Evidence for the neurotransmitter role of norepinephrine in the ventral thalamic nucleus of the guinea pig: Localization, uptake and release

The localization, uptake and release of norepinephrine (NE) by the isolated guinea pig ventral thalamic nucleus (VT) were studied to determine the possible neurotransmitter role of this catecholamine. The NE content in the VT was 356 +/- 24 ng/g wet weight and intraneuronal localization of NE was demonstrated in the VT by histofluorescence. [3H]NE accumulation into the VT slices was about 3 times that of [3H]NE (10(-7) M) in the medium 10 min after incubation. Kinetic analyses indicated 2 components of [3H]NE accumulation, one representing a high (Km1 4 X 10(-7) M and Vmax1 1.5 pmol/mg/10 min) and a low (Km2 1.8 X 10(-6) M and Vmax2 5 pmol/mg/10 min) affinity uptake system. Desmethylimipramine (10(-5) M) reduced the high affinity uptake of [3H]NE by 30% of the control value. Electrical stimulation of the slices increased the efflux of [3H]NE from tissues preloaded with [3H]NE, in a current- and frequency-dependent fashion. The release of [3H]NE induced by stimulation of up to 1 mA was inhibited by tetrodotoxin (10(-6) M) or Ca-free medium containing EGTA (10(-4) M). These findings provide strong evidence for the neurotransmitter role of NE in the VT.     

Regional changes in monoamine content and uptake of the rat brain during postnatal development.

Regional norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) contents in the developing rat brain were estimated. The rate of increase in NE content was the highest in diencephalon, followed by the lower brain stem, limbic-striatum, neocortex and cerebellum. With postnatal aging, DA concentration increased markedly in limbic-striatum, slightly in the neocortex and negligibly in other regions. In each region except cerebellum, 5-HT content increased gradually but the rate of increase in diencephalon was relatively high. Comparison of the kinetics of high affinity uptake of L-[3H]NE and [3H]5-HT between the neonatal and the adult brain indicated that Km values of L-[3H]NE and [3H]5-HT uptake were 2.9 X 10(-7) M and 1.7 X 10(-7) M respectively in neocortex, diencephalon and lower brain stem and 4.3 X 10(-7) M and 2.3 X 10(-7) M in limbic-striatum in the neonate as well as in the adult. Vmax values of both amines uptake differed regionally and the values in the neonate were lower than those in the adult in all regions. Limbic-striatum showed a higher Vmax value than other regions in uptake of both amines. These results suggested that innervation of monoaminergic neurons in the brain progressed with increasing age, that projections of both NE and 5-HT neurons were relatively high into hypothalamus and limbic-striatum and that DA neuron projections concentrated at striatum. Although the brain, except for limbic-striatum, showed neither regional nor developmental differences in affinity of L-[3H]NE and [3H]5-HT to synaptosomes, the density of nerve terminal of both monoaminergic neurons increased in all regions of the brain during postnatal development. In limbic-striatum, higher Km and Vmax values of both amines, uptake suggest the existence of both amines' uptake into DA terminal to some extent.     

Presynaptic modulation of sympathetic neurotransmission in rat left ventricle slices: effect of pressure overload

Summary. The purpose of this study was to establish the rat left ventricle (LV) tissue slice system for examination of norepinephrine (NE) release from sympathetic nerve terminals. Moreover, initial experiments were performed to use the LV tissue slice system to examine differences in NE uptake and release following cardiac pressure overload induced by abdominal aortic constriction (AC). Kinetic parameters (Vmax, Km) for the specific uptake of [³H]-NE demonstrated high affinity (Km, 1.94 ± 0.83 μM) and moderate capacity uptake (Vmax, 182 ± 6 fmol/mg/weight/min). Following 10 days of pressure overload, the Vmax for [³H]-NE uptake was significantly reduced (by 46%) in LV slices from AC rats compared to sham-operated (SO) controls. In control rat LV slices preloaded with [³H]-NE, electrically evoked [³H]-overflow was calcium- and stimulus pulse number-dependent. The neuronal uptake inhibitor, desipramine (DMI), increased (by 60%) evoked [³H]-overflow from LV slices. The α₂-agonist, UK14304, decreased evoked [³H]-overflow from LV slices in a concentration-dependent manner (maximal reduction of 75%). The β₂-agonist, salbutamol, increased evoked [³H]-overflow from LV slices in a concentration-dependent manner (maximal increase of 200%). In separate experiments, the LV tissue slice system was used to examine the effect of pressure overload on evoked [³H]-overflow. Following 10 days of pressure overload, evoked [³H]-overflow from LV slices of AC rats was increased (by 50%) compared to SO control. Increases in evoked [³H]-overflow from LV slices of AC rats compared to SO controls remained evident in the presence of DMI. These results demonstrate the relative importance of NE release and uptake using an in vitro LV tissue slice system. Sympathetic nerve terminals innervating rat LV were demonstrated to possess functional presynaptic α₂- and β₂-adrenergic receptors. Finally, using this LV tissue slice system, reductions in the uptake velocity and increases in evoked NE release were demonstrated in response to acute cardiac pressure overload. Received August 12, 1999; accepted January 12, 2000     

3H]glycine uptake in rat hippocampus: kinetic analysis and autoradiographic localization.

The uptake of [3H]glycine by rat hippocampal tissue in vitro has been characterized. [3H]Glycine transport into a crude synaptosomal (P2) fraction was resolved into two components. The high affinity component (Km = 21 +/- 5.4 microM, Vmax = 490 +/- 234 pmol/3 min/mg protein) was almost completely sodium dependent whereas the low affinity component (Km = 2.214 +/- 0.958 mM, Vmax = 13.9 +/- 0.5 nmol/3 min/mg protein) was partially dependent on sodium ions. Amongst a range of amino acids, only L-serine, L-glutamate, L-proline, histidine and glycine itself inhibited [3H]glycine uptake at 1 mM. The autoradiographic localization of [3H]glycine uptake in rat hippocampal slices revealed a general pattern of labeling in dendritic regions with a sparing of pyramidal and granule neuron cell bodies. However, a laminar distribution was apparent since the amino acid was preferentially accumulated in the hilus of the dentate gyrus, in the stratum lacunosum-moleculare, in the alveus and in the molecular layer of the lower blade of the dentate gyrus. A diffuse pattern of accumulation was apparent in these areas along with dense clusters of silver grains. The clusters were associated with small cell bodies and might represent glycine uptake into astrocytes. Glycine transport mechanisms may influence the modulatory effects of this amino acid on N-methyl-D-aspartate receptor-mediated neurotransmission in the hippocampus.     

Mechanisms for termination of the action of dopamine in carotid body chemoreceptors

The possible presence of a high affinity uptake mechanism for dopamine and the metabolism of this amine was investigated in the rabbit carotid body. The type I cells of this sensory organ contain high levels of dopamine and share with sympathetic nerve endings (which possess a high affinity uptake mechanism for catecholamines) the properties of being presynaptic catecholaminergic elements, but also have in common with chromaffin cells from intact adrenal medulla (which lack a high affinity catecholamine uptake) a similar embryological origin and ultrastructural appearance. Our experiments revealed only a low affinity uptake process (Km = 6.76 X 10(-4) M [3H]dopamine; Vmax = 1.84 X 10(-9) mol [3H]dopamine/mg protein/min) in the rabbit carotid body. In agreement with our kinetic findings, the uptake of [3H]dopamine was found to be independent of the Na+ concentration in the incubation media. The efflux from the tissue of incorporated [3H]dopamine was very fast (88% wash-out with a half-time of 4 min). DOPAC was the principal catabolite of dopamine in the carotid body, and the organ exhibited a very low capacity for norepinephrine synthesis. Neither chronic carotid sinus nerve section nor chronic sympathectomy modified the uptake of [3H]dopamine, suggesting that the lack of expression of a high affinity uptake was not a consequence of trophic suppression by the neural innervation of the organ. We conclude that overflow, or wash-out, of released dopamine may be quantitatively the most important mechanism for the inactivation of this putative neurotransmitter in the rabbit carotid body.     

PEPT2-mediated transport of 5-aminolevulinic acid and carnosine in astrocytes

5-aminolevulinic acid (ALA) and carnosine have important physiological and pathophysiological roles in the CNS. Both are substrates for the proton-coupled oligopeptide transporter PEPT2. The purpose of the current study was to determine the importance of PEPT2 in the uptake of ALA and carnosine in rat and mouse (PEPT2+/+ and PEPT2-/-) cultured neonatal astrocytes. Although neonatal astrocytes are known to express PEPT2, its quantitative importance in the transport of these compounds is not known. [14C]ALA uptake in neonatal rat astrocytes was inhibited by dipeptides, an alpha-amino containing cephalosporin (which is a PEPT2 substrate) but was not affected by a non-amino containing cephalosporin (which is not a PEPT2 substrate). Uptake was pH sensitive as expected from a proton-coupled transporter and was saturable (Vmax=715+/-29 pmol/mg/min, Km=606+/-14 microM). [3H]Carnosine uptake in neonatal rat astrocytes was inhibited by dipeptides but not by histidine (a substrate for the peptide/histidine transporters PHT1 and PHT2) and also showed saturable transport (Vmax=447+/-23 pmol/mg/min, Km=43+/-5.5 microM). Neonatal astrocytes from PEPT2-/- mice had a 62% reduction in [14C]ALA uptake and a 92% reduction in [3H]carnosine uptake compared to PEPT2+/+ mice. These results demonstrate that PEPT2 is the primary transporter responsible for the astrocytic uptake of ALA and carnosine.     

Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth cones isolated from developing rat forebrain: uptake and release of GABA and noradrenaline

A growth cone-enriched fraction isolated from neonatal rat forebrain was shown to accumulate gamma-amino [3H]butyric acid ([3H]-GABA) and [3H]noradrenaline ([3H]NA). Uptake of both neurotransmitters was sodium- and temperature-dependent and exhibited saturation kinetics with Km values of 17.7 microM and 4.5 microM respectively and Vmax values of 114 pmol/min/mg protein and 59 pmol/min/mg protein respectively. Electron microscopic autoradiography showed that about 50% of isolated growth cones can accumulate [3H]GABA. Inhibitor studies showed that beta-alanine was a relatively weak inhibitor of [3H]GABA uptake compared to nipecotic acid and diamino-butyric acid. Growth cone fractions preloaded with [3H]GABA and [3H]NA demonstrated a K+ (25 mM) -induced release of both neurotransmitters. Of the K+-stimulated release of [3H]GABA 50% was Ca2+-dependent, whereas the release of [3H]NA was entirely Ca2+-independent.     

Effects of 4-aminopyridine on synaptic transmission in the cat spinal cord

The accumulation of [3H] catecholamines from [3H] tyrosine in frontal cortical, septal, striatal and hippocampal slices was examined following intracerebroventricular (i.c.v.) injections of ACTH 1-24, lysine vasopressin (LVP) and saline. Both ACTH 1-24 and LVP (1 microgram) selectively increased the accumulation of [3H] dopamine (DA) in frontal cortical slices, but did not affect that of [3H] norepinephrine (NE). LVP but not ACTH 1-24 also inhibited the accumulation of [3H] DA in striatal slices. ACTH 1-24 did not alter the accumulation of [3H] NE in hippocampal slices, nor did LVP alter the accumulation of either catecholamine (CA) in septal slices. In vitro incubations with ACTH analogs of LVP failed to alter the rate of accumulation of [3H] CAs in striatal, substantia nigral and frontal cortical slices, except for an inhibitory effect at high doses. This effect is believed to be an artifact of precursor dilution caused by release of tyrosine following degradation of the peptides. Neither peptide modified the increased [3H] CA accumulation stimulated by 26 mM K+, nor did ACTH 1-24 modify the inhibition of [3H] CA accumulation caused by 3 X 10 -6 M haloperidol or 3 X 10 -7 M apomorphine. Selective activation of the mesocortical DA system has also been reported ot occur in response to footshock, suggesting the possibility that endogenous ACTH and/or LVP might mediate the stress-induced activation of mesocortical DA synthesis. Alternatively, i.c.v. injections of these peptides may themselves be stressful and thus indirectly elicit the response.     

Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; [3H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [3H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [3H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [3H]-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of [3H]-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. We estimated that this system enabled the cells to concentrate [3H]-5-HT up to 44-fold at an external [3H]-5-HT concentration of 10(-7) M. Inhibition of [3H]-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of [3H]-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on [3H]-5-HT uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of physical stress on 5-hydroxytryptamine uptake kinetics (Km and Vmax) in human platelets.

Platelet 5-HT uptake is possibly one mechanism by which plasma level of 5-HT is physiologically controlled. This mechanism may be of importance in the vascular 5-HT mediated tone and protect against thrombolysis and cardiovascular events. Physical stress is known to affect various platelet functions. The aim of this study was to investigate whether or not physical stress has any influence on platelet 5-HT uptake kinetics. In healthy adult persons (n = 13) the maximal rate (Vmax) and Michaelis-Menten constant (Km) of platelet 5-HT uptake (in vitro) were determined before (a), immediately after (b) and 1 hour after 50 min of squash playing (c). Immediately after squash Vmax and Km were elevates significantly as compared to the values before squash [Vmax(a): 77.0 +/- 31.0 (SD) pmol 5-HT/10(8) pl./min, Vmax(b): 109.9 +/- 48.4 (SD) pmol 5-HT/10(8) pl./min, p less than 0.01; Km(a): 5.16 +/- 1.73 (SD) x 10(-7) mol/1, Km(b): 7.59 +/- 2.37 (SD) x 10(-7) mol/1, p less than 0.05]. The percent elevations of Vmax (47%) and Km (42%) were of the same range. One hour after squash the values of Vmax(c) and Km(c) were found to be normalized. It is concluded that physical stress, like squash playing, does not affect net platelet 5-HT uptake in healthy persons at physiological plasma 5-HT concentrations. The significant increases of Vmax and Km of platelet 5-HT uptake are suggested to be adaptation reactions to keep the platelet net 5-HT uptake constant.     

PSA-NCAM expression in the piriform cortex of the adult rat. Modulation by NMDA receptor antagonist administration

Uptake of biocytin and biotin was investigated in cultured transformed variants of neuronal (NB2a neuroblastoma) and glial (C6 astrocytoma) CNS cells. NB2a cells took up both compounds but biocytin was transported more efficiently than biotin in the nanomolar concentration range. In NB2a cells a single transport mechanism was found for biocytin with different kinetic parameters in the presence of high extracellular Na+ (Km 0.4 microM, Vmax 20 pmol/min/mg), K+ (Km 1.7 microM, Vmax 32 pmol/min/mg), or choline+ (Km 0.1 microM, Vmax 5 pmol/min/mg). Two transport systems (Km1 17 microM, Vmax1 53 pmol/min/mg; Km2 314 microM, Vmax2 360 pmol/min/mg) were identified for biotin with only system 1 being Na+-dependent. Biocytin uptake was competitively inhibited by excess biotin but not vice versa. Inhibition studies with structural analogs indicated different specificities for biotin and biocytin uptake. Biocytin uptake into C6 cells was hardly detectable whereas biotin was taken up by diffusion (kD 0.6 microl/min/mg) and a single saturable mechanism (Km 70 microM, Vmax 119 pmol/min/mg) at high extracellular Na+. High extracellular K+ enhanced biotin diffusion into C6 cells. Inhibition studies with structural analogs revealed a less specific biotin uptake mechanism in C6 than in NB2a cells. Biocytin normalized deficient biotin-dependent propionyl-CoA carboxylase activity within 4 h in biotin-deficient NB2a cells whereas in C6 cells reactivation was <20% thereby confirming that biocytin is only poorly transported into C6 cells. Specific biocytin uptake into NB2a cells is to our knowledge the first demonstration of a carrier-mediated transport mechanism for this compound. Neuronal biocytin uptake might contribute to the pathogenesis of biotinidase deficiency where biocytin is present in elevated levels.     

Uptake, metabolism, and release of [3H]-histamine by glial cells in primary cultures of chicken cerebral hemispheres

Labelled histamine was taken up into cultured glial cells of chick embryonic brain by a system with high affinity for histamine and diffusion. The active uptake, occurring at low concentrations of the amine, was Na+ dependent and gave an apparent Km of 0.24 microM and a Vmax of 0.31 pmol x mg protein-1 x min-1. The uptake was completely blocked by desmethylimipramine (Ki = 2.5 microM) and partially by the histamine agonists and histamine-N-methyltransferase blockers 4-methylhistamine and 2-methylhistamine (I30 values obtained were 2 microM and 5 microM). Other psychoactive drugs were either ineffective (imipramine) or they showed moderate inhibitory effects (amitriptyline and cocaine). Ouabain (100 microM) inhibited uptake by approximately 50%. Diffusion occurred at high concentrations of the amine, was insensitive to extracellular Na+, and was proportional to histamine concentration up to 1 mM. [3H]-Histamine, taken up into the cells, was metabolized and/or released. The spontaneous efflux of the radioactivity measured after 10 min of exposure to [3H]-histamine (when most of it was still unmetabolized), was moderately Ca++ dependent, accelerated by both reduced concentrations of extracellular Na+ and enhanced concentrations of K+ and inhibited by desmethylimipramine. After prolonged (60 min) incubation, histamine metabolites detected in the cells presented 78% of the chromatogram radioactivity and consisted of N tau-methylhistamine and N tau-methylimidazole acetic acid. These results indicate that at low nM concentrations, histamine is taken up and metabolized by (and released from) glial cells by an Na(+)-dependent system, and the intracellular metabolism seems to serve an increased uptake of the amine.     

Platelet serotonin uptake in panic disorder patients: a replication study

Platelet serotonin uptake was measured in 29 patients with DSM-III panic disorder or agoraphobia with panic attacks and compared to values obtained in 23 controls. Both the affinity constant (Km) and the maximal rate of uptake (Vmax) were determined in a buffered medium using 14C-serotonin. Patients and controls did not differ significantly with respect to age or Km values. A statistically significant difference was observed for Vmax (mean +/- SD = 65 +/- 22 pmol/10(8) platelet/min in patients vs. 44 +/- 13 pmol/10(8) platelets/min in controls). This finding suggests an overactivity of peripheral serotonergic function in panic disorder, which may also imply a similar dysfunction centrally.     

Pre- and postsynaptic muscarinic receptors in surgical samples from human cerebral cortex

The present study was carried out using fresh surgical material from human cerebral cortex of patients who were not medicated with atropine or other drugs known to affect the cholinergic system. The concentration of [3H]L-quinuclidinyl benzilate binding sites was 0.45 /+- 0.05 pmol/mg protein and the Kd-value of the receptor-[3H]L-QNB-complex was 0.038 /+- 0.005 nM. Agonist binding was studied by varying the concentration of carbamylcholine (10(-8) to 10(-2) M) in the presence of a constant concentration (0.2nM) of [3H]L-quinuclidinyl benzilate. The data revealed the existence of two populations of binding sites for carbamylcholine with different affinities and capacities. Presynaptic muscarinic receptors were studied in slices of the cerebral cortex, which were loaded with [3H]choline. The muscarinic antagonist, atropine (10(-6) and 10(-7) M) acting at the presynaptic muscarinic receptors enhanced the release of [3H] acetylcholine. It was also shown that muscarinic stimulation leads to elevation of cyclic GMP levels in the human cerebral cortical slices.     

ATP-dependent [3H]Met-enkephalin uptake by bovine adrenal chromaffin granule membrane

Uptake of [3H]Met-enkephalin by purified chromaffin granule membrane isolated from bovine adrenal medulla was investigated. Addition of a single divalent cation, such as Mg2+ or Ca2+, did not affect the uptake. The presence of 3 mM ATP in the incubation medium stimulated the uptake to two times that of the control. The same stimulation effect was also found in the presence of ATP plus a single divalent cation. The ATP-dependent uptake reached its half-maximal level within 5 min after initiation of the reaction at 25 degrees C, and reached a plateau within 10 min. The apparent Km for [3H]Met-enkephalin uptake by the chromaffin granule membrane was about 2.3 X 10(-6) M. GTP, CTP, UTP and ADP did not stimulate enkephalin uptake. Several calcium inhibitors such as trifluoperazine, verapamil and 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octylester (TMB-8) markedly inhibited enkephalin uptake. The inhibition exceeded 80%, although these chemicals inhibited Ca2+-ATPase activity in chromaffin granule membrane only 30% under the same conditions. Ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), at 3 mM also inhibited uptake about 30%. The results indicate that uptake of Met-enkephalin by chromaffin granule membrane ghosts was driven not only by ATPase activity, but also by some other Ca2+-ATP-mediated mechanism(s).     

Topographical distribution of dopaminergic innervation and of dopaminergic receptors in the rat striatum. I. Mictoestimation of [3H] dopamine uptake and dopamine content in microdiscs.

Topographical variations in the uptake of [3H] dopamine (DA) and in the endogenous content of DA were estimated in the striatum of the rat. For this purpose, microdiscs were punched out in serial 500 mum sections. [3H] DA uptake was measured in 0.25 M sucrose homogenates prepared from microdiscs punched out from frozen slices (--7C). This uptake was similar to that observed in fresh tissues. It was unaffected by desmethylimipramine (5 X 10(-7) M), inhibited by benztropine (10(-6) M) and no longer detectable after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopaminergic pathway. Both [3H] DA uptake and DA content decreased regularly from the rostral to the caudal part of the structure. In contrast, no important differences could be found in the dorso-ventral plane. These results suggest that the extent of dopaminergic innervation is heterogenous within the structure.     

Castration differentially alters [3H]nisoxetine binding to norepinephrine uptake sites in olfactory bulb and frontal cortex of male rats

In the present study, [3H]nisoxetine binding to norepinephrine (NE) uptake sites and [3H]norepinephrine uptake were investigated within olfactory bulb (OB) and frontal cortex homogenates from intact and castrated male rats. Statistically significant reductions in the number of [3H]nisoxetine binding sites (Bmax) were found in OB from the castrates, while significantly increased Bmax values were obtained in the frontal cortex. Castration also significantly altered the affinity (Kd) of [3H]nisoxetine binding in the frontal cortex, but not in the OB. Assessment of [3H]norepinephrine uptake showed that in neither brain regions were there any statistically significant differences in Km nor Vmax between the castrated and intact male rats, indicating that the basal uptake process is not changed following castration in either of these brain areas. These results demonstrate the differential effects of castration upon [3H]nisoxetine binding sites between the OB and frontal cortex. Such findings provide new evidence for one of the mechanisms by which androgens may modulate central noradrenergic activity.     

Release of endogenous N-acetylaspartylglutamate (NAAG) and uptake of [3H]NAAG in guinea pig cerebellar slices

For the purpose of obtaining chemical information about the physiological role of N-acetylaspartylglutamate (NAAG), the release of endogenous NAAG from and the uptake of [3H]NAAG by Guinea pig cerebellar slices were investigated in comparison with L-aspartate (Asp) and L-glutamate (Glu). Although endogenous NAAG was found to be released spontaneously from the slices as is endogenous Asp and Glu, high-K+-induced facilitation of release occurred only for endogenous Asp and Glu in a Ca2+-dependent manner, but not for NAAG. It was confirmed that [3H]NAAG itself was taken up in a Na+-dependent manner by the slices by two low-affinity processes with small Vmax values, and labeled Glu and glutamine were detected as the metabolites of [3H]NAAG in the slices. The [3H]NAAG uptake was slower than that of labeled Glu and was significantly depressed by NAAG, Asp, Glu and D-aspartate, but not affected by gamma-aminobutyrate, suggesting that NAAG may share a common uptake carrier with excitatory amino acids. These results suggest that endogenous NAAG may act extracellularly, but the amount of endogenous NAAG released from nerve terminals by presynaptic depolarization may be very small if any, and also that spontaneously liberated NAAG can be inactivated by low-affinity uptake systems, at least, in the Guinea pig cerebellum.     

Red blood cell triiodothyronine uptake in unipolar major depression: effect of a chronic antidepressant treatment

The evolution of kinetic parameters (Vmax, maximal velocity, and Km, Michaelis constant) of red blood cell (RBC) triiodothyronine (L-T3) initial uptake was followed in 19 inpatients suffering from unipolar depression after 1 week (D7) and 4 weeks (D28) of a chronic administration of fluvoxamine, in relation with the clinical efficacy of the drug. In a drug-free state (DO), Vmax (in pmol/min/10(8) cells) and Km (in nM) were significantly increased in depressed patients (Vmax +/- S.D.= 1.02 +/- 0.29, p< 0.01 and Km +/- S.D.= 68.8 +/-15.4, p< 0.05; n=19) compared to healthy volunteers matched for age and sex (Vmax +/- S.D.= 0.82 +/- 0.15 and Km S.D.= 58.8 +/- 9.0; n= 19). When patients were dichotomized on the basis of their treatment response, responders had kinetic parameters significantly increased (Vmax +/-S.D.= 1.03 +/- 0.26, p< 0.01 and Km +/- S.D.= 71.7 +/- 18.7, p< 0.05, n= 10) compared to controls, whereas non-responders had not (Vmax +/- S.D.= 1.00 +/- 0.33, NS and Km +/- S.D.= 65.7 +/- 10.9, NS, n= 9). At D7, Vmax differed from the one of controls only in the responders (Vmax +/- S.D.= 1.03 +/-0.26, p< 0.01). In addition, the percentage of variation of the individual Vmax values during the first week of treatment was significantly lower in responders than in non-responders (deltaVmax(D7-D0) +/- S.D. in % = 10.7 +/- 6.0 and 22.0 +/- 11. 1, p< 0.05, respectively). At D28, kinetics of L-T3 uptake normalized only in the responders (Vmax +/- S.D.= 0.91 +/- 0.13, NS; Km+/-S.D.= 65.7 +/- 7.4, NS). The results indicate that both RBC L-T3 uptake at the pretreatment level and its change during the first week of fluvoxamine treatment were related to the further clinical response to the antidepressant. RBC L-T3 uptake seems to be a biological correlate of the depressive symptomatology since the disturbances disappear only with the clinical remission.