bfzp. Biochem. Physiol., 1973, Vol. 46A, pp. 639 to 645. Pergamon Press. Printed in Great Britain

ROLE OF ALKALINE PHOSPHATASE IN INTESTINAL WATER ABSORPTION BY EELS ADAPTED TO SEA WATER

MOMOKO OIDE

Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan

(Received 22 January 1973)

Abstract-l. Water movement from mucosa to serosa was measured in ever-ted intestines isolated from sea water-adapted or fresh water-adapted eels.

2. Water movement was enhanced by raising the pH of the mucosal medium, and reduced when inhibitors of alkaline phosphatase were added to the mucosal surface.

3. The role of intestinal alkaline phosphatase in salt and water absorption is discussed in relation to sea water adaptation of the eel.

INTRODUCTION

WHEN eels are transferred from fresh water to sea water, they drink sea water and absorb it from the intestine, and the rate of water absorption is particularly high during the first week of sea water adaptation both in oiero (H. Oide & Utida, 1968) and in witro (M. Oide & Utida, 1967). C oncomitantly, the alkaline phosphatase activity of the intestinal mucosa increases (Utida & Isono, 1967; Utida et al,, 1968; Oide, 1970). Since there is always a considerable quantity of clear, alkaline fluid in the intestine of teleosts living in sea water (Smith, 1930; Hickman, 1968), it is conceivable that alkaline phosphatase is involved in salt and water absorption in sea water-adapted eels.

In the present work, the effects of pH and of inhibitors of alkaline phosphatase on water movement were examined in the isolated intestine of the eel in order to elucidate the role of alkaline phosphatase in water absorption.

MATERIALS AND METHODS

Japanese cultured eels, Anguilla juporzicu, weighing about 200 g, were obtained from a commercial fishpond and kept without feeding in a fresh water tank at 20°C for about a week before use. Some eels were then transferred to a sea water tank (20°C) and kept there for 7 days (sea water eels), unless otherwise mentioned, and the others were kept in the fresh water tank for the same period (fresh water eels).

Sampling and determination of ionic concentration and pH of body fluids

Eels were lightly anaesthetized with 0.05% MS 222 (Sandoz). Blood was drawn by cardiac puncture and serum was obtained as described elsewhere (M. Oide & Utida, 1967). After opening the abdomen, a sample of abdominal fluid was taken with a pipette and the intestine was isolated. The intestinal fluid was then collected in a test-tube. Concentrations

639

640 MOMOKO OIDE

of sodium, potassium and calcium in these samples were estimated with a Coleman flame photometer (Model 21). The pH was determined with a pH meter using a glass electrode (Toa Electronics Ltd., Japan) in the case of intestinal fluid, and with pH indicator paper (Merck) in the case of serum and abdominal fluid.

Preparation and incubation of everted intestine

Eels were sacrificed by decapitation. The intestine was dissected out, freed from the surrounding tissues and everted using a wire with a hook at the tip. Both ends of the intestine were tied with a thread. The intestine was weighed at the beginning of each experiment (IV,) and transferred to a flask containing 20 ml of diluted (one-third) artificial sea water (ASW; 460.9 mM NaCl, 9.4 mM KCl, 10.9 mM CaCl, and 22.6 mM MgCl,) gassed with 9.5% O,-5o/0 COz, and incubated by shaking at 20°C for 1 hr. The pH of each medium was adjusted by 50 mM (final concentration) Tris-HCl. At the end of the experi- ment, the intestine was weighed again (IV,) and its surface area was measured by spreading on section paper. Net water flux (from mucosa to serosa) was calculated as (IV, - IV,)/ (surface area), and expressed as ~1 H,0/cm2 per hr.

Inhibitors and hormones

Tetrasodium ethylenediamine tetra-acetate dihydrate (EDTA), sodium borate and L- cysteine (Wako Pure Chemical Industries Ltd., Tokyo) were added to the incubation medium as inhibitors of intestinal alkaline phosphatase. Ouabain (Merck) was used as an inhibitor of Na-K ATPase. A single injection of ACTH (Schering; 1 U/O.05 ml saline per 100 g B.W.) or cortisol acetate (Schering; 0.5 mg/0.02 ml suspension per 100 g B.W.) was given intraperitoneally to fresh water eels, which were sacrificed 2 days after the injection.

Hypophysectomy

Fresh water eels were hypophysectomized by the method described by Hirano et al. (1967). Four days after the operation, they were transferred to sea water and kept for 5 days.

RESULTS

Ionic concentration and pH of serum, abdominal and intestinalJluid in sea water eels

As shown in Table 1, abdominal fluid was lower in Na and Ca, and higher in K concentration compared with serum. Na concentration in intestinal fluid was one-quarter the serum value, while Ca concentration almost doubled. The intestinal fluid was alkaline (pH S-7), in contrast to serum and abdominal fluid which were neutral (pH 7.2).

TABLE 1-10~ CONCENTRATION AND pH OF SERUM, ABDOMINAL AND INTESTINAL FLUIDS IN SEA WATER EELS (MEAN + s.E.; N = 3)

Ion concentration (m-equiv/l.)

Na K Ca PH

Serum 162.7 + 4.3 3.53 + 0.10 5.70 f 0.14 7.2 Abdominal fluid 147.7 f O-2 5.18 f 0.42 3.50 * 0.18 7.2 Intestinal fluid 40.3 f 2.9 3.03 f 0.02 9.56 f. 0.95 8.7 f 0.07

WATBRABSORPTIONBYBELSADAPTRD TO SEAWATER 641

Effects of $H of mucosal medium on water movement across the intestine

The water movement across the everted intestine was estimated by using mucosal medium at pH 7.2, 9-O and 10.0, in sea water and fresh water eels.

In sea water eels, the net water flux at pH 7.2 was 7.7 $/cm2 per hr. When the pH values were changed to alkaline, the flux increased nearly 5*5-fold (pH 9.0) to six-fold (pH 10-O), and the differences were highly significant (PC 0.001). In fresh water eels, although the flux also enhanced five-fold when the pH was changed from 7.2 to 9-O (P-C O-01), the flux at each pH was significantly (P-C 0.01 at pH 7.2, and PC 0.001 at pH 9.0) less than that in sea water eels (Table 2).

TABLE%--EFFECTOF pH OFMUCOSALMEDIUM(ONE-THIRDARTIFICIALSBAWATBR)ONWATER MOVEMENT (MuCOSA ~0 SEROSA) IN EVERTED INTESTINE (MEAN+ S.E.)

Adaptation Net water flux of eels PH No. (~~l/cm~ per hr)

Sea water 7.2 5 7.7 f 0.8 9.0 5 43.1 f 3.0

10.0 5 48.1 + 5.3

Fresh water 7.2 3 3.0 + 0.7 9.0 3 15.5 f 2.3

Eflects of enzyme inhibitors on water mwement across the intestine

Inhibitors of alkaline phosphatase and Na-K ATPase were added to the mucosal medium (pH 9.0) and the water movement was estimated in sea water eels.

EDTA (10 mM), d so ium borate (40 mM) and L-cysteine (0.8 n&I), which are inhibitors of intestinal alkaline phosphatase, reduced the water flux by 68.8, 93.4 and 36.8 per cent, respectively. Ouabain, an inhibitor of Na-K ATPase, reduced the water flux by 46.4 per cent, whereas ouabain plus EDTA decreased it by 88.5 per cent (Table 3).

TABLE ~--EFFECT OF INHIBITORS IN MUCOSAL MEDIUM (ONE-THIRD ARTIFICIAL SEA WATER,

pH9.0) ONWATERMOVBh%ENT(MIJCOSATOSEROSA)INEVERTEDINTESTINBFROMSBAWATEREELS

(MEANT S.E.)

Inhibitors

None EDTA (10 mM) Sodium borate (40 mM) L-Cysteine (0.8 mM) Ouabain (0.1 mM) Ouabain (0.1 mM)

+ EDTA (10 mM)

No. Net water flux @l/cm* per hr) o/o Inhibition

7 42.6 f 2.3 0 4 13.3 f 1.9 68.2 3 2.7 + 0.4 93.4 4 26.9 f 1 *l 36.8 5 22.8 f 3.4 46.4 4 4.9fl.O 88.5

642 MOMOKO OIDE

Eflects of hypophysectomy and injection of ACTH and cortisol on water movement

The water movement of everted intestine with mucosal medium at pH 9.0 was estimated in hypophysectomized (sea water) eels and ACTH or cortisol-treated (fresh water) eels.

In intact eels, the water flux was significantly higher in sea water-adapted than in fresh water eels (P< 0.001 at pH 9*0), as shown in Table 2, whereas no such

increase was observed when eels were hypophysectomized and kept in sea water for 5 days; the flux was almost the same as that in fresh water eels and significantly lower than that in intact sea water eels (PC 0.001). A single injection of ACTH

(1 U/100 g B.W.) or cortisol acetate (0.5 mg/lOO g B.W.) into intact fresh water eels enhanced the water flux significantly (PC 0.05 or P< 0.01, respectively), up to the level of intact sea water eels (Table 4).

TABLE ~-EFFECT OF HYPOPHYSECTOMY AND INJECTION OF ACTH OR CORTISOL ON WATER MOVEMENT (M~c0s~ TO SERO~A) IN EVERTED INTESTINE (MEAN+S.E.)

Adaptation

Sea water *

Type of eels

Intact Hypophy-

sectomized

Net water flux Injection No. ($/cm2 per hr)

-

Saline 3 51.4 f 2.5 Saline 3 16.1 + 2.0

Fresh water Intact Saline 3 13.3 t 1.6 Intact ACTH (1 U/100 g) 3 39.2 + 7.7 Intact Cortisol (0.5 mg/lOO g) 4 54.8 f 7.4

* Adapted to sea water for 5 days.

DISCUSSION

It has been shown by experiments in vivo (Skadhauge & Maetz, 1967; Skad- hauge, 1969) and in vitro (Oide, 1967; M. Oide & Utida, 1967; Utida et al., 1967) that intestinal absorption of salt and water is augmented during sea water adapta- tion of the eel. When the hypophysis is removed, an augmentation in sea water- adapted eels is no longer observed (Hirano, 1967; Hirano et al., 1967), and a single injection of ACTH or cortisol into fresh water eels produces a significant increase in intestinal absorption (Hirano & Utida, 1968). Ouabain inhibits the salt and water movement across the intestine (Oide, 1967), and the activity of Na-K ATPase in the intestinal mucosa increases when the eels are adapted to sea water (Oide, 1967; Jampol & Epstein, 1970). Injections of cortisol to fresh water eels also increase intestinal Na-K ATPase activity (Epstein et al., 1971). Therefore, it has been suggested that the increase in Na-K ATPase activity induced by the ACTH-cortisol system is involved in the augmentation of the salt and water absorption in sea water-adapted eels.

On the other hand, it has been shown that sea water adaptation of the trout and the eel increases the intestinal alkaline phosphatase activity, and an activity

WATER ABSORPTION BY BBL5 ADAPTED TO SEA WATER 643

ratio with/without NaCl (500 mM) is greater in sea water-adapted or marine teleosts than in fresh water teleosts (Utida, 1967; Utida & Isono, 1967; Utida et al., 1968; Oide, 1970). Little attention has been paid so far to the fact that intestinal fluid of teleosts in sea water is alkaline (Smith, 1930). Recently, Hickman (1968) estimated that the pH value of rectal fluid taken from flounders was 8.24. The pH of intestinal fluid of sea water eels was 8.7, and the net water flux across the isolated intestine increased five- to six-fold by increasing the pH of the incubation media from neutral (pH 7.2) to alkaline (pH 9-O or 10-O). Not only water, but also sodium flux from mucosa to serosa increased when mucosal pH was changed from 7.2 to 9.0 ; from 13.2 + 0.9 to 22.7 f 0.7 p-equiv/cm2 per hr (N= 4), in non-everted intestines from sea water eels (M. Oide, unpublished). The solute-linked water flow is suggested by the mechanism of water absorption in the eel intestine (Skadhauge, 1969; Maetz, 1970).

When inhibitors of alkaline phosphatase such as EDTA, sodium borate and L-cysteine were added to the incubation medium at pH 9.0, the water flux across the intestine of sea water eels was inhibited by 68.8, 93.4 and 36.8 per cent, respectively. In previous work (Oide, 1970), these inhibitors decreased the activity of purified alkaline phosphatase from intestinal mucosa of sea water eels by 87.6, 77.6 and 94.8 per cent, respectively. In the case of L-cysteine, the degree of inhibition of water flux was much lower than that of enzyme activity. This may be due to the difference of pH value (10-O in the enzyme esti- mation and 9-O in water flux), since the inhibitory rate of alkaline phosphatase by L-cysteine depends upon the pH of the incubation medium (Agus et aZ., 1966). At any rate, the fact that water movement is suppressed by inhibitors of alkaline phosphatase suggests strongly the involvement of this enzyme in salt and water absorption from the intestine.

It is well known that intestinal alkaline phosphatase in teleosts is localized in the luminal border of intestinal epithelial cells (Al-Hussaini, 1949; Hollands & Smith, 1964). Water movement across the non-everted intestine from mucosa to serosa was higher when the mucosal medium was alkaline, as compared with the case that the serosal medium was alkaline (M. Oide, unpublished). On the contrary, the degree of inhibition by ouabain on salt and water movement in the eel intestine is much higher when it is in the serosal medium than in the mucosal medium (Oide, 1967), similar to what occurs in mammalian intestine (Schultz & Zalusky, 1964). In goldfish intestine, the same effect has also been reported (Smith, 1964), and the ATPase has been localized on the serosal side of the epithelial cells (Hollands & Smith, 1964). Since the degree of inhibition by ouabain in mucosal medium on water flux is around 45 per cent, whether its pH is 7.4 or 9.0, changes in pH of mucosal medium do not seem to influence the intestinal Na-K ATPase mechanism. When ouabain and EDTA were added to the mucosal medium (pH 9.0) at the same time, water flux was decreased to 88.5 per cent. The fact that this value is higher than the degree of inhibition by ouabain or EDTA alone indicates that sodium and water absorption of the intestine is accelerated by both Na-K ATPase and alkaline phosphatase.

644 MOMOKO OIDE

In the present study, the water flux at pH 7.2 was very low in fresh water eels (3.0 &cm2 per hr), and increased to 7.7 &cm2 per hr in sea water eels. This increase in water flux at pH 7.2 seems to depend on Na-K ATPase. However, the intestine of sea water eels contains alkaline fluid (around pH 9) and, in this condi-

tion, the water flux increased greatly up to 43 $/cm2 per hr. Since this large increase is conceived as dependent upon alkaline phosphatase activity, the participation of alkaline phosphatase in the enhancement of salt and water absorption seems to be more important than that of Na-K ATPase. In this connection, the increase in

intestinal Na-K ATPase in sea water eels (adapted for 7 days) is 2.5 times (Oide, 1967) or 2.0 times (Jampol & Epstein, 1970), whereas in alkaline phosphatase it is

5.5 times (Utida et al., 1968). In the present study using alkaline media, hypophysectomy abolished the

increase in intestinal water flux during adaptation of the eels to sea water. A single injection of ACTH or cortisol into fresh water eels enhanced the water flux up to the level of intact sea water eels. These results are similar to the results using isolated, non-ever-ted intestines incubated in Ringer solution at pH 7.4 (Hirano,

1967; Hirano & Utida, 1968). When the hypophysis is removed, an increase in the activity of intestinal alkaline phosphatase is no longer observed, presumably due to

the absence of ACTH and, hence, cortisol (Oide, 1973).

Acknowledgements-The author wishes to express her gratitude to Professor Seiitiro Utida, Ocean Research Institute, University of Tokyo, for valuable advice throughout this investigation, and also to Professor Howard A. Bern, University of California, for reading the manuscript.

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WATER ABSORPTION BY m ADAPTED TO SEA WATER 645

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Key Word Index--Alkaline phosphatase; intestinal mucosa; role in water absorption; eel; sea-water adaptation.