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.     

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.     

Molecular and functional characterization of an Na+-independent choline transporter in rat astrocytes

In this study, we examined the molecular and functional characterization of choline uptake into cultured rat cortical astrocytes. Choline uptake into astrocytes showed little dependence on extracellular Na+. Na+-independent choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (Km) of 35.7 +/- 4.1 microm and a maximal velocity (Vmax) of 49.1 +/- 2.0 pmol/mg protein/min. Choline uptake was significantly decreased by acidification of the extracellular medium and by membrane depolarization. Na+-independent choline uptake was inhibited by unlabeled choline, acetylcholine and the choline analogue hemicholinium-3. The prototypical organic cation tetrahexylammonium (TEA), and other n-tetraalkylammonium compounds such as tetrabutylammonium (TBA) and tetrahexylammonium (THA), inhibited Na+-independent choline uptake, and their inhibitory potencies were in the order THA > TBA > TEA. Various organic cations, such as 1-methyl-4-tetrahydropyridinium (MPP+), clonidine, quinine, quinidine, guanidine, N-methylnicotinamide, cimetidine, desipramine, diphenhydramine and verapamil, also interacted with the Na+-independent choline transport system. Corticosterone and 17beta-estradiol, known inhibitors of organic cation transporter 3 (OCT3), did not cause any significant inhibition. However, decynium22, which inhibits OCTs, markedly inhibited Na+-independent choline uptake. RT-PCR demonstrated that astrocytes expressed low levels of OCT1, OCT2 and OCT3 mRNA, but the functional characteristics of choline uptake are very different from the known properties of these OCTs. The high-affinity Na+-dependent choline transporter, CHT1, is not expressed in astrocytes as evidenced by RT-PCR. Furthermore, mRNA for choline transporter-like protein 1 (CTL1), and its splice variants CTL1a and CTL1b, was expressed in rat astrocytes, and the inhibition of CTL1 expression by RNA interference completely inhibited Na+-independent choline uptake. We conclude that rat astrocytes express an intermediate-affinity Na+-independent choline transport system. This system seems to occur through a CTL1 and is responsible for the uptake of choline and organic cations in these cells.     

Detection of choline transporter-like 1 protein CTL1 in neuroblastoma × glioma cells and in the CNS, and its role in choline uptake

Choline is an essential nutrient necessary for synthesis of membrane phospholipids, cell signalling molecules and acetylcholine. The aim of this study was to detect and characterize the choline transporter-like 1 (CTL1/SLC44A1) protein in CNS tissues and the hybrid neuroblastoma × glioma cell line NG108-15, which synthesizes acetylcholine and has high affinity choline transport but does not express the cholinergic high affinity choline transporter 1. The presence of CTL1 protein in NG108-15 cells was confirmed using our antibody G103 which recognizes the C-terminal domain of human CTL1. Three different cognate small interfering RNAs were used to decrease CTL1 mRNA in NG108-15 cells, causing lowered CTL1 protein expression, choline uptake and cell growth. None of the small interfering RNAs influenced carnitine transport, demonstrating the absence of major non-specific effects. In parental C6 cells knockdown of CTL1 also reduced high affinity choline transport. Our results support the concept that CTL1 protein is necessary for the high affinity choline transport which supplies choline for cell growth. The presence of CTL1 protein in rat and human CNS regions, where it is found in neuronal, glial and endothelial cells, suggests that malfunction of this transporter could have important implications in nervous system development and repair following injury, and in neurodegenerative diseases.     

Acid secretion by mitochondrion-rich cells of medaka (Oryzias latipes) acclimated to acidic freshwater

In the present study, medaka embryos were exposed to acidified freshwater (pH 5) to investigate the mechanism of acid secretion by mitochondrion-rich (MR) cells in embryonic skin. With double or triple in situ hybridization/immunocytochemistry, the Na(+)/H(+) exchanger 3 (NHE3) and H(+)-ATPase were localized in two distinct subtypes of MR cells. NHE3 was expressed in apical membranes of a major proportion of MR cells, whereas H(+)-ATPase was expressed in basolateral membranes of a much smaller proportion of MR cells. Gill mRNA levels of NHE3 and H(+)-ATPase and the two subtypes of MR cells in yolk sac skin were increased by acid acclimation; however, the mRNA level of NHE3 was remarkably higher than that of H(+)-ATPase. A scanning ion-selective electrode technique was used to measure H(+), Na(+), and NH(4)(+) transport by individual MR cells in larval skin. Results showed that Na(+) uptake and NH(4)(+) excretion by MR cells increased after acid acclimation. These findings suggested that the NHE3/Rh glycoprotein-mediated Na(+) uptake/NH(4)(+) excretion mechanism plays a critical role in acidic equivalent (H(+)/NH(4)(+)) excretion by MR cells of the freshwater medaka.     

Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

Synthesis of acetylcholine (ACh) by non‐neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na⁺‐dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non‐neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non‐neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter‐like proteins, a five gene family choline‐transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na⁺‐independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non‐neuronal cell lines and presents a mechanism to target non‐neuronal ACh synthesis without affecting neuronal ACh synthesis.     

Calcitonin receptor-stimulating peptide-1 regulates ion transport and growth of renal epithelial cell line LLC-PK1

Calcitonin receptor-stimulating peptide-1 (CRSP-1) is a peptide recently identified from porcine brain by monitoring the cAMP production through an endogenous calcitonin (CT) receptor in the renal epithelial cell line LLC-PK(1). Here we investigated the effects of CRSP-1 on the ion transport and growth of LLC-PK(1) cells. CRSP-1 inhibited the growth of LLC-PK(1) cells with a higher potency than porcine CT. CRSP-1 enhanced the uptake of (22)Na(+) into LLC-PK(1) cells more strongly than did CT and slightly reduced the (45)Ca(2+) uptake. The enhancement of the (22)Na(+) uptake was abolished by 5-(N-ethyl-N-isopropyl) amiloride, a strong Na(+)/H(+) exchanger (NHE) inhibitor for NHE1, even at a concentration of 1x10(-8)M, although other ion transporter inhibitors did not affect the (22)Na(+) uptake. These results indicate that CRSP-1 enhances the (22)Na(+) uptake by the specific activation of NHE1. Taken together, CRSP-1 is considered to be a new regulator for the urinary ion excretion and renal epithelial cell growth.     

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V_max values from 1,973 ± 118 to 380 ± 18 nmol·mg^–1·min^–1, when the binding affinity did not change significantly (K_m values 56 ± 8 and 53 ± 6 µM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages. protein trafficking     

Increased colonic sodium absorption in rats with chronic renal failure is partially mediated by AT1 receptor agonism

To test the hypothesis that colonic Na(+) transport is altered in the 5/6 nephrectomized rat model of chronic renal failure (CRF), we measured Na(+) fluxes across distal colon from control (CON), CRF, and CRF rats treated with the angiotensin II (ANG II) receptor antagonist losartan (+LOS). We also evaluated overall fluid and Na(+) balance and compared colonic protein and mRNA expression profiles for electroneutral [sodium-hydrogen exchanger (NHE)] and electrogenic Na(+) transport [epithelial sodium channel (ENaC)] in these groups. Consistent with a 60% enhancement in colonic Na(+) absorption in CRF, urinary Na(+) excretion increased by about 50% while serum Na(+) homeostasis was maintained. These CRF-induced changes in Na(+) handling were normalized by treatment with LOS. Net Na(+) absorption was also stimulated in in vitro tissues from CON rats following acute serosal addition of ANG II (10(-7) M), and this increase was blocked by AT(1) antagonism but not by an AT(2) antagonist. In CRF, colonic protein and mRNA expression variably increased for apical NHE2, NHE3, and ENaC alpha-, beta-, gamma-subunits, whereas expression of basolateral NHE1 and Na(+)-K(+)-ATPase (alpha-isoform) remained unaltered. Upregulation of the ENaC subunit mRNA was attenuated somewhat by LOS treatment. Previously, we showed that colonic AT(1) receptor protein is upregulated twofold in CRF, and here we find that AT(1) and AT(2) mRNA and AT(2) protein abundance is unchanged in CRF. We conclude that Na(+) absorption in CRF rat distal colon is increased due to elevated expression of proteins mediating electroneutral and electrogenic uptake and that it is partially mediated by AT(1) receptors.     

Coupling of angiotensin II AT1 receptors to neuronal NHE activity and carrier-mediated norepinephrine release in myocardial ischemia

In ischemia, cardiac sympathetic nerve endings (cSNE) release excessive amounts of norepinephrine (NE) via the nonexocytotic Na(+)-dependent NE transporter (NET). NET, normally responsible for NE reuptake into cSNE, reverses in myocardial ischemia, releasing pathological amounts of NE. This carrier-mediated NE release can be triggered by elevated intracellular Na(+) levels in the axoplasm. The fact that ischemia activates the intracellular pH regulatory Na(+)/H(+) exchanger (NHE) in cSNE is pivotal in increasing intraneuronal Na(+) and thus activating carrier-mediated NE release. Angiotensin (ANG) II levels are also significantly elevated in the ischemic heart. However, the effects of ANG II on cSNE, which express the ANG II receptor, AT(1)R, are poorly understood. We hypothesized that ANG II-induced AT(1)R activation in cSNE may be positively coupled to NHE activity and thereby facilitate the pathological release of NE associated with myocardial ischemia. We tested this hypothesis in a cSNE model, human neuroblastoma cells stably transfected with rat recombinant AT(1A) receptor (SH-SY5Y-AT(1A)). SH-SY5Y-AT(1A) constitutively expresses amiloride-sensitive NHE and the NET. NHE activity was assayed in BCECF-loaded SH-SY5Y-AT(1A) as the rate of the Na(+)-dependent alkalinization in response to an acute acidosis. ANG II activation of AT(1)R markedly increased NHE activity in SH-SY5Y-AT(1A) via a Ca(2+)-dependent pathway and promoted carrier-mediated NE release. In addition, in guinea pig cSNE expressing native AT(1)R, ANG II elicited carrier-mediated NE release. In SH-SY5Y-AT(1A) and cSNE, amiloride inhibited the ANG II-mediated release of NE. Our results provide a link between AT(1)R and NHE in cSNE, which can exacerbate carrier-mediated NE release during protracted myocardial ischemia.     

Transport and toxic mechanism for aluminum citrate in human neuroblastoma SH-SY5Y cells

Aluminum (Al) is thought to be a risk factor for neurodegenerative disorders, but the molecular mechanism has been not clarified yet. In this study, we examined how a transport system handled transport of Al citrate, the major Al species in brain, and effect of Al citrate treatment on expression of the transporter and on susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells. Uptake of Al citrate by the cells was temperature- and concentration-dependent, and inwardly-directed Na(+)-gradient-independent. Simultaneous application and preloading of L-cystine or L-glutamate inhibited and stimulated, respectively, the Al citrate uptake by SH-SY5Y cells, demonstrating kinetically that Na(+)-independent L-cystine/L-glutamate exchanger, system Xc(-), is involved in its uptake. When the cells were treated with Al citrate, but not citrate, for 2 weeks, but not a day, the expression of the transporter was decreased. Although the cell viability and glutathione content of the cells were not altered by the treatment with Al citrate alone, the number of dead cells among the Al citrate-treated cells increased on exposure to oxidative stress caused by a glucose deprivation/reperfusion treatment. These findings demonstrate that Al citrate is a substrate for system Xc(-), and that chronic treatment with Al citrate causes downregulation of the transporter and increases the vulnerability of the cells to oxidative stress without a direct effect on the viability or GSH content.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

TRPC1-mediated inhibition of 1-methyl-4-phenylpyridinium ion neurotoxicity in human SH-SY5Y neuroblastoma cells

Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca(2+) channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+)) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP(+) showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP(+). MPP(+)-induced alpha-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP(+) was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP(+) neurotoxicity, which was partially dependent on external Ca(2+). Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP(+) treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson's disease-inducing agents.     

Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni

A cDNA encoding a high-affinity Na(+)-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na(+)-dependent [(3)H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [(3)H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [(3)H]-choline in the absence of Na(+). The Na(+)-dependent component of [(3)H]-choline uptake by TrnCHT-infected cells was saturable with a K(m) for choline transport of 8.4 microM. Several compounds reported to be potent blockers of [(3)H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (K(i)=4.1 microM) and two oxoquinuclidium analogues of choline, quireston-A (K(i) approximately 10 microM) and quireston (K(i) approximately 100 microM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na(+)-independent uptake of [(3)H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.     

Choline transport for phospholipid synthesis

Choline is an essential nutrient for all cells because it plays a role in the synthesis of the membrane phospholipid components of the cell membranes, as a methyl-group donor in methionine metabolism as well as in the synthesis of the neurotransmitter acetylcholine. Choline deficiency affects the expression of genes involved in cell proliferation, differentiation, and apoptosis, and it has been associated with liver dysfunction and cancer. Abnormal choline transport and metabolism have been implicated in a number of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. Therefore, the study of choline transport and the characteristics of choline transporters are of central importance to understanding the mechanisms that underlie membrane integrity and cell signaling in such disorders. Kinetic studies with radiolabeled choline and inhibitors distinguish three systems for choline transport: (i) low-affinity facilitated diffusion, (ii) high-affinity, Na+-dependent transport, and (iii) intermediate-affinity, Na+-independent transport. It is only recently, however, that the proteins having transport characteristics of at least one of these systems have been identified. They include (i) polyspecific organic cation transporters (OCTs) with low affinity for choline, (ii) high-affinity choline transporters (CHT1s), and (iii) intermediate-affinity choline transporter-like (CTL1) proteins. CHT1 and CTL1 but not OCT transporters are selectively inhibited with hemicholinium-3 and essentially display characteristics of specialized transporters for targeted choline metabolism. CHT1 is abundant in neurons and almost exclusively supplies choline for acetyl-choline synthesis. The focus here is more on newly-discovered CTL1 choline transporters. They are expressed in different organisms and cell types, apparently not for the biosynthesis of acetylcholine but for the production of the most abundant metabolite of choline, the membrane lipid phosphatidylcholine.     

Compared Effects of Two Vesicular Acetylcholine Uptake Blockers, AH5183 and Cetiedil, on Cholinergic Functions in Torpedo Synaptosomes: Acetylcholine Synthesis, Choline Transport, Vesicular Uptake, and Evoked Acetylcholine Release

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.