Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH-SY5Y

Abstract: We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K⁺]_o (100 m M ) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [³H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K⁺-evoked release was unaffected by hypoxia (P o ₂≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca²⁺_o with 1 m M EGTA abolished NA release. K⁺-evoked release was also dramatically reduced in the presence of 200 µ M Cd²⁺ to block voltage-gated Ca²⁺ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd²⁺-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca²⁺ influx through the nAChR pore, or by activating an unidentified Cd²⁺-resistant Ca²⁺-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

Turgor- dependent membrane permeability in relation to calcium level

The relationship between the inhibiting effect of Ca²⁺ and of low turgor pressure on K⁺ release from fresh-cut discs of carrot ( Daucus carota var. Nantes) storage tissue was studied. A range of Ca²⁺ concentrations in the tissue was obtained by adding 0.5 m M EDTA or CaSO₄ at different concentrations to the medium. Calcium inhibited K⁺ release in fully turgid cells (2.5 μmol K⁺ g⁻¹ h⁻¹ in 0.5 m M EDTA vs 0.4 μmol K⁺ g⁻¹ h⁻¹ in 10 m M CaSO₄). Less turgid cells, obtained by equilibration with 0.2 M mannitol, released K⁺ at only 30% of the rate of the turgid cells, yet the pattern of K⁺ release as a function of Ca²⁺ level was similar in both turgid and non-turgid cells. Removal of calcium by EDTA occasionally injured cell membranes in the fully turgid discs but never in the less turgid ones. In view of the additive effect of Ca²⁺ and low turgor on K⁺ release regardless of the treatment order, it is suggested that the two factors exert their effect on membrane permeability independently of each other.     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

Characterization of the Exocytotic Release of Glutamate from Guinea-Pig Cerebral Cortical Synaptosomes

Abstract: A continuous enzyme-linked fluorometric assay was used for determining the characteristics for glutamate exocytosis from guinea-pig cerebrocortical synaptosomes. Ca²⁺-dependent release can be induced not only by K⁺, but also by the Na⁺ channel activator veratridine and the Ca²⁺ ionophore ionomycin. K⁺-induced release can be inhibited by the Ca²⁺ channel inhibitor verapamil. Sr²⁺ and Ba²⁺ substitute for Ca²⁺ in promoting K⁺-induced release. Agents that would be predicted to transform the transvesicular pH gradient into a membrane potential are without effect on glutamate release. However, the protonophore carbonylcy-anide p -trifluoromethoxyphenylhydrazone causes a time-dependent loss of exocytosis that is oligomycin insensitive and may be due to depletion of vesicular glutamate. The Ca²⁺-independent release of glutamate from the cytosol on depolarization is unchanged or promoted by metabolic inhibitors that lower the ATP/ADP ratio. In contrast, Ca²⁺-dependent release is ATP dependent and is blocked by the combined inhibition of oxidative phosphorylation and glycolysis.     

Extracellular Ionic Composition Alters Kinetics of Vesicular Release of Catecholamines and Quantal Size During Exocytosis at Adrenal Medullary Cells

Abstract: The temporal resolution of carbon-fiber microelectrodes has been exploited to examine the plasticity of quantal secretory events at individual adrenal medullary cells. The size of individual quantal events, monitored by amperometric oxidation of released catecholamines, was found to be dependent on the extracellular ionic composition, the secretagogue, and the order of depolarization delivery. Release was observed with either exposure to 60 m M K⁺ in the presence of Ca²⁺ or exposure to 3 m M Ba²⁺ in solutions of different pH, with and without external Ca²⁺. Ba²⁺ was demonstrated to induce Ca²⁺-independent exocytotic release for an extended period of time (>4 min) relative to release induced by K⁺ (∼30 s), which is Ca²⁺ dependent. In all cases, simultaneous changes of intracellular divalent cations, monitored by fura-2 fluorescence, accompanied quantal release and had a similar time course. Exocytosis caused by Ba²⁺ in Ca²⁺-free medium had a larger mean spike area at pH 8.2 than at pH 7.4. When Ba²⁺-induced spikes measured at pH 7.4 were compared, the spikes in Ca²⁺-free medium were found to be broader and shorter but had the same area. Release induced by K⁺ after exposure to Ba²⁺ was comprised of larger quantal events when compared with preceding K⁺ stimulations. Finally, spikes obtained with Ba²⁺ exposure at an extracellular pH of 5.5 had a different shape than those obtained in more basic solutions. These changes in spike size and shape are consistent with the interactions between catecholamines and other intravesicular components.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

Compartments of Labeled and Endogenous γ-Aminobutyric Acid Giving Rise to Release Evoked by Potassium or Veratridine in Rat Cortical Slices

Abstract— To establish compartments involved in depolarization-induced release of γ-aminobutyric acid (GABA) in rat brain slices, the amount of exogenous labeled and endogenous GABA released and retained was followed during 48 min exposure to 50 m m -K⁺ or to 50 μ m -veratridine. Endogenous GABA was measured with high performance liquid chromatography. The presence of 10 μ m -aminooxyacetic acid throughout prevented both the metabolism of GABA and the formation of endogenous GABA due to depolarization. During super-fusion with 50 m m -K⁺ and 2.6 m m -Ca²⁺ the efflux of labeled and endogenous GABA after an initial large increase declined to 10% of the highest value with constant and identical rates. Kinetic analysis of efflux showed that 10% of endogenous and 25% of labeled GABA present is available for release by high K⁺ and Ca²⁺. In the absence of Ca²⁺, release by high K⁺ of both labeled and endogenous GABA was nearly suppressed. Veratridine, unlike high K⁺, caused an efflux which declined with an initial fast and late very slow phase. The slow efflux by veratridine was doubled in the absence of Ca²⁺. Exposure to veratridine in the absence of Ca²⁺ during 120 min released nearly 70% of labeled and endogenous GABA present. Results suggest that only about 0.25 μmol g⁻¹ endogenous GABA is the source of physiological Ca²⁺-dependent release, while much of the remaining GABA present is released only under unphysiological conditions.     

Glycine release from Y79 retinoblastoma cells

Abstract: Glycine release, induced by a high concentration of potassium chloride (K⁺), was investigated in cultured human Y79 retinoblastoma cells. The cells were labeled by incubation with [2-³H]glycine prior to K⁺ depolarization. Depolarization with 55 m M K⁺ caused an immediate, Ca²⁺-dependent release of approximately 20% of the cellular radiolabeled glycine content. Chemical analysis of the intracellular free glycine content also showed that approximately 20%, 2.4 nmol/mg protein, was released after K⁺ depolarization. Glycine release from labeled Y79 cells was not stimulated by incubation with 55 mM choline chloride. Based on measurements with an amino acid analyzer, it is concluded that of the free amino acids contained in the Y79 cell, only glycine is specifically released into the extracellular fluid by K⁺ depolarization. Although the intracellular content of serine and glutamate decreased, these amino acids were not released from the cells. Further studies with [U-¹⁴C]serine suggest that serine is converted into glycine in Y79 cells. Veratridine also caused an immediate release of [2-³H]glycine from the cells, and this was blocked by tetrodotoxin. This suggests that the Y79 cells possess voltage-dependent Na⁺ channels. These results indicate that K ⁺ - and veratridine-stimulated glycine release occurs in Y79 retinoblastoma cells, providing additional evidence that this continuously cultured line may be a useful model for certain human retinal and central nervous system functions.     

A Toxin Fraction (FTX) from the Funnel-Web Spider Poison Inhibits Dihydropyridine-Insensitive Ca2+ Channels Coupled to Catecholamine Release in Bovine Adrenal Chromaffin Cells

Abstract: In adrenal chromaffin cells, depolarization-evoked Ca²⁺ influx and catecholamine release are partially blocked by blockers of L-type voltage-sensitive Ca²⁺ channels. We have now evaluated the sensitivity of the dihydropyridine-resistant components of Ca²⁺ influx and catecholamine release to a toxin fraction (FTX) from the funnel-web spider poison, which is known to block P-type channels in mammalian neurons. FTX (1:4,000 dilution, with respect to the original fraction) inhibited K⁺-depolarization-induced Ca²⁺ influx by 50%, as monitored with fura-2, whereas nitrendipine (0.1–1 μ M ) and FTX (3:3), a synthetic FTX analogue (1 m M ), blocked the [Ca²⁺]_i transients by 35 and 30%, respectively. When tested together, FTX and nitrendipine reduced the [Ca²⁺]_i transients by 70%. FTX or nitrendipine reduced adrenaline and noradrenaline release by ∼80 and 70%, respectively, but both substances together abolished the K⁺-evoked catecholamine release, as measured by HPLC. The ω-conotoxin GVIA (0.5 μ M ) was without effect on K⁺-stimulated ⁴⁵Ca²⁺ uptake. Our results indicate that FTX blocks dihydropyridine- and ω-conotoxin-insensitive Ca²⁺ channels that, together with L-type voltage-sensitive Ca²⁺ channels, are coupled to catecholamine release.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Difference in the Effectiveness of Ca2+ to Evoke Catecholamine Secretion Between Adrenaline-and Noradrenaline-Containing Cells of Bovine Adrenal Medulla

Abstract: Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca²⁺ (0.8-160 μM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 μM), high K⁺ (14-56 μM), and bradykinin (0.1-1,000 μM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca²⁺-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K⁺ in perfused adrenal glands. These results indicate that the effectiveness of Ca²⁺ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Adenosine Receptor Agonists Inhibit K+-Evoked Ca2+ Uptake by Rat Brain Cortical Synaptosomes

Abstract: The uptake of Ca²⁺ by a K⁺-depolarized rat brain cerebral cortical crude synaptosomal preparation (P₂ fraction) was investigated. The characteristics of the Ca²⁺ uptake system are similar to those observed by other investigators. The preparation is also a suitable model with which to study the effects of adenosine on Ca²⁺ uptake and neurotransmitter release, as it is generally accepted that K⁺-evoked Ca²⁺ uptake is intimately related to depolarization-induced release of neurotransmitters. We have demonstrated that an extracellular receptor is involved in mediating the adenosine-evoked inhibition of K⁺-evoked Ca²⁺ uptake. The pharmacological properties of the receptor suggest that it may be similar in some respects to the A₂-receptor associated with adenylate cyclase. The adenosine uptake inhibitor, dipyridamole, potentiated the action of adenosine, suggesting that re-uptake is important in controlling the extracellular adenosine concentration and thus in the regulation of the adenosine receptor. The adenosine receptor antagonist theophylline inhibited the effects of adenosine. Calmodulin inhibited K⁺- evoked uptake of Ca²⁺ by the synaptosomal fraction.     

Basic and Regulatory Mechanisms of In Vitro Release of Met-Enkephalin from the Dorsal Zone of the Rat Spinal Cord

Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met-enkephalin-like material (MELM) that behaved as authentic Met-enkephalin under two different chromatographic procedures (Bio-gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μ M ) or to an excess of K⁺ (28 and 56 m M instead of 5.6 m M ). The K ⁺ -evoked release was totally dependent on the presence of Ca²⁺ in the super-fusing fluid whereas the spontaneous efflux of MELM was only partially Ca²⁺-dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met-enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μ M ), but not its desulphated derivative, substance P-sulphoxide (10 μ M ), and to a lesser extent substance P, enhanced the K⁺-evoked MELM release. In contrast, γ-aminobutyric acid (10 μ M ) and (–)-baclofen (1 μ M ) partially prevented the stimulatory effect of K⁺ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K⁺-evoked release of MELM.     

Ethylenediamine as a Specific Releasing Agent of γ-Aminobutyric Acid in Rat Striatal Slices

Abstract: Following incubation with [¹⁴C]y-aminobutyric acid (GABA) or [³H]dopamine, slices of rat striatum were superfused with media containing 36 mM K⁺ or ethylenediamine (EDA), 1 or 5 mM. Both K⁺ and EDA induced a release of [¹⁴C]GABA, the K⁺-induced release being largely Ca²⁺-dependent, while the EDA-induced release was not. Whereas K⁺ also evoked a Ca²⁺-dependent release of [³H]dopamine, EDA evoked no release of dopamine. EDA may therefore have potential as a specific GABA releasing agent.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Presynaptic Inhibition by Concanavalin A: Are α-Latrotoxin Receptors Involved in Action Potential-Dependent Transmitter Release?

Abstract: Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [³H]noradrenaline release by up to 30% with half-maximal inhibition at 0.16 µ M . Concanavalin A also reduced the release triggered by extracellular Ca²⁺ in neurons depolarized by 25 m M K⁺ or rendered Ca²⁺-permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the α₂-adrenergic agonist UK 14,304. Inactivation of G_s and G_i/G_o type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage-dependent K⁺ and Ca²⁺ currents. In contrast, the lectin efficiently blocked both the Ca²⁺-dependent and -independent α-latrotoxin-induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as α-latrotoxin-evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl α- d -mannopyranoside. The dimeric derivative, succinyl-concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to α-latrotoxin, concanavalin A failed to alter K⁺-evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross-linking of α-latrotoxin receptors may reduce action potential-dependent transmitter release.     

Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on ⁴⁵Ca²⁺ uptake and glutamate release. ⁴⁵Ca²⁺ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK-801. K⁺ depolarization-induced ⁴⁵Ca²⁺ uptake was also reduced by MK-801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the ⁴⁵Ca²⁺ uptake induced by depolarization. Opioid agonists, selective for μ-, κ-, and δ-receptors, inhibited the ⁴⁵Ca²⁺ uptake induced by K⁺ depolarization. The combination of low concentrations of MK-801 and opioid agonists resulted in additive inhibition of K⁺- induced ⁴⁵Ca²⁺ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca²⁺ uptake under acute, and perhaps also chronic, opiate treatment.