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Neuroblastoma-glioma hybrid cells contain clonidine-displacing substance
European Journal of Pharmacology, 174 (1989) 135-138 Elsevier
135
EJP 20523
Short communication
Ne
obi
toma-glioma hybrid cells contain cionidine-displacing substance P a u l E r n s b e r g e r , M a r y P. M e e l e y a n d D o n a l d J. Reis
Division of Neurobiology, Cornell University Medical College, 411 E. 69th Street, New York, N Y 10021, U.S.A. Received 23 October 1989, accepted 31 October 1989
Cionidine-displacing substance (CDS) isolated from bovine brain potently inhibits clonidine binding and elicits contraction of gastric smooth muscle. We sought to determine if CDS was contained in neuron-like clonal cells (neuroblastoma × glioma hybrid NG108-15). Extracts were prepared from osmotically shocked P2 fractions of NG108-15 cells. One unit of CDS, as defined by a [3H]p-aminoclonidine radioreceptor assay using bovine frontal cortex membranes, was obtained from each 1.3 million cells processed. CDS isolated from NG108-15 cells was biologically active on gastric smooth muscle. NG108-15 cells may serve as a model system for the study of this endogenous clonidine-like ligand. [3 H]p-Aminoclonidine; Clonidine-displacing substance; Clonidine; Imidazole binding sites; (Radioligand binding)
1. Introduction
The NG108-15 cell line is a hybrid of mouse neuroblastoma and rat glioma that expresses m a n y neuronal traits - far more, in fact, than does the parent neuroblastoma line (Hamprecht, 1977). These cells express imidazole binding sites specific for clonidine and its analogs (Feinland et al., 1988). The putative endogenous ligand for these receptors is an endogenous clonidine-displacing substance (CDS) (Ernsberger et al., 1987; 1988). This substance has been isolated from bovine brain and potently displaces the binding of [3H]clonidine (Atlas et al., 1987) and [3H]p-aminoclonidine (Meeley et al., 1986; Ernsberger et al., 1988; in press) to brain and kidney membranes. CDS appears to be biologically active in the brain (Meeley et al., 1986; Atlas et al., 1987) and in the periphery (Atlas et al., 1987; Felsen et al., 1987). The struc-
Correspondence to: P. Ernsberger, Division of Neurobiology, Cornell University Medical College, 411 E. 69th Street, New York, NY 10021, U.S.A.
ture of CDS is unknown. Because the brain is highly heterogeneous, the use of a neuron-like clonal cell line as an alternative source of CDS may facilitate its characterization. We therefore sought to determine whether NG108-15 cells may be a source of bioactive CDS.
2. Materials and methods 2.1. Cell culture
Cells of the mouse neuroblastoma x rat glioma clonal cell line NG108-15 (Hamprecht, 1977) were cultured as monolayers at an initial plating density of 4000 cells/cm z in Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal calf serum, 44 m M N a H C O 3, 0.1 m M hypoxanthine, 1.0 /xM aminopterin and 16 # M thymidine. The medium was changed every 48 h beginning 3 days after plating. The cells were incubated at 36.5 ° C in a humidified atmosphere of 90% air-10% CO 2. Cells from confluent cultures (passages 38-42) were harvested by vigorous shaking in calcium-free
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
136 H a n k ' s balanced salt solution. The harvested cells were counted in a hemocytometer, centrifuged at 250 x g for 5 min at 25°C, flash-frozen in a mixture of dry ice and acetone, and stored at - 7 0 ° C for up to one month.
2.2. Isolation of CDS Since CDS can be obtained from a P2 fraction of brain following osmotic shock (Meeley et al., 1989), P2 pellets were homogenized in hypotonic medium, and the soluble extract was processed and tested for CDS content. NG108-15 cells were slowly thawed and homogenized in ice-cold Trissucrose (270 m M sucrose buffered with 50 mM Tris-HCl, p H 7.4; 1-4 x 106 cells/ml) with six strokes of a Teflon pestle rotating at 1000 rpm. Homogenates were centrifuged at 500 x g for 10 min at 4 ° C, and the resulting P1 pellet was resuspended in Tris-sucrose and centrifuged as before. The combined supernatants were centrifuged at 3 7 0 0 0 X g for 15 min, and the pellet (P2) was osmotically shocked by rehomogenizing in 4 ml distilled water (Polytron, setting 5, 10 s), and incubating on ice for 15 min. The lysate was recentrifuged at 37000 x g for 15 min to remove the membrane fraction. The supernatant was centrifuged at 100000 X g for 60 min, and the soluble fraction was lyophilized overnight, then extracted by resuspending in 20 ml methanol and bath-sonicating and re-extracted with an additional 10 ml methanol. Extracts were combined, filtered, aliquoted, dried under vacuum, and stored at - 7 0 ° C . Dried extract was reconstituted in water immediately prior to assay.
2.3. [ 3H]p-Aminoclonidine radioreceptor assay [3H]p-Aminoclonidine binding to bovine frontal cortex membranes was assayed as described previously (Ernsberger et al., 1987; 1988). Washed P2 membrane fractions were incubated with 1 nM [3H]p-aminoclonidine for 40 min at 25 ° C. Nonspecific binding was defined in the presence of 10 /~M phentolamine. Inhibition curves were analyzed by computerized curve-fitting (McPherson, 1983). One unit of CDS was defined as the amount of
material required to inhibit the specific binding of [ 3H]p-aminoclonidine by 50%.
2.4. Superfusion bioassay Gastric fundus strips were suspended on force transducers and superfused with Krebs-Henseleit medium at 10 m l / m i n for bioassay of CDS according to previously established procedures (Felsen et al., 1987).
2.5. Drugs and reagents [3H]p-Aminoclonidine was obtained from New England Nuclear (46 C i / m m o l ) . Phentolamine was a gift from Ciba-Geigy.
3. Results
3.1. Effect of NG108-15 cell extracts on [3H]paminoclonidine binding CDS is defined by displacement of [3H]paminoclonidine from its binding sites on brain membranes (Ernsberger et al., 1988). Extracts prepared from osmotically shocked P2 fractions of NG108-15 cells potently inhibited [3H]p-aminoclonidine binding to bovine frontal cortex membranes (fig. 1). The interaction of CDS with [3H]p_aminoclonidine binding sites appeared to be homogeneous, since the Hill slope (nil) did not differ from that for unlabeled p-aminoclonidine (0.71 + 0.08 vs. 0.7 + 0.06; N = 4). A total of 162 ___24 units of CDS, as defined by this radioreceptor assay, were isolated from 2.4 x 108 cells. The radioreceptor activity per mg dry weight was not determined, since the dry weight of the total extract was too small to be accurately measured; however, this indicated that activity per unit weight exceeded that obtained with whole brain extracts (3 units/mg; Meeley et al., 1986).
3.2. Action of NG108-15 cell extracts on gastric fundus smooth muscle Extracts from P2 fractions of NG108-15 cells elicited robust contractions of superfused rat
137 % Total Specific
a p e a k tension of 1.1 + 0.3 g. Thus, b o t h C D S p r e p a r a t i o n s gave a p p r o x i m a t e l y 1 g p e a k tension p e r unit. T h e average m a x i m a l c o n t r a c t i o n of the s a m e gastric strips was 3.4 + 0.7 g, as d e t e r m i n e d b y a d m i n i s t r a t i o n of a 200 p m o l dose of p r o s t a g l a n d i n E 2. T h e m a x i m u m c o n t r a c t i o n elicited b y N G 1 0 8 - 1 5 extracts c o u l d n o t be d e t e r m i n e d d u e to insufficient material.
3H-PAC Binding 100
-
///
75
50
f
4. Discussion
25
0
I 1.5
I 2
I 2.5
[ 3
I 3.5
10g (dilutionCDS) Fig. 1. Inhibition of [3H]p-aminoclonidine ([3H]PAC) binding to frontal cortex membranes by CDS isolated from NG108-15 cells. Data are expressed as percent of total specific [3H]paminoclonidine binding as defined by 10 ~M phentolamine, and represent the mean of four separate experiments. The quantity of CDS-containing extract is given on the x axis; this was normalized for an extract prepared from 2.4× 108 cells. Thus, at the dilutions showed, full-strength corresponds to the total preparation, and a 103 dilution represents 0.1% of the total preparation. Two independent preparations were tested, one obtained with 2.4× l0 s cells and the other with 108 cells. CDS was isolated by osmotically shocking P2 fractions, lyophilizing the soluble fraction, and extracting with methanol. The CDS isolated from as few as 100000 cells (a 1:2500 (10-3.4) dilution of the original extract) produced a significant inhibition of [3H]p-aminoclonidine binding (P < 0.05, t-test).
gastric strip when a p p l i e d as a bolus. T h e response to N G 1 0 8 - 1 5 extracts was similar to that previously r e p o r t e d for extracts p r e p a r e d from bovine b r a i n ( F e l s e n et al., 1987). A n aliquot c o n t a i n i n g 1.8 units, as d e f i n e d b y the [ 3 H ] p - a m i n o c l o n i d i n e r a d i o r e c e p t o r assay, elicited a p e a k tension of 1.6 + 0.3 g (n = 8 gastric strips). W h e n tested in parallel, 1.0 unit of b o v i n e b r a i n extract p r o d u c e d
This s t u d y d e m o n s t r a t e s that cells of the N G 1 0 8 - 1 5 clonal line c o n t a i n C D S , a b i o a c t i v e substance d e f i n e d b y its d i s p l a c e m e n t of l a b e l e d clonidine f r o m m e m b r a n e b i n d i n g sites a n d b y its elicitation of c o n t r a c t i o n s of the rat gastric fundus. N G 1 0 8 - 1 5 cells also express i m i d a z o l e r e c e p t o r s ( F e i n l a n d et al., 1988), which have b e e n s h o w n to selectively b i n d C D S ( E r n s b e r g e r et al., 1988). This cell line m a y c o n s t i t u t e a m o d e l s y s t e m expressing b o t h a novel r e c e p t o r a n d its e n d o g e n o u s ligand. I n an a n a l o g o u s m a n n e r , N G 1 0 8 - 1 5 cells synthesize e n k e p h a l i n s a n d acetylcholine, a n d express the c o r r e s p o n d i n g 3 o p i a t e a n d m u s c a r i n i c acetylcholine receptors ( H a m p r e c h t , 1977). Thus, expression of r e c e p t o r s t o g e t h e r with their e n d o g e nous h g a n d s a p p e a r s to be a c o n s i s t e n t p r o p e r t y of this cell line. Since N G 1 0 8 - 1 5 cells are a h y b r i d of t u m o r cell lines d e r i v e d f r o m n e u r o n s a n d f r o m glia ( H a m p r e c h t , 1977), it is c o n c e i v a b l e that the presence of C D S in these cells results f r o m c h a r a c t e r istics c o n t r i b u t e d b y the glial p a r e n t cell line. However, the specific regional d i s t r i b u t i o n of C D S in b r a i n shows that it is n e a r l y a b s e n t f r o m white m a t t e r ( M e e l e y et al., 1989). This i n d i r e c t evidence suggests that C D S is a c o m p o n e n t of neurons. Moreover, i m i d a z o l e r e c e p t o r s are n o t p r e s e n t in glia ( F e i n l a n d et al., 1988). T h e question arises as to the site of synthesis for the C D S c o n t a i n e d in N G 1 0 8 - 1 5 cells. W h i l e it is p r o b a b l y synthesized within the cells t h e m selves, C D S has been d e t e c t e d in s e r u m ( H e n s l e y et al., in press). Thus, the p o s s i b i l i t y that N G 1 0 8 15 cells take up C D S f r o m the culture m e d i u m and, b y s o m e m e c h a n i s m , sequester it until released b y o s m o t i c shock, c a n n o t b e e x c l u d e d
138
without additional experiments using serum-free media. The present study demonstrates that cultured NG108-15 cells contain a substance similar to CDS, based on two independent assays. NG108-15 cells may represent a model system for the study of CDS and its receptor.
Acknowledgements Supported by HL18974. We thank Dr. Diane Felsen for guidance and assistance with the bioassay. We acknowledge Ms. Liubov Lyandvert for maintaining the NG108-15 ceils in culture under the direction of Dr, Lorraine Iacovitti, and we thank Dr. Jose Musacchio (Dept. of Pharmacology, New York University) for providing starting stocks of the cells. We also acknowledge the technical assistance of Mr. Phillip McCauley.
References Atlas, D., S. Diamant, H.M. Fales and L. Pannell, 1987, The brain's own clonidine: purification and characterization of endogenous clonidine-displacing substance from brain, J. Cardiovasc. Pharmacol. 10 (Suppl. 12), S122. Ernsberger, P., G. Feinland, M.P. Meeley and D.J. Reis, Characterization and visualization of clonidine-sensitive imidazole sites in rat kidney which recognize clonidine-displacing substance, Am. J. Hypert. (in press).
Ernsberger, P., M.P. Meeley, J.J. Mann and D.J. Reis, 1987, Clonidine binds to imidazole binding sites as well as a 2adrenoceptors in the ventrolateral medulla, European J. Pharmacol. 134, 1. Ernsberger, P., M.P. Meeley and D.J. Reis, 1988, An endogenous substance with clonidine-like properties: selective binding to imidazole sites in the ventrolateral medulla, Brain Res. 441, 309. Feinland, G., P. Ernsberger, M.P. Meeley, M.J. Evinger and D.J. Reis, 1988, Differential expression of imidazole and alpha2-adrenergic receptors and clonidine-displacing substance in NG108-15, glial, and chromaffin cells, Soc. Neurosci. Abstr. 14, 412. Felsen, D., P. Ernsberger, M.P. Meeley and D.J. Reis, 1987, Clonidine-displacing substance is biologically active on smooth muscle, European J. Pharmacol. 142, 453. Hamprecht, B., 1977, Structural, electrophysiological, biochemical and pharmacological properties of neuroblastomaglioma cell hybrids in cell culture, Int. Rev. Cytol. 49, 99. Hensley, M.L., M.P. Meeley, P.M. McCauley, P. Ernsberger and D.J. Reis, Clonidine-displacing substance is present in peripheral tissues of the rat, Am. J. Hypert. (in press). McPherson, G.A., 1983, A practical computer-based approach to the analysis of radioligand binding experiments, Comput. Prog. Biomed. 17, 107. Meeley, M.P., P. Ernsberger, L.K. Char, M. Noponen and D.J. Reis, 1989, Regional and subcellular distribution of clonidine-displacing substance in brain, Soc. Neurosci. Abstr. 15, 1179. Meeley, M.P., P.R. Ernsberger, A.R, Granata and D.J. Reis, 1986, An endogenous clonidine-displacing substance from bovine brain: receptor binding and hypotensive actions in the ventrolateral medulla, Life Sci. 38, 1119.
135
EJP 20523
Short communication
Ne
obi
toma-glioma hybrid cells contain cionidine-displacing substance P a u l E r n s b e r g e r , M a r y P. M e e l e y a n d D o n a l d J. Reis
Division of Neurobiology, Cornell University Medical College, 411 E. 69th Street, New York, N Y 10021, U.S.A. Received 23 October 1989, accepted 31 October 1989
Cionidine-displacing substance (CDS) isolated from bovine brain potently inhibits clonidine binding and elicits contraction of gastric smooth muscle. We sought to determine if CDS was contained in neuron-like clonal cells (neuroblastoma × glioma hybrid NG108-15). Extracts were prepared from osmotically shocked P2 fractions of NG108-15 cells. One unit of CDS, as defined by a [3H]p-aminoclonidine radioreceptor assay using bovine frontal cortex membranes, was obtained from each 1.3 million cells processed. CDS isolated from NG108-15 cells was biologically active on gastric smooth muscle. NG108-15 cells may serve as a model system for the study of this endogenous clonidine-like ligand. [3 H]p-Aminoclonidine; Clonidine-displacing substance; Clonidine; Imidazole binding sites; (Radioligand binding)
1. Introduction
The NG108-15 cell line is a hybrid of mouse neuroblastoma and rat glioma that expresses m a n y neuronal traits - far more, in fact, than does the parent neuroblastoma line (Hamprecht, 1977). These cells express imidazole binding sites specific for clonidine and its analogs (Feinland et al., 1988). The putative endogenous ligand for these receptors is an endogenous clonidine-displacing substance (CDS) (Ernsberger et al., 1987; 1988). This substance has been isolated from bovine brain and potently displaces the binding of [3H]clonidine (Atlas et al., 1987) and [3H]p-aminoclonidine (Meeley et al., 1986; Ernsberger et al., 1988; in press) to brain and kidney membranes. CDS appears to be biologically active in the brain (Meeley et al., 1986; Atlas et al., 1987) and in the periphery (Atlas et al., 1987; Felsen et al., 1987). The struc-
Correspondence to: P. Ernsberger, Division of Neurobiology, Cornell University Medical College, 411 E. 69th Street, New York, NY 10021, U.S.A.
ture of CDS is unknown. Because the brain is highly heterogeneous, the use of a neuron-like clonal cell line as an alternative source of CDS may facilitate its characterization. We therefore sought to determine whether NG108-15 cells may be a source of bioactive CDS.
2. Materials and methods 2.1. Cell culture
Cells of the mouse neuroblastoma x rat glioma clonal cell line NG108-15 (Hamprecht, 1977) were cultured as monolayers at an initial plating density of 4000 cells/cm z in Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal calf serum, 44 m M N a H C O 3, 0.1 m M hypoxanthine, 1.0 /xM aminopterin and 16 # M thymidine. The medium was changed every 48 h beginning 3 days after plating. The cells were incubated at 36.5 ° C in a humidified atmosphere of 90% air-10% CO 2. Cells from confluent cultures (passages 38-42) were harvested by vigorous shaking in calcium-free
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
136 H a n k ' s balanced salt solution. The harvested cells were counted in a hemocytometer, centrifuged at 250 x g for 5 min at 25°C, flash-frozen in a mixture of dry ice and acetone, and stored at - 7 0 ° C for up to one month.
2.2. Isolation of CDS Since CDS can be obtained from a P2 fraction of brain following osmotic shock (Meeley et al., 1989), P2 pellets were homogenized in hypotonic medium, and the soluble extract was processed and tested for CDS content. NG108-15 cells were slowly thawed and homogenized in ice-cold Trissucrose (270 m M sucrose buffered with 50 mM Tris-HCl, p H 7.4; 1-4 x 106 cells/ml) with six strokes of a Teflon pestle rotating at 1000 rpm. Homogenates were centrifuged at 500 x g for 10 min at 4 ° C, and the resulting P1 pellet was resuspended in Tris-sucrose and centrifuged as before. The combined supernatants were centrifuged at 3 7 0 0 0 X g for 15 min, and the pellet (P2) was osmotically shocked by rehomogenizing in 4 ml distilled water (Polytron, setting 5, 10 s), and incubating on ice for 15 min. The lysate was recentrifuged at 37000 x g for 15 min to remove the membrane fraction. The supernatant was centrifuged at 100000 X g for 60 min, and the soluble fraction was lyophilized overnight, then extracted by resuspending in 20 ml methanol and bath-sonicating and re-extracted with an additional 10 ml methanol. Extracts were combined, filtered, aliquoted, dried under vacuum, and stored at - 7 0 ° C . Dried extract was reconstituted in water immediately prior to assay.
2.3. [ 3H]p-Aminoclonidine radioreceptor assay [3H]p-Aminoclonidine binding to bovine frontal cortex membranes was assayed as described previously (Ernsberger et al., 1987; 1988). Washed P2 membrane fractions were incubated with 1 nM [3H]p-aminoclonidine for 40 min at 25 ° C. Nonspecific binding was defined in the presence of 10 /~M phentolamine. Inhibition curves were analyzed by computerized curve-fitting (McPherson, 1983). One unit of CDS was defined as the amount of
material required to inhibit the specific binding of [ 3H]p-aminoclonidine by 50%.
2.4. Superfusion bioassay Gastric fundus strips were suspended on force transducers and superfused with Krebs-Henseleit medium at 10 m l / m i n for bioassay of CDS according to previously established procedures (Felsen et al., 1987).
2.5. Drugs and reagents [3H]p-Aminoclonidine was obtained from New England Nuclear (46 C i / m m o l ) . Phentolamine was a gift from Ciba-Geigy.
3. Results
3.1. Effect of NG108-15 cell extracts on [3H]paminoclonidine binding CDS is defined by displacement of [3H]paminoclonidine from its binding sites on brain membranes (Ernsberger et al., 1988). Extracts prepared from osmotically shocked P2 fractions of NG108-15 cells potently inhibited [3H]p-aminoclonidine binding to bovine frontal cortex membranes (fig. 1). The interaction of CDS with [3H]p_aminoclonidine binding sites appeared to be homogeneous, since the Hill slope (nil) did not differ from that for unlabeled p-aminoclonidine (0.71 + 0.08 vs. 0.7 + 0.06; N = 4). A total of 162 ___24 units of CDS, as defined by this radioreceptor assay, were isolated from 2.4 x 108 cells. The radioreceptor activity per mg dry weight was not determined, since the dry weight of the total extract was too small to be accurately measured; however, this indicated that activity per unit weight exceeded that obtained with whole brain extracts (3 units/mg; Meeley et al., 1986).
3.2. Action of NG108-15 cell extracts on gastric fundus smooth muscle Extracts from P2 fractions of NG108-15 cells elicited robust contractions of superfused rat
137 % Total Specific
a p e a k tension of 1.1 + 0.3 g. Thus, b o t h C D S p r e p a r a t i o n s gave a p p r o x i m a t e l y 1 g p e a k tension p e r unit. T h e average m a x i m a l c o n t r a c t i o n of the s a m e gastric strips was 3.4 + 0.7 g, as d e t e r m i n e d b y a d m i n i s t r a t i o n of a 200 p m o l dose of p r o s t a g l a n d i n E 2. T h e m a x i m u m c o n t r a c t i o n elicited b y N G 1 0 8 - 1 5 extracts c o u l d n o t be d e t e r m i n e d d u e to insufficient material.
3H-PAC Binding 100
-
///
75
50
f
4. Discussion
25
0
I 1.5
I 2
I 2.5
[ 3
I 3.5
10g (dilutionCDS) Fig. 1. Inhibition of [3H]p-aminoclonidine ([3H]PAC) binding to frontal cortex membranes by CDS isolated from NG108-15 cells. Data are expressed as percent of total specific [3H]paminoclonidine binding as defined by 10 ~M phentolamine, and represent the mean of four separate experiments. The quantity of CDS-containing extract is given on the x axis; this was normalized for an extract prepared from 2.4× 108 cells. Thus, at the dilutions showed, full-strength corresponds to the total preparation, and a 103 dilution represents 0.1% of the total preparation. Two independent preparations were tested, one obtained with 2.4× l0 s cells and the other with 108 cells. CDS was isolated by osmotically shocking P2 fractions, lyophilizing the soluble fraction, and extracting with methanol. The CDS isolated from as few as 100000 cells (a 1:2500 (10-3.4) dilution of the original extract) produced a significant inhibition of [3H]p-aminoclonidine binding (P < 0.05, t-test).
gastric strip when a p p l i e d as a bolus. T h e response to N G 1 0 8 - 1 5 extracts was similar to that previously r e p o r t e d for extracts p r e p a r e d from bovine b r a i n ( F e l s e n et al., 1987). A n aliquot c o n t a i n i n g 1.8 units, as d e f i n e d b y the [ 3 H ] p - a m i n o c l o n i d i n e r a d i o r e c e p t o r assay, elicited a p e a k tension of 1.6 + 0.3 g (n = 8 gastric strips). W h e n tested in parallel, 1.0 unit of b o v i n e b r a i n extract p r o d u c e d
This s t u d y d e m o n s t r a t e s that cells of the N G 1 0 8 - 1 5 clonal line c o n t a i n C D S , a b i o a c t i v e substance d e f i n e d b y its d i s p l a c e m e n t of l a b e l e d clonidine f r o m m e m b r a n e b i n d i n g sites a n d b y its elicitation of c o n t r a c t i o n s of the rat gastric fundus. N G 1 0 8 - 1 5 cells also express i m i d a z o l e r e c e p t o r s ( F e i n l a n d et al., 1988), which have b e e n s h o w n to selectively b i n d C D S ( E r n s b e r g e r et al., 1988). This cell line m a y c o n s t i t u t e a m o d e l s y s t e m expressing b o t h a novel r e c e p t o r a n d its e n d o g e n o u s ligand. I n an a n a l o g o u s m a n n e r , N G 1 0 8 - 1 5 cells synthesize e n k e p h a l i n s a n d acetylcholine, a n d express the c o r r e s p o n d i n g 3 o p i a t e a n d m u s c a r i n i c acetylcholine receptors ( H a m p r e c h t , 1977). Thus, expression of r e c e p t o r s t o g e t h e r with their e n d o g e nous h g a n d s a p p e a r s to be a c o n s i s t e n t p r o p e r t y of this cell line. Since N G 1 0 8 - 1 5 cells are a h y b r i d of t u m o r cell lines d e r i v e d f r o m n e u r o n s a n d f r o m glia ( H a m p r e c h t , 1977), it is c o n c e i v a b l e that the presence of C D S in these cells results f r o m c h a r a c t e r istics c o n t r i b u t e d b y the glial p a r e n t cell line. However, the specific regional d i s t r i b u t i o n of C D S in b r a i n shows that it is n e a r l y a b s e n t f r o m white m a t t e r ( M e e l e y et al., 1989). This i n d i r e c t evidence suggests that C D S is a c o m p o n e n t of neurons. Moreover, i m i d a z o l e r e c e p t o r s are n o t p r e s e n t in glia ( F e i n l a n d et al., 1988). T h e question arises as to the site of synthesis for the C D S c o n t a i n e d in N G 1 0 8 - 1 5 cells. W h i l e it is p r o b a b l y synthesized within the cells t h e m selves, C D S has been d e t e c t e d in s e r u m ( H e n s l e y et al., in press). Thus, the p o s s i b i l i t y that N G 1 0 8 15 cells take up C D S f r o m the culture m e d i u m and, b y s o m e m e c h a n i s m , sequester it until released b y o s m o t i c shock, c a n n o t b e e x c l u d e d
138
without additional experiments using serum-free media. The present study demonstrates that cultured NG108-15 cells contain a substance similar to CDS, based on two independent assays. NG108-15 cells may represent a model system for the study of CDS and its receptor.
Acknowledgements Supported by HL18974. We thank Dr. Diane Felsen for guidance and assistance with the bioassay. We acknowledge Ms. Liubov Lyandvert for maintaining the NG108-15 ceils in culture under the direction of Dr, Lorraine Iacovitti, and we thank Dr. Jose Musacchio (Dept. of Pharmacology, New York University) for providing starting stocks of the cells. We also acknowledge the technical assistance of Mr. Phillip McCauley.
References Atlas, D., S. Diamant, H.M. Fales and L. Pannell, 1987, The brain's own clonidine: purification and characterization of endogenous clonidine-displacing substance from brain, J. Cardiovasc. Pharmacol. 10 (Suppl. 12), S122. Ernsberger, P., G. Feinland, M.P. Meeley and D.J. Reis, Characterization and visualization of clonidine-sensitive imidazole sites in rat kidney which recognize clonidine-displacing substance, Am. J. Hypert. (in press).
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