Size Of Chloride Cells Research Articles

Hydrocortisone treatment may enhance survival and stocking of Beluga sturgeon (Huso huso Linnaeus, 1758) in estuaries of the Caspian Sea

Beluga sturgeon ( Huso huso Linnaeus, 1758) fingerlings are released into the Caspian Sea for recruiting and enhancing commercial and recreational fishing purposes. These fingerlings are reared in fresh water, but released to the estuaries that may be caused mortalities due to acute osmotic stress. In this study, the fingerlings in whole ( in vivo ) or their gill tissue ( in vitro ) were exposed to three different levels of ‘the stress hormone’ cortisol (3, 5, 7 mg L -1 hydrocortisone sodium phosphate) for 24 hrs. The effects of treatments on blood cortisol levels and the size and numbers of gill chloride cells were monitored. In each case, hormonal treatment significantly increased blood cortisol levels and also the number but decreased the size of the chloride cells. We conclude that bathing in hydrocortisone could promote the survival rate of the fingerlings in brackish water and may be have a positive effect on their osmoregulation potentiality.

Cytological comparison of gill chloride cells and blood serum ion concentrations in kutum (Rutilus frisii kutum) spawners from brackish (Caspian Sea) and fresh water (Khoshkrood River) environments

Abstract The size and number of chloride cells and serum ion concentrations in kutum, Rutillus frisii kutum Nordman, from brackish (Caspian Sea) and fresh water (Khoshkrood River) environments were studied to gain a better understanding of osmoregulation in this species. Twenty mature kutum specimens were collected from the Caspian Sea (Anzali coasts, 8.49 ppt salinity and 12.4°C temperature) and 20 specimens from Khoshkrood River (0.18 ppt salinity and 18°C temperature). Gill samples were analyzed histologically and concentrations of Na+, Cl- , K+, and Mg2+ ions were determined in the blood serum. Concentrations of Na+, Cl- , K+, and Mg2+ ions and osmotic pressure in mature kutum from brackish water were significantly higher than in specimens from fresh water. The average size and number of chloride cells in the fish from seawater were considerably larger than those from fresh water. The mean size of chloride cells was 6.89 ± 1.16 μm in brackish water samples and 5.1 ± 0.81 μm in river samples. The average number of chloride cells in brackish and river water samples were 16.92 and 6.57, respectively. The density and size of chloride cells increased with increases in salinity

Changes in growth and osmoregulation during acclimation to saltwater in juvenile Amur sturgeon Acipenser schrenckii

We evaluated the ability of juvenile Amur sturgeon (Acipenser schrenckii) to osmoregulate and grow in saltwater. Hatchery-reared juveniles (mean weight 106.8 g, 5-month old) were transferred from freshwater to 10, 20, and 25 salinity saltwater over a period of 20 d. We measured the growth, serum osmolality, ion concentrations, and Na+/K+-ATPase activity. In addition, we prepared samples of gill tissue to quantify morphological changes in gill ultrastructure. Rearing in up to 25 saltwater for 30 d had no significant effect on growth. Similarly, serum osmolality and ion concentrations were similar to levels reported in other teleosts following acclimation to saltwater. Serum osmolality and Na+, Cl− concentrations increased significantly with the initial increase in salinity. Afterwards, levels tended to stabilize and then decrease. Serum K+ levels did not change during acclimation to saltwater. Gill Na+/K+-ATPase activity increased initially as salinity was increased. However, the activity later decreased and, finally stabilized at 3.7±0.1 μmol Pi/mg·prot·h in 25 saltwater (1.6 times higher than the level in those in freshwater). In fish that were held only in freshwater, the chloride cells were located in the interlamellar regions of the filament and at the base of the lamella. Following acclimation to 25 saltwater for 30 d, the number and size of chloride cells increased significantly. Our results suggest that juvenile Amur sturgeon is able to tolerate, and grow in, relatively high concentrations of saltwater.

Chloride cell morphometrics of Common carp, Cyprinus carpio, in response to different salinities

Salinity plays an important role in the survival, metabolism, and distribution of fish during development. The successful establishment of species in a given habitat depends upon the ability of each developmental stage to cope with salinity via osmoregulation. The present study was conducted to assess the effect of salinity on number and size of chloride cells or mitochondria rich cells and their relation to osmoregulatory ability in Cyprinus carpio, a freshwater (FW) fish. No mortality occurred when the Common carp was gradually transferred into brackish water. Branchial chloride cell numbers decreased after brackish water exposure, whereas a gradual increase was observed in chloride cell size. Chloride cells exist in both FW and brackish water. The number of chloride cell fish in 9 ppt salinity was greatly significant (p < 0.05) when compared with other treatments, while chloride cells with larger size were observed in 12 ppt salinity (p < 0.05). Hence, our data indicate that in culture conditions, adult Common carp can survive successfully in brackish water with salinity of up to 9 ppt.

Growth performance and osmoregulation in the shi drum ( Umbrina cirrosa) adapted to different environmental salinities

In order to investigate the ability of shi drum ( Umbrina cirrosa) to be reared at diverse locations, growth and osmoregulatory performance were assessed at full-strength seawater (40 psu), nearly iso-osmotic water (10 psu) and low salinity water (4 psu). At the end of the 84-day experimental period, fish reared at 4 psu displayed shorter mean fork length, lower mean body weight, lower specific growth rate and higher food conversion efficiency than fish reared at 10 or 40 psu. The effect of salinity on growth performance was also reflected by changes in plasma triglycerides and cholesterol, with fish reared at 4 psu exhibiting the lowest mean concentrations, while there was no significant difference in mean plasma glucose concentrations among treatments. Plasma osmolality was lower at 4 psu from day 42 onwards, while there was no significant difference in mean plasma K + and Cl − concentrations. Plasma sodium and gill Na +/K +-ATPase activity showed minimum values on day 42 at 4 psu, but at the end of the experiment there was no difference among groups. Pavement cells, mucus cells and chloride cells were identified by histology on the gill epithelium. In shi drum reared at full seawater, mucus cells contained a mixture of acid and neutral mucins, whereas in fish adapted to hypo-osmotic environment neutral mucins were mainly observed. There was a significant increase in chloride cell number over the course of the study in all fish, but there was no difference among the three experimental salinities. Finally, in fish reared at 40 psu salinity, chloride cells increased in size significantly compared to fish adapted to 4 psu, whereas at 10 psu after 42 d there was a significant reduction in chloride cell size. These results indicate that shi drum reared from full-strength seawater to iso-osmotic salinity do not face any osmoregulatory imbalance, while fish reared in hypo-osmotic water displayed osmoregulatory impairment and low growth performance.

Ontogenic change of gill chloride cells in leptocephalus and glass eel stages of the Japanese eel, Anguilla japonica

The development of gill chloride cells was examined in premetamorphic larvae (leptocephali) and juveniles (glass eels) of the Japanese eel, Anguilla japonica. Branchial chloride cells were detected by immunocytochemistry using an antiserum specific for Na+,K+-ATPase. The specificity and availability of the antiserum for the detection of Japanese eel chloride cells were confirmed by Western blot analysis. The chloride cells first appeared on the developing gill filaments in a mid larval stage of leptocephalus (32.2 mm). Both immunoreactivity and the number of chloride cells gradually increased as the fish grew to a late stage of leptocephalus over 54 mm. In glass eels just after metamorphosis, gill lamellae developed from the gill filaments, and a rich population of chloride cells was observed in the gill filaments. In glass eels collected at a coastal area, chloride cells were extensively distributed in the gill filaments. The chloride cell size decreased progressively in glass eels transferred from seawater (SW) to freshwater (FW), whereas there was no difference in cell number. In contrast, some Na+,K+-ATPase immunoreaction distinct from typical chloride cells was observed in the gill lamellae throughout FW-transferred fish, but disappeared in control fish maintained in SW for 14 days. These findings indicate that the gill and gill chloride cells developed slowly during the extremely long larval stage, followed by rapid differentiation during a short period of metamorphosis. The excellent euryhalinity of glass eels may be due to the presence of the filament chloride cells and lamellar Na+,K+-ATPase-immunoreaction, presumably being responsible for SW and FW adaptation, respectively.

Characterization of ion channel and transporter mRNA expressions in isolated gill chloride and pavement cells of seawater acclimating eels

Ion channels and transporters (i.e. cystic fibrosis transmembrane regulator (CFTR), inward rectifier potassium channel (eKir), Na/K-ATPase, Na/K/Cl 2 co-transporter (NKCC), aquaporin-3 (AQP-3), and Na/H exchanger-1 (NHE-1)) are known to be expressed in gill epithelia of teleost fish. Owing to the anatomical complexity of gill structures, their temporal expression profile in seawater acclimating gill pavement (PVCs) and chloride cells (CCs) are limited. In this study, we isolated the gill PVCs and CCs from seawater acclimating Japanese eels to address the issue. In the gill epithelia of freshwater adapted eels, CCs expressed the highest mRNA and/or protein levels of Na/K-ATPase, NKCC, and eKir as demonstrated by real-time PCR and/or immunohistochemical staining. AQP-3 mRNA was highly expressed in freshwater PVCs and its protein was in general expressed in all gill cells. The NHE-1 transcripts were expressed in similar levels in both PVCs and CCs. CFTR mRNA transcript was almost undetectable in all the freshwater gill cell samples. Seawater acclimation induced the transcript and/or protein levels of Na/K-ATPase, NKCC, CFTR, and eKir in CCs. The upregulation and the coexpression of these transporters in CCs suggested their cohort function in mediating Na +, K +, and Cl − transport. The expression of CFTR was found to be tightly regulated as its expression was restricted only in “seawater CCs”. AQP-3 transcript and protein levels in PVCs reduced significantly during the acclimation. Interestingly immunocytochemical (ICC) staining of seawater gill epithelia revealed that AQP-3 immunoreactivities were mainly localized in seawater CCs. In the acclimation, there was no significant reduction of NHE-1 mRNA in both PVCs and CCs, however its protein level dropped significantly in the seawater condition. The present study is the first to demonstrate the activation of the mRNA transcripts for the ion channels and transporters in isolated gill CCs during seawater acclimation. The activating mechanism is found to be confined primarily in CCs. These results indicated that in addition to the increase in size and number of CCs, the molecular remodeling and the functional plasticity of CCs were essential in the ion transport process during seawater acclimation.

Branchial osmoregulatory response to salinity in the gilthead sea bream,Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.

Chloride cells and pavement cells in gill epithelia of Acipenser naccarii: ultrastructural modifications in seawater‐acclimated specimens

Modifications in the chloride (mitochondria‐rich) and pavement cells of the gill epithelia of the Adriatic sturgeon Acipenser naccarii after their transfer under hypertonic environmental conditions (salinity 35) were examined by light and electron microscopy. In contrast to freshwater specimens, seawater‐acclimated fish showed a marked increase in the number and size of chloride cells. Ultrastructural modifications included: presence of a slightly invaginated apical crypt, a darker cytoplasm, a more compact tubular system, a major increase in cisternae from Golgi apparatus stacks and flattened‐out sacs with dilated ends that produced an increase in lateral and basal cell surfaces. All these changes indicated enhanced cellular activity. Pavement cells, which largely covered the chloride cells on the gill filament and lamella, exhibited a complex system of microridges on their apical surface. Typical features included numerous desmosomes that characterized the intercellular junction, and the presence in the apical cytoplasm of bundles of filaments and of electro‐dense vesicles in freshwater fish or clear vesicles in seawater‐acclimated animals.

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.

Developmental and environmental regulation of chloride cells in young American shad, Alosa sapidissima.

Location, abundance, and morphology of gill chloride cells were quantified during changes in osmoregulatory physiology accompanying early development in American shad, Alosa sapidissima. During the larval-juvenile transition of shad, gill chloride cells increased 3.5-fold in abundance coincident with gill formation, increased seawater tolerance, and increased Na(+),K(+)-ATPase activity. Chloride cells were found on both the primary filament and secondary lamellae in pre-migratory juveniles. Chloride cells on both the primary filament and secondary lamellae increased in abundance (1.5- to 2-fold) and size (2- to 2.5-fold) in juveniles held in fresh water from August 31 to December 1 (the period of downstream migration) under declining temperature. This proliferation of chloride cells was correlated with physiological changes associated with migration (decreased hyperosmoregulatory ability and increased gill Na(+),K(+)-ATPase activity). Increases in chloride cell size and number of fish in fresh water were delayed and of a lower magnitude when shad were maintained at constant temperature (24 degrees C). When juveniles were acclimated to seawater, chloride cell abundance on the primary filament did not (though size increased 1.5- to 2-fold), but cells on the secondary lamellae disappeared. Na(+),K(+)-ATPase was immunolocalized to chloride cells in both fresh water and seawater acclimated fish. The disappearance of chloride cells on the secondary lamellae upon seawater acclimation is evidence that their role is confined to fresh water. The proliferation of chloride cells in fresh water during the migratory-associated loss of hyperosmoregulatory ability is likely to be a compensatory mechanism for increasing ion uptake. J. Exp. Zool. 290:73-87, 2001.

Evidence for an Osmoregulatory Role of Thyroid Hormones in the Freshwater Mozambique Tilapia Oreochromis mossambicus

The existing equivocal reports on the osmoregulatory role of triiodothyronine (T3) and thyroxine (T4) in teleosts prompted a reinvestigation of their osmoregulatory function in the euryhaline teleost Oreochromis mossambicus. Evidence is presented for thyroidal involvement in hydromineral balance in freshwater tilapia. Dose- and tissue-related responses to various T3 and T4 concentrations were observed in the branchial and renal tissues. The branchial Na+,K+-ATPase activity, known to reflect sodium pump dynamics, increased significantly after the administration of low doses of T3 (20 and 40 ng · g−1) or T4 (40 and 80 ng · g−1). Higher doses of T3 and T4 (>160 ng · g−1) did not change the enzyme activity, compared to sham-injected fish. Conversely, the specific activity of renal Na+,K+-ATPase decreased significantly at all doses of T3 or T4. Further, immunoreactive Na+,K+-ATPase in T4-treated fish increased in branchial chloride cells and this was coupled with a significant increase in the size of chloride cells. T4 treatment, however, did not change branchial chloride cell density. Plasma osmolality, [Na+], and [Cl−] increased, whereas [K+] decreased following low doses of T3 or T4. As expected, plasma levels of T3 and T4 increased significantly in a dose-dependent manner after a single injection of either T3 or T4. The basal levels of T3 and T4 were 4.45 ± 0.49 and 1.25 ± 0.26 nmol · L−1, respectively. This study shows that physiological concentrations of T3 (<10.57 nmol · L−1) and T4 (<6.64 nmol · L−1) enhance branchial Na+ pump activity and chloride cell morphometric dynamics, favoring hyperosmoregulatory capacity in freshwater tilapia. These data are consistent with the hypothesis that thyroid hormones perform a role in hydromineral regulation in freshwater teleosts.

Changes in salinity tolerance and branchial chloride cells of newborn guppy during freshwater and seawater adaptation

To clarify an osmoregulatory role of branchial chloride cells in successful seawater adaptation of newborn guppies Poecilia reticulata, the present study examined the functional and morphological changes in branchial chloride cells during freshwater and seawater adaptation using immunohistochemical localization of Na+,K+-ATPase. In the newborn guppies of the freshwater-adapted strain, chloride cells were located on the primary lamellae and showed strong immunoreactivity for Na+,K+-ATPase. During adaptation of the newborn guppies to freshwater, their salinity tolerance decreased within five days after birth. Although the total number of chloride cells did not change, the immunoreactive chloride cells decreased in number and size and the immunonegative chloride cells appeared on the primary lamellae. In the seawater-adapted strain, on the other hand, the newborn guppies had larger chloride cells and higher salinity tolerance than those of the freshwater-adapted strain. Acclimation of the mothers to freshwater caused reductions in the chloride cell size and the salinity tolerance of their newborn guppies, indicating that the mother's environmental salinity enhanced the chloride cell size and the salinity tolerance of the newborn guppies in the seawater-adapted strain. During adaptation of the newborn guppies to seawater, their salinity tolerance increased within five days after birth. All of the chloride cells showed a strong immunoreactivity and the cell size increased with no changes in cell number. During the freshwater and seawater adaptation, the salinity tolerance significantly correlated with the sectional area of the immunoreactive chloride cells in the gills. These results indicate that the osmoregulatory activity of the chloride cells, expressed as the immunoreactivity for Na+,K+-ATPase and the cell number and size, is important for the successful seawater adaptation of newborn guppies. J. Exp. Zool. 284:137–146, 1999. © 1999 Wiley-Liss, Inc.

Changes in gill morphology of Atlantic salmon ( Salmo salar) smolts due to addition of acid and aluminum to stream water

One-year-old Atlantic salmon smolts were held in three artificial channels adjacent to a softwater (mean sp. cond. 30 μS cm −1, circumneutral stream. Water in one channel was untreated (mean pH 6.25); the others received additions of acid (to mean pH 5.6), or acid plus aluminum (to mean pH 5.5; mean exchangeable Al 158 μg litre −1). Gills were sampled after 16 and 23 days of exposure for morphometric examination. On primary lamellae, chloride cells were more numerous in both experimental treatments than in controls. In contrast, numbers of chloride cells on secondary lamellae were elevated only in fish exposed to acid without added Al. Chloride cell size and shape also varied with time and treatment. Fewer gill mucous cells were found in fish exposed to acid plus Al than in controls. Chloride cell proliferation and structural changes may represent an attempt to compensate for increased ionic effluxes with low pH stress by increasing uptake. However, if Al concentrations are high, chloride cells do not proliferate along the secondary lamellae, or proliferating cells are damaged and lost. This may limit the potential to increase ionic uptake.

Physiology of seawater acclimation in the striped bass, Morone saxatilis (Walbaum)

Several experiments were performed to investigate the physiology of seawater acclimation in the striped bass, Morone saxatilis. Transfer of fish from fresh water (FW) to seawater (SW; 31-32 ppt) induced only a minimal disturbance of osmotic homeostasis. Ambient salinity did not affect plasma thyroxine, but plasma cortisol remained elevated for 24h after SW transfer. Gill and opercular membrane chloride cell density and Na(+),K(+)-ATPase activity were relatively high and unaffected by salinity. Average chloride cell size, however, was slightly increased (16%) in SW-acclimated fish. Gill succinate dehydrogenase activity was higher in SW-acclimated fish than in FW fish. Kidney Na(+), K(+)-ATPase activity was slightly lower (16%) in SW fish than in FW fish. Posterior intestinal Na(+),K(+)-ATPase activity and water transport capacity (Jv) did not change upon SW transfer, whereas middle intestinal Na(+),K(+)-ATPase activity increased 35% after transfer and was correlated with an increase in Jv (110%). As salinity induced only minor changes in the osmoregulatory organs examined, it is proposed that the intrinsic euryhalinity of the striped bass may be related to a high degree of "preparedness" for hypoosmoregulation that is uncommon among teleosts studied to data.

Effects of salinity on chloride cells and Na + K +-ATPase activity in the teleost Gillchthys mirabilis

1. 1. Longjawed mudsuckers, Gillichthys mirabilis, in 30ppt seawater (SW) were transferred to 1.5, 30 and 60ppt SW. 2. 2. In the first 1–3 days after transfer, plasma chloride level and plasma osmolarity rose in the 60ppt SW fish, and decreased in the 1.5ppt SW fish. 3. 3. By day 21, however, plasma chloride and osmolarity were at or near the levels seen in the controls (30ppt). 4. 4. Branchial and jawskin Na +, K +-ATPase activities were high in all salinities, and did not differ significantly among treatments. 5. 5. The vital fluorescent stains DASPEI and anthroylouabain were used to detect mitochondria and Na +, K +-ATPase, respectively, in chloride cells. 6. 6. Both stains indicated that jawskin chloride cell density did not differ among treatment groups. 7. 7. In contrast, chloride cell size increased significantly with increasing salinity. 8. 8. The chloride cells of fish in 60 ppt SW were noticeably angular in outline, whereas those of both the 1.5 and 30ppt SW fish were circular. 9. 9. The results are discussed in relation to the ion transport requirements encountered in the intertidal habitat of the mudsucker.

Effects of prolactin on chloride cells in opercular membrane of seawater-adapted tilapia

Effects of prolactin on morphology and numbers of chloride cells in the opercular membrane of seawater-adapted tilapia ( Oreochromis mossambicus) have been examined. Following five daily injections of ovine prolactin at a dose of 10 μg · g body wt −1, blood samples were taken and opercular membranes were removed and stained with a fluorescent mitochondrial dye (dimethylaminostyrylethylpyridiniumiodine), a fluorescent derivative of ouabain (anthroylouabain), and a histological stain specific for the extensive tubular system of chloride cells (zinc-osmium-iodine). Mean plasma osmolarity and sodium increased 23–24% following prolactin injection. An increase in the relative frequency of chloride cells between 20 and 180 μm 2 in cross-sectional area and a decrease in the relative frequency of chloride cells greater than 180 μm 2 were observed following prolactin injections. Average cell size decreased 46–70% and cell height decreased 26–38% following prolactin injections. There was no significant change in cell density. Anthroylouabain staining was observed in both prolactin- and saline-injected fish, and no significant effect on Na +,K +-adenosinetri-phosphatase activity was seen in either opercular membrane or gill tissue. The results demonstrate an effect of prolactin on chloride cell size and provide a morphological correlate for decreased secretory activity of chloride cells following prolactin injections.

Cortisol directly stimulates differentiation of chloride cells in tilapia opercular membrane

Opercular membranes from freshwater tilapia (Oreochromis mossambicus) were maintained in vitro for 4 days and exposed to several concentrations of cortisol (0, 0.01, 0.1, 1, and 10 micrograms/ml). Chloride cell size, number, and Na(+)-K(+)-ATPase content were examined using a fluorescent mitochondrial dye (dimethylaminostyrylethylpyridiniumiodine), a fluorescent analogue of ouabain (anthroylouabain) that binds specifically to Na(+)-K(+)-ATPase, and a cytological stain specific for plasma and tubular membranes. In the absence of cortisol, chloride cell density of the freshwater tilapia opercular membrane decreased (from initial levels of 6,114 +/- 451 to 18 +/- 9 cells/cm2) and was restored by cortisol in a dose-dependent manner. Chloride cell height (5.5 +/- 0.3 microns initially and 7.8 +/- 0.5 microns after 4 days in vitro) increased twofold (13.1 +/- 0.7 microns) after exposure to 1 microgram/ml cortisol. Initially and after 4 days in control medium, there was no detectable staining with anthroylouabain; exposure to 1 microgram/ml cortisol resulted in the appearance of numerous anthroylouabain-positive chloride cells. Without cortisol, Na(+)-K(+)-ATPase activity of the opercular membrane remained constant through 4 days of culture (0.4-0.6 mumol ADP.mg protein-1.h-1); addition of cortisol caused a dose-dependent increase to a maximum of 1.2 +/- 0.1 mumol Pi.mg protein-1.h-1. In vitro cortisol also maintained the size, density, and appearance of chloride cells from opercular membrane of seawater-adapted tilapia. The results indicate that in vitro cortisol exposure causes morphological and biochemical differentiation of the seawater form of the chloride cell.