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Light and ultrastructural characteristics of neuroblastoma glioma hybrid NG 108-15 cells
Life Sciences, Vol. 33, Sup. I, 1983, pp. 719-722 Printed in the U.S.A.
Pergamon Press
LIGHT AND ULTRAST~3CIURAL C}LARACI~RISTICS CF NEUROBLASTCMAGLICMAHYBRID NG 108-15 C F ~ S Arnold Schecter, Department of Preventive Medicine, Upstate M,edical Center, Clinical Canpus, S.U.N.Y., Binghamton, New York 13901 USA (Received in final form June 26, 1983) Sunmary "Undifferentiated"neuroblastoma glic~a hybrid NG 108-15 cells have not previously been characterized at a light and ultrastructural level despite their use in opiate receptor studies and their possession of one or more opioids. Three cell types rather than one were found. Type A is the classic round hybridc~a cell. Type B is a neuron like cell and Type C is a giant cell similar to Type A cells but larger and with dense cytoplasm. Dense core vesicles, 800-1200 A ° in di~neter were found as were virus particles in most Type A cells. Multivesicular bodies with clear vesicles which are interpreted as glutaraldehyde artefacts were found but no clear neurosecretory vesicles were found in any A cells--Etorphine at 10 -6 M for 48 hours and naloxone 10 -4 M were e~ployed to product dependence and precipitated abstinence but no clear cut morphologic differences were noted between control, opiate dependent and precipitated abstinent cells. Neuroblastoma glicrna hybrid NG 108-15 cells have been used as a neuronal model because of their relative simplicity cempared to brain, (1,2,3). The cells are believed to contain delta and episilon opioid receptors, (1,4). Binding of morphine and other opiates to receptors results in an inhibition of adenylate cyclase activity and a decrease in cyclic AMP levels in intact cells, (2). Under certain conditions these cells may be manipulated to increase levels of specific neurotransmitter enzyme activities such as acetylcholinesterase and choline acetyltransferase, as well as stimulus dependent release of acetylcholine, (5,6). Glaser, et.al~7,8) demonstrated that NG 108-15 cells also contained met-enkephalin and leu-enkephalin. Braas, et.al. (9) confirmed the presence of enkephalins by immunocytochemical and biochemical means and localized enkephalin like /xmaznoreactivity to the dense core vesicles. Schultz, et.al. (i0) found dynorphin and alpha neoendorphin like material in these cells and noted considerable variability in the amount of dynorphin and alpha neoendorphin activity in various samples. Although morphologically related neuroblastoma glicrna cells were previously described, (ii), the NG 108-15 line has never been morphologically characterized. Methods Cells were grown in the laboratory of Werner Klee and then processed in our laboratory. Cells were grown in flasks and no attempt was made to produce cell differentiation in the series reported here. Cells were grown in Dulbeccos modified Eagles medium contain/_ng glucose. Fixation for electron microscopy was performed in the usual fashion using phosphate buffered glutaraldehyde, osmium, and Epon 812 6~edding. Cells were fixed and embedded in pellet form. A Philips 201 electron microscope was used to exanine ~ e specimens. This ~P_search was funded b y a Sandez Fellowship frc~nthe Sandoz Foundation, Inc. of New York. 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
720
Neuroblastoma Glioma Hybrid NG 108-15 Cells
Vol. 33, Sup. I, 1983
FIG. i. Light Micrograph (IM) of NG 108-15 with A cells predcrainating but with one large C or giant cell at the top of the illustration. 240X. FIG. 2. IM of Type A cells with one, Type B cell in lower right. 375X. FIG. 3. IM of Type B cell surrounded by Type A cells. 375X. FIG. 4. IM Type A cells with prGminent peripheral extensions. 375X. FIG. 5. Electron micrograph (~M) of Type A cells with nucleus N and peripheral extension PE labell~d. 3,150X. FIG. 6. ~M of portion of presumably a Type C or giant cell with nucleus N labelled. Dense cytoplasmic matrix is apparent. 3,900X.
Vol. 33, Sup. I, 1983
Neuroblastoma Glioma Hybrid NG 108-15 Cells
721
FIG. 7. E~i A cell with dense core vesicles (DC) near cell surface as well as a coated vesicle CV opeining at the cell surface. Clusters of ribosomes R are seen as are filaments F in peripheral extensions and elsewhere. 34,950X. FIG.8. ~4 an artefactual multivesicular body MVB, dense core vesicles (DC), and peripheral extensions are seen. 18,525X. FIG. 9. EM Mitochondria (M) with characteristic inclusions and a myelin whorl (~W) are labelled. Ribosomes and endoplasmic reticulum are seen also. 27,000X. FIG. 10. ~4 Virus like inclusions are shown within the cisternae of endoplasmic reticultm~. 57,000X. FIG. ii. Isolated vesicles DC prepared in the laboratory of Brian Cox. 32,500X. FIG. 12. EM mouse posterior pituitary shc~ing typical clear as ~ i i as large grey or dense core neurosecretory granules. 55,575X. FIG.13. EM of mouse adrenal medulla illustrating other dense core vesicles morphologically similar to those found in NG 108-15 cells. 24,000X.
722
Neuroblastoma Glioma Hybrid NG 108-15 Cells
Vol. 33, Sup. I, 1983
Results Three morphologically distinct cell types are distinquishable by light microscopy. The predcminant cell type is the "undifferentiated" or A cells. These cells are round or oval in profile, contain a large nucleus with prominent nucleoli and heterchrcmatin, a moderate anount of cytopla~n wit/~ ntlnerous mitochondria, much rough endoplas~nic reticulum and ribosomal content. Filaments and microtubules are abundent. Some ~nooth endoplasmic reticul~n is noted as are occasicnal golgi ccmplexes. Autophages and myelin whorls are frequently encountered. Finger like peripheral extensions are frequent. The less frequent second or B cell is nerve like in shape. The third infrequent cell type, or C cell, is--th-e-giant cell which resembles A cells except for its large size and dense cytoplamnic matrix. The remainder of this paper is devoted to a description of the predc~dnant A cells where we found only one type of neurosecretory granule, a dense core granule. These vary between 800 and 1,200 A ° in diameter and morphologically resemble dense granules as those in cells of the pituitary or adrenal medulla. Clear vesicles were not found in A cells in our preparations despite expectations that clear acetylcholine type vesicles would be present. However, blebs or blisters with multivesicular protrusions were observed which we interpret as glutaraldehyde artefacts described by Hasty and Hay, (12) . Virus like particles were found in many of the preparations. Conclusions The previously unrecognized virus may alter the genetic makeup of the cells and, along with the heterogeneity of cell types, account for variation in opioid content and receptor type and anount. Ultrastructural characterization of membranes used from these cells is desirable. Microscopic characterization of cell morphology is indicated to determine the percentage of the three cell types, which may have markedly different receptor and opioid characteristics, in any given preparation. Isolation of each of the three cell types and cloning of each could permit pharmacological and biochemical characterization of each cell type. Isolation of the dense core vesicles with analysis of content is in progress (Fig. ll) . Further studies will describe the ultrastructure of Type B and C cells as well as differentiated cells. No striking differences were ~bserv--ed at either a light or ~M level between treated and control groups. Thanks are given for the expert technical assistance of Ms. Nancy Hesler, EM technician, the extreme generosity of Dr. Werner Klee, and the collaboration of Drs. Brian Cox and Christopher Malineux. References i. W.A. KLFF and M. NIRI~BERG, Proc.Natl.Acad.Sci.USA 71 3474-3477(1974). 2. S.K. SHARMA, W.A. KT.RE, M. NIRENBERG, Proc.Natl.Acad--.Sci.USA 72 3092-3096 (1975). 3. W.A. ET.~E,Cell Membrane Receptor s for Viruses,Antigens and Antilxxlies, polypeptide Hormones and Small Molecules,Ed.R.F. Beers,Jr. and E.G. Bassett, Raven Press 451-466i1976). 4. R.G. HAMMONDS,JR., P. FERRARA,and C.H. LI, Proc.Natl.Acad.Sci.USA 78 2218-2220 (1981). 5. B. HAMPRE~T, Int.Rev.Cytol. 49 99-170 (1977). 6. R. MCGEE, P. SIMPSON, C. CHRISTIAN, M. MATA, P. NELSON and M.NIR~qBERG(1978) 7. T.D. GLASER, K. VANCALKER, C. HUBNER, Stadtkus and Hamprecht, Eur.J. Pharmacol 65 319-320 (1980). 8. T. GLASER, K. HUBNER and B.HAMPPZCHT, J.Neurochem. 39 59-69 (1982). 9. K.M. BRAAS, S.R. CHILDERS, D.C. U'PRICHARD, J.Neurosci.(in press)(1983). i0. R. SCHULZ, C. GRAMSCH, M. WUSTER, Neuropeptides 3 271-283 (1983). ii. M.P. DANIEI~, B. HAMPRECHT, J. C.B. 63 691-699 (T974). 12. D.L. HASTY and E.D. HAY, J. C.B. 78 756-768 (1978).
Pergamon Press
LIGHT AND ULTRAST~3CIURAL C}LARACI~RISTICS CF NEUROBLASTCMAGLICMAHYBRID NG 108-15 C F ~ S Arnold Schecter, Department of Preventive Medicine, Upstate M,edical Center, Clinical Canpus, S.U.N.Y., Binghamton, New York 13901 USA (Received in final form June 26, 1983) Sunmary "Undifferentiated"neuroblastoma glic~a hybrid NG 108-15 cells have not previously been characterized at a light and ultrastructural level despite their use in opiate receptor studies and their possession of one or more opioids. Three cell types rather than one were found. Type A is the classic round hybridc~a cell. Type B is a neuron like cell and Type C is a giant cell similar to Type A cells but larger and with dense cytoplasm. Dense core vesicles, 800-1200 A ° in di~neter were found as were virus particles in most Type A cells. Multivesicular bodies with clear vesicles which are interpreted as glutaraldehyde artefacts were found but no clear neurosecretory vesicles were found in any A cells--Etorphine at 10 -6 M for 48 hours and naloxone 10 -4 M were e~ployed to product dependence and precipitated abstinence but no clear cut morphologic differences were noted between control, opiate dependent and precipitated abstinent cells. Neuroblastoma glicrna hybrid NG 108-15 cells have been used as a neuronal model because of their relative simplicity cempared to brain, (1,2,3). The cells are believed to contain delta and episilon opioid receptors, (1,4). Binding of morphine and other opiates to receptors results in an inhibition of adenylate cyclase activity and a decrease in cyclic AMP levels in intact cells, (2). Under certain conditions these cells may be manipulated to increase levels of specific neurotransmitter enzyme activities such as acetylcholinesterase and choline acetyltransferase, as well as stimulus dependent release of acetylcholine, (5,6). Glaser, et.al~7,8) demonstrated that NG 108-15 cells also contained met-enkephalin and leu-enkephalin. Braas, et.al. (9) confirmed the presence of enkephalins by immunocytochemical and biochemical means and localized enkephalin like /xmaznoreactivity to the dense core vesicles. Schultz, et.al. (i0) found dynorphin and alpha neoendorphin like material in these cells and noted considerable variability in the amount of dynorphin and alpha neoendorphin activity in various samples. Although morphologically related neuroblastoma glicrna cells were previously described, (ii), the NG 108-15 line has never been morphologically characterized. Methods Cells were grown in the laboratory of Werner Klee and then processed in our laboratory. Cells were grown in flasks and no attempt was made to produce cell differentiation in the series reported here. Cells were grown in Dulbeccos modified Eagles medium contain/_ng glucose. Fixation for electron microscopy was performed in the usual fashion using phosphate buffered glutaraldehyde, osmium, and Epon 812 6~edding. Cells were fixed and embedded in pellet form. A Philips 201 electron microscope was used to exanine ~ e specimens. This ~P_search was funded b y a Sandez Fellowship frc~nthe Sandoz Foundation, Inc. of New York. 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
720
Neuroblastoma Glioma Hybrid NG 108-15 Cells
Vol. 33, Sup. I, 1983
FIG. i. Light Micrograph (IM) of NG 108-15 with A cells predcrainating but with one large C or giant cell at the top of the illustration. 240X. FIG. 2. IM of Type A cells with one, Type B cell in lower right. 375X. FIG. 3. IM of Type B cell surrounded by Type A cells. 375X. FIG. 4. IM Type A cells with prGminent peripheral extensions. 375X. FIG. 5. Electron micrograph (~M) of Type A cells with nucleus N and peripheral extension PE labell~d. 3,150X. FIG. 6. ~M of portion of presumably a Type C or giant cell with nucleus N labelled. Dense cytoplasmic matrix is apparent. 3,900X.
Vol. 33, Sup. I, 1983
Neuroblastoma Glioma Hybrid NG 108-15 Cells
721
FIG. 7. E~i A cell with dense core vesicles (DC) near cell surface as well as a coated vesicle CV opeining at the cell surface. Clusters of ribosomes R are seen as are filaments F in peripheral extensions and elsewhere. 34,950X. FIG.8. ~4 an artefactual multivesicular body MVB, dense core vesicles (DC), and peripheral extensions are seen. 18,525X. FIG. 9. EM Mitochondria (M) with characteristic inclusions and a myelin whorl (~W) are labelled. Ribosomes and endoplasmic reticulum are seen also. 27,000X. FIG. 10. ~4 Virus like inclusions are shown within the cisternae of endoplasmic reticultm~. 57,000X. FIG. ii. Isolated vesicles DC prepared in the laboratory of Brian Cox. 32,500X. FIG. 12. EM mouse posterior pituitary shc~ing typical clear as ~ i i as large grey or dense core neurosecretory granules. 55,575X. FIG.13. EM of mouse adrenal medulla illustrating other dense core vesicles morphologically similar to those found in NG 108-15 cells. 24,000X.
722
Neuroblastoma Glioma Hybrid NG 108-15 Cells
Vol. 33, Sup. I, 1983
Results Three morphologically distinct cell types are distinquishable by light microscopy. The predcminant cell type is the "undifferentiated" or A cells. These cells are round or oval in profile, contain a large nucleus with prominent nucleoli and heterchrcmatin, a moderate anount of cytopla~n wit/~ ntlnerous mitochondria, much rough endoplas~nic reticulum and ribosomal content. Filaments and microtubules are abundent. Some ~nooth endoplasmic reticul~n is noted as are occasicnal golgi ccmplexes. Autophages and myelin whorls are frequently encountered. Finger like peripheral extensions are frequent. The less frequent second or B cell is nerve like in shape. The third infrequent cell type, or C cell, is--th-e-giant cell which resembles A cells except for its large size and dense cytoplamnic matrix. The remainder of this paper is devoted to a description of the predc~dnant A cells where we found only one type of neurosecretory granule, a dense core granule. These vary between 800 and 1,200 A ° in diameter and morphologically resemble dense granules as those in cells of the pituitary or adrenal medulla. Clear vesicles were not found in A cells in our preparations despite expectations that clear acetylcholine type vesicles would be present. However, blebs or blisters with multivesicular protrusions were observed which we interpret as glutaraldehyde artefacts described by Hasty and Hay, (12) . Virus like particles were found in many of the preparations. Conclusions The previously unrecognized virus may alter the genetic makeup of the cells and, along with the heterogeneity of cell types, account for variation in opioid content and receptor type and anount. Ultrastructural characterization of membranes used from these cells is desirable. Microscopic characterization of cell morphology is indicated to determine the percentage of the three cell types, which may have markedly different receptor and opioid characteristics, in any given preparation. Isolation of each of the three cell types and cloning of each could permit pharmacological and biochemical characterization of each cell type. Isolation of the dense core vesicles with analysis of content is in progress (Fig. ll) . Further studies will describe the ultrastructure of Type B and C cells as well as differentiated cells. No striking differences were ~bserv--ed at either a light or ~M level between treated and control groups. Thanks are given for the expert technical assistance of Ms. Nancy Hesler, EM technician, the extreme generosity of Dr. Werner Klee, and the collaboration of Drs. Brian Cox and Christopher Malineux. References i. W.A. KLFF and M. NIRI~BERG, Proc.Natl.Acad.Sci.USA 71 3474-3477(1974). 2. S.K. SHARMA, W.A. KT.RE, M. NIRENBERG, Proc.Natl.Acad--.Sci.USA 72 3092-3096 (1975). 3. W.A. ET.~E,Cell Membrane Receptor s for Viruses,Antigens and Antilxxlies, polypeptide Hormones and Small Molecules,Ed.R.F. Beers,Jr. and E.G. Bassett, Raven Press 451-466i1976). 4. R.G. HAMMONDS,JR., P. FERRARA,and C.H. LI, Proc.Natl.Acad.Sci.USA 78 2218-2220 (1981). 5. B. HAMPRE~T, Int.Rev.Cytol. 49 99-170 (1977). 6. R. MCGEE, P. SIMPSON, C. CHRISTIAN, M. MATA, P. NELSON and M.NIR~qBERG(1978) 7. T.D. GLASER, K. VANCALKER, C. HUBNER, Stadtkus and Hamprecht, Eur.J. Pharmacol 65 319-320 (1980). 8. T. GLASER, K. HUBNER and B.HAMPPZCHT, J.Neurochem. 39 59-69 (1982). 9. K.M. BRAAS, S.R. CHILDERS, D.C. U'PRICHARD, J.Neurosci.(in press)(1983). i0. R. SCHULZ, C. GRAMSCH, M. WUSTER, Neuropeptides 3 271-283 (1983). ii. M.P. DANIEI~, B. HAMPRECHT, J. C.B. 63 691-699 (T974). 12. D.L. HASTY and E.D. HAY, J. C.B. 78 756-768 (1978).