[3H]Noradrenaline Release from Brain Slices Induced by an Increase in the Intracellular Sodium Concentration: Role of Intracellular Calcium Stores

Rat brain slices, prelabeled with [3H]noradrenaline, were superfused and exposed to K+ depolarization (10-120 mM K+) or to veratrine (1-25 microM). In the absence of extracellular Ca2+ veratrine, in contrast to K+-depolarization, caused a substantial release of [3H]noradrenaline, which was completely blocked by tetrodotoxin (0.3 microM). The Ca2+ antagonist Cd2+ (50 microM), which strongly reduced K+-induced release in the presence of 1.2 mM Ca2+, did not affect release induced by veratrine in the absence of extracellular Ca2+. Ruthenium red (10 microM), known to inhibit Ca2+-entry into mitochondria, enhanced veratrine-induced [3H]noradrenaline release. Compared with K+ depolarization in the presence of 1.2 mM Ca2+, veratrine in the absence of Ca2+ caused a somewhat delayed release of [3H]noradrenaline. Further, in contrast to the fractional release of [3H]noradrenaline induced by continuous K+ depolarization in the presence of 1.2 mM Ca2+, that induced by prolonged veratrine stimulation in the absence of Ca2+ appeared to be more sustained. The data strongly suggest that veratrine-induced [3H]noradrenaline release in the absence of extracellular Ca2+ is brought about by a mobilization of Ca2+ from intracellular stores, e.g., mitochondria, subsequent to a strongly increased intracellular Na+ concentration. This provides a model for establishing the site of action of drugs that alter the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

The Suppression of Stimulus-Evoked Release of Amino Acid Neurotransmitters from Synaptosomes by Verapamil

Verapamil at 200 microM, prevented the respiratory stimulation, K+ loss, transmitter release, and 45Ca2+ entry into incubated synaptosomes evoked by veratrine (25 to 75 microM) or by high K+ (56 mM). Verapamil (100 microM) also blocked gamma-aminobutyric acid homoexchange, whilst tetrodotoxin was ineffective. Much lower concentrations of verapamil (less than 1 microM) blocked the 45Ca2+ entry caused by veratrine, but not its action in releasing neurotransmitter or K+. It is concluded that verapamil, at 30 to 200 microM, blocks active Na+ channels, thereby preventing depolarization. At greater than 1 microM, verapamil blocks Ca+ channels selectively.     

Acetylation of Synaptosomal Protein: Inhibition by Veratridine

Abstract: Incubation of synaptosomes with [³H]acetate results in rapid labeling of protein. Labeling is decreased in the presence of veratridine, and the effect of veratridine is blocked by tetrodotoxin. Most of the radioactivity can be removed by base or acid hydrolysis, and is probably incorporated as acetate; it is this fraction that is affected by the veratridine. The data suggest that veratridine stimulates deacetylation of synaptosomal protein. This raises the question whether acetylation-deacetylation is involved in membrane function.     

Characterization of Delta-Sleep-Inducing Peptide-Evoked Release of Met-Enkephalin from Brain Synaptosomes in Rats

Delta-sleep-inducing peptide (DSIP) stimulates the release of Met-enkephalin (Met-ENK) from superfused slices of the rodent lower brainstem in vitro. In our present study, DSIP (10(-10)-10(-9) M) induced a significant release of Met-ENK from medullary synaptosomes of rats. This DSIP-evoked release of Met-ENK was Ca2+ dependent and tetrodotoxin (TTX) insensitive. Furthermore, DSIP (10(-11)-10(-9) M) significantly increased 45Ca2+ uptake in medullary synaptosomes. These results demonstrate that DSIP acts directly on the nerve endings of Met-ENK-containing neurons to release this pentapeptide by generating a Ca2+ influx into these neurons. Effects of DSIP on Met-ENK release in other discrete brain regions were also studied. Significant DSIP-evoked Met-ENK release from synaptosomes was observed in the cortex, hypothalamus, and midbrain (at concentrations of 10(-10) and 10(-9) M) and in the hippocampus and thalamus (only at 10(-9) M), but not in the striatum. In the hypothalamus, the release of Leu-enkephalin from its synaptosomes was slightly, but not significantly, enhanced by DSIP (10(-10)-10(-8) M). Our findings demonstrate that DSIP triggered a Ca2+ influx in nerve endings to induce a subsequent release of Met-ENK from neurons in only certain brain regions.     

Glutamatergic Control of Dopamine Release in the Rat Striatum: Evidence for Presynaptic N-Methyl-D-Aspartate Receptors on Dopaminergic Nerve Terminals

The N-methyl-D-aspartate (NMDA) receptor-mediated regulation of the release of newly synthesized [3H]dopamine [( 3H]DA) was studied in vitro, both on rat striatal slices using a new microsuperfusion device and on rat striatal synaptosomes. Under Mg2(+)-free medium conditions, the NMDA (5 X 10(-5) M)-evoked release of [3H]DA from slices was found to be partly insensitive to tetrodotoxin (TTX). This TTX-resistant stimulatory effect of NMDA was blocked by either Mg2+ (10(-3) M) or the noncompetitive antagonist MK-801 (10(-6) M). In addition, the TTX-resistant NMDA-evoked response could be potentiated by glycine (10(-6) M) in the presence of strychnine (10(-6) M). The coapplication of NMDA (5 X 10(-5) M) and glycine (10(-6) M) stimulated the release of [3H]DA from striatal synaptosomes. This effect was blocked by Mg2+ (10(-3) M) or MK-801 (10(-5) M). These results indicate that some of the NMDA receptors involved in the facilitation of DA release are located on DA nerve terminals. These presynaptic receptors exhibit pharmacological properties similar to those described in electrophysiological studies for postsynaptic NMDA receptors.     

Characterization of the Effect of Monensin on γ-Amino-n-Butyric Acid Release from Isolated Nerve Terminals

The action of the polyether antibiotic monensin on the release of gamma-[3H]amino-n-butyric acid [( 3H]GABA) from mouse brain synaptosomes is characterized. Monensin enhances the release of this amino acid transmitter in a dose-dependent manner and does not modify the efflux of the nontransmitter amino acid alpha-[3H]aminoisobutyrate. The absence of external Ca2+ fails to prevent the stimulatory effect of monensin on [3H]GABA release. Furthermore, monensin is less effective in stimulating [3H]GABA release in the presence of Ca2+. The releasing response to monensin is absolutely dependent on external Na+. The blockade of voltage-sensitive Na+ or Ca2+ channels does not modify monensin-induced release of the transmitter. Also, the blockade of the GABA uptake pathway fails to prevent the stimulatory effect of monensin on [3H]GABA release. Although monensin markedly increases Na+ permeability in synaptosomes, these data indicate that the Ca2+-independent monensin-stimulated transmitter release is not mediated by the Na+-dependent uptake pathway. It is concluded that the entrance of Na+ through monensin molecules inserted in the presynaptic membrane might be sufficient to initiate the intraterminal molecular events underlying transmitter release.     

Neural Regulation of Muscle Glucose 6-Phosphate Dehydrogenase: Effect of Batrachotoxin and Tetrodotoxin

Abstract: We tested the hypothesis that glucose 6-phosphate dehydrogenase (G6PD) activity in the rat skeletal muscle is regulated by putative axonally derived neurotrophic factors. This was accomplished by comparing the effects of nerve section and subperineural injection of batrachotoxin (BTX) or tetrodotoxin (TTX) on G6PD in rat extensor digitorum longus (EDL) muscle. BTX, an agent known to block nerve impulse conduction and axonal transport, increased G6PD activity to 155% and 163% of control by days 2 and 4 after injection. Denervation of the EDL muscle by section of the peroneal nerve 10–20 mm from its entrance to the muscle caused G6PD activity to increase to 170% of control by day 1 and to 200% and 180% of control by days 2 and 4, respectively. The increase in enzyme activity after denervation and after subperineural injection of BTX was due in part to muscle inactivity resulting from blockade of nerve impulses. This conclusion is based upon the observation that subperineural injection of TTX at an identical site in the peroneal nerve caused a small but significant (30%) increase in G6PD activity after 4 days. Choline acetyltransferase (CAT) activity was assessed as a measure of the efficacy of blockade of slow axonal transport. Decreases in CAT activity following denervation or injection of BTX or TTX were parallel to increases in G6PD activity observed under these conditions. These results argue for a role of axonal transport in neural regulation of muscle G6PD, with a small contribution by neuromuscular activity.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Small-cell Lung Cancer (Human): Potentiation of Endocytic Membrane Activity by Voltage-gated Na<Superscript>+</Superscript> Channel Expression in Vitro

The possible functional role of voltage-gated Na⁺ channel (VGSC) expression in controlling endocytic membrane activity in human small-cell lung cancer (SCLC) cell lines (H69, H209, H510) was studied using uptake of horseradish peroxidase (HRP). The normal human airway epithelial (16HBE14o) cell line was used in a comparative approach. Uptake of HRP was vesicular, strongly temperature-sensitive and suppressed by cytoskeletal poisons (cytochalasin D and colchicine), consistent with endocytosis. Compared with the normal cells, HRP uptake into SCLC cells was kinetically more efficient, resulting in more than four-fold higher uptake under optimized conditions. Importantly, HRP uptake into SCLC cells was inhibited significantly by the specific VGSC blocker tetrodotoxin, as well as lidocaine and phenytoin. These effects were dose-dependent. None of these drugs had any effect on the uptake into the 16HBE14o cells. Uptake of HRP into SCLC cells was reduced by ∼66% in Na⁺-free medium and was partially (∼30%) dependent on extracellular Ca²⁺. The possibility that the endocytic activity in the H510 SCLC cells involved an endogenous cholinergic system was investigated by testing the effects of carbachol (a cholinergic receptor agonist) and eserine (an inhibitor of acetylcholinesterase). Both drugs inhibited HRP uptake, thereby suggesting that basal cholinergic activity occurred. It is concluded that VGSC upregulation could enhance metastatic cell behavior in SCLC by enhancing endocytic membrane activity.     

Cysteine Mapping in the Ion Selectivity and Toxin Binding Region of the Cardiac Na+ Channel Pore

Aqueous exposure of critical residues in the selectivity region of voltage gated Na⁺ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3) and IV (D4) of the human heart Na⁺ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate (MTSEA⁺) and (2-sulfonatoethyl)methanethiosulfonate (MTSES⁻) in all six of the Cys-substituted channels, including Trp → Cys substitutions at homologous positions in D3 and D4 that were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification, each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >10². Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C (the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even though both were exposed. Neither position responded to MTSES⁻ suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES⁻ markedly increased, and MTSEA⁺ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp¹⁷¹³ acts electrostatically in an unrestricted region. These results suggest that Lys¹⁴¹⁸ lies in a restricted region favorable to cations, whereas Asp¹⁷¹³ is at a more peripheral location in the Na⁺ channel pore. Received: 8 May 1996/Revised: 15 August 1996