AJP - Heart Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 273: H1737-H1744, 1997;
0363-6135/97 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, X.
Right arrow Articles by Westfall, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, X.
Right arrow Articles by Westfall, T. C.
Vol. 273, Issue 4, H1737-H1744, October 1997

Autoreceptor-induced inhibition of neuropeptide Y release from PC-12 cells is mediated by Y2 receptors

Xiaoli Chen, Debora A. Dimaggio, Song Ping Han, and Thomas C. Westfall

Department of Pharmacological and Physiological Science, Saint Louis University Health Sciences Center, St. Louis, Missouri 63104

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Pheochromocytoma (PC)-12 cells express Y1, Y2, and Y3 neuropeptide Y (NPY) receptors when differentiated with nerve growth factor (NGF). The present work evaluated NGF-differentiated PC-12 cells as a model system to study modulation of NPY release by NPY autoreceptors. We demonstrated that both K+ and nicotine stimulated concomitant release of NPY and dopamine from differentiated PC-12 cells. We also showed in this study that NPY release from PC-12 cells was attenuated in a concentration-dependent manner by peptide YY (PYY)-(13---36), a selective agonist for the Y2 type of NPY receptors. This result demonstrated that NPY release could be modulated by NPY autoreceptors of the Y2 subtype. The inhibitory action of PYY-(13---36) may be mediated at least in part by inhibition of N-type Ca2+ channels, because PYY-(13---36) could not produce further inhibitory effects in the presence of a maximum effective concentration of omega -conotoxin, an N-type Ca2+-channel blocker. The inhibition by PYY-(13---36) could be blocked by pretreatment of cells with pertussis toxin, suggesting that an inhibitory GTP-binding protein was involved. Furthermore, the function of NPY autoreceptors could be modulated by other receptors such as beta -adrenergic and ATP receptors. The evoked release of NPY was also attenuated by ATP and adenosine, which have been shown to be colocalized and coreleased with NPY from sympathetic nerve terminals. These results suggest that PC-12 cells differentiated with NGF may be an ideal model to study regulatory mechanisms of NPY release and that autoreceptor-mediated regulation of NPY release appears to act through the Y2 subtype of the NPY receptor.

catecholamines; dopamine; adenosine 5'-triphosphate; adenosine

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NEUROPEPTIDE Y (NPY) is a tyrosine-rich 36-amino acid peptide initially isolated from porcine brain by Tatemoto et al. (35). NPY belongs to a pancreatic polypeptide family of peptides that includes peptide YY (PYY), to which it has ~70% homology, and pancreatic polypeptide (PPY), to which it has ~50% homology. NPY has a wide distribution in the central and peripheral nervous systems. It is colocalized with catecholamines in certain populations of central adrenergic and noradrenergic neurons as well as in the peripheral sympathetic nerves and the adrenal medulla (9). NPY is also coreleased with norepinephrine from perivascular sympathetic nerves (21).

Although initially NPY receptors were classified according to differences in binding and rank order of the pharmacological potency of NPY fragments and analogs (41), at present, at least five distinct NPY receptors have been established by molecular cloning approaches. These include Y1 (12, 19, 20, 29), Y2 (11, 32), Y4 (12, 22), Y5 (12), and Y6 (13, 37). Another potential subtype, the Y3 receptor, has not yet been cloned but has been suggested based on functional and biochemical studies (24, 25, 27). Of these receptor subtypes, the pharmacological profiles for Y1, Y2, and Y3 are the best established, whereas those for types Y4, Y5, and Y6 need further investigation.

At the sympathetic neuroeffector junction, the Y1 subtype of NPY receptors is primarily located on the postsynaptic membrane of vascular smooth muscle cells. Activation of the Y1 receptor with NPY or selective Y1 agonist [Leu31,Pro34]NPY produces potentiation of the contraction of a variety of vasoactive agents in vitro (29, 45) and marked increases in systemic blood pressure in vivo (17). The excitatory action of Y1-receptor activation is usually accompanied by an increase in the intracellular calcium concentration in vascular muscle and other cells (33, 34). Y2 receptors are primarily located on the presynaptic membrane of sympathetic nerve terminals and have been shown to have inhibitory actions on norepinephrine release. Y2 receptors are also located on other neurons such as the dorsal root ganglion. In this preparation, Y2 agonists attenuate the evoked-release substance P and calcium influx (36). A pertussis toxin (PTX)-sensitive GTP-binding protein appears to couple the Y2 receptor to the intracellular events in this preparation. The action of NPY on neurotransmitter or hormone release, however, appears to vary with the tissue preparations used. For example, NPY potentiates the stimulatory effect of luteinizing hormone releasing hormone on luteinizing hormone release from rat anterior pituitary cells (4), whereas the Y2 agonist decreases glutaminergic transmission in rat hippocampal neurons (3). Catecholamine release is enhanced by NPY in the perfused bovine adrenal gland (17), whereas it is attenuated in chromaffin cells (18) and in pheochromocytoma (PC)-12 cells (6, 7). These differences may be due to the differential distribution of Y1, Y2, or Y3 receptors as well as by the heterogeneity of the cell population in these tissues. These variations in action may be reduced if a cell line with controlled Y1, Y2, or Y3 receptor expression is used in release studies.

The regulation of NPY release by various neurotransmitter receptors has been previously studied in several laboratories. Unfortunately, most of these studies have been carried out in tissue preparations containing more than one cell type. Therefore, it is sometimes difficult to demonstrate the direct action of the neurotransmitter on NPY-containing neurons. There are advantages to having a homogeneous cell population that both releases NPY and has receptors for the modulatory neurotransmitters being evaluated. There are also advantages to using a homogeneous cell population when biochemical techniques are used to study the intracellular events following receptor activation.

Pernow and Lundberg (28) have reported that NPY release from pig kidney was attenuated by PYY, suggesting a mechanism for autoregulation of NPY release. It is not clear which subtype of NPY receptor mediates this regulatory action, because PYY is a nonselective agonist for both Y1 and Y2 receptors. The intracellular mechanisms for the inhibitory action of PYY have also not been studied.

Our previous work (6, 7) has established PC-12 cells as a model system in which to study the regulation of catecholamine release. The phenotype of PC-12 cells is altered by treatment with nerve growth factor (NGF) or glucocorticoid. Whereas the undifferentiated PC-12 cell does not have functional NPY receptors, treatment with either dexamethasone or NGF results in expression of such receptors. Dexamethasone induces functional Y1-receptor expression, whereas Y1-, Y2-, and Y3-receptor expression can be induced by NGF. The purpose of the present study was threefold: 1) to establish PC-12 cells as a model system in which to study NPY release, 2) to determine whether NPY release is subject to inhibitory autoreceptor inhibition, and 3) to examine the effects after activation of other transmitters during NPY release and the interaction between these receptors and NPY autoreceptors during NPY release.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Cultures

PC-12 cells with a low passage number (<30) were a generous gift from Dr. Steven Sabol (National Institutes of Health, Bethesda, MD). PC-12 cells were cultured in cell culture inserts (25 mm, 0.45-µm pore size, Falcon Plastics, Oxnard, CA), housed in six-well plates (Costar, Cambridge, MA), and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum and 10% heat-inactivated horse serum and incubated in a humidified atmosphere containing 5% CO2 in air at 37°C for 5-7 days. PC-12 cells were also differentiated with NGF (50 ng/ml). Medium changes and NGF additions were repeated every other day.

NPY-Immunoreactivity Release Study

PC-12 cells were grown on cell culture inserts with NGF for 5-7 days. NPY (and also dopamine)-release experiments were performed according to the method described by DiMaggio et al. (6, 7). The inserts were placed into wells containing low (2.5 mM)- or high (50 mM)-K+ Krebs bicarbonate buffer with appropriate drugs. Preliminary experiments were carried out to examine the time course of NPY immunoreactivity (ir; NPYir) release in the presence of a high-K+ buffer. Samples were collected at 3, 5, 10, 15, and 20 min of exposure to the high-K+ (50 mM) buffer and measured for NPYir. It was observed that the peak release of NPYir occurred after incubation with a high-K+ buffer for 15 min. A 20-min incubation showed no further increase. Therefore, we decided to carry out all additional experiments with a 15-min incubation period. After a 15-min incubation period, the inserts were then removed from the wells. Aliquots of the release medium were assayed immediately for NPYir. Cells were still intact at the end of the incubation period, and cells on the inserts were extracted with acid for determining total NPYir.

Quantitation of NPYir

NPYir was determined directly in acid extracts of tissue and release medium by radioimmunoassay using a specific antiserum that was raised in rabbits against porcine NPY, as described by DiMaggio and co-workers (6, 7). Radioimmunoassay was performed using a 5-day disequilibrium method. Duplicated samples were incubated with the NPY antiserum. Twenty-four hours later, 125I-NPY was added to each tube. After a 72-h incubation period (at 4°C), antibody-bound 125I-NPY was separated from free 125I-NPY by centrifugation after addition of a second antibody (goat anti-rabbit serum) and polyethylene glycol, and radioactivity was measured in a gamma -counter. The antiserum was examined for cross-reactivity with homologous peptides and peptide fragments by incubating antisera with several dilutions of unlabeled NPY-(1---36) or the appropriate peptide. It was observed that the NPY antisera did not recognize heterologous and homologous peptide sequences including rat beta -endorphin, PYY, PPY and the COOH-terminal hexapeptide of human PPY, as well as a variety of COOH-terminal fragments including NPY-(14---36), NPY-(18---36), PYY-(13---36), and peptide YX2 (PYX2). The NPYir in acid extracts of tissue and release medium was characterized and compared with authentic rat and porcine NPY-(1---36) by passage over a high-performance liquid chromatography (HPLC) column followed by assay. The elution profiles of NPYir were compared with profiles of authentic NPY eluted under similar conditions. It was observed that NPYir material eluted as a single peak and coeluted with synthetic human NPY (6, 7). The antisera recognized mature NPY-(1---36).

Quantitation of Dopamine

When the inserts were removed from the wells containing buffer and agents, perchloric acid (0.4 N) containing 0.1% cysteine was added to the aliquots of the release medium. The cells on the inserts were extracted with acid for determining total dopamine content. Dopamine was determined by HPLC column and quantified by electrochemical detection (23). The HPLC system consists of a Varian (model 2510) solvent delivery system and an autosampler (model 9090; Varian, Walnut Creek, CA) coupled to a C18 column and an ESA Coulochem II detector. Separations were performed isocratically using a filtered and degassed mobile phase consisting of 10% methanol, 0.1 M sodium phosphate, 0.2 mM sodium octyl sulfate, and 0.1 mM EDTA, adjusted to pH 2.8 with phosphoric acid. The HPLC system was coupled to a 386 SX computer with which chromatograms were recorded and analyzed with Varian Star workstation software.

Statistics

Data were expressed as means ± SE of the percentage of fractional release, which was the amount of NPYir released in the buffer divided by the amount of NPYir present in the tissue before depolarization stimulation times 100. Statistical analyses were carried out using two-way analysis of variance followed by Newman-Keuls multiple-range test.

Materials

Porcine NPY, PYY-(13---36), and PYX2 were purchased from Peninsula Laboratories (Belmont, CA). Benextramine, isoproterenol, clonidine, ATP, alpha ,beta -methylene ATP, nifedipine, and omega -conotoxin were obtained from Sigma Chemical (St. Louis, MO). 125I-NPY was purchased from Amersham (Arlington Heights, IL). Goat anti-rabbit serum was obtained from Linco Research (St. Louis, MO). PEG-8000 was obtained from Fisher Scientific (St. Louis, MO). NGF was purchased from Collaborator Biomedical Products (Bedford, MA). DMEM, fetal bovine serum, and horse serum were purchased from JRH Biosciences (Lenexa, KS).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Differentiated PC-12 Cell as a Sympathetic Neuronal Model to Study NPY Release

Release of NPYir and dopamine from NGF-differentiated PC-12 cells. PC-12 cells in inserts were incubated in the buffer containing stimulants for 15 min. Aliquots of the same sample were then measured simultaneously for NPYir and dopamine by radioimmunoassay and HPLC-electrochemical detection, respectively (see METHODS). The basal release of NPYir and dopamine from NGF-treated PC-12 cells was 0.04 ± 0.003 and 2.04 ± 0.06 ng/well, respectively. The percent basal fractional release of NPYir and dopamine was 7.03 ± 0.09 and 2.08 ± 0.08%, respectively. KCl (50 mM) or nicotine (100 uM) produced significant increases in release of NPYir and dopamine from NGF-treated PC-12 cells (Fig. 1).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1.   Corelease of neuropeptide Y (NPY) and dopamine (DA) from nerve growth factor (NGF)-treated PC-12 cells. NPY and DA were concomitantly released from pheochromocytoma (PC)-12 cells during 15-min KCl (50 mM) depolarization and nicotine (Nic, 100 µM) stimulation. Results are expressed as percent basal release (basal release = 100). All data are expressed as means ± SE (n = 5-6).

Concentration-dependent effect of PYY-(13---36) on K+-evoked NPYir release from NGF-treated PC-12 cells. K+ (50 mM) increased NPYir release about one- to twofold over that of basal. The selective Y2 agonist PYY-(13---36) (10-8-10-6 M) inhibited K+-evoked NPYir release in a concentration-dependent manner (Fig. 2). The maximum inhibitory effect was achieved by 10-7 M PYY-(13---36) (35% inhibition).


View larger version (51K):
[in this window]
[in a new window]
  		Fig. 2.   Concentration-response curve for peptide YY (PYY)-(13---36) on K+-evoked NPY release. Basal NPY release was 37% of KCl (50 mM)-induced release. KCl-induced NPY release was expressed as 100%. An NPY Y2-receptor agonist PYY-(13---36) (10-10-10-7 M) inhibited K+-evoked NPY release in a concentration-dependent manner, suggesting presence of NPY autoreceptors. All data are expressed as means ± SE (n = 5-6). Significant difference as indicated: * P < 0.05.

Mechanisms Underlying NPY Autoreceptor-Mediated Inhibition During NPYir Release

Antagonism of PYY-(13---36)-induced inhibition of NPYir release. PYX2 is a synthetic decapeptide amide and NPY analog that has been shown to specifically inhibit the binding of 3H-labeled NPY to its receptors (35) and to inhibit the NPY-induced increases in intracellular calcium in human erythroleukemia cells. In the present study, as shown in Fig. 3, the inhibition of K+-evoked NPYir release induced by PYY-(13---36) (200 nM) was abolished in the presence of PYX2 (200 nM), suggesting that the effect of PYY-(13---36) is mediated by NPY receptors.


View larger version (56K):
[in this window]
[in a new window]
  		Fig. 3.   Blockade of inhibitory effect of PYY-(13---36) (PYY13) on NPY release by peptide YX2 (PYX2). Inhibition by PYY-(13---36) (200 nM) in K+ (50 mM)-evoked NPY release was antagonized in presence of an NPY receptor antagonist, PYX2 (200 nM). Results are expressed as %fractional release (%FR). All data are expressed as means ± SE (n = 5-6). Significant difference as indicated: * P < 0.05, ** P < 0.01.

Benextramine, an alpha -adrenoceptor antagonist, has been shown to inhibit 3H-labeled NPY specific binding in rat brain membranes and irreversibly binds to what appears to be the Y2 receptor in the bovine hippocampus (8). Benextramine (1 µM) also attenuated the PYY-(13---36)-induced inhibition of the K+-evoked NPYir release in the present study (Fig. 4).


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 4.   Blockade of inhibitory effect of PYY-(13---36) on NPY release by benextramine. Benextramine (1 µM) blocked PYY-(13---36) (200 nM) inhibition of K+ (50 mM)-evoked NPY release. Results are expressed as %FR. All data are expressed as means ± SE (n = 5-6). Significant difference as indicated: * P < 0.05, ** P < 0.01.

Effect of PTX on PYY-(13---36)-induced inhibition of NPYir release. Pretreatment of PC-12 cells with PTX (50 ng/ml) for 18 h did not significantly change basal or K+-evoked NPYir release. However, the PYY-(13---36)-induced inhibition of K+-evoked NPYir release was abolished by PTX pretreatment (Fig. 5).


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of pertussis toxin (PTX) on inhibitory effect of PYY-(13---36). Pretreatment of PC-12 cells with PTX (50 ng/ml, hatched bars) for 18 h did not significantly change basal and K+ (50 mM)-evoked NPY release. However, PYY-(13---36) (200 nM) inhibition of K+-evoked NPY release was abolished by PTX pretreatment. Results are expressed as %FR. All data are expressed as means ± SE (n = 5-6). Open bars, non-PTX-treated PC-12 cells. Significant difference as indicated: * P < 0.05.

Effect of omega -conotoxin on NPYir release and PYY-(13---36)-induced inhibition of NPYir release. We examined the effect of omega -conotoxin on the K+-evoked release of NPYir. The fractional basal release of NPYir was 25 ± 3% and in the presence of omega -conotoxin was 15 ± 2, 12 ± 2, and 13 ± 2% at 10-8, 10-7, and 10-6 M, respectively. All three concentrations produced a significant inhibition of NPYir release with a P value P <0.055. A combination of PYY-(13---36) and omega -conotoxin (at maximum effective concentration, 10-7 M) did not produce an additional inhibitory effect on NPYir release (Fig. 6).


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 6.   Effect of omega -conotoxin (CgTx) on inhibitory effect of PYY-(13---36). Attenuation of NPY release produced by CgTx (100 nM) and PYY-(13---36) (100 nM) was not additive. Solid line represents basal NPY release. Results are expressed as %FR. All data are expressed as means ± SE (n = 5-6). +, Presence of; -, absence of. Significant difference as indicated: * P < 0.05.

Action of Cotransmitters on NPYir Release and Interactions Between Regulatory Transmitter Receptors and NPY Autoreceptors

Effect of adenosine and ATP during NPY release. It has been shown that the adenosine analog cyclohexyladenosine reduced the stimulated overflow of both norepinephrine and NPY from perfused guinea pig heart (14). Moreover, it is well established that both ATP and adenosine can inhibit norepinephrine release from a variety of preparations (38). The effects of adenosine and ATP on NPYir release were therefore next examined. As shown in Fig. 7, adenosine (1 µM) or ATP (3 mM) significantly inhibited the K+-evoked NPYir release. The ATP-induced inhibition of the evoked NPY release was antagonized by alpha ,beta -methylene ATP, an ATP receptor antagonist (100 nM).


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of adenosine (Adenos) and ATP on evoked release of NPY. Adenos (1 µM) or ATP (3 mM) significantly inhibited K+-evoked NPY release. Effect of ATP was antagonized by alpha ,beta -methylene ATP (alpha beta mATP, 100 nM), an ATP receptor antagonist. Results are expressed as %FR. All data are expressed as means ± SE. Significant difference as indicated: * P < 0.05.

Effect of ATP on PYY-(13---36)-induced inhibition of NPYir release: antagonism by alpha ,beta -methylene ATP. ATP (3 mM) was also shown to block the inhibitory effect of PYY-(13---36) on the K+-evoked NPY release (Fig. 8). This effect of ATP was antagonized by alpha ,beta -methylene ATP (100 nM).


View larger version (51K):
[in this window]
[in a new window]
  		Fig. 8.   Effect of ATP on inhibitory effect of PYY (13---36) ATP (3 mM) blocked inhibitory effect of PYY (13---36) (200 nM) on K+ (50 mM)-evoked NPY release. This effect of ATP was antagonized by alpha beta mATP (100 nM). Results are expressed as %FR. All data are expressed as means ± SE (n = 5-6). Significant difference as indicated: * P < 0.05, ** P < 0.01.

Effect of isoproterenol on NPY release and PYY-(13---36)-induced inhibition of NPYir release. Isoproterenol, a beta -adrenergic receptor agonist, has been shown to enhance nerve stimulation-evoked NPYir and norepinephrine release from the pithed guinea pig (5). In the present study, isoproterenol (200 nM) was shown to block the inhibitory effect of PYY-(13---36) on the K+-evoked NPYir release, although it did not significantly change the K+-evoked NPYir release (Fig. 9).


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 9.   Effect of isoproterenol (Iso) on inhibitory effect of PYY (13---36). Iso (200 nM), a beta -adrenoceptor agonist, blocked inhibitory effect of PYY (13---36) (200 nM) on K+-evoked NPY release, although it did not significantly change K+-evoked NPY release. Results are expressed as %FR. All data are expressed as means ± SE. Significant difference as indicated: * P < 0.05, ** P < 0.01.

Effect of clonidine on the PYY 13-36-induced inhibition of NPY release. Clonidine, an alpha 2-adrenergic receptor agonist (10 µM), failed to alter the K+-evoked release of NPYir (Fig. 10). Higher concentrations also failed to inhibit K+-evoked NPYir release (data not shown). In addition, clonidine did not significantly change the inhibitory effect of PYY-(13---36) on the K+-evoked NPYir release.


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 10.   Effect of clonidine (Clon) on inhibitory effect of PYY 13-36 on NPY release. Clon (10 µM), an alpha -adrenoceptor agonist, did not significantly change inhibitory effect of PYY (13---36) on K+-evoked NPY release. Results are expressed as %FR. All data are expressed as means ± SE. Significant difference as indicated: * P < 0.05.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

PC-12 cells have an immature adrenal chromaffin cell phenotype, and they synthesize and store both catecholamines and NPY (6, 7). Undifferentiated PC-12 cells express only the Y1 subtype of NPY receptors (2, 6, 7). The phenotype of PC-12 cells can be altered to resemble sympathetic neurons by differentiation with NGF. Previous studies from our laboratory (2, 6, 7) have shown that differentiated PC-12 cells express both functional Y1 and Y2 subtypes of NPY receptors. Recent studies also suggest the presence of Y3 receptors in NGF-differentiated PC-12 cells (24, 25). Activation of both Y1 and Y2 receptors decreases evoked adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. In addition, there is evidence that activation of both Y1 and Y2 receptors may also inhibit calcium channels, resulting in a decrease in intracellular calcium levels in various preparations (2, 26). We previously observed (2) that activation of Y2 but not Y1 receptors in NGF-differentiated PC-12 cells resulted in an inhibition of calcium entry and simultaneous decrease in dopamine release. Because NPY is also synthesized and stored in PC-12 cells, the present study was designed to further examine PC-12 cells differentiated with NGF as a sympathetic neuronal model to study NPYir release and its regulation by NPY receptors and by other neurotransmitter receptors.

As a cell membrane depolarizing agent, 50 mM K+ induced a significant increase in NPYir release that was also accompanied by a significant increase in dopamine release. This observation, together with our previous fura 2 study in differentiated PC-12 cells (2), suggests that an immediate increase in intracellular calcium transient appears to be responsible for the stimulatory effect of K+ on NPYir and dopamine release. We also obtained results showing that nicotine increased the release of both NPYir and dopamine. Numerous studies have demonstrated that nicotine stimulates neurotransmitter release by activating nicotinic cholinergic receptors, resulting in an increase in calcium influx through N-type calcium channels, activation of protein kinase C, and an increase in cAMP production (15).

Activation of Y2 receptors by the Y2-type selective agonist NPY-(13---36) has been shown to attenuate the release of neurotransmitters, such as catecholamine and substance P (2, 6, 7, 36). NPY receptor activation also attenuated the release of NPY from sympathetic nerves in the pig kidney (28). Results obtained in the present study, in which we observed that the Y2-selective ligand PYY-(13---36) inhibited the evoked release of NPYir, are consistent with these observations. Thus the induced release of NPYir as well as the presence of functional Y2 receptors in the differentiated PC-12 cells provided an opportunity to study the modulation of NPYir release in a homogeneous cell population.

The inhibitory action of PYY-(13---36) on the K+-evoked NPYir release was completely abolished by PYX2. This decapeptide amide is an NPY analog that has been reported to displace [3H]NPY binding from its receptors (35). The antagonism of the inhibitory effect of PYY-(13---36) on NPYir release by PYX2 suggests therefore that the inhibition of PYY-(13---36) on NPYir release is a receptor-mediated process.

The NPY autoreceptor present in PC-12 cells differentiated with NGF seems to be of the Y2 subtype based on the following observations: 1) the Y2-selective ligand PYY-(13---36) attenuated the K+-induced NPYir release; 2) NPY-(13---36), another selective Y2-receptor agonist, attenuated the K+-induced calcium influx in NGF-differentiated PC-12 cells (2); and 3) NPY-(13---36) did not affect the K+-induced calcium influx in dexamethasone-differentiated PC-12 cells (2), which only express the Y1 subtype of NPY receptors (6, 7).

The inhibitory Y2 receptor seems to be coupled to intracellular events through an inhibitory GTP-binding protein of the Gi or Go type, because the inhibitory effect on NPYir release was prevented by pretreatment with PTX. A similar PTX sensitivity was seen for the Y2-mediated inhibition of the nicotine-induced release of dopamine (2).

Numerous studies have demonstrated calcium-dependent release of NPY in a variety of preparations. Our previous studies measured intracellular calcium transients using fura 2 spectrofluorometry as well as dopamine release from differentiated PC-12 cells. These studies showed that Y2-receptor agonists inhibited K+-induced increases in intracellular calcium concentration by reducing the calcium influx with simultaneous inhibition of the evoked catecholamine release (2). NPY Y2 autoreceptor-induced inhibition of NPYir release seen in the present study may also be mediated by the inhibition during calcium influx. Therefore, the effects of specific calcium channel blockers on NPYir release and PYY (13---36)-induced inhibition of NPYir release was examined. omega -Conotoxin, an N-type of calcium channel blocker, was shown to mimic the inhibitory effect of PYY-(13---36) on NPYir release. Moreover, in the presence of the maximum effective concentration of omega -conotoxin, PYY-(13---36) did not produce further inhibition on NPYir release. The lack of synergism or additivity between PYY-(13---36) and omega -conotoxin in inhibiting NPYir release suggests that the PYY-(13---36)-attenuated NPYir release may be mediated through an inhibition of N-type calcium channels, although inhibition of other types of Ca2+ channels may also occur.

Norepinephrine, epinephrine, ATP, and NPY are colocalized in the adrenal gland. In the present study, we observed that NPYir release from PC-12 cells can be modulated by ATP as well as adenosine. The adenosine-induced inhibition of the evoked release of NPYir is consistent with the observation in the guinea pig heart that the adenosine analog cyclohexyladenosine significantly reduced the stimulated overflow of NPY (14). Adenosine and ATP are well known to reduce the electrical field stimulation-induced overflow of norepinephrine in rat vas deferens (16) and in blood vessels (38). In isolated myocytes obtained from the right ventricles of ferrets, ATP decreased L-type calcium currents in a concentration-dependent manner (30). However, ATP has not previously been observed to inhibit NPYir release. Thus this appears to be the first demonstration that ATP and adenosine can modulate NPYir release. This is of particular interest because of the fact that ATP is colocalized and coreleased with NPY and catecholamines from sympathetic neurons and the adrenal medulla.

Isoproterenol has been shown to enhance nerve stimulation-evoked NPYir and norepinephrine release from the pithed guinea pig (5). In the present study, isoproterenol did not produce a significant effect on the K+-evoked release of NPYir. However, the inhibition by PYY-(13---36) on NPYir release was attenuated by isoproterenol. The blockade induced by isoproterenol therefore suggests an interaction between Y2 and beta -adrenergic receptors. The interaction may occur at a receptor level or intracellular second messenger level. The existence of a catecholamine-NPY receptor interaction has previously been proposed in that NPY reduced the density of alpha 2-adrenergic binding sites and clonidine decreased the binding of iodinated NPY in the medulla oblongata (16). This kind of reciprocal modulation may occur between NPY receptors and other types of receptors. As discussed above, ATP was shown to attenuate NPYir release from PC-12 cells; however, ATP did not enhance the inhibitory effect of PYY-(13---36) on NPYir release. In contrast, ATP blocked the inhibitory effect of PYY-(13---36), and this effect was reversed by desensitization of the ATP receptor with alpha ,beta -methylene ATP. These results suggest that there might be an interaction between the Y2 receptor and the ATP receptor. The possible interaction between autoreceptors and other transmitter receptors may represent a new mechanism regulating transmitter release. Clonidine has been shown to inhibit the release of NPY from sympathetic nerve terminals, whereas alpha 2-antagonists enhance the evoked release (14). However, in this study, we did not observe any effect of clonidine on NPYir release. One possible explanation is suggested by the observation that adrenal chromaffin cells express imidazoline but not alpha 2-adrenergic receptors (31).

In conclusion, this study has demonstrated, using NGF-differentiated PC-12 cells as a model, that NPYir release and its regulation mechanisms can be studied. NPYir release can be downregulated by its autoreceptors. The autoregulation of NPYir release may be mediated by Y2 receptors, N-type Ca2+ channels, and a PTX-sensitive GTP-binding protein. NPYir release can also be modulated by purine receptors and beta -adrenergic receptors. The interaction may occur at the level of the receptor as well as at the level of intracellular signaling.

    ACKNOWLEDGEMENTS

This research was supported by National Institutes of Health Grants HL-26319, HL-35202, and T32-NS07254.

    FOOTNOTES

Address for reprint requests: T. C. Westfall, Dept. of Pharmacological and Physiological Science, Saint Louis Univ. School of Medicine, Health Sciences Center, 1402 S. Grand Blvd., St. Louis, MO 63104.

Received 3 December 1996; accepted in final form 5 June 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bleakman, D., W. F. Colmers, A. Fournier, and R. J. Miller. Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurons of the rat via a Y2 receptor. Br. J. Pharmacol. 103: 1781-1789, 1991[Medline].

2.   Chen, X., and T. C. Westfall. Modulation of intracellular calcium transients and dopamine release by neuropeptide Y in PC-12 cells. Am. J. Physiol. 266 (Cell Physiol. 35): C784-C793, 1994[Abstract/Free Full Text].

3.   Colmers, W. F., G. J. Klapstein, A. Fournier, S. St-Pierre, and K. A. Treherne. Presynaptic inhibition by neuropeptide Y in rat hippocampal slice in vitro is mediated by a Y2 receptor. Br. J. Pharmacol. 102: 41-44, 1991[Medline].

4.   Crowley, W. R., G. V. Shah, B. L. Carroll, D. Kennedy, M. E. Dockter, and S. P. Kalra. Neuropeptide Y enhances luteinizing hormone-releasing hormone-induced LH release and elevation in cytosolic Ca2+ in rat anterior pituitary cells: evidence for involvement of extracellular Ca2+ influx through voltage-sensitive channels. Endocrinology 127: 1487-1494, 1990[Abstract].

5.   Dahlöf, P., V. H. Tarizzo, J. M. Lundberg, and C. Dahlöf. alpha - and beta -Receptor mediated effects on nerve stimulation evoked release of neuropeptide Y-like immunoreactivity in the pithed guinea pig. J. Auton. Nerv. Syst. 35: 199-210, 1991[Medline].

6.  DiMaggio, D. A. Neuropeptide Y Function in Sympathoadrenomedullary Models (PhD thesis). St. Louis, MO: St. Louis University Press, 1992.

7.   DiMaggio, D. A., J. M. Farah, Jr., and T. C. Westfall. Effects of differentiation on neuropeptide Y receptors and responses in rat pheochromocytoma cells. Endocrinology 134: 719-727, 1994[Abstract].

8.   Doughty, M. B., S. S. Chu, D. W. Miller, L. Ke, and R. E. Tessel. Benextramine: a long-lasting neuropeptide Y receptor antagonist. Eur. J. Pharmacol. 185: 113-114, 1990[Medline].

9.   Everitt, B. J., Y. Hökfelt, L. Terenius, V. Tatemoto, V. Mutt, and M. Goldstein. Differential coexistence of neuropeptide Y-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11: 443-462, 1984[Medline].

10.   Fuhlendorff, J., U. Gether, and L. Aakerlund. [Leu31, Pro34]neuropeptide Y: a specific Y1 agonist. Proc. Natl. Acad. Sci. USA 87: 182-186, 1990[Abstract/Free Full Text].

11.   Gehlert, D. R., L. S. Beavers, O. Johnson, S. Gackenheimer, D. A. Schober, and R. A. Gadski. Expression cloning of a human brain neuropeptide Y2 receptor. Mol. Pharmacol. 49: 224-228, 1996[Abstract].

12.   Gerald, C., M. W. Walker, L. Criscione, E. L. Gustofson, C. Batzl-Hartmann, K. E. Smith, O. Vaysse, M. M. Durkin, T. M. Loz, D. L. Linemeyer, A. O. Schoffhauser, S. Whitebread, K. G. Hofbauer, R. I. Tober, T. A. Branchek, and R. L. Weinshank. A receptor subtype involved in neuropeptide Y-induced food intake. Nature 382: 168-171, 1996[Medline].

13.   Gregor, P., Y. Feng, L. B. DeCarr, L. J. Cornfield, and M. L. McCalebi. Molecular characterization of a second mouse pancreatic polypeptide receptor and its inactivated human homologue. J. Biol. Chem. 271: 27776-27781, 1996[Abstract/Free Full Text].

14.   Haass, M., B. Cheng, G. Richardt, R. E. Lang, and A. Schomig. Characterization and presynaptic modulation of stimulation-evoked exocytotic co-release of noradrenaline and neuropeptide Y in guinea pig heart. Naunyn Schmiedebergs Arch. Pharmacol. 339: 71-78, 1989[Medline].

15.   Haass, M., G. Richard, T. Brenn, E. Schomig, and A. Schomig. Nicotine-induced release of noradrenaline and neuropeptide Y in guinea-pig heart: role of calcium channels and protein kinase C. Naunyn Schmiedebergs Arch. Pharmacol. 344: 527-531, 1991[Medline].

16.   Härfstrand, A., K. Fuxe, L. Agnati, and B. Fredholm. Reciprocal interactions between alpha 2-adrenoceptor agonist and neuropeptide Y binding sites in the nucleus tractus solitarius of the rat. J. Neural Transm. 75: 83-99, 1989.

17.   Hexum, T. D., and L. R. Russett. Stimulation of cholinergic receptor mediated secretion from the bovine adrenal medulla by neuropeptide Y. Neuropeptides 13: 35-41, 1989[Medline].

18.   Higuchi, H., E. Costa, and H.-Y. Y. Yang. Neuropeptide Y inhibits the nicotine-mediated release of catecholamines from bovine adrenal chromaffin cells. J. Pharmacol. Exp. Ther. 244: 468-474, 1988[Abstract/Free Full Text].

19.   Krause, J., C. Eva, P. H. Seeburg, and R. Sprengel. Neuropeptide Y1 subtype pharmacology of a recombinantly expressed neuropeptide receptor. Mol. Pharmacol. 41: 817-821, 1992[Abstract].

20.   Larhammar, L., A. G. Bloomqvist, F. Yee, E. Jazin, H. Yoo, and C. Wahlestedt. Cloning and functional expression of human neuropeptide Y/peptide YY receptor of the Y1 type. J. Biol. Chem. 267: 10935-10938, 1992[Abstract/Free Full Text].

21.   Lundberg, J. M., A. Rudehill, A. Sollevi, G. Fried, and G. Wallin. Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation, and resupply by axonal transport. Neuroscience 28: 475-486, 1989[Medline].

22.   Lundell, I., A. G. Bloomqvist, M. M. Berglung, D. A. Schober, D. Johnson, M. A. Statnick, R. A. Gadski, D. R. Gehlert, and D. Larhammar. Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY. J. Biol. Chem. 270: 29123-29128, 1995[Abstract/Free Full Text].

23.   McAuley, M. A., and T. C. Westfall. Possible location and function of neuropeptide Y receptor subtypes in the rat mesenteric arterial bed. J. Pharmacol. Exp. Ther. 261: 863-868, 1992[Abstract/Free Full Text].

24.   McCullough, L. A., and T. C. Westfall. Neuropeptide Y inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma cells. Eur. J. Pharmacol. 287: 271-272, 1995[Medline].

25.   McCullough, L. A., and T. C. Westfall. Mechanisms of catecholamine synthesis inhibition by neuropeptide Y: role of Ca2+ channels and protein kinases. J. Neurochem. 67: 1090-1099, 1996[Medline].

26.   McQuiston, A. R., J. J. Petrozzino, J. A. Connor, and W. F. Colmers. Neuropeptide Y receptors inhibit N-type calcium currents and reduce transient calcium increases in rat dentate granule cells. J. Neurosci. 16: 1422-1429, 1995[Abstract/Free Full Text].

27.   Nörenberg, W., M. Bek, N. Limberger, K. Takeda, and P. Illes. Inhibition of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells by Y3-type neuropeptide Y receptors via the adenylate cyclase/protein kinase A septum. Naunyn Schmiedebergs Arch. Pharmacol. 351: 337-347, 1995[Medline].

28.   Pernow, J., and J. M. Lundberg. Modulation of noradrenaline and neuropeptide Y (NPY) release in the pig kidney in vivo: involvement of alpha 2, NPY, and angiotensin II receptors. Naunyn Schmiedebergs Arch. Pharmacol. 340: 379-385, 1989[Medline].

29.   Petitto, J. M., Z. Huang, and D. B. McCarthy. Molecular cloning of a NPY-Y1 receptor cDNA from rat splenic lymphocytes: evidence of low levels of mRNA expression and [125I]NPY binding sites. N. Neuroimmunol. 54: 81-86, 1994[Medline].

30.   Qu, Y., D. L. Campbell, H. H. Himmel, and H. C. Strauss. Neuromodulation of calcium current by extracellular ATP in isolated ventricular myocytes. Adv. Exp. Med. Biol. 346: 11-18, 1993[Medline].

31.   Regunathan, S., M. P. Meeley, and D. J. Reis. Expression of non-adrenergic imidazoline sites in chromaffin cells and mito-chondrial membranes of bovine adrenal medulla. Biochem. Pharmacol. 45: 1667-1675, 1993[Medline].

32.   Rose, P. M., P. Fernandes, J. S. Lynch, S. T. Frazier, S. M. Fisher, K. Koduknla, B. Kienzle, and R. Seethala. Cloning and functional expression of a cDNA enclosing a human type 2 neuropeptide receptor. J. Biol. Chem. 270: 22661-22664, 1995[Abstract/Free Full Text].

33.   Scheenen, W. J., H. G. Yntema, P. H. Willems, E. W. Roubos, L. R. Lieste, and B. G. Jenks. Neuropeptide Y inhibits Ca2+ oscillations, cyclic AMP and secretion in melanotrope cells of Xenopus laevis via a Y1 receptor. Peptides 16: 889-895, 1995[Medline].

34.   Shigeri, Y., S. Nakajima, and M. Fujimoto. Neuropeptide Y1 receptors mediated increases in intracellular Ca2+ concentrations via phospholipase C-dependent pathway in porcine aortic smooth muscle cells. J. Biochem. (Tokyo) 118: 515-520, 1995[Abstract/Free Full Text].

35.   Tatemoto, K., M. Carlquist, and V. Mutt. Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296: 659-660, 1982[Medline].

36.   Walker, M. W., D. A. Ewald, T. M. Perney, and R. J. Miller. Neuropeptide Y modulates transmitter release and Ca2+ currents in rat sensory neurons. J. Neurosci. 8: 2438-2446, 1988[Abstract].

37.   Weinberg, D. H., J. S. Sirinatsinghji, C. P. Tau, L.-L. Shiao, N. Morin, M. R. Rigby, R. H. Heavens, D. R. Rapoport, M. I. Bayne, M. A. Cascieri, C. D. Strader, D. L. Linemeyer, and B. J. MacNeil. Cloning and expression of a novel neuropeptide Y receptor. J. Biol. Chem. 271: 16485-16488, 1966[Abstract/Free Full Text].

38.   Westfall, D. P., F. Shinozuka, K. M. Forsyth, and R. A. Bjur. Presynaptic purine receptors. Ann. NY Acad. Sci. 604: 130-135, 1990[Medline].

AJP Heart Circ Physiol 273(4):H1737-H1744
0363-6135/97 $5.00 Copyright © 1997 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
W. H. Roden, J. B. Papke, J. M. Moore, A. L. Cahill, H. Macarthur, and A. B. Harkins
Stable RNA interference of synaptotagmin I in PC12 cells results in differential regulation of transmitter release
Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1742 - C1752.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
L. Pinilla, R. Fernandez-Fernandez, J. Roa, J. M. Castellano, M. Tena-Sempere, and E. Aguilar
Selective role of neuropeptide Y receptor subtype Y2 in the control of gonadotropin secretion in the rat
Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1385 - E1392.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
D. Kohno, H.-Z. Gao, S. Muroya, S. Kikuyama, and T. Yada
Ghrelin Directly Interacts With Neuropeptide-Y-Containing Neurons in the Rat Arcuate Nucleus: Ca2+ Signaling via Protein Kinase A and N-Type Channel-Dependent Mechanisms and Cross-Talk With Leptin and Orexin
Diabetes, April 1, 2003; 52(4): 948 - 956.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Hallden, M. Hadi, H. T. Hong, and G. W. Aponte
Y Receptor-mediated Induction of CD63 Transcripts, a Tetraspanin Determined To Be Necessary for Differentiation of the Intestinal Epithelial Cell Line, hBRIE 380i Cells
J. Biol. Chem., September 24, 1999; 274(39): 27914 - 27924.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
T. Kawada, M. Sugimachi, T. Shishido, H. Miyano, T. Sato, R. Yoshimura, H. Miyashita, T. Nakahara, J. Alexander Jr., and K. Sunagawa
Simultaneous identification of static and dynamic vagosympathetic interactions in regulating heart rate
Am J Physiol Regulatory Integrative Comp Physiol, March 1, 1999; 276(3): R782 - R789.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, X.
Right arrow Articles by Westfall, T. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, X.
Right arrow Articles by Westfall, T. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online