Physiol. Rev. 85: 97-177, 2005;
doi:10.1152/physrev.00050.2003
0031-9333/05 $18.00
The Multifunctional Fish Gill: Dominant Site of Gas Exchange, Osmoregulation, Acid-Base Regulation, and Excretion of Nitrogenous Waste
David H. Evans,
Peter M. Piermarini and
Keith P. Choe
Department of Zoology, University of Florida, Gainesville, Florida; Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; and Mt. Desert Island Biological Laboratory, Salisbury Cove, Maine
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ABSTRACT
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The fish gill is a multipurpose organ that, in addition to providing for aquatic gas exchange, plays dominant roles in osmotic and ionic regulation, acid-base regulation, and excretion of nitrogenous wastes. Thus, despite the fact that all fish groups have functional kidneys, the gill epithelium is the site of many processes that are mediated by renal epithelia in terrestrial vertebrates. Indeed, many of the pathways that mediate these processes in mammalian renal epithelial are expressed in the gill, and many of the extrinsic and intrinsic modulators of these processes are also found in fish endocrine tissues and the gill itself. The basic patterns of gill physiology were outlined over a half century ago, but modern immunological and molecular techniques are bringing new insights into this complicated system. Nevertheless, substantial questions about the evolution of these mechanisms and control remain.
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I. INTRODUCTION
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Fishes are aquatic vertebrates that are members of the largest and most diverse vertebrate taxon (
25,000 species), that dates back over 500 million years. They have evolved into three major lineages: Agnatha (hagfish and lampreys), Chondrichthyes (sharks, skates, and rays; usually referred to as elasmobranchs), and Actinopterygii (bony fishes, with teleosts being the most prevalent). Regardless of lineage, the majority of fish species uses the gill as the primary site of aquatic respiration. Aerial-breathing species may use the gill, swim bladder, or other accessory breathing organs (including the skin). The fish gill evolved into the first vertebrate gas exchange organ and is essentially composed of a highly complex vasculature, surrounded by a high surface area epithelium that provides a thin barrier between a fish's blood and aquatic environment (Fig. 1). The entire cardiac output perfuses the branchial vasculature before entering the dorsal aorta and the systemic circulation. The characteristics of the gill that make it an exceptional gas exchanger are not without trade-offs. For example, the high surface area of the gills that enhances gas exchange between the blood and environment can exacerbate water and ion fluxes that may occur due to gradients between the fish's extracellular fluids and the aquatic environment. In the past 50 years, it has become clear that the branchial epithelium is the primary site of transport processes that counter the effects of osmotic and ionic gradients, as well as the principal site of body fluid pH regulation and nitrogenous waste excretion. Thus the branchial epithelium in fishes is a multipurpose organ that plays a central role in a suite of physiological responses to environmental and internal changes. Despite the fact that fishes do have kidneys, the gill actually performs most of the functions that are controlled by pulmonary and renal processes in mammals. The purpose of this review is to integrate the latest morphological, biochemical, and molecular data (with appropriate references to historical studies) in an effort to delimit what is known and unknown about this interesting organ.
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II. EVOLUTIONARY ORIGIN AND EXTERNAL STRUCTURE
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A. Gills of Protovertebrates and Modern Fishes
One of the defining characteristics of vertebrates is the presence of pharyngeal gill slits during some life stage (e.g., embryo, juvenile, or adult). In protovertebrates (i.e., urochordates, cephalochordates), the gills are relatively simple structures compared with those of extant fishes. For example, in amphioxus (a cephalochordate), the gill slits are formed by numerous, vertically oriented gill bars in the pharynx. These gill bars contain an internal support rod (made of collagen), blood vessels, and neuronal processes (33); externally they possess a ciliated epithelium. Despite the presence of branchial blood vessels that connect the ventral aorta to the dorsal aorta (502), the gill bars of amphioxus do not have a large diffusing capacity for oxygen uptake and are not a likely site of respiratory gas exchange (669). Instead, the gill bars play a prominent role in feeding by filtering food particles from water that is passed through the gill slits by the ciliated gill bar epithelium.
In contrast to the specialized feeding device of protovertebrates, the gills of modern fishes have evolved into an anatomically complex, multifunctional tissue with discrete external (i.e., epithelial) and internal (i.e., circulatory and neural) elements. The gills of fishes are located near the head region and are composed of several paired gill arches on the pharynx (Figs. 1 and 2). Anchored to the gill arches is a complex arrangement of epithelial, circulatory, and neural tissues, which will be described in the next two sections.
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FIG. 2. Generalized schematics of gills and associated pouches/arches in hagfishes (A and E), lampreys (B and F), elasmobranchs (C and G), and teleosts (D and H). All schematics are oriented anterior to posterior, with oral opening at top. [Modified from Wilson and Laurent (805).]
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B. External Structure of Fish Gills
1. Gross anatomy
The general anatomy of the gills varies among the three extant evolutionary lineages of fishes, but a simplified arrangement can be generalized for elasmobranchs and teleosts (agnathans will be described separately). Typically, cartilaginous or bony support rods (gill rays) radiate laterally from the medial (internal) base of each gill arch, and connective tissue between the gill rays forms an interbranchial septum. The interbranchial septum supports several rows of fleshy gill filaments (termed hemibranchs) that run parallel to the gill rays on both the cranial and caudal sides of a gill arch (Fig. 2). Gill filaments are the basic functional unit of gill tissue, and their structure is described in section IIB2.
A set of cranial and caudal hemibranchs from the same arch is referred to as a holobranch. In most elasmobranchs, four pairs of holobranchs are present in the branchial chamber, with only a pair of caudal hemibranchs on the first gill arch (Fig. 2C). The interbranchial septum of elasmobranch holobranchs extends from the base of each respective gill arch to the skin and forms distinct external gill slits. The typical path of water flow through the elasmobranch gills is for water to enter the pharynx via the mouth or spiracles (cranial valves for water entry), then pass over the gill filaments and follow the interbranchial septum until the water exits via the gill slits (Fig. 2, C and G).
In teleosts, only four pairs of holobranchs are present, and the interbranchial septum is much reduced compared with elasmobranchs. The septum usually only extends to the base of the filaments (Fig. 2H), and thus the filaments of teleosts are much more freely moving than those of elasmobranchs. No distinct external gill slits are found in teleosts, but a thin, bony flap called the operculum externally protects the branchial chamber (Figs. 1 and 2D). In teleosts, water enters the pharynx from the mouth, then passes over the filaments and follows the inner wall of the operculum until it exits via a caudal opening of the operculum (Figs. 1 and 2H).
Among agnathans, the gills of adult, parasitic lampreys have a similar overall organization to elasmobranch gills, except no gill rays are present in lampreys, and the skeletal base of the gill arch in lampreys is situated external (lateral) to the hemibranchs; in elasmobranchs (and teleosts), the base of the gill arch is medial to the hemibranchs. In addition, the interbranchial septa of lampreys are slightly concave in shape, which results in the cranial and caudal hemibranchs of adjacent holobranchs forming pouchlike structures with discrete internal and external branchiopores or ducts that open to the pharynx and environment, respectively (Fig. 2, B and F). Lampreys have six pairs of holobranchs, with only a caudal and cranial hemibranch on the first and last gill arch, respectively; this results in a total of seven paired gill pouches (Fig. 2B). Water flow through lamprey gills is rather diverse compared with other fishes, because, during feeding, lampreys attach to their prey by oral suction, and therefore the typical mouth to gill water path is not available. Through contractions of the extensive musculature that surrounds the pouches, water is moved into and out of the external ducts in a tidal manner to irrigate the gill filaments (e.g., Fig. 2F), which allows branchial respiration to occur while feeding. The flow of water through lamprey gills can also be modified to pump water from the external ducts, through the gills, and into the pharynx via the internal ducts. This water path may be used to clean the pharynx (see Ref. 632), or it may assist in the removal of the lamprey from its prey.
The gills of hagfish (the other agnathan lineage) have a rather unique gill anatomy and organization compared with those of other fishes. The gills are composed of 5–14 pairs of lens-shaped pouches that have discrete incurrent and excurrent ducts that connect to the pharynx and the environment (either directly or indirectly via a common pore, depending on species), respectively (Figs. 2A and 3A and Ref. 25). Unlike other fishes, no well-developed skeletal structures (e.g., arches) are associated with the pouches. From the internal wall of each pouch, several extensive epithelial folds arise that span the lateral dimension of the gill pouch and extend towards the center of the pouch (Fig. 3A). The folds are radially arranged around the mediolateral axis of the pouch. Despite their atypical arrangement and morphology, these folds are considered to be comparable to the gill filaments of other fishes (25, 432). The path of water through hagfish gills resembles that of other fishes, except that water enters the pharynx through a nasal opening instead of the mouth. Water then enters the pouches via their incurrent ducts, passes the gill filaments, and exits the pouches via excurrent ducts (Fig. 2E). In addition to this path, hagfish can irrigate the pharynx via a pharyngocutaneous duct on the left side of the animal that is caudal to the last gill pouch and connects the pharynx directly to the environment (Fig. 2A). This unique duct may be used to irrigate the pharynx and gill pouches while the animal is feeding, which usually involves burying the head region within a prey item (see Ref. 805).
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FIG. 3. Anatomy of hagfish gills. A: schematic of a longitudinal cut through a gill pouch from the Atlantic hagfish, with a lateral perspective of a primary gill fold (filament) and its lamellae (boxed area). Note radial arrangement of additional filaments around the pouch. Large arrows indicate direction of water flow; small arrows indicate direction of blood flow. [Modified from Elger (169).] B: scanning electron micrographs of a gill filament from the Pacific hagfish, comparable to boxed area in A. Top micrograph (x30) shows an overview of a filament, with afferent (AF) and efferent (EF) regions, and respiratory lamellae (R) with second-order folds (2). MU indicates muscular layer around the pouch. Arrow indicates flow of water through pouch from incurrent duct (ID) to excurrent duct (ED); arrowheads indicate flow of blood across filament. Bottom panel (x70) reveals higher order folds of the lamellae, i.e., third- (3), fourth- (4), fifth- (5), and sixth-order (6) folds. [Modified from Mallatt and Paulsen (432).]
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2. Gill filament anatomy
In most fishes, gill filaments are long and narrow projections lateral to the gill arch that taper at their distal end (Fig. 4). Each filament is supplied with blood from an afferent filamental artery (AFA) that extends along the filament. Blood in this vessel also travels across the filament's breadth through numerous folds on the dorsal and ventral surfaces of the filament-termed lamellae, which are perpendicular to the filament's long axis (Figs. 1 and 4). Blood that crosses the lamellae drains into an efferent filamental artery (EFA) that runs along the length of the filament and carries blood in the opposite direction to that in the AFA. Blood flow through the gills and gill filaments is described in more detail in section IIIA.
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FIG. 4. Scanning electron micrographs of gill filaments. A: filaments from a teleost (gulf toadfish, Opsanus beta), which radiate off a gill arch. Leaflike lamellae branch off the filaments (e.g., boxed area). Note the efferent edge (Ef.E) of the filaments, which is facing up; the afferent edge is in the depth of the figure. Bar = 100 µm. [From Evans (181).] B: high magnification view of filaments from an agnathan (pouched lamprey, Geotria australis), with the Ef.E of the filaments facing up. An asterisk indicates a lamella on each filament. Bar = 100 µm. [From Bartels et al. (29).] C: side profile of a filament from an elasmobranch (ocellated river stingray, Potamotrygon motoro) showing the afferent edge (Af.E), Ef.E, and lamellae (L). Arrow indicates the Ef.E of an underlying filament. Bar = 100 µm. (From P. M. Piermarini and D. H. Evans, unpublished micrograph.)
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Lamellae are evenly distributed along a filament's length, and the spaces between lamellae are channels through which water flows. A closer look at an individual lamella reveals that it is essentially composed of two epithelial sheets, held apart by a series of individual cells, termed pillar cells (Fig. 5). The spaces around the pillar cells and between the two epithelial layers are perfused with blood, flowing as a sheet, not through vessels per se (Fig. 5). Lamellae dramatically increase the surface area of the gill filament epithelium and result in a small diffusion distance between the blood that perfuses each lamella and the respiratory water. Moreover, blood flow through the lamellae is countercurrent to water flow between them (Fig. 1). Therefore, the lamellae are well-suited for gas exchange, but are also well-suited for diffusive losses or gains of ions and water to/from the environment (see sects. IV and V).
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FIG. 5. Scanning electron micrograph of a longitudinal cut through a gill filament from an elasmobranch (Atlantic stingray, Dasyatis sabina). Asterisks indicate lamellar blood spaces formed by pillar cells (arrows). Bar = 50 µm. (From Piermarini and Evans, unpublished micrograph.)
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The above description of the gill filament (and lamellae) is applicable to most fish groups (i.e., elasmobranchs, teleosts, and lampreys), but not hagfish. As with their gill gross anatomy, the gill filament anatomy of hagfish is unique compared with other fishes. Each filament is supplied with blood from an afferent radial artery that begins near the excurrent duct of a pouch. Blood traverses medially across the filament where it encounters lamellae (Fig. 3) that are oriented in the same plane as the filamental blood flow; lamellae are oriented perpendicular to filamental blood flow in other fishes. Blood that flows through the lamellae is collected by an efferent radial artery near the incurrent duct of a pouch. Blood flow through hagfish gills and gill filaments is described in more detail in section IIIA. Lamellae of hagfish gills are much more complex than those of other fishes. For example, lamellae begin as secondary folds of the gill filament and continue to fold to form several higher order folds (Fig. 3B). These folds increase the surface area of the filament and, similar to lamellae in other fishes, the folds are relatively thin structures composed of epithelial sheets separated by pillar cells. Regardless of the degree of folding, blood flow through the lamellae remains in the same direction (i.e., lateral to medial), which is countercurrent to water flow through the pouch. Thus, like other fishes, the lamellae of hagfish gills are well-suited for gas exchange.
In all fishes, the region of the filament that contains the afferent blood supply is commonly referred to as the afferent edge, whereas the region that collects efferent blood is referred to as the efferent edge. These two terms are synonymous with trailing edge and leading edge, respectively, relative to water flow across the filament. In hagfish, the additional term of respiratory region or respiratory zone is sometimes given to the lamellae and their higher order folds.
3. The gill epithelium
The epithelium that covers the gill filaments and lamellae provides a distinct boundary between a fish's external environment and extracellular fluids and also plays a critical role in the physiological function of the fish gill. The gill epithelium is composed of several distinct cell types (reviewed in Refs. 386, 805), but primarily consists of pavement cells (PVCs) and mitochondrion-rich cells (MRCs), which comprise >90% and <10% of the epithelial surface area, respectively.
A) PAVEMENT CELLS.
Although PVCs cover the vast majority of the gill filament surface area, they are largely considered to play a passive role in the gill physiology of most fishes (see below for exceptions). PVCs are assumed to be important for gas exchange because they are thin squamous, or cuboidal, cells with an extensive apical (mucosal) surface area and are usually the primary cell type that covers the sites of branchial gas exchange, i.e., the lamellae (386, 390, 805).
The apical membrane of PVCs is characterized by the presence of microvilli and/or microplicae (microridges), which often have elaborate arrangements that vary between species (e.g., Figs. 9, 11, and 13A). These apical projections likely increase the functional surface area of the epithelium and may also play a role in anchoring mucous to the surface. Typically, PVCs do not contain many mitochondria but may be rich in cytoplasmic vesicles or have a distinct Golgi apparatus (see Refs. 386, 390). In agnathans and elasmobranchs, PVCs possess subapical secretory granules or vesicles that contain mucous and fuse with the apical membrane (24, 25, 169, 432, 805) (Fig. 13A). The intercellular junctions between PVCs and adjoining cells are extensive or multistranded (e.g., Figs. 7B, 10B, 12B, and 13A), which makes the junctions "tight" and presumably relatively impermeable to ions (26, 31, 344, 664).
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FIG. 9. Scanning electron and transmission electron micrographs of gill filaments from killifish in seawater (A and C) and killifish transferred from seawater to fresh water after 30 days (B and D). Note transformation of the apical region of MRCs (arrows) from a smooth, concave crypt that is recessed below the pavement cells (PVCs) (A and C) to a convex surface studded with microvilli that extend above the surrounding PVCs (B and D). Also note that an accessory cell (AC) is not associated with the MRC from fresh water-acclimated killifish (C and D) and that the distinct, whorl-like microridges on the surfaces of PVCs do not change with salinity (A and C). Bar = 1 µm, except where noted. [Modified from Katoh and Kaneko (352).]
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FIG. 11. Scanning electron micrograph of the gill epithelium from a freshwater elasmobranch (Atlantic stingray). Note the expansive, flat apical surface of PVCs that is sparsely populated with microvilli, relative to the constricted, uneven surfaces of MRCs that are characterized by dense clusters of microvilli (long arrows) and microplicae (short arrows). Bar = 10 µm. (From Piermarini and Evans, unpublished micrograph.)
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FIG. 13. Transmission electron micrographs of the gill epithelium from freshwater (FW) and seawater (SW) lampreys. A (x3,740) shows a PVC and a FW-MRC from a FW brook lamprey (Lampetra appendix). In the PVC, note the subapical secretory vesicles (asterisks) and relatively flat apical membrane. In the FW-MRC, note the numerous mitochondria (m), subapical vesicular system (vs), and extensive apical membrane microprojections (short arrow). Intercellular junctions between PVCs and FW-MRCs are extensive (long arrows). [Unpublished micrograph generously provided by Drs. Helmut Bartels and John Youson (Univ. of Toronto).] B shows cross sections through the gill filament of FW (x2,750) and SW (x5,000) pouched lampreys to show the SW-MRCs lined up next to one another. Note the more extensive and organized tubular system (asterisks) between mitochondria in the SW-MRCs of SW lampreys, relative to FW lampreys. (Unpublished micrograph generously provided by Dr. Helmut Bartels.)
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FIG. 7. Transmission electron micrographs of MRCs from the gills of seawater teleosts. A: MRC from the sole (Solea solea) containing numerous mitochondria (m), a tubular system (ts), a subapical tubulovesicular system (tvs), and an apical crypt. [From Evans (185).] B: apical region of a MRC from the killifish (Fundulus heteroclitus). This MRC forms deep tight junctions (**) with surrounding PVCs and shallow tight junctions (*) with surrounding accessory cells (ACs), which share an apical crypt with the MRC. [Modified from Evans et al. (202).]
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FIG. 10. Transmission electron micrographs of the gill epithelium from a seawater elasmobranch (spiny dogfish). In A, note the large size of the MRC with its numerous mitochondria (m), complex basolateral membrane infoldings (white arrow), and extensive tubulovesicular system (tvs), relative to the neighboring PVC. The apical membrane of this MRC is not visible, but B shows the apical surface of another MRC, which forms deep intercellular junctions (black arrows) with adjacent PVCs, and has an extensive subapical tubulovesicular system. In A, bar = 5 µm and RBC indicates a red blood cell. In B, bar = 0.5 µm. [Modified from Wilson et al. (809).]
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FIG. 12. Transmission electron micrographs of the gill epithelium from a hagfish (Atlantic hagfish). In A (x3,740), note the large MRCs intercalated between PVCs. Asterisks indicate exposed apical membranes of MRCs. (Unpublished micrograph generously provided by Dr. Helmut Bartels, München.) B (x6,630) shows a higher magnification micrograph of a MRC, with a basolateral tubular system (ts) that closely associates with mitochondria (m). Also note the subapical tubulovesicular system (tvs) and the deep intercellular junctions (black arrows) between the MRC and neighboring PVCs. [Modified from Bartels (25).]
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In freshwater teleost gills, evidence suggests that some PVCs may play an active role in ion uptake and acid-base transport by the gills. For example, an ultrastructural study of the fresh water, brown bullhead catfish (Ictalurus nebulosus) gill epithelium demonstrated the presence of studded vesicles near the apical membrane of PVCs that resembled vacuolar-proton-ATPase (V-ATPase)-rich vesicles found in other ion regulatory epithelia (e.g., turtle urinary bladder and mammalian renal tubules) (391). Subsequent immunohistochemical (405, 716, 806) and biochemical (378) studies in rainbow trout (Onchorynchus mykiss) and tilapia (Oreochromis mossambicus) have verified the presence of V-ATPase in PVCs (see sects. VB1 and VID1). Moreover, recent studies have isolated a subpopulation of mitochondrion-rich PVCs (MR-PVCs) from rainbow trout gills that are V-ATPase-rich (248, 262). These MR-PVCs appear to play an active role in sodium uptake and acid extrusion (248, 639) (see sect. VB1).
B) MITOCHONDRION-RICH CELLS.
In contrast to PVCs, MRCs occupy a much smaller fraction of the branchial epithelial surface area, but they are considered to be the primary sites of active physiological processes in the gills. Whereas PVCs are found in all regions of gill filaments, MRCs are usually more common on the afferent (trailing) edge of filaments, as well as the regions that run between individual lamellae, termed the interlamellar region (Fig. 6). Also, MRCs are usually not found on the epithelium covering the lamellae; however, certain environmental conditions are associated with the presence of lamellar MRCs in some species (see below).
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FIG. 6. Distribution of mitochondrion-rich cells (MRCs) in gill filaments. A: scanning electron micrograph (left) and a corresponding confocal laser scanning micrograph (right) of a filament from a teleost (tilapia, Oreochromis mossambicus). The filament in the confocal micrograph is stained with DASPMI, a vital mitochondrial fluorescent dye. In this micrograph, the MRCs are stained red; note their increased abundance between the lamellae, which are stained green, and on the Af.E, compared with the Ef.E. Bar in scanning electron and confocal micrographs = 100 and 250 µm, respectively. [Modified from Van Der Heijden et al. (769).] B: confocal laser scanning micrograph of a filament from an agnathan (Atlantic hagfish) stained with DASPEI (another vital mitochondrial fluorescent dye). Filament is in similar orientation as the one shown in Figure 3B. Note similar distribution of MRCs as found in the tilapia, i.e., increase in MRC abundance from Ef.E to Af.E. [Modified from Choe et al. (94).]
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The ultrastructure and function of MRCs are highly variable among the three extant lineages of fishes, and therefore specific details on MRCs in agnathans, elasmobranchs, and teleosts will be described separately. However, a few general trends exist that are applicable to all groups. For example, MRCs are large ovoid-shaped cells, and as their name suggests, they have high densities of mitochondria in their cytoplasm, relative to PVCs. In addition, MRCs are highly polarized cells, with their apical and basolateral cell membranes having distinct morphologies (see below) and transport-protein expression profiles (see sects. V, B and C, VID, and VIIB).
I) Teleosts.
The ultrastructure of teleost MRCs has been studied extensively in several species, and numerous, extensive reviews exist on the subject (e.g., Refs. 343, 386, 390, 586, 617, 805). Teleost MRCs are often referred to as "chloride cells" in the literature, which is attributed to their NaCl secretory function in seawater teleosts. In freshwater teleosts, MRCs are also present, but they are characterized by different forms and functions than MRCs of seawater teleosts, and will be described separately.
One of the most striking characteristics of MRCs in seawater teleosts is the presence of an intricate tubular system that is formed by extensive invaginations of the basolateral membrane (601). This tubular/membranous labyrinth extends throughout most of the cytoplasm, where it closely associates with mitochondria (see Refs. 386, 390, 601, 805) (Fig. 7A) and is the site of expression for the active transport enzyme Na+-K+-ATPase, which indirectly energizes NaCl secretion by these cells (349) (see sects. VB4 and VC2A). The apical membrane of MRCs is concave and recessed below the surface of surrounding PVCs to form an apical pore or crypt that is shared with other MRCs (Fig. 7). These crypts (e.g., Fig. 9A) have been described as "potholes" in a "cobblestone street" of PVCs that expose the apical surface of MRCs to the ambient environment (343). The apical membrane itself is sparsely populated with short microvilli (387), and overall is morphologically unspecialized. However, the subapical cytoplasm contains a tubulovesicular system comprised of numerous vesicles and tubules that shuttle to and fuse with the apical membrane (Fig. 7A) (393, 805); these vesicles may be responsible for trafficking of the cystic fibrosis transmembrane regulator (CFTR; see sect. VC3) chloride channel to the apical membrane of MRCs, as seen by Marshall et al. (453). Intercellular junctions between MRCs and adjacent PVCs are extensive (multistranded) and are considered to be "tight," forming a relatively impermeable barrier to ions (344, 664) (Fig. 7B).
In seawater teleosts, MRCs exist in multicellular complexes with other MRCs and accessory cells (ACs). Similar to MRCs, ACs contain numerous mitochondria, but ACs are much smaller (e.g., Fig. 9C), with a less-developed tubular system and lower expression of Na+-K+-ATPase, relative to MRCs (307). The true nature of ACs is debatable; for example, in some species (e.g., brown trout; Salmo trutta) ACs appear to be a well-defined, discrete cell type (615), whereas in others (e.g., tilapia) evidence suggests that ACs are just a developmental stage of MRCs (796, 799). Regardless, in seawater teleosts, the multicellular complexes form an apical crypt shared by the apical membranes of ACs and MRCs (Fig. 7C). Cytoplasmic processes of ACs extend into the apical cytoplasm of MRCs to form complex interdigitations (387, 805). Importantly, these interdigitations form junctions between MRCs and accessory cells that are not extensive (Fig. 7B) and are considered to be leaky to ions (307, 344, 664), thus providing a paracellular route for Na+ extrusion (see sect. VC).
It should be noted that high densities of MRCs that are ultrastructurally and functionally identical to MRCs of the seawater teleost gill epithelium are found in the opercular epithelium of killifish (Fundulus heteroclitus) (350) and tilapia (232) and the jawskin epithelium of the longjaw mudsucker (Gillichthys mirabilis; Ref. 454). These flat epithelial sheets have been extremely valuable model systems for deciphering the mechanisms of ion transport in seawater teleost MRCs (reviewed by 343, 441) (see sect. VC).
In freshwater teleosts, MRCs are primarily found on the afferent edge and interlamellar region of the gill filament, but lamellar MRCs are also present in some species (e.g., Refs. 682, 764). In general, the MRCs of freshwater teleosts share some ultrastructural characteristics with those from seawater teleosts, such as the extensive basolateral membrane infoldings that form a tubular system associated with mitochondria, and the subapical tubulovesicular system (see Refs. 393, 586, 590). However, important differences exist. For example, the ACs and multicellular complexes associated with MRCs in seawater teleosts are not common in freshwater teleosts; the MRCs often occur singly in freshwater teleost gill epithelium, intercalated between PVCs. Also, freshwater teleost MRCs form extensive, multistranded intercellular junctions with surrounding PVCs or other MRCs to form a relatively impermeable barrier to ions. The apical membrane of freshwater MRCs is usually flush with or noticeably protruded above adjacent PVCs (e.g., Ref. 353) and contains distinct patterns of microvilli or microridges (586, 590). However, in some species (e.g., tilapia, mangrove killifish, Rivulus marmoratus) apical crypts still exist (361, 586, 590, 614). Although the basolateral tubular system still occurs in MRCs of freshwater teleosts, this structure appears to be less developed compared with seawater MRCs (e.g., Ref. 610).
In several species of freshwater teleosts, distinct morphological MRC subtypes have been detected in the gills (e.g., Atlantic salmon, Salmo salar; brown trout; guppies, Poecilia reticulata; loaches, Cobitis taenia; grudgeon, Gobio gobio; and Nile tilapia, O. niloticus). Pisam and colleagues (612–616) have described two MRC subtypes (
and 
) based on the degree of osmium staining (dark or light) in the cytoplasm, apical membrane morphology and associated subapical structures, extent of tubular system, cell shape, and anatomic location on the filament. The -MRCs (Fig. 8A) have light cytoplasmic staining, usually a smooth apical membrane associated with numerous subapical vesicles, a well-developed tubular system, an elongated shape, and are found at the base of lamellae where they may associate with an accessory cell (e.g., in Atlantic salmon and tilapia). In contrast, -MRCs (Fig. 8B) have dark cytoplasmic staining, usually have complex apical membrane projections associated with an extensive subapical tubulovesicular network, a less-developed tubular system, an ovoid shape, and are found in the interlamellar region. In the brown trout, -MRCs are associated with an accessory cell (615). Upon acclimation to seawater, the -MRCs proliferate into the typical seawater teleost MRC, whereas the -MRCs degenerate. Specific functional roles of - and -MRCs in freshwater teleosts have yet to be identified.
Recent studies using immunohistochemistry and cell isolation techniques have corroborated and extended some of Pisam and colleagues' findings. For example, immunohistochemical studies of Na+-K+-ATPase in guppy and chum salmon (Oncorhynchus keta) gills (682, 680) corroborated the proliferation of -MRCs upon seawater acclimation and also demonstrated that the - and -MRCs are rich and poor in Na+-K+-ATPase, respectively, which is consistent with the less-developed tubular system of -MRCs. With the use of a Percoll density gradient and subsequent cell sorting to isolate MRCs from gill suspensions in Japanese eels (Anguilla japonica), two MRC populations have been identified in freshwater eels that correspond to the - and -MRCs, and a decreased percentage of -MRCs (with a corresponding increased percentage of -MRCs) has been described upon seawater acclimation (816, 817).
This cell sorting technique has been used more recently to isolate, purify, and characterize two populations of MRCs (based on binding to peanut lectin agglutinin) in the gill epithelium of freshwater rainbow trout (248, 262). The lectin-binding cells (PNA+) have ultrastructural characteristics typical of teleost MRCs (e.g., a tubular system and tubulovesicular system), and the cells that did not bind lectin (PNA–) are actually MR-PVCs (see above). Reid et al. (639) have referred to the PNA+ MRCs and MR-PVCs as - and -MRCs, respectively, based on functional analogy to type B and type A intercalated cells of the mammalian nephron, respectively. However, it is not known if either of the above MRCs in the rainbow trout directly correspond to the morphological - and -MRCs described by Pisam and colleagues in other teleosts.
A recent ultrastructural and immunohistochemical study on the gills of a euryhaline killifish (F. heteroclitus) (353) has detailed the acute and chronic changes in MRCs associated with transfer from seawater to freshwater environments (Fig. 9). In this study, it was found that MRCs from seawater killifish first morphologically and immunochemically convert to freshwater MRCs and then are gradually replaced by newly generated freshwater MRCs. Among the acute changes noted were 1) a transformation of a recessed, concave apical membrane with few microvilli to a protrusive, convex apical membrane with extensive microvilli, within 7 days of freshwater exposure; 2) degeneration of ACs and multicellular complexes within 12 h of exposure to fresh water; and 3) loss of apical CFTR immunoreactivity within 24 h of exposure to freshwater.
II) Elasmobranchs.
Despite a few recent studies in the spiny dogfish (Squalus acanthias) (807, 809), the ultrastructure of elasmobranch MRCs in general has been little studied. All ultrastructural descriptions of elasmobranch MRCs have been conducted on primarily marine species, and the ultrastructure of MRCs in the gills of freshwater elasmobranchs has not been examined to date. Elasmobranch MRCs are usually found on the afferent edge of the filament and between lamellae and exist singly in the epithelium. However, numerous MRCs have been identified on gill lamellae of an elasmobranch living in fresh water (Atlantic stingray, Dasyatis sabina) (604, 605). The tight junctions between elasmobranch MRCs and adjacent PVCs are multistranded (Fig. 10), which suggests that the junctions are relatively impermeable to ions (807, 809). In elasmobranch MRCs, the tortuous basolateral tubular system seen in marine teleost MRCs is lacking. However, the basolateral membrane of elasmobranch MRCs does have moderate infoldings (Fig. 10), and they are likely the site of Na+-K+-ATPase expression (604, 807) in some MRCs and V-ATPase expression in other MRCs (605) (see sect. VB1).
The subapical cytoplasm of elasmobranch MRCs is characterized by a tubulovesicular system that contains numerous vesicles (Fig. 10), and the apical membrane is characterized by dense clusters of microvilli that can exist in different morphologies (127, 387, 807) (Piermarini and Evans, unpublished data) (Fig. 11). This may suggest that different functional MRC subtypes are present in the elasmobranch gill epithelium, and recent data suggest that at least two immunohistochemically distinct populations of MRCs exist in the Atlantic stingray (605, 607) and spiny dogfish (201) gills and that these cells are likely involved with distinct ion regulatory functions (see sects. VB4 and VC2).
III) Agnathans.
The ultrastructure of MRCs in agnathans varies considerably between hagfish and lampreys. In two hagfish species that have been examined (Atlantic hagfish, Myxine glutinosa; Pacific hagfish, Eptatretus stouti), MRCs are primarily found on the afferent edge of gill filaments (25, 94, 169, 432) (e.g., Fig. 6B). In contrast to marine teleost MRCs, hagfish MRCs exist singly in the epithelium and the tight junctions between MRCs and adjacent PVCs are extensive, which suggests they are impermeable to ions (26) (Fig. 12). Similar to marine teleost MRCs, hagfish MRCs have an extensive intracellular tubular system that is continuous with the basolateral membrane and is closely associated with mitochondria (25, 169, 432) (Fig. 12B). This tubular system is likely the site of Na+-K+-ATPase expression (see Refs. 32, 94), but high-resolution localization data are still lacking. The apical membrane of hagfish MRCs contains microvilli, and the subapical region of the cytoplasm has a tubulovesicular system with densely packed vesicles that fuse with the apical membrane (25, 169, 432) (Fig. 12B).
In the gills of adult, anadromous lamprey species (e.g., pouched lamprey, Geotria australis; sea lamprey, Petromyzon marinus; river lamprey, Lampetra fluviatilis) ultrastructural studies have identified two distinct types of MRCs that are associated with freshwater and seawater life-stages. When in fresh water, lamprey gills possess a MRC subtype that is only found in freshwater individuals (intercalated MRC or FW-MRCs) and another MRC subtype that is found in both freshwater and seawater individuals (chloride cells or SW-MRCs) (Fig. 13). The FW-MRCs are found in the interlamellar region and the afferent edge of filaments where they occur singly in the epithelium, intercalated between PVCs and SW-MRCs (29, 32). The basolateral membrane of FW-MRCs is not highly convoluted and is not associated with an elaborate tubular system (32, 507). The FW-MRC apical membrane is characterized by numerous microridges (515) that form distinct patterns (29, 32), and the subapical cytoplasm is rich in vesicles that can fuse with the apical membrane (32, 507, 515) (Fig. 13A). Additionally, freeze-fracture studies have found particles in the apical membrane of FW-MRCs that are associated with the presence of V-ATPase in other epithelia (32). Given these morphological properties and the occurrence of FW-MRCs only in freshwater lampreys, it has been suggested that this cell is involved with active ion uptake (32, 507).
The SW-MRCs (or chloride cells) of lampreys are primarily found in the interlamellar region, but also occur on the afferent edge of the filament. They are laterally compressed in the longitudinal plane of the filament and line up next to one another for almost the entire length of the interlamellar region (29, 31) (Figs. 13B and 14). Ultrastructurally, SW-MRCs share more similarities to marine teleost MRCs than to FW-MRCs of lampreys. For example, SW-MRCs have extensive basolateral membrane infoldings that form an intracellular tubular system, which closely associates with mitochondria (29, 31, 507, 847). Additionally, SW-MRCs have a subapical tubulovesicular system composed of vesicles. In contrast to marine teleost MRCs, the apical membrane of lamprey SW-MRCs does not form an apical pit, but it does have relatively dense or sparse clusters of microvilli compared with surrounding cells, depending on salinity (see below).
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FIG. 14. Scanning electron micrographs of a fracture through the interlamellar region of a FW (x2,625) and SW (x1,950) pouched lamprey gill epithelium. Note the SW-MRCs stacked along the interlamellar regions and the constricted apical surface (between arrows) of SW-MRCs from the FW lamprey, relative to the expansive apical surfaces (between arrows) of SW-MRCs from the SW lamprey. [Modified from Bartels et al. (29).]
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Ultrastructural differences exist between the SW-MRCs of freshwater and seawater lampreys. For example, the tight junctions between SW-MRCs in freshwater lampreys are extensive, but upon acclimation to seawater the junctions become less extensive and are presumably leaky to ions. The apical membrane morphology of SW-MRCs also varies with salinity (29). In freshwater lampreys the apical membrane surface area is restricted due to surrounding pavement cells that cover much of SW-MRC's apical surface, which results in a dense packing of apical microvilli on SW-MRCs (Fig. 14A). However, in seawater lampreys or freshwater lampreys recently acclimated to seawater, the PVCs appear to retract and expose more of the SW-MRCs apical surface, which becomes relatively flat with few microvilli (Fig. 14B). Finally, the tubular system of SW-MRCs appears to be more organized and robust in seawater lampreys compared with freshwater lampreys (31) (Fig. 13B). All of these differences are consistent with a more prominent and active SW-MRC in the gills of seawater lampreys, which suggests they are involved with active NaCl secretion.
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III. INTERNAL STRUCTURE: VASCULAR AND NEURAL
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A. Vascular
Blood enters the gills through afferent branchial arteries (ABAs), which receive the entire cardiac output via the ventral aorta. The blood that flows through an ABA feeds the two hemibranchs of an arch, where the blood is oxygenated at the lamellae of the filaments. Oxygenated blood from the filaments of an arch is collected by an efferent branchial artery (EBA), which directs blood to the dorsal aorta for systemic distribution. Upon closer examination of blood flow through the filaments, two distinct, yet interconnected, circulatory systems are apparent: the arterio-arterial and arteriovenous vasculature. Because of the large variations in the anatomy of these systems among species (especially in the arteriovenous vasculature), it is difficult to make generalizations. Therefore, the description that follows is not intended to be an all-encompassing description of the gill circulatory pathways, but aims to introduce some of the more important anatomical features of the vasculature that relate to functional aspects of the physiological processes discussed later in this review. For more thorough accounts of gill vascular anatomy, see References 386, 542, 547.
1. Arterio-arterial vasculature
The arterio-arterial vasculature is often referred to as the respiratory pathway, because it is responsible for the exchange of gases between a fish's blood and its environment. Because of the general similarities in this circulatory system among elasmobranchs, lampreys, and teleosts, these groups will be described together; the respiratory pathway of hagfishes will be described separately. In the arterio-arterial system, blood from an ABA feeds filaments on the hemibranchs of an arch via afferent filamental arteries (AFAs), which travel along the length of a filament (Fig. 15). In elasmobranchs and lampreys, the AFA regularly feeds the corpus cavernosum (CC) or cavernous body (Fig. 16), which is an extensive network of interconnected vascular sinuses in the afferent portion of the filament, e.g., the spiny dogfish (149a, 556); little skate, Raja erinacea (556); lesser-spotted dogfish, Scyliorhinus canicula (487); Endeavour dogfish, Centrophorus scalpratus (123); sparsely spotted stingaree, Urolophus paucimaculatus (153); Western shovelnose stingaree, Trygonoptera mucosa (153); and Arctic lamprey, Lampetra japonica (516, 517). Possible functions of the CC are as a hydrostatic support device for the gill filament (123), a blood pressure regulator (516, 835), and a site of erythrocyte phagocytosis (516, 835).
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FIG. 15. Generalized schematic of blood flow through the major vessels of a gill arch and filament. Arterio-arterial pathway: blood travels (thin dotted arrows) from the afferent branchial artery (ABA) to an afferent filamental artery (AFA), which runs along the length of a filament. The AFA distributes blood to lamellae (L) via afferent lamellar arterioles (ALAs), and the lamellar blood is received by an efferent filamental artery (EFA) via efferent lamellar arterioles (ELAs). Blood flow through the lamellae is countercurrent to water flow across the lamellae (large white-on-black dotted arrow). The EFA returns blood from the filament to the efferent branchial artery (EBA), which distributes blood to the dorsal aorta for systemic circulation. Arteriovenous pathway: blood in the EFA can be distributed to the arteriovenous circulation, i.e., interlamellar vessels (ILVs), via postlamellar arteriovenous anastomoses (arrowheads) or by nutrient arteries (NA), which arise from the EFA or EBA. The ILVs are presumably drained by branchial veins (BV), which return blood to the heart. [Modified from Olson (547).]
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FIG. 16. Scanning electron micrograph (x45) of a vascular cast from the gills of an elasmobranch (Western shovelnose stingaree, Trygonoptera mucosa), showing the afferent filamental artery (AF) and corpus cavernosum (CC). [Modified from Donald (153).]
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In teleosts, a CC is not present, but in some species the AFAs contain blebs or dilations, e.g., the channel catfish, Ictalurus punctatus (51); European perch, Perca fluviatilis (244, 389); rainbow trout (389); and skipjack tuna, Katsuwonus pelamis (552). It has been hypothesized that these blebs are vestigial structures derived from the CC of elasmobranchs and agnathans (386) or are functional structures that play a role in dampening pulsatile blood flow (244).
Regularly spaced along the length of the CC (elasmobranchs and lampreys) or AFA (teleosts) are afferent lamellar arterioles (ALAs) that feed one or more lamella(e) (Figs. 15 and 17). Blood flow through the lamellae is consistent with "sheet flow" theory (208) and occurs through narrow vascular spaces delineated by pillar cells, termed the lamellar sinusoids (see sect. IIB2 and Figs. 5, 18, and 19). Pillar cells are composed of a central trunk that houses the main body of the cell and thin cytoplasmic extensions (flanges) that spread from the trunk and connect to adjacent pillar cells (see Refs. 547, 805), thereby lining the lamellar blood space (Fig. 19). In rainbow trout and another teleost, the roach (Rutilus rutilus), the lamellar sinusoids lead to deformation, deceleration, and redirection of erythrocytes during their passage through lamellae. This phenomenon is hypothesized to enhance lamellar gas exchange (528).
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FIG. 19. Schematic and transmission electron micrograph of a cross section through an outer portion of a teleost (rainbow trout) lamella. Note the spool-shaped pillar cells (PC) with their cytoplasmic flanges (PF) that line the lamellar blood spaces, which are filled with red blood cells (RBC) in the micrograph. The pillar cells envelope extracellular bundles of collagen (C), which connect to the basement membrane (BM) that underlies the lamellar epithelium (e.g., PVC). Within the pillar cells are microfilaments (MF) that may be involved with pillar cell contraction (see text). The outer marginal channel (OMC) is formed by pillar cells on its inner boundary and endothelial cells (ECs) on its outer boundary. [Modified from Olson (547).]
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Contractile microfilaments are found in pillar cells of several teleosts (43, 523), lesser-spotted dogfish (835), and sea lampreys (847), and evidence for smooth muscle myosin has been detected in pillar cells from the Australian snapper (Chrysophrys auratus), a teleost (699). More recently, FHL5, a novel actin fiber-binding protein, has been localized in pillar cells in the Japanese eel, and its expression increased subsequent to acclimation to fresh water or volume expansion (499). These findings are suggestive of a contractile nature of pillar cells to possibly regulate blood flow through the lamellae (see Ref. 386). Recently, in the rainbow trout (724) and Atlantic cod (Gadus morhua; Ref. 711), in vivo videomicroscopy has provided direct evidence for regulation of blood flow through the lamellar sinusoids via pillar cell contraction (see sect. VIIB).
In teleosts (e.g., Refs. 312, 523), elasmobranchs (e.g., Ref. 835), and lampreys (e.g., Refs. 517, 847), pillar cells also envelop extracellular collagen bundles, which connect to collagen fibrils of the basement membranes that underlie the lamellar epithelial sheets (Fig. 19). The collagen bundles may play a role in anchoring the opposing epithelial sheets to one another, and in maintaining structural integrity of a lamella during increases of blood pressure.
In addition to the lamellar sinusoids, blood flow across the lamellae may take a less convoluted route via the inner marginal channel (IMC) and outer marginal channel (OMC) (Figs. 18–20). The IMC is a discontinuous channel at the base of lamellae that is interrupted by pillar cells (Fig. 20A). This structure is embedded within the body of the filament body, which suggests it does not contribute to gas exchange. Moreover, evidence suggests that the IMC may allow blood to cross lamellae without exchanging gases with the respiratory medium, thus acting as a nonrespiratory shunt (571, 762). The IMC may also be involved with the collection and distribution of blood within the lamellar sinusoids (see Ref. 547).
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