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NEUROPHARMACOLOGY
Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska
Received April 15, 2003; accepted July 30, 2003.
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Abstract |
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-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino}hexyl)-1H-pyrrole-2,5-dione]. In conclusion, short-term exposure of bovine chromaffin cells to NPY results in a long-lasting increase in the subsequent stimulation of InsP formation by ATP.
; Zhu et al., 1992). It enhances ATP-stimulated inositol phosphate (InsP) (Zheng et al., 1997b) and cAMP (Zhang et al., 2001) formation in bovine chromaffin cells. NPY exerts its effects through the Y1 receptor; ATP exerts its effects through a purinergic receptor (P2Y2-like), which is coupled to Gq (unpublished observation). NPY also inhibits forskolin-stimulated cAMP formation through a pertussis toxin-sensitive process establishing the link between the Y1 receptor and Gi/o (Zhu et al., 1992).
Persistent stimulation of receptors coupled to Gi/o has been shown to produce enhancement rather than the expected inhibition of ligand-stimulated cAMP formation. This phenomenon was first observed during prolonged stimulation of the opioid receptor responsive to morphine (Sharma et al., 1975). Several laboratories have demonstrated similar effects through activation of Gi/o-coupled receptors, including opiate, muscarinic, and 
2-adrenergic receptors, resulting in enhanced ligand-stimulated cAMP formation involving Gs (Watts, 2002). This heterologous sensitization has also been observed when InsP formation has been measured in cells that have been pretreated with agonists acting on receptors coupled to Gi/o (Schmidt et al., 1996). We examined this phenomenon in bovine chromaffin cells using the Gi- and Gq-coupled receptor agonists NPY and ATP, respectively. Here, we provide the first report that NPY can participate in heterologous sensitization and that preincubation with NPY does not increase InsP formation unless there is the subsequent addition of a Gq-coupled P2Y receptor agonist such as ATP.
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Materials and Methods |
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; Zhu et al., 1992). The cells were plated in 35-mm plastic culture dishes at a density of 3 x 106 cells/dish in an atmosphere of 5% CO2 at 37°C. Viability and purity as determined by trypan blue exclusion and neutral red staining, respectively, were greater than 95%.
Inositol Phosphate Determination. Assays were essentially as described previously (Nakahata et al., 1986) with minor modifications. Cells plated on 35-mm dishes were labeled for 18 h with 2 µCi of [3H]inositol in 1 ml of inositol-free high-glucose DMEM. After labeling, cells were rinsed twice with DMEM, 20 mM HEPES, pH 7.4 (DMEM-HEPES) before being subjected to further treatments. DMEM-HEPES was used to wash cells; LiCl (10 mM) was added to the DMEM-HEPES when it was used as the preincubation medium or the dissolving medium for the stimulating agents. The inositol phosphates were then separated from the phospholipids using the chloroform/methanol/H2O (1:1:0.9) extraction procedure. The resulting aqueous methanol phase was added to a Dowex 1-X8 (formate form) column and the inositol phosphates eluted with 8 ml of 1 M ammonium formate containing 0.1 M formic acid. Radioactivity in a 3-ml aliquot (8-ml total volume) of the eluate (a) and a 0.375-ml aliquot (1-ml total volume) of the organic phase containing the inositol phospholipids (b) were determined by liquid scintillation counting. The percent conversion of inositol phospholipids to inositol phosphates was calculated by the formula a/(a + b) x 100.
cAMP Determination. Cyclic AMP accumulation was measured as previously described (Zhu et al., 1992). In brief, chromaffin cells were loaded with 5 µCi of [3H]adenine in DMEM containing DMEM-HEPES at 37°C for 90 min. After washing, cells were stimulated with forskolin (30 µM) containing 3-isobutyl-1-methylxanthine (200 µM) for 10 min. The [3H]cAMP was extracted and separated from other tritiated nucleotides by sequential ion exchange chromatography (Zhu et al., 1992). The radioactivity of the samples was determined by liquid scintillation spectroscopy. Values are expressed as percent conversion of [3H]ATP to [3H]cAMP.
Materials. Peptides (except NPY) were purchased from Peninsula Laboratories Inc. (Belmont, CA) or Bachem California (Torrance, CA). [3H]Myo-inositol and inositol-free DMEM were purchased from ICN Biomedicals Inc. (Irvine, CA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Data Analysis. Data were analyzed by Student's t test or two-way ANOVA and plotted using GraphPad Prism, version 3.02 (GraphPad Software Inc., San Diego, CA).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) occurs in bovine chromaffin cells.
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The onset of the response was determined by incubating cells with NPY (1 x 10-7 M) for various times up to 60 min followed by two washes and stimulation (20 min) with ATP (3 x 10-5 M) (Fig. 2). The ATP-stimulated InsP formation in these cells was significant (p < 0.05) and nearly maximal at the earliest time point examined (0.5 min) with no significant increase over the next 30 min; InsP formation at 60 min was slightly but significantly different from the 0.5-min point (p < 0.05). Cells preincubated with buffer only for 60 min and washed, followed by stimulation with ATP, produced the same amount of InsP as did cells that were not preincubated.
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The duration of the increase in InsP formation in cells preincubated with NPY was examined by incubating the cells with NPY (20 min), followed by two washes and additional incubations in buffer for 30 or 60 min (the reversal period) before ATP stimulation. The NPY enhancement of InsP formation was sustained for 60 min after NPY removal and washing of the cells. InsP formation at 60 min was still greater (
30%; p < 0.05) than InsP formation after cells were incubated for the same time but with no NPY added (Fig. 3). Next, we examined whether NPY is acting through the Y1 receptor and whether the duration of the enhancing effect could be due to another source of NPY such as through basal secretion from chromaffin cells during the reversal period. The inclusion of the Y1-selective antagonist HU-404 (Hutzler et al., 2001) (1 x 10-7 M) during the preincubation with NPY only, shows that NPY sensitization is not observed when HU-404 is included during the preincubation period (Fig. 3).
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Preincubation (20 min) of chromaffin cells with NPY (1 x 10-7 M) and then stimulation with increasing concentrations of ATP resulted in a concentration-dependent enhancement of InsP formation (EC50 = 1.20 x 10-5 M) (Fig. 4A). A similar experiment where cells were preincubated with increasing concentrations of NPY followed by washing and stimulation (20 min) with ATP (3 x 10-5 M) resulted in a concentration-dependent enhancement of InsP formation (EC50 = 2.0 x 10-8 M) (Fig. 4B). The NPY receptor subtype mediating the enhancing effect was further characterized using various NPY analogs with differing receptor subtype preferences. NPY and the Y1-preferring agonist [Leu31Pro34]NPY were equally effective at enhancing ATP-stimulated InsP formation and significantly different from ATP alone, whereas the effect of peptide YY (which is ineffective at the putative chromaffin cell Y3 receptor; Wahlestedt et al., 1992) is significant although somewhat weaker than that of NPY (Fig. 5). The enhancement of InsP formation by the Y2 receptor-preferring agonist NPY13-36 was not significantly different from ATP alone. Further characterization of the receptor as a Y1 subtype was provided by the selective antagonist HU-404, which completely prevented the NPY-sensitizing effect (Figs. 3 and 6).
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Incomplete NPY removal from cells by the washing process after preincubation could produce an apparent sensitization effect. The possibility of residual NPY binding contributing to the enhancement of ATP-stimulated InsP formation was examined in three ways. First, cells were incubated (20 min) at either a high (saturating) or a low (EC50) NPY concentration (1 x 10-7 M or 3 x 10-8 M), followed by two washes and stimulation with ATP (3 x 10-5 M) (20 min) containing either buffer or HU-404 (1 x 10-6 M). The sensitizing effect of the peptide occurred regardless of whether HU-404 was present during ATP stimulation of cells preincubated with either concentration of NPY (Fig. 6, A and B). When HU-404 was present during preincubation, the antagonist completely prevented NPY sensitization (Figs. 3 and 6).
The second approach was to wash the cells multiple times after incubating cells (20 min) with either a high (saturating) or a low (EC50) NPY concentration (1 x 10-7 or 3 x 10-8 M) followed by stimulation with ATP (3 x 10-5 M) (20 min). The sensitizing effect of the peptide was statistically significant after each of four washes regardless of the concentration of NPY used during the preincubation period (Fig. 7, A and B).
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The third approach was to determine whether a response that depends on receptor occupancy, e.g., NPY inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity is altered by preincubation with NPY. Chromaffin cells were preincubated with NPY (1 x 10-7 M) (20 min), followed by two washes and stimulation with forskolin (30 µM) (5 min). There was no statistical significance (Student's t test) between cAMP accumulation in cells preincubated with NPY and stimulated with FSK (percent conversion of [3H]ATP to [3H]cAMP 1.00 ± 0.18 S.D.) compared with cells stimulated with FSK only (percent conversion 1.16 ± 0.19 S.D.; basal percent conversion 0.035 ± 0.014 S.D.). Incubation of NPY with FSK produced a significant inhibition (p < 0.01) of cAMP accumulation (percent conversion 0.80 ± 0.17 S.D.).
NPY, acting through the Y1 receptor, inhibits forskolin-stimulated cAMP (Zhu et al., 1992) and tyrosine hydroxylase phosphorylation (Zheng et al., 1997a) in bovine chromaffin cells through a pertussis toxin-sensitive (PTX) process, demonstrating involvement of Gi/Go. Accordingly, PTX-treated cells were preincubated with NPY (20 min) followed by stimulation with ATP (Fig. 8). The enhancing effect of NPY in PTX-treated cells was only 23% of that observed in non-treated cells. By comparison, the NPY-enhancing effect was similarly reduced (80%) in PTX-treated cells not preincubated with NPY, i.e., NPY was present during ATP stimulation (not shown). Notably, a portion of the increase in InsP formation due to ATP alone, as well as the basal amount of InsP, was sensitive to PTX treatment, suggesting that a P2Y receptor coupled to Gi/Go (Ralevic and Burnstock, 1998) contributes to InsP formation.
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The selective phospholipase C inhibitor U73122
[GenBank]
(Bleasdale et al., 1990) completely inhibits the NPY enhancement of both ATP-stimulated cAMP (Zhang et al., 2001) and InsP formation (Fig. 9). Inclusion of U73122
[GenBank]
(1 x 10-5 M) along with NPY during the preincubation period also completely prevented the enhancing effect of NPY as well as the stimulation by ATP (Fig. 9). InsP formation in cells preincubated with U73122
[GenBank]
, washed twice, and stimulated (20 min) with ATP was similarly inhibited (data not shown). The inactive analog U73343
[GenBank]
(Bleasdale et al., 1990) had no effect on either the ATP-induced InsP formation or the enhancement by NPY (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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) and has been implicated in several physiological processes, including increased blood pressure (Boublik et al., 1989), stimulation of food intake (Clark et al., 1985), decreased anxiety (Kask et al., 2002), anti-seizure activity (Erickson et al., 1996), and reduced ethanol preference (Thiele et al., 1998). The mechanism(s) by which it produces these effects is unknown; what is known is that NPY acts through a variety of G protein-coupled receptor subtypes to alter the concentration of intracellular messengers (Michel et al., 1998). For example, NPY can increase (Motulsky and Michel, 1988) or decrease (Bleakman et al., 1991) intracellular Ca2+ concentrations, increase (Zhang et al., 2001) or decrease cAMP (Motulsky and Michel, 1988; Lobaugh and Blackshear, 1990; Zhu et al., 1992) levels, and increase InsP formation (Hinson et al., 1988; Zheng et al., 1997b). These changes in intracellular messengers undoubtedly influence immediate as well as long-term cellular processes such as secretion and protein synthesis, which are integral to the functioning of the above-mentioned processes.
Bovine chromaffin cells provide a well characterized model with which to investigate the signaling mechanisms through which NPY acts. This peptide, acting through its Y1 receptor, enhances both ATP-stimulated InsP and cAMP formation in chromaffin cells even though it has no ability to increase the intracellular concentration of either of these messengers on its own (Zheng et al., 1997b; Zhang et al., 2001). The mechanism of this effect is unknown but may involve the enhancement of Gq activation of phospholipase C by 
subunits released from Gi/o (Rhee, 2001). Other agents acting through Gi/o have been shown to enhance the effects of Gq-linked receptor stimulation (Selbie et al., 1997; Quitterer and Lohse, 1999). The effects of ATP and NPY are through Gq (Zhang et al., 2001) and Gi-linked (Zhu et al., 1992) receptors, respectively, supporting the feasibility of this notion.
The enhancing effect of a ligand on a Gq-linked receptor does not necessarily require the simultaneous presence of both ligands but can be demonstrated when a ligand, acting through a Gi-linked receptor, is preincubated with the cell of interest and removed before stimulation with the second ligand (Schmidt et al., 1998). This phenomenon is referred to as heterologous sensitization (Watts, 2002). Accordingly, incubation of bovine chromaffin cells with NPY (1 x 10-7 M) followed by washing the cells and stimulation with ATP (3 x 10-5 M) resulted in enhancement of InsP formation that was similar in magnitude to that seen when NPY and ATP were present simultaneously. The effect is nearly optimal at the earliest time measured (0.5 min) with no significant increase over the next 30 min. In contrast, reversal of sensitization followed a slower time course as InsP formation was still enhanced 60 min after NPY removal. Moreover, endogenously released NPY did not contribute to the NPY-sensitizing effect.
The EC50 (2.0 x 10-8 M) for NPY sensitization is similar to that reported previously for both enhancement of ATP-stimulated InsP formation (EC50 = 3.5 x 10-8 M) (unpublished observation) and cAMP formation (EC50 = 4.1 x 10-8 M) (Zhang et al., 2001). Moreover, the EC50 (EC50 = 1.20 x 10-5 M) for ATP stimulation postsensitization with NPY is similar to that for ATP when NPY is present during stimulation (EC50 = 3.9 x 10-5 M) (D. A. Drakulich and T. D. Hexum, unpublished data). The NPY-sensitizing effect is mediated by the Y1 receptor since the Y1-preferring agonist, [Leu31Pro34]NPY, is equally effective with NPY, the Y2-preferring agonist, NPY13-36, is ineffective and the selective Y1 receptor antagonist, HU-404, blocks the effect of NPY. The same receptor subtype has been shown to mediate other NPY actions on bovine chromaffin cells (Zheng et al., 1997a,b; Zhang et al., 2000, 2001).
NPY contains several hydrophobic amino acid residues and as such interacts with the lipid component of cell membranes. This partitions the residues into the correct compartment and preorients NPY for binding (Bader et al., 2001). Thus NPY has high affinity for its chromaffin cell receptor (IC50 = 0.26 x 10-9 M, [125I]NPY displacement)(Zhu et al., 1992) and binding may persist in spite of wash out procedures as used in the present studies. At least four separate pieces of information demonstrate that the sensitizing effect of NPY is not due to an action resulting from residual NPY binding. First, the enhancing effect of NPY still persisted after incubating cells with either of two different NPY concentrations, one at a saturating concentration and one equal to the EC50, followed by two washes and ATP stimulation in the presence of HU-404, a selective Y1 antagonist. The effect of any NPY remaining after the washes would be expected to be antagonized by HU-404 since the IC50 for this compound is 2.8 x 10-8 M (D. A. Drakulich and T. D. Hexum, unpublished data). Second, the NPY enhancement of InsP formation remained greater than 30% 60 min after NPY removal and washing of the cells even though the rate of [125I]NPY dissociation (k-1) is 0.071/min with a t1/2 of 9 min. The t1/2 can be considered a maximum time for dissociation since the binding studies were carried out at 4°C (Cherdchu et al., 1989) and NPY preincubation to demonstrate sensitization was performed at 37°C. Third, the sensitizing effect of the peptide was essentially undiminished after four washes regardless of the concentration of NPY (saturating or equal to the EC50) used during the preincubation period. These data strongly suggest that residual NPY binding is not responsible for NPY sensitization of ATP-stimulated InsP formation. Fourth, NPY preincubation does not inhibit FSK-stimulated cAMP accumulation.
NPY, acting through the Y1 receptor, inhibits bovine chromaffin cell forskolin-stimulated cAMP (Zhu et al., 1992), and nicotinic receptor-stimulated tyrosine hydroxylase phosphorylation (Zheng et al., 1997a) through a PTX-sensitive process. PTX treatment of chromaffin cells reduced the enhancement of ATP-stimulated InsP formation, resulting from NPY preincubation by 87%, which was similar to the reduction observed in PTX-treated cells when ATP and NPY were present concurrently. Thus, NPY sensitization of ATP-stimulated InsP formation is mediated by Gi/Go. A portion of the increase in InsP formation due to ATP alone was sensitive to PTX treatment, suggesting that a P2Y receptor coupled to Gi/Go contributes to InsP formation. P2Y receptors have been shown to be coupled to Gi/Go (Ralevic and Burnstock, 1998), and this coupling has been demonstrated in bovine chromaffin cells by previous studies that showed the PTX sensitivity of ATP modulated Ca2+ currents (Diverse-Pierluissi et al., 1991; Gandia et al., 1993).
We previously reported that the enhancing effect of NPY on InsP formation was not PTX-sensitive (Zheng et al., 1997b), results that are at apparent odds with our current data. We derived our conclusion from the percentage of changes before and after PTX treatment. However, reexamination of InsP formation (expressed as percentage of control) reveals that the NPY-enhancing effect on ATP-stimulated InsP formation is significantly reduced by PTX treatment compared with NPY enhancement of ATP-stimulated InsP formation in untreated cells (Zheng et al., 1997b). Moreover, NPY-enhanced InsP formation relative to ATP alone is less in PTX-treated cells than in untreated cells. The data presented here are unequivocal in terms of the PTX sensitivity of the enhancing effect of NPY. Thus, NPY enhancement of ATP-stimulated InsP formation is mediated by Gi/Go in either event.
The mechanism by which P2Y receptor signaling in bovine chromaffin cells is sensitized by prior stimulation of Y1 receptors is not revealed by these studies. Since phospholipase C can be activated by subunits (Rhee, 2001) and Y1 receptor activation results in an increase in the presence of subunits, one possibility is that NPY preincubation provides subunits, which enhance phospholipase C activity. However, this cannot be the sole mechanism for the effect since subunits reassociate with G upon GTP hydrolysis (t1/2 = 0.2-7 min) (Chidiac and Ross, 1999), which is inconsistent with the duration of NPY sensitization (
60 min). Studies with U73122
[GenBank]
, the phospholipase C inhibitor, did not provide any information on the mechanism of sensitization since this agent inhibited ATP-stimulated InsP formation when included in the preincubation media in the absence of NPY. Other possibilities include changes in the activity or subcellular location of Gq similar to that proposed for the mechanism of dopamine D2 receptor-induced sensitization of adenylyl cyclase (Watts et al., 2001) and/or activation of protein kinase C as proposed for the sensitization of phospholipase C signaling by muscarinic receptor-linked activation of Gi (Schmidt et al., 1998). Studies in our laboratory are underway to investigate these and other possibilities.
The response of the chromaffin cell to NPY sensitization is unknown. Since ATP has been shown to modify [Ca2+]i, resulting in alterations in catecholamine secretion (Diverse-Pierluissi et al., 1991), it can be speculated that NPY sensitization primes the cell for a continual high level of ATP-stimulated InsP formation that is not subject to NPY receptor desensitization. Thus, maximal levels of InsP formation can be maintained even in the absence of further NPY release, which might be an expected outcome of receptor cross talk. Our data further characterize the process of heterologous sensitization by demonstrating that NPY, an important and abundant neuropeptide that is costored with catecholamines, participates in this phenomenon and that NPY sensitization requires the presence of a second agent to reveal its effect on InsP formation.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: NPY, neuropeptide Y; InsP, inositol phosphate; Y1, NPY receptor subtype; P2Y, purinergic receptor subtype; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; FSK, forskolin; PTX, pertussis toxin; U73122
[GenBank]
, 1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino}hexyl)-1H-pyrrole-2,5-dione; U73343
[GenBank]
, 1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]-amino}hexyl,-2,5-pyrrolidinedione; HU-404, (R)-N-diphenylacetyl-N G-{[2-(ethoxycarbonyl)methyl]aminocarbonyl}-N-[(4-hydroxyphenyl)-methyl]argininamide hydrobromide.
Address correspondence to: Dr. Terry D. Hexum, Department of Pharmacology, University of Nebraska Medical Center, 986260 Nebraska Medical Center, Omaha, NE 68198-6260. E-mail: thexum{at}unmc.edu
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, NPY preincubation; 
, no added NPY. *, p < 0.05 (relative to no NPY); +, p < 0.05 (relative to ATP + NPY at 0.5 min).
, NPY; 
, NPY + HU-404; 
, no NPY;
, HU-404, no NPY. *, p < 0.05 (relative to NPY + HU-404).
