0013.7227/92/130%0825$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. Society
Nerve Growth Factor a Nicotinic Cholinergic THELMA
C. MADHOK
AND BURT
130, No. 2
Printed
Enhances [3H]Nicotine Binding Receptor on PC 12 Cells*
in U.S.A.
to
M. SHARP
Endocrine-Neuroscience Laboratory, Minneapolis Hennepin County Medical Center and University
Medical Research Foundation, Department of Medicine, Minnesota, Minneapolis, Minnesota 55415
of
an average Kd value of 207 nM (n = 2) at 4 C. By saturation binding analysis of intact cells, an average Kd of 292 nM (n = 2) and a binding capacity (B,,,) of 15,118 molecules/cell were obtained. [3H]Nicotine binding was inhibited on an equimolar basis by L-(-)nicotine and N-methylcarbamylcholine. D-(+) Nicotine was 7-fold less potent, whereas oc-bungarotoxin, mecamylamine, and atropine showed no significant inhibition. [3H]Nicotine binding was also inhibited quantitatively by monospecific polyclonal antibodies raised against the predicted as-subunit sequence (amino acids 130-139) of the rat neuronal nicotinic cholinergic receptor. This study represents the first biochemical characterization of NGF-stimulated nicotine-binding sites on PC 12 cells and confirms previous evidence of the presence of functional nicotinic cholinergic receptors on these cells. (Endocrinology 130: 825-830, 1992)
ABSTRACT. Although nicotinic cholinergic agonists have functional effects on PC 12 cells, radioligand-binding sites have not been detected. We, therefore, studied PC 12 cells incubated in the presence of nerve growth factor (NGF) and determined that specific [“HInicotine-binding sites were induced approximately 2.5fold in the presence of NGF (50 or 100 rig/ml). Specific binding was maximal between the first (100 rig/ml NGF) and seventh (50 rig/ml NGF) days of treatment and was stable for 2 weeks with addition of NGF every 3 days. Using intact cells, average association and dissociation rates for [“HInicotine were 0.00021 min-’ nM-’ (n = 2) and 0.048 min.’ (n = 2), respectively, at 4 C, yielding an average apparent Kd of 229 nM. At 22 C, stable equilibrium was not attained during association studies. A similar Kd value for broken cell preparations was obtained by kinetic analysis (i.e. an average association rate of 0.00042 min-’ nM-’ and dissociation rate of 0.087 min.‘), yielding
N
ICOTINIC cholinergic responses have been reported using the PC 12 cell line, derived from rat pheochromocytoma, which develops a neuronal morphology upon exposure to nerve growth factor (NGF). Nicotinic cholinergic receptors (nAcChR) have been identified functionally on these cells by measuring the effect of nicotinic agonists on 45Na+ influx (1) or @jRb+ efflux (2,3). These ion fluxes were inhibited by nicotinic receptor antagonists such as mecamylamine. Binding sites on PC 12 cells have also been characterized using [1251]bungarotoxin (4,5), which labels receptors that are distinctly different from the high affinity neuronal sites for [3H]nicotine. To our knowledge, however, no report has used [3H]nicotine to demonstrate and characterize nicotine-binding sites on PC 12 cells. Herein, we have demonstrated the presence of [3H] nicotine-binding sites on PC 12 cells that were inducible by NGF. Binding constants were defined by both equilibrium and kinetic analyses on intact and broken PC 12 Received July 26, 1991. Address all correspondence and requests for reprints to: Dr. Thelma Madhok, Minneapolis Medical Research Foundation, 825 South 8th Street, Suite M50, Minneapolis, Minnesota 55404. * This work was supported by USPHS Grants DA-04446 (to T.C.M.) and DA-03977 (to B.M.S.).
cells. A monospecific antibody that inhibits [3H]nicotine binding to rat brain membrane was also shown to inhibit binding to PC 12 cells. These studies will permit comparison of the nAcChR on PC 12 cells with the family of nAcChRs in rat brain.
Materials Growth
and harvesting
Cells (American
of
Type
and Methods PC 12 cells Culture
Collection,
Rockville,
MD)
were maintained in proliferative growth phase, using RPMI1640 (Sigma, St. Louis, MO) supplemented with penicillinstreptomycin (Sigma), 10% heat-inactivated horse serum, and 5% fetal bovine serum (Hyclone, Logan, UT), in a 5% CO, atmosphereat 37 C. Passageswere at lo- to 14-day intervals. In preparation for each experiment, cells (1 X lo’/20 ml) were plated on poly-L-lysine-coated flasks (Costar, Cambridge,MA) and allowedto attach for 24 h, followed by the addition of NGF (2.5s; UBI, Lake Placid, NY; 50 or 100 rig/ml), which produced neurite outgrowth within 8 h. When the cultures were not
replenished routinely with NGF, a reduction in [“HInicotine binding was observed,in addition to detachment and reversion to a roundedmorphology (our unpublishedobservation). Therefore, cells were routinely cultured in 50 rig/ml NGF for 1 week, with NGF replenished at 3-day intervals and/or 1 day before
harvest. Cells were dislodgedwith 0.02% EDTA-Hanks’ Buffered Salt Solution, followed by the turbulent addition of serum825
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 18:54 For personal use only. No other uses without permission. . All rights reserved.
NGF ENHANCES [“HINICOTINE
826
supplementedmedium. Cells were spun at 200 x g for 5 min, and then resuspendedtwice in cold phosphate buffer (50 mM phosphatecontaining 0.5 mM phenylmethylsulfonylfluoride, 5 mM EDTA, 10 mg/liter bacitracin, 25mg/liter trypsin inhibitor, 157 mg/liter benzamidine, 500 mg/liter aprotinin, 1 mg/liter leupeptin, and 0.2% azide), pH 7.5, at 4 C. Broken cell preparations were obtained by disrupting cells in cold phosphate buffer, using a motor-driven Teflon pestle homogenizer. rH1Nicotin.e
binding
assays
[“HINicotine-binding siteswere measuredon PC 12 cells, as previously describedin rat brain (6), using either intact cells or a broken cell preparation. Briefly, 50-80 nM [‘lH]nicotine (SO-SOCi/mmol; DuPont-New England Nuclear Corp., Boston, MA) was incubated with 3 x lo6 intact or broken cells in a total volume of 120 ~1phosphatebuffer, pH 7.5, for 60 min at 4 C. Nonspecific binding was measuredusing 1.0 mM L(-)nicotine (Sigma). Bound [“HI nicotine was separated from free ligand by filtration under vacuum through Whatman GF/ B filters (Clifton, NJ) that had been presoaked for 30 min at room temperature in 0.4% BSA containing 0.01%polylysine in phosphatebuffer. This was followed by three washeswith icecold buffer. Specifically bound nicotine representsthe difference between the total bound (disintegrations per min) and nonspecificbinding. D-(+)Nicotine, asa (+)di-p-toluoyltartrate salt (Sigma), was used for competition studies. [“HINicotine binding assayswere performed in triplicate in two or three separateexperiments. Measurements
of Kd and binding
capacity (B,,.J
The association rate (K,) for [“HInicotine was calculated using linear regressionanalysis of the relationship between In B,,/(B,, - B,) us. time, where B,, and B, constituted specific binding at equilibrium and that at other time intervals, respectively. The slope of this line was the experimentally observed apparent rate constant (K,,,,,). Similarly, the dissociation rate, K.,, was calculated from the slopeof the relationship between In B,/B, us. time, where B, represented the concentration of ligand-receptor complex just before initiation of dissociationin the presenceof excessunlabeledligand. K, wasestimatedusing the equation K1 = K,,ha- K.1/L, where L was the ligand concentration. The equilibrium constant (Kd) was obtained from the relationship Kd = K-,/K, (7). Analysis of saturation data obtained under equilibrium conditions was performed using a nonlinear curve-fitting program (EBDA/SCAFIT) (8). Interaction of PC 12 cell with a monospecific antibody against the an-subunit of the rat brain nAcChR
BINDING
Endo. Voll30.
DEAE chromatography, and affinity chromatography, using thyroglobulin-Sepharose 4B, followed by Sr peptide-Sepharose 6B. It was then concentrated using either lyophilization or hydroxyapatite (designated S3 antibody). PC I2 cells were preincubated in the presence of buffer, nonimmune rabbit immunoglobulin G, or the monospecificS3 antibody for 1 h at 22 C before binding with [“HInicotine (80 nM), as described above. Statistical analysisof antibody inhibition wasperformed using one-way analysis of variance with Scheffe’s contrast; P C 0.05 was consideredsignificant.
Results In the initial experiments, NGF (50 or 100 rig/ml) was added to cultures of PC 12 cells that were harvested 0, 24, or 36 h and 3, 7, 10, or 14 days thereafter. As shown in Fig. 1, concomitant with neurite outgrowth induced by NGF [assessedvisually in accord with previous reports (lo)], specific [3H]nicotine binding increased approximately 2.5-fold over the baseline. Using intact cells in the binding assay, specific binding was maximal 24 h after administration of 100 rig/ml NGF or by 7 days after treatment with 50 rig/ml NGF and was stable for 2 weeks. Cells grown without NGF were maintained for only 7 days, since detachment from the flask and eventual cell death were observed. Specific [3H]nicotine-binding sites on NGF-stimulated cells appeared to increase linearly with the number of cells in the assay, as shown in Table 1, since doubling the number of cells resulted in an
2000
-E- 1500 e u c 2 P 0 z.E it
1000
500
raised
The decapeptideantigen, designated S3 decapeptide, comprised amino acids 130-139 of the predicted cu,
Vol. Society
Nerve Growth Factor a Nicotinic Cholinergic THELMA
C. MADHOK
AND BURT
130, No. 2
Printed
Enhances [3H]Nicotine Binding Receptor on PC 12 Cells*
in U.S.A.
to
M. SHARP
Endocrine-Neuroscience Laboratory, Minneapolis Hennepin County Medical Center and University
Medical Research Foundation, Department of Medicine, Minnesota, Minneapolis, Minnesota 55415
of
an average Kd value of 207 nM (n = 2) at 4 C. By saturation binding analysis of intact cells, an average Kd of 292 nM (n = 2) and a binding capacity (B,,,) of 15,118 molecules/cell were obtained. [3H]Nicotine binding was inhibited on an equimolar basis by L-(-)nicotine and N-methylcarbamylcholine. D-(+) Nicotine was 7-fold less potent, whereas oc-bungarotoxin, mecamylamine, and atropine showed no significant inhibition. [3H]Nicotine binding was also inhibited quantitatively by monospecific polyclonal antibodies raised against the predicted as-subunit sequence (amino acids 130-139) of the rat neuronal nicotinic cholinergic receptor. This study represents the first biochemical characterization of NGF-stimulated nicotine-binding sites on PC 12 cells and confirms previous evidence of the presence of functional nicotinic cholinergic receptors on these cells. (Endocrinology 130: 825-830, 1992)
ABSTRACT. Although nicotinic cholinergic agonists have functional effects on PC 12 cells, radioligand-binding sites have not been detected. We, therefore, studied PC 12 cells incubated in the presence of nerve growth factor (NGF) and determined that specific [“HInicotine-binding sites were induced approximately 2.5fold in the presence of NGF (50 or 100 rig/ml). Specific binding was maximal between the first (100 rig/ml NGF) and seventh (50 rig/ml NGF) days of treatment and was stable for 2 weeks with addition of NGF every 3 days. Using intact cells, average association and dissociation rates for [“HInicotine were 0.00021 min-’ nM-’ (n = 2) and 0.048 min.’ (n = 2), respectively, at 4 C, yielding an average apparent Kd of 229 nM. At 22 C, stable equilibrium was not attained during association studies. A similar Kd value for broken cell preparations was obtained by kinetic analysis (i.e. an average association rate of 0.00042 min-’ nM-’ and dissociation rate of 0.087 min.‘), yielding
N
ICOTINIC cholinergic responses have been reported using the PC 12 cell line, derived from rat pheochromocytoma, which develops a neuronal morphology upon exposure to nerve growth factor (NGF). Nicotinic cholinergic receptors (nAcChR) have been identified functionally on these cells by measuring the effect of nicotinic agonists on 45Na+ influx (1) or @jRb+ efflux (2,3). These ion fluxes were inhibited by nicotinic receptor antagonists such as mecamylamine. Binding sites on PC 12 cells have also been characterized using [1251]bungarotoxin (4,5), which labels receptors that are distinctly different from the high affinity neuronal sites for [3H]nicotine. To our knowledge, however, no report has used [3H]nicotine to demonstrate and characterize nicotine-binding sites on PC 12 cells. Herein, we have demonstrated the presence of [3H] nicotine-binding sites on PC 12 cells that were inducible by NGF. Binding constants were defined by both equilibrium and kinetic analyses on intact and broken PC 12 Received July 26, 1991. Address all correspondence and requests for reprints to: Dr. Thelma Madhok, Minneapolis Medical Research Foundation, 825 South 8th Street, Suite M50, Minneapolis, Minnesota 55404. * This work was supported by USPHS Grants DA-04446 (to T.C.M.) and DA-03977 (to B.M.S.).
cells. A monospecific antibody that inhibits [3H]nicotine binding to rat brain membrane was also shown to inhibit binding to PC 12 cells. These studies will permit comparison of the nAcChR on PC 12 cells with the family of nAcChRs in rat brain.
Materials Growth
and harvesting
Cells (American
of
Type
and Methods PC 12 cells Culture
Collection,
Rockville,
MD)
were maintained in proliferative growth phase, using RPMI1640 (Sigma, St. Louis, MO) supplemented with penicillinstreptomycin (Sigma), 10% heat-inactivated horse serum, and 5% fetal bovine serum (Hyclone, Logan, UT), in a 5% CO, atmosphereat 37 C. Passageswere at lo- to 14-day intervals. In preparation for each experiment, cells (1 X lo’/20 ml) were plated on poly-L-lysine-coated flasks (Costar, Cambridge,MA) and allowedto attach for 24 h, followed by the addition of NGF (2.5s; UBI, Lake Placid, NY; 50 or 100 rig/ml), which produced neurite outgrowth within 8 h. When the cultures were not
replenished routinely with NGF, a reduction in [“HInicotine binding was observed,in addition to detachment and reversion to a roundedmorphology (our unpublishedobservation). Therefore, cells were routinely cultured in 50 rig/ml NGF for 1 week, with NGF replenished at 3-day intervals and/or 1 day before
harvest. Cells were dislodgedwith 0.02% EDTA-Hanks’ Buffered Salt Solution, followed by the turbulent addition of serum825
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 18:54 For personal use only. No other uses without permission. . All rights reserved.
NGF ENHANCES [“HINICOTINE
826
supplementedmedium. Cells were spun at 200 x g for 5 min, and then resuspendedtwice in cold phosphate buffer (50 mM phosphatecontaining 0.5 mM phenylmethylsulfonylfluoride, 5 mM EDTA, 10 mg/liter bacitracin, 25mg/liter trypsin inhibitor, 157 mg/liter benzamidine, 500 mg/liter aprotinin, 1 mg/liter leupeptin, and 0.2% azide), pH 7.5, at 4 C. Broken cell preparations were obtained by disrupting cells in cold phosphate buffer, using a motor-driven Teflon pestle homogenizer. rH1Nicotin.e
binding
assays
[“HINicotine-binding siteswere measuredon PC 12 cells, as previously describedin rat brain (6), using either intact cells or a broken cell preparation. Briefly, 50-80 nM [‘lH]nicotine (SO-SOCi/mmol; DuPont-New England Nuclear Corp., Boston, MA) was incubated with 3 x lo6 intact or broken cells in a total volume of 120 ~1phosphatebuffer, pH 7.5, for 60 min at 4 C. Nonspecific binding was measuredusing 1.0 mM L(-)nicotine (Sigma). Bound [“HI nicotine was separated from free ligand by filtration under vacuum through Whatman GF/ B filters (Clifton, NJ) that had been presoaked for 30 min at room temperature in 0.4% BSA containing 0.01%polylysine in phosphatebuffer. This was followed by three washeswith icecold buffer. Specifically bound nicotine representsthe difference between the total bound (disintegrations per min) and nonspecificbinding. D-(+)Nicotine, asa (+)di-p-toluoyltartrate salt (Sigma), was used for competition studies. [“HINicotine binding assayswere performed in triplicate in two or three separateexperiments. Measurements
of Kd and binding
capacity (B,,.J
The association rate (K,) for [“HInicotine was calculated using linear regressionanalysis of the relationship between In B,,/(B,, - B,) us. time, where B,, and B, constituted specific binding at equilibrium and that at other time intervals, respectively. The slope of this line was the experimentally observed apparent rate constant (K,,,,,). Similarly, the dissociation rate, K.,, was calculated from the slopeof the relationship between In B,/B, us. time, where B, represented the concentration of ligand-receptor complex just before initiation of dissociationin the presenceof excessunlabeledligand. K, wasestimatedusing the equation K1 = K,,ha- K.1/L, where L was the ligand concentration. The equilibrium constant (Kd) was obtained from the relationship Kd = K-,/K, (7). Analysis of saturation data obtained under equilibrium conditions was performed using a nonlinear curve-fitting program (EBDA/SCAFIT) (8). Interaction of PC 12 cell with a monospecific antibody against the an-subunit of the rat brain nAcChR
BINDING
Endo. Voll30.
DEAE chromatography, and affinity chromatography, using thyroglobulin-Sepharose 4B, followed by Sr peptide-Sepharose 6B. It was then concentrated using either lyophilization or hydroxyapatite (designated S3 antibody). PC I2 cells were preincubated in the presence of buffer, nonimmune rabbit immunoglobulin G, or the monospecificS3 antibody for 1 h at 22 C before binding with [“HInicotine (80 nM), as described above. Statistical analysisof antibody inhibition wasperformed using one-way analysis of variance with Scheffe’s contrast; P C 0.05 was consideredsignificant.
Results In the initial experiments, NGF (50 or 100 rig/ml) was added to cultures of PC 12 cells that were harvested 0, 24, or 36 h and 3, 7, 10, or 14 days thereafter. As shown in Fig. 1, concomitant with neurite outgrowth induced by NGF [assessedvisually in accord with previous reports (lo)], specific [3H]nicotine binding increased approximately 2.5-fold over the baseline. Using intact cells in the binding assay, specific binding was maximal 24 h after administration of 100 rig/ml NGF or by 7 days after treatment with 50 rig/ml NGF and was stable for 2 weeks. Cells grown without NGF were maintained for only 7 days, since detachment from the flask and eventual cell death were observed. Specific [3H]nicotine-binding sites on NGF-stimulated cells appeared to increase linearly with the number of cells in the assay, as shown in Table 1, since doubling the number of cells resulted in an
2000
-E- 1500 e u c 2 P 0 z.E it
1000
500
raised
The decapeptideantigen, designated S3 decapeptide, comprised amino acids 130-139 of the predicted cu,
