The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.     

Involvement of reduced acetylcholine release in Delta9-tetrahydrocannabinol-induced impairment of spatial memory in the 8-arm radial maze

To clarify the mechanism by which Delta9-tetrahydrocannabinol, a major psychoactive component of marijuana, impairs spatial memory in the 8-arm radial maze in rats via the cholinergic system, we used two acetylcholinesterase inhibitors, physostigmine and tetrahydroaminoacridine. Moreover, we examined the effect of Delta9-tetrahydrocannabinol on acetylcholine release in the frontal cortex and dorsal and ventral hippocampus using in vivo microdialysis. Physostigmine (0.01-0.05 mg/kg, i.p.) and tetrahydroaminoacridine (1-5 mg/kg, p.o.) improved the impairment of spatial memory induced by Delta9-tetrahydrocannabinol (6 mg/kg, i.p.) in the 8-arm radial maze. Delta9-tetrahydrocannabinol (6 mg/kg, i.p.) produced a significant decrease in acetylcholine release in the dorsal hippocampus as assessed by microdialysis. Moreover, tetrahydroaminoacridine at a dose of 1 mg/kg, which improved the impairment of spatial memory, reversed the decrease in acetylcholine release induced by Delta9-tetrahydrocannabinol in the dorsal hippocampus during 60-120 min after the Delta9-tetrahydrocannabinol injection. These findings suggest that inhibition of the cholinergic pathway by reduced acetylcholine release is one of the means by which Delta9-tetrahydrocannabinol impairs spatial memory in the 8-arm radial maze.     

Conformational aspects of the biological effect of functional fragments of the p21ras oncoprotein family. 2. Fragment 1-16, various sequences]

The conformational analysis data on active ([Val12-Gly13], [Asp12-Gly13] and [Gly12-Asp13]) and passive ([Gly12-Gly13] and [Pro12-Gly13]) modifications of the p21ras family oncoproteins are presented. The activating amino acid substitutions are shown to be accompanied by essential changes in the secondary structure, resulted in the 9-16 fragment spiralization. The spatial structure of the 1-9 fragment does not vary for all the predominant forms of the active and passive analogues. The results of the conformational analysis have been used for studying the structural-functional relationships.     

Proteolysis of semax analogues with different N-terminal amino acids by aminopeptidases

Proteolysis of semax (Met-Glu-His-Phe-Pro-Gly-Pro, Sem) and its analogues ([Ala1]Sem, [Gly1]Sem, [Thr1]Sem, [Trp1]Sem) that are differ from semax in substitution of N-terminal Met residue were studied. It is shown that such replacement changes the rate of peptides degradation by N-aminopeptidases (EC 3.4.11.2, Sigma, Type VI, 9.2 units. Akt. / mg). [Ala1]Sem, [Gly1]Sem and [Thr1]Sem semax analogues proved to be more stable to proteolysis than semax (Sem), and their initial product of proteolysis is His-Phe-Pro-Gly-Pro (Sem-5). For triptophan analogue both Glu-His-Phe-Pro-Gly-Pro (Sem-6) and Sem-5 product are formed in similar quantities. It is found that all investigated analogues can be used as inhibitors in Sem proteolysis.     

CCK-8 Inhibits Acute Morphine-induced Spatial Reference Memory Impairment in Mice

Administration of morphine may impair learning and memory processes. Cholecystokinin has been reported to be involved in various types of memory, and our previous study found that Cholecystokinin octapeptide attenuates spatial memory impairment in chronic morphine-treated mice. However, the effect of CCK-8 on acute morphine-induced memory impairment is not clear. In this study, effect of acute CCK-8 and morphine on spatial reference memory was evaluated using Morris water maze in KM mice. Acetylcholine (Ach) content was measured using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). Pre-training with morphine (5, 10 mg/kg, i.p.) significantly impaired spatial reference memory acquisition without disturbing the performance in the visible platform task, while pre-test morphine has no effect on memory retrieval. Pre-training (0.01, 0.1 and 1 μg, i.c.v.) or pre-test (0.1 and 1 μg, i.c.v.) of CCK-8 facilitated spatial reference memory acquisition and retrieval, respectively. CCK-8 (0.1 and 1 μg) significantly attenuated memory loss by pre-training morphine. Furthermore, CCK-8 (1 μg, i.c.v) increased acetylcholine contents of hippocampus in saline or morphine-treated mice. Our study identifies CCK-8 reversed spatial reference memory loss induced by acute morphine, and the mnemonic effect could be related to the facilitation of CCK-8 on memory acquisition and retrieval through accelerating acetylcholine release in hippocampus.     

Spatial working memory in rats: the effect of the dipeptide gamma-L-glutamyl-taurine and haloperidol

To assess the possible involvement of the dipeptide gamma-L-glutamyl-taurine (Litoralon) and some of its analogues in the maintenance of spatial working memory, rats were treated with the dipeptides immediately or 2 hours after completing the first four choices in an 8 arm radial maze, or 3 hours before the test to exclude proactive effects of the compounds. Treatment with Litoralon, SZJ 3381 and 3361 at doses of 100 and 500 micrograms/kg (i.p.) did not impair spatial memory in rats, regardless of when these substances were injected during the session. By contrast, haloperidol (500 micrograms/kg, i.p.) treatment resulted in a dramatic decrease of performance.     

Dehydroevodiamine.HCl prevents impairment of learning and memory and neuronal loss in rat models of cognitive disturbance

We previously reported that dehydroevodiamine.HCl (DHED) has anticholinesterase and antiamnesic activities. To verify the effects of DHED on cognitive deficits further, we tested it on the scopolamine-induced amnesia model of the rat using the passive avoidance and eight-arm radial maze tests. A single (20 mg/kg p.o.) and repeated (10 mg/kg p.o.) administrations of DHED could significantly reverse the latency time shortened by scopolamine (1 mg/kg i.p.) to control level. The impaired spatial working memory induced by scopolamine (1 mg/kg i.p.) was also improved significantly by a single injection (6.25 mg/kg i.p.) and repeated administrations of DHED (10 mg/kg p.o.) in the eight-arm radial maze test. In addition, we examined the effects of DHED on the memory impairment and the histological changes of the brain after unilateral electrolytic lesion of the entorhinal cortex (EC) and middle cerebral artery occlusion in rats. The cognitive deficits caused by EC lesion and middle cerebral artery occlusion were improved significantly by repeated administrations of DHED (6.25 mg/kg i.p.) after EC lesion or ischemic insult once a day for 7 days in the passive avoidance test. Histological analysis showed that the neuronal loss in the DHED-treated group was notably reduced in the hippocampal area (CA1) of ischemic rats and in the dentate gyrus and hippocampal area (CA1 and CA3) of EC-lesioned rats compared with the nontreated group. The infarction area was decreased significantly by a single administration of DHED (6.25 mg/kg i.p.) 30 min before ischemic insult for 6 h. These results suggest that DHED might be an effective drug for not only the Alzheimer's disease type, but also the vascular type of dementia.     

Quetiapine attenuates spatial memory impairment and hippocampal neurodegeneration induced by bilateral common carotid artery occlusion in mice

Quetiapine, a new atypical antipsychotic drug, has beneficial effects on cognitive impairment and neuropathological changes in treating chronic neurodegenerative diseases. Our previous studies have demonstrated that quetiapine may have neuroprotective properties. In the present study, we investigated the effects of a 2-week pre-administration of quetiapine (10 mg/kg/day, i.p.) on spatial memory impairment and hippocampal neurodegeneration induced by 60-minute bilateral common carotid artery occlusion (CCAO). Following a 7-day recovery phase from CCAO, the spatial memory of the mice was tested using a modified water maze test. After the behavioural test, the mice were sacrificed and brain sections were stained with NeuN (a neuron-specific soluble nuclear antigen), cresyl violet (Nissl), and Fluoro-Jade B. CCAO significantly induced spatial memory impairment and caused neurodegeneration in the hilus of hippocampus, while quetiapine significantly attenuated these changes. This is the first study showing that quetiapine significantly attenuates CCAO-induced spatial memory impairment and this improvement parallels the alleviative effects of quetiapine on CCAO-induced neurodegeneration in the hilus of hippocampus. The results suggest that quetiapine may have defending effects on the impairments induced by cerebral ischemia, which enhances our understanding about the mechanisms of quetiapine.     

Induction of cognitive impairment by scopolamine and noncholinergic agents in rhesus monkeys.

In primates, treatment with scopolamine impairs performance of a spatial delayed response task in a way which mimics deficits seen spontaneously in aged primates and demented patients. Despite their efficacy in reversing scopolamine induced disruption, the effects of cholinergic agonists on cognition in aged primates and dements are unimpressive, suggesting that other neurotransmitter systems are also involved in this type of deficit. We have induced a scopolamine-like impairment of spatial delayed response performance in rhesus monkeys using phencyclidine (0.1-0.2 mg/kg i.m.), lorazepam (0.4-0.6 mg/kg s.c.) or tetrahydrocannabinol (1-4 mg/kg p.o.), but not amphetamine (0.1-0.4 mg/kg i.m.), yohimbine (0.1-1.0 mg/kg i.m.) or morphine (2-4 mg/kg i.m.). Our findings suggest that disruption of specific neurotransmitter systems other than acetylcholine may contribute importantly to cognitive decline in aging and dementia.     

The influence of NMDA receptor agonist and antagonist on morphine state-dependent memory of passive avoidance in mice

The measurement of step-down latency in passive avoidance has been used to study memory in laboratory animals. The pre-training injection of 5 mg/kg morphine impaired memory, which was restored when 24 h later the same dose of the drug was administered. To explore the possible involvement of NMDA modulators on morphine-induced memory impairment, we have investigated the effects of intracerebroventricular (i.c.v.) administration of NMDA and the competitive NMDA antagonist, DL-AP5, on morphine-induced memory impairment or recall, on the test day. Morphine (5 mg/kg, s.c.) was administered 30 min before training to induce impairment of memory and 24 h later, 30 min before test to improve it. Pre-test administration of NMDA (0.00001, 0.0001 and 0.001 microg/mouse, i.c.v.) did not alter the retention latency compared to the saline-treated animals. But restored the memory impairment induced by pre-training morphine (5 mg/kg, s.c.). Pre-test administration of DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) by itself decreased the retention latencies. The same doses of DL-AP5 increased pre-training morphine-induced memory impairment. Co-administration of NMDA (0.0001 and 0.001 microg/mouse, i.c.v.) and morphine (5 mg/kg, s.c.) on the test day increased morphine memory improvement. Conversely, DL-AP5 (1, 3.2 and 10 microg/mouse, i.c.v.) inhibited morphine-induced memory recall. It is concluded that NMDA receptors may be involved, at least in part, in morphine state-dependent learning in mice.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Antagonists of oxytocin featuring replacement with modified beta-mercaptopropionic acids at position 1.

Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O. Since the more hydrophilic NH and C=O substitutions showed a sharply decreased antagonistic potency (rat uterotonic test in vitro), additional modifications were made to reduce their hydrophilicity. Acylation of the NH group with various acyl groups, and ketalization or thioketalization of C=O with more or less bulky substituents led to a partial restoration of potency, the N-carbamyl- and the 2-mercapto-2-adamantaneacetyl analogues being equipotent with PA. Internal cyclization by amidation of the NH-group with Gly-9, resulted in a bicyclic analogue, (cyclo 1-9)[(HN)Pmp1, Gly9]PA which was equipotent with PA. When Pen-6 was introduced into the bicyclic derivative instead of Cys-6, to reduce the flexibility of the rings, the resulting (cyclo 1-9)[(HN)Pmp1, Pen6, Gly9]PA had somewhat better potency (pA2 = 8.17) in the uterotonic test and no detectable activity in the antidiuretic assay. In the case of substitution of PA with beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, (S)Pmp, there was also an increase in inhibitory potency in the uterotonic test (pA2 = 8.08): the analogue had extremely weak antidiuretic activity. To establish the importance of the steric effects of the Pen-6 substitution, analogues [Pen6]PA and [(S)Pmp1, Pen6]PA were made and found to be very potent, with a pA2 of 8.72 and 8.86, respectively. The high potency of the latter analogue and its extremely weak action in the diuretic assay makes it an attractive candidate for studies on the inhibition of the biological effects of oxytocin and for the prevention of preterm labour.     

Synthesis and study of cyclic and linear analogs of neurotensin

New cyclic analogues of neurotensin (NT): [cyclo (13----8), Gly8]NT-(8-13), [cyclo (13----7), Gly7]NT-(7-13), [cyclo (13----5 epsilon), Lys5]NT-(5-13), [cyclo (13----4 epsilon), Lys4]NT-(4-13), and their linear precursors have been synthesized. The latter (protected linear compounds) were prepared by solid-phase peptide synthesis, and cyclization was attained by using diphenylphosphoryl azide. Cyclization of C-terminal hexa- and octapeptide fragments of NT was found to lead to cycloanalogues possessing high depressor activity. As judged by CD spectral data in aqueous solution, the cyclohexapeptide analogue has a relatively rigid conformation different from its linear counter-part and the NT-(9-13) fragment, whereas NT, its cyclohepta- and cyclononapeptides have random structure.     

Interleukin-1 beta analogues with markedly reduced pyrogenic activity can stimulate secretion of adrenocorticotropic hormone in rats

We examined the adrenocorticotropic hormone-releasing activities of several human interleukin-1 beta analogues that have markedly reduced pyrogenic activities in rats. Among the analogues tested, [Gly4]-, [Leu93]- and [1-148]-interleukin-1 beta increased the plasma adrenocorticotropic hormone level to almost that induced by authentic human interleukin-1 beta. Modifications of the N-terminus of the authentic molecule, i.e., [7-153]- and [Des-Ala1, Asp4]-interleukin-1 beta, significantly reduced the hormone-releasing activity. These data suggest that the adrenocorticotropic hormone-releasing activity of human interleukin-1 beta resides in the N-terminal structure of the authentic peptide and can be separated from its pyrogenic activity.     

The effect of amino acid substitutions on the 3-dimensional structure of fragment 1--16 of the oncoprotein p21 ras

Using the method of theoretical conformational analysis, spatial structure of fragment 1-16 of active [( Val12-Gly13], [Asp12-Gly13], [Gly12-Asp13]) and passive [( Gly12-Gly13] and [Pro12-Gly13]) modifications of oncoproteins family p21 ras have been investigated. The activation of these proteins has been shown to be accompanied by reorganization of three-dimensional structure of the polypeptide chain.     

Neuroprotective effect and brain receptor binding of taltirelin,a novel thyrotropin-releasing hormone (TRH) analogue,in transient forebrain ischemia of C57BL/6J mice

Thyrotropin-releasing hormone (TRH) and some of its stable analogues have been shown to improve neurologic dysfunctions such as brain trauma in both animals and humans. Our previous study revealed that taltirelin, a novel orally active TRH analogue, binds to rat brain TRH receptors in vivo. The present study was undertaken to investigate whether taltirelin has neuroprotective effects in transient brain ischemia of C57BL/6J mice induced by bilateral carotid artery occlusion (2VO). Neuronal cell density in the hippocampal CA1 region of C57BL/6J mice was significantly (39.9%) decreased 1 week after 2VO-reperfusion, compared to the case of the sham group, and this reduction of hippocampal neuronal density was significantly suppressed by an intravenous (i.v.) injection of taltirelin (0.3 mg/kg). The i.v. injection of taltirelin at this dosage produced a significant increase in the dissociation constant (Kd) of specific [3H]MeTRH binding in sham and 2VO-reperfusion groups (33.6 and 51.4%, respectively) compared with the vehicle-treated group. These results indicate that the intravenously injected taltirelin bound to TRH receptors in the ischemic brain. There was little difference in the brain-to-plasma concentration ratio (Kp) of [14C]sucrose between the sham and 2VO groups of C57BL/6J mice, indicating that the tight junction of the blood-brain barrier may be intact in the ischemic brain.In conclusion, the study has shown that taltirelin may have a significant neuroprotective effect on the ischemic brain.     

Evidence that the hypothermic response of mice to delta-9-tetrahydrocannabinol is not mediated by changes in thermogenesis in brown adipose tissue.

Delta 9-Tetrahydrocannabinol (20 mg/kg i.p.) and propranolol (20 and 50 mg/kg i.p.) produced marked falls in the rectal temperatures of mice kept at an ambient temperature of 22 degrees C. Propranolol (50 mg/kg i.p.) also decreased the thermogenic activity of brown fat, as measured by a decrease in the level of [3H]GDP binding to mitochondria obtained from mouse interscapular brown adipose tissue. In contrast, delta 9-tetrahydrocannabinol (20 mg/kg i.p.) did not affect mitochondrial GDP binding even though the dose used was one shown previously to depress heat production. GDP binding was also unaffected by this cannabinoid in brown adipose tissue taken from mice that had been kept at 13 degrees C instead of 22 degrees C. In mice kept at 34 degrees C, isoprenaline (0.25 and 1.0 mg/kg s.c.) induced a marked rise in rectal temperature and increased the level of GDP binding to brown fat mitochondria. Propranolol (50 mg/kg i.p.) prevented the hyperthermic response to isoprenaline, the mice becoming hypothermic instead. Delta 9-Tetrahydrocannabinol (20 mg/kg i.p.) had no effect on isoprenaline-induced hyperthermia. We conclude from these data that there is no significant involvement of brown adipose tissue in the hypothermic response of mice to delta 9-tetrahydrocannabinol.     

The effects of DHEA, 3β-hydroxy-5α-androstane-6,17-dione, and 7-amino-DHEA analogues on short term and long term memory in the mouse

Neurosteroids have been reported to modulate memory processes in rodents. Three analogues of dehydroepiandrosterone (DHEA), two of them previously described (7β-aminoDHEA and 7β-amino-17-ethylenedioxy-DHEA), and a new one (3β-hydroxy-5α-androstane-6,17-dione) were synthesized, and their effects were evaluated on memory. This study examined their effects on long term and short term memory in male (6 weeks old) NMRI mice in comparison with the reference drug. Long term memory was assessed using the passive avoidance task and short term memory (spatial working memory) using the spontaneous alternation task in a Y maze. Moreover, the effects of DHEA and its analogues on spontaneous locomotion were measured. In all tests, DHEA and analogues were injected at three equimolar doses (0.300–1.350–6.075 μM/kg). DHEA and its three analogues administered immediately post-training at the highest doses (6.075 μM/kg, s.c.) improved retention in passive avoidance test. Without effect per se in the spatial working memory task, the four compounds failed to reverse scopolamine (1 mg/kg, i.p.)-induced deficit in spontaneous alternation. These data suggested an action of DHEA and analogues in consolidation of long term memory particularly when emotional components are implied. Moreover, data indicated that pharmacological modulation of DHEA as performed in this study provides derivatives giving the same mnemonic profile than reference molecule.     

Effects of novel 5-HT1A receptor antagonists on measures of postsynaptic 5-HT1A receptor activation in vivo

The effects of two putative 5-HT_1A antagonists, 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-iodobenzamido]ethyl]piperazine (p-MPPI) and 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-flourobenzamido]ethyl]piperazine (p-MPPF), were examined in vivo in two tests of postsynaptic 5-HT_1A receptor activation, hypothermia and reciprocal forepaw treading, in the rat. Both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (10 mg/Kg, I.p.) antagonized the hypothermia induced by the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (0.5 mg/Kg, S.c.). Neither p-MPPI nor p-MPPF administered alone at a dose of 10 mg/kg (i.p.) induced hypothermia. Similarly, both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (2.5 mg/Kg, I.p.) completely antagonized 8-OH-DPAT (2 mg/Kg, S.c.)-induced forepaw treading in rats pretreated with reserpine (1 mg/Kg, S.c., 18–24 hours prior to the experiment). p-MPPI and p-MPPF, at doses of 10 mg/kg (i.p.) did not induce forepaw treading in reserpine pretreated animals. The results of the present study demonstrate that p-MPPI and p-MPPF act as 5-HT_1A receptor antagonists in these measures of postsynaptic 5-HT_1A a receptor activation.     

Binding and Active Transport of Large Analogues of Acetylcholine by Cholinergic Synaptic Vesicles In Vitro

A previous structure-activity investigation of acetylcholine (ACh) revealed a positive correlation between additional hydrophobic bulk and increased potency for inhibition of active transport of [3H]ACh by synaptic vesicles isolated from the electric organ of Torpedo. In the current study, several ACh analogues that are significantly larger than previously studied "false transmitters" were synthesized in the tritiated form by chemical means and tested for active transport. These are analogue 14 [(+/-)-(cis,trans)-1-benzyl-1-methyl-3-acetoxypyrrolidinium iodide], analogue 15 [(+/-)-1,1-dimethyl-3-benzoyloxypyrrolidinium iodide], and analogue 16/17 [(+/-)-(cis,trans)-1-benzyl-1-methyl-3-benzoyloxypyrrolidinium iodide]. These analogues place significant additional hydrophobic bulk on one or the other (analogues 14 and 15) or both (analogue 16/17) of the two pharmacophores of a small, conformationally constrained analogue of ACh. [3H]Analogue 14 and [3H]analogue 15 are actively transported, with Vmax values the same as or less than that of ACh, depending on the vesicle preparation. The observation that Vmax is the same for an analogue and ACh in some vesicle preparations suggests that the rate-limiting step does not involve ACh bound to the transporter. [3H]Analogue 16/17 is actively transported very poorly. Km values for ACh and for transported ACh analogues vary by up to two- to threefold in different vesicle preparations. The ACh transporter is much less selective for transported substrates than anticipated.