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B Collier

Publications and source records attributed to B Collier.

At least 19 recordsLinked to original sources
PubMed1999

Enzyme activity and protein of multiple forms of choline acetyltransferase: effects of calyculin A and okadaic acid.

Choline acetyltransferase (ChAT) appears to exist in multiple forms, three of which can be isolated biochemically as cytosolic (cChAT), ionically-membrane bound (ibChAT) and non-ionic membranous (mChAT). In this study, we first examined whether the quantitative distribution of enzyme protein and enzyme activity was the same. Enzyme activity and ChAT protein distributed similarly: the majority of ChAT activity and protein were found in cChAT followed by mChAT and least activity and amount were in ibChAT. Our second objective was to investigate the effects of calyculin A or okadaic acid on the subcellular distribution of ChAT activity and amount from rat hippocampal formation. Calyculin A and okadaic acid decreased significantly (p < 0.01) cytosolic and membranous ChAT activity; ionically-bound ChAT was not significantly (p > 0.67) different from control. Removal of calyculin A or okadaic acid restored cytosolic ChAT activity (p > 0.9 as compared to control), but not membranous enzyme activity (p < 0.05 as compared to control). The immunoreactive cytosolic ChAT was reduced significantly (p < 0.01) by calyculin A and okadaic acid. Enzyme amount of membranous ChAT was decreased significantly by calyculin A (p < 0.01) and okadaic acid (p < 0.001). Enzyme amount of ionically-bound ChAT was not changed (p > 0.99) by either of these two phosphatase inhibitors. This investigation demonstrates that alterations in ChAT activity of each subfraction parallel changes in enzyme amounts in the same fractions.

Animals↗
PubMed1999

Effects of calyculin A and okadaic acid on acetylcholine release and subcellular distribution in rat hippocampal formation.

The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P3 fraction, but cytoplasmic ACh contained in the S3 fraction was not significantly affected. The P3 fraction is known to be heterogeneous; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K+-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [3H]ACh from [3H]choline, the ratio of [3H]ACh in the S3 to P3 fractions was not much changed by exposure of tissue to calyculin A or okadaic acid; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.

Acetylcholine↗
PubMed1998

Translational inhibition in vitro of human papillomavirus type 16 L2 mRNA mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2.

Human papillomavirus (HPV) type 16 belongs to the group of "high risk" HPV types that are frequently detected in anogenital cancers. The expression of HPV-16 late genes encoding the virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We have previously identified negative elements in the 3' end of L2 RNA that act in cis to reduce mRNA utilization without substantially affecting mRNA levels. The experiments reported here demonstrate the interaction of cellular proteins with an inhibitory sequence present in the coding region of the L2 mRNA. Using RNA gel shift assays and UV cross-linking, we have detected three cellular proteins interacting specifically with the sense strand of the L2 mRNA, two of which were identified as heterogeneous ribonucleoprotein K (hnRNP K) and the poly(rC) binding- protein (PCBP). Recombinant hnRNP K, PCBP-1, and PCBP-2 that were over expressed in bacteria and partially purified bound to the HPV-16 L2 mRNA in a sequence-specific manner. Interestingly, PCBP-1, PCBP-2, and hnRNP K specifically and efficiently inhibited translation of the HPV-16 L2 mRNA in vitro. Therefore, these proteins may play an important role in the regulation of HPV-16 late gene expression and virus production in vivo.

Base Sequence↗
PubMed1998

Synaptic inhibitory effects of edrophonium on sympathetic ganglionic transmission.

PURPOSE: To evaluate the effect of edrophonium on synaptic transmission in the superior cervical ganglion. METHODS: In anaesthetized rats the effect of edrophonium on synaptic transmission was studied in vitro by testing whether it blocks the compound action potential recorded from postganglionic fibres evoked by stimulation of preganglionic axons. The superior cervical ganglion was excised and the cervical sympathetic trunk and internal carotid nerve were used for stimulating and recording, respectively. Drugs superfused included edrophonium (0.1-500 microM), neostigmine (0.1-10 microM), and muscarinic M1 and M2 antagonists pirenzepine and AFDX-116 (200 nM-10 microM), respectively. To evaluate a presynaptic action, the effect of edrophonium on basal and high-K+ (35 mM) evoked release of [3H]ACh from the superior cervical ganglion was studied in vitro. To evaluate a postsynaptic action, edrophonium's effect on postganglionic nerve discharge in response to arterial injection of ACh (100 micrograms) into the superior cervical ganglion was determined in vivo. RESULTS: Edrophonium (10-500 microM) decreased the compound action potential amplitude (ED50 163.5 microM). A decrease was not produced by neostigmine, nor was it reversed by pirenzepine or AFDX-116. Edrophonium blocked postganglionic cell firing in response to exogenously administered ACh. Although edrophonium did not affect basal or high-K+ evoked ACh release, when the evoked increase was calculated as a multiple of the basal release, it caused approximately a 30% (P < 0.005) reduction. CONCLUSIONS: Edrophonium blocks ganglionic cholinergic transmission postsynaptically and, possibly, presynaptically. The mechanism(s) by which this occurs does not appear to involve inhibition of cholinesterase, or activation of M1 or M2 receptor subtypes.

Acetylcholinesterase↗
PubMed1998

Amyloid beta-peptide inhibits high-affinity choline uptake and acetylcholine release in rat hippocampal slices.

The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid beta-peptide (A beta) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that A beta accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which A beta peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that A beta-related peptides, under acute conditions, potently inhibit K+-evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized A beta-mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. A beta(1-40) (10(-8) M) potently inhibited veratridine-evoked endogenous ACh release from rat hippocampal slices and also decreased the K+-evoked release potentiated by the nitric oxide-generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of A beta(1-40). The activity of the enzyme ChAT was not altered by A beta peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various A beta peptides (10(-14) to 10(-6) M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of A beta (10(-6) M). Taken together, these results indicate that A beta peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that A beta peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to A beta peptides.

Acetylcholine↗
PubMed1997

Bradycardia produced by pyridostigmine and physostigmine.

PURPOSE: The bradycardia produced by pyridostigmine and physostigmine in an animal model of acute cardiac denervation was examined according to its relation to cholinesterase inhibition and sensitivity to block by cholinergic receptor antagonists. METHODS: Cats were anaesthetised, vagotomised and propranolol-treated. Heart rate was continuously recorded. Erythrocyte cholinesterase activity of arterial blood was measured using a radiometric technique. Nicotinic and muscarinic M1 receptors were blocked with hexamethonium and pirenzepine, respectively. M2 receptors were blocked with gallamine, pancuronium and AFDX-116. RESULTS: With pyridostigmine and physostigmine the dose-response relationship for the decrease in heart rate (ED50 1.05 +/- 0.25 and 0.198 +/- 0.03 mg.kg-1, respectively) was shifted to the right of that for the inhibition of cholinesterase activity (ED50 0.094 +/- 0.03 and 0.032 +/- 0.01 mg.kg-1, respectively). The decrease in cholinesterase activity reached a plateau at a cumulative dose of 0.56 +/- 0.08 and 0.32 +/- 0.08 mg.kg-1, respectively. In contrast, there did not appear to be a plateau in the bradycardic effect. The bradycardia produced by pyridostigmine and physostigmine was blocked by hexamethonium (ED50 10 +/- 1.3 and 15.3 +/- 2.4 mg.kg-1, respectively), pirenzepine (ED50 68 +/- 16 and 138 +/- 32 micrograms.kg-1, respectively), gallamine (56 +/- 11 and 67 +/- 17 micrograms.kg-1, respectively), pancuronium (32 +/- 10 and 30 +/- 4 micrograms.kg-1, respectively), and AFDX-116 (31 +/- 4 and 28 +/- 4 micrograms.kg-1, respectively). CONCLUSION: The bradycardia produced by reversible anticholinesterase drugs containing a carbamyl group is not clearly related to the degree of cholinesterase activity, and has a low sensitivity to nicotinic and muscarinic M1 and a high sensitivity to muscarinic M2 receptor antagonists.

Animals↗
PubMed1996

GRBase, a database linking information on proteins involved in gene regulation.

The Gene Regulation Database (GRBase) is a compendium of information on the structure and function of proteins involved in the control of gene expression in eukaryotes. These proteins include transcription factors, proteins involved in signal transduction, and receptors. GRBase is now accessible via the World Wide Web (http://www.access.digex.net/regulate). A key feature of this database is the linking of each entry to data in other databases. The database is also available by anonymous ftp (URL ftp://ftp.trevigen.com/pub/Tfactors/) in both text and Filemaker pro formats.

Computer Communication Networks↗
PubMed1996

Different properties of the bradycardia produced by neostigmine and edrophonium in the cat.

PURPOSE: The bradycardia produced by neostigmine and edrophonium was examined according to its relation to cholinesterase inhibition and to its sensitivity to block by muscarinic receptor antagonists. For comparison, the ability of muscarinic antagonists to block the bradycardia produced by electrical stimulation of the vagus nerve was determined. METHODS: Cats were anaesthetized, vagotomized and propranolol-treated. Heart rate was continuously recorded. Erythrocyte cholinesterase activity of arterial blood was measured using a radiometric technique. The right vagus nerve was isolated for electrical stimulation. The muscarinic antagonists used were atropine, glycopyrrolate, pancuronium, gallamine, and AFDX-116. RESULTS: Neostigmine produced a dose-dependent decrease in cholinesterase activity which reached a plateau at a cumulative dose of 0.16 mg.kg-1 (ED50 0.009 +/- 0.003 mg.kg-1). Neostigmine produced a dose-dependent decrease in heart rate with the dose-response relationship (ED50 0.1 +/- 0.01 mg.kg-1; P = 0.0006) shifted to the right of that for the inhibition of cholinesterase activity. In contrast to the anticholinesterase effect, the bradycardic effect did not reach a plateau and continued to increase even at doses at which the cholinesterase inhibition was maximal. The maximal decrease in heart rate when the heart was still in sinus rhythm was by 81 +/- 13 bpm (49 +/- 7% of baseline), which was produced by a dose of 0.32 mg.kg-1. Edrophonium produced dose-dependent decreases in cholinesterase activity and heart rate, which were highly correlated (correlation coefficient r = 0.99, P < 0.0001). The ED50 of the reduction in heart rate (0.9 +/- 0.18 mg.kg-1) and cholinesterase activity (0.89 +/- 0.12 mg.kg-1) produced by edrophonium were similar. Moreover, the reduction in heart rate and cholinesterase activity produced by edrophonium reached a plateau at the same dose (6.4 mg.kg-1). At this dose, heart rate decreased by 22 +/- 2 bpm (14.6 +/- 0.9% of baseline). Compared to the bradycardia produced by stimulation of the vagus nerve, that produced by neostigmine was blocked by muscarinic antagonists at significantly lower doses while that produced by edrophonium was blocked at similar doses. CONCLUSIONS: The neostigmine-induced bradycardia is poorly correlated with cholinesterase inhibition compared to that produced by edrophonium, and has a higher sensitivity to muscarinic receptor antagonists compared to that produced by edrophonium or vagus nerve stimulation. These results are consistent with the hypothesis that the neostigmine-induced bradycardia is, in part, the result of neostigmine directly activating cholinergic receptors within the cardiac parasympathetic pathway. The bradycardia produced by edrophonium may be accounted for solely by an anticholinesterase action.

Acetylcholine↗
PubMed1996

Vasoactive intestinal peptide and impotence in experimental diabetes mellitus.

OBJECTIVE: To determine whether a defect in vasoactive intestinal peptide (VIP)-mediated vasodilatation underlies diabetic impotence. MATERIALS AND METHODS: Rats treated with streptozotocin for 8 weeks developed diabetes, as shown by hyperglycaemia and glycosuria, and had significant impairment of sexual function, as determined by tests of sexual behavior. The VIP content of the penis and major pelvic ganglion, the VIP release by the penis in vitro and the responsiveness of the vasculature of the penis in vivo to intracavernous VIP injection were determined. RESULTS: In diabetic rats, the VIP content of the major pelvic ganglion and penis was markedly increased, while the acetylcholine content of the penis was normal. The amount of VIP released in vitro by high potassium concentration or veratridine was similar for penile tissue slices of normal and diabetic rats. Intracavernous injection of VIP induced erection in the control rats but not in diabetic rats, whereas intracavernous injection of the adenylate-cyclase activator forskolin produced erection in both control and diabetic rats. CONCLUSION: Because VIP induces vasodilatation by activating adenylate cyclase, and forskolin produced erection in the diabetic rats, the failure of VIP to produce erection in these rats is unlikely to be due to a defect in the second-messenger mechanism or in the properties of vascular smooth muscle. Thus, a defect at the level of the VIP receptor or of the associated G-protein possibly explains the failure of intracavernous VIP to produce erection in the diabetic rats. Hence, an abnormality in VIP is a component of sexual dysfunction in the diabetic rat and the defect is at the level of the VIP receptor or associated G-protein.

Acetylcholine↗
PubMed1996

Effects of colchicine application to preganglionic axons on choline acetyltransferase activity and acetylcholine content and release in the superior cervical ganglion.

These experiments investigate the effect of block, by colchicine, of fast axonal transport in the cat's cervical sympathetic trunk (CST) on the superior cervical ganglion's choline acetyltransferase (ChAT) enzyme activity, acetylcholine (ACh) content, and ACh release. Electron microscopy on the segment of the CST exposed to colchicine 1 or 4 days earlier showed disappearance of microtubules and accumulation of vesicles and smooth membrane tubules but no disruption of the axonal cytomatrix. At 4 days following colchicine treatment, the number and size of synaptic boutons per grid square in the ganglion ipsilateral to the colchicine-treated CST were similar to those in the control ganglion. At 2 and 4 days following exposure of the CST to colchicine, ChAT activity in the ipsilateral ganglion was reduced to 78 +/- 8 and 54 +/- 8% of control values, respectively. ACh stores in the ganglia were also reduced (to 81 +/- 6% of control values at 2 days and to 51 +/- 5% of control values at 4 days). Ganglionic transmission and its sensitivity to blockade by hexamethonium during 2-Hz CST stimulation were not impaired at day 4 postcolchicine. ACh release evoked by 2-Hz stimulation of colchicine-treated axons was similar to release from untreated axons, despite the decrease in the ganglionic ACh content. In contrast, ACh release evoked by 20-Hz stimulation was depressed. The amount of ACh released during 5-Hz stimulation in the presence of vesamicol by the terminals of colchicine-treated axons was similar to that released by the terminals of untreated axons. These results suggest the following conclusions: (a) Colchicine-sensitive fast axonal transport contributes significantly to maintaining ChAT stores in preganglionic axon terminals. (b) The half-life of ChAT in sympathetic preganglionic terminals is approximately 4 days. (c) One consequence of colchicine-induced block of axonal transport is a reduced ACh content of preganglionic nerve terminals. (d) This decrease in ACh content appears to be the result of a loss in a reserve transmitter pool, whereas the size of the readily releasable compartment is maintained.

Acetylcholine↗
PubMed1996

Effects of the phosphatase inhibitors calyculin A and okadaic acid on acetylcholine synthesis and content of rat hippocampal formation.

The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 nM) and okadaic acid (50 nM) reduced significantly (p < 0.01) the synthesis of [3H]ACh from [3H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner (p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly (p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner (p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly (p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [3H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced Vmax values for high- and low-affinity choline uptake (p < 0.01) with no significant change in Km values (p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.

Acetylcholine↗
PubMed1995

Inhibition of potassium-stimulated acetylcholine release from rat brain cortical slices by two high-affinity analogs of vesamicol.

In this work, we investigated the effects of two structural analogs of the drug vesamicol, which inhibits the vesicular acetylcholine (ACh) transport, on the potassium-stimulated release of ACh from rat brain cortical slices. These vesamicol analogs, 4-aminobenzovesamicol (ABV) and (trans)-cyclohexovesamicol (transDec), were almost as potent as vesamicol in inhibiting the evoked release of ACh from cortex slices. Similar to vesamicol, the presence of these analogues inhibited the ability of ACh newly-synthesized from [3H]choline to become releasable. However, vesamicol's action was reversible, while ABV and transDec caused a persistent block of this [3H]ACh release. In addition, vesamicol did not affect the release of pre-stored [3H]ACh, but ABV and transDec partially inhibited the release of [3H]ACh in this condition, suggesting that the two latter drugs may alter some of the steps posterior to the entry of [3H]ACh into synaptic vesicles. The rank order of potency for these drugs to reduce ACh release (vesamicol = transDec > ABV) is close to the rank order for inhibition of ACh vesicular transport (transDec > vesamicol > ABV), but is completely different from the order of affinities of these drugs for the vesamicol receptor (ABV > transDec > > vesamicol). These results suggest that although these two vesamicol analogs are able to block ACh release due to their effects on the vesicular transport system, they may have other unexpected actions not shared by vesamicol.

Acetylcholine↗
PubMed1995

The site of the inhibitory action of endogenous opioids in the superior cervical ganglion of the cat.

A low-frequency stimulus train to the preganglionic input inhibits synaptic transmission in the superior cervical ganglion (SCG) of the cat. The inhibition is blocked by naloxone as well as by selective antagonists at mu and delta opiate receptors, which suggests that the mediator is an endogenous opioid [27,29]. Exogenous opioid peptides, including methionine-enkephalin (Met-Enk), which is present in preganglionic axons of the SCG, inhibit ganglionic transmission by a naloxone-sensitive mechanism. In the present study we test, in the anesthetized cat, whether the naloxone-sensitive synaptic inhibition is mediated by a pre- and/or post-synaptic mechanism. As a test of presynaptic inhibition, we measured the acetylcholine (ACh) released by preganglionic stimulation into the venous effluent of the perfused SCG. As a test of post-synaptic inhibition, we measured the effect of a preganglionic conditioning train on the ganglion cell firing evoked by ganglion-stimulant drugs injected into the arterial supply of the ganglion. In presence of naloxone (3 microM), which blocked the synaptic inhibition, the amount of ACh released by stimulated preganglionic axons did not change. Thus, the endogenous opioid which mediates the naloxone-sensitive inhibition does not act by depressing ACh release. In contrast, the ganglion cell firing evoked by ganglion-stimulant drugs was markedly depressed by a conditioning train, and naloxone blocked the depression, which suggests that the endogenous mediator of the naloxone-sensitive inhibition acts postsynaptically to decrease the excitability of ganglion cells. Exogenous Met-Enk depressed both ACh release by preganglionic stimulation and the firing of ganglion cells evoked by ganglion-stimulant drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗
PubMed1995

Increased acetylcholine content induced by antidromic stimulation of a sympathetic ganglion: a possible retrograde action of adenosine.

Prolonged high-frequency orthodromic stimulation of superior cervical ganglia is known to result in increased acetylcholine (ACh) synthesis and ACh content after the period of stimulation. In a previous study, we provided evidence to suggest that adenosine acts as an extracellular signal to activate this increased ACh synthesis and we proposed that the source of that adenosine might be postsynaptic. Thus, the purpose of the present study was to test whether direct stimulation of the post-ganglionic nerves could affect ganglionic ACh content. Antidromic conditioning of ganglia (15 Hz, 45 min) did not affect significantly their ACh content. However, if ganglia were allowed a 15-min rest period after this antidromic conditioning, their ACh stores were increased by 20%; a similar increase was induced by 4-Hz stimulation before the rest period. During the 15-Hz antidromic stimulation, ACh release was not clearly increased above the basal level, suggesting that preganglionic nerve endings were not stimulated to an extent that could explain the increased ACh content. Orthodromic stimulation (5 Hz) of ganglia 15 min after they had been subjected to antidromic conditioning (15 Hz, 45 min) showed increased ACh release in comparison with that from control unconditioned ganglia. Moreover, the extra ACh released by the conditioned ganglia was quantitatively similar to the increase in the ACh stores, as if most, or all, of the additional ACh was released by preganglionic stimulation. If the antidromic conditioning and the rest period were done during perfusion with Ca(2+)-free medium, the ganglia did not accumulate extra ACh.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Stereoselectivity of the inhibition of [3H]hemicholinium-3 binding to the sodium-dependent high-affinity choline transporter by the enantiomers of alpha- and beta-methylcholine.

In a previous report, we showed that the enantiomers of alpha- and beta-methylcholine inhibited choline uptake with stereoselectivity, but that their transport by the choline carrier of nerve terminals showed stereospecificity. The present experiments used the same choline analogues to determine if either of the above characteristics pertains to their ability to interact with the [3H]-hemicholinium-3 binding site present on striatal membranes and synaptosomes. [3H]Hemicholinium-3 binding to striatal membranes could be inhibited stereoselectively by the enantiomers of beta-methylcholine, but R(+)-alpha-methylcholine was little better than its enantiomer in this test. However, [3H]hemicholinium-3 binding to striatal synaptosomes was inhibited stereoselectively by the enantiomers of both alpha- and beta-methylcholine. This difference between the properties of [3H]hemicholinium-3 binding to membranes or to synaptosomes appears related to the presence of two ligand binding states. The [3H]hemicholinium-3 binding site could be shifted to a low-affinity state by ATP treatment and to a high-affinity state by EDTA washing. When the [3H]hemicholinium-3 binding site existed in its low-affinity state, binding was inhibited stereoselectively by the enantiomers of both alpha- and beta-methylcholine, but when shifted to its high-affinity state, it was inhibited stereoselectively only by the enantiomers of beta-methylcholine. We conclude that hemicholinium-3 interacts with the substrate recognition site of the high-affinity choline transporter, but that the stereoselectivity of this site changes depending on its affinity state.

Animals↗