John J. O'Neill

Inhibition of High‐Affinity Choline Uptake and Acetylcholine Synthesis by Quinuclidinyl and Hemicholinium Derivatives

Abstract: Choline uptake into cholinergic neurons for acetylcholine (ACh) synthesis is by a specific, high‐affinity, sodium‐ and temperature‐dependent transport mechanism (HAChU). To assess the role of choline availability in regulation of ACh synthesis, the structure‐activity relationships of several hemicholinium (HC) and quinuclidinyl analogs were evaluated in a dose response manner. As confirms previous studies, the HCs, e.g., HC‐3, acetylsecohemicholinium, and HC‐15 are potent inhibitors of HAChU, HC‐3 being the most potent (I50= 6.1 × 10−8M). In the present study, the most potent quinuclidinyl derivative was the N‐methyl‐3‐quinuclidinone (I50= 5.6 × 10−7M). This compound had approximately 100‐fold greater inhibitory activity than the corresponding racemic alcohol, suggesting that the 3‐hydroxyl functional group is not absolutely essential for activity. Increasing the size of the N‐functional group from a methyl to an allyl in the alcohol led to a 10‐fold increase in activity. However, removal of the quaternizing N‐methyl group yielding the tertiary amine, 3‐quinuclidinol hydrochloride, greatly reduced its capacity to inhibit HAChU. Of the 2‐benzylidene‐3‐quinuclidinone derivatives studied, only the m‐chloro derivative significantly reduced HAChU.

Non-Cholinesterase Effects of Anticholinesterases

The presence in the nervous tissue of extremely potent peptides arises from the enzymatic degradation of larger molecular weight proteins. These smaller units are thought to exert modulatory actions on known transmitters. Their short half-life when injected is due to the degradative action of specific EXO and ENDOPEPTIDASES. It is suggested from the meager evidence available that organophosphates may adversely affect the processes of formation and/or degradation to account for many of the adverse effects of organophosphate anticholinesterases which cannot be attributed to acetylcholine accumulation.

Effect of Inorganic Lead on Rat Brain Mitochondrial Respiration and Energy Production

Abstract: Toxicologically significant amounts of inorganic lead were added to rat brain mitochondrial preparations that did not contain EDTA or Pi. The binding of the lead to the mitochondria was measured by anodic stripping voltometry. In the presence of lead, the respiratory control ratios decreased, implying a decrease in the degree of dependence of respiration on a phosphate acceptor. Nucleotide contents were also measured, and in the presence of inorganic lead the actual amounts of ATP formed from ADP were found to be significantly decreased as well.

In vivo protection against soman toxicity by known inhibitors of acetylcholine synthesis in vitro

Soman inhibits the enzyme acetylcholinesterase, essentially irreversibly, producing an accumulation of acetylcholine (ACh) which is responsible for many of its toxic effects. Current approaches to treatment include: (1) atropine, a muscarinic receptor blocker; (2) pyridine-2-aldoxime methylchloride (2-PAM), an enzyme reactivator; and (3) carbamate protection of the enzyme. However, no fully satisfactory regimen has been found, primarily because of the rapid aging process. In this study, compounds known to inhibit ACh synthesis in vitro were evaluated in combination with atropine and 2-PAM so as to assess their potential utility in protection against soman toxicity in rats. Acetylsecohemicholinium (100 micrograms/kg, i.c.v.t., 30 min prior to soman), an inhibitor of high affinity choline uptake (HAChU) and cholineacetyltransferase (ChAT) activity in vitro, enhanced the protective effects of atropine and 2-PAM, reducing the mortality within the first 2 hr following soman. N-Hydroxyethylnaphthylvinylpyridine (NHENVP), a quaternary ChAT inhibitor (1.7 mumol/kg, i.m.), significantly reduced the overall percent mortality due to soman from 80% to 20%. The compound was most effective when administered 2-3 min prior to soman and was effective only by the intramuscular route. N-Allyl-3-quinuclidinol, a potent HAChU inhibitor (1 mumol/kg, i.m.) was the most effective quinuclidine analog evaluated, also reducing the percent mortality for a 24-hr period. Unlike NHENVP, it was most effective when given 30-60 min prior to soman. It is suggested from the data that compounds that disrupt presynaptic ACh synthesis in vitro may prove effective in treating organophosphate poisoning. The results demonstrate interesting differences among the compounds studied and provide insight for the design of protectants against soman toxicity. These findings further underscore the need to examine the structure activity and pharmacokinetic properties of these compounds, i.e. comparison of routes of administration, dose-response relationships, and time to effect.

Effect of Chronic Lead Ingestion by Rats on Glucose Metabolism and Acetylcholine Synthesis in Cerebral Cortex Slices

Abstract: The effect of chronic low‐level lead (Pb2+) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb2+‐exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs‐Ringer bicarbonate buffer with 10 mM glucose and tracer amounts of [6‐3H]glucose and either [6‐14C]glucose or [3‐14C]β‐hydroxybutyrate. Altering the concentration of Pb2+ in the drinking water produced a dose‐related increase in blood and brain lead levels. When tissue from Pb2+‐exposed rats was incubated with mixed‐labeled glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the 3H/14C ratios. Similar effects of Pb2+ were found when 14C‐labeled β‐hydroxy‐butyrate was substituted for the [14C]glucose. It appears from these data that Pb2+ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb2+‐induced changes observed in cholinergic function.

β‐Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine

Abstract— Rat brain cortex slices were incubated with 10 mm‐glucose and trace amounts of [6‐3H]glucose and [3‐14C]β‐hydroxybutyrate. The effects of (‐)‐hydroxycitrate, an inhibitor of ATP‐citrate lyase; methylmalonate, an inhibitor of β‐hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and 3H:14C ratios calculated. Incorporation of [14C]β‐hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of 14C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [3H‐,14C]‐glucose. (–)‐Hydroxycitrate (5.0 mM) reduced incorporation of [3H]glucose and [14C]β‐hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm) showed a decrease in 14C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm) also produced a decreased 14C incorporation into ACh and its precursors. At 10 mm, acetoacetate reduced 3H and 14C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β‐hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.

Canine spinal cord energy state after experimental trauma

INJURY to the spinal cord causes tissue changes which become irreversible with time, usually resulting in permanent loss of neurological function below the level of the trauma site. Little is presently known about the biochemical events occurring after spinal cord trauma. Changes in tissue metabolism such as a severe depletion of tissue energy stores may be responsible for the ultimate failure of neurological function after injury. KHAN et al. (1974) reported that tissue ATP and phosphocreatine declined rapidly and found a significant increase in lactic acid in traumatized cat spinal cord. Locm et al. (1971) also observed increased concentrations of lactic acid at the trauma site after injury of monkey spinal cord. In the present paper we report the effects on tissue energy metabolites of controlled experimental injury to the spinal cord of the canine as a function of time after trauma. A preliminary report of these findings has appeared in the literature (WALKER et al., 1976).

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 (IC50 = 25 and 29 microM, respectively). The R-isomer of CAB was also a more potent inhibitor of high-affinity choline uptake (IC50 = 4.8 microM) than S-CAB (IC50 = 63 microM). When R-CAB (10 mumol/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 post-soman, the compound significantly reduced lethality up to 5 LD50S. This represents enhanced protection when compared to NAQ (up to 100 mumol/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.

Vesamicol, an inhibitor of acetylcholine vesicle packaging, increases synaptophysin phosphorylation in rat cortical synaptosomes

Vesamicol (AH5183) is an inhibitor (IC50, 50 nM) of acetylcholine (ACh) vesicle packaging. Vesamicol increases the phosphorylation pattern of synaptophysin (p38), identified as a vesicle-specific phosphoprotein involved in vesicle-mediated neurotransmitter release. Percoll fractionation of the rat cortex yielded a cholinergic-enriched synaptosomal Fraction 4. Fraction 4 contained the highest enrichment of cholineacetyl-transferase activity (86 +/- 4.6 mumole AcCh/g protein/hr.) in the Percoll gradient. Fraction 4 demonstrated oxygen consumption (108 +/- 23.4 nmole/mg protein), levels of adenosine triphosphate, ATP, (10.29 +/- 0.45 nmole/mg protein) and adenosine diphosphate, ADP, (10.54 +/- 2.72 nmole/mg protein), energy potential (ATP/[ADP] [Pi], (0.49) phosphate uptake (65-80 nmoles phosphate/mg tissue), 32Pi labelling (130 +/- 12 x 10(5) DPM/mg tissue; 74 +/- 9.8 x 10(2) nmoles phosphate/mg tissue). Synaptophysin was identified by Western blotting and confirmed by qualitative immunoprecipitation. Synaptophysin phosphorylation was confirmed by autoradiograph. Synaptophysin phosphorylation increased (225%) in the presence of vesamicol (ED50, 1 nM) in Fraction 4. Vesamicol (50 nM) and vanadate (54 microM) were compared for their effects on synaptophysin. This study suggests that during the inhibition of acetylcholine packaging by vesamicol that synaptophysin is phosphorylated. Therefore, the phosphorylation and dephosphorylation of synaptophysin may be involved in the transport of acetylcholine in or out of the synaptic vesicle.

SYNAPTOPHYSIN IS PHOSPHORYLATED IN RAT CORTICAL SYNAPTOSOMES TREATED WITH BOTULINUM TOXIN A

Phosphorylation and dephosphorylation of neuronal proteins have been implicated in regulation of synaptic transmission. Studies were performed to determine if synaptophysin was phosphorylated or dephosphorylated during exposure of synaptosomes to botulinum toxin A (BoTX/A). Cholinergic-enriched synaptosomes were preincubated in the presence of 3H-choline to label newly synthesized acetylcholine (3H-ACh). This was followed by incubation with low or high potassium to stimulate release of newly synthesized 3H-ACh. BoTX/A inhibited total Ach release by 15-19% and inhibited release of newly synthesized 3H-ACh by 35%. A 165% increase in synaptophysin phosphorylation occurred in a dose-dependent manner over a range of doses (0.2 nM, 2 nM, 20 nM, 100 nM) of BoTX/A. When 4-Aminopyridine was added to synaptosomes that were BoTX/A treated, synaptophysin was dephosphorylated to control levels. Synaptosomes incubated with BoTX/A exhibited an inhibition of potassium stimulated ACh release and an increase in synaptophysin phosphorylation. Synaptophysin phosphorylation may be involved in inhibition of acetylcholine release.