Effects of calmodulin antagonists on sodium-dependent high-affinity choline uptake.

The effects of calmodulin (CaM) antagonists were investigated on the sodium-dependent high-affinity choline uptake (SDHACU) as assessed by the specific binding of [3H]hemicholinium-3 ([3H]HCh-3) and high-affinity [3H]choline uptake. Potassium depolarization caused a significant 2-fold increase in the specific binding of [3H]HCh-3 in slices of rat striatum in vitro. CaM antagonists, including trifluoperazine (TFP), W-5, W-7, promethazine and haloperidol, dose-dependently inhibited potassium depolarization-stimulated [3H]HCh-3 binding with IC50s of 20, 40, 70, 30 and 48 (microM), respectively. Scatchard analysis revealed that the inhibitory effect of TFP resulted from a decrease in Bmax but no change in Kd of [3H]HCh-3 binding. Potassium depolarization of slices also stimulated high-affinity [3H]choline uptake, which was completely inhibited by 10 microM TFP. These results are discussed in relation to the regulatory mechanisms of SDHACU.

Therapeutic effect of THA on hemicholinium-3-induced learning impairment is independent of serotonergic and noradrenergic systems.

Tetrahydroaminoacridine (THA: Tacrine) has previously been shown to reverse deficits in spatial discrimination learning induced by hemicholinium-3 (HC-3). In the present experiments the effects of prior depletion of serotonin (5-HT) or noradrenaline (NA) on this reversal were examined. In the first experiment 5-HT lesions were made by injecting 5,7-DHT (2 x 50 micrograms/5 microliters) into the lateral ventricles of rats pretreated with desmethylimipramine (DMI 25 mg/kg IP). A permanently indwelling guide tube was then implanted over the right lateral ventricle. Subsequent testing under drug-free conditions, revealed no effect of the lesion on the number of trials needed to attain criterion (nine consecutive correct choices) in two-platform spatial discrimination learning in a watermaze. Using a latin square design rats were then tested for the effects of HC-3 and THA. HC-3 (5 micrograms/5 microliters ICV) or placebo (CSF) were injected 60 min before the start of a 30-trial training session. THA (4.6, 10 mg/kg SC) or placebo were then injected 15 min before training. Choice accuracy but not choice latency was significantly impaired by HC-3 and the effect was reversed by THA in both sham operated and 5-HT lesioned rats. In the second experiment two injections of DSP-4 (50 mg/kg IP) were given, following cannulation, to deplete forebrain NA. The lesion had no effect on spatial learning under drug-free conditions and failed to block the THA-induced reversal of spatial discrimination learning deficits following HC-3. These results confirm that forebrain Ach depletion by HC-3 impairs spatial discrimination learning and that the deficit is reversed by THA.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemicholinium-3 impairs spatial learning and the deficit is reversed by cholinomimetics.

The effects of hemicholinium-3 (HC-3) on spatial discrimination learning were studied. Rats were equipped with indwelling cannulae in the right lateral ventricle and, following recovery, were trained on a two platform spatial discrimination task in a water maze. In this task a visible escape platform remains in a fixed position in the pool during a single training session, whilst the location of an identical "float" (which affords no escape) is randomly varied. For each session the location of the fixed escape platform was changed and the rats were retrained to criterion following pretreatment either with artificial cerebrospinal fluid (CSF) or HC-3 (2.5, 5.0 micrograms/rat/ICV) 1 h before training. Each rat received every treatment according to a latin square design. The results showed that spatial learning was dose dependently impaired by HC-3, choice accuracy being reduced to chance levels by the higher dose. There was no evidence of motoric difficulty, as choice latencies were not significantly increased. Experiments were then conducted to test for reversal of the deficit using a range of psychotropic drugs. Rats were treated with CSF or HC-3 (5 micrograms/rat ICV) 60 min prior to testing and test drugs were injected 15 min before testing. Some doses of physostigmine (46-460 micrograms/kg/SC) and tetrahydroaminoacridine (THA) (2.2-10 mg/kg/SC) reversed the spatial learning deficit. The muscarinic agonists arecoline (0.046-1 mg/kg/SC), aceclidine (1-10 mg/kg/SC), oxotremorine (30-100 micrograms/kg/SC) and RS-86 (0.46, 1.0 microgram/kg/SC) were also effective. Pilocarpine (0.22-2.2 mg/kg/SC) showed marginal activity and isoarecoline (4.6-10 mg/kg/SC) was inactive. Nicotine (0.32, 1, 3.2 mg/kg/SC) and piracetam (10, 30, 100 mg/kg IP) were also inactive. The alpha 2 agonist, clonidine (46, 100 micrograms/kg SC) and the antagonist idazoxan (32, 100 micrograms/kg SC) were also inactive. Learning deficits were not reversed by haloperidol (20, 60 micrograms/kg), amphetamine (0.1, 0.46 mg/kg), the selective 5-HT1A agonist 8-OH-DPAT (30, 100 micrograms/kg) or by the benzodiazapine antagonist ZK-93426 (1, 3.2, 10 mg/kg). The results show that forebrain Ach depletion by HC-3 impairs spatial discrimination learning and these deficits are reversed by cholinesterase inhibitors and some muscarinic receptor agonists. Some degree of pharmacological selectivity is indicated by the failure of a range of other drugs to reverse the impairments.

Methylpiperidine analog of hemicholinium-3: a selective, high affinity non-competitive inhibitor of sodium dependent choline uptake system.

The potency of hemicholinium-3 (HC-3) and its analogs to inhibit sodium dependent high affinity choline uptake were evaluated in rat striatal synaptosomal preparation. Hemicholinium-3 inhibited sodium dependent high affinity choline uptake (IC50 = 18 nM) while the half molecule of HC-3, HC-15, was inactive. The order of potency for choline uptake inhibition of piperidine substituted HC-3 molecule was as follows: 4-methylpiperidine (A-5 and CA-5) much greater than HC-3 much greater than unsubstituted piperidines (CA-1 and A-1) much greater than 2- or 3-methylpiperidine (A-2 and A-3) and 4-hydroxypiperidine (A-7). The tertiary amine derivative of 4-methylpiperidine substituted HC-3 (A-4) was nearly 10-fold less potent than its corresponding quaternary derivative (A-5). Choline uptake was inhibited competitively by HC-3 and non-competitively by A-5. The inhibition of choline uptake by A-5 was readily reversible by washing. A-5 did not inhibit the uptake of dopamine and gamma-aminobutyric acid. These findings suggest that the N-methyl,4-methylpiperidine analog of HC-3 (A-5) is the most potent of all known inhibitors of sodium dependent high affinity choline uptake and that the inhibition of choline uptake by this compound is mediated through a mechanism distinct from a simple competitive one.

Amnesia produced by intracerebroventricular injections of hemicholinium-3 in mice was prevented by pretreatment with piracetam-like compounds.

Intracerebroventricular (ICV) injections of hemicholinium-3 (HC-3) to mice before the training trial in a passive avoidance task produced an amnesic effect at the 24-hour retention test. Pretreatment by IP injection of piracetam, etiracetam, or pramiracetam, 30 minutes before HC-3 injections antagonized the amnesic effects of HC-3. Pretreatment with choline was not effective. The depletion of cerebral acetylcholine by the HC-3 injection was not prevented by piracetam or etiracetam.

Depletion of total acetylcholine by hemicholinium-3 in isolated rat diaphragm is less in the presence of dexamethasone.

Low concentrations of dexamethasone (Dex) stimulate the initial rate of radioactive choline (Ch) accumulation in the endplate-rich area (EPA) of indirectly stimulated hemidiaphragms, while higher concentrations (greater than 0.6 microM) inhibit. This biphasic concentration-effect curve is found even in the presence of 26 microM hemicholinium-3 (HC-3), an inhibitor of Ch accumulation. In incubations (3 min) where the total hemidiaphragm acetylcholine (ACh) content is not altered by 26 microM HC-3, the inhibition by HC-3 of both the Ch accumulation rate and the incorporation of radioactive Ch into ACh in the EPA of stimulated tissues is less in the presence of 0.2 microM Dex. In 120 min incubations with 15 microM HC-3 and without added Ch, the tissue ACh content is depleted in both stimulated and unstimulated hemidiaphragms. In both cases the depletion of ACh is significantly less in the presence of 0.2 microM Dex. In stimulated tissues a comparable depletion of ACh due to 15 microM HC-3 is also found with 1 and 10 microM Ch added to the medium. It is significantly less when 0.2 microM Dex and 1 microM Ch are added to the medium. In 120 min incubations with stimulated tissue, the amount of "bound' ACh is increased by addition of 30 microM Ch to the medium, decreased in the presence of 0.2 microM Dex, and greatly decreased in the presence of 15 microM HC-3. In the presence of Dex plus HC-3, the decrease in the amount of "bound' ACh due to either Dex or HC-3 alone, is abolished provided that 30 microM Ch is also present.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of corticosteroids on sciatic nerve-tibialis anterior muscle of rats treated with hemicholinium-3. An experimental approach to a possible mechanism of action of corticosteroids in myasthenia gravis.

We studied the effect of intraperitoneally administered corticosteroids on the neuromuscular transmission in the sciatic nerve-tibialis anterior muscle preparation of the anesthetized rat stimulated at a rate of 10 Hz. Administered simultaneously with hemicholinium-3 (HC-3), 80 mug per kilogram (that is, half the lethal dose for 50 percent survival), prednisolone and dexamethasone cause a marked reversal of the block of the neuromuscular transmission caused by HC-3. The effect of aldosterone is very small. The blocking action of d-tubocurarine is not antagonized by either prednisolone or dexamethasone. Choline provides total protection against the HC-3 blockade, whereas physostigmine, in a just sublethal dose, is ineffective. We tentatively conclude that in myasthenia gravis the carrier-mediated transport of choline into the nerve endings may be deficient and that the beneficial effect of corticosteroids in this condition is based on their ability to ameliorate the deficient choline transport.

The effect of corticosteroids and hemicholinium-3 on choline uptake and incorporation into acetylcholine in rat diaphragm.

The glucocorticoids prednisolone and dexamethasone antagonize the inhibition by hemicholinium-3 of both the rate of choline uptake and the incorporation of choline into acetylcholine in the rat diaphragm. Aldosterone has no such effects. It is concluded that the beneficial effect of glucocorticoids in the treatment of myasthenia gravis may be due not only to immunosuppression, but also to some direct effect on presynaptic events perhaps via a choline carrier or an enzyme of choline metabolism.

Effect of prednisolone on neuromuscular blocking in mice in vivo.

The working capacity of inbred female Swiss mice on the rotating rod was tested in an investigation of the effect of previously injected prednisolone, 1.2 mg, on the neuromuscular blocking effect of two different doses of d-tubocurarine and of low-dose hemicholinium-3. The effect of both drugs could be partially or wholly antagonized by prednisolone. The consequences of this finding are discussed in relation to the beneficial effect of prednisone in patients with myasthenia gravis.

Antagonism of corticosteroids against the lethal effects of hemicholinium-3 in rats and mice.

The corticosteroids prednisolone or dexamethasone administered simultaneously with hemicholinium-3 (HC-3) by i.p. injection, increased the LD50 of HC-3 in rats and mice about twofold. Daily treatment with prednisolone for 7 days also increased the LD50 of HC-3 in rats. Choline completely protected rats and mice against HC-3. A sublethal dose of physostigmine did not change the LD50 of HC-3 in rats but increased the LD50 in mice to a small extent. The possible implications of these experiments for the explanation of the beneficial effects of corticosteroids in myasthenia gravis are discussed.

Hydrocortisone and the concentration of choline in the plasma of rodents.

Cortisone has been reported to reduce significantly the plasma choline concentration in dogs within 30 min after its injection. However, in the present experiments hydrocortisone had no effect on the plasma choline concentration of mice, guinea-pigs and rabbits. The protective effect of hydrocortisone against the toxic effects of hemicholinium in mice which was described previously cannot, therefore, be attributed to changes in the concentration of plasma choline.