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Immunocytochemical localization of a kallikrein-like serin protease (Esterase A) in rat salivary glands.

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THE ANATOMICAL RECORD 221:475-481(1988)
Immunocytochemical Localization of
a Kallikrein-like Serine Protease (Esterase A)
in Rat Salivary Glands
Department of Anatomy (J.A.V.S., R.F.), Department of Pharmacology (J.C.),and Department
of Biochemistry (L.C.), Medical University of South Carolina, Charleston, SC 29425
Light and electron microscopic (EM) immunocytochemical methods
have been used to localize arginine esterase A, a kinin-generating enzyme immunologically similar to tissue kallikrein, in rat salivary glands. Both polyclonal and
monoclonal antibodies to arginine esterase A were used in these studies. By means
of a polyclonal antiserum, esterase A was found in granular tubules of submandibular glands and in striated ducts of all three major salivary glands, in a distribution
similar to that of tissue kallikrein. With recently developed specific monoclonal
antibodies to esterase A, this enzyme was localized in the granules of some (but not
all) granular convoluted tubule cells (GCT) and along the basal membranes (but not
in apical granules) of striated ducts. By an EM immunoperoxidase method, esterase
A was localized subcellularly in granules of some GCT cells and along the basal cell
membranes of the tubule and duct system. Thus, this enzyme is found in some sites
(GCT granules) shared with tissue kallikrein, but in some unique sites, i.e., basal
membranes of striated ducts. The polyclonal antibody used in the present study
cross-reacted with tissue kallikrein, but when absorbed with kallikrein, it gave the
staining pattern characteristic of monoclonal antibody to esterase A.
Rodent submandibular glands contain several esteropeptidases that are immunologically cross-reactive (Ekfors et al., 1967; Bothwell et al., 1979; Brandtzaeg et al.,
1976; see also Barka, 1980). Kallikrein, the most intensively studied of these enzymes, is present in the granular convoluted tubules of rat and mouse submandibular
glands and at the apex of rat striated duct cells of all
three major salivary glands (Prstavik et al., 1976; Simson et al., 1979,1983).Arginine esterase A, a member of
the kallikrein multigene family of enzymes, is also a
kinin-generating enzyme isolated from rat urine (Chao,
1983)and is present in several tissues (Chao et al., 1984).
It differs from tissue kallikrein in molecular weight and
charge as well as in its response to inhibitors. Hence,
although esterase A and kallikrein are immunologically
cross-reactive and cleave similar substrates, the two
molecules are structurally and functionally distinct. Important questions are: How do these related enzymes
function in the glands, and where are they localized? It
is clear that, although kallikrein and esterase A are
often present in the same tissues, their relative concentrations and hormone responsiveness differ (Chao et al.,
1983, 1984), suggesting functional differentiation and
The present study has been undertaken to localize
esterase A at the light and electron microscopic levels
in rat salivary glands. The underlying assumption is
that the cellular localization of a molecule reflects synthesis, storage, or site of action of that molecule. Both
polyclonal and monoclonal antibodies to arginine esterase A have been utilized to determine the specific cellular localization of esterase A. Several fixation procedures
0 1988 ALAN R. LISS, INC.
were used in initial studies to optimize preservation and
immunostaining of the antigen. Postembedding immunocytochemistry has been performed on material prepared for transmission electron microscopy, in order to
investigate the subcellular localization of esterase A.
Female Sprague-Dawley rats, weighing about 200 gm,
were used for the immunocytochemical studies.
Rat Urinary Esterase A isolation and Antiserum Production
Rat urinary esterase A was purified with ammonium
sulfate fractionation and DEAE-cellulose, aprotinin-affinity, and Sephacryl 5-200 column chromatography
(Chao, 1983).The homogeneity of rat urinary esterase A
used as antigen was determined by polyacrylamide slabgel electrophoresis. The polyclonal antiserum against
purified rat urinary esterase A was raised in rabbits as
follows. Purified rat urinary esterase A in Dulbecco's
phosphate-buffered saline, pH 7.3 (Ca2+, M 2 + free),
was emulsified with an equal volume of Freund's complete adjuvant, and 0.1-ml aliquots were injected subcutaneously at 10 dorsal sites in each of three 6- to 8-lb
male New Zealand rabbits. Then 100 pg of esterase A
was administered to each rabbit. The first booster was
given 1month later, followed by additional boosters at
2-week intervals. Precipitating antibody was found after
Received June 1, 1987; accepted November 4, 1987.
the second booster injection by Ouchterlony double-immunodiffusion analysis. The antibody was shown to have
a Keq of 6.99 x lolo M-' for purified 1251-labeledrat
urinary esterase A and cross-reacted with rat urinary
kallikrein (Chao et al., 1984).
Monoclonal antibodies to rat urinary esterase A were
developed and characterized as described previously for
rat and human tissue kallikrein (Woodley et al., 1985;
Chao et al., 1985). The hybrid cells were selected and
screened for esterase A antibody production by enzymelinked immunosorbent assay (ELISA) as described previously (Chao et al., 1985). Clones producing antibody
were subcloned and ascitic fluid was produced in syngeneic mice. Control ascitic fluid was harvested a s peritoneal fluid from parental myeloma, Sp210-Ag14(Chao et
al., 1983). Monoclonal antibodies from the anti-esterase
A clones (Chao and Chao, 1987) exhibited no cross-reactivity with rat tissue kallikrein or another kallikreinrelated enzyme, tonin (Shih et al., 1986) in radioimmunoassay, ELISA, and Western blot analyses.
Light microscopy
Initial experiments with a variety of fixatives indicated that preservation of both antigenicity and localization of esterase A was optimal with 0.1 M sodium
phosphate-buffered 4% formaldehyde. Some of the tissues were fixed by immersion; others were fixed by
perf'usion via the ascending aorta (Simson et al., 1978).
Fixation times were 30 min or 1hr. Following fixation,
tissues were rinsed in two changes of 70% alcohol, dehydrated through absolute alcohol, cleared in xylene,
and embedded in paraffin. Sections were cut at 5 pm.
Electron microscopy
Tissues for electron microscopy were fixed in either
calcium acetate-buffered 4% formaldehyde (CAF) or a
0.1 M sodium phosphate-buffered, 3% paraformaldehyde-1% glutaraldehyde mixture, pH 7.4-7.6. Tissues
were dehydrated in graded alcohols and propylene oxide, and embedded in epoxy resin. Thin sections (100
nm) of embedded tissue were cut on a Sorvall-Blum MT2 ultramicrotome.
Light microscope immunostaining with polyclonal
antiserum raised in rabbit against rat urinary esterase
A employed a modification of the immmunoperoxidase
bridge technique (Mason et al., 1969). The primary
antibody (rabbit-anti-esterase A) was used at dilutions
ranging from 1:lOO to 1:500. The secondary antiserum
was goat anti-rabbit IgG (1:20 dilution), and the tertiary
antiserum was rabbit anti-horseradish peroxidase (1:20
dilution) (Cappel Laboratories, CooperNorthington,
Malvern, PA). Horseradish peroxidase (HRP, grade IV,
Sigma Chemicals, St. Louis) was added subsequently to
the tertiary antibody. Specific mouse monoclonal antibodies to esterase A were 6C11,5Al0, or F3B9 (Chao and
Chao, 1987),used a t dilutions ranging from 1:25 to 1:250.
The secondary antibody was HRP-conjugated rabbit antimouse immunoglobulin. A color reaction for peroxidase
was demonstrated with 3,3'-diaminobenzidine (Sigma
Chemicals) and HzO2 a s substrates. To enhance and
preserve the immunostaining, sections were subsequently dipped briefly (5-10 sec) in 1%aqueous Os04.
The method used for EM immunostaining was similar
to that described previously for the ultrastructural localization of kallikrein in salivary glands (Simson et al.,
1983). The dilution of the primary antiserum was either
1:1,000 or 1:10,000. The secondary antibody was a goat
anti-rabbit IgG (1:40 dilution) and the third step of the
bridge was a 1:40 dilution of a rabbit peroxidase-antiperoxidase complex (PAP) (Sternberger, 1979). The PAP
complex was obtained from CooperNorthington (Malvern, PA).
Both normal serum controls and absorption controls
were performed. In the normal serum controls, normal
rabbit serum (NRS) was substituted for the primary
antiserum at the same dilution. Absorption controls were
performed in which the primary polyclonal antiserum
was absorbed with esterase A a t several antigen-to-antibody ratios. The ratios of antigen:antibody (Ag:Ab)
were 1:2, 1:1,2:1,4:1, and 1 O : l (pg:pl). The final antibody
concentration was 1:500. The antigen-antibody complexes were allowed to form at 4°C overnight then centrifuged (12,OOOg for 20 min) before use in
immunostaining. Since kallikrein and esterase A are
antigenically similar, the polyclonal anti-esterase A
antiserum was also absorbed with kallikrein at various
Ag:Ab ratios and used for immunostaining.
Light Microscopy
Using the polyclonal antiserum to esterase A, immunostaining was observed primarily in the duct and tubular system of the major salivary glands. In the
submandibular gland, immunostaining was present in
Fig. 1. Sequential sections of paraffin-embedded rat submandibular granular convoluted tubule (GCT) cells and in striated
gland. a: Immunostained with polyclonal antibody to esterase A (1:500
dilution). Granular convoluted tubules (GCT) are all stained, although duct ceIls (Figs. 1, 2). Most GCT cells were stained, but
some cells exhibit greater staining intensity than others. Striated the staining intensity was variable from cell to cell;
ducts (arrows) exhibit apical staining. b: Normal rabbit serum (NRS) some cells exhibited intense staining, whereas others
(1500) in place of the primary antiserum. Striated ducts (arrows) were almost devoid of immunoreactivity. The apical
exhibit low-level, nonspecific staining. c: Anti-esterase A absorbed
with esterase A (1pl antibody: 1 pg antigen; final antibody dilution = granular zones and the basal regions of striated ducts
1500).RBCs in vascular channels around the ducts (arrows) exhibit were both stained. Immunostaining was absent in norsome endogenous peroxidase activity. X 120.
mal serum controls (Fig. lb). In the absorption controls,
which the anti-esterase A antiserum was preabsorbed
Fig. 2. Higher-magnification light micrographs of submandibular
esterase A at antigen:antibody ratios between 1:1
glands immunostained for esterase A. a: Polyclonal anti-esterase A
(1:500). Note the variable staining in GCT cells. Arrow indicates a and 10:1 (pg;pl), the immunostaining was completely
striated duct. b: Polyclonal anti-esterase A absorbed with kallikrein (1 absorbed (Fig. lc). When rat urinary kallikrein was used
pl antibody: 10 pg antigen; final antibody dilution = 1500).GCT cells as the absorbing antigen at the same ratios, immunoare still faintly stained, especially along membranes. c: Monoclonal
antibody to esterase A (1:lOO).Some GCT cells are intensely stained. staining of granular tubules and striated ducts was diminished at a 2:l (pg/pl) kallikrein-to-antiserum ratio,
Striated ducts (arrow) are stained only basolaterally. ~400.
Fig. 3.Striated ducts of parotid glands. a: Parotid, polyclonal antibody to esterase A (1:lOO). b: Parotid, monoclonal antibody to esterase
A (1.100). Apical staining is not seen with the monoclonal antibody.
x 1,200.
Fig. 4. Electron micrographs of submandibular GCT cells. a: Immunostained with polyclonal antiserum to esterase A (1:1,000).The cell
with large granules (arrow) in the lower right exhibits considerable
immunostaining, whereas other cells (arrowhead) show little staining
above background (cf. panel b). b: Control, with NR!3 (1:1,000)in place
of the primary antiserum. ~ 2 0 , 0 0 0 .
but was not abolished, even at a 1O:l kallikrein-to-antibody ratio (Fig. 2b). Interestingly, kallikrein absorption
abolished the immunostaining in the apex of striated
ducts but not in granular tubules. When monoclonal
antibodies to esterase A were used for immunostaining
(Fig. 2c), immunoreactivity was present in some GCT
cells and in the basal regions of striated duct cells but
not in the apical granules. This and the kallikrein ab-
Fig. 5. Electron micrographs of the base of striated duct cells in
submandibular glands. a-c: Immunostained for esterase A (1:1,000);
NRS (1:1,000) control for nonspecific background. a: Dense staining
(arrowheads) of the basal cell membrane at its junction with the basal
lamina. b A basal “pool” of immunostained material (*I is segregated
from the rest of the cytoplasm, apparently by a membrane. C: Some
immunostaining is discernible at focal sites along invaginations of the
basal cell membrane (arrows), characteristic of this cell type, as well
as at the very base of the cell (arrowheads). d: NRS (1:1,000)
in place
of the primary antibody. Basal membranes do not exhibit staining
above the fine stippling in the cytoplasmic background. (magnifications: a, X 18,200. b.c,d, ~24,000).
Striated ducts of parotid glands were a190 immunosorption data suggest that the apical imunoreactivity of
striated ducts seen with the polyclonal antiserum re- reactive with polyclonal antiserum (Fig. 3a). With monoflects cross-reactivity with kallikrein as observed bio- clonal antibodies, the apical immunoreactivity was not
present and only a faint rim of immunostaining was
chemically (Chao et al., 1984).
seen at the base of striated duct cells (Fig. 3b). A com- but not the apical granules. This differential pattern of
parable ductal staining was seen in the sublingual gland localization of kallikrein and esterase A in salivary
(not shown).
gland striated ducts (i.e., kallikrein at the apex and
esterase A at the base) suggests that an important difElectron Microscope Localization of Esterase A in the
ference exists in the function of these two arginine esterSubmandibular Gland
opeptidases that may be related to ion andor water
Granules of GCT cells stained for esterase A, as ex- transport, since this is the primary function of striated
pected from light microscope observations. The granules ducts in salivary glands. An important role for kalliin some cells exhibited intense immunoreactivity (Fig. krein andlor kinins in ion transport has been previously
4a), whereas in other cells staining was indistinguish- suggested in studies on other systems (Cuthbert and
able from control background level (cf. Fig. 4b). Small Margolius, 1982; Tomita et al., 1985). The difference in
apical granules rarely exhibited distinct immunoreac- distribution of kallikrein and esterase A has been striktivity. The basal region of cells of both the GCT and ingly confirmed in this site as well as in kidney tubules
striated ducts exhibited staining along the basal mem- in current studies using non-cross-reactivemonoclonal
branes (Fig. 5a,c) and in basal “pools” that were difficult antibodies against both kallikrein and esterase A (Simto identify (Fig. 5b). Staining on the basal membrane son and Chao, in preparation).
per se was more intense than on the lateral membrane,
or on basal invaginations of the basal membrane, which
exhibited only patchy staining (Fig. 512).Although some
This work has been supported in part by National
background staining in controls seemed inevitable with Institutes of Health grants HL29566, HL29397, and
this method (see Figs. 4b, 5d), the specific staining of HL 33552, and by NIH grant #RR05767 to the College
GCT granules, ductal basal membranes, and basal cyto- of Dental Medicine. The skilled secretarial assistance of
plasmic “pools” was distinguishable from nonspecific Ms. Marion Hinson is gratefully acknowledged.
background staining.
Two alkaline esteropeptidases, rat urinary kallikrein
and rat urinary esterase A, are present in rat urine
(Nustad and Pierce, 1974). Rat urinary kallikrein has
been purified and characterized in detail (Chao and Margolius, 1979)and has been localized at the cellular level
in salivary glands (Qrstavik et al., 1976; Simson et al.,
1979, 1983). The properties of arginine esterase A are
distinct from those of kallikrein. Esterase A is clearly
demonstrable in plasma; antiserum against arginine esterase A formed an immunoprecipitin arc with rat
plasma at positions similar to urinary esterase A in
immunoelectrophoresis(Chao, 1983). Rat urinary esterase A is also a kinin-releasing enzyme (Chao, 1983);
however, its cellular origin and functional roles are not
Recently, esterase A was separated from kallikrein in
rat urine and purified to homogeneity, and an antiserum
was generated against the pure esterase A (Chao, 1983).
In addition, a specific direct radioimmunoassay for esterase A has been developed for measuring the enzyme
levels in urine and various organs (Chao et al., 1984).
Rat urinary esterase A, measured by direct radioimmunoassay, coexists with kallikrein in rat kidney, salivary
gland, pancreas, intestine, and colon (Chao et al., 1984).
The present study is the first report on the cellular
localization of esterase A.
It is clear from these studies that kallikrein and esterase A overlap in their localization in salivary gland
ducts and tubules. However, there are some intriguing
differences in their patterns of localization that suggest
differences in their sites of origin, storage, andlor action.
The fact that the apical staining of striated ducts was
abolished by absorption of the polyclonal antibody with
tissue kallikrein suggests that the enzyme at this site is
primarily tissue kallikrein. Moreover, esterase A localization in the granules of GCT cells was more uneven
than previously observed for kallikrein (Simson et al.,
1979, 1983).Finally, the monoclonal antibodies to esterase A stained the basal portions of striated duct cells
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like, serine, gland, localization, esterase, salivary, rat, kallikrein, protease, immunocytochemical
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