Effects of hemicholinium-3 and sodium ions on choline uptake system in excised superior cervical sympathetic ganglia of rats

Active choline uptake by rat superior cervical sympathetic ganglia (SCG), which contain abundant cholinergic nerve terminals, was studied with respect to sensitivity to inhibition by hemicholinium-3 (HC-3) and dependence on extracellular Na⁺ under standard conditions of assay. Choline was taken up by a single saturable process with apparentK _m=3.07×10^–5 M and Vmax=286 pmoles/min/mg protein. Neither denervation followed by degeneration of cholinergic nerve terminals nor axotomy with successive neuronal degeneration significantly decreased in choline uptake by the ganglia in vitro. HC-3 dose-dependently inhibited ganglionic choline uptake more effectively at lower than at higher choline concentrations. HC-3 sensitive inhibition of ganglionic choline uptake was not seen in young rats one week after birth but appeared with maturity, attaining approximately 50% maximal inhibition in adult SCG. Extent of inhibition by HC-3 and Na⁺ dependence of ganglionic choline uptake was not altered by denervation or axotomy.Abbreviations used (HC-3) hemicholinium-3 - (HAChU) high affinity choline uptake - (LAChU) low affinity choline uptake - (SCG) superior cervical ganglia - (Ch) choline - (ACh) acetylcholine     

High-Affinity Choline Transporter

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na⁺- and Cl^–-dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.     

Isotopic labeling of synaptosomes that accumulate choline and the effect of narcotic drugs

Subcellular studies of choline uptake of rat striatum indicated a correspondence between the Na⁺-dependent uptake and choline acetyltransferase (ChAc), whereas there was a lack of correspondence between the Na⁺-independent uptake and ChAc. Subcellular studies also showed a correspondence between the Na⁺-dependent uptake and hemicholinium-3 inhibition, and more important, particles that accumulate choline were shown to consist of at least two subcellular populations. A comparison was made of kinetic data from three areas of the rat brain: corpus striatum, cerebral cortex, and hypothalamus. Taken together, our data on choline uptake give added support to the idea that the Na⁺-dependent choline transport is concentrated in the striatum and specifically related to cholinergic nerve endings. Morphine and methadone in vitro inhibited the Na⁺-dependent choline uptake. In vivo morphine induced a significant lowering of theV _max in the rat cerebral cortex, but not in the striatum. This finding is consistent with the known action of morphine on acetylcholine turnover.Preliminary reports of this work were presented at the Fifth Meeting of the American Society for Neurochemistry in New Orleans, March 1974, and the Fall ASPET Meeting in Montreal, August 1974 (1,2).     

CHOLINE UPTAKE BY CHOLINERGIC NEURON CELL SOMAS

The cellular compartments of ciliary ganglia take up choline by a single, saturable process with K_m=7.1 × 10^?5 M and V_max= 4.66 pmol/min per ganglion: Denervation of the ganglia and the resultant degeneration of nerve terminals caused no significant decrease of the rate of accumulation of choline by the ganglia. This indicates that the measured uptake is by the postganglionic ncurons and nonneural elements (NNE: glial and connective tissue cells) in the ganglia. This uptakc is not dependent on metabolic energy and is not affectcd by lowcring Na⁺ or raising K⁺ concentrations in the incubating mcdia but is depressed in the presence of ouabain and hemicholinium-3. The presence or Na⁺-dependent. rapidly saturable uptake in the preganglionic nerve terminals which is not detectablc kinetically is, however, inferred from a decrease in ACh synthesis in dcncrvatcd prcparations and a similar decrcasc in intact ganglia incubated in low Na+ solution.     

Cation-induced asymmetry of choline flux across presynaptic plasma membranes

Highly cholinergic synaptosomes from the optic lobes of Sepia officinalis retain their ability to concentrate K⁺ and extrude Na⁺ and to synthesise acetylcholien in vitro. Choline uptake is hemicholinium-3 and Na⁺ sensitive but is not obligatorily coupled to choline metabolism, or an energy supply as shown by the action of metabolic and ion pump inhibitors. The influx and efflux and/or steady-state distributions of choline in the presence of Na⁺, Li⁺, Rb⁺, Cs⁺ and mannitol were studied. The influx studies at different cis-choline concentrations revealed two systems for choline influx with different monovalent cation sensitivity and suggested a 1 : 1 interaction of choline with both mechanisms. Choline efflux was stimulated by trans-choline. Calculations of the internal/external concentration ratio expected if choline transport were coupled to the Na⁺ gradient gave a maximal value of about 10². A secondary active transport of choline, where Na⁺ is the driver solute provides an explanation for the cation sensitivity of the mechanism as well as for the method of coupling of choline transport to the varying demands of the nervous system for acetylcholine.     

Evaluation of acetate uptake and its relative conversion to acetylcholine in Torpedo electric organ synaptosomes under different ionic and metabolic conditions

The uptake of acetate and its incorporation into acetylcholine were measured under various conditions in nerve terminals isolated from the electric organ in order to characterize acetate uptake and to study the relationship between acetate uptake and acetylcholine synthesis in a pure cholinergic preparation. It was found that increasing extracellular choline up to 10^?4 M had no effect on either acetate uptake or the conversion of acetate to ACh, while the addition of hemicholinium-3 to the incubation medium led to decreases in both parameters. Hence, it appears that endogenous levels of choline are sufficient to support ongoing acetylcholine synthesis in this preparation and that this synthesis depends to some extent on the uptake of extracellular choline. Nonetheless, in the absence of choline uptake, both the uptake of acetate and the conversion of acetate to acetylcholine remained substantial, indicating that internal sources of choline as well can be used for acetylcholine synthesis.Acetate uptake displayed a marked requirement for external Na⁺ and was decreased following depolarization of the synaptosomes by an elevated K⁺ concentration. The conversion of acetate to acetylcholine followed a similar pattern, except that a small reduction in acetylcholine synthesis was observed in the absence of external Ca²⁺, while acetate uptake was unaffected. The addition of ATP, AMP-PNP or phosphate to the incubation medium caused an increase in both the uptake and incorporation of acetate, but adenosine had no effect on either of these functions. Choline uptake, meanwhile, was unchanged in the presence of ATP, phosphate or adenosine. Acetate uptake appears to be more closely linked to its intracellular metabolism than to the transmembrane movement of choline itself.The mechanism by which acetate crosses the nerve terminal membrane has not been established, but the possibility that acetate is a substrate for a monocarboxylate transport system such as has been described in other systems can be ruled out as inhibitors of anion permeability do not block acetate uptake in this preparation.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Uptake of neurotransmitters and precursors by clonal lines of astrocytoma and neuroblastoma: III. Transport of choline

Clonal lines of glial, neuronal, and nonneural origin accumulate choline via a high-affinity carrier-mediated transport system withK _m in the range of 10–14 M. These cell lines also accumulate choline by a second system that is not saturable at 10 mM choline, and that may represent diffusion. The transport of choline in glial cells differs from that seen in neuronal cells with respect to its Na⁺ requirement. The omission of Na⁺ from the incubation medium reduces high-affinity choline transport in neuronal cells and enhances it in glial cells. Kinetic analysis of the data indicates that reversible cholinesterase inhibitors and hemicholinium-3 (HC-3) inhibit the high-affinity transport system for choline. On the other hand, the diffusional or low-affinity component of choline transport in either cell type appears to have no Na⁺ requirement and is unaffected by either cholinesterase inhibitors or 10^–4 M HC-3. The neuronal-glial differences in the Na⁺ requirement of choline transport may be related to the coupling of transport to choline metabolism, which differs in the two cell types. The presence of a high-affinity transport system for choline in clonal glial lines used as models of normal glia suggest that glia may modulate the availability of choline for acetylcholine synthesis at cholinergic synapses.     

Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells

We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na⁺ from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na⁺-free conditions was inhibited by a Na⁺/H⁺ exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.     

Identification of a high affinity taurine transporter which is not dependent on chloride

Taurine transport by lactating gerbil mammary tissue has been examined. Taurine uptake is, mediated by a high-affinity system which is specific for -amino acids. The uptake of taurine is Na⁺-dependent but appears not to be obligatorly dependent upon Cl^–. Thus, replacing Na⁺ with choline almost abolished taurine uptake. Substituting Cl^– with NO ₃ ^– had no effect whereas SCN^– induced a small but significant increase in taurine influx. Taurine uptake was Na⁺-dependent under conditions where Cl^– had been replaced with NO ₃ ^– . However, it is apparent that the Na⁺-dependent taurine transport system requires the presence of a permeable anion because replacing Cl^– with gluconate markedly reduced taurine uptake. Cell-swelling, induced by a hyposmotic challenge, increased the efflux of taurine from gerbil mammary tissue via a pathway sensitive to niflumic acid.Abbreviations Tris (Tris(hydroxymethyl)aminomethane - BES (N,N-bis[2-hydroxyethyl]-2-aminoethane sulphonic acid)     

Regulation of Rat Brain Synaptosomal [3H]Hemicholinium-3 Binding and [3H]Choline Transport Sites Following Exposure to Choline Mustard Aziridinium Ion

Abstract: Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the “ground-state” (basal) high-affinity choline transport but only 50% of “ground-state” hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K⁺-stimulated increases in choline transport and of [³H]hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [³H]hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca²⁺ or presence of high Mg²⁺ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg²⁺ levels removed the ability of the mustard to inhibit K⁺-stimulated increases of either [³H]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [³H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extracellular Ca²⁺.     

Quantitative analysis of acetylcholine release in depolarized hippocampal slices

Time course of the hippocampal slice acetylcholine content and the rate of acetylcholine release were studied during high K⁺-induced depolarization for 4 to 60 min. At the end of the potassium exposure, both the acetylcholine remaining in the tissue and appearing in the incubation medium were quantitatively determined by gas chromatography using a nitrogen-sensitive detector. During prolonged K⁺ incubation, the acetylcholine content of the slices decreased by 60%, reaching a steady state after 16 min. The increase in the acetycholine concentration of the depolarizing medium showed a biphasic pattern, with rate constants of 1.40 and 0.69 nmol/min/g in the early (0–16 min) and late (16–60 min) phase, respectively. K⁺-evoked acetylcholine release was Cal⁺-dependent, but addition of choline did not alter tissue levels of acetylcholine or the pattern of K⁺-evoked acetylcholine release. The rate of acetylcholine release was markedly decreased by inhibition of choline uptake with hemicholinium-3 or by addition of 4-(1-naphthylvinyl)pyridine which inhibits both ACh producing enzyme, choline acetyltransferase and choline uptake mechanism. These data confirm the essential role during depolarization of extracellular choline transport into the cholinergic terminals utilizing choline released by the slices during the incubation. It is concluded that drugs which can influence the processes of choline uptake and acetylcholine sythesis can alter the rate of acetylcholine release measured under similar conditions.     

Taurine transport by rabbit kidney brush-border membranes: Coupling to sodium,chloride, and the membrane potential

Summary Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na⁺-d-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na⁺ gradient (K⁺ diffusion potential) and could be completely inhibited by other -amino acids. It displayed a specific anion requirement (Cl^–Br^–SCN^–>I^–>NO ₃ ^– ). At chemical Na⁺ equilibrium, Cl^– gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl^– gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl^– as well as Br^–. The Cl^– stoichiometry was found to be one, whereas taurine transport, in the presence of Cl^–, was sigmoidally related to the Na⁺ concentration, resulting in a coupling ratio of 2 to 3 Na⁺: 1 taurine. Upon Cl^– replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na⁺ concentration (up to 150mm). These results suggest a 2 to 3 Na⁺:1 Cl^–:1 taurine cotransport mechanism driven mainly by the Na⁺ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl^– appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

Ionic mechanisms underlying acetylcholine-, nicotine-, and muscarine-induced depolarization of Helix lucorum neuron RPa4

Research was carried out into the ionic aspects of depolarization potentials produced inHelix lucorum neuron RPa4 by injecting three cholinomimetics into the soma: acetylcholine, nicotine, and muscarine. Substances were used suppressing Na⁺, K⁺, Ca²⁺, and Cl^– conductance at the membrane. Acetylcholine brought about increased Na⁺, Ca²⁺; and Cl^– conductance. As the choline component was only slight, due to the similarity of membrane and resting potential for chloride, it might be deduced that the prevailing response to acetylcholine is associated with chemically controlled input of Na⁺ and Ca²⁺ into the cell. Nicotine and muscarine induced mainly sodium and calcium conductance respectively.M. V. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 305–314, May–June, 1989.     

The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na⁺/l-glutamate (l-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K⁺ and Cl^?. The presence of 100 mM K⁺ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K⁺ concentration and that in the absence of any sodium gradient, an outward K⁺ gradient was unable to influence the Na⁺/acidic amino acid transport system. It was also found that Cl^? could specifically activate the Na⁺-dependent l-glutamate (l-aspartate) uptake either in the presence or in the absence of K⁺. Also the effect of Cl^? was observed only in the presence of an inward Na⁺ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na⁺ gradient and in the presence of chloride gradient. l-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K⁺ or Cl^? did not show any translocation of net charge.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Effects of ouabain on chloride movements across the seawater eel intestine

Summary Simultaneous measurements of transepithelial potential difference (PD) and net water flux were made in the stripped intestine of seawater eels, and the effects of ouabain on these two parameters were examined in normal Ringer solution or under a chloride concentration gradient. Ouabain reduced the serosa-negative PD and the net water flux in normal Ringer solution with a linear relationship between the PD and the net water flux. Removal of K⁺ from the Ringer solution on both serosal and mucosal sides also reduced the PD and the net water flux to approximately zero. On the other hand, blocking the Na⁺–K⁺ pump by ouabain, K⁺-free or Na⁺-free Ringer solution increased the diffusion potential for Cl^–. Inhibition of Cl^– transport and increment in Cl^– permeability by ouabain occurred almost simultaneously. It is likely, therefore, that Cl^– transport as well as Cl^– permeability is dependent on Na⁺–K⁺ pump activity. A possible mechanism of dependence of Cl^– transport on the Na⁺–K⁺ pump is discussed in relation to the increment in Cl^– permeability.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.