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SH-SY5Y Cells. SH-SY5Y cells were cultured in 96-well plates until confluent. After removal of culture medium, [3H]noradrenaline 0.07 M, 2.5 Ci ml ; was added 60 l well ; in oxygenated Krebsbicarbonate buffer 118 mM NaCl, 2.4 mM KCl, 2.4 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 10 mM D-glucose, 1 mM ascorbic acid, and 10 M pargyline, pH 7.4 ; , and the cells were incubated for 1 h at 37C. After loading the cells with [3H]noradrenaline, they were washed twice with Krebs buffer with added nomifensine 0.5 M ; to prevent reuptake of released [3H]noradrenaline. Cells were then incubated for 5 min in the same buffer, with or without mecamylamine. The buffer was replaced with Krebs buffer plus nomifensine containing nicotine and or galantamine 80 l well ; . After 5 min, the medium containing released [3H]noradrenaline was transferred to a 96-well Optiplate PerkinElmer Life Sciences, Zaventem, Belgium ; . Microscint-40 170 l; PerkinElmer ; was added to each well and radioactivity was counted using a microbeta liquid scintillation counter Wallac MicroBeta TriLux 1450; PerkinElmer Life Sciences, Espoo, Finland; counting efficiency, 31% ; . Radioactivity remaining in the cultures was determined by addition of 80 l 0.5 M perchloric acid and incubation for 1 h at 37C, followed by scintillation counting. The total amount of [3H]noradrenaline present in the cells at the point of stimulation was equivalent to the tritium released plus tritium remaining. Released [3H]noradrenaline was calculated as a percentage of total radioactivity in the corresponding wells, and results were then expressed as a percentage of basal release buffer stimulation ; . The average values of basal release and corresponding total radioactivity were 3189 80 and 39, 424 809 cpm mean S.E.M., n 60 ; respectively. Therefore, basal release corresponds to 8.0% of the radioactivity present in culture wells at the beginning of the experiment. Hippocampal Slices. The measurement of [3H]noradrenaline release from rat hippocampal slices was based on the method of Anderson et al. 2000 ; . Rats 250350 g ; were killed by cervical dislocation, and hippocampi were rapidly dissected. Hippocampal slices 0.25 mm ; were prepared using a McIlwain tissue chopper, washed twice with Krebs-bicarbonate buffer as above ; , and loaded with [3H]noradrenaline 0.07 M, 2.5 Ci ml ; for 30 min at 37C. After four washes with Krebs buffer plus nomifensine 0.5 M ; , slices were dispersed into 96-well Multiscreen filter plates Millipore, Bedford, MA ; . Hippocampi from two animals were sufficient for one 96-well plate. Buffer was removed using a vacuum filtration unit.
Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1006, New York NY 10029 Dr. Aledort is a consultant for Baxter and Immunex, and is on the speakers' bureaus for Nabi and Aventis. A potential dependency on the pernicious tobacco smoking breathing exercises which deliver the tobacco smoke into your mouth or lungs so that its nicotine content can be absorbed into your bloodstream in order to maintain your habitual concentration. PURPOSE. To examine the effects of acetylcholine ACh ; on glutamate-induced neurotoxicity in embryonic rat retinal neurons. METHODS. Primary cultures were obtained from rat retinas at embryonic days 17 to 19. Cultured cells were exposed to glutamate for 10 minutes, followed by incubation in glutamate-free medium for 1 hour. Drugs were added to the incubation medium for 1 to 24 hours until immediately before glutamate exposure and were removed from culture medium during glutamate exposure and the postincubation period. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method. RESULTS. Cell viability was markedly reduced by 10-minute exposure to 500 M glutamate followed by a 1-hour incubation in glutamate-free medium. Incubating the cultures with 1 M ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol 1 M ; . The protective effect of ACh against glutamate neurotoxicity was inhibited by a nicotinic acetylcholine receptor nAChR ; antagonist, mecamylamine 0.5 M ; , whereas a muscarinic acetylcholine receptor mAChR ; antagonist, atropine 0.5 M ; did not affect ACh-induced protection. In addition, a similar protection was induced by application of nicotine 1 M ; , but not by muscarine 1 M ; . Pretreatment with nicotine induced a protective effect in a time-dependent manner, ranging from 1 to 12 hours. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 M induced dose-dependent protection against glutamate neurotoxicity. Furthermore, the protective action of nicotine was inhibited by simultaneous application of dopamine D1 receptor antagonist, SCH23390 1 M ; , with nicotine, whereas a dopamine D2 receptor antagonist, domperidone 1 M ; , did not affect nicotine-induced protection. CONCLUSIONS. These results suggest that pretreatment of cultured rat retinal neurons with ACh or the nAChR agonists, nicotine and carbachol, has a protective action against glutamate neurotoxicity through nAChRs and that the dopamine release induced by nicotinic stimulation subsequently protects the retinal neurons by way of dopamine D1 receptors. Invest Ophthalmol Vis Sci. 2002; 43: 446 ; ells are closely connected with surrounding cells including neurons. Neurons respond to neurotransmitters released by adjacent neurons and take part in the formation of neuronal networks, whereas neurons maintain homeostasis and survive within the networks among surrounding neurons. In our previous study, we showed the involvement of glutamate in ischemia-reperfusion-induced retinal injury in vivo.1 Glutamate is an excitatory neurotransmitter in the retina, 2 4 yet it has a toxic action57 on postsynaptic neurons by stimulating its receptors when it is present in excess under pathologic conditions such as retinal ischemia.8 10 The N-methyl-Daspartate NMDA ; receptor, a subtype of glutamate receptors, plays a predominant role in the delayed retinal neuronal death induced by glutamate.7, 1116 Furthermore, we have demonstrated that dopamine, one of the chemical neuromodulators in the retina, has a protective action on cultured embryonic rat retinal neurons against NMDA receptormediated glutamate neurotoxicity through dopamine D1 receptors.15 Based on these findings, we suggest that neurotransmitters such as glutamate and dopamine may contain some signals affecting neuronal cells' survival and death in addition to their signal informations of the neuronal network. Acetylcholine ACh ; , which is released from cholinergic amacrine cells, is one of the major endogenous neurotransmitters in the retina. ACh receptors AChRs ; are subdivided into two main types, nicotinic AChRs nAChRs ; and muscarinic AChRs mAChRs ; , and both nAChRs and mAChRs are prevalently distributed in the ganglion cell layer GCL ; and the inner nuclear layer INL ; of the rat retina.17, 18 Therefore, to elucidate whether or not ACh is involved in neuronal cells' survival and death in the retina, we examined the effect of ACh on glutamate-induced neurotoxicity mediated through NMDA receptors in cultured rat retinal neurons.
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From the Department of Obstetrics and Gynecology, University of Oklahoma, Oklahoma City J.C.C. the National Institute of Child Health and Human Development, Bethesda, Md. M.A.K., R.P.N. the Department of Obstetrics and Gynecology, University of Alabama at Birmingham, Birmingham J.C.H. the Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh S.L.H., R.P.H. the Biostatistics Center, George Washington University, Rockville, Md. E.A.T., M.L.F. the Department of Obstetrics and Gynecology, Wake Forest University School of Medicine, Winston-Salem, N.C. J.M.E. the Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas K.J.L. the Department of Obstetrics and Gynecology, Thomas Jefferson University, Philadelphia R.W. and the Department of Obstetrics and Gynecology, University of Utah, Salt Lake City M.V. ; . Address reprint requests to Dr. Klebanoff at NICHD, NIH, 6100 Executive Blvd., Rm. 7B03 MSC 7510, Bethesda, MD 20892-7510, or at mk90h nih.gov. Other authors were Wayne Trout, M.D., Department of Obstetrics and Gynecology, Ohio State University, Columbus; Atef Moawad, M.D., Department of Obstetrics and Gynecology, University of Chicago, Chicago; Baha M. Sibai, M.D., Department of Obstetrics and Gynecology, University of Tennessee, Memphis; Menachem Miodovnik, M.D., Department of Obstetrics and Gynecology, University of Cincinnati, Cincinnati; Mitchell Dombrowski, M.D., Department of Obstetrics and Gynecology, Wayne State University, Detroit; Mary J. O'Sullivan, M.D., Department of Obstetrics and Gynecology, University of Miami, Miami; J. Peter VanDorsten, M.D., Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston; Oded Langer, M.D., Department of Obstetrics and Gynecology, University of Texas at San Antonio, San Antonio; and James Roberts, M.D., Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh, Pittsburgh. * Other members of the Network of MaternalFetal Medicine Units are listed in the Appendix and orinase. Is the person's day centered around taking medication? If so, consultation with the health care provider may clarify long-term risks and benefits of the medications and identify other treatment options. Does the person take pain medication only on occasion, perhaps three or four pills per week? If this is the case, then the likelihood of dependency is low. Have there been any other chemical alcohol or drug ; abuse problems in the person's life? If so, then it is important to inform the health care provider, who will need to take that into consideration when prescribing. Does the person in pain spend most of the day resting, avoiding activity, or feeling depressed? If so, that suggests the pain medication is failing to promote rehabilitation. Daily activity is necessary for the body to produce its own pain relievers, to maintain strength and flexibility, and to keep life full and meaningful. Encourage the pain person to request recommendations from a physician for an exercise program. Is the pain person able to function work, household chores, and play ; with pain medication in a way that is clearly better than without? Chances are that the pain medication is contributing to wellness. Most people who are addicted to pain medications or other substances excluding nic0tine and caffeine ; do not function well. They are undependable and forgetful. Dr. Townley is chief cardiology resident at the Queen Elizabeth II Health Sciences Centre, Dalhousie University, Halifax, Nova Scotia. Dr. Howlett is associate professor of medicine, Dalhousie University and medical director, Queen Elizabeth II Heart Function and Transplantation Clinic, Halifax, Nova Scotia and tolbutamide. In prenatal toxicity, including teratogenicity, the occurrence of the sometimes very pronounced species and strain differences is well known. The reasons for the differences in response or in susceptibility to the action of 'teratogens' are manifold, and not all of the sources of variability in outcome are clearly understood." Dr D Neubert, Institute of Toxicology & Embryopharmacology, Free University Berlin, Germany, writing in the book Advances in Applied Toxicology, ed. A D Dayan & A J Paine, publ. Taylor & Francis, p 203, 1989, for example, pro smoking.
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