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Neuropeptide Y secretion from bovine chromaffin cells inhibits cyclic amp accumulation
Life Sciences 67 (2000) 617Ð625
Neuropeptide Y secretion from bovine chromafÞn cells inhibits cyclic amp accumulation Jialin Zheng, Guimei Zhou, Terry D. Hexum* University of Nebraska Medical Center, Department of Pharmacology, 986260 Nebraska Medical Center, Omaha, NE 68198-6260, USA
Abstract Neuropeptide Y (NPY) is secreted from bovine chromafÞn cells in response to nicotinic receptor stimulation and may exhibit autocrine, paracrine or endocrine effects. Stimulation of bovine chromafÞn cells with nicotine followed by the addition of forskolin (FSK) to the media results in a decrease in cyclic AMP accumulation compared to that seen in the absence of nicotine. Pertussis toxin (PTX) treatment or the addition of BIBP 3226, a selective NPY Y1 receptor antagonist prevents the inhibitory effect of nicotine. Fractionation of media obtained from cells stimulated with nicotine reveals an NPYlike substance that inhibits FSK-stimulated cAMP accumulation. Thus, an NPY-like substance can be secreted from bovine chromafÞn cells in quantities sufÞcient to inhibit FSK-stimulated cAMP accumulation. These results suggest that NPY can act in an autocrine fashion to regulate chromafÞn cell function. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptide Y; cAMP; ChromafÞn cell; Nicotine
Introduction NPY is a 36 amino acid member of the pancreatic polypeptide family that acts through G-protein coupled receptors (1). It is distributed throughout the body and potently increases food intake and blood pressure and decreases anxiety (1). NPY is co-localized with catecholamines in many tissues including the bovine adrenal medulla where it is co-stored with norepinephrine (2,3). ChromafÞn cells from the bovine adrenal medulla contain NPY receptors classiÞed as Y1 (4). It was previously reported that NPY can inhibit chromafÞn cell catecholamine secretion (5) suggestive of a physiological role for NPY in these cells. We examined this phenomenon in more detail and reported that NPY can inhibit the secretion of [3H]norepinephrine but only when NPY is added to culture media in concentrations greater than those secreted by nico* Corresponding author. Tel.: (402) 559-4562; fax: (402) 559-7495. E-mail address: [email protected] (T.D. Hexum) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 6 6 -4
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tinic receptor stimulation (6). Our studies concluded that the inhibitory effect is non-receptor mediated and due to the ability of NPY and NPY fragments to inhibit ion movement through the nicotinic receptor ion channel through an undetermined mechanism (7). Thus, inhibition of secretion does not appear to be a mechanism by which NPY regulates chromafÞn cell activity. Another approach to determine the signiÞcance of NPY in chromafÞn cell physiology would be to determine whether NPY secreted from chromafÞn cells can alter chromafÞn cell second messenger production. If secreted NPY can produce changes in cellular second messenger levels this would demonstrate that: a) NPY can act in an autocrine fashion on chromafÞn cells; and b) quantities of NPY sufÞcient to produce physiologically important effects are secreted. Moreover by revealing the second messenger system(s) responsive to NPY, insight into the mechanism by which this peptide alters chromafÞn cell function may be provided. For example, changes in cAMP levels may suggest an effect of NPY on catecholamine synthesis or changes in intracellular Ca21 may suggest an inßuence on gene expression. In this report we demonstrate that an NPY-like substance, secreted from chromafÞn cells in response to nicotinic receptor stimulation can inhibit forskolin-stimulated cyclic AMP (cAMP) accumulation. Methods Cell culture The isolation and culture of bovine adrenal chromafÞn cells wereas performed with modiÞcations as previously described (8). The cells were plated in 6-well or 12-well plastic culture plates (4 3 106 cells/well) in an atmosphere of 5% CO2 at 378C. One-half of the medium was changed every other day. ChromafÞn cells used in studies were kept in culture for 3Ð7 days. Cyclic AMP assay The assay for cAMP accumulation was as described previously with minor modiÞcations (9). Cultured chromafÞn cells were washed once with pre-warmed serum-free DMEM containing 20 mM HEPES, pH 7.4, and loaded with 5 mCi [3H]adenine in 0.5 ml/well DMEM with HEPES at 378C for 120 min. Cells were then washed twice with the same buffer and exposed to the additives (pretreatment and treatment) for the times indicated. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (1.25 3 1026 M), was always included in the assay. Intracellular [3H]cAMP was extracted overnight with 1 ml of ice-cold 5% trichloroacetic acid containing 1 mM unlabeled cAMP. [3H]cAMP was separated from other tritiated nucleotides by sequential ion-exchange chromatography over Dowex and alumina columns. The ATP and cAMP fractions (3 ml each) were collected in scintillation vials to which 10 ml Budget Solve was added. The radioactivity of the samples was determined by liquid scintillation spectroscopy with an efÞciency of 40%. Data is calculated from the percentage conversion of [3H]ATP to [3H]cAMP by the various treatment conditions. High performance liquid chromatography Reversed phase liquid chromatography was performed using an Altex 420 Controller/ Programer, Model 155 Variable wavelength detector and two 110A solvent metering pumps.
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Solvent A was 0.1% trißuoroacetic acid (TFA, Pierce) in chromatographic grade water; solvent B was 80% CH3CN in 0.1% TFA. Samples were partially puriÞed using C18 reversed phase cartridges (Alltech Extract Clean) [wash solution: 2 volumes 1% formic acid adjusted to pH 3 with triethylamine (TEAF); eluant: 2 volumes 75% CH3CN/25% TEAF] and injected (200 ml sample loop) onto a Rainin Microsorb-MV (4.6 mm 3 25 cm) C18 5 mm 100A reversed phase column. A linear gradient from 15 to 70% CH3CN was applied over a period of 45 min (ßow rate: 1 ml/min) and 1 ml fractions collected. Solvent was removed under reduced pressure and the residue assayed for NPY immunoreactive material (NPY-IR). Recovery of NPY from an Extract Clean cartridge or HPLC column was 30% or 50%, respectively. Radioimmunoassay Radioimmunoassays (RIA) were carried out in buffer containing 0.05 M sodium phosphate, 0.15 M sodium chloride, 0.025 M Na2EDTA, 0.25% BSA (Crystalline, Pentex, Miles) and 0.1% sodium azide at pH 7.4. After addition of 100 ml of standard (porcine NPY) or test sample, 100 ml of antibody (C-terminal directed antibody to NPY, Ab3-5 provided by M. Brown, UCSD), at a Þnal dilution of 1:400,000 was added to siliconized (SurfaSil, Pierce) glass tubes. Tubes were mixed and incubated at 48 for 48 h. [125ITyr36]NPY (10,000 cpm) in 100 ml of RIA buffer containing 0.1% Triton X-100 was added and allowed to incubate (48) overnight. Separation of free from bound tracer was accomplished by adding 100 ml of normal rabbit serum as co-precipitant (1:200), 100 ml of goat anti-rabbit serum (1:40) and 500 ml of 10% polyethylene glycol. After incubation at 48 for 15 min and centrifuging at 3,000 rpm for 20 min, supernatants were aspirated and the pellets counted in a TM Analytical gamma counter. Data analysis Data were analyzed by One-way ANOVA followed by the Tukey-Kramer Multiple Comparisons Test using GraphPad Instatª. Curves were Þt and analyzed using GraphPad Prismª. Materials Peptides were either purchased from Peninsula Laboratories, Inc. (Belmont, CA) and Bachem California (Torrance, CA) or provided by Jean Rivier, Ph.D., The Salk Institute for Biological Studies (La Jolla, CA). Culture medium was purchased from Life Technologies (Grand Island, NY). All other items were purchased from Sigma (St. Louis, MO). [125I]-Tyr36NPY was obtained from DuPont NEN Products (Boston, MA). Results Inhibition of forskolin-stimulated cAMP accumulation by conditioned media Forskolin (3 3 1025 M) induced a 120-fold increase in cAMP accumulation in bovine chromafÞn cells during a 5 min incubation (Fig. 1). If cells were preincubated with nicotine (1 3 1025 M) for various times followed by forskolin (FSK) stimulation, a progressive decrease in cAMP accumulation occurred. After 2 minutes of incubation with nicotine, cAMP accumulation was 85% of control values (p , 0.02) progressing to 65% after 10 min (p ,
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Fig. 1. Conditioned media from nicotine-stimulated chromafÞn cells inhibit FSK-stimulated cAMP accumulation. Bovine chromafÞn cells were preincubated for various time periods at 378 with buffer, nicotine in buffer (1 3 1025 M) or authentic NPY in buffer (1 3 1029). FSK (3 3 1025 M) was then added to the media for an additional 5 min at 378. Cyclic AMP accumulation is calculated as the percent [3H]ATP converted to [3H]cAMP. No FSK: 0.031%; with FSK: 4.283%. FSK stimulation of cells in the presence of buffer only is deÞned as control and set at 100%. * p , 0.02; ** p , 0.005, relative to control (FSK alone).
0.005). No further change in cAMP accumulation was seen with extended nicotine incubation (34 min). Preincubation with authentic NPY (1 3 1029 M) decreased cAMP accumulation to approximately 55% of control values regardless of incubation time. Nicotine effect on cAMP accumulation is concentration dependent and PTX-sensitive Increasing nicotine concentrations produced a concentration dependent inhibition of cAMP accumulation (60% of control at 1 3 1025 M) (Fig. 2). The involvement of Gi (or Go) in the effect of nicotine treatment of chromafÞn cells was assessed by treating cells with pertussis toxin (PTX) (100 ng for 18 h). PTX pretreatment completely abolished the inhibitory effect of nicotine on cAMP accumulation (Fig. 2). BIBP blocks the effect of nicotine on cAMP accumulation Addition of the selective NPY Y1 receptor antagonist BIBP 3226 ((R)-N2-(diphenacetyl)N-[(4-hydroxyphenyl)methyl]-argininamide) (BIBP) (3 3 1027 M) to the culture media prior to incubation with nicotine (1 3 1025 M for 30 min) blocked the inhibitory effect of nicotine on FSK-stimulated cAMP accumulation in chromafÞn cells (Fig. 3). BIBP also blocked the effect of authentic NPY (1 3 1029 M) incubated under the same conditions but in the absence of nicotine. The nicotinic receptor antagonist, hexamethonium (3 3 1024 M), also blocked the inhibitory effect of nicotine on cAMP accumulation (data not shown).
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Fig. 2. The inhibitory effect of nicotine on chromafÞn cell cAMP accumulation is PTX-sensitive. Bovine chromafÞn cells were preincubated for 15 min at 378 with increasing concentrations of nicotine in control cells (j) or cells treated with PTX (u) (100 ng/ml for 18 h at 378). Cells were then stimulated with FSK (3 3 1025 M) for 9 min at 378 and the percent conversion of [3H]ATP to [3H]cAMP determined (see Fig. 1).
Fig. 3. BIBP antagonizes the effect of nicotine on chromafÞn cell cAMP accumulation. Bovine chromafÞn cells were preincubated for 30 min at 378 with buffer, nicotine in buffer (1 3 1025 M) with and without BIBP (3 3 10 27 M) or authentic NPY in buffer (1 3 1029) with and without BIBP (3 3 1027 M). Cells were then stimulated with FSK (3 3 1025 M) for 5 min at 378 and the percent conversion of [3H]ATP to [3H]cAMP determined (see Fig. 1). * p ,0.05; ** p,0.01 relative to control.
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HPLC analysis of conditioned chromafÞn cell media shows presence of NPY-like material Determination of the agent responsible for inhibition of FSK-stimulated cAMP accumulation was performed by HPLC. Media was obtained from chromafÞn cells that were either incubated with nicotine (1 3 1025 M) or authentic NPY (1 3 1029 M). Incubation of cells with nicotine (1 3 1025 M) for 30 min produced a single peak detected by an NPY antibody at 26 min (Fig. 4). Incubation of authentic NPY with chromafÞn cells under the same conditions yielded a co-eluting peak. Controls were obtained under two sets of conditions. One set consisted of HPLC fractionation of media obtained from cells ncubated for 30 min but not stimulated with nicotine. The second control consisted of samples from a gradient run obtained after no sample injection using the same reversed phase column used to fractionate condition media. No measurable immunoreactive peaks were obtained from either condition.
Fig. 4. Media from chromafÞn cells stimulated with nicotine contain NPY-like material. Bovine chromafÞn cells were either stimulated with nicotine (1 3 1025 M) (j) or authentic NPY (1 3 1029 M) (h) for 30 min at 378. Controls consisted of media from unstimulated cells (d) and fractions obtained in the absence of sample injection (s). Media was removed from the cells, hydrophobic material partially puriÞed using a reversed phase cartridge (see Methods) and the residue suspended in HPLC solvent and fractionated by reversed phase chromatography. Solvent was removed under reduced pressure, the samples resuspended in buffer and assayed radioimmunochemically using a speciÞc NPY antibody.
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HPLC fractions inhibit FSK-stimulated cAMP accumulation HPLC fractions of conditioned media were examined for their ability to inhibit FSKstimulated cAMP accumulation (see previous section). Incubation of the fractions obtained from the chromatography of media obtained from nicotine-treated (1 3 1025 M for 30 min) cells with chromafÞn cells subsequently revealed one peak at 26 min capable of inhibiting 50% of FSK-stimulated cAMP accumulation (Fig. 5). This peak co-eluted with authentic NPY (see Fig. 4). In addition, several minor peaks capable of inhibiting FSK-stimulated cAMP accumulation to the extent of 20% or less of control were also observed. HPLC fractions obtained from the chromatography of authentic NPY (see Fig. 4) produced a similar pattern of inhibition with a peak also at 26 min. Examination of fractions from controls (see Fig. 4) showed a minor inhibitory peak at 28 min in media from non-stimulated cells. Discussion Bovine chromafÞn cells contain NPY Y1 receptors. Physiologically the ligand for these receptors may be provided by the splanchnic nerve in a paracrine fashion or by the chromafÞn
Fig. 5. ChromafÞn cell HPLC fractions inhibit FSK-stimulated cAMP accumulation. Bovine chromafÞn cells were incubated with aliquots of HPLC fractions (Fig. 4) followed by FSK (3 3 1025 M) stimulation for 5 min at 378. Cyclic AMP accumulation is measured as described above (Fig. 1). Nicotine (j); NPY (u); unstimulated cells (d); no sample injection (s).
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cells in an autocrine fashion. NPY is secreted from chromafÞn cells in response to nicotinic receptor stimulation or membrane depolarization with high K1 concentrations. The physiological consequences to these cells of NPY secretion is unknown although we have recently shown that the addition of low NPY concentrations to culture media inhibits catecholamine synthesis by inhibiting tyrosine hydroxylase phosphorylation and subsequently enzyme activity (4). Here we provide further support for the notion that NPY secreted from bovine chromafÞn cells is involved in the regulation of chromafÞn cell function. Our approach is to examine the effect of NPY secreted from chromafÞn cells on cAMP accumulation in these cells. NPY secretion from chromafÞn cells occurs in response to the addition of nicotinic receptor agonists to culture media (10,11). Moreover, FSK stimulation increases cAMP accumulation in these cells (8). Thus the effectiveness of secreted NPY on cAMP accumulation can be examined by Þrst stimulating chromafÞn cells with nicotine followed by the measurement of cAMP accumulation in the presence of FSK (8). The incubation of nicotine with chromafÞn cells for increasing time periods should result in the proportionate accumulation of secreted material. Accordingly stimulation of chromafÞn cells with nicotine for varying time periods results in increasing inhibition of FSK-stimulated cAMP accumulation. The degree of inhibition is similar to that produced by NPY (1 3 1029 M) in the absence of nicotine stimulation. Treatment of cells with PTX to ADP-ribosylate a G protein completely prevented the effect of nicotine. The time dependent nature of the inhibition of cAMP accumulation by nicotine and the effect of PTX demonstrate that a secreted product rather than nicotine is responsible for the inhibition of cAMP accumulation. Preliminary identiÞcation of the responsible inhibitory agent is provided by the effect of BIBP, a selective Y1 receptor antagonist (12). The addition of this agent to culture media prior to FSK stimulation prevented the inhibitory effect of nicotine on chromafÞn cell cAMP accumulation. Moreover, BIBP prevented the effect of NPY added to the culture media under similar conditions. This data demonstrates that an agent acting on the Y1 receptor is responsible for the inhibition of cAMP accumulation. Further characterization of the agent responsible for the inhibition of cAMP accumulation was provided by HPLC fractionation of conditioned media. Fractionation of media after nicotine stimulation of chromafÞn cells revealed the presence of a peak co-eluting with authentic NPY. Examination of this fraction as well as others obtained from conditioned media for their ability to inhibit FSK-stimulated cAMP accumulation showed that the peak co-eluting with NPY to be the most effective inhibitor. Taken together these data demonstrate than an NPY-like substance is secreted from bovine chromafÞn cells in response to nicotinic receptor stimulation. Moreover, this NPY-like substance is an effective inhibitor of FSK-stimulated cAMP accumulation. Thus an NPY-like substance is secreted in sufÞcient quantities to act in a autocrine fashion to alter chromafÞn cell production of cAMP. These data provide further support for the notion that NPY regulates chromafÞn cell function probably by a processes which uses cAMP such as tyrosine hydroxylase activation (13) or synthesis (14). Acknowledgments We thank Dr. Jean Rivier, The Salk Institute, San Diego, CA, for providing the NPY. This work was supported by a grant from the American Heart Association #9650472N.
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References 1. C. WAHLESTEDT and D.J. REIS, Annu Rev Pharmacol Toxicol. 33 309Ð352 (1993). 2. E.A. MAJANE, H. ALHO, Y. KATAOKA, C.H. LEE, and H.Y. YANG, Endocrinology. 117 1162Ð1168 (1985). 3. I.M. VARNDELL, J.M. POLAK, J.M. ALLEN, G. TERENGHI, and S.R. BLOOM, Endocrinology. 114 1460Ð1462 (1984). 4. J.L. ZHENG, P.J. ZHANG, and T.D. HEXUM, Mol. Pharmacol. 52 1027Ð1033 (1997). 5. H. HIGUCHI, E. COSTA, and H.Y. YANG, J Pharmacol Exp Ther. 244 468Ð474 (1988). 6. T.D. HEXUM, J. ZHENG, and J. ZHU, J Pharmacol Exp Ther. 271 61Ð66 (1994). 7. J. ZHENG, R.A. MORRISETT, J. ZHU, and T.D. HEXUM, J Pharmacol Exp Ther. 274 891Ð897 (1995). 8. J. ZHU, W. LI, M.L. TOEWS, and T.D. HEXUM, J Pharmacol Exp Ther. 263 1479Ð1486 (1992). 9. R.A. JOHNSON and M.L. TOEWS, Mol Pharmacol. 37 296Ð303 (1990). 10. Y. KATAOKA, E.A. MAJANE, and H.Y. YANG, Neuropharmacology. 24 693Ð695 (1985). 11. T.D. HEXUM and L.R. RUSSETT, Neuropeptides. 13 35Ð41 (1989). 12. H.A. WIELAND, K.D. WILLIM, M. ENTZEROTH, W. WIENEN, K. RUDOLF, W. EBERLEIN, W. ENGEL, and H.N. DOODS, J Pharmacol Exp Ther. 275 143Ð149 (1995). 13. R.E. ZIGMOND, M.A. SCHWARZSCHILD, and A.R. RITTENHOUSE, Annu Rev Neurosci. 12 415Ð461 (1989). 14. M.K. STACHOWIAK, J.S. HONG, and O.H. VIVEROS, Brain Res. 510 277Ð288 (1990).
Neuropeptide Y secretion from bovine chromafÞn cells inhibits cyclic amp accumulation Jialin Zheng, Guimei Zhou, Terry D. Hexum* University of Nebraska Medical Center, Department of Pharmacology, 986260 Nebraska Medical Center, Omaha, NE 68198-6260, USA
Abstract Neuropeptide Y (NPY) is secreted from bovine chromafÞn cells in response to nicotinic receptor stimulation and may exhibit autocrine, paracrine or endocrine effects. Stimulation of bovine chromafÞn cells with nicotine followed by the addition of forskolin (FSK) to the media results in a decrease in cyclic AMP accumulation compared to that seen in the absence of nicotine. Pertussis toxin (PTX) treatment or the addition of BIBP 3226, a selective NPY Y1 receptor antagonist prevents the inhibitory effect of nicotine. Fractionation of media obtained from cells stimulated with nicotine reveals an NPYlike substance that inhibits FSK-stimulated cAMP accumulation. Thus, an NPY-like substance can be secreted from bovine chromafÞn cells in quantities sufÞcient to inhibit FSK-stimulated cAMP accumulation. These results suggest that NPY can act in an autocrine fashion to regulate chromafÞn cell function. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Neuropeptide Y; cAMP; ChromafÞn cell; Nicotine
Introduction NPY is a 36 amino acid member of the pancreatic polypeptide family that acts through G-protein coupled receptors (1). It is distributed throughout the body and potently increases food intake and blood pressure and decreases anxiety (1). NPY is co-localized with catecholamines in many tissues including the bovine adrenal medulla where it is co-stored with norepinephrine (2,3). ChromafÞn cells from the bovine adrenal medulla contain NPY receptors classiÞed as Y1 (4). It was previously reported that NPY can inhibit chromafÞn cell catecholamine secretion (5) suggestive of a physiological role for NPY in these cells. We examined this phenomenon in more detail and reported that NPY can inhibit the secretion of [3H]norepinephrine but only when NPY is added to culture media in concentrations greater than those secreted by nico* Corresponding author. Tel.: (402) 559-4562; fax: (402) 559-7495. E-mail address: [email protected] (T.D. Hexum) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 6 6 -4
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tinic receptor stimulation (6). Our studies concluded that the inhibitory effect is non-receptor mediated and due to the ability of NPY and NPY fragments to inhibit ion movement through the nicotinic receptor ion channel through an undetermined mechanism (7). Thus, inhibition of secretion does not appear to be a mechanism by which NPY regulates chromafÞn cell activity. Another approach to determine the signiÞcance of NPY in chromafÞn cell physiology would be to determine whether NPY secreted from chromafÞn cells can alter chromafÞn cell second messenger production. If secreted NPY can produce changes in cellular second messenger levels this would demonstrate that: a) NPY can act in an autocrine fashion on chromafÞn cells; and b) quantities of NPY sufÞcient to produce physiologically important effects are secreted. Moreover by revealing the second messenger system(s) responsive to NPY, insight into the mechanism by which this peptide alters chromafÞn cell function may be provided. For example, changes in cAMP levels may suggest an effect of NPY on catecholamine synthesis or changes in intracellular Ca21 may suggest an inßuence on gene expression. In this report we demonstrate that an NPY-like substance, secreted from chromafÞn cells in response to nicotinic receptor stimulation can inhibit forskolin-stimulated cyclic AMP (cAMP) accumulation. Methods Cell culture The isolation and culture of bovine adrenal chromafÞn cells wereas performed with modiÞcations as previously described (8). The cells were plated in 6-well or 12-well plastic culture plates (4 3 106 cells/well) in an atmosphere of 5% CO2 at 378C. One-half of the medium was changed every other day. ChromafÞn cells used in studies were kept in culture for 3Ð7 days. Cyclic AMP assay The assay for cAMP accumulation was as described previously with minor modiÞcations (9). Cultured chromafÞn cells were washed once with pre-warmed serum-free DMEM containing 20 mM HEPES, pH 7.4, and loaded with 5 mCi [3H]adenine in 0.5 ml/well DMEM with HEPES at 378C for 120 min. Cells were then washed twice with the same buffer and exposed to the additives (pretreatment and treatment) for the times indicated. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (1.25 3 1026 M), was always included in the assay. Intracellular [3H]cAMP was extracted overnight with 1 ml of ice-cold 5% trichloroacetic acid containing 1 mM unlabeled cAMP. [3H]cAMP was separated from other tritiated nucleotides by sequential ion-exchange chromatography over Dowex and alumina columns. The ATP and cAMP fractions (3 ml each) were collected in scintillation vials to which 10 ml Budget Solve was added. The radioactivity of the samples was determined by liquid scintillation spectroscopy with an efÞciency of 40%. Data is calculated from the percentage conversion of [3H]ATP to [3H]cAMP by the various treatment conditions. High performance liquid chromatography Reversed phase liquid chromatography was performed using an Altex 420 Controller/ Programer, Model 155 Variable wavelength detector and two 110A solvent metering pumps.
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Solvent A was 0.1% trißuoroacetic acid (TFA, Pierce) in chromatographic grade water; solvent B was 80% CH3CN in 0.1% TFA. Samples were partially puriÞed using C18 reversed phase cartridges (Alltech Extract Clean) [wash solution: 2 volumes 1% formic acid adjusted to pH 3 with triethylamine (TEAF); eluant: 2 volumes 75% CH3CN/25% TEAF] and injected (200 ml sample loop) onto a Rainin Microsorb-MV (4.6 mm 3 25 cm) C18 5 mm 100A reversed phase column. A linear gradient from 15 to 70% CH3CN was applied over a period of 45 min (ßow rate: 1 ml/min) and 1 ml fractions collected. Solvent was removed under reduced pressure and the residue assayed for NPY immunoreactive material (NPY-IR). Recovery of NPY from an Extract Clean cartridge or HPLC column was 30% or 50%, respectively. Radioimmunoassay Radioimmunoassays (RIA) were carried out in buffer containing 0.05 M sodium phosphate, 0.15 M sodium chloride, 0.025 M Na2EDTA, 0.25% BSA (Crystalline, Pentex, Miles) and 0.1% sodium azide at pH 7.4. After addition of 100 ml of standard (porcine NPY) or test sample, 100 ml of antibody (C-terminal directed antibody to NPY, Ab3-5 provided by M. Brown, UCSD), at a Þnal dilution of 1:400,000 was added to siliconized (SurfaSil, Pierce) glass tubes. Tubes were mixed and incubated at 48 for 48 h. [125ITyr36]NPY (10,000 cpm) in 100 ml of RIA buffer containing 0.1% Triton X-100 was added and allowed to incubate (48) overnight. Separation of free from bound tracer was accomplished by adding 100 ml of normal rabbit serum as co-precipitant (1:200), 100 ml of goat anti-rabbit serum (1:40) and 500 ml of 10% polyethylene glycol. After incubation at 48 for 15 min and centrifuging at 3,000 rpm for 20 min, supernatants were aspirated and the pellets counted in a TM Analytical gamma counter. Data analysis Data were analyzed by One-way ANOVA followed by the Tukey-Kramer Multiple Comparisons Test using GraphPad Instatª. Curves were Þt and analyzed using GraphPad Prismª. Materials Peptides were either purchased from Peninsula Laboratories, Inc. (Belmont, CA) and Bachem California (Torrance, CA) or provided by Jean Rivier, Ph.D., The Salk Institute for Biological Studies (La Jolla, CA). Culture medium was purchased from Life Technologies (Grand Island, NY). All other items were purchased from Sigma (St. Louis, MO). [125I]-Tyr36NPY was obtained from DuPont NEN Products (Boston, MA). Results Inhibition of forskolin-stimulated cAMP accumulation by conditioned media Forskolin (3 3 1025 M) induced a 120-fold increase in cAMP accumulation in bovine chromafÞn cells during a 5 min incubation (Fig. 1). If cells were preincubated with nicotine (1 3 1025 M) for various times followed by forskolin (FSK) stimulation, a progressive decrease in cAMP accumulation occurred. After 2 minutes of incubation with nicotine, cAMP accumulation was 85% of control values (p , 0.02) progressing to 65% after 10 min (p ,
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Fig. 1. Conditioned media from nicotine-stimulated chromafÞn cells inhibit FSK-stimulated cAMP accumulation. Bovine chromafÞn cells were preincubated for various time periods at 378 with buffer, nicotine in buffer (1 3 1025 M) or authentic NPY in buffer (1 3 1029). FSK (3 3 1025 M) was then added to the media for an additional 5 min at 378. Cyclic AMP accumulation is calculated as the percent [3H]ATP converted to [3H]cAMP. No FSK: 0.031%; with FSK: 4.283%. FSK stimulation of cells in the presence of buffer only is deÞned as control and set at 100%. * p , 0.02; ** p , 0.005, relative to control (FSK alone).
0.005). No further change in cAMP accumulation was seen with extended nicotine incubation (34 min). Preincubation with authentic NPY (1 3 1029 M) decreased cAMP accumulation to approximately 55% of control values regardless of incubation time. Nicotine effect on cAMP accumulation is concentration dependent and PTX-sensitive Increasing nicotine concentrations produced a concentration dependent inhibition of cAMP accumulation (60% of control at 1 3 1025 M) (Fig. 2). The involvement of Gi (or Go) in the effect of nicotine treatment of chromafÞn cells was assessed by treating cells with pertussis toxin (PTX) (100 ng for 18 h). PTX pretreatment completely abolished the inhibitory effect of nicotine on cAMP accumulation (Fig. 2). BIBP blocks the effect of nicotine on cAMP accumulation Addition of the selective NPY Y1 receptor antagonist BIBP 3226 ((R)-N2-(diphenacetyl)N-[(4-hydroxyphenyl)methyl]-argininamide) (BIBP) (3 3 1027 M) to the culture media prior to incubation with nicotine (1 3 1025 M for 30 min) blocked the inhibitory effect of nicotine on FSK-stimulated cAMP accumulation in chromafÞn cells (Fig. 3). BIBP also blocked the effect of authentic NPY (1 3 1029 M) incubated under the same conditions but in the absence of nicotine. The nicotinic receptor antagonist, hexamethonium (3 3 1024 M), also blocked the inhibitory effect of nicotine on cAMP accumulation (data not shown).
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Fig. 2. The inhibitory effect of nicotine on chromafÞn cell cAMP accumulation is PTX-sensitive. Bovine chromafÞn cells were preincubated for 15 min at 378 with increasing concentrations of nicotine in control cells (j) or cells treated with PTX (u) (100 ng/ml for 18 h at 378). Cells were then stimulated with FSK (3 3 1025 M) for 9 min at 378 and the percent conversion of [3H]ATP to [3H]cAMP determined (see Fig. 1).
Fig. 3. BIBP antagonizes the effect of nicotine on chromafÞn cell cAMP accumulation. Bovine chromafÞn cells were preincubated for 30 min at 378 with buffer, nicotine in buffer (1 3 1025 M) with and without BIBP (3 3 10 27 M) or authentic NPY in buffer (1 3 1029) with and without BIBP (3 3 1027 M). Cells were then stimulated with FSK (3 3 1025 M) for 5 min at 378 and the percent conversion of [3H]ATP to [3H]cAMP determined (see Fig. 1). * p ,0.05; ** p,0.01 relative to control.
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HPLC analysis of conditioned chromafÞn cell media shows presence of NPY-like material Determination of the agent responsible for inhibition of FSK-stimulated cAMP accumulation was performed by HPLC. Media was obtained from chromafÞn cells that were either incubated with nicotine (1 3 1025 M) or authentic NPY (1 3 1029 M). Incubation of cells with nicotine (1 3 1025 M) for 30 min produced a single peak detected by an NPY antibody at 26 min (Fig. 4). Incubation of authentic NPY with chromafÞn cells under the same conditions yielded a co-eluting peak. Controls were obtained under two sets of conditions. One set consisted of HPLC fractionation of media obtained from cells ncubated for 30 min but not stimulated with nicotine. The second control consisted of samples from a gradient run obtained after no sample injection using the same reversed phase column used to fractionate condition media. No measurable immunoreactive peaks were obtained from either condition.
Fig. 4. Media from chromafÞn cells stimulated with nicotine contain NPY-like material. Bovine chromafÞn cells were either stimulated with nicotine (1 3 1025 M) (j) or authentic NPY (1 3 1029 M) (h) for 30 min at 378. Controls consisted of media from unstimulated cells (d) and fractions obtained in the absence of sample injection (s). Media was removed from the cells, hydrophobic material partially puriÞed using a reversed phase cartridge (see Methods) and the residue suspended in HPLC solvent and fractionated by reversed phase chromatography. Solvent was removed under reduced pressure, the samples resuspended in buffer and assayed radioimmunochemically using a speciÞc NPY antibody.
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HPLC fractions inhibit FSK-stimulated cAMP accumulation HPLC fractions of conditioned media were examined for their ability to inhibit FSKstimulated cAMP accumulation (see previous section). Incubation of the fractions obtained from the chromatography of media obtained from nicotine-treated (1 3 1025 M for 30 min) cells with chromafÞn cells subsequently revealed one peak at 26 min capable of inhibiting 50% of FSK-stimulated cAMP accumulation (Fig. 5). This peak co-eluted with authentic NPY (see Fig. 4). In addition, several minor peaks capable of inhibiting FSK-stimulated cAMP accumulation to the extent of 20% or less of control were also observed. HPLC fractions obtained from the chromatography of authentic NPY (see Fig. 4) produced a similar pattern of inhibition with a peak also at 26 min. Examination of fractions from controls (see Fig. 4) showed a minor inhibitory peak at 28 min in media from non-stimulated cells. Discussion Bovine chromafÞn cells contain NPY Y1 receptors. Physiologically the ligand for these receptors may be provided by the splanchnic nerve in a paracrine fashion or by the chromafÞn
Fig. 5. ChromafÞn cell HPLC fractions inhibit FSK-stimulated cAMP accumulation. Bovine chromafÞn cells were incubated with aliquots of HPLC fractions (Fig. 4) followed by FSK (3 3 1025 M) stimulation for 5 min at 378. Cyclic AMP accumulation is measured as described above (Fig. 1). Nicotine (j); NPY (u); unstimulated cells (d); no sample injection (s).
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cells in an autocrine fashion. NPY is secreted from chromafÞn cells in response to nicotinic receptor stimulation or membrane depolarization with high K1 concentrations. The physiological consequences to these cells of NPY secretion is unknown although we have recently shown that the addition of low NPY concentrations to culture media inhibits catecholamine synthesis by inhibiting tyrosine hydroxylase phosphorylation and subsequently enzyme activity (4). Here we provide further support for the notion that NPY secreted from bovine chromafÞn cells is involved in the regulation of chromafÞn cell function. Our approach is to examine the effect of NPY secreted from chromafÞn cells on cAMP accumulation in these cells. NPY secretion from chromafÞn cells occurs in response to the addition of nicotinic receptor agonists to culture media (10,11). Moreover, FSK stimulation increases cAMP accumulation in these cells (8). Thus the effectiveness of secreted NPY on cAMP accumulation can be examined by Þrst stimulating chromafÞn cells with nicotine followed by the measurement of cAMP accumulation in the presence of FSK (8). The incubation of nicotine with chromafÞn cells for increasing time periods should result in the proportionate accumulation of secreted material. Accordingly stimulation of chromafÞn cells with nicotine for varying time periods results in increasing inhibition of FSK-stimulated cAMP accumulation. The degree of inhibition is similar to that produced by NPY (1 3 1029 M) in the absence of nicotine stimulation. Treatment of cells with PTX to ADP-ribosylate a G protein completely prevented the effect of nicotine. The time dependent nature of the inhibition of cAMP accumulation by nicotine and the effect of PTX demonstrate that a secreted product rather than nicotine is responsible for the inhibition of cAMP accumulation. Preliminary identiÞcation of the responsible inhibitory agent is provided by the effect of BIBP, a selective Y1 receptor antagonist (12). The addition of this agent to culture media prior to FSK stimulation prevented the inhibitory effect of nicotine on chromafÞn cell cAMP accumulation. Moreover, BIBP prevented the effect of NPY added to the culture media under similar conditions. This data demonstrates that an agent acting on the Y1 receptor is responsible for the inhibition of cAMP accumulation. Further characterization of the agent responsible for the inhibition of cAMP accumulation was provided by HPLC fractionation of conditioned media. Fractionation of media after nicotine stimulation of chromafÞn cells revealed the presence of a peak co-eluting with authentic NPY. Examination of this fraction as well as others obtained from conditioned media for their ability to inhibit FSK-stimulated cAMP accumulation showed that the peak co-eluting with NPY to be the most effective inhibitor. Taken together these data demonstrate than an NPY-like substance is secreted from bovine chromafÞn cells in response to nicotinic receptor stimulation. Moreover, this NPY-like substance is an effective inhibitor of FSK-stimulated cAMP accumulation. Thus an NPY-like substance is secreted in sufÞcient quantities to act in a autocrine fashion to alter chromafÞn cell production of cAMP. These data provide further support for the notion that NPY regulates chromafÞn cell function probably by a processes which uses cAMP such as tyrosine hydroxylase activation (13) or synthesis (14). Acknowledgments We thank Dr. Jean Rivier, The Salk Institute, San Diego, CA, for providing the NPY. This work was supported by a grant from the American Heart Association #9650472N.
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