response by 55% and its inhibitory effect was additive with that of ω-conotoxin GVIA but not with that of nifedipine. An equimolar concentration of paraoxon, an irreversible inhibitor of cholinesterases, had no influence on [Ca²⁺]_i response. Tacrine exhibited the same inhibitory effect when paraoxon was present. In conclusion, our data indicate that high-voltage-activated calcium channels of the L-type and the N-type are both present in the SN56 cells but that they are fully expressed only after 6–7 days in culture. Tacrine attenuates the influx of calcium by inhibiting the L-type calcium channels. This inhibitory effect is not a consequence of the anticholinesterase activity of tacrine. The finding that low micromolar concentrations of tacrine may interfere with calcium-dependent events is likely to be of importance for the evaluation of the therapeutic potential of the drug.     

Model of potassium dynamics in the central nervous system

A one-dimensional numerical model of potassium dynamics in the central nervous system is developed. The model incorporates the following physiological processes in computing spatial and temporal changes in extracellular K+ concentration, [K+]o: 1) the release of K+ from K+ sources into extracellular space, 2) diffusion of K+ through extracellular space, 3) active uptake of K+ into cells and blood vessels, 4) passive uptake of K+ into a cellular distribution space, and 5) the transfer of K+ by K+ spatial buffer current flow in glial cells. The following tissue parameters can be specified along the single spatial dimension of the model: 1) the volume fraction and tortuosity of extracellular and glial cell spaces, 2) the volume fraction of the cellular distribution space, 3) rate constants of active uptake and passive uptake processes, and 4) glial cell membrane conductance. The model computes variations in [K+]o and current flow through glial cells for three tissue geometries: 1) planar geometry (the retina and the surface of the brain), 2) cylindrical geometry (tissue surrounding a blood vessel), and 3) spherical geometry (tissue surrounding a point source of K+). For simple sources of K+, the performance of the model matches that predicted from analytical equations. Simulations of previous ion dynamics experiments indicate that the model can accurately predict ion diffusion and K+ current flow in the brain. Simulations of electroretinogram generation and K+ siphoning onto blood vessels suggest that unanticipated K+ dynamics mechanisms may be operating in the central nervous system.     

Acuity perimetry: Estimation of neural channels

Measurements of peripheral visual acuity allow quantitative estimations of retino-cortical neural channels. Analysis of results from high-pass resolution perimetry revealed that about 2/3 of all channels are contained within 30 degrees of visual field eccentricity and that loss of 1/3 raises the average threshold level about 1 decibel. The analytical procedure can be applied to any type of visual field defect.     

Flurazepam: Effects on sodium and potassium currents in myelinated nerve fibres

The effect of flurazepam-HCl on single myelinated nerve fibres of the frog Rana esculenta was investigated. Flurazepam affected both Na and K currents: 0.25 mM of the drug decreased the peak Na inward current to about 50%. The initial increase and subsequent decay of the Na current was slowed down by a factor of 1.5 independent of membrane potential. The drug induced a slow phase in the recovery from Na inactivation and frequency dependence of the Na current block. The K current rose at a normal rate and was then inactivated to a sustained outward current. The time constant of block development (π_k) and the steady state block were potential-dependent. With 1 mM flurazepam, π_k decreased from 2.9 ms at E = 10 mV to 1.5 ms at E =90 mV, and the steady state block increased from 65% at E = - 20 mV to 81% at E = 90 mV. Recovery from the block proceeded faster at E =- 70 mV (π = 27 ms) than at E = - 120 mV (π = 89 ms). The effects of the drug on the K current were interpreted in terms of the reaction scheme proposed by Armstrong (J. Gen. Physiol. 54, 553; 1969).     

Effect on Mn2+ on permeability properties of frog skin

Summary Mn²⁺ added to the inner bathing solution of frog skin caused a transient increase in potential difference (PD) and a decrease in total skin conductance and mannitol influx. Net Na flux and short-circuit current (Is. c.) were also reduced, the isotopic net flux being reduced more than Is. c. This observed discrepancy appears to be the result of Cl^– retention in the outer medium since it was not observed when the skin was bathed in a sulfate-substituted chloridefree solution. The effect of Mn²⁺ on the inner side of the frog skin appears to be due to a reduced permeation of Na⁺ and Cl^– through the outer barrier of the skin.Addition of Mn²⁺ to the outer solution bathing the frog skin caused an increase in PD and a smaller increase in Is. c. These changes were not associated with alterations in the fluxes of Na⁺ or mannitol and were observed only when chloride was present in the bathing solutions. The effect of Mn²⁺ on this side of the frog skin may therefore be due to a net retention of Cl^– in the outer solution.     

Effects of McN-A-343, a cholinomimetic drug,on endplate currents in the frog

Summary The muscarinic ganglion stimulating agent, McN-A-343 has unusual blocking actions on endplate currents (EPCs) at frog neuromuscular junctions. McN-A-343 caused depolarization by a curaresensitive process, blocked neuromuscular transmission, depressed EPCs and reduced the time for EPC decay. These results are explained best by a nicotinic agonist action of McN-A-343 on the acetylcholine receptor to cause ion flow and the blockade by McN-A-343 of the open ion channels. The actions of McN-A-343 are similar to those of decamethonium (C-10) described by others. Unlike C-10, however, McN-A-343 did not alter the exponential character of the EPC or alter the voltage dependency of the EPC. The prototypical nicotinic agonist, dimethylphenylpiperazinium had no effect on EPC parameters of endplate clamped at-90 mV.This work was supported by Grants NS 07540-12 and NS 12563-03, Institute of Neurological and Communicative Disorders and Stroke, N.I.H.     

Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells

Membrane transporters play important roles in mediating chemosensitivity and -resistance of tumor cells. ABC transporters, such as ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP, are frequently associated with decreased cellular accumulation of anticancer drugs and multidrug resistance of tumors. SLC transporters, such as folate, nucleoside, and amino acid transporters, commonly increase chemosensitivity by mediating the cellular uptake of hydrophilic drugs. Ion channels and pumps variably affect sensitivity to anticancer therapy by modulating viability of tumor cells. A pharmacogenomic approach, using correlations between drug potency and transporter gene expression in multiple cancer cell lines, has shown promise for identifying potential drug-transporter relationships and predicting anticancer drug response, in an effort to optimize chemotherapy for individual patients.     

Risk factors and prevention of cardiotoxicity induced by 5-fluorouracil or capecitabine

Aim: 5-Fluorouracil (5-FU) and its prodrug capecitabine are cardiotoxic. This retrospective study aimed to identify risk factors and to give practical measures to make such chemotherapy feasible if cardiotoxicity occur. Method: Review of cardiotoxicity among 668 patients treated with 5-FU or capecitabine for gastrointestinal cancers. Results: Cardiotoxicity occurred in 29 cases (4.3%). The number of cases according to cardiotoxicity CTC grades 2–4 for patients with and without pre-existing cardiovascular disease were none, 10, and 2 cases, and 3, 14, and no cases, respectively (P=0.16). In three patients intercurrent decrease of renal clearances to <30, 48 and 71 ml min^-1 led to markedly increased cardiotoxicity. Chemotherapy dose reduction to 70 or 50%, either alone or in addition to antiangina medication prevented cardiotoxicity during subsequent chemotherapy in nine (60%) and three (20%) cases out of 15 assessable patients (P=0.001), respectively. To abolish symptoms of cardiotoxicity, sublingual nitroglycerine was efficient for 15 patients and inefficient for two (P=0.001). Conclusion: Cardiac and renal co-morbidity are risk factors for 5-FU induced cardiotoxicity. In this situation, rechallenge with modified 5-FU-based chemotherapy regimen supported by symptomatic medical treatment is feasible.