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Chronic treatment with nicotine or potassium attenuates depolarisation-evoked noradrenaline release from the human neuroblastoma SH-SY5Y
Neuroscience Letters 331 (2002) 167–170 www.elsevier.com/locate/neulet
Chronic treatment with nicotine or potassium attenuates depolarisation-evoked noradrenaline release from the human neuroblastoma SH-SY5Y Angel Agis-Torres a,*, Stephen G. Ball b, Peter F. T. Vaughan a a
Seccio´n Departamental de FisiologI´a, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. b Academic Unit of Cardiovascular Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, UK Received 7 June 2002; received in revised form 2 August 2002; accepted 2 August 2002
Abstract Chronic treatment, of SH-SY5Y cells, with KCl (20 mM) for 4 days decreased 100 mM KCl-evoked noradrenaline (NA) release by 50% and nicotine (100 mM)-evoked NA release by 55%. Pretreatment with the L-type calcium channel antagonist, nifedipine, prevented this inhibitory effect of chronic exposure to 20 mM KCl on NA release. In contrast pretreatment with 10 mM nicotine for 4 days had no effect on 100 mM KCl -evoked secretion and decreased nicotinic -evoked NA release by only 25%. Inclusion of nifedipine prevented the inhibition of NA release by chronic nicotine treatment. These data are discussed in relation to effects of chronic moderate, depolarisation by either K 1 or nicotine on influx of Ca 21 via L-type voltage sensitive calcium channels. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nicotine; Noradrenaline release; L-type calcium channels; Neuroblastoma SH-SY5Y
Previous studies have found that nicotinic acetylcholine receptors are upregulated by chronic exposure to nicotinic agonists. Thus not only is the density of high affinity [ 3H]nicotinic binding sites increased in post mortem samples of tobacco smokers [2] but upregulation of nicotinic acetylcholine receptor (nAChR) has been observed following chronic exposure to nicotine in vivo in rodent brain [19]. More recently upregulation of a-bungarotoxin (a-Bgt) binding sites following chronic nicotine treatment has been observed in primary cultures of hippocampal neurones [1] and SH-SY5Y cells [12]. In addition De Koninck and Cooper [4], in sympathetic neurones and Ridley et al. [11], in primary cultures of hippocampal neurones and the cell line SH-SY5Y found that low levels of chronic depolarisation with KCl upregulated a7 nAChR subunits by a mechanism involving influx of Ca 21 via L-type voltage sensitive Ca 21-channels (VSCCs) and activation of Ca 21-calmodulin dependent kinase (CAM-kinase II). In contrast, the upregulation of a-Bgt binding sites, by nicotine, was not affected by these inhibitors. Thus these authors concluded that chronic, moderate depolarisation with KCl upregulates a7 nAChR by a mechanism involving entry of Ca 21 via L-type VSCCs and activation of CAM kinase * Corresponding author. Tel.: 134-91-394-1838; fax: 134-91394-1838. E-mail address: [email protected] (A. Agis-Torres).
II. In contrast, a different mechanism is involved in the upregulation of a7 nAChR subunits by nicotine. Ridley et al. [11] also provide data to show that chronic treatment of SH-SY5Y cells with nicotine, but not KCl, also upregulated the a3 * nAChR subunit by a mechanism not involving L-type VSCCs. Although there have been several studies (see above) on the effect of chronic exposure to nicotine on expression levels of nAChR rather fewer studies have looked at the effect of chronic exposure to nicotine on the activity of nAChRs. Previous studies have reported that acute activation of nAChR in SH-SY5Y cells leads to depolarisation [5], increases in [Ca 21]i [12,14] and release of [ 3H]noradrenaline (NA) [15,19]. Recently Ridley et al. [12] have reported that exposure of SH-SY5Y cells to nicotinic agonists or 20mM KCl led to significant decreases in [Ca 21]i, evoked by 10 mM nicotine. Thus rather paradoxically conditions which lead to the upregulation of nAChR result in a decrease in nAChR-evoked increases in [Ca 21]i. In contrast De Koninck and Cooper [4] reported that exposure of sympathetic neurones to 40 mM KCl for 48 h, which upregulates nAChR a7 subunits has no effect on the magnitude nor kinetics of acetylcholine-evoked current densities. They thus concluded that nAChR a7 subunits do not contribute to the macroscopic AChR-induced currents in rat sympathetic neurones. In contrast Molinari et al. [9] reported that chronic
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treatment, of HEK293 cells transfected with human a7nAChR subunits, with nicotine or the nicotinic antagonist methyllcaconitine (MLA) resulted in a 2-fold increase in whole cell currents evoked by nicotine. The aim of the present study was to investigate the effect of treating SH-SY5Y cells with either 10 mM nicotine or 20 mM KCl (conditions reported to upregulate nAChR) on depolarisation-evoked release of NA triggered by either 100 mM nicotine or 100 mM KCl. Previous studies by our group (see Vaughan et al. [15,17] for reviews) have shown that SH-SY5Y cells express depolarisation-evoked release of NA stimulated by KCl [10], dimethylphenylpiperazinium (DMPP) acting at nAChR [14,18], and barium [16]. In the present study the effect of exposure of SH-SY5Y cells to nicotine (10 mM) and KCl (20 mM) for 4 days on the stimulation of NA release by nicotine (100 mM) and KCl (100 mM) was examined. SH-SY5Y cells were cultured in 1:1 mixture of Hams F12 and minimum essential medium containing 1% non-essential amino acids and 10% fetal calf serum in 2.5% CO2 at 378C in 50 cm 2 culture flasks as described previously [10]. After 4 days in culture either 10 mM nicotine or 20 mM KCl was added, to the culture medium, in the presence or absence of 5 mM nifedipine for a further 4 days prior to assaying for depolarisation-evoked NA release. Release of NA was determined using a perfusion assay based on Wade et al. [18]. Confluent flasks were washed extensively in warm phosphate buffered saline (PBS), to remove the added drugs, then incubated for 1 h with 50 nM [ 3H]NA (20 mCi) in N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid] (HEPES)-buffered saline (HBS; 135 mM NaCl, 5 mM KCl, 0.6 mM MgCl2·7H2O, 2.5 mM CaCl2·2H2O, 10 mM HEPES, 6 mM d-glucose, 0.2 mM ascorbic acid and 0.2 mM pargyline) (pH 7.4). The cell layer was then washed twice with PBS minus Ca 21 and Mg 21 followed by incubation at 378C for 10 min in PBS minus calcium and magnesium to detach the cell layer. The detached cells were collected by centrifugation at 600 rev./min (Centra-4R, International Equipment Company). The cell pellet was resuspended in 1 ml HBS and kept on ice until required for experimentation. Aliquots (100 ml) were placed on a filter paper disc in a Swinnex perfusion chamber and washed with HBS (1 ml/min) using a peristaltic pump until a low, stable basal level of [ 3H]NA release was achieved. Release was evoked by exposing the cells to either 100 mM KCl or 100 mM nicotine in HBS for 2 min at 12 min (s1) and 29 min (s2) of perfusion. The outflow from the Swinnex chamber was collected at 30 s intervals, in scintillation vials, 1 min before and 4 min after the stimulus. At the end of this period the filter paper, containing cells, was removed from the Swinnex chamber and placed in a scintillation vial containing 0.5 ml of 0.4 M perchloric acid. Scintillation fluid (4 ml) was added to the vials and [ 3H]NA content determined by liquid scintillation counting (Tri Carb 2000CA, Packard). As indicated in the relevant figure the effect of co-incubation with nifedipine (5 mM) was
Fig. 1. Effect of exposure of SH-SY5Y cell layers to 20 mM K 1 or 10 mM nicotine on 100 mM K 1-evoked NA release. [ 3H]NA release was evoked by 100 mM K 1 in perfusates of untreated cells (untreated); cells pretreated with 5 mM nifedipine (nifed.); 20 mM K 1 (K); 20 mM K 1 and 5 mM nifedipine (K 1 nifed.); 10 mM nicotine (nic.); or 10 mM nicotine and 5 mM nifedipine (nic. 1 nifed.). Release is expressed as the percentage of the radioactivity released by K 1 compared to the total radioactive content of each aliquot of cells. Each column represents the mean ^SEM for 10–19 separate experiments as indicated.
examined on the release of NA from cells which had been preincubated with either 20 mM KCl or 10 mM nicotine. The amount of [ 3H]NA release during s1 (release during s2 was not used in this study) was expressed as a percentage (mean values ^SEM; n is at least 10) of the total radioactivity present in the cell aliquot before release was evoked and from which basal release was subtracted, determined from the stable base line obtained before adding the stimulus. Levels of significance were determined using the Student unpaired t-test. Incubation of SH-SY5Y confluent cell layers with culture
Fig. 2. Effect of exposure of SH-SY5Y cell layers to 20 mM K 1 or 10 mM nicotine on 100 mM nicotine-evoked NA release. [ 3H]NA release was evoked by 100 mM nicotine in perfusates of untreated cells (untreated); cells pretreated with 5 mM nifedipine (nifed.); 20 mM K 1 (K); 20 mM K 1 and 5 mM nifedipine (K 1 nifed.); 10 mm nicotine (nic.) or 10 mM nicotine and 5 mm nifedipine (nic. 1 nifed.). Release is expressed as the percentage of the total radioactive content of each aliquot of cells. Each column represents the mean ^ SEM for 10–28 separate experiments as indicated.
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Fig. 3. Release of [ 3H]NA evoked by 100 mM nicotine (s1 and s2) in perfusates of untreated cells or pretreated with 20 mM veratridine for 1 day. Depicted points are means ^ SEM (n ¼ 3). Periods of stimulation are indicated by horizontal bars located upon the x-axis. Release during s2 was not used in this study.
medium to which 20 mM KCl had been added for 4 days led to a decrease in [ 3H]NA release evoked by 100 mM KCl, in HBS, from 4.44 ^ 0.4% (n ¼ 19) to 2.37 ^ 0.2% (n ¼ 16). This represents a highly significant decrease (46%; P , 0:001) of NA secretion (Fig. 1). Similarly chronic exposure of SH-SY5Y cells to 20 mM KCl, for 4 days, inhibited nicotine-evoked release of [ 3H]NA from 4.57 ^ 0.3% (n ¼ 29) to 2.01 ^ 0.2% (n ¼ 18). This inhibition of NA release by 56% was also highly significant (Fig. 2, P , 0:005). Both these effects were prevented if 5 mM nifedipine was included with the chronic exposure to 20 mm KCl (Figs. 1 and 2). In contrast exposure of SH-SY5Y cell layers to 10 mM nicotine for 4 days had no significant effect on the release of [ 3H]NA evoked by 100 mM KCl (Fig. 1) but did decrease nicotine-evoked release of [ 3H]NA by 25% from 4.57 ^ 0.3% (n ¼ 29) to 3.42 ^ 0.3% (n ¼ 25; Fig. 2, P , 0:05). This relatively modest decrease in nicotine-evoked NA release was also prevented if nifedipine (5 mM) was present during chronic exposure to nicotine (Fig. 2). Interestingly, exposure of confluent layers of SHSY5Y cells to 20 mM veratridine for 1 day led to a 42.08 ^ 6.1% (n ¼ 3) decrease in nicotine-evoked NA secretion (Fig. 3). Initial studies found that treatment of confluent layers of SH-SY5Y cells for 1 day with 10 mM 1-[N,O-Bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl] -4-phenylpiperazine (KN-62) inhibited 100 mM K 1-evoked NA release by 63.05 ^ 9.1% (n ¼ 3) respectively. Thus it
was not possible to study the role of CAM-kinase II in the inhibition of depolarisation-evoked release by chronic exposure to 20 mM KCl. In addition exposure of cell layers to 20 mM ionomycin, which leads to a generalised increase in [Ca 21]i inhibited 100 mM nicotine-evoked NA release by 66.01 ^ 2.5% (n ¼ 3). Basal release resulted unaffected by any of the treatments. These data suggest that chronic exposure of SH-SY5Y cells to low levels of K 1 inhibit depolarisation-evoked release of NA by a mechanism that depends on the influx of Ca 21 through L-type VSS Ca 21 channels. This suggests another mechanism by which cells exposed to elevated extracellular K 1 following moderate ischaemia suffer a dysfunction. These studies suggest that upregulation of a7 subtypes nAChR by KCl and nicotine or a3 nAChR by nicotine, reported by Ridley et al. [11], do not enhance depolarisation-evoked NA secretion in SH-SY5Y cells. The failure of chronic exposure to nicotine to enhance NA secretion was unexpected in view of the observation that chronic exposure to nicotine enhances a3 expression and increases in this subtype have been correlated to AChevoked currents in sympathetic neurones [4]. The relatively modest decrease (25%) in nicotine-evoked NA release following chronic exposure to nicotine is possibly due to the partial desensitisation of nAChR. This observation should be compared with the report of Ridley et al. [12] that chronic exposure to 10 mM nicotine decreased Ca 21
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uptake evoked by 10 mM but not 100 mM nicotine. Our observation that chronic exposure to 20 mM KCl leads to a 50% inhibition of nicotine-evoked NA release is in agreement with the data of Ridley et al. [12] which show a decrease in nicotine-evoked Ca 21 uptake under these conditions. The effect of chronic pre-treatment with KCl on KClevoked release of NA agrees less well with the data of these authors who reported very little effect on KCl-evoked Ca 21 uptake under these conditions. Although it has been reported that KN-62 may inhibit Ca 21-influx in some preparations [7], De Koninck and Cooper [4] reported that this compound did not inhibit uptake of Ca 21 via L-type VSCCs. Thus the inhibition of KCl-evoked NA release following exposure to KN-62, reported in the present study, is unlikely to be due to an inhibition of L-type VSCCs by this compound. However De Koninck and Cooper [4] found that KN-62 inhibited Ca 21-uptake via other high-voltage activated Ca 21-channels. In view of our previous study [8] showing that almost 40% of NA release evoked by 100 mM K 1 in SH-SY5Y cells is mediated via N-type Ca 21- channels the possibility that KN-62 is inhibiting K 1-evoked release by this mechanism cannot be excluded at present. An alternative possibility is a role for CAM kinase II either in the replenishment of a pool of readily releasable of secretory granules (e.g. in pancreatic cells [6]) or at a step between influx of Ca 21 and fusion of vesicles with the plasma membrane in PC12 cells [13]. This would suggest a role for CAM kinase II in secretion of NA from large dense core vesicles analogous to its role on phosphorylation of synapsin and the movement of small synaptic vesicles from reserve stores to release site at the plasma membrane [3]. P.F.T.V. would like to acknowledge that the studies carried out in this project were supported by the EU Biomed ProjectBMH4-CT96-1586. [1] Barrantes, G.E., Rogers, A.T., Lindstrom, J. and Wonnacott, S., a-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with a 7 and up-regulation by chronic nicotine treatment, Brain Res., 672 (1995) 228–236. [2] Benwell, M.E.M., Balfour, D.J.K. and Anderson, J.M., Evidence that tobacco smoking increases the density of (2) [ 3H] nicotine binding sites in human brain, J. Neurochem., 50 (1988) 1243–1247. [3] De Camilli, P., Benfenati, F., Valtorta, F. and Greengard, P., The synapsins, Annu. Rev. Cell Biol., 6 (1990) 433–460. [4] De Koninck, P. and Cooper, E., Differential regulation of neuronal nicotinic ACh receptor subunit genes in cultured neonatal rat sympathetic neurons: specific induction of a7 by membrane depolarisation through a Ca 21/ calmodulindependent kinase pathway, J. Neurosci., 15 (1995) 7966– 7978.
[5] Gould, J., Reeve, H.L., Vaughan, P.F.T. and Peers, C., Nicotinic acetylcholine receptors in human neuroblastoma (SHSY5Y) cells, Neurosci. Lett., 145 (1992) 201–204. [6] Gromada, J., Hoy, M., Renstrom, E., Bokvist, K., Eliasson, L., Gopel, S. and Rorsman, P., CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells, J. Physiol. (London), 518 (1999) 745–759. [7] Li, G.D., Hidaka, H. and Wollenheim, C.B., Inhibition of voltage-gated Ca 21-channels and insulin secretion in HIT cells by the Ca 21-calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca 21 channels, Mol. Pharmacol., 42 (1992) 489–498. [8] McDonald, R.L., Vaughan, P.F.T. and Peers, C., Muscarinic (M1) receptor-mediated inhibition of K 1-evoked [ 3H]-noradrenaline release from human neuroblastoma (SH-SY5Y) cells via inhibition of L- and N- type Ca 21 channels, Br. J. Pharmacol., 113 (1994) 621–627. [9] Molinari, E.J., Delbono, O., Messi, M.L., Renganathan, M., Arneric, S.P., Sullivan, J.P. and Gopalakrishnan, M., Upregulation of human alpha 7 nicotinic receptors by chronic treatment with activator and antagonist ligands, Eur. J. Pharmacol., 347 (1998) 131–139. [10] Murphy, N.P., Ball, S.G. and Vaughan, P.F.T., Potassiumand carbachol- evoked release of [ 3H]noradrenaline from human neuroblastoma cells, SH-SY5Y, J. Neurochem., 56 (1991) 1810–1815. [11] Ridley, D.L., Rogers, A. and Wonnacott, S., Differential effects of chronic drug treatment on a3* and a7 nicotinic receptor binding sites, in hippocampal neurones and SHSY5Y cells, Br. J. Pharmacol., 133 (2001) 1286–1295. [12] Ridley, D.L., Pakkanen, J. and Wonnacott, S., Effects of chronic drug treatment on increases in intracellular calcium mediated by nicotinic acetylcholine receptors in SH-SY5Y cells, Br. J. Pharmacol., 135 (2002) 1051–1059. [13] Schweitzer, E.S., Sanderson, M.J. and Wasterling, C.G., Inhibition of regulated catecholamine secretion from PC12 cells by the Ca 21/calmodulin kinase-II inhibitor KN-62, J. Cell Sci., 108 (1995) 2619–2628. [14] Vaughan, P.F.T., Kaye, D.F., Reeve, H.L., Ball, S.G. and Peers, C., Nicotinic receptor-mediated release of noradrenaline in the human neuroblastoma SH-SY5Y, J. Neurochem., 60 (1993) 2159–2166. [15] Vaughan, P.F.T., Peers, C. and Walker, J.H., The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release, Gen. Pharmacol., 26 (1995) 1191–1201. [16] Vaughan, P.F.T., Kaye, D.F., Ball, S.G., Reeve, H.L. and Peers, C., The effect of barium on [ 3H]noradrenaline release from the human neuroblastoma SH-SY5Y, Eur. J. Neurosci., 7 (1995) 875–880. [17] Vaughan, P.F.T., Walker, J.H. and Peers, C., The regulation of neurotransmitter secretion by protein kinase C, Mol. Neurobiol., 18 (1998) 125–155. [18] Wade, J.A., Vaughan, P.F.T. and Peers, C., Hypoxia enhances [ 3H]-noradrenaline release evoked by nicotinic receptor activation from the human neuroblastoma SHSY5Y, J. Neurochem., 71 (1998) 1482–1489. [19] Wonnacott, S., The paradox of nicotinic acetylcholine receptor upregulation by nicotine, Trends Pharmacol. Sci., 11 (1990) 216–219.
Chronic treatment with nicotine or potassium attenuates depolarisation-evoked noradrenaline release from the human neuroblastoma SH-SY5Y Angel Agis-Torres a,*, Stephen G. Ball b, Peter F. T. Vaughan a a
Seccio´n Departamental de FisiologI´a, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. b Academic Unit of Cardiovascular Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, UK Received 7 June 2002; received in revised form 2 August 2002; accepted 2 August 2002
Abstract Chronic treatment, of SH-SY5Y cells, with KCl (20 mM) for 4 days decreased 100 mM KCl-evoked noradrenaline (NA) release by 50% and nicotine (100 mM)-evoked NA release by 55%. Pretreatment with the L-type calcium channel antagonist, nifedipine, prevented this inhibitory effect of chronic exposure to 20 mM KCl on NA release. In contrast pretreatment with 10 mM nicotine for 4 days had no effect on 100 mM KCl -evoked secretion and decreased nicotinic -evoked NA release by only 25%. Inclusion of nifedipine prevented the inhibition of NA release by chronic nicotine treatment. These data are discussed in relation to effects of chronic moderate, depolarisation by either K 1 or nicotine on influx of Ca 21 via L-type voltage sensitive calcium channels. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nicotine; Noradrenaline release; L-type calcium channels; Neuroblastoma SH-SY5Y
Previous studies have found that nicotinic acetylcholine receptors are upregulated by chronic exposure to nicotinic agonists. Thus not only is the density of high affinity [ 3H]nicotinic binding sites increased in post mortem samples of tobacco smokers [2] but upregulation of nicotinic acetylcholine receptor (nAChR) has been observed following chronic exposure to nicotine in vivo in rodent brain [19]. More recently upregulation of a-bungarotoxin (a-Bgt) binding sites following chronic nicotine treatment has been observed in primary cultures of hippocampal neurones [1] and SH-SY5Y cells [12]. In addition De Koninck and Cooper [4], in sympathetic neurones and Ridley et al. [11], in primary cultures of hippocampal neurones and the cell line SH-SY5Y found that low levels of chronic depolarisation with KCl upregulated a7 nAChR subunits by a mechanism involving influx of Ca 21 via L-type voltage sensitive Ca 21-channels (VSCCs) and activation of Ca 21-calmodulin dependent kinase (CAM-kinase II). In contrast, the upregulation of a-Bgt binding sites, by nicotine, was not affected by these inhibitors. Thus these authors concluded that chronic, moderate depolarisation with KCl upregulates a7 nAChR by a mechanism involving entry of Ca 21 via L-type VSCCs and activation of CAM kinase * Corresponding author. Tel.: 134-91-394-1838; fax: 134-91394-1838. E-mail address: [email protected] (A. Agis-Torres).
II. In contrast, a different mechanism is involved in the upregulation of a7 nAChR subunits by nicotine. Ridley et al. [11] also provide data to show that chronic treatment of SH-SY5Y cells with nicotine, but not KCl, also upregulated the a3 * nAChR subunit by a mechanism not involving L-type VSCCs. Although there have been several studies (see above) on the effect of chronic exposure to nicotine on expression levels of nAChR rather fewer studies have looked at the effect of chronic exposure to nicotine on the activity of nAChRs. Previous studies have reported that acute activation of nAChR in SH-SY5Y cells leads to depolarisation [5], increases in [Ca 21]i [12,14] and release of [ 3H]noradrenaline (NA) [15,19]. Recently Ridley et al. [12] have reported that exposure of SH-SY5Y cells to nicotinic agonists or 20mM KCl led to significant decreases in [Ca 21]i, evoked by 10 mM nicotine. Thus rather paradoxically conditions which lead to the upregulation of nAChR result in a decrease in nAChR-evoked increases in [Ca 21]i. In contrast De Koninck and Cooper [4] reported that exposure of sympathetic neurones to 40 mM KCl for 48 h, which upregulates nAChR a7 subunits has no effect on the magnitude nor kinetics of acetylcholine-evoked current densities. They thus concluded that nAChR a7 subunits do not contribute to the macroscopic AChR-induced currents in rat sympathetic neurones. In contrast Molinari et al. [9] reported that chronic
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treatment, of HEK293 cells transfected with human a7nAChR subunits, with nicotine or the nicotinic antagonist methyllcaconitine (MLA) resulted in a 2-fold increase in whole cell currents evoked by nicotine. The aim of the present study was to investigate the effect of treating SH-SY5Y cells with either 10 mM nicotine or 20 mM KCl (conditions reported to upregulate nAChR) on depolarisation-evoked release of NA triggered by either 100 mM nicotine or 100 mM KCl. Previous studies by our group (see Vaughan et al. [15,17] for reviews) have shown that SH-SY5Y cells express depolarisation-evoked release of NA stimulated by KCl [10], dimethylphenylpiperazinium (DMPP) acting at nAChR [14,18], and barium [16]. In the present study the effect of exposure of SH-SY5Y cells to nicotine (10 mM) and KCl (20 mM) for 4 days on the stimulation of NA release by nicotine (100 mM) and KCl (100 mM) was examined. SH-SY5Y cells were cultured in 1:1 mixture of Hams F12 and minimum essential medium containing 1% non-essential amino acids and 10% fetal calf serum in 2.5% CO2 at 378C in 50 cm 2 culture flasks as described previously [10]. After 4 days in culture either 10 mM nicotine or 20 mM KCl was added, to the culture medium, in the presence or absence of 5 mM nifedipine for a further 4 days prior to assaying for depolarisation-evoked NA release. Release of NA was determined using a perfusion assay based on Wade et al. [18]. Confluent flasks were washed extensively in warm phosphate buffered saline (PBS), to remove the added drugs, then incubated for 1 h with 50 nM [ 3H]NA (20 mCi) in N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid] (HEPES)-buffered saline (HBS; 135 mM NaCl, 5 mM KCl, 0.6 mM MgCl2·7H2O, 2.5 mM CaCl2·2H2O, 10 mM HEPES, 6 mM d-glucose, 0.2 mM ascorbic acid and 0.2 mM pargyline) (pH 7.4). The cell layer was then washed twice with PBS minus Ca 21 and Mg 21 followed by incubation at 378C for 10 min in PBS minus calcium and magnesium to detach the cell layer. The detached cells were collected by centrifugation at 600 rev./min (Centra-4R, International Equipment Company). The cell pellet was resuspended in 1 ml HBS and kept on ice until required for experimentation. Aliquots (100 ml) were placed on a filter paper disc in a Swinnex perfusion chamber and washed with HBS (1 ml/min) using a peristaltic pump until a low, stable basal level of [ 3H]NA release was achieved. Release was evoked by exposing the cells to either 100 mM KCl or 100 mM nicotine in HBS for 2 min at 12 min (s1) and 29 min (s2) of perfusion. The outflow from the Swinnex chamber was collected at 30 s intervals, in scintillation vials, 1 min before and 4 min after the stimulus. At the end of this period the filter paper, containing cells, was removed from the Swinnex chamber and placed in a scintillation vial containing 0.5 ml of 0.4 M perchloric acid. Scintillation fluid (4 ml) was added to the vials and [ 3H]NA content determined by liquid scintillation counting (Tri Carb 2000CA, Packard). As indicated in the relevant figure the effect of co-incubation with nifedipine (5 mM) was
Fig. 1. Effect of exposure of SH-SY5Y cell layers to 20 mM K 1 or 10 mM nicotine on 100 mM K 1-evoked NA release. [ 3H]NA release was evoked by 100 mM K 1 in perfusates of untreated cells (untreated); cells pretreated with 5 mM nifedipine (nifed.); 20 mM K 1 (K); 20 mM K 1 and 5 mM nifedipine (K 1 nifed.); 10 mM nicotine (nic.); or 10 mM nicotine and 5 mM nifedipine (nic. 1 nifed.). Release is expressed as the percentage of the radioactivity released by K 1 compared to the total radioactive content of each aliquot of cells. Each column represents the mean ^SEM for 10–19 separate experiments as indicated.
examined on the release of NA from cells which had been preincubated with either 20 mM KCl or 10 mM nicotine. The amount of [ 3H]NA release during s1 (release during s2 was not used in this study) was expressed as a percentage (mean values ^SEM; n is at least 10) of the total radioactivity present in the cell aliquot before release was evoked and from which basal release was subtracted, determined from the stable base line obtained before adding the stimulus. Levels of significance were determined using the Student unpaired t-test. Incubation of SH-SY5Y confluent cell layers with culture
Fig. 2. Effect of exposure of SH-SY5Y cell layers to 20 mM K 1 or 10 mM nicotine on 100 mM nicotine-evoked NA release. [ 3H]NA release was evoked by 100 mM nicotine in perfusates of untreated cells (untreated); cells pretreated with 5 mM nifedipine (nifed.); 20 mM K 1 (K); 20 mM K 1 and 5 mM nifedipine (K 1 nifed.); 10 mm nicotine (nic.) or 10 mM nicotine and 5 mm nifedipine (nic. 1 nifed.). Release is expressed as the percentage of the total radioactive content of each aliquot of cells. Each column represents the mean ^ SEM for 10–28 separate experiments as indicated.
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Fig. 3. Release of [ 3H]NA evoked by 100 mM nicotine (s1 and s2) in perfusates of untreated cells or pretreated with 20 mM veratridine for 1 day. Depicted points are means ^ SEM (n ¼ 3). Periods of stimulation are indicated by horizontal bars located upon the x-axis. Release during s2 was not used in this study.
medium to which 20 mM KCl had been added for 4 days led to a decrease in [ 3H]NA release evoked by 100 mM KCl, in HBS, from 4.44 ^ 0.4% (n ¼ 19) to 2.37 ^ 0.2% (n ¼ 16). This represents a highly significant decrease (46%; P , 0:001) of NA secretion (Fig. 1). Similarly chronic exposure of SH-SY5Y cells to 20 mM KCl, for 4 days, inhibited nicotine-evoked release of [ 3H]NA from 4.57 ^ 0.3% (n ¼ 29) to 2.01 ^ 0.2% (n ¼ 18). This inhibition of NA release by 56% was also highly significant (Fig. 2, P , 0:005). Both these effects were prevented if 5 mM nifedipine was included with the chronic exposure to 20 mm KCl (Figs. 1 and 2). In contrast exposure of SH-SY5Y cell layers to 10 mM nicotine for 4 days had no significant effect on the release of [ 3H]NA evoked by 100 mM KCl (Fig. 1) but did decrease nicotine-evoked release of [ 3H]NA by 25% from 4.57 ^ 0.3% (n ¼ 29) to 3.42 ^ 0.3% (n ¼ 25; Fig. 2, P , 0:05). This relatively modest decrease in nicotine-evoked NA release was also prevented if nifedipine (5 mM) was present during chronic exposure to nicotine (Fig. 2). Interestingly, exposure of confluent layers of SHSY5Y cells to 20 mM veratridine for 1 day led to a 42.08 ^ 6.1% (n ¼ 3) decrease in nicotine-evoked NA secretion (Fig. 3). Initial studies found that treatment of confluent layers of SH-SY5Y cells for 1 day with 10 mM 1-[N,O-Bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl] -4-phenylpiperazine (KN-62) inhibited 100 mM K 1-evoked NA release by 63.05 ^ 9.1% (n ¼ 3) respectively. Thus it
was not possible to study the role of CAM-kinase II in the inhibition of depolarisation-evoked release by chronic exposure to 20 mM KCl. In addition exposure of cell layers to 20 mM ionomycin, which leads to a generalised increase in [Ca 21]i inhibited 100 mM nicotine-evoked NA release by 66.01 ^ 2.5% (n ¼ 3). Basal release resulted unaffected by any of the treatments. These data suggest that chronic exposure of SH-SY5Y cells to low levels of K 1 inhibit depolarisation-evoked release of NA by a mechanism that depends on the influx of Ca 21 through L-type VSS Ca 21 channels. This suggests another mechanism by which cells exposed to elevated extracellular K 1 following moderate ischaemia suffer a dysfunction. These studies suggest that upregulation of a7 subtypes nAChR by KCl and nicotine or a3 nAChR by nicotine, reported by Ridley et al. [11], do not enhance depolarisation-evoked NA secretion in SH-SY5Y cells. The failure of chronic exposure to nicotine to enhance NA secretion was unexpected in view of the observation that chronic exposure to nicotine enhances a3 expression and increases in this subtype have been correlated to AChevoked currents in sympathetic neurones [4]. The relatively modest decrease (25%) in nicotine-evoked NA release following chronic exposure to nicotine is possibly due to the partial desensitisation of nAChR. This observation should be compared with the report of Ridley et al. [12] that chronic exposure to 10 mM nicotine decreased Ca 21
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uptake evoked by 10 mM but not 100 mM nicotine. Our observation that chronic exposure to 20 mM KCl leads to a 50% inhibition of nicotine-evoked NA release is in agreement with the data of Ridley et al. [12] which show a decrease in nicotine-evoked Ca 21 uptake under these conditions. The effect of chronic pre-treatment with KCl on KClevoked release of NA agrees less well with the data of these authors who reported very little effect on KCl-evoked Ca 21 uptake under these conditions. Although it has been reported that KN-62 may inhibit Ca 21-influx in some preparations [7], De Koninck and Cooper [4] reported that this compound did not inhibit uptake of Ca 21 via L-type VSCCs. Thus the inhibition of KCl-evoked NA release following exposure to KN-62, reported in the present study, is unlikely to be due to an inhibition of L-type VSCCs by this compound. However De Koninck and Cooper [4] found that KN-62 inhibited Ca 21-uptake via other high-voltage activated Ca 21-channels. In view of our previous study [8] showing that almost 40% of NA release evoked by 100 mM K 1 in SH-SY5Y cells is mediated via N-type Ca 21- channels the possibility that KN-62 is inhibiting K 1-evoked release by this mechanism cannot be excluded at present. An alternative possibility is a role for CAM kinase II either in the replenishment of a pool of readily releasable of secretory granules (e.g. in pancreatic cells [6]) or at a step between influx of Ca 21 and fusion of vesicles with the plasma membrane in PC12 cells [13]. This would suggest a role for CAM kinase II in secretion of NA from large dense core vesicles analogous to its role on phosphorylation of synapsin and the movement of small synaptic vesicles from reserve stores to release site at the plasma membrane [3]. P.F.T.V. would like to acknowledge that the studies carried out in this project were supported by the EU Biomed ProjectBMH4-CT96-1586. [1] Barrantes, G.E., Rogers, A.T., Lindstrom, J. and Wonnacott, S., a-Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with a 7 and up-regulation by chronic nicotine treatment, Brain Res., 672 (1995) 228–236. [2] Benwell, M.E.M., Balfour, D.J.K. and Anderson, J.M., Evidence that tobacco smoking increases the density of (2) [ 3H] nicotine binding sites in human brain, J. Neurochem., 50 (1988) 1243–1247. [3] De Camilli, P., Benfenati, F., Valtorta, F. and Greengard, P., The synapsins, Annu. Rev. Cell Biol., 6 (1990) 433–460. [4] De Koninck, P. and Cooper, E., Differential regulation of neuronal nicotinic ACh receptor subunit genes in cultured neonatal rat sympathetic neurons: specific induction of a7 by membrane depolarisation through a Ca 21/ calmodulindependent kinase pathway, J. Neurosci., 15 (1995) 7966– 7978.
[5] Gould, J., Reeve, H.L., Vaughan, P.F.T. and Peers, C., Nicotinic acetylcholine receptors in human neuroblastoma (SHSY5Y) cells, Neurosci. Lett., 145 (1992) 201–204. [6] Gromada, J., Hoy, M., Renstrom, E., Bokvist, K., Eliasson, L., Gopel, S. and Rorsman, P., CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells, J. Physiol. (London), 518 (1999) 745–759. [7] Li, G.D., Hidaka, H. and Wollenheim, C.B., Inhibition of voltage-gated Ca 21-channels and insulin secretion in HIT cells by the Ca 21-calmodulin-dependent protein kinase II inhibitor KN-62: comparison with antagonists of calmodulin and L-type Ca 21 channels, Mol. Pharmacol., 42 (1992) 489–498. [8] McDonald, R.L., Vaughan, P.F.T. and Peers, C., Muscarinic (M1) receptor-mediated inhibition of K 1-evoked [ 3H]-noradrenaline release from human neuroblastoma (SH-SY5Y) cells via inhibition of L- and N- type Ca 21 channels, Br. J. Pharmacol., 113 (1994) 621–627. [9] Molinari, E.J., Delbono, O., Messi, M.L., Renganathan, M., Arneric, S.P., Sullivan, J.P. and Gopalakrishnan, M., Upregulation of human alpha 7 nicotinic receptors by chronic treatment with activator and antagonist ligands, Eur. J. Pharmacol., 347 (1998) 131–139. [10] Murphy, N.P., Ball, S.G. and Vaughan, P.F.T., Potassiumand carbachol- evoked release of [ 3H]noradrenaline from human neuroblastoma cells, SH-SY5Y, J. Neurochem., 56 (1991) 1810–1815. [11] Ridley, D.L., Rogers, A. and Wonnacott, S., Differential effects of chronic drug treatment on a3* and a7 nicotinic receptor binding sites, in hippocampal neurones and SHSY5Y cells, Br. J. Pharmacol., 133 (2001) 1286–1295. [12] Ridley, D.L., Pakkanen, J. and Wonnacott, S., Effects of chronic drug treatment on increases in intracellular calcium mediated by nicotinic acetylcholine receptors in SH-SY5Y cells, Br. J. Pharmacol., 135 (2002) 1051–1059. [13] Schweitzer, E.S., Sanderson, M.J. and Wasterling, C.G., Inhibition of regulated catecholamine secretion from PC12 cells by the Ca 21/calmodulin kinase-II inhibitor KN-62, J. Cell Sci., 108 (1995) 2619–2628. [14] Vaughan, P.F.T., Kaye, D.F., Reeve, H.L., Ball, S.G. and Peers, C., Nicotinic receptor-mediated release of noradrenaline in the human neuroblastoma SH-SY5Y, J. Neurochem., 60 (1993) 2159–2166. [15] Vaughan, P.F.T., Peers, C. and Walker, J.H., The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release, Gen. Pharmacol., 26 (1995) 1191–1201. [16] Vaughan, P.F.T., Kaye, D.F., Ball, S.G., Reeve, H.L. and Peers, C., The effect of barium on [ 3H]noradrenaline release from the human neuroblastoma SH-SY5Y, Eur. J. Neurosci., 7 (1995) 875–880. [17] Vaughan, P.F.T., Walker, J.H. and Peers, C., The regulation of neurotransmitter secretion by protein kinase C, Mol. Neurobiol., 18 (1998) 125–155. [18] Wade, J.A., Vaughan, P.F.T. and Peers, C., Hypoxia enhances [ 3H]-noradrenaline release evoked by nicotinic receptor activation from the human neuroblastoma SHSY5Y, J. Neurochem., 71 (1998) 1482–1489. [19] Wonnacott, S., The paradox of nicotinic acetylcholine receptor upregulation by nicotine, Trends Pharmacol. Sci., 11 (1990) 216–219.