Characterization of a Selective Antagonist of Neuropeptide Y at the Y2 Receptor
SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF A Y2 ANTAGONIST*

(Received for publication, September 19, 1996, and in revised form, December 4, 1996)

Eric Grouzmann , Thierry Buclin §, Maria Martire , Clara Cannizzaro , Barbara Dörner par , Alain Razaname par and Manfred Mutter par

From the Division d'Hypertension and § Division de Pharmacologie Clinique, Centre Hospitalier Universitaire Vaudois, Lausanne, the par  Institut de Chimie Organique, Université de Lausanne, CH-1011 Lausanne, Switzerland, and the  Institute of Pharmacology, Catholic University of Sacred Heart, Rome, 00168 Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Neuropeptide Y (NPY) is a potent inhibitor of neurotransmitter release through the Y2 receptor subtype. Specific antagonists for the Y2 receptors have not yet been described. Based on the concept of template-assembled synthetic proteins we have used a cyclic template molecule containing two beta -turn mimetics for covalent attachment of four COOH-terminal fragments RQRYNH2 (NPY 33-36), termed T4-[NPY(33-36)]4. This structurally defined template-assembled synthetic protein has been tested for binding using SK-N-MC and LN319 cell lines that express the Y1 and Y2 receptor, respectively. T4-[NPY(33-36)]4 binds to the Y2 receptor with high affinity (IC50 = 67.2 nM) and has poor binding to the Y1 receptor. This peptidomimetic tested on LN319 cells at concentrations up to 10 µM shows no inhibitory effect on forskolin-stimulated cAMP levels (IC50 for NPY = 2.5 nM). Furthermore, we used confocal microscopy to examine the NPY-induced increase in intracellular calcium in single LN319 cells. Preincubation of the cells with T4-[NPY(33-36)]4 shifted to the right the dose-response curves for intracellular mobilization of calcium induced by NPY at concentrations ranging from 0.1 nM to 10 µM. Finally, we assessed the competitive antagonistic properties of T4-[NPY(33-36)]4 at presynaptic peptidergic Y2 receptors modulating noradrenaline release. the compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of NPY 13-36, a Y2-selective fragment, yielding a pA2 value of 8.48. Thus, to our best knowledge, T4-[NPY(33-36)]4 represents the first potent and selective Y2 antagonist.


INTRODUCTION

Neuropeptide Y (NPY)1 is a 36-amino acid peptide amide distributed widely in the central and peripheral nervous system (1-3). NPY exerts many biological effects, especially on cardiovascular, metabolic, food intake, behavior, anxiety, and endocrine regulation (4). Several lines of evidence suggest potential roles for NPY in the pathophysiology of hypertension, obesity, diabetes, and psychiatric disorders (5). NPY acts through a number of G-protein-coupled receptors termed Y1, Y2, Y4/PP, Y3, and Y1-like receptors (4). Only Y1, Y2, and Y4/PP receptors have been cloned (6-10). Y1 receptors are present in the sympathetic nervous system mainly postsynaptically and mediate vasoconstriction. Those of the Y2 subtype are present prejunctionally and inhibit the release of catecholamines (11). Furthermore, it has been demonstrated that NPY Y2 receptors are located on noradrenergic nerve terminals within the hypothalamus and other brain regions, exerting an inhibitory action on [3H]norepinephrine release evoked by appropriate concentrations of potassium ions (12). Y2 receptors are also involved in endocrine control; NPY has been reported to inhibit through the Y2 receptor potassium-stimulated glutamate release (13), alpha -melanocyte-stimulating hormone release (14), release of luteinizing hormone in a steroid-free environment (15), prolactin release (16), and to potentiate the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland (17).

NPY belongs to the pancreatic polypeptide family characterized by a common helical structure termed PP fold (18). NPY consists of a polyproline type II helix for residues 1-8 followed by a beta -turn through positions 9-14, an amphipathic alpha -helix at 15-32, and a flexible COOH terminus at 33-36 (18). The complete sequence of NPY is needed for binding to the Y1 receptor, whereas COOH-terminal fragments are selective for the Y2 receptor (19). Alanine scan studies performed on the NPY molecule have shown that the COOH-terminal part of NPY is essential for its biological activity on the Y1 and the Y2 receptors (20); the COOH-terminal pentapeptide amide is important for both receptors and probably represents the binding site (20). However, Arg-33 and Arg-35 may not be exchanged by L-alanine in the Y1 system, whereas Arg-35 and Tyr-36 are the most critical residues for the Y2 receptor. Based on these observations, specific peptidic and non-peptidic compounds have been designed providing potent Y1-selective antagonists (21-23). Earlier, we generated a cyclic truncated analog of NPY, [Ahx5-24,gamma -Glu2-beta Lys30] NPY, which acts as a specific agonist for the Y2 receptor (24); however, no antagonists specific for the Y2 receptor have been described yet. NPY fragments shorter than NPY 27-36 are no longer able to bind to the Y2 receptor.

The assembly of bioactive peptides on topological template molecules according to the template-assembled synthetic proteins (TASP) concept has been shown to induce or stabilize specific conformations of various peptides and consequently, to modify their biological and pharmacokinetic properties (25). We have, for example, synthesized a TASP molecule able to bind and to stimulate selectively the angiotensin II AT2 receptor (26). Here, we design a molecule composed of four truncated NPY peptide fragments (NPY 33-36) attached to a cyclic carrier molecule via their NH2 termini. This TASP molecule was investigated for binding by NPY Y1 and Y2 receptors, and its antagonistic activity was established by its ability to prevent the increase in intracellular calcium induced by NPY using a new methodology based on the analysis of Ca2+ increase in single cells. The biological antagonistic properties of the compound were confirmed by measuring the blockade of the inhibitory action of a Y2 agonist on K+-induced [3H]norepinephrine release from perfused rat hypothalamic synaptosomes.


MATERIALS AND METHODS

Reagents

Porcine NPY, NPY 13-36, and Leu-31, Pro-34 NPY were purchased from Novabiochem (Läufelfingen, Switzerland). Fluo-3/AM and pluronic acid were obtained from Molecular Probes (Eugene, OR), the Ca2+ ionophore 4-bromo A-23187 was from Sigma. Tween 20 was from Pierce.

Synthesis of the TASP T4-[NPY(33-36)]4

For the effective synthesis of the TASP (27) T4-[NPY(33-36)]4 (III in Fig. 1) chemoselective ligation methods were applied. Oxime bond formation (28) was used to attach the functionally modified NH2-terminal (aminoxy group) peptide fragments to the cyclic peptide template, T4 (I). T4 contains four lysine residues acting as attachment sites and the beta -turn mimic 8-aminomethyl-2-naphthoic acid (29). The epsilon -amino groups of lysine were transformed to aldehydic functions by reaction with glyoxylic acid 1,1-diethylacetal and subsequent hydrolysis (30) to yield I. Solid phase synthesis of the partial NPY sequence Arg-Gln-Arg-Tyr (II) was performed on Rink amide resin using N-(9-fluorenylmethoxycarbonyl) chemistry and 2,2,5,7,8-pentamethylchroman-6-sulfonyl, trityl, and tert-butyl protection for the functional groups of the Arg, Gln, and Tyr side chains (31). The ligation reaction proceeded as follows. The tetrakis aldehyde T4 was dissolved in 1 M sodium acetate, and the pH was adjusted to 5 with acetic acid. A 1.2-fold excess of the tetrapeptide II (with respect to the aldehyde groups) in M sodium acetate (pH 5) was added, and the mixture was stirred at room temperature for 15 h. The crude product was purified directly by semipreparative reverse phase HPLC, and the isolated TASP III was characterized by electrospray-mass spectrometry (Fig. 2) and amino acid analysis.


Fig. 1. Synthesis of the TASP molecule T4-[NPY(33-36)]4. T4, cyclic template (I); 4 denotes the number of attachment sites (i.e. the epsilon -amino groups of the Lys residues transformed to aldehydic functions) for the neuropeptide Y fragment NPY(33-36) (II).
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Fig. 2. Reverse phase HPLC and electrospray mass spectrum (inset) of T4-[NPY(33-36)]4. Molecular mass, 3,920.37 Da. The TASP molecule was eluted on a Vydac 218 TP 54 (5 µm, C18, 4.6 × 250 mm) column, using a buffer gradient of 5-40% buffer B (buffer A = 0.9% trifluoroacetic acid in H2O; buffer B = 0.9% trifluoroacetic acid in CH3CN), at 1.0 ml/min over 30 min, monitoring at 214 nm. The TASP molecule eluted at 24.6 min.
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Cell Cultures

SK-N-MC cells were derived from a human neuroblastoma and were cultured according to the American Type Cell Culture recommendations.

LN319 cells, obtained from a human glioblastoma, were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, glutamine, 100 IU of penicillin, and 100 µg/ml streptomycin in a 5% CO2, 95% air incubator at 37 °C. Tissue culture media were purchased from Life Technologies, Inc. (Basel, Switzerland), and fetal calf serum was obtained from Seromed (Berlin, Germany). The cells were used for experiments from passage 190 to 210. To obtain reproducible data over 2 years of experiments, 70% confluent cells were washed with phosphate-buffered saline and harvested using 0.15% trypsin containing 0.4 mM EDTA. Cells were further diluted 1:3 and plated onto either 60-mm cell culture dishes (Nunc, Denmark) or 12-mm glass coverslips (Huber and Co, AG, Reinach, Switzerland). Media were changed every 3 days.

Binding Assays

Y1 Binding Assay

Binding of iodinated NPY (Amersham, Buckingamshire, UK, 74 TBq/mmol) was performed by incubation at 37 °C for 1 h of various peptide dilutions in Eagle's minimum essential medium containing 0.5% bovine serum albumin, 4 mM MgCl2, and 10 mM Hepes with SK-N-MC cells that exhibit exclusively Y1 receptors. Cells were then washed three times with buffer and lysed in 1% Nonidet P-40 (Fluka, Neu-Ulm, Germany), 8 M urea, 3 M acetic acid. Nonspecific binding was estimated by adding 1 µM NPY to the incubation mixture. Displacement curves were obtained by incubation of various concentrations of competitive peptides together with a nonsaturating dose of iodinated NPY. At the end of the incubation period, cells were washed and lysed. Bound radioactivity was determined by gamma -counting. Half-maximal inhibition of the binding, obtained with 125I-NPY, is given as the IC50. Each point represents the mean ± S.D. of at least four experiments.

Y2 Binding Assay

We used a human glioblastoma cell line, LN319, for Y2 binding studies (24). Prior to performing the binding experiments, adhering LN319 cells were harvested in 50 mM Tris (pH 7.5), which contained 100 mM NaCl, 4 mM MnCl2, 1 mM EGTA, 0.1% bovine serum albumin, 0.25 mg/ml bacitracin, and incubated at room temperature for 45 min. Bound radioactivity was determined after separating the unbound fraction by centrifugation.

Determination of cAMP

Six-well plates, containing confluent LN319 cell cultures, were washed and incubated at 37 °C for 1 h in Eagle's minimum essential medium containing 0.5% bovine serum albumin, 4 mM MgCl2, 10 mM Hepes, 100 µM papaverin, and 10 µM forskolin and one of the peptides to be tested in varying dilutions. Cells were washed once in 100 mM sodium phosphate buffer (pH 7.5) and lysed with 0.75 ml of 0.1 M HCl. After centrifugation, the supernatant was recovered and lyophilized. cAMP concentration was measured by a radioimmunoassay using a commercially available kit (Amersham).

Antagonistic Properties of T4-]NPY(33-36)]4 on the Free Cytosolic Calcium Response to NPY

LN319 cells were plated on glass coverslips 48 h before intracellular free calcium measurements. Intracellular free calcium concentration [Ca2+]i was determined using the fluorescent probe fluo-3/AM. The dye was loaded into the cells by adding the acetoxymethyl ester fluo-3/AM (2.5 µM) from a 1 mM stock in dry dimethyl sulfoxide to the culture medium (Dulbecco's modified Eagle's medium or Eagle's minimum essential medium without serum or complements) and incubating the cells for 30 min at room temperature in the dark. Pluronic acid (2 µl/ml) in 25% dimethyl sulfoxide was added to fluo-3/AM to disperse the dye. After loading, the cells were washed three times with medium and placed in a chamber with 0.5 ml of physiological saline solution containing 140 mM NaCl, 2 mM CaCl2, 4.6 mM KCl, 1.0 mM MgCl2, 10 mM glucose, and 10 mM Hepes (pH 7.4). Tween 20 (Pierce) 0.0008% was present in the medium to prevent NPY from sticking to the walls of the exposed surfaces. Fluorescence images of the intracellular calcium localization were obtained with a laser-scanned confocal microscope (MRC 500 confocal imaging system, Bio-Rad) equipped with an argon ion laser and a fluorescein (488 nm) or rhodamine (514 nm) filter cartridge. The scanner and detectors were attached to an inverted microscope (Diaphot, Nikon).

The confocal microscopy technique with video recording provides serial readings, at 5-s intervals, of the individual fluorescence for a set of cells. The changes in Ca2+ were evaluated in single cells on whole images containing 5-15 cells using the NIH image analyzer program (29-385 cells were used to test each concentration of peptides). In each experiment it uses a fixed delineation of the cell borders, entered with a pointer device on the image screen. For each cell, five intensity readings were recorded. The base-line fluorescence was determined by averaging two consecutive images (Fbase); the signal induced by adding the T4-[NPY(33-36)]4 solution (Fantag); the peak response induced by adding the NPY solution (FNPY); the maximal response observed after adding the nonfluorescent Ca2+ ionophore A-23187 (10 µM) to saturate the intracellular dye with calcium and thereby obtain maximal fluorescence (Fmax); and the minimal fluorescence (Fmin) was measured after addition of an excess of EDTA (5 mM). The Fantag values were not used for further analysis, after it had been demonstrated that T4-[NPY(33-36)]4 did not induce any significant response.

The intracytoplasmic calcium concentrations at the peak of the NPY effect were derived from fluorescence readings by using the formula (32) in nmol/liter: CaNPY = 320 (FNPY - Fmin)/(Fmax - FNPY).

Attempts were made to correct CaNPY value for the base-line level; however, as the peak response was poorly correlated with the base-line value (r2 = 0.13, rlog/log2 = 0.24), this only added noise without modifying the results; so CaNPY was retained as the response variable. It was transformed to logarithmic functions to normalize its distribution. Means and standard deviations of log10(CaNPY) were computed from the individual cell responses for each level of NPY and T4-[NPY(33-36)]4 (their antilog reflecting, respectively, the geometric means and coefficients of variation of the original CaNPY values).

A dual response Hill model was used to relate the log10(CaNPY) values to the NPY concentrations, to account for the observed fading of the response at very high doses of the agonist (33). The T4-[NPY(33-36)]4 was considered as a competitive antagonist, affecting the apparent EC50 of NPY. Thus the mathematical expression of the pharmacodynamic model was as follows,
<UP>log</UP><SUB><UP>10</UP></SUB>(<UP>Ca<SUB>NPY</SUB></UP>)=E<SUB><UP>min</UP></SUB>+E<SUB><UP>max</UP></SUB> · <FENCE><FR><NU><FENCE><FR><NU><UP>NPY</UP></NU><DE><UP>EC</UP><SUB><UP>50</UP></SUB></DE></FR></FENCE><SUP>&ggr;</SUP></NU><DE><FENCE><FR><NU><UP>NPY</UP></NU><DE><UP>EC</UP><SUB><UP>50</UP></SUB></DE></FR></FENCE><SUP>&ggr;</SUP>+<FENCE><FR><NU><UP>Antag</UP></NU><DE><UP>AC</UP><SUB><UP>50</UP></SUB></DE></FR></FENCE><SUP>&dgr;</SUP>+1</DE></FR>−<FR><NU><FENCE><FR><NU><UP>NPY</UP></NU><DE><UP>FC</UP><SUB><UP>50</UP></SUB></DE></FR></FENCE><SUP>&ggr;</SUP></NU><DE><FENCE><FR><NU><UP>NPY</UP></NU><DE><UP>FC</UP><SUB><UP>50</UP></SUB></DE></FR></FENCE><SUP>&ggr;</SUP>+<FENCE><FR><NU><UP>Antag</UP></NU><DE><UP>AC</UP><SUB><UP>50</UP></SUB></DE></FR></FENCE><SUP>&dgr;</SUP>+1</DE></FR></FENCE>
<UP><SC>Model</SC></UP><UP> 1</UP>
where Emin is the base-line (no effect) value; Emax, the maximal response; EC50, the concentration of NPY associated with a half-maximal response; FC50, the concentrations associated with a one-half fading of the maximal response; AC50 the concentration of T4-[NPY(33-36)]4 inducing a doubling of the apparent EC50 and FC50 of NPY. The last parameters were expressed in nM. The exponents gamma  and delta  modulate the sigmoidicity of the concentration-effect relationships of NPY and T4-[NPY(33-36)]4, respectively. This model was fitted to the whole set of individual cell responses by means of nonlinear regression module of the SYSTAT software, version 5.02 (SPSS Inc., Chicago, IL 60611), which provides least square estimates with asymptotic standard errors using the simplex algorithm.

Antagonistic Properties of T4-[NPY(33-36)]4 at Presynaptic Peptidergic Y2 Receptors Modulating Norepinephrine Release

Preparation of Synaptosomes

Nerve endings were prepared from the hypothalamus of adult Wistar rats (200-250 g) according to the method of Gray and Whittacker with minor modifications (34). Briefly, the rat hypothalamus was homogenized using a Teflon-glass tissue grinder (clearance 0.25 mm) in 40 volumes of 0.32 M sucrose buffered at pH 7.40 with phosphate. The homogenate was centrifuged (5 min, 1,000 × g) to remove nuclei and debris, and the supernatant was again centrifuged (20 min, 12,000 × g) to isolate crude synaptosomes. The synaptosomal pellet (at a protein concentration of 0.6-0.8 mg/ml) was then resuspended in a physiological medium with the following composition (in mM): 125 NaCl, 3 KCl, 1.2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 22 NaHCO3, 10 glucose, aerated with 95% O2 and 5% CO2 at 37 °C (pH 7.40). Protein was measured by the method of Lowry et al. (35).

Norepinephrine Release Experiments

Synaptosomes were incubated with [3H]norepinephrine (Amersham Radiochemical Center, Buckinghamshire, UK; specific activity, 39 Ci/mmol; 0.05 µM final concentration) for 15 min at 37 °C. The labeled particles were then distributed in several parallel superfusion chambers and superfused at 37 °C with continuously oxygenated medium, at a rate of 0.6 ml/min (36). After 10 min, to equilibrate the system and to reach a constant spontaneous efflux, T4-[NPY(33-36)]4 was added and perfusion continued for a further 15 min. The synaptosomes were depolarized with 15 mM KCl for 90 s (substituting for an equimolar concentration of NaCl). The NPY fragment, NPY 13-36 (Peninsula Laboratories, Merseyside, U. K.) was added concomitantly with K+. Fractions were collected every min, and the radioactivity (present as [3H]norepinephrine) in each fraction and in the filters was determined after separation on Biorex 70 columns (37).

Evaluation of Results

The [3H]norepinephrine found in each fraction collected was calculated as a percentage of the total [3H]norepinephrine recovered (fractions plus filters). The concentration-dependent effects of NPY 13-36 were calculated as follows. The area of base-line efflux curve was subtracted from the area of the total release curve obtained in the absence and in the presence of the compound tested. The areas under the release curves of the time course were recorded for each experiment according to the Newton-Cotes integration formula. Data obtained according to this method were used to calculate the percentage inhibition of the K+-evoked release of [3H]norepinephrine in the presence of NPY 13-36 or in the presence of NPY 13-36 plus T4-[NPY(33-36)]4. The apparent pA2 value for the antagonist was calculated by means of the Schild regression analysis according to the following formula: pA2 = log(E'/E - 1) - logB, where E' and E are those concentrations of the agonist which caused half-maximum effects in the presence and in the absence of the antagonist, respectively. B is is the concentration of the antagonist.


RESULTS

Binding Assays

As described above, the SK-N-MC and LN319 cells express Y1 and Y2 receptor subtypes, respectively. For competitive binding studies, in addition to the native NPY, we used peptides with differential selectivity for Y1 and Y2 binding. The Leu-31- and Pro-34-substituted NPY has been shown to be a Y1 agonist (38), whereas the NPY 13-36 has been reported to bind preferentially to the NPY Y2 receptor subtype.

Fig. 3 depicts the results of binding experiments obtained with the two cell lines. SK-N-MC cells (Fig. 3A) bind NPY and Leu-31, Pro-34 NPY equally well as shown by the similar competition displacement curves (Fig. 3A). In contrast, NPY 13-36 binding was 2,000-fold less as this cell line does not express Y2 receptors. Neither the template nor NPY33-36 bound to SK-N-MC cells (IC50 > 10 µM), and T4-[NPY(33-36)]4 shows only a poor affinity for the Y1 receptor (IC50 = 6.6 µM). The LN319 cells (Fig. 3B) exhibited a comparable high affinity for NPY and NPY 13-36 with IC50 of 0.085 and 0.126 nM, respectively. In contrast, Leu-31, Pro-34 NPY bound poorly to the Y2 receptor. Similar to the observation for the Y1 receptor,neither the template nor NPY 33-36 exhibited affinity for LN319 cells (IC50>10 µM), but good binding to the Y2 receptor was obtained with T4-[NPY(33-36)]4 (IC50 = 67.2 nM). T4-[NPY(33-36)]4 was also added to angiotensin II type 1 and type 2 and muscarinic receptor preparations at concentrations up to 10 µM, and there was no binding. A slight interaction was observed at a high concentration of the antagonist in binding experiments with the alpha 1B-adrenergic receptor (42% inhibition at 20 µM).


Fig. 3. Representative concentration-response curves of the displacement of 125I-NPY by selective peptides for the Y1 (panel A) and Y2 (panel B) receptors in SK-N-MC cells (Y1) and LN319 cells (Y2). Four experiments were performed with each analog. The percentage inhibition of 125I-NPY binding to the receptor, which is caused by the increasing concentrations of competitors, is shown on the y axis. High affinity binding to the Y1 receptor on SK-N-MC cells was found for NPY (open circle ) and Leu-31, Pro-34 NPY (down-triangle). On LN319 cells NPY and NPY 13-36 (triangle ) exhibited high affinity binding, and T4-[NPY(33-36)]4 (bullet ) slightly reduced affinity. NPY 33-36 (diamond ) and the template (star ) did not bind to either the Y1 or Y2 receptor.
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cAMP Measurements

To assess whether this peptidomimetic had intrinsic agonist properties we tested its ability to inhibit cAMP accumulation in LN319 cells. Whereas NPY inhibits forskolin-stimulated cAMP accumulation in LN319 cells with an IC50 of 2.5 nM, T4-[NPY(33-36)]4 at concentrations up to 10 µM was devoid of effect.

Calcium Measurements in LN319 Cells

During the study, 3,674 individual cell responses were measured, with an average number of 131 responses (range 30-385) for each concentration of NPY and T4-[NPY(33-36)]4. The distribution of individual peak cytosolic calcium responses for each concentration of NPY and T4-[NPY(33-36)]4 is shown on Fig. 4. Preincubation of the cells for 1 min with T4-[NPY(33-36)]4 had no agonistic effect. Logarithmic expression conferred a quite symmetrical bell-shaped distribution. The variances were large, without evidence of significant nonhomogeneities. Preliminary studies showed that the addition of physiological saline solution to the cells elicited a negligible increase in the fluorescence corresponding to a Delta [Ca2+]i of 4.1 ± 5 nM (mean ± S.D., n = 73). Table I contains the geometric means and coefficients of variations of the intracellular calcium responses, which are plotted in Fig. 5. In the absence of T4-[NPY(33-36)]4, NPY induced a dose-dependent rise in calcium response between 1 and 100 nmol/liter, which declined progressively at 0.5 and 5 µmol/liter. Pretreatment with T4-[NPY(33-36)]4 shifted this dose-response relationship to the right, in a dose-dependent manner. A concentration of T4-[NPY(33-36)]4 at 1,490 nM doubled the apparent EC50 and FC50 of NPY (Table II). The fitting of the general pharmacodynamic model provided estimates of parameters, which are indicated in Table II. The half-fading concentration of NPY, FC50, was estimated with poor precision, in accordance with the few points on the descending part of the dose-response curve. This model explained most of the variance in the data (r2 = 0.91). The intercorrelations between the parameter estimates were acceptable (highest value r = 0.77). The Hill coefficient gamma  departed significantly from unity, based on its 95% confidence interval; this was not the case for delta .


Fig. 4. Frequency distribution of the logarithm of the peak intracellular calcium responses of single cells to NPY challenges at different concentrations, after pretreatment by vehicle only or T4-[NPY(33-36)]4 at three concentrations. See Table I for the corresponding number of cells and geometric means for each NPY and T4-[NPY(33-36)]4 level.
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Table I.

Geometric means of the peak intracellular calcium level after NPY challenge at various concentrations in individual cells pretreated with vehicle only or T4-[NPY(33-36)]4 at three concentrations

The number of cells in each experiment (N) and the geometric coefficient of variation (CV) are shown in parentheses.
NPY concentration T4-[NPY(33-36)]4 concentration
0 1,000 5,000 10,000

nM nM
0 15.0
(N = 73, CV = 2.1)
0.1 15.3 20.6 6.8 15.1
(N = 154, CV = 1.7) (N = 76, CV = 1.4) (N = 30, CV = 4.3) (N = 46, CV = 0.9)
0.2 25.4
(N = 124, CV = 1.1)
1 28.3 28.4 16.0 15.8
(N = 202, CV = 2) (N = 147, CV = 1.1) (N = 66, CV = 2.1) (N = 67, CV = 2)
2 27.1
(N = 186, CV = 2.4)
10 39.7 35.8 25.0 22.8
(N = 368, CV = 2.5) (N = 161, CV = 1.7) (N = 68, CV = 2.1) (N = 72, CV = 2.1)
20 52.5
(N = 302, CV = 2.2)
100 63.4 32.7 34.5 22.3
(N = 327, CV = 2.4) (N = 163, CV = 1.6) (N = 29, CV = 0.9) (N = 89, CV = 2.7)
500 50.2 62.7 34.9 25.1
(N = 385, CV = 2.5) (N = 161, CV = 1.3) (N = 79, CV = 2.4) (N = 55, CV = 3.2)
5,000 41.9 58.3 69.2 44.5
(N = 52, CV = 1.2) (N = 43, CV = 2.3) (N = 63, CV = 1.7) (N = 76, CV = 0.9)


Fig. 5. Geometric means and S.E. of the intracellular calcium level observed after adding various concentrations of NPY, in individual cells pretreated by vehicle only (open circle ), by T4-[NPY(33-36)]4 at 1,000 (black-triangle), 5,000 (bullet ), and 10,000 nM (black-square). The lines represent the predicted response according to the pharmacokinetic model fitted to the individual data, for the same values of T4-[NPY(33-36)]4 (r2 = 0.91). Vehicle, --; T4-[NPY(33-36)]4: 1 µM (- -), 5 µM (-·), 10 µM (-··)
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Table II.

Estimates of the seven parameters of the pharmacokinetic model

For the parameter description, see "Materials and Methods." The results were obtained by nonlinear regression across the whole experimental data set.
Parameter Unit Estimate Asymptotic standard error

Emin log of Ca in nM 1.15 0.04
Emax log of Ca in nM 0.97 0.15
EC50 NPY nM 6 2
FC50 NPY nM 9,720 7,490
 gamma Without dimension 0.39 0.05
AC50 nM 1,490 540
T4-[NPY(33-36)]4
 delta Without dimension 1.1 0.2

NPY also increased in a dose-dependent manner the number of cells that get a rise in free calcium (18 and 71% at 0.1 and 100 nM concentrations, respectively), whereas T4-[NPY(33-36)]4 decreased the number of cells that responded to NPY (data not shown). To express the calcium response in an all-or-none fashion, cells were considered as responding if their peak Ca2+ was higher than the mean ± 2 S.D. observed after vehicle only (see Table I). The dose-response curve in the cells that responded to NPY provided similar results when compared with those observed for the mean of responding + nonresponding cells (see Fig. 5).

Antagonistic Properties of T4-[NPY(33-36)]4 at Presynaptic Peptidergic Y2 Receptors Modulating Norepinephrine Release

Fig. 6 shows the dose-response inhibition curve of NPY 13-36 on the release of [3H]norepinephrine evoked by depolarization with 15 mM KCl from hypothalamic nerve endings. Assuming that inhibition of 40% represented the maximum response, then a 6.5 nM concentration of NPY 13-36, which caused 20% inhibition, roughly produced the half-maximum effect. Therefore these IC20 values were used to calculate the pA2 value of the compound T4-[NPY(33-36)]4. The concentration-response curve of the NPY 13-36 fragment was shifted to the right by the addition of the T4-[NPY(33-36)]4 in a concentration-dependent manner (IC50 values in the presence of 1, 10, and 100 nM were 9, 22, and 109 nM, respectively). The concentration-response curve of the NPY 13-36 fragment was shifted to the right by the addition of 10 nM antagonist (Fig. 7). The antagonist appeared competitive for the Y2 receptor that modulated norepinephrine release because the effect of each antagonist concentration could be overcome by high doses of the agonist NPY 13-36. To assess competitive antagonism of T4-[NPY(33-36)]4, the apparent pA2 value for this compound was determined using three different concentrations (1, 10, and 100 nM). The half-maximum inhibitory concentrations of the NPY 13-36 fragment on norepinephrine release obtained in the presence of various concentrations of the antagonist have been used in the Schild regression analysis. The compound T4-[NPY(33-36)]4 caused a marked shift to the right of the concentration-response curve of the NPY 13-36 fragment, yielding a pA2 value of 8.48. The pA2 value of the antagonist T4-[NPY(33-36)]4 was calculated from concentration-response curves corresponding to those shown in Fig. 7.


Fig. 6. Inhibition effect of NPY 13-36 on K+-evoked [3H]norepinephrine release. The concentrations of the NPY 13-36 fragment are plotted versus the percentage of inhibition of the [3H]norepinephrine release, assuming that the 40% inhibition represented the maximum effect. Each point presented is the average (±S.E.) of four experiments run in triplicate. Fractions of the superfusate were collected every min, and the radioactivity present as [3H]amine in each fraction and in the filters was measured as described under "Materials and Methods." The [3H]norepinephrine present in each fraction is expressed as a percentage of the total [3H]norepinephrine recovered (fractions plus filter). The area below the curve of the time course was calculated for each experiment according to the Newton Cotes integration formula. Values obtained from these calculation have been used as a parameter to evaluate inhibition effect of NPY 13-36 on K+-evoked [3H]norepinephrine release.
[View Larger Version of this Image (15K GIF file)]



Fig. 7. Concentration-response curve of the NPY 13-36 fragment on [3H]norepinephrine release in the presence of vehicle (open circle ), 1 (black-triangle), 10 (bullet ), and 100 nM (black-square) of the antagonist. The same methodology as in Fig. 6 was used.
[View Larger Version of this Image (15K GIF file)]



DISCUSSION

The present sudy shows that T4-[NPY(33-36)]4 is a potent and selective ligand for the NPY Y2 receptor and exhibits in vitro antagonistic properties in two models of Y2-mediated effects. Several NPY Y1 receptor antagonists have been characterized and demonstrated to inhibit the pharmacological vasopressor effect of NPY. This compound displays a high affinity for the Y2 receptor (IC50 = 62 nM) but exhibits 2 orders of magnitude less interaction with the Y1 receptor. The antagonistic properties of the molecule have been confirmed by demonstrating inhibition of function.

For these functional studies, we used an original experimental approach involving single-cell recordings of cytosolic calcium responses to a biochemical stimulus. Other investigators have reported a similar technique on a limited number of cells (39-41). The ability to perform hundreds or thousands of single-cell measurements required the development of appropriate statistical methods to cope with the large amount of data. The pharmacological characterization of an antagonist with this new methodology cannot rely on simple Shild plots drawn across average points. Thus, we used computer-based nonlinear regression methods, with special attention to the variability structure displayed by the data.

Preliminary explorations led us to use the intracytoplasmic calcium concentrations at the peak of the NPY effect as the response variable. Correction of this value for the base-line calcium level could be theoretically justified. However, this only added noise to the data, as the peak response was poorly correlated with the base-line value, and this correction was discarded. The response variable showed a strongly skewed distribution. A logarithmic transformation brought the results closer to a Gaussian distribution, allowing the application of a least square approach to estimate pharmacodynamic parameters. This observation indicates a log-normal behavior of calcium concentrations, consistent with its contamination by multiplicative randomness and warrants the description of results by geometric rather than arithmetic means. Biologically, the NPY stimulus is indeed linked to the intracytoplasmic calcium response by a cascade of amplification steps. The high response variability must be emphasized: most coefficients of variation associated with the geometric means exceeded 100%. A pharmacodynamic study could have recorded the average response of a cluster of cells, a piece of tissue, or a whole organ. In that situation, however, only a global effect would have been measured, without consideration for the heterogeneity of individual cell responses. Moreover, averaging would have distorted the signal in many ways. Thus, the results shown here may not reflect what might be observed in multiple cell or tissue preparations. On the other hand, the individual cell behavior itself can be considered as the integration of many subcellular quantal responses (42, 43). Interestingly, when the same procedure of calculation was used for only the responding cells, we obtained an almost identical mathematical model. This finding may indicate that the free calcium response to NPY does not obey an all-or-none rule. Thus, the shape of the dose-effect relationship at the cellular level not only reflects the dose-effect relationship at the subcellular scale but also its variable nature (42).

Despite the high number and variability of the single-cell responses, a pharmacodynamic model was fitted satisfactorily to the whole data set by nonlinear regression. We have chosen a dual Hill model, able to account for the fading of the response at high doses, which is frequent in the field of peptide pharmacology. This model included a competitive antagonism, which adequately describes the pharmacodynamics of T4-[NPY(33-36)]4 in this experimental setting. However, all terms of the model must be interpreted with caution. As this was not a binding study, the EC50, FC50, and AC50 cannot simply be equated with receptor affinities. Neither can they be considered equivalent to values measured in a living organism. This may account for the apparent differences observed between the pharmacodynamic parameters of the two functional assays we have used. Nevertheless, we cannot exclude the possibility that LN319 cells and hypothalamic synaptosomes exhibit different Y2 receptors (44). The slope coefficients gamma  and delta  have also no straightforward interpretation; they rely strongly on the variance structure of the underlying phenomena (42).

Second, T4-[NPY(33-36)]4 also inhibited in a dose-dependent manner the action of a Y2 agonist on K+-induced [3H]norepinephrine release from perfused rat hypothalamic synaptosomes. This effect was prevented by a large dose of the Y2 agonist NPY 13-36, demonstrating that T4-[NPY(33-36)]4 acts as a fully competitive antagonist.

No intrinsic agonistic properties either on calcium or cAMP transduction systems were observed with T4-[NPY(33-36)]4 using LN319.

The NPY gene has recently been disrupted in mice (45). These NPY-deficient mice exhibit seizures, an effect that may be due to inhibition of glutamate release which is known to be mediated by Y2 receptors present on the endings of presynaptic excitatory neurons of NPY (13). Therefore, this compound may be a useful tool for the study of the role of NPY in various disorders such as seizures or abnormalities in the reproductive axis.


FOOTNOTES

*   This work was supported by Grant 31.43.169.95 from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Centre Hospitalier Universitaire Vaudois, Division d'Hypertension, CH-1011 Lausanne, Switzerland. Tel.: 41-21-314-0741; Fax: 41-21-314-0761; E-mail: Eric.Grouzmann{at}chuv.hospvd.ch.
1   The abbreviations used are: NPY, neuropeptide Y; TASP, template-assembled synthetic protein; HPLC, high performance liquid chromatography.

Acknowledgments

We thank Prof. H. R. Brunner for continuous encouragement and support in providing an excellent work environment. We gratefully acknowledge Prof. Trefor Morgan for carefully reading the manuscript. We thank Ali Maghraoui for creating the figures.


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