Various species of teleostean fishes were adapted to fresh or salt water and their gill surface epithelium was examined using several techniques of electron microscopy.In both fresh and salt water the branchial epithelium is mostly covered by flat respiratory cells. They are characterized by unusual outer membrane fracture faces containing intramembranous particles and pits in various stages of ordered aggregation. Freeze fracture studies showed that the tight junctions between respiratory cells are made of several interconnecting strands, probably representing high resistance junctions. The organization of intramembranous elements and the morphological characteristics of the junctions do not vary in relation to the external salinity. Towards the base of the secondary gill lamellae, the layer of respiratory cells is interrupted by mitochondria-rich cells ("chloride cells"), also linked to respiratory cells by multistranded junctions.There is a fundamental reorganization of the chloride cells associated with salt water adaptation. In salt water young adjacent chloride cells send interdigitations into preexisting chloride cells. The apex of the seawater chloride cell is therefore part of a mosaic of sister cells linked to surrounding respiratory cells by multistranded junctions. The chloride cells are linked to each other by shallow junctions made of only one strand and permeable to lanthanum. It is therefore suggested that salt water adaptation triggers a cellular reorganization of the epithelium in such a way that leaky junctions (a low resistance pathway) appear at the apex of the chloride cells.Chloride cells are characterized by an extensive tubular reticulum which is an extension of the basolateral plasma membrane. It is made of repeating units and is the site of numerous ion pumps. The presence of shallow junctions in sea wateradapted fish makes it possible for the reticulum to contact the external milieu. In contrast in the freshwater-adapted fish the chloride cell's tubular reticulum is separated by deep apical junctions from the external environment.Based on these observations we discuss how solutes could transfer across the epithelium.t Deceased 16 August I977. 96J. CELL BIOLOGY 9 The Rockefeller University Press 9

A~STRaCT The addition of ammonium ions to the external medium results in an inhibition of the sodium influx and net uptake in Carassius auratus, while intraperitoneal injection of ammonium produces the opposite effect. The simultaneous chloride balance is not significantly affected by these treatments. The addition of bicarbonate ions to the external medium results in a reduction of the influx and net flux of chloride, while injection of bicarbonate produces the opposite effect. The simultaneous sodium balance is not significantly altered. The effects of the external additiom are reversible after elimination of the excess ammonium or bicarbonate ions by rinsing. Inhibition of carbonic anhydrase in the gill by injection of acetazoleamide produces a simultaneous inhibition of both sodium and chloride exchanges. These results confirm the hypothesis of an exchange of sodium for ammonium, and of bicarbonate for chloride across the gill. A tentative schematic representation of the ionic absorption mechanisms in the branchial cell of the fresh-water teleosts is given. Similarities with other biological membranes and especially with the renal tubule are pointed out.In the preceding publication (Garcla Romeu and Maetz, 1964) substantial evidence was advanced to show that in Carassius auratus the absorption of sodium and chloride ions by the gill is independent. Such independence can only be explained by the assumption that these ions are independently exchanged with endogenous ions. Krogh (1937Krogh ( , 1939 has suggested that the NH + and HCO~ ions might play this role. The work presented here is an attempt to verify this hypothesis.In support of the postulated sodium-ammonium exchange is the fact that the gill is the principal site for nitrogenous excretion in teleosts (Smith, 1929; I~o 9

The teleostean gill is a multi-purpose organ, specialized for respiratory gas exchanges, clearance of waste products of nitrogenous metabolism and maintenance of acid-base and mineral balances. Structural studies reveal a complex epithelium. The ‘chloride-cells are almost certainly the site of ion exchange in relation to salt balance. Functional studies show that the gill is responsible for the net absorption of Na + and Cl - occurring in fresh water and extrusion of these ions in sea water. In fresh water, a coupling between endogenous NH 4 + or H + and HCO 3 - excretion and Na + and Cl - absorption is observed. In sea water active Na + excretion is linked with K + absorption from the external medium. In parallel, active Cl - excretion occurs. The gill is also the site of Na + /Na + and Cl - /Cl - exchanges which involve 25 to 75 % of the internal NaCl per hour. The relative importance of simple diffusion and exchange-diffusion in these exchanges is assessed. Biochemical studies reveal two enzymes playing important roles in the ionic pumps: carbonic anhydrase and Na-K activated ATPase. Studies involving transfer of euryhaline fishes from low to high salinity, show that the switch from freshwater to seawater types of gill function is far from instantaneous. Synthesis or destruction of functional sites and renewal of specialized cells are involved. The role of external or internal NaCl concentration changes as stimuli for these ‘inductive processes’ and the endocrine control of these functional changes are briefly discussed.

The net sodium extrusion rate by the gill of the seawater-adapted euryhaline flounder is identical to the potassium influx. The excretion of sodium is blocked in K(+)-free seawater solutions. The instantaneous sodium outflux readjustment pattern of flounders transferred from seawater to solutions of various sodium chloride or potassium chloride concentrations is consistent with the hypothesis of a linkage between Na(+) outflux and K(+) influx through a common exchange carrier. External Na(+) and K(+) compete for this comnmonz carrier. It is suggested that the exchange diffusion mechanism (linkage of sodium influx and outflux) and the high internal sodium turnover rate which characterizes all seawater teleosts are the results of this competitive process. The sodium-potassium dependent adenosine triphosphatase system occurring in the gill of the seawater teleosts may play a central role in this sodium-potassium exchange pump.

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