Cations differentially affect subpopulations ofl-glutamate receptors in rat synaptic plasma membranes

Several cations were examined for their ability to specifically affect one of the 3l-glutamate (l-Glu) binding sites in rat forebrain synaptic plasma membranes (i.e. Na⁺-dependent, Cl⁻-dependent and Cl⁻-independent). Na⁺-dependent binding was potently inhibited by K⁺ and NH₄⁺ ions. Other monovalent cations testedd (Cs⁺, Li⁺, triethylammonium) had no effect on this binding site. Polyvalent cations (Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Cr³⁺) also had little effect on the Na⁺-dependentl-Glu binding site. Cl⁻-dependentl-Glu binding was potently inhibited by Na⁺ ions but was not affected by other monovalent ions. All of the divalent cations were potent inhibitors of both Cl⁻-dependent and -independent binding. The results show that these binding sites ofl-Glu can be distinguished by their response to cations and suggest possible novel modes of regulation in vivo.     

Receptor-mediated uptake of labeled transferrin by embryonic chicken dorsal root ganglion neurons in culture

Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM ¹²⁵I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized ¹²⁵I-transferrin was quantified with a gamma counter. ¹²⁵I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 μM unlabeled transferrin. No competition for the uptake of ¹²⁵I-transferrin was observed in the presence of 1.25 μM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment (‘half-site’) of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment (‘half-site’) of transferrin (1.25 μM) or in the presence of concanavalin A (1.25 μM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons.     

High-affinity uptake system for cysteine in crude synaptosomal fractions of rat cerebral cortex

The in vitro uptake of [³⁵S] cysteine was studied in crude synaptosomal preparation of the cerebral cortex of rat. The accumulation of cysteine was found to be temperature- and time-dependent. It was linear at least for four minutes at 37 C with characteristics of saturable kinetics. Uptake of cysteine was Na⁺- and K⁺-dependent. Increasing the Na⁺ ion concentration increased the accumulation of cysteine in synaptosomal preparations; unlike the Na⁺ ion, an increase in the K⁺ ion, an increase in the K⁺ ion inhibited cysteine uptake. Cysteine was accumulated against concentration gradients by a saturable mechanism. Double reciprocal plot of the cysteine uptake suggests two types of affinity systems, with K_m values for the high-affinity uptake of about 12.2 μM and for the low-affinity uptake of about 4 mM. The high-affinity uptake was also significantly inhibited by ouabain, a potent inhibitor of the Na⁺-K⁺-dependent ATPase, and other metabolic inhibitors. The results of the effects of cysteine analogues on uptake also suggested that it is a substrate-specific high-affinity uptake system for cysteine.     

Sodium-dependent glutamate uptake as an activator of oxidative metabolism in primary astrocyte cultures from newborn rat

In the present report we describe the effect of glutamate on respiratory activity in primary cultures of astrocytes, derived from cerebral cortex of newborn rat. Glutamate (100 μM) caused an increased oxygen consumption. This effect could not be inhibited by antagonists to the NMDA or AMPA/kainate receptors. Neither trans-ACPD (an agonist to the metabotropic glutamate receptor) nor the Krebs cycle intermediate α-ketoglutarate had any effect on the respiratory rate. An uncontrolled influx of Na⁺, caused by gramicidin, could mimic the glutamate effect on respiratory activity. In addition, the glutamate effect was abolished by addition of ouabain or replacement of Na⁺ by Li⁺ in the perfusion buffer. We conclude that the co-transport of Na⁺, in the Na⁺ -dependent high-affinity glutamate uptake system, mediated the glutamate-induced increase in oxygen consumption through an increased activity of Na⁺/K⁺-ATPases. © 1995 Wiley-Liss, Inc.     

Glial plasmalemmal vesicles: A subcellular fraction from rat hippocampal homogenate distinct from synaptosomes

By a Percoll density-gradient centrifugation of rat hippocampal ho-mogenate, We found a novel subcellular fraction (specific gravity ≈ 1.046 g/ml), besides synaptosomes (≈1.060 g/ml), which showed a high activity of Na⁺-dependent glutamate uptake. The initial rate of the glutamate uptake in this fraction was as high as twice that in synaptosomes. Activities of choline acetyltransferase and high affinity choline uptake were, on the other hand, much lower. γ-Aminobutyric acid uptake activity was nearly equivalent in both fractions. Electron microscopic observations revealed that the fraction was morphologically different from synaptosomal or myelin fractions, but mainly consisted of two different types of empty membrane vesicles; irregular (0.3?0.8 μm in diameter) and spheroid type (0.2 μm). The immunoreactivity to glial fibrillary acidic protein was appreciably high in this fraction. The marker enzyme analysis showed the fraction was rich in plasma membranes. On the basis of these results, the fraction is termed glial plasmalemmal vesicles(GPV). We analyzed kinetically the reaction of Na⁺ -dependent glutamate uptake by GPV comparing with that by synaptosomes. Km values for glutamate in GPV was 4.7 μM and V_max was 33 nmol/mg/min, while in synaptosomes 11 μM and 17 nmol/mg/min, respectively. Hill coefficients of Na⁺ activation in GPV and synaptosome were 1.1 and 2.0, respectively. Thus, the mechanism or transporter molecule in glial cells for Na⁺-dependent glutamate transport is likely to be different from that in neurons. © 1993 Wiley-Liss, Inc.     

Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes

The [³H]resiniferatoxin (RTX) binding assay using membrane preparations has been used to identify and characterize the vanilloid receptors in the central and peripheral nervous system of different species. In the present study, using cultured adult rat dorsal root ganglion neurons either in suspension or attached to the tissue culture plates, we developed an assay to measure specific [³H]RTX binding by the intact cells. We were able to characterize the vanilloid binding characteristics of the neurons and compared those to the properties of vanilloid binding sites present in rat dorsal root ganglia membrane preparations. We found that [³H]RTX bound with similar affinity and positive cooperatively to attached neurons (cultured for 5 days before being assayed), neurons in suspension (using a filtration assay) and dorsal root ganglion membrane preparations. Dissociation constants obtained in the three assays were 47.6 ± 3.5 pM, 38.4 ± 3.1 pM and 42.6 ± 3.1 pM, respectively. The cooperativity indexes determined by fitting the data to the Hill equation were 1.73 ± 0.11, 1.78 ± 0.12 and 1.78 ± 0.09, respectively. The maximal binding capacity was 0.218 ± 0.026 fmol/10³ cells and 0.196 ± 0.021 fmol/10³ cells in the case of the attached cells and cells in suspension, respectively. Nonradioactive RTX, capsaicin, capsazepine and resiniferonol 20-homovanillylamide fully displaced specifically bound [³H]RTX from cells in suspension with K_i and Hill coefficient values of 42.5 ± 5.3 pM, 2.06 ± 0.16 μM, 3.16 ± 0.21 μM and 32.4 ± 4.1 nM and 1.79 ± 0.17, 1.68 ± 0.06, 1.72 ± 0.11 and 1.81 ± 0.12, respectively. Structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and inducing ⁴⁵Ca uptake in rat dorsal root ganglion neurons. Affinities for receptor binding and stimulation of ⁴⁵Ca uptake of RTX, resiniferonol 20-homovanillylamide, RTX-thiourea, tinyatoxin, phorbol 12,13-dibenzoate 20-homovanillylamide and capsaicin were 38.5 ± 2.9 pM, 25.7 ± 3.0 nM, 68.5 ± 3.8 nM, 173 ± 25 pM, 7.98 ± 0.83 μM and 4.93 ± 0.35 μM as compared to 0.94 ± 0.12 nM, 26.5 ± 3.5 nM, 149 ± 30 nM, 1.46 ± 0.25 nM, 1.41 ± 0.48 μM and 340 ± 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperatively of receptor binding; however, stimulation of ⁴⁵Ca uptake appeared to follow a non-cooperative mechanism of action. The competitive capsaicin antagonist capsazepine inhibited specific binding of [³H]RTX by rat dorsal root ganglion membrane preparations with K_i and Hill coefficient values of 3.89 ± 0.38 μM and 1.74 ± 0.11. On the other hand it inhibited the induction of ⁴⁵Ca uptake into the cells induced by capsaicin and RTX in a non-cooperative fashion with K_i values of 271 ± 29 nM and 325 ± 47 nM. Our results show that the membrane binding assay relates to the reality of receptor function in the intact, cultured neurons, both in terms of affinity and positive cooperatively. However the different vanilloid derivatives displayed markedly distinct structure-activity relations for high affinity receptor binding and stimulation of ⁴⁵Ca uptake into rat dorsal root ganglion neurons. Among various explanations for this discrepancy, we favor the possibility that the two assays detect distinct classes of the vanilloid (capsaicin) receptor present in primary sensory neurons.     

Effects of Na gradient on the intracellular Ca oscillation in the sympathetic ganglion cell: Na---Ca exchange in the neurone cell soma?

(+)-Tubocurarine ((+)-Tc:10–100 μM) reduced the duration of the afterhyperpolarization, which was induced by the activation of Ca²⁺-dependent K⁺-conductance (G_K,Ca) following an action potential in the bullfrog sympathetic ganglion cell, but did not affect the maximum rates of rise and fall of Na⁺- and Ca²⁺-dependent action potentials. The amplitudes of slow rhythmic membrane hyperpolarizations produced by rhythmic rises in the G_K,Ca were also decreased by (+)-Tc without a change in their intervals. Thus, (+)-Tc appears to block the Ca²⁺-dependent K⁺-channel of the bullfrog sympathetic ganglion cell.     

Specific interaction of glutamate with membranes from cultured retinal pigment epithelium

Excitatory amino acids (EAA) have been shown to induce phagocytosis in retinal pigment epithelial (RPE) cells. In order to explore if this action is receptor-mediated, we have identified and characterized receptors for L-glutamate through the binding of [³H]L-glutamate to membranes from chick RPE cells in primary culture. Specific binding was found saturable, with K_B = 333nM and B_max = 3.2 pmol/mg protein in frozen/thawed membranes. Na⁺-independent binding was present in cultures of 16 and 25 days in vitro, and was not affected by temperature. Pharmacological profile of analogues of EAA at different receptor types suggests the presence of a metabotropic type receptor (L-glutamate > S-2-amino-3-phosphonopropionate > 2-amino-4-phosphonobutyrate = trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > quisqualate). Excitatory amino acid analogues acting at the NMDA-receptor also displaced bound L-glutamate, and a noticeable stimulation of specific binding of this ligand by glycine was shown; this effect was mimicked by D-serine and 1-hydroxy-3-aminopyrrolidone-2 (HA-966) but not by 7-chlorokynurenate, and was not inhibited by strychnine. Since taurine and GABA also increased specific binding, it is likely that modulation of EAA receptors in RPE differs from that in neurons. © 1993 Wiley-Liss, Inc.     

Characterization of Na-dependent binding sites of [H]glutamate in synaptic membranes from rat brain

Some biochemical characteristics of Na⁺-dependent binding of [³H]l-glutamic acid (Glu) were studied using crude synaptic membrane preparations from the rat brain as compared with Na⁺-independent binding. In vitro addition of sodium chloride (1–100 mM) exhibited a significant enhancement of [³H]Glu binding to synaptic membranes in a concentration-dependent manner independent of the incubation temperature employed (2 or 30 °C). In contrast, sodium acetate elicited a concentration-dependent augmentation of the binding at 2 °C to a significantly greater extent than that found at 30 °C. It was found that the binding found in the presence of 100 mM sodium acetate reached its maximal value within 10 min of incubation followed by a rapid decline up to 60 min at 30 °C, while gradually increasing up to 60 min at 2 °C. The Na⁺-independent basal binding was significantly activated by the alteration of incubation temperature from 2 to 30 °C and reached equilibrium within 10 min of incubation at both incubation temperatures. The Na⁺-dependent binding was more promptly attenuated by the addition of excess of non-radiactive Glu (1 mM) at 30 °C than that at 2 °C, whereas the Na⁺-independent binding was greatly suppressed by the addition at 2 °C in comparison with that at 30 °C. Quisqualic acid induced a considerably less-potent inhibition of the Na⁺-dependent binding than that of the Na⁺-independent binding. Neither N-methyl-l-aspartic acid nor kainic acid had such a significant effect on each binding.d-Aspartic acid exerted a significant elimination of the Na⁺-dependent binding in a concentration-dependent manner without significantly affecting the Na⁺-independent binding. In vitro addition of sodium acetate (1–100 mM) exhibited a concentration-dependent suppression of [³H]Glu binding in the presence ofd-aspartate. Pretreatment of synaptic membranes with various sulphhydryl reagents such asp-chloromercuribenzoic acid (PCMB), N-ethylmaleimide (NEM) and5, 5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) invariably resulted in a significant deterioration of the Na⁺-independent binding in a concentration-dependent manner, whereas the Na⁺-dependent binding was significantly abolished exclusively by 1 mM PCMB. These results suggest that the Na⁺-dependent binding sites may be distinctly different from the Na⁺-independent binding sites in nature and the former sites may be closely related to the Na⁺-dependent sites of Glu uptake with a high affinity for this putative neurotransmitter.     

In Vivo and In Vitro Effects of Phenytoin (PHT) on ATPases and [14C]-PHT Binding in Synaptosomes and Mitochondria from Rat Cerebral Cortex

Summary: The effect of phenytoin (PHT) on Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities and on [¹⁴C]-PHT binding in vitro to synaptosomal and mitochondrial sub cellular fractions from rat cerebral cortex was studied after chronic PHT treatment. Synaptosomal and mitochondrial fractions were characterized with plasma membrane and mitochondrial enzymatic markers. Synaptoso-mal Na⁺-K⁺-ATPase was not affected in vitro by PHT 1–200 μM or by chronic treatment with 2–50 mg/kg/day of the unlabeled drug for 8 days. Mitochondria1 Mg²⁺-ATPase was significantly stimulated by PHT after chronic treatment with 5 mg/kg/day for 8 days; reaching maximal effect (76%), at 10–25 mg/kg. PHT had no effect on mitochondrial Mg²⁺-ATPase when added in vitro. [¹⁴C]-PHT binding in vitro to the subcellular fractions was determined by dialysis to assess in vivo binding of the unlabeled PHT during chronic treatment. Indeed, [¹⁴C]-PHT bound to synaptosomes was significantly reduced by chronic PHT treatment from 218 ±10 to 119 & 11 pmol/mg protein after 1 week of treatment; a similar effect was obtained after 2–3 weeks with 10 mg/kg/day. Mitochondrial fraction bound 117 ±10 pmol/mg protein labeled PHT. Chronic treatment with unlabeled PHT also reduced the amount of [¹⁴C]-PHT bound to 19.9 ± 2.2 pmol/mg protein. These results show slow reversible PHT in vivo binding to synaptosomes and mitochondrias from rat cerebral cortex, supporting the idea that the modulatory action of PHT on Na+ and Ca2+ permeabilities are mediated through these slow reversible binding proteins. The data also suggest a possible role of intra synaptosomal mitochondria in [Ca²⁺]_i buffering.     

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

A comparison of sodium-dependent glutamate binding with high-affinity glutamate uptake in rat striatum

The specific sodium-dependent binding of [³H]glutamate to membranes of the rat striatum was examined and a comparison made with high affinity glutamate uptake. In the presence of sodium, [³H]glutamate binding was saturable and of high affinity. No binding could be detected in the absence of sodium. Removal of the cortical afferents to the striatum resulted in a parallel decrease in Na⁺-dependent glutamate binding and in high-affinity glutamate uptake. After the injection of the neurotoxin kainic acid into the striatum, the density of Na⁺-dependent glutamate binding sites was reduced by 40%, while high-affinity uptake showed no significant decrease. Drugs which inhibit high-affinity uptake were also effective at inhibiting Na⁺-dependent binding. The results suggest that about half the Na⁺-dependent glutamate binding sites in the striatum represent high-affinity uptake sites on the corticostriatal terminals. The remainder of the binding sites are located on striatal neurons and may also be uptake sites.     

Neuronal-like features of TE671 cells: Presence of a functional nicotinic cholinergic receptor

The human medulloblastoma cell line TE671 has been investigated and found to have several ‘neuron-like’ properties, including the presence of a functional nicotinic receptor. The cell line TE671 is composed of at least 5 stable morphologic cell types. Resting potentials recorded with intracellular microelectrodes were low (−17 mV to −31 mV) but all cell types were capable of generating Na⁺-dependent action potentials following anode-brake stimulation. Rare spontaneous hyperpolarizing potentials, suggestive of synaptic activity, were also observed.TE671 cells were completely unresponsive to iontophoresed GABA but did respond to acetylcholine (ACh). The most common response to ACh was a depolarization accompanied by an increase in membrane conductance. When large amounts of ACh were delivered, depolarization followed by hyperpolarization was frequently observed. Depolarizing responses to ACh are abolished in Na⁺-free solution while hyperpolarizing responses to ACh were still present following the removal of both Na⁺ and Cl⁻ from the bathing solution.The depolarization response to ACh is mediated through a nicotinic cholinergic receptor. Depolarization was completely blocked in the presence of 10⁻⁶ M α-bungarotoxin,4.4 × 10⁻⁵Md-tubocurarine, or10⁻⁴M decamethonium. Atropine was only 50% effective at 10⁻⁴ M and hexamethonium was ineffective at 10⁻⁴ M.In vitro binding of receptor ligands to membranes prepared from TE671 cells revealed high levels of [¹²⁵I]α-bungarotoxin (α-BuTx) binding sites, in addition to lower levels of other ligand binding sites.[¹²⁵I]α-BuTx bound to a single, saturable high affinity site in either membrane preparations or intact TE671 cells. Binding was potently inhibited by the classical nicotinic acetylcholine receptor antagonistsd-tubocurarine and decamethonium. Nicotine and carbamylcholine showed intermediate potencies in inhibiting binding while atropine and hexamethonium showed little ability to inhibit [¹²⁵I]α-BuTx binding.The data obtained from [¹²⁵I]α-BuTx binding studies agree qualitatively with the electrophysiological data on the depolarizing ACh response and together they provide strong evidence that TE671 cells possess a functional nicotinic acetylcholine receptor. This cell line may therefore be useful as a stable source with which to characterize mammalian neuronal nicotinic acetylcholine receptors and membrane events related to its activation.     

Vulnerability of Medium Spiny Striatal Neurons to Glutamate: Role of Na+/K+ ATPase

In Huntington's disease neuronal degeneration mainly involves medium-sized spiny neurons. It has been postulated that both excitotoxic mechanisms and energy metabolism failure are implicated in the neuronal degeneration observed in Huntington's disease. In central neurons, >40% of the energy released by respiration is used by Na⁺/K⁺ ATPase to maintain ionic gradients. Considering that impairment of Na⁺/K⁺ ATPase activity might alter postsynaptic responsivity to excitatory amino acids (EAAs), we investigated the effects of the Na⁺/K⁺ ATPase inhibitors, ouabain and strophanthidin, on the responses to different agonists of EAA receptors in identified medium-sized spiny neurons electrophysiologically recorded in the current- and voltage-clamp modes. In most of the cells both ouabain and strophanthidin (1–3 μM) did not cause significant change in the membrane properties of the recorded neurons. Higher doses of either ouabain (30 μM) or strophanthidin (30 μM) induced, per se, an irreversible inward current coupled to an increase in conductance, leading to cell deterioration. Moreover, both ouabain (1–10 μM) and strophanthidin (1–10 μM) dramatically increased the membrane depolarization and the inward current produced by subcritical concentrations of glutamate, AMPA and NMDA. These concentrations of Na⁺/K⁺ ATPase inhibitors also increased the membrane responses induced by repetitive cortical activation. In addition, since it had previously been proposed that dopamine mimics the effects of Na⁺/K⁺ ATPase inhibitors and that dopamine agonists differentially regulate the postsynaptic responses to EAAs, we tested the possible modulation of EAA-induced membrane depolarization and inward current by dopamine agonists. Neither dopamine nor selective dopamine agonists or antagonists affected the postsynaptic responses to EAAs. Our experiments show that impairment of the activity of Na⁺/K⁺ ATPase may render striatal neurons more sensitive to the action of glutamate, lowering the threshold for the excitotoxic events. Our data support neither the role of dopamine as an ouabain-like agent nor the differential modulatory action of dopamine receptors on the EAA-induced responses in the striatum.     

Membrane-bound enzymes of erythrocytes in human muscular dystrophy: (Na+ + K+-ATPase, Ca2+-ATPase, K+- and Ca2+-p-nitrophenylphosphatase.

Four enzyme activities were studied in erythrocyte membranes from patients with Duchenne and congenital myotonic muscular dystrophy. (Na⁺ + K⁺)-stimulated, Mg²⁺-dependent adenosinetriphosphatase, measured in two different media, showed normal activity and ouabain inhibition, as did K⁺-stimulated p-nitrophenylphosphatase. The specific activity of Ca²⁺-stimulated p-nitrophenylphosphatase was twice normal in Duchenne membranes. Ca²⁺-stimulated, Mg²⁺-dependent adenosine-triphosphatase was augmented in membranes from both Duchenne and congenital myotonic muscular dystrophic patients. The cause of the increased activities may be the necessity for compensating an alteration in the calcium metabolism in the dystrophic erythrocytes.Several kinetic parameters of the two Ca²⁺-stimulated enzyme activities were studied in Duchenne and control membranes. Most were not changed, with the exception of the Na⁺-stimulation of Ca²⁺-ATPase. In Duchenne membranes two affinity sites were present with half maximal activating concentrations of 58 ± 4 and 4 ± 1 mM Na⁺. In control membranes only one affinity site was found with K_a = 26 ± 9 mM Na⁺.     

Differential inhibition of catecholamine secretion by amitriptyline through blockage of nicotinic receptors,sodium channels,and calcium channels in bovine adrenal chromaffin cells

We investigated the effects of amitriptyline, a tricyclic antidepressant, on [³H]norepinephrine ([³H]NE) secretion and ion flux in bovine adrenal chromaffin cells. Amitriptyline inhibited [³H]NE secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and 70 mM K⁺. The half maximal inhibitory concentration (IC₅₀) was 2 μM and 9 μM, respectively. Amitriptyline also inhibited the elevation of cytosolic calcium ([Ca²⁺]_i) induced by DMPP and 70 mM K⁺ with IC₅₀ values of 1.1 μM and 35 μM, respectively. The rises in cytosolic sodium ([Na⁺]_i) and [Ca²⁺]_i induced by the Na⁺ channel activator veratridine were also inhibited by amitriptyline with IC₅₀ values of 7 μM and 30 μM, respectively. These results suggest that amitriptyline at micromolar concentrations inhibits both voltage-sensitive calcium (VSCCs) and sodium channels (VSSCs). Furthermore, submicromolar concentrations of amitriptyline significantly inhibited DMPP-induced [³H]NE secretion and [Ca²⁺]_i rise, but not veratridine- or 70 mM K⁺-induced responses, suggesting that nicotinic acetylcholine receptors (nAChR) as well as VSCCs and VSSCs can be targeted by amitriptyline. DMPP-induced [Na⁺]_i rise was much more sensitive to amitriptyline than the veratridine-induced rise, suggesting that the influx of Na⁺ and Ca²⁺ through the nAChR itself is blocked by amitriptyline. Receptor binding competition analysis showed that binding of [³H]nicotine to chromaffin cells was significantly affected by amitriptyline at submicromolar concentrations. The data suggest that amitriptyline inhibits catecholamine secretion by blocking nAChR, VSSC, and VSCC. Synapse 29:248–256, 1998. © 1998 Wiley-Liss, Inc.     

High-affinity hemicholinium-3 binding to brain membranes: reduction by treatment witphysostigmine in vivo

We examined high-affinity, sodium-dependent binding of hemicholinium-3 to brain membranes. Binding, withK_d of about 3 nM, was highest in corpus striatum, intermediate in cerebral cortex and hippocampus, and low in cerebellum. Treatment witphysostigmine in vivo reduced binding in all regions without affecting apparent affinity.     

α-Bungarotoxin binding sites on sensory neurones and their axonal transport in sensory afferents

The autoradiographic localization of [¹²⁵I]α-bungarotoxin binding sites on primary sensory fibres was investigated. Nicotinic α-bungarotoxin binding sites were localized to a small sub-population of large dorsal root ganglion cells in the rat, monkey, cat and human dorsal root ganglia. Ligation of the sciatic nerve or dorsal root in the rat resulted in an anterograde accumulation of binding sites proximal to the dorsal root ganglion, and a small retrograde accumulation. Unilateral dorsal root section in the rat produced a loss of toxin binding sites mainly within lamina III of the dorsal horn. These results suggest that nicotinic α-bungarotoxin binding sites manufactured in large dorsal root ganglion cell bodies are transported both centrally to the spinal cord and also peripherally.     

Alterations in the dopaminergic receptor system after chronic administration of cocaine

Several studies suggest that one of the most important factors contributing to cocaine dependence is an alteration in the actions of the neurotransmitter dopamine in the central nervous system. In order to understand some of the neuroreceptor consequences of cocaine administration, groups of rats were injected with cocaine (2 daily doses of 15 mg/kg) for 1 to 21 days. Binding of [³H]cocaine, [³H]SCH23390, [³H]raclopride, and [³H]BTCP in striatal and cortical tissue from the treated animals was compared to controls. [³H]Cocaine binding was increased by the drug in the striatum and cortex at days 14 and 21, respectively. The binding of [³H]SCH23390 to D₁ dopamine receptors was significantly increased at day 3 of cocaine exposure. In striatal membranes, [³H]BTCP binding to dopamine uptake sites was significantly increased after day 7, whereas binding in cortical membranes was increased from day 1. [³H]Raclopride binding to D₂ dopamine receptors remained unchanged throughout the study in both cortical and striatal tissues. These results indicate that repeated exposure to cocaine produces an upregulation (possible supersensitivity) in cortical D₁, cocaine, and DA-uptake sites which occurs in a time-dependent manner. These increases are coupled with an upregulation in striatal D₁, cocaine, and DA-uptake sites, without simultaneous changes in D₂ receptors. Thus, cocaine's effects are not uniformly distributed across all brain regions, but rather are focused within areas of the dopamine system. © 1993 Wiley-Liss, Inc.     

SHORT COMUNICATION Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons

We investigated the effects of oxygen (O₂)/glucose deprivation on intracellular sodium concentration ([Na⁺]_i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na⁺]_i using the Na⁺-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O₂/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na⁺]_i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (d -amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na⁺/Ca²⁺-exchanger, affected these responses to O₂/glucose. The present results demonstrate that, in cortical neurons, O₂/glucose deprivation induces an early rise in [Na⁺]_i which cannot be ascribed to the activity of voltage gated Na⁺-channels, glutamate receptors or of the Na⁺/Ca²⁺-exchanger.