Surface Area Of Chloride Cells Research Articles

In vitro effects of environmental salinity and cortisol on chloride cell differentiation in embryos of Mozambique tilapia, Oreochromis mossambicus, measured using a newly developed 'yolk-ball' incubation system.

To examine the functional differentiation of chloride cells in the yolk-sac membrane of tilapia (Oreochromis mossambicus) embryos, we developed a 'yolk-ball' incubation system in which the yolk sac was separated from the embryonic body and subjected to incubation in vitro. The yolk-ball preparation consists of the yolk and the covering yolk-sac membrane, which contains a rich population of chloride cells. After appropriate cutting, the incision on the yolk ball healed during incubation in balanced salt solution for 3h, so that the yolk-sac membrane completely enclosed the yolk. Yolk balls prepared from freshwater-acclimated embryos were transferred either to fresh water or to sea water and incubated for 48 and 96 h to elucidate the morphological changes in the chloride cells in response to environmental salinity. The chloride cells in the yolk-sac membrane were larger in sea water than in fresh water. In yolk balls transferred to sea water, chloride cells often formed multicellular complexes characteristic of seawater-type chloride cells. In those transferred to fresh water, however, the cells were small and rarely formed such complexes. These responses of chloride cells were identical to those observed in intact embryos. Thus, chloride cells in the yolk-sac membrane could differentiate into the seawater type independent of the embryonic body. To examine the possible effects of exogenous cortisol on chloride cell differentiation, the yolk balls were incubated for 48 h in fresh water or sea water containing different doses of cortisol (0.1-10 microg x ml(-1)). Although chloride cells were consistently larger in sea water than in fresh water in all experimental groups, cortisol administration had no effect on chloride cell surface area in either medium. These findings indicate that the chloride cells in the yolk-sac membrane are equipped with an autonomous mechanism of functional differentiation that is independent of the embryonic endocrine and nervous systems. The yolk-ball incubation system established here is an excellent experimental model for further studies on chloride cell differentiation and function.

The effect of highly alkaline water (pH 9.5) on the morphology and morphometry of chloride cells and pavement cells in the gills of the freshwater rainbow trout: relationship to ionic transport and ammonia excretion

Exposure of rainbow trout (Oncorhynchus mykiss) to alkaline water (pH 9.5) impairs ammonia excretion (JAmm) and gill-mediated ion-exchange processes, as characterized by decreased Cl-(JC1in) and Na+influx (JNain) across the gill. Scanning electron microscopy suggested that the depression of JC1inwas concomitant with an early decrease in the population of the most active chloride cells (CCs), partly compensated for by an increasing number of immature CCs. However, within 72 h after the onset of exposure to alkaline water, there was a 2-fold increase in the fractional apical surface area of CCs that paralleled complete recovery of the maximal Cl-influx rate (JC1max). These results suggest that recovery of JC1maxwas associated with greater CC surface area, resulting in more transport sites on the gill epithelium. Morphometric analysis of the outermost layer of pavement cells on the lamellar epithelium showed a greater density of microvilli during exposure to alkaline water, which may have contributed to partial restoration of the number of Na+transport sites (JNamax). Finally, the blood-to-water gill-diffusion distance decreased by 27% after 72 h at pH 9.5, and likely contributed to progressive restoration of ammonia excretion in alkaline water.

Effect of cadmium on the cytoskeleton and morphology of gill chloride cells in parr and smolt Atlantic salmon (Salmo salar)

This study documented the effect of cadmium on salmon parr and smolt gill morphology. Cadmium-induced changes in chloride cell (CC) cytoskeletal elements were investigated, as well as the modifications of CC surface area and density. In cadmium-treated parr (10 µg Cd l-1 for 2 days), immunofluorescent light microscopy revealed the appearance of an intense actin staining located in the CC apical part. Transmission electron microscopic observations revealed a change in the organization of the microfilaments at the CC apex, with the appearance of numerous aggregates of filamentous actin. Higher cadmium concentrations (30 and 50 µg l-1) and prolonged treatment times (7 to 14 days) did not modify such reorganisation. Microtubules were not significantly affected by similar treatments. Further, scanning electron microscopic analysis revealed that cadmium induces a significant increase of parr CC surface area as early as the second day of exposure. After 2 days, mature CC density had also increased. In smolt, a rise in CC surface area was observed, although CC density did not significantly increase.

Branchial and renal calcium fluxes in rainbow trout ( Oncorhynchus mykiss) during metabolic alkalosis

Rainbow trout ( Oncorhynchus mykiss) were infused with NaHCO 3, inducing a metabolic alkalosis, and renal and branchial Ca 2+ fluxes were monitored. Because such treatment is known to alter gill epithelial morphology by increasing exposed chloride cell surface area, it was hypothesized that Ca 2+ uptake would be increased in these fish. Whole body Ca 2+ influx was significantly increased after 6 hr of NaHCO 3 infusion and remained elevated throughout the duration of the experiment. Branchial and renal Ca 2+ effluxes were largely unaffected by NaHCO 3 infusion. Plasma total Ca 2+ concentrations were significantly decreased after 6 hr of NaHCO 3 infusion and remained so until 48 hr. Such results suggest relocation of Ca 2+ from the plasma to other body compartments, such as bone. Analysis of the kinetics of whole body Ca 2+ uptake revealed that infusion of NaHCO 3 for 48 hr caused a significant increase in the maximal uptake rate of Ca 2+; the affinity constant of Ca 2+ uptake was unchanged. Measurement of various enzymatic activities from gill basolateral membranes revealed that although Na +, K +-ATPase activity was significantly increased in NaHCO 3-infused fish, neither Ca 2+-ATPase activity nor ATP-dependent Ca 2+ transport was affected. These findings suggest that the basolateral membrane Ca 2+ transporter does not alter its capacity to move Ca 2+ under alkalotic conditions. We suggest that the chloride cell apical membrane is the principle regulator of branchial Ca 2+ uptake in rainbow trout under alkalotic conditions. Such a suggestion concurs with the original hypothesis that an increase in apical surface area would lead to increased Ca 2+ uptake.

The chloride cell: structure and function in the gills of freshwater fishes.

This review focuses on the structure and function of the branchial chloride cell in freshwater fishes. The mitochondria-rich chloride cell is believed to be the principal site of trans-epithelial Ca2+ and Cl- influxes. Though currently debated, there is accruing evidence that the pavement cell is the site of Na+ uptake via channels linked electrically to an apical membrane vacuolar H(+)-ATPase (proton pump). Chloride cells perform an integral role in acid-base regulation. During conditions of alkalosis, the surface area of exposed chloride cells is increased, which serves to enhance base equivalent excretion as the rate of Cl-/HCO3- exchange is increased. Conversely, during acidosis, the chloride cell surface area is diminished by an expansion of the adjacent pavement cells. This response reduces the number of functional Cl-/HCO3- exchangers. Under certain conditions that challenge ion regulation, chloride cells proliferate on the lamellae. This response, while optimizing the Ca2+ and Cl- transport capacity of the gill, causes a thickening of the blood-to-water diffusion barrier and thus impedes respiratory gas transfer.

The effects of hypercapnia on branchial and renal calcium fluxes in the rainbow trout ( Oncorhynchus mykiss )

Whole body calcium influx, branchial calcium efflux, and renal Ca2+ excretion were measured in rainbow trout (Oncorhynchus mykiss) exposed to hypercapnia. These experiments were performed to assess the potential impact on Ca2+ balance of the changes in gill morphology known to accompany respiratory acidosis in this species. After 48 h of hypercapnia, gill filamental chloride cell fractional area was significantly reduced. Despite this reduction and the presumed involvement of the chloride cell in calcium influx, whole body calcium influx was increased after 12 h of hypercapnia and remained elevated for 48 h. Branchial calcium efflux was unaltered during hypercapnia exposure, whereas renal Ca2+ excretion was elevated over preflux values only at 6 h of hypercapnia. Measurement of the kinetics of whole body calcium influx after 48 h of hypercapnia revealed a significant increase in the maximal uptake rate of Ca2+, yet the affinity constant of Ca2+ uptake was unaffected. Measurements of high-affinity Ca2+ -ATPase activities and ATP-dependent Ca2+ transport of gill basolateral membrane vesicles revealed that the ATP-dependent Ca2+ extrusion mechanism of the gills was not affected by hypercapnia. The results of the present study clearly show that the reduced chloride cell surface area that accompanies hypercapnia in trout does not impair calcium homeostasis. Although adjustments to the basolateral membrane high affinity Ca2+ transporter do not appear to play a role, the mechanism(s) underlying the maintenance of calcium homeostasis under hypercapnic conditions are unresolved.

THE EFFECTS OF BRANCHIAL CHLORIDE CELL PROLIFERATION ON RESPIRATORY FUNCTION IN THE RAINBOW TROUT ONCORHYNCHUS MYKISS

The objectives of this study were to induce chloride cell (CC) proliferation on the gill lamellae of rainbow trout Oncorhynchus mykiss and to evaluate the consequences for respiratory function. Chronic elevation of hormone levels was used to induce CC proliferation; fish were injected with a combination of cortisol (8 mg kg-1 intramuscularly every day for 10 days) and ovine growth hormone (2 mg kg-1 intraperitoneally every second day for 10 days). The extent of CC proliferation was quantified using scanning electron microscopy and a two-dimensional analysis. An extracorporeal preparation in combination with environmental hypoxia was used to assess the effects of CC proliferation on respiratory function. Arterial blood was routed from the coeliac artery through an external circuit in which pH (pHa), partial pressure of oxygen (PaO2) and partial pressure of carbon dioxide (PaCO2) were monitored continuously. Environmental hypoxia was imposed by gassing a water equilibration column supplying the experimental chamber with N2. The hormone treatment increased the average CC surface area by 2.7-fold and CC density by 2.2-fold; the combined effect was a fivefold increase in CC fractional area. While the PaO2 values of hormone-treated and control fish were similar at PwO2>12.0 kPa, the arterial O2 tensions of treated fish were significantly lower than those of the control group for PwO2¾12.0 kPa. In comparison with control fish at all environmental O2 tensions, the hormone-treated fish exhibited elevated PaCO2 values and a significant acidosis. The effects of CC proliferation on blood gas variables in hormone-treated fish were accompanied by a significantly elevated ventilation amplitude and a lowered ventilation frequency. The results of this study demonstrated (i) that impairment of respiratory gas transfer coincides with CC proliferation, (ii) that O2 and CO2 transfer are influenced differently and (iii) that partial compensation is achieved through physiological adjustments.

Gill morphology during hypercapnia in brown bullhead (Ictalurus nebulosus): role of chloride cells and pavement cells in acid‐base regulation

The role of gill chloride cells (CCs) and pavement cells (PVCs) in acid‐base regulation was evaluated in brown bullhead catfish (Ictalurus nebulosus) subjected to acute hypercapnia (water Pco2=15 torr). Chronic (10 day) cortisol treatment was used as a tool to cause CC proliferation to permit a comparison of the regulatory capacities in groups of fish with widely different gill CC populations. Cortisol (4mg kg−1 day−1) caused a pronounced increase (170%) in the surface area of CCs exposed to the water based on scanning and transmission electron microscope analysis. The density of PVC apical membrane microvilli was significantly increased (20%) by cortisol treatment. Exposure of either group of fish to hypercapnia caused similar changes in gill epithelial morphology including: (i) a marked reduction in the surface area of exposed CCs (52 and 78% reduction in the control and cortisol‐treated fish, respectively); and (ii) pronounced increases in PVC apical membrane microvilli density (21 and 27% in the control and cortisol‐treated fish, respectively).The rates of Cl− uptake (Jincl−) and Na+ uptake (JinNa+) were elevated (150 and 262%, respectively) in the cortisol‐treated fish. Regardless of treatment, Jincl− was markedly reduced to approximately the same levels after 6 h of hypercapnia, JinNa+ was stimulated in the control group and reduced in the cortisol‐treated group and thus, after 6 h of hypercapnia, JinNa+ was equal in each group. The similar morphological responses in fish possessing different initial populations suggests that the predominant mechanism of acid‐base regulation during hypercapnia, reduction of C1−/HCO3− exchange, is accomplished by removal of the CC‐associated C1‐/HCO3− exchange sites from the water. The increase in PVC microvilli density during hypercapnia suggests a role for the PVC in acid‐base regulation.

The effects of experimentally altered gill chloride cell surface area on acid-base regulation in rainbow trout during metabolic alkalosis

To evaluate the role of the gill chloride cells in regulating metabolic alkalosis in rainbow trout (Oncorhynchus mykiss), the surface area of branchial chloride cells was altered experimentally using combined cortisol/ovine growth hormone injections. Long-term (10-day) treatment of fish with cortisol/ovine growth hormone caused an increase in the two-dimensional chloride cell fractional surface area when compared to uninjected fish (from 8.4 to 29.7%). This was the combined result of an increase in the size of individual cells (from 34.6 to 59.2 μm2) and increased numbers of cells (from 2368 to 5006 cells · mm-2). Metabolic alkalosis was induced by intra-arterial infusion of 140 mmol · l-1 NaHCO3; control fish were infused with 140 mmol · l-1 NaCl. Blood pH and plasma [HCO3-] increased in both the untreated and the cortisol/ovine growth hormone-treated fish. However, the increases in pH (from 8.05 to 8.53) and [HCO3-] (from 5.9 to 22.2 mmol · l-1) in the untreated fish were significantly greater than in the cortisol/ovine growth hormone-treated fish (pH increased from 7.78 to 8.11; [HCO3-] increased from 5.5 to 13.9 mmol · l-1). In all fish, NaHCO3 infusion elicited an increase in the rate of branchial basic equivalent excretion (acidic equivalent uptake) which, in turn, was caused by decreases and increases in branchial Na+ uptake and Cl- uptake, respectively. In the untreated fish, there was a pronounced increase (75%) in chloride cell surface area during NaHCO3 infusion. The attenuation of the metabolic alkalosis during HCO3- infusion in the cortical/ovine growth hormone-treated fish was caused, at least in part, by an enhancement of branchial basic equivalent excretion. In these fish that already displayed a proliferation of chloride cells, there was no further increase in chloride cell surface area. The changes in Na+ influx and Cl- influx were quantitatively similar during NaHCO3 infusion in both groups. This suggests that the greater rate of base excretion in the cortisol/ovine growth hormone-treated fish was caused by a greater percentage of Cl- uptake being coupled to HCO3- excretion and less to Cl- excretion (Cl- exchange diffusion).

Branchial chloride cell proliferation in the rainbow trout, Oncorhynchus mykiss: implications for gas transfer

The effects of branchial chloride cell proliferation on ion transport capability and gill morphometry were evaluated in the rainbow trout, Oncorhynchus mykiss, to test the hypothesis that chloride cell (CC) proliferation benefits ionic regulation at the expense of efficient gas transfer. The extent of hormone-induced CC proliferation (using ovine growth hormone (oGH), cortisol, or a combination of both) on the gill filament epithelium was assessed by determining the fractional surface area of exposed cells using scanning electron microscopy. Cortisol and oGH were equally effective in increasing CC fractional surface area (~2×), owing to the enlargement of individual CCs. The combined cortisol/oGH treatment resulted in an even greater increase in CC fractional area (~6×), as both the size and number of CCs increased. Sham injections were without effect on CC surface area or number. Significant increases in Na+ (Jin Na+) and Cl− uptake (Jin Cl−) were observed after all hormone treatments and were correlated positively with the increases in the CC fractional surface area. These findings support the view that CC proliferation enhances branchial ion transport capability. Lamellar epithelial thickness (measured by transmission electron microscopy) was increased in hormone-treated fish, while lamellar surface area (measured using light microscopy) was unaffected. The area of the interlamellar water channels (calculated from light micrographs) was significantly reduced in hormone-treated fish. These results suggest that, in trout, a compromise is made between the efficiency of ion regulation and gas transfer in which the enlargement/proliferation of CCs may impede gas transfer.

Interrelationships between gill chloride cell morphology and calcium uptake in freshwater teleosts

The involvement of the freshwater fish gill chloride cells (CCs) in trans-branchial calcium uptake (JinCa(2+)) was investigated. This was accomplished by assessing the interspecific relationships between the apical surface area of CCs exposed to the external environment and JinCa(2+). Three species of freshwater teleosts, the rainbow trout (Oncorhynchus mykiss), the American eel (Anguilla rostrata) and the brown bullhead catfish (Ictalurus nebulosus), were used. Chronic (ten-day) treatment with cortisol in each species was used as a tool to evoke variations in both JinCa(2+) and gill CC morphology in order to assess intraspecific relationships between CC surface area and JinCa(2+). The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that catfish possessed the lowest fractional area of exposed CC (CCFA) on the gill filament epithelium (12,744 ± 2248 μm(2)/mm(2)) and was followed, in increasing order, by American eel (21,355 ± 981 μm(2)/mm(2)) and rainbow trout (149,928 ± 26,545 μm(2)/mm(2)). With the exception of catfish, chronic treatment with cortisol caused significant increases in CCFA owing to proliferation of CCs and/or enlargement of individual CCs (eel only). The rates of JinCa(2+) closely reflected the CC fractional area in each species. The results of correlation analysis revealed significant correlations between CC fractional area and JinCa(2+) in trout and eel. Owing to the absence of an effect of cortisol treatment, there was no significant correlation in catfish because of insufficient variation in CC fractional area in this species. CC fractional area was significantly correlated with JinCa(2+) among the three species examined. These results suggest that CC is involved in calcium uptake in freshwater teleosts and that both intra- and interspecific differences in the rates of calcium uptake can be accounted for by variability in the surface area of exposed CCs on the gill epithelia.

The interrelationships between gill chloride cell morphology and ionic uptake in four freshwater teleosts

We have investigated the role of the gill chloride cell in transbranchial Na+ and Cl− uptake in four species of freshwater teleost maintained in water of identical ionic composition. The basic experimental protocol was to determine whether interspecific variability in the rates of whole body Na+ or Cl− uptake could be accounted for by similar interspecific variability in the fractional area of branchial chloride cells exposed to the external environment. To investigate the underlying cause(s) of intraspecific variability, chronic (10 day) treatment with cortisol in each species was used as a tool to evoke variations in both the rates of ionic uptake and chloride cell morphology. Examination of transmission and scanning electron micrographs revealed distinctive chloride cell and pavement cell morphology in each species. The results of quantitative morphometry, based on analysis of scanning electron micrographs, demonstrated that European eel (Anguilla anguilla) possessed the lowest chloride cell fractional area on the filament epithelium (11 288 ± 2133 μm2/mm2) followed, in increasing order, by brown bullhead catfish (Ictalurus nebulosus; 48 341 ± 7694 μm2/mm2), tilapia (Oreochromis mossambicus; 85 194 ± 10 326 μm2/mm2), and rainbow trout (Oncorhynchus mykiss; 146 333 ± 31 356 μm2/mm2). With the exception of rainbow trout, chronic treatment with cortisol caused significant increases in the chloride cell fractional area of filament epithelium owing to enlargement of the surface area of individual chloride cells and (or) proliferation of chloride cells. Both the inter- and intra-specific differences in chloride cell fractional area were reflected by similar differences in whole body Cl− and Na+ uptake. The results of correlation analysis revealed (with the exception of whole body Na+ uptake in A. anguilla) significant correlations between chloride cell fractional area and the rates of ionic uptake within and among the four species that were examined. These data suggest that the chloride cell is a significant site of ionic uptake in freshwater teleosts and that both inter- and intra-specific differences in the rates of ionic uptake can be explained by variability in the surface area of chloride cells on the gill epithelia.