Uptake by central nervous tissues as a mechanism for the regulation of extracellular adenosine concentrations

The various facets of the uptake of adenosine by central nervous tissues are described. The uptake process includes the transport of nucleoside across neuronal and glial plasma membranes and its metabolism within the cell. Much of the transported adenosine is phosphorylated into adenosine nucleotides. Inhibitors of adenosine uptake increase extracellular levels of adenosine and can thus potentiate its pharmacological actions. This may be an important component in the actions of various groups of psychoactive drugs.     

Do benzodiazepines bind at adenosine uptake sites in CNS?

Benzodiazepines inhibit adenosine uptake into rat cerebral cortical synaptosomes and their potency as inhibitors of adenosine uptake is closely correlated with therapeutic efficacy. Agents which possess “benzodiazepine like” activities such as CL218,872, zopiclone and fominoben and which displace benzodiazepine binding to brain cell membranes, are also inhibitors of adenosine uptake into brain synaptosomes. The IC₅₀ values of all these compounds as inhibitors of adenosine uptake are in close agreement with the IC₅₀ values obtained for the displacement of benzodiazepine binding to the brain receptors. Adenosine uptake inhibitors (dipyridamole, hexobendine, papaverine, 6-(2-hydroxy-5-nitrobenzyl)thioguanosine) which competitively inhibit adenosine uptake, presumably by blocking adenosine binding to its carrier-protein, are competitive inhibitors of diazepam binding to the brain membrane receptors. The finding of a pronounced correlation between inhibition of benzodiazepine binding and inhibition of adenosine uptake further supports the proposal that benzodiazepines may exert part of their pharmacological action through the inhibition of adenosine uptake.     

CFTR mutations in Turkish and North African cystic fibrosis patients in Europe: implications for screening

AIMS: To obtain more insight into the variability of the CFTR mutations found in immigrant cystic fibrosis (CF) patients who are living in Europe now, and to estimate the test sensitivity of different frequently used methods of DNA analysis to detect CF carriers or patients among these Turkish or North African immigrants. METHODS: A survey among 373 European CF centers asking which CFTR mutations had been found in Turkish and North African CF patients. RESULTS: 31 and 26 different mutations were reported in Turkish and North African patients, identifying 64.2% (113/176) and 87.4% (118/135) alleles, respectively (p < 0.001). The mean sensitivity (detection rate) of three most common CFTR mutation panels to detect these mutations differed between Turkish and North African people, 44.9% (79/176) versus 69.6% (94/135) (p < 0.001), and can be increased to 57.4% (101/176) and 79.3% (107/135) (p < 0.001), respectively, by expanding these panels with 13 mutations which have been found on two or more alleles. CONCLUSION: 35.8% and 12.6%, respectively, of CF alleles in Turkish and North African patients living in Europe now had not been identified. Among these populations, the test sensitivity of common CFTR mutation panels is insufficient for use in screening programs in Europe, even after expansion with frequent Turkish and North African mutations. This raises questions about whether and how to implement CF carrier and neonatal screening in a multiethnic society.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

The role of calcium in monoamine-induced depression of cerebral cortical neurones

The calcium antagonists, lanthanum, verapamil and manganese, have been shown to antagonize the depressant actions of ionto-phoretically applied monoamines (noradrenaline, 5-hydroxytryptamine, dopamine and histamine), but not of γ-aminobutyric acid, on cerebral cortical neurones. Cocaine and ethanol, which also affect membrane calcium fluxes, have a similar antagonistic action. These findings suggest that calcium ions are essential for, and perhaps mediate, the characteristic depressant actions of the monoamines on cerebral cortical neurones.     

Vasoactive intestinal polypeptide excitation of cerebral cortical neurons: lack of synergism with adenosine and noradrenaline

Vasoactive intestinal polypeptide (VIP), applied iontophoretically, excited 40% of the spontaneously firing rat cortical neurons tested. No neurons were depressed by VIP. When applied simultaneously with adenosine or noradrenaline, VIP depressed the firing of cortical neurons, but this depression could be reproduced by the passage of similar positive currents through a 50 mM NaCl-containing barrel of the multiple barrelled micropipette. VIP, therefore, excited rat cortical neurons and no depressant actions were apparent when VIP was applied together with adenosine or noradrenaline. Leakage of adenosine or noradrenaline during iontophoretic applications of the peptide may account for the reported inhibitory actions of VIP.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.     

Biological staining: mechanisms and theory

New staining techniques continue to be introduced, and older ones continue to be used and improved. Several factors control specificity, selectivity and visibility of the end product in any procedure using dyes, fluorochromes, inorganic reagents or histochemical reactions applied to sections or similar preparations. Local concentration of the tissue target often determines the intensity of the observed color, as does the fine structure within the object being stained, which may facilitate or impede diffusion of dyes and other reagents. Several contributions to affinity control the specificity of staining. These include electrical forces, which result in accumulation of dye ions in regions of oppositely charged tissue polyions. Weaker short-range attractions (hydrogen bonding, van der Waals forces or hydrophobic bonding, depending on the solvent) hold dyes ions and histochemical end products in contact with their macromolecular substrates. Nonionic forces can also increase visibility of stained sites by causing aggregation of dye molecules. Covalent bonds between dye and tissue result in the strongest binding, such as in methods using Schiff's reagent and possibly also some mordant dyes. The rate at which a reagent gains access to or is removed from targets in a section or other specimen affect what is stained, especially when more then one dye is used, together or sequentially. Rate-controlled staining is greatly influenced by the presence and type of embedding medium, such as a resin, that infiltrates the tissue. The rates of chemical reactions are major determinants of outcome in many histochemical techniques. Selective staining of different organelles within living cells is accomplished mainly with fluorochromes and is controlled by mechanisms different from those that apply to fixed tissues. Quantitative structure-activity relations (QSAR) of such reagents can be derived from such molecular properties as hydrophilic-hydrophobic balance, extent of conjugated bond systems, acid-base properties and ionic charge. The QSAR correlates with staining of endoplasmic reticulum, lysosomes, mitochondria, DNA, or the plasma membranes of living cells.