Studies on the cornea. V. Electron microscopic
localization of adenosine triphosphatase
activity in the rabbit cornea
in relation to transport
Gordon I. Kaye* and Lois W. Tice*
The distribution of ATPase activity in adult and 7-day-old rabbit corneas has been studied
by electron microscopic-histochemical techniques. In the cornea! endothelium, the lead phosphate end product of the ATPase reaction is localized at the lateral margins of the cells and
in the intercellular spaces. This activity is first demonstrable at 7 days. Pinocytotic vesicles,
even those clearly containing ThOs marker, show no nucleotide phosphatase activity. Epithelial
ATPase is less fixation resistant than endothelial ATPase, but, after short glutaraldehyde fixation, end product is localized in the intercellular spaces.
T he frequent finding of histochemically
ity and vesicular function has remained
obscure, however, primarily because most
of the morphologically recognizable pinocytotic vesicles studied have not yet been
shown to be physiologically active in transport. In the corneal endothelium, pinocytosis has been demonstrated to be a true
transport process, and the morphological
aspects of this process have been intensively studied.7"11 In the experiments reported here, investigation of the histochemical distribution of phosphatases in corneal
endothelium was carried out in the hope
that the results might clarify the relationships among pinocytosis, phosphatase activity, and transport.
JLi
demonstrable
nucleoside phosphatase activity in pinocytotic vesicles has led some
investigators to suggest that this activity
may be involved in transport by these
vesicles.1"0 The relation between this activFrom the F. Higginson Cabot Laboratory of Electron Microscopy, Division of Surgical Pathology
of the Departments of Surgery and Pathology;
and the Department of Pathology, College of
Physicians and Surgeons, Columbia University,
New York, N. Y.
Supported in part by Grants GM-11343, NB02343, and NB-03448 of the National Institute
of Health, United States Public Health Service,
and by Grant P-362 of the American Cancer
Society.
"Recipient of a Career Scientist Award of the
Health Research Council of the City of New
York under Contract 1-320.
° "Present address: Laboratory of Physical Biology,
National Institute of Arthritis and Metabolic
Diseases, National Institute of Health, Bethesda,
Md.
Materials and methods
Corneas of adult and 6- and 7-day-old
rabbits were fixed for 5, 30, and 120 minutes in 1 to 6 per cent glutaraldehyde in
0.1M cacodylate buffer, pli 7.2, with or
without addition of 0.2M sucrose. Other
adult corneas were fixed for 2 hours in
22
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Number 1
4 per cent glyoxal in 0.2M cacodylate buffer, pH 6.5, containing 0.4 sucrose and for
one hour in 6.5 per cent hydroxyadipaldehyde in 0.1M cacodylate buffer, pH 7.0,
also containing 0.4M sucrose.
After appropriate buffer rinses, corneal
slices, or segments were incubated in the
Wachstein-Meisel medium12 for the demonstration of ATPase activity.
In control experiments, corneas were incubated in the medium from which substrate was omitted, or in which equimolar
amounts of ADP, AMP, CTP, IDP, or
/3-glyceropho.sphate were substituted for
the ATP normally used as substrate. The
reaction was followed in the light microscope by periodically examining thin slices
of cornea which had been treated with
dilute ammonium sulfide to convert precipitated lead phosphate to black lead sulfide; when a visible reaction was noted
(10 to 30 minutes), incubation of the material to be used for electron microscopy
was stopped.
Approximately half of the adult and
ATPase activity in cornea 23
newborn corneas studied were exposed to
ThO,° for 10 to 30 minutes prior to fixation by injection of the marker into the
anterior chamber. In addition, some ThOL.
injected corneas either were not incubated
in the ATPase medium or were used as
substrate-free controls.
At the end of the incubation, corneas
were rinsed thoroughly in buffer and postfixed in buffered 1 to 2 per cent OsO4 for
1 to 2 hours. Dehydration and embedding
in Epon 812 were carried out as described
previously.10 Sections were cut on a PorterBlum microtome using diamond knives and
were routinely stained with 1 per cent
uranyl acetate in 50 per cent alcohol and
in lead citrate13 prior to examination in an
RCA EMU 3-G electron microscope.
Results
Corneas fixed in glutaraldehyde and incubated in the histochemical medium containing ATP as substrate showed precipi°Thorotrast, Testagnr and Co., Detroit, Mich.
Fig. 1. In this portion of rabbit cornea] endothelium reacted for ATPase activity in the
Wachstein-Meisel medium,1- lead phosphate end product is localized in the intercellular space.
(Original magnification x32,000.)
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24
Kmje and Ticv
Fig. 2. For legend see opposite page.
Fig. 3. For legend see opposite page.
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Inoestigativu
Ophthalmology
February H)flG
ATPasa activity in cornea 25
Volume 5
Nmnhtir I
Fig. 2. In a ThO- control, incubated in substrate-free medium, only ThO; is in the endothelium.
The marker is adsorbed at the apical surface and is in vesicles in the apical cytoplasm. The
fusion of vesicles with the lateral cell membrane (arrows) is particularly well demonstrated
in the glutaraldehyde-fixed material. (Original magnification x34,000.)
Fig. 3. In this corneal endothelium which was reacted for ATPase activity after injection of
ThOa into the anterior chamber, both ThO: and lead phosphate are found in the intercellular
space. Vesicles and vacuoles in the apical cytoplasm which contain ThO : have no lead phosphate end product (arrows). (Original magnification x38,000.)
V
t
V
t
Fig. 4. In this unstained section of a cornea treated in the same way as that shown in Fig. 3,
the denser particles of ThO; are discernible within the lead phosphate deposits in the intercellular space. Whereas vesicles free in tlie cytoplasm (V) have no lead phosphate associated
with them, two vesicles which have fused with the lateral plasma membrane (arrows) show
an ATPase reaction. (Original magnification x32,000.)
tated final product, lead phosphate, in the
corneal endothelium (Fig. 1) closely related to the lateral plasma membranes of
the endothelial cells and in the intercellular
spaces. Pinocytotic vesicles in the apical
cytoplasm contained no precipitated lead
phosphate (Figs. 1, 3, 4).
Corneas which had been exposed to
ThCX in the anterior chamber for 30 min-
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utes prior to fixation showed uptake of
the marker into vesicles. These specimens
also demonstrated the apparent movement
of marker-filled vesicles around the apical
complex, their fusion with the lateral cell
membrane (Fig. 2), and the extrusion of
their contents into the intercellular space,
a sequence of processes previously reported.7"11 The fusion of vesicles with the
26 Kaye and Tice
V
Investigative Ophthalmology
February 1966
Figs. 6-10. In these portions of cornea] endothelium incubated with ADP, AMP, CTP, IDP, and
/3-glycerophosphate, respectively, slight reaction,
or no reaction at all, is seen in the intercellular
space. (Figs. 6; 8, original magnification xl7,lOO.
Figs. 7, 9, 10, original magnification x32,700.)
Fig, 5. This portion of corneal endothelium of a
7-day-old rabbit shows a weak positive reaction
for ATPase in the intercellular space. This is the
earliest stage at which this reaction has been
found. A vesicle (V) contains ThO-j, which is first
taken up by the corneal endothelial cells at 4 to
8 days. (Original magnification x39,700.)
lateral membrane is particularly well demonstrated in all glutaraldehyde-fixed corneas (Fig. 2; arrows).
Corneas exposed to ThCX. and subsequently incubated in the ATPase medium
also showed marker in pinocytotic vesicles
and in the intercellular space, indicating
transport by the vesicles. In such doubly
marked preparations (ThO-. and lead phosphate), the results obtained with each
marker were equivalent to those obtained
with the markers used separately. Despite
the unequivocal identification of pinocytotic vesicles by clearly visible ThO- particles within them (arrows, Fig. 3), no lead
phosphate was seen associated with the
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vesicles but was again restricted to the
intercellular space (Figs. 3 and 4). The
great electron scattering ability of the
ThO. allowed it to be identified even within deposits of lead phosphate. The differences in appearance of the ThO- and lead
phosphate were particularly apparent in
unstained preparations (Fig. 4).
Corneas from 6- and 7-day-old rabbits,
the age at which pinocytotic transport can
first be demonstrated,1"'115 showed a slight
reaction along the lateral cell margins,
particularly in the basal half of the cells
(Fig. 5). Vesicles and vacuoles which contained ThO- in these preparations again
contained no ATPase activity.
Vtilmne 5
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ATPase activity in cornea. 27
28
Kaije and Tice
hiucsl.igcit.ivo
Ophthalmology
February 1966
11
Fig, 11. In the basal portion of the corneal epithelium, fixed for only 5 minutes in glutaraldehyde prior to incubation, lead phosphate end product is only in the intercellular spaces of the
basal and intermediate layers. The basal membrane of the basal cells is negative, as is the
basement lamina. (Original magnification xl0 ; 300.)
In control preparations, no lead phosphate was seen after incubation without
substrate. In preparations in which other
substrates were substituted for ATP, slight
reaction was seen in the lateral margins of
endothelial cells. The reaction was identical
with ADP, AMP, CTP, I DP, or 0-glycerophosphate as substrate (Figs. 6 to 10).
Under altered fixation conditions (i.e.,
when fixation was carried out for 5 minutes in 2 per cent glutaraldehyde or when
glyoxal or hydroxyadipaldehyde was used
rather than glutaraldehyde), the same localization of final product was seen with
ATP as substrate.
After brief glutaraldehyde fixation, the
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adult corneal epithelium showed ATPase
activity along the margins of all the cells
except the apical surface of the outermost
squamous layer and the basal surface of
the basal layer (Fig. 11). Longer fixation
apparently destroyed this activity in the
adult cornea. Seven-day-old corneas, fixed
for as long as 30 minutes, showed some
ATPase activity in the epithelium (Fig.
12).
Discussion
The biochemical demonstration in many
tissues of ATPases synergistically activated
by Na+ + K+, together with studies showing
that activity of these enzymes was cor-
Volume 5
Nmtil/ar 1
ATPase activity in cornea 29
Fig. 12. In this portion of the intermediate layer of a 7-day-old rabbit corneal epithelium,
lead phosphate is localized in the intercellular spaces. (Original magnification x24,100.)
related with rates of cation transport and
was inhibited by ouabain,17"" stimulated
numerous attempts to localize sites of these
enzymes by histochemical methods. An
interesting finding in some of the histochemical studies was that the so-called
pinocytotic vesicles of certain capillaries/• smooth muscle cells,'1 some serosal cells/'
and Schwann cells4 demonstrated an intense nucleoside phosphatase activity. This
activity, seen with a variety of nucleoside
di- and triphosphate substrates, appeared
to be localized on the inner surface of the
vesicles, From these results, it was suggested that the phosphatase activity seen
in such locations might be due to the activity of the so-called pump enzymes or
might be related in some other way to the
"transport" activity of the vesicles.1"*'c
The first of these suggestions has always
been somewhat controversial. As early as
1961, Novikoff reported-0 that histochemical phosphatase activity was insensitive
to ouabain, and, with few exceptions,-T-2S
most other workers have confirmed that
histochemical activity is not activated by
monovalent cations or inhibited by ouabain.1'"' 21)'30 Although activation by monovalent cations was not demonstrable under
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histochemical conditions of incubation, it
was possible that the magnesium-dependent activity of this enzyme might still
be demonstrated. Even this possibility appears unlikely in view of the relative sensitivity of the Mg-dependent activity to
inhibition by -SH reagents and the fact
that both aldehyde fixatives and lead ions
are sulfhydryl inhibitors. The limited data
available suggest that activity of the enzyme is strongly inhibited by lead and by
brief exposures to dilute formaldehyde."'
Such an argument against the identity
of the enzyme demonstrated histochemically with the so-called pump enzymes is
not necessarily conclusive, since only
traces of chemically demonstrable activity
need remain after fixation for activity to
be demonstrated in situ, if the histochemical method used is sufficiently sensitive.
Unfortunately, the sensitivity of the Wachstein-Meisel technique is not known. In the
end, perhaps the strongest argument against
identification of the histochemical activity
with pump enzymes is that, chemically, regardless of activating ions, the pump enzyme is quite specific for ATP as substrate"'
and is strongly inhibited by calcium. The
histochemical activity, on the other hand,
30 Kaye and Tice
is relatively nonspecific in its substrate requirements and, in at least one site, appears
insensitive to calcium inhibition.3While pinocytosis has been shown to be
an energy-dependent process,™- :u the surface attachment and initial uptake phases
may not require energy.*'1'3r> Certainly, energy-dependent electrolyte transport has
never been shown to be directly associated
with pinocytotic vesicles even though
changes in electrolyte concentration at the
cell surface can stimulate vesicle formation.33' 3(; Moreover, the over-all importance
of vesicular activity to cell transport processes remains problematic, for rapid, directed, net transport activity by pinocytotic
vesicles has been clearly demonstrated in
only one site, the corneal endothelium.7'9
Although the vesicles of the myocardial
capillary endothelium have been shown to
take up colloidal markers and move them
across the cell,37"39 the loading and transit
times for these vesicles, although incompletely determined, appear to be too slow
to account for significant net fluid transport.
The present study, combining the use of
colloidal tracers, previously used to study
transport in the corneal endothelium,711
and histochemical techniques to localize
ATPase activity in this cell layer, clearly
demonstrates that the pinocytotic vesicles
engaged in transport exhibit no nucleoside
phosphatase activity. However, the localization of activity in the lateral margins of
the endothelial cells is identical with that
found in the gall bladder,10 colon,'11 kidney,1- toad urinary bladder,(! ciliary epithelium,13 and other sites in which electrolyte
transport occurs.*0 Although it seems highly
unlikely that the histochemical activity
seen is due to surviving cation-activated
ATPase, it is interesting that in all sites examined in which active electrolyte transport (and, therefore, concomitant fluid
transport) occurs there is a localization of
ATPase activity on the lateral cell margins.
Unless these findings are taken to be coincidental, it may be inferred that the enzyme so demonstrated has some connection,
as yet undefined, with transport processes.
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lmycstigalioc
February
I960
Since the present study demonstrates that
pinocytotic vesicles engaged in transport
show no ATPase activity, it is difficult to
explain the results obtained by Barrnett
and co-workers1"1' (: and by Carasso and coworkers/' There certainly is, with the possible exception of the myocardial capillaries
discussed above,37'39 a distinct lack of data
indicating that the vesicles studied by
Barrnett and co-workers'"1' (i and Carasso
and co-workersr> have any connection with
transport. It is most likely that vesicles
which exhibit such relatively nonspecific nucleoside phosphatase activity have a digestive or permanently absorptive function, as
in the invaginations of the cell surface for
protein absorption described by Roth and
Porter.41 There is little doubt that the vesicles in the ciliate Campanella umbellaria
studied by Carasso and co-workers" are
pinocytosing material from the food vacuole for subsequent digestion. Indeed, Rostgaard and Barrnett themselves suggest the
possibility of such a digestive or absorptive
function for the fixation-resistant phosphatase of intestinal smooth muscle.1
It appears that the corneal endothelium,
because of its peculiar avascular condition,
has adapted the general cellular absorptive
process of pinocytosis to a specific transport
function. From the data presented here and
from the studies of Kaye and Donn10 on
ouabain effects on pinocytosis, it seems
that the pinocytotic transport activity in the
corneal endothelium may be dependent
upon a functioning electrolyte transport
system in the lateral margin of the cells.
The authors are indebted for their help in various phases of this study to Mrs. Jeanne D. Cole
and Miss Mary Rayburn.
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