(Received for publication, September 19, 1996, and in revised form, December 4, 1996)
From the Division d'Hypertension and § Division de
Pharmacologie Clinique, 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 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),
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 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.
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
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
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 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 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,
Institut de Chimie Organique,
-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.
-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).
-turn
through positions 9-14, an amphipathic
-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,
-Glu2-
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.
-turn mimic
8-aminomethyl-2-naphthoic acid (29). The
-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 1 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 -amino groups of the Lys residues
transformed to aldehydic functions) for the neuropeptide Y fragment
NPY(33-36) (II).
[View Larger Version of this Image (22K GIF file)]
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.
[View Larger Version of this Image (17K GIF file)]
-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.
Fmin)/(Fmax
FNPY).
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
and
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 SynaptosomesNerve 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 ExperimentsSynaptosomes 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 ResultsThe [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.
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 1B-adrenergic
receptor (42% inhibition at 20 µM).
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 CellsDuring 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
[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
departed significantly from unity, based on its 95% confidence interval; this was not the case for
.
|
|
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 ReleaseFig.
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.
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 and
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.
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.