0022-1554/91/$3.30
The Journal
of Histochemistry
Copyright
©
1991
by The
and Cytochemistry Histochemical
Vol. 39,
Society,
SUGIYAMA,2
Department
of Pathology
Received
for
SAMUEL
and
publication
Laboratory
August
in Ion Transport
S. SPICER,
Medicine,
1990
2,
and
PAUL D. MUNYER,
Medical
in revised
University
form
tery
cells
staining N-linked
with PSA and LCA showed a high content of oligosaccharides in transport cell GCs. Reactivity with PHA-L and PHA-E identified GC with triantennary and with bisected biantennary N-linked oligosaccharides, respectively, in these cells. High affinity for DSA and PWM demonstrated abundant N-acetyl lactosamine in N-linked side chains. Ion transporting epitheial cells reacting with lectins specific for N-linked oligosaccharides induded strial marginal cells and outer sulcus cells of the cochlea and dark cells, transitional cells, and planum semilunatum cells of the vestibular system. In general, all of the inner ear transport epitheial cells revealed a similarlectin binding profile, with
types
that
mediate
identified
in large
important
transport
enzymes,
method
method
offers
ATPase from
isoform
in neural
kidney
and
submandibular
Immunostaining in several
types
marginal
and
sulcus transitional
cell
types
2
cells
and
exhibit
cells
by National
Correspondence
and Laboratory Medicine, Icy Avenue, Charleston,
with The
former
the
1990;
high
to: Dr. Setsuo
[3H]-ouabain
of Na,Kt
inner
ear differs
in epithelium
of ger-
in the in
and
ear,
cochlea the
histochemistry;
and
Sugiyama,
dark,
of the
Grants
cells (26),
plas-
P50 DC00422
Carolina,
Ex-
these
basolateral
Department of South
planum
system.
and
of Pathology 171 Ash-
26,
1990
A. SCHULTE Camlina
29425.
(0A2055).
Gerbil.
characterizing
these
cells
(suprastrial
and
transport.
Such in
In addition,
has been localized in a found in two distinct populations attachment ofReissner’s membrane
are
to the lateral
cells)
in ion
data
Na,K-ATPase
which
just superior
ofmitochondria(22,34,50). with the cytochemical
as active
immunoreactive of fibrocytes
lying
in the inferior
portion
of the spiral
ligament
(41).
Histochemical tins
has
provided
ofglycoconjugates prominent ion
strial
October
South
as well as an abundance specializations concur
transport
K-ATPase
including
vestibular
transitional
of Health
Medical University SC 29425.
levels ofNa,
inner
amplification
Institutes
of
1991
in USA.
the one exception that SBA reacted strongly with ion transporting cells in the vestibular system but only wealdy with those in the cochlea. Fibrocytes specialized for ion transport located in distinct areas in the suprastrial and inferior regions of the spiral ligament also stained with lectins that demonstrate N-glycosylation. However, transport fibrocytes differed from transport epithelial cells in two ways. First, they reacted with HPA, DBA, VVA, and SJA specific for 0-linkages and second, they failed to react with UEA I. The staining pattern forN-glycosylated GC resembled that for Na,K-ATPase in inner ear, suggesting a relationship between these constituents. (J Histoc.bem Cytochem 39:425-434, 1991) KEY WORDS: Cochlea; Vestibular system; Ion transport; Lectin
subset
means
(41,45).
in the
425-434,
Cells
BRADLEY
Charleston,
accepted
abundant
localized
by
isoform
gerbil
one
most
immunocytochemtwo methods in dis-
the
cells ofthe and
cells
semilunatum elaborate
ear
been
ofthe
been
inner
that
gland
outer
for the planum
Supported DCOO7fl.
the
has demonstrated
cept
1
cells
of epithelial
semilunatum,
ROl
over showing
and stromal
the
has
of the
(23,41,45). and
ear have One
autoradiography
advantages
isoenzymes
inner
methods.
types
(30,56),
in epithelial
in the
Na,K-ATPase, cell
and immunocytochemistry
tinguishing
bil
transport
by histochemical
in several
an enzymatic ical
ion
part
histochemically
(25),
19,
and
Carolina,
malemma structural
Introduction Cell
of South
October
in gerbil inner ear were differentiated by staining glycoconjugates (GCs) with a batof horseradish peroxidase-conjugated lectins. Strong
Ion transport histochemically
pp.
Artide
of Glycoconjugates Inner Ear’
SETSUO
4,
Printed
Original
Distribution of Gerbil
No.
Inc.
staining
of tissues
a means
of exploring
(GCs)
GCs
in situ.
in a number
transporting
cells
of the
the
Lectin
ofcells
(20, 44). The questions
with a battery cytochemistry
that
function
addressed inner
ear
chemical
are
of labeled
has disclosed primarily
here concerned similarly
1cc-
composition
endowed
in ion
whether with
GCs and, so, what type of GCs the cells produce. These questions were investigated histochemically with a battery of peroxidaselabeled lectins that exhibit widely different sugar affinities and provide a sensitive in
means
ofdetecting
and partially
characterizing
GCs
situ.
Materials
and Methods
Tissue Preparation.
Inner ears were obtained from 24 Mongolian gerbils (Meriones unguiculatus) 3-6 months of age. The gerbils were maintamed in a low-noise room on food and water ad libitum and had normal
425
Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on April 12, 2015
SUGIYAMA,
426
Table Lectin (common Lens
used
1 . Lectins
in histochemical
name)
culinaris
Pisum
sativum
Nominal binding
carbohydrate specificitya
GIcNAc
aMan
>
aGlc
aMan
>
aGlc
>
simplicifolia
ajGlcNAc
(unknown) Datura
GSA
apple)
Phytolacca
DSA
americana
(pokeweed)
Triticum (wheat
GIcNAc
=
PWM
vulgare germ)
and glycogen
to
aMan
(4)
>
GIcNAc
(2)
aMan
(4)
>
GlcNAc
(2)
GlcNAc
(4)
GlcNAc(3 1 ,4G1cNAc)1_3 = Ga1131 ,4GIcNAc
GlcNAc
(2)
GlcNAc(31
GIcNAc
(2)
GlcNAc(3 1 ,4GlcNAc)i_2 > 3GlcNAc > NeuAc
aNeuAc
(4) > GIcNAc
GlcNAc(31
,4GlcNAc)12
GIcNAc
(1)
,2Man triantennate oligosaccharide
GalNAc
(2)
GaINAc
(2)
GalNAc
(4)
GaINAc
(4)
GaINAc
(1)
GalNAc
(4)
>>
(1)
GaINAc
(4)
=
Gal
(4)
GaINAc
(1)
=
Gal
(1)
-
WGA
,4GlcNAc)i_5
(Gal31
,4GlcNAc)2_5
Succinyl WGA (wheat germ)
sWGA
Phaseolus vulgaris (leukoagglutinin)
PHA-L
GlcNAcf3l complex
PHA-E
Bisected complex oligosaccharide
Phaseolus
vulgaris
(erythroagglutinin)
Dolichos
GalNAcal,3Gal GalNAcal,3GaINAc aj3GalNAc
tree)
Arachis
hypogaea
> aj3Gal
Gal (4)
a,3GalNAc
>
a,3GaI
SBA
GalNAcal,6Gal
Maclura pom:fera (hedge apple
tree)
>
aGalNAc
> aGal
MPA
3Gal
communis
bean)
RCA
uGal
>
>>
GaINAc
Gal
>
Fuca1,2Gal31,4GlcNAc
Fuc (4)
LTA
Fuc (3)
a-Fuc
eumpaeus
seed)
UEA
I aNeuAc
Jiavus
(slug)
aNeuAc
> aNeuGc
LFA
‘ Affinity
Inhibition
for oligomers of binding
each sugar indicate of partial inhibition.
(3)
I
a-Fuc
Lotus tetragonolobus (asparagus pea) (gorse
a,3Gal
GalI3l,3GaINAc
max
(castor
>
PNA
(soy bean)
Ricinus
aGalNAc
SJA
(peanut) Glycine
>>
DBA
japonica
(pagoda
> aGalNAc
VVA
gram)
Sophora
> aGalNAc
(1)
HPA
biflorus
(horse
type
GalNAcal,3GaINAc
Helix pomatia (snail) Vicia vilosa (hairy vetch)
hearing
found statii
II
stramonium
(thorn
b
Sugar(s) isthibit
PSA
Griffonth
Limax
SCHULTE
LCA
(pea)
Ulex
MUNYER,
studies
Abbreviation
(lentil)
SPICER,
or monosaccharides in the presence
(1)
(4) >> GaINAc GlcNAc (1)
in solution. of a
0. 1 M
concentration
its degree of inhibition as judged by reduced Sugars not shown were not inhibitory.
as assessed by evoked potentials
=
ofthe
brainstem
of six different staining
and auditory
intensity,
nerve
( 32,40). Gerbils were exsanguinated under urethane anesthesia by transcardial perfusion with 0.9% saline containing 0.1% sodium nitrite and were then perfused with fixative containing 6% mercuric chloride, 1.0% sodium acetate, and 0.3% glutaraldehyde. The bullae were then opened and the stapes removed. The round window was perforated and the same fixative was gently injected into the scala vestibuli via the oval window. The ears were removed and immersed in the fixative for 1 hr at room temperature. After a saline
monosaccharides with
(4) being
was assessed complete
for each lectin-HRP
inhibition
and (3).
conjugate. (2),
and
(1)
Numbers denoting
associated decreasing
with levels
rinse, the ears were decalcified in daily changes of 0.12 M EDTA (pH 7.0) under slow stirring for 48-72 hr at room temperature. The specimens were then dehydrated through a graded series of ethanols, cleared in xylenes, and embedded in paraffin. Lectin Histochemistry. Twenty lectins with a wide range of carbohydrate binding specificities (Thble 1) were purchased from Sigma (St Louis, MO) or Calbiochem (Lajolla, CA). References to the sugar specificity of the lectins are as follows: LCA and PSA (8,16); GSA II (11,19); DSA (6,10);
Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on April 12, 2015
GLYCOCONJUGATES
Table
2.
ION
IN
TRANSPORT
Staining
ofion
transport
Strial
marginal cells
Outer
CELLS
427
cells in inner
ear with
lectin-HRP
conjugates’
Suprastrial
Inferior
Cochlea
jjd
Ve stibular
Supralimbal
b
spiral
Dark
ligamentc
system
cells ampullae and sacculus
cells
cellsb
4 4
2
2
3
2-3
4
2
2
3
2-3
4
1/0
0/0
0/0
1/0
LCA PSA GSA
sulcus
2-3/0-1
Transitional
and
semilunatum
cells
2 2
1-2/0
0/0
DSA
3
2
1
2
1-2
3
2
PWM WGA
3 4
1-2
1
1
1-2
4
3
2
3
4
4
4
3
sWGA
1
0
1
2
1-2
1
1
PHA-L
4
2
1
2
1-2
4
2
PHA-E HPA
4
3
3
4
4
4
4
0
0
0
1
2
0
0
WA
0
0
0
2
2-3
0
0
DBA
0
0
0
1
0-1
0
0
SJA
0
0
0
1
0-1
0
0
PNA
0-1
0
0
1
0-1
1
0
SBA
1
1
2
3
3
4
3
MPA RCAI LTA
2
0
2
2-3
2-3
2
2
3
1
2
3
2-3
4
4
0
0
0
0
0
0
0
UEA-I
4
4
0
0
0
4
2
LFA
4
2
2
4
2-4
4
a
4
,
Numbers
indicate
degree
(21);
HPA
and
WGA DBA
RCA I (3,8); After
succinyl
VVA (52);
ITA (2,35);
UEA
peroxidase
by various
conjugate
sections
lectins strate
cells based
medium
amine
without
lectin;
(Gal),
on visual
assessment
of relative
staining
intensity
were
or DBA-HRP
conjugate
penultimate
to sialic solution
in an attempt acid.
This
ofsialidase
before for which
incubation this
with
lectin
shows
The
and
mm
10
to HRP and
sg/ml
solution acid
(Fuc),
entailed
in
The
glands.
Other
HRP-conjugated affinity
staining
of each
Gal or GaINAc
tivity PNA-
ofthe
enzyme
eliminate
sections GSA
lectin
with
with glyco-
Cochlea Marginal
cells of the stria vascularis
showed
moderate
ing with
LCA,
WGA,
PHA-L,
PWM,
sparing
and
II, sWGA, SBA, to the basolateral the
apical
surface
7a).
ofepithelial
cells lining
cells in lectin
of supralimbal
PWM,
and
offibrocytes
ic) and
la and intensity cells
the outer
binding
but
id). UEA I, (Table 2).
lining
the scala
the
inferior
a lectin
The
(Table
2).
in the suprastrial
portion
binding
cells.
PHA-L lying
of the
profile
spiral
region
(Figures
ligament
(Figure
essentially
fibrocytes,
identical
however,
differed
to that from
the
supralimbal cells as well as the strial marginal cells in showing aSfinity for a diverse group ofGalNAc binding lectins including HPA, VVA, DBA, SJA, SBA, and MPA (l#{224}ble2).
Vestibular
DSA,
cells,
marginal
profile
to
unstained,
(19).
Results
PSA,
with GSA waslocalized
less intense staining (Figures both cell types with equal
DSA,
of supralimbal
was estab-
were digested
ofstrial
0.
membrane.
5a, 6a,
plasmalemma
binding
la) exhibited
alcianophilia
II to eliminate
marginal
3a, 4a,
that
showed stained
with
la and
residues
for 24 hr at 37*C
and
basolateral
generally however, The
(NeuAc),
with
2a,
resembled
Populations
before
activity
affinity
id,
from
vestibuli just above the medial attachment of Reissner’s membrane coincided closely with that of strial marginal cells. However, the supralimbal cells failed to bind UEA I and expressed weaker reac-
sub-
N-acetyl-D-galactos-
incubation
la,
sulcus
(GlcNAc). sialidase
PNA
MnCl2,
neuraminic
to demonstrate
(42).
lished by its capacity to impart in sections of mouse sublingual diastase
to a 5-10
L-fucose
with
sections
for
the sites on a scale
of Reissner’s
of strial
(Figures
of unconjugated
ofsections
0.1 M N-acetyl (Man),
plasmalemma
(18).
(b) exposure
digested
MgCl2,
(a) substitution
(c) exposure
conjugated
of lectin-HRP
and incubated
medium
(39);
(42). thick
3
among
UEA I, and LFA, and light reactivity and MPA (‘EthIc 2). The lectin affmity
(55):
MPA
were
solution
0.1 mM
in PBS
PHA-E
(16);
4-sm
.tg/ml
or N-acetyl-D-glucosamine
sections
Lectins
previously
containing
included:
containing D-mannose
LFA (31).
a 5-10
rinsed
conjugates;
(GalNAc),
IU/ml
7.2,
then
substrate
conjugate
Some
and
as described
(24); SBA
demercurialization,
staining
for lectin-HRP
D-galactose
I (36);
pH
were
for lectin
lectin-HRP
PHA-L (28);
and
H2O2-diaminobenzidine Controls
(33); PNA
for 24 hr at 4’C with
in 0.1 M PBS, The
.
WGA
SJA (54);
(HRP)
deparaffinization
were incubated CaCl2
(8,29); (4);
to horseradish
gen,
binding
most reactive. Each value was obtained from examination of ten or more stained sections. 6 Cells lining scale vestibuli immediately superior to the medial (supralimbal) or lateral (suprastrial) attachments C The fibrillar staining pattern is attributed to the Type II fibrocytes filling this area of the spiral ligament. d Results of GSA II staining are shown without/or with diastase pre-digestion.
PWM
0.2
of lectin
planum
to strong PHA-E,
stainRCA
Dark
cells
that
ofstrial
7b),
except
(Figure I,
pullae
System disclosed for
4). Dark and
a lectin
marginal showing
much
cells stained
utriculus.
Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on April 12, 2015
staining
cells (Figures
Lectin
profile ib,
greater
identically affinity
strikingly
similar
le, 2b, 3b, 4b, affinity
with
for
SBA
all lectins
of transitional
to
Sb, 6b, and
cells
(Table
2)
in the amand
pla-
UM.
sty
--.‘-
#{149}1
I ..
,!
SM
sv
R
UM
i.cstV#{233}___ Figure
1. Paraffin
.
of gerbil
if
inner ear stained with PHA-L--HRP conjugate. (a) Middle turn of cochlea. Stria vascularis (StV) stains strongly. Outer sulcus in the suprastrial region (55) and inferior spiral ligament (ISL) show moderate reactivity, as does the basilar membrane (BM). Staining in the tectorial membrane (TM) is confined to part of the undersurface in the middle zone and the tip. (b) Vestibular system. Dark cells of ampulla and utriculus overlying intrinsically pigmented melanocytes (arrows) are intensely stained. Transitional cells (arrowheads) interposed between dark cells and neurosensory epithelium in a utricular macula (UM) and crista ampullaris (CA) stain with moderate intensity. (C) Fibrocytes bordering the scala vestibuli (SV) in the suprastrial region (55) show moderate reactivity. R, Reissner’s membrane. StV, stria vascularis. (d) The basolateral plasmalemma of strial marginal cells is strongly reactive, but the apical surface bordering scala media (SM) lacks lectin affinity. (a) The basolateral plasmalemma of ampullar dark cells overlying melanocytes (arrows) is stained intensely, but the apical membrane bordering endolymph (E) is unreactive. (1) The basolateral plasmalemma of transitional cells (arrows) adjacent to a utricular macula (UM) shows moderate reactivity. Original magnifications: a,b x 100; c,d x 1000; e,f x 400. Bars: a,b = 100 tm; c-f - 10 tm.
(OS) epithelial
sections
1Id
cells and fibrocytes
428
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I
.
pm
‘I.-
%_
-
E
E
#{149}.
E
,
E
--
. ‘3_
.
j:
a
1-
.;
2b
I
.__
3a
3b
E
E
4a
5b
IfE
E.
E
L6c1
6b
71
Figure pigment.
2-7.
In all figures,
E, endolymph.
a shows Original
marginal
cells of stria vascularis
magnifications
Figure 2. (a) Strial marginal to marginal cells. DSA-HRP
cells show moderate conjugate.
Figure
3. (a) Marginal
and (b) dark
Figure
4. (a) The basolateral
plasmalemma
Figure
5. (a) Marginal
and (b) dark
Figure
6. (a)In contrasttostrong
Figure
7. (a) Marginal
cells
cells
cells
staining and (b) dark
cells
Bars
at their basolateral
show
show
with UEAI(cf. cells
react
-
no reactivity. cells shows strong
intensely
VVA-HRP
with
plasmalemma
Arrows
in all figures
point
to intrinsically
dense
melanin
UEA which LFA.
but not at their apical
surface.
(b) Vestibular
dark cells stain similarly
conjugate.
only faint staining,
reactivity.
Figure5)LTA,
dark cells of ampullae.
5 tm.
x 1000. reactivity
of marginal cells
and b shows
l-HRP
whereas
dark cells stains
intensely.
SBA-HRP
conjugate.
conjugate.
also bindsto LFA-HRP
(b) that of vestibular
Fuc, shows
no affinityformarginalcellsor(b)dark
conjugate.
Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on April 12, 2015
cells. LTA-HRP
conjugate.
SUGIYAMA,
430
Table
3. Summary
of nom inal
class
ofca
rbohydrate
in e ach type
i dentifled
ofion
SPICER,
MUNYER,
transpo rt cella
Epitheium Carbohydrate
marginal
Strial
moiety
Fibrocytes
Vestibular dark cells
Transitional and planum semilunatum cells
Supralimbal
Fibrocytes
Suprastrial
cells
cells
2
2
3
3
4
3
1
1-2
1-2
3
4
4
3
4
4
4
2
4
2
1
2
1-2
0
0
0
0
0
1
1-2
3
1
4
4
2
3
2-3
4
4
4
2
0
0
0
4
2
4
3
2
4
2-4
1
0
1-2
0
0
1
2-3
cells
cells
4
2
4
3
2
4
Man/GIc (LCA,
sulcus
Outer
SCHUITE
spiral
in
ligament
PSA)
Lactosaminic
type
(DSA,
PWM
Bisected,
sWGA)
>>
biantennary
(PHA-E)
Triantennary (PHA-L)
GalNAc (HPA,
DBA,
VVA,
SJA)
Gal (RCA
I)
(UEA
I)
Fuc
NeuAc (LEA,
WGA)
Glycogen (GSA
II)
Numbers
a
num
denote
staining
semilunatum
cells
intensity
the designated
with
in the
ampullae
and
lectins.
of transitional
cells
cells
have
provided
information
on sorting
of constituents
in the utriculus closely resembled that ofdark cells(’l#{224}ble2) (Figures lb and lf). The staining in all of these cells was localized to the
the enzyme Na,K-ATPase to the basolateral Epithelial cells lining the inner ear function
basolateral
approximately
also
the
plasmalemma for strial
case
and
excluded
marginal
cells.
all lectins in marginal cells and tional and planum semilunatum Sections digested with in PNA or DBA affinity
Method
the
apical
However,
dark cells cells.
neuraminidase in any site
surface,
the affinity exceeded
failed in inner
as was for nearly
that
to show ear.
in transian increase
Controls
the
ent monosaccharides generally conformed termined
lectin
lectin-HRP resulted to that
exposed in place
conjugates
to HRP and of conjugated with
each
substrate lectins.
of six differ-
in a staining inhibition pattern expected from the biochemically
specificities
(lhble
that de-
lectin
histochemical
studies
on
the
inner
ear
knowledge,
however,
there
are no published
binding to GCs in other cell types Epithelial cells generally perform facilitated cal and
by the organization basolateral
domains
systems using cultured composition of apical rized
dolymph The GCs
mM
Na
concentration
char-
and in establishing the difference across the coch-
the pumping
of K from
the interstitial
and establishing its ion content. lectin binding profile observed here
among
ginal and lunatum,
staining
(13-15,37,
38,47-49,51) have shown the presence of specific GCs in the tectonal membrane and at the surface of hair cells in the cochlea, and in the cupula and otolithic membranes ofthe vestibular organ. To our
0-20
several
inner
ear epithelial
cells,
space
to the en-
revealed
similarity
including
strial
of mar-
outer sulcus cells in the cochlea and dark, planum semiand transitional cells in the vestibular system (Table 3).
epithelia
with
similar
GCs
also have
in common
an abun-
dance of the enzyme Na,K-ATPase and consequently function similarly in transporting ions across the cell. Therefore, these cells exhibit comparably strong lectin staining for GCs and immuno-
Discussion Recent
K and
as
(5). of the
lear and vestibular epithelial cells, respectively. The unique ion content of endolymph and associated transepithelial potential are essential to transduction of mechanical stimuli to electric signals of auditory nerves. The enzyme Na,K-ATPase located in the basolateral plasmalemma of several types of cells in the inner ear
These
1).
150 mM
of the inner ear’s endolymph mV and +0-20 mV potential
facilitates
No staining was evident in sections medium or to unconjugated lectins Pre-incubating
acteristic ±80-100
such
plasmalemma in maintenance
epithelial
cells.
Such
of the inner ear. vectorial functions
of their of differing
cells have been and basolateral studies
reports
plasma
membrane
compositions.
on lectin are
into
api-
Recently,
developed to investigate the plasma membrane in pola-
on Madin-Darby
canine
strial marginal semilunatum positive
which
kidney
for Na,K-ATPase
(41,45).
Quantitative
corresponded with that in ATPase content both the lectin receptors and the ATPase
GCs
plasmalemma
variability
cells and vestibular dark cells and and transitional cells. Moreover, and
Na,K-ATPase
in reactive
The physiological but three possibilities
were
epithelial
localized cell
in GCs
among these cells, since were most abundant in
types
role of the lectin-reactive can be considered. First,
less so in planum both the lectinat the (Figure
basolateral 8).
GCs is unknown, the lectin reactivity
could be attributable to the Na,K-ATPase per se. The 3-subunit of mammalian Na,K-ATPase is a glycoprotein and is known to possess
three
probable
Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on April 12, 2015
sites
for
N-linked
glycosylation
in the
GLYCOCONJUGATES
IN
ION
TRANSPORT
CELLS
431
p
I
..
. -
-
.
s...-
‘
8a(
8b
I
8c
I
:-..
Figure 8. Marginal cells of stria vascularis and vestibular dark cells immunostained for Na,K-ATPase (immunostaining graphs taken from a paraffin section show intense immunoreactivity at the basolateral plasmalemma of (a) marginal and Figures 2-7). (C) A 1-tm thick epoxy section at higher resolution shows staining of Na,K-ATPase atthe basolateral brain Na’,K’-ATPase. Original magnifications: a,b x 1000; c x 1600. Bars = 5 tm.
methods are described in ref. 41). Microcells and (b) dark cells. (cf. Figures id, le, plasmalemma of marginal cells. Anti-bovine
predicted
PWM,
amino
are thought in their
acid
localization
could
facilitate
fluencing It has
dark
cells
modulated
(9).
basolateral
these
associations
The
role of the into
knowledge
the
depend
lectin-reactive
GCs of the
of its localization
not
would
ultrastructurally
investigations
have
in specificity
in
tissue
the same
for the type
can be obtained
Information sites
by
examination
terminal
For example,
about
sugar
DSA,
PWM,
residue and
terminal sWGA,
GIcNAc. However, DSA bind strongly to lactosaminic
present
finding
that
strial
different
marginal
WGA
infrom
to the
lectins
in stearic
of GCs
that
bind
to
requirements.
to sialoconjugate
which
with
Notably,
LFA equalled for
ion
WGA
than
with
sWGA
with
WGA.
cells
by
binding attributing
to sialic
acid sequences cells
strongly,
could
cells.
across The
all transport
abolishment
NeuNAc
more
of
than
by
only by GlcNAc in the stainthe stronger staining with
acid.
(12). LCA and indicating
stained
PSA
the abundant
the
core
region
homology
all inner
presence
of
in their
ear trans-
of N-linked
oligosaccharides. The comparable binding ofLCA and PSA among the several epithelial sites of ion transport and the similar sugar inhibition profiles observed here for LCA and PSA affirm the biochemical
evidence
for the comparable
tins. LCA and PSA binding Fuc al,6 on the core GIcNAc
affinity
for
binding
is attributable
WGA (8,21).
and The
lower chains
affinity for a Fuc as compared with
dark
cells
of the
vestibular
sWGA,
in the ion transport
and PSA show similar specificity for GCs (1,8) and also exhibit considerable
PWM, unlike oligosaccharides and
with
the staining
that
transport
high
cells
but faintly
affinity in transport cells for WGA, which binds and GIcNAc residues (29), compared with sucreacts only with GIcNAc (33), adds further cvi-
GlcNAc and ofsuccinyl WGA ing solution further supports
port
residues.
attests
affinity
LCA N-linked
but have recsugar while
composition
all possess
and
cell types
and
of affinity
to adjacent
and
of sialoconjugate.
amino knowledge
the diverse
but differ
WGA,
(53),
in relation
of linkage
sWGA
cinyl
dence
between lectin-positive a relevance of the GC
provided
with
are
but
DSA
The much stronger to both sialic acid
expressed
result
for specific terminal sugar residues for many lectins, ognized also that they often bind to the same terminal differing
in cell
at present,
with
LFA staining
oligosaccharides. perhaps
strongly
then be explained on the basis of the presence of lactosaminic-type chains in the strial and the dark cells (Table 3). The presence of lactosaminic-type side chains is also supported by the strong staining in these sites with RCA I, which binds preferentially to lactosamine.
mar-
cells
epithelia
is not clear
Na,K-ATPase. The close correspondence GCs and Na,K-ATPase, however, suggests to the transport activity of the cells. Biochemical
all
the
a role
epithelial
on N-linked GC
in
play
react
or other
preferentially
but
in-
of sodium
ofglucose
could
GCs
plasmalemma.
membrane
GCs
the
through
a recycling
of certain
of some
significance
the
uptake
a glycoprotein
reportedly
Second,
possibly
for
here
to Na,K-ATPase
across
basolateral
Third,
surfaces
the
or water
associations
by uvomorulin,
at the
demonstrated
variability.
a mechanism
at the
Cell-cell
GCs
ion transport,
sodium-coupled
is localized and
sight
that
entailing
recognition.
The
quantitative
of ions
suggested
possibly
ginal
and
permeability
substrates,
(46).
and also bear similarity
enzyme-directed
been
ions,
sequence
to be N-linked
LCA
and
PSA
Downloaded from jhc.sagepub.com at UNIV OF VIRGINIA on April 12, 2015
specificity
to such
a Fuc
residue on the ITA and because
positive
of these
two 1cc-
to N-linked chains is augmented by (27). It appears unlikely that UEA I
sites
residue
because
UEA
I has
core GlcNAc of N-linked UEA I did not stain three
(Table
2).
432
SUGIYAMA,
The nominal GlcNAc31,2Man
inhibitory (24), and
this
unit for PHA-L is the disaccharide lectin binds preferentially to trian-
tennary chains containing this reacts with the bisected complex
internal disaccharide (7). type N-linked oligosaccharide
prefers
The
cal
biantennary
results
nate
chains
with
and
PHA-L
bisected
(55).
and
strongly
PHA-E
biantennate
chains,
in
PHA-L
and
can
staining
staining
be attributed
SJA,
taming
with
failed
Supralimbal
and
cells
inhibition here
chains
GCs
stained
ion
lectins
such
affinity
to and
react Type
with
with the for GCs
other
cells
as HPA,
for 0-linked
VVA,
chains
con-
cells.
II fibrocytes
PHA-L.
These
cells are then
type side chains
in the
less abundant
considered
suprastrial
to contain
with which
GlcNAc1,2Man
and
fewer
DSA and PWM
to which
PHA-L
pared
binds
of the
react or
potential.
investigation
histochemically
cells
tennary
N-acetyl
unique
morphology
differed further ing for GalNAc
(50)
and
high
content
from transport epithelial with HPA, VVA, DBA,
port
cells
chains
to be located
in the fibrocytes
and
their
The
specificity
ofUEA
I resembles
I reacts
with fucose Type I and Type
of both
hand,
residues II blood
lacks affinity
that
elaborate
location
ofITA
can-
(2), except
in the oligosaccharide group H substance.
that
side chain LTA, on the
for the Fuca1,2Gal31,3G1cNAc
We wish
to thank
editorial
strial
and
the presence and possessing II chains below
in lacking
cells
of inner
ear might
oligosaccharides
UEA
fibrocytes
I affinity.
differed
The
presence
exclusively in the basolateral and not fibrocytic cells would
for the
as an anchoring
Strikingly,
resembled
the
those
ion
in the
on
I chain
or of a low level of Type provided by hA. Such stain-
fucoconjugate thelial cells GC
depend
the Type
of either 0- or N-linked oligosaccells and dark cells of inner ear.
with content strial marginal
transport
then
resembling
terminal Gafl3l,3GlcNAc the detection sensitivity
ing is compatible charides in the Notably,
dark
ofeither
from
transport
of a UEA
1. Allen
2.
vestibular
epithelial
system
epithelia
I-recognizable
plasmalemma of epibe consistent with a role cells
in
the
affinity
typing
reacted such
with
as the
ion bian-
Consequently,
attest
to
type carbohydrates
the
presence
in ion trans-
Heape
skills
for
ofMs
her
Leslie
excellent
technical
Harrelson
assistance.
are also greatly
ap.
Cited
cochlea
for all
AK,
Desai
Allen
NN,
HJ,Johnson
ficities
of lectins
munol
Commun
Neuberger
A: The
purification ofthe glycoprofaba) and a comparison of its specificity. Biochem 3 155:127, 1976
( Vicia
EAZ,
Matta
KL: A comparison and Lotus
from Ulex europaeus 6:585,
ofthe
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speciIm-
tetragonolobus.
1977 determinants ofRicinus for oligosaccharides.
Fiete D: Structural and toxin specificity
communis
J Biol Chem
1979
4. Baker DA, Sugii 5, Kabat EA, Ratcliffe RM, Hermentin P, Lemieux RU: Immunochemical studies on the combining sites ofForssman hapten reactive hemagglutins from Dolichos bifiorus, Helix pomatia, and Wisteria floribunda. Biochemistry 22:2741, 1983 5. Caplan
MJ, Anderson
ing and polarized nous component 46:623,
HC,
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GE,)amiesonJD:
Intracellular
sort-
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1986
6. CrowleyJF, Goldstein U’ ArnarpJ, studies on the lectin from Datura Biophys 231:524, 1984 7. Cummings minants
L#{246}nngrenJ: Carbohydrate stramonium seeds. Arch
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RD. required
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9. Drescher carbonic
DG, 1985,
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Ms Sharon
and
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glycoprotein.
transporting
lectins
of deterof a compo-
that GCs,
coch-
genera-
ear.
tein lectin from the broad bean properties with lectins of similar
sequence
of the Type I hapten (36) and shows weaker binding than UEA I to the Fuc present in Type II chains, which differ only in their 3i,4 linkage to G1cNAc (17). The staining of UEA I-positive and LTAnegative
inner
problem
of glycopeptide.
these
complex
gerbil
lectins
Possibly
in the
in the
structure
N-linked
type
mY).
Acknowledgments
of the also of
intercellular fibers, and other extracellular matrix con(50). Accordingly, GC stained for 0- as well as N-linked appears
other
light stainindicating
The two regions are found consist
processes infiltrating the stroma, but an intercellular not be excluded by light microscopic examination. UEA
(41),
cells in showing SJA, and PNA,
of vestibular
not
difference
by the
most for
with
ofN-linked of the
cochlea
with their similar K concentration.
and
chemical
However, affinity
results
predominantly
to this
exact
lactosaminic
histochemical
dark,
to
of their
ofNa,K-ATPase
the presence of 0-linked oligosaccharides. spiral ligament where Type II fibrocytes capillaries, stituents
basis
sites.
not
(+ 0-20
vestibular
is limited
the
possess
but
GC content
system
in the relates
present
Literature on the
vestibular
present
electrical
they
so classified
of the
in a reactive
to vestibular
cells,
cells in the cochlea establish a much greater 80-100 mV) across the epithelium as com-
epithelium
transport
strongly
cells. The similar
of
mining
bound
semilunatum
The
preciated
with PHA-E. fibrocytes,
SBA
SCHUITE
MUNYER,
epithelial cells concurs the endolymph’s high
(+
GC
transport
and dark cells (Table 3). The fibrocytes appear to possess instead a greater proportion of bisected, biantennary N-linked chains, since stain strongly Type II transport
those
SBA-reactive
lear
The
in marginal
ion transport difference with
the
the
regions of the spiral ligament differed from the epithelial and dark cells in showing weaker staining with DSA, PWM,
lactosaminic
However, potential
nent
almost
DBA,
SBA.
and cochlea ion transport function in maintaining
1ccwith
except
and planum and outer sulcus
marginal
tion
these lectins
transport
these
of
1). Nevertheless,
cells with
because
several
ear’s
by GalNAc
(Table
ion transport
a well-established
GaINAc,
inferior marginal
observed
to N-linked
trianten-
GalNAc in solution the affinity of PHA
partial
ofepithelial
affinity only for N-linked identically and because and
the
with
PHA-E
the present tins
agreement
histochemi-
in the inner
tested
transitional,
PHA-E and
to abundant
respectively,
transport cells (Table 3). However, lectin has been reported to inhibit (10,43),
positive
testify
lectins
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