piperidines and 1-1--((1-1--biphenyl)-4-4--diylbis(2-hydroxy-2-1-ethanediyl))bis(1-4-dimethylpiperidinium)

The quaternary ammonium salt (E)-4-(1-naphthylvinyl)pyridine hydroxyethyl bromide (B111) and the tertiary amine salt (E)-1-methyl-4-(1-naphthylvinyl)-1,2,3,6-tetrahydropyridine hydrochloride (B115), both previously shown to protect against organophosphate (OP) toxicity, were examined in vivo for effects on rat brain choline acetyltransferase (CAT) activity and acetylcholine (ACh) levels. When administered iv, but not when given ip, B111 was able to inhibit brain CAT 29% and reduce brain ACh levels 25%, yet was unable to prevent soman-induced increases in ACh. B115, which may serve as a depot form of a quaternary ammonium analogue, was able to decrease CAT activity as much as 80% upon multiple ip administration. This CAT inhibitory potency was unprecedented for a tertiary amine salt of its structure. However, ACh levels were reduced by no more than 25% and B115 was ineffective in preventing soman- and sarin-induced increases in ACh. Since the degree of inhibition of CAT activity produced by B111 and B115 was not accompanied by a corresponding decrease in ACh levels, the protection afforded by these compounds against OP toxicity is most likely not related to CAT inhibition. B115 was also tested for its ability to affect cholinergic receptor binding. B115 was administered to rats ip, twice daily, at low doses throughout a 3-week period. Analysis of cortex tissue revealed a 45% increase in nicotinic receptor binding with no change in either total muscarinic receptor binding (M-1 and M-2) or high-affinity muscarinic receptor binding (M-2 alone).

Topics: Acetylcholine; Animals; Atropine Derivatives; Biphenyl Compounds; Brain; Choline O-Acetyltransferase; Delayed-Action Preparations; Drug Administration Schedule; Injections, Intraperitoneal; Injections, Intraventricular; Male; Naphthylvinylpyridine; Organophosphorus Compounds; Parasympatholytics; Piperidines; Rats; Rats, Inbred Strains; Soman

A-4, A-5 and HC-3 are experimental bis tertiary and quaternary amines which have been shown to be potent inhibitors of the sodium-dependent, high affinity choline uptake system. When incubated with neuroblastoma cells, experimental compounds A-4, A-5 and HC-3 inhibit choline metabolism. Over a 24-hr incubation, A-4, A-5 and HC-3 produced a significant decrease in total choline accumulation, choline incorporation into phospholipid and free choline content. However, despite decreases in choline incorporation into phospholipid, no change occurred in content of phosphatidylcholine. Treatment of cells with A-4, A-5 and HC-3 resulted in an increase in the incorporation of S-adenosyl-methionine into phosphatidylcholine. However, the incorporation of ethanolamine or serine into phosphatidylcholine was not increased. Phosphatidylcholine turnover was decreased in cells treated with A-4 and A-5. A-4, A-5 and HC-3 produce significant decreases in choline metabolism; however, the cells are able to maintain membrane integrity by decreasing turnover of phosphatidylcholine and increasing phosphatidylcholine synthesis through the methylation pathway. These studies suggest that the biological effects of A-4 and A-5 are independent of membrane perturbations.

Topics: Animals; Biphenyl Compounds; Choline; Ethanolamine; Ethanolamines; Mice; Neuroblastoma; Phosphatidylcholines; Phospholipids; Piperidines; S-Adenosylmethionine; Tumor Cells, Cultured

The neuroblastoma-glioma hybrid cell (NG108-15) has a sodium-dependent, high-affinity choline transport system with a Km of 16.0 +/- 3.4 microM and a Vmax of 214.5 +/- 27.7 pmol/min/mg protein. A-4, A-5 and HC-3 produce dose-dependent inhibition of high-affinity choline transport in NG108-15 cells. Following 24 h exposure to approximately the EC50 of each inhibitor, no significant decrease was found in total choline accumulation or in choline incorporation into phosphatidylcholine. However, when additional inhibitor was added during the 24 h incubation, significant decreases in choline accumulation were produced by A-4 and A-5. Following 24 h exposure to each compound, only A-4 was able to significantly affect free choline content. In contrast, each inhibitor was able to significantly decrease acetylcholine content following 24 h exposure. Possible reasons for consistent decreases in acetylcholine versus minimal changes in choline metabolism will be discussed.

Topics: Acetylcholine; Animals; Biological Transport, Active; Biphenyl Compounds; Choline; Hemicholinium 3; Neurons; Parasympatholytics; Piperidines; Tumor Cells, Cultured

A-4 and A-5 are tertiary and N-methyl quaternary 4-methylpiperidine analogs of hemicholinium-3 (HC-3). Previous work in this laboratory has shown A-4 and A-5 to be inhibitors of the sodium-dependent, high affinity choline uptake system (SDHACU). Their effects on choline transport were characterized further using neuroblastoma 41A3 cells. These cells rapidly take up choline through two separate mechanisms: a SDHACU system and a sodium-independent, low affinity uptake system (SILACU). A-4, A-5 and HC-3 decreased 5 microM choline transport in a dose-dependent fashion. The compounds were unable to decrease choline transport at 250 microM choline suggesting that they are inactive with respect to SILACU. All three compounds significantly increased the Km but not the Vmax for the SDHACU system, suggesting a competitive mechanism of inhibition. Ki values ranged from 18 to 25 microM for A-4, 20 to 26 microM for A-5 and 68 to 75 microM for HC-3. Dose-response curves for inhibition of choline transport by A-5 and HC-3 were not changed by a 24-hr pre-exposure of the cells to each inhibitor. However, after a 24-hr pre-exposure to A-4, a significantly different dose-response curve was obtained compared to the dose-response curve for A-4 in untreated cells. After a 24-hr pre-exposure, a 4-hr recovery period was sufficient to remove the effect of each compound. These data suggest that A-4 and A-5, like HC-3, inhibit the SDHACU, competitively and reversibly.

Topics: Animals; Biological Transport; Biphenyl Compounds; Choline; Dose-Response Relationship, Drug; Mice; Neuroblastoma; Parasympatholytics; Piperidines; Sodium; Tumor Cells, Cultured

The blood-brain barrier (BBB) transport system for choline and basic drugs was characterized by the in vivo carotid artery injection technique (Oldendorf, Brain Res., 24, 372-376, 1970) and in vitro uptake into isolated bovine brain capillaries (Pardridge et al., J. Neurochem., 44, 1178-1184, 1985). Basic drugs such as eperisone, thiamine and scopolamine significantly inhibited choline uptake by the BBB with the half inhibitory concentration, IC50 value of 1.45, 2.06, and 0.47 mM, respectively. On the contrary, the uptake of choline was not inhibited by amino acids (L-phenylalanine and L-arginine) and acidic drugs (nicotinic acid, salicylic acid and valproic acid). Choline was taken up by the isolated brain capillaries in concentration and temperature dependent manners. The uptake of choline by the isolated bovine brain capillaries was significantly inhibited by eperisone, scopolamine and thiamine in consistent with the in vivo results. Furthermore, eperisone inhibited competitively the uptake of choline with the inhibition constant, Ki value of 455 microM. According to these results it was suggested that in the BBB choline and basic drugs would share a common carrier-mediated transport system.

Topics: Animals; Biological Transport; Biphenyl Compounds; Blood-Brain Barrier; Capillaries; Choline; Drug Carriers; Isoproterenol; Kinetics; Male; Parasympatholytics; Piperidines; Propiophenones; Rats; Scopolamine; Thiamine; Tritium

Rats were injected with either saline; A-4 (40 mg/kg, i.p.), a bis tertiary amine derivative of hemicholinium-3; or A-5 (50 micrograms/kg, i.p.), a bis quaternary amine derivative of hemicholinium-3, 1 h prior to moderate fluid percussion brain injury. A variety of reflexes and responses were measured up to 60 min following injury, and body weight and several neurological measures were taken daily up to 10 days following injury. Pretreatment with either A-4 or A-5 significantly attenuated components of transient behavioral suppression, as well as more enduring deficits in body weight and beam walk and beam balance performance. A-4 administered prior to fluid percussion was found to reduce striatal, but not pontine, acetylcholine content. A-5 did not significantly reduce acetylcholine content in either area. Both A-4 and A-5 pretreatment prevented a significant increase in acetylcholine content in the cerebrospinal fluid following fluid percussion injury; however, only A-5 significantly reduced plasma acetylcholine content. These results confirm cholinergic involvement in the production of both transient and longer-lasting behavioral deficits following traumatic brain injury. Furthermore, traumatic brain injury may allow plasma constituents to gain access to the central nervous system.

Topics: Acetylcholine; Animals; Biphenyl Compounds; Brain Injuries; Cholinergic Fibers; Male; Movement Disorders; Piperidines; Rats; Rats, Inbred Strains

Previous studies have shown that quaternary and tertiary 4-methyl piperidine derivatives of hemicholinium-3 (A-5 and A-4, respectively) are potent inhibitors of choline uptake. The d-, l-, and mesostereoisomers of A-5 and A-4 were separated and the potency and reversibility were compared. Isomeric forms of each compound were found to be approximately equipotent inhibitors in the following preparations: inhibition of rabbit neuromuscular transmission using the sciatic nerve-gastrocnemius muscle preparation, reductions in acetylcholine content in rat caudate tissue slices and inhibition of choline uptake in neuroblastoma cells, line NB41A3. Because these results show no difference in potency or reversibility for the stereoisomers of A-5 or A-4, these studies indicate that hydroxyl substitutions in these agents do not play a role in their biologic activity. Perhaps only 2-point attachment is required for inhibition of choline transport by hemicholinium-like compounds.

Topics: Acetylcholine; Animals; Biphenyl Compounds; Caudate Nucleus; Cells, Cultured; Choline; Hemicholinium 3; Male; Piperidines; Rabbits; Rats; Rats, Inbred Strains; Sciatic Nerve; Stereoisomerism