In vitro estrogenic activity of formononetin by two bioassay systems.

AIM: To study the estrogenic activity of formononetin in vitro. METHODS: We have established a highly sensitive bioassay system by placing estrogen-responsive elements upstream of the luciferase reporter gene, and used this assay to determine the estrogenic activity of formononetin. Cell growth was measured by the MTT (3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and MG-63 cell function was studied by measuring alkaline phosphatase activity. RESULTS: Formononetin activated expression of the estrogen-responsive reporter gene in human breast cell line MCF-7 in a concentration-dependent manner (0.5-500 microM), and this activation was inhibited by estrogen antagonist (ICI 182780 at 100 nM). Furthermore, it induced the proliferation of MCF-7 breast cancer cells and MG-63 osteosarcoma cells, and it also increased the alkaline phosphatase activity in MG-63 cells. CONCLUSION: Formononetin is a phytoestrogen that exhibits variable degrees of estrogen receptor agonism in different test systems.

Ginsenoside Re attenuate beta-amyloid and serum-free induced neurotoxicity in PC12 cells.

AIM: To study the effect of ginsenoside Re on PC12 cell damage induced by serum deprivation and beta-amyloid peptide. METHODS: PC 12 cell survival was measured by MTT and lactate dehydrogenase (LDH) assay. Results Serum-free medium and beta-amyloid peptide (10-100 microM) induced cytotoxicity in PC 12 cells. Ginsenoside Re (0.1-100 microM) attenuated the cytotoxic effects of serum-free medium and beta-amyloid peptide (50 microM) in a concentration-dependent manner. CONCLUSION: Ginsenoside Re prevented PC 12 cells from lesion induced by serum-free medium and beta-amyloid peptide.

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

The cyclic AMP-mediated expression of acetylcholinesterase in myotubes shows contrasting activation and repression between avian and mammalian enzymes.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterase (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from approximately 1.8- to approximately 2.5-fold, but the luciferase activity was decreased by over 60%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. In addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

The promoter of human acetylcholinesterase is activated by a cyclic adenosine 3',5'-monophosphate-dependent pathway in cultured NG108-15 neuroblastoma cells.

Different transcription elements have been proposed to play a role in the regulation of acetylcholinesterase (AChE) in muscle and neuron, and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway is one of them. In order to test the possible role of cAMP in regulating the expression of human AChE, an approximately 2.2 kb DNA fragment of human AChE promoter was linked up stream to a luciferase reporter. The chimeric DNA was transfected into cultured NG108-15 neuroblastoma cells. Application of Bt(2)-cAMP and forskolin increased the promoter driven luciferase activity over 2-fold in the transfected NG108-15 cells; the increase was parallel to the activation of endogenous AChE protein and enzymatic activity. The intracellular cAMP level was increased in the Galpha(sQL) (constitutively active mutant of Galpha(s)) cDNA transfected NG108-15 cells. The Galpha(sQL) cDNA transfected cells showed an increase of over 10-fold in the luciferase activity. In addition, a constitutively active mutant of activating transcription factor-1 (ATF-1) was able to turn on human AChE promoter by approximately 4-fold when they were co-expressed in the neuroblastoma cells. These results support the involvement of a cAMP-dependent pathway in regulating the expression of human AChE.

The cAMP-dependent protein kinase mediates the expression of AChE in chick myotubes.

Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, stimulates the expression of AChR and AChE at the vertebrate neuromuscular junctions. The signaling mechanism of CGRP-induced AChE expression in muscle was determined both in vitro and in vivo. In cultured chick myotubes, the intracellular cAMP-dependent protein kinase (PKA) activity increased to approximately 2-fold after the application of CGRP or PKA activators; the induction was blocked by PKA inhibitors. in vivo transfection analysis on chick gastrocnemius muscles showed that the transfection of cDNA encoding constitutively active mutant Galphas increased the expression of AChE mRNA and protein to approximately 2-fold, while the constitutively active mutant Galphai cDNA transfection showed an opposite effect. The induced catalytic subunit of AChE at approximately 105 kDa was determined by specific antibody. These findings indicate that the CGRP-induced AChE expression in chick muscle is mediated by a PKA-dependent pathway.

A cysteine-rich form of Xenopus neuregulin induces the expression of acetylcholine receptors in cultured myotubes.

Neuregulin-1 (NRG-1) has diverse functions in neural development, and one of them is to up regulate the expression of acetylcholine receptors (AChRs) at muscle fibers during the formation of neuromuscular junctions. NRG-1 has two prominent alternative splicing sites at the N-terminus; it could be an immunoglobulin (Ig)-like domain named Ig-NRG-1 or an apolar cysteine-rich domain (CRD) named CRD-NRG-1. cDNAs encoding Xenopus CRD-NRG-1 were isolated by cross-hybridization with Xenopus Ig-NRG-1 cDNA fragment. The amino acid sequence of Xenopus CRD-NRG-1 is 45 to 70% identical to the human, rat, and chick homologs. Similar to Ig-NRG-1, two variation sites within CRD-NRG-1 were identified at the spacer domain with 0 or 43 amino acids inserted and at the C-terminus of the EGF-like domain to derive either alpha or beta isoform. Two transcripts encoding CRD-NRG-1, approximately 7.5 and approximately 9.0 kb, were revealed in adult brain and spinal cord, but the expression in muscle was below the detectable level. The recombinant Xenopus CRD-NRG-1 when applied onto cultured myotubes was able to induce the tyrosine phosphorylation of ErbB receptors and the expression of AChR. The AChR-inducing activity of CRD-NRG-1 was precipitated by anti-NRG-1 antibody but not by heparin. In situ hybridization showed a strong expression of CRD-NRG-1 mRNA in developing brain, spinal cord, and myotomal muscles of Xenopus embryo. Similar to the results in other species, both CRD-NRG-1 and Ig-NRG-1 may play a role in the developing Xenopus neuromuscular junctions.

The calcitonin gene-related peptide-induced acetylcholinesterase synthesis in cultured chick myotubes is mediated by cyclic AMP.

In vertebrate neuromuscular junctions, post-synaptic specialization includes aggregation of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE). The motor nerve provides soluble factors and electrical activity to achieve this striking localization of AChRs/AChE. Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, is able to stimulate the expression of AChR in cultured myotubes. Similar to AChR regulation, synthesis of AChE in cultured chick myotubes is also stimulated by CGRP. Application of CGRP onto cultured myotubes stimulated the accumulation of intracellular cyclic AMP (cAMP) as well as the expression of AChE mRNA and protein. However, the enzymatic activity of AChE remained unchanged. In cultured myotubes, various drugs affecting the intracellular level of cAMP, such as N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, cholera toxin, and forskolin, could mimic the effect of CGRP in stimulating the expression of AChE. When myotubes were transfected with cDNA encoding constitutively active mutant Galpha(s), the intracellular cAMP synthesis was increased. The increase in cAMP level was in parallel with an increase in the expression of AChE, whereas transfection of active mutant Galpha(i) cDNA decreased the cAMP level as well as the AChE expression. In addition, expression of collagen-tailed AChE was up-regulated by the cAMP pathway. These findings indicated that CGRP-induced AChE regulation is mediated by the cAMP pathway and represented the first evidence to suggest that the regulation of mRNA synthesis of AChR and AChE can be mediated by the same neuron-derived factor.

Over-expression of acetylcholinesterase stimulates the expression of agrin in NG108-15 cells.

Several lines of evidence suggest the non-cholinergic functions of acetylcholinesterase (AChE) in promoting neurite outgrowth of cultured neurons and in inducing the postsynaptic specializations of developing neuromuscular junctions. In order to support the hypothesis, a cholinergic synapse-forming cell line NG108-15 was over-expressed with chick AChE by cDNA transfection. The transfected NG108-15 cells secreted a approximately 105-kDa protein, recognized by anti-AChE antibody in Western blot analysis, corresponding to the chick AChE catalytic subunit. Over 80% of the recombinant enzyme were secreted into the conditioned medium and they were enzymatically active. In the NG108-15 cell-muscle co-cultures, the AChR-aggregating activity of NG108-15 cells was increased by the over-expression of AChE. The increase in AChR-aggregating activity of the transfected NG108-15 cells paralleled with the increase in agrin and neurofilament expression of the transfected cells as determined by their corresponding antibodies. However, the intracellular cAMP level remained unchanged in the AChE over-expressed NG108-15 cells. These results support the hypothesis that AChE could play a role in promoting neuron differentiation.

NG108-15 cells induce the expression of muscular acetylcholinesterase when co-cultured with myotubes.

Although muscular activity has been demonstrated to regulate the expression of acetylcholinesterase (AChE) in cultured myotubes, the exact role of the presynaptic terminus in regulating AChE expression at the neuromuscular junctions is not known. A chimeric co-culture of neuroblastoma x glioma hybrid NG108-15 cells with chick myotubes was established. By using chick-specific anti-AChE antibody, a protein of approximately 105 kDa in size corresponding to chick AChE catalytic subunit was revealed by Western blot analysis from the extracts of neuron-muscle co-cultures. In the co-cultures, NG108-15 cells induced the up regulation of muscle AChE expression by approximately 2.5-fold, while the control protein, chick muscle alpha-actinin at approximately 100 kDa, remained relatively unchanged. The NG108-15 cell-induced muscle AChE expression in the co-cultures was persistent when the muscular activity was blocked by alpha-bungarotoxin. In order to determine the AChE-inducing activity derived from NG108-15 cells, the cultured chick myotubes were treated with either conditioned medium of NG108-15 cells or its cell lysate. However, the muscle AChE, both in protein and activity levels, remained relatively unchanged. Our finding suggests that an AChE-inducing factor(s) is derived from the neuroblastoma cells in the co-cultures, but that may require the nerve-muscle contacts in culture.

Denervation decreases the ipsilateral expression of AChE in chick lumbaric motor neurons.

In vertebrate neuromuscular junctions, acetylcholinesterase (AChE; EC 3.1.1.7) is highly concentrated at the synaptic basal lamina and the postsynaptic muscle fiber. The postsynaptic muscle cell is the primary source of AChE. However, several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE monoclonal antibody in immunohistochemical staining, we found that the AChE-positive cells were labeled only at the motor neurons of the chick spinal cords. When the protein extract of chick spinal cords was analyzed by a Western blot analysis, a protein band of approximately 105 kDa was recognized. In denervated chicks, the expression of motor neuron AChE, as recognized on a Western blot, decreased by approximately 50% 4 days after denervation. The AChE expression in denervated chick spinal cords, however, was restored to control level 10 days after denervation. The decreased AChE expression was restricted to the ipsilateral side of the denervated chick spinal cord while the contralateral side was relatively unchanged. In comparison with the contralateral side, the level of AChE protein and enzymatic activity expressed in the ipsilateral spinal cord was approximately 50% lower. This is the first demonstration to show that the ipsilateral and contralateral sides of chick spinal cords respond differently after nerve denervation.

A globular, not asymmetric, form of acetylcholinesterase is expressed in chick motor neurons: down-regulation toward maturity and after denervation.

In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE catalytic subunit, anti-butyrylcholinesterase (BuChE) catalytic subunit, and anti-AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was approximately 105 kDa, which was similar to that of the globular enzyme from low-salt extracts of muscle; both of them were approximately 5 kDa smaller than the asymmetric AChE from high-salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation.

Calcitonin gene-related peptide increases the expression of acetylcholinesterase in cultured chick myotubes.

Calcitonin gene-related peptide (CGRP), a neuropeptide may play a role in the formation of neuromuscular junctions is synthesized by the motor neurons and is able to stimulate the expression of acetylcholine receptor (AChR) in cultured myotubes. By using antibody and DNA probe that are specific for acetyl cholinesterase (AChE), we reported the expression of AChE could also be stimulated by CGRP in cultured chick myotubes. After CGRP application, the amount of AChE protein, that showed a molecular weight of approximately 105 kDa as recognized by the anti-AChE monoclonal antibody, was increased by approximately 1.7-fold. Two transcripts encoding AChE, approximately 4.8 and approximately 6.0 kb, were identified and their levels of expression were increased to approximately 3-fold after treatment with CGRP. However, the total AChE enzymatic activity in the CGRP-treated myotubes was unchanged. These evidences suggest that most of the CGRP-induced AChE proteins in the cultured chick myotubes are the inactive pool of enzyme.