AMER. ZOOL., 26:209-224 (1986)
Structure and Function of the Chloride Cell of
Fundulus heteroclitus and Other Teleosts1
KARLJ. KARNAKY, JR.
Department of Physiology and Cell Biology, University of Texas Medical School,
P.O. Box 20708, Houston, Texas 77225
SYNOPSIS. Teleosts, the bony fishes, inhabit both freshwater and seawater environments.
Some euryhaline fish, such as Fundulus heteroclitus, alternate between the two milieux
several times daily. Regardless of adaptation, the gills of these animals possess a highly
specialized cell type called the chloride cell. This cell contains numerous mitochondria
and exhibits a greatly amplified basolateral cell surface richly endowed with Na,K-ATPase.
Recent studies on isolated opercular epithelia containing chloride cells have demonstrated
active chloride secretion and passive transepithelial sodium movements, and have established the chloride secretory role of this cell type in seawater-adapted teleosts. Current
models suggest that chloride transport occurs via a transcellular route. Seawater chloride
cells exist in multicellular units and share simple, shallow tight junctions which are thought
to be the route for passive sodium movement. Freshwater chloride cells, whose function
remains to be elucidated, are generally described as existing in a unicellular configuration.
However, recent observations in Fundulus heteroclitus adapted to salinities as low as 1%
sea water reveal that chloride cells persist in multicellular complexes with apical crypts.
Strikingly, tight junctions between chloride cells in this freshwater environment are deep.
The topic of teleost osmoregulation and
the chloride cell has been extensively
reviewed in the last several years (Evans,
1979; Karnaky, 1980; Degnan and Zadunaisky, 1982; Evans et al, 1982; Foskett et
al, 1983; Degnan, 1984a). Two published
symposia honoring Jean Maetz give extensive coverage of this topic (Lahlou, 1980;
plus a special issue of the Am. J. Physiol.,
238 Regulatory Integrative Comp. Physiol. 7:R139-R276, 1980). This review
focuses on the structure and function of
the teleost chloride cell, particularly as
revealed by the study of isolated epithelia
such as the operculum of Fundulus hetero-
via the gills. The seawater fish is in the
opposite situation. Its body fluids are much
less salty than the external environment
and it is faced with the problem of exosmosis and constant influx of ions across
permeable body membranes. The solution
of the seawater fish is to drink sea water,
absorb NaCl and water from the ingested
fluid, and to excrete the extra NaCl
through the gills. Most teleosts can tolerate
only a very narrow range of salinities (the
stenohaline teleosts). Remarkably, some
species can tolerate a very wide range of
external salinities (the euryhaline teleosts).
clitus.
THE TELEOST GILL AND CHLORIDE CELL
The cell type of primary interest in this
review is the chloride cell. This cell was
Teleosts, the bony fishes, face rather first identified and described over 50 years
severe osmotic problems whether they live ago in a classic work by Keys and Willmer
in fresh water or in sea water. In fresh (1932). Only two years prior to this diswater, the fish's body fluids are salty com- covery, Homer Smith (1930) had prepared to the external environment and the sented compelling evidence for an extrafish is faced with endosmosis. The fish's renal site for NaCl excretion in seawater
answer to this challenge is to drink little, teleosts. A year later Keys (1931a, b), using
excrete large quantities of dilute urine, and a heart-gill perfusion preparation from the
absorb ions from the external environment European eel, discovered that chloride ions
were transported from the perfusion solution into the external sea water bathing the
1
From the Symposium on The Biology of Fundulus gills. When Keys and Willmer (1932)
heteroclitus presented at the Annual Meeting of the examined the gills of seawater teleosts they
American Society of Zoologists, 27-30 December
discovered a large, ovoid cell in the gills of
1983, at Philadelphia, Pennsylvania.
SALT HOMEOSTASIS IN TELEOSTS
209
FIG. 1. Seawater-adapted Fundulus heteroclztus Transverse section of a gill filament showing the afferent
blood vessel (A) and the cartilaginous shaft (C). A respiratory leaflet is marked RL. Even at this relatively low
magnification, chloride cells are conspicuous because of their columnar shape and granular cytoplasm. In
several of these chloride cells the apical crypt is prominent (arrows). Fixed with glutaraldehyde, postfixed
with osmium tetroxide, stained with methylene blue and azure 11. Micrograph by Hal Church, Mt. Desert
Island Biological Laboratory, Maine. x 560.
these fish. It resembled the acid-secreting
cell in the stomach and they named the cell
the "chloride-secreting" cell. They also
found this cell type in the gills of freshwater teleosts, and they suggested that this
cell may perform an absorptive function in
this environment. For abbreviation, in the
remainder of this review I will call this cell
the "chloride cell," regardless of the environment to which the fish is adapted.
Teleosts possess four gill arches on each
side of the body. Projecting from each of
these gill arches are hundreds of gill filaments (Fig. 1). Cross sections of gill filaments show a central cartilaginous shaft
and other connective tissue surrounded by
an epithelium. T h e respiratory leaflets, sites
of oxygenation of red blood cells, protrude
from these gill filaments. At the light
microscopic level the gill chloride cell
exhibits some rather distinguishing characteristics (Fig. 1). It is a large cell, generally columnar-shaped, with a single
nucleus, and generally extends from the
basal lamina to the external world. T h e gill
is covered by a single layer of cells called
pavement cells in an arrangement much
like that of a cobblestone street: where
t h e r e a r e potholes, chloride cells a r e
directly exposed to the external world.
TELEOST CHLORIDE CELL
211
FIG. 2. Electron micrograph of the apical region of a gill chloride cell from seawater-adapted Cypnnodon
variegatus. The apical crypt (AC) with its mucous content is visible in the top center of the micrograph. A
small portion of the nucleus (N) is shown in the lower portion of this micrograph. The supranuclear zone is
filled with a host of organelles. Besides mitochondria (M), which are especially prominent, the Golgi complex
(G) and elements of the rough endoplasmic reticulum (RER) may also be seen. Note the secretory droplets
(arrowheads) surrounding the Golgi complex, and between this organelle and the apical crypt. Note that this
apical crypt is actually shared by two chloride cells. A second chloride cell is represented in this micrograph
by a small finger interdigitating with the major chloride cell of the micrograph. Arrows point to the tight
junctions connecting the two chloride cells. Fixed with osmium tetroxide and stained with uranyl and lead
salts, x 28,600.
212
K A R L J . KARNAKY, JR.
i
FIG. 3. A relatively large field of chloride cell cytoplasm from gill tissue of seawater-adapted Cyprinodon
variegatus. Note that the cytoplasmic ground substance isfilledwith an extensive, anastomosing tubular system
and that tubules are often in intimate association with mitochondria. Since the tubular system is an invagination
of the basolateral cell surface, it follows that the contents of these tubular elements represent interstitial fluid.
Fixed with osmium tetroxide and stained with uranyl and lead salts, x 42,000.
At the electron microscopic level the
chloride cell exhibits numerous mitochondrial profiles (Fig. 2). Perhaps most interesting, an elaborate anastomosing tubular
system (tubular reticulum) pervades the
cytoplasm except for the Golgi region and
a region adjacent to the apical plasma
membrane (Fig. 3). These tubules are
approximately 60-80 nm in diameter and
are continuous with the plasma membrane
213
TELEOST CHLORIDE CELL
TABLE 1. Net sodium flux and external salinity in teleosts.
Species
Medium
Sarotherodon mossambicus
(formerly Tilapia mossambica)
Fundulus heteroclitus
200% SW
100% SW
40% SW
100% SW
40% SW
200% SW
100% SW
Anguilla anguilla
9FiW, CW
FW
(0.1 mM)
Net flux
-1,340
-470
-75
-835
-340
-670
- 8 5 to -170
— 10
+ 1.5
Na turnover rate
116
55.5
9.9
46.2
13.5
61.2
27.2
±
+
±
±
±
±
+
0.1
22
3.95
2.3
2.4
2.7
7.9
3.5
Reference
1
2
3
4
5
5
Notes: Flux in ^M'h~'-(100g) '. Na turnover rate in %-h"1. Negative sign denotes excretion; positive sign
denotes absorption. SW = sea water; FW = fresh water.
References: (1) Potts et al., 1967; (2) Potts and Evans, 1967; (3) Maetz and Skadhuage, 1968; (4) Maetz et al.,
1967; (5) Maetz, 1970.
at the basal and lateral surfaces of the chloride cell. Electron opaque tracers demonstrate this continuity unequivocally (Philpott, 1966). The tubular system thus
represents a tremendous amplification of
the basal and lateral, but not the apical,
surface of the chloride cell. A hallmark of
the seawater chloride cell is an apical invagination called the apical crypt or apical pit.
This structure is about 3 nm in diameter
and often contains a polyanionic mucus
(Philpott, 1968). Interestingly, this crypt is
shared by at least two chloride cells. One
of these chloride cells is generally a fully
developed cell, while the second may be
either another fully developed chloride cell
or what has been termed an accessory or
adjacent cell (Sardet et al, 1979; Hootman
and Philpott, 1980; Laurent and Dunel,
1980; Lacy, 1983). This latter cell type has
a less-well developed tubular system which
is non-reactive in Na,K-ATPase cytochemical localization studies (Hootman and
Philpott, 1980). The accessory cell has less
cytochemically demonstrable carbonic
anhydrase than do fully developed chloride cells (Lacy, 1983). Chloride cell morphology has been reviewed in great detail
recently (Hootman and Philpott, 1980;
Karnaky, 1980; Laurent and Dunel, 1980;
Philpott, 1980; Sardet, 1980).
AN EARLY MODEL OF CHLORIDE
CELL SECRETION
The 1960s represented a decade in which
there was an increasing awareness of the
importance of the plasmalemmal enzyme,
Na,K-ATPase, in epithelial ion transport.
In 1967, Epstein et al. reported that the
activity of Na,K-ATPase was several times
as great in the gills of seawater-adapted
specimens of Fundulus heteroclitus as compared to freshwater-adapted specimens.
Maetz (1969) showed that the concentration of K in the external environment
influenced the Na excretion rate; i.e., the
more K in the external environment, the
greater the Na efflux. Also, a number of
workers had shown in the 1960s that the
greater the external salinity, the greater
the Na efflux rate (Table 1). Importantly,
whole-animal studies showed that ouabain
in the external solution decreased sodium
efflux from the animal (Evans et al., 1973).
Maetz (1969) synthesized this data into an
elegant model for chloride cell function.
He proposed that Na,K-ATPase located on
the apical crypt membrane of the chloride
cell exchanged cellular Na for K in the sea
water. The provocative ideas of Maetz's
model stimulated a number of studies on
fish osmoregulation and chloride cell function.
I was interested in testing whether there
is a correlation between Na,K-ATPase
activity and Na efflux rates and between
the Na,K-ATPase activity and the surface
area of the chloride cell apical crypt, the
proposed site of Na,K-ATPase. We assayed
Cyprinodon variegatus for the activity of gill
Na,K-ATPase and found that this activity
increased with increasing external salinity
214
K A R L J . KARNAKY, JR.
TABLE 2.
Tissue
Human RBC
Flounder RBC
Rabbit blastocyst
Cultured guinea pig kidney
Hela cell
Rabbit renal medulla
Avian salt gland
Teleost chloride cell
Ouabain binding in several transport systems.
Number of ouabain molecules
bound per cell
4.5
6.5
3-9
7.5
8.0
4.1
2.6
1.5
x 10*
x 1O<
x 10 5
x 10 5
x 10 s
x 10 6
x 10 7
x 1OB
(Karnaky et al., 1976a). The activity in
200%-seawater-adapted fish was roughly 4
times greater than the activity in 100%seawater-adapted fish. In our light microscopic examination, we found that although
chloride cells of 200%-seawater adapted
fish were larger than chloride cells from
100%-seawater-adapted fish, the size of
apical crypts had not changed. If more
Na,K-ATPase had been inserted in this
apical crypt membrane it must have been
at a greater density than before the stimulation of the higher external salinities.
Furthermore, the only real change in
membrane surface area was found in the
tubular system. In 200%-seawater-adapted
fish, this system had amplified tremendously, so that seemingly little cytoplasm
remained. These results indicated that
Na,K-ATPase is located mainly at the basolateral plasma membrane of chloride cells.
I was able to pursue this study in greater
detail at the Mt. Desert Island Biological
Laboratory with C. Stirling, W. Kinter, and
L. Kinter. We used Stirling's powerful highresolution [3H]ouabain autoradiographic
technique to localize Na,K-ATPase. This
technique has many attractive features;
namely, the tritiated form of this cardiac
glycoside can be used to modify physiological and biochemical properties of a tissue,
the time course and amount of binding can
be assayed, and the isotope can be subsequently localized by high-resolution autoradiography. We adapted specimens of
Fundulus heteroclitus to 10%-, 100%-, and
200% sea water and observed that gill ouabain binding and Na,K-ATPase activity
increased with increasing external salinity
(Karnaky et al, 19766). When [3H]ouabain
was perfused from the blood side of the
Reference
Lauf and Joiner (1976)
Cala(1974)
Benos(1981)
Baker and Willis (1972)
Baker and Willis (1972)
Shaver and Stirling (1978)
Hootman and Ernst (1981)
Karnaky et al. (1976*)
chloride cells, the autoradiographic silver
grains were over the areas of chloride cell
cytoplasm containing the tubular system.
When we irrigated the gills with
[3H]ouabain, we found no label on the apical crypt membrane. Thus there was no
indication that the Na,K-ATPase was
located on the apical crypt plasmalemma.
In fact, quantification of the bound ouabain demonstrated that there are 1.5 x 108
Na,K-ATPase sites on each chloride cell,
all located on the tubular system (basolateral) membrane. This number is very large
and can be compared in Table 2 with the
number of sites in a variety of cell types. I
am not aware of a higher estimate for the
number of Na,K-ATPase sites per cell than
is exhibited by the teleost chloride cell.
Further corroboration of the tubular system location of Na,K-ATPase was presented by Hootman and Philpott (1979),
using Ernst's nitrophenyl phosphatase
cytochemical technique (Ernst, 1972).
THE CHLORIDE CELL IN THE
USSING CHAMBER
In the short-circuit current technique an
epithelium is mounted between two hemichambers containing identical solutions of
physiological saline. Thus chemical and
osmotic gradients across the epithelium are
eliminated. Electrical potential sensing
electrodes are placed next to the epithelium to measure the transepithelial potential difference (P.D.). An additional circuit
is used to pass current across the membrane of correct polarity and magnitude to
"clamp" the potential difference across the
epithelium to zero, thus eliminating transepithelial electrical gradients. This current is called the short-circuit current (1^).
215
TELEOST CHLORIDE CELL
TABLE 3. Electrical properties of isolated epithelia from seawater- and freshwater-adapted teleosts.
Species
(epithelium)
Adaptation
(time)
Fundulus
heteroclitus
1% sea water
(3 months)
OiA/cm')
P.D.
(mV)
(ficrn')
94.1 ± 10.4
(20)
14.8 ± 1.9
(20)
169.0 ± 14.0
(20)
Degnan et al. (1977)
136.5 ± 11.1
(64)
1.2 ± 0.2
(19)
18.7 ± 1.2
(64)
1.4 ± 0.2
173.7 ± 12.1
(64)
3,709 ± 629
Degnan e( a/. (1977)
(9)
(9)
100.6 ± 7.5
(16)
13.7 ± 3.0
21.5 ± 1.5
(16)
16.9 ± 5.8
259 ± 25
(16)
1,149
(8)
(8)
R
Reference
(operculum)
Sarotherodon
mossambicus
(operculum)
Gillichthys
mirabilis
Sea water
(several weeks)
Dechlorinated tap
water
(several weeks)
Sea water
(2-3 weeks)
5% sea water
(>10 days)
(8)
Foskett e< a/. (1981)
Foskett e* a/. (1981)
Marshall (1977)
(jaw)
714
Marshall (1977)
20.8 ± 7.2
15.2 ± 3.6
(11)
(11)
(11)
Number of observations in parentheses beneath value of electrical property. Values expressed as mean ±
standard error of the mean. Polarity of the potential difference across these epithelia is seawater side electronegative to the blood. R = resistance. Resistance values from Gillichthys calculated from conductance values.
Sea water
(> 10 days)
Under these conditions of no chemical,
temperature, or electrical gradients, any
ion which moves across the membrane in
a net fashion does so by active transport
mechanisms. The movement of these ions
is detected by isotope flux studies, in which
paired membranes are used to measure
mucosa to serosa and serosa to mucosa
fluxes. Since these ions are charged particles, their transport rates (jiEquivhr"1cm"2) can be converted using Faraday's
constant, to currents (/uAmp-cm"2). If chloride cells could be studied with the shortcircuit current technique, important
insights could be gained into the function
of this presumptive chloride secreting cell.
The first attempt to apply this technique
to the sea raven opercular epithelium was
hampered by the fact that very few chloride cells are present in this epithelium
(Karnaky, 1972). Burns and Copeland
(1950) had reported the presence of a large
population of chloride cells in the opercular epithelium of Fundulus heteroclitus. I
found that this epithelium is very easy to
dissect from the fish, and could be mounted
in an Ussing-type chamber. The Fundulus
heteroclitus opercular epithelium provided
a nearly ideal membrane for this technique
(Degnan et al, 1977; Karnaky et al., 1977).
In this same year, Marshall (1977) introduced the jaw epithelium of Gillichthys
mirabilis as a membrane amenable to the
short-circuit current technique. More
recently Foskett et al. (1981) have introduced the use of the opercular epithelium
from the euryhaline Sarotherodon mossambicus. Electrophysiological data obtained
from several isolated teleost epithelia which
contain chloride cells are presented in
Table 3. Significantly, ion flux studies
under short-circuit current conditions show
for each of these epithelia that chloride
transport is active (Karnaky et al., 1977;
Marshall and Bern, 1980; Foskett et al.,
1981). There is no net flux of sodium under
short-circuit current conditions in epithelia from Fundulus heteroclitus (Degnan et al.,
1977), Gillichthys mirabilis (Marshall and
Bern, 1980), and Sarotherodon mossambicus
(Foskett et al., 1983).
OPERCULAR AND JAW EPITHELIUM
MORPHOLOGY
One of the attractive features of this epithelium studied in vitro is that blood circulation is no longer an important consideration, as it is in the intact perfused gill.
Although vascular flow is undoubtedly an
important component of the control of
chloride cell function in vivo, with the isolated opercular epithelium the investigator
can examine direct effects of various agents
and environmental conditions on the chlo-
216
KARLJ. KARNAKY, JR.
density in this epithelium, up to 4 x 105
cells per cm2. Between these chloride cells
are several other cell types, mucous cells
and non-differentiated cells. The mucous
cells touch the external environment of the
epithelium. Recently, Lacy (1983) has
reported that in Fundulus heteroclitus, the
non-differentiated cells can actually be subdivided into two classes of cells, supporting
cells and vesicular cells.
At the electron microscopic level the
chloride cells of the opercular epithelium
are identical in structure to those of the
gills (Karnaky and Kinter, 1977). Thus the
opercular epithelium resembles a flattened
gill filament without the respiratory leaflets and without complicated vasculature.
Morphological observations on seawateradapted teleosts have revealed that the histology and fine structure of chloride cells
in the opercular epithelium of Sarotherodon
mossambicus (Foskett et al., 1981) and in the
jaw epithelium of Gillichthys mirabilis (Marshall and Nishioka, 1980) are essentially
identical to descriptions given for Fundulus
FIG. 4. Electron micrograph of two chloride cells in
heteroclitus
(Degnan et al., 1977; Karnaky
the mouth roof epithelium of seawater-adapted Funand
Kinter,
1977; Lacy, 1983).
dulus heteroclitus. The two chloride cells can be distinguished by the difference in staining intensity of their
cytoplasm. The smaller, darker cell appears to be surrounded by the larger, light-staining cell. Actually,
these two cells interdigitate to share an apical crypt
(AC) as do the chloride cells in Figure 2. An arrow
points to a finger of the lighter cell interdigitated with
the darker cell. Two pavement cells partially overlap
the chloride cells. Fixed with glutaraldehyde, postfixed with osmium ferrocyanide, and stained with uranyl and lead salts. Micrograph by Leon Garretson.
University of Texas Medical School at Houston,
x 10,260.
ride cell. Histologically the opercular epithelium is similar to the gill with one very
important exception: the opercular epithelium lacks the respiratory leaflets (Burns
and Copeland, 1950; Karnaky and Kinter,
1977), the location for oxygenation of red
blood cells and presumably the site of passive ion fluxes. The opercular epithelium
is a flat sheet of cells, with chloride cells
extending the whole distance from the basal
lamina to the external world. The external
surface is again the cobblestone-like pattern with pavement cells forming the outer
layer. Chloride cells can reach a very high
THE CHLORIDE CELL IS RESPONSIBLE
FOR CHLORIDE SECRETION
Opercular and jaw epithelia have allowed
researchers to answer a critical question
concerning the role of the chloride cell in
chloride secretion in teleosts adapted to sea
water. Several studies have taken advantage of fluorescent dyes which stain mitochondria and can easily be applied to flat
epithelia. In the case of Fundulus heteroclitus we were able to utilize epithelia which
contained a high density of chloride cells
(operculum) and a low density of chloride
cells (mouth roof epithelium, Fig. 4).
Briefly, the procedure is first to mount
an epithelium in the Ussing chamber and
record the steady-state 1^. Next the epithelium is exposed to the mitochondrial
dye and the number of chloride cells in a
unit area of epithelium is counted. In two
species of teleosts, Fundulus heteroclitus and
Gillichthys mirabilis, linear regression analysis has revealed a significant correlation
between I^. and chloride cell number (Karnaky et al., 1979; Marshall and Nishioka,
TELEOST CHLORIDE CELL
2 If
FIG. 5. Fluorescence-microscope photograph of unfixed opercular epithelium from seawater-adapted Fundulus heteroclitus. The epithelium was incubated with DASPMI dye. Presumptive mitochondria-rich chloride
cells appear as large, ovoid cells against a background of non-staining (mitochondria-poor) cells. The nucleus
of each presumptive chloride cell stands out as a dark area. See text for description of method to demonstrate
that DASPMI-positive cells are chloride cells. Micrograph by Leon Garretson. University of Texas Medical
School at Houston. X490.
firm the identity of chloride cells at the end
of each experiment. This study showed
conclusively that current is carried across
the opercular epithelium only at discrete
points directly over chloride cell apical
crypts. The surface of the apical crypt is
50 times more conductive than that of
pavement cells.
Table 4 summarizes the values for I,,, per
chloride cell and 1^. per cm2 of chloride cell
apical membrane area from the above
cular epithelium of Sarotherodon mossambi- mentioned studies. Since crypt diameters
cus, Foskett and Scheffey (1982) combined are measured from electron micrographs
a vibrating probe technique with bright- of tissue which has been fixed, dehydrated,
field optics to determine which cells are and embedded, procedures which cause
carrying current across the epithelium. some shrinkage, the calculated area is
Chloride cells were identified visually dur- undoubtedly an underestimate. It should
ing experiments on the basis of their size be noted that the 1^ of the opercular epi(diameter about 20 >im), as no other cell thelium of Fundulus heteroclitus (Degnan et
type in this opercular membrane is this al., 1977) and the jaw epithelium of Gilllarge (Foskett et al., 1981). Chloride cells ichthys mirabilis (Marshall and Bern, 1979)
are the only cell type with an appreciable can be stimulated by pharmacological
number of mitochondria in this opercular agents, whereas the opercular epithelium
membrane and the specific mitochondrial of Sarotherodon mossambicus normally transprobe, DASPEI (dimethylaminostyryl- ports chloride at maximal rates and cannot
ethylpyridiniumiodine) was used to con- be further stimulated (Foskett et al., 1982a).
1980; Karnaky et al., 1984). We have
recently demonstrated that a fluorescent
mitochondrial dye, DASPMI (dimethylaminostyrylmethylpyridiniumiodine),
stains only chloride cells by comparing cellular staining of this dye in the fluorescent
microscope (Fig. 5) with the staining pattern of cells in which the mitochondrial
enzyme, cytochrome oxidase, was localized
at the electron microscopic level (Karnaky
et al., 1984). In a recent study on the oper-
218
KARL J. KARNAKY, JR.
TABLE 4.
Epithelium
Species
I^/chloride
cell
(nA)
Crypt
diameter
(Mm)
0.50
3.8
1.00*
Short-circuit current generated by chloride cells.
I^/chloride
cell apical
surface
area
(mA/cm 1 )
Method
Reference
2.3
DASPMI—Ussing Chamber
Karnaky et al. (1979)
3.8
4.5
DASPMI—Ussing Chamber
Karnaky et al. (1984)
2.50
3.0
17.7
Vibrating Probe
Foskett et al. (1983)
0.65
3.5
3.4
DASPEI—Ussing Chamber
Marshall and
Nishioka(1980)
Opercular
Fundulus
heleroclitus
Fundulus
heteroclitus
Sarotherodon
mossambicus
Jaw
Gillichthys
mirabilis
All data from seawater-adapted animals, except Gillichthys data, which include animals adapted to sea water
and to double-strength sea water. Crypt shape is assumed to be hemispheric. Unlike Sarotherodon mossambicus,
IK values for the other species are sub-maximal, and can be stimulated with secretagogues.
* This report has a greater number of cell counts than the author's previous report.
for the chloride cell (Fig. 6). This general
model has been proposed for several chloride-secreting epithelia (Frizzell et al.,
1979). The primary driving force of this
model is the tubular system Na,K-ATPase,
which creates a large Na gradient, with high
concentrations in the tubular system lumen
and low concentrations in the chloride cell
cytoplasm. This pump represents primary
A MODEL FOR CHLORIDE SECRETION
active transport. A second mechanism, a
BY THE CHLORIDE CELL
NaCl carrier, is driven by the Na gradient
There is no doubt that the chloride cell created by the Na,K-ATPase. This latter
is responsible for secretion in the opercular carrier is a secondary active transport
epithelium. How does this cell function to mechanism, and is sensitive to loop diureteffect this important osmoregulatory ion ics. Chloride enters the cell via this carrier
movement? A consideration of this func- and diffuses to the apical crypt membrane,
tion must include further information on where it exits by electrical forces. Na exits
the morphology of the chloride cell. Chlo- passively to the seawater side via the "leaky"
ride cells in seawater-adapted fish share tightjunction between chloride cells, driven
apical crypts with at least one other chlo- by the transepithelial potential gradient
ride cell. Freeze-fracture morphological established by the chloride movement.
studies by Sardet et al. (1979) in gills and
Data from two studies of Fundulus hetErnst et al. (1980) in the opercular epithe- eroclitus chloride cells (Karnaky et al., 1976t;
lium have revealed that tight junctions Karnaky et al., 1984) can be combined to
between chloride cells and pavement cells examine an important feature of this
and between adjacent pavement cells are model, the NaCl carrier. Using Faraday's
deep (300-500 nm) and elaborate, whereas constant, our value of 1 nA per chloride
those between adjacent chloride cells are cell (Table 4) translates to 6.2 x 109 charges
shallow (25 nm) and simple. This junc- (chloride ions) per sec per cell. If we divide
tional morphology, coupled with the by the number of Na,K-ATPase sites per
observation that Na fluxes across the oper- chloride cell (1.5 x 108 sites/cell) estimated
cular epithelium are passive (Degnan et al., by quantitative high-resolution 3H-auto1977) has suggested the following model radiography of chloride cells, we find that
Consequently, for Fundulus and Gillichthys
the values for I^ per chloride cell and IK
per chloride cell apical surface area are
submaximal. Remarkably, the Isc values
given in Table 4 (which are derived from
two different methods and three species)
expressed either per cell or per membrane
area, are in excellent general agreement.
219
TELEOST CHLORIDE CELL
each Na,K-ATPase site is responsible for
41 chloride ions per sec. If we assume a
turnover number of 148 sec"1 for Na,KATPase (Lane et al., 1979; data from lamb
kidney assayed at 37°C), a temperature correction for 23°C of 1/3.2 x (Dixon and
Hokin, 1974; data from electric organ), and
a stoichiometry of 3Na's and 2K's (Sen and
Post, 1964), then approximately 46 net
positive charges move to the blood side per
sec. Results of these calculations imply that
the stoichiometry of the putative NaCl
cotransport carrier is approximately 1 Na:
1 Cl. However, these calculations may need
further modification. First, unidirectional
isotopic flux measurements in opercular
epithelia of Fundulus heteroditus (Karnaky
et al., 1977) have demonstrated that there
is an appreciable Cl backflux which is about
14% of the forward flux. Thus the 1 nA
per chloride cell may actually be 1.14 nA
flowing through the NaCl carrier. Correspondingly, each Na,K-ATPase may be
responsible for 47 Cl ions rather than 41.
This larger number is also consistent with
the stoichiometry of 1 Na:Cl. Second, the
value for the Na,K-ATPase turnover used
in this calculation may be an overestimate.
This value is based on assays run with a Na
concentration of 105 mM, a value which is
probably higher than the Na concentration of chloride cell cytoplasm. If the turnover number is one-half of that used above
then the stoichiometry would be closer to
1 Na: 2 Cl. Interestingly, a number of loop
diuretic-sensitive NaCl cotransport systems appear to be 1 Na: 1 K: 2 Cl (Greger
and Schlatter, 1983). The results of these
calculations are consistent with the concept
of a NaCl cotransport mechanism but with
an as yet uncertain stoichiometry.
ION ABSORPTION IN FRESHWATER
TELEOSTS
Most of our knowledge of teleost osmoregulation in both seawater and freshwater
environments has been derived from
whole-animal studies. Though one of the
physiologist's ultimate goals is to understand osmoregulation at the organismal
level, results from intact fish studies can be
clouded by several uncertainties. In a critical review of methodology used to study
INTERNAL
FIG. 6. A schematic model for the movement of Na
and Cl by chloride cells of seawater-adapted teleosts.
A chloride cell (CC) is joined to a special chloride cell
called an accessory cell (AC) by a shallow tight junction. In contrast, deep tight junctions join chloride
cells to pavement cells (PC) and pavement cells to
pavement cells. The seawater compartment is electronegative with respect to the internal compartment
of the animal. The primary driving force for the
secretion is the tubular system Na,K-ATPase (primary active transport) which creates a large Na gradient, with high concentrations in the tubular system
lumen and low concentrations in the chloride cell
cytoplasm. This Na gradient drives a NaCl carrier
(secondary active transport), also located in the tubular system membrane. Cl enters the cell via this carrier
and diffuses to the apical crypt membrane, where it
exits to the seawater compartment by electrical forces.
Na exits passively to the seawater side via the "leaky"
tight junction between chloride cells, driven by the
trans-epithelial potental gradient extablished by the
Cl movement. N denotes the nucleus.
teleost osmoregulation, Evans et al. (1982)
listed the pitfalls of whole-animal studies
in which the investigator attempts to vary
the ionic composition or pH of the external
and internal environments. The major
problems noted were: a) experimental
manipulation may not actually have a direct
effect on ion exchange mechanisms, but
rather may alter the electrical gradient
across the body; b) it is nearly impossible
to alter the composition of the blood in
intact animals; c) it is difficult to separate
cardiovascular effects from epithelial
effects; d) a stress response (with accom-
220
KARL J. KARNAKY, JR.
panying neuroendocrine changes) can be
elicited. Nevertheless, studies with intact
fish have revealed the broad outlines of
freshwater osmoregulation.
Kerstetter et al. (1970) using the irrigated trout gill, showed an active, saturable
Na transport and presented evidence for a
Na/H exchange. These investigators monitored the transepithelial potential and
found that changes in this parameter were
insufficient to account for flux changes.
Amiloride, the potent inhibitor of Na
transport, inhibits active transport of Na
across trout gills (Kirschner et al., 1973)
where it appears to compete with Na for a
saturable component on the apical membrane (Greenwald and Kirschner, 1976).
Kerstetter and Kirschner (1972) and De
Renzis (1975) have shown active chloride
absorption in rainbow trout and goldfish,
respectively. De Renzis (1975) showed that
thiocyanate inhibited chloride influx. In
these experiments, transepithelial potential was monitored and, again, changes in
this parameter could not account for
changes in fluxes.
The classic work by Keys (1931a, b) using
the heart-gill preparation served to establish active chloride secretion across the
teleost gill. However, this preparation suffers the disadvantage that the vascular
effects, branchial versus cardiac, of hemodynamic agents cannot be easily differentiated (Evans et al., 1982). This specific
problem is eliminated in the isolated, perfused head preparation, in which cannulae
are placed into the ventral aorta proximal
to the heart, and into the mouth. The
efferent perfusate drains from the dorsal
aorta and the open body cavity. A further
refinement of this preparation can be
achieved if the efferent perfusate is collected via a cannula in the dorsal aorta.
This allows partitioning of the efferent
perfusate into dorsal arterial and venous
components (Girard and Payan, 1976). In
the trout, arterial blood moves from the
afferent filamental artery into the secondary lamellae, which are lined by respiratory
cells. The blood then flows into the efferent filamental artery and may, via arteriovenous anastomoses, take a venous route
through the filamental sinus, which is in
close approximation to chloride cells. Using
this preparation, Girard and Payan (1977)
presented evidence that all of the Na and
Cl influx was across the lamellar epithelium in the trout gill. Since most chloride
cells are located not on the lamellar epithelium, but on the filamental epithelium,
this finding has been taken as evidence that,
in freshwater fish, ionic absorption does
not involve chloride cells. Evans et al. (1982)
have noted some of the pitfalls of this technique, e.g., some preparations (but not all)
are subject to rapid and serious hemodynamic degradation. Generally workers have
not measured the transepithelial potential
simultaneously with ion fluxes during ion
substitution and drug studies. Also, workers have not generally focused attention on
the ratio of the perfusate inflow to outflow.
Even slight leakage will produce spurious
determinations of ion flux rates.
In summary, both Na and Cl are actively
transported from medium to blood in all
freshwater animals which have been studied (Kirschner, 1979). As noted above, isolated opercular and jaw epithelia from several species of fish (Karnaky et al., 1977;
Marshall, 1977; Foskett et al, 1981) have
been helpful in elucidating mechanisms of
teleost osmoregulation in seawater teleosts.
However, to date relatively little information concerning freshwater osmoregulation has been gained from these in vitro
preparations. Table 3 lists several studies
which focus on epithelia from freshwateradapted teleosts. In one of the initial publications on the isolated opercular epithelium of Fundulus heteroclitus (Degnan et al.,
1977) we were surprised by observations
on the opercular epithelia from specimens
adapted to 1 % sea water for three months.
When mounted in Ussing chambers in the
normal teleost Ringer used for studying
opercular epithelia from seawater-adapted
specimens, these freshwater epithelia
exhibited electrical properties very similar
to those exhibited by seawater-adapted epithelia, with slightly lower chloride secretion rates. At the time we suggested that
these in vitro experimental conditions could
have interrupted the inhibitory influence
of a quick acting and transitory hormone
which had been present in the freshwater
TELEOST CHLORIDE CELL
221
fish. Recently, however, Degnan (19846), hormonal agent which inhibits chloride
using opercular epithelia of seawater- secretion in the freshwater state, as sugadapted Fundulus heteroclitus, has shown gested in one of our original publications
that mucosal Na or Cl substitution almost on the opercular epithelium (Degnan et al.,
completely inhibits Cl efflux, suggesting a 1977). Alternatively, activation may involve
possible direct regulatory influence by the ionic or osmotic stimulation. None of these
external salinity on the Cl secretory rate. in vivo studies on freshwater-adapted
In the opercular epithelium of seawater- teleosts offers direct evidence for or against
adapted Fundulus heteroclitus (Mayer-Go- chloride cell involvement in ion absorptive
stan and Zadunaisky, 1978) and of sea- mechanisms which characterize freshwawater-adapted Sarotherodon mossambicus ter-adapted teleosts in vivo.
(Foskett et al., 19826) repeated prolactin
injections lowered chloride efflux com- MORPHOLOGY OF THE CHLORIDE CELL IN
FRESHWATER-ADAPTED TELEOSTS
pared to sham-injected seawater-adapted
controls. Also, freshwater adaptation in
The fine structure of chloride cells of
Fundulus heteroclitus resulted in a lowered fish adapted to sea water differs dramatichloride efflux rate. Similar differences cally from that of fish adapted to fresh
between freshwater and seawater adapta- water. As noted above, seawater chloride
tion are found in another species, Gilli- cells exist in multicellular complexes and
chthys mirabilis. Marshall (1977) compared share shallow (25 nm) tight junctions.
jaw epithelia of Gillichthys mirabilis adapted Recent electron microscopic studies, howto 5% and 100% sea water. He described ever, have described the freshwater chloa statistically significant reduction in short- ride cell in a unicellular form, sharing deep
circuit current and conductance when the junctions with adjacent pavement cells
fish were adapted to the lower salinity. Fos- (Sardet et al, 1979; Laurent and Dunel,
kett et al., (1981) have studied the effects 1980). Thus, euryhaline teleosts undergoof varying salinities with Sarotherodon mos- ing adaptation from fresh water to sea water
sambicus, a species which normally lives in must make the transition from the unicelfresh water, but which can adapt to sea lular form to a multicellular complex. Prewater. Transfer from sea water to fresh sumably this transition involves cell movewater results in dramatic changes: the ment and/or cell morphogenesis, events
operculum has negligible short-circuit cur- which could easily take place in the time
rent, no net chloride flux, and chloride cells frame of several days to several weeks used
are small and poorly developed, exhibiting in these studies. However, a euryhaline telittle contact with the surface, and no asso- leost species such as Fundulus heteroclitus
ciation with accessory cells. Following must possess the ability to adapt to fresh
transfer to sea water, the chloride current water and to sea water several times per
is activated within 24 hours and continues day in the estuarine enviroment. Do chloto increase to fully-adapted levels after 1- ride cells in this species rapidly change from
2 weeks. Chloride cells differentiate over the unicellular configuration to the multhis period and exhibit the fully developed ticellular configuration? Lacy (1983)
seawater morphology.
reported that opercular epithelial chloride
cells persist in multicellular complexes in
In summary, the studies on isolated epi- fish adapted to fresh water (8.6% sea water).
thelia from freshwater-adapted Fundulus We recently examined gill and opercular
heteroclitus and Gillichthys mirabilis demon-epithelial chloride cells of Fundulus heterostrate chloride secretion, albeit at lesser clitus adapted for several weeks to 1 % sea
rates than observed in seawater-adapted water and confirmed that chloride cells
animals. Since, a priori, this secretion would remain in multicellular complexes and
be harmful to the freshwater-adapted ani- share apical crypts (Karnaky and Garretmal in vivo, I can only conclude that the son, unpublished observations). We also
method of investigation somehow activates discovered a new type of junction between
the chloride secretion mechanism. This chloride cells. This junction is deep,
activation may involve the removal of some
222
K A R L J . KARNAKY, JR.
approximately 600 nm as measured in
transmission electron micrographs of sectioned material. We have also found that
when opercular epithelia from 1% seawater-adapted Fundulus heteroclitus are placed
in Ussing chambers in normal teleost
Ringer solution, they develop a strong
secretory short-circuit current within 3050 min. In opercular epithelia fixed at these
times, chloride cells exist in multicellular
complexes but the deep tight junction has
disassembled to the shallow junction which
characterizes chloride cells from seawateradapted fish. These preliminary observations suggest that part of the mechanism
by which Fundulus heteroclitus can achieve
rapid euryhalinity is by maintaining chloride cells in multicellular complexes with
apical crypts, even in salinities as low as 1 %
sea water. A major morphological change
in chloride cells in freshwater to seawateradaptation is in the complexity of the tight
junction between these cells.
CONCLUDING REMARKS
Our knowledge of teleost osmoregulation has been advanced considerably by the
study of the remarkable euryhaline species,
Fundulus heteroclitus. It is clear that many
exciting discoveries in teleost osmoregulation await the curious in future years. In
particular this species may provide an
important model system for studying the
quite interesting phenomenon of rapid
euryhalinity with its accompanying morphological changes.
ACKNOWLEDGMENTS
Support by NIH (GM24766; GM29099)
and NSF (DCB-8409165) grants and an
American Heart Association Established
Investigatorship. The assistance of L. Garretson and R. Cintron is gratefully
acknowledged.
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