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WelChol ® (colesevelam hydrochloride), a bile acid sequestrant for the treatment of hypercholesterolemia, was evaluated for adverse effects on reproduction and fetal development using standard preclinical tests. During gestation, Sprague–Dawley rats used in the developmental toxicity study received feed, feed/control article or feed plus 300, 1000 or 3000 mg/kg/day colesevelam whereas rats in the pre- and postnatal toxicity study received vehicle or 100, 300 or 1000 mg/kg/day colesevelam via gavage. New Zealand white rabbits received control or 100, 500 or 1000 mg/kg/day colesevelam via gavage. No deaths, premature deliveries or gross pathologic lesions were observed up to gestation day (GD) 20 for rats and GD 28 for rabbits. No significant differences in the number of pregnant animals, average litter size, percentage of viable fetuses, fetal body weights, number of corpora lutea, fetal viability, or gross malformations were observed versus controls. Pre- and postnatal effects were assessed in pregnant rats receiving 100, 300 or 1000 mg/kg/day colesevelam from GD 6 to postpartum day 22. Gestation, parturition and lactation in F 0 generation dams were similar between treatment and control groups. Colesevelam did not affect physical or neurological development or induce gross pathological changes in F 1 generation rats. Colesevelam does not produce developmental toxicity in rats or rabbits, nor does it exhibit pre- or postnatal toxicity in rats at the tested doses.

Deferitrin, a novel, orally available iron chelator, is in the early stage of clinical development for the treatment of chronic iron overload due to transfusional therapy. Preclinical pharmacology studies demonstrate iron excretion largely by the fecal route. Initial clinical studies have shown deferitrin to be well absorbed. Further clinical studies are ongoing to determine the efficiency and safety of deferitrin.

The relative abilities of a series of local anesthetics in which either or both the ester oxygens had been replaced with sulfur or selenium to block axonal or synaptic preparations are compared with their abilities to displace 3 H-nicotine from lobster axon plasma membrane fragments. The relative affinities of a series of cholinergic agonists and antagonists for synaptic and axonal membranes are discussed as is the salt sensitivity and reversibility of their interactions. The utility of a conjugate of α-bungarotoxin and horse-radish peroxidase for the histochemical visualization of binding sites of intact axons or axonal vesicles is discussed. Labelling of a peptide isolated from axon plasma fragments with MBTA is compared with labelling of peptides isolated from synaptic membranes.

An aerobic microorganism isolated from deep sea thermal vents in the Gulf of California and grown with the model coal compound dibenzothiophene (DBT) was found to produce DBT oxidation products, including DBT-sulfoxide and DBT-sulfone. The organism can utilize DBT as sole carbon and sulfur source, but grows better when the medium is supplemented with yeast extract. After five to seven days of incubation with DBT, some DBT-sulfoxide and, to a lesser extent, DBT-sulfone, intermediates in the desulfurization of DBT, were detected. When the culture was grown in medium with DBT-sulfoxide as substrate, little oxidation occurred, but when DBT-sulfone was used as substrate, it was rapidly degraded. The extracellular extract is also active against DBT, producing sulfur oxidation products.

We have previously reported that the 10s molecular form (G4) of acetylcholinesterase (AChE) is selectively lost from several cortical areas of Alzheimer's disease (AD) brain. In the current follow-up study, we microdissected several areas of nondemented and AD brain, including the hippocampus, amygdala, and cingulate gyrus. Tissue homogenates were separated on sucrose density gradients and the resulting fractions were analyzed for AChE activity in order to define the ratios of the predominant AChE molecular forms (G4/G1). Both the hippocampus and amygdala exhibited distinct patterns of alterations in the G4/G1 ratio which correlate with the known distribution of histopathological changes in AD brain. In order to further define the major pool of AChE that is depleted in AD, we separated fractionated tissue homogenates into salt-soluble and detergent-soluble fractions. The G4/G1 ratios were only altered in the detergent-soluble fractions, indicating that the loss of the G4 AChE molecular form involves a selective depletion of the membrane pool. Available evidence would suggest that this form is the AChE molecular form physiologically relevant at the cholinergic synapse.

Colesevelam hydrochloride (HCl) (WelChol; Sankyo Pharma) is a novel, highly potent, bile acid-binding polymer used for the treatment of hypercholesterolemia. The primary aim of this study was to determine the effects of dietarily administered colesevelam HCl on fertility and reproductive performance parameters. To assess these effects, sexually mature Sprague-Dawley rats were randomized to one of five treatment groups: feed alone, feed plus control article (SigmaCell), or feed plus colesevelam HCl 200, 1000, or 2000 mg/kg/day. Male and female rats were administered the appropriate group agent for 28 and 15 days, respectively, and were subsequently paired together for cohabitation and mating. Females continued to receive the test agent in their dietary formulation through presumed gestation day (GD) 7. Presumed pregnant females underwent cesarean section on GD 20. Food consumption rate, body weight, gross necropsy, and standard preclinical tests for reproduction and fertility were performed for each test animal. No statistically significant differences were found between control and drug-treated groups for any tested endpoints of reproduction. All animals placed in cohabitation successfully mated. Uterine and litter end points were unaffected by dosages of colesevelam HCl as high as 2000 mg/kg/day. There were no significant differences between treatment group litter averages in the number of corpora lutea, implantation sites, litter size, live fetuses, body weights, early/late resorptions, and the number of dams with viable fetuses. In addition, no external alterations of fetal morphology were attributable to treatment with colesevelam HCl when administered up to the embryo implantation stage. In male animals, no significant differences were found between the colesevelam HCl and control study groups in the average caudal epididymal sperm count or sperm concentration, total number of motile and nonmotile sperm, and the total percentage of motile sperm. Based on these data, colesevelam HCl does not have any significant adverse reproductive or fertility effects in rats, even when administered at doses approximately 30 times greater than the approved clinical dose.

Tetrahydroaminoacridine (THA; Tacrine) is a potent, non-competitive inhibitor of the neuronal enzyme acetylcholinesterase (AChE) and, consequently, a potent modulator of central cholinergic function. The compound reportedly improves the memory deficits of Alzheimer's dementia. Experiments were run with purified bovine caudate AChE to examine the kinetic properties of THA-AChE interaction within the scheme of multiple binding sites on the enzyme and a proposed "map" of the enzyme surface. The kinetic analyses were also designed to determine whether chemical modification of peripheral anionic sites on AChE may provide insight into mechanism for selective pharmacological alteration of cholinergic function in the brain. The studies demonstrated that THA is a reversible, non-competitive inhibitor with an I50 of 160 +/- 10 nM. THA bound primarily at a hydrophobic area outside of the catalytic sites, and binding of THA enhanced the effect of Ca2+ binding to a separate group of "accelerator" sites. Experiments with Al3+ demonstrated non-competitive inhibitor effects that were additive with THA inhibition and consistent with a model suggesting interaction of THA and Al3+ at the enzyme surface. In vitro enzyme inhibition studies also provide evidence for THA "protection" of the catalytic site against inhibition by the high-affinity phosphorylating agent, DFP (isoflurophate).

Experimental and clinical evidence continues to accumulate regarding the beneficial effects of omental placement upon the brain and spinal cord for repair of apparently irreversible injuries. Of particular interest have been the rapid neurological changes observed 24 h to several days following omental transposition in some stroke patients with long-standing motor and speech deficits (1). The observation that these changes could occur years after a stroke raised the possibility that omental tissue might contain neurotrophic factors that facilitate regenerative and possibly synaptogenic events in the central nervous system.

Giant axons isolated from the circumesophageal connectives of the lobster were used to determine the effects of the thiol reagent, mercurochrome, on the excitability of the axonal membrane. Intracellular microelectrode recordings demonstrated that mercurochrome depolarized the resting membrane potential only slightly but completely blocked conduction of the propagated action potential. Partial conduction block was markedly potentiated by repeated electrical stimulation. To investigate whether mercurochrome produced a specific shift in membrane ion selectivity, the compound was tested in low Na + and high K + media. Calculation of transference numbers demonstrated that mercurochrome lacked any marked Na or K ion channel specificity. Thiol group protection experiments suggest that some of the effects of mercurochrome may not necessarily involve thiols. Treatment of the axons with a disulphide reducing agent prior to the application abolished the potentiating effects of electrical stimulation, but decreased the sensitivity of the nerve fibres to mercurochrome possibly through the involvement of non-specific disulphide groups.

The attachment of 125 I-α-bungarotoxin (BgTx) which is reportedly bound exclusively to “nicotinic” acetylcholine receptors, as well as 3 H-atropine and 3 H-3-quinuclidinyl benzilate (QNB), which reportedly bind exclusively to “muscarinic” receptors, was measured in isolated lobster axon plasma membrane fragments and in the soluble axonal protein fraction. 125 I-α-BgTx binding was also measured in lysolecithin-solubilized fragments. Binding assays were adapted for these studies and are described in detail. High affinity, saturable binding of all three ligands to membrane fragments was observed, as well as binding of BgTx to a macromolecule present in both the soluble fraction and the membrane fragments. These experiments provide the first evidence for the very tight binding of both “nicotinic” and “muscarinic” ligands in peripheral nerve.

A conjugate of alpha-bungarotoxin with horseradish peroxidase was used to visualize alpha-bungarotoxin binding sites at the fine structural level in isolated axonal membrane vesicles from lobster walking leg nerve. These plasma membrane vesicles have previously been shown to exhibit saturable binding of [3H]nicotine and [3H]acetylcholine. Binding of the toxin was identified in the axon plasma membrane and could be blocked by pretreatment with excess free alpha-bungaratoxin or d-tubocurarine. Binding sites for alpha-bungarotoxin were identified by the same technique in sections of intact nerve fibers from both lobster and spider crab and were found to be localized primarily in the axolemma rather than in the Schwann cell membrane.

Choline acetyltransferase (ChAT), the enzyme responsible for the formation of ACh from choline and acetyl-coenzyme A, is a marker of cholinergic function and is significantly depressed in the brains of Alzheimer patients. It has been shown that omental tissue contains several neuroactive substances and causes revascularization when placed upon the brain of stroke patients. In this study, it was demonstrated that omental tissue exhibits specific ChAT activity. This activity was choline-dependent, inhibited by N-ethylmaleimide (a known ChAT inhibitor), and was characterized by kinetic parameters consistent with values for the neuronal enzyme. It is suggested that omental placement to the brain together with oral choline administration might prove to be useful for increasing ACh synthesis in Alzheimer's disease.

Although it has been shown that Ca 2+ can inhibit the conduction blocking action of local anesthetic agents in lobster peripheral nerve, it is not known whether Ca 2+ directly competes for anionic membrane sites or indirectly alters the drug-membrane interaction. The present experiments were carried out during a sufficiently long time course to determine whether the blocking action of lidocaine, procaine, or tetracaine was reduced by Ca 2+ or whether Ca 2+ only delayed the onset of local anesthesia. Lidocaine HCl (5 m m ) reduced the amplitude of the compound action potential by 50% in 10 min in low-Ca 2+ (5 m m ) lobster physiologic saline, whereas in high-Ca 2+ (75 m m ) saline, 5 m m lidocaine reduced the action potential amplitude 50% only after 25 min external bath perfusion. Similar results were seen with 2 m m procaine and 0.2 m m tetracaine. The data confirm the suggestion that Ca 2+ merely slows the passage of local anesthetic agents across glial membranes and are not compatible with a model that describes competition of Ca 2+ and local anesthetic agents for a specific binding site in nerve membranes.

Conformationally defined enantiomeric local anesthetics and fluorescent dyes were synthesized. Neither the local anesthetics nor the fluorescent probes showed stereospecificity in interacting with nerve membranes. The fluorescence signals generated by the dyes showed excellent correlation with the time course and shape of the nerve action potential.

Measurements of altered activity of soluble acetylcholinesterase from E. electricus electric organ by the inorganic cations aluminum, scandium and yttrium demonstrate that these ions are noncompetitive enzyme inhibitors. Al3+ inhibited enzyme activity at all substrate and inhibitor concentrations studied. Inhibition by Al3+ did not appear to be sensitive to the active site-specific, competitive ligand physostigmine or to calcium, a peripheral site-binding activator cation. Inhibition by another peripheral site-binding noncompetitive inhibitor, decamethonium, was not altered by Al3+. Al3+ appears thus to have interacted with a class of peripheral anionic sites on AChE distinct from the beta- or P1 peripheral anionic sites that bind Ca2+ and C-10 and may be a useful probe of a subclass of gamma- or P2-4 peripheral anionic sites. A possible mechanism for Al3+ neurotoxicity, via alterations of the enzymes of cholinergic neurotransmission, is also suggested.

Mean levels of the two hydrolases angiotensin-converting enzyme (ACE) and acetylcholinesterase (AChE), the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and total protein concentration were examined in cerebrospinal fluid (CSF) samples from a group of patients with dementia of the Alzheimer's type, a group of comparably demented patients with Parkinson's disease, and a neurologically healthy elderly control group. Both pathological groups exhibited a significant decrease in the mean levels of ACE activity and DOPAC per milliliter and were distinguishable from one another based on mean CSH HVA levels. Unlike the Parkinson's disease group, whose mean concentration of HVA was lower than, but not significantly different from that of the control group, the mean HVA concentration of the Alzheimer's disease group was significantly elevated. In contrast, comparisons of the mean CSF AChE activity (expressed per milliliter or per milligram of protein) and CSF total protein concentration did not reveal significant differences for any of the groups. Independent of CSF protein concentration, ACE activity per milliliter exhibited a positive correlation with AChE activity per milliliter within the control and Parkinson's disease groups, whereas a statistically significant correlation for these CSF hydrolases was not observed within the Alzheimer's disease group. Thus, the CSF profiles for patients with mild dementias associated with Alzheimer's or Parkinson's disease differed by at least two neurochemical criteria. Based on the levels of ACE activity, DOPAC, and HVA per milliliter of CSF, two discriminant functions were derived and resulted in the correct classification of 71% of all subjects (n = 38) into Alzheimer's disease, Parkinson's disease, and neurologically healthy control groups.

Choline acetyltransferase and acetylcholinesterase enzymatic activities were measured in 33 cytoarchitectonic subregions of the cerebral cortex in two rhesus monkeys. As expected, the hippocampus and amygdala were rich in these enzymes. In addition, the paralimbic (mesocortical) regions of the brain (e.g., parahippocampal, insular, caudal orbitofrontal, and temporopolar areas) also contained high levels of both enzymes. In contrast, the concentration of these cholinergic markers was the lowest within all frontal and temporoparietal association areas. As a group, the primary sensory and motor regions contained an intermediate level of choline acetyltransferase activity. Both cholinergic markers also showed a gradual increase from the isocortical toward the more primitive periallocortical subsectors of paralimbic areas. These anatomical patterns have potential implications for the role of cholinergic pathways in the memory process and in the pathogenesis of Alzheimer's disease.

The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl3 produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl3, initial activity was markedly increased. LaCl3 exerted a marked, concentration-dependent inhibition of intact cell AChE.

1. Acetylcholinesterase (AChE) activity of axonal membrane vesicles prepared from lobster (Homarus americanus) walking leg nerve is about 200 mumol ACh/hr/mg protein. 2. The enzyme is a membrane-bound glycoprotein that can be solubilized by lysolecithin and digitonin but is rapidly inactivated by Triton X-100 and other nonionic detergents. 3. The walking leg nerve enzyme contrasts most markedly with postsynaptic AChE in its lack of inhibition by excess substrate and its poor susceptibility to peripheral anionic site ligands. 4. Axonal AChE is competitively blocked by eserine sulfate (1 microM) and by low concentrations of procaine (0.1-0.5 mM). 5. Lidocaine, a tertiary amine, and its quaternary ammonium derivative QX-314 are equipotent non-competitive enzyme inhibitors (Kiapp 2-3 mM), while the primary amine compound GX-HCl is totally inactive. 6. Calcium (10 50 mM), propidium (1 microM) and decamethonium (1 microM), peripheral anionic site ligands in the synaptic enzyme, do not protect the axonal enzyme against inhibition by local anesthetics. 7. Although it is possible that this enzyme is an isozyme of the true AChE of excitable membranes, or that it is an incomplete enzyme fragment, it is also possible that there is a unique membrane-bound AChE present in large quantities in crustacean peripheral nerve.

Cholinesterases, including pseudocholinesterase (BChE) of human plasma and acetylcholinesterase (AChE) of erythrocytes and cerebrospinal fluid (CSF), have been considered as possible markers in dementia of the Alzheimer type (DAT). Reported data, however, are widely varied, and no significant pattern emerges when total enzyme activity is assayed. In the present studies, we have reexamined the relationship of ChE activities in DAT and control patients. ChE activity was measured in plasma, erythrocytes, and CSF from DAT patients and compared with normal controls as well as with samples from patients with a diagnosis other than DAT. Early age onset (presenile) and late age onset (senile) DAT were also compared. No significant differences in total enzyme activity were found in any of the comparisons. Calculations of AChE/BChE ratios in CSF also provided no significant indication of any changes in ChE activities in DAT. It is suggested that measurements of total AChE or BChE activity in these biological materials do not provide a useful index of alterations in central cholinergic function in patients with DAT.

125I-alpha-Bungarotoxin (alpha-BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of alpha-BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible (KappD congruent to 1.7 +/- 0.32 X 10(-7) M; Bmax congruent to 249 +/- 46 pmol/mg protein) at pH 7.8, 10 mM-Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca2+ and Mg2+) in the medium. 125I-alpha-BGT binding could be inhibited by cholinergic drugs (atropine congruent to d-tubocurarine greater than nicotine greater than carbamylcholine congruent to choline) and local anesthetics (procaine greater that tetracaine congruent to lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4-aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to alpha-BGT binding sites in other tissues are discussed.

Summary Fluorescence techniques have been used to measure the binding of mercurochrome to walking leg nerve bundles of the spider crab. Reversibility of binding of mercurochrome by 2-mercaptoethanol was also measured. Both parameters were increased by electrical stimulation of the nerve bundle.

1. Studies are in progress to characterize the nature of ligand interactions at peripheral anionic sites on mammalian brain AChE, including the β-anionic or “accelerator” anionic sites where enzyme activity is increased upon Ca 2+ binding. 2. Terbium was studied as a fluorescence probe of Ca 2+ binding sites in partially purified AChE from whole rat brain. 3. Scatchard analysis of Tb 3+ binding in low ionic strength (2 mM) Pipes buffer revealed at least two populations of sites: high affinity sites with K d (app) ∼ 7.6 μ M and low-affinity sites with a K d (app) ∼ 49.6 μ M. 4. Low-affinity binding was selectively inhibited by 50 mM NaCl; high-affinity binding was completely inhibited by 2mM CaCl 2 ; and all the bound Tb 3+ could be displaced by 1 mM EDTA. 5. The heterogeneity of Tb 3+ binding sites is consistent with the multiple, concentration-dependent effects of Tb 3+ on enzyme activity.

Kinetic analysis of the interaction of trivalent cations with mammalian brain acetyl-cholinesterase revealed at least three distinct concentration-dependent effects on enzyme activity. Acetylcholinesterase was purified from bovine caudate nucleus by affinity chromatography to a specific activity of 1.1 mmoles acetylthiocholine · hr −1 · (mg protein) −1 . The cations studied included the chloride salts of lanthanum, terbium, yttrium and scandium in low and high ionic strength buffers (2mM Pipes ± 0.1 M NaCl). At low ionic strength, high affinity noncompetitive or allosteric activation was observed at very low cation concentrations (1–10 μm); at higher concentrations (50–200 μM) these cations were noncompetitive inhibitors; and at 200–500 μM they exerted a mixed competitive-noncompetitive inhibition. Activation by low cation concentrations was not evident in high ionic strength buffers, while enzyme inhibition by all the trivalent cations was similar at low and high ionic strength. Inhibition by all of the multivalent cations was fully reversed by a 10-fold excess of EDTA or by a 100-fold dilution of the inhibited enzyme. The water-soluble carboxyl group affinity reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, was shown to specifically block the activating effect of the multivalent cations supporting the suggestion that the β- or “activator” peripheral anionic sites (P 1 ) involve a carboxyl group outside the enzyme active site.

Acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, is a heterogeneous enzyme that can be separated into multiple molecular forms. A tetrameric membrane-bound form (G4) and a monomeric soluble form (G1) are the two predominant enzyme species in mammalian brain. The distribution of AChE molecular forms was defined by sucrose density gradients of 11 anatomical regions of postmortem brains from 10 patients with dementia of the Alzheimer type (DAT) and 14 nondemented controls of similar ages. The results demonstrate an overall loss of protein and enzyme activity in all areas of the DAT brains studied and a selective loss of the G4 form of AChE in Brodmann areas 9, 10, 11, 21, 22, and 40, and the amygdala. There was no change in the G4/G1 ratio in areas 17 and 20, in the hippocampus, or in the cerebellum. There was a high regional correlation of the G4/G1 ratios with published values for choline acetyltransferase activity but lower correlation with total AChE activity. We propose that there is a predominant loss of the G4 form of AChE in DAT and that this loss is correlated with the degeneration of presynaptic elements.

It has previously been shown that terbium binds to membrane vesicles prepared from the walking leg nerve of the lobster (Homarus americanus) with a high affinity Kd of 2.2 microM. Fluorescence of bound Tb3+ occurs via energy transfer from the aromatic residues of proteins (gamma ex = 280 nm; gamma em = 546 nm), and calcium inhibits Tb3+ binding competitively with a Ki of 1.8 mM. Displacement studies with EDTA demonstrate that more than 95% of the bound Tb3+ is at the vesicle exterior and is not being taken up by the vesicles. To investigate the putative role of Ca2+ in the interaction of local anesthetics with axonal membranes, lidocaine and the analogs GX-HCl and QX-314 were tested as inhibitors of Tb3+ binding. Inhibition by lidocaine is seen only at considerably higher doses (25 mM) than are required for conduction block of intact nerves (5 mM). Inhibition by lidocaine and the primary amine analog GX-HCl is entirely noncompetitive, whereas the quaternary ammonium derivative QX-314 appears to be a mixed competitive-noncompetitive inhibitor of Tb3+ binding. These data are not compatible with the hypothesis that there is a functionally essential cation binding site on the axonal membrane surface for which Ca2+ and local anesthetics compete, although local anesthetic action may be modified indirectly by altered calcium concentrations. Evidence is presented for a mechanism by which local anesthetics indirectly displace Tb3+ by altering the physical state of the axonal membrane.

Tb 3+ , a fluorescent trivalent cation with physicochemical properties similar to Ca 2+ , binds to peripheral nerve membrane vesicles prepared from the walking leg nerve bundle of the lobster ( Homarus americanus ). Saturable binding is measured for at least two classes of binding site. Bound Tb 3+ can be displaced by other cations in the order: Ca 2+ > Mg 2+ = Zn 2+ > NH 4 + . The binding of Tb 3+ to the lower affinity site ( K D (app) = 6.0 μ M) is inhibitable by Na + , Mg 2+ and Ca 2+ , whereas the higher affinity site ( K D (app) = 2.2 μ M) is only sensitive to Ca 2+ . Using this spectral probe the role of Ca 2+ in peripheral nerve membrane function can be investigated.

• We examined the relationship of disease laterality to neuropsychological and neurochemical features in patients with idiopathic Parkinson's disease (PD). We tested patients with PD, patients with Alzheimer's type of senile dementia, and a control group neuropsychologically, and we determined their CSF levels of homovanillic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindolacetic acid, serotonin, and acetylcholinesterase. The patients with PD were divided into two groups depending on the side of the body with greater disease involvement. Both parkinsonian groups, those more affected on the left (group L) and those more affected on the right (group R), were otherwise similar in all other clinical and historical features. Group L patients showed greater neuropsychological impairments than group R patients. Group L also had significantly higher CSF levels of homovanillic acid and acetylcholinesterase than group R. These findings of neuropsychological and neurochemical differences between groups L and R suggest functional or anatomic asymmetries of dopaminergic systems in the CNS.

Conformationally defined enantiomeric local anesthetics and fluorescent dyes were synthesized. Neither the local anesthetics nor the fluorescent probes showed stereospecificity in interacting with nerve membranes. The fluorescence signals generated by the dyes showed excellent correlation with the time course and shape of the nerve action potential.

Isologous local anesthetics containing the ester, thiolester, or selenolester grouping and their quaternary ammonium analogs were studied for their ability to displace [3H]nicotine from plasma membrane fragments of lobster nerve. Tertiary and quaternary analogs were equiactive. The relative ability of oxo, thio, and seleno analogs to displace nicotine was the same as their relative ability to block axonal conduction and synaptic transmission. Among cholinergic ligands, choline and aminocholine, previously shown to be inactive as depolarizing agents, were uniquely unable to displace nicotine. The findings are compatible with the presence of a biopolymer capable of binding cholinergic ligands in axonal membranes and support the postulate that the relative inactivity of quaternary compounds in intact axons is due to permeability barriers.

1. Using terbium ions as fluorescence probes of calcium-binding sites and osmotic shock to induce trapping of Tb3+ in the vesicle interior, direct binding assays have been developed to study the competition between calcium and local anesthetics for binding sites at the cytoplasmic surface of axonal membrane vesicles. 2. Pharmacologically active concentrations of the membrane-permeable local anesthetic, lidocaine, competitively displace bound Tb3+ in the vesicles, while QX-314, a quaternary ammonium analog of lidocaine that has poor access to the vesicle interior, exhibits no significant displacement of osmotically-loaded, internally-bound Tb3+. 3. These experiments support the hypothesis that local anesthetics may function by displacing Ca2+ from a functionally specific binding site in nerve membranes.

The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ of E. electricus and on the activity of AChE in intact electroplaques from the same species were studied. 0.1 mM LaCl3 produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl3, initial activity was markedly increased. LaCl3 exerted a marked, concentration-dependent inhibition of intact cell AChE. La3+ and Ca2+ appear to interact competitively. In the presence of both 10 mM CaCl2 and 0.1 mM LaCl3, the initial activitity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 X 10(-4) M. Inhibition of intact cell enzyme by 0.1 mM LaCl3 was relieved by increasing the CaCl2 concentration to 10 mM at ACh concentrations less than 2 X 10(-4) M. The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.

Lysolecithin-solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)-sepharose. Bound glycoproteins were eluted with α-methyl-D- mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A-binding glycoprotein fraction. A 5-fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [3H]quinuclidinyl benzilate (QNB), [3nicotine (Nic), and [1251]α-bung arotoxin (BgTx) in an attempt to distinguish possible “muscarinic” and “nicotinic” binding sites in axonal membranes. All of the high-affinity “muscarinic” [3H]QNB binding activity appeared in the non-Con A-binding protein fractions, while binding of the two “nicotinic” ligands, [3Nic and 125I-BgTx, was found in both the glycoprotein and non-Con A-binding protein fractions. BgTx interaction with the Con A-binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non-Con A-binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.

In vitro binding assays to measure muscarinic receptor number and affinity were carried out with isolated synaptic plasma membranes from bovine caudate nuclei. Diethyl- p -nitrophenyl phosphate (paraoxon; PX), dichlorvos (DDVP), and tetraethyl pyrophosphate (TEPP) were demonstrated to be weak noncompetitive receptor inactivators. PX, DDVP, and TEPP decreased the number of [ 3 H ]quinuclidinyl benzilate (QNB) binding sites by 16 to 31% and exhibited no significant effect on the affinity of central muscarinic receptors for QNB ( K d ⋍ 0.3 n m ). QNB binding was inhibited by PX at 5 n m , DDVP at 50 n m , and TEPP at 50 n m , concentrations with no effect on acetylcholinesterase activity. Similar measurements with two carbamates, physostigmine and neostigmine (1 n m ), showed no effect of neostigmine on QNB binding, while physostigmine both decreased the number of receptors and enhanced their affinity for QNB. The modulation of muscarinic receptors by 5 n m PX was abolished by carbodiimide modification of the membrane preparation. It is suggested that a decrease in the number of muscarinic cholinergic receptors in the brain may underlie some of the neurological and affective disorders observed after chronic exposure of humans to organophosphate insecticides.

Homogenates of calf caudate nuclei were found to contain at least three distinct subclasses of cholinergic, muscarinic receptors. These subtypes, labeled with [3H]quinuclidinyl benzilate (QNB), can be separated by rapid filtration with the use of the selective ligands, pirenzepine, AF-DX116, and 4-DAMP which have high affinity for the M1, M2, and M3 subtypes, respectively. Paraoxon was found to modulate [3H]QNB binding in a noncompetitive manner at concentrations below those needed to affect acetylcholinesterase activity. Pretreatment of the membrane protein with high concentrations of both the M2 selective antagonist, AF-DX116, and the M3 selective antagonist, 4-DAMP, protected against paraoxon inhibition of [3H]QNB binding, while the M1 selective antagonist pirenzepine did not. Paraoxon sensitive sites, M2 and M3, are found predominantly on presynaptic neurons in the central nervous system. It is postulated that blockade of these sites may interfere with negative feedback inhibition of acetylcholine release and facilitate the development of behavioral and motor deficits that may be associated with chronic exposure to low levels of organophosphates.

Conduction block by thiol reagents is potentiated by repeated, brief electrical stimulation. These studies have been quantitated with N-ethylmaleimide and mercurochrome showing a nonlinear relationship between dose and number of stimuli required to produce inexcitability.p-Chloromercuribenzoate, mercurochrome and fluorescein mercuric acetate block conduction and are reversible with β-mercaptoethanol and exhibit the “stimulation effect.” N-Ethylmaleimide, Ellman's reagent (DTNB), and 2-dimethylaminoethyl selenolbenzoate exhibit the “stimulation effect”, but blockade is irreversible. In a series of local anesthetics, procaine, 2-diethylaminoethyl thiolbenzoate, and 2-dimethylaminoethyl selenolbenzoate, only the selenolester reacts with SH groups and shows a “stimulation effect”. Iodoacetate and iodoacetamide block nerve conduction without a stimulation effect. Possible interpretations of this effect include: altered permeability, unmasking of buried SH groups in the membrane, or electrolytic reduction of disulfides.