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.

Differential CSF butyrylcholinesterase levels in Alzheimer's disease patients with the ApoE epsilon4 allele, in relation to cognitive function and cerebral glucose metabolism.

Butyrylcholinesterase (BuChE) is increased in the cerebral cortex of Alzheimer's disease (AD) patients, particularly those carrying epsilon4 allele of the apolipoprotein E gene (ApoE) and certain BuChE variants that predict increased AD risk and poor response to anticholinesterase therapy. We measured BuChE activity and protein level in CSF of eighty mild AD patients in relation to age, gender, ApoE epsilon4 genotype, cognition and cerebral glucose metabolism (CMRglc). BuChE activity was 23% higher in men than women (p<0.03) and 40-60% higher in ApoE epsilon4 negative patients than in those carrying one or two epsilon4 alleles (p<0.0004). CSF BuChE level correlated with cortical CMRglc. Patients with high to moderate CSF BuChE showed better cognitive function scores than others. We hypothesize that CSF BuChE varies inversely with BuChE in cortical amyloid plaques. Thus, low BuChE in a patient's CSF may predict extensive incorporation in neuritic plaques, increased neurotoxicity and greater central neurodegeneration.

Cholinesterase reactivation in vivo with a novel bis-oxime optimized by computer-aided design.

Recently, several bis-pyridiniumaldoximes linked by a variable-length alkylene chain were rationally designed in our laboratories as cholinesterase reactivators. Extensive in vitro tests of these oximes with acetylcholinesterase inhibited by two different organophosphate agents, echothiophate and diisopropylfluorophosphate, revealed one compound with particularly good reactivation kinetics and affinity for phosphorylated acetylcholinesterase (AChE). This compound, designated "ortho-7", with a heptylene chain bridging two aldoximes ortho to a pyridinium ring nitrogen, was chosen for detailed comparison with the classic reactivator pyridine-2-aldoxime methochloride (2-PAM). In vitro, ortho-7 reactivated AChE selectively, without restoring activity of the related enzyme butyrylcholinesterase (BChE). For in vivo studies, rats were injected with ortho-7 or 2-PAM before or after organophosphate exposure, and the activities of AChE and BChE were determined at multiple intervals in blood and solid tissues. Ortho-7 behaved nearly as well in the animal as in vitro, reactivating AChE to the same extent as 2-PAM in all peripheral tissues studied (serum, red blood cell, and diaphragm), but at doses up to 100-fold smaller. Like other oxime reactivators, ortho-7 did not reactivate brain AChE after systemic administration. Nonetheless, this agent could be useful in combination therapy for organophosphate exposure, and it may provide a platform for development of additional, even more effective reactivators.

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

Effect of extrinsic denervation on muscarinic neurotransmission in the canine ileocolonic region.

To explore the hypothesis that denervation hypersensitivity increases ileocolonic motor activity after extrinsic denervation, we compared muscarinic neurotransmission in canine ileocolonic loops that were isolated and either extrinsically innervated or extrinsically denervated. We recorded ileal, ileocolonic sphincter (ICS) and colonic pressures, and colonic tone, compliance and relaxation during ileal distention. Muscarinic effects were probed by neostigmine, and minimally effective doses of muscarinic receptor antagonists. Denervation augmented ileal, ICS and colonic contractile activity; colonic high-amplitude propagating contractions (HAPCs) were also augmented; colonic relaxation during ileal distention was abolished. Neostigmine induced HAPCs in both loop preparations. Pirenzipine (M1 antagonist) reduced ileal contractile activity in all loops and reduced colonic relaxation during ileal distention in innervated loops. Pirenzipine also reduced colonic tone and colonic HAPCs, more in denervated loops. Darifenacin (M3 antagonist) reduced ileocolonic contractile activity and tone more than did AF-DX 116 (M2 antagonist) in all loops. Cholinergic receptor subtypes modulate different facets of ileocolonic motor activity in the canine ileocolonic region. Increased sensitivity at M1 muscarinic receptors may partly account for the effects of extrinsic denervation.

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.

Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth.

Acetylcholinesterase (AChE) can promote neurite outgrowth through a mechanism that is independent of its role in hydrolyzing the neurotransmitter acetylcholine. It has been proposed that this neuritogenic capacity of AChE may result from its intrinsic capacity to function in adhesion. In this study we directly tested this hypothesis using neuroblastoma cell lines that have been engineered for altered cell-surface expression of AChE. Using a microtiter-plate adhesion assay and the electrical cell-substrate impedance-sensing (ECIS) method, we demonstrate that the level of cell-substratum adhesion of these cells directly correlates with their level of AChE expression. Furthermore, this adhesion is blocked by either an anti-AChE antibody or a highly specific AChE inhibitor (BW284c51), both of which have also been shown to block neurite outgrowth. In addition, cells that overexpress AChE showed enhanced neurite initiation. By employing cell lines with different levels of AChE expression in two types of cell-substratum adhesion assays, our current studies provide evidence for an adhesive function for AChE. These results, together with the fact that AChE shares sequence homology and structural similarities with several known cell adhesion molecules, support the hypothesis that AChE promotes neurite outgrowth, at least in part, through an adhesive function.

Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase. Engineering effective butyrylcholinesterase mutants for cocaine detoxication.

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.

Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse.

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.

Novel messenger RNA and alternative promoter for murine acetylcholinesterase.

A portion of the 5'-flanking region of murine acetylcholinesterase was cloned from genomic DNA by 5'-rapid amplification of genomic ends, identified in a mouse genomic library, and sequenced. Multiple potential binding sites for universal and tissue-specific transcription factors were suggestive of a promoter region within this DNA sequence. Potential promoter activity was confirmed by coupling the new sequence to the open reading frame of a luciferase reporter gene in transient expression experiments with nerve and muscle cells. 5'-Rapid amplification of cDNA ends with templates from multiple sources revealed a novel transcription start site (at position -626, relative to translation start), located 32 bases downstream from a TATAA sequence. This start site appeared to mark a novel exon (1a) comprising 291 base pairs between positions -335 and -626, relative to the translation start. Supporting this conclusion, polymerase chain reactions with cDNA from mouse brain, heart, and other tissues, consistently amplified a transcript containing the exon 1a sequence fused to the invariant sequence beginning at position -22 in exon 2, but lacking exon 1. Northern blot analyses confirmed the in vivo expression of exon 1a-containing transcripts, especially in heart, brain, liver, and kidney. These results indicate that the murine acetylcholinesterase gene has a functioning alternative promoter that may influence expression of acetylcholinesterase in certain tissues.

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.

Cholinesterases in neural development: new findings and toxicologic implications.

Developing animals are more sensitive than adults to acute cholinergic toxicity from anticholinesterases, including organophosphorus pesticides, when administered in a laboratory setting. It is also possible that these agents adversely affect the process of neural development itself, leading to permanent deficits in the architecture of the central and peripheral nervous systems. Recent observations indicate that organophosphorus exposure can affect DNA synthesis and cell survival in neonatal rat brain. New evidence that acetylcholinesterase may have a direct role in neuronal differentiation provides additional grounds for interest in the developmental toxicity of anticholinesterases. For example, correlative anatomic studies show that transient bursts of acetylcholinesterase expression often coincide with periods of axonal outgrowth in maturing avian, rodent, and primate brain. Some selective cholinesterase inhibitors effectively suppress neurite outgrowth in model systems like differentiating neuroblastoma cells and explanted sensory ganglia. When enzyme expression is altered by genetic engineering, acetylcholinesterase levels on the outer surface of transfected neurons correlate with ability to extend neurites. Certain of these "morphogenic" effects may depend on protein-protein interactions rather than catalytic acetylcholinesterase activity. Nonetheless, it remains possible that some pesticides interfere with important developmental functions of the cholinesterase enzyme family.

1,25-Dihydroxyvitamin D3 receptors in the central nervous system of the rat embryo.

We have mapped areas within the central nervous system (CNS) of the developing fetal rat which immunostain for the 1,25-dihydroxyvitamin D3 receptor (VDR). The VDR was detected from days 12 to 21 of gestation throughout the CNS; immunostaining was particularly intense in the neuroepithelium and within the differentiating fields of various areas of the brain. Cells within the spinal cord, dorsal root, and other ganglia exhibited positive staining for the VDR. The intensity of staining for the VDR diminished or disappeared in the neuroepithelium throughout the CNS during the later days of development, while in the differentiating fields single VDR immunoreactive cells were observed. The presence of the VDR in the CNS was confirmed by in situ hybridization and RNA-based polymerase chain reaction methods with di-deoxy sequencing of the resultant DNA product. These results support the hypothesis that 1, 25-dihydroxyvitamin D3, through interactions with the VDR, may play a role in the development of the CNS.

Acetylcholinesterase immunolesioning: regional vulnerability of preganglionic sympathetic neurons in rat spinal cord.

Rats given antibodies against acetylcholinesterase (AChE) develop sympathetic dysfunction stemming from losses of preganglionic neurons in spinal cord. Central effects of AChE antibodies are surprising since IgG does not readily cross the blood-brain barrier, and lesions of peripheral terminals should not cause cell death. This study was designed to explore the distribution of central neural damage and to investigate features that might account for vulnerability. Rat spinal cord and brainstem were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity. Four months after administration of AChE antibodies, ChAT-positive neurons in the intermediolateral nucleus (IML) were 61-66% fewer throughout the thoracolumbar cord (T1, T2, T8, T12, L1). NOS-positive neurons in these loci were affected to the same extent by antibody-treatment, although they were only two-thirds as numerous. By contrast, neurons in the central autonomic nucleus of the thoracolumbar cord were scarcely affected. These results point to immunochemical differences in the central autonomic outflow, which may partially explain the puzzling selectivity of neural damage in AChE immunolesioning. Different results were obtained after guanethidine sympathectomy, which ablated nearly all neurons in the superior cervical ganglion without any effect on preganglionic neurons in the IML. Therefore, if the central effects of antibodies are indirectly mediated by loss of trophic support from the periphery, this support cannot arise from adrenergic neurons but must come from other ganglionic cells.

Developmental expression of acetyl- and butyrylcholinesterase in the rat: enzyme and mRNA levels in embryonic dorsal root ganglia.

Dorsal root ganglia (DRG) in the adult rat contain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), enzymes implicated in neural morphogenesis. We used quantitative histochemistry, reverse transcription-PCR (RT-PCR), and in situ hybridization histochemistry to study cholinesterase expression during embryogenesis. Longitudinal sections of rat embryos, embryonic day 9 (E9), E11-E17, and E19, were studied by video microscopy of the stained enzyme reaction products. Both enzymes were detectable in the early DRG (E11-E12), with BChE being most prominent. There was a spatiotemporal change in expression of each cholinesterase within the DRG. From E13 on, AChE expression predominated, especially in the neuronal cell bodies, while BChE was more highly expressed in the surrounding neuropil and the ganglionic roots. This distribution resembled the pattern in adult DRG. AChE mRNA levels, as determined by RT-PCR from DRG collected at days E12-E17, and E19, varied in parallel with the intensity of enzyme stain in the DRG. Overall, these results demonstrate temporally regulated ganglionic expression of cholinesterases, which may be important in the development of the sensory nervous system.

Comparison of the in vitro sensitivity of rat acetylcholinesterase to chlorpyrifos-oxon: what do tissue IC50 values represent?

The toxicological literature is replete with studies which have attempted to correlate differences in in vivo sensitivity to anticholinesterases with a common in vitro measure: acetylcholinesterase (AChE) IC50 values. Generally, it is assumed that these IC50 values reflect the intrinsic sensitivity of the AChE molecule to the inhibitor. Our goal was to ascertain whether differences in AChe sensitivity to an organophosphate (i.e., IC50 values) are due to varying properties of the enzyme molecule (i.e., present assumption) or to extrinsic factors. Tissue samples were obtained from immature and adult Long-Evans rats. AChE IC50 values were determined by incubating tissue homogenates with chlorpyrifos-oxon (active metabolite of chlorpyrifos, a common organophosphate insecticide) for 30 min at 26 degrees C, and then measuring residual AChE activity. The following IC50 values were noted for postnatal day 4 and adult animals, respectively: brain, 10 nM for both ages; liver, 96 and 527 nM; plasma, 18 and 326 nm. Thus, the "apparent" sensitivity of AChe was prone to vary dramatically with age and tissue type. In contrast, when AChE was isolated from the same tissues by immunoprecipitation, there were no age- or tissue- related differences (IC50 approximately equal to 3 nM in every case). These data show clearly that IC50 values from a crude homogenate do not measure the true sensitivity of AChE to the inhibitor. Presumably, for chlorpyrifos-oxon, at least, the tissue IC50 values depend greatly on a tissue's propensity to sequester or hydrolyze chlorpyrifos-oxon.

Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression.

Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level (p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.

Effects of 1,25-dihydroxyvitamin D3 on growth of mouse neuroblastoma cells.

Epitopes of the 1,25-dihydroxyvitamin D(1,25(OH)2D3) receptor have been shown in developing dorsal root ganglia in fetal mice, as well as in cells maintained in culture [Johnson, J.A., Grande, J.P., Windebank, A.J. and Kumar, R., 1,25-Dihydroxyvitamin D3 receptors in developing dorsal root ganglia of fetal rats, Dev. Brain Res., 92 (1996) 120-124]. To investigate a possible role for 1,25(OH)2D3 in neural cell growth and development, a murine neuroblastoma cell line that expresses 1,25(OH)2D3 receptors, was treated with 1,25(OH)2D3. Treatment with 1,25(OH)2D3 resulted in a decrease in cell proliferation, a change in cell morphology, and the expression of protein markers of mature neuronal cells. The decrease in cell proliferation was accompanied by an increase in the expression of nerve growth factor (NGF). Anti-NGF monoclonal antibody added to the growth medium blocked the decrease in cell proliferation caused by 1,25(OH)2D3 treatment. Our results show that the sterol hormone 1,25(OH)2D3, causes a decrease in the proliferation of mouse neuroblastoma cells through alterations in the expression of NGF.

Selective disruption of neurotransmission by acetylcholinesterase antibodies in sympathetic ganglia examined with intracellular microelectrodes.

Antibodies to acetylcholinesterase (AChE) induce adrenergic dysfunction in rats by selective, complement-mediated destruction of preganglionic sympathetic nerve terminals. To analyze this phenomenon at the neuronal level, monoclonal antibodies to AChE (1.6 mg) were injected via the tail vein, and superior cervical ganglia (SCG) or inferior mesenteric ganglia (IMG) were studied in vitro. In control SCG, all impaled neurons generated action potentials during direct injection of depolarizing current or indirect stimulation through the preganglionic nerve. Current injection remained effective in ganglia from treated rats, but preganglionic stimulation was greatly impaired: at 12 h and 3 d, less than 10% of the neurons responded, even to a maximal stimulus (150 V); at 9 d, only 25% responded. By contrast, in IMG, synaptic transmission was much less affected by antibody exposure: 60% or more of examined neurons responded to preganglionic stimulation. Differences in antibody access did not explain differing sensitivities of SCG and IMG since immunohistochemistry showed rapid accumulation of IgG deposits in both ganglia. These results are believed to reflect widespread but subtotal preganglionic sympathectomy by AChE antibodies. Current information indicates that paravertebral ganglia are all antibody-sensitive, but some prevertebral ganglia are resistant, suggesting immunochemical differences between them.