Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 189-194 (1993) Kugler Publications, Amsterdam/New York
Arginine vasotocin and fish osmoregulation R.J. Balment, J.M. Warne, M. Tierney* and N. Hazon* Department of Physiological Sciences, University of Manchester, ManchesterM13 9PT and Gatty Marine Laboratory, School of Biological and Biomedical Sciences, University of St. Andrews, Fife KY16 8LB U.K.
Keywords: arginine vasotocin, teleost fish, euryhaline, osmoregulation, neurohypophysis, kidney, gill, angiotensin
Abstract Pituitary arginine vasotocin (AVT) secretion is sensitive to the osmotic challenge associated with transfer of euryhaline teleosts between sea water (SW) and fresh water (FW). Pituitary AVT content in FW-adapted flounders greatly exceeds that in SW-adapted fish. Plasma AVT concentrations are in the range 10 - 2-10- 11 M (1-100 pg/ml). In euryhaline species, like the eel, flounder and trout, there were no consistent, marked differences in plasma AVT concentrations between FW- and SW-adapted fish. In SW- but not FW-adapted flounders plasma AVT and sodium concentrations are correlated. During the initial period of acclimation from FW to SW eels, show a transitory rise in plasma AVT concentration which is associated with a transitory increase in plasma Angiotensin II. In view of the range of plasma AVT concentration observed in SW- and FW-adapted fish, it is evident that of the described dose-dependent effects of AVT on urine production, only the antidiuretic responses are likely to be of physiological significance. In addition to the presence of a Vl-type vascular receptor for AVT, the nephron also possesses a V2 -type receptor, coupled to adenylate cyclase. In the gill tissue AVT receptors are also present, but in this tissue receptor occupancy leads to inhibition of cAMP production rather than the stimulation observed in renal tissue. The functional significance of the gill AVT receptor remains to be established.
Resume La scr6tion d'arginine vasotocine (AVT) hypophysaire est sensible aux modifications de la pression osmotique observees lors du transfert d'eau douce en eau de mer de tl1ost6ens euryhalins. Les contenus hypophysaires en AVT chez des flets adapts a l'eau douce sont largement sup6rieurs a ceux des poissons adapts a l'eau de mer. Les concentrations plasmatiques en AVT se situent entre 10- 12 et 10-11 M. Chez les especes euryhalines comme l'anguille, le flet et la truite, iln'y a pas de differences marques dans les concentrations plasmatiques d'AVT en eau douce et en eau de mer. Chez des poissons adapts a l'eau de mer, les niveaux plasmatiques d'AVT et de sodium sont correl6s ce qui n'est pas le cas chez les poissons adapts l'eau douce. Pendant la periode initiale d'aclimatation a l'eau de mer, une augmentation transitoire des niveaux plasmatiques d'AVT est observe chez l'anguille, associ6e a une augmentation de l'angiotensine II plasmatique. Compte tenues des concentrations plasmatiques d'AVT mesur6es chez des poissons adapts a l'eau douce ou a l'eau de mer, il est evident que parmi les effets dose-dependants de I'AVT sur la production d'urine decrits dans la litterature, seules les r6ponses antidiur6tiques ont vraissemblablement une signification physio-
190 logique. En plus de la presence de rcepteurs vasculaires pour I'AVT de type Vl, le nephron possede aussi des rcepteurs de type V2 couples a l'ad6nylate cyclase. Dans les branchies, les rcepteurs a I'AVT sont aussi presents, mais dans ce tissu, l'occupation des rcepteurs conduit a une inhibition de la production d'AMPc contrairement a la stimulation observe dans le tissu renal. La signification fonctionnelle du rcepteur branchial I'AVT reste a tablir.
Introduction The role of AVT in teleostean osmoregulation is unclear, though dose-dependent pressor actions and effects on renal fluid loss have been described in the eel (Henderson and Wales 1974; Babiker and Rankin 1978). The physiological significance of such observations is, however, unknown as the normal range of plasma AVT concentrations in teleosts has not been established. The neurohypophysial secretion of AVT does appear to be sensitive to osmotic challenge. Histological studies have demonstrated depletion of neurosecretory material during acclimation to hypertonic media in both the trout, Salmo gairdneri (Holmes and McBean 1963) and the eel Anguilla anguilla (Sharratt et al. 1964). Using the rat antidiuretic bioassay to measure AVT biological activity in pituitary extracts, we have also recently shown pituitary AVT content to be elevated in fresh water (FW) compared with sea water (SW) acclimated flounder, Platichthys flesus (Perrott et al. 1991). Hyodo and Urano (1991) have also examined the effect of altered environmental salinity on the expression of vasotocin precursor genes in the trout using in situ hybridisation techniques. The pro vasotocin hybridisation signals in magnocellular neurones were markedly decreased after transfer of fish from FW to 80% SW. By contrast, in the european eel after long term adaptation to SW, pituitary AVT content was higher than in FW-adapted fish (Holder 1968, 1970). Although there is now general agreement that neurohypophysial secretion of AVT is responsive to osmotic stimuli, their precise nature remains unclear. In our preliminary attempts to measure plasma AVT concentrations in flounder, it was evident that plasma AVT concentration and plasma osmolality were closely correlated (Perrott et al. 1991). Curiously, however, this relationship, comparable
with that reported for tetrapods, was demonstrable only in SW-adapted fish, no such relationship was apparent in FW-adapted flounder.
Measurement of plasma AVT The key to understanding the physiological contribution of AVT to osmoregulatory adaptation will in part result from a better understanding of the circulating AVT levels in euryhaline fish as they are adapting to hypo- and hypertonic media. Recently we have measured plasma AVT concentrations in flounder by radioimmunoassay using the antiserum and protocol previously developed by Gray and Simon (1983) for measurement of AVT in birds. Blood samples were collected rapidly by direct needle puncture of caudal blood vessels and the AVT in separated plasma was extracted using SepPak C 18 cartridges prior to assay (Perrott et al. 1991). The assay limit of detection was 0.75 pg/ml. As shown in Fig. 1 there was an apparent increase in plasma AVT concentration as fish were slowly adapted (10 days in each salinity) from SW to FW. These higher levels in FW- than SW-adapted fish concur with our previous observations using a less sensitive assay system for both flounder and rainbow trout (Perrott et al. 1991). However, no such clear difference in SW- and FW-adapted eel plasma AVT concentrations were apparent (FW 1.03 + 0.09 pg/ml, n = 9 and SW 0.9 + 0.08 pg/ml, n = 9). Previous measures in the eel, after very long periods in SW, had suggested considerable elevation in plasma AVT concentration (Henderson et al. 1985). Plasma AVT concentrations in the lamprey, Lampetrafluviatlis, adapted to FW, 6.52 + 0.88 pg/ml, n = 14 and SW 5.99 1.81 pg/ml, n = 10 were also comparable. Notably, the plasma AVT concentration in SW-adapted flounder was once again correlated with plasma Na + concentra-
191 *I
15 -
E
I
12-
F///i
-
I--
9g
E
6-
U
I
i/
3 -
,\ v
100
67
00
33
EXTERNAL SALINITY
% SW
Fig. 1. Plasma AVT concentrations in flounders adapted to 100%, 67o7o, 33070 or 0°o SW (i.e., FW). Fish were held in each media for at least 10 days prior to rapid blood sampling. Values are means + SEM for the number of fish indicated at the base of each column. *p < 0.05 for t-test comparison of 1000% SW and FW fish.
tion, though no such relationship was evident in FW-adapted fish (see Fig. 2). Measures of plasma AVT concentrations in fish adapted to media for long periods may differ from those in fish during the process of acclimation. Accordingly, we have examined plasma hormone concentrations in groups of eels sacrificed at 2 day intervals following transfer from FW to SW. In addition to AVT, plasma angiotensin II (AII) concentrations were also determined by RIA. As shown in Figure 3 there was a small transitory rise in plasma AVT concentrations around the fourth day after transfer to SW, though AVT levels after 14 days in SW were comparable with those in FW animals. The SW transfer was also associated with a transitory increase in plasma AII concentration. In view of the reported contribution of AII to the stimulation of AVT and vasopressin secretion in tetrapods (Ramsay et al. 1978), it is tempting to speculate that this may also be effective in fish. In a variety of fish species, both marine and FW, plasma AVT concentrations fell in the range 10- 12 to 10- 1 M which is comparable with levels reported in tetrapods. These measures are also comparable with the bioassay estimates for plasma AVT ac-
tivity (10.9 1.2 pg/ml) in FW eels by Holder et al. (1982). It seems reasonable, therefore, to suggest that the physiological range of plasma AVT concentration is 10-12 - 10 - 1l M. This definition allows us to reassess some of the earlier observations of the biological actions of AVT and to identify those which may be of physiological significance.
Renal actions of AVT Manipulation, in vivo, of plasma AVT levels in FW-adapted euryhaline fish has profound effects on kidney function. Doses of AVT (10- 100 ng/kg), sufficient to produce a systemic pressor effect, are accompanied by a diuresis in the european eel (Henderson and Wales 1974; Babiker and Rankin 1978). Similarly, in vitro, in the perfused trout trunk preparation maintained under constant infusion, AVT produces a pressor response accompanied by a diuresis. In this preparation the use of the antagonist KBIV 24, which antagonises the vascular V 1 receptor type action of AVT, abolishes both the pressor action and diuresis caused by AVT (Pang et al. 1983). This suggests that it is the vascu-
192 15-
[FW
12E 0)tCL
A
9-
C
A
AA
A
6-
A
A
0 ,m
A
A
A A
3-
A
A
4-,
M C U a 0
I-U
E In M 03 EL
015-
S
12-
sO
*
* 8.
9-
0 9
6-
0
3II II
I v
0
I I
110
I
I
I
I
I
130 140 150 160 120 170 Plasma sodium concentration (mmol/l)
I
1
180
190
Fig. 2. Plasma AVT concentration plotted against plasma sodium concentration in blood samples taken from 16 flounders adapted to FW ( ) and 18 flounders adapted to SW ( ). Fish were held in each medium for at least 10 days prior to blood sampling. Linear regression analysis indicated positive correlation between plasma AVT and sodium concentrations in SW (r2 = 0.38; p < 0.05) but not FW fish.
lar pressor action of AVT that is driving the diuresis. Lower doses of AVT, which do not produce a pressor response, can elicit an antidiuresis (Henderson and Wales 1974; Babiker and Rankin 1978). It is possible to estimate the likely change in circulating concentrations of AVT produced by the AVT injection regime employed in these earlier studies. On the assumption that fish blood volume is approximately 3 ml per 100 g body weight (Holmes and Donaldson 1969), intravenous AVT injection of 10 ng/kg body weight will maximally increase AVT concentration in the blood by around 300 pg/ml (10-10 M), and a 100 ng/kg dose by 3000 pg/ml (10- 9 M). This calculation does not, of course, take into account the eventual distribution of the peptide in other extracellular fluid compartments. Accordingly, when these previously reported renal responses to AVT are considered in relation to the physiological range of plasma AVT concentrations we have measured, it seems likely that plasma AVT concentrations induced to achieve the
pressor responses are several orders of magnitude greater than the physiological range. The accompanying diuresis is, therefore, likely to be only of pharmacological significance. However, the lower AVT doses (0.001-0.01 ng/kg), which are 'nonpressor' are likely to raise plasma AVT concentrations (0.03-0.3 pg/ml; 10-14 - 10- 13 M) within the physiologically realistic range and the observed antidiuretic response is thus likely to represent a physiologically significant action of AVT. The antidiuresis induced by AVT may also be occurring at higher 'diuretic' doses of AVT but the effect would be overridden by the AVT-induced rise in systemic blood pressure. Indeed, experiments employing the perfused trout trunk preparation maintained at a constant pressure (Pang et al. 1983; Dunne 1992), which precludes the influence of AVT pressor action, indicate that an antidiuretic response to AVT is achieved even at very high doses. The renal mechanism responsible for AVTinduced changes in urine flow would appear to de-
193 80 E E
T
60 I*
40
E[
20.
E_1
0.
-Li
2.0
1.5 . E
R
1.0 0.5. 0.
I FW 2 l
FW
2
8 6 4 6 Time (days) in S W
T 14
Fig. 3. Plasma AVT and angiotensin II (AII) concentrations in eels during transfer of animals from FW to SW. Blood samples were taken from separate groups of animals (n value indicated at the base of each column) adapted to FW or 2, 4, 6, and 14 days after transfer to SW. **p < 0.01; ***p < 0.001 for comparisons with FW values by t-test. Values are means SEM.
pend on alteration in the rate of glomerular filtration, which in turn involves changes in the numbers of nephrons filtering, as the transport maxima for glucose shows direct proportionality to inulin clearance (Henderson and Wales 1974; Babiker and Rankin 1978). Nephrons are 'shutting down' or derecruiting during the AVT-induced antidiuresis, due to changes in the numbers of glomeruli being perfused. This suggests that there is a vascular AVT receptor within the kidney to mediate this response. Presumably this involves vascular smooth muscle, and a receptor similar to the tetrapod V1 type. This is supported by observations in the perfused trout trunk, maintained at constant pressure, that AVTinduced antidiuresis is abolished by an analogue of AVT, the V1 receptor antagonist KBIV 24 (Pang et al. 1983). The effects of AVT at high doses in SW-adapted
eels are broadly similar to those of FW-adapted fish, with a diuresis being observed. At doses in SW eels, which would have been non-pressor antidiuretic doses in FW-adapted animals, no noticeable change in urine production was reported (Babiker and Rankin 1978). This absence of an antidiuretic action in SW animals is perhaps not surprising as the animals are already exhibiting a marked antidiuresis. Although it may be possible to largely ascribe the antidiuretic action of AVT to a vascular V1-type receptor within the kidney, we have recently examined the possible presence of the tetrapod V2-type receptor, which is more normally associated with antidiuresis. Using isolated nephron preparations it has been possible to demonstrate the presence of a receptor similar to the adenylate cyclase coupled 'V2 -type' receptor of higher vertebrates (Sainsbury and Balment 1991). In nephron preparations from FW- and SW-adapted trout, AVT (10-11-10-7 M) produced dose-dependent stimulation of cAMP production (Perrott et al. 1992). This type of receptor was previously considered restricted to tetrapod groups (Pang 1983) and to be linked to altered tubular rather than glomerular function. From a comparative perspective it is interesting to speculate that it could be linked to the antidiuresis in fish, although there is, as yet, no evidence to support a direct tubular action of AVT.
AVT and gill function The presence of AVT receptors has been demonstrated in the gills (Guibollini et al. 1988) and in view of the characteristics of the responses to V1 and V2 receptor type agonists and antagonists, this gill receptor was a designated new type 'NHF' (Guibollini and Lahlou 1990). Occupation of the gill AVT receptor alters adenylate cyclase activity, inhibiting cAMP production by isolated gill cell membrane preparations (Guibollini and Lahlou 1987) and isolated whole gill cell preparations (Sainsbury and Balment 1991). This effect was more pronounced in gill tissue taken from SWrather than FW-adapted trout. There is, however, as yet, no established link between the gill receptor
194 second messenger system for AVT and the mechanisms controlling ion or water fluxes across the gill in FW and SW. The physiological function, if any, of AVT at the gill is still not clear, though gill vasculature is sensitive to AVT, which causes an increase in resistance to blood flow through the gill (Bennett and Rankin 1986). Examination of the factors which influence pituitary AVT secretion clearly implicate an osmoregulatory role for AVT in fish. Definition of the physiological circulatory levels of AVT now provides an insight into which of the described biological actions of AVT may contribute to such a physiological role. From the restricted studies of few species of euryhaline teleosts, it appears that, physiologically, AVT may act on the kidney to restrict urine production. This contrasts with the long held notion that AVT was a hormone to promote water excretion. It is also now evident that there are probably 2 types of renal receptors for AVT, though the functional significance of the V2 -type remains to be established. For obvious practical reasons the majority of studies of the renal actions of AVT have been carried out in FW fish. Nonetheless, AVT is present in SW-adapted animals, though whether its target tissue actions are similar in the two media remains to be elucidated.
References cited Babiker, M.M. and Rankin, J.C. 1978. Neurohypophysial hormone control of kidney function in the european eel Anguilla anguilla adapted to sea water or fresh water. J. Endocrinol. 76: 347-358. Bennett, M.B. and Rankin, J.C. 1986. The effect of neurohypophysial hormones on the vascular resistance of the isolated perfused gill of the european eel Anguilla anguilla L. Gen. Comp. Endocrinol. 64: 60-66. Dunne, B. 1992. Control of renal salt excretion in teleost fish.
Ph. D. Thesis, Bangor University, U.K. Gray, D.A. and Simon, E. 1983. Mammalian and avian antidiuretic hormone: studies related to possible species variation in osmoregulatory systems. J. Comp. Physiol. Biochem. B 151: 241-246. Guibollini, M.E., Henderson, I.W., Mosley, W. and Lahlou, B. 1988. Arginine vasotocin binding to isolated branchial cells of the eel: Effect of salinity. J. Mol. Endocrinol. 1: 125-130. Guibollini, M.E. and Lahlou, B. 1987. Neurohypophysial peptide inhibition of adenylate cyclase activity in fish gills. FEBS Letters 220: 98-102.
Guibollini, M.E. and Lahlou, B. 1990. Evidence for the presence of a new type of neurohypophysial hormone receptor in fish gill epithelium. Am. J. Physiol. 258: R3-9. Henderson, I.W., Hazon, N. and Hughes, K. 1985. Hormone, ionic regulation and kidney function in fishes. J. Exp. Biol. 39: 245-265. Henderson, I.W. and Wales, N.A.M. 1974. Renal diuresis and antidiuresis after injections of arginine vasotocin in the fresh water eel Anguilla anguilla L. J. Endocrinol. 61: 487-500. Holder, F.C., Schroeder, M.D., Pollatz, M., Guerne, J.M., Vivien-Roels, B., Pevet, P., Buijs, R.M., Dogterom, J. and Meiniel, A. 1982. A specific and sensitive bioassay for arginine-vasotocin: Description, validation and some applications in lower and higher vertebrates. Gen. Comp. Endocrinol. 47: 483-491. Holder, F.C. 1968. Mise en evidence et dosage de l'activite de type ocytocique d'homogenats totaux et de fractions granulaires du system preoptico-hypophysaire de l'anguille Anguilla anguilla L. Gen. Comp. Endocrinol. 11: 235-242. Holder, F.C. 1970. Separation chromatographique et dosage de l'arginine-vasotocin et de l'isotocin de l'hypophyse et du noyau pr6optique chez l'anguilla de mer du Nord Anguilla anguilla L. Experimentia 26: 1199-2000. Holmes, W.N. and Donaldson, E.M. 1969. The body compartments and the distribution of electrolytes. In Fish Physiology. Vol. 1. pp 1-89. Edited by W.S. Hoar and D.J. Randall. Academic Press, New York. Holmes, W.N. and McBean, R.L. 1963. Studies on the glomerular filtration rate of rainbow trout, Salmo gairdneri.J. Exp. Biol. 40: 335-341. Hyodo, S. and Urano, A. 1991. Changes in expression of provasotocin and proisotocin genes during adaptation to hyper- and hypo-osmotic environments in rainbow trout. J. Comp. Physiol. B 161: 549-556. Pang, P.K.T. 1983. Evolution of control of epithelial transport in vertebrates. J. Exp. Biol. 106: 283-299. Pang, P.K.T., Furspan, P.B. and Sawyer, W.H. 1983. Evolution of neurohypophysial hormone action in vertebrates. Am. Zool. 23: 655-662. Perrott, M.N., Carrick, S. and Balment, R.J. 1991. Pituitary and plasma arginine vasotocin levels in teleost fish. Gen. Comp. Endocrinol. 83: 68-74. Perrott, M.N., Sainsbury, R.J. and Balment, R.J. 1993. Peptide hormone-stimulated second messenger production in the teleostean nephron. Gen. Comp. Endocrinol. (In press). Ramsay, D.J., Keil, L.C., Sharpe, M.C. and Shinsako, J. 1978. Angiotensin II infusion increases vasopressin, ACTH and IIhydroxycorticosteroid secretion. Am. J. Physiol. 234: R66-R71. Sainsbury, R.J. and Balment, R.J. 1991. Arginine vasotocin (AVT) modulation of cAMP production in isolated renal tubules and dispersed gill cells in rainbow trout. J. Endocrinol. 131: suppl. 123. Sharratt, B.M., Bellamy, D. and Chester Jones, I. 1964. Adaptation of the silver eel Anguilla anguilla to sea water and to artificial media together with observations on the role of the gut. Comp. Biochem. Physiol. 11: 19-30.
Arginine vasotocin and fish osmoregulation R.J. Balment, J.M. Warne, M. Tierney* and N. Hazon* Department of Physiological Sciences, University of Manchester, ManchesterM13 9PT and Gatty Marine Laboratory, School of Biological and Biomedical Sciences, University of St. Andrews, Fife KY16 8LB U.K.
Keywords: arginine vasotocin, teleost fish, euryhaline, osmoregulation, neurohypophysis, kidney, gill, angiotensin
Abstract Pituitary arginine vasotocin (AVT) secretion is sensitive to the osmotic challenge associated with transfer of euryhaline teleosts between sea water (SW) and fresh water (FW). Pituitary AVT content in FW-adapted flounders greatly exceeds that in SW-adapted fish. Plasma AVT concentrations are in the range 10 - 2-10- 11 M (1-100 pg/ml). In euryhaline species, like the eel, flounder and trout, there were no consistent, marked differences in plasma AVT concentrations between FW- and SW-adapted fish. In SW- but not FW-adapted flounders plasma AVT and sodium concentrations are correlated. During the initial period of acclimation from FW to SW eels, show a transitory rise in plasma AVT concentration which is associated with a transitory increase in plasma Angiotensin II. In view of the range of plasma AVT concentration observed in SW- and FW-adapted fish, it is evident that of the described dose-dependent effects of AVT on urine production, only the antidiuretic responses are likely to be of physiological significance. In addition to the presence of a Vl-type vascular receptor for AVT, the nephron also possesses a V2 -type receptor, coupled to adenylate cyclase. In the gill tissue AVT receptors are also present, but in this tissue receptor occupancy leads to inhibition of cAMP production rather than the stimulation observed in renal tissue. The functional significance of the gill AVT receptor remains to be established.
Resume La scr6tion d'arginine vasotocine (AVT) hypophysaire est sensible aux modifications de la pression osmotique observees lors du transfert d'eau douce en eau de mer de tl1ost6ens euryhalins. Les contenus hypophysaires en AVT chez des flets adapts a l'eau douce sont largement sup6rieurs a ceux des poissons adapts a l'eau de mer. Les concentrations plasmatiques en AVT se situent entre 10- 12 et 10-11 M. Chez les especes euryhalines comme l'anguille, le flet et la truite, iln'y a pas de differences marques dans les concentrations plasmatiques d'AVT en eau douce et en eau de mer. Chez des poissons adapts a l'eau de mer, les niveaux plasmatiques d'AVT et de sodium sont correl6s ce qui n'est pas le cas chez les poissons adapts l'eau douce. Pendant la periode initiale d'aclimatation a l'eau de mer, une augmentation transitoire des niveaux plasmatiques d'AVT est observe chez l'anguille, associ6e a une augmentation de l'angiotensine II plasmatique. Compte tenues des concentrations plasmatiques d'AVT mesur6es chez des poissons adapts a l'eau douce ou a l'eau de mer, il est evident que parmi les effets dose-dependants de I'AVT sur la production d'urine decrits dans la litterature, seules les r6ponses antidiur6tiques ont vraissemblablement une signification physio-
190 logique. En plus de la presence de rcepteurs vasculaires pour I'AVT de type Vl, le nephron possede aussi des rcepteurs de type V2 couples a l'ad6nylate cyclase. Dans les branchies, les rcepteurs a I'AVT sont aussi presents, mais dans ce tissu, l'occupation des rcepteurs conduit a une inhibition de la production d'AMPc contrairement a la stimulation observe dans le tissu renal. La signification fonctionnelle du rcepteur branchial I'AVT reste a tablir.
Introduction The role of AVT in teleostean osmoregulation is unclear, though dose-dependent pressor actions and effects on renal fluid loss have been described in the eel (Henderson and Wales 1974; Babiker and Rankin 1978). The physiological significance of such observations is, however, unknown as the normal range of plasma AVT concentrations in teleosts has not been established. The neurohypophysial secretion of AVT does appear to be sensitive to osmotic challenge. Histological studies have demonstrated depletion of neurosecretory material during acclimation to hypertonic media in both the trout, Salmo gairdneri (Holmes and McBean 1963) and the eel Anguilla anguilla (Sharratt et al. 1964). Using the rat antidiuretic bioassay to measure AVT biological activity in pituitary extracts, we have also recently shown pituitary AVT content to be elevated in fresh water (FW) compared with sea water (SW) acclimated flounder, Platichthys flesus (Perrott et al. 1991). Hyodo and Urano (1991) have also examined the effect of altered environmental salinity on the expression of vasotocin precursor genes in the trout using in situ hybridisation techniques. The pro vasotocin hybridisation signals in magnocellular neurones were markedly decreased after transfer of fish from FW to 80% SW. By contrast, in the european eel after long term adaptation to SW, pituitary AVT content was higher than in FW-adapted fish (Holder 1968, 1970). Although there is now general agreement that neurohypophysial secretion of AVT is responsive to osmotic stimuli, their precise nature remains unclear. In our preliminary attempts to measure plasma AVT concentrations in flounder, it was evident that plasma AVT concentration and plasma osmolality were closely correlated (Perrott et al. 1991). Curiously, however, this relationship, comparable
with that reported for tetrapods, was demonstrable only in SW-adapted fish, no such relationship was apparent in FW-adapted flounder.
Measurement of plasma AVT The key to understanding the physiological contribution of AVT to osmoregulatory adaptation will in part result from a better understanding of the circulating AVT levels in euryhaline fish as they are adapting to hypo- and hypertonic media. Recently we have measured plasma AVT concentrations in flounder by radioimmunoassay using the antiserum and protocol previously developed by Gray and Simon (1983) for measurement of AVT in birds. Blood samples were collected rapidly by direct needle puncture of caudal blood vessels and the AVT in separated plasma was extracted using SepPak C 18 cartridges prior to assay (Perrott et al. 1991). The assay limit of detection was 0.75 pg/ml. As shown in Fig. 1 there was an apparent increase in plasma AVT concentration as fish were slowly adapted (10 days in each salinity) from SW to FW. These higher levels in FW- than SW-adapted fish concur with our previous observations using a less sensitive assay system for both flounder and rainbow trout (Perrott et al. 1991). However, no such clear difference in SW- and FW-adapted eel plasma AVT concentrations were apparent (FW 1.03 + 0.09 pg/ml, n = 9 and SW 0.9 + 0.08 pg/ml, n = 9). Previous measures in the eel, after very long periods in SW, had suggested considerable elevation in plasma AVT concentration (Henderson et al. 1985). Plasma AVT concentrations in the lamprey, Lampetrafluviatlis, adapted to FW, 6.52 + 0.88 pg/ml, n = 14 and SW 5.99 1.81 pg/ml, n = 10 were also comparable. Notably, the plasma AVT concentration in SW-adapted flounder was once again correlated with plasma Na + concentra-
191 *I
15 -
E
I
12-
F///i
-
I--
9g
E
6-
U
I
i/
3 -
,\ v
100
67
00
33
EXTERNAL SALINITY
% SW
Fig. 1. Plasma AVT concentrations in flounders adapted to 100%, 67o7o, 33070 or 0°o SW (i.e., FW). Fish were held in each media for at least 10 days prior to rapid blood sampling. Values are means + SEM for the number of fish indicated at the base of each column. *p < 0.05 for t-test comparison of 1000% SW and FW fish.
tion, though no such relationship was evident in FW-adapted fish (see Fig. 2). Measures of plasma AVT concentrations in fish adapted to media for long periods may differ from those in fish during the process of acclimation. Accordingly, we have examined plasma hormone concentrations in groups of eels sacrificed at 2 day intervals following transfer from FW to SW. In addition to AVT, plasma angiotensin II (AII) concentrations were also determined by RIA. As shown in Figure 3 there was a small transitory rise in plasma AVT concentrations around the fourth day after transfer to SW, though AVT levels after 14 days in SW were comparable with those in FW animals. The SW transfer was also associated with a transitory increase in plasma AII concentration. In view of the reported contribution of AII to the stimulation of AVT and vasopressin secretion in tetrapods (Ramsay et al. 1978), it is tempting to speculate that this may also be effective in fish. In a variety of fish species, both marine and FW, plasma AVT concentrations fell in the range 10- 12 to 10- 1 M which is comparable with levels reported in tetrapods. These measures are also comparable with the bioassay estimates for plasma AVT ac-
tivity (10.9 1.2 pg/ml) in FW eels by Holder et al. (1982). It seems reasonable, therefore, to suggest that the physiological range of plasma AVT concentration is 10-12 - 10 - 1l M. This definition allows us to reassess some of the earlier observations of the biological actions of AVT and to identify those which may be of physiological significance.
Renal actions of AVT Manipulation, in vivo, of plasma AVT levels in FW-adapted euryhaline fish has profound effects on kidney function. Doses of AVT (10- 100 ng/kg), sufficient to produce a systemic pressor effect, are accompanied by a diuresis in the european eel (Henderson and Wales 1974; Babiker and Rankin 1978). Similarly, in vitro, in the perfused trout trunk preparation maintained under constant infusion, AVT produces a pressor response accompanied by a diuresis. In this preparation the use of the antagonist KBIV 24, which antagonises the vascular V 1 receptor type action of AVT, abolishes both the pressor action and diuresis caused by AVT (Pang et al. 1983). This suggests that it is the vascu-
192 15-
[FW
12E 0)tCL
A
9-
C
A
AA
A
6-
A
A
0 ,m
A
A
A A
3-
A
A
4-,
M C U a 0
I-U
E In M 03 EL
015-
S
12-
sO
*
* 8.
9-
0 9
6-
0
3II II
I v
0
I I
110
I
I
I
I
I
130 140 150 160 120 170 Plasma sodium concentration (mmol/l)
I
1
180
190
Fig. 2. Plasma AVT concentration plotted against plasma sodium concentration in blood samples taken from 16 flounders adapted to FW ( ) and 18 flounders adapted to SW ( ). Fish were held in each medium for at least 10 days prior to blood sampling. Linear regression analysis indicated positive correlation between plasma AVT and sodium concentrations in SW (r2 = 0.38; p < 0.05) but not FW fish.
lar pressor action of AVT that is driving the diuresis. Lower doses of AVT, which do not produce a pressor response, can elicit an antidiuresis (Henderson and Wales 1974; Babiker and Rankin 1978). It is possible to estimate the likely change in circulating concentrations of AVT produced by the AVT injection regime employed in these earlier studies. On the assumption that fish blood volume is approximately 3 ml per 100 g body weight (Holmes and Donaldson 1969), intravenous AVT injection of 10 ng/kg body weight will maximally increase AVT concentration in the blood by around 300 pg/ml (10-10 M), and a 100 ng/kg dose by 3000 pg/ml (10- 9 M). This calculation does not, of course, take into account the eventual distribution of the peptide in other extracellular fluid compartments. Accordingly, when these previously reported renal responses to AVT are considered in relation to the physiological range of plasma AVT concentrations we have measured, it seems likely that plasma AVT concentrations induced to achieve the
pressor responses are several orders of magnitude greater than the physiological range. The accompanying diuresis is, therefore, likely to be only of pharmacological significance. However, the lower AVT doses (0.001-0.01 ng/kg), which are 'nonpressor' are likely to raise plasma AVT concentrations (0.03-0.3 pg/ml; 10-14 - 10- 13 M) within the physiologically realistic range and the observed antidiuretic response is thus likely to represent a physiologically significant action of AVT. The antidiuresis induced by AVT may also be occurring at higher 'diuretic' doses of AVT but the effect would be overridden by the AVT-induced rise in systemic blood pressure. Indeed, experiments employing the perfused trout trunk preparation maintained at a constant pressure (Pang et al. 1983; Dunne 1992), which precludes the influence of AVT pressor action, indicate that an antidiuretic response to AVT is achieved even at very high doses. The renal mechanism responsible for AVTinduced changes in urine flow would appear to de-
193 80 E E
T
60 I*
40
E[
20.
E_1
0.
-Li
2.0
1.5 . E
R
1.0 0.5. 0.
I FW 2 l
FW
2
8 6 4 6 Time (days) in S W
T 14
Fig. 3. Plasma AVT and angiotensin II (AII) concentrations in eels during transfer of animals from FW to SW. Blood samples were taken from separate groups of animals (n value indicated at the base of each column) adapted to FW or 2, 4, 6, and 14 days after transfer to SW. **p < 0.01; ***p < 0.001 for comparisons with FW values by t-test. Values are means SEM.
pend on alteration in the rate of glomerular filtration, which in turn involves changes in the numbers of nephrons filtering, as the transport maxima for glucose shows direct proportionality to inulin clearance (Henderson and Wales 1974; Babiker and Rankin 1978). Nephrons are 'shutting down' or derecruiting during the AVT-induced antidiuresis, due to changes in the numbers of glomeruli being perfused. This suggests that there is a vascular AVT receptor within the kidney to mediate this response. Presumably this involves vascular smooth muscle, and a receptor similar to the tetrapod V1 type. This is supported by observations in the perfused trout trunk, maintained at constant pressure, that AVTinduced antidiuresis is abolished by an analogue of AVT, the V1 receptor antagonist KBIV 24 (Pang et al. 1983). The effects of AVT at high doses in SW-adapted
eels are broadly similar to those of FW-adapted fish, with a diuresis being observed. At doses in SW eels, which would have been non-pressor antidiuretic doses in FW-adapted animals, no noticeable change in urine production was reported (Babiker and Rankin 1978). This absence of an antidiuretic action in SW animals is perhaps not surprising as the animals are already exhibiting a marked antidiuresis. Although it may be possible to largely ascribe the antidiuretic action of AVT to a vascular V1-type receptor within the kidney, we have recently examined the possible presence of the tetrapod V2-type receptor, which is more normally associated with antidiuresis. Using isolated nephron preparations it has been possible to demonstrate the presence of a receptor similar to the adenylate cyclase coupled 'V2 -type' receptor of higher vertebrates (Sainsbury and Balment 1991). In nephron preparations from FW- and SW-adapted trout, AVT (10-11-10-7 M) produced dose-dependent stimulation of cAMP production (Perrott et al. 1992). This type of receptor was previously considered restricted to tetrapod groups (Pang 1983) and to be linked to altered tubular rather than glomerular function. From a comparative perspective it is interesting to speculate that it could be linked to the antidiuresis in fish, although there is, as yet, no evidence to support a direct tubular action of AVT.
AVT and gill function The presence of AVT receptors has been demonstrated in the gills (Guibollini et al. 1988) and in view of the characteristics of the responses to V1 and V2 receptor type agonists and antagonists, this gill receptor was a designated new type 'NHF' (Guibollini and Lahlou 1990). Occupation of the gill AVT receptor alters adenylate cyclase activity, inhibiting cAMP production by isolated gill cell membrane preparations (Guibollini and Lahlou 1987) and isolated whole gill cell preparations (Sainsbury and Balment 1991). This effect was more pronounced in gill tissue taken from SWrather than FW-adapted trout. There is, however, as yet, no established link between the gill receptor
194 second messenger system for AVT and the mechanisms controlling ion or water fluxes across the gill in FW and SW. The physiological function, if any, of AVT at the gill is still not clear, though gill vasculature is sensitive to AVT, which causes an increase in resistance to blood flow through the gill (Bennett and Rankin 1986). Examination of the factors which influence pituitary AVT secretion clearly implicate an osmoregulatory role for AVT in fish. Definition of the physiological circulatory levels of AVT now provides an insight into which of the described biological actions of AVT may contribute to such a physiological role. From the restricted studies of few species of euryhaline teleosts, it appears that, physiologically, AVT may act on the kidney to restrict urine production. This contrasts with the long held notion that AVT was a hormone to promote water excretion. It is also now evident that there are probably 2 types of renal receptors for AVT, though the functional significance of the V2 -type remains to be established. For obvious practical reasons the majority of studies of the renal actions of AVT have been carried out in FW fish. Nonetheless, AVT is present in SW-adapted animals, though whether its target tissue actions are similar in the two media remains to be elucidated.
References cited Babiker, M.M. and Rankin, J.C. 1978. Neurohypophysial hormone control of kidney function in the european eel Anguilla anguilla adapted to sea water or fresh water. J. Endocrinol. 76: 347-358. Bennett, M.B. and Rankin, J.C. 1986. The effect of neurohypophysial hormones on the vascular resistance of the isolated perfused gill of the european eel Anguilla anguilla L. Gen. Comp. Endocrinol. 64: 60-66. Dunne, B. 1992. Control of renal salt excretion in teleost fish.
Ph. D. Thesis, Bangor University, U.K. Gray, D.A. and Simon, E. 1983. Mammalian and avian antidiuretic hormone: studies related to possible species variation in osmoregulatory systems. J. Comp. Physiol. Biochem. B 151: 241-246. Guibollini, M.E., Henderson, I.W., Mosley, W. and Lahlou, B. 1988. Arginine vasotocin binding to isolated branchial cells of the eel: Effect of salinity. J. Mol. Endocrinol. 1: 125-130. Guibollini, M.E. and Lahlou, B. 1987. Neurohypophysial peptide inhibition of adenylate cyclase activity in fish gills. FEBS Letters 220: 98-102.
Guibollini, M.E. and Lahlou, B. 1990. Evidence for the presence of a new type of neurohypophysial hormone receptor in fish gill epithelium. Am. J. Physiol. 258: R3-9. Henderson, I.W., Hazon, N. and Hughes, K. 1985. Hormone, ionic regulation and kidney function in fishes. J. Exp. Biol. 39: 245-265. Henderson, I.W. and Wales, N.A.M. 1974. Renal diuresis and antidiuresis after injections of arginine vasotocin in the fresh water eel Anguilla anguilla L. J. Endocrinol. 61: 487-500. Holder, F.C., Schroeder, M.D., Pollatz, M., Guerne, J.M., Vivien-Roels, B., Pevet, P., Buijs, R.M., Dogterom, J. and Meiniel, A. 1982. A specific and sensitive bioassay for arginine-vasotocin: Description, validation and some applications in lower and higher vertebrates. Gen. Comp. Endocrinol. 47: 483-491. Holder, F.C. 1968. Mise en evidence et dosage de l'activite de type ocytocique d'homogenats totaux et de fractions granulaires du system preoptico-hypophysaire de l'anguille Anguilla anguilla L. Gen. Comp. Endocrinol. 11: 235-242. Holder, F.C. 1970. Separation chromatographique et dosage de l'arginine-vasotocin et de l'isotocin de l'hypophyse et du noyau pr6optique chez l'anguilla de mer du Nord Anguilla anguilla L. Experimentia 26: 1199-2000. Holmes, W.N. and Donaldson, E.M. 1969. The body compartments and the distribution of electrolytes. In Fish Physiology. Vol. 1. pp 1-89. Edited by W.S. Hoar and D.J. Randall. Academic Press, New York. Holmes, W.N. and McBean, R.L. 1963. Studies on the glomerular filtration rate of rainbow trout, Salmo gairdneri.J. Exp. Biol. 40: 335-341. Hyodo, S. and Urano, A. 1991. Changes in expression of provasotocin and proisotocin genes during adaptation to hyper- and hypo-osmotic environments in rainbow trout. J. Comp. Physiol. B 161: 549-556. Pang, P.K.T. 1983. Evolution of control of epithelial transport in vertebrates. J. Exp. Biol. 106: 283-299. Pang, P.K.T., Furspan, P.B. and Sawyer, W.H. 1983. Evolution of neurohypophysial hormone action in vertebrates. Am. Zool. 23: 655-662. Perrott, M.N., Carrick, S. and Balment, R.J. 1991. Pituitary and plasma arginine vasotocin levels in teleost fish. Gen. Comp. Endocrinol. 83: 68-74. Perrott, M.N., Sainsbury, R.J. and Balment, R.J. 1993. Peptide hormone-stimulated second messenger production in the teleostean nephron. Gen. Comp. Endocrinol. (In press). Ramsay, D.J., Keil, L.C., Sharpe, M.C. and Shinsako, J. 1978. Angiotensin II infusion increases vasopressin, ACTH and IIhydroxycorticosteroid secretion. Am. J. Physiol. 234: R66-R71. Sainsbury, R.J. and Balment, R.J. 1991. Arginine vasotocin (AVT) modulation of cAMP production in isolated renal tubules and dispersed gill cells in rainbow trout. J. Endocrinol. 131: suppl. 123. Sharratt, B.M., Bellamy, D. and Chester Jones, I. 1964. Adaptation of the silver eel Anguilla anguilla to sea water and to artificial media together with observations on the role of the gut. Comp. Biochem. Physiol. 11: 19-30.
