Cellular composition and ultrastructure of the gill epithelium of larval and adult lampreys : Implications for osmoregulation in fresh and seawater
1 Anatomische Anstalt, Ludwig-Maximilians-Universität München,
Pettenkoferstr. 11, 80336 München, Germany
2 School of Biological Sciences and Biotechnology, Murdoch University,
Murdoch 6150, Western Australia
* Author for correspondence (e-mail: bartels{at}anat.med.uni-muenchen.de)
Accepted 21 June 2004
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Summary |
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Key words: lamprey, Agnatha, osmoregulation, gill epithelium, mitochondria-rich cell, chloride cell, pavement cell
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Introduction |
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Hagfishes, which are found only in marine habitats, are osmoconformers
(McFarland and Munz, 1965;
Cholette et al., 1970
). The
concentrations of Na+ and Cl in their sera
approximate those in full-strength seawater, a situation unique amongst
vertebrates and which results in their internal milieu being essentially
iso-osmotic with that of their marine environment
(Robertson, 1974
). These
characteristics imply that hagfishes have always lived in marine habitats
(Robertson, 1957
,
1974
;
Lutz, 1975
).
In contrast to hagfishes, the anadromous species of lampreys spend a
substantial part of their life cycle in freshwater
(Hardisty and Potter, 1971;
Hardisty et al., 1989
). The
efficient osmoregulatory mechanisms evolved by lampreys enable the
concentrations of Na+ and Cl in their internal
milieu to be maintained at levels well above that of freshwater, when the
animal is living in rivers, and well below that of full-strength seawater,
when the animal is residing in marine environments
(Morris, 1972
;
Beamish et al., 1978
). Since
the osmolality of their serum is far lower than that of seawater, it has been
concluded that lampreys have an ancient freshwater history and that the marine
parasitic phase of their life cycle was developed relatively late in their
evolution (Hardisty et al.,
1989
). However, the recent discovery of a lamprey-like fossil in
lower Cambrian marine deposits (Shu et
al., 1999
) strongly suggests that the initial evolution of
lampreys occurred in a marine environment at a very early date, i.e. over 545
million years ago, and thus prior to the time when this group invaded
freshwater.
The marked differences between both the ionic composition and osmolality of the body fluids of hagfishes and lampreys are consistent with the long period that these two agnathan groups are believed to have been separated. Indeed, as long ago as 1932, Homer Smith stated that "these two groups lead back to a parting of the ways in the evolution of body fluids".
The teleost fishes are found in a wide range of fresh, brackish and marine
habitats and thus, as a group, are faced with the same variety of osmotic
problems as those experienced by the anadromous species of lampreys during the
course of their life cycle. The diversity and abundance of teleosts account
for this group having been the subject of the majority of the studies aimed at
elucidating the mechanisms by which fish regulate the ionic composition and
osmolality of their serum (Smith,
1930,
1932
;
Krogh, 1939
; Karnaky,
1980
,
1986
;
Zadunaisky, 1984
;
Perry, 1997
; Wilson et al.,
2000a
,b
).
Although lampreys are not closely related to teleosts, their gills and kidneys
likewise constitute the main organs responsible for osmoregulation, and their
overall mechanisms for regulating the concentrations of Na+ and
Cl in their body fluids are similar
(Morris, 1972
;
Beamish, 1980
;
Hardisty et al., 1989
).
However, ultrastructural studies have demonstrated that, particularly when
lampreys are in freshwater, the cellular composition of their gill epithelium
differs from that of teleosts (Bartels et al.,
1996
,
1998
). These studies have also
revealed that certain epithelial cells in the lamprey gill possess highly
distinctive ultrastructural features, which they share with particular cell
types in specific ion-transporting epithelia in other vertebrates and for
which a function has been determined.
In the present review, the ultrastructural characteristics of each of the cell types present in the gill epithelium at the different stages in the life cycle of lampreys are described. The characteristics of each cell type are then considered in the context of both the type of environment in which the animal containing that cell is found, i.e. freshwater or seawater, and the roles played by analogous cells in other vertebrates. This then enables each of these various cell types to be assigned a presumptive role in lamprey osmoregulation.
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Osmoregulatory mechanisms in freshwater |
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In contrast to the indirect coupling of Na+ uptake to
H+ secretion, as described above, the uptake of
Cl from an hypotonic environment by all epithelia studied
thus far occurs directly by means of an
HCO3/Cl antiport
(Garcia-Romeu and Ehrenfeld,
1975; Larsen,
1991
; Marshall et al.,
1997
). The cells engaged in Cl uptake are
further characterised by the presence of cytosolic carbonic anhydrase and a
Cl channel in their basolateral membrane
(Larsen, 1991
).
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Osmoregulatory mechanisms in seawater |
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Concomitant morphological and electrophysiological studies using the
opercular epithelium have confirmed that, when teleosts are in seawater,
Cl is secreted by this cell type through a secondary active
transport that provides the driving force for the passive transport of
Na+ through leaky pathways between adjacent chloride cells
(Karnaky et al., 1977;
Ernst et al., 1980
;
Foskett and Scheffey, 1982
;
Foskett and Machen, 1985
). The
channel through which Cl is secreted by the teleost chloride
cell has now been identified as a homologue of the mammalian cystic fibrosis
transmembrane conductance regulator (CFTR;
Singer et al., 1998
;
Wilson et al., 2000b
;
Marshall et al., 2002
).
Identical mechanisms for hypertonic salt secretion are also present in the
secretory cells of both the rectal gland of elasmobranch fishes and the nasal
salt gland of some species of marine birds (Kirschner,
1977
,
1980
;
Riddle and Ernst, 1979
;
Ernst et al., 1981
). It is
thus assumed that this mechanism is universally employed by vertebrates for
osmoregulation in hypertonic environments.
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Osmoregulation during the life cycle of lampreys |
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Ammocoetes of the sea lamprey Petromyzon marinus are unable to
osmoregulate in environments in which the osmolality exceeds that of their own
serum, i.e. 225 mosmol kg1
(Beamish et al., 1978
;
Morris, 1980
). Although
50% of larval P. marinus died within 24 h of being placed in
water of 350 mosmol kg1, i.e. about one-third of
full-strength seawater, the recently metamorphosed young adults of anadromous
species can readily be acclimated to full-strength seawater and are then able
to maintain their serum osmolality at
260 mosmol kg1
(Beamish et al., 1978
). This
ability to change from hyper-osmotic regulation in freshwater to hypo-osmotic
regulation in seawater is so effective that more than 80% of young adult
P. marinus can even survive direct transfer from fresh to
full-strength seawater (Potter and
Beamish, 1977
). Similar results have been obtained with young
adults of the anadromous lampreys Lampetra fluviatilis and
Geotria australis (Potter and
Huggins, 1973
; Potter et al.,
1980
). The ability to osmoregulate in hypertonic environments is
lost by adult lampreys soon after they have completed their marine trophic
phase and embarked on their upstream spawning migration (Morris,
1956
,
1958
;
Pickering and Morris,
1970
).
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The gill epithelium during the life cycle of lampreys |
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The presence or absence of the different cell types at the surface of the gills at different phases in the life cycle of anadromous lampreys can now be used to propose which cells are involved in osmoregulation in hypo- and hyper-osmotic environments. For example, since the intercalated MR cell is the only cell type present throughout all freshwater phases, and is absent during the marine phase, it presumably plays a crucial role in osmoregulation when lampreys are in hypo-osmotic media. Likewise, since the chloride cell develops just prior to the marine phase and disappears soon after the completion of this phase, it can only be involved in osmoregulation when lampreys are in an hyper-osmotic medium. Although the pavement cell is the only cell type that is present on the gill surface throughout the entire life cycle of lampreys and is thus a potential candidate as an osmoregulatory cell in both fresh and seawater, there is currently no evidence that this cell type is required for osmoregulation in seawater (see below). The fact that the ammocoete MR cell is never found after the completion of the larval phase demonstrates that any role that it plays in osmoregulation in ammocoetes must be undertaken by other cell type(s) during the freshwater phases of post-larval life.
The freshwater phases in the life cycle
The intercalated MR cell
The cell in the lamprey gill epithelium that we have termed the
intercalated MR cell (Bartels et al.,
1998) corresponds to that designated by Youson and Freeman
(1976
) and Mallatt and
Ridgway (1984
) as a chloride
cell in the ammocoete gill. Our choice of the term intercalated MR cell is
based on the fact that, while these cells differ from the chloride cells in
the gills of adult lampreys in that they lack a membranous tubulous system
(see above), they do have the same ultrastructural characteristics as the MR
cells that, for example, are intercalated in the epithelium of both the skin
and urinary bladder of amphibians, of the urinary bladder of reptiles and of
the collecting duct of the amphibian and mammalian kidney (Figs
2A,
3;
Brown and Breton, 1996
). Thus,
the branchial intercalated MR cell of lampreys is characterized by the
presence of numerous membranous vesicles and tubules between the mitochondria
and also often immediately beneath the apical membrane
(Fig. 2A). Moreover, the apical
surface of the intercalated MR cell is enlarged by slender, branching
microfolds that, from scanning electron microscopy, can be seen to produce a
honeycomb appearance (Figs 2A,
3C). As primarily shown in the
epithelium of the turtle urinary bladder, the extent of such enlargements,
which varies markedly amongst intercalated MR cells, is inversely related to
the number of membranous tubules and vesicles in the apical cytoplasm, which
are incorporated into and removed from the apical membrane by exo- and
endocytosis, respectively (Stetson and
Steinmetz, 1983
; Brown,
1989
; Brown and Breton,
2000
). A coat of studs projects about 12 nm outwards from the
cytoplasmic side of the apical membrane
(Fig. 2B). In freeze-fracture
replicas, the cell membrane and the membranes of the cytoplasmic vesicles and
tubules of the intercalated MR cell are characterized by the presence of
rod-shaped particles on the protoplasmic (P) face and by complementary pits on
the exoplasmic (E) face (Fig.
3A,B). These particles, which are either 1618 nm or
2427 nm in length and 89 nm in width, consist of two or three
globular subunits. They are located in the apical membrane of almost all of
the intercalated MR cells and in the basolateral membrane of a few of these
cells (Bartels and Welsch,
1986
; Bartels et al.,
1998
).
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In the lamprey gill epithelium, these cuboidal intercalated MR cells are
generally confined to the filaments, where they typically occur singly at
their base and between the lamellae
(Bartels et al., 1998). They
are intercalated between ammocoete MR cells in larval lampreys
(Fig. 3C) and either between
chloride and pavement cells or between pavement cells in downstream migrating
lampreys and between pavement cells in upstream migrating lampreys.
The intercalated MR cells in urinary epithelia and the amphibian epidermis
are rich in cytosolic carbonic anhydrase (CA II) and contain, within their
plasma membrane and the membranes of cytoplasmic vesicles, a vesicular type of
proton pump (Brown and Breton,
1996,
2000
). The peripheral
cytoplasmic subunit V1 of this pump has been immunolocalized to the studs on
the cytoplasmic side of the MR cell membrane
(Brown et al., 1987
).
Furthermore, a combination of physiological and morphological studies in
various urinary epithelia indicates that the rod-shaped particles are either
the transmembrane portion or an intimate associate of this pump
(Stetson and Steinmetz, 1986
;
Brown et al., 1987
;
Kohn et al., 1987
). This view
is supported by the presence of H+ V-ATPase activity in all of
those membranes that have been shown by freeze-fracture replicas to contain
rod-shaped particles (Brown and Breton,
1996
).
On the basis of differences in the locations of the H+ V-ATPase,
rod-shaped particles and a bicarbonate exchanger, two subtypes of intercalated
MR cells (A and B) were initially distinguished in the collecting duct of the
mammalian kidney and turtle urinary bladder
(Stetson and Steinmetz, 1985;
Brown et al., 1988
;
Brown and Breton, 1996
). The
subtype A contains the H+ V-ATPase and rod-shaped particles in its
apical and cytoplasmic vesicular membranes and possesses, in its basolateral
membrane, the anion exchanger, identified as an alternatively spliced kidney
form of the band 3 protein AE-1 (Fig.
4A). This subtype is responsible for electrogenic H+
secretion. The subtype B of the intercalated MR cell is characterized by an
apical membrane that possesses a bicarbonate exchanger, which, although
functionally detectable, does not immunoreact with antisera against AE-1 or
any other anion exchanger (Fig.
4B). This subtype is responsible for
HCO3 secretion. Although the AE-1-negative
subtype B was originally distinguished from subtype A by the presence of
H+ V-ATPase and rod-shaped particles in its basolateral membrane,
immunocytochemical studies have now shown that the H+ V-ATPase can
occur in various locations in these AE-1-negative MR cells
(Brown and Breton, 1996
). This
has led to the identification of a third subtype (C) of the intercalated MR
cell in the amphibian epidermis (Fig.
4C), which is characterised by the presence of an anion exchanger
and the H+ V-ATPase in its apical membrane and thus provides a
mechanism for the uptake of Cl from a dilute solution
(Larsen et al., 1992
).
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The ultrastructural and functional characteristics of intercalated MR cells
have been conserved in certain ion-transporting epithelia of vertebrates as
diverse as amphibians, reptiles and mammals
(Brown and Breton, 1996).
Since the intercalated MR cells in the lamprey gill epithelium have the same
unique ultrastructural characteristics as the other members of this group of
cells, they presumably perform the same basic functions as those cells. The
vast majority of the intercalated MR cells in the lamprey gill epithelium
contains rod-shaped particles in their apical membrane and thus belong to the
subtypes A or C of these cells. It is assumed that both subtypes, which cannot
be distinguished on the basis of the location of the rod-shaped particles (and
H+ V-ATPase) in their apical membranes alone, are present in the
lamprey gill epithelium. Subtype A is envisaged as actively secreting
H+, whereas subtype C is responsible for taking up
Cl. The presence of an anion exchanger in parallel with the
H+ V-ATPase in the apical membrane, as is the case in subtype C
cells, would enable the actively secreted hydrogen ions to bind the
HCO3 as it leaves the cell and thereby establish
an HCO3 gradient across this membrane, which
would drive the uptake of Cl. The low external pH generated
by nearby subtype A intercalated MR cells would further enhance this effect
and help overcome the unfavourable Cl gradient, with the
intracellular Cl concentration being approximately
1020-fold greater than the extracellular concentration. Thus, the
characteristics of subtype C make it much more efficient for taking up
Cl from the environment than would those of subtype B, whose
apical membrane contains the anion exchanger but not the H+
V-ATPase. It is assumed that the primary role of the subtype B of the
intercalated MR cell is HCO3 secretion during
alkalosis, e.g. in urinary epithelia where these cells frequently occur
(Alper et al., 1989
), rather
than the uptake of Cl from a dilute solution. This
conclusion would explain why the subtype B is very rare in the lamprey gill
epithelium.
Finally, there is the question of whether epithelial Na+
channels are present in the apical membrane of intercalated MR cells and
provide a route for the uptake of Na+ through these cells.
Ehrenfeld et al. (1989)
proposed that, in the frog skin under `natural' conditions, i.e. low external
Na+ concentrations and open circuit, a significant amount of
Na+ is taken up through the MR cells and that only under
Ussing-like conditions, i.e. high mucosal Na+ concentration and
short circuit, is Na+ taken up through the granular cells. By
contrast, Nagel and Dörge
(1996
) concluded that, even
under natural conditions, the uptake of Na+ via MR cells
is negligible and occurs almost exclusively through the granular cells.
The pavement cell
The cells that form the surface of the lamellae in adult lampreys were
called pavement cells by Bartels
(1989) to be consistent with
the terminology used for comparable cell types in the gills of teleosts and
other fishes (Laurent, 1984
;
Wilson and Laurent, 2002
).
These cells correspond to those which, in ammocoetes, were designated as
mitochondria-poor cells by Youson and Freeman
(1976
), mucous-platelet cells
by Mallatt and Ridgway (1984
)
and mucous pavement cells by Mallatt et al.
(1995
). In ammocoetes, the
pavement cells are restricted to the apex of the lamellae and thus, as will be
described later, beyond the region occupied by the ammocoete MR cells
(Youson and Freeman, 1976
),
whereas in adults they cover the entire lamellar surface and, at least in
those in freshwater, also part of the interlamellar region of the filament.
They are squamous on the lamellae and columnar in the interlamellar region,
particularly in upstream migrants.
The pavement cell is ultrastructurally very similar to the granular cell in
the amphibian urinary bladder (Wade et
al., 1975; Bartels,
1989
). It is thus characterized by the presence of numerous ovoid
mucous granules, which are located in the apical cytoplasm, a rough
endoplasmic reticulum with few cisternae, a well-developed Golgi apparatus and
less mitochondria than are present in the two types of MR cells
(Fig. 5A). The apical surface
bears microplicae or short microvilli. Freeze-fracture replicas have shown
that the majority of the intramembranous particles of the apical membrane are
located on its E face and that few particles are present on its P face
(Fig. 5B,C). This pattern of
distribution of intramembrane particles differs from that found in the plasma
membrane of most other vertebrate cells, including the basolateral membrane of
both the lamprey pavement cell and the granular cell of the amphibian urinary
bladder. Most of the particles on the E face of the apical membrane are
relatively large (diameter 1013 nm) and of greater size than those on
the corresponding P face (diameter 68 nm).
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The granular cell in the toad urinary bladder epithelium facilitates ionic
and osmotic regulation by acting as the effector cell through which water and
Na+ are taken up independently. These two functions are hormonally
controlled by the antidiuretic hormone (ADH) and the mineralocorticosteroid
aldosterone. The permeability of the apical membrane of the granular cell to
water is very low, unless this cell has been stimulated by ADH
(DiBona et al., 1969;
Harris et al., 1991
). This low
permeability has been related to the presence of only a few particles on the P
face of the apical membrane (Bourguet et
al., 1976
), which in turn may reflect an unusual composition or
arrangement of lipids in the exoplasmic half of this membrane
(Harris et al., 1991
). Since
this unusual and highly specialised membrane structure is shared by the
pavement cell in the lamprey gill epithelium, the apical membrane of the
latter cell is likewise assumed to be relatively impermeable to water. This
conclusion is supported by the observation by Bentley
(1962
) that the permeability
of the body surface of adult Lampetra fluviatilis to water is far
lower than that of either the isolated toad urinary bladder or even the frog
skin. It thus appears relevant that, particularly in adult lampreys, the
contribution made by pavement cells to the area of the body surface that is
exposed to the environment is far greater than that of any other cell type.
Thus, when adult lampreys are in freshwater, the possession by these cells of
a relatively impermeable apical membrane helps protect these animals against
an osmotic influx of water across the gills.
During periods of water shortage in toads, ADH increases the permeability
of the apical membrane of the granular cells to water by recruiting water
channels (aquaporin 2) into this membrane from a cytoplasmic pool
(Wade et al., 1981; Brown et
al., 1989). Thus, since lampreys are not threatened by dehydration when in
freshwater and thus do not require an ADH-mediated mechanism for either
conserving or taking up water, it is hardly surprising that this hormone does
not elicit an hydro-osmotic response in these animals
(Bentley and Follett,
1962
).
The uptake of Na+ from the urine by the granular cell in the
toad bladder, which is regulated by aldosterone and ADH, occurs through ENac
in the apical membrane and is driven by Na+/K+-ATPase
activity in the basolateral membrane
(Macknight et al., 1980;
Garty, 1986
;
Garty and Palmer, 1997
).
Although lampreys do not produce aldosterone
(Bentley and Follett, 1962
),
the administration of this hormone (but not of neurohypophyseal hormones) to
these agnathans likewise increases the extrarenal uptake of Na+
(Bentley and Follett, 1962
,
1963
). This finding raises the
possibility that, as with the granular cells of Na+-resorbing
epithelia such as the toad urinary bladder and epidermis, ENac are present in
the lamprey pavement cell. The pavement cell thus becomes the main candidate
for the uptake of Na+ and the target of unidentified
mineralocorticoid hormone(s) in lampreys.
The ammocoete MR cell
The cell that we term the ammocoete MR cell
(Bartels et al., 1998)
corresponds to the mitochondria-rich cell of Morris and Pickering
(1975
), the mitochondria-rich
platelet cell of Youson and Freeman
(1976
) and the ion-uptake
cell of Mallatt and Ridgway
(1984
). The latter three
groups of authors considered that this cell is responsible for taking up ions
from the environment.
The ammocoete MR cells represent up to 60% of the cells at the surface of
the gill lamellae of larval lampreys. They are arranged in large groups at the
base of the lamellae and in the region between the lamellae. Their
mitochondria, which occupy about one-third of the cell volume
(Mallatt et al., 1995), have
an unusually electron-dense matrix (Fig.
2C). Elongated mucous granules, which are smaller than those of
pavement cells, lie directly beneath the apical membrane of some of these
cells. In freeze-fracture replicas, the apical membrane of the ammocoete MR
cell exhibits the typical characteristics of vertebrate cell membranes, i.e.
the P face of the cleaved membrane contains most of the globular particles
(diameter 89 nm) whereas the E face contains few particles but numerous
pits (Bartels et al.,
1998
).
The presence of numerous mitochondria and a positive histochemical reaction
for carbonic anhydrase by the ammocoete MR cell
(Conley and Mallatt, 1988) are
consistent with the view that, in larval lampreys, this cell is responsible
for exchanging Na+ for H+ and/or Cl
for HC03
(Morris and Pickering, 1975
;
Youson and Freeman, 1976
;
Mallatt and Ridgway, 1984
).
However, our preliminary studies show that, when ammocoetes of G.
australis are held in distilled water, in which the uptake of
Na+ and Cl is maximally stimulated, the
characteristics of the ammocoete MR cells (and pavement cells) are unaffected
whereas the density of the intercalated MR cells increases significantly
beyond that found with these cells in ammocoetes held in 10% seawater (H.
Bartels, J. Rosenbruch and I. C. Potter, unpublished observations).
Furthermore, adult lampreys in freshwater still possess the osmoregulatory
capacity to overcome the same osmotic challenges as ammocoetes even though
they do not possess ammocoete MR cells. The ammocoete MR cell may thus have a
function additional to or other than osmoregulation. Since lampreys feed in
freshwater only during their larval phase, it is possible that the ammocoete
MR cell, which apparently has no morphological counterpart in other vertebrate
epithelia, may be involved in excreting ions and/or waste products that have
been derived from the digestion of their algal and detrital food
(Bartels et al., 1998
). The
ability to take up ions via the branchial epithelium would be
particularly valuable during the upstream spawning run since, during that
migration, the lamprey ceases feeding and its gut degenerates
(Youson, 1981
) and thus no
longer has the potential to acquire ions from food.
Models for Na+ uptake by the branchial epithelium of lampreys in freshwater
Since the intercalated MR cell and pavement cell are the only two cell
types that are present on the gill surface of both larval and adult lampreys
in freshwater, it has been assumed that Na+ and
Cl must be taken up through either one or both of these cell
types (Bartels, 1989;
Bartels et al., 1998
). Since
the putative role of the subtype C of the intercalated MR cell in the uptake
of Cl has been discussed above, we will now focus on the
uptake of Na+.
The arrangement and ultrastructure of the intercalated MR cell and pavement
cell in the gill epithelium of adult lampreys in freshwater bear a striking
resemblance to those of the intercalated MR cells and granular cells in the
amphibian skin and urinary bladder, respectively. This led to the proposal
that, in the lamprey gill, the intercalated MR cells (subtypes A and C)
likewise facilitate the uptake of Na+ by active H+
secretion and that the pavement cell is the primary candidate for the uptake
of Na+ (Fig. 6;
Bartels, 1989;
Bartels et al., 1998
). Except
in `very tight' epithelia, such an indirect coupling of H+
secretion and Na+ uptake requires a close spatial relationship
between the cell types involved. This is the case in both of the freshwater
stages of adult lampreys, i.e. during their downstream and upstream migration,
when the MR cells are intercalated between (or directly neighbouring) the
granular cells. However, in larval lampreys, the pavement cells are always
separated from the intercalated MR cells by large groups of ammocoete MR
cells. The distance between these two cell types would thus presumably be too
large to produce an effect on the electrical potential generated by the active
H+ secretion through intercalated MR cells on the pavement cells.
Although the electrical resistance of the gill epithelium of larval lampreys
has not yet been determined, the tight junctions, as revealed in
freeze-fracture replicas, do not possess the structural characteristics
considered responsible for epithelial tightness, e.g. a large number of
superimposed junctional strands, exhibiting a high degree of branching and
anastomosing and consisting of solid fibrils rather than particles (see fig.
3A in Bartels et al., 1998
;
Claude and Goodenough, 1973
;
Claude, 1978
;
Cerejido et al., 1989
).
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Provided that the gill epithelium of larval lampreys is not very tight and
the ammocoete MR cells do not contribute to the uptake of Na+,
there are two possible alternative mechanisms for this transport in
ammocoetes. The first hypothesis is based on the assumption that, in
ammocoetes, Na+ uptake is coupled to H+ secretion. Under
this condition, only the intercalated MR cell can be responsible for the
uptake of Na+. This model, which is consistent with that proposed
for the MR cells in the frog skin under natural conditions (see above;
Ehrenfeld et al., 1989;
Ehrenfeld and Klein, 1997
),
has the advantage of being equally applicable to larval and adult lampreys. It
requires the presence both of the H+-ATPase and the ENac in the
apical membrane and of significant amounts of
Na+/K+-ATPase in the basolateral membrane of the
intercalated MR cell. However, there is as yet no direct evidence from
patchclamp or immunocytochemical studies that the ENac is present in
intercalated MR cells of the amphibian epidermis or in any other location in
which these cells occur. Furthermore, studies on the cellular distributions of
Na+/K+-ATPase in the toad and turtle urinary bladders
and frog skin have shown that the vast majority of Na+ pumps are
localised in the granular cells and that the intercalated MR cell possesses
only a few of these pumps. This distribution pattern is consistent with the
model that the granular cell compartment is responsible for Na+
uptake (Rick et al., 1978
;
Durham and Nagel, 1986
;
Nagel and Dörge, 1996
)
and suggests that the few Na+ pumps present in the intercalated MR
cells are sufficient to maintain the balance of the Na+ and
K+ gradients across the basolateral membrane but do not
significantly contribute to Na+ uptake.
The alternative model assumes that Na+ is taken up by pavement cells and is energized only by the activity of the Na+ pump, located in the basolateral membrane of this cell, and is not facilitated by H+ secretion by intercalated MR cells. The question then arises as to whether the rate of this branchial Na+ uptake, possibly in concert with intestinally resorbed dietary Na+, is sufficient to substitute the passive loss of Na+. Under such a condition, H+ secretion might provide the energy for Cl uptake through the subtype C of the intercalated MR cells (see above). By contrast, since the requirements for a coupling of Na+ uptake and H+ secretion are met in adults, H+ secretion through the subtypes A and C could also facilitate Na+ uptake in the adult stages of the life cycle in addition to driving the uptake of Cl. This model implies that the mechanisms for regulating the Na+ concentration are not the same in larval and adult lampreys. It also takes into account the fact that the composition and arrangement of the branchial epithelial cells in larval and adult lampreys differ and that the larval lamprey feeds whereas the adult lamprey in freshwater does not.
The marine phase in the life cycle
The chloride cell
Lamprey chloride cells are disc-like and form long continuous rows that
extend throughout the interlamellar region of the filament and into the basal
region of the filament where lamellae are absent (Figs
7A,
8;
Bartels et al., 1996). The
cytoplasm of these cells contains numerous membranous tubules, between which
large mitochondria are intercalated (Fig.
7A), thereby paralleling the situation with the chloride cells of
teleost fishes. Since these tubular membranes represent a vast intracellular
amplification of the basolateral cell membrane, the lumen of the tubules
constitutes part of the extracellular space
(Philpott and Copeland, 1963
;
Philpott, 1966
;
Nakao, 1974
;
Karnaky et al., 1976a
;
Peek and Youson, 1979a
). The
particles in the membranes of these tubules have been shown by freeze
fractures to be located mainly on their P face and to be arranged in linear
and helicoidally twisted arrays (Fig.
7B; Hatae and Benedetti,
1982
; Bartels and Welsch,
1986
).
|
|
When young adult lampreys are still in freshwater, the surface of all but a
small circular central region of their chloride cells is covered by the
flanges of adjacent pavement cells (Fig.
8A). After young adults have entered the marine environment, their
pavement cell flanges retract, with the result that the surface of each
chloride cell that is exposed to the environment then occupies a rectangle,
the length of which corresponds to the entire width of the interlamellar
region (Fig. 8B). In addition,
the apical surface loses its short microvilli and thus becomes relatively
smooth (Fig.
8B;Bartels et al.,
1996). Freeze-fracture replicas demonstrate that, when these
lampreys are still in freshwater, the particles in the apical membrane of the
chloride cells are randomly distributed on the P face. However, after the
lamprey has entered seawater, most of the particles present on both fracture
faces are hexagonally arranged into clusters with a periodic spacing of
approximately 19 nm (Fig. 7C,D;
Bartels et al., 1993
).
As a consequence of the changes at the gill epithelium surface, the length
of the paracellular pathway between adjacent chloride cells is increased by
four to five times. At the same time, the tight junctions between chloride
cells become greatly reduced in depth and their strands decline in number from
four to either one or two (Fig.
7C), whereas those between chloride and pavement cells remain deep
and the number of their strands declines only from four to three
(Bartels and Potter, 1991).
Finally, the membranous tubules in the cytoplasm, which are convoluted when
the lamprey is still in freshwater, become aligned and organised into tight
bundles when the animal enters seawater. After the fully grown lamprey has
left the sea and embarked on its spawning run, the chloride cells become
covered by the flanges of adjacent pavement cells and undergo apoptosis.
In the gills of marine teleosts, the site of
Na+/K+-ATPase activity lies predominantly in the
membranous tubules in the cytoplasm of their chloride cells
(Karnaky et al., 1976b), which
contains intramembranous particles that form a tight and regular
(`cobblestone') arrangement. Since these particles are considered to
correspond to the Na+ pumps
(Sardet et al., 1979
;
Sardet, 1980
), it has been
proposed that the particles of the helicoidally twisted linear arrays in the
corresponding membrane of lamprey chloride cells are also the sites of
Na+/K+-ATPase activity
(Hatae and Benedetti, 1982
;
Bartels and Welsch, 1986
).
Furthermore, in teleosts, this membrane contains a furosemide-sensitive
Na+/K+/2Cl cotransporter
(Eriksson et al., 1985
),
through which Cl enters the cell on its basolateral side, a
process driven by the steep Na+ gradient maintained across this
membrane by the activity of the Na+ pump
(Karnaky, 1986
).
The type of clusters of intramembranous particles, which are present in the
apical membrane of the chloride cells of lampreys in seawater, has not been
observed in any of the Cl secretory cell types engaged in
osmoregulation in marine environments. However, `crystalline-like plaques',
whose particles are of a similar size and spacing as those in the lamprey
chloride cell membrane, are present in the apical membrane of the principal
cells in the renal collecting tubule of the salamander Amphiuma means
(Biemesderfer et al., 1989),
i.e. in a cell engaged in taking up rather than secreting Na+ and
Cl (Hunter et al.,
1987
). Since the changes in the apical membrane of the lamprey
chloride cell described earlier take place at the time when the animal becomes
faced with the need to excrete monovalent ions in a hypertonic environment
(and when an extended leaky paracellular pathway is developed between the
chloride cells), the clusters of particles in the apical membrane of lamprey
chloride cells are likely to be associated with the transport of
Cl across this membrane. The occurrence of these clusters of
particles in cell membranes, across which Cl is transported
in opposite directions, implies that the direction of Cl
movement depends on the electrochemical gradient.
The above description of the lamprey chloride cell demonstrates that, when
lampreys are in seawater, this cell possesses the morphological
characteristics that, in ion-secretory cells in epithelia such as those of the
teleost gill and operculum, the elasmobranch rectal gland and the avian nasal
gland, have been shown to be involved in secreting excess monovalent ions when
the animal is in hypertonic environments
(Kirschner, 1980). In this
regard, the three most salient features are: (1) the secretory cells are
organised into multicellular units, (2) there is an extended leaky
paracellular pathway (shunt) between the secretory cells and (3) the
(baso)lateral membrane is greatly amplified, which thereby provides space for
a large number of Na+ pumps. It is thus concluded that, as in the
gills of marine teleosts, the chloride cell of lampreys in hypertonic
environments is responsible for secreting the excess component of the
Na+ and Cl that has been absorbed through the
alimentary canal (Fig. 9). The
mechanisms used for such secretion by lamprey chloride cells are assumed to be
essentially the same as those employed by the chloride cells of teleosts in
seawater. They thus involve a secondary active transport of
Cl through the chloride cells, which drives the passive
movement of Na+ through the leaky pathways between these cells
(Bartels and Potter, 1991
;
Bartels et al., 1996
).
|
The pavement cell
The only morphological change observed in the pavement cells of young adult
lampreys as these animals migrate from fresh to seawater was a reduction from
five to four in the number of strands in their tight junctions
(Bartels and Potter, 1993).
Since the number of strands of the tight junctions between pavement and
chloride cells also declined, in this case from four to three
(Bartels and Potter, 1991
), the
paracellular pathway in the lamprey gill epithelium becomes more leaky during
the transition from fresh to seawater. Although such a conclusion is
consistent with the results of studies on teleosts, which have shown that the
permeability of their gills to Na+ is greater in sea than
freshwater (Potts, 1984
), any
such increase in the permeability to Na+, when young adult lampreys
enter seawater, would be due mainly to the pronounced changes that occur to
the structure and length of the occluding junctions between the chloride
cells. Since, as in marine teleosts, the chloride cell of lampreys apparently
possesses all of the structural characteristics required to fulfil the
osmoregulatory function of gills in seawater, there are at present no
indications that the osmoregulation of lampreys in seawater requires an
involvement of the pavement cell.
![]() |
Comparisons between epithelial cell types in the gills of lampreys and teleosts |
---|
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---|
The freshwater chloride cell of teleosts differs from the intercalated MR
cell of lampreys through its possession of a tubular system, which is less
elaborate than that of the chloride cell in seawater, and by the absence of
rod-shaped particles in its plasma and cytoplasmic membranes. Although the
term `MR cell' has been proposed for the freshwater chloride cell of teleosts
(Pisam and Rambourg, 1991),
the gills of teleosts in freshwater do not contain cells with the cytological
characteristics of either the intercalated MR cells of lampreys and higher
vertebrates or the ammocoete MR cell. Experimental studies indicate that, in
teleosts, the freshwater chloride cell exchanges Cl for
HCO3 while the pavement cell exchanges
Na+ for H+ (Goss et
al., 1992
; Perry,
1997
). The latter conclusion is supported by the results of
immunocytochemical studies, which showed that the pavement cell is the only
cell in the teleost gill epithelium that contains both H+-ATPase
and ENac (Sullivan et al.,
1995
; Wilson et al.,
2000a
). The freshwater chloride cell of teleosts thus performs the
role that we consider is carried out in lampreys by the subtype C of the
intercalated MR cell. The function performed by the pavement cell of teleosts
in freshwater, in turn, is ascribed in lampreys to the activities of the
subtypes A and C of the intercalated MR cell and the pavement cell, coupled in
adults and possibly uncoupled in ammocoetes.
The differences between the gill epithelium of teleosts and lampreys in
marine environments are less pronounced than those described above for
freshwater and are mainly associated with chloride cells, which in both groups
are responsible for excreting excess Cl and Na+.
They thereby constitute the main effector cells for osmoregulation in
seawater. Although the chloride cells of teleosts and lampreys are both
located close to the afferent filament artery and form multicellular
complexes, the arrangement and size of these complexes in the two groups
differ. Thus, the chloride cells in the gills of teleosts form small groups of
24 cells whereas those of lampreys form long rows. Furthermore, while
accessory cells, which lack an extensive tubular system and associated
Na+/K+-ATPase, also frequently contribute to the groups
of chloride cells in teleosts (Hootman and
Philpott, 1980), such cells are not found in lampreys
(Bartels and Potter, 1991
).
Moreover, in teleosts, each group of chloride cells and of chloride and
accessory cells share an apical crypt
(Karnaky, 1986
), a structure
not found in association with these cells in lampreys
(Nakao, 1974
;
Peek and Youson, 1979a
;
Bartels and Potter, 1991
;
Bartels et al., 1993
,
1996
). The paracellular
pathways between the chloride cells in both lampreys and teleosts and between
chloride and accessory cells in teleosts contain leaky occluding junctions
through which Na+ enters the environment passively
(Sardet et al., 1979
;
Ernst et al., 1980
;
Bartels and Potter, 1991
).
However, the increase that occurs in the length of this shunt, following the
migration of individuals from fresh to seawater, is achieved in teleosts
through the development of interdigitations amongst the cells that form
multicellular complexes (Sardet et al.,
1979
; King et al.,
1989
) and in lampreys by the retraction of pavement cells
(Bartels et al., 1996
).
Finally, the apical membrane of the chloride cells of teleosts in seawater
does not contain the prominent clusters of particles that are found in this
membrane in lampreys (Sardet et al.,
1979
; Ernst et al.,
1980
; Bartels et al.,
1993
).
As mentioned earlier, the fact that the osmolality of the sera of all
stages in the life cycle of lampreys is far less than that of seawater argues
that this agnathan group spent a considerable period in freshwater
(Hardisty et al., 1989).
However, the discovery of a lamprey-like fossil in Cambrian marine deposits
from 545 million years ago strongly indicates that the Petromyzontiformes
evolved in marine environments (Janvier,
1999
; Shu et al.,
1999
). If this is the case, the specialised parasitic phase in the
life cycle of contemporary anadromous lampreys represents a secondary return
to a marine environment. By contrast, the teleosts are believed to have
evolved in freshwater and then undergone extensive adaptive radiation in the
sea during the Mesozoic, with some groups subsequently reinvading freshwater
(Lutz, 1975
). The possession
of the same basic mechanisms for osmoregulation by lampreys and teleosts thus
presumably represents the result of convergent evolution. Such independent
evolution of the same mechanisms would account for differences between the
characteristics of the cell types used for osmoregulation by these two
divergent groups.
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Acknowledgments |
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