Expression, pharmacological, and functional evidence for
PACAP/VIP receptors in human lung
Rebeca
Busto1,
Isabel
Carrero1,
Luis G.
Guijarro1,
Rosa M.
Solano1,
José
Zapatero2,
Fernando
Noguerales3, and
Juan C.
Prieto1
1 Department of Biochemistry
and Molecular Biology and
3 Department of Morphological
Sciences and Surgery, University of Alcalá, 28871 Alcalá de
Henares; and 2 Department of
Thoracic Surgery, Ramón y Cajal Hospital, 28034 Madrid, Spain
 |
ABSTRACT |
Pituitary adenylate
cyclase-activating peptide (PACAP) type 1 (PAC1) and common
PACAP/vasoactive intestinal peptide (VIP) type 1 and 2 (VPAC1 and
VPAC2, respectively) receptors
were detected in the human lung by RT-PCR. The proteins were identified
by immunoblotting at 72, 67, and 68 kDa, respectively. One class of
PACAP receptors was defined from
125I-labeled PACAP-27 binding
experiments (dissociation constant = 5.2 nM; maximum binding
capacity = 5.2 pmol/mg protein) with a specificity:
PACAP-27
VIP > helodermin
peptide
histidine-methionine (PHM)
secretin. Two classes of VIP receptors
were established with 125I-VIP
(dissociation constants of 5.4 and 197 nM) with a specificity: VIP
helodermin
PACAP-27
PHM
secretin. PACAP-27 and VIP were
equipotent on adenylyl cyclase stimulation
(EC50 = 1.6 nM), whereas other
peptides showed lower potency (helodermin > PHM
secretin).
PACAP/VIP antagonists supported that PACAP-27 acts in the human lung
through either specific receptors or common PACAP/VIP receptors. The
present results are the first demonstration of the presence of
PAC1 receptors and extend our
knowledge of common PACAP/VIP receptors in the human lung.
pituitary adenylate cyclase-activating peptide; vasoactive
intestinal peptide; signal transduction; pituitary adenylate
cyclase-activating peptide type 1 receptor; adenylyl cyclase
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INTRODUCTION |
VASOACTIVE INTESTINAL PEPTIDE (VIP) and pituitary
adenylate cyclase-activating peptide (PACAP) are widely distributed
neuropeptides belonging to the same family of peptides as secretin and
glucagon (1). VIP is a 28-amino acid peptide with a 68% sequence
homology with PACAP-27 and a widespread distribution in the peripheral nervous system. VIPergic fibers are present in the upper airways and
tracheobronchial tree and are particularly abundant around seromucous
glands and smooth muscle (15). VIP appears to be the major peptide
transmitter of the nonadrenergic noncholinergic inhibitory component of
autonomic innervation of the lung where it acts as a potent smooth
muscle relaxant and induces bronchodilation and vasodilation (27). VIP
also exerts an antiproliferative effect on human airway smooth muscle
cells (16). Because airway smooth muscle proliferation is a major
feature of bronchial asthma, the observed VIP deficiency in the airways
of severe asthmatic patients could explain the hyperresponsiveness and
hyperplasia of airway smooth muscle cells in that disease (22).
Interestingly, VIP has recently been shown to suppress the growth and
proliferation of human small cell lung cancer (SCLC) as studied on
tumor cell implants in athymic nude mice (17). On the other hand, nerve fibers displaying PACAP immunoreactivity have been found in the respiratory tract of different species (rat, guinea pig, ferret, pig,
sheep, and squirrel monkey) (36). In humans, PACAP-containing nerve
fibers are abundant in the tracheal and bronchial walls, located among
smooth muscle bundles and around glands and small blood vessels (15).
PACAP has been described as a potent relaxant of tracheal smooth
muscle, and it also possesses anti-inflammatory activity (3).
The cDNAs for three distinct PACAP/VIP receptors have been cloned (10):
the PACAP type 1 (PAC1) receptor
(which stimulates both adenylyl cyclase and phospholipase C) has a
higher affinity for PACAP (27 or 38) than for VIP, whereas the common
PACAP/VIP type 1 and 2 (VPAC1 and
VPAC2, respectively) receptors
have equal affinity for these two peptides (4, 34).
VPAC2 probably corresponds to the
"helodermin-preferring" VIP binding site. Specific receptors for
VIP have been identified in membrane preparations from rat and human
lungs (23, 24) as well as in non-small cell lung cancer (NSCLC) and
SCLC cell lines (13, 29). PACAP binds to a lung receptor that until now
is believed to be a VIP receptor that recognizes with nearly equal
affinity VIP, PACAP-27, and PACAP-38. PACAP receptors have been
described in the rat lung (11) and also in human SCLC and NSCLC cell
lines (19, 38) where they can regulate tumor cell growth and differentiation.
Despite the existence of PACAP-containing fibers innervating human
airways, there are no reports at present on the existence of specific
PACAP receptors in the human lung and the functional role of PACAP in
the respiratory system. The close structural and functional
relationship between PACAP and VIP and the coexistence of both peptides
in lung nerve fibers (15) increase the interest in these peptides
because of the possibility of their concomitant action at this level.
In the present report, we studied the expression and characteristics of
PACAP receptors and the corresponding signal transduction step involved
in the action of this peptide in the human lung. For comparative
purposes, we also ran experiments in parallel on rat lung membranes.
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MATERIALS AND METHODS |
Reagents. VIP, peptide
histidine-methionine (PHM), and secretin were purchased from Neosystem
(Strasbourg, France). PACAP-27 and anti-rabbit IgG, heavy and light
chain (goat) peroxidase conjugated, were from Calbiochem-Novabiochem
(San Diego, CA). PACAP-(6
38) was from American Peptide (Sunnyvale,
CA). Helodermin was from Peninsula (St. Helens, UK).
[Lys1,Pro2,5,Arg3,4,Tyr6]VIP,
creatine phosphate, phosphocreatine kinase, IBMX, Triton X-100, and
chemicals for SDS-PAGE were from Sigma (Alcobendas, Spain). Protein
markers for SDS-PAGE were from Bio-Rad (Hercules, CA). The specific
antisera for VIP and PACAP receptors were generous gifts from Dr. E. J. Goetzl (University of California, San Francisco, CA) and Dr. A. Arimura
(Tulane University, Belle Chasse, LA), respectively. The
peroxidase system Supersignal substrate for Western blotting was from
Pierce (Rockford, IL). The Ultraspec kit for RNA extraction was from
Biotecx (Houston, TX). The first-strand cDNA synthesis kit for RT-PCR
[avian myeloblastosis virus (AMV)] was from Boehringer
Mannheim (Barcelona, Spain). Oligonucleotides for PCR were synthesized
by Pharmacia Biotech (San Cugat, Spain); deoxynucleotide
triphosphates and pGEM DNA markers were from Promega (Madison, WI), and
EcoTaq enzyme was from Ecogen
(Barcelona, Spain).
Membrane preparation. Normal lung
tissue from eight patients (40-60 yr old) suffering from
bronchopulmonary cancer was obtained during segmentectomy or lobectomy.
The tissue was rapidly frozen in liquid nitrogen and then stored at
80°C. Human lung membranes were prepared with a method (28)
that overcomes the problem of lung contamination by coal particles.
Approximately 5-10 g of lung were used at a time for membrane
preparation. For comparative purposes, lung membranes were obtained in
the same manner from male Wistar rats weighing 300-350 g.
PACAP/VIP binding studies. Human lung
(0.04-0.06 mg protein/ml) or rat lung (0.04-0.08 mg
protein/ml) membranes were incubated for 45 min at 15°C with 150 pM
125I-labeled PACAP-27 or 30 pM
125I-VIP in 0.25 ml of 25 mM
Tris · HCl buffer (pH 7.4) containing 5 mM
MgCl2, 0.1% bacitracin, 0.5%
BSA, and increasing concentrations of PACAP-27, VIP, PHM, helodermin,
or secretin. At the end of incubation, the samples were filtered under
vacuum with Whatman GF/C filters pretreated with 0.5%
polyethylenimine, washed twice with 5 ml of ice-cold 50 mM
Tris · HCl buffer (pH 7.4) containing 0.5 mM EDTA and
0.2% BSA, and counted for radioactivity. Nonspecific binding
(determined in the presence of 1 µM VIP or PACAP-27) averaged ~4%
of the total radioactivity in both cases and was subtracted from the
total binding to obtain the specific binding.
125I-VIP and
125I-PACAP-27 were prepared with
the chloramine T method at a specific activity of ~250 Ci/g (31).
Adenylyl cyclase assay. As previously
described (22), human or rat lung membranes (0.01-0.02 mg
protein/ml) were incubated in 25 mM triethanolamine-HCl buffer (pH 7.4)
containing 1.5 mM ATP, an ATP-regenerating system (7.4 mg/ml of
creatine phosphate and 1 mg/ml of creatine kinase), 5 mM
MgSO4, 1 mM IBMX, 1 mM EDTA, 1 mg/ml of bacitracin, and the test substances (VIP, PACAP-27, helodermin, PHM, secretin, and forskolin) in 0.1-ml final volume. After
30 min of incubation at 30°C, the reaction was stopped by heating
the mixture for 3 min at 100°C. After the addition of 0.2 ml of an
alumina slurry (0.75 g/ml in triethanolamine-HCl buffer, pH 7.4) and
centrifugation at 3,300 g for 10 min
at 4°C, the supernatant was taken for the assay of cAMP.
Immunodetection of PACAP/VIP
receptors. Human lung membranes were solubilized in 50 mM Tris · HCl buffer (pH 7.4), 1% (vol/vol) Triton
X-100, and 0.01% trypsin inhibitor for 30 min at 4°C. After centrifugation at 38,000 g for 15 min
at 4°C, the supernatant was mixed with the same volume of 50 mM
Tris · HCl buffer containing 20% glycerol, 6% SDS,
10%
-mercaptoethanol, and 0.05% bromphenol blue, and the proteins
were run on 10% SDS-PAGE. After transfer of the proteins to
nitrocellulose sheets, PACAP/VIP receptors were immunodetected with
rabbit anti-human PAC1 (30),
VPAC1, and
VPAC2 (7) receptor sera. To allow
for the recognition of nonspecific staining, the primary antibodies
were also used after preabsorption with the corresponding synthetic
peptides used for immunization. The immunoreactive proteins were
revealed with peroxidase-conjugated goat anti-rabbit IgG and analyzed
by luminescence according to a standard protocol from Pierce.
RNA extraction and RT-PCR. Total RNA
was isolated from cryopreserved lung fragments by extraction with the
Ultraspec kit. The cDNA was obtained by retrotranscription of 5 µg of
total RNA with the first-strand cDNA synthesis kit for RT-PCR (AMV). An amount of cDNA corresponding to 1.25 µg of total RNA was used for PCR
with the EcoTaq polymerase from Ecogen
according to the manufacturer's instructions. The primers used for
amplification of PACAP/VIP receptors are described in Ref. 31. PCR was
carried out with a Hybaid OmniGene thermal cycler at 45 cycles (45 s at 72°C, 30 s at 95°C, and 30 s at 60°C) in a mixture with the
following final concentration: 1 µM primers, 250 µM deoxynucleotide
triphosphates, 1.25 U EcoTaq, and
1.5-3 mM MgCl2 in
EcoTaq buffer.
-Actin cDNA was used
as a control. Ten microliters of each PCR were submitted to
electrophoresis on a 1.2% agarose gel, stained with ethidium bromide,
and visualized under ultraviolet light. Validity of the PCR products
was assessed on the basis of the specificity of the primers and the
size and restriction enzyme digestion of the amplification products.
Statistical analysis. Unless otherwise
stated, data are reported as means ± SE. The statistical analysis
was performed by Student's t-test and
the Mann-Whitney test. Testing was performed with Instat (GraphPad
Software, San Diego, CA). P < 0.05 was considered significant.
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RESULTS |
Identification of PACAP/VIP receptor
mRNAs. We used the RT-PCR methodology to determine the
expression of the different PACAP/VIP receptors in the human lung. With
the appropriate cDNA (i.e., cDNA giving a positive signal for
-actin
amplification) from four different samples studied, RT-PCR identified
PAC1-,
VPAC1-, and
VPAC2-receptor mRNAs. RT-PCR
products had the expected sizes, and two bands were detected for
PAC1 receptor in three samples, corresponding to the normal (null) PACAP receptor (304 bp) and the SV-1
or SV-2 splicing variants (386 bp) (Fig.
1).

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Fig. 1.
RT-PCR analysis of pituitary adenylate cyclase-activating peptide
(PACAP) type 1 (PAC1;
lane 1) and common PACAP/vasoactive
intestinal peptide (VIP) type 1 and 2 [VPAC1
(lane 2) and
VPAC2 (lane
3), respectively] receptor mRNAs in human lung.
cDNA generated from total RNA was amplified with specific primers. Nos.
at left, DNA standard molecular-mass
markers (lane M) shown as base
pairs. Data correspond to a representative experiment of 4 patients
tested.
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Immunodetection of PAC1,
VPAC1, and VPAC2
receptors.
A protein of ~72-kDa molecular mass was detected by
means of a rabbit antiserum against a synthetic peptide corresponding to positions 411-453 (COOH-terminal intracellular domain) of the PAC1 receptor (30). However, the
use of nonreducing conditions showed proteins with greater
electrophoretic mobility (molecular mass of 58 kDa; data not shown),
supporting the presence of intramolecular disulfide bridges in the
PAC1-receptor molecule.
Immunodetection of the human PACAP/VIP receptors
VPAC1 and
VPAC2 was carried out by using the
anti-human VPAC1-receptor peptide A (which recognizes the first extracellular loop, amino acids 191-222) and B (which binds to the carboxy-terminal cytoplasmic tail, positions 391-457) antibodies and
VPAC2-receptor peptide 4 (which
recognizes the amino terminus, positions 54-70), 6 (against the
first extracellular loop, amino acids 174-195), and 22 and 23 (which bind to the extracellular amino terminus, positions 19-37
and 67-83, respectively) antibodies (7). Peptide A and B
antibodies detected a protein of ~67 kDa, whereas peptide 4, 6, 22, and 23 antibodies detected proteins of 68 kDa. The specificity of the
antibodies was established by their preincubation with the
corresponding immunizing peptides: the preabsorption step completely
abolished the immunostaining. Figure 2
shows the results from representative experiments with the anti-human
PAC1-receptor peptide,
VPAC1-receptor peptide B, and
VPAC2-receptor peptide 22 antibodies.

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Fig. 2.
Immunoblot detection of PAC1,
VPAC1, and
VPAC2 receptors in human lung
membranes. Lanes 2, membranes (50 µg of protein) were
resolved and immunoblotted with antisera to
PAC1,
VPAC1 (peptide B), and
VPAC2 (peptide 22) receptors as
described in MATERIALS AND METHODS.
Lanes 1, specificity controls of
preincubation of each antibody with corresponding immunizing peptide.
No. at left, reference protein-size marker. Experiments are
representative of 5 performed with different patients.
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Receptor binding and specificity of peptide
binding. We studied the specific binding of
125I-PACAP-27 and
125I-VIP as well as the
pharmacological profile of binding for different structurally related
peptides. 125I-PACAP binding was
competitively inhibited by unlabeled PACAP-27 and VIP, which were
equipotent, whereas helodermin and PHM exhibited a 15 times lower
potency and secretin displaced
125I-PACAP with a very low
affinity (it did not achieve the 50% of displacement at the highest
concentration used; Fig. 3, Table 1). Scatchard analysis of the data gave
a straight line, indicating that PACAP binds to a single population of
sites. The data were analyzed with the least-squares nonlinear
regression computer program LIGAND (20). From five distinct human lung
samples, the dissociation constant
(Kd) was
estimated at 5.2 ± 1.4 nM and the maximum binding capacity
(Bmax) at 5.2 ± 1.1 pmol/mg
membrane protein.

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Fig. 3.
Pharmacology of 125I-labeled
PACAP-27 binding to lung membranes. A:
human lung membranes were incubated for 45 min at 15°C with
125I-PACAP-27 and increasing
concentrations of unlabeled PACAP-27 ( ), VIP ( ), helodermin
( ), peptide histidine-methionine (PHM; ), and secretin ( ).
[peptide], Peptide concentration. Values are means ± SE
of experiments performed in duplicate with samples from 5 different
patients. B: similar studies with rat
lung membranes. Values are means ± SE of 7 experiments performed in
duplicate.
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Similar studies were carried out in the rat lung where the potency of
the different peptides to inhibit
125I-PACAP binding gave the
results shown in Fig. 3 and Table 1: PACAP
VIP
PHM
helodermin
secretin. In the rat, Scatchard analysis of PACAP
binding gave a curvilinear plot that was interpreted by the LIGAND
program in terms of two classes of PACAP receptors possessing different
affinities: a first site with high affinity (Kd = 0.65 ± 0.13 nM) and a low-binding capacity (6.3 ± 1.3 pmol/mg protein) and
a second site with low affinity
(Kd = 185 ± 15.3 nM) and a high-binding capacity (83.2 ± 7.4 pmol/mg protein).
Stoichiometric experiments were also performed on human and rat lung
membranes with a fixed concentration of
125I-VIP and increasing doses of
unlabeled ligand (Fig. 4). The
corresponding Scatchard analysis for VIP binding gave upwardly concave
curves. The data in human samples (n = 4) were interpreted as above and resolved into high-affinity
(Kd = 5.4 ± 3.3 nM; Bmax = 7.1 ± 2.8 pmol/mg protein) and low-affinity
(Kd = 197 ± 30.8 nM; Bmax = 138 ± 15.5 pmol/mg protein) binding sites. Rat preparations also resolved into
high-affinity (Kd = 0.44 ± 0.11 nM; Bmax = 5.3 ± 1.9 pmol/mg protein) and low-affinity
(Kd = 124 ± 21.3 nM; Bmax = 110 ± 11.5 pmol/mg protein) binding sites. The pharmacological features of
125I-VIP binding to human and rat
lung membranes were analyzed in competitive experiments with various
VIP structurally related peptides (Fig. 4). As shown by the mean
IC50 values (Table
2), there were similar patterns in the
specificity of these ligands for VIP receptors because, in both the
human and rat, the order of potency of the different peptides was VIP = helodermin
PACAP-27
PHM
secretin but with a higher affinity
in the rat than in the human samples.

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Fig. 4.
Inhibition of 125I-VIP binding to
lung membranes by increasing concentrations of unlabeled PACAP-27
( ), VIP ( ), helodermin ( ), PHM ( ), and secretin ( ).
A: human lung membranes. Values are
means ± SE of experiments performed in duplicate with samples from
4 different patients. B: rat lung
membranes. Values are means ± SE of 12 experiments
performed in duplicate.
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Functionality of PACAP/VIP receptors in adenylyl
cyclase stimulation. The signaling pathway of the
different peptides studied is functionally coupled to the adenylyl
cyclase system as shown in experiments with increasing doses of these
agonists (Fig. 5). VIP and PACAP had the
same efficacy and potency (EC50
~1.65 nM), whereas other related peptides were clearly less potent
and/or efficacious in enzyme stimulation: helodermin
EC50 = 14.7 nM, PHM
EC50 = 100 nM, and secretin
EC50 = 775 nM. Maximal stimulation of adenylyl cyclase activity was achieved with forskolin, whereas the
PACAP/VIP highest level of activity was ~50% of this maximum. No
additivity was found between VIP and PACAP on the stimulation of
adenylyl cyclase (data not shown).

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Fig. 5.
Stimulation of adenylyl cyclase activity in human lung membranes.
Membranes were incubated with increasing concentrations of forskolin,
PACAP-27, VIP, helodermin, PHM, and secretin. [agent],
Agent concentration. Values are means ± SE of experiments performed
in duplicate with samples from 3 different patients.
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Effects of VIP and PACAP antagonists on VIP- and
PACAP-induced adenylyl cyclase stimulation. To clarify
whether PACAP and VIP have an independent pathway in the stimulation
of this enzyme, we tested the effects of the VIP and PACAP
antagonists
[Lys1,Pro2,5,Arg3,4,Tyr6]VIP
(9) and PACAP-(6
38) (26), which by themselves had no effect on cAMP
(data not shown), on the PACAP- and VIP-induced adenylyl cyclase
activity. As shown in Fig. 6, when VIP
stimulated adenylyl cyclase, both antagonists produced a 25% decrease
in the enzyme activity. However, when PACAP was used to activate this
system, PACAP-(6
38) exerted a clear inhibition, whereas the VIP
antagonist did not have any effect on PACAP stimulation.

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Fig. 6.
Effect of VIP and PACAP antagonists [1.5 × 10 6 M VIP antagonist and
10 7 M PACAP-(6 38)]
on adenylyl cyclase activity stimulated by 1.5 × 10 9 M ( EC50) VIP or PACAP-27. Values
are means ± SE expressed as percentage of control activity (after
subtraction of basal levels) of experiments performed in duplicate with
samples from 3 different patients.
** P < 0.02 vs. control value
by Student's t-test.
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DISCUSSION |
VIP exerts important biological effects on lung structures, including
relaxation of airways and vascular smooth muscle, regulation of
microvascular tone and permeability, regulation of mucus secretion, and
inhibition of the release of macromolecules from mucus-secreting glands
(16, 27). A VIP deficiency in the airways of severe asthmatic patients
has been reported (22). Moreover, recent data show inhibitory activity
of VIP on SCLC proliferation, indicating the potential usefulness of
this peptide as an antineoplastic agent, at least in some forms of lung
cancers (17). However, the role of the new members of the VIP family
remains to be established at this level. The existence of a rich supply
of PACAP-immunoreactive fibers in human airways (15) gives a
morphological basis for PACAP-mediated control of the physiology of
human lung, but in contrast with VIP, only sparse data are available on
PACAP in the human lung.
RT-PCR experiments showed the mRNA expression of the three classes of
PACAP/VIP receptors (PAC1,
VPAC1, and
VPAC2) in the human lung.
Previously, Northern blot analysis revealed the expression of a
VPAC1 transcript (32), and
VPAC2 has also been detected by
the RNase protection assay (37) in this human tissue. We used normal,
cancer-free lung tissue that was carefully dissected out from pieces
obtained during segmentectomy or lobectomy in human patients with
bronchopulmonary cancer. However, we cannot absolutely discard the
possibility that PACAP receptors could be induced in lung cancer
patients and may not be present in the healthy human lung. Studies by
our laboratory are in progress to show the distribution of these
peptide receptors in different human lung cancers as well as in pieces
of normal lung obtained from organs that did not present this pathology.
The human PACAP receptor, similar to the rat PACAP receptor, can be
expressed as different splice variants as a result of the variable
expressión of two cassettes of 28 amino acids within the third
cytoplasmic loop encoded by two exons that are alternatively spliced
(25). The position of PAC1 primers
used in our experiments includes the site of insertion of the SV-1
and/or SV-2 exons and may detect the different splice variants of
the PACAP receptor. Hence, two splice variants were obtained for
PAC1 (null and SV-1 and/or SV-2),
in agreement with previous observations on the human retina (21).
In immunodetection experiments with a specific antibody, we identified
the PAC1 receptor in human lung as
a protein of ~72-kDa molecular mass, which is comparable to the
molecular mass reported from other human tissues (2). Immunodetection
of VPAC1 and VPAC2 gave the molecular masses
previously described in a cross-linking study (5).
The study of the pharmacological profile of the specific PACAP receptor
(PAC1) by
125I-PACAP-27 binding to membranes
from human lungs showed that PACAP-27 and VIP were nearly equipotent.
It has been established that PAC1 receptors present a greater affinity for PACAP than for VIP, which does
not agree with present pharmacological data on the displacement of
125I-PACAP-27. On the other hand,
the PACAP receptor appears to be a single class of high-affinity
binding sites, as indicated by Scatchard analysis, although the
possibility of the existence of low-affinity binding sites cannot be
discarded. The high-affinity PACAP-specific binding site represents
only a small fraction (3.5%) of the total PACAP/VIP binding sites, but
in addition to the variable action of PACAP via
PAC1-receptor splice variants,
PACAP has the further potential for inducing a variety of responses
through its ability to interact with the
VPAC1 and
VPAC2 receptors.
The VIP receptor has been previously characterized in the human lung
(5, 28) as a functional protein coupled to the adenylyl cyclase system
(35). The present study defines a high-affinity binding site with a
Kd of 5.4 nM and
a density of 7.1 pmol/mg protein. These data and the observed
IC50 values for VIP, PACAP, and
secretin agree with previous observations in the NSCLC NCI-H592 cell
line (18).
The experiments performed for comparative purposes in rat lung
membranes indicated that the
Kd of the
high-affinity sites was 0.44 nM for VIP and 0.65 nM for PACAP, whereas
the number of receptors was not far from that seen for the
corresponding human sites. The pharmacological profile of rat versus
human receptors was similar for VIP but showed different affinities for
helodermin and PHM in displacing
125I-PACAP-27. In the rat, we
found two classes of binding sites (with high and low affinity) for VIP
and PACAP as interpreted by Scatchard analysis.
The results obtained in the experiments with PACAP/VIP antagonists
agree with those showing a higher affinity of PACAP-(6
38) for
PAC1 and
VPAC2
(IC50 = 40 nM) than for
VPAC1
(IC50 = 600 nM) receptors (8).
They are also compatible with previous data on osteoblast-like cells
with the same antagonists (33) because the competitive antagonist
PACAP-(6
38) markedly inhibited PACAP-induced adenylyl cyclase
activity but exerted little effect on VIP-induced enzyme stimulation,
whereas the VIP antagonist caused a low level of inhibition when VIP
stimulated adenylyl cyclase and it practically did not modify the PACAP
effect at this level. These findings strongly support that PACAP-27
stimulates adenylyl cyclase via specific receptors as well as via the
common VIP receptors. An apparent discrepancy was seen when the binding
profiles of the various peptides tested were compared with the
corresponding activity profiles on adenylyl cyclase stimulation in
human lung membranes: helodermin was about equipotent to VIP and
PACAP-27 on adenylyl cyclase activity as well as on the displacement of
125I-VIP binding, but it was less
potent than these two peptides on the displacement of
125I-PACAP-27 binding. In fact,
these observations may be interpreted as other evidence of the presence
and functionality of the VPAC2 receptor in the human lung. The
VPAC2 receptor corresponds to the
helodermin-preferring receptor previously characterized on the basis of the relative potency of natural and synthetic VIP analogs
in various systems including lung cancer-derived cell lines (14).
Functional coupling to the adenylyl cyclase system also showed a lack
of additivity for VIP and PACAP action, but we cannot discard the
possibility of the coupling of the PACAP receptor to another
transduction system (i.e., phospholipase C) or the involvement of a
limiting adenylyl cyclase (PACAP and VIP only achieve 50% of maximum
stimulation with forskolin) interacting with different receptors. In
this context, SCLC proliferation appears to be sustained by multiple
autocrine and paracrine circuits involving
Ca2+-mobilizing neuropeptides
because Ca2+ liberation may
increase transcription of genes regulating cell proliferation
(c-myc and
c-fos) (12). It is known that PACAP can regulate tumor cell growth and differentiation through
c-fos-mediated pathways, and
PACAP-(6
38) has been shown to inhibit a PACAP-induced increase in
c-fos mRNA (6).
The results reported in this study show the complexity of PACAP/VIP
receptors in the human lung. It is necessary to discern whether some
effects of VIP in the human lung could be accomplished by PACAP through
specific receptors because the development of more potent
PACAP/VIP-related analogs or peptide mimetics will permit a potential
antiasthma or antitumor strategy, with more selective compounds having
therapeutical possibilities.
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ACKNOWLEDGEMENTS |
R. Busto and I. Carrero contributed equally to this study.
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FOOTNOTES |
We are greatly indebted to Dr. A. Arimura (Tulane University, Belle
Chasse, LA) and Dr. E. J. Goetzl (University of California, San
Francisco, CA) for generously supplying the specific peptides against
the human pituitary adenylate cyclase-activating peptide/vasoactive intestinal peptide receptors.
This work was supported by Dirección General de
Investigación Científica y Técnica (Grant
PB94-0360), Universidad de Alcalá (Grant E037/97), and
Consejería de Educación y Cultura de la Comunidad de
Madrid (predoctoral fellowship to R. Busto).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. C. Prieto,
Unidad de Neuroendocrinología Molecular, Departamento de
Bioquímica y Biología Molecular, Universidad de
Alcalá, E-28871 Alcalá de Henares, Spain (E-mail:
bqjcp{at}bioqui.alcala.es).
Received 9 December 1998; accepted in final form 11 March 1999.
 |
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