Butyrylcholinesterase regulates central ghrelin signaling and has an impact on food intake and glucose homeostasis.

BACKGROUND: Ghrelin is the only orexigenic hormone known to stimulate food intake and promote obesity and insulin resistance. We recently showed that plasma ghrelin is controlled by butyrylcholinesterase (BChE), which has a strong impact on feeding and weight gain. BChE knockout (KO) mice are prone to obesity on high-fat diet, but hepatic BChE gene transfer rescues normal food intake and obesity resistance. However, these mice lack brain BChE and still develop hyperinsulinemia and insulin resistance, suggesting essential interactions between BChE and ghrelin within the brain. METHODS: To test the hypothesis we used four experimental groups: (1) untreated wild-type mice, (2) BChE KO mice with LUC delivered by adeno-associated virus (AAV) in combined intravenous (i.v.) and intracerebral (i.c.) injections, (3) KO mice given AAV for mouse BChE (i.v. only) and (4) KO mice given the same vector both i.v. and i.c. All mice ate a 45% calorie high-fat diet from the age of 1 month. Body weight, body composition, daily caloric intake and serum parameters were monitored throughout, and glucose tolerance and insulin tolerance tests were performed at intervals. RESULTS: Circulating ghrelin levels dropped substantially in the KO mice after i.v. AAV-BChE delivery, which led to normal food intake and healthy body weight. BChE KO mice that received AAV-BChE through i.v. and i.c. combined treatments not only resisted weight gain on high-fat diet but also retained normal glucose and insulin tolerance. CONCLUSIONS: These data indicate a central role for BChE in regulating both insulin and glucose homeostasis. BChE gene transfer could be a useful therapy for complications linked to diet-induced obesity and insulin resistance.

Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats.

We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 microl/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at <0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed these concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 microM, 1.5 microl/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 microM and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 microM paraoxon but was transiently elevated (0.5-1.5 h) with 10 microM paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very different views into enzyme-inhibitor interactions. Furthermore, the proliferation/migration of cells containing high amounts of cholinesterase just adjacent to a dialysis probe could affect the recovery and thus detection of extracellular acetylcholine in microdialysis studies.

Axonal transport of dopamine- -hydroxylase by human sural nerves in vitro.

Dopamine-beta-hydroxylase activity accumulated above a ligature on biopsy samples of normal human sural nerves incubated in vitro. The rate of accumulation indicated that this enzyme was transported distally at a velocity of 2 millimeters per hour. Axoplasmic transport of dopamine-beta-hydroxylase was greatly reduced in sural nerves from a few patients with peripheral neuropathies.

Immunochemical differences among molecular forms of acetylcholinesterase in brain and blood.

Molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) differ in their solubility properties as well as in the number of their catalytic subunits. We used monoclonal antibodies to investigate the structure of acetylcholinesterase forms in brain, erythrocytes and serum of rats, rabbits and other mammals. Two antibodies were found to bind tetrameric acetylcholinesterase in preference to the monomeric enzyme. These antibodies also displayed lower affinity for certain forms of 'soluble' brain acetylcholinesterase than for the 'membrane-associated' counterparts. Furthermore, one of them was virtually lacking in affinity for the membrane-associated enzyme of erythrocytes. The basis for the antibody specificity was not fully determined. However, the immunochemical results were supported by measurements of enzyme thermolability, which showed that the catalytic activity of 'soluble' acetylcholinesterase was comparatively heat-resistant. These observations point toward structural differences among the solubility classes of acetylcholinesterase.

Thermal inactivation of the molecular forms of acetylcholinesterase and butyrylcholinesterase.

To compare acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), we utilized the physical parameter of thermolability. In serum or muscle extracts from mouse and rat, butyrylcholinesterase was inactivated as a unimodal function of temperature. Inactivation began at 51 degrees C and was complete at 54-57 degrees C (depending upon time of incubation). Acetylcholinesterase was inactivated in two stages. A 60% decrease in activity from 42 to 48 degrees C was followed by a plateau. The second stage of inactivation began at 51 degrees C and was complete at 57-60 degrees C (depending upon time of incubation). Sucrose density gradients revealed that the partial loss of acetylcholinesterase activity at 48 degrees C was due to inactivation of the monomeric 4 S enzyme, which was the most thermolabile molecular form in each tissue examined. When heated after isolation on density gradients, most of the forms of acetylcholinesterase and butyrylcholinesterase lost activity as a single exponential function of time. The monomers of both enzymes were inactivated fastest. Inactivation of the larger froms was slower and required higher temperatures. Tetrameric 10 S acetylcholinesterase was unique in following a time course that could only be fitted by a double exponential equation (i.e., when this form was heated to 55 degrees C, almost 60% of the activity showed a short half-life while the remainder showed a long half-life). This behavior did not reflect differences in the thermolability of soluble and membrane-derived tetramers.

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies.

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.

Neurofibrillary tangles have no obligatory predilection for acetylcholinesterase-rich neurons.

Parts of the brain that are prone to NFT formation normally contain many neurons that are intensely acetylcholinesterase (AChE)-positive. In this study, we used thioflavin-S immunofluorescence, AChE histochemistry, and AChE immunocytochemistry to investigate the possibility that intense AChE positivity may act as a perikaryal marker for the vulnerability to NFT formation. Our observations in entorhinal and motor cortices and in the subthalamic nucleus demonstrate major mismatches between the distribution of AChE-rich neurons in normal brains and the distribution of NFT in AD. There is therefore no obligatory relationship between intense AChE positivity in the premorbid period and subsequent vulnerability to tangle formation.

Acetylcholinesterase promotes beta-amyloid plaques in cerebral cortex.

Studies in vitro have suggested that acetylcholinesterase (AChE) may interact with beta-amyloid to promote deposition of amyloid plaques in the brain of patients with Alzheimer's disease. To test that hypothesis in vivo, we crossed Tg2576 mice, which express human amyloid precursor protein and develop plaques at 9 months, with transgenic mice expressing human AChE. The resulting F1 hybrids (FVB/N x [C57B6 x SJL/J]) expressed both transgenes in brain. By 6 months of age, their cerebral cortex showed authentic plaques that stained both by thioflavin S and by beta-amyloid 1-40 and 1-42 immunohistochemistry. The plaques also stained positively for other components including Cd11b, GFAP, and AChE. Plaque onset in the hybrids occurred 30-50% sooner than in the parental lines. Plaque numbers increased with age and plaques remained more numerous in the doubly transgenic animals at 9 and 12 months. Quantitative immunoassay via ELISA also showed an increase of total amyloid content in brain at 9-12 months. These histological and biochemical results support the conclusion that AChE may play a role in pathogenesis of Alzheimer's disease

A paradigm for examining toxicant effects on viability, structure, and axonal transport of neurons in culture.

N1E.115 murine neuroblastoma cells differentiating in serum-free medium were used to develop a paradigm for testing neurotoxicity in vitro. The paradigm was designed to test the effects of toxicants on four different aspects of cell function or structure: 1. Viability as shown by the retention of cellular radiolabel (51Cr); 2. Growth and maintenance of neurites as reflected by the incidence and average length of these processes; 3. Gross structure of neurites; and 4. Velocity and flux of rapid anterograde and retrograde axonal transport as judged by video-enhanced differential interference contrast microscopy. To evaluate this paradigm, colchicine and vinblastine were used as neurotoxicants with a well-understood mechanism of action. These agents were only weakly cytotoxic according to the Cr-release assay, but were able to interfere with neurite outgrowth at nanomolar concentrations. Neurites that were elaborated in the presence of vinblastine and colchicine were often disfigured by numerous swellings packed with organelles. In established neurites, micromolar concentrations of vinblastine inhibited organellar motility with great rapidity, blocking all signs of transport within 20 min. The effect of colchicine was slower and less complete, but still impressive. We suggest that this four-part analysis represents a highly sensitive in vitro test for neurotoxicity, and a means of analyzing the relation between abnormalities of transport and structural damage of nerve cells.

Complement regulatory proteins and selective vulnerability of neurons to lysis on exposure to acetylcholinesterase antibody.

Systemic injection of antibodies against acetylcholinesterase (AChE) induces complement-mediated destruction of preganglionic nerve terminals in paravertebral sympathetic ganglia, but spares other AChE-rich structures, such as nerve terminals in prevertebral sympathetic ganglia, parasympathetic ganglia, and the neuromuscular junction. This pattern of differing sensitivity to "AChE immunolesion" might be explained by a differing expression of proteins that serve to protect host cells from complement activation. Two major complement regulatory proteins in rats are Crry, which interferes with the assembly of C3 convertase, and CD59, which blocks formation of the terminal cytolytic membrane attack complex. The present study used immunohistochemistry to demonstrate an inverse relation between levels of CD59 and Crry expression and sensitivity to AChE immunolesion in several AChE-rich targets. Thus, the most sensitive structures, i.e., preganglionic nerve terminals in the adrenal gland and superior cervical ganglion (SCG), expressed undetectable levels of CD59 and Crry immunoreactivities. By contrast, AChE-rich, but antibody-resistant, cholinergic nerve terminals in the inferior mesenteric ganglia (IMG) and diaphragm muscle expressed significant amounts of CD59 and Crry. Such expression was functionally important because, after membrane-anchored CD59 was removed from explanted IMG with phosphatidylinositol phospholipase C, exposure to AChE antibody and complement caused greater immunolesion. It was concluded that differential expression of regulatory proteins in different parts of the nervous system influences regional vulnerability to complement mediated damage.

Complement-mediated lesion of sympathetic ganglia in vitro with acetylcholinesterase antibodies.

When administered to rats, antibodies against acetylcholinesterase (AChE) selectively destroy presynaptic inputs to sympathetic ganglia. To investigate the mechanism of this immunolesion, we created an in vitro system in which relevant components could be manipulated. Freshly dissected rat superior cervical ganglia (SCG) were incubated 15-20 h at 37 degrees C in fresh human serum (a potent source of complement) with continuous oxygenation. More than 96% of neurons in six control ganglia retained synaptic inputs, as defined by action potentials or excitatory postsynaptic potentials (EPSP) upon stimulation of the preganglionic trunk. However, when anti-AChE antibodies were present (0.16 mg/ml), none of 61 neurons from six incubated ganglia showed synaptic responses although membrane potential and input resistance remained normal. Staining for AChE and synaptophysin (a synaptic vesicle marker) was also disrupted in ganglia exposed to AChE antibodies in complement-sufficient serum. When complement was eliminated by substituting serum that was heat-inactivated or deficient in C3, synaptic input was retained in 60-90% of neurons incubated with AChE antibodies. Choline acetyltransferase activity (ChAT), an enzymatic marker of cholinergic cytoplasm in sympathetic ganglia, was largely lost after incubation with AChE antibodies and serum. However, incubation with AChE antibodies in heat-inactivated serum, or serum that was deficient in C3 or C8, caused no measurable loss of ganglionic ChAT activity. These findings strongly implicate the complement cascade in the destruction of preganglionic sympathetic terminals that follows binding of AChE antibodies.

Transient expression of acetylcholinesterase messenger RNA and enzyme activity in developing rat thalamus studied by quantitative histochemistry and in situ hybridization.

The molecular basis for transient expression of acetylcholinesterase in noncholinergic regions of the early postnatal rat brain was studied by in situ hybridization histochemistry. A 33P-labelled 63-mer DNA oligonucleotide was used to probe acetylcholinesterase messenger RNA in the brains of rat pups at one, two, six, nine, 12, 16 and 21 days of age (birth = day 0). Cryostat brain-sections were hybridized with probe and exposed to X-ray film or emulsion coatings. Acetylcholinesterase messenger RNA was quantitated by counting silver grains and by measuring X-ray film density with video imaging and computer-based densitometry. Adjacent sections were stained histochemically for acetylcholinesterase activity, also quantitated by video densitometry. Overall there was a significant correlation between apparent levels of acetylcholinesterase activity and acetylcholinesterase messenger RNA. Increases in message tended to accompany the surges of acetylcholinesterase activity that marked the maturation of thalamocortical sensory relay pathways. Acetylcholinesterase expression in the youngest rats was generally sparse but it increased markedly during the first postnatal week, especially in the sensory relay nuclei of the thalamus. Levels of message and enzyme activity in the medial and dorsolateral geniculate and the ventral posteromedial and ventral posterolateral nuclei rose to a peak, typically about day 9. Beyond this time there was a gradual decline. By day 21 the staining and in situ hybridization patterns resembled those in adult brains, whose thalamic relay nuclei are impoverished in acetylcholinesterase activity and messenger RNA. Thus, acetylcholinesterase expression is strongly modulated in certain thalamic systems as they undergo neural morphogenesis.

Lesion of central cholinergic systems by systemically administered acetylcholinesterase antibodies in newborn rats.

To determine if systemically administered antibodies could reach antigenic targets and cause immunologic lesions in brains of newborn rats, murine monoclonal antibodies against rat acetylcholinesterase were injected i.p. on the first postnatal day. As early as 24 h after injection, antibodies were detected immunocytochemically in brain parenchyma, along with punctate debris that showed intense cholinesterase activity. Total acetylcholinesterase activity in the brain dropped by 30%, and 10S activity was almost undetectable at day 3, implying true enzyme loss since the antibodies did not directly impair catalytic function. At day 7, 10S acetylcholinesterase began to recover but the activity remained only half that of controls. At day 12, total acetylcholinesterase activity was still reduced (30% in whole brain, 40% in cerebral cortex), consistent with lasting damage to cholinesterase-expressing cortical neurons. This conclusion was confirmed by histochemical experiments showing a nearly complete disappearance of acetylcholinesterase fiber-staining in cerebral cortex and basal ganglia at days 4 and 8, with residual deficits at day 12. Choline acetyltransferase activity decreased in the cerebral cortex, implying a loss of cholinergic terminals, but specifically immunoreactive perikarya remained abundant in the basal forebrain. Immunocytochemistry showed no obvious changes in three non-cholinergic markers: tyrosine hydroxylase, tryptophan hydroxylase, and glutamic acid decarboxylase. Overall, it appeared that acetylcholinesterase antibodies induced widespread but reversible damage of cholinergic fibers and terminals, while sparing cholinergic cell bodies and many other neural systems.

Effects of antibodies against acetylcholinesterase on the expression of peptides and catecholamine synthesizing enzymes in the rat adrenal gland.

In the rat, systemic administration of murine monoclonal antibodies against acetylcholinesterase caused rapid piloerection and ptosis (within 30-60 min after the injection). Using indirect immunohistochemistry the effect of these antibodies on peptides and enzyme expression was studied in the rat adrenal gland. Four days after antibody administration a total disappearance of acetylcholinesterase-immunoreactive fibers was observed. However, groups of acetylcholinesterase-immunoreactive chromaffin cells and intramedullary ganglion cells, both cell types showing acetylcholinesterase immunoreactivity also in the control adrenal medulla, expressed increased immunoreactivity. Analysis revealed that the acetylcholinesterase-immunoreactive chromaffin cell groups lacked phenylethanolamine-N-methyltransferase staining both in controls and treated rats. Antibody administration also affected levels of several peptides present in nerve fibers and chromaffin cells. Thus, the number of cells expressing enkephalin, calcitonin gene-related peptide and galanin was dramatically increased compared to the very few cells observed containing these three peptides in the normal gland. The majority of cells expressing enkephalin after antibody treatment also showed phenylethanolamine-N-methyltransferase immunoreactivity. In contrast, the few chromaffin cells expressing strong enkephalin-like immunoreactivity in controls were phenylethanolamine-N-methyltransferase negative. The sparse networks of calcitonin gene-related peptide- and galanin-positive fibers found in control adrenals were unchanged after the antibody treatment. However, the dense network of enkephalin varicose fibers totally disappeared after the antibody injection. A few substance P- and somatostatin-immunoreactive cells, not present in the normal gland, appeared after administration of the antibodies, whereas no changes were encountered with regard to immunoreactive nerve fibers. No clear differences between normal and treated animals could be observed in chromaffin cells with regard to immunoreactivity for neuropeptide Y or any of the four catecholamine-synthesizing enzymes, tyrosine hydroxylase, aromatic 1-amino acid decarboxylase, dopamine beta-hydroxylase or phenylethanolamine-N-methyltransferase. The present findings demonstrating a disappearance of acetylcholinesterase- and enkephalin-immunoreactive nerve fibers in the adrenal gland after intravenous injection of acetylcholinesterase antibodies support earlier reports showing that these antibodies cause degeneration of preganglionic fibers, and that neuronal decentralization of the adrenal gland induces marked increases in the levels of several peptides in chromaffin cells.

Death of intermediolateral spinal cord neurons follows selective, complement-mediated destruction of peripheral preganglionic sympathetic terminals by acetylcholinesterase antibodies.

Systemically injected anti-acetylcholinesterase antibodies in rats cause selective lesions of preganglionic sympathetic neurons. Adult rats were examined up to four months after a single i.v. injection of murine monoclonal acetylcholinesterase antibodies or normal immunoglobulin G (1.5 mg). Within 4 h, antibody-treated rats developed ptosis, a sign of sympathetic dysfunction that was never reversed. Persistent pupillary constriction reflected preserved and unopposed parasympathetic function. Weight gain was depressed, but locomotor activity, excitability, and sensorimotor responses were normal, and gross neuromuscular performance was near normal. These findings were supported by biochemical evidence for selective sympathetic damage. Acetylcholinesterase activity was reduced for the whole period of observation in sympathetic ganglia and adrenal glands but fell only transiently in muscle and serum. At all times, choline acetyltransferase activity (a marker of presynaptic terminals) was unaffected in muscle but grossly depleted in ganglia. Light and electron microscopy showed that preganglionic sympathetic terminals of superior cervical ganglia were severely damaged while parasympathetic ganglia were less affected and motor endplates of skeletal muscle were apparently spared. Immunocytochemistry revealed punctate deposits of murine immunoglobulin G and complement component C3 in ganglionic neuropil 12 h after antibody injection. This finding was consistent with complement-mediated lysis of preganglionic terminals. Morphometric analysis of preganglionic neurons in the intermediolateral nucleus of the spinal cord showed progressive loss of cholinergic perikarya over several months. We conclude that antibody-induced destruction of ganglionic terminals leads to death of preganglionic sympathetic neurons and, hence, permanent dysautonomia.

Immunologically induced sympathectomy of preganglionic nerves by antibodies against acetylcholinesterase: increased levels of peptides and their messenger RNAs in rat adrenal chromaffin cells.

Systemic administration of murine monoclonal acetylcholinesterase antibodies to rats has been shown to cause selective degeneration of sympathetic preganglionic neurons. In the present study rats were subjected to a single i.v. injection of these acetylcholinesterase antibodies, or to normal IgG or saline for control. Exophthalmos, piloerection and eyelid-drooping (ptosis) were observed within 1 h after administration of the antibodies. Rats were killed at different time-points after antibody administration, and the adrenal glands were analysed by means of indirect immunohistochemistry and in situ hybridization histochemistry. As soon as 3 h after the antibody treatment, a marked increase in the number of chromaffin cells expressing mRNA encoding, respectively, enkephalin, calcitonin gene-related peptide, galanin, neurotensin and substance P was seen. At 12 h the peptide mRNA levels were still elevated and there was a concomitant increase in the number of peptide-immunoreactive cells. All peptide levels remained high for at least 48 h; however, 77 days after the antibody treatment only enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-immunoreactive cells could be encountered. A disappearance of acetylcholinesterase- and enkephalin-positive fibers was already seen 3 h after the antibody treatment, and after 24 h no fibers were encountered. In contrast, up until 48 h there was no apparent change in the number or intensity of immunofluorescent fibers expressing calcitonin gene-related peptide, galanin, neurotensin or substance P. However, 77 days after the antibody treatment the number of calcitonin gene-related peptide- and substance P-immunoreactive fibers was increased as compared to controls. In addition, reappearance of acetylcholinesterase- and enkephalin-immunoreactive fibers was seen 77 days after antibody administration, although their number was still low as compared to controls. Double-labeling immunohistochemistry revealed that the chromaffin cells expressing peptides after the antibody treatment preferentially were adrenaline storing cells (noradrenaline-negative). The majority of these cells expressed only one peptide. Both surgical transection of the splanchnic nerve as well as treatment with acetylcholine receptor antagonists mimicked the effects seen after the acetylcholinesterase-antibody treatment, although changes were less pronounced. The present results show that interruption of splanchnic transmission induces fast, marked, and selective increases in peptide expression in rat adrenal chromaffin cells.

C-terminals on motoneurons: electron microscope localization of cholinergic markers in adult rats and antibody-induced depletion in neonates.

C-terminals on motoneurons are defined as those accompanied by characteristic postsynaptic specializations termed subsurface cisterns. We have previously shown, by light microscope immunolabelling methods, that subsurface cisterns occur regularly beneath choline acetyltransferase- and acetylcholinesterase-containing boutons on motoneurons. In the present study, the cholinergic nature of C-terminals suggested by these results was further investigated by immunohistochemistry and electron microscopy in adult rats and in neonates treated with a murine monoclonal acetylcholinesterase antibody which was previously shown to cause immunological lesions of central cholinergic systems. In both the facial nucleus and lumbar segment of spinal cord of adult rats, C-terminals were seen intensely immunostained for the cholinergic markers choline acetyltransferase and acetylcholinesterase. Immunolabelled terminals made contact with either neuronal somata or large calibre dendrites, which were positive for the cholinergic markers, and exhibited club-shaped or thin elongated morphologies suggestive of terminal or en passant type synaptic interactions. The close relationship found between cholinergic markers and immunolabelled subsurface cisterns in adults was maintained on motoneurons of eight-day-old rats. While subcutaneous treatment of newborn rat with acetylcholinesterase antibody appeared to have no effect on the distribution of immunopositive subsurface cisterns in motoneurons when examined on postnatal day 8, the density of labelling for the two cholinergic markers around these neurons was reduced. Areas of neuropil immediately surrounding motoneurons in treated animals often showed signs of extensive swelling and deterioration indicative of a lesion event, and these motoneurons frequently displayed subsurface cisterns unapposed to C-terminals. These results support our earlier conclusion, based on light microscope investigation, that the majority if not all C-terminals are cholinergic in the areas investigated and demonstrate the potential utility of immunolesion methods in the study of C-terminal function.

Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were examined in several tissues of normal and hypophysectomized male and female rats. Significant sex differences in the mean AChE activities of normal rats were observed in the superior cervical ganglion (three times more activity in males) and in serum (50% more activity in females). Sex differences in the BuChE activity of serum and liver were even larger (ten times more activity in females), but the activity of other tissues was similar in both sexes. Hypophysectomy had little effect on the mean activity of AChE but did alter BuChE activity in certain tissues. Most of the effects of hypophysectomy on mean BuChE activity were opposite in direction in the two sexes. For example, in males hypophysectomy caused increases in the BuChE activity of serum (300%) and liver (43%), while in females it caused decreases in both tissues (25 and 30% respectively). In rats of a given group, the AChE activity of each tissue appeared to be regulated independently of the activity in other tissues. By contrast, BuChE activity showed statistically significant correlations in more than half of the tissue-pairs examined in control rats of either sex. These correlations can be considered to reflect a tendency toward body-wide regulation. In female rats, the cross-tissue correlations were largely eliminated by hypophysectomy. This finding indicates that the regulation of BuChE may be strongly affected by hormones under the control of the pituitary gland. However, in male rats, only the correlations involving atria were altered by hypophysectomy. Therefore, the effects of hormones on BuChE are probably both sex and tissue dependent.

Dansylarginine N-(3-ethyl-1.5-pentanediyl)amide. A potent and selective fluorescent inhibitor of butyrylcholinesterase.

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 x 10(-7) M) but not of acetylcholinesterase (IC50 = 4 x 10(-4) M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate Ki unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a Kd of 4.5 x 10(-7) M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.

Slow accumulation of acetylcholinesterase in rat brain during enzyme inhibition by repeated dosing with chlorpyrifos.

When given to rats, O,O'-diethyl-O-[3,5,6-trichloro-2-pyridyl]- phosphorothionate (chlorpyrifos), a common insecticide, causes an unusually lengthy dose-dependent fall in the activity of brain acetylcholinesterase (AChE; EC 3.1.1.7). To determine whether the slow recovery involves impaired AChE synthesis, experiments were designed to measure AChE activity, immunoreactive AChE protein (AChE-IR) and AChE mRNA. Male, Long-Evans rats, maintained at 350 +/- 5 g, were dosed (s.c.) weekly for 4 weeks with 0, 15, 30, or 60 mg/kg chlorpyrifos in peanut oil. Brain tissue was harvested 1, 3, 5, 7 and 9 weeks after treatment began. AChE activity was measured by Ellman assay, and AChE-IR was estimated by two-site ELISA using monoclonal antibodies to rat brain AChE. While AChE activity fell significantly at all times and doses, AChE-IR increased at 3 and 5 weeks in the two higher dosage groups. Larger increases of AChE-IR were observed after chlorpyrifos was administered for 4 weeks by the oral route. Northern blots quantified with reference to cyclophilin were consistent with stable levels of AChE mRNA. Overall, it appears that chronically reduced brain AChE activity after chlorpyrifos reflects sustained enzyme inhibition, not loss of enzyme protein or suppression of AChE message.