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.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

High-affinity sodium-dependent uptake of ascorbic acid by rat osteoblasts

Summary Ascorbic acid is essential for the formation of bone by osteoblasts, but the mechanism by which osteoblasts transport ascorbate has not been investigated previously. We examined the uptake ofl-[¹⁴C]ascorbate by a rat osteoblast-like cell line (ROS 17/2.8) and by primary cultures of rat calvaria cells. In both systems, cells accumulatedl-[¹⁴C]ascorbate during incubations of 1–30 min at 37°C. Unlike propionic acid, which diffuses across membranes in protonated form, ascorbic acid did not markedly alter cytosolic pH. Initial ascorbate uptake rate saturated with increasing substrate concentration, reflecting a high-affinity interaction that could be described by Michaelis-Menten kinetics (apparentK _m =30±2 m andV _max=1460±140 nmol ascorbate/g protein/min in ROS 17/2.8 cells incubated with 138mm extracellular Na⁺). Consistent with a stereoselective carrier-mediated mechanism, unlabeledl-ascorbate was a more potent inhibitor (IC₅₀=30±5 m) ofl-[¹⁴C]ascorbate transport than wasd-isoascorbate (IC₅₀=380±55 m). Uptake was dependent on both temperature and Na⁺, since it was inhibited by cooling to 4°C and by substitution of K⁺, Li⁺ or N-methyl-d-glucamine for extracellular Na⁺. Decreasing the external Na⁺ concentration lowered both the affinity of the transporter for ascorbate and the apparent maximum velocity of transport. We conclude that osteoblasts possess a stereoselective, high-affinity, Na⁺-dependent transport system for ascorbate. This system may play a role in the regulation of bone formation.     

Neurochemical,pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids

Specific binding ofl-[³H]glutamate ([³H]Glu) andl-[³H]asparate ([³H]Asp) to cerebellar membranes represented a time-, temperature- pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K _off/K _on=K _d) for [³H]Glu binding to this fraction indicated high-affinity (443 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for [³H]Glu and [³H]Asp as receptors with differential pharmacological, specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37°C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents andl-Glu (50–1000 M) provided protection, against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8–12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8–12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously.     

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.     

Measurement of local rates of brain protein synthesis by quantitative autoradiography: Validation with L-[3H]valine

Following the injection of 4-day old rats with 150 mMl-[3,4-³H]valine (10mol/g, IP) the incorporation of³H into protein was linear 2 hours. Valine specific activity in the brain acid-soluble fraction was constant between 30 and 120 min after injection with a mean value of 82.3% of the injectate. Significant amounts of tritated metabolites accumulated in the brain acid-soluble fraction (41.4% of radioactivity at 120 min) but do not prove an impediment to measuring rates of protein synthesis. The rate of protein synthesis in cerebral cortex of the 4-day old rat was measured by quantitative autoradiography using [³H]valine and³H-sensitive film. The measured rate shows excellent agreement with that found previously usingl-[1-¹⁴C]valine. Our results suggest that [³H]valine can be a useful precursor to measure local rates of brain protein synthesis by quantitative autoradiography.     

L-lysine is a barbiturate-like anticonvulsant and modulator of the benzodiazepine receptor

Our earlier observations showed thatl-lysine enhanced the activity of diazepam against seizures induced by pentylenetetrazol (PTZ), and increased the affinity of benzodiazepine receptor binding in a manner additive to that caused by -aminobutyric acid (GABA). The present paper provides additional evidence to show thatl-lysine has central nervous system depressant-like characteristics.l-lysine enhanced [³H]flunitrazepam (FTZ) binding in brain membranes was dose-dependent and stimulated by chloride, bromide and iodide, but not fluoride. Enhancement of [³H]FTZ binding byl-lysine at a fixed concentration was increased by GABA but inhibited by pentobarbital between 10^–7 to 10^–3M. While GABA enhancement of [³H]FTZ binding was inhibited by the GABA mimetics imidazole acetic acid and tetrahydroisoxazol pyridinol, the enhancement by pentobarbital andl-lysine of [³H]FTZ binding was dose-dependently increased by these two GABA mimetics. The above results suggest thatl-lysine and pentobarbital acted at the same site of the GABA/benzodiazepine receptor complex which was different from the GABA binding site. The benzodiazepine receptor antagonist imidazodiazepine Ro15-1788 blocked the antiseizure activity of diazepam against PTZ. Similar to pentobarbital, the anti-PTZ effect ofl-lysine was not blocked by Ro15-1788. Picrotoxinin and the GABA, receptor antagonist bicuculline partially inhibitedl-lysine's enhancement of [³H]FTZ binding with the IC₅₀s of 2 M and 0.1 M, respectively. The convulsant benzodiazepine Ro5-3663 dose-dependently inhibited the enhancement of [³H]FTZ binding byl-lysine. This article shows the basic amino acidl-lysine to have a central nervous system depressant characteristics with an anti-PTZ seizure activity and an enhancement of [³H]FTZ binding similar to that of barbiturates but different from GABA.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

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     

Presynaptic Modulation of K+-Evoked [3H]Dopamine Release in Striatal and Frontal Cortical Synaptosomes of Normotensive and Spontaneous-Hypertensive Rats

Regional differences in presynaptic [³H]dopamine ([³H]DA) release and its modulation by D₂ DA-receptors between the frontal cortex and striatum obtained from Wystar-Kyoto (WKY) and spontaneous-hypertensive rats (SHR) have been evaluated using superfused synaptosomes. Synaptosomal tritium content was significantly lower in the frontal cortex than in the striatum in both SHR and WKY (45% and 48%, respectively), but no differences in tritium content were obtained between strains. However, the 15 mM K⁺-evoked [³H]DA overflow was lower in the SHR as compared to WKY rats in both brain regions (striatum 23%, frontal cortex 21). Concentration-response curves for quinpirole (1nM-10 M)-mediated inhibition of 15mM K⁺-evoked [³H]DA release showed no differences between SHR and WKY. These results suggest that SHR has less ability to release [³H]DA as compared to WKY rats, but SHR did not show differences in the autoregulation of such release in both the frontal cortex and striatum.     

Interactions between valproate,glutamate, aspartate,and GABA with respect to uptake in astroglial primary cultures

Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na⁺-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and -aminobutyric acid (GABA) into astrocytes were examined. The [³H]valproate transport into the astrocytes was increased after exposure tol-glutamate but notl-aspartate. On the other hand, after acute exposure for the drug, the transport of [³H]l-glutamate and [³H]l-aspartate decreased, as also did the affinity but not the transport capacity for the [³H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics ofl-glutamate orl-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes.     

l-[3H]lysine binding to rat retinal membrane: II. effect of kainic acid,d,l-α-aminoadipic acid,iodoacetic acid,and modification by dark-exposure

The rat retina and the different brain regions contain membranes sites that bindl-lysine in the nanomolar range. These binding sites undergo changes in different experimental conditions, thus: I) intraocular injection of kainic acid induces a reduction of the density ofl-lysine binding sites, II)d,l--aminoadipic acid injected into the eye enhances both kinetic parameters (B _max andK _d) ofl-[³H]lysine binding sites, III) the intraperitoneal injection of iodoacetic acid decreases the sensitivity for its ligand binding sites, and IV) the exposure to darkness of the rats reducesl-[³H]lysine binding in the retina, thalamus, hypothalamus and superior colliculus, but not in the occipital cortex; such a decrease appears to be characterized, at least in the retina, by a lower sensitivity of the binding sites forl-lysine after the exposure to darkness. The results show thatl-lysine binding sites are located on kainic acid-sensitive cells and can be involved in the physiological mechanism of vision.     

Effects of local anesthetics on phospholipid topology and dopamine uptake and release in rat brain synaptosomes

Summary The effects of local anesthetics on the topology of aminophospholipids and on the release and uptake of dopamine in rat brain synaptosomes have been examined. A metabolically intact preparation of synaptosomes was prepared which maintains aminophospholipid asymmetry and the capacity for sodium-driven uptake and depolarization-dependent release of dopamine. Incubation of synaptosomes with local anesthetics at 37°C induced perturbations in the topology of aminophospholipids as determined by their reactivities to the covalent probe trinitrobenzenesulfonic acid. The reaction of trinitrobenzenesulfonate with phosphatidylethanolamine and phosphatidylserine was inhibited 10–20% by low concentrations of tetracaine (1–100 m) and enhanced by high concentrations (0.3–1.0mm). Other local anesthetics showed a similar biphasic effect with a potency order of dibucaine>tetracaine>lidocaineprocaine. K⁺-stimulated, Ca²⁺-dependent release of [³H]dopamine was inhibited significantly at low concentrations of tetracaine (1–10 m) but enhanced at higher concentrations (0.1–1.0mm). Dibucaine and procaine had a similar biphasic effect on the dopamine release. For each of the local anesthetics tested, the inhibition of the reaction of phosphatidylethanolamine and phosphatidylserine with trinitrobenzenesulfonate occurred at concentrations which were shown also to inhibit the release of [³H]dopamine. Local anesthetics were shown to inhibit uptake of [³H]dopamine with a potency order which reflects their potency in producing anesthesia. The inhibition of dopamine uptake by dibucaine, tetracaine, lidocaine, or procaine was characterized by inhibitory constants (K _I ) of 1.8±0.4 m, 27±5 m, 190 m and 0.5mm, respectively.Abbreviations TNBS 2,4,6-trinitrobenzene sulfonate - PE phosphatidylethanolamine - PS phosphatidylserine - ESR electron spin resonance - TLC thin-layer chromatography - DA dopamine     

Functional and biochemical characteristics of a putative quisqualate-type receptor in rat striatum: Effect of brain lesions

Excitatory amino acids such asl-glutamate (Glu) and quisqualate (QUIS) markedly potentiated K⁺-evoked release of exogeneous [³H]dopamine (DA) from rat striatal slices. Intranstriatal kainic acid injections resulted in a total disappearance of the stimulatory effects of Glu on evoked-release of [³H]DA as well as in a parallel reduction in the maximal number (B_max) of ad-aspartate-insensitivel-[³H]Glu binding site in striatal particulate fractions. Following cortical ablation, the potentiating effect of Glu on [³H]DA release in decorticated striatal slices lasted longer, compared to normal slices, and occured during the 2nd min following K⁺-depolarization. However, the extent (%) of Glu stimulation on [³H]DA release remained the same in decorticated and normal striatal slices. Cortical ablation produced also a significant decrease in the B_max and in theK _d of thed-aspartateinsensitive binding site towardsl[³H]Glu. These results support the proposal that thed-aspartate-insensitive Glu binding site is somehow related to an amino acid receptor-mediated modulation of dopaminergic transmission in the rat corpus striatum.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Uptake of glycine froml-alanylglycine into renal brush border vesicles

Summary Isolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel froml-Ala · [³H]Gly andd-Ala · [³H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg²⁺. The microvilli vesicles transiently accumulated radiolabel froml-Ala · [³H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular > intravesicular) Na⁺ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all³H uptake froml-Ala · [³H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate ofl-ala · [³H]Gly hydrolysis to free alanine and [³H] glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel froml-Ala · [³H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate thatl-Ala · [³H]Gly is hydrolyzed at the external surface of the microvilli with the [³H]glycine released being transported into the vesicles by a Na⁺ gradient-dependent system identical to that employed for free glycine.Microvilli vesicle uptake of radiolabel fromd-Ala · [³H]Gly exhibited no Na⁺ dependent overshoot effect.d-Ala · [³H]Gly was completely resistant to microvilli-catalyzed hydrolysis.Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range ofl-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli.Renal dipeptidase catalyzed hydrolysis ofl-Ala · Gly but notd-Ala · Gly, as was the case with microvilli-catalyzed hydrolysis of these dipeptides.With its location in the renal brush border microvilli and its hydrolytic action againstl-dipeptides, renal dipeptidase may act at the luminal surface of the proximal tubule cell to hydrolyzel-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na⁺ gradient-dependent processes.     

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

Lithium: Effect on [3H]spiperone binding,ionic content,and amino acid levels in the brain of rats

After prolonged treatment of rats with lithium (pellets, 0.21% lithium carbonate, or 0.5 mg/ml lithium chloride in drinking water) for three months, the level of lithium in plasma was 0.87 meq/liter; in several brain regions, between 1.06–1.39 eq/g wet weight. The content of sodium and potassium in the plasma was normal. The level of potassium in the brain regions tested increased by 13–30% and that of sodium by about 10%. Glycine levels increased significantly in all the regions (cerebral cortex, midbrain, cerebellum, and spinal cord). In the cerebellum GABA was also increased, while glutamine was decreased. In midbrain, apart from increases in glycine levels, alamine, valine, GABA and lysine were also increased. In the spinal cord, glutamic acid was also increased. Changes were largely in the putative neurotransmitters. Long-term treatment with lithium also influenced the high-affinity binding of [³H]spiperone in the cerebral cortex and corpus striatum. Two specific binding sites were found in both brain regions; the main change was the reduction in the lower affinity binding site (B _max2).