The gill epithelium undergoes marked changes during the life cycle of anadromous lampreys and, in particular, as the animal migrates from fresh to seawater and vice versa (Table 1). These changes include alterations to the composition and spatial relationships of the cells and to the structure of their tight junctions. The outer cellular layer of the gill filaments and lamellae of ammocoetes comprises two types of MR cell (i.e. the ammocoete MR cell and the intercalated MR cell) and pavement cells (Bartels et al., 1998). During metamorphosis, the ammocoete MR cells disappear and the chloride cells develop (Peek and Youson, 1979b). Thus, following the completion of metamorphosis, the gill epithelium of the downstream migrating young adult possesses chloride cells, pavement cells and intercalated MR cells. The last of these cells disappears after the young adult has entered seawater and does not reappear until the adult has finished growing and has re-entered freshwater on its upstream spawning migration (Bartels et al., 1998). By contrast, the chloride cells gradually disappear during the spawning run (Morris, 1957; Morris and Pickering, 1976).
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 16–18 nm or 24–27 nm in length and 8–9 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).
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).
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 10–20-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 10–13 nm) and of greater size than those on the corresponding P face (diameter 6–8 nm).
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 8–9 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).
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 patch–clamp 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.