Biomedical subjects

W Ma

Publications and source records attributed to W Ma.

At least 109 records · Page 6Linked to original sources

PubMed2000

Regulation of cyclooxygenase-2 induction in the mouse uterus during decidualization. An event of early pregnancy.

The infertility phenotype of cyclooxygenase-2 (Cox-2)-deficient female mice establishes the important role of Cox-2 in pregnancy. Cox-2 deficiency results in defective ovulation, fertilization, implantation, and decidualization; the latter of which can be restored in part by the prostacyclin analog carbaprostacyclin. Uterine Cox-2 expression during early pregnancy shows distinct localization and kinetics in the uterine luminal epithelium and underlying stromal cells, suggesting that expression is tightly regulated. Several intracellular signaling cascades including ERK, p38, and JNK are implicated in vitro as critical components of regulated Cox-2 expression in response to mitogens, growth factors, and cytokines. We investigated the involvement of these signaling pathways during Cox-2 induction in vivo by monitoring uterine kinase activity after intraluminal application of a deciduogenic stimulus. Our results show that the ERK and p38 pathways are activated in uterine preparations as early as 5-min post-stimulation. ERK activation was sustained for several hours with a return to baseline levels by 4 h. p38 activation was rapid with a peak at 5-min post-stimulation and returned to near baseline levels after 45 min. Systemic administration of a MEK inhibitor completely inhibited ERK activation, but did not affect early (2 h) luminal epithelial or late (24 h) stromal Cox-2 expression and only modestly affected decidualization. In contrast, administration of a p38 inhibitor modestly inhibited early Cox-2 expression in the luminal epithelium, while dramatically diminishing late stromal expression. In parallel, induced stromal peroxisomal proliferator activated receptor-delta (PPARdelta) expression is blunted by p38 inhibition. p38 inhibition also significantly inhibited decidualization. These results suggest that p38, but not ERK, activation is required for induced Cox-2 and PPARdelta expression during decidualization. In addition, inhibition of p38 led to decreased decidualization suggesting that an intracrine prostanoid pathway consisting of Cox-2, prostacyclin, and PPARdelta is required for maintenance of early pregnancy.

Animals↗

PubMed2000

Primary neural precursor cell expansion, differentiation and cytosolic Ca(2+) response in three-dimensional collagen gel.

To investigate the ability to culture neural precursor cells in a three-dimensional (3D) collagen gel, neuroepithelial cells were isolated from embryonic day 13 rat cortex, dispersed within type I collagen and maintained for up to 30 days in vitro. Cultured in Neuorobasal medium supplemented with B27 containing basic fibroblast growth factor, the collagen-entrapped precursor cells actively expanded and formed clone-like clusters. Many cells in the center of the cluster were proliferating as revealed by 5-bromo-2'-deoxyuridine uptake. Some cells began to migrate away from the center at 5 days and were labeled by either neuronal marker neuron-specific beta-tubulin (TuJ1) or astrocytic marker glial fibrillary acidic protein. The differentiated neurons (TuJ1(+)) exhibited characteristic cytosolic Ca(2+) oscillations in response to excitatory neurotransmitter glutamate. These findings suggest the suitability of the 3D culture system for the proliferation and differentiation of neural precursor cells.

Animals↗

PubMed2000

Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells.

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic transmission in mature neurons and may play critical roles in neuronal development. However, the developmental stage at which neuronal cells begin to express functional receptors and their roles in lineage progression remain unclear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum-free medium in response to basic fibroblast growth factor. RT-PCR revealed the presence of mRNAs encoding AMPA(A), AMPA(C), KA(1), KA(2), NMDA(1), and NMDA(2D) subunits after 3 days in culture. The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca(2+) imaging and whole-cell patch-clamp recording techniques in cells pulse-labeled with bromodeoxyuridine (BrdU) for 1-4 hr. The recorded cells were then double-immunostained for BrdU incorporation and neuron-specific beta-tubulin (TuJ1). The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca(2+) and inward currents only in differentiating neurons. In contrast, proliferating (BrdU(+)TuJ1(-)) cells failed to respond to any ionotropic glutamate receptor agonists. Interestingly, Ca(2+) imaging revealed that a subpopulation of BrdU(+)TuJ1(+) cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro results were supported by flow cytometric sorting of AMPA-responsive cells pulse-labeled with BrdU for 1 hr in vivo, which revealed that functional AMPA receptors appear in BrdU(+)TuJ1(+) cells under physiological conditions and may play a role in terminal cell division.

Animals↗

PubMed2000

GCIP, a novel human grap2 and cyclin D interacting protein, regulates E2F-mediated transcriptional activity.

Regulation of mammalian cell growth and proliferation is governed through receptor-mediated signaling networks that ultimately converge on the cell cycle machinery. Adaptor proteins play essential roles in the formation of intracellular signaling complexes, relaying extracellular signals from the plasma membrane to the nucleus of a cell. The leukocyte-specific adaptor protein Grap2 is a central linker protein in immune cell signaling and activation. Using Grap2 as bait protein, we identified a novel human protein, GCIP (Grap2 cyclin-D interacting protein). We found that GCIP bound to Grap2 in both yeast two-hybrid assays and in mammalian cells through binding to the COOH-terminal unique domain and SH3 domain (designated QC domain) of Grap2. GCIP also associated with cyclin D both in vitro and in vivo. The expression of GCIP was found in all human tissues examined with the highest level of expression in the heart, muscle, peripheral blood leukocytes, and brain. Furthermore, phosphorylation of retinoblastoma protein by cyclin D-dependent protein kinase was reduced and E2F1-mediated transcription activity was inhibited in cells transfected with GCIP. High level expression of GCIP in terminally differentiated tissues and the inhibition of E2F1 transcription activation suggest that GCIP could play an important role in controlling cell differentiation and proliferation.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Ultrasensitive pheromone detection by mammalian vomeronasal neurons.

The vomeronasal organ (VNO) is a chemoreceptive organ that is thought to transduce pheromones into electrical responses that regulate sexual, hormonal and reproductive function in mammals. The characteristics of pheromone signal detection by vomeronasal neurons remain unclear. Here we use a mouse VNO slice preparation to show that six putative pheromones evoke excitatory responses in single vomeronasal neurons, leading to action potential generation and elevated calcium entry. The detection threshold for some of these chemicals is remarkably low, near 10(-11) M, placing these neurons among the most sensitive chemodetectors in mammals. Using confocal calcium imaging, we map the epithelial representation of the pheromones to show that each of the ligands activates a unique, nonoverlapping subset of vomeronasal neurons located in apical zones of the epithelium. These neurons show highly selective tuning properties and their tuning curves do not broaden with increasing concentrations of ligand, unlike those of receptor neurons in the main olfactory epithelium. These findings provide a basis for understanding chemical signals that regulate mammalian communication and sexual behaviour.

Animals↗

PubMed2000

Partial sciatic nerve ligation induced more dramatic increase of neuropeptide Y immunoreactive axonal fibers in the gracile nucleus of middle-aged rats than in young adult rats.

Neuropeptide changes in primary sensory neurons caused by partial nerve injury are likely involved in the development of neuropathic pain. In this study, using immunocytochemistry, we examined neuropeptide Y (NPY) expression in lumbar dorsal root ganglion (DRG) cells of young adult (2-3 months old) and middle-aged (8-10 months old) rats 4 weeks after partial sciatic nerve ligation (PSNL). Significantly higher NPY immunoreactivity was induced in the injured side DRG neurons, the dorsal horn and the gracile nuclei in middle-aged rats than in young rats. Using combined fluorescent dye tracing and NPY immunostaining, we found in middle-aged rats that 46% injured DRG neurons projected to the gracile nucleus and 45% of injured neurons were also NPY-IR, whereas 42% spared DRG neurons projected to the gracile nucleus and 18% of spared neurons were also NPY-IR. Thus PSNL induces NPY up-regulation in spared as well as injured DRG neurons, both contribute to the increased NPY immunoreactivity in the gracile nucleus in the middle-aged rats. The more dramatic increase of NPY in DRG neurons of middle-aged rats after PSNL shows that the responses to partial nerve injury are age-dependent, that suggests a possible relevance to the higher incidence of neuropathic pain in human middle age.

Aging↗

PubMed2000

Synaptic connectivity in hippocampal neuronal networks cultured on micropatterned surfaces.

Embryonic rat hippocampal neurons were grown on patterned silane surface in order to organize synapse formations in a controlled manner. The surface patterns were composed of trimethoxysilylpropyl-diethylenetriamine (DETA) lines separated by tridecafluoro-1,1,2,2-tetrahydrooctyl-1-dimethylchlorosilane (13F) spaces. Pre- and post-synaptic specializations were identified by immunostaining for synapsin I and microtubule-associated protein-2 (MAP-2). Functional synaptic connections were examined by recording simultaneously from pairs of neurons using the whole-cell configuration of the patch-clamp technique. Spontaneous and evoked synaptic currents were recorded in neurons cultured for 2-14 days. The formation of functional connections was accompanied by the appearance of spontaneous synaptic currents (SSCs), which could be detected after approximately 3 days in culture in the absence of evoked synaptic currents (ESCs). ESCs were detected only after approximately 7 days in culture, mostly in the form of unidirectional synaptic connections. Other forms of synaptic connectivity, such as bidirectional and autaptic connections, were also identified. Both transient GABAergic and glutamatergic signals mediated the transmissions between communicating cells. These results demonstrate the combination of various types of synaptic connections forming simple and complex networks in neurons cultured on line (DETA)-space (13F) patterns. Finally, precisely synchronized SSCs were recorded in neuron pairs cultured on pattern indicating the existence of a fast-acting feedback mechanism mediated by pre-synaptic GABA(A) receptors.

Alkanes↗

PubMed2000

Large-scale culture and selective maturation of human Langerhans cells from granulocyte colony-stimulating factor-mobilized CD34+ progenitors.

Dendritic cells (DCs) play a critical role as APCs in the induction of the primary immune response. Their capacity for Ag processing and presentation is tightly regulated, controlled by a terminal developmental sequence accompanied by striking changes in morphology, organization, and function. The maturation process, which converts DCs from cells adapted for Ag accumulation to cells adapted for T cell stimulation, remains poorly understood due in part to difficulties in the culture and manipulation of DCs of defined lineages. To address these issues, we have devised conditions for the culture of a single DC type, Langerhans cells (LCs), using CD34+ cells from G-CSF-mobilized patients. Homogenous populations of LCs, replete with abundant immunocytochemically demonstrable Birbeck granules, could be stably maintained as immature DCs for long periods in culture. Unlike other human DC preparations, the LCs remained fully differentiated after cytokine removal. Following exposure to TNF-alpha, LPS, or CD40 ligand, the LCs could be synchronously induced to mature. Depending on the agent used, distinct types of LCs emerged differing in their capacity for T cell stimulation, IL-12 production, intracellular localization of MHC products, and overall morphology. Most interestingly, the expression of different sets of Toll family receptors is induced or down-regulated according to the maturation stimulus provided. These results strongly suggest that different proinflammatory stimuli might drive distinct developmental events.

Adult↗

PubMed2000

Investigation of the mechanism of action of microperoxidase-11, (MP11), a potential anti-cataract agent, with hydrogen peroxide and ascorbate.

The interaction of hydrogen peroxide, ascorbate and microperoxidase-11 (MP11), a ferriheme undecapeptide derived from cytochrome c, has been investigated using spectrophotometry, oxymetry, electron paramagnetic resonance (EPR), and mass spectroscopy techniques. It is shown that in 50 m M phosphate pH 7. 0-7.4 in the absence of other reactants H(2)O(2)induces a concentration-dependent decrease in absorption at the Soret band (399 nm) of the microperoxidase, with concomitant H(2)O(2)decomposition and oxygen evolution. The reaction causes irreversible heme degradation, concomitant with loss of enzymatic activity. Ascorbate effectively protects MP11 from degradation and inhibits oxygen evolution. At ascorbate concentrations greater than that of H(2)O(2), microperoxidase degradation is almost completely prevented. Mass spectrometry showed that H(2)O(2)oxidizes the microperoxidase to a monooxygenated product, which did not form if ascorbate was included in the reaction system. There appears to be a 1:1 relationship between H(2)O(2)degradation and ascorbate oxidation. EPR experiments revealed that an ascorbate radical was formed during the reaction. These reactions may be described by a scheme where a putative 'compound I' of the microperoxidase is reduced by ascorbate back to the original redox state (ferric) of the peroxidase in two one-electron steps, concomitantly with oxidation of the ascorbate to an ascorbate radical or in one two-electron transfer step forming dehydroascorbate. In the absence of ascorbate, the 'compound I' reacts further with the peroxide causing microperoxidase degradation and partial oxygen evolution. These observations are relevant to the interaction of ferrihemes with H(2)O(2)and ascorbic acid and may be pertinent for the potential application of MP11 as an anti-cataract agent.

Ascorbic Acid↗

PubMed2000

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons.

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342-2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 microM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 microM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments. These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.

Age Factors↗

PubMed2000

Photoaging of the skin from phenotype to mechanisms.

The skin is increasingly exposed to ambient UV-irradiation thus increasing its risk for photooxidative damage with longterm detrimental effects like photoaging, which is characterized by wrinkles, loss of skin tone, and resilience. Photoaged skin displays prominent alterations in the cellular component and the extracellular matrix of the connective tissue with an accumulation of disorganized elastin and its microfibrillar component fibrillin in the deep dermis and a severe loss of interstitial collagens, the major structural proteins of the dermal connective tissue. The unifying pathogenic agents for these changes are UV-generated reactive oxygen species (ROS) that deplete and damage non-enzymatic and enzymatic antioxidant defense systems of the skin. As well as causing permanent genetic changes, ROS activate cytoplasmic signal transduction pathways in resident fibroblasts that are related to growth, differentiation, senescence, and connective tissue degradation. This review focuses on the role of UV-induced ROS in the photodamage of the skin resulting in biochemical and clinical characteristics of photoaging. In addition, the relationship of photoaging to intrinsic aging of the skin will be discussed. A decrease in the overall ROS load by efficient sunscreens or other protective agents may represent promising strategies to prevent or at least minimize ROS induced photoaging.

Animals↗

PubMed2000

PCR-based subtractive hybridization identifies repressed genes in growth-arrested human dermal fibroblasts following combined treatment with 8-methoxypsoralen and UVA irradiation (PUVA).

To identify genes which are repressed in growth-arrested human dermal fibroblasts upon a single treatment with 8-methoxypsoralen and UVA irradiation (PUVA) we have used a PCR-based subtractive hybridization protocol resulting in cloning of four PUVA-repressed genes. Sequence analysis and homology searches identified three known genes related to growth control, lipid and connective tissue metabolism. One cDNA clone represented a novel gene. Northern blot analyses confirmed a PUVA-dependent reduction in mRNA expression in fibroblasts in vitro. The identification of growth arrest related repressed genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these genes in the control and regulation of the postmitotic phenotype of fibroblasts on a molecular and cellular level.

Cell Division↗

PubMed2000

Immobilization of neural cells in three-dimensional matrices for biosensor applications.

To overcome logistical difficulties with current designs of cell- or tissue-based biosensors which have individual cells or tissue slices immobilized on membranes or microelectrode arrays, we have proposed a system that uses three-dimensional cultures of neural cells immobilized in hydrogel matrices. In this design, immobilized cells would be maintained in a reservoir and then transferred to a detector platform when needed for analysis. The development of such a system relies upon a renewable supply of cells and the ability to culture cells for long periods of time in three-dimensions while maintaining their physiological function. To investigate the ability to culture neural cells in 3D matrices, embryonic rat cortical neurons and astrocytes were immobilized by matrix entrapment in a novel sugar poly(acrylate) hydrogel and collagen gels. The sugar poly(acrylate) hydrogel does not appear to support neural cell growth as a result of a lack of cell adherence, small pore size and, possibly, harshness of synthesis conditions. In contrast, collagen gels support the growth of cortical neurons, astrocytes, as well as neural progenitor cells. Evidence is also presented from immunocytochemistry and patch-clamp measurements which shows that neural progenitor cells proliferate in culture and can be induced to differentiate into neural cell types. Thus, they potentially represent a renewable cell source.

Animals↗

PubMed2000

Formulation development and antitumor activity of a filter-sterilizable emulsion of paclitaxel.

PURPOSE: Paclitaxel is currently administered i.v. as a slow infusion of a solution of the drug in an ethanol:surfactant:saline admixture. However, poor solubilization and toxicity are associated with this drug therapy. Alternative drug delivery systems, including parenteral emulsions, are under development in recent years to reduce drug toxicity, improve efficacy and eliminate premedication. METHODS: Paclitaxel emulsions were prepared by high-shear homogenization. The particle size of the emulsions was measured by dynamic light scattering. Drug concentration was quantified by HPLC and in vitro drug release was monitored by membrane dialysis. The physical stability of emulsions was monitored by particle size changes in both the mean droplet diameter and 99% cumulative distribution. Paclitaxel potency and changes in the concentration of known degradants were used as chemical stability indicators. Single dose acute toxicity studies were conducted in healthy mice and efficacy studies in B 16 melanoma tumor-bearing mice. RESULTS: QW8184, a physically and chemically stable sub-micron oil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drug loading (8-10 mg/mL) having a mean droplet diameter of <100 nm and 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both in the presence and absence of human serum albumin. Based on single dose acute toxicity studies, QW8184 is well tolerated both in mice and rats with about a 3-fold increase in the maximum-tolerated-dose (MTD) over the current marketed drug formulation. Using the B16 mouse melanoma model, a significant improvement in drug efficacy was observed with QW8184 over Taxol. CONCLUSIONS: QW8184, a stable sub-micron o/w emulsion of paclitaxel has been developed that can be filter-sterilized and administered i.v. as a bolus dose. When compared to Taxol, this emulsion exhibited reduced toxicity and improved efficacy most likely due to the composition and dependent physicochemical characteristics of the emulsion.

Animals↗

PubMed2000

Calcitonin gene-related peptide, substance P and protein gene product 9.5 immunoreactive axonal fibers in the rat footpad skin following partial sciatic nerve injuries.

Chronic constriction injury (CCI) and partial ligation (PSNL) of the sciatic nerve induce a similar neuropathic pain syndrome in rats. We examined calcitonin gene-related peptide (CGRP), substance P (SP) and protein gene product (PGP) 9.5 immunoreactive (IR) axons in the footpad skin after the two types of injury. Four and 14 days after CCI, CGRP- and SP-IR axons in the ipsilateral footpad skin disappeared in most rats, but in one third, sparse CGRP- and SP-IR fibers remained. PGP-IR axons dramatically decreased, but some thick fiber fascicles appeared. At the ultrastructural level, these PGP-IR thick fiber fascicles were characterized as unmyelinated axons surrounded by non-IR Schwann cells. Some of these axons were swollen and irregular in shape. In contrast, 4 days after PSNL, CGRP-, SP-, and PGP-IR axons in the ipsilateral footpad skin were present, though significantly reduced in density, in all rats, and by 14 days all IR fiber densities in the footpad skin partially recovered. The loss of CGRP and SP axons in the footpad skin of the CCI model suggests that sensory nerves containing neuropeptides are not essential in transducing stimuli applied to the footpad skin into neuropathic pain, but the abnormal PGP-IR unmyelinated axons in thick fiber fascicles might play a role. The partial loss and rapid recovery of IR axons in the footpad skin after PSNL shows that the two injury models, causing similar behaviors, are associated with very different patterns of cutaneous innervation at the time when the pain syndrome is well developed.

Animals↗

PubMed2000

Pidd, a new death-domain-containing protein, is induced by p53 and promotes apoptosis.

The p53 tumour suppressor promotes cell-cycle arrest or apoptosis in response to cellular stress, such as DNA damage and oncogenesis. This role of p53 is important for its tumour-suppression function and depends, at least in part, on its ability to bind to specific DNA sequences and activate the transcription of target genes. The pathway through which p53 promotes apoptosis is not fully understood. Here we describe a new gene regulated by p53 that encodes a predicted protein of 915 amino acids in mice (910 amino acids in humans), which we have named Pidd. The mouse Pidd cDNA contains a p53 consensus DNA binding sequence upstream of the Pidd-coding region. This sequence element bound to p53 and conferred p53-dependent inducibility on a heterologous reporter gene. Moreover, Pidd RNA was induced by ionizing radiation in a p53-dependent manner and the basal level of Pidd RNA was dependent on Trp53 status. Overexpression of Pidd inhibited cell growth in a p53-like manner by inducing apoptosis. Antisense inhibition of Pidd expression attenuated p53-mediated apoptosis. Our data suggest that Pidd is an effector of p53-dependent apoptosis.

Amino Acid Sequence↗

PubMed2000

Blockade and reversal of spinal morphine tolerance by peptide and non-peptide calcitonin gene-related peptide receptor antagonists.

This study examined the effects of the peptide CGRP receptor antagonist CGRP(8-37) and the newly-developed non-peptide CGRP receptor antagonist BIBN4096BS for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. Repeated administration of intrathecal morphine (15 microg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED(50) value in the tailflick and paw pressure tests. Co-administration of CGRP(8-37) (4 microg) or BIBN4096BS (0.05, 0.1 microg) with morphine (15 microg) prevented the decline of antinociceptive effect and increase in ED(50) value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP(8-37) also did not potentiate the acute actions of spinal morphine. In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP(8-37) (4 microg) with morphine (15 microg) partially restored the antinociceptive effect and ED(50) value of acute morphine, reflecting the reversal of tolerance. Animals tolerant to intrathecal morphine expressed increased CGRP and substance P-like immunostaining in the dorsal horn of the spinal cord. The increase in CGRP, but not substance P-like immunostaining, was blocked by a co-treatment with CGRP(8-37) (4 microg). In animals already tolerant to morphine, the increase in CGRP but not substance P-like immunostaining was partially reversed by CGRP(8-37) (4 microg). These data suggest that activation of spinal CGRP receptors contributes to both the development and expression of spinal opioid tolerance. CGRP receptor antagonists may represent a useful therapeutic approach for preventing as well as reversing opioid tolerance.

Animals↗

PubMed2000

Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

Acetylcholine↗