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     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in cytoplasmic and vesicular acetylcholine content in insect ganglia during tetanic stimulation

Acetylcholine (ACh) content was reduced by about 30 pmol or 20% of the initial ACh content in the cockroach sixth abdominal ganglion in response to prolonged (30 min) tetanic stimulation at 40 Hz of the cercal nerves in the presence of 10⁻³ M hemicholinium-3 (HC-3). The reduction in ACh content in ganglia occurred in the cytoplasmic rather than the vesicular ACh fraction. The latter showed instead a transient increase followed by a gradual decrease to the previous level. Similar changes in ACh in the fractions were produced also by the stimulation, although the ACh content in ganglia did not change in a calcium-free saline, but was reduced in the presence of 50 μM dantrolene or 1–5 mM cobalt chloride. Synaptic transmission at the cercal nerve-giant nerve fiber synapses rapidly decreased and was abolished within a few minutes during tetanic stimulation at 40 Hz, but recovered on reducing the frequency to 0.1 Hz. The decline in transmission was not affected by HC-3, but a significant delay was observed in the recovery following 30 min of tetanic stimulation in the presence of HC-3.These results may suggest that the depletion of ACh as a functional store occurs in the cytoplasmic ACh fraction, rather than in the vesicular one, after prolonged stimulation in the presence of HC-3. The latter fraction shows and increase in the uptake of cytoplasmic ACh that depend on the presence of intracellular calcium ions during stimulation.     

Peripheral and central actions of AF64A (ethylcholine mustard aziridinium ion) on acetylcholine release, in vitro: Comparison with hemicholinium

The acute effects of ethylcholine mustard aziridinium ion (AF64A) and hemicholinium-3 (HC-3) on the release of endogenous acetylcholine (ACh) from isolated tissues were examined. Whereas addition of HC-3 (10⁻⁶–10⁻⁵ M) significantly reduced the output of ACh from isolated guinea-pig ileum longitudinal muscle strip elicited by 10 Hz stimulation, AF64A had no effect and even enhanced the release of radiolabel elicited by 1 Hz stimulation when this tissue was pre-loaded with [³H]choline. Similarly, HC-3 (10⁻⁵ M) reduced ouabain-induced endogenous ACh release from isolated rat hippocampus. Addition of AF64A (10⁻⁵−5 × 10⁻⁵ M) caused a slight increase in ACh release. In isolated rat cortex, however, AF64A did not affect ACh release. Moreover, AF64A caused a decrease in ouabain-stimulated ACh release from striatum. The present study indicates that: (a) the in vitro actions of AF64A differ from those of HC-3 and (b) the acute effects of AF64A on endogenous ACh release vary, depending on the tissues studied and the stimulation parameters used.     

Muscarinic stimulation of submucosal glands in swine trachea

The properties of muscarinic acetylcholine receptors (mAChR) on tracheal explants and isolated submucosal gland cells were determined using [3H]quinuclidinyl benzilate ([3H]QNB) and N-[3H]methylscopolamine ([3H]NMS) as ligands. Analysis of competitive displacement of ([3H]NMS binding by pirenzepine demonstrated the presence of M1- (27 +/- 2%) and M2G- (73 +/- 2%) receptors on isolated tracheal submucosal gland cells (TSGC's) in control. Daily administration of diisopropylfluorophosphate (DFP) inhibited cholinesterase activity by greater than 95%. After 7 days of DFP treatment, [3H]QNB binding to intact TSGC's decreased from 14.2 +/- 0.6 to 6.3 +/- 0.8 fmol/10(6) cells; similarly, [3H]NMS binding fell from 8.1 +/- 1.9 to 2.0 +/- 0.8 fmol/10(6) cells. The loss of mAChR's was predominantly of the M2G subtype with the relative proportion dropping to 33%. In addition, 90% of the receptors assumed the high-affinity state for carbachol displacement of [3H]NMS. Mucus secretion was quantitated by measuring the release of 3H-labeled mucus macromolecules from explants of tracheal submucosal glands and isolated cells. Acetylcholine (ACh), 2 X 10(-5) M, stimulated mucus secretion by 2.5 and 2.3 times the basal rate, respectively. Elimination of acetylcholinesterase (AChe) by DFP increased the ACh sensitivity by 18- and 5-fold. Tracheal explants or TSGC's obtained 2 h after an in vivo DFP treatment showed a 6- and 3-fold ACh stimulation. This ACh sensitivity decreased during the continued daily dosing with DFP such that only a 1.3- and 1.1-fold ACh stimulation was apparent after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of hemicholinium-3 on choline and acetylcholine levels in a sympathetic ganglion.

Pregangliaaonic stimulation of the cat's superior cervical ganglion in the presence of hemicholinium-3 (HC-3) produced the expected depletion of acetylcholine (ACh) stores, but failed to cause a corresponding reduction in the choline content. These results suggest that either HC-3 possesses an intracellular site of action or that in lower doses it selectively inhibits a specialized choline transport system in cholinergic nerves. At a dose of 2 mg/kg, HC-3 probably blocked ACh synthesis completely in ganglia stimulated at 20 Hz. Under these conditions, there was a rapid depletion of ACh to about 50% of control levels during the first 5 min of stimulation and thereafter the rate of decline in ACh levels proceeded at a much slower pace. Since the 2 mg/kg dose of HC-3 did not raise plasma choline concentrations, it may be assumed that non-specialized choline transport systems in other tissues were not significantly inhibited by this dose of HC-3. However, when the dose of HC-3 was increased to 4 mg/kg, plasma choline levels increased by 58%.     

Relations Between the Extracellular Concentrations of Choline and Acetylcholine in Rat Striatum

Abstract: Changes in extracellular levels of acetylcholine (ACh) and choline (Ch) in the striatum of rats were examined by in vivo microdialysis after intraperitoneal injections of drugs. A dopamine D₂ antagonist, sulpiride (20 mg/kg), and a muscarinic antagonist, atropine (3.5 mg/kg), increased ACh levels and decreased Ch levels. On the contrary, the D₂ agonist (±)-2-( N -phenylethyl- N -propyl)amino-5-hydroxytetralin (N-434; 5 mg/kg) and an anesthetic, pentobarbital (50 mg/kg), decreased ACh levels and increased Ch levels. Perfusion of 10 µ M hemicholinium-3 (HC-3), a Ch uptake inhibitor, through the striatum induced a complete inhibition of ACh release and increased Ch levels in all drug-treated groups. The degree of relative increase in the level of Ch induced by HC-3 differed among the drug-pretreated groups; compared with the control group, the relative increase was larger in the sulpiride- and atropine-treated groups and smaller in the N-434 and pentobarbital-treated groups. Thus, we demonstrated reciprocal relations between extracellular concentrations of Ch and ACh after treatments by drugs. The data suggest that in the striatum, which is rich in cholinergic innervation, the extracellular Ch concentration is to a large extent determined by activity of the cholinergic transmission reflected in high-affinity choline uptake.     

The effect of atropine upon acetylcholine release from cat superior cervical ganglia and rat cortical slices: measurement by a radio-enzymic method.

Atropine is known to increase the release of acetylcholine (ACh) from cerebral cortex, and the present experiments tested the effect of this drug upon ACh release in the superior cervical ganglion of the cat. The release of ACh was measured by a radio-enzymic method, which was shown to provide an estimate of the ACh content of samples collected from perfused ganglia that was similar (102%) to that obtained by the method of bioassay more usually used . Atropine (3 X 10(-6) M) increased (3.5 to 4-fold) the amount of ACh released by rat's sliced cerebral cortex incubated in a high (23 mM) potassium medium. However atropine (3 X 10(-6)-3 X 10(-5) M) did not change the amount of ACh released by ganglia during preganglionic nerve stimulation (5-10 Hz). It is concluded that cholinergic nerve terminals in different tissues appear to have different pharmacological properties.     

Inhibition of choline efflux results in enhanced acetylcholine synthesis and release in the guinea-pig corticocerebral synaptosomes.

Synthesis and release of [3H]acetylcholine ([3H]ACh) were measured in synaptosomes from the guinea pig cerebral cortex after preloading with [3H]choline ([3H]Ch). We demonstrate here that inhibition of choline (Ch) efflux results in an increase in acetylcholine (ACh) synthesis and release. Our findings are as follows: (1) inhibition of [3H]Ch efflux by hemicholinium-3 (HC-3) (100 microM), increased the levels of both the released (116% of control) and the residing (115% of control) [3H]ACh. (2) The muscarinic agonist, McN-A-343 (100 microM), which was previously shown to inhibit Ch efflux, also increased the released (121% of control) and the residing (109% of control) [3H]ACh. (3) Omission of Na+ ions (which are required for Ch transport) from the incubation medium had similar effects to those observed with McN-A-343 and HC-3. These results suggest inverse relationships between Ch efflux on one hand, and ACh synthesis and release on the other hand. (4) Depolarization with 50 mM K+, or with the K+ channel blocker, 4-aminopyridine (100 microM), also increased the total level of [3H]ACh (113 and 107% of nondepolarized synaptosomes, respectively). However, whereas conditions that inhibit Ch transport such as HC-3, McN-A-343 and "no sodium" increased both the residing and the released [3H]ACh depolarization with high K+ or 4-aminopyridine reduced the residing (79 and 87% of control, respectively) and increased only the released [3H]ACh (182 and 148% of control, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in purity of commercial samples of hemicholinium-3: effects on high affinity choline uptake, neuromuscular transmission, acetylcholinesterase and carbachol and acetylcholine contractions.

Commercial samples of hemicholinium-3 (HC-3) have been found to vary in colour (from white to a sandy-yellow colour) and chemical composition. There were no significant differences between the various HC-3 samples with regards to inhibition of high affinity choline uptake into synaptosomes or inhibition of neuromuscular transmission in the chick biventer cervicis (CBC) preparation. Yellow HC-3 inhibited acetylcholinesterase more than white HC-3 with I₅₀ of 4.8 × 10^?5 M and 3.3 × 10^?4 M, respectively. Carbachol-induced contractions of the CBC preparation were inhibited more by yellow HC-3 than white HC-3; the opposite was true for acetylcholine- induced contractions. The results indicated that there is a minor contaminant in yellow HC-3 other than deanol which was a potent inhibitor of acetylcholinesterase and the carbachol response.     

Inhibition of cholinergic neurotransmission in human airways by beta 2-adrenoceptors

The purpose of the study was to determine whether catecholamines modulate cholinergic neurotransmission in isolated human airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension, and contractions were induced by either electrical field stimulation (EFS) or exogenous acetylcholine (ACh). Isoproterenol, epinephrine, and norepinephrine in that order of potency produced concentration-dependent inhibition of comparable responses to EFS and ACh. However a potency difference of 100-fold for isoproterenol (IC50 = 4.80 X 10(-8) M for EFS and 3.70 X 10(-6) M for ACh) and 10-fold for both epinephrine and norepinephrine was observed for inhibition of responses to EFS compared with responses to ACh. The inhibitory effects of isoproterenol on responses to EFS were prevented by propranolol and ICI 118551 (a beta 2-antagonist) but not by betaxolol (a beta 1-antagonist). Tyramine had no effect on contractions elicited by EFS. These experiments demonstrate that beta-agonists inhibit cholinergic nerve-induced contractions of human bronchi more potently than contractions induced by exogenous ACh, suggesting modulation of cholinergic neurotransmission by prejunctional beta 2-receptors.     

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals

The effects of hemicholinium-3 (HC-3) or 4-(1-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 μg HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP resulted in a further reduction in ACh. Pretreatment with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR) against soman was increased from 0.8 to 7.3. These data are consistent with the hypothesis that excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.     

Antagonism of relaxation to isoproterenol caused by agonist interactions

The interaction of contractile agonists on the relaxation elicited with isoproterenol (ISO) was studied in 112 tracheal smooth muscle (TSM) strips from 20 dogs in vitro. Strips were contracted to the same active target tension (TT) with acetylcholine (ACh), histamine (HIS), serotonin (5-hydroxytryptamine, 5-HT), potassium chloride (KCl), or the combinations of ACh + HIS, ACh + 5-HT, HIS + KCl, HIS + 5-HT (50% TT from each agonist). Although a less potent agonist, adding HIS to cause 50% of the TT reduced the concentration of ACh to elicit the remaining 50% TT and substantially altered relaxation by ISO compared with HIS alone [concentration required to achieve 50% relaxation (RC50) = 9.2 +/- 2.4 X 10(-8) vs. 9.0 +/- 4.4 X 10(-9) M to HIS alone; P less than 0.003]. Relaxation for TSM strips contracted with ACh + HIS was comparable to that elicited from the same TT with ACh alone, although concentrations required in combination were lower than for either agonist alone. Trachealis strips contracted equivalently with KCl + HIS also had augmented contraction and attenuated relaxation (RC50 = 3.7 +/- 0.8 X 10(-8) M; P less than 0.015 vs. HIS alone). However, combinations of 5-HT + ACh and 5-HT + HIS did not alter relaxation to ISO from that elicited by the weaker agonist alone. We demonstrate that TSM relaxation depends on the combination of agonists eliciting contraction and may be inhibited substantially by interactions among contractile agonists.     

Plant phenols as in vitro inhibitors of glutathione S-transferase(s)

Ellagic acid, a commonly occurring plant phenol, was shown to be a potent in vitro inhibitor of GSH-transferase(s) activity. Other plant phenols such as ferrulic acid, caffeic acid and chlorogenic acid also showed a concentration dependent inhibition of GSH-transferase(s) activity. The I50 values of ellagic acid, caffeic acid, chlorogenic acid and ferrulic acid were 8.3 X 10(-5)M, 14.0 X 10(-5)M, 20.0 X 10(-5)M and 22.0 X 10(-5)M respectively, suggesting that ellagic acid is the most potent inhibitor of all the four studied plant phenols. At 55 microM concentration of ellagic acid, a significant inhibition (35-47%) was observed on GSH-transferase activity towards CDNB, p-nitrobenzyl chloride and 1,2-epoxy-3-(p-nitrophenoxy)propane as substrates. Ellagic acid inhibited GSH-transferase(s) activity in a non-competitive manner with respect to CDNB while with respect to GSH it inhibited the enzyme activity in a competitive manner. Other phenolic compounds purpurogallin , quercetin, alizarin and monolactone also showed a concentration dependent inhibition of the enzyme activity with a I50 of 0.8 X 10(-5)M, 1.0 X 10(-5)M, 8.0 X 10(-5)M and 16.0 X 10(-5)M respectively. These inhibitors of GSH-transferase(s) activity should be useful in studying the in vitro enzyme mediated reactions of exogenous and endogenous compounds.     

THE RELATIONSHIP BETWEEN ACETYLCHOLINE RELEASE FROM BRAIN SLICES AND THE ACETYLCHOLINE CONTENT OF SUBCELLULAR FRACTIONS PREPARED FROM BRAIN

Abstract— Acetylcholine (ACh) release from sliced cerebral cortex of rats was measured when the tissue was incubated in a high K⁺ (46 m m ) medium containing eserine. In the absence of hemicholinium (HC-3), ACh release was well maintained, but in the presence of HC-3, ACh release declined within 15–20 min. Subcellular fractions representing nerve-ending free (cytoplasmic) ACh and nerve-ending bound ACh were prepared from slices that had been stimulated to release ACh in the presence of HC-3. Both nerve-ending stores of ACh were depleted when their content was compared to tissue that had not been stimulated and there was no demonstrable difference in the rate of depletion of either of the two fractions. Stimulating slices with K⁺ in the absence of HC-3 also depleted cytoplasmic and vesicle-bound ACh. It is concluded that, under these experimental conditions, both nerve ending stores of ACh are available for release and that, in the absence of HC-3, ACh synthesis can maintain ACh release, but cannot maintain tissue ACh content.     

Effects of LF-14, THA and physostigmine in rat hippocampus and cerebral cortex

The effects of physostigmine, tetrahydroaminoacridine (THA) and LF-14 [3,3-dimethyl-1(4- amino-3-pyridyl)urea], a 3,4-diaminopyridine derivative, were compared on inhibition of acetyl- cholinesterase (AChE) activity, and release of [³H]acetylcholine (ACh) from rat brain cortical and hippocampal slices. All three compounds caused a concentration dependent inhibition of AChE, with an order of potency physostigmine > THA > LF-14. The electrically stimulated release of ACh from hippocampal and cortical slices was decreased by 10⁻⁵M physostigmine, although the effect was significant only in cortex. THA (5 × 10⁵M) caused a slight, but not significant, decrease in ACh release from both tissues. In contrast, LF-14 (5 × 10⁻⁵ M) caused an approx. 3-fold enhancement of stimulated release. When AChE was inhibited by prior addition of physostigmine, THA caused only a slight enhancement of ACh release, whereas LF-14 greatly increased release. ACh release was also reduced by stimulation of presynaptic muscarinic receptors with oxotremorine. In this case, THA had no effect on ACh release, while LF-14 was able to reverse the inhibition. This study suggests that LF-14 acts to promote ACh release through blocking K⁺ channels, and has a less potent AChE inhibitory effect. It is possible that a compound like LF-14 could be useful in treating diseases of cholinergic dysfunction such as Alzheimer's disease, by both promoting the release of ACh and inhibiting its breakdown.     

Developmental alterations in guanine nucleotide regulation of the beta-adrenergic receptor-adenylate cyclase system of skeletal muscle

The postnatal development of skeletal muscle is accompanied by an increased capacity for glycogenolysis and anaerobic glycolysis. In the present study, regulatory features of cAMP synthesis were examined in neonatal and adult rabbit sarcolemmal membranes. Adult sarcolemma exhibited a 3-, 6-, and 10-fold greater adenylate cyclase activity than neonate for basal, NaF, and isoproterenol plus GTP, respectively. The Km for activation by isoproterenol was 1.4 X 10(-8) M and 6 X 10(-8) M for GTP. The number of beta-receptors was similar (0.9-1.2 pmol/mg). 10 microM GTP shifted isoproterenol EC50 from 1 X 10(-8) M to 1 X 10(-7) M in adult; neonatal agonist affinity was unaffected by GTP. Cholera toxin stimulated adenylate cyclase activity 2-fold and catalyzed 32P ribosylation of a Mr = 42,000 peptide in adult sarcolemma; both activities were low or absent in neonate. Isoproterenol-stimulated GTPase activity was elevated 4-fold in adult compared to neonatal sarcolemma. Mn2+ ion-stimulated basal activity, an indicator of catalytic function of adenylate cyclase, was also elevated in adult. Together, these findings suggest that the development of catecholamine-sensitive cAMP synthesis in muscle is governed by the coordinate expression of the regulatory and catalytic proteins of adenylate cyclase, but not the beta-receptor.     

Ca2+-dependent facilitated shortening in isotonic contraction of trachealis muscle

We compared isotonic shortening with isometric force generation as a function of external Ca2+ in 166 tracheal smooth muscle (TSM) strips from 27 mongrel dogs in vitro. Concentration-response curves were generated with muscarinic stimulation (acetylcholine, ACh), alpha-adrenergic receptor activation (norepinephrine after beta-adrenoceptor blockade, NE), serotonin (5-HT), and KCl-substituted Krebs-Henseleit solution. The concentrations of 5-HT causing half-maximal shortening (ECS50, 1.54 +/- 0.14 X 10(-7) M) and half-maximal active isometric tension (ECT50, 1.72 +/- 0.30 X 10(-7) M) were similar (P = NS). Likewise, ECS50 (21.9 +/- 0.7 mM) and ECT50, (22.0 +/- 0.9 mM) were similar for KCl. In contrast, facilitated isotonic shortening (i.e., greater isotonic shortening for comparable degrees of force generation) was elicited with ACh and NE for all levels of force generation between 15 and 85% of maximum and for all concentrations of ACh from 3 X 10(-8) to 3 X 10(-5) M (P less than 0.05 for all points). Facilitated isotonic shortening also was elicited for all concentrations of NE from 10(-8) to 10(-6) M (P less than 0.05 for all points). Removal of Ca2+ from the perfusate substantially reduced the potency of ACh (P less than 0.001) and abolished differences between ECS50 (2.23 +/- 0.28 X 10(-5) M) and ECT50 (2.50 +/- 0.46 X 10(-5) M, P = NS). We demonstrate that for comparable degrees of force generation, muscarinic and alpha-adrenergic receptor activation cause greater isotonic shortening than KCl or 5-HT and that this facilitated shortening is associated with the concentration of external Ca2+.     

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.     

Effects of Acetylethylcholine Mustard on [3H]Quinuclidinyl Benzilate Binding and Acetylcholine Release in Rat Brain Synaptosomes

The effects of acetylethylcholine mustard and its aziridinium derivative (AMMA) on acetylcholine (ACh) release and [3H]quinuclidinyl benzilate (QNB) binding were studied in rat cortical synaptosomes. After incubation for 5 min at 37 degrees C, AMMA reduced [3H]QNB binding with an IC50 of 9 microM. Following incubation for 5 min with 50 microM AMMA and washing, there was a 62% reduction in the [3H]QNB binding capacity with no change in the KD value for the remaining receptors, a result indicating the irreversibility of the AMMA binding. AMMA and oxotremorine both reduced the basal and 30 mM K+-induced release of newly synthesized [3H]ACh in dose-dependent manners over a 2.5-min period. At identical 50 microM concentrations, AMMA produced a much longer inhibition of basal [3H]ACh release than oxotremorine did. The inhibition of basal and 30 mM K+-induced [3H]ACh release by AMMA (10-250 microM) was blocked by 2 microM atropine during a 2.5-min release incubation, but not during a 30-min release incubation. After synaptosomes were treated with 50 microM AMMA for 5 min and the unbound drug was washed out from the tissue, [3H]ACh release (basal and K+-induced) was reduced. AMMA (50 microM) reduced high-affinity choline uptake and ACh synthesis by greater than 90% in this tissue, but these effects did not account for the [3H]ACh release inhibition, because they were not atropine sensitive and hemicholinium-3 had no effect on [3H]ACh release under the conditions used in these studies, i.e., after extracellular [3H]choline was washed out. Taken together, these results suggest that AMMA may be an irreversible agonist at presynaptic muscarinic autoreceptors.