THE EFFECT OF DELTA-AMINOLAEVULINIC ACID ON THE UPTAKE AND EFFLUX OF [3H]GABA IN RAT BRAIN SYNAPTOSOMES

§-Aminolaevulinic acid (§-ALA) is an omega amino acid which can be considered as an analogue of γ-aminobutyric acid (GABA). We have examined the effect of §-ALA on [³H]GABA uptake and release in the synaptosome fraction of rat cerebral cortex and report: (1) High concentrations of §-ALA (0.75-5 mM) stimulated [³H]GABA release very markedly, the stimulation with 1mM and 5mM-§-ALA exceeding the maximum obtainable with unlabelled GABA; (2) Low concentrations of §-ALA (0.1-0.5 mM) produced little stimulation of [³H]GABA efflux, less than that produced by similar concentrations of unlabelled GABA; (3) 0.1 mM-§-ALA reduced the stimulation of [³H]GABA efflux elicited by 55 mM-K⁺ and the combination of 1 mM-§-ALA and 55mM-K⁺ produced a lower stimulation of efflux than 1 mM-§-ALA alone; (4) §-ALA inhibits [³H]GABA uptake in a linearly competitive fashion and inhibition is maximal at 0.5 mM-§-ALA. These results are discussed in relation to the neuronal high affinity GABA transport mechanism and inhibition of the synaptosomal Na⁺ and K⁺ -dependent ATPase. It is also postulated that §-ALA increases the chloride conductance of the synaptosomal membrane, possibly by acting on presynaptic GABA receptors.     

EFFECT OF STRESS ON THE DISPOSITION OF CATECHOLAMINES LOCALIZED IN VARIOUS INTRANEURONAL STORAGE FORMS IN THE BRAIN STEM OF THE RAT

Abstract— The utilization of [³H]norepinephrine newly taken up or newly synthesized from [³H]tyrosine was studied in the brain stem of normal and stressed rats up to 5 h after the intracistemal injection of [³H]norepinephrine or [³H]tyrosine. The biphasic disappearance of the exogenous as well as of the endogenously synthesized [³HJnorepinephrine revealed that the amine is localized in at least two main compartments (A and B). The half-life of the amine newly taken up or newly synthesized, mainly localized in compartment A, is of short duration (between 15 and 30 min); the amine stored for a longer period of time and mainly distributed in compartment B is utilized more slowly (half-life, 180 to 260 min). A stress of short duration (15 min) induced by electric shocks applied to the feet increased the utilization of [³HJnorepinephrine newly taken up or newly synthesized from [³H]dopamine or [³H]tyrosine, but has no effect on the [³H]norepinephrine stored for a longer time period, indicating that the amine in compartment A is released in preference to that stored in compartment B. A stress of longer duration (180 min) increased the utilization of [³H] norepinephrine in both compartments and induced a sustained increased in norepinephrine synthesis as shown by the enhanced formation of [³H]norepinephrine from [³H]tyrosine in brain stem slices in vitro. The electrical stress was without effect on [³H]norepinephrine uptake. As for [³H]norepinephrine, the 15 min of stress enhanced the utilization of [³H] dopamine newly taken up or newly synthesized from [³H]tyrosine and had no effect on [³H]dopamine stored for a longer time period. These results suggest an increased release of both [³H]dopamine and [³H]norepinephrine from noradrenergic terminals of the rat brain stem. Finally, the 15 min of stress appeared to have no effect on the utilization of [³H] serotonin newly synthesized from [³H]tryptophan in serotonergic neurons of the brain stem, whereas the 180 min of stress increased the utilization of 5-HT in this structure.     

FACTORS INFLUENCING THE EFFLUX OF [3H]GAMMAAMINOBUTYRIC ACID FROM SATELLITE GLIAL CELLS IN RAT SENSORY GANGLIA

The spontaneous efflux of [³H]GABA from the satellite glial cells of rat dorsal root ganglia and the efflux evoked by 64 mM-K⁺ were studied in the presence of 10^-5M-amino-oxyacetic acid and found not to be affected by 10^-4M-D 600 or by elevated (9.6mM) Ca²⁺ in the absence of Mg²⁺. [³H]GABA efflux was increased by replacing sodium ions in the washing medium by choline ions and 64 mM-K⁺ failed to increase the efflux further. The drugs veratridine (10^-6 and 10^-4M) and batrachotoxin (10^-8 and 10^-6 M) failed to alter the spontaneous efflux of [³H]GABA from the glial cells. A variety of compounds, including amino acids, a GABA analogue and a GABA antagonist were tested for their ability to affect [³H]GABA efflux. The results indicated that compounds which inhibit GABA uptake into glial cells were also able to stimulate [³H]GABA efflux from these cells. The results are discussed with reference to possible mechanisms involved in the release of GABA from glial cells.     

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [³H]NE uptake capacity, an effect characterized by a robust decrease in the V_max of the transport of [³H]NE. As expected, reserpine did not displace the binding of [³H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [³H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [³H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca²⁺/Ca²⁺–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [³H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [³H]NE uptake and eliminated the inhibitory effects of reserpine on [³H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca²⁺-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.     

AN ADAPTATION OF THE PUSH-PULL CANNULA METHOD TO STUDY THE IN VIVO RELEASE OF [3H]DOPAMINE SYNTHESIZED FROM [3H]TYROSINE IN THE CAT CAUDATE NUCLEUS: EFFECTS OF VARIOUS PHYSICAL AND PHARMACOLOGICAL TREATMENTS

Abstract— The release of [³H]dopamine ([³H]DA) continuously synthesized from l-[3,5–³H]tyrosine from the caudate nucleus of the cat was estimated in halothane anaesthetized or‘encéphale isolé’animals. For this purpose, an improved superfusion cannula, avoiding tissue damage, was used. The best localization for the tip of the superfusion cannula was found first by determining the topographical distribution of endogenous DA within the caudate nucleus. A rostro-caudal heterogenous distribution of the transmitter was detected. In perfusion experiments, l-[3,5–³H]tyrosine was introduced continuously at a rate of 33μl/min. [³H]DA was the only catecholamine found in serial 15 min fractions as revealed by cochromatography. The spontaneous release of [³H]DA was greater in anaesthetized than in ‘encéphale isolé’ cats; it represented 150 and 100 times the blank value, respectively. Depolarization by K⁺ (30 mm) applied locally in the striatum or by electrical or mechanical stimulation of the substantia nigra caused a transitory increase in [³H]DA release. Conversely, a decrease in nerve activity induced by tetrodotoxin (5 × 10^?-⁷ m) or by electrocoagulation of the substantia nigra was associated with a decline in the amounts of [³H]DA in superfusates. A temporary reduction in [³H]DA release could also be obtained by a short-lasting cooling block of the substantia nigra. As expected, d-amphetamine (10^?-⁵ m) and benzotropine(10^?-⁷ m) added to the superfusing medium increased [³H]DA release. These pharmacological results, as well as the changes in [³H]DA release observed after various manipulations of the activity of dopaminergic neurones, confirms the validity and the high sensitivity of this approach.     

CALCIUM-DEPENDENT RELEASE OF EXOGENOUSLY LOADED γ-AMINO-[U-14C]BUTYRATE FROM SYNAPTOSOMES: TIME COURSE OF STIMULATION BY POTASSIUM, VERATRIDINE, AND THE CALCIUM IONOPHORE, A23187

—Synaptosomes which had taken up [¹⁴C]GABA were applied to a filter and rapidly perfused with various solutions in order to study the time course of release of this putative transmitter and the characteristics of its release. Depolarization of the synaptosomes with veratridine or 56mM-K⁺ or pretreatment with the Ca²⁺ ionophore, A23187, increased the calcium-dependent efflux of [¹⁴C]GABA. Release of [¹⁴C]GABA was increased by Ca²⁺ within 0.3 s of exposure, and the maximal release rate was not maintained for longer than 0.6 s. The reduction in the rate of release was not attributable to a decrease in calcium influx, but rather appeared to reflect fatigue at some subsequent stage in release. Stimulation by 56mM-K⁺ also elicited a calcium-independent increase in the efflux of radioactive GABA, which appeared to arise in part from subcellular particles other than synaptosomes.     

Characterization of Xylamine Binding to Proteins of PC12 Pheochromocytoma Cells

PC12 pheochromocytoma cells take up 3,4-dihydroxyphenylethylamine (dopamine) and norepinephrine by a Na+-dependent, cocaine-sensitive system. The kinetics suggest that the same transporter functions for both substrates. Xylamine, a nitrogen mustard that blocks catecholamine uptake into neurons, irreversibly inhibited norepinephrine uptake into PC12 (IC50 = 15 microM). Pretreatment with 10 microM xylamine did not inhibit norepinephrine transport if 10 microM cocaine or 100 microM norepinephrine was also present during the pretreatment period or if Na+ was absent. These results indicate that xylamine must interact with the norepinephrine transporter to inhibit norepinephrine uptake. PC12 accumulated [3H]xylamine; this uptake had Na+-dependent and Na+-independent components. The Na+-dependent uptake was saturable (Km = 13 microM), and it was inhibited by cocaine (IC50 = 0.6 microM), desipramine (IC50 less than 1 nM), and norepinephrine (IC50 = 1 microM). Several proteins became prominently labeled when intact PC12 cells were incubated with [3H]xylamine; these proteins were enriched in a plasma membrane fraction and have molecular weights of 17,000, 24,000, 31,000, 33,000, 41,000, 42,000, 52,000, and 80,000. Other proteins were labeled less prominently. The labeling of all proteins was markedly decreased when the incubation with [3H]xylamine occurred in the presence of cocaine, desipramine, gramicidin D, or in a Na+-free buffer. These results indicate that xylamine must be transported into the cells for covalent binding to proteins to occur. [3H]Xylamine labeled essentially the same proteins when incubated with cell homogenates, but competition experiments with bretylium, desipramine, and cocaine failed to reveal which of the [3H]xylamine-labeled proteins is associated with the norepinephrine transporter.     

Accumulation of noradrenaline and its oxidation products by cultured rodent astrocytes

The accumulation of [³H]noradrenaline ([³H]NA) and its oxidation products was studied in primary cultures of cerebral astrocytes. Astroglial accumulation of radiolabeled catecholamine ([³H] NA and oxidation products) was enhanced by manganese or iron, but it was inhibited by unlabeled NA, dopamine or ascorbate. Tissue:medium ratios of radioactivity increased as extracellular [³H]NA was oxidized. When extracellular oxidation was prevented by ascorbate, as confirmed by high performance liquid chromatography with electrochemical detection, either ouabain pretreatment or nominally Na⁺-free incubation medium inhibited approximately one-half of specific [³H]NA accumulation by rat (but not mouse) astrocytes. These observations suggest that neurological responses to trace metals and ascorbate may arise from the effects of these agents on the clearance of extracellular catecholamines. Astrocytes can accumulate oxidation products of NA more rapidly than they take up NA itself, but ascorbate at physiological concentrations prevents the oxidation process in extracellular fluid. Furthermore, in the presence of ascorbate, Na⁺-dependent transport mediates a significant component of NA accumulation in rat astrocytes.     

Transport of dopamine and levodopa and their interaction in COS-7 cells heterologously expressing monoamine neurotransmitter transporters and in monoaminergic cell lines PC12 and SK-N-SH

The present study investigated the effects of levodopa, a precursor of dopamine (DA) therapeutically used for the treatment of Parkinson's disease, on DA transport in the two different systems, COS-7 cells heterologously expressing rat monoamine transporter cDNA and in monoaminergic cell lines PC12 and SK-N-SH. Levodopa enhanced uptake of [3H]DA and [3H]norepinephrine (NE) but not [3H]serotonin in the transfected COS-7 cells in a concentration-dependent manner. On the other hand, in PC12 and SK-N-SH cells where NET is functionally expressed, levodopa enhanced [3H]DA and [3H]NE uptake at low concentrations and inhibited the uptake at higher concentrations. The effects of levodopa on catecholamine transporters in the opposite direction suggest a different mechanism at the intra- and extracellular sites in a levodopa transport-dependent and independent manner.     

RELEASE OF NOREPINEPHRINE FROM NEURONS IN DISSOCIATED CELL CULTURES OF CHICK SYMPATHETIC GANGLIA VIA STIMULATION OF NICOTINIC AND MUSCARINIC ACETYLCHOLINE RECEPTORS

Abstract— Dissociated cell cultures of chick embryo sympathetic ganglia were incubated with [³H]nor-epinephrine ([³H]NE) which was taken up and stored in reserpine-sensitive sites. Exposure of the cultures to cholinergic agonists for 5 min intervals resulted in the releaseof a significant proportion (2–20%) of the intracellular stores of [³H]NE. Studies with specific cholinergic agonists and antagonists indicated that release of [³H]NE could be evoked by stimulation of either nicotinic or muscarinic receptors. Release evoked by both nicotinic and muscarinic agonists was totally blocked in the presence of 3 μM-tetrodotoxin. thus indicating that release was mediated via active electrical responses. Release by both types of agonists was also blocked in the presence of elevated Mg²⁺ or when free Ca²⁺ was removed from the extracellular medium. These findings are consistent with the presence of a stimulus-secretion coupling mechanism. Release evoked by nicotine was optimal in the presence of 1.2 mM-Ca²⁺, whereas release evoked by the muscarinic agonist methacholine increased by about 2-fold when the Ca²⁺ concentration was decreased from 1.2 to 0.3 mM. The latter observation may be due to a lowered threshold for evocation of active responses at low concentrations of Ca²⁺. Finally, no evidence was observed for interaction between the two types of receptors. These findings (a)indicate that cultured chick sympathetic neurons possess functional nicotinic and muscarinic cholinergic receptors as well as the ability to release NE via a stimulus-secretion coupling mechanism; (b) suggest that such cultures may be particularly useful for studying the molecular events which link stimulation of cholinergic receptors to neurotransmitter release; and (c) provide further evidence that muscarinic receptors may play aphysiological role in ganglionic transmission.     

Dynamic Storage of Dopamine in Rat Brain Synaptic Vesicles In Vitro

Abstract: The dynamics of catecholamine storage were studied in highly purified, small synaptic vesicles from rat brain both during active uptake or after inhibiting uptake with reserpine, tetrabenazine, or removal of external dopamine. To assess turnover during active uptake, synaptic vesicles were allowed to accumulate [³H]dopamine ([³H]DA) for ～10 min and then diluted 20-fold into a solution containing unlabeled DA under conditions such that active uptake could continue. After dilution, [³H]DA was lost with single exponential kinetics at a half-time of ～4 min at 30°C in 8 mM Cl^? medium, in which both voltage and H⁺ gradients are present in the vesicles. In 90 mM Cl^? medium, in which high H⁺ and Cl^? gradients but no voltage gradient are present, [³H]DA escaped at a half-time of ～7 min. In both high and low Cl^? media, ～40% of [³H]DA efflux was blocked by reserpine or tetrabenazine. The residual efflux also followed first-order kinetics. These results indicate that two efflux pathways were present, one dependent on DA uptake (and thus on the presence of external DA) and the other independent of uptake, and that both pathways function regardless of the type of electrochemical H⁺ gradient in the vesicles. The presence of both uptake-dependent and -independent efflux was observed in experiments using DA-free medium, instead of uptake inhibitors, to prevent uptake. Uptake-independent efflux showed molecular selectivity for catecholamines; [¹⁴C]DA was lost about three times faster than [³H]norepinephrine after adding tetrabenazine directly (without dilution) to vesicles that had taken up comparable amounts of each amine. In addition, the first-order rate constant for uptake-independent efflux showed little change over a 60-fold range of internal DA concentrations, which suggests that this pathway had a high transport capacity. All efflux was blocked at 0°C, suggesting that efflux did not occur through a large pore. There was little or no change in the proton gradient in synaptic vesicles, monitored by [¹⁴C]methylamine equilibration, during the experimental manipulations used here. Thus, the driving force for catecholamine uptake remained approximately constant. The physiological role of uptake-independent efflux could be to allow the monoamine content of synaptic vesicles to be regulated over a time range of minutes and, thereby, control the amount released by exocytosis. These results imply that catecholamines turn over with a half-time of minutes during active uptake by brain synaptic vesicles in vitro.     

Implication of Ca2+-Dependent Protein Tyrosine Phosphorylation in Carbachol-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract: The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [³H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca²⁺. PLD activity was assessed by measuring the formation of [³H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65–70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44^MAPK, and 42^MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca²⁺-free buffer, CCh-induced [³H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca²⁺ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca²⁺. Extracellular Ca²⁺ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca²⁺-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Nerve Growth Factor Stimulates the Production of Inositol 1,3,4-and 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate in PC 12 Cells

Abstract: In PC12 cells, preincubated with [³H]inositol, nerve growth factor (NGF) stimulated an ～ 100% increase in the levels of [³H]inositol 1,3,4-trisphosphate {[³H]-Ins(1,3,4)P₃}, [³H]inositol 1,4,5-trisphosphate {[³H]lns(1,4,5)P₃}, and [³H]inositol 1,3,4,5-tetrakisphosphate {[³H]-Ins(1,3,4,5)P₄} as early as 5–15 s after addition of NGF. This NGF-mediated response was apparent only when the cells had been cultured in the absence of fetal bovine serum (FBS). PC12 cells cultured in FBS-containing medium did not display NGF-mediated increases in [³H]-Ins(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]-Ins(1,3,4,5)P₄ levels. Using cells cultured in the absence of FBS, epidermal growth factor (EGF) and fibroblast growth factor also stimulated production of [³H]lns(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]lns(1,3,4,5)P₄. Lavendustin A, a tyrosine kinase inhibitor, inhibited both the EGF-and NGF-stimulated increases in the levels of these tritiated inositol phosphates. These results suggest that NGF stimulates the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ and that this response is dependent on tyrosine kinase activity. Furthermore, although the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ may be a common response to factors stimulating neuronal differentiation, it is not sufficient for stimulation of neuronal differentiation.     

Flux of catecholamines through chromaffin vesicles in cultured bovine adrenal medullary cells

Primary cultures of bovine adrenal medullary chromaffin cells were pulse-labeled with [3H]dopamine or [3H]norepinephrine and examined for radioactive and total catecholamine contents by high performance liquid chromatography after additional incubations of 15 min to 10 days. [3H]Dopamine was rapidly taken up by chromaffin vesicles in situ and converted to norepinephrine with a half-time of approximately 6 h. [3H] Norepinephrine taken up by the cells was metabolized in three phases. 1) During its brief transit through the cytoplasm, 20 to 35% of this amine was converted to [3H]epinephrine. 2) Following vesicular accumulation, 65 to 70% of the remaining [3H]norepinephrine was methylated to form [3H]epinephrine with a half-time of approximately 30 h, corresponding to the rate of vesicular catecholamine loss from reserpine-treated cells. 3) The residual [3H]norepinephrine decreased with a half-time of 5 days, probably representing loss from norepinephrine-storing cells. [3H]Epinephrine formed endogenously had a half-life in the cultures of approximately 15 days. These data suggest that leakage of norepinephrine from chromaffin vesicles into the cytoplasm limits the rate of dopamine conversion to epinephrine in the adrenal medulla. The kinetic data indicate that approximately 18% of the endogenous norepinephrine and 73% of the endogenous dopamine are present in epinephrine cells.     

COMPARISON OF LEVELS OF ADENOSINE 3'',5''-MONOPHOSPHATE IN HIGHLY PURIFIED AND MIXED PRIMARY CULTURES OF NEURONS AND NON-NEURONAL CELLS FROM EMBRYONIC CHICK SYMPATHETIC GANGLIA

Primary cultures containing ≥99% neurons, ≥99% non-neuronal cells (glia), or both cell types were prepared from the sympathetic ganglia of 12-day chick embryos. Levels of cyclic AMP in the non-neuronal cells (～14 pmol/mg protein) were approximately 3-fold higher than levels in the neurons (～4 pmol/mg protein). Mixed cultures had concentrations of cyclic AMP which fell between the values measured for pure neuronal and pure non-neuronal cultures. The measured cyclic AMP values of mixed cultures were indistinguishable from values predicted by summing the expected contributions of the neurons and non-neuronal cells. Thus, contact between the neurons and non-neuronal cells in these mixed cultures did not appear to alter the level of cyclic AMP in either cell type. Neuronal-glial interactions, such as the specific neuronal stimulation of non-neuronal cell proliferation, occurred independently of any changes in the level of cyclic AMP in the mixed cultures. Cell density was varied in both pure and mixed cultures, and both cyclic AMP concentrations and amounts of [³H]thymidine incorporation into DNA were measured. The cyclic AMP content of the non-neuronal cells varied inversely with cell density. [³H]Thymidine incorporation was independent of cell density in both neuronal and non-neuronal cultures. Parallel density-dependent decreases in cyclic AMP concentration and [³H]thymidine incorporation were observed in mixed cultures as cell density was increased. The data suggest that there is no relationship between changes in rate of non-neuronal cell proliferation and cyclic AMP levels in these cultures.     

High-Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants

Abstract: Primary astrocyte cultures from neonatal rat brains show uptake of [³H]norepinephrine ([³H]NE). This uptake has a high-affinity component with an apparent K_m of approximately 3 × 10^?7 M. At 10^?7 M [³H]NE both the initial rate of uptake and steady-state content of [³H]NE is inhibited by up to 95% by omission of external Na⁺. The Na⁺-dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC₅₀ values of 2 × 10^?9 and 4 × 10^?8 M, respectively. Inhibition of [³H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high-affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not exclusively neuronal.     

Interleukin-2 Modulates Evoked Release of [3H]Dopamine in Rat Cultured Mesencephalic Cells

Abstract: Mesencephalic cell cultures were used as a model to investigate the effects of interleukin-2 (IL-2) on evoked release of [³H]dopamine ([³H]DA) and γ-[³H]-aminobutyric acid ([³H]GABA). At low concentrations (10^?13-10^?12M), IL-2 potentiated [³H]DA release evoked by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate, whereas higher IL-2 concentrations (10^?9-10^?8M) had no effect. IL-2 (10^?14-10^?8M) modulated K⁺-evoked [³H]DA release in a biphasic manner, with low concentrations (10^?12-10^?11M) of IL-2 potentiating and higher concentrations (10^?9-10^?8M) inhibiting K⁺-induced [³H]DA release. IL-2 (10^?14-10^?8M) by itself failed to alter spontaneous [³H]DA release. The inhibition by IL-2 of K⁺-evoked [³H]DA release was reversible and not due to neurotoxicity, as preexposure to IL-2 (10^?8M) had no significant effect on the subsequent ability of dopaminergic cells to take up and to release [³H]DA. Under our experimental conditions, IL-2 (10^?8 M) did not alter Ca²⁺-independent [³H]GABA release evoked by either K⁺ or NMDA. The results of this study indicate that IL-2 is able to potentiate [³H]DA release evoked by a number of different stimuli, including K⁺ depolarization and activation of both NMDA and non-NMDA receptor subtypes in mesencephalic cell cultures. IL-2 is active at very low concentrations, a finding that indicates a potent effect of IL-2 on dopaminergic neurons and implicates a physiological role for this cytokine in the modulation of DA release.     

Evidence that Inhibition of Nicotine-Mediated Catecholamine Secretion from Adrenal Chromaffin Cells by Enkephalin, β-Endorphin, Dynorphin (1-13), and Opiates is not Mediated via Specific Opiate Receptors

Abstract: The opioid peptides Met- and Leu-enkephalin, dynorphin (1-13), and β-endorphin and the narcotic analgesics, morphine, levorphanol, and dextrorphan all produced a dose-dependent inhibition of nicotine (5 × 10^?6m )-mediated release of [³H]norepinephrine ([³H]NE) from bovine adrenal chromaffin cells in culture. None of these agents affected [³H]NE release induced by high K⁺ (56 mm ). Although the above results suggest that the opioid peptides and narcotic analgesics inhibit catecholamine release from adrenal chromaffin cells in culture, we suggest that these effects are not mediated by specific opiate binding sites, since (1) the inhibition was only produced with high concentrations of the agents—the threshold concentrations were 10^?7 to 10^?5m and higher; (2) the inhibition produced by the narcotic analgesics did not display stereospecificity, because the (d-isomer, dextrorphan, was slightly more active than the l-isomer, levorphanol; (3) the narcotic antagonists naloxone, naltrexone, and levallorphan did not reverse the inhibition produced by either the narcotic analgesics (e.g., morphine) or the opioid peptides (e.g., dynorphin). These three antagonists themselves inhibited the nicotine-mediated release of [³H]NE from the adrenal chromaffin cells in culture. Finally (4), the I₂-Tyr¹ substituted analogues of β-endorphin and dynorphin that are biologically less active than the parent compounds produced an inhibition of the nicotine-mediated [³H]NE release similar to that of their parent compounds. These results do not support the idea that high-affinity stereospecific opiate binding sites are involved in the inhibitory modulation of nicotinic evoked catecholamine release from bovine adrenal chromaffin cells in culture.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

The Action of Black Widow Spider Venom on Cholinergic Mechanisms in Synaptosomes

Abstract: Black widow spider venom (BWSV) promoted the massive release of labeled acetylcholine from synaptosomes and in addition, inhibited high-affinity choline uptake into the preparation. Both activities occurred in the absence of [Ca²⁺]₀. When Na⁺ in Krebs-Ringer was replaced isotonically by sucrose, BWSV did not cause any release of [³H]ACh. On the other hand, BWSV was still effective if Na⁺ was replaced by lithium, glucosamine, or Tris. Tetrodotoxin (10^?5 M) failed to prevent the ACh-releasing action of the venom. The uptake of [³H]norepinephrine and [³H]tyrosine into the P₂ fraction was significantly inhibited by BWSV pretreatment. However, the effect of the venom on the uptake of [³H]deoxyglucose was slight. In addition, the venom-induced release of [³H]norepinephrine was much higher than that of [³H]deoxyglucose. The change in membrane potential of the preparation in duced by BWSV as examined using the voltage-sensitive fluorescence probe, 3, 3′-dipentyl-2, 2′-oxacarbocyanine. BWSV pretreatment markedly increased the synaptosomal fluorescence, indicating a depolarization of the preparation. This action of the venom was also observed in a Ca²⁺ -free or K⁺ -free medium, but could be blocked by pretreatment with antivenom. Pretreatment of the P₂ fraction with concanavalin A completely blocked the action of BWSV. Also, the BWSV failed to promote the release of transmitter if the venom was prein-cubated with a low concentration of purified gangliosides. Even after prolonged treatment with high concentrations of BWSV, an electron microscopic study showed no depletion of the synaptic vesicles in presynaptic terminals of the cortical P₂ preparations or striatal slices. It is suggested that the venom expresses its activity by binding to glycoproteins and/or gangliosides on the synaptic membrane, opening a cation channel. The subsequent depolarization then inhibits uptake processes and promotes transmitter release that is independent of external calcium.