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Ultrastructure of a new cell in the gills of the air-breathing fish Helostoma temmincki
© 1970 by Academic Press, Inc.
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J. ULTRASTRUCTURERESEARCH31, 312-322 (1970)
U l t r o s t r u c t u r e of a N e w Cell in the Gills of t h e A i r - B r e a t h i n 9 Fish H e l o s t o m a t e m m i n c k i ~ ZIEDONIS SKOBE,2 PHILIAS R. GARANT,z a n d JOHN T. ALBRIGHT
Biology Department, Boston University, Boston, Massachusetts 02215 Received September 26, 1969 A cell which has not been previously described was observed in the gills of
Helostoma temmincki. This cell contains long mitochondria, over 3 #, in parallel array, surrounded by an extensive system of coated channels. There is a 200 A. space between the membranes of the mitochondria and the channels. The particles coating the channels are seen in the center ol this space. These particles are about 80 A, in diameter with 60 • spaces between them. A connection is sometimes seen between the membranes of the channels and the particles. The close association between mitochondria and the coated channels is a characteristic of cells thought to be functioning in the active transport of ions and water. This morphological similarity to other transport cells, and additional experiments in which the specimens were subjected to gradients of hypertonic and hypotonic water, suggest that this cell also functions in the active transport of a substance.
E x a m i n a t i o n of the gills of Helostoma temmincki with the electron m i c r o s c o p e revealed the presence of cells which, to our knowledge, have n o t been previously described. A survey of several specimens of this species i n d i c a t e d t h a t these cells, c o n t a i n i n g long filamentous m i t o c h o n d r i a in close a p p o s i t i o n to an intracellular system of c o a t e d channels, are often situated a d j a c e n t to the b l o o d vessels of the gill. It is the p u r p o s e of this r e p o r t to describe the ultrastructure of these cells, which are n o t to be confused with the chloride cells (5, 17, 24, 25, 28, 29) that c o n t a i n n u m e r ous m i t o c h o n d r i a a n d a n e t w o r k of s m o o t h e n d o p l a s m i c reticulum. A l t h o u g h we have n o t yet d e t e r m i n e d their functional significance, the fine structure of these cells z Supported in part by Research Grant HE06214 from the National Institute of Health, Public Health Service. 2 Recipient of a N.A.S.A. fellowship. 3 Recipient of a Career Development Award from N.I.H., U.S. Public Health Service: Presently at the Forsyth Dental Center, Boston, Massachusetts.
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indicates that they are very likely concerned with the active transport of some substance. Since these cells represent the second cell type with apparent specializations for transport and/or concentrating activities to have been thus far described in teleost gills (the chloride cell being the first), we shall refer to them as beta cells until such time as a more appropriate name with a functional connotation is determined from future studies.
MATERIALS AND METHODS
Helostoma temmincki, commonly called "kissing gourami," were purchased from a local tropical fish store. These fish, ranging in size from 5 cm to 12 cm, were kept in the laboratory for several days in an aquarium filled with tap water. During that time some of the fish were transferred to a tank in which the salinity was slowly increased by adding salts of artificial seawater until the tank contained about 40% seawater. The water in another tank was diluted with distilled water until the tank contained 50% distilled water. The osmolarity of the original tank remained unchanged. Specimens from each tank were killed by decapitation and the gills were dissected out in a dish containing a formaldehyde-glutaraldehyde solution buffered to pH 7.4 with cacodylate (14). The gills were then cut into small pieces and placed in fresh fixative for 30 minutes, rinsed in cacodylate buffer, and postfixed in 1% osmium tetroxide at 4°C. The tissues were rapidly dehydrated in a graded series of ethanol, brought to room temperature, and embedded in Epon (18). Ultrathin sections were cut on a Porter-Blum microtome equipped with a diamond knife. The sections were stained with uranyl acetate and lead citrate (30) and examined with an RCA EMU-3G electron microscope operating at 50 kV.
RESULTS The respiratory epithelial lining of the secondary gill filaments of HeIostoma temmincki was observed to be multilayered and composed of four different cell types. The predominant cells were the typical squamous cells which covered the entire gill surface. The surface cells contained numerous cytoplasmic filaments, free ribosomes, small sparsely distributed surface microvilli, and the external surface of the plasma membrane was coated by a filamentous substance. Numerous mucus-secreting goblet cells and round chloride cells were intermingled among the flat surfce cells. Both chloride and mucous cells were frequently observed between cells of the superficial layer, thereby in contact with the external environment. A very conspicuous fourth cell type, the beta cell, was clearly observed in thick Epon sections stained with toluidine blue for light microscopy by a characteristic configuration of alternating long mitochondria and intracellular channels. These cells were commonly located beneath a cover of lining cells and in close proximity to the underlying blood vessels.
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A single basal lamina, 0.05 to 0.1 #, separated the beta cells from the endothelium. The remaining surfaces of the beta cells abutted on chloride cells, mucous cells, or epithelial lining cells. Occasionally, however, they opened to thesurface of the gill. In those instances tight junctions closed the intercellular space betWeen the beta cell and the surface lining c e l l s . . : : The characteristic feature of the beta cell was an association of numerous filamentous mitochondria with a system of coated intracytoplasmic channels. The long and narrow mitochondria of the beta cells, sometimes reaching a length of over 3 #, were especially unusual in that:: their cristae were frequently observed to run lengthwise from one end of the mitochondrion to the other, (Fig. 6). Electron dense inclusions, about 0.04 # in diameter, 'were present in the mitochondria of both'the beta cells and the chloride cells. A 200-A Space was delimited by the outer mitochondrial membrane and the limiting membrane of the intracellular channel. Extending along the center of this space was a row of particles about 80-100 A in diameter. I t w a s evident that these particles formed an outer coating of the channel wall when,: as in Fig. 9, the channels were not in such Close contact with the mitochondria. In such instances the particles appeared as small spikes radiating from the cytoplasmic surface of the channel. The internal or luminal leaflet of the three-layered limiting membrane of the channel was also associated with a surface coat. This luminal coat consisted of a globular substance approx!mately 30-40 A in diameter in direct contact with the membrane. A small amount of filamentous material was present within the lumen of the channel. The channels of varying diameter were closely wrapped around the full length of the mitochondria. An impression was gained by frequent observations of interconnecfions between adjacent Channels that long, continuous, convoluted channels existed in each cell rather than many short isolated ones (Figs. 2 and 5). A puzzling feature of beta cell channels is that they did not appear to open to the surface of the cell. EVer/when the cell was in contact with the surface of the gill (Fig. 10), the channels remained intracellular. Nor was there an opening to these channels along the cell surface adjacent t o the blood vessels (Figs. 5, 7, and 8). This portion of the cell, however, was usually modified by a folding back of the plasma membrane upon itself, making it difficult to follow the Continuity of the narrow extrace llular space. FIG. 1. Low magnification elect[roJnmicrograph of a secondary gill lamella. At the center of the lamellar a so-called '~pillar celi~!~(P)lines a blood vessel:containing red blood cells (RBC). Above the vesselthere are two chloride:)~l.ls(Cl) and below it three are two beta cells (B) with typically long mitochondria and extensive systems of coated channels. Epithelial cells (Ep) line the lamellae. × 7200.
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The beta cells of those fish which were acclimated to 50 % distilled water (4 days) were altered in three ways. First, the cristae of the m i t o c h o n d r i a were often pleated (Fig. 9). Second, the channels no longer extended completely a r o u n d m i t o c h o n d r i a , b u t rather, they appeared as a series of oval a n d r o u n d vesicles. M a n y of these vesicles were n o longer in such close association with m i t o c h o n d r i a as were the long convoluted channels in the control fish (Fig. 9). The third difference is that multivesicular bodies became quite a b u n d a n t (Figs. 9 a n d 10). N o ultrastructural changes were noted between the beta cells of the control fishes a n d those in 40 % water (Fig. 8).
DISCUSSION It is k n o w n that the gills of teleosts, in a d d i t i o n to providing a thin well vascularized surface to gaseous exchange, also have i o n regulatory functions
(2, 16, 32).
The site of gaseous exchange, the secondary lamellae of gill filaments, consists, of one or more layers of epithelial cells separated by a b a s e m e n t m e m b r a n e f r o m a rich capillary bed (11, 20). The same basic a r r a n g e m e n t has also been observed in the gills a n d l a b y r i n t h i n e organ of the a n a b a n t i d , the climbing perch (12). Acidophilic cells present i n the surface epithelium of teleost gills have been t e r m e d chloride cells by Keys a n d Willmer
(17) because they are believed to f u n c t i o n in the
secretion of chloride ions in a hypertonic e n v i r o n m e n t . Chloride cells were first FIG. 2. A chloride cell (Cl) is seen adjacent to a beta cell (B). Both are neai the surface epithelium (Ep) which is coated by "fuzz" and both line a blood vessel (BV). Note the branching of the channels in the beta cell (arrows). x 12,900. FIc. 3. High magnification of the channel system of a chloride cell. x 52,000. FIG. 4. High magnification of the channels of a beta cell. The plasma membrane lining the channels is modified on both surfaces. The surface next to the lumen (L) of the channels is coated with "fuzz", and there is a row of particles (arrows), about 80 ~ in diameter, in the 200 A space between the mitochondria (M) and the outer channel wall. x 83,000. FIa. 5. A beta cell lies between a blood vessel (BV) and surface epithelium (Ep). N, Nucleus; G, Golgi apparatus. × 16,500. FIG. 6. Often only a basement lamina (Bl) is between the beta cell and the blood vessel (BV). The mitochondria of the beta cell are almost completely enveloped by the channels. L, Lumen of the channels; N, nucleus. × 18,500. FIG. 7. High magnification of mitochondria from the beta cell shown in Fig. 6. Note that several of the cristae are parallel to the longitudinal axis of the mitochondria. L, Lumen of the channels; M, mitochondria, x 36,000. FIG. 8. The tonicity of the water from which this fish was taken was increased by adding salts of 20 % seawater. The increase in tonicity did not alter the appearance of the beta cells from those taken from control fish (Figs. 1-7). BV, Blood vessel; Bl, basement lamina; L, lumen of channels. × 19,500. FIG. 9. A beta cell of a fish which was adapted to 50 % distilled water. The channels no longer wrap around the mitochondria as in Fig. 7. The cristae of the mitocbondria are often pleated. The particles shown in Fig. 4 can now be seen coating the outside of the channels (arrows). MVB, Multivesciular bodies. × 23,800. FI~. 10. A beta cell in contact with the surface of the gill. This is also from a fish in distilled water. Ep, Surface epithelium; L, lumen of channels; MVB, multivesciular bodies.
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described in saltwater fish and in fish adapted to salt water (5, 9, 17, 23, 31), and recently in the gills of freshwater teleosts (19, 20, 21). In the electron microscope these cells have been observed to contain many mitochondria and an extensive system of smooth endoplasmic reticulum (15, 22, 25, 28, 29). Such chloride cells are also very abundant in the gills of H. temrninicki (Figs. 1-3). The literature pertaining to the function of the chloride cell has been reviewed by Potts (26). It is thought that these cells actively secrete chloride ions in seawater and may also function to absorb these ions in a hypotonic environment. The main ultrastructural features of the chloride cell, namely many mitochondria in close association with a system of channels whose walls are lined by repeating particles (28), are also the outstanding features of cells from other tissues which function in electrolyte transport. Two such transport tissues are of particular interest because they have similar cytological features to the beta cell. The involuted plasma membranes forming the channel walls in rectal papillae of the blow fly (10) and the channel walls of "goblet cells" of Hyalophora cecropia midgut (1) are coated by repeating subunits. These subunits have the same dimensions as the particles between the channel wall and the mitochondrial membranes in the beta cell. The function of the rectal papillae is believed to be the active transport of water and ions into the hemolymph (33). The '"goblet cells" are thought to be active in potassium transport from the hemolymph to the lumen of the gut (1). The functioning of ion and water transport systems has been explained in the following three ways. The first is that an ion pump, located in the plasma membranes, is in close association with mitochondrial membranes which supply the energy. This is Copeland's mitochondrial pump (6). The second explanation has been offered by Berridge and Gupta (3) and is based on Curran's model of three compartments separated by a selective membrane and a nonselective membrane (7, 8). The solute flows from the first compartment to the third. This model for transporting water is also dependent on the active transport of an ion and requires energy supplied by mitochondria. In the cecropia goblet cell, the mitochondria are directly involved as an ion storage unit. Therefore these mitochondrial membranes play a more active role in transport than only to provide energy (1). Most likely, the beta cell of the gills also actively transports an ion in some manner, utilizing its coated channels and its abundant mitochondria. On the basis of morphological similarity the beta cell is most like the "goblet cell" of the cecropia midgut (1). Both have an extensive system of channels. Both have a similar coating associated with the walls of these channels. The "'goblet cell" and possibly the beta cell transport material to the outside; to the lumen of the gut and to the gill chamber, respectively. Both have long, filamentous mitochondria.
R E P O R T OF A N E W C E L L I N G I L L S OF H E L O S T O M A
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In the beta cell the cristae are often oriented in the direction of the long axis, an observation which has previously been reported in the adreno-cortex (34), the nephron of a starved summer frog (13), and cells of other tissues reviewed by Novikoff (21). The evidence that the beta cell transports material outward is somewhat conjectural because it is based on static electron micrographs of the cell from fish subjected to various salinities. However, if distilled water was added to the tank of fish, the channels of the beta cell appeared shorter, giving the impression of a series of elongate vesicles. The mitochondria are somewhat reduced in size, possibly causing the cristae to become pleated, an occurrence observed in a variety of tissues taken from normal animals (27). The space between the mitochondria and the channel wall is larger. Therefore, the mitochondria are no longer in such extremely close contact with the channels as they were in the control condition. These changes indicate reduced surface area for transport and increased distance for ATP diffusion from mitochondria to the channel wall. This would result in a general decrease of transport activity of the cell. There was some evidence of increased activity in the beta cells from those fish in water of higher salinity. In addition to those cytological features associated with transport already described, these cells also had large numbers of pinocytotic vesicles. These vesicles seemingly provide an additional mechanism for the movement of ions. There are two outstanding problems that remain completely unsolved. The first is the significance of the channels if they are not in direct contact with the outside of the cell. In typical transport cells the channels are continuous with the plasma membrane. However, the continuity of the channels in the beta cell may have been obscured by the method of fixation used. Such fixation dependency of channel membranes has been observed in the nephrons of the toadfish (4). The second problem concerns the material to be transported. An interesting feature of this problem is that not all fish have beta cells. Hughes and Munshi (12) did not report them in the climbing perch, in which case they would not exist in all the air-breathing fish. Further investigation must reveal more about the distribution of these cells among fish in order to facilitate the formulation of meaningful physiological experiments to determine the functional significance of these cells. REFERENCES 1. 2. 3. 4. 5. 6.
ANDERSON,E. and HARVEY,W. R., 3". Cell Biol. 31, 107 (1966). BEVELANDER,G., J. Morphol. 57, 335 (1935). BERRIDGE,M. J. and GUPTA, B. L., J. Cell Sei. 2, 89 (1967). BULGER,R. E., Am. J. Anat. 117, 171 (1965). COVELAND,D. E., J. Morphol. 82, 201 (1948). -Protoplasma 63, 363 (1966).
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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
CURRAN, P. F., J. Gen. Physiol. 43, 1137 (1960). CURRAN, P. F. and MCINToSH, J. R., Nature 193, 347 (1962). GETMAN,H. C., Biol. Bull. 99, 439 (1950). GUVTA,B. L. and BERRIDGE,M. J., J. Cell Biol. 29, 376 (1966). HUGHES,G. M. and GRIMSTONE,A. V., Quart. J. Mieroscop. Sci. 106, 343 (1965). HUGHES,G. M. and MUNSHI, J. S. D., Nature 219, 1382 (1968). KARNOVSKY,M. J., Exptl. Mol. Pathol. 2, 347 (1963). -Y. Cell Biol. 27, 137a (1965). KESSEL,R. G. and BEAMS, H. W., J. Ultrastruct. Res. 6, 77 (1962). KEYS, A., Z. Vergleich. Physiol. 15, 364 (1931). KEYs, A. and WILLMER,E. N., J. Physiol (London) 76, 368 (1932). LUVT, J. H., J. Biophys. Biochem. Cytol. 9, 409 (1961). MUNSHI,J. S. D., Quart. J. Mieroscop. Sei. 105, 79 (1964). NEWSTEAD,J. D., Z. Zellforsch. Mikroseop. Anat. 69, 396 (1967). NOVIKOFF,A. B., in BRACKET,J. and MIRSKY, A. E. (Eds.), The Cell, Vol. II, pp~. 299421. Academic Press, New York, 1961. PETRm, P. and BOCHER, 0., Z. Zellforsch. Mikroscop. Anat. 96, 66 (1969). PETTENGmL,O. and CO~ELAND, D. E., J. ExptI. Zool. 108, 235 (1948). PmLPOTT, C. W., Anat. Record 142, 267 (1962). PHILVOTT,C. W. and COVELAND,D. E., J. Cell Biol. 18, 389 (1963). POTTS, W. T. W., Ann. Rev. Physiol. 30, 73 (1968). REVEL, J. P., FAWCETT, D. W., and PmLVOTT,C. W., J. Cell Biol. 16, 187 (1963) RITCH, R. and PmLVOTT, C. W., Exptl. Cell Res. 55, 17 (1969). TrtREADOOLD,L. T. and HousToN, A. H., Exptl. Cell Res. 34, 1 (1964). V~NABLE,J. H. and COGGESHALL,R. A., J. Cell Biol. 25, 407 (1965). VICKERS,T., Quart. J. Microseop. Sci. 104, 507 (1961). VIRABHADRACHARI,V., Quart. J. Microscop. Sei. 102, 361 (1961). WlGGLESWORTH,V. B., Quart. J. Microscop. Sci. 75, 131 (1932). ZELANDER,T., J. Ultrastruct. Res., Suppl. 2, 1 (1959).
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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J. ULTRASTRUCTURERESEARCH31, 312-322 (1970)
U l t r o s t r u c t u r e of a N e w Cell in the Gills of t h e A i r - B r e a t h i n 9 Fish H e l o s t o m a t e m m i n c k i ~ ZIEDONIS SKOBE,2 PHILIAS R. GARANT,z a n d JOHN T. ALBRIGHT
Biology Department, Boston University, Boston, Massachusetts 02215 Received September 26, 1969 A cell which has not been previously described was observed in the gills of
Helostoma temmincki. This cell contains long mitochondria, over 3 #, in parallel array, surrounded by an extensive system of coated channels. There is a 200 A. space between the membranes of the mitochondria and the channels. The particles coating the channels are seen in the center ol this space. These particles are about 80 A, in diameter with 60 • spaces between them. A connection is sometimes seen between the membranes of the channels and the particles. The close association between mitochondria and the coated channels is a characteristic of cells thought to be functioning in the active transport of ions and water. This morphological similarity to other transport cells, and additional experiments in which the specimens were subjected to gradients of hypertonic and hypotonic water, suggest that this cell also functions in the active transport of a substance.
E x a m i n a t i o n of the gills of Helostoma temmincki with the electron m i c r o s c o p e revealed the presence of cells which, to our knowledge, have n o t been previously described. A survey of several specimens of this species i n d i c a t e d t h a t these cells, c o n t a i n i n g long filamentous m i t o c h o n d r i a in close a p p o s i t i o n to an intracellular system of c o a t e d channels, are often situated a d j a c e n t to the b l o o d vessels of the gill. It is the p u r p o s e of this r e p o r t to describe the ultrastructure of these cells, which are n o t to be confused with the chloride cells (5, 17, 24, 25, 28, 29) that c o n t a i n n u m e r ous m i t o c h o n d r i a a n d a n e t w o r k of s m o o t h e n d o p l a s m i c reticulum. A l t h o u g h we have n o t yet d e t e r m i n e d their functional significance, the fine structure of these cells z Supported in part by Research Grant HE06214 from the National Institute of Health, Public Health Service. 2 Recipient of a N.A.S.A. fellowship. 3 Recipient of a Career Development Award from N.I.H., U.S. Public Health Service: Presently at the Forsyth Dental Center, Boston, Massachusetts.
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indicates that they are very likely concerned with the active transport of some substance. Since these cells represent the second cell type with apparent specializations for transport and/or concentrating activities to have been thus far described in teleost gills (the chloride cell being the first), we shall refer to them as beta cells until such time as a more appropriate name with a functional connotation is determined from future studies.
MATERIALS AND METHODS
Helostoma temmincki, commonly called "kissing gourami," were purchased from a local tropical fish store. These fish, ranging in size from 5 cm to 12 cm, were kept in the laboratory for several days in an aquarium filled with tap water. During that time some of the fish were transferred to a tank in which the salinity was slowly increased by adding salts of artificial seawater until the tank contained about 40% seawater. The water in another tank was diluted with distilled water until the tank contained 50% distilled water. The osmolarity of the original tank remained unchanged. Specimens from each tank were killed by decapitation and the gills were dissected out in a dish containing a formaldehyde-glutaraldehyde solution buffered to pH 7.4 with cacodylate (14). The gills were then cut into small pieces and placed in fresh fixative for 30 minutes, rinsed in cacodylate buffer, and postfixed in 1% osmium tetroxide at 4°C. The tissues were rapidly dehydrated in a graded series of ethanol, brought to room temperature, and embedded in Epon (18). Ultrathin sections were cut on a Porter-Blum microtome equipped with a diamond knife. The sections were stained with uranyl acetate and lead citrate (30) and examined with an RCA EMU-3G electron microscope operating at 50 kV.
RESULTS The respiratory epithelial lining of the secondary gill filaments of HeIostoma temmincki was observed to be multilayered and composed of four different cell types. The predominant cells were the typical squamous cells which covered the entire gill surface. The surface cells contained numerous cytoplasmic filaments, free ribosomes, small sparsely distributed surface microvilli, and the external surface of the plasma membrane was coated by a filamentous substance. Numerous mucus-secreting goblet cells and round chloride cells were intermingled among the flat surfce cells. Both chloride and mucous cells were frequently observed between cells of the superficial layer, thereby in contact with the external environment. A very conspicuous fourth cell type, the beta cell, was clearly observed in thick Epon sections stained with toluidine blue for light microscopy by a characteristic configuration of alternating long mitochondria and intracellular channels. These cells were commonly located beneath a cover of lining cells and in close proximity to the underlying blood vessels.
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A single basal lamina, 0.05 to 0.1 #, separated the beta cells from the endothelium. The remaining surfaces of the beta cells abutted on chloride cells, mucous cells, or epithelial lining cells. Occasionally, however, they opened to thesurface of the gill. In those instances tight junctions closed the intercellular space betWeen the beta cell and the surface lining c e l l s . . : : The characteristic feature of the beta cell was an association of numerous filamentous mitochondria with a system of coated intracytoplasmic channels. The long and narrow mitochondria of the beta cells, sometimes reaching a length of over 3 #, were especially unusual in that:: their cristae were frequently observed to run lengthwise from one end of the mitochondrion to the other, (Fig. 6). Electron dense inclusions, about 0.04 # in diameter, 'were present in the mitochondria of both'the beta cells and the chloride cells. A 200-A Space was delimited by the outer mitochondrial membrane and the limiting membrane of the intracellular channel. Extending along the center of this space was a row of particles about 80-100 A in diameter. I t w a s evident that these particles formed an outer coating of the channel wall when,: as in Fig. 9, the channels were not in such Close contact with the mitochondria. In such instances the particles appeared as small spikes radiating from the cytoplasmic surface of the channel. The internal or luminal leaflet of the three-layered limiting membrane of the channel was also associated with a surface coat. This luminal coat consisted of a globular substance approx!mately 30-40 A in diameter in direct contact with the membrane. A small amount of filamentous material was present within the lumen of the channel. The channels of varying diameter were closely wrapped around the full length of the mitochondria. An impression was gained by frequent observations of interconnecfions between adjacent Channels that long, continuous, convoluted channels existed in each cell rather than many short isolated ones (Figs. 2 and 5). A puzzling feature of beta cell channels is that they did not appear to open to the surface of the cell. EVer/when the cell was in contact with the surface of the gill (Fig. 10), the channels remained intracellular. Nor was there an opening to these channels along the cell surface adjacent t o the blood vessels (Figs. 5, 7, and 8). This portion of the cell, however, was usually modified by a folding back of the plasma membrane upon itself, making it difficult to follow the Continuity of the narrow extrace llular space. FIG. 1. Low magnification elect[roJnmicrograph of a secondary gill lamella. At the center of the lamellar a so-called '~pillar celi~!~(P)lines a blood vessel:containing red blood cells (RBC). Above the vesselthere are two chloride:)~l.ls(Cl) and below it three are two beta cells (B) with typically long mitochondria and extensive systems of coated channels. Epithelial cells (Ep) line the lamellae. × 7200.
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The beta cells of those fish which were acclimated to 50 % distilled water (4 days) were altered in three ways. First, the cristae of the m i t o c h o n d r i a were often pleated (Fig. 9). Second, the channels no longer extended completely a r o u n d m i t o c h o n d r i a , b u t rather, they appeared as a series of oval a n d r o u n d vesicles. M a n y of these vesicles were n o longer in such close association with m i t o c h o n d r i a as were the long convoluted channels in the control fish (Fig. 9). The third difference is that multivesicular bodies became quite a b u n d a n t (Figs. 9 a n d 10). N o ultrastructural changes were noted between the beta cells of the control fishes a n d those in 40 % water (Fig. 8).
DISCUSSION It is k n o w n that the gills of teleosts, in a d d i t i o n to providing a thin well vascularized surface to gaseous exchange, also have i o n regulatory functions
(2, 16, 32).
The site of gaseous exchange, the secondary lamellae of gill filaments, consists, of one or more layers of epithelial cells separated by a b a s e m e n t m e m b r a n e f r o m a rich capillary bed (11, 20). The same basic a r r a n g e m e n t has also been observed in the gills a n d l a b y r i n t h i n e organ of the a n a b a n t i d , the climbing perch (12). Acidophilic cells present i n the surface epithelium of teleost gills have been t e r m e d chloride cells by Keys a n d Willmer
(17) because they are believed to f u n c t i o n in the
secretion of chloride ions in a hypertonic e n v i r o n m e n t . Chloride cells were first FIG. 2. A chloride cell (Cl) is seen adjacent to a beta cell (B). Both are neai the surface epithelium (Ep) which is coated by "fuzz" and both line a blood vessel (BV). Note the branching of the channels in the beta cell (arrows). x 12,900. FIc. 3. High magnification of the channel system of a chloride cell. x 52,000. FIG. 4. High magnification of the channels of a beta cell. The plasma membrane lining the channels is modified on both surfaces. The surface next to the lumen (L) of the channels is coated with "fuzz", and there is a row of particles (arrows), about 80 ~ in diameter, in the 200 A space between the mitochondria (M) and the outer channel wall. x 83,000. FIa. 5. A beta cell lies between a blood vessel (BV) and surface epithelium (Ep). N, Nucleus; G, Golgi apparatus. × 16,500. FIG. 6. Often only a basement lamina (Bl) is between the beta cell and the blood vessel (BV). The mitochondria of the beta cell are almost completely enveloped by the channels. L, Lumen of the channels; N, nucleus. × 18,500. FIG. 7. High magnification of mitochondria from the beta cell shown in Fig. 6. Note that several of the cristae are parallel to the longitudinal axis of the mitochondria. L, Lumen of the channels; M, mitochondria, x 36,000. FIG. 8. The tonicity of the water from which this fish was taken was increased by adding salts of 20 % seawater. The increase in tonicity did not alter the appearance of the beta cells from those taken from control fish (Figs. 1-7). BV, Blood vessel; Bl, basement lamina; L, lumen of channels. × 19,500. FIG. 9. A beta cell of a fish which was adapted to 50 % distilled water. The channels no longer wrap around the mitochondria as in Fig. 7. The cristae of the mitocbondria are often pleated. The particles shown in Fig. 4 can now be seen coating the outside of the channels (arrows). MVB, Multivesciular bodies. × 23,800. FI~. 10. A beta cell in contact with the surface of the gill. This is also from a fish in distilled water. Ep, Surface epithelium; L, lumen of channels; MVB, multivesciular bodies.
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described in saltwater fish and in fish adapted to salt water (5, 9, 17, 23, 31), and recently in the gills of freshwater teleosts (19, 20, 21). In the electron microscope these cells have been observed to contain many mitochondria and an extensive system of smooth endoplasmic reticulum (15, 22, 25, 28, 29). Such chloride cells are also very abundant in the gills of H. temrninicki (Figs. 1-3). The literature pertaining to the function of the chloride cell has been reviewed by Potts (26). It is thought that these cells actively secrete chloride ions in seawater and may also function to absorb these ions in a hypotonic environment. The main ultrastructural features of the chloride cell, namely many mitochondria in close association with a system of channels whose walls are lined by repeating particles (28), are also the outstanding features of cells from other tissues which function in electrolyte transport. Two such transport tissues are of particular interest because they have similar cytological features to the beta cell. The involuted plasma membranes forming the channel walls in rectal papillae of the blow fly (10) and the channel walls of "goblet cells" of Hyalophora cecropia midgut (1) are coated by repeating subunits. These subunits have the same dimensions as the particles between the channel wall and the mitochondrial membranes in the beta cell. The function of the rectal papillae is believed to be the active transport of water and ions into the hemolymph (33). The '"goblet cells" are thought to be active in potassium transport from the hemolymph to the lumen of the gut (1). The functioning of ion and water transport systems has been explained in the following three ways. The first is that an ion pump, located in the plasma membranes, is in close association with mitochondrial membranes which supply the energy. This is Copeland's mitochondrial pump (6). The second explanation has been offered by Berridge and Gupta (3) and is based on Curran's model of three compartments separated by a selective membrane and a nonselective membrane (7, 8). The solute flows from the first compartment to the third. This model for transporting water is also dependent on the active transport of an ion and requires energy supplied by mitochondria. In the cecropia goblet cell, the mitochondria are directly involved as an ion storage unit. Therefore these mitochondrial membranes play a more active role in transport than only to provide energy (1). Most likely, the beta cell of the gills also actively transports an ion in some manner, utilizing its coated channels and its abundant mitochondria. On the basis of morphological similarity the beta cell is most like the "goblet cell" of the cecropia midgut (1). Both have an extensive system of channels. Both have a similar coating associated with the walls of these channels. The "'goblet cell" and possibly the beta cell transport material to the outside; to the lumen of the gut and to the gill chamber, respectively. Both have long, filamentous mitochondria.
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In the beta cell the cristae are often oriented in the direction of the long axis, an observation which has previously been reported in the adreno-cortex (34), the nephron of a starved summer frog (13), and cells of other tissues reviewed by Novikoff (21). The evidence that the beta cell transports material outward is somewhat conjectural because it is based on static electron micrographs of the cell from fish subjected to various salinities. However, if distilled water was added to the tank of fish, the channels of the beta cell appeared shorter, giving the impression of a series of elongate vesicles. The mitochondria are somewhat reduced in size, possibly causing the cristae to become pleated, an occurrence observed in a variety of tissues taken from normal animals (27). The space between the mitochondria and the channel wall is larger. Therefore, the mitochondria are no longer in such extremely close contact with the channels as they were in the control condition. These changes indicate reduced surface area for transport and increased distance for ATP diffusion from mitochondria to the channel wall. This would result in a general decrease of transport activity of the cell. There was some evidence of increased activity in the beta cells from those fish in water of higher salinity. In addition to those cytological features associated with transport already described, these cells also had large numbers of pinocytotic vesicles. These vesicles seemingly provide an additional mechanism for the movement of ions. There are two outstanding problems that remain completely unsolved. The first is the significance of the channels if they are not in direct contact with the outside of the cell. In typical transport cells the channels are continuous with the plasma membrane. However, the continuity of the channels in the beta cell may have been obscured by the method of fixation used. Such fixation dependency of channel membranes has been observed in the nephrons of the toadfish (4). The second problem concerns the material to be transported. An interesting feature of this problem is that not all fish have beta cells. Hughes and Munshi (12) did not report them in the climbing perch, in which case they would not exist in all the air-breathing fish. Further investigation must reveal more about the distribution of these cells among fish in order to facilitate the formulation of meaningful physiological experiments to determine the functional significance of these cells. REFERENCES 1. 2. 3. 4. 5. 6.
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