Noradrenaline, a Transmitter Candidate in the Retina

The occurrence, metabolism, uptake, and release of noradrenaline were studied in the bovine retina with the following results. (1) Small amounts of noradrenaline occur in the retina and are restricted to the area corresponding to the inner nuclear and plexiform layers. (2) Retinal tissue can metabolise [¹⁴C]dopamine to form quantities of [¹⁴C]noradrenaline. (3) [¹⁴C]Noradrenaline can also be partly metabolised to form [¹⁴C]normetanephrine. (4) When bovine retinas were incubated with 5 × 10^-7 M-[³H]noradrenaline for 20 min and processed for autoradiography, most of the label was associated with apparent nerve processes in the inner plexiform layer. Biochemical analysis showed that more than 95% of the label was noradrenaline. (5) [¹⁴C]Noradrenaline uptake saturated with increasing noradrenaline concentrations and followed Michaelis-Menten kinetics. This uptake could be accounted for by two processes, a high-affinity system with a K_m1 of 5 × 10^-8 M and a V_max1 of 0.193 pmol/mg/10 min and a low-affinity system with a K_m2 of 6.3 × 10^-5 M and a V_max2 of 0.109 nmol/mg/10 min. (6) Noradrenaline uptake was strongly dependent on temperature and sodium, less dependent on potassium, and independent of calcium and magnesium ions. (7) Centrally acting drugs, such as desipramine, imipramine, desmethylimipramine, and amitriptyline, inhibited noradrenaline uptake by more than 55% at the concentration of 5 × 10^-5 M. These drugs at the same concentration diminished dopamine uptake by less than 30%. (8) Noradrenaline uptake is stereospecific, the (-) isomer having a greater affinity for the uptake sites than the (+) isomer. (9) [¹⁴C]Noradrenaline in the retina could be released by increasing the external potassium concentration. This release was calcium-dependent and was blocked by 20 mM-cobalt chloride. The present studies could be interpreted as supporting the idea that noradrenaline acts as a transmitter in the retina.     

THE UPTAKE AND RELEASE OF [3H]DOPAMINE IN THE GOLDFISH RETINA

Abstract— The uptake and release of [³H]dopamine was studied in the goldfish retina with the following results: (1) when goldfish retinas were incubated with 2 ± 10^-7m -[³H]dopamine for less than 20min and processed for autoradiography. most of the label was associated with dopaminergic terminals that contact certain horizontal cells. Biochemical analysis showed that > 93% of this label was [³H]-dopamine. (2) [³H]dopamine uptake saturated with increasing dopamine concentration and followed Michaelis-Menten kinetics. This uptake could be explained by a single ‘high-affinity’ mechanism with a K_m of 2.61 ± 0.41 ± 10^-7m and a V_max of 66 ± 12 ± 10^-12 mol/min/mg protein. (3) [³H]dopamine uptake was temperature-dependent with a temperature coefficient of 1.7 and an energy of activation of 11.4 kcal/mol. (4) The initial rate of uptake was unaffected by the absence of Ca²⁺ or the presence of Co²⁺; however, more than 85, uptake was blocked in the absence of external Na⁺. (5) Neither 1 mm -cyanide nor 5 mm -iodoacetate blocked more than 30% of uptake individually; however, in combination > 70% of uptake was blocked. (6) Centrally acting drugs benztropine and diphenylpyraline inhibited at least 60–70% of [³H]dopamine uptake. (7) [³H]dopamine in the retina could be released by increasing the external K⁺ concentration. This release was Ca²⁺ -dependent and was blocked by 10mm -Co²⁺ or 2Omm -Mg²⁺. The amount of [³H]dopamine released was not affected by the presence of benztropine, diphenylpyraline or fluphenazine in the incubation medium. These studies add further support for dopamine as a neurotransmitter used by interplexiform cells of the goldfish retina.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

DEVELOPMENTAL CHANGES IN THE KINETICS OF γ-AMINOBUTYRIC ACID TRANSPORT BY CHICK RETINA

—Isolated retinas from chick embryos and mature animals were incubated in [³H]GABA at 25°C for 10 min in order to investigate kinetic properties of the amino acid uptake system. Embryo retina accumulated [³H]GABA by two distinct kinetic systems with K_m values of the order 10⁻⁴m and 10⁻⁵m for the low- and high-affinity mechanisms respectively. However, as the retina matured, the high-affinity process disappeared and only the low-affinity system was detectable. No obvious explanation can be offered for this phenomenon although a similar observation has previously been made in chick brain by other workers.     

SYNAPTIC VESICLES ISOLATED FROM RAT HEART: l-[3H]NOREPINEPHRINE UPTAKE PROPERTIES1

A fraction containing synaptic vesicles was isolated from rat heart by differential centrifugation, and the uptake of l-[³H]norepinephrine was studied in vitro., Uptake was highly dependent upon time and temperature, and was linear for 6 min at 30° or 4 min at 37°C. About 80% of the measured uptake required both ATP and Mg²⁺ and was inhibited by nanomolar concentrations of reserpine; no inhibition was obtained with cocaine. These properties are characteristic of storage vesicle uptake as opposed to synaptic membrane uptake. Uptake of norepinephrine was saturable and displayed a single K_m value of 2 μM. The uptake was completely stereospecific, as unlabeled dl-norepinephrine was less than half as effective as unlabeled l-norepinephrine in reducing uptake of l-[³H]norepinephrine. Norepinephrine uptake could be inhibited by various phenethylamines and indoleamines following the rank order: reserpine > harmaline > 5-hydroxytryptamine > dopamine > norepinephrine. The vesicle preparation also incorporated [³H]5-hydroxytryptamine and [³H]dopamine. 5-Hydroxytryptamine uptake displayed a K_m of 0.5 μM and a maximal uptake equivalent to that seen with norepineph-rine; dopamine uptake followed complex kinetics. Administration of reserpine in vivo or destruction of sympathetic neurons by long-term guanethidine treatment both eliminated the ability of the preparation to take up norepinephrine. Synaptic vesicles of cardiac sympathetic neurons thus resemble vesicles prepared from other central and peripheral catecholaminergic tissues; this method may be used readily to examine drug effects on rat heart synaptic vesicle function.     

Adult development of the hippocampal-serotonin system of C57BL/6N mice; analysis of high-affinity uptake of 3H-5HT in slices and synaptosomes

The saturable and specific high-affinity uptake of [³H]serotonin ([³H]5HT) (5 × 10^?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [³H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [³H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P₂ suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10^?5 M cold 5-HT (83 and 78% respectively), 10^?5 M fluoxetine (85 and 82% respectively) and 10^?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [³H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent K_m for serotonin at both ages (6.7 x 10^?8 M), but a 44% decrease in V_max in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.     

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

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

KINETICS OF THE SODIUM-DEPENDENT TRANSPORT OF GAMMA-AMINOBUTYRIC ACID BY SYNAPTOSOMES

The kinetics of transport of gamma-aminobutyric acid [2,3-³H] by synaptosomes from rat brain was studied by means of a rapid filtration technique. The rate of uptake was proportional to the protein concentration over the range 0.05—0.2 mg of synaptosomal protein per ml. Although apparent allosteric kinetics were observed with sodium, transport followed simple saturation kinetics with respect to GABA and no heterotropic, cooperative effects of GABA on sodium on kinetics were observed. A minimum of three interacting sodium sites is suggested the basis of Hill plots of the sodium data. Both the apparent K_m and V_max for GABA were functions of the sodium ion concentration but the effect of sodium was considerably greater on V_max than on the apparent K_m The V_max for GABA was 1.1 ± 0.5 nmol.min^?1 mg^?1 of protein at 95 mm sodium and decreased to 12 per Cent of this value at 19 mm sodium. The apparent K^m for GABA increased from 4.0 ± 1.0 μm at 95 mm sodium to 8.4 ± 2.0 μm at 19 mm sodium. Potassium was a noncompetitive inhibitor with respect to GABA and did not affect the apparent cooperativity observed with sodium. These findings are discussed in terms of models of GABA transport.     

Dopamine Uptake by Rat Striatal Synaptosomes: Time-and Temperature-Dependent Decay and Protection by Dithiothreitol and Dopamine

The uptake of [³H]dopamine (DA) into rat striatal synaptosomes in the presence of a monoamine oxidase inhibitor was studied using a filtration technique. After a 10-min preincubation period, a fast initial uptake of [³H] DA was seen. Uptake reached a maximum after 4 min of incubation. If incubation was continued for more than 7 min, a gradual decrease in synaptosomal [³H]DA levels was found. Uptake was dependent on preincubation time; initial uptake velocity and maximal uptake decreased irreversibly with increasing preincubation periods. Moreover, the capacity of the synaptosomes to retain the [³H]DA during longer incubation times was progressively affected. The decrease in initial uptake activity was due to a decrease in the V_max of the transport system. Dithiothreitol (2.8 mM) protected synaptosomal uptake activity against deterioration at 37°C. Also, DA itself (10^-7M) stabilized the uptake mechanism if added to the suspension before preincubation was started. Since [³H]DA uptake observed after loading the synaptosomes with labeled DA was similar to the uptake seen if the synaptosomes were not previously loaded with DA, it was concluded that under these conditions synaptosomal DA is completely exchangeable with exogenous substrate. Prolonged storage of the synaptosomes at 0°C also resulted in a time-dependent decrease in uptake activity (t_1/2= 116 min). The addition of unlabeled DA or dithiothreitol to the suspension did not affect instability at 0°C.     

Evidence for the Sequential Formation of Two Complexes Between an Uptake Inhibitor, GBR 12783 [1-[2-(Diphenylmethoxy)ethyl]-4-(3-Phenyl-2-Propenyl)piperazine], and the Neuronal Transporter of Dopamine

Abstract : Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37°C produced an inhibition of the specific uptake of [³H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC₅₀ = 1.85 ± 0.1 nM) instead of incubation alone (IC₅₀ = 25 ± 3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V_o) decreased for increasing concentrations of inhibitor (K_i≥ 20 nM). TI slowly isomerized to a more stable complex TI^* (K^*_i≤ 5 nM) with a value of t_1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (V_S) is set to zero were generally preferred, suggesting that formation of TI^* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V_o, and V_S tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in K_M and a decrease in V_max ; these effects were improved for 10 nM GBR 12783 and at 20°C. These results are discussed in relation to previous data concerning [³H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition ; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.     

Characterization of Inhibitor-Sensitive and -Resistant Adenosine Transporters in Cultured Human Fetal Astrocytes

Abstract: The kinetic characteristics of [³H]adenosine uptake, the extent to which accumulated [³H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of K_T and V_max, and the sensitivities with which nucleoside transport inhibitors blocked [³H]adenosine accumulations were determined in cultured human fetal astrocytes. K_T and V_max values for accumulations of [³H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′-iodotubercidin (ITU) were 6.2 µM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components respectively. In the presence of EHNA and ITU, where <4% of accumulated [³H]adenosine was metabolized, transport per se was measured, and kinetic values for K_T and V_max were 179 µM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [³H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [³H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [³H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [³H]adenosine transport; for the inhibitor-sensitive components the IC₅₀ values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC₅₀ values were 2.5 µM for NBI, 5.1 µM for dilazep, and 39.0 µM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.     

Functional Identification and Molecular Cloning of a Human Brain Vesicle Monoamine Transporter

A vesicle monoamine transporter was functionally identified, molecularly cloned, and characterized from a human substantia nigra cDNA library. The ATP-dependent transport of 5-[³H]hydroxytryptamine ([³H]5-HT) by digitonin-permeabilized fibroblasts expressing the vesicle monoamine/H^± antiporter in culture exhibited a K_m of 0.55 μM. Reserpine and tetrabenazine, inhibitors of two monoamine binding sites, effectively blocked [³H]5-HT accumulation with K₁ values of 34 and 78 nM, respectively. Pretreatment of cells with as little as 10 nM reserpine in the presence of ATP abolished uptake. The rank order for substrate inhibition of [³H]5-HT uptake for both the previously reported rat vMAT1 and the human transporter clone followed the order 5-HT > dopamine > epinephrine > norepinephrine > 1 -methyl-4-phen- ylpyridinium > 2-phenylethylamine > histamine. The virtually identical transport characteristics of rvMATI and hvMAT1 confirm the relevance of neuropharmacological studies of rat brain biogenic amine uptake and storage to human brain neurochemistry.     

IN VITRO EXPERIMENTS ON THE METABOLISM, ACCUMULATION AND RELEASE OF 5-HT IN THE NERVOUS SYSTEM OF THE SNAIL HELIX POMATIA

Abstract— The accumulation, metabolism and stimulated-induced release of 5-HT in the nervous system of the snail was studied. When nervous tissue was incubated at 24°C in a medium containing [¹⁴C]5-HT or [³H]tryptophan, tissue: medium ratios of about 25:1 and 4:1 respectively were obtained after 45 min incubation. The process responsible for [¹⁴C]5-HT accumulation showed properties of an active transport system: it was temperature sensitive and was greatly inhibited by dinitrophenol and ouabain. Furthermore, the accumulation process was inhibited by imipramine and desipramine. Of a number of analogues of indole, N-acetyl-5-HT and 5-hydroxytryptophan were the most potent in the inhibition of the accumulation of [¹⁴C]5-HT. The presence of a large molar excess of amino acids had little effect. A small amount (less than 14 per cent) of the accumulated [¹⁴C]5-HT was metabolized to form 5-hydroxyindole acetic acid, even after long periods (2 h) of incubation. The accumulated [³H]tryptophan was metabolized to form 5-hydroxytryptophan and 5-HT; the content of formed [³H]5-HT increased with incubation time whilst the [³H]5-hydroxytryptophan remained more or less constant. The presence of p-chlorophenylalanine in the incubation medium did not interfere with the accumulation of [³H]tryptophan, though it inhibited the formation of [³H]5-hydroxytryptophan and to a greater extent [³H]5-HT. A rapid efflux of the accumulated [¹⁴C]5-HT from snail nervous tissue was observed on electrical stimulation. Slower release resulted when the Ca²⁺ ion content of the incubation medium was replaced by Mg²⁺ ions. There is also a slight efflux of radioactive substances following electrical stimulation in tissues previously incubated in [³H]tryptophan. Most of this radioactivity was attributed to the formed [³H]5-HT. The data support the idea that 5-HT is a transmitter-substance in the snail Helix pomatia, and that re-uptake of the substance is a method of inactivating the released amine.     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.     

Release and Uptake Rates of 5-Hydroxytryptamine in the Dorsal Raphe and Substantia Nigra Reticulata of the Rat Brain

Abstract: Fast scan cyclic voltammetry with carbon fiber electrodes has been used to investigate the dynamics of the neurotransmitter 5-hydroxytryptamine (5-HT) in the extracellular fluid of two brain regions: the dorsal raphe and the substantia nigra reticulata. The method used previously was shown to be optimized to allow the time course of 5-HT concentration changes to be measured rapidly. Measurements were made in slices prepared from the brains of rats with the carbon fiber electrode inserted into the tissue and a bipolar stimulating electrode placed on the slice surface. Identification of 5-HT as the detected substance in both regions was based on voltammetric, anatomical, physiological, and pharmacological evidence. Autoradiography using [³H]paroxetine revealed highest 5-HT transporter binding densities in the regions in which voltammetric measurements were made. Evaluation of the pharmacological actions of tetrodotoxin and tetrabenazine, as well as the effects of calcium removal, suggested that 5-HT storage was vesicular and that the release process was exocytotic. The effects of fluoxetine (0.5 µM) were typical of a competitive uptake inhibitor, changing K_m with little effect on V_max. Release of 5-HT was found to be maximal with wide (2-ms) stimulus pulses in both regions, as expected for release from small unmyelinated processes, and to increase linearly with the number of pulses when high frequencies (100 Hz) were used. At lower frequencies, the concentration observed was a function of both release and uptake. Kinetic simulations of the data revealed that the major difference in 5-HT neurotransmission between the two regions was that release and uptake rates are twice as large in the dorsal raphe ([5-HT] per pulse = 100 ± 20 nM, V_max = 1,300 ± 20 nM/s for dorsal raphe; [5-HT] per pulse = 55 ± 7 nM, V_max = 570 ± 70 nM/s for substantia nigra reticulata). When normalized to tissue content, uptake rates in both regions were identical and similar to rates previously reported for dopamine in dopamine terminal regions. Nonetheless, compared with dopaminergic transmission in terminal regions such as the striatum, the absolute clearance rates in the substantia nigra reticulata and dorsal raphe were lower, resulting in a longer lifetime of 5-HT in the extracellular fluid and allowing long-range interactions.     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

NEUROCHEMICAL PROPERTIES OF ADRENERGIC NERVES REGENERATED AFTER 6-HYDROXYDOPAMINE

Abstract— The uptake-storage properties and synthesis of noradrenaline, and fluorescence morphology of adrenergic nerves which have been allowed to regenerate for 4 weeks after a chemical sympathectomy produced by 6-hydroxydopamine have been investigated in mouse iris and atrium. The regenerated nerve terminals displayed a lower formaldehyde-induced fluorescence intensity whereas the non-terminal axons exhibited a stronger fluorescence intensity and a more beaded appearance compared with mature nerves. The endogenous noradrenaline concentration after 6-hydroxydopamine was 30% in iris and 45% in atrium compared to control values. Recovery of [³H]noradrenaline uptake was found to be more rapid than that of endogenous noradrenaline concentration after the 6-hydroxydopamine treatment. [³H]Noradrenaline uptake in regenerating and adult mature nerves both obeyed Michaelis-Menten kinetics having identical K_m values. There was a close correlation between [³H]noradrenaline uptake and nerve density of adrenergic nerves regenerated after 6-hydroxydopamine. These results show that [³H]noradrenaline uptake is a better index for the number of regenerated nerve terminals than is the endogenous noradrenaline concentration. The retention of [³H]noradrenaline taken up and accumulated in vitro was about the same in regenerated and mature nerves, although a slight tendency to less effective retention was observed in the regenerated nerves. Subcellular distribution studies showed that relatively less [³H]noradrenaline was recovered in the microsomal fraction after 6-hydroxydopamine treatment. The formation of ¹⁴C-labelled catecholamines from [¹⁴C]DOPA was higher in regenerating nerves than indicated by the endogenous noradrenaline concentration but lower than that indicated by the [³H]noradrenaline. It is concluded that the regenerating nerves contain less endogenous noradrenaline than adult mature nerves and that the uptake mechanism develops promptly, whereas the development of the storage mechanism lags behind.     

THE UPTAKE OF 36Cl INTO ASTROCYTES IN TISSUE CULTURE BY A POTASSIUM-DEPENDENT, SATURABLE PROCESS

Abstract— Previous suggestions that the K⁺-dependent uptake of chloride by mammalian cerebral cortex is localized to cortical astrocytes has been investigated in two lines of neural cells in tissue culture. Hamster astrocytes and mouse neuroblastoma cells were each cultured on small glass cover slips in Eagle's (astrocytes) or Dulbecco's (neuroblastoma) supplemented media. At 48 h, cultures were incubated at 37°C for 1–60 min in the presence of ³⁶Cl plus [³H]inulin (as a measure of extracellular space). In media containing approximately 140 mm chloride, the rate of uptake of ³⁶Cl by the astrocytes was a function of the concentration of K⁺ (range 5–54 mm ) in the medium, and the uptake was characterized by saturation kinetics and an apparent K_m of 38·5 mm . The uptake was temperature and apparently energy dependent, significantly inhibited by 5 or 10 mm Br^-, I^-, SCN^- or ClO₄^-, and competitively inhibited by 10 mm acetazolamide (K_i= 27·1 mm ). None of these characteristics were observed with neuroblastoma cultures studied under similar conditions. In chloride-free media, the cellular K/Na ratio of the astrocytes shifted from the usual value of 4·55 to a value of 0·29, the culture medium became more alkaline than normal, and the cells spontaneously sloughed from the cover slips but remained normally viable. Our observations are the first to provide direct support for previous suggestions that there is a mediated, K⁺-dependent coupled cation, chloride and fluid uptake by mammalian cerebrocortical glia and that this uptake is an enzymatically catalysed process. The observations have been discussed in terms of a presumed central role for astrocytes in modulating the external ionic milieu of the neurons they surround and in terms of implications for epilepsy, stroke and cortical edema.     

ACTIVE UPTAKE OF [3H]5-HT BY SYNAPTIC VESICLES FROM RAT BRAIN

The question of whether synaptic vesicles accumulate [³H]5-HT by an active process was investigated in a mixed population of vesiclcs from whole rat brain. The temperature dependence and the effect of metabolic inhibitors were studied in synaptosomal suspensions and vesicular fractions. Arrhenius plots for synaptosomes differed from those for vesicles as did the temperature coefficients for these two fractions. For synaptosomes the Q₁₀ was 7 and for vesicles 1.6. However, if ATP was added to the incubation, the temperature dependence of vesicular amine accumulation became manifest; the Arrhenius plot resembled that of synaptosomes and the Q₁₀ was greater than 20 indicating strong temperature dependence. In the presence of ATP, vesicular uptake was stimulated approx 8-fold. Ouabain, dinitrophenol and NEM inhibited synaptosomal uptake but failed to affect [³H]5-HT accumulation by vesicles in the absence of ATP. When ATP was added, vesicular uptake was also blocked by NEM but was unaffected by either ouabain or DNP. Total observed uptake consisted of two components, one ATP-dependent and one nonsaturable and ATP-independent. The active process had a K_m= 1.25 × 10^?7 M and could be completely blocked by either 10^?3 M or 10^?7 M-reserpine. Active vesicular [³H]5-HT uptake was magnesium dependent and was inhibited by sodium and potassium. Cation effects on uptake were specific and could not be accounted for by either changes in osmotic pressure or ionic strength. It was concluded that synaptic vesicles from whole rat brain accumulate [³H]5-HT by an active process.