Similarities of adenosine uptake systems in astrocytes and neurons in primary cultures

Uptake of extracellular adenosine was studied in primary cultures of astrocytes or neurons. Both cell types showed a high affinity uptake. TheK _m values were not significantly different (6.5±3.75 M in astrocytes and 6.1±1.86 M in neurons), but the intensity of the uptake was higher in astrocytes than in neurons (V _max values of 0.16±0.030 and 0.105±0.010 nmol×min^–1×mg^–1 protein, respectively). The temperature sensitivity was similar in the two cell types. Adenosine uptake inhibitors and benzodiazepines inhibited the adenosine uptake systems in both astrocytes and neurons with IC₅₀ values in the high nanomolar or the micromolar range and the rank order of potency was similar in the two cell types. In both cell types the (–) isomers of two sets of benzodiazepine stereoisomers were more potent than the (+) isomers. Dixon analysis showed that dipyridamole, papaverine, hexobendine and chlordiazepoxide inhibited the adenosine uptake competitively and clonazepam noncompetitively in both cell types.     

Uptakes of iodide and chloride by primary cultures of mouse astrocytes and neurons

Primary cultures of both mouse astrocytes and neurons accumulate more¹²⁵I^– than³⁶Cl^– from the medium. The average cell/medium ratio of¹²⁵I^– of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of³⁶Cl^– of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both¹²⁵I^– and³⁶Cl^– calculated from the cell/medium ratios in astrocytes and neurons are significantly lower than their corresponding resting transmembrane potentials which suggest that both iodide and chloride are actively transported into both cell types. With respect to different transport inhibitors, thiocyanate is more effective in inhibiting¹²⁵I^– uptake whereas furosemide is more effective in inhibiting³⁶Cl^– uptake. Radioiodide uptake by mouse astrocytes was directly proportional to the [Na⁺]_o but was not significantly affected by changes of [Cl^–]_o or [HCO ₃ ^– ]_o, except that it is low in bicarbonate-free medium. Radiochloride uptake by astrocytes was inversely related to [Cl^–]_o and [HCO ₃ ^– ]_o and was not affected [Na⁺]_o, except that it was low in sodium-free medium. Radioiodide uptake by neurons was directly related to [Na⁺]_o between 60 and 140 mM and inversely related to [HCO ₃ ^– ]_o between 10 and 40 mM, but it was not affected by [Cl^–]_o. Radiochloride uptake by neurons was directly related to [Cl^–]_o and to [Na⁺]_o between 60 and 140 mM and was not affected by [HCO ₃ ^– ]_o. However, in sodium-free medium both¹²⁵I^– and³⁶Cl^– uptakes into neurons were higher than those in [Na⁺]_o between 5 and 60 mM. These results indicate that uptake of¹²⁵I^– and³⁶Cl^– into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.Special issue dedicated to Dr. Paola S. Timiras     

Intracellular activity of morphologically identified neurons of the grass frog's optic tectum in response to moving configurational visual stimuli

Summary In the grass frogRana temporaria, various classes of tectal neurons were identified by means of intracellular recording and iontophoretic staining using potassium-citrate/Co³⁺-lysine-filled micropipettes, which have been defined previously by extracellular recording methods. Class T5(1) neurons had receptive fields (RF) of 33°±5° diameter. In response to a moving 8°×8° square (S), a 2°×16° worm-like (W), or a 16°×2° antiworm-like (A) moving stripe, these cells showed excitatory postsynaptic potentials (EPSPs) and spikes which were interrupted occasionally by small inhibitory postsynaptic potentials (IPSPs). The excitatory responses (R) were strongest towards the square (R_S) and less to the worm (R_W). For the antiworm (R_A) the responses were smallest or equal to the worm stimulus yielding the relationship R_S>R_WR_A. Some of these cells were identified as pear-shaped or large ganglionic neurons, whose somata were located in the tectal cell layer 8. The somata of other large ganglionic neurons were found in layer 7 and the somata of other pear-shaped neurons at the top of layer 6, both displaying T5(1) properties. Class T5(2) neurons (RF=34°±3°) responded with large EPSPs and spikes, often interrupted by small IPSPs, when their RF was traversed by the square stimulus. The excitatory activity was somewhat less to the worm stimulus, whereas no activity at all, or only IPSPs, were recorded in response to the antiworm-stimulus; thus yielding the relationship for the excitatory activity R_S>R_W>R_A 0. Such a cell was identified as pyramidal neuron; the soma was located at the top of layer 6, with the long axon travelling into layer 7 to the medulla oblongata. Class T5(3) neurons (RF=29°±6°) showing EPSPs and spikes according to the relationship R_S>R_A>R_W have been identified as large ganglionic neurons. Their somata were located in layer 8. Class T5(4) neurons (RF=24±7°) responded only to the square stimulus with EPSPs and spikes, sometimes interrupted by IPSPs and yielding the relationship R_S>R_AR_W0. The somata of these large ganglionic or pear-shaped neurons were located in layer 8. Class T1(1) neurons (RF=30°–40°) were most responsive to stimuli moving at a relatively long distance in the binocular visual field, and have been identified as pear-shaped neurons. Their somata were located in layer 6.Further neurons are described and morphologically identified which have not yet been classified by extracellular recording methods. For example,IPSP neurons (RF=20°–30°) responded (R) with IPSPs only according to the relationship R_S>R_A R_W. The somata of these pear-shaped neurons were located in layer 6.The properties of tectal cells in response to electrical stimulation of the optic tract and to brisk changes of diffuse illumination suggest certain neuronal connectivity patterns. The results support the idea ofintegrative functional units (assemblies) of connected cells which are involved in various perceptual processes, such as configurational prey selection expressed by T5(2) prey-selective neurons.Abbreviations A antiworm-like 16°×2° stripe stimulus with long axis perpendicular to the direction of movement - W wormlike 2°×16° stripe stimulus with long axis oriented parallel to the direction of movement - S square 8°×8° moving stimulus - ERF excitatory receptive field - IRF inhibitory receptive field - RF receptive field - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential     

(4S)-4-amino-5,6-heptadienoic acid (MDL 72483): A potent anticonvulsant GABA-T inhibitor

(4S)-4-Amino-5,6-heptadienoic acid ((S)--allenyl-GABA; MDL 72483) is a potent inactivator of brain GABA-T in mice; (ED₅₀ (i.p.)=60 mg·kg^–1; ED₅₀ (oral)=70 mg·kg^–1). Its anticonvulsant effects against 3-mercaptopropionic acid (MPA)-induced seizures in mice is related to the elevation of whole brain GABA concentrations: The mentioned doses of MDL 72483 which cause a decrease of GABA-T activity by 50%, produce within 5 h after dosing an increase of GABA concentration by about 3 mol·g^–1, and protect 50% of the mice against seizures in this model of presynaptic GABA deficit. When given orally MDL 72483 is about five times more potent than vigabatrin ((4R/S)-4-amino-5-hexenoic acid) a known antiepileptic GABA-T inhibitor. Complete protection was achieved with a dose of 150 mg·kg^–1. Similar to vigabatrin, MDL 72483 does not protect significantly against metrazol-induced convulsions. However, at a dose of 300 mg·kg^–1, the time elapsing between metrazol administration and onset of convulsions was prolonged by a factor of 3.4. Oral administration of MDL 72483 for up to 19 days at a daily dose of 91–96 mg·kg^–1 did not produce any obvious behavioral changes in mice, nor was the ED₅₀ of the drug in MPA-seizure tests significantly altered by the pretreatment. These observations indicate that MDL 72483 is a promising drug for the treatment of certain epilepsies.Special issue dedicated to Dr. Eugene Roberts.     

Molecular recognition between 4a<Emphasis Type="Italic">S/R</Emphasis>-galanthamine diastereoisomers and α-cyclodextrin

Molecular recognition between 4aS/R-galanthamine diastereoisomers (1: 4aS-galanthamine; 2: 4aR-galanthamine) and -cyclodextrin (-CD) were studied by use of docking and molecular dynamics (MD) simulation approaches. The binding energy of constructed 2···-CD complexes is ~17 kcal mol^–1 lower than that of 1···-CD, implying a stronger binding ability of 2 with -CD than that of 1. The theoretical modeling result is consistent with our previous CZE result, which demonstrated that -CD is an efficient chiral additive for separating 1 and 2. The modeling result also indicates that both hydrophobic interaction and H-bond force may work as major factors for molecular recognition between the galanthamine diastereoisomers and -CD. Figure Chemical structures of 4aS-galanthamine (left) and 4aR-galanthamine (right)Abbreviations Galanthamine 4aS,6R,8aS-4a,5,9,10,11,12-Hexahydroxy-3-methoxy-11-methyl-6H-benzofuro[3a,3,2-e,f]benzazepin-6-ol     

Uptake of acetyl-l-carnitine in the brain

Analysis in mouse brain slices of the uptake of acetyl-l-[N-methyl-¹⁴C]carnitine with time showed it to be concentrative, and kinetic analysis gave aK _m of 1.92 mM and aV _max of 1.96 mol/min per ml, indicating the presence of a low-affinity carrier system. The uptake was energy-requiring and sodium-dependent, being inhibited in the presence of nitrogen (absence of O₂), sodium cyanide, low temperature (4°C), and ouabain, and in the absence of Na⁺. The uptake of acetyl-l-carnitine was not strictly substrate-specific; -butyrobetaine,l-carnitine,l-DABA, and GABA were potent inhibitors, hypotaurine andl-glutamate were moderate inhibitors, and glycine and -alanine were only weakly inhibitory. In vivo, acetyl-l-carnitine transport across the blood-brain barrier had a brain uptake index of 2.4±0.2, which was similar to that of GABA. These results indicate an affinity of acetyl-l-carnitine to the GABA transport system.     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

Temperature insensitive O2 in blood of the tree frogChiromantis petersi

Summary Respiratory gas exchange and blood respiratory properties have been studied in the East-African tree frogChiromantis petersi. This frog is unusually xerophilous, occupies dry habitats and prefers body temperatures near 40°C and direct solar exposure. Total O₂ uptake was low at 81 l O₂·g^–1·h^–1±19.0 (SD) at 25°C increasing to 253.5 l O₂·g^–1·h^–1±94.8 (SD) at 40°C giving aQ ₁₀ value of 2.1. Skin O₂ uptake at 25°C was 38.5% of total. The gas exchange ratio was 0.71 for whole body gas exchange, 0.61 for the lungs and 1.02 for the skin at 25°C.Blood O₂ affinity was low with aP ₅₀ of 47.5 mmHg at 25°C and pH 7.65. Then _H-value at 25°C increased from 2.7 aroundP ₅₀ to 5.0 at O₂ saturations exceeding 70–80%. Surprisingly, blood O₂ affinity was nearly insensitive to temperature expressed by a H value of ±1.0 kcal·mole between 25 and 40°C.The adaptive significance of the low O₂ affinity, the increase ofn _H with O₂ saturation and the temperature insensitive O₂-Hb binding is discussed in relation to the high and fluctuating body temperatures ofChiromantis.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Transepithelial transport in cell culture: Stoichiometry of Na/phlorizin binding and Na/d-glucose cotransport. A two-step,two-sodium model of binding and translocation

Summary The renal cell line LLC-PK₁ cultured on a membrane filter forms a functional epithelial tissue. This homogeneous cell population exhibits rheogenic Na-dependentd-glucose coupled transport. The short-circuit current (I _sc) was acccounted for by net apical-to-basolaterald-glucose coupled Na flux, which was 0.53±0.09(8) eq cm^–2hr^–1, andI _sc, 0.50±0.50(8) eq cm^–2hr^–1. A linear plot of concurrent net Na vs. netd-glucose apical-to-basolateral fluxes gave a regression coefficient of 2.08. As support for a 21 transepithelial stoichiometry, sodium was added in the presence ofd-glucose and the response ofI _sc analyzed by a Hill plot. A slope of 2.08±0.06(5) was obtained confirming a requirement of 2 Na for 1d-glucose coupled transport. A Hill plot ofI _sc increase to addedd-glucose in the presence of Na gave a slope of 1.02±0.02(5). A direct determination of the initial rates of Na andd-glucose translocation across the apical membrane using phlorizin, a nontransported glycoside competitive inhibitor to identify the specific coupled uptake, gave a stoichiometry of 2.2 A coupling ratio of 2 for Na,d-glucose uptake, doubles the potential energy available for Na-gradient coupledd-glucose transport. In contrast to coupled uptake, the stoichiometry for Na-dependentphlorizin binding was 1.1±0.1(8) from Hill plot analyses of Na-dependent-phlorizin binding as a function of [Na]. Although occurring at the same site the process of Na-dependent binding of phlorizin differs from the binding and translocation ofd-glucose. Our results support a two-step, two-sodium model for Na-dependentd-glucose cotransport; the initial binding to the cotransporter requires a single Na andd-glucose, a second Na then binds to the ternary complex resulting in translocation.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans

The present investigation examined the relationship between CO₂ sensitivity [at rest (S _R) and during exercise (S _E)] and the ventilatory response to exercise in ten elderly (61–79 years) and ten younger (17–26 years) subjects. The gradient of the relationship between minute ventilation and CO₂ production ( _E/ CO₂) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P<0.01]. At rest, S _R was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) 1 · min^–1 · kPa^–1; 1.44 (0.23) vs 2.26 (0.28) 1 · min^–1 · mmHg^–1; P<0.05], but S _E was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l · min^–1 · kPa^–1; 2.38 (0.33) vs 2.56 (0.21) 1 · min^–1 · mmHg^–1]. There were significant correlations between both S _R and S _E, and _E/ CO₂ (P<0.05; P<0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that _E/ CO₂ is not an appropriate index of the ventilatory response to exercise for elderly humans.     

Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Osmotic regulation ofmyo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H₂O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H₂O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H₂O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 g/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.     

Arterial hypoxemia and performance during intense exercise

In order to determine the level of hypoxemia which is sufficient to impair maximal performance, seven well-trained male cyclists [maximum oxygen consumption (VO_2max)51·min^–1 or 60 ml·kg^–1·min^–1] performed a 5-min performance cycle test to exhaustion at maximal intensity as controlled by the subject, under three experimental conditions: normoxemia [percentage of arterial oxyhemoglobin saturation (%S _a O₂)>94%], and artificially induced mild (%S _aO₂=90±1%) and moderate (%S _aO₂=87±1%) hypoxemia. Performance, evaluated as the total work output (Work_tot) performed in the 5-min cycle test, progressively decreased with decreasing %S _aO₂ [mean (SE) Work_tot=107.40 (4.5) kJ, 104.07 (5.6) kJ, and 102.52 (4.7) kJ, under normoxemia, mild, and moderate hypoxemia, respectively]. However, only performance in the moderate hypoxemia condition was significantly different than in normoxemia (P=0.02). Mean oxygen consumption and heart rate were similar in the three conditions (P=0.18 andP=0.95, respectively). End-tidal partial pressure of CO₂ was significantly lower (P=0.005) during moderate hypoxemia compared with normoxemia, and ventilatory equivalent of CO₂ was significantly higher (P=0.005) in both hypoxemic conditions when compared with normoxemia. It is concluded that maximal performance capacity is significantly impaired in highly trained cyclists working under an %S _aO₂ level of 87% but not under a milder desaturation level of 90%.     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Characterization of separated cell types from developing rat cerebellum

The characteristics of [³H]GABA transport were investigated in preparations greatly enriched in different classes of cerebellar cells. In contrast to observations in situ, isolated Purkinje cells readily accumulated [³H]GABA. In comparison with astrocytes, theV _max of the high-affinity uptake process was sixfold higher (0.31 vs. 0.05 nmol/min/10⁶ cells) and the apparentK _t twofold greater (2 vs. 1 M). In contrast to these cell types, uptake was very low in granule cell-enriched preparations.cis-1,3-Aminocyclohexane carboxylic acid was a potent inhibitor of [³H]GABA uptake by the Purkinje cells and a weak blocker in astrocytes, while the converse was the case for -alanine. Diaminobutyric acid strongly inhibited uptake in both cell types. [³H]GABA transport was Na⁺ dependent in both cell classes. However, veratridine and ouabain selectively blocked [³H]GABA accumulation in the Purkinje cells, which were also more sensitive than the astrocytes to the glycolysis inhibitor, NaF. The results indicated, therefore, marked differences between Purkinje cells and astrocytes in the properties of both the [³H]GABA transport systems and the underlying metabolic processes.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.