Inhibitor Of High-affinity Choline Uptake Research Articles

The antihyperalgesic effect of cytidine-5′-diphosphate-choline in neuropathic and inflammatory pain models

This study was designed to test the effects of intracerebroventricularly (i.c.v.) administered CDP-choline (cytidine-5'-diphosphate-choline; citicoline) and its metabolites in rat models of inflammatory and neuropathic pain. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 µmol) produced a dose and time-dependent reversal of mechanical hyperalgesia in both carrageenan-induced inflammatory and chronic constriction injury-induced neuropathic pain models in rats. The antihyperalgesic effect of CDP-choline was similar to that observed with an equimolar dose of choline (1 µmol). The CDP-choline-induced antihyperalgesic effect was prevented by central administration of the neuronal high-affinity choline uptake inhibitor hemicholinium-3 (1 µg), the nonselective nicotinic receptor antagonist mecamylamine (50 µg), the α7-selective nicotinic ACh receptor antagonist, α-bungarotoxin (2 µg) and the γ-aminobutyric acid B receptor antagonist CGP-35348 (20 µg). In contrast, i.c.v. pretreatment with the nonselective opioid receptor antagonist naloxone (10 µg) only prevented the CDP-choline-induced antihyperalgesic effect in the neuropathic pain model while the nonselective muscarinic receptor antagonist atropine (10 µg) did not alter the antihyperalgesic effect in the two models. These results indicate that CDP-choline-elicited antihyperalgesic effect in different models of pain occurs through mechanisms that seem to involve an interaction with supraspinal α7-selective nicotinic ACh receptors, and γ-aminobutyric acid B receptors, whereas central opioid receptors have a role only in the neuropathic pain model.

The antinociceptive effects of centrally administered CDP-choline on acute pain models in rats: The involvement of cholinergic system

This study investigates the antinociceptive effect of intracerebroventricular (i.c.v.) injection of cytidine-5′-diphosphate choline (CDP-choline; citicoline) and the involvement of cholinergic mechanisms in rats. Three different pain models were utilized: thermal paw withdrawal test, mechanical paw pressure test and acetic acid writhing test. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 μmol) produced dose and time-dependent antinociception. Equimolar dose of choline (1 μmol; i.c.v.) produced antinociceptive response similar to the one observed in CDP-choline given animals. On the other hand, cytidine (1 μmol; i.c.v.) failed to produce response in the thermal paw withdrawal test and the mechanical paw pressure test but in the writhing test in which it produced significant antinociceptive effect. CDP-choline-induced antinociception was prevented by the neuronal high affinity choline uptake inhibitor HC-3 (1 μg; i.c.v.), the nonselective nicotinic receptor antagonist mecamylamine (50 μg; i.c.v.) and by the α 7-selective nicotinic receptor antagonist, MLA (25 μg; i.c.v.). However, it was not changed by the nonselective muscarinic receptor antagonist atropine (10 μg; i.c.v.) in the thermal paw withdrawal test and mechanical paw pressure test. In the writhing test, all antagonist pretreatments produced blockade similar to that obtained from CDP-choline injected animals. CDP-choline did not impair the motor performance of rats as evaluated by a rota-rod test. Therefore, it can be postulated that CDP-choline exerts an antinociceptive effect mediated by a central cholinergic mechanism. Activation of specific α 7-nicotinic cholinergic receptors through the activation of presynaptic cholinergic mechanisms appears to be involved in the antinociceptive effect of this drug.

Centrally injected CDP-choline increases plasma vasopressin levels by central cholinergic activation.

In the present study, both the effects of intracerebroventricular (i.c.v.) injection of cytidine-5'-diphosphate choline (CDP-choline) on plasma vasopressin levels and the choline involvement of these effects were investigated. I.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 micromol) increased plasma vasopressin levels dose- and time-dependently. I.c.v. injection of equimolar dose of choline (1 micromol) produced similar vasopressin response. However equimolar dose of cytidine (1 micromol; i.c.v.), the other hydrolysis product of CDP-choline, did not affect plasma vasopressin levels. Pretreatment of rats with hemicholinium-3, neuronal high affinity choline uptake inhibitor (20 microg; i.c.v.) blocked the vasopressin response to i.c.v. CDP-choline (1 micromol). Pretreatment of rats with mecamylamine (50 microg; i.c.v.), a nonselective nicotinic receptor antagonist, abolished the increase in plasma vasopressin induced by CDP-choline while atropine (10 microg; i.c.v.), nonselective muscarinic receptor antagonist, failed to change the response. In conclusion, intracerebroventricularly injected CDP-choline can increase plasma vasopressin levels by activating central nicotinic cholinergic receptors through the activation of presynaptic cholinergic mechanisms.

Assessment of choline uptake for the synthesis and release of acetylcholine in brain slices by a dynamic autoradiographic technique using [ 11C]choline

The uptake of choline for the synthesis and release of acetylcholine was investigated in brain slices by dynamic positron autoradiography using [ 11C]choline. Brain slices (330 μm) were incubated with [ 11C]choline in oxygenated Krebs–Ringer medium at 34 °C and serial two-dimensional time-resolved images of the uptake and release of radioactivity were recorded on Storage Phosphor screens. [ 11C]Choline uptake increased with the period of incubation and was 1.9 times higher in the striatum than cerebral cortex. The uptake in the striatum was significantly diminished by hemicholinium-3 (HC-3), an inhibitor of high-affinity choline uptake. Pretreatment of brain slices with 50 mM K + for 20 min enhanced the uptake in striatum. The uptake of [ 11C]choline in brain slices was saturable using nonlabeled choline. Two uptake systems, a high-affinity and a low-affinity system, were confirmed to exist by kinetic analysis using Lineweaver–Burk plots. The 11C radioactivity that had accumulated in the striatum disappeared on treatment with veratridine, a depolarization agent, in the presence of HC-3. This pattern of disappearance was consistent with that of the appearance of unlabeled and labeled acetylcholine in the medium. These results indicate that this method is useful for obtaining information regarding the uptake of choline for the synthesis and release of acetylcholine in live brain tissues.

The effects of continuous and single-dose radiation on choline uptake in organotypic tissue slice cultures of rabbit hippocampus

The objective of the present study was to determine the time-dependent course of choline uptake in mature organotypic slice cultures of rabbit hippocampal formation and to assess the effects of continuous and single high-dose irradiation on choline uptake in cultivated slices in vitro. Transverse slices of hippocampus were dynamically incubated in a cerebrospinal fluid-like culture medium for 72 h. To study the changes in choline uptake longitudinally, the slice cultures were processed with 0.1 µ M [3H]-choline, and tritium accumulation was counted. Two different gamma irradiation sources (125I seeds and a clinical 60Co source) were used as representative models of interstitial radiosurgery and other radiosurgical techniques. A total dose of approximately 6000 cGy was delivered to the brain slices in one session or in a continuous, relatively low-dose rate fashion, and their effects on high-affinity choline uptake were examined. In another set of experiments with 125I, 5 µM hemicholinium-3 was used in choline uptake procedures as a competitive high-affinity choline uptake inhibitor. The results can be summarized as follows: (1) in the control group of the hippocampal tissue culture, there was a significant increase in tritium accumulation values from 0 to 48 h and a decrease thereafter; (2) continuous 125I irradiation caused a highly significant depression of the accumulation of tritium compared to that observed in the control group throughout its application for 72 h; (3) there was no significant change in the accumulation of tritium in the slices after single high-dose rate irradiation with a 60Co source; and (4) 5 µM hemicholinium significantly depressed the accumulation of tritium in both the control and the 125I-irradiated groups, and there was no longer a difference between 125I-irradiated and control groups when both groups were treated with hemicholinium. These results demonstrate that the delivery of continuous but relatively low-dose rate gamma irradiation is more efficacious than single high-dose external irradiation on high-affinity choline uptake in hippocampal nervous tissue. The results also indicate that continuous irradiation specifically affected the high-affinity energy-dependent choline uptake mechanism, whereas nonspecific choline uptake did not seem to be disturbed. [Neurol Res 2001; 23: 669-675]

COMPARATIVE PRESYNAPTIC NEUROCHEMICAL CHANGES IN RAT STRIATUM FOLLOWlNG EXPOSURE TO CHLORPYRIFOS OR PARATHION

Organophosphorus pesticides (OPs) exert acute toxicity through inhibition of acetylcholinesterase (AChE) in target tissues. Previous studies in our laboratory have demonstrated, however, that dosages of the OPs chlorpyrifos (CPF) or parathion (PS), which cause similar degrees of brain AChE inhibition in adult male rats, can produce marked differences in toxicity. While compensatory changes in postsynaptic receptors can modulate the clinical expression of AChE inhibition and lead to tolerance to these toxicants, we propose that OP-selective changes in presynaptic cholinergic processes can also regulate the ultimate consequences of AChE inhibition. The relative effects of either vehicle (peanut oil, 2 ml/ kg, sc), CPF (280 mg/kg), or PS (6.6 mg/ kg) on clinical signs of toxicity and AChE activity, high-affinity choline uptake (HACU), and potassium evoked acetylcholine (ACh) release in striatum were examined for a 7-d period after exposure in adult female Sprague-Dawley rats. In vitro effects of CPF-oxon or paraoxon, the active oxidative metabolites of CPF and PS, on HACU were also examined in comparison with the prototype inhibitor hemicholinium-3 (HC-3) . Similar to our previous findings in male rats, female rats treated with dosages of CPF or PS causing similar maximal degrees of AChE inhibition (82-96%) exhibited marked differences in response; that is, PS produced more extensive signs of acute toxicity (salivation, lacrimation, urination and/or defecation, i.e., SLUD signs and involuntary movements). CPF reduced striatal synaptosomal HACU at 1, 2, and 7 d after exposure, whereas PS only decreased HACU at 2 d posttreatment. While CPF-oxon was a weak inhibitor of HACU (IC50 >200 mu M), paraoxon had no effect on this process in vitro. Potassium-evoked ACh release in the presence of physostigmine (20 mu M) was not affected by either OP at 1 d but was increased 2 d after either CPF or PS treatment and remained elevated at 7 d after exposure in CPF-treated rats only. ACh release in the presence of both physostigmine and the muscarinic antagonist atropine (1 mu M) was decreased by both OPs as early as 1 d after exposure and remained lower at 2 d posttreatment. By 7 d, however, ACh release in response to atropine was decreased in CPF-treated animals only, sug gesting that both CPF and PS affected muscarinic autoreceptor function but with somewhat different time courses. These results suggest that different OPs may selectively modify presynaptic cholinergic processes and that early, OP-selective changes in HACU/ACh synthesis may contribute to the differential toxicity noted following extensive AChE inhibition by either CPF or PS.

Centrally administered choline increases plasma prolactin levels in conscious rats

Intracerebroventricular (i.c.v.) administration of choline, a precursor of acetylcholine (ACh) increased plasma prolactin levels in a time and dose-dependent manner in conscious rats. Pretreatment of rats with the cholinergic muscarinic antagonist, atropine (10 μg, i.c.v.), blocked the increase in plasma prolactin level. The increase was not influenced by pretreatment with the cholinergic nicotinic antagonist, mecamylamine (50 μg, i.c.v.). Pretreatment with hemicholinium-3 (HC-3; 20 μg, i.c.v.), a high affinity choline uptake inhibitor, attenuated the choline-induced increase of plasma prolactin levels. These results show that choline increases plasma prolactin levels by activating muscarinic receptors via presynaptic mechanisms.

Effect of neostigmine on concentration and extraction fraction of acetylcholine using quantitative microdialysis

A quantitative microdialysis method was used to determine the effect of local perfusion of 0, 100, 200, and 300 nM neostigmine (NEO) on acetylcholine (ACh) extracellular concentration and microdialysis extraction fraction ( E d) in the striatum of the rat. Because of the efficiency of AChE, the inhibition of this enzyme is expected to result in a substantial increase in ACh levels and a decrease in the E d of ACh. The extracellular concentration of ACh increased linearly with increasing concentrations of NEO. The control ACh concentration was determined to be 18.4±11.8 nM ( n=10; mean±S.E.M.) The ACh extracellular concentration for the remaining groups was determined to be 173±14 nM ( n=5), 329±52.5 nM ( n=13), and 581±109 nM ( n=10) for the 100, 200, and 300 nM NEO groups, respectively. Perfusion with 300 nM NEO resulted in a significant reduction in the E d of ACh (64.5±3.5% vs. 43.6±7.5%, P<0.05). In contrast to ACh, perfusion with 0, 1, and 10 μM hemicholinium-3, an inhibitor of high-affinity choline uptake, increased choline levels but did not affect the E d of choline. The effects on E d are consistent with E d being influenced by rapid clearance mechanisms.

Intracerebroventricular choline reverses hypotension induced by acute chemical sympathectomy.

1. The effect of centrally administered choline on blood pressure was investigated in rats made hypotensive by chemical sympathectomy. Chemical sympathectomy was produced by intravenous (i.v.) injection of 50 mg kg-1 of 6-hydroxydopamine (6-OHDA). Intracerebroventricular (i.c.v.) administration of choline (50-150 micrograms) 2 h after 6-OHDA treatment increased blood pressure and reversed the hypotension in a dose-dependent manner without affecting the heart rate. The pressor response was associated with an increase in plasma vasopressin levels. 2. Pretreatment of rats with the nicotinic receptor antagonist, mecamylamine (50 micrograms, i.c.v.), but not the muscarinic receptor antagonist atropine (10 micrograms, i.c.v.), blocked both the pressor and vasopressin responses to choline (150 micrograms). Pretreatment of rats with hemicholinium-3 (HC-3), a high affinity choline uptake inhibitor, greatly attenuated the pressor response to i.c.v. choline (150 micrograms). 3. The vasopressin V1 receptor antagonist, beta-mercapto-beta,beta-cyclopentamethylenepropionyl-O-Me-Try,Arg) - vasopressin (10 micrograms kg-1; i.v.) given 5 min after i.c.v. choline, decreased the blood pressure but failed to return it to the pre-choline levels. Prazosine (0.5 mg kg-1; i.p.), an antagonist of alpha-adrenoceptors, also decreased blood pressure. Administration of both antagonists together eliminated the pressor response to choline, and the blood pressure was reduced further to below the pre-choline levels. 4. It is concluded that i.c.v. choline can increase blood pressure in rats made hypotensive by acute chemical sympathectomy through the activation of central nicotinic receptors by presynaptic mechanisms. An elevation in plasma levels of both vasopressin and catecholamines (possibly released from the adrenal medulla) is involved in the pressor response to choline.

Synthesis and Release of Acetylcholine by the Isolated Perifused Trout Caudal Neurosecretory System

The neurons of the caudal neurosecretory system of teleosts contain, in addition to urotensin I and urotensin II, a high concentration of acetylcholine (T. Ichikawa, 1978,Gen. Comp. Endocrinol.35, 226–233). The isolated urophysis (and attached terminal spinal cord region) of the rainbow troutOncorhynchus mykisswas incubated with [3H]choline (0.2 MBq/ml) for 45 min at 22° in the presence of the cholinesterase inhibitor, physostigmine. Unreacted choline was removed by perifusion with fish Ringer solution. Incorporation of radioactivity into newly synthesized [3H]acetylcholine was 4.9 ± 2.1 × 105Bq/g wet tissue wt. When incubations were carried out in the presence of hemicholinium-3, an inhibitor of high-affinity choline uptake, or when physostigmine was omitted from the incubation buffer and/or when [3H]inulin was substituted for [3H]choline, the incorporation of radioactivity was greatly reduced (<0.5 × 105Bq/g). The release of [3H]acetylcholine from the preparation increased to 338 ± 59% of basal (P< 0.05) when the concentration of K+in the perifusion buffer was raised to 41 mM,but neither urotensin I (10−7M) nor urotensin II (10−6M) had a significant effect on release. The data indicate that the trout caudal neurosecretory system possesses a high-affinity uptake system for choline and that newly synthesized acetylcholine is released in response to a depolarizing stimulus.

3-carbamyl-N-allylquinuclidinium bromide: Effects on cholinergic activity and protection against soman

3-Carbamyl- N-allylquinuclidinium bromide (CAB) was synthesized and evaluated for its pharmacological effects on cholinergic activity and for protection in vivo against soman toxicity in guinea pigs. This carbamylated derivative of N-allyl-3-quinuclidinol (NAQ), a potent inhibitor of high-affinity choline uptake, demonstrated stereospecific alterations of cholinergic function as well as protection against soman. The R-isomer, but not the S-isomer, of CAB inhibited erythrocyte acetylcholinesterase (AChE) and plasma pseudocholinesterase (pChE) in a concentration-response manner ( ic 50 = 25 and 29 μM, respectively). The t R-isomer of CAB was also a more potent inhibitor of high-affinity choline uptake ( ic 50 = 4.8 μM) than S-CAB ic 50 = 63 μM). When IRR-CAB (10 μmol kg , i.m.) was administered to guinea pigs 30 min prior to soman in conjunction with atropine (16 mg kg , i.m.) given 1 min postsoman, the compound significantly reduced lethality up to 5 ld 50s. This represents enhanced protection when compared to NAQ (up to 100 μmol kg ); the S-isomer of CAB failed to protect against soman intoxication. The results demonstrate that reversible inhibition of AChE with suppression of acetylcholine synthesis into a single compound, CAB, enhances the protection against organophosphates.

Neuropeptide modulation of muscarinic receptors and function in cerebral cortex of young and senescent rats

The possible influence of several neuropeptides on muscarinic receptor binding and function in fronto-parietal cortex of young and senescent Fischer 344 rats was examined. Low concentrations (100 nM) of cholecystokinin, neurotensin and vasoactive intestinal polypeptide (VIP), added in vitro, enhanced carbachol-stimulated phosphoinositide metabolism in cortical miniprisms from both young and senescent rats, while somatostatin was ineffective. Interestingly, the VIP receptor antagonist [d-parachloro-Phe 6, Leu 17]VIP shifted the dose-response curve for carbachol significantly to the right, indicating inhibition of phosphoinositide hydrolysis. No direct actions of neuropeptides on the number or affinity of [ 3H]-quinuclidinyl benzilate binding sites nor on agonist conformation states of the muscarinic receptor were noted in cortex from young animals. The neuropeptide modulation of phosphoinositide metabolism was selective for muscarinic systems, as norepinephrine-stimulated phosphoinositide hydrolysis was not altered. Pretreatment with hemicholinium-3, an inhibitor of high-affinity choline uptake, did not prevent the neuropeptide effects, indicating the interaction was probably postsynaptic. It is possible that pharmacologic manipulation of peptidergic processes could improve cholinergic neurotransmission in brain.

Cholinergic regulation of arterial pressure by the C1 area of the rostral ventrolateral medulla

In anesthetized, paralyzed rats intravenous administration of the acetylcholinesterase inhibitor physostigmine (PHY) (100 micrograms/kg) evoked a dose-related rise in arterial pressure (AP) and heart rate (HR) and an associated increase in sympathetic nerve activity (SNA). The responses to PHY were blocked by electrolytic lesions of, or microinjection of kainic acid into, a specific site in the rostral ventrolateral medulla containing a cluster of neurons immunoreactive for the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase and corresponding to the C1 area of the nucleus reticularis rostroventrolateralis (RVL). The C1 area and its surround contain a heretofore unrecognized network of varicose neuronal processes and perikarya labeled immunocytochemically with a monoclonal antibody to the ACh-synthesizing enzyme, choline acetyltransferase (CAT). PHY increased, by over 3-fold, the spontaneous activity of reticulospinal cardiovascular neurons in the C1 area which excite preganglionic sympathetic neurons in the intermediolateral spinal column. The effects of PHY on AP, SNA, and the discharge of reticulospinal neurons were blocked by systemic administration of the muscarinic antagonist scopolamine. Microinjections within the C1 area of the RVL of scopolamine, the M2-selective muscarinic receptor antagonist AF-DX 116, or the high-affinity choline uptake inhibitor hemicholinium-3 blocked the pressor effects of PHY. The nicotinic antagonist hexamethonium and the M1-selective muscarinic receptor antagonist pirenzepine were without effect. We conclude that (1) the increases in AP, HR, and SNA elicited by the systemic administration of PHY result from the augmented action of ACh released from cholinergic terminals within the C1 area of the RVL; (2) the locally released ACh acts through muscarinic receptors of the M2 subtype within the C1 area to produce excitation of intrinsic reticulospinal sympathoexcitatory neurons, thereby increasing the activity of sympathetic preganglionic neurons and consequently elevating AP and HR; and (3) while the specific function of the cholinergic innervation of the C1 area in cardiovascular regulation is unknown, it may contribute to the tonic regulation of AP.

Induction of cortical cholinergic hypofunction and memory retention deficits through intracortical AF64A infusions

Ethylcholine mustard aziridinium ion (AF64A), an irreversible inhibitor of high-affinity choline uptake on cholinergic nerve terminals, appears to selectively decrease presynaptic cholinergic markers after intracerebral injection. To restrict AF64A's action to cholinergic terminals within the frontoparietal (FP) cortex, the present study utilized multiple-site cortical infusions of the agent. Following an extensive histological analysis, a dose of 1 nmol AF64A/1 μl was selected for determining AF64A's effects on acetylcholinesterase (AChE) staining, cortical cholinergic/non-cholinergic markers, and passive avoidance behavior. Adult rats given two infusions of AF64A into the right FP cortex had reduced AChE staining throughout 75% of the ipsilateral FP cortex at 10 days following infusion, thus suggesting an extensive cortical diffusion of the agent; minimal non-specific damage was seen (totalling only 4% of the ipsilateral FP cortex for both infusion sites) and no effects on AChE staining were observed in the striatum or hippocampus. Three weeks after bilateral AF64A infusions into the FP cortex (two injections on each side), significant frontal cortex deficits were observed in high-affinity choline uptake, acetylcholine synthesis, acetylcholine release, and hemicholinium-3 binding compared to vehicle-infused controls. However, choline acetyltransferase activity within the anterior cortex did not appear to be consistently affected by AF64A infusion. Cortical glutamic acid decarboxylase activity, as well as cortical monoaminergic markers, and neuropeptide levels were also unaffected. Moreover, animals that received bilateral AF64A infusions and were tested two weeks afterwards showed marked memory retention deficits during both the 24-h and 48-h postshock trials of passive avoidance testing. These results indicate that cortical AF64A infusion induces a specific, long-term cholinergic hypofunction of presynaptic markers within the cortex, resulting in a significant long-term memory impairment. Since the primary cholinergic innervation to the FP cortex, originating in the nucleus basalis of Meynert, appears to become dysfunctional (but not totally degenerative) in Alzheimer's disease, cortical AF64A infusions may closely reflect this cholinergic dysfunction by ‘functionally’ eliminating cortical cholinergic terminals.