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Nicotine rescues PC12 cells from death induced by nerve growth factor deprivation
ELSEVIER
Neuroscience Letters 213 (1996) 145-147
Nicotine rescues PC12 cells from death induced by nerve growth factor deprivation Hiroshi Yamashita, Shigenobu Nakamura* Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 KasuraL Minami-ku, Hiroshima 734, Japan Received 16 May 1996; revised version received 18 June 1996; accepted 19 June 1996
Abstract We examined the protective effect of nicotine against neuronal cell death in the model system of serum- and nerve growth factor (NGF)-free cultures of PC12 cells. Serum deprivation induced massive death of undifferentiated PC12 cells, which was inhibited by the addition of NGF, but not by the addition nicotine (100 taM). Even after PCI2 cells had been differentiated by the treatment with NGF, serum and NGF deprivation induced rapid and massive death of these cells., The addition of nicotine (1-100 taM) to the deprivation system prevented such cell death almost completely. The protective effect of nicotine was abolished by hexamethonium or meeamylamine, inhibitors of nicotinic acetylcholine (nACh) receptors. Treatment with reserpine to deplete intracellular catecholamines did not influence the effect. The results suggest that the protective effect of nicotine against neuronal cell death is exerted through nACh receptors.
Keywords: Nicotine; Nicotinic acetylcholine receptor; PC12 cell; Nerve growth factor; Cell death
Parkinson's disease is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC), which possess nicotine-binding sites on both cell bodies and axon terminals [1]. Several epidemiological studies suggest a negative relationship between the cigarette smoking and the incidence of Parkinson's disease, independent of other factors [2]. Nicotine, the major component in cigarette, accelerates the turnover of brain dopamine [3], facilitates its release in the striatum [4], and inhibits dopamine reuptake via nicotinic acetylcholine (nACh) receptors [5]. Although nicotine exerts various pharmacological actions on dopaminergic neurotransmission, these effects are not long-lasting and fail to explain the protective effect of nicotine against the neuronal cell death, resulting in the decreased incidence of Parkinson's disease. Several studies have shown that serum-free culture of PC12 cells is a useful model system for studying the neuronal cell death. When cultured in serum-free medium, PC12 cells die [6] and exhibit the characteristic pattern
* Corresponding author. Tel.: +81 82 2575201; fax: +81 82 5050490.
of DNA fragmentation associated with apoptosis [7]. After these cells have been differentiated by long-term nerve growth factor (NGF) treatment, cell death is evoked by withdrawal of both serum and NGF [7,8]. In this system NGF, basic fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl-2 rescue PC12 cells from death [6,7,9]. Nicotine has been demonstrated to inhibit death of human histiocytic lymphoma cells induced by diverse stimuli including tumor necrosis factor, ultra violet light, chemotherapeutic drugs, and calcium ionophore [10]. In the present study, we examined the protective effect of nicotine against death of PC12 cells. PC12 cells were cultured on collagen-coated dishes in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum [11]. For the experiments on promotion of cell survival, cells were washed in serum-free RPMI 1640 medium [7] and resuspended in RPMI 1640 medium and plated in collagencoated 96-well plastic plates (1.6-2.0 x 104 cells/well). Cultures were pretreated for 7 days with 50 ng/ml NGF (Toyobo Co., Japan) in RPMI 1640 medium supplemented with 1% heat-inactivated horse serum for the use of differ-
0304-3940f)6/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII S0304-3940(96) 12829-9
146
H. Yamashita et al. / Neuroscience Letters 213 (1996) 145-147
140"
[]
120"
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1"7 Nie
i~_ 100-
~_120-
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~ 100-
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;~ 80-
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Nicotine
20-
Fig. I. Effect of nicotine on survival of PCI2 cells in serum-free medium. Cells were washed and plated in RPMI 1640 medium for 2 days in the presence or absence of 50 ng/ml NGF or in the pre~nce of 100 pM nicotine. At the end of each growth period, WST assay was performed to determine the relative number of living cells. Values are the mean + SD of five experiments. entiated PC I2 cells. The cells were washed and passaged into serum-free RPMI 1640 medium as described above. The number of living cells in the 96-well plates was determined by the W S T assay [ 12]. For the W S T assay 16.5 mg of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt ( W S T - I ) (Dojin, Japan) were dissolved in 4.5 ml o f phosphate-buffered saline (PBS), then 0.5 ml o f the l-methoxy 5-methylphenazinium methylsulfate solution (0.7 mg/ml) (Dojin, Japan) was added in this solution. From the stock solution, 10/zl/100/zl medium were added to each well and plates were incubated at 37°C. After 1 h the optical absorption was measured with a m i c r o - E L I S A reader at a test wavelength of 405 nm and a reference wavelength of 620 nm. Data are presented as percentages o f cells relative to their corresponding control. 140-
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Fig. 2. Effect of nicotine on survival of differentiated PCI2 cells in NGF- and serum-free medium. Cells were washed and plated in RPMI 1640 medium for I, 2 and 5 days in the presence (11) or absence (O) of 50 ng/ml NGF or in the presence of nicotine at concentrations of I /~M (A), 10 #M (©) and 100/~M (O). At the end of each growth period, WST assay was performed to determine the relative number of living cells. Values are the mean + SD of five experiments.
Res+Ni¢
il
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_~ 2 0 @ nO
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o
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Fig. 3. Effect of nicotinic ACh receptor antagonists and reserpine on survival of differentiated PCI2 ceils in NGF- and serum-free medium. Cells were washed and plated in RPMI 1640 medium for ! and 2 days in the presence or absence of 100 ~M nicotine (Nic) or in the presence of 100/zM nicotine plus 3/zM mecamylamine (Mec), 500 p.M hexamethonium (Hex) or 10/~M reserpine (Res). At the end of each growth period, WST assay was performed to determine the relative number of living cells. Values are the mean + SD of five experiments.
To examine the ability of nicotine to protect PC12 cells from death induced by serum deprivation, cells were deprived of serum and treated with nicotine. Serum deprivation for 2 days caused a massive cell death, which was inhibited by the addition of 50 ng/ml N G F as previously described [8] (Fig. 1). However, addition of 100 # M nicotine failed to protect P C I 2 cells from death induced by serum deprivation (Fig. 1). The effect of nicotine on differentiated PC12 cells was also examined. PC12 cells were pretreated with 50 ng/ml N G F for 7 days and were then deprived of N G F and serum. N G F deprivation of these cells led to rapid and massive cell death, and addition of N G F rescued the cells from death (Fig. 2). Addition of nicotine at 1 - 1 0 0 # M inhibited the death of NGF-deprived PC12 cells almost completely on days 1 and 2 (Fig. 2). However, more than 50% of cells in the presence of nicotine at 1 and 10/zM died by day 5, and cells in the presence of 100 # M nicotine completely died by day 9. To examine whether the protective effect of nicotine is mediated by nACh receptors, the effect of nACh receptor antagonists on cell survival was studied. The protective effect of nicotine disappeared, when 3/zM mecamylamine, an antagonist of nACh receptor, was added (Fig. 3). The protective effect o f nicotine was also prevented by 500/xM hexamethonium, another nACh receptor antagonist (Fig. 3). Mecamylamine and hexamethonium alone neither protected nor accelerated the cell death. The results suggest that protective effect of nicotine is mediated by nACh receptors. To elucidate whether the protective effect of nicotine is mediated by catecholamines which are released
H. Yamashita et al. / Neuroscience Letters 213 (1996) 145-147
from cells by nicotinic stimulation, cells were exposed to 10 /~M reserpine to deplete intracellular dopamine and norepinephrine for 24 h prior to NGF deprivation. After this treatment, 100/~M nicotine stimulation for 10 min did not evoke a significant dopamine or norepinephrine release, determined by HPLC-ECD. Treatment with reserpine did not affect protective effect of nicotine on days 1 and 2 (Fig. 3), indicating that the effect might not be mediated by catecholamines released by nicotinic stimulation. The present study demonstrated that nicotine promoted survival of differentiated PC 12 cells after NGF deprivation in a mecamylamine- and hexamethonium-sensitive manner. However, the protective effect of nicotine on undifferentiated PC12 cells was distinctly different from that on differentiated PC12 cells. There is an evidence that NGF increases both the conductance capacity of nACh receptors [13] and the number of PC12 ceils expressing functional nACh receptor [14]. Taken together, we assume that an increase of functional nACh receptors might have brought about the protective effect of nicotine. Activation of ml muscarinic ACh (mACh) receptors also inhibited the death of both serum and NGF deprived PC12M1 cells, stably expressing ml mACh receptors [15]. Therefore, it is considered that activation of both nicotinic and muscarinic ACh receptors enhance the survival of cells in serumNGF deprived system. Concentrations of nicotine used in the present study are comparable to the plasma nicotine level observed in smokers [16]. The low incidence of Parkinson's disease among smokers might be associated with in vitro protective effect of nicotine against death of neuronal cells in SNPC, which may be exerted through nACh receptors. The functional interaction between nicotine and the nigrostriatal dopaminergic system would provide a possible clue for novel treatment or prevention of Parkinson's disease. This work was supported by a grant-in-aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health and Welfare of Japan. [1] Clarke, P.B. and Pert, A., Autoradiographic evidence for nicotine receptors on nigrostriatal and mesolimbic dopaminergic neurons, Brain Res., 348 (1985) 355-358.
147
[2] Baron, J.A., Cigarette smoking and Parkinson's disease, Neurology, 36 (1986) 1490-1496. [3] Anderson, K., Fuxe, K., Agnati, L.F. and Eneroth, P., Effects of acute central and peripheral administration of nicotine on the ascending dopamine pathways in the male rat brain. Evidence of nicotine-induced increases of dopamine turnover in various telencephalic dopamine nerve terminal systems, Med. Biol., 59 (1981) 170-176. [4] Rapier, C., Lunt, G.G. and Wonnacon, S., Stereoselective nicotineinduced release of dopamine from striatal synaptosomes: concentration dependence and repetitive stimulation, J. Neurochem., 50 (1988) 1123-1130. [5] Yamashita, H., Kitayama, S., Zhang, Y.X., Takabashi, T., Dohi, T. and Nakamura, S., Effect of nicotine on dopamine uptake in COS cells possessing the rat dopamine transporter and in PCI2 cells, Biochem. Pharmacol., 49 (1995) 742-745. [6] Rukenstein, A., Rydel, R.E. and Greene, L.A., Multiple agents rescue PCI2 ceils from serum-free cell death by translation- and transcription-independent mechanisms, J. Neurosci., 11 (1991) 2552-2563. [7] Batistatou, A. and Greene, L.A., Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity, J. Cell Biol., 115 (1991) 461-471. [8] Greene, L.A., Nerve growth factor prevents the death and stimulates neuronal differentiation of clonal PCI2 pheochromocytoma ceils in serum-free medium, J. Cell Biol., 78 (1978) 747-755. [9] Mab, S.P., Zhong, L.T., Liu, Y., Roghani, A., Edwards, R.H. and Bredesen, D.E., The protooncogene bcl-2 inhibits apoptosis in PCI2 cells,. J. Neurochem., 60 (1993) t 183-1186. [10] Wright, S.C., Zhong, J., Zheng, H. and Larrick, J.W., Nicotine inhibition of apoptosis suggests a role in tumor promotion,. FASEB J., 7 (1993) 1045-1051. [11] Greene, L.A. and Tischler, A.S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. USA, 73 (1976) 2424-2428. [12] Ishiyama, M., Shiga, M. and Sasamoto, K., A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye, Chem. Pharm. Bull., 41 (1993) 1118-1122. [13] Amy, CM. and Bennet, E.L., Increased sodium ion conductance through nicotinic ACh receptor channels in PC12 cells exposed to nerve growth factors, J. Neurosci., 3 (1983) 1547-1553. [14] Rogers, S.W., Mandelzys, A., Deneris, E.S., Cooper, E. and Heinemann, S., The expression of nicotinic ACh receptors by PC12 cells treated with NGF, J. Neurosci., 12 (1992) 4611-4623. [15] Lindenboim, L., Pinkas-Kramarski, R., Sokolovsky, M. and Stein, R., Activation of muscarinic receptors inhibits apoptosis in PC12MI cells, J. Neurochem., 64 (1995) 2491-2499. [16] US Department of Health and Human Services, The Health Consequences of Smoking, A Report of the Surgeon General, US Government Printing Office, Washington, DC, 1988.
Neuroscience Letters 213 (1996) 145-147
Nicotine rescues PC12 cells from death induced by nerve growth factor deprivation Hiroshi Yamashita, Shigenobu Nakamura* Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 KasuraL Minami-ku, Hiroshima 734, Japan Received 16 May 1996; revised version received 18 June 1996; accepted 19 June 1996
Abstract We examined the protective effect of nicotine against neuronal cell death in the model system of serum- and nerve growth factor (NGF)-free cultures of PC12 cells. Serum deprivation induced massive death of undifferentiated PC12 cells, which was inhibited by the addition of NGF, but not by the addition nicotine (100 taM). Even after PCI2 cells had been differentiated by the treatment with NGF, serum and NGF deprivation induced rapid and massive death of these cells., The addition of nicotine (1-100 taM) to the deprivation system prevented such cell death almost completely. The protective effect of nicotine was abolished by hexamethonium or meeamylamine, inhibitors of nicotinic acetylcholine (nACh) receptors. Treatment with reserpine to deplete intracellular catecholamines did not influence the effect. The results suggest that the protective effect of nicotine against neuronal cell death is exerted through nACh receptors.
Keywords: Nicotine; Nicotinic acetylcholine receptor; PC12 cell; Nerve growth factor; Cell death
Parkinson's disease is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC), which possess nicotine-binding sites on both cell bodies and axon terminals [1]. Several epidemiological studies suggest a negative relationship between the cigarette smoking and the incidence of Parkinson's disease, independent of other factors [2]. Nicotine, the major component in cigarette, accelerates the turnover of brain dopamine [3], facilitates its release in the striatum [4], and inhibits dopamine reuptake via nicotinic acetylcholine (nACh) receptors [5]. Although nicotine exerts various pharmacological actions on dopaminergic neurotransmission, these effects are not long-lasting and fail to explain the protective effect of nicotine against the neuronal cell death, resulting in the decreased incidence of Parkinson's disease. Several studies have shown that serum-free culture of PC12 cells is a useful model system for studying the neuronal cell death. When cultured in serum-free medium, PC12 cells die [6] and exhibit the characteristic pattern
* Corresponding author. Tel.: +81 82 2575201; fax: +81 82 5050490.
of DNA fragmentation associated with apoptosis [7]. After these cells have been differentiated by long-term nerve growth factor (NGF) treatment, cell death is evoked by withdrawal of both serum and NGF [7,8]. In this system NGF, basic fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl-2 rescue PC12 cells from death [6,7,9]. Nicotine has been demonstrated to inhibit death of human histiocytic lymphoma cells induced by diverse stimuli including tumor necrosis factor, ultra violet light, chemotherapeutic drugs, and calcium ionophore [10]. In the present study, we examined the protective effect of nicotine against death of PC12 cells. PC12 cells were cultured on collagen-coated dishes in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal calf serum [11]. For the experiments on promotion of cell survival, cells were washed in serum-free RPMI 1640 medium [7] and resuspended in RPMI 1640 medium and plated in collagencoated 96-well plastic plates (1.6-2.0 x 104 cells/well). Cultures were pretreated for 7 days with 50 ng/ml NGF (Toyobo Co., Japan) in RPMI 1640 medium supplemented with 1% heat-inactivated horse serum for the use of differ-
0304-3940f)6/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII S0304-3940(96) 12829-9
146
H. Yamashita et al. / Neuroscience Letters 213 (1996) 145-147
140"
[]
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~_120-
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~ 100-
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20-
Fig. I. Effect of nicotine on survival of PCI2 cells in serum-free medium. Cells were washed and plated in RPMI 1640 medium for 2 days in the presence or absence of 50 ng/ml NGF or in the pre~nce of 100 pM nicotine. At the end of each growth period, WST assay was performed to determine the relative number of living cells. Values are the mean + SD of five experiments. entiated PC I2 cells. The cells were washed and passaged into serum-free RPMI 1640 medium as described above. The number of living cells in the 96-well plates was determined by the W S T assay [ 12]. For the W S T assay 16.5 mg of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt ( W S T - I ) (Dojin, Japan) were dissolved in 4.5 ml o f phosphate-buffered saline (PBS), then 0.5 ml o f the l-methoxy 5-methylphenazinium methylsulfate solution (0.7 mg/ml) (Dojin, Japan) was added in this solution. From the stock solution, 10/zl/100/zl medium were added to each well and plates were incubated at 37°C. After 1 h the optical absorption was measured with a m i c r o - E L I S A reader at a test wavelength of 405 nm and a reference wavelength of 620 nm. Data are presented as percentages o f cells relative to their corresponding control. 140-
~120p 100~
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~ 60E " 40.~_ "~ 2 0 n" O
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Fig. 2. Effect of nicotine on survival of differentiated PCI2 cells in NGF- and serum-free medium. Cells were washed and plated in RPMI 1640 medium for I, 2 and 5 days in the presence (11) or absence (O) of 50 ng/ml NGF or in the presence of nicotine at concentrations of I /~M (A), 10 #M (©) and 100/~M (O). At the end of each growth period, WST assay was performed to determine the relative number of living cells. Values are the mean + SD of five experiments.
Res+Ni¢
il
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_~ 2 0 @ nO
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o
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2 Day
Fig. 3. Effect of nicotinic ACh receptor antagonists and reserpine on survival of differentiated PCI2 ceils in NGF- and serum-free medium. Cells were washed and plated in RPMI 1640 medium for ! and 2 days in the presence or absence of 100 ~M nicotine (Nic) or in the presence of 100/zM nicotine plus 3/zM mecamylamine (Mec), 500 p.M hexamethonium (Hex) or 10/~M reserpine (Res). At the end of each growth period, WST assay was performed to determine the relative number of living cells. Values are the mean + SD of five experiments.
To examine the ability of nicotine to protect PC12 cells from death induced by serum deprivation, cells were deprived of serum and treated with nicotine. Serum deprivation for 2 days caused a massive cell death, which was inhibited by the addition of 50 ng/ml N G F as previously described [8] (Fig. 1). However, addition of 100 # M nicotine failed to protect P C I 2 cells from death induced by serum deprivation (Fig. 1). The effect of nicotine on differentiated PC12 cells was also examined. PC12 cells were pretreated with 50 ng/ml N G F for 7 days and were then deprived of N G F and serum. N G F deprivation of these cells led to rapid and massive cell death, and addition of N G F rescued the cells from death (Fig. 2). Addition of nicotine at 1 - 1 0 0 # M inhibited the death of NGF-deprived PC12 cells almost completely on days 1 and 2 (Fig. 2). However, more than 50% of cells in the presence of nicotine at 1 and 10/zM died by day 5, and cells in the presence of 100 # M nicotine completely died by day 9. To examine whether the protective effect of nicotine is mediated by nACh receptors, the effect of nACh receptor antagonists on cell survival was studied. The protective effect of nicotine disappeared, when 3/zM mecamylamine, an antagonist of nACh receptor, was added (Fig. 3). The protective effect o f nicotine was also prevented by 500/xM hexamethonium, another nACh receptor antagonist (Fig. 3). Mecamylamine and hexamethonium alone neither protected nor accelerated the cell death. The results suggest that protective effect of nicotine is mediated by nACh receptors. To elucidate whether the protective effect of nicotine is mediated by catecholamines which are released
H. Yamashita et al. / Neuroscience Letters 213 (1996) 145-147
from cells by nicotinic stimulation, cells were exposed to 10 /~M reserpine to deplete intracellular dopamine and norepinephrine for 24 h prior to NGF deprivation. After this treatment, 100/~M nicotine stimulation for 10 min did not evoke a significant dopamine or norepinephrine release, determined by HPLC-ECD. Treatment with reserpine did not affect protective effect of nicotine on days 1 and 2 (Fig. 3), indicating that the effect might not be mediated by catecholamines released by nicotinic stimulation. The present study demonstrated that nicotine promoted survival of differentiated PC 12 cells after NGF deprivation in a mecamylamine- and hexamethonium-sensitive manner. However, the protective effect of nicotine on undifferentiated PC12 cells was distinctly different from that on differentiated PC12 cells. There is an evidence that NGF increases both the conductance capacity of nACh receptors [13] and the number of PC12 ceils expressing functional nACh receptor [14]. Taken together, we assume that an increase of functional nACh receptors might have brought about the protective effect of nicotine. Activation of ml muscarinic ACh (mACh) receptors also inhibited the death of both serum and NGF deprived PC12M1 cells, stably expressing ml mACh receptors [15]. Therefore, it is considered that activation of both nicotinic and muscarinic ACh receptors enhance the survival of cells in serumNGF deprived system. Concentrations of nicotine used in the present study are comparable to the plasma nicotine level observed in smokers [16]. The low incidence of Parkinson's disease among smokers might be associated with in vitro protective effect of nicotine against death of neuronal cells in SNPC, which may be exerted through nACh receptors. The functional interaction between nicotine and the nigrostriatal dopaminergic system would provide a possible clue for novel treatment or prevention of Parkinson's disease. This work was supported by a grant-in-aid from the Research Committee of CNS Degenerative Diseases, the Ministry of Health and Welfare of Japan. [1] Clarke, P.B. and Pert, A., Autoradiographic evidence for nicotine receptors on nigrostriatal and mesolimbic dopaminergic neurons, Brain Res., 348 (1985) 355-358.
147
[2] Baron, J.A., Cigarette smoking and Parkinson's disease, Neurology, 36 (1986) 1490-1496. [3] Anderson, K., Fuxe, K., Agnati, L.F. and Eneroth, P., Effects of acute central and peripheral administration of nicotine on the ascending dopamine pathways in the male rat brain. Evidence of nicotine-induced increases of dopamine turnover in various telencephalic dopamine nerve terminal systems, Med. Biol., 59 (1981) 170-176. [4] Rapier, C., Lunt, G.G. and Wonnacon, S., Stereoselective nicotineinduced release of dopamine from striatal synaptosomes: concentration dependence and repetitive stimulation, J. Neurochem., 50 (1988) 1123-1130. [5] Yamashita, H., Kitayama, S., Zhang, Y.X., Takabashi, T., Dohi, T. and Nakamura, S., Effect of nicotine on dopamine uptake in COS cells possessing the rat dopamine transporter and in PCI2 cells, Biochem. Pharmacol., 49 (1995) 742-745. [6] Rukenstein, A., Rydel, R.E. and Greene, L.A., Multiple agents rescue PCI2 ceils from serum-free cell death by translation- and transcription-independent mechanisms, J. Neurosci., 11 (1991) 2552-2563. [7] Batistatou, A. and Greene, L.A., Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity, J. Cell Biol., 115 (1991) 461-471. [8] Greene, L.A., Nerve growth factor prevents the death and stimulates neuronal differentiation of clonal PCI2 pheochromocytoma ceils in serum-free medium, J. Cell Biol., 78 (1978) 747-755. [9] Mab, S.P., Zhong, L.T., Liu, Y., Roghani, A., Edwards, R.H. and Bredesen, D.E., The protooncogene bcl-2 inhibits apoptosis in PCI2 cells,. J. Neurochem., 60 (1993) t 183-1186. [10] Wright, S.C., Zhong, J., Zheng, H. and Larrick, J.W., Nicotine inhibition of apoptosis suggests a role in tumor promotion,. FASEB J., 7 (1993) 1045-1051. [11] Greene, L.A. and Tischler, A.S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. Natl. Acad. Sci. USA, 73 (1976) 2424-2428. [12] Ishiyama, M., Shiga, M. and Sasamoto, K., A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye, Chem. Pharm. Bull., 41 (1993) 1118-1122. [13] Amy, CM. and Bennet, E.L., Increased sodium ion conductance through nicotinic ACh receptor channels in PC12 cells exposed to nerve growth factors, J. Neurosci., 3 (1983) 1547-1553. [14] Rogers, S.W., Mandelzys, A., Deneris, E.S., Cooper, E. and Heinemann, S., The expression of nicotinic ACh receptors by PC12 cells treated with NGF, J. Neurosci., 12 (1992) 4611-4623. [15] Lindenboim, L., Pinkas-Kramarski, R., Sokolovsky, M. and Stein, R., Activation of muscarinic receptors inhibits apoptosis in PC12MI cells, J. Neurochem., 64 (1995) 2491-2499. [16] US Department of Health and Human Services, The Health Consequences of Smoking, A Report of the Surgeon General, US Government Printing Office, Washington, DC, 1988.