THE DEVELOPMENT OF SURFACE SPECIALIZATION
IN THE SECRETORY EPITHELIUM OF THE
AVIAN SALT GLAND IN RESPONSE TO OSMOTIC STRESS
STEPHEN A. ERNST and RICHARD A. ELLIS
From the Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island
02912. Dr. Ernst's present address is Department of Biology, Rice University, Houston, Texas 77001
ABSTRACT
Cell surface specialization, a characteristic common to most ion-transporting epithelia, was
studied in the salt (nasal) gland of the domestic duck in relation to osmotic stress. Three
days after hatching, experimental ducklings were given 1 % NaCI to drink for 12 hr and
freshwater for the remainder of each day. Control ducklings were maintained exclusively
on freshwater. The fine structure of the secretory epithelium was examined on various days
of the regimen. The nasal gland epithelium of the secretory lobule is composed of several
types of cells. Peripheral cells, lying at the blind ends of the branched secretory tubules, are
similar in both control and experimental animals at all stages of glandular development.
These generative cells contain few mitochondria and have nearly smooth cell surfaces.
Partially specialized secretory cells predominate in the secretory tubules of control animals
and appear as transitional cells in the tubular epithelium of salt-stressed animals. These cells
contain few mitochondria and bear short folds along their lateral cell surfaces. Fully specialized cells dominate the secretory epithelium of osmotically stressed ducklings. The
lateral and basal surfaces of these cells are deeply folded, forming complex intra- and
extracellular compartments. This vast increase in absorptive surface area is paralleled by
an increase in the number of mitochondria that pack the basal compartments. The development of this fully specialized cell is correlated with the marked increase in (Na+-K+)ATPase activity in the glands of osmotically stressed birds.
INTRODUCTION
Cells involved primarily in ion-transport share
similar morphological and enzymatic patterns.
The most striking similarities in the secretory
cells of such osmoregulatory organs as the avian
salt gland (9, 13, 30), the reptilian lacrimal gland
(1, 11), the elasmobranch rectal gland (7), and
the mammalian kidney (39) are the presence of
abundant mitochondria and the amplification of
the cell surface to form complex intra- and extracellular compartments. Likewise, the most striking similarities in enzymatic activity in these
tissues are the particularly high quantities of oxidative enzymes and adenosine triphosphatases.
Of particular interest is an ATPase, first demonstrated by Skou (49) in crab nerves, which re+
+.
quires Mg2+ and is activated by Na and K
The presence of this enzyme, (Na+-K+)-ATPase,
in considerable amounts in nervous and in ionsecreting tissues (see reviews 27 and 50) suggests
that it plays an integral role in ion-transport.
This hypothesis is supported by the observation
that active transport of Na + and K+ across the
305
plasma membrane of intact erythrocytes and
(Na+-K+)-ATPase activity in erythrocyte ghosts
share at least nine characteristics (37). In particular, both systems have an obligatory requirement
for Mg 2+ , Na + , and K+ and both of them are
inhibited by the cardiac glycoside, ouabain. These
common properties
characterize
(Na+-K+) ATPase in a large number of tissues from various
species (29).
Although the morphological and enzymatic
similarities between ion-concentrating epithelia
are well established, less is known about the onset
of, and the relationship between, structural and
enzymatic specializations within the context of
developing systems. The avian salt gland, during
early development, is an excellent organ for such
a study, since the gland undergoes hypertrophy
(4, 10, 23, 46) and enzymatic differentiation (2, 3,
6, 10, 14, 22) in response to osmotic stress. These
glands function in extrarenal excretion in several
species of marine birds (16, 42-46). The nasal
glands of salt-loaded birds secrete an almost pure
NaCl solution that is hypertonic to plasma and
seawater (e.g., 590 mEq/l in the domestic duck
[22]). This nasal secretion, which is under the
control of adrenocorticosteroids (36) and cholinergic nerve fibers (51), is a response to a general
osmotic load since the intravenous injection of
sucrose can elicit a secretory response (42).
Because nasal excretion is inhibited by a retrograde injection of ouabain (51), a (Na+-K+)ATPase appears necessary for secretory function
of the gland; several investigators have demonstrated the enzyme in homogenates of avian salt
glands (6, 14, 22, 24). Recently, Ernst et al. (14)
demonstrated a direct relationship between
osmotic stress and the level of (Na+-K+)-ATPase
in the salt gland of developing ducklings. This
response to physiological demand was confirmed
and extended by Fletcher et al. (22) who showed
that the salt-induced increase in the level of
(Na+-K+)-ATPase was correlated with an increased competence of the gland to concentrate
and eliminate ingested salts. As the present study
shows, the increased levels of glandular (Na + K+)-ATPase activity and the
concomitant
increase in secretory competence are paralleled
by the amplification of the surface of the secretory
cells at the ultrastructural level.
FIGURE 1 This histological section shows a secretory lobule from a salt gland of a duckling maintained on a saltwater regimen for 14 days. The main duct (MD), embedded in
the dense interlobular connective tissue (ICT), branches into a central canal (arrows).
The radially arranged branched secretory tubules radiate flom the central canal. Small,
dark peripheral cells are restricted to the blind ends (arrowheads) of the secretory tubules
adjacent to the interlobular connective tissue. Principal secretory cells comprise the remainder of the tubular epithelium. 7-gu paraffin section stained with hematoxylin and eosin.
X 44.
FIGURE 2 Epon-Araldite thick (I-,u) section of the salt gland of a control duckling on
day 11 stained with borax-toluidine blue. There is no clear distinction between the pelipheral cells lying near the interlobular connective tissue (ICT) and the principal secretory
cells. X 160.
FIGiURE
Epon-Araldite thick (1-jz) section of the salt gland of an experimental duckling
on day 11 stained with borax-toluidine blue. The pale peripheral cells near the interlobular
connective tissue (ICT) are distinguished easily from the mitochondlia-rich, dark principal
secretory cells lining the remainder of the secretory tubules. Three peripheral cells are in
mitotic mretaphase (arrows). X 160.
FIGURE 4
In this section from the salt gland of a 2-day-old duckling, the secretary tubule
abuts on the dense interlobular connective tissue (ICT) and is invested by a thin layer of
peritubular connective tissue (PCT). The cytoplasm of these peripheral cells contains
abundant free ribosomles and few initochondria. The cell surface is unfolded. Several
darkly staining cells (*) are present which are in contact with the basement membrane,
but do not apparently extend to the lumenal surface. The function and fate of these cells
is not known. X 3,400.
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VOLUME 40, 1969
S. A. ERNST AND R. A. ELLIS
Cell Specialization in Salt Gland
307
MATERIAL AND METHODS
RESULTS
Animals
Gross Morphology
Domestic ducklings (Anas platyrhynchus), received 2
days after hatching, were placed on a diet consisting
of duck starting mash and fresh water. 2 days later
(day 0) and for the next 31 days, half of the ducklings
(experimental group) were given a 1% solution of
NaCI for 12 hr starting at 10 A.M. and fresh tap water
for the remainder of the day, while the remaining
ducklings (control group) received fresh tap water
throughout the day. All birds were raised under an
artificial photoperiod of 12 hr, and heat was supplied
continuously by infrared heat lamps. Both the biochemical assays for salt gland (Na+-K+)-ATPase
activity described previously (14) and the ultrastructural studies to be described in this communication were derived from the same group of newly
hatched ducklings.
The nasal glands are paired crescent-shaped
structures that lie in the supraorbital depressions
of the frontal bone (32). Secretions elaborated by
each gland pass through paired ducts into the
anterior nasal cavity (32). From the nasal cavity,
the secretion flows out through the nares.
Histological Organization
Embryologically, each nasal gland develops
from two ducts (main ducts) which grow posteriorly as invaginations of the nasal epithelium.
Thus, each gland consists of two distinct but
closely applied parts. The anterior portion, comprising two-thirds of the salt gland, arises from
PreparativeProceduresfor
Electron Microscopy
On each day of the regimen that a biochemical
assay for ATPase activity was performed, one control
and one experimental animal were sacrificed so as to
provide salt gland tissue for electron microscopy. A
number of glutaraldehyde fixatives, over a wide range
of osmolalities, were tested. The best results were
obtained either with a glutaraldehyde fixative of
relatively low osmotic strength (250 milliosmols) or
with the formaldehyde-glutaraldehyde fixative recommended by Karnovsky (26). The latter gave the most
consistent preservation of fine structure and was the
principal fixative used in these experiments.
Small pieces (less than 3 mm3) of excised gland
were fixed for 1 hr at room temperature on a reciprocal shaker in formaldehyde-glutaraldehyde. The tissue was then rinsed for 2-1/A-12 hr in cold 0.1 M
sodium cacodylate buffer, pH 7.4, and postfixed with
cold unbuffered 1' osmium tetroxide for 1 hr.
Following fixation, the tissue was dehydrated rapidly
and embedded in Epon-Araldite (52). Thick sections
(1-2 A) and thin sections (silver-grey) were cut with
glass knives on a Porter-Blum MT-2 ultramicrotome.
At least four tissue blocks were sectioned from each
animal on each day of the experiment in order to
reduce sampling error to a minimum. Thick sections
were stained with borax-toluidine blue (1% toluidine
blue and 0.5% sodium borate in 35%,7 ethanol) for
light microscopy. Thin sections were placed on
uncoated copper grids and stained for 3-5 min with
saturated uranyl acetate in a 1:1 mixture of 70%
ethanol and 100% methanol and for 5 10 min with
lead citrate (38) diluted 1:2 with 0.01 N NaOH.
Stained sections were examined with an RCA-3D
electron microscope.
.308
FIGURE 5 The apical surfaces of the secretory cells
from the salt gland of a 2-day-old duckling have
short microvilli (Mv) extending into the lumen (L) of
the tubule. The cytoplasm contains abundant free
ribosomes and a few mitochondria (M). The opposed
lateral surfaces of the cells are usually flat, but some
bear short folds. Intermittently, they are joined by
desmosomes (arrows). The junctional complexes (J)
block direct passage from the intercellular spaces to
the lumen. Multivesicular bodies (MB) are found
commonly near the apical surface, and profiles of
rough surface endoplasmic reticulum (RER) and
smooth vesicles (v) are scattered randomly throughout
the cytoplasm. X 12,400.
TIE JOUAtNAL OF CELL BIOLOGY . VOI.Ml 40, 1969
the median duct, while the more posterior portion is derived from the more lateral duct (32).
Lateral branchings of the main ducts over the
frontal bone form the central canals. Radial outgrowths from each central canal give rise to
branched secretory tubules with closed terminal
ends. The secretory tubules which radiate from
each central canal are lined by simple cuboidalcolumnar epithelium and form a lobule that is
surrounded by a thick layer of connective tissue
(interlobular connective tissue). Each secretory
tubule is invested with a thin connective tissue
sheath (peritubular connective tissue), and the
central canals are, in turn, embedded in a relatively thick intralobular connective tissue sheath.
The general histological organization of a secretory lobule is shown in Fig. 1.
The vascularization and innervation of the
gland are similar to those described for the salt
gland of the herring gull (16, 17).
The Effect of Salt Regimen on Cell Surface
Specialization of the Secretory Epithelium of
the Salt Gland
Based on differential affinity for basic dyes and
varying reactivity for several cytochemical procedures at the light microscopic level, two types
of cells comprise the secretory epithelium: periph-eral cells and principal secretory cells (9, 10, 47).
The peripheral cells (Figs. 1-3) lie at the blind
ends of the secretory tubules and are active in
mitosis (12). The remainder of the epithelium is
composed of principal secretory cells (Figs. 1-3).
On the basis of distribution of mitochondria, the
difference between peripheral and principal cells
in the secretory epithelium of control animals is
not readily apparent at the level of the light microscope (Fig. 2), even after 9 days on the freshwater regimen. In contrast, after 9 days on the
saltwater regimen, the principal secretory cells are
FIGURE 6
The morphology of the peripheral cells from the salt gland of a control duckling on day 9 is
similar to that seen in the preceding micrograph. The basal cell surface is flat and rests on a continuous
basal lamina (arrows). A prominent Golgi complex (G) is usually adjacent to the nucleus. An amorphous
osmiophilic substance fills the lumen (L). This material is found occasionally in the lumen of the secretary
tubules of both control and experimental animals, but its significance is unknown. X 10,800.
S. A. ERNST AND R. A. ELLIS
Cell Specialization in Salt Gland
309
FIGURE 7 The peripheral cells from the salt gland of an experimental duckling on day 5 are similar in
morphology to peripheral cells in control animals (Figs. 5 and 6). The lateral and basal surfaces are nearly
flat with only a few folds, and some cells contain more mitochondria than others. The tip of a secretory cell
protrudes into the lumen (L), and nuclear pores (arrows) are obvious where a nucleus is sectioned tangentially. X 6,500.
packed with mitochondria (Fig. 3) and are distinguished easily from the large, clear peripheral
cells.
PeripheralCells
The general structure of the secretory tubule of
the nasal gland from a newly hatched duckling
(2 days old) is shown in Fig. 4. At this early stage
in development, the lobulation of the gland is not
well developed and the secretory tubules are
generally short, unbranched outgrowths from the
elongating ducts. However, regardless of the
stage of differentiation, the peripheral cells in the
glands of both control (Figs. 4-6) and salt-stressed
(Fig. 7) ducklings maintain a basically simple,
unspecialized architecture. The cytoplasm is
moderately dense and contains abundant free
ribosomes but only a few mitochondria (Figs.
4-7). The apical surfaces of the epithelial cells
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TIIE JOURNAL OF CELL BIOLOGY
bear short microvilli extending into the lumen
(Fig. 5), whereas the lateral cell surfaces are
nearly flat with only a few minor folds (Figs. 4-7).
Near the luminal surfaces of adjacent cells,
apposed plasma membranes are united by typical
junctional complexes (Fig. 5), as described in
other epithelia (18). In addition to the junctional
complexes, desmosomes intermittently join the
opposed lateral surfaces of adjacent cells (Fig. 5).
The flat basal surface of the epithelium rests on a
conspicuous basal lamina (Figs. 6 and 7). A
prominent Golgi apparatus is usually found in the
perinuclear region and profiles of rough surface
endoplasmic reticulum and smooth vesicles are
scattered randomly throughout the cytoplasm
(Figs. 5-7).
The general descriptive term of principal
secretory cell used on the light microscopic level
is not applicable when the secretory epithelium is
VOLUME 40, 1969
examined with the electron microscope. These
cells, which comprise the bulk of the nasal epithelium, may be placed in two arbitrary classes
based on cell surface amplification and distribution of mitochondria: partially specialized secretory cells and fully specialized secretory cells.
Partially Specialized Secretory Cells
During the development of the nasal glands of
ducklings maintained on freshwater, the partially
specialized secretory cells eventually predominate
in the elongating tubules. These cells are characterized by short, interdigitating villous folds along
the lateral cell surfaces (Fig. 8). They contain
few mitochondria and the basal cell surfaces are
nearly flat. Variations of this partially specialized
cell relate to the depth of the plications along the
lateral surfaces, and many variations are present
within the gland regardless of the age of the
animal.
Partially specialized secretory cells are present
as a transitional cell type in the nasal glands of
birds maintained on saltwater (Fig. 9) and, as in
the control animal, all stages of differentiation are
present in the tubular epithelium. The lateral cell
surfaces of the partially specialized cells shown in
Fig. 9 are elaborately folded so that the cytoplasm
is divided into numerous thin leaflets. Desmosomes are often found intermittently between the
interdigitating plications of adjacent cells. The
mitochondria, which are generally excluded from
the intracellular compartments formed by these
lateral cytoplasmic extensions, are usually found
in the small, unmodified perinuclear region (Figs.
8 and 9). Infoldings of the basal surface are
extremely sparse.
The partially specialized secretory cells are
derived from the mitotically active peripheral
cells (12). As the daughter cells migrate from the
periphery and as the secretory tubules elongate,
FIGURE 8
The lateral cell surfaces of these partially specialized secretory cells from the salt gland of a
control duckling on day 3 form short, villous folds which interdigitate with the lateral projections from
adjacent cells. Mitochondria are excluded from the lateral processes and are restricted to the perinuclear
zone. Basal surfaces of these cells are nearly flat, with few folds, and rest on a continuous basal lamina
(arrows). X 9,700.
S. A. ERNST AND R. A. E.LIS
Cell Specialization in Salt Gland
311
These partially specialized cells from the salt gland of an experimental duckling on day 1
resemble those in control birds seen in the preceding micrograph (Fig. 8) except that the lateral processes
are somewhat longer. Desmosomes (arrows) intermittently join the interdigitating folds. A portion of the
lumen (L) is shown. X 10,500.
FIGURE 9
the lateral cell surfaces become progressively
more specialized. Since the secretory tubules at
early stages of development are still short, it is
possible, in favorable sections, to show several
stages in cell specialization in a single electron
micrograph (Fig. 10).
Fully Specialized Secretory Cells
The basal cell surface remains nearly flat in
the secretory cells of birds maintained on fresh-
water. In the experimental animals, however, the
basal surface is further amplified and the number
of mitochondria is greatly increased, giving rise
to the fully specialized secretory cell type (Fig.
10). The sequence of events leading to this form
can be demonstrated in a series of micrographs
(Figs. 11 14). Basal folds begin as short invaginations (Fig. 11). As the folds become deeper, mitochondria are occasionally trapped within the cytoplasmic leaflets (Figs. 12 and 13). Like the basal
This section, showing peripheral cells, partially specialized secretory cells
and fully specialized secretory cells from te salt gland of an experimental duckling on
day 3, passes nearly longitudinally through a tubule. The peripheral cells lying next to
the dense interlobular connective tissue (ICT) have the typical form described in Figs.
4 7. One of the peripheral cells is shown in mitosis. The secretory cells comprising the
balance of the tubule become increasingly specialized along both the lateral (X) and basal
(arrows) cell surfaces as they traverse the lobule toward the central canal (upper left).
In addition, the number of mitochondria in these secretory cells increase correspondingly.
Distribution of mitochondria at the light microscopic level (Fig. 3) parallels that shown
here at the electron miicroscopic level. Portions of the tubular lumen (L) are shown.
X 8,500.
FIGtRE 10
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THE JOUIINAL OF1 CELL BIOLOGY - VOLU.TME 40, 19)69
S. A.
ERNST AND
R. A. E.IS
Cell Specialization in Salt Gland
313
lamina of the distal convoluted tubule of the kidney, the basal lamina remains uninterrupted and
does not invaginate with the plasma membrane
(Fig. 13). While the basal compartments elongate,
there is a concomitant increase in the number
of mitochondria which pack them (Figs. 12-14).
In many cases, the basal folds of adjacent cells
mesh with one another forming an elaborate
complex of extracellular spaces and intracellular
compartments (Fig. 14). The basal folds generally
invest the cytoplasm to the level of the nucleus
and, in conjunction with lateral folds, vastly
increase the total absorptive surface of the secretory cell (Fig. 15). In contrast, the apex of the
cell has a limited surface area and is only slightly
modified by the presence of a few microvilli
(Fig. 15). Although the fully specialized secretory
cells are occasionally found in the secretory epithelium as early as the 2nd day on the salt regimen, this cell type is not dominant until after
day 4. Extensive folding of the basal surface in
the secretory epithelium in the glands of unstressed animals, however, is rarely seen, even in
the later days of the experiment.
The distribution of peripheral cells and partially and fully specialized secretory cells in the
secretory tubules of the salt gland of ducklings
maintained on freshwater and on saltwater for
various periods of time is diagrammed in Fig. 16.
DISCUSSION
The salt glands of marine birds are extraordinary
structures for studying the functional relationship
+)
ATPase and active transport
between (Na+-K
on the level of the whole organ (14, 22) and,
moreover, since their development and differentiation is stimulated by ingested salts (4, 10, 23,
46), the functional competence of the transport
system may be correlated with the morphology of
the secretory cells. The nasal gland provides an
additional advantage in such a study because its
primary and perhaps only function is the concen-
FIGURE 11 This micrograph shows secretory cells from the salt gland of an experimental duckling on
day 1 at the first stage in the specialization of the basal surface in which there are only short plications
of the plasma membrane. X 14,900.
The basal folds in these secretory cells from the salt gland of an experimental duckling on
day 5 protrude some distance into the cytoplasm, and mitochondria are occasionally trapped in the
intracellular compartments formed by the plicated basal membrane (arrows). X 8,400.
FIGURE 12
FIGURE 13 The basal compartments in these secretory cells from the salt gland of an experimental
duckling on day 5 are more highly developed, and the cytoplasmic processes contain mitochondria (arrowheads). The basal lamina (BL) does not invaginate with the plasma membrane. X 10,800
314
THE JOURNAL OF CELL BIOLOGY
VOLUME 40, 1969
FIGURE 14 The basal compartments in these secretory cells from the salt gland of an experimental
duckling on day 5 are fully developed and contain abundant mitochondria. The basal folds of adjacent
cells mesh with one another forming an elaborate pattern of intra- and extracellular compartments. In
this micrograph, the basal folds of a cell with dense cytoplasm (arrows) interleaf with the basal plications
of another less dense cell (arrowheads). The intracellular compartments may be as narrow as 50-80 m,
but are considerably broader at points where they accommodate mitochondria. X 18,900.
tration and secretion of electrolytes (42-46). In
the present investigation, the changes in the level
of glandular (Na+-K+)-ATPase activity in response to osmotic stress (14, 22), and the capacity of the salt gland to elaborate and concentrate
an effluent (15, 22) are correlated with cell differentiation in the gland.
We showed previously that osmotic stress
effects a fourfold increase in the specific activity
of glandular (Na+-K+)-ATPase over the basal
levels found in the nasal glands of unstressed
ducklings (14). Furthermore, the elevated level of
the enzyme, which reached a maximum after 9
days of osmotic stress, fell to control levels when
osmotically stressed birds were placed on a fresh-
water regimen. These results were confirmed by
Fletcher et al. (22) in adult ducks. In addition,
Fletcher et al. showed that the increased levels of
)
(Na+-K + ATPase in salt-stressed adult birds is
accompanied by an increase in both the rate of
secretion (by a factor of 3.2) and the concentration of Na+ in the secretion (by a factor of 1.3).
Similar increments also occur in osmotically
stressed, newly hatched ducklings (15). Osmotic
stress, therefore, results in a physiologically more
efficient transport system. This increased efficiency is related directly to the amplification of
the surface of the secretory cells of the nasal gland
epithelium.
Nasal glands of control ducklings, even at very
S. A. ERNST ANU R. A. ELLIS
Cell Specialization in Salt Gland
315
FIGURE 15
This micrograph of fully specialized secretory cells from the salt gland of an experimental
duckling on day 11 demonstrates, at low magnification, the vast increase in surface area resulting from
the complex folding at the lateral (X) and basal (arrows) cell surfaces. In contrast, the area of the apical
interface is very small, and only a few microvilli protrude into the lumen (L). X 6,600.
316
16 This diagram shows the effect of salt and fresh water regimens on the development of cellular
specialization in the secretory epithelium of the salt gland. Peripheral cells (A) exhibit little specialization of their cell surfaces and contain few mitochondria. Partially specialized secretory cells (B) are characterized by short folds along their lateral surfaces and flat basal plasma membranes. The distribution of
mitochondria in this cell type is similar to that of the peripheral cells. Two stages in the development of
the fully specialized secretory cell type (C and D) are shown. In the first stage (C), the cells exhibit some
folding at the basal surface in addition to the lateral plications. Mitochondria, more numerous in this cell
type than il the partially specialized secretory cell, are quite evenly distributed in the cytoplasm. In the
second stage (D), this specialized secretory cell is fully developed. Both the lateral and the basal plasma
membranes are extensively folded, forming complex intracellular compartments and extracellular spaces.
The mitochondria are increased dramatically in number and pack the basal labyrinths. The distribution
of these cell types in freshwater- and saltwater-adapted glands on various days of the regimen is shown
for a single secretory tubule as it grows out from the central canal. In addition to the development of
cellular specialization due to osmotic stress, the secretory tubules in the salt glands of experimental animnals elongate more rapidly and exhibit more branching than in the control glands.
FIGURE
early stages in their development (e.g., day I),
have low levels of (Na+-K+)-ATPase activity (14)
and are able to concentrate and eliminate salt
(15). Morphologically, the only cells in the secretory epithelium at this time, other than the generative peripheral cells (Figs. 4-7), are the partially
specialized secretory cells with short surface folds
of varying length along the lateral cell surfaces
(Fig. 8). Later in the development of glands in
unstressed ducklings, this cell type predominates
(Fig. 16). Although there is some increase in the
total absorptive surface area of the epithelium
during this period, there is neither an increase in
the specific activity of (Na+-K+)-ATPase (14)
nor an increase in the concentration of Na+ in
the effluent or in the rate of secretion of the effluent (15) in response to an injected salt load.
Although the lateral surfaces of the secretory
cells from the nasal glands of control ducklings
become more specialized during the course of
development, the basal surface is virtually unmodified (Fig. 8). In contrast, the basal surfaces
of the secretory cells of osmotically stressed birds
undergo progressive differentiation (Figs. 10-14)
until the cytoplasm of the cell is divided into what
Fawcett (21) has termed basal compartments.
Furthermore, the basal folds of adjacent cells
interdigitate (Fig. 14), thereby creating a complex system of intra- and extracellular spaces. As
the basal compartments develop, abundant
mitochondria become trapped within them (Fig.
14). The basal invaginations, coupled with the
lateral plications, vastly increase the cell surface
area (Fig. 15). The distribution of this fully
specialized cell type in secretory tubules of the
salt gland is shown diagrammatically in Fig. 16.
The gradual predominance of this cell type with
successive days of salt stress is paralleled by a
dramatic increase in the specific activity of
(Na+-K+)-ATPase activity (14), and by an
increase in the capacity to concentrate and
eliminate an injected salt load (15). Thus, the
increased competence of the salt gland at the biochemical and the physiological levels relates
specifically to the development of this fully
specialized cell. This hypothesis is further supported by the absence of the fully specialized cell
from the secretory epithelium of control animals.
Attempts to localize a ouabain-sensitive (Na + K+)-ATPase by modifications of the WachsteinMeisel procedure (53) for the demonstration of
318
THE JOURNAL OF CELL BIOLOGY
Mg2+-activated ATPase (20, 31, 34) have failed
because all of the divalent cations capable of precipitating liberated phosphate are inhibitory to
the enzyme (5). Furthermore, the validity of this
cytochemical method has recently been questioned (33, 40). Cell fractionation techniques
have, however, shown the enzyme to be associated
with the membranes of the cell (see review 27).
The enzyme is probably bound to the plasma
membrane, although there is little information to
indicate which surface of the cell is involved.
Bonting et al. (6) suggested that, in the avian salt
gland, (Na+-K+)-ATPase is localized along the
apical surfaces of the secretory cells since, in
several other tissues, ouabain inhibits Na + transport on the surface of the cell where Na+ is extruded. However, this interpretation is questionable when cytomorphogenetic changes during
salt stress are considered. Throughout the course
of cell differentiation in osmotically stressed
animals, the apical surface of the secretory cells
undergoes little, if any, change and remains
extremely small in area when compared to the
lateral and basal surfaces. In contrast, both the
lateral and the basal surfaces are modified greatly
to increase the cell surface area. Moreover, the
mitochondria are preferentially distributed in the
cell so that the majority are packed within the
basal compartments. The vast increase in absorptive area along the basal and lateral surfaces (at a
time when the specific activity of (Na+-K+) ATPase is rapidly increasing) and the close association of the folded membranes with a readily
available supply of ATP (from the mitochondria)
suggest that the transport system is localized
along the lateral and, particularly, the basal
surfaces of the cell. Hokin (25) showed that salt
gland slices stimulated by acetylcholine maintain
an intracellular Na + concentration higher than
that of the external medium, indicating that a
concentrating mechanism may be acting at the
basal surface. In these experiments, however, this
mechanism was not sensitive to ouabain. Additional evidence by Hokin (25) indicates that
transport across the luminal surface may be
passive since the intracellular concentration of
Na + was approximately equivalent to the concentration of Na + in the effluent. In summary,
Na+ may be transported actively from the extracellular (basal and lateral) compartments to the
intracellular compartments of the secretory cells.
VOLUME 40, 1969
+
Subsequent movement of Na across the luminal
surface of the secretory cells may be active or
passive.
It is evident that cell membrane specialization,
particularly at the lateral and the basal surfaces,
is characteristic of cells involved in ion transport,
although the specific form of the specialization
varies with each epithelium. In amphibian skin
(19), the gallbladder (28), the urinary bladder
(35), and the salt (lacrimal) glands of marine
turtles (1, 11), the cells are specialized by elaborate folding of the lateral surfaces. In the convoluted tubules of the kidney (39) and the striated
duct of salivary glands (41, 48), the cells are
deeply plicated at their basal surface. In two
cases of extreme specialization for ion transport,
the avian salt gland (9, 13, 30) and the elasmobranch rectal gland (7), both the basal and the
lateral surfaces of the secretory cells are folded.
The degree of development of the basal plications
also varies with different ion-secreting epithelia
(21). Thus, Fawcett (21) showed that the basal
compartments are less well developed in the
ciliary body, the choroid plexus, and the proximal
convoluted tubule of the frog kidney than they
are in the distal convoluted kidney tubules and
the striated ducts of the salivary gland. The basal
folds of the secretory cells in the avian salt gland
and the rectal gland are highly developed and, at
least in the salt gland, the level of membrane
differentiation is directly related to osmotic stress
(Fig. 16). Invariably, the development of basal
compartments in ion-concentrating cells accompanies an increase in the number of mitochondria
which fill the intracellular compartments and are
apposed closely to the folded membranes. Thus,
secretory cells involved in the active transport of
electrolytes show striking similarities in membrane specialization and in the distribution of
mitochondria.
Finally, the presence of basal and/or lateral
compartments in ion-transporting systems may
have functional significance in permitting the
coupling of water transport to active solute
transport. A standing-gradient flow system, described by Diamond and Bossert (8), may operate
in 'backwards" fluid-transporting epithelia (i.e.,
epithelia which actively transport solute into the
cell from the extracellular channels), such as the
avian salt gland, to produce a hypertonic secretion. In the avian salt gland, the development of
highly compartmentalized secretory cells (Figs.
11-15) and elevated levels of (Na+-K+)-ATPase
(14, 22) are directly affected by osmotic stress and
result in the increased capacity of the salt gland
to concentrate and to eliminate ingested salts
(15, 22). Such a system, therefore, emphasizes the
importance of ultrastructural geometry in iontransporting epithelia and, as such, lends indirect
support to this hypothesis.
The authors wish to thank Dr. R. J. Goss and Dr.
E. H. Leduc for their valuable help and criticism
during the course of this work, and Mrs. L. A. Farr
for her excellent technical assistance. The illustrations
were prepared by Mr. R. E. Wall.
This research forms part of a dissertation submitted
by S. A. Ernst in partial fulfillment of the requirements for the Ph.D. degree at Brown University,
1968 and was supported by a training grant from the
United States Public Health Service GM-00582-07.
Received for publication 9 July 1968.
REFERENCES
1. ABEL, J. H., JR., and R. A. ELLIS. 1966. Histochemical and electron microscopic observations
on the salt secreting lacrymal glands of marine
turtles. Amer. J. Anat. 118:337.
2. BALLANTYNE, B., and J. FOURMAN. 1966. Cholinesterases and the secretory activity of the
duck supraorbital gland. J. Physiol. 188:32P.
3. BALLANTYNE,
B., and W. G.
WOOD.
supraorbital (nasal) glands from saline-fed and
freshwater-fed domestic ducks (Anas platyrhynchus). J. Anat., London. 98:571.
5. BONTING, S. L., L. L. CARAVAGGIO, and N. M.
1967. A
histochemical and biochemical investigation ot
/-glucuronidase activity in the quiescent and
secreting supra-orbital gland of Anas domesticus.
J. Physiol. 191:89P.
4. BENSON, G. K., and J. G. PHILLIPS. 1964. Observations on the histological structure of the
6.
HAWKINS. 1962. Studies on sodium-potassiumactivated adenosinetriphosphatase. IV. Correlation with cation transport sensitive to
cardiac glycosides. Arch. Biochem. Biophys. 98:
413.
BONTING, S. L., L. L. CARAVAGGIO, M. CANADY,
and N. M. HAWKINS. 1964. Studies on sodium-
adenosintriphosphatase.
potassium-activated
XI. The salt gland of the herring gull. Arch.
Biochem. Biophys. 106:49.
S. A. ERNST AND R. A. ELLIS
Cell Specialization in Salt Gland
319
7. BULGER, R. E. 1963. Fine structure of the rectal
(salt-secreting) gland of the spiny dogfish,
Squalus acanthias. Anat. Rec. 147:95.
J.
8. DIAMOND,
M., and W.
H.
BOSSERT.
1968.
Functional consequences of ultrastructural geometry in "backwards" fluid-transporting epithelia. J. Cell Biol. 37:694.
9. DOYLE, W. L. 1960. The principal cells of the
salt gland of marine birds. Exp. Cell Res. 21:386.
10. ELLIS,
R.
A., C
C.
GOERTEMILLER, JR., R.
DELELLIs, and Y. A. KABLOTSKY.
25.
A.
1963. The
effect of a salt water regimen on the development of the salt glands of domestic ducklings.
Develop, Biol. 8:286.
11. ELLIS, R. A., and J.
24.
26.
H. ABEL, JR. 1964. Inter-
cellular channels in the salt-secreting glands
of marine turtles. Science. 144:1340.
12. ELLIS, R. A. 1965. DNA labelling and X-irradiation studies of the phosphatase-positive peripheral cells in the nasal (salt) glands of ducks.
Amer. Zool. 5:648.
27.
28.
13. ELLIS, R. A., and S. A. ERNST. 1967. The effect
of salt water intake on the specialization of the
surface of secretory cells in the nasal (salt)
glands of domestic ducks. Anat. Rec. 157:239.
1966. Specificity of sodium chloride in the
stimulation of growth in the salt glands of
ducklings. Z. Mikrosk. Anat. Forsch. 74:296.
+HOKIN, M. R. 1963. Studies on a Na' + K
dependent, ouabain-sensitive adenosine triphosphatase in the avian salt gland. Bliochinm.
Biophys. Acta. 77:108.
+
HOKIN, M. R. 1967. The Nat, K , and C1content of goose salt gland slices and the
effects of acetylcholine and ouabain. J. Gen.
Physiol. 50:2197.
KARNOVSKY, M. J. 1965. A formialdehyde-glutaraldehyde fixative of high osmolality for use
in electron microscopy. J. Cell Biol. 27:137A.
(Abstr.)
KATZ, A. I., and F. H. EPsrTEIN. 1967. The
physiological role of sodium-potassium activated adenosine triphosphatase in the active
transport of cations across biological membranes. Israel J. Mied. Sci. 3:155.
KAYE, G. I., H. O. WHEELER, R. T. WHITLOCK,
and N. LANE. 1966. Fluid transport in the
rabbit gallbladder. A combined physiological
and electron microscopic study. J. Cell Biol.
30:237.
C.
R.,
R.
L. POST,
and D. L.
14. ERNST, S. A., C. C. GOERTEMILLER, JR., and R. A.
29. KINSOLVING,
ELLIS. 1967. The effect of salt regimens on the
development of (Na+-K+)-dependent ATPase
activity during the growth of salt glands of
ducklings. Biochim. Biophys. Acta. 135:682.
15. ERNST, S. A., and B. BROMBERG. 1967. Unpublished observations.
16. FANGE, R., K. SCHMIDT-NIELSEN, and H. OSAKI.
1958. The salt gland of the herring gull. Biol.
Bull. 115:162.
BEAVER. 1963. Sodium plus potassium transport adenosine triphosphatase activity in the
kidney. J. Cell. Comp. Physiol. 62:85.
30. KOMNICK, H. 1963. Elecktronenmickroskopische
Untersuchungen zur functionellen Morphologie des lonentransportes in der Salzdriise von
Larus argentatuas (III. Funktionelle Morphologie
der Tubulusepithelzellen). Ptoplasma. 56:605.
31. MARCHESI, V. T., and G. E. PALADE. 1967. The
localization of Mg-Na-K-activated adenosine
triphosphatase on red cell ghost membranes.
J. Cell Biol. 35:385.
32. MARPLES, B. J. 1932. The structure and development of the nasal glands of birds. Proc. Zool.
Soc., (London). 2:829.
17. FANOE, R., K. SCHMIDT-NIELsEN, and M.
ROBINSON. 1958. Control of secretion from the
avian salt gland. Amer. J. Physiol. 195:321.
18. FARQUHAR,
M. G., and G. E. PALADE.
1963.
Junctional complexes in various epithelia. J.
Cell Biol. 17:375.
19. FARQUHAR,
M. G., and G. E. PALADE.
1965.
33. MosEs,
H. L., A. S. ROSENTHAL, D. L. BEAVER,
Cell junctions in amphibian skin. .1. Cell Biol.
26:263.
20. FARQUHAR, M. G., and G. E. PALADE. 1966.
Adenosine triphosphatase localization in amphibian epidermis. J. Cell Biol. 30:359.
21. FAWCETT, D. F. 1962. Physiologically significant
specializations of the cell surface. Circulation.
26:1105.
and S. S. SCHUFFMAN. 1966. Lead ion and
phosphatase histochemistry. II. Effect of adenosine triphosphate hydrolysis by lead ion on
the histochemical localization of adenosine
triphosphatase activity. J. Histochem. Cytochem.
14:702.
34. NOVIKOFF, A. B., J. DRUCKER, W. Y. SHIN, and
22. FLETCHER, G. L., I. M. SAINER, and W. N.
apparent adenosinetriphosphatase activity of
cell membranes in formol-calcium-fixed tissues.
J. Histochem. Cytochem. 9:434.
35. PEACHEY, L. D., and H. RASMUSSEN. 1961.
Structure of the toad's urinary bladder as
related to its physiology. J. Biophys. Biochem.
Cytol. 10:529.
HOLMES. 1967. Sequential changes in the
adenosinetriphosphatase activity and the electrolyte excretory capacity of the nasal glands
of the duck (Anas platyrhynchos) during the
period of adaptation to hypertonic saline. J.
Exp. Biol. 47:375.
23. GOERTEMILLER,
320
C. C., JR.,
and R.
THE JOUItNAL OF CELL BIOLOGY
A. ELLIs.
S. GOLDFISChIER. 1961. Further studies of the
36. PHILLIPS, J.
VOLUMIE 40, 1969
G., W. N.
HOLMES,
and D. G.
BUTLER. 1961. The effect of total and subtotal
adrenalectomy on the renal and extra-renal
response of the domestic duck (Anas platyrhynchus) to saline loading. Endocrinology. 69:958.
function of the salt gland in the brown pelican.
Auk. 75:282.
45. SCHMIDT-NIELSEN,
K.
1960. The salt-secreting
gland of marine birds. Circulation. 21:955.
37. POST, R. L., C. R. MERRITT, C. R. KINSOLVING,
46. SCHMIDT-NIELSEN, K., and Y. T. KIM. 1964. The
and C. D. ALBRICHT. 1960. Membrane adenosine triphosphatase as a participant in the
active transport of sodium and potassium in
the human erythrocyte. J. Biol. Chem. 235:
1796.
38. REYNOLDS, E. S- 1963. The use of lead citrate at
high pH as an electron-opaque stain in electron
microscopy. J. Cell Biol. 17:208.
39. RHODIN, J. 1958. Anatomy of kidney tubules.
Int. Rev. Cytol. 7:485.
effect of salt intake on the size and function of
the salt gland of ducks. Auk. 81:160.
40. ROSENTHAL,
A\.S.,
H. L. MOSES, D. L. BEAVER,
and S. S. SCHUFFMAN. 1966. Lead ion and
phosphatase histochemistry. I. Nonenzymatic
hydrolysis of nucleoside phosphates by lead
ion. J. Histochem. Cytochem. 14:698.
41. RUTBERG, V. 1961. Ultrastructure and secretory
mechanism of the parotid gland. Acta Odontol.
Sand. Suppl. 30 19:1.
42. SCHMIDT-NIELSEN, K., C. B. JORGENSEN, and
H. OSAKI. 1958. Extrarenal salt secretion in
birds. Amer. J. Physiol. 193:101.
43. SCHMIDT-NsELsEN, K., and W. J. L. SLADEN.
1958. Nasal salt secretion in the Humboldt
penguin. Nature. 181:1217.
44. SCHMIDT-NIELSEN, K., and R. FANGE. 1958. The
47. SCOTHORNE, R. J. 1959. The nasal glands of
birds: a histological and histochemical study
of the inactive gland in the domestic duck.
J. Anat. 93:246.
48. SCOTT, B. L., and D. C. PEASE. 1959. Electron
microscopy of the salivary and lacrimal glands
of the rat. Amer. J. Anat. 104:115.
49. SKOU, J. C. 1957. The influence of some cations
on an adenosine triphosphatase from peripheral
nerves. Biochim. Biophys. Acta. 23:394.
5(.
SKOU, J. C. 1965. Enzymatic basis for active
+
+
transport of Na and K across cell membrane.
Physiol. Rev. 45:596.
51. THESLEFF, S., and K. SCHMIDT-NIELSEN. 1962.
An electrophysiological study of the salt gland
of the herring gull. Amer. J. Physiol. 202:597.
52. VOELZ, H., and M. DWORKIN. 1962. Fine structure of Myxococcus xanthus during morphogenesis. J. Bacteriol. 84:943.
53. WACHSTEIN, M., and E. MEISEL. 1957. Histochemistry of hepatic phosphatases at a physiologic pH. With special reference to the
demonstration of bile canaliculi. Amer. J. Clin.
Pathol. 27:13.
S. A. ENST AND R. A. ELLIS
(ell Specialization in Salt Gland
321