Effects of vinca alkaloids on uptakes of amino acids by rat brain synaptosomes

Synaptosomal uptakes of a number of amino acids were strongly inhibited by vinblastine (I₅₀ = 30–50 μM). Vincristine was a less effective inhibitor and colchicine had virtually no effect. The inhibitory effects of vinblastine on uptakes of alanine, leucine and serine were modified by cations and by nucleotides. The degree of inhibition was reduced by Mg²⁺ Ca²⁺ and Na⁺; nucleotides antagonized the inhibitory effect of vinblastine but this effect required the presence of divalent cations. The inhibitory effects of vinblastine were almost abolished when the incubations were carried out at 5° instead of 37°. The efflux of amino acids from synaptosomal particles was retarded by vinblastine; this effect could be observed at a concentration as low as 15μM. By analogy with the effects of vinca alkaloids on biological processes in other tissues, our present results suggest that neurotubular protein participates in the initial rapid uptake and the subsequent efflux of amino acids in synaptosomal fractions.     

Artefactual inhibition of dopamine uptake by psychotropic drugs on striatal synaptosomal preparation

On striatal synaptosomal preparations, using a double labelling test, numerous antidepressant drugs demonstrated an inhibitory effect on [³H]DA uptake at the same high concentrations producing a [¹⁴C]DA release. This releasing effect was also shared by non-antidepressant agents and was observed on synaptosomes preloaded with [³H]5HT. The imipramine-induced release of DA was not modified by increasing concentration of K⁺, by decreasing concentration of Na⁺, by deleting Ca²⁺ from the incubation medium, or by blocking the catecholamine uptake systems with nomifensine or cocaine. The imipramine effect was reversible and was possibly initiated by a transient physico-chemical modification of the synaptosomal membrane. It was concluded that the DA uptake carrier is probably not involved in the effect of these drugs.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na⁺-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na⁺-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na⁺-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na⁺ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na⁺-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A₂ (cPLA₂). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA₂ specific inhibitor AACOCF₃. ATP depletion-induced alterations in Na⁺-dependent phosphate uptake and cell viability were prevented by AACOCF₃. Inhibition of Na⁺-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA₂ activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.     

Transport of NG-Nitro-L-Arginine Across Intestinal Brush Border Membranes by Na+-Dependent and Na+ Independent Amino Acid Transporters

Purpose. To clarify the transport mechanism of N^G-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). Methods. Dog intestinal BBM vesicles were used. Results. The time course of L-NNA uptake showed a Na⁺-dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na⁺, indicating participation of Na⁺-dependent and Na⁺-independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na⁺. N^G-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. Conclusions. The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na⁺ differently from that across plasma membranes of nonepithelial cells, because B^{0, +} and b^{0, +} amino acid transporters function partly in L-NNA transport across intestinal BBM.     

The inhibition of Ca uptake in cardiac membrane vesicles by verapamil

Cardiac membrane vesicles take up Ca²⁺ in response to Na⁺ gradient (high inside) and negative inside membrane potential. Both components of the Ca²⁺ uptake, the Na⁺ gradient dependent uptake and the membrane potential dependent uptake are inhibited by verapamil; the action is dose-dependent and the concentrations of verapamil required to inhibit the Ca²⁺ uptake to 50% of its maximal value are 50 and 60 μM respectively. In the concentration ranges tested (50–750 μM Ca²⁺), the inhibitory effect of verapamil could not be antagonized by increasing the Ca²⁺ concentration of the medium. Introducing verapamil into the vesicles by rapid freezing and slow thawing of the vesicles had the same inhibitory effect as adding the same concentration of verapamil on the outside of the vesicles. Adding verapamil to both sides of the vesicle membrane led to higher inhibition of Ca²⁺ uptake. It is proposed that addition of verapamil can cause a change in cardiac membranes which is manifested by a decrease in the driving membrane potential and Ca²⁺ transport.     

Thyrotropin-Releasing Hormone (TRH) Uptake in Intestinal Brush-Border Membrane Vesicles: Comparison with Proton-Coupled Dipeptide and Na+-Coupled Glucose Transport

The mechanism of thyrotropin-releasing hormone (pGlu-His-Pro-NH₂; TRH) uptake across the luminal membrane of intestinal enterocytes was investigated using brush-border membrane vesicles (BBMV) from rabbit duodenum and jejunum and rat upper small intestine. [¹⁴C]Glucose accumulated within the intestinal vesicles (at 10 sec), in the presence of an inwardly directed Na⁺ gradient, 7- to 14-fold higher than equilibrium values (65 min). The vesicles also accumulated the dipeptide [¹⁴C]Gly-Sar. Dipeptide uptake was greatest in the presence of both an inwardly directed proton gradient and an inside negative membrane potential. The H⁺-dependent Gly-Sar transport was not affected by the presence of an excess (46-fold) of cold TRH. In contrast to the observations with glucose and Gly-Sar, the uptake of [³H]TRH after 10 or 60 sec (in each of the vesicle preparations) was not enhanced by either Na⁺ or H⁺ gradient conditions. The absence of vesicular accumulation of TRH was not due to peptide hydrolysis. For example, after a 60-sec incubation with rabbit jejunal BBMV no degradation of the tripeptide was evident. After 65 min, 6% of [³H]TRH had undergone degradation, by deamidation, to form TRH-OH. These studies provide no evidence for the oral absorption of TRH by a Na⁺- or H⁺-dependent carrier system in the brush-border membrane. Previous observations of TRH absorption in vivo may be accounted for by passive absorption of the peptide combined with its relative resistance to luminal hydrolysis.     

Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na+-channel α-toxin

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200–4500 Da, and 6000–7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.     

Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters

Transporters for the monoamine neurotransmitters, including noradrenaline, 5-hydroxytryptamine [5-HT] and dopamine, have twelve transmembrane spanning regions and cotransport Na⁺ and Cl^– ions. Another family of Na⁺-dependent transporters is that containing the Na⁺/glucose and Na⁺/proline cotransporters that are found in the epithelial cells of renal and intestinal brush border membranes. It has been shown that various trivalent lanthanides can substitute for Na⁺ for transport of glucose and proline. The aim of this study was to determine the effects of lanthanides on the activities of the human noradrenaline, 5-HT and dopamine transporters. Cultured cells were incubated for 2min with 10nM ³H-noradrenaline (SK-N-SH-SY5Y human neuroblastoma cells), ³H-5-HT (JAR human placental choriocarcinoma cells) or ³H-dopamine (COS-7 cells transfected with the cDNA of the human dopamine transporter). Specific amine uptake was determined as the difference between accumulation of the amine in the cells in the absence and presence of a corresponding uptake inhibitor. Under both isotonic (150mM NaCl or LiCl or 90mM lanthanide salt) and hypertonic (150mM NaCl + 100mM LiCl, 250mM LiCl or 150mM lanthanide salt) conditions, replacement of Na⁺ by Li⁺, La³⁺, Eu³⁺ or Sm³⁺ abolished the specific uptake of noradrenaline in SK-N-SH-SY5Y cells and replacement of Na⁺ by Li⁺ or Eu³⁺ decreased the specific uptake of 5-HT in JAR cells by 94–100% and that of dopamine in transfected COS-7 cells by 95–99%. The direct effects of Eu³⁺ (with Na⁺ present) on the human noradrenaline transporter in SK-N-SH-SY5Y cells were also examined. Eu³⁺ inhibited noradrenaline uptake into the cells (IC₅₀ 2.6mM) and nisoxetine binding to crude membranes of SK-N-SH-SY5Y cells (IC₅₀ 4.7mM) with similar potencies. Further experiments showed that 4.5mM EuCl₃ in the presence of 150mM Na⁺ caused a 3.5-fold increase in the K_m of noradrenaline and no change in the maximal rate of noradrenaline uptake. EuCl₃ (4.5mM) also caused a pronounced inhibition of the Na⁺-dependent stimulation of noradrenaline uptake by SK-N-SH-SY5Y cells. It can be concluded from these data that, in contrast with the Na⁺/glucose and Na⁺/proline cotransporters, the lanthanides cannot substitute for Na⁺ in the transport of substrates by the monoamine neurotransmitter transporters and that the lanthanides inhibit the latter transporters by interacting with sites of the transporters involved in amine and Na⁺ binding. Received: 15 July 1996 / Accepted: 28 January 1997     

Increase in Na+−Ca2+ exchange activity in sarcolemma isolated from mesenteric arteries of spontaneously hypertensive rats

Na⁺?Ca²⁺ exchange process in sarcolemmal vesicles isolated from mesenteric arteries of Wistar-Kyoto normotensive(WKY) and spontaneously hypertensive rats(SHR) was investigated. The sarcolemmal fractions isolated after homogenization and sucrose density gradient centrifugation were enriched with 5′-nucleotidase and ouabain sensitive, K⁺-dependent phosphatase activities. When the vesicles were loaded with Na⁺, a time dependent Ca²⁺ uptake was observed. However, very little Ca²⁺ uptake was observed when the vesicles were loaded with K⁺, or Ca²⁺ uptake of the Na⁺-loaded vesicles was carried out in high sodium medium so that there was no sodium gradient. When the vesicles loaded with Ca²⁺ by Na⁺?Ca²⁺ exchange were diluted into potassium medium containing EGTA, Ca²⁺ was rapidly released from the vesicles. Na⁺-dependent Ca²⁺ uptake was increased in SHR compared to WKY, but passive efflux of preaccumulated Ca²⁺ from the vesicles was decreased in SHR. The data indicate that the membrane vesicles of rat mesenteric arteries exhibit Na⁺?Ca²⁺ exchange activity. It is also suggested that changes of this process in vascular smooth muscle cell membrane of SHR may be involved in higher intracellular Ca²⁺ concentration and higher basal tone in SHR.     

Effect of Interaction of Heavy Metals on (Na+-K+) ATPase and the Uptake of 3H-DA and 3H-NA in Rat Brain Synaptosomes

Abstract: The effect of interaction of Mn²⁺, Pb²⁺ and Cd²⁺ on (Na⁺-K⁺) ATPase and uptake of labelled dopamine (³H-DA) and labelled noradrenaline (³H-NA) were studied in vitro in rat brain synaptosomes. The inhibition of (Na⁺-K⁺) ATPase by Pb²⁺ and Cd²⁺ alone was concentration dependent, however, Mn²⁺ had almost no effect on the activity of this enzyme. Interaction of Cd²⁺ with either Pb²⁺ or Mn²⁺ was most powerful in inhibiting the activity of synaptosomal transport ATPase. Lower concentrations of Pb²⁺ increased while higher concentrations inhibited synaptosomal uptake of ³H-DA and ³H-NA. Lower concentrations of Cd²⁺ increased the uptake of ³H-DA while at concentrations of 100 μM, the uptake was inhibited, this metal had strong inhibitory effect on the uptake of ³H-NA. Mn²⁺ had inhibited the uptake of labelled amines. Interaction of Mn²⁺ with Pb²⁺ or Cd²⁺ produced inhibition on the uptake of ³H-DA and ³H-NA. The results of the uptake of biogenic amines in the presence of metal ions apparently had no correlation with the activity of (Na⁺-K⁺) ATPase which is involved in the active transport of cations across cell membranes.     

Nine novel precursors of Buthus martensii scorpion α-toxin homologues

The cDNAs encoding nine novel α-toxin homologues were isolated from the venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch (BmK). They are rich in AAAA and TTTT elements at the 5′ UTRs. The flanking region of the translation initiation codon ATG is AAAATGAA, which is highly conserved in scorpion Na⁺, K⁺ and Cl⁻ channel toxin genes. These putative scorpion α-toxins shared 45.5–98.4% homology with the characterized BmK α-toxins, and were completely conserved in the positions of all eight cysteines. This showed, together with higher homology at nucleotide level than that at amino acid level, that these toxins may originate from a common ancestor. The discovery of a series of homologues of scorpion α-toxin with a different degree of natural mutation in the primary structure will provide us with a valuable system for studying the structure–function relationship of scorpion toxins.     

Chlorphentermine uptake by isolated perfused rabbit lung

Studies have shown that many amines accumulate in the lung both in vivo and in vitro. Chlorphentermine (CP) is a phenylethanolamine type of anorexigenic agent in use, known to be accumulated in the lung and to cause pulmonary phospholipidosis. The present study was undertaken to characterize the uptake and accumulation processes of [¹⁴C]-CP in the lung. These processes were examined in artificially ventilated, recirculating, isolated perfused rabbit lung preparations using either autologous blood or artificial perfusate at initial CP concentrations of 0.125 to 2.0 mm. Perfusate and lung homogenate samples were analyzed for the parent compound and possible metabolite(s). No metabolism of CP was observed. Steady state uptake was reached after 25 min of perfusion. Higher pulmonary extraction of CP was observed from artificial medium than from whole blood. Lung uptake was concentration dependent and was saturable at a concentration of 1.0 mm CP in the perfusate. Higher concentrations resulted in edematous and deteriorating preparations. Total CP uptake by the lungs could be represented by a combination of a non-linear saturable and a linear non-saturable process. Experiments with partial or complete depletion of Na⁺ from the perfusate, inclusion of harmaline (10^?3 and 10^?4m) or ouabain (10^?3m), inhibitors of Na⁺-dependent processes, suggest that CP uptake is partially Na⁺ dependent. In vitro lung slice experiments with Na⁺-free medium and medium containing harmaline (1 mm) or iodoacetate (1 mm) yielded similar results. Preloading the lung with an equivalent concentration of imipramine, which is known to be concentrated in the lung, resulted in partial inhibition of CP uptake. However, preloading with CP was without effect on imipramine uptake. These results are consistent with a Na⁺-dependent, carrier-mediated transport process for the uptake of CP by the lung tissue.     

Phenolphthalein- and harmaline-induced disturbances in the transport functions of isolated brush border and basolateral membrane vesicles from rat jejunum and kidney cortex

Phenolphthalein and harmaline were examined with respect to their effects on the transport functions of purified brush border and basolateral membrane vesicles from rat jejunum and kidney cortex. Phenolphthalein (0.5 mM) inhibited Na⁺-coupled d-glucose uptake by intestinal brush border membrane vesicles without affecting Na⁺-coupled l-alanine transport, Na⁺ transport or Na⁺-independent d-glucose transport. In renal brush border membrane vesicles, the same concentration of this drug did not even affect Na⁺-coupled d-glucose uptake. At a concentration of 1 mM or higher, however, Phenolphthalein rendered both intestinal and renal vesicles leaky to these solutes. In intestinal and renal basolateral membrane vesicles, phenolphthalein at a concentration of 0.5 mM noticeably inhibited (Na⁺-K⁺)-ATPase activity, but showed no effect on phloretin-sensitive Na⁺-independent d-glucose uptake. At 1 mM this drug also inhibited ouabain-insensitive ATPase activity. Harmaline, at concentrations greater than 2 mM, inhibited not only Na⁺-coupled d-glucose and l-alanine uptake by both intestinal and renal brush border membrane vesicles, but also Na⁺ translocation. The drug, however, affected neither Na⁺-independent d-glucose uptake nor the general permeability of these membranes. Harmaline also inhibited (Na⁺-K⁺)-ATPase activity of intestinal and renal basolateral membrane vesicles without affecting ouabain-insensitive ATPase. It did not influence, however, phloretin-sensitive Na⁺-independent d-glucose uptake by these vesicles. These observations suggest that harmaline acts as an inhibitor of Na⁺ and Na⁺-dependent transport mechanisms in intestinal as well as renal brush border membranes. Phenolphthalein at the lower concentration selectively inhibited certain transport processes in these membranes as well as in basolateral membranes, whereas at the higher concentration it caused widespread structural disturbances, possibly through its chaotropic action on membranes.     

Inhibitory characteristics of the mycotoxin penicillic acid on (Na+-K+)-activated adenosine triphosphatase

Penicillic acid, a cardioactive mycotoxin produced by various Penicillium molds, is a potent and selective inhibitor of membrane (Na⁺-K⁺)-adenosine triphosphatase (ATPase). A broad range of inhibition of activity by the toxin was demonstrated with a half-maximal concentration equal to 1.8 × 10^?8M. Inhibition was time and pH dependent and complete after 20–30 min preincubation within a narrow range of physiological pH. Kinetic evaluation of cationic substrate activation of (Na⁺-K⁺⁾-ATPase indicated competitive inhibition with regard to Na⁺ concentration and noncompetitive inhibition with regard to K⁺ concentration. Also K⁺ -dependent p-nitrophenyl phosphatase activity was not significantly altered by penicillic acid, and uncompetitive inhibition with regard to ATP activation of the enzyme was demonstrated. Preliminary binding studies indicated that inhibition of ATPase activity could be partially restored by repeated washing and by incubation with dithiothreitol and cysteine. Penicillic acid (high concentrations) impaired [³H]ouabain binding to membrane preparations but this effect was noncompetitive, indicating different sites of action for the two inhibitors. A significant linear correlation between reactive enzyme sulfhydryl content [SH] and ATPase activity in the presence of varying concentrations of toxin also was noted. It is postulated that penicillic acid inhibition of (Na⁺-K⁺) -ATPase occurs via critically accessible membrane thiol receptors regulating Na⁺-dependent phosphorylation of the transport enzyme.     

Transport Characteristics of Ceftibuten,a New Oral Cephem,in Rat Intestinal Brush-Border Membrane Vesicles: Relationship to Oligopeptide and Amino β-Lactam Transport

Ceftibuten undergoes H⁺-coupled uphill transport across rat small intestinal brush-border membrane vesicles. The effects of amino acids, peptides, folate, and -lactams on the uptake of ceftibuten were examined. Uptake of ceftibuten was competitively inhibited by dipeptides or tripeptides. A counter-transport effect on ceftibuten uptake was observed in the vesicle preloaded with these peptides, and the transport was temporarily against a concentration gradient (overshooting). On the other hand, ceftibuten uptake was not changed by amino acids and a tetrapeptide. Therefore, ceftibuten is predominantly transported via the oligopeptide transport system in the brush-border membranes. The relationship of ceftibuten transport to folate and other oral antibiotics was also investigated. Cyclacillin, cephradine, and cefadroxil exhibited both inhibitory and countertransport effects, but folate, cefaclor, and cephalexin showed only a slight inhibitory effect. As the transport of cefaclor showed no uphill uptake in the presence of a H⁺ gradient and its H⁺ stimulated uptake was small, a H⁺ gradient-independent carrier-mediated system seems to participate in its transport. These findings suggest that two different carrier-mediated transport systems, H⁺ gradient dependent and independent, may exist for oral cephems.     

Interaction of central nervous system drugs with synaptosomal transport processes.

Interaction of a wide range of central nervous system (CNS) drugs with the transport of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) into synaptosomes from cerebral cortex was investigated. By determining the effect on Na,K-ATPase, the interaction of these drugs with the synaptic transport of Na ⁺ and K⁺ was also assessed. Although of different numerical value, the order of potency of the drugs in inhibiting the uptakes of both biogenic amines was comparable. Furthermore, similar inhibition constants were obtained for the low affinity uptake of 5-HT and the uptake of NE. Desipramine and amphetamine strongly inhibited both uptake processes. The narcotic drugs displayed a wide range of inhibitory potency. An exceptionally strong, but not stereospecific, effect was obtained with levorphanol, which inhibited 5-HT uptake with a K_i of 0.7 μM. While methadone and pentazocine had inhibition constants between 20 and 50 μM, morphine, codeine and naloxone were markedly weak inhibitors with K_i, values extending into the mM range. The uptake of both 5-HT and NE was strongly inhibited by quinacrine. Among the drugs investigated, only methadone showed substantial inhibition of synaptosomal Na,K-ATPase. The reversible inhibition of the enzyme by the drug was diminished at concentrations of Na ⁺ and K ⁺ reflecting synaptic discharge. The drug partially bound to the ouabain site of Na,K-ATPase and also inhibited the Mg-activated component. The feasibility and experimental conditions were established for using a modified crude mitochondrial fraction as synaptosomal preparation to study transport processes in general and the uptake of biogenic amines in particular.     

Effect of low concentrations of K+ and Cl− on the Na+-dependent neuronal uptake of [3H] dopamine

The specific uptake of [³H] dopamine (DA) was studied using a crude synaptosomal fraction obtained from rat striatum. In a medium containing a 10 mM NaHC03/NaH2PO4 buffer and no added K⁺ ions, addition of NaCl elicited an increase in DA uptake for Na⁺ concentrations from 10 to 60 mM, and then a decrease of uptake for Na⁺ concentrations up to 130 mM. These data confirm that rather low NaCl concentrations produce a maximal DA uptake. This biphasic curve of uptake resulted from significant changes in the V _max of the DA uptake. Except for 10 mM Na⁺, this curve was not significantly modified when 9 mM NaHCO₃/NaH₂PO₄ were replaced by 9 mM NaCl. This result indicates that the Cl^– dependence of the DA uptake is mainly secondary to the Na⁺ dependence. Addition of KCl up to 3 mM did not modify the ascending part of the NaCl-dependent uptake curve. In contrast, the reduction in uptake produced by high Na⁺ concentrations was prevented in a concentration-dependent manner by KCl; this effect resulted from a decrease in the K_m and an increase in the V _max for the uptake.Measurements of membrane potential, with the help of the fluorescent probe 3,3-diethylthiadicarbocyanine iodide [DiSC₂(5)] and purified synaptosomes prepared from rat striatum and cerebral cortex, revealed that addition of 3 mM KCl to a medium containing a high Na⁺ concentration and no K⁺ ions produced a marked and stable decrease in the fluorescence level. This decrease which corresponds to an increase in membrane polarization was blocked by 0.1 mM ouabain. These data suggest that low K⁺ concentrations are likely to prevent the decrease in uptake elicited by high Na⁺ concentrations by restoration, via a Na⁺/K⁺ ATPase-mediated mechanism, of the membrane potential and/or a transmembrane electrochemical Na⁺ gradient more favourable to DA uptake.