Glutamate Neurotoxicity in Culture Depends on the Presence of Glutamine: Implications for the Role of Glial Cells in Normal and Pathological Brain Development

Glutamate toxicity was studied in neuronal (SC9), glial (WC5), and neuroblastoma-glioma hybrid cell lines. In all three cell types, glutamate had a dual effect, depending on the concentration of glutamine in the culture medium. An expected dose-dependent cytotoxicity of the amino acid was observed when cells were cultured in medium containing the standard glutamine concentration (1-4 mM), but when the culture's glutamine content was decreased to 0.15-0.5 mM, glutamate had an apparent opposite, growth-promoting effect. The specificity of glutamate effect was indicated by the following: (a) it was stereospecific, with the L and not the D isomer being active; (b) monosodium aspartate was inactive in the presence of either high or low glutamine; and (c) monosodium glutamate and monopotassium glutamate had a similar dual effect. Furthermore, the glutamate receptor antagonist gamma-glutamylglycine blocked the amino acid cytotoxicity in a dose-dependent fashion. As glial cells are a major source of glutamine in the brain, neuronal-glial co-cultures were used to analyze the possible role of glial cells in glutamate neurotoxicity. It was found that SC9 cells were more sensitive to glutamate when co-cultured with WC5 cells. Continuous depolarization of the SC9 cells with KCl decreased cell number, but glutamate had no additive neurotoxic effect when added with KCl. We suggest that glutamine, glial cells, and neuronal activation play roles in modulating glutamate neurotoxicity, in developing as well as aged brains. It is tempting to speculate also that alterations in the glutamate/glutamine ratio under pathological conditions may take part in the etiology of some neurodegenerative diseases.     

Effects of Pro-Leu-Gly-NH2 and cyclo (Leu-Gly) on the binding of 3H-quinuclidinyl benzilate to striatal cholinergic muscarinic receptors

The effects of Pro-Leu-Gly-NH2 (melanotropin release inhibiting factor, MIF) and its analog, cyclo (Leu-Gly) on the mouse and rat striatal cholinergic muscarinic receptors labeled with 3H-quinuclidinyl benzilate (QNB) were investigated. 3H-QNB bound to the rat striatal muscarinic receptors at a single high affinity site with receptor density (Bmax value) of 1200 fmol per mg protein and an apparent dissociation constant (Kd value) of 53.5 pM. At 140 pM concentration of 3H-QNB, the specific binding to the receptors was 724 fmol per mg protein. MIF in a concentration range of 10(-9) to 10(-4) M did not alter the binding of 3H-QNB but at 10(-3) M decreased the binding by 25%. Cyclo (Leu-Gly), on the other hand, in the concentration range of 10(-9) to 10(-3) M had no effect on the binding of 3H-QNB. A single injection of MIF (3 or 10 mg/kg IP) to rats did not alter the Bmax or the Kd value of 3H-QNB to bind to the striatal membranes. 3H-QNB bound to the mouse striatal muscarinic receptors at a single high affinity site with a Bmax value of 991 fmol/per mg protein and a Kd value of 21 pM. Neither acute administration of MIF (3 or 10 mg/kg IP) nor chronic treatment of the peptide (2, 8 or 32 mg/kg IP, daily for 5 days) to mice could influence the binding of 3H-QNB to the striatal muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolated rat gastric parietal cells: Cholinergic response and pharmacology

Isolated, partially purified or enriched rat gastric muscosal parietal cells were shown to respond to carbamycholine (EC₅₀ = 2 μM) and other muscarinic cholinergic agonists as measured by an increased accumulation of ¹⁴C-aminopyrine, an indirect measure of acid secretion. The secretory response to carbamylcholine was shown to be inhibited stereoselectively and reversibly by nanomolar concentrations of muscarinic cholinergic antagonists. Non-muscarinic antagonists, including cimetidine, were either ineffective or very weak inhibitors. The affinity constants calculated for cholinergic antagonist inhibition of ¹⁴C-aminopyrine accumulation induced by carbamylcholine were similar to those previously calculated from direct binding studies on purified parietal cell particulate fractions using ³H-QNB (1). These studies support the existence of specific parietal cell muscarinic cholinergic receptors with which the natural secretagogue acetylcholine interacts to regulate gastric acid secretion.     

Neuronal Activation Regulates the Expression of Opioid Receptors: Possible Role of Glial-Derived Factors and Voltage-Dependent Ion Channels

The previous observation that a continuous chemical depolarization of aggregating rat brain cells with KCl alters the expression of opioid receptors was examined in more detail. In contrast to its significant and converse effect on forebrain and hindbrain cells cultured in serum-containing medium, KCl had only a small and transient effect in serum-free cultures of both types. The basal receptor density in serum-free cultures was similar to the receptor density in KCl-treated serum-containing cultures, but medium conditioned by glial cells restored partially the effect of KCl in serum-free cultures. The effect of KCl in serum-containing forebrain cultures was enhanced by the voltage-dependent calcium channel blocker verapamil, and magnesium and cadmium had a similar, though smaller, effect. The sodium channel activator veratridine had a profound and dose-dependent inhibitory effect on the expression of the receptors in forebrain and hindbrain cultures, and tetrodotoxin blocked the veratridine effect. Information about the selectivity of the effect of neuronal activation on the various opioid receptor subtypes was obtained with the neuroblastoma X glioma hybrid M8 cells that possess only delta type opioid receptors. A Scatchard analysis of [3H]etorphine binding to these cells has shown that depolarization increased the Bmax, but had little, if any, effect on the affinity (KD) of the ligand to the receptors. The significance of depolarization and voltage-dependent sodium and calcium channels on the expression of different opioid receptor subtypes is discussed.     

Antilaminin IgG releases TXB2 through activation of the cholinergic system

Antilaminin IgG was bound to cholinergic muscarinic receptors of normal mice heart and released TXB2, simulating the biological effect of a cholinergic agonist. Antilaminin IgG interfered with the binding of the radiolabelled muscarinis antagonist (-)3H-QNB in a noncompetitive fashion. Following the interaction of the antibody with the cholinergic receptor, an increased production of TXB2 occurred. This effect required the activation of the muscarinic cholinergic system, because it was blunted by atropine and mimicked by acetylcholine.     

Circulating antibodies against neonatal cardiac muscarinic acetylcholine receptor in patients with Sjögren's syndrome

Isolated congenital heart block may be associated with Primary Sjogren's Syndrome. In this work we demonstrated that IgG present in the sera ofpatients with Primary Sjogren's Syndrome (PSS) could bind and activate muscarinic acetylcholine receptors of rat neonatal atria. These antibodies were able to inhibit in a irreversible manner the binding of ³H-QNB to muscarinic cholinergic receptors of purified rat atria membranes. Moreover, IgG from PSS individuals could modify biological effects mediated by muscarinic cholinoceptors activation, i.e. decrease contractility and cAMP and increase phosphoinositide turnover and cGMP. Atropine blocked all of these effects and carbachol mimicked them; confirming muscarinic cholinergic receptors-mediated PSS IgG action. Neither binding nor biological effect were obtained using adult instead of neonatal rat atria. IgG from sera of normal women were not effective in the studied system. The prevalence of cholinergic antibody was 100% in PSS and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against muscarinic cholinergic receptors may be another serum factor to be considered in the pathophysiology of the development of congenital heart block.     

电离辐射后大鼠离体胰腺腺泡淀粉酶释放及M受体数量变化

我们曾观察到大鼠经γ-射线照射后胰淀粉酶活性降低和分泌减少[1],为进一步探讨照射后胰酶分泌减少的机制,本研究制备出分散的大鼠胰腺腺泡悬液并以不同浓度的~3H-二苯羟乙酸-3-喹咛环酯(~3Hquinuclidinyll benzilatc,简称~3H-QNB)进行M受体结合测定,同时观察胆碱能介质氨甲酰胆碱刺激腺泡所引起的淀粉酶释放反应。结果表明,γ-射线10Gy照射后3天,大鼠分散的胰腺腺泡在氨甲酰胆碱刺激时淀粉酶释放量减少到对照的50％,腺泡M受体与~3H-QNB最大结合量(Bmax)减少到对照的38％,伋M受体与~3H-QNB结合的解离常数(K_D)无改变,说明胰腺腺泡细胞M受体数量的减少可能是照射后胰腺腺泡分泌淀粉酶减少的原因之一。     

Increase in muscarinic receptors in rat intestine by thyrotropin releasing hormone (TRH)

The effect of Thyrotropin Releasing Hormone (TRH) on the contractile activity elicited by acetylcholine and electric stimulation in the rat ileus terminalis was investigated. TRH did not show any intrinsic contractile activity but, after a 30 minute latency period, the peptide caused a shift to the left of the dose-response curve for both acetylcholine and electric stimulation. The binding of 3H-quinuclidinylbenzilate (3H-QNB) assayed on ileum slices disclosed that the addition of TRH increased the number of muscarinic cholinergic receptors without changes in affinity when incubation was performed at pH 7.8, but no effect TRH was demonstrated at pH 7.4. Therefore, in spite of its neural and direct actions on intestine motor activity, TRH may affect the acetylcholine induced contraction by increasing the number of muscarinic receptors at a specific pH.     

The Cu-Zn superoxide dismutase (SOD1) inhibits ERK phosphorylation by muscarinic receptor modulation in rat pituitary GH3 cells

The Cu-Zn superoxide dismutase (SOD1) belongs to a family of isoenzymes that are able to dismutate the oxygen superoxide in hydrogen peroxide and molecular oxygen. This enzyme is secreted by many cellular lines and it is also released trough a calcium-dependent depolarization mechanism involving SNARE protein SNAP 25. Using rat pituitary GH3 cells that express muscarinic receptors we found that SOD1 inhibits P-ERK1/2 pathway trough an interaction with muscarinic M1 receptor. This effect is strengthened by oxotremorine, a muscarinic M agonist and partially reverted by pyrenzepine, an antagonist of M1 receptor; moreover this effect is independent from increased intracellular calcium concentration induced by SOD1. Finally, P-ERK1/2 inhibition was accompanied by the reduction of GH3 cell proliferation.These data indicate that SOD1 beside the well studied antioxidant properties can be considered as a neuromodulator able to affect mitogen-activated protein kinase in rat pituitary cells trough a M1 muscarinic receptor.     

Modulation of neuritogenesis by astrocyte muscarinic receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.     

Interrelation of immune and mediator lymphocyte receptors in mice

A study was made of the action of a specific muscarinic antagonist 3H-quinuclidinyl benzylate on the immune rosette formation in BALB/c mice. It was shown that treatment of mouse spleen lymphocytes by 3H-QNB at a concentration of 10(-9) M-10(-14) M brought about rosette formation inhibition. The process was dose-dependent. Atropine reversed the action of 3H-QNB.     

Reduced muscarinic receptor plasticity in frontal cortex of aged rats after chronic administration of cholinergic drugs

Age-related differences in muscarinic receptor plasticity were observed in young, adult and senescent Fischer 344 rats (3, 9 and 27 months old, respectively) following the chronic, intracerebroventricular (ivt) administration of a cholinergic agonist, oxotremorine, or antagonist, methylatropine. After three weeks treatment of young rats with ivt oxotremorine, the maximum number (Bmax) of 3H-QNB binding sites in frontal cortex, determined by saturation experiments, was reduced by 27%, with no apparent change in the affinity (Kd) of 3H-QNB for the muscarinic receptor. Conversely, chronic ivt methylatropine administered to 3 month old animals caused a 29% increase in Bmax with no significant change in Kd. Adult animals showed a somewhat lesser degree of muscarinic receptor plasticity (16% down-regulation after oxotremorine, 22% up-regulation after methylatropine). However, 3H-QNB binding parameters in frontal cortex from senescent rats were not significantly altered following identical treatments with oxotremorine or methylatropine. Thus, muscarinic receptor adaptation to chronic, cholinergic drug administration was impaired in aged animals. This reduced receptor plasticity with aging could have important implications for the long-term drug treatment of elderly patients and for the therapeutic efficacy of cholinergic drugs in age-related neurological disorders, such as Alzheimer's disease.     

Muscarinic Receptor Subclasses in the Chick Embryo Retina: Influence of Corticosterone Treatment

The present study was performed on retinas of chick embryos receiving at day 8 of incubation an intracerebral injection of 0.02 microgram of corticosterone. We had previously shown with the use of [3H]quinuclidinylbenzilate [( 3H]QNB) that such treatment induced the appearance of two muscarinic binding sites in the treated retinas, whereas only one was detectable in the controls. In the present study we investigated muscarinic cholinergic receptor subclasses with agonist and antagonist binding. Agonist binding was studied by varying the concentrations of carbachol and acetylcholine (10(-9) M-10(-5) M) in the presence of a constant concentration (0.2 nM) of [3H]QNB. Two subpopulations of receptors were revealed, a high- and a low-affinity receptor, in both treated and control retinas. However, in the hormone-treated retinas, the two subpopulations significantly differed from the controls in their affinity and in their relative percentage among the total receptor population. Moreover, using pirenzepine, an antagonist known to have the capacity to distinguish between muscarinic cholinergic subclasses, two receptor subpopulations were found to be present in the hormone-treated retinas but a single one in the controls. It is suggested that hormone treatment can either induce the appearance of a new subclass of muscarinic cholinergic receptors or favor the maturation of a population of retinal cells having these receptors. Pirenzepine binding in retinas from intact embryos of 7, 9, and 11 days of incubation revealed one receptor subpopulation. Thus, these findings are more consistent with the hypothesis that corticosterone effects the target cells, either inducing changes in muscarinic receptor and/or modifying the receptor environment.     

Depolarizing stimuli and neurotransmitters utilize separate pathways to activate protein kinase C in sympathetic neurons.

Several types of extracellular signals affect the function of peripheral neurons. Depolarizing stimuli cause sudden increases in permeability to various ions leading to propagation of nerve impulses and release of transmitter substances. Neurons also receive external signals via neurotransmitter receptors located on the membrane. Different types of receptors present on sympathetic neurons are believed to modulate stimulation-evoked release of norepinephrine. We have investigated the effects of depolarizing stimuli and neurotransmitters on different signaling pathways in homogeneous cultures of chick sympathetic neurons. Depolarizing stimuli (35 mM KCl; electrical stimulation, 1 Hz for 5 min) and neurotransmitters (acetylcholine and 5-hydroxytrypatmine) enhanced membrane binding of protein kinase C by 2-5-fold. 35 mM KCl increased formation of 1,2-diacylglycerol and hydrolysis of [3H]phosphatidycholine without affecting [3H] phosphoinositide hydrolysis. Neurotransmitters increased [3H]inositol phosphates and 1,2-diacylglycerol without affecting the hydrolysis of [3H]phosphatidylcholine. 5-Hydroxytryptamine and acetylcholine (muscarinic component) did not increase Ca2+ concentration in the Indo-1-loaded neuronal cell body or the growth cone, but 35 mM KCl and electrical stimulation caused a marked increase in Ca2+ concentration in both regions of sympathetic neurons. We believe this to be the first demonstration of these two types of signalling mechanisms co-existing in sympathetic neurons; depolarization activate the phosphatidylcholine pathway and neurotransmitters activate the phosphatidylinositol pathway. The importance of two pathways in controlling neuronal Ca2+ concentration and the release of transmitter is discussed.     

Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

Changes in brain muscarinic acetylcholine receptors and behavioral responses to atropine and apomorphine in chronic atropine-treated rats

Chronic blockade of cholinergic transmission with atropine resulted in a decrease in atropine-induced activity in the rats, whereas apomorphine - induced locomotion was enhanced. Maximal binding of ³H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, to homogenate of cerebral cortex, striatum and hippocampus was significantly higher in chronic atropine-treated rats than in control animals. No difference was observed in K_D value of the specific ³H-QNB binding or in ID₅₀ value of oxotremorine in inhibiting ³H-QNB binding. No change in the specific binding of ³H-spiroperidol, a dopaminergic antagonist, was observed in those three regions of brains of chronic atropine-treated rats when it was compared with that of control animals. The role of brain muscarinic acetylcholine receptors in behavioral responses is discussed relating an effect of dopaminergic neurons on cholinergic activities.     

Bombesin and calcitonin secretion by pulmonary carcinoma is modulated by cholinergic receptors

Three established cell lines derived from human small cell carcinoma of the lung, and known to produce significant amounts of peptide hormones were used to evaluate the regulation of hormone secretion by cholinergic agonists. In two of the cell lines (DMS 53, DMS 153) acetylcholine chloride, bethanechol chloride, and carbamylcholine at the concentrations of 10(-3)M to 10(-5)M stimulated secretion of bombesin and calcitonin as measured by RIA. The third cell line, DMS 406, was not significantly stimulated. Inhibition of induced stimulation by the cholinergic antagonist atropine, but not hexamethonium, indicated the presence of muscarinic rather than the nicotinic type of cholinergic receptors on the stimulatable cells. These receptors appear to mediate hormone secretion comparably to normal endocrine cells.     

Differences in the functional responses of two cell lines each expressing Pi-hydrolysis-coupled muscarinic receptors

Fluorescent oxonol dyes were used to measure changes in the membrane potential of two different cell lines each expressing Pi-hydrolysis coupled muscarinic receptors. Both SK-N-SH human neuroblastoma cells and m1-transfected A9 L cells express muscarinic receptors, which, when stimulated, elicit a large increase in intracellular calcium, and release of inositol phosphates. Despite the similarity in this second-messenger response, muscarinic stimulation resulted in a hyperpolarization in the transfected A9 L cells whereas a small depolarization was observed in the neuroblastoma cells. The carbachol-mediated hyperpolarization of the transfected A9 L cells could be mimicked by increasing intracellular calcium with the ionophore A23187, suggesting, that it may be mediated by calcium-activated potassium channels. Exposure of SK-N-SH cells to A23187, on the other hand, had no effect on the membrane potential. These studies demonstrate that the activation of a second messenger system does not solely dictate the electrophysiological response of a cell, but that other factors such as the expression of ion-channels is critical in the determination of that response.     

The distribution of cholinergic enzymes and 3H-QNB binding in smooth muscles of the cow stomach (Bos taurus)

1. The activity of choline acetyltransferase (CAT) and cholinesterases (ChEs), and the binding of 3H-quinuclidinyl benzilate (3H-QNB) were measured in the longitudinal and circular muscle layers from the four compartments of the cow stomach. 2. CAT and ChEs activity were unevenly distributed in the various regions but distributional pattern of activity of both enzymes was very similar. 3. The maximum binding sites of 3H-QNB were also unequal in the various regions and the distribution of 3H-QNB binding was also similar to that of the activity of two enzymes. 4. These results suggest that cholinergic fibers and muscarinic receptors are not distributed symmetrically throughout the smooth muscle layers of the four compartments of the cow stomach. There may be a positive correlation between the innervation and the density of the receptors on the smooth muscle.     

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.