| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Laboratory Medicine and
Pathology,*
Mayo Clinic and Mayo Foundation, Rochester,
Minnesota; the Departments of Neurosurgery
and
Pathology,
Kitasato University School of
Medicine, Kanagawa, Japan; and the Division of Cell Biology and
Experimental Cancer Research,§
Institute of
Pathology, University of Berne, Berne, Switzerland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PACAP and VIP share binding sites in a variety of tissue types.8,9 These polypeptides bind to two major sites: type I sites, which show preferential binding to PACAP-38 and PACAP-27 over VIP, and type II sites, which have nearly equally high affinity for PACAP-38, PACAP-27, and VIP. cDNAs for three distinct human PVR subtypes, including PVR-1, PVR-2 (also known as VIP1R), and PVR-3 (also known as VIP2R) have been recently cloned, and represent seven transmembrane-spanning G-protein-coupled receptors that belong to the secretin/glucagon family of receptors.10,11 The type I binding site of PACAP is similar to PVR-1, and type II binding sites share PVR-2 and PVR-3.12
The identification of oncogenic mutations that constitutively activate adenylyl cyclase and cAMP formation in pituitary adenomas has provided further support for the view that pituitary cells proliferate in response to cAMP.13,14 In fact, both VIP and PACAP seem to be general activators of pituitary cell function, being able to activate adenylyl cyclase in pituitary adenomas.15
PACAP types I and II binding sites were previously investigated by
binding assays in human pituitary adenomas16
and were found
in most types of adenomas. PACAP-38 had a modest role in the regulation
of GH, ACTH, and 
-subunit secretion from human tumorous pituitary
corticotrophs and somatotrophs in hormone assays.17
In situ hybridization (ISH) is useful in demonstrating gene expression in individual cells but is limited in its ability to detect low copy numbers of mRNAs. Recent studies have used biotinylated tyramide in a catalyzed reporter deposition ISH (CARD-ISH) amplification system to increase the sensitivity of assays detecting protein by immunohistochemistry18 and mRNA by ISH.19
We used reverse transcription polymerase chain reaction (RT-PCR) with Southern hybridization, conventional ISH, and CARD-ISH to analyze the expression of PACAP, PVR-1, -2, and -3 mRNAs, and PACAP binding sites in human pituitary tumors. Our studies localized PVR-1, -2, and -3 in pituitary adenomas and showed for the first time the in situ localization of PACAP receptors.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Pituitary adenomas included 15 GH tumors, 10 prolactin (PRL) adenomas, 9 ACTH adenomas, and 36 clinically nonfunctioning adenomas with no evidence of hormone hypersecretion and serum PRL levels less than 100 µg/L. Fourteen tumors, which stained for follicle-stimulating hormone (FSH) or luteinizing hormone (LH) ß-subunits, were classified as gonadotroph adenomas. The remaining 22 tumors, which did not show hormone immunoreactivity or focal staining in which less than 25% of cells for gonadotropin ß-subunits, were classified as null cell adenomas. Ultrastructural studies were done on some of the null cell and gonadotroph adenomas to confirm the immunohistochemical classification.
Portions of normal and neoplastic pituitary tissues frozen at -70°C were used for RNA extraction, immunohistochemistry, ISH studies, and PACAP binding studies. Frozen sections of both pituitary adenomas and non-neoplastic autopsy pituitaries were cut at 10 µm, fixed in 4% paraformaldehyde, washed in 2X standard saline citrate (SSC), dehydrated in alcohol, stored at -70°C, and then used for immunohistochemistry, ISH, and CARD-ISH experiments.
Receptor Autoradiograph with 125I-Labeled VIP and 125I-Labeled PACAP Radioligands
125I-labeled VIP (2000 Ci/mmol; Anawa, Wangen, Switzerland) was used as the radioligand. Only the mono [125iodo-Tyr10]-VIP, eluted as single peak from high-pressure liquid chromatography and analyzed by mass spectrometry, was used. The slide-mounted tissue sections were incubated for 90 minutes in a solution of 50 mmol/L Tris/HCL (pH 7.4) containing 2% bovine serum albumin, 2 mmol/L EGTA, 0.1 mmol/L bacitracin, and 5 mmol/L MgCl2 to inhibit endogenous proteases in the presence of 30 pmol/L 125I-labeled VIP at room temperature as described previously.20,21 To estimate nonspecific binding, paired serial sections were incubated as described above, except that 20 nmol/L VIP or PACAP-1-27 (Bachem, Bubendorf, Switzerland) were added to the incubation medium. After this incubation, the slides were washed twice in ice-cold 50 mmol/L Tris/HCL (pH 7.4) containing 0.25% bovine serum albumin, then in buffer alone, and quickly dried under a stream of cold air. The sections were subsequently exposed to a 3H-labeled hyperfilm (Amersham, Little Chalfont, UK) for 1 week. The autoradiograms were quantified using a computer-assisted image processing system previously described.22 Normally, a tissue was defined as receptor positive when the optical density measured in the total binding section was at least twice the optical density of the nonspecific binding section.
The same pituitary tumors were also evaluated with [125I-Ac-His1]PACAP-1-27 (2000 Ci/mmol; Anawa) for their receptor subtype specificity; displacement experiments under the same conditions as for VIP receptor autoradiography using increasing concentrations of unlabeled VIP and PACAP-1-27 were performed to differentiate PACAP type I and type II binding sites.8,23
Oligonucleotide Primers and Probes
Oligonucleotide primers and hybridization probes were produced on
a DNA oligonucleotide synthesizer (Applied Biosystems, Foster City, CA)
(Table 1)
. Both primers and probes for
human PACAP,24
PVR-1,25
PVR-2,26,27
and PVR-327,28
were synthesized on the basis of published
sequences and GenBank sequences. The sequences of the oligonucleotides
were checked against the EMBL/GenBank sequence database, and no
significant homology with other published sequences was found.
|
Total RNA extraction was performed by the single-step methods (TRIzol reagent kit, Life Technologies) from 3 nontumorous pituitaries and 35 cases of pituitary adenomas.29,30
First-strand complementary DNA (cDNA) was prepared from total RNA by using a first-stand synthesis kit (Stratagene, La Jolla, CA). The RT reaction was performed at 37°C for 60 minutes in a final volume of 50 µl with 5 µg of total RNA, 300 ng of oligo dT primer, 1X RT buffer, 1.0 mmol/L each deoxyribonucleotide (dATP, dCTP, dTTP, and dGTP), 40 U of RNAse inhibitor, and 50 U of Moloney murine leukemia virus reverse transcriptase. The reaction product was then heated at 95°C for 5 minutes and immediately placed on ice.
The PCR was performed in 100-µl final reaction volumes containing 5 µl of RT reaction product as template DNA, corresponding to cDNA synthesized from 500 ng of total RNA, 1X PCR buffer (Promega, Madison, WI), 1.5 mmol/L MgCl2, 0.2 mmol/L each deoxynucleotide (Boehringer Mannheim, Indianapolis, IN), 300 ng of each sense and antisense primer for PACAP and PVR-1, -2, -3, and 2.5 U of Taq DNA polymerase (Promega). Programmable temperature cycling (Perkin-Elmer/Cetus 480, Norwalk, CT) was performed with the following cycle profile: 95°C for 5 minutes, followed by 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes (30 cycles) for GAPDH and PACAP and 94°C for 1 minute, 60°C for 1 minute and 72°C for 2 minutes (40 cycles) for PVR-1, -2, and -3, respectively. After the last cycle, the elongation step was extended at 72°C for 10 minutes.
A 20-µl aliquot of PCR product was analyzed by gel electrophoresis using a 2% agarose gel and was stained with ethidium bromide. PHx174 DNA/HaeIII digest (Boehringer Mannheim) was used as the standard. The separated PCR products were transferred to nylon membrane filters. Southern hybridization, with a single internal probe that hybridized to regions within the amplified sequences, was performed. Hybridization was performed with 1 x 106 cpm/ml [33P]deoxyadenosine-diphosphate-labeled probe at 42°C for 18 hours. After washing with 6X SSC/0.1% SDS at 23°C for 20 minutes and at 42°C for 20 minutes, autoradiography was performed at -70°C with Kodak Omat-AR film (Eastman Kodak, Rochester, NY) with intensifying screens. In RT-PCR experiments, total RNAs from the human autopsy hypothalamus and non-neoplastic pituitaries were included as respective positive and negative controls for PACAP and PVR-1, -2, and -3.
Immunohistochemistry
Immunostaining for anterior pituitary hormones used the
avidin-biotin peroxidase complex method (Vector Laboratories,
Burlingame, CA). Primary antibodies against human anterior pituitary
hormones included GH (1:1000 dilution), PRL (1:1000), LH-ß (1:500),
FSH-ß (1:500), and thyroid-stimulating hormone (TSH)-ß (1:1000),
all rabbit polyclonal and obtained from the National Pituitary Agency,
Bethesda, MD. Rabbit polyclonal ACTH (1:1000) was from Dako Corp.,
Santa Barbara, CA. The monoclonal antibody to the -subunit of
glycoprotein hormones (1:250) was purchased from Biogenex (San Ramon,
CA). Chromogranin A antibody (LK2H 10, 1:1000) was produced in our
laboratory, as previously described.31
The reaction
products were visualized by 3,3'-diaminobenzidine tetrahydrochloride.
ISH
A cocktail of oligonucleotide probes for PACAP and for PVRs were labeled with digoxigenin-deoxyuridine 5-triphosphate (Boehringer Mannheim) by terminal deoxyribonucleotidyl transferase reaction, as previously reported.31 The ISH procedure was performed as described previously.31,32 In brief, the sections were treated with 1 µg/ml proteinase K (Boehringer Mannheim) at 23°C for 10 minutes, followed by heat treatment, hydrochloride treatment, acetylation, and then prehybridization. Thereafter, the sections were hybridized with 1 ng/ml cocktail probe at 42°C for 18 hours. After hybridization, immunodetection was performed using antidigoxigenin at a 1:500 dilution (Boehringer Mannheim). The reaction product was visualized by nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Life Technologies). Control experiments were carried out using internal sense probes.
CARD-ISH
The CARD-ISH technique was modified (GenPoint kit, Dako, Carpinteria, CA) to detect mRNA and performed according to a modified protocol developed in our laboratory. The PACAP and PVR-1, -2, and -3 probes were the same as those used for ordinary ISH. A cocktail of oligonucleotide probes for PACAP and for PVR were labeled with biotin-11-dUTP (Boehringer Mannheim) by terminal deoxyribonucleotidyl transferase reaction, as previously reported.31 Target retrieval was performed by heating frozen tissue sections in 10 mmol/L citric acid (pH 6.0) in a microwave oven for 5 minutes (up to 95°C) and digesting with 1 µg/ml proteinase K at 23°C for 10 minutes. Sections were then treated with 0.2 N HCL for 20 minutes followed by incubation with 0.25% (v/v) acetic anhydride in triethanolamine for 10 minutes.
To reduce background staining, slides were immersed in 3% H2O2 in methanol for 30 minutes and covered with prehybridization buffer for 1 hour at room temperature. Thereafter, the sections were hybridized with 1 ng/µl cocktail probe at 42°C for 18 hours. After stringent washing for 10 minutes at 42°C, the complexes were amplified with primary streptavidin/horseradish peroxidase complex (1/400) for 15 minutes, biotinyl/tyramide solution (1/2), for 15 minutes, and secondary streptavidin/horseradish peroxidase (1/2) for 20 minutes with fresh 1X Tris-buffered saline/Tween 20 washes being performed after each step. The reaction product was visualized by developing the slides in diaminobenzidine chromogen/H2O2 solution for 5 minutes. Frozen sections of human hypothalamus were used as positive controls. Additional controls included 1) using sense probes for negative control and 2) omission of the biotinyl-tyramide amplification during CARD-ISH.
Grading of the ISH and CARD-ISH was based on signal intensity as follows: -, negative; 1+, weak; 2+, moderate; 3+, strong, with more than 5% of the cells staining to be considered as positive.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The results of the in vitro receptor autoradiography
are summarized in Table 2
. Comparison of
the same tumors analyzed by receptor autoradiography and for mRNA
expression using aliquots of the same tumors showed similar results.
Other than PRL adenomas, the majority of pituitary adenomas had PACAP
binding sites. 125I-labeled VIP binding was particularly
high in gonadotroph and null cell adenomas, GH adenomas, and ACTH
adenomas. 125I-labeled VIP binding was characterized by
high-affinity displacement by VIP and by the 27-amino-acid form of
PACAP (PACAP-1-27), suggesting the presence of the PACAP type II
binding site. Figure 1
shows the high
density of those receptors in a GH tumor, an ACTH tumor, and a
gonadotroph and null cell tumor. In all cases, 125I-labeled
PACAP receptor autoradiography was performed to evaluate the presence
of the PACAP type I binding sites as well. Whereas a high-affinity
displacement by PACAP-1-27 was seen in all cases,
125I-labeled PACAP was displaced by VIP in most instances
in a biphasic manner, with a high- and a low-affinity component,
suggesting the presence of both PACAP types I and II binding sites.
Figure 2
shows a GH tumor and gonadotroph
and null cell tumor with 125I-labeled PACAP binding fully
displaced by 100 nmol/L PACAP but only partly displaced by 100 nmol/L
VIP. Competition curves with 125I-labeled PACAP showed the
high- and low-affinity site of VIP binding compared with the single
high-affinity site of PACAP binding (data not shown).
|
|
|
Results of RT-PCR and Southern hybridization studies are shown in
Tables 2 and 3
as well as in Figure 3
. Analysis of PACAP mRNA and PVR-1, -2,
and -3 mRNAs demonstrated the expected 317-bp, 303-bp, 324-bp, and
584-bp PCR products in all positive specimens tested. PACAP mRNA was
detected in the hypothalamus and in three non-neoplastic pituitaries
but not in any of the 35 pituitary adenomas analyzed (Figure 3)
. PVR-1,
-2, and -3 mRNAs were detected in the hypothalamus and in three
non-neoplastic pituitaries. PVR-1 mRNA was detected in
gonadotropin-secreting adenomas, null cell adenomas, GH adenomas, and
ACTH adenomas. However, it was detected in only one of six
prolactinomas (Table 3)
. PVR-2 mRNA was demonstrated in most adenomas
(Table 3)
whereas PVR-3 mRNA was detected in most adenomas except for
prolactinomas where it was lacking in five of six tumors (Table 3)
.
|
|
Conventional ISH with a digoxigenin-labeled PACAP oligonucleotide probe showed strong staining for PACAP in hypothalamic neurons and weak focal staining in the posterior but not in the anterior pituitary gland. All adenomas examined for PACAP were negative.
On conventional ISH, PVR mRNAs was detected in the cytoplasm of both
non-neoplastic pituitary cells and hypothalamic neurons (data not
shown). PVR-1, -2, and -3 mRNAs were weakly positive in some adenomas
(Table 3)
. To enhance detection of these receptor mRNAs, CARD-ISH was
used. The specificity of the CARD-ISH was checked by using a sense
probe (Figure 4)
and by omitting the
biotinylated tyramide, both of which resulted in absent or weak
staining.
|
. PVR
mRNAs were detected in many adenomas that were negative by conventional
ISH. The signal intensity was increased for PVR mRNAs by CARD-ISH;
moderate to strong signals were observed in tissues that expressed only
weak or absent signal by conventional ISH. A positive signal for PVR
mRNAs was usually present in most adenoma cells (Figure 4)
. PVR-1 and
PVR-3 were usually negative in prolactinomas, but PVR-2 mRNA was
moderately to strongly positive by CARD-ISH. In GH, ACTH, gonadotropin,
and null cell adenomas, PVR-1, -2, and -3 mRNAs were consistently
present by CARD-ISH. The results with RT-PCR were similar to those with
CARD-ISH (Table 3)
. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
VIP binding sites have been designated as PACAP type II binding sites, and these sites share two receptors, including PVR-2 and PVR-3.12 PVR-3 (VIP2R) was recently cloned.27,28,33 PVR-2 (VIP1R) and PVR-3 (VIP2R) have distinct distributions in the central nervous system, with high levels of PVR-2 (VIP1R) mRNA in cortex, hippocampus, hypothalamus, and cerebellum34 and PVR-3 (VIP2R) in the hippocampus, thalamus, and the suprachiasmatic nucleus. The distribution of VIP binding sites in the central nervous system, determined by autoradiography, is consistent with the combined distributions of the PVR-2 (VIP1R) and PVR-3 (VIP2R) mRNAs.33 In previous studies using binding assay and adenylate cyclase stimulation,16 it was suggested that VIP reacted mainly with PVR-3 (VIP2R) and that either VIP is inactive in prolactinomas or PVR-2 mRNA in prolactinomas is not a functional receptor. As VIP has been shown to play a role in the regulation of PRL release from lactotrophs,35,36 our results suggest that PRL release from lactotrophs may be regulated mainly via PVR-2, but not PVR-3.
Solution RT-PCR and Southern hybridization analyses showed PACAP mRNA to be expressed in the hypothalamus, only weakly in non-neoplastic pituitary, and not at all in pituitary adenomas. These results were confirmed by ISH, in which hypothalamic neurons were strongly positive for PACAP mRNA, posterior pituitaries were only focally and weakly positive, and the anterior pituitary and the spectrum of pituitary adenomas were negative for PACAP. Our results are in agreement with previously published immunoassay studies.37,38
In the present study, RT-PCR and Southern hybridization analyses showed PVR-1 mRNA to be strongly expressed in gonadotroph and null cell adenomas and variably expressed in GH and ACTH adenomas but not expressed in prolactinomas. PVR-2 mRNA was strongly expressed in all pituitary adenomas, including prolactinomas. PVR-3 mRNA was strongly expressed in most adenomas but was usually negative in prolactinomas. There was generally good agreement between the receptor autoradiography binding studies and RT-PCR and ISH studies. Detection of mRNA for PVR-2 in prolactinomas was more frequent than protein binding by receptor autoradiography. This may reflect low levels of mRNA amplified by RT-PCR, which may not translate into functional receptor proteins, as has been shown previously for somatostatin receptors in exocrine pancreatic cancers.39
PACAP and VIP have multiple functions in a variety of tissues.9,40 For instance, it has been suggested that they act as hypothalamic hormones controlling anterior pituitary cell function. In support of this notion is the demonstration of PACAP- and VIP-immunoreactive neurons within the median eminence41,42 and specific binding sites for PACAP have been found on anterior pituitary cell membranes.43
To date, only a few studies of PVRs in human pituitary adenomas have
been published.16,17,44
One study using RT-PCR indicated
that PVR-1 mRNA was highly expressed in all adenomas except
prolactinomas.44
Another report of the effects of PACAP on
hormone secretion demonstrated that PACAP-38 had a modest role in the
regulation of GH, ACTH, and -subunit secretion from some tumorous
pituitary corticotrophs and somatotrophs.17
Using a binding
assay and adenylate cyclase assays, Robberecht et al reported that
PACAP-27 and PACAP-38 stimulated adenylate cyclase activity equally
well and in all pituitary adenomas except prolactinomas.16
The expression of PVR-3 in human pituitary adenomas has not been
previously reported, so our study links the expression of PVR-3 mRNA to
specific pituitary adenoma subtypes. A schematic summary of our
findings on the differential expression of PACAP and PACAP binding and
PVR-1, -2, and -3 is shown in Figure 5
.
|
With the CARD-ISH technique, mRNAs were readily detected in individual cells, and the signal intensity was increased when compared with conventional ISH. One advantage of performing CARD-ISH with nonisotopic probes is the excellent resolution obtained with biotin labeling after amplification by tyramide. This technique may approach the sensitivity of RT-PCR. With this increase in sensitivity, an in situ CARD technique has some advantage over RT-PCR or in situ RT-PCR, including reproducibility and ease of performance of the assay.18,19
In summary, these studies show a differential distribution of PACAP binding sites and PVR-1, -2, and -3 mRNA expression in pituitary adenomas. The differences observed, especially with regard to prolactinomas, probably reflect different regulatory roles of PVR in these tumors. The high levels of PVR in gonadotroph and null cell adenomas indicate the importance of the cAMP regulatory system in these tumors.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported in part by National Institutes of Health grant CA 42951, by grants-in-aid for scientific research (07670219 and 08671611) from the Ministry of Education, Science, and Culture, Japan, and by a Parents' Association grant from Kitasato University, School of Medicine, Japan.
Accepted for publication September 3, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain of Gs and stimulate adenylyl cyclase in human pituitary tumors. Nature 1989, 340:692-696[Medline]
This article has been cited by other articles:
 |
|
 |
|
  A. O. L. Wong, W. Li, C. Y. Leung, L. Huo, and H. Zhou Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp. I. Functional Coupling of Cyclic Adenosine 3',5'-Monophosphate and Ca2+/Calmodulin-Dependent Signaling Pathways in PACAP-Induced GH Secretion and GH Gene Expression in Grass Carp Pituitary Cells Endocrinology, December 1, 2005; 146(12): 5407 - 5424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Schulz, C. Rocken, C. Mawrin, W. Weise, V. Hollt, and S. Schulz Immunocytochemical Identification of VPAC1, VPAC2, and PAC1 Receptors in Normal and Neoplastic Human Tissues with Subtype-Specific Antibodies Clin. Cancer Res., December 15, 2004; 10(24): 8235 - 8242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Reubi Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy Endocr. Rev., August 1, 2003; 24(4): 389 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Mazzocchi, L. K. Malendowicz, P. Rebuffat, L. Gottardo, and G. G. Nussdorfer Expression and Function of Vasoactive Intestinal Peptide, Pituitary Adenylate Cyclase-Activating Polypeptide, and Their Receptors in the Human Adrenal Gland J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2575 - 2580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Reubi, U. Laderach, B. Waser, J.-O. Gebbers, P. Robberecht, and J. A. Laissue Vasoactive Intestinal Peptide/Pituitary Adenylate Cyclase-activating Peptide Receptor Subtypes in Human Tumors and Their Tissues of Origin1 Cancer Res., June 1, 2000; 60(11): 3105 - 3112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Vaudry, B. J. Gonzalez, M. Basille, L. Yon, A. Fournier, and H. Vaudry Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: From Structure to Functions Pharmacol. Rev., June 1, 2000; 52(2): 269 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Oka, L. Jin, E. Kulig, B. W. Scheithauer, and R. V. Lloyd Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Transforming Growth Factor-{beta}1-Induced Apoptosis in a Human Pituitary Adenoma Cell Line Am. J. Pathol., December 1, 1999; 155(6): 1893 - 1900. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
