Vol. 102A,
Camp. Biochem. Physiol.
No. 4, pp. 637-643,
0300-9629/92 $5.00 + 0.00
1992
0 1992Pergamon Press Ltd
Printed in Great Britain
MINI REVIEW
GILL (Na+ + K+)-ATPase INVOLVEMENT AND REGULATION DURING SALMONID ADAPTATION SALT WATER
TO
A. R. BORGATTI,A. PAGLIARANI and V. VENTRELLA Dipartimento di Biochimica “G. Moruzzi”-Universita di Bologna, Via Belmeloro, I/2-Bologna, Tel.: 051-243019; Fax: 051-243053 (Received
Italy.
16 January 1992)
Abstract-l. The involvement of gill (Na+ + K+ )-ATPase in salmonid adaptation to salt water (SW) is discussed. 2. Gill (Na+ + K+)-ATPase increase during SW adaptation is mainly related to the increased number and complexity of chloride cells deputed to salt extrusion. 3. The temporal relationships between serum peaks of thyroid hormones, cortisol, growth hormone, prolactin and gill (Na+ + K+ )-ATPase rise during salmonid smoltification, suggest a hormonal involvement in the enzyme stimulation and thus in the acquirement of SW tolerance. 4. Literature on gill (Na+ + K+ )-ATPase response to hormonal treatments is reviewed. The effects produced on gill (Na+ + K+ )-ATPase and chloride cells by exogenous hormones point out a complex inter-relationship between the hormones considered. The mechanisms involved in hormonal regulation of the enzyme remain a matter of debate
chemical mechanisms so far hypothesized for both the NaCl uptake in FW and the NaCl extrusion in SW via the gills are based on (Nat + K+)-ATPase (De Renzis and Bomancin, 1984; Payan et al., 1984; Zadunaisky, 1984).
INTRODUCfION Teleosts cope with external osmotic pressures ranging
from cu 0 mOsm/l in fresh water (FW) to as much as to 1100 mOsm/l and even more in salt water (SW). Nevertheless, each species is able to maintain the osmolality of body fluids to a quite constant value, generally between 250 and 450 mOsm/l. FW species, whose osmolality is around 25&300 mOsm/l are hy perosmotic with respect to the environment, whereas SW species exhibit osmolalities of ca 400 mOsm/l and are hypoosmotic (Evans, 1979). By 1930 Smith had stressed the importance of branchial epithelium in teleost osmoregulation (Smith, 1930). In FW teleosts the gills are the site of active NaCl absorption compensating for outward diffusion, whereas in SW teleosts the gills actively excrete the salt absorbed through the gut (Payan et al., 1984). Gill epithelium exhibits distinct morphological features related to the habitat. The differences are mainly shown by the so-called chloride cells localized on the primary lamellae (De Renzis and Bornancin, 1984; Zadunaisky, 1984). These cells are small and isolated in FW fish, whilst they are large, abundant, ~tochond~a-~ch and clustered in grapes of at least two cells sharing a common apical crypt in SW fish. In the latter case, adjacent chloride cells are joined by ion-permeable junctions and the basolateral membrane, where the (Na+ + K+)-ATPase is typically localized, is amplified by many infoldings that form a densely branched network of tubules. These structural changes of the chloride cell complex are well known to occur when an euryhaline species is transferred from FW to SW (Karnaky, 1986). The bio-
(Na+ + K+ )-A TPase and NaCi extrusion in salt water fNa+ + K + )-ATPase, an almost ubiquitous enzyme in the animal world, plays a key role in Na+ transport and serves a variety of essential functions. The enzyme complex, basolaterally localized in oriented cells, spans the plasmalemma and acts as a cation pump. The complex consists of a and B subunits whose association is reputed essential for the catalytic activity. The energy obtained from the hydrolysis of 1 ATP is utilized for the uphill transport of 3 Na+ outwards and 2 K+ inwards, thus maintaining a low intra~llular concentration of Nat. These ion movements contribute to the onset of a negative intracellular transmembrane potential (Skou, 1988). During SW adaptation, an overall increase of gill (Nat + K+ )-ATPase is generally observed (De Renzis and Bornancin, 1984) both in salmonids and in non-salmonids (Table 1). On the other hand, SW fish are reported to exhibit higher gill (Na+ + K+ )-ATPase activities with respect to FW ones (Table 1). Such ~ha~our is related to gill mo~hological adaptations and especially to the proliferation of the chloride cells, as well as to the simultaneous enlargement of basolateral membranes enriched in enzyme units (Zadunaisky, 1984). According to the model originally brought about by Silva et al. (1977) and still substantially accepted with some modifications (Zadunaisky, 1984; Karnaky, 1986), in the chloride cells of SW teleosts, the Nat gradient generated by
637
638
A. R. BORGATTI er
al.
Table 1. Gill (Na+ + K+)-ATPase activities @mol P,/mg protein/hr) in freshwater- (FW) and saltwater(SW) adapted teleosts (Na + + K + )-ATPase FW SW 2.9
2.1 10.1 8.8 12.4 1.5
.____--l-12.6
(‘Z, 30
11.0 37.3 45.2 84.4 3.5 41.6 53.1 33.6
;; 25 37 26 25 2s 25
IS.6 20.5
30 30 25 25
11.0
9.4
_~
Reference ___._.
._____~
Pagliarani el rrl. (1991) McCormick er crl. (1989a) Sargent and Thomson (1974) Ho and Chan (1980) Epstein et al. (1967) Dhe~amba ef al. (1975) Kamiya and Utida (1969) Kamiya and Utida (1969) Kamiya and Utida (1969) Trigari er al. (1985) Ventrella er ul. (1990) Kamiya and Utida (1969) Kamiya and Utida (1969)
T, Assay temperature; nr, not reported.
the enzyme activity drives the spontaneous Na+ entry in the cell which in turn energizes the sjmultaneous uphill Cl- influx via a Na+/K’/2 Cl- cotransporter located in the same basolateral membrane. Once entered, Cl- diffuses out of the cell through specific CAMP-modulated channels in the apical crypt. The Na+ extruded by the (Na+ + K+ )-ATPase diffuses to the sea-water side through leaky junctions between adjacent chloride cells, driven by the transepithelial potential difference. In sea-water adapted fish, the sea-water compartment is negative with respect to the internal milieu (Zadunaisky, 1984). Significance of salt water adaptation in salmonids Among teleosts, Salmonidae comprise many euryhaline species, able to tolerate conditions of widely differing salinity. In anadromous species, euryhalinity is truly confined to limited portions of the life cycle, including the obligatory seaward migration of FWborn juveniles. This migration, according to the species, occurs within the first l-4 years and it is immediately preceded or accompanied by a series of morphological, behavioral, physiological and biochemical changes from the FW parr to the smolt prepared for the transition into SW (Folmar and Dickoff, 1980). Many studies have been focused on the search for biochemical or hormonal factors that may be used as predictive criteria for the seaward migration and subsequent survival in the SW milieu. The applied interest of these studies lies in the perspective of using these factors to trigger or anticipate the socalled smoltification in species that undergo this process, or at least to preadapt non-smolting FW-born fish to successful life in SW. The advantages of the precocious SW transfer of salmonid fish reared in FW, including the observed improved growth and food conversion (Usher et al., 1991) and the cheapness of the residence in SW (Higgs ez ai., 1982) are, however, ~ounterbalan~d by the high mortality produced by osmotic stress which especially hits juveniles (Jackson, 1981). In anadromous species, maximal survival is attained when SW transfer is performed during the smolting phase (Zaugg and Beckman, 1990). Modulation of gill (Na+ + KC)-ATPase activity during hyporegulation The increased gill (Nat + Kc >ATPase activity represents one of the most striking biochemi~l events during the smoltification (Folmar and DickofT, 1980).
The enzyme rise was long recognized as clearly essential for survival in hyperosmoti~ environments. Therefore, the factors that can stimulate the enzyme activity in FW have been considered potentially pre-adaptive to SW entry (Zaugg and Beckman, 1990). The processes involved in the acquirement of SW tolerance and in the modulation of gill (Na+ + K+)-ATPase activity during FW smoltification seem to be ruled by a variety of hormones (Folmar and Dickoff, 1980; Young et al., 1989a,b). As first pointed out by Hoar (1965) and currently accepted (Langhorne and Simpson, 1986), the endocrine system is believed to be involved in the control of the timing of the changes occurring during the smoltification, acting as a link between environmental fluctuations and physiological events. Though osmoregulatory mechanisms are known to be under endocrine control (Bisbal and Specker, 1991), so far the investigation of the possible relations between hormone levels, salinity adaptation and gill (Na+ + K+ )-ATPase increase has yielded contradictory results. They often appeared to be strictly dependent on the experimental timing, fish age and size, as well as on whether the species belonged to Salmonidae or not and, in the former case, on whether it smoltified or not. As serum levels of thyroid hormones, cortisol (Folmar and Dickoff, 1980; Langhorne and Simpson, 1986; Young et al., 1989b) and growth hormone (GH) (Bouef et al., 1989; Young et al., 1989a) peak during the Parr-smolt transfo~ation, which precedes SW entry, these hormones were considered to be directly involved in hypoosmoregulation. On the contrary, the overall decrease of prolactin after SW entry suggested that this hormone might mainly play a FW-adapting role (Young et al., 198917). Some hormones peaking during smoltification, e.g. thyroid hormones, have been extensively used to accelerate the acquirement of salinity tolerance in FW-adapted salmonids (Saunders et ai., 1985; Boeuf et al., 1989). One of the mechanisms involved in the known stimulation of mammalian (Nat f K’ )ATPase by thyroid hormones, namely the activation of the biosynthesis of new enzyme units via regulation of the induction and translation of mRNAs that code for u and /? subunits of the (Na+ + K+ )-ATPase (Ismail-Beigi, 1988), may act also on teleosts. In this case, (Na+ + K+ )-ATPase enhanced biosynthesis would be required by the chloride cell prolife~tion during SW adaptation (Madsen and Korsgaard,
+++ =
+
+++
t+t = +
s4
mykiss
Oncorhynchw
++
-k-I-
FW FW FW 4
myki.wb kisutch
Oncorhynchus O~corhyn~h~
3
FW
4
z FW and SW FW FW FW FW
2 FW FW FW and SW SW
FW FW FW FW and SW FW FW FW FW FW FW
4
5
3
4
4
4
4
3
4
4
3
3
5
5
4
4
4
4
5
4
4
4
2
4
E FW
(presmolt) Salmo trutta trutta (Parr) Salmo trutta(presmolt)
kisutch
Oncorhynehur
=
kinrlch
Oncorhynchus
(Parr)
myki.&’
Oncorhynchtu
tnrtta
S&w
f
trutta
(ma.)
mossumbicusC
ki&ich
Oreochromis
~ssa~i~usc
Oncorhy~ch~
mossombicusc
Oreochromis
Oreochromis
(presmolt)
(molt)
(n.a.)
(n.a.)
moss~bi~sc
kisutch
Oncorhynchuf Oreochromis
kinrtch
i”
-!-
WC
-k6
kisutch
mykiss
Oncorhynchus Onrorhynchtls
mykiss
Oncorhynchus
mykiss
I 3
FW
Reference Saunders et al. (19SS) Omeljaniuk and Eales (1986) Pagliarani et ul. (1990) McCormick et ai. (1989b) Madsen and Korsgaard (1989) DickofT et al. f 1977) Madsen et af. (1990b) Dan@ (1986) Langdon et al. (1984) Bisbal and Specker (1991) Madsen (199Od) Madsen (t99Ob) Madsen (1990a) Eib and Hossner (1985) Redding et al. (1984) Richman III and Zaugg (1987) McCormick et al, (1989b) McCormick (1990) Dangb (1986) Herndon et al. (1991) McCormick er al. {1989b) Madsen (199ob) Dang& ( 1986) Madsen (199M) Madsen ( 1990~) McCormick et al. (1989b) Richman 111 and Zaugg (1987) Madsen (199Od) Madsen and Korsgaard (1991) Madsen (I 99Oc) McCormick er al, (1989b)
En~n salmonid species; din opercular membranes from FW fish; ‘in SW fish. I Oral; 2 immersion in hormone containing water; 3 hormone dispersed in the in vitro epithelium culture medium; 4 intraperitoneal injection; 5 subcutaneous implants of hormone-containing pelt& or capsules. = Indicates no difference; - decrease; + increase; + + strong increase with respect to the control; f + + synergism between two hormones.
c c e + -tc f = + cc+ + +++ c
i-d + -t
+
+
+
=
zz
(n.a.)
@i-r)
Oncorhynchus
f
+
trutta
Oncorhynchus
trwtta
Mm0
+
I+
s&r
Satmo
+
@molt)
rno~gornbieus’
mykiss
Oreockromis
Oncorhynchus
+
es
=
(part) kisutch
salar
~~eorhyn~h~
Salmo
salar
=
+
kisutch
Oncorhynchus
2
mykissb mykissb
Oncorhynchus Oncorhynchus
I
Administration Route Phase
s&m s&r
Species
Salmo
s
=
se
f +
f
Chloride cells”
FW Fresh water; SW salt water; “Evaluated in FW fish gills, except when differently stated; boriginally named Salmo guirdneri;
Cortisol + GH
GH
ProIaetin Cork01 + T, Cotiisol + Td
cortisoi
T4
=
+
(Na* t K+)-ATPase FW SW ~________
Table 2. Effect of thyroid hormones, cortisoi. prolactin and growth hormone (GH) on gill (Na+ + K’ f-ATPase and chloride cell number in salmonids and other teleosts
Administered hormone -~ -~
640
A. R. BORGATTI et al.
1989). However, while there is general agreement on the thyroid hormone capability of increasing SW tolerance (Saunders et al., 1985), the stimulatory effect on gill (Na+ + K+)-ATPase is still controversial (Table 2). Accordingly, thyroid hormone administration to euryhaline teleosts during the FW phase was reported to be ineffective with regard to gill (Na+ + K+)-ATPase activity (Saunders et al., 1985; Dangt, 1986; Madsen, 1990b; Pagliarani et al., 1990), stimulatory (Dickoff et al., 1977; Madsen and Korsgaard, 1989) and even inhibitory (Omeljaniuk and Eales, 1986). When assayed after SW entry, the enzyme activity was shown to be unaltered with respect to the control (Dangit, 1986) or conversely stimulated (Pagliarani et al., 1990). These contradictory results seem hardly interpretable, since the studies reported often differed in the species, route of hormone administration, hormone doses and experimental design. Though many studies were carried out using thyroxine (T4), 3,5,3’-L-triiodothyronine (T,), which fish obtain from hepatic monodeiodination of T, (Eales et al., 1990), is considered as the active thyroid hormones in teleosts (Higgs et al.. 1982) as in higher vertebrates. As reported in our previous work (Pagliarani et al., 1990) and here illustrated in Fig. 1, the oral administration of T, to rainbow trout during FW residence did not affect gill (Na+ + K+)-ATPase activity, in spite of the quantitative increase of chloride cells, especially immature (M. Galeotti, personal communication). After transfer to an average 2% salinity and treatment suspension, T, serum level, previously raised by hormone administration, became quickly similar to the control; in the meanwhile, a dramatic enhancement of gill (Na+ + K+ )-ATPase activity,
u i-
Fresh
1
O’ 27 MARCH
Fig.
: salt
water
2’8 1
’
b
20
wateii I,
27
1
,;
dare
(IPIIL
Changes gill + )-ATPase (I), cell (II) Galeotti, communiand T, (III) rainbow fed containing ppm T, (T) or 10% NaCl (N) or untreated (C) during the freshwater phase. On 20 April, all trout were transferred to saltwater and the treatments suspended. Each point represents mean of at least three determinations.
parallel to a further prompt increase of chloride cell number, overwhelmed the expected salinity driven increase of enzyme activity shown by the untreated group. The administration of a hypersaline diet, mimicking the salt input typical of the SW habitat during the FW phase, to a parallel group, allowed us to enlighten a putative T, role during SW adaptation. In FW, the salt diet promoted a gradual synchronous increase of both the (Na+ + K+)-ATPase activity and chloride cell number. These parameters, both higher in the salt group than in the T, group during the FW residence, after transfer to brackish water further increased, though to a delayed and limited extent, in the salt group with respect to T,-treated group. The final number of chloride cells after 20 days in brackish water was apparently unaffected by the FW experimental treatments. On the contrary, in comparison to the control, gill (Na+ + K+ )-ATPase activity was strikingly enhanced by T, and also, though to a lower extent, by salt administration. Assuming that NaCl input triggers the activation of the hypo-osmoregulatory branchial apparatus deputed to NaCl extrusion, it seems reasonable to conceive that T, may play a preparatory role by activating the generation of immature chloride cells and/or the synthesis of mRNAs of (Nat + K+)ATPase units with a similar role to that played during vertebrate metamorphosis (Miwa and Inui, 1987). In this case, other events, e.g. the increased environmental salinity, may be required to switch on the prepared apparatus. On the other hand, Petronini et al. (1989) reported that intracellular Na+ concentration changes, probably similar to those occurring immediately after SW transfer, directly affect the rate of protein synthesis by acting on mRNA translation. A more direct effect on gill (Na+ + K+)-ATPase activity was then ascribed to cortisol (McCormick et al., 1989b), the major corticosteroid in salmonids (Folmar and Dickoff, 1980; Langhorne and Simpson, 1986). Cortisol, a primary stress hormone in vertebrates, has been considered as a hypo-osmoregulatory hormone in teleosts (Folmar and Dickoff, 1980; Langhorne and Simpson, 1986; Madsen, 1990a,b,c,d) as it was shown to promote ion excretion and water conservation in hyperosmotic media (Redding et al., 1984). The increased salt secretion via the gills probably occurs via direct stimulation of (Na+ + K+ )ATPase activity (McCormick et al., 1989b; Madsen, 1990b). As summarized in Table 2, in most cases after cortisol administration, an increased gill (Na+ + K+)-ATPase activity was observed. In FW, the enzyme activation was generally related to chloride cell proliferation, both in salmonids (Richman III and Zaugg, 1987; Madsen, 1990b,d) and in nonsalmonids (McCormick, 1990). However, as other studies on salmonids reported no effect of the hormone (Langdon et al., 1984; Eib and Hossner, 1985) or even gill (Nat + K+)-ATPase depression (Redding et al., 1984), the involvement of endogenous factors such as interspecific differences (Langdon et al., 1984), changes in the hormonal susceptivity during the life cycle and the interference of other hormones (Madsen, 1990a,b,c,d) were suggested. After SW entry, the cortisol treatment constantly produced higher gill (Na+ + K+)-ATPase activities with respect to untreated subjects, either when the
Gill (Na+ + K+)-ATPase and salmonid SW entry
hormone administration was confined to the FW phase (Madsen, 1990a) or performed throughout the experiment in both habitats (DangC, 1986). While cortisol was found to promote chloride cell differentiation, an opposite effect was ascribed to prolactin (Foskett et al., 1983). In SW-adapted tilapia, prolactin treatment did not affect gill (Na+ + K+)-ATPase activity, nor total chloride cell number whereas it decreased the average size of these cells, probably because the hormone prevented young chloride cells from growing and differentiating (Herndon et al., 1991). It is still unknown if prolactin acts similarly on salmonids. As prolactin has long been reported to antagonize SW adaptive mechanisms (Foskett et al., 1983), the hormone rise observed during salmonid smoltification, prior to gill (Na+ + K+)-ATPase and thyroid hormone peaks, was interpreted as a device to prevent FW ion loss consequent to the development of SW-adaptive mechanisms, whereas the subsequent decrease may be necessary to allow gill (Na+ + K+)-ATPase activity to increase (Young et al., 1989a). However, the inverse relationship generally observed between plasma prolactin and gill (Na+ + K+)-ATPase activity remains so far unexplained. The observed effects of cortisol and thyroid hormone treatments during the FW phase, as well as the asynchronism between T, and cortisol serum peaks respectively, found prior to (Folmar and Dickoff, 1980; Boeuf et al., 1989) and almost contemporary to (Langhorne and Simpson, 1986) the gill (Nat + K+ )-ATPase rise during salmonid smoltification, suggested the existence of a mutual relationship between cortisol and thyroid hormones during SW adaptation. A complementary role of these hormones in hypo-osmotic regulation was also indicated by the observed synergism of cortisol and T, on tilapia gill (Na+ + K+)-ATPase activity and chloride cell number. Therefore, while thyroid hormones promote an overall stimulation of protein synthesis, cortisol may act as a driving factor during the (Na+ + K+)ATPase synthesis (DangC, 1986). This mechanism is similar to that hypothetically ascribed in the present review to the salinity increase to explain the striking enhancement of gill (Na+ + K+)-ATPase activity observed in T,-pretreated trout after brackish water transfer (Pagliarani et al., 1990). On the other hand, the failed synergism of cortisol and thyroid hormones in salmonid species (McCormick et al., 1989b; Madsen, 1990b) hints that different euryhaline teleosts may differently regulate the osmoregulatory processes. More recently, GH, another hormone peaking during salmonid smoltification (Folmar and Dickoff, 1980; Boeuf et al., 1989), was implicated in SW adapatation. However, in this case, studies carried out by administering exogenous hormone to FWadapted fish resulted into controversial data (Table 2). In the trout gill (Na+ + K+)-ATPase activity and chloride cell proliferation were shown to be stimulated by GH with a similar efficiency to cortisol and, in certain cases, an even higher efficiency (Madsen, 1990d). The similarity of effects of the two hormones, as well as the demonstration of a regulatory role of GH on salmonid inter-renal (Young, 1989b), are consistent with the hypothesis that the
641
GH effect is mediated by the stimulation of cortisol release by inter-renal. On the other hand, the more powerful effect on (Na+ + K+)-ATPase activity and chloride cell number produced in the same species by a combination of GH and cortisol with respect to the individual administration of the two hormones (Madsen, 1990c,d), suggested that cortisol and GH may act on the same mechanisms in uiuo. In coho salmon, however, GH stimulated gill (Na+ + K+)ATPase activity, but was powerless on chloride cell number (Richman III and Zaugg, 1987). Moreover, in vitro studies carried out on the branchial epithelium from the same species thoroughout the course of Parr-smolt transformation showed no significant effect on gill (Na+ + K+)-ATPase activity of GH, either alone or in combination with cortisol, in spite of the stimulation exerted by the latter alone on the enzyme activity (McCormick et al., 1989b). Therefore, the observed in vivo GH effects appear to be at least partially mediated by the endocrine system. However, as stressed by Madsen (1990b), the involvement of thyroid gland on the observed GH effects seems to be excluded, in spite of the thyreotropic nature in teleosts of mammalian GH used in most studies. Finally, the different effect (Table 2) of a combination of GH and cortisol, administered under similar experimental conditions, to the same species but in different stages of life cycle (Madsen, 1990d; Madsen and Korsgaard, 1991), stresses the involvement of the endogenous hormonal status on the response to hormonal treatments. CONCLUSION
The analysis of the effects produced by thyroid hormones cortisol, prolactin and GH, namely the hormones mainly implicated in teleost osmoregulatory mechanisms, on gill (Na+ + K+)-ATPase activity points out contradictory results. The data reported often appear to be related to a wide variety of elements. Therefore, as first stressed by Folmar and Dickoff (1980), the events that accompany gill (Na+ + K+ )-ATPase rise during salmonid smoltification and generally SW adaptation of euryhaline teleosts appear to depend on a variety of hormones whose roles and mutual relationships so far remain partly obscured, despite considerable literature on the subject. The existence of a complex inter-relationship between these hormones must be taken into account whenever the possibility of using hormonal treatments to pre-adapt FW-born species to SW is considered. REFERENCES
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642
A. R. BOAGATT1
De Renzis G. and Bornancin M. (1984) Ion transport and gill ATPases. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. X, Part B, pp. 65-104. Academic Press, London. Dharmamba M., Bornancin M. and Maetz J. (1975) Environmental salinity and sodium and chloride exchanges across the gills of Tilapia mossamhica. J. Physiol. 70, 627436. Dickoff W. W., Folmar L. and Gorbman A. (1977) Relationship of thyroxine and gill Na + + K+ adenosine triphosphatase (ATPase) in coho salmon Oncorhynchus kisutch. Am. 2001. 17, 857. Eales J. G., Higgs D. A., Uin L. M.. MacLatchy D. L., Bres O., McBride J. R. and Dosanjh B. S. (1990) Influence of dietary lipid and carbohydrate levels and chronic 3,5,3’triiodo-L-thyronine treatment on thyroid function in immature rainbow trout Oncorhynchus mykiss. Gen. camp. Endocr. 80, 146--154. Eib D. W. and Hossner K. L. (1985) The effects of cortisol and actinomycin D injections on chloride cells and branchial Na+ + K+-ATPase in rainbow trout (Salmo gairdneri). Gen. romp. Endow. 59, 449-452. Epstein F. H., Katz A. J. and Pickford G. E. (1967) Sodium and potassium-activated adenosine triphosphatase of gill: role in adaptation of teleosts to saltwater. Science 156, 1245-1247. Evans D. H. (1979) Fish. In Comparatice Physiology) of Osmoregulation in Animals (Edited by Maloiy G. M. O.), Vol. 1, pp. 305-390. Academic Press, London. Folmar L. C. and Dickoff W. (1980) The parrsmolt transformation (smoltification) and sea-water adaptation in salmonids. A review of selected literature. Aquaculture 21, l-37. Foskett J. K., Bern H. A., Machen T. E. and Conner M. (1983) Chloride cells and the hormonal control of teleost fish osmoregulation. J. exp. Eiol. 106, 255-28 I. Herndon T. M., McCormick S. and Bern H. A. (1991) Effects of prolactin on chloride cells in opercular membrane from seawater-adapted tilapia. Gen. camp. Endocr. 83, 283-289. Higgs D. A., Fagerlund U. H. M., Eales J. G. and McBride J. R. (1982) Application of thyroid and steroid hormones as anabolic agents in fish culture. Camp. Biochem. Physiol. 73B, 143-176. Ho S. M. and Chan D. K. 0. (1980) Branchial ATPases and ionic transport in the eel Anguilla japonica. 1. Na+ + K+ATPase. Comp. Biochem. Physiol. 66B, 255-260. Hoar W. S. (1965) The endocrine system as a chemical link between the organism and its environment. Trans. R. Sot. Can. 3, 1755200. Ismail-Beigi F. (1988) Thyroid thermogenesis: regulation of (Na+ + K+)-adenosine triphosphatase and active Na, K transport. Am. 2001. 28, 363-371. Jackson A. J. (1981) Osmotic regulation in rainbow trout (Salmo gairdneri) following transfer to sea-water. Aquaculture 24, 143-151. Kamiya M. and Utida S. (1969) Sodium-potassiumactivated adenosine triphosphatase activity in gills of freshwater, marine and euryhahne teleosts. Camp. Biochem. Physiol. 31, 671674. Karnaky Jr K. J. (1986) Structure and function of the chloride cell of Fundulus heteroclifus and other teleosts. Am. Zool. 26, 209-224. Langdon J. S., Thorpe J. E. and Roberts R. J. (1984) Effects of cortisol and ACTH on gill Na+ + K+-ATPase in juvenile Atlantic salmon Salmo salar. Camp. Biochem. Physiol. VA, 9-12. Langhorne P. and Simpson T. H. (1986) The interrelationship of cortisol, gill (Na+ + K+ )-ATPase and homeostasis during the Parr-smolt transformation of Atlantic salmon (Salmo salar L.). Gen. camp. Endocr. 61,203-213. Madsen S. S. (1990a) Cortisol treatment improves the development of hypoosmoregulatory mechanism in the
et ai.
euryhaline rainbow trout Salmo gairdneri. Fish Physiol. Biochem. 8, 45-52. Madsen S. S. (1990b) Effect of repetitive cortisol and thyroxine injections on chloride cell number and Na+/K+-ATPase activity in gills of freshwater acclimated rainbow trout Salmo gairdneri. Comp. Biochem. Physiol. 95A, 171-175. Madsen S. S. (1990~) Enhanced hypoosmoregulatory response to growth hormone after cortisol treatment in immature rainbow trout Salmo gairdneri. Fish Physiol. Biochem. 8, 271-279. Madsen S. S., (1990d) The role of cortisol and growth hormone in seawater adaptation and development of hypoosmoregulatory mechanisms in sea trout parr (Salmo trutta trurta). Gen. camp. Endocr. 79, I- 11. Madsen S. S. and Korsgaard B. (1989) Time course of repetitive oestradiol- 178 and thyroxine injections on the natural spring smolting of Atlantic salmon. Salmo salar L. J. Fish Biol. 35, 119.-128. Madsen S. S. and Korsgaard B. (1991) Opposite effects of l7b-oestradiol and combined growth hormone-cortisol treatment on hypo-osmoregulatory performance in sea trout presmolts Salmo trutta. Gen. camp. Endow. 83, 276-282. McCormick S. D. (1990) Cortisol directly stimulates differentiation of chloride cells in tilapia opercular membrane. Am. J. Physiol. 259, R857-R863. _ McCormick S. D.. Moves C. D. and Ballantvne J. S. (1989a) Influence of salinity-on the energetics of gill and ‘kidney of Atlantic salmon (Salmo salar j. Fish Physial. Biochem. 6, 253-254. McCormick S. D., Nishioka R. S. and Bern H. A. (1989b) In tiitro hormonal stimulation of Na+ + KC-ATPase activity and ouabain binding in gill tissue of coho salmon. Aquaculture 82, 379. Miwa S. and Inui Y. (1987) Effect of various doses of thyroxine and triiodothyronine on the methamorphosis of flounder (Paralichtys olipaceus). Gen. camp. Endocr. 67, 356-363. Omeljaniuk R. J. and Eales J. G. (1986) The effect of 3,5,3’-triiodo-L-thyronine on gill Na+ + K+-ATPase of rainbow trout Salmo gairdneri in fresh water. romp. Biochem. Ph ysiol. &IA, 427429. Pagharani A., Ventrella V., Ballestrazzi R., Trombetti F., Pirini M. and Triaari G. (1991) Salinitv denendence of the properties of gill(Na+ + K’ j-ATPase in* rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. MOB, 229-236. Pagliarani A, Ventrella V., Trombetti F., Ballestrazzi R.. Trigari G. and Borgatti A. R. (1990) Response of gill (Na+ + K+ )-ATPase to oral administration of thyroid hormone (3,5,3’ triiodo-L-thyronine) and NaCl as a tool of adaptation of freshwater rainbow trout to seawater. It. J. Biochem. 39, 139A-l41A. Payan P., Girard J. P. and Mayer-Gostan N. (1984) Branchial ion movements in teleosts. The roles of respiratory and chloride cells. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. X, Part B, pp. 39-64. Academic Press, London. Petronini P. G., Tramacere M. R., Mazzini A., Kay J. E. and Borghetti A. F. (1989) Control of protein synthesis by extracellular Na+ in cultured fibroblasts. J. Cell. Physiol. 140, 202-21 I. Redding J. M., Schreck C. B., Birks E. K. and Ewing R. D. (1984) Cortisol and its effects on plasma thyroid hormone and electrolyte concentrations in freshwater and during in yearling coho salmon seawater acclimation Oncorhynchus kisutch. Gen. camp. Endocr. 65, 189-198. Richman III N. H. and Zaugg W. S. (1987) Effects of cortisol and growth hormone on osmoregulation in preand desmoltified coho salmon (Oncorhynchus kisutch). Gen. camp. Endocr. 65, 189-198. Sargent J. R. and Thompson A. J. (1974) The nature and
Gill (Na + + K+ )-ATPase and salmonid SW entry properties of the inducible sodium-plus-potassium iondependent adenosine triphosphatase in the gills of eels (Anguiila an~j~~a) adapted to freshwater and sea-water. Biochem. J. 144, 69-75.
Saunders R. L., McCormick S. D., Henderson E. B., Eales J. G. and Johnston C. E. (1985) The effect of orally administered 3,5,3’-triiodo-t_-thyronine on growth and salinity tolerance of Atlantic salmon (Salmo salar L.). Aquaculture 45, 143-l 56.
Silva P., Solomon R., Spokes K. and Epstein F. H. (1977) Ouabain inhibition of gill NaK-ATPase: relations to active chloride transport, J. exp. Zoo/. 199, 419-426. Skou J. C. (1988) Overview: the Na, K-pump. method Enzymot. 156, I-25. Smith H. (1930) The absorption and excretion of water and salts by marine teleosts. Am. J. Physiol. 93, 480-505. Trigari G., Borgatti A. R., Pagliarani A. and Ventrella V. (1985) Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.). Camp. Biochem. Physiol. SOB, 23-33.
Usher M. L., Talbot C. and Eddy F. B. (1991) Effects of transfer to seawater on growth and feeding in Atlantic salmon smolts (Salmo salar L.). Aquacuiture 94,309-326. Ventrella V., Trombetti F., Pagliarani A., Trigari G. and
643
Borgatti A. R. (1990) Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.). Camp. 3~oehem. Physiol. %B, 95-105.
Young G., Bjornsson B. Th., Prunet P., Lin R. J. and Bern H. A. (1989a) Plasma levels of prolactin and growth hormone during Parr-smelt transformation of coho salmon Oncorhynchus kisutch. Aquaculture 82, 383.
Young G., Bjiimsson B. Th., Prunet P., Lin R. J. and Bern H. A. (1989b) Smoitification and sea-water adaptation in coho salmon (Oncorhyn~h~ kisutch): plasma proiactin, growth hormone, thyroid hormones and cortisol. Gen. camp. Endocr. 74, 335-345.
Zadunaisky J. A. (1984) The chloride cell: the active transport of chloride and the paracellular pathways. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. X, Part B, pp. 130-176. Academic Press, London. Zaugg W. S. and Beckman B. R. (1990) Saltwater-induced decreases in weight and length relative to seasonal gill changes in Na+ + K+-ATPase coho salmon (Oneorhynch~ kisutch): a test for saltwater adaptability. Aqua~uiiure 86, 19-23.
Camp. Biochem. Physiol.
No. 4, pp. 637-643,
0300-9629/92 $5.00 + 0.00
1992
0 1992Pergamon Press Ltd
Printed in Great Britain
MINI REVIEW
GILL (Na+ + K+)-ATPase INVOLVEMENT AND REGULATION DURING SALMONID ADAPTATION SALT WATER
TO
A. R. BORGATTI,A. PAGLIARANI and V. VENTRELLA Dipartimento di Biochimica “G. Moruzzi”-Universita di Bologna, Via Belmeloro, I/2-Bologna, Tel.: 051-243019; Fax: 051-243053 (Received
Italy.
16 January 1992)
Abstract-l. The involvement of gill (Na+ + K+ )-ATPase in salmonid adaptation to salt water (SW) is discussed. 2. Gill (Na+ + K+)-ATPase increase during SW adaptation is mainly related to the increased number and complexity of chloride cells deputed to salt extrusion. 3. The temporal relationships between serum peaks of thyroid hormones, cortisol, growth hormone, prolactin and gill (Na+ + K+ )-ATPase rise during salmonid smoltification, suggest a hormonal involvement in the enzyme stimulation and thus in the acquirement of SW tolerance. 4. Literature on gill (Na+ + K+ )-ATPase response to hormonal treatments is reviewed. The effects produced on gill (Na+ + K+ )-ATPase and chloride cells by exogenous hormones point out a complex inter-relationship between the hormones considered. The mechanisms involved in hormonal regulation of the enzyme remain a matter of debate
chemical mechanisms so far hypothesized for both the NaCl uptake in FW and the NaCl extrusion in SW via the gills are based on (Nat + K+)-ATPase (De Renzis and Bomancin, 1984; Payan et al., 1984; Zadunaisky, 1984).
INTRODUCfION Teleosts cope with external osmotic pressures ranging
from cu 0 mOsm/l in fresh water (FW) to as much as to 1100 mOsm/l and even more in salt water (SW). Nevertheless, each species is able to maintain the osmolality of body fluids to a quite constant value, generally between 250 and 450 mOsm/l. FW species, whose osmolality is around 25&300 mOsm/l are hy perosmotic with respect to the environment, whereas SW species exhibit osmolalities of ca 400 mOsm/l and are hypoosmotic (Evans, 1979). By 1930 Smith had stressed the importance of branchial epithelium in teleost osmoregulation (Smith, 1930). In FW teleosts the gills are the site of active NaCl absorption compensating for outward diffusion, whereas in SW teleosts the gills actively excrete the salt absorbed through the gut (Payan et al., 1984). Gill epithelium exhibits distinct morphological features related to the habitat. The differences are mainly shown by the so-called chloride cells localized on the primary lamellae (De Renzis and Bornancin, 1984; Zadunaisky, 1984). These cells are small and isolated in FW fish, whilst they are large, abundant, ~tochond~a-~ch and clustered in grapes of at least two cells sharing a common apical crypt in SW fish. In the latter case, adjacent chloride cells are joined by ion-permeable junctions and the basolateral membrane, where the (Na+ + K+)-ATPase is typically localized, is amplified by many infoldings that form a densely branched network of tubules. These structural changes of the chloride cell complex are well known to occur when an euryhaline species is transferred from FW to SW (Karnaky, 1986). The bio-
(Na+ + K+ )-A TPase and NaCi extrusion in salt water fNa+ + K + )-ATPase, an almost ubiquitous enzyme in the animal world, plays a key role in Na+ transport and serves a variety of essential functions. The enzyme complex, basolaterally localized in oriented cells, spans the plasmalemma and acts as a cation pump. The complex consists of a and B subunits whose association is reputed essential for the catalytic activity. The energy obtained from the hydrolysis of 1 ATP is utilized for the uphill transport of 3 Na+ outwards and 2 K+ inwards, thus maintaining a low intra~llular concentration of Nat. These ion movements contribute to the onset of a negative intracellular transmembrane potential (Skou, 1988). During SW adaptation, an overall increase of gill (Nat + K+ )-ATPase is generally observed (De Renzis and Bornancin, 1984) both in salmonids and in non-salmonids (Table 1). On the other hand, SW fish are reported to exhibit higher gill (Na+ + K+ )-ATPase activities with respect to FW ones (Table 1). Such ~ha~our is related to gill mo~hological adaptations and especially to the proliferation of the chloride cells, as well as to the simultaneous enlargement of basolateral membranes enriched in enzyme units (Zadunaisky, 1984). According to the model originally brought about by Silva et al. (1977) and still substantially accepted with some modifications (Zadunaisky, 1984; Karnaky, 1986), in the chloride cells of SW teleosts, the Nat gradient generated by
637
638
A. R. BORGATTI er
al.
Table 1. Gill (Na+ + K+)-ATPase activities @mol P,/mg protein/hr) in freshwater- (FW) and saltwater(SW) adapted teleosts (Na + + K + )-ATPase FW SW 2.9
2.1 10.1 8.8 12.4 1.5
.____--l-12.6
(‘Z, 30
11.0 37.3 45.2 84.4 3.5 41.6 53.1 33.6
;; 25 37 26 25 2s 25
IS.6 20.5
30 30 25 25
11.0
9.4
_~
Reference ___._.
._____~
Pagliarani el rrl. (1991) McCormick er crl. (1989a) Sargent and Thomson (1974) Ho and Chan (1980) Epstein et al. (1967) Dhe~amba ef al. (1975) Kamiya and Utida (1969) Kamiya and Utida (1969) Kamiya and Utida (1969) Trigari er al. (1985) Ventrella er ul. (1990) Kamiya and Utida (1969) Kamiya and Utida (1969)
T, Assay temperature; nr, not reported.
the enzyme activity drives the spontaneous Na+ entry in the cell which in turn energizes the sjmultaneous uphill Cl- influx via a Na+/K’/2 Cl- cotransporter located in the same basolateral membrane. Once entered, Cl- diffuses out of the cell through specific CAMP-modulated channels in the apical crypt. The Na+ extruded by the (Na+ + K+ )-ATPase diffuses to the sea-water side through leaky junctions between adjacent chloride cells, driven by the transepithelial potential difference. In sea-water adapted fish, the sea-water compartment is negative with respect to the internal milieu (Zadunaisky, 1984). Significance of salt water adaptation in salmonids Among teleosts, Salmonidae comprise many euryhaline species, able to tolerate conditions of widely differing salinity. In anadromous species, euryhalinity is truly confined to limited portions of the life cycle, including the obligatory seaward migration of FWborn juveniles. This migration, according to the species, occurs within the first l-4 years and it is immediately preceded or accompanied by a series of morphological, behavioral, physiological and biochemical changes from the FW parr to the smolt prepared for the transition into SW (Folmar and Dickoff, 1980). Many studies have been focused on the search for biochemical or hormonal factors that may be used as predictive criteria for the seaward migration and subsequent survival in the SW milieu. The applied interest of these studies lies in the perspective of using these factors to trigger or anticipate the socalled smoltification in species that undergo this process, or at least to preadapt non-smolting FW-born fish to successful life in SW. The advantages of the precocious SW transfer of salmonid fish reared in FW, including the observed improved growth and food conversion (Usher et al., 1991) and the cheapness of the residence in SW (Higgs ez ai., 1982) are, however, ~ounterbalan~d by the high mortality produced by osmotic stress which especially hits juveniles (Jackson, 1981). In anadromous species, maximal survival is attained when SW transfer is performed during the smolting phase (Zaugg and Beckman, 1990). Modulation of gill (Na+ + KC)-ATPase activity during hyporegulation The increased gill (Nat + Kc >ATPase activity represents one of the most striking biochemi~l events during the smoltification (Folmar and DickofT, 1980).
The enzyme rise was long recognized as clearly essential for survival in hyperosmoti~ environments. Therefore, the factors that can stimulate the enzyme activity in FW have been considered potentially pre-adaptive to SW entry (Zaugg and Beckman, 1990). The processes involved in the acquirement of SW tolerance and in the modulation of gill (Na+ + K+)-ATPase activity during FW smoltification seem to be ruled by a variety of hormones (Folmar and Dickoff, 1980; Young et al., 1989a,b). As first pointed out by Hoar (1965) and currently accepted (Langhorne and Simpson, 1986), the endocrine system is believed to be involved in the control of the timing of the changes occurring during the smoltification, acting as a link between environmental fluctuations and physiological events. Though osmoregulatory mechanisms are known to be under endocrine control (Bisbal and Specker, 1991), so far the investigation of the possible relations between hormone levels, salinity adaptation and gill (Na+ + K+ )-ATPase increase has yielded contradictory results. They often appeared to be strictly dependent on the experimental timing, fish age and size, as well as on whether the species belonged to Salmonidae or not and, in the former case, on whether it smoltified or not. As serum levels of thyroid hormones, cortisol (Folmar and Dickoff, 1980; Langhorne and Simpson, 1986; Young et al., 1989b) and growth hormone (GH) (Bouef et al., 1989; Young et al., 1989a) peak during the Parr-smolt transfo~ation, which precedes SW entry, these hormones were considered to be directly involved in hypoosmoregulation. On the contrary, the overall decrease of prolactin after SW entry suggested that this hormone might mainly play a FW-adapting role (Young et al., 198917). Some hormones peaking during smoltification, e.g. thyroid hormones, have been extensively used to accelerate the acquirement of salinity tolerance in FW-adapted salmonids (Saunders et ai., 1985; Boeuf et al., 1989). One of the mechanisms involved in the known stimulation of mammalian (Nat f K’ )ATPase by thyroid hormones, namely the activation of the biosynthesis of new enzyme units via regulation of the induction and translation of mRNAs that code for u and /? subunits of the (Na+ + K+ )-ATPase (Ismail-Beigi, 1988), may act also on teleosts. In this case, (Na+ + K+ )-ATPase enhanced biosynthesis would be required by the chloride cell prolife~tion during SW adaptation (Madsen and Korsgaard,
+++ =
+
+++
t+t = +
s4
mykiss
Oncorhynchw
++
-k-I-
FW FW FW 4
myki.wb kisutch
Oncorhynchus O~corhyn~h~
3
FW
4
z FW and SW FW FW FW FW
2 FW FW FW and SW SW
FW FW FW FW and SW FW FW FW FW FW FW
4
5
3
4
4
4
4
3
4
4
3
3
5
5
4
4
4
4
5
4
4
4
2
4
E FW
(presmolt) Salmo trutta trutta (Parr) Salmo trutta(presmolt)
kisutch
Oncorhynehur
=
kinrlch
Oncorhynchus
(Parr)
myki.&’
Oncorhynchtu
tnrtta
S&w
f
trutta
(ma.)
mossumbicusC
ki&ich
Oreochromis
~ssa~i~usc
Oncorhy~ch~
mossombicusc
Oreochromis
Oreochromis
(presmolt)
(molt)
(n.a.)
(n.a.)
moss~bi~sc
kisutch
Oncorhynchuf Oreochromis
kinrtch
i”
-!-
WC
-k6
kisutch
mykiss
Oncorhynchus Onrorhynchtls
mykiss
Oncorhynchus
mykiss
I 3
FW
Reference Saunders et al. (19SS) Omeljaniuk and Eales (1986) Pagliarani et ul. (1990) McCormick et ai. (1989b) Madsen and Korsgaard (1989) DickofT et al. f 1977) Madsen et af. (1990b) Dan@ (1986) Langdon et al. (1984) Bisbal and Specker (1991) Madsen (199Od) Madsen (t99Ob) Madsen (1990a) Eib and Hossner (1985) Redding et al. (1984) Richman III and Zaugg (1987) McCormick et al, (1989b) McCormick (1990) Dangb (1986) Herndon et al. (1991) McCormick er al. {1989b) Madsen (199ob) Dang& ( 1986) Madsen (199M) Madsen ( 1990~) McCormick et al. (1989b) Richman 111 and Zaugg (1987) Madsen (199Od) Madsen and Korsgaard (1991) Madsen (I 99Oc) McCormick er al, (1989b)
En~n salmonid species; din opercular membranes from FW fish; ‘in SW fish. I Oral; 2 immersion in hormone containing water; 3 hormone dispersed in the in vitro epithelium culture medium; 4 intraperitoneal injection; 5 subcutaneous implants of hormone-containing pelt& or capsules. = Indicates no difference; - decrease; + increase; + + strong increase with respect to the control; f + + synergism between two hormones.
c c e + -tc f = + cc+ + +++ c
i-d + -t
+
+
+
=
zz
(n.a.)
@i-r)
Oncorhynchus
f
+
trutta
Oncorhynchus
trwtta
Mm0
+
I+
s&r
Satmo
+
@molt)
rno~gornbieus’
mykiss
Oreockromis
Oncorhynchus
+
es
=
(part) kisutch
salar
~~eorhyn~h~
Salmo
salar
=
+
kisutch
Oncorhynchus
2
mykissb mykissb
Oncorhynchus Oncorhynchus
I
Administration Route Phase
s&m s&r
Species
Salmo
s
=
se
f +
f
Chloride cells”
FW Fresh water; SW salt water; “Evaluated in FW fish gills, except when differently stated; boriginally named Salmo guirdneri;
Cortisol + GH
GH
ProIaetin Cork01 + T, Cotiisol + Td
cortisoi
T4
=
+
(Na* t K+)-ATPase FW SW ~________
Table 2. Effect of thyroid hormones, cortisoi. prolactin and growth hormone (GH) on gill (Na+ + K’ f-ATPase and chloride cell number in salmonids and other teleosts
Administered hormone -~ -~
640
A. R. BORGATTI et al.
1989). However, while there is general agreement on the thyroid hormone capability of increasing SW tolerance (Saunders et al., 1985), the stimulatory effect on gill (Na+ + K+)-ATPase is still controversial (Table 2). Accordingly, thyroid hormone administration to euryhaline teleosts during the FW phase was reported to be ineffective with regard to gill (Na+ + K+)-ATPase activity (Saunders et al., 1985; Dangt, 1986; Madsen, 1990b; Pagliarani et al., 1990), stimulatory (Dickoff et al., 1977; Madsen and Korsgaard, 1989) and even inhibitory (Omeljaniuk and Eales, 1986). When assayed after SW entry, the enzyme activity was shown to be unaltered with respect to the control (Dangit, 1986) or conversely stimulated (Pagliarani et al., 1990). These contradictory results seem hardly interpretable, since the studies reported often differed in the species, route of hormone administration, hormone doses and experimental design. Though many studies were carried out using thyroxine (T4), 3,5,3’-L-triiodothyronine (T,), which fish obtain from hepatic monodeiodination of T, (Eales et al., 1990), is considered as the active thyroid hormones in teleosts (Higgs et al.. 1982) as in higher vertebrates. As reported in our previous work (Pagliarani et al., 1990) and here illustrated in Fig. 1, the oral administration of T, to rainbow trout during FW residence did not affect gill (Na+ + K+)-ATPase activity, in spite of the quantitative increase of chloride cells, especially immature (M. Galeotti, personal communication). After transfer to an average 2% salinity and treatment suspension, T, serum level, previously raised by hormone administration, became quickly similar to the control; in the meanwhile, a dramatic enhancement of gill (Na+ + K+ )-ATPase activity,
u i-
Fresh
1
O’ 27 MARCH
Fig.
: salt
water
2’8 1
’
b
20
wateii I,
27
1
,;
dare
(IPIIL
Changes gill + )-ATPase (I), cell (II) Galeotti, communiand T, (III) rainbow fed containing ppm T, (T) or 10% NaCl (N) or untreated (C) during the freshwater phase. On 20 April, all trout were transferred to saltwater and the treatments suspended. Each point represents mean of at least three determinations.
parallel to a further prompt increase of chloride cell number, overwhelmed the expected salinity driven increase of enzyme activity shown by the untreated group. The administration of a hypersaline diet, mimicking the salt input typical of the SW habitat during the FW phase, to a parallel group, allowed us to enlighten a putative T, role during SW adaptation. In FW, the salt diet promoted a gradual synchronous increase of both the (Na+ + K+)-ATPase activity and chloride cell number. These parameters, both higher in the salt group than in the T, group during the FW residence, after transfer to brackish water further increased, though to a delayed and limited extent, in the salt group with respect to T,-treated group. The final number of chloride cells after 20 days in brackish water was apparently unaffected by the FW experimental treatments. On the contrary, in comparison to the control, gill (Na+ + K+ )-ATPase activity was strikingly enhanced by T, and also, though to a lower extent, by salt administration. Assuming that NaCl input triggers the activation of the hypo-osmoregulatory branchial apparatus deputed to NaCl extrusion, it seems reasonable to conceive that T, may play a preparatory role by activating the generation of immature chloride cells and/or the synthesis of mRNAs of (Nat + K+)ATPase units with a similar role to that played during vertebrate metamorphosis (Miwa and Inui, 1987). In this case, other events, e.g. the increased environmental salinity, may be required to switch on the prepared apparatus. On the other hand, Petronini et al. (1989) reported that intracellular Na+ concentration changes, probably similar to those occurring immediately after SW transfer, directly affect the rate of protein synthesis by acting on mRNA translation. A more direct effect on gill (Na+ + K+)-ATPase activity was then ascribed to cortisol (McCormick et al., 1989b), the major corticosteroid in salmonids (Folmar and Dickoff, 1980; Langhorne and Simpson, 1986). Cortisol, a primary stress hormone in vertebrates, has been considered as a hypo-osmoregulatory hormone in teleosts (Folmar and Dickoff, 1980; Langhorne and Simpson, 1986; Madsen, 1990a,b,c,d) as it was shown to promote ion excretion and water conservation in hyperosmotic media (Redding et al., 1984). The increased salt secretion via the gills probably occurs via direct stimulation of (Na+ + K+ )ATPase activity (McCormick et al., 1989b; Madsen, 1990b). As summarized in Table 2, in most cases after cortisol administration, an increased gill (Na+ + K+)-ATPase activity was observed. In FW, the enzyme activation was generally related to chloride cell proliferation, both in salmonids (Richman III and Zaugg, 1987; Madsen, 1990b,d) and in nonsalmonids (McCormick, 1990). However, as other studies on salmonids reported no effect of the hormone (Langdon et al., 1984; Eib and Hossner, 1985) or even gill (Nat + K+)-ATPase depression (Redding et al., 1984), the involvement of endogenous factors such as interspecific differences (Langdon et al., 1984), changes in the hormonal susceptivity during the life cycle and the interference of other hormones (Madsen, 1990a,b,c,d) were suggested. After SW entry, the cortisol treatment constantly produced higher gill (Na+ + K+)-ATPase activities with respect to untreated subjects, either when the
Gill (Na+ + K+)-ATPase and salmonid SW entry
hormone administration was confined to the FW phase (Madsen, 1990a) or performed throughout the experiment in both habitats (DangC, 1986). While cortisol was found to promote chloride cell differentiation, an opposite effect was ascribed to prolactin (Foskett et al., 1983). In SW-adapted tilapia, prolactin treatment did not affect gill (Na+ + K+)-ATPase activity, nor total chloride cell number whereas it decreased the average size of these cells, probably because the hormone prevented young chloride cells from growing and differentiating (Herndon et al., 1991). It is still unknown if prolactin acts similarly on salmonids. As prolactin has long been reported to antagonize SW adaptive mechanisms (Foskett et al., 1983), the hormone rise observed during salmonid smoltification, prior to gill (Na+ + K+)-ATPase and thyroid hormone peaks, was interpreted as a device to prevent FW ion loss consequent to the development of SW-adaptive mechanisms, whereas the subsequent decrease may be necessary to allow gill (Na+ + K+)-ATPase activity to increase (Young et al., 1989a). However, the inverse relationship generally observed between plasma prolactin and gill (Na+ + K+)-ATPase activity remains so far unexplained. The observed effects of cortisol and thyroid hormone treatments during the FW phase, as well as the asynchronism between T, and cortisol serum peaks respectively, found prior to (Folmar and Dickoff, 1980; Boeuf et al., 1989) and almost contemporary to (Langhorne and Simpson, 1986) the gill (Nat + K+ )-ATPase rise during salmonid smoltification, suggested the existence of a mutual relationship between cortisol and thyroid hormones during SW adaptation. A complementary role of these hormones in hypo-osmotic regulation was also indicated by the observed synergism of cortisol and T, on tilapia gill (Na+ + K+)-ATPase activity and chloride cell number. Therefore, while thyroid hormones promote an overall stimulation of protein synthesis, cortisol may act as a driving factor during the (Na+ + K+)ATPase synthesis (DangC, 1986). This mechanism is similar to that hypothetically ascribed in the present review to the salinity increase to explain the striking enhancement of gill (Na+ + K+)-ATPase activity observed in T,-pretreated trout after brackish water transfer (Pagliarani et al., 1990). On the other hand, the failed synergism of cortisol and thyroid hormones in salmonid species (McCormick et al., 1989b; Madsen, 1990b) hints that different euryhaline teleosts may differently regulate the osmoregulatory processes. More recently, GH, another hormone peaking during salmonid smoltification (Folmar and Dickoff, 1980; Boeuf et al., 1989), was implicated in SW adapatation. However, in this case, studies carried out by administering exogenous hormone to FWadapted fish resulted into controversial data (Table 2). In the trout gill (Na+ + K+)-ATPase activity and chloride cell proliferation were shown to be stimulated by GH with a similar efficiency to cortisol and, in certain cases, an even higher efficiency (Madsen, 1990d). The similarity of effects of the two hormones, as well as the demonstration of a regulatory role of GH on salmonid inter-renal (Young, 1989b), are consistent with the hypothesis that the
641
GH effect is mediated by the stimulation of cortisol release by inter-renal. On the other hand, the more powerful effect on (Na+ + K+)-ATPase activity and chloride cell number produced in the same species by a combination of GH and cortisol with respect to the individual administration of the two hormones (Madsen, 1990c,d), suggested that cortisol and GH may act on the same mechanisms in uiuo. In coho salmon, however, GH stimulated gill (Na+ + K+)ATPase activity, but was powerless on chloride cell number (Richman III and Zaugg, 1987). Moreover, in vitro studies carried out on the branchial epithelium from the same species thoroughout the course of Parr-smolt transformation showed no significant effect on gill (Na+ + K+)-ATPase activity of GH, either alone or in combination with cortisol, in spite of the stimulation exerted by the latter alone on the enzyme activity (McCormick et al., 1989b). Therefore, the observed in vivo GH effects appear to be at least partially mediated by the endocrine system. However, as stressed by Madsen (1990b), the involvement of thyroid gland on the observed GH effects seems to be excluded, in spite of the thyreotropic nature in teleosts of mammalian GH used in most studies. Finally, the different effect (Table 2) of a combination of GH and cortisol, administered under similar experimental conditions, to the same species but in different stages of life cycle (Madsen, 1990d; Madsen and Korsgaard, 1991), stresses the involvement of the endogenous hormonal status on the response to hormonal treatments. CONCLUSION
The analysis of the effects produced by thyroid hormones cortisol, prolactin and GH, namely the hormones mainly implicated in teleost osmoregulatory mechanisms, on gill (Na+ + K+)-ATPase activity points out contradictory results. The data reported often appear to be related to a wide variety of elements. Therefore, as first stressed by Folmar and Dickoff (1980), the events that accompany gill (Na+ + K+ )-ATPase rise during salmonid smoltification and generally SW adaptation of euryhaline teleosts appear to depend on a variety of hormones whose roles and mutual relationships so far remain partly obscured, despite considerable literature on the subject. The existence of a complex inter-relationship between these hormones must be taken into account whenever the possibility of using hormonal treatments to pre-adapt FW-born species to SW is considered. REFERENCES
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