Sodium-dependent High-affinity Uptake Research Articles

Up-regulation of P2X7 receptor-mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex.

Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP-mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow-desensitizing P2X7 receptor (P2X7R). Modulation of [(3) H]GABA and [(14) C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug-resistant epilepsy (non-MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy. The P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-100 μm) decreased [(3) H]GABA and [(14) C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 μm) was prevented by the selective P2X7R antagonist, A-438079 (3 μm). Down-modulation of [(14) C]glutamate uptake by BzATP (100 μm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [(3) H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples. High-frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low-affinity slowly desensitizing ionotropic P2X7R, leads to down-modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high-frequency firing, endure GABA signaling and increase GABAergic rundown, thereby unbalancing glutamatergic neuroexcitation. This study highlights the relevance of the ATP-sensitive P2X7R as an important negative modulator of GABA and glutamate transport and prompts for novel antiepileptic therapeutic targets.

Na+-d-glucose cotransporter in the kidney of Leucoraja erinacea: molecular identification and intrarenal distribution

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb d-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts.

The Transport of Choline

ABSTRACTCholine has many physiological functions throughout the body that are dependent on its available local supply. However, since choline is a charged hydrophilic cation, transport mechanisms are required for it to cross biological membranes. Choline transport is required for cellular membrane construction and is the rate-limiting step for acetylcholine production. Transport mechanisms include: (1) sodium-dependent high-affinity uptake mechanism in synaptosomes, (2) sodium-independent low-affinity mechanism on cellular membranes, and (3) unique choline uptake mechanisms (e.g., blood–brain barrier choline transport). A comprehensive overview of choline transport studies is provided. This review article examines landmark and current choline transport studies, molecular mapping, and molecular identification of these carriers. Information regarding the choline-binding site is presented by reviewing choline structural analog (hemicholinium-3 and 15, and other nitrogen/methyl-hydroxyl compounds) inhibition studies. Choline transport in Alzheimer's disease, brain ischemic events, and aging is also discussed. Emphasis throughout the article is placed on targeting the choline transporter in disease and use of this carrier as a drug delivery vector.

Glutamate-induced swelling of single astroglial cells in primary culture

Glutamate induced an increase in cell volume within one minute and evoked cytosolic Ca 2+ transients in type 1 astroglial cells in primary culture obtained from the cerebral cortex of newborn rat. Even the metabotropic glutamate receptor agonists (1S,3R)-1-aminocyclopentane-1,3-dicar☐ylic acid (1S-3R-ACPD) and L(+)-2-amino-4 phosphonobutyric acid (L-AP4) induced a cell swelling with ACPD inducing a parallel Ca 2+ transient while L-AP4 did not. A new method was used where rapid changes in relative cell volume could be followed at the single cell level. Relative volume changes in cultured single astroglial cells were examined by microspectrofluorimetry after loading the cells with the highly fluorescent intracellular probe fura-2/AM. At its isosbestic point, 358 nm, fura-2 is ion-insensitive and the fluorescent signals emitted are related only to the intracellular dye concentration. By varying the excitation wavelengths, changes in intracellular Ca 2+ transients could be recorded simultaneously with the relative volume variations of the individual cells. Thus, as rapid changes in cell volume were followed, the results from this method could be of physiological significance. Glutamate-induced cell swelling was blocked by BaCl 2 and by tetraethylammonium, suggesting that K + channels are operative in glutamate-induced cell swelling. Furthermore, the glutamate-induced swelling was blocked by the Na +; K +, and 2Cl − co-transport inhibitor furosemide. The glutamate-induced swelling was partially blocked by pertussis toxin and partially blocked also by the glutamate carrier-blocker dihydroaspartate. When the ionotropic glutamate receptor α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid was blocked with the antagonist 2,3-dihydroxy-6-nitro7-sulfamoyl-benzo(F)quinoxaline, glutamate still induced a swelling, suggesting that this receptor was not directly involved in the glutamate-induced volume increase. Even in situations of blocked or partially blocked swelling, intracellular Ca 2+ transients could be obtained. Furthermore, the glutamate-induced swelling was evoked even in low extracellular Ca 2+ concentrations. Our data suggest that glutamate-induced rapid swelling is a complex process at the molecular level. One hypothetical mechanism might be that glutamate interacts with metabotropic glutamate receptors and induces a release of Ca 2+ from internal stores. Furthermore glutamate interacts with K + channels, and probably at least one co-transporter and the sodium-dependent high-affinity uptake glutamate carrier, resulting in cell swelling.

The glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate depresses excitatory synaptic transmission via a presynaptic mechanism in cultured hippocampal neurons

Sodium-dependent high-affinity uptake of glutamate is thought to play a major role in the maintenance of very low extracellular concentrations of excitatory amino acids (EAA), and may modulate the actions of released transmitter at G-protein-coupled receptors and extrasynaptic receptors that are activated over a longer distance and time course. We have examined the effects of the recently developed uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) on monosynaptically evoked excitatory postsynaptic currents (EPSCs) in very-low-density cultures of hippocampal neurons. L-Trans-PDC produced a decreased amplitude of both the non-NMDA and NMDA receptor-mediated components of monosynaptically evoked EPSCs. Examination of miniature EPSCs (mEPSCs) indicated that changes in the sensitivity of postsynaptic non-NMDA receptors did not underline the decrease in evoked EPSC amplitudes. The metabotropic receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) also depressed both components of the EPSC. The competitive metabotropic receptor antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the depression of EPSC amplitude induced by 1S,3R-ACPD and also blocked the effects of L-trans-PDC. Finally, low concentrations of L-glutamate (2 microM) mimicked the effects of L-trans-PDC on EPSC amplitude. From these results we conclude that the application of L-trans-PDC to cultured hippocampal neurons results in the activation of presynaptic metabotropic receptors, leading to a decrease in synaptic transmission. We propose that this effect is due to an increase in ambient glutamate concentrations following inhibition of glutamate uptake, resulting in presynaptic inhibition of excitatory synaptic transmission.

Regional differences in the inhibition of l-glutamate and l-aspartate sodium-dependent high affinity uptake systems in rat CNS synaptosomes by l- trans-pyrrolidine-2,4-dicarboxylate, threo-3-hydroxy- d-aspartate and d-aspartate

The sodium-dependent high affinity transport of l-[ 3H]glutamate and l-[ 3H]aspartate into synaptosomal fractions prepared from three different regions was employed to investigate the inhibitors l- trans-pyrrolidine-2,4-dicarboxylate, threo-3-hydroxy- d-aspartate and d-aspartate. These substances showed regional heterogeneity as inhibitors of sodium-dependent high affinity uptake of l-glutamate and l-aspartate. l- trans-Pyrrolidine-2,4-dicarboxylate was a more potent inhibitor of the uptake of l-glutamate than of l-aspartate in the cortex (IC 50 8 μM vs l-glutamate and 13 μM vs l-aspartate) and cerebellum (IC 50 4 μM v l-glutamate and 8 μM vs l-aspartate). threo-3-Hydroxy- d-aspartate was a more potent inhibitor of the uptake of l-glutamate than of l-aspartate in the cortex (IC 50 9 μM vs l-glutamate and 13 μM vs l-aspartate) and hippocampus (IC 50 6 μM v l-glutamate and 11 μM v l-aspartate). d-Aspartate was a more potent inhibitor of the uptake of l-glutamate than of l-aspartate only in the cortex (IC 50 8 μM vs l-glutamate and 15 μM vs l-aspartate). These results thus support other evidence that there is regional heterogeneity in sodium-dependent high affinity acidic amino acid uptake sites in the brain.

Effect of tianeptine on the central cholinergic system: involvement of serotonin.

The effect of tianeptine on in vivo acetylcholine (ACh) release from brain hemispheric regions of freely moving rats was investigated using the microdialysis technique coupled with a sensitive radioenzymatic method. Tianeptine, at the dose of 30 mg/kg i.p., reduced ACh release from dorsal hippocampi by 40% in 40 min, and induced a 30% decrease of ACh output from frontal cortices while at the doses of 10 and 20 mg/kg it had no effect. In striata the drug did not significantly affect ACh release although it showed a tendency to increase it. The ACh content in the three areas considered was not affected by tianeptine at above doses. The drug did not alter choline-o-acetyltransferase and acetylcholinesterase activities suggesting that it did not influence the cholinergic system through direct action on the ACh metabolism; furthermore, it did not influence the sodium-dependent high-affinity uptake of choline in striatum, cortex and hippocampus. Impairment of serotonergic (5-HT) neurotransmission by chemical lesion of the median raphe nucleus or by metergoline, a blocker of 5-HT receptors, antagonized the cholinergic effect of tianeptine. The involvement of the serotonergic system is specific because lesions of the noradrenergic dorsal bundle failed to prevent the inhibitory action of tianeptine. The present data suggest that 5-HT may mediate the effect of tianeptine on the cholinergic system in dorsal hippocampi.

A comparison of cholinergic effects of HI-6 and pralidoxime-2-chloride (2-PAM) in soman poisoning

The effects of HI-6 and pralidoxime chloride (2-PAM) on soman-induced lethality, time to death and several cholinergic parameters in rats were compared to understand the beneficial action of HI-6. Treatment with atropine sulfate (ATS) or HI-6 alone protected against 1.2 and 2.5 LD 50s of soman respectively, whereas 2-PAM or methylated atropine (AMN) alone afforded no protection. Addition of ATS, but not AMN, to HI-6-treated rats enhanced the protection from 2.5 to 5.5 LD 50s. HI-6 increased the time-todeath, while 2-PAM had no effect; a combination of HI-6 and ATS provided the most significant increase in time-to-death. Cholinesterase (ChE) activity was not altered in any tissue by ATS, HI-6 or 2-PAM treatment individually, but was markedly inhibited in all tissues by 100 μg/kg of soman. In soman-poisoned rats, the HI-6, but not the 2-PAM, group had significantly higher levels of ChE in blood and other peripheral tissues than did the group given soman alone. Neither HI-6 nor 2-PAM affected soman-inhibited ChE in the brain. Additional ATS treatment had no effect on ChE activity. HI-6 and 2-PAM neither modified baseline brain acetylcholine (ACh) or choline (Ch) levels nor protected against soman-induced ACh or Ch elevation. 2-PAM exhibited a 4-fold more potent in vitro inhibition of 3H-quinuclidinyl benzilate ( 3H-QNB) binding and sodium-dependent high-affinity Ch uptake (HACU) than did HI-6 in brain tissues. The findings that 2-PAM is a more potent in vitro inhibitor of muscarinic receptor binding and HACU than HI-6, and yet neither elevates ChE activity in the periphery nor protects rats against soman poisoning, indicate the importance of higher ChE activity in the periphery of HI-6-treated rats. Maintenance by HI-6 of a certain amount of active ChE in the periphery appears to be important for survival after soman exposure.

Age-related alteration in excitatory amino acid neurotransmission in rat brain

The excitatory amino acids as neurotransmitters in the neocortex, hippocampus, striatum, thalamus, amygdala, nucleus basalis of Meynert and cerebellum from rats aged 4 months, 12 months and 24 months have been examined by measuring sodium-dependent high affinity uptake of D-[ 3H]-aspartate into preparations containing synaptosomes. Calcium-dependent K +-stimulated release of endogenous glutamate from the nucleus basalis was also measured. The hippocampus and cerebellum failed to show significant age-related changes in uptake of D-[ 3H]-aspartate. D-[ 3H]-aspartate uptake decreased significantly in the neocortex (29%), striatum (29%), nucleus basalis (26%), amygdala (19%) and thalamus (16%) in the middle-aged rats as compared to the young rats, but the changes were not progressive with age. The release of glutamate from the nucleus basalis was unaltered during the aging process.

Treatment with Oxiracetam or Choline Restores Cholinergic Biochemical and Pharmacological Activities in Striata of Decorticated Rats

Interruption of the corticostriatal pathway by undercutting the frontal cortex resulted after 2 weeks in a 40% reduction of basal acetylcholine (ACh) release in vivo, and in inhibition of the striatal sodium-dependent high-affinity uptake of choline (SDHACU) to the same extent. The lesion, too, completely prevented the rise (about 35%) in striatal ACh content induced by oxotremorine and apomorphine acting at muscarine and dopamine receptors, respectively. Acute intraperitoneal injections of 100 mg/kg of either oxiracetam or choline chloride resulted in time-dependent recovery of ACh output from the striata of decorticated rats to control levels. Oxiracetam also normalized the ex vivo striatal SDHACU activity of decorticated rats 2 h after administration without any effect in sham-operated rats. Oxiracetam or choline chloride administered before oxotremorine (0.8 mg/kg, i.p.) or apomorphine (1 mg/kg, i.p.) reinstated the ACh-increasing effect of these agonists. It is suggested that choline chloride acts directly simply by being the precursor for ACh, whereas oxiracetam may act indirectly, possibly by increasing the availability of choline chloride for ACh synthesis. Furthermore, the frontally decorticated rat could constitute a useful model for studying means to restore the deficit in striatal cholinergic neurotransmission.

Methylmercury‐Induced Movement and Postural Disorders in Developing Rat: High‐Affinity Uptake of Choline, Glutamate, and γ‐Aminobutyric Acid in the Cerebral Cortex and Caudate‐Putamen

Subcutaneous administration of methylmercuric chloride to neonatal rats resulted in movement and postural disorders during the fourth postnatal week. Sodium-dependent high-affinity uptake of radiolabeled choline, glutamate, and gamma-aminobutyric acid (GABA) was measured in homogenates of cerebral cortex and caudate-putamen. There was a significant decrease in the uptake of [3H]choline in the cerebral cortex, but not in the caudate-putamen, at the onset of neurological impairment (73-75%) and at one subclinical stage of toxicity (58-64%). No significant differences in [3H]glutamate uptake were detected in either region. The uptake of [3H]GABA in the presence of 1 mM beta-alanine, which was employed to inhibit the glial uptake process, was reduced significantly in both the cerebral cortex and caudate-putamen at the onset of neurological impairment (50-62%) and at one subclinical stage (40-51%). This decrease in [3H]GABA uptake is consistent with the results of previous studies using this animal model, which demonstrated a preferential degeneration of GABAergic neurons in the cerebral cortex and caudate-putamen of methylmercury-treated animals. Because the high-affinity uptake of choline is the rate-limiting step for acetylcholine synthesis by cholinergic neurons, the decrease in [3H]choline uptake may reflect an abnormal development of cholinergic innervation of the cerebral cortex.

Chelation of endogenous membrane calcium inhibits γ‐aminobutyric acid uptake in synaptosomes

In a previous work, we have demonstrated that calcium chelators induce the release of gamma-aminobutyric acid (GABA) from synaptosomes in a Na+ -dependent manner and that this release is blocked by cations such as Mg2+, La3+, and ruthenium red. In the present study, we show that treatment of synaptosomes with 0.1 mM EGTA in the absence of both Ca2+ and Mg2+ inhibits the sodium-dependent high-affinity uptake of [3H]GABA by about 50%. This inhibition increased to about 65% with 1.5 mM EGTA, and it was completely prevented by an excess of Ca2+ or by 1.2 mM Mg2+. In contrast, when EDTA was used as a chelator, Mg2+ was unable to reverse the inhibition. The inhibitory effect of 0.1 mM EGTA was also prevented by 250 microM La3+ or by 20 microM ruthenium red. In the absence of chelators and the presence of Ca2+ and Mg2+, 50 microM and 200 microM La3+ inhibited GABA uptake by about 20 and 50%, respectively, whereas 20 microM ruthenium red produced a nonsignificant 25% inhibition and nifedipine was without effect. It is concluded that the membrane-surface negative charges, probably those of the sialic acid molecules that have been implicated in the functioning of the GABA carrier, must be neutralized by endogenous Ca2+ or by another cation in order to permit the adequate function of the transporter. The inhibition by La3+ in the absence of the chelators could be explained by a binding of this cation to the Na+ sites on the GABA carrier.

Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [ 3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx ( V max) and half-saturation concentration ( K m) corresponded to 1.06 · 10 −4 μmol/min per mg protein and 27.5 μM, respectively. Competition experiments in the presence of sodium ion showed that [ 3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as β-alanine and hypotaurine as well as unlabeled taurine, γ-Aminobutyric acid (GABA) (0.1 MM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, l-methionine, l-2,4-diaminobutyric acid, glycine, l-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na + gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.

Fentanyl produces cholinergically-mediated analeptic and EEG arousal effects in rats

Fentanyl (20 μg/kg i.p.), administered to naltrexone-pretreated, pentobarbital-anesthetized rats, produced a shortening of the duration of narcosis. This analeptic effect was blocked by atropine, but not by methylatropine, indicating that a central cholinergic mechanism was involved. Fentanyl also increased sodium-dependent high affinity uptake of choline activity in the hippocampus and cortex that had been depressed by the barbiturate. Injection of 0.8 ng of fentanyl into the pontis oralis in the pontine reticular formation also produced analepsis in naltrexone-pretreated, pentobarbitalized rats. Hippocampal EEG recordings also showed the appearance of cholinergically-mediated theta activity, which was indicative of arousal activity in the hippocampus. These results suggest that fentanyl, in addition to possessing potent opiate activity, also activates a nonopioid-mediated central cholinergic arousal system.

The role of calcium in the regulation of [ 3H]hemicholinium-3 binding sites in rat brain

The role of calcium in the regulation of sodium-dependent high-affinity uptake of choline was assessed in vitro in slices of the rat brain, by measuring the specific binding of [ 3H]hemicholinium-3 ([ 3H]HCh-3) and the uptake of [ 3H]choline. Depolarization with potassium of slices of hippocampus, cortex, or striatum significantly increased the specific binding of [ 3H]HCh-3 when compared to control slices. However, the observed potentiation of specific binding of [ 3H]HCh-3 was markedly inhibited by the removal of calcium from the incubation medium in cortex or hippocampus, but not in slices of striatum. Alterations in the uptake of [ 3H]choline directly paralleled the observed changes in the specific binding of [ 3H]HCh-3 in striatum of the rat and were unaffected by the reduction of calcium in the incubation medium. The inorganic calcium channel antagonists, cadmium and cobalt, but not magnesium, zinc, manganese or lanthanum, significantly inhibited the 40 mM potassium chloride-induced stimulation of the binding of [ 3H]HCh-3 in the striatum. Finally, the calcium ionophore A23187 significantly increased the binding of [ 3H]HCh-3 in slices of striatum, either in the presence or absence of calcium in the bathing medium. This study demonstrates regional differences in the role of extracellular calcium in the regulation of the uptake of choline and suggests the involvement of intracellular release of calcium in the in vitro regulation of the sodium-dependent high-affinity uptake of choline in the striatum

Modulatory effect of dopamine on high-affinity glutamate uptake in the rat striatum.

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.

Quisqualate-sensitive, chloride-dependent transport of glutamate into rat brain synaptosomes.

A chloride-dependent transport process for glutamate has been identified in partially purified rat brain synaptosomes. This process shares many characteristics with the chloride-dependent sequestration process for glutamate in brain sonicates, which was previously thought to represent a quisqualate receptor, such as sensitivity to specific inhibitors and regulation by anions. Increasing the concentrations of chloride led to an increase in the apparent Vmax without affecting the KT. Synaptosomes preincubated with [3H]-L-glutamate exhibit an efflux of the radiolabel, which was stimulated by a substrate for the carrier in the incubating medium, indicating the bidirectional nature of the transport. The chloride-dependent transfer process is restricted to the brain, and regional and developmental profiles clearly distinguish it from the sodium-dependent high-affinity uptake process for glutamate. Nevertheless, the effects of excitotoxic lesions strongly suggest a neuronal localization of the chloride-dependent transport.

The effects of an in vivo administration of phencyclidine on sodium-dependent high affinity choline uptake in rat hippocampus and striatum in vitro

The effect of phencyclidine and other drugs on sodium-dependent high-affinity uptake of choline in the rat hippocampus and/or striatum was investigated and related to the behavioral changes induced by these agents. In contrast to atropine (40.0 mg/kg), which increased the uptake of choline in synaptosomes from both the rat hippocampus and striatum, the administration of phencyclidine (54.4 mg/kg), 30 min prior to sacrifice, caused a significant decrease in the uptake of choline in synaptosomes from rat striatum (but not hippocampus). This effect of phencyclidine could be seen up to 1 hr after administration of drug, but by 3.5 hr the uptake of choline was essentially back to normal. The inhibition of striatal uptake of choline occurred at a time when brain levels of phencyclidine and its metabolites were at their highest, and the animals were essentially immobile; it did not appear to correlate with behavioral changes (including stereotypy and catalepsy) seen at later times after this dose of phencyclidine (54.4 mg/kg), or at earlier times after smaller doses. Amphetamine (6.0 mg/kg) also decreased the uptake of choline in the striatum. Haloperidol (2.0 and 3.0 mg/kg) blocked both the phencyclidine and amphetamine-induced inhibition of the uptake of choline but only the behavioral effects of amphetamine. The data suggest that phencyclidine may be exerting an indirect effect on the uptake of choline in the striatum via its interaction with the dopaminergic system. However, this neurochemical effect of phencyclidine could not be related in any simple way to the immobility, stereotypy or catalepsy caused by this drug.

Topographic changes in high-affinity glutamate uptake in the cat red nucleus, substantia nigra, thalamus, and caudate nucleus after lesions of sensorimotor cortical areas

The distribution of presumed glutamatergic projections from sensorimotor cortical areas to the red nucleus (RN), the substantia nigra (SN), the ventrolateral thalamic complex, and the caudate nucleus (CN) was investigated in the cat. For this purpose, the changes in the sodium-dependent high-affinity glutamate uptake (HAGU) rate were measured in homogenates of tissue microdissected from various parts of these subcortical structures after chronic surgical ablation of sensorimotor cortical areas. After 8 to 10 days survival, significant reductions in HAGU activity were noted in the structures studied on the side ipsilateral to the operated cortex. Within each structure, various quantitative or qualitative changes were observed. Higher decreases in HAGU activity were found in the caudal part of the RN, the ventrolateral thalamic nucleus, and the dorsolateral part of the CN than in the other parts of these structures. The lateral part of the SN showed a large decrease in HAGU rate and its medial part a small but significant increase. Referring to the anatomic data concerning the organization of cortical projections from sensorimotor areas to the structures studied, it was shown that our results support the view that glutamate could act as a neurotransmitter along various corticosubcortical pathways.

The effect of choline mustard on the rat superior cervical ganglia.

The effect of choline mustard aziridinium ion (ChM) on the isolated superior cervical ganglion of the rat was investigated. In the presence of ChM (22.5 microM), stimulation at 1 Hz resulted in a slowly developing blockade of transmission, which did not occur at a stimulus frequency of 0.1 Hz. Choline (0.1 mM) slowed the onset of the blockade produced by stimulation at 1 Hz in the presence of ChM. The presence of excess thiosulphate ions prevented the action of ChM on the transmission in the superior cervical ganglion. Treatment of the ganglion with ChM (22.5 microM) only slightly inhibited the depolarization produced by carbachol (dose ratio 1.3), suggesting the drug produced a small degree of receptor blockade. [3H]-choline accumulation in the rat superior cervical ganglia displays several components: (a) sodium-dependent high affinity uptake (SDHAU) that can be activated further by preincubation in a high concentration of K+ ions; (b) sodium-dependent low affinity uptake (SILAU); (c) linear diffusional accumulation which does not saturate. Hemicholinium-3 selectively inhibits the activated sodium-dependent high affinity uptake, but is a weak inhibitor of resting sodium-dependent high affinity uptake and sodium-independent low affinity uptake. ChM inhibits both activated and resting sodium-dependent high affinity uptake, but is a very weak inhibitor of sodium-independent low affinity uptake. Homocholine shows similar selectivity. ChM inhibition of activated sodium-dependent high affinity uptake is very much more persistent than that of hemicholinium-3. Hemicholinium-3 and ChM both inhibit [3H]-acetylcholine synthesis.