Immunocytochemical Finding of the Amidating Enzymes in Mouse

Volume 50(10): 1401–1415, 2002
The Journal of Histochemistry & Cytochemistry
http://www.jhc.org
ARTICLE
Immunocytochemical Finding of the Amidating Enzymes
in Mouse Pancreatic A-, B-, and D-cells: A Comparison with
Human and Rat
Oihana Garmendia, Maria P. Rodríguez, Maria A. Burrell, and Ana C. Villaro
Department of Cytology and Histology, University of Navarra, Pamplona, Spain
S U M M A R Y -Amidation is catalyzed by two enzymatic activities, peptidyl-glycine -hydroxylating mono-oxygenase (PHM) and peptidyl- -hydroxyglycine -amidating lyase (PAL), denoted collectively as peptidyl-glycine -amidating mono-oxygenase (PAM), which also may
include transmembrane and cytoplasmic domains. PAM is present in mammalian pancreas,
where it appears to be abundant in the perinatal period. Nevertheless, there is no agreement on the cell type(s) that produces PAM or even on its presence in adults. In the present
study we found PAM (PHM and cytoplasmic domain) immunoreactivity (IR) in A-, B-, and
D-cells of adult mouse pancreas. In contrast to previous reports, PAM IR was found in B-cells
of human and rat. Most of the B/D-cells were PAM immunoreactive, although with variable
intensity, whereas less than half of A-cells displayed IR. Immunocytochemistry and Western
blotting suggested the existence of different PAM molecules. Differences in the cellular
distribution of IR for PAM domains were also observed. Whereas PHM-IR was extended
throughout the cytoplasm in the three cell types, presumably in the secretory granules, IR
for the cytoplasmic domain in A/D-cells was restricted to a juxtanuclear region, perhaps indicating its cleavage in Golgi areas. Although glucagon, insulin, and somatostatin are nonamidated, amidated peptides (glucagon-like peptide 1, adrenomedullin, proadrenomedullin N-terminal 20 peptide) were found in the three cell types.
(J Histochem Cytochem 50:1401–1415, 2002)
Regulatory peptides are generated from larger precursors via a variety of post-translational modifications (Sossin et al. 1989), one of which is the amidation of the peptide carboxy-terminal amino acid.
More than half of the known peptide hormones of
mammals are amidated (Eipper et al. 1992b; Prigge et
al. 2000). In addition to resistance to carboxypeptidase degradation, amidation frequently confers biological activity to the peptide (Cuttitta 1993; Merkler
1994).
The formation of an amidated peptide from its glycine-extended propeptide requires an enzyme complex
denoted collectively as peptidyl-glycine -amidating
Correspondence to: Oihana Garmendia, Dept. of Cytology and
Histology, University of Navarra, 31080 Pamplona, Spain. E-mail:
[email protected]
Received for publication September 4, 2001; accepted May 15,
2002 (1A5630).
© The Histochemical Society, Inc.
0022-1554/02/$3.30
KEY WORDS
amidating enzyme
PAM
mouse
human
rat
pancreas
immunocytochemistry
Western blotting
mono-oxygenase (PAM) (Eipper et al. 1983). -Amidation is a two-step process catalyzed by two separate
enzyme activities, peptidyl-glycine -hydroxylating
mono-oxygenase (PHM) and peptidyl--hydroxyglycine -amidating lyase (PAL), both encoded by the
same gene, which has been cloned and appears to be
highly conserved among different species (Eipper et al.
1987). The PHM enzyme is contained in the amino-terminal third of the PAM precursor (Figure 1), followed
by the PAL enzyme (Glauder et al. 1990; Stoffers et al.
1991; Milgram et al. 1992). The carboxy-terminal third
of the molecule encodes a transmembrane domain and
a hydrophilic cytoplasmic domain (Eipper et al. 1987).
The diversity found in PAM enzymes is due to tissuespecific PAM mRNA alternative splicing and/or posttranslational proteolysis, which yields different bifunctional and monofunctional enzymes as well as soluble
and membrane-bound forms (Eipper et al. 1992a; Milgram et al. 1992).
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Garmendia, Rodríguez, Burrell, Villaro
Figure 1 PAM precursor and antisera
used in the study.
The amidating enzymes have been identified in diverse tissues of mammalian species, such as heart, neurons, and glial cells, and in several exocrine and endocrine glands (Sakata et al. 1986; Eipper et al. 1988;
Ouafik et al. 1989; May et al. 1990; Rhodes et al.
1990; Braas et al. 1992; Schafer et al. 1992; Martinez
et al. 1993a).
In relation to the presence of PAM in endocrine tissue, only a few studies refer to the pancreas. Most of
them show evidence of PAM existence in pancreatic
tissue extracts, and only two studies, carried out in
human (Martinez et al. 1993b) and rat (Braas et al.
1992), used immunocytochemical methods to detect
the amidating enzymes in situ. PAM activity has been
described biochemically in the pancreas of several
mammalian species, including rat (Sakata et al. 1986;
Ouafik et al. 1987; Scharfmann et al. 1988; Maltese et
al. 1989; Zhou and Thorn 1990), sheep (Kapuscinski
and Shulkes 1995), and human (Tateishi et al. 1994).
In such biochemical studies, a maximum of PAM enzymatic activity is generally reported in neonatal specimens. Moreover, some of the studies point out that in
adult pancreas the enzyme PAM is almost undetectable in rat (Maltese et al. 1989). Nevertheless, the two
Table 1 PAM antisera used in the study
rendering immunolabelinga
Domain
Code
PHM(1)b 1764c
PHM(3)b CC
32233 “4”d
PAL
PAL2
32240 “4”d
CD
6E6c
a P,
Specificity Type Sequence Dilution Microwave
Rat
Human
P
P
37–382
288–310
1:2000
1:1000
No
No
Human
P
527–546
1:1000
Yes
Rat
M
928–945
1:2000
Yes
polyclonal; M, monoclonal; CD, cytoplasmic domain.
Eipper (Johns Hopkins University School of Medicine, Baltimore, MD).
c A.M. Treston (National Cancer Institute, Rockville, MD).
d Internal numbering per Guembe et al. (1999).
b B.A.
immunocytochemical studies that detected PAM immunoreactivity (IR) in human and rat pancreas were
carried out in adult specimens (Braas et al. 1992; Martinez et al. 1993b).
On the other hand, there is no agreement concerning
the cell types that express the amidating enzyme. Some
of the biochemical studies locate PAM activity in endocrine islets and suggest its presence in -cells (Ouafik et
al. 1987; Scharfmann et al. 1988; Maltese et al. 1989).
Immunocytochemical studies have described the presence of PAM in pancreatic islets, namely in human
A-cells (Martinez et al. 1993b) or in rat “peripheral”
(interpreted as A/PP) cells (Braas et al. 1992).
The aim of the present study was the immunocytochemical study of the enzyme PAM in a third mammalian species (mouse), to address the two aboveTable 2
Antisera against pancreatic and amidated peptidesa
Peptide
Glucagon
Insulin
Somatostatin
Somatostatin
PP
GLP1
AM
PAMP
Gastrin
PHI
Pancreastatin
BET
Met-ENK
Amylin
Type
M
M
P
M
P
P
P
P
M
P
P
P
P
P
Source
Sigmab
Sigma
RPMSc
Biogenesis
ICN
RPMS
NCI
NCI
CURE/UCLA
RPMS
RPMS
RPMS
Incstard
RPMS
Code
G2654
I2018
1460
8330–0496
G4711-1
1167
2075
2336
93
1250
1671
2086
20065
1973
Dilution IR Microwave
1:4000
1:2000
1:2000
1:1000
1:1600
1:2000
1:1000
1:1000
1:60000
1:500
1:1000
1:2000
1:5000
1:500
No
No
No
Yes
No
No
Yes
Yes
Yes
No
No
No
No
No
a PP, pancreatic polypeptide; GLP, glucagon-like peptide; AM, adrenomedullin; PAMP, pro-adrenomedullin N-20 terminal peptide; PHI, peptide histidine
isoleucine; BET, big endothelin; ENK, enkephalin; P, polyclonal; M, monoclonal.
b Sigma Biosciences (St Louis, MO).
c RPMS (Royal Postgraduate Medical School, Hammersmith Hospital, UK).
d Incstar (Stillwater, MN).
Amidating Enzymes (PAM) in Mammalian Pancreas
mentioned controversial aspects: the presence of the
enzyme PAM in adult mammalian pancreas and the
cell type(s) that produces the enzyme.
Materials and Methods
For immunocytochemical methods, 24 female Swiss mice
were sacrificed by cervical dislocation and their pancreata
fixed, embedded in paraffin or resin, and cut into sections.
Because immunocytochemical staining may vary according
Figure 2 (A) In mouse pancreas,
PAM-IR cells are mostly found in endocrine islets. Occasionally they are
present in acini (arrow, detail in B)
and ducts (arrowhead, detail in C).
Original magnifications A 125; B,C
1250.
Figures 3 and 4 Absorption controls
of PAM in serial reversed-face paraffin sections of adult mouse pancreas.
PHM (3A) and cytoplasmic domain
(4A) immunolabeling is abolished using the preabsorbed antisera (3B,4B).
Original magnifications 125, 250.
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to tissue processing, different processing protocols were carried out. Pancreata embedded in paraffin were previously
fixed in either Bouin for 24 hr (10 pancreata), Zamboni for
24 hr (two pancreata), or PAF 4% for 4 hr (two pancreata).
Before resin embedding, either Zamboni fixative for 24 hr
(five pancreata) or PAF 4%–glutaraldehyde 1% fixative for
2 hr (five pancreata) was used. Paraffin- and resin-embedded
material was cut into 4-m and 1-m sections, respectively.
Bouin- and formol-fixed rat and human pancreata were used
as controls.
For Western blotting analysis, five female Swiss mice
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were sacrified by cervical dislocation and their pancreata
frozen in liquid nitrogen and stored at 80C.
Immunocytochemical staining was performed using the
avidin–biotin complex (ABC) (Hsu et al. 1981) and EnVision Plus complex (DAKO; Carpinteria, CA) techniques.
Both PAM (Table 1) and various pancreatic and amidated
peptides (Table 2) antisera were used. To analyze co-localization of the amidating enzymes and pancreatic hormones,
two immunocytochemical methods were employed: the ABC
complex technique in serial sections and double immunostaining on the same section using the ABC and alkaline
phosphatase–anti-alkaline phosphatase (APAAP; Mason
and Sammons 1978) techniques.
ABC Complex Technique
Endogenous peroxidase was blocked by treatment with 3%
hydrogen peroxide for 10 min. To avoid nonspecific background, the sections were incubated with normal swine or
rabbit serum (1:20; University of Navarra) for 30 min before
overnight incubation with the specific primary antibody at
4C. Sections were then incubated for 30 min with biotinylated
swine anti-rabbit (K353; Dakopatts, Glostrup, Denmark) or
rabbit anti-mouse (K354; Dakopatts) immunoglobulins, followed by incubation with the avidin–biotin–peroxidase
complex (K355, Dakopatts) for 30 min. Sections were
washed in TBS (Tris buffer 0.05 M, NaCl 0.5 M, pH 7.36)
after each incubation. The bound antibodies were visualized
with 3-3 diaminobenzidine tetrahydrochloride (DAB, D-3637;
Sigma, St Louis, MO) in sodium acetate/acetic acid 0.1 M pH
5.6, containing 2.5% nickel ammonium sulfate, 0.2% -d-glucose, 0.04% ammonium chloride, and 0.001% glucose oxidase (Shu et al. 1988). The sections were then counterstained
with hematoxylin, dehydrated, and mounted in DPX.
EnVision Complex Technique
Endogenous peroxidase was blocked with the DAKO EnVision system reagent 1. Primary antibody was added and incubated overnight at 4C. Sections were rinsed in TBS and incubated for 30 min with the labeled polymer (HRP, reagent
2). The bound antibodies were visualized as explained for
the ABC method.
Garmendia, Rodríguez, Burrell, Villaro
Microwave Pretreatment
When required for antigen retrieval, sections were incubated
for 15 min in citrate buffer 0.01 M, pH 6.0, at full microwave heating power, followed by a second incubation in the
same buffer at half power (Tables 1 and 2).
Specificity Controls
Human and rat pancreata were used as positive controls.
Absorption controls for PAM and pancreatic peptide-immunoreactive antibodies were performed in mouse. In human
and rat, absorption tests for PAM antisera were also carried
out. Antisera that rendered immunoreaction were preincubated overnight with their respective synthetic antigens (Table 3). Preabsorbed antibodies were then used for immunocytochemistry.
Western Blotting
Pancreata were homogenized in a buffer containing 10 mM
Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxicholate, 0.1% SDS, and 5 mM EDTA. Tissue final protein
concentration was determined (BCA kit; Pierce, Rockford,
IL) after ultracentrifugation. Thirty g protein was heated
to 70C for 10 min and loaded into the sample well. Tissue
protein extracts were electrophoretically separated on a gradient Nu PAGE 3–8% Tris-acetate gel (Novex; San Diego,
CA) and run at 150 V for 1 hr under reducing (5% -mercaptoethanol) and non-reducing conditions. Transfer blotting was accomplished in the same apparatus equipped with
a titanium plate electrode and transferred to a polyvinyldifluoride membrane (Inmovilon PVDF; Millipore, Bedford,
MA) at 30 V for 1 hr. The blots were saturated with 5% (w/v)
non-fat milk in PBS for 1 hr and incubated overnight in a
1:1000 dilution of rabbit anti-PHM or rabbit anti-CD in
PBS. After washing three times in PBS–Tween, they were incubated for 1 hr with peroxidase-conjugated anti-rabbit or
anti-mouse antibody at 1:5000 (Amersham Life Science; Arlington Heights, IL) and washed again in PBS–Tween. The
immune complexes were detected with Lumi-Light Plus
Western Blotting substrate (Roche Diagnostics; Indianapolis, IN) according to the supplier’s instructions.
Results
Distribution of PAM IR in Mouse Pancreas
Double Immunostaining
Sections were incubated overnight with a mixture of the two
primary antibodies. The second layer consisted of a mixture
of biotinylated swine anti-rabbit and non-biotinylated goat
anti-mouse immunoglobulins. After rinsing in TBS, optimally diluted ABC and APAAP complexes were added for
30 min and then re-incubated in non-biotinylated rabbit
anti-mouse immunoglobulins and APAAP complexes for 10
more min each. The APAAP bound antibodies were visualized in red with previously hexazoted new fuchsin (Sigma;
638) in 4% HCl 2 M and 1 mg/ml naphthol solution in 0.2 M
Tris-HCl (pH 9.2). The ABC bound antibodies were visualized in black as we have previously described, after rinsing
the sections in water and sodium acetate/acetic acid 0.1 M,
pH 5.6. The sections were finally mounted in PBS:glycerol
(1:1).
PAM-IR was abundant in the pancreas of adult mice.
Immunolabeling was observed mostly in islet cells, but
immunostained cells were also found scattered within
acini and ducts (Figure 2).
Table 3
Antisera and peptides used in absorption controls
Antiserum
PHM1
PHM3
CD
GLP1
AM
PAMP
a Internal
Dilution
Peptidea
Source
Concentration (nmol/ml)
1:1000
1:1000
1:2000
1:2000
1:2000
1:2000
P61
P66
P130–131
P20
P53
P60
RPMS
NCI
UNb
Sigma
NCI
NCI
1–10
2
1–10
10
1–10
1–10
numbering.
of Navarra, Spain.
b University
Amidating Enzymes (PAM) in Mammalian Pancreas
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Figure 5 (A) PAM (PHM1)-IR in human endocrine islets. (B) Absorption control in serial reversed-face paraffin section. Original magnifcation 125.
Figure 6 Human pancreas. PHM1 immunostaining (A) co-localizes with insulin-IR (B). Original magnification 250.
Figure 7 (A) PAM (PHM1)-IR in rat endocrine islets. (B) Absorption control in serial reversed-face paraffin section. Original magnification 125.
Figure 8 Rat pancreas. PHM1-immunostaining (A) co-localizes with insulin-IR (B). Original magnification 250.
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Figures 9–11 PAM-IR in mouse B-cells.
Garmendia, Rodríguez, Burrell, Villaro
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Amidating Enzymes (PAM) in Mammalian Pancreas
Immunostaining was obtained with antisera raised
against the PHM enzyme region and the hydrophilic
cytoplasmic domain of PAM (Table 1; Figures 3A and
4A). As will be made clear, immunolabeling for the
cytoplasmic domain of PAM was variable according
to the method of fixation. Preabsorption of the immunoreactive antisera against PHM and the cytoplasmic
domain of PAM with their corresponding antigens
(Table 3) abolished immunoreactivity (Figures 3 and
4). Although IR was also observed with the antisera
against the PAL domain (data not shown), absorption
tests did not confirm the specificity of immunoreaction.
Human and rat pancreata, used as positive controls, also displayed abundant PAM-IR (Figures 5–8).
Preabsorption tests confirmed the specificity of staining (Figures 5 and 7).
Identification of the PAM-producing Cell Types
In mouse pancreas, co-localization studies of PAM and
the classical pancreatic regulatory peptides showed that
three cell types [B-cells (insulin), A-cells (glucagon),
and D-cells (somatostatin)] displayed PAM-IR. For
D-cells, immunoreaction was found only in a few specimens. Co-localization of the three peptides and PAMIR was observed in serial sections of both paraffinand plastic-embedded material (B-cells, Figures 9–11;
A-cells, Figures 12–14; D-cells, Figures 11 and 12) and
by double immunolabeling techniques (Figures 18–
20). On the contrary, no IR for PAM was found in PP
cells (Figure 17).
Most, if not all, mouse B-cells were positive for
PAM (Figures 9–11 and 14), although with different
degrees of intensity (Figure 9). When obtained, PAMIR was also present in most D-cells (Figure 15). On
the contrary, only a subpopulation of A-cells was
PAM-immunoreactive (Figure 12).
In human and rat pancreata, in addition to the expected PAM IR in A-cells, immunostaining was also
demonstrated in B-cells (Figures 6 and 8).
Cellular Distribution and Pattern of PAM Staining
PAM Staining Pattern of Mouse A-, B-, and D-cells.
Mouse endocrine cells positive for PAM were not immunoreactive for the same PHM antisera (Table 4). In
addition, differences concerning the cellular distribution of CD staining were also observed.
PHM Staining. In the three cell types, PHM immunolabeling extended throughout the cell (Table 5). Un-
expectedly, although A-, B-, and D-cells were stained
with PHM antisera, the three cell types did not show
the same IR pattern. A-cells were labeled with the antiserum PHM3 but not with PHM1, whereas B- and
D-cells showed the opposite IR pattern, (PHM1/
PHM3) (Figures 1 and 20). Such staining patterns
did not change with different processing procedures.
As discussed below, the existence of different molecular forms of PAM in mouse A- and B-cells might explain the differences in immunoreaction.
Cytoplasmic Domain Staining. In addition to extended PHM-IR, A- and D-cells showed cytoplasmic
areas immunoreactive for the cytoplasmic domain of
PAM near the nucleus (Figures 14 and 16). Both positive and negative A- and D-cells were observed for this
antiserum, but bearing this localized staining pattern
in mind, the lack of IR in some A/D-cells might be due
to sectioning artifact. Whereas in A- and D-cells immunolabeling was obtained with all fixatives used, B-cells
rendered only weak IR for the cytoplasmic domain of
PAM in Zamboni-fixed material. In this case, the
staining pattern was not juxtanuclear but extended
throughout the cytoplasm of B-cells (Figure 11; Tables
4 and 5).
PHM Western Blotting. Pancreatic extracts were analyzed by Western blotting using both PHM1, PHM3,
and cytoplasmic domain antisera (Figure 23). Western blotting analysis revealed immunoreactive bands
from about 47 to 96 kD. Most of the mouse bands
were immunoreactive for a single PHM antiserum:
PHM1 (three bands of 62, 66 and 74 kD) or PHM3
(four bands of 53, 57, 60, and 96 kD). Only a 50-kD
band displayed immunoreactivity for the two PHM
antisera. Nevertheless, Western blotting does not discriminate whether such immunoreactive bands correspond to the same molecule or to different proteins of
the same weight. In relation to CD, a band of approximately 46 kD was also immunoreactive for CD.
PHM3 bands of 57 and 96 kD also seem to coincide
in weight with a CD-immunoreactive band.
Co-localization of PAM and Amidated Peptides
Because neither glucagon, insulin, or somatostatin are
amidated, some amidated peptides (Table 2) were immunocytochemically investigated to determine the
possible functional significance of PAM enzymes in
mouse A-, B-, and D-cells. The study revealed that, in
the three cell types, at least one amidated peptide is
Figures 9 and 10 PHM-IR (A) is present in most B-cells (B), although displaying different intensities (arrows).
Figure 11 Cytoplasmic domain-IR (A) is also found in some B-cells (B) in Zamboni-fixed pancreas. Figures 9 and 11, serial reversed-face paraffin sections. Original magnification 500, 250. Figure 10, serial plastic sections. Original magnification 500.
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Garmendia, Rodríguez, Burrell, Villaro
Figures 12–14 PAM-IR in mouse A-cells (arrows). PHM-IR (Figures 12A and 13A) and cytoplasmic domain-IR (Figure 14A) are present in a
subpopulation of glucagon-IR A-cells (Figures 12B, 13B and 14B). Note that cytoplasmic domain-IR is restricted to a juxtanuclear area (arrowheads, Figure 14A). Figures 12 and 14, serial reversed-face paraffin sections. Original magnification 500, 1250. Figure 13, serial plastic sections. Original magnification 500.
Amidating Enzymes (PAM) in Mammalian Pancreas
Figures 15–17 PAM-IR in D-cells.
Figure 15 PHM-IR in somatostatin-immunoreactive D-cells (arrows). Serial plastic semithin sections. Original magnification 500.
Figure 16 Cytoplasmic domain-IR (A) is also present in somatostatin-IR cells (B) but is restricted to a juxtanuclear area (arrows).
Figure 17 PHM (A) and PP (B) do not co-localize.
Figures 16 and 17 Serial reversed-face paraffin sections. Original magnification 500.
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Table 4
Garmendia, Rodríguez, Burrell, Villaro
PAM immunoreactivity pattern in mouse islet cells
Pancreatic cell type
PHM1
PHM3
CD
A
B
D
PP
/d
/a
/a, b
c
/a, c
and cells exist.
D-cells may be due to section artifact (see text).
in Zamboni-fixed tissue.
d Only in some specimens.
a Both
b Negative
c Only
present: GLP1 and AM in A-cells, AM and PAMP in
B-cells, and AM in D-cells (Figures 21, 22, and 24).
These amidated peptides co-localize not only with the
corresponding classical pancreatic peptides but also
with the amidating enzymes (Figures 21 and 25). In
the case of the amidated peptide AM, we found different IR intensities throughout the AM population.
Discussion
Presence of PAM in Mammalian Adult Pancreas
Studies about the presence of PAM in mammalian
pancreas, both neonatal and adult, have been carried
out in very few species, with no agreement on its presence in adult specimens. The present study reports the
existence of PAM in adults of a third mammalian species (mouse) by both immunocytochemistry and Western blotting. Although the present report focuses
mainly on mouse pancreas, we also show the presence
of amidating enzymes in more pancreatic mammalian
cell types than were previously described in human
and rat.
In concordance with previous immunocytochemical
studies in human and rat (Braas et al. 1992; Martinez
et al. 1993b), our results show that in mouse the enzyme PAM is produced by the pancreatic endocrine
tissue of adults. Nevertheless, whereas in previous
studies of human and rat few PAM-immunoreactive
cells were reported, in the present study PAM immu-
nolabeling was demonstrated in many cells of mouse,
human, and rat endocrine pancreas. Such differences
are probably due to the different antisera used (see below). The abundance of pancreatic PAM-immunoreactive cells appears to contradict the low levels of
PAM mRNA reported in adult rat pancreas (Maltese
et al. 1989). However, both findings might reflect a
shorter lifespan of PAM mRNA compared with the
translated enzyme, which would remain in the secretory granules for a longer period. In mouse, PAM-IR
is predominantly observed at the islets of Langerhans,
and only occasional scattered immunostained cells
within acini and ducts have been found.
Identification of the PAM-producing Pancreatic
Cell Types
As indicated, previous immunocytochemical studies
localized the enzyme PAM in human A-cells (Martinez
et al. 1993b) and in rat “peripheral” (A/PP) islet cells
(Braas et al. 1992). We have equally demonstrated the
presence of PAM in A-cells of the mouse. However, in
partial disagreement with such previous studies, we
have identified the enzyme not only in mouse A-cells,
but also in B- and D-cells. In fact, concerning the presence of PAM in mammalian B-cells, one of our antisera (PHM1) also rendered immunostaining in B cells
of both human and rat pancreata (Figures 6 and 8).
This difference in present and previous immunocytochemical results (Braas et al. 1992; Martinez et al.
1993b) is probably because of the different antisera
used. In fact, although biochemical studies do not
identify clearly the PAM-producing cell type(s), they
also suggest that B-cells could be responsible for PAM
activity in rat pancreas (Ouafik et al. 1987; Maltese et
al. 1989).
As occurs in human pancreas (Martinez et al.
1993b), PP cells of mouse lacked PAM staining. This
result was unexpected because PP is the only amidated
peptide out of the four classic pancreatic hormones.
However, mouse A-, B-, and D-cells do contain other
Figures 18–20 PAM in A/B-cells.
Figure 18 B-cells (purple) displaying both PHM-1 (black) and insulin (red)-IR
Figure 19 Double IHC was performed in serial reversed-face paraffin sections to demonstrate different IR patterns for PHM antisera of
mouse glucagon (arrow) and insulin (arrowhead) cells. Insulin-IR cells (red in A) are PHM3 (black in A) but PHM1 (black in B). GlucagonIR cells (red in B) are PHM1 but PHM3 (black in A).
Figure 20 A-cells (purple) display both PHM3 (black) and glucagon (red) IR.
Figures 21 and 22 Amidated peptides in mouse endocrine islets.
Figure 21 In A-cells (purple in B) there is a complete co-localization of glucagon (red) and GLP-1 (black). Some of them are also PHM3 (A).
Serial reversed-face paraffin sections.
Figure 22 Some D-cells (arrow) immunoreact for both somatostatin (red) and AM (black). Other types of AM-producing cells are also
present (arrowhead). Double immunohistochemistry in paraffin. Original magnification 500.
Amidating Enzymes (PAM) in Mammalian Pancreas
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Garmendia, Rodríguez, Burrell, Villaro
Table 5
PAM-IR depending on processing methods
CD-IRa
PHM-IR
A
B
D
PP
a Results
All methods
All methods
Plastic
Paraffin
All methods
All fixatives
Zamboni
Bouin
Cell localization
Other fixatives
No data available
All methods
PHM
CDa
Extended
Extended
Extended
Juxtanuclear
Extended
Juxtanuclear
in paraffin; antiserum against the cytoplasmic domain of PAM did not render IR in plastic sections.
amidated peptides. The absence of IR in PP-producing
cells could be explained by amidation taking place in
blood by PAM after secretion, as has already been
suggested (Martinez and Treston 1996) or by the existence of another PAM molecular form that could not
be detected with our antisera. The existence of a different, yet-undescribed amidating enzyme has also
been proposed as an explanation for the lack of PAM
enzymes in this cell type (Martinez and Treston 1996).
Subpopulations of PAM-producing Mouse Pancreatic
Endocrine Cells
The enzyme PAM has been identified in less than half
of mouse A-cells. Conversely, most, if not all, B- and
probably D-cells appear to be immunoreactive for PAM
antisera, although with different degrees of intensity. A
similar description of PAM-producing subpopulations
was reported in pituitary by Steel et al. (1994).
The existence of a PAM-immunoreactive A-cell
subpopulation does not necessarily imply that the rest
of the A-cells cannot synthesize the enzyme. PAM
might be produced in certain functional moments, responding to nutritional or other physiological situations. This might also account for the absence of PAM
in D-cells of certain specimens. In the case of B-cells,
there appears to be basal production of PAM in the
entire population, but the differences observed in the
intensity of staining might also be related to higher or
lower enzymatic production phases. Therefore, in
mouse pancreatic endocrine cells, functional PAMproducing stages may be suggested, which would give
rise to dynamic, changing subpopulations. Such PAMproducing stages may be related to the production of
amidated peptides, as is suggested by the different degree of intensities found in IR for the amidated peptide adrenomedullin (Figures 22 and 25).
chemical assays are needed to ascertain the presence of
monofunctional (only PHM) or bifunctional (PHM/
PAL) amidating enzymes in mouse pancreas. Nevertheless, Western blotting analysis showed the existence of an immunoreactive band around 96 kD (Figure 23), a size large enough to correspond to a
bifunctional amidating enzyme estimated to be at least
75 kD (Eipper et al. 1992a,b). The smaller forms
should correspond to monofunctional PHM proteins.
In the case of the 74-kD PHM1-IR band, it could also
account for a bifunctional enzyme, because molecular
weights are only estimates.
Immunocytochemistry and Western blotting also
showed the existence of CD-IR in mouse pancreas,
suggesting the existence of membrane-bound molecules. According to molecular weights, CD-IR bands
could correspond either to monofunctional or bifunctional enzymes. In addition, soluble molecules (lacking
CD) also appear to exist.
The existence of different molecular forms of PAM
enzymes in mouse pancreatic islets could involve different catalytic properties of each PAM molecular
form, e.g., the specificity for different substrates, as
has been previously described in Lymnaea (Spijker et
al. 1999).
Differences in PHM Immunostaining Pattern of
Pancreatic Endocrine Cell Types
PHM-IR was observed in mouse A-, B-, and D-cells
with all the technical procedures. However, the IR to
different PHM antisera found (PHM3/PHM1 in
Diversity of PAM Enzymes in Mouse Endocrine
Pancreatic Cell Types
Immunocytochemistry and Western blotting analysis
have suggested the existence of different PAM molecules in mouse endocrine pancreas. Immunocytochemistry detected specific IR for PHM and for the
cytoplasmic domain of PAM. However, PAL immunoreactivity is uncertain because preabsorbed PAL antisera still stained the cells. Therefore, further bio-
Figure 23 PHM1, PHM3, and CD Western blotting in mouse pancreas.
1413
Amidating Enzymes (PAM) in Mammalian Pancreas
Figure 24 PAMP-IR (A) is present in
B-cells (B).
Figure 25 AM cells (A) also display
PAM-IR (B). Serial reversed-face paraffin sections. Original magnifications 250, 500.
A-cells; PHM1/PHM3 in B- and D-cells; Table 4)
suggests the existence of different PAM molecular
forms in insular cells, which would be recognized by
different antisera.
Both PHM antisera were expected to recognize the
same cells by immunocytochemistry, given that the sequence against which the PHM3 antibody was raised
(PAM 288–310) is enclosed in the sequence against
which PHM1 was produced (PAM 37–382). However, PHM1 recognized B-cells, whereas PHM3 recognized A-cells (Table 4). This could be explained if
PAM molecules present in A- and B-cells were somehow different, so that each form could only be recognized by one of the antisera but not by the other. Such
molecular differences between mouse A- and B-cell’s
PAM molecules could be due either to different posttranslational processing (e.g., proteolysis, glycosylation, phosphorylation), giving rise to conformational
changes of the protein, or to alternative splicing, as
previously reported in pancreatic carcinoma cells
(Tateishi et al. 1994) and in other tissues (Stoffers et
al. 1989,1991; Zhang et al. 1997).
Furthermore, Western blots showed the presence of
PAM-IR bands with different molecular weights depending on the antiserum used. These differences might
also be explained by alternative splicing of the PAM
gene product or by post-translational processing that
would cause PAM proteins to run differently in the
electrophoresis.
In relation to the cytoplasmic domain of PAM, im-
munolabeling was different in A- and D-cells (obtained with all fixatives) compared to B-cells (weaker
and only in Zamboni-fixed tissue). Although differences in the amounts of enzymes containing the cytoplasmic domain may be considered, the reason for
these differences is unknown. In addition, a different
cellular localization of the IR for the cytoplasmic domain of PAM was obtained (restricted in A/D-cells
and extended in B-cells, as discussed below).
PAM in A- and D-cells
In both cell types, PHM-IR was extended throughout
the cytoplasm (presumably in the secretory granules),
whereas the staining for the cytoplasmic domain of
PAM was localized in a restricted juxtanuclear area.
Such a morphological pattern suggests that the immunostained zones correspond to biosynthetic, presumably Golgi, areas. In view of the different immunolocalization patterns of PHM and the cytoplasmic
domain regions, we suggest that proteolytic cleavage
of cytoplasmic and transmembrane domains may occur in Golgi stacks, yielding soluble PAM enzymes in
mouse A- and D-cells. A similar immunolabeling pattern in Golgi areas of PAM integral proteins (including the cytoplasmic domain of PAM) has been shown
in neuroendocrine transfected AtT-20 cells (Milgram
et al. 1993,1997; Milgram and Mains 1994). These authors also suggested that proteolytic cleavage of integral
domains takes place, yielding soluble PAM enzymes.
1414
PAM in B-cells
Because in this case immunolabeling of both cytoplasmic and PHM domains was extended throughout the
cytoplasm, differences in the biosynthetic pathway of
PAM in mouse B- and A/D-cells appear to exist. Given
that the cytoplasmic domain staining in B-cells is
weaker than that of PHM, both soluble (PHM) and
membrane-bound (PHM/cytoplasmic domain) PAM
molecular forms could exist in the secretory granules.
This would occur if proteolytic cleavage of cytoplasmic and transmembrane domains affected only part of
the enzymatic pool. In fact, Western blotting showed
the existence of a PHM1-IR band with a similar size
(46 kD) to a cytoplasmic domain immunoreactive
one, together with other PHM1-IR bands, presumably
soluble, lacking a cytoplasmic domain.
Functional Significance of PAM in Mouse
Endocrine Islets
Little is known about the significance of PAM in the
endocrine pancreas. Insulin, glucagon and somatostatin
are non-amidated peptides, and therefore the presence
of the enzyme should be related to the presence of
other peptides.
PAM in A-cells. According to our results, the production of GLP-1 and AM could account for the presence of amidating enzymes in mouse A-cells. We have
found a total co-localization of GLP-1 with glucagon,
as previously described in rat (Fridolf et al. 1991).
Consequently, GLP-1 and PHM4 co-exist only in a
subpopulation of A-cells. As indicated, they probably
correspond not to static but to changing PAM-producing groups of cells.
PAM in B- and D-cells. In mouse B-cells, both AM
and PAMP might be substrates for PAM enzymatic activity. Most B (insulin/PAM)-cells are immunoreactive for AM and PAMP, although displaying different
intensities for AM. Our results also show the presence
of AM in D-cells.
The presence of AM has already been reported in
pancreata of both mammalian (Washimine et al.
1995; Martinez et al. 1996), including a short reference in mouse (Cameron and Fleming 1998), and in
non-mammalian (Lopez et al. 1999) species. Its presence in all vertebrate groups appears to indicate that
AM plays an important role in the regulation of pancreatic function, including inhibition of insulin, response to septic shock, and amylase release (Martinez
et al. 1996; Elsasser et al. 1999; Tsuchida et al. 1999).
Previous studies in rat pancreas reported strong
AM-IR in PP cells and a low intensity of AM staining
in the rest of the pancreatic endocrine cell types (Martinez et al. 1996,1998). In the present study, although
we also found different immunostaining intensities in
Garmendia, Rodríguez, Burrell, Villaro
the islets of Langerhans, A-, B-, and D-cells that
stained strongly for AM and also for the amidating
enzyme PAM were observed.
In summary, PAM-IR has been localized to adult
mouse endocrine pancreatic A-, B-, and D-cells, colocalizing with amidated peptides such as GLP-1, AM,
and PAMP. Subpopulations of PAM-producing cells
have been found. Different immunostaining and Western blotting patterns suggest the existence of diverse
PAM molecular forms in mouse pancreatic cells.
Acknowledgments
Supported by the PIUNA (University of Navarra).
We wish to thank Prof F. Cuttitta (National Cancer Institute; Rockville, MD) and Prof B.A. Eipper (Johns Hopkins
University; Baltimore, MD) for the generous gift of some of
the antisera and peptides used in this work.
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