A New Technique for Measuring Water Transport Across the Seawater Eel Intestine

It is well known that marine teleosts ingest sea water and absorb water together with monovalent ions from the intestine to compensate for water loss from the body (Smith, 1930). Such solute-linked water transport has been demonstrated in the isolated seawater eel intestine in vitro (Oide & Utida, 1967; Utida et al. 1972; Ando, 1975, 1980, 1981, 1983, 1985), where the net water flux was measured gravimetrically using an intestinal sac (Wilson & Wiseman, 1954). However, for elucidating mechanisms of water transport more precisely by altering ionic composition, this method has the defects that the inside of the intestinal sac is not stirred and the ionic concentration of the inside solution tends to change gradually with time.

In highly water-permeable epithelia such as small intestine, the relatively small net water flux can hardly be obtained from the difference between larger unidirectional fluxes of labelled water. Besides the gravimetric method, two other methods have been employed. One method involves monitoring the water level inside a small capillary tube connected to one side of the Ussing chamber, using either optical measurements (Rüphi, De Sousa, Favrod-Coune & Posternak, 1972; Van Os, Michels & Siegers, 1976; Eldrup, Frederiksen, Møllgård & Rostgaard, 1982) or changes in electrical capacitance (Wiedner, 1976; Van Os, Wiedner & Wright, 1979). However, in these systems oxygen supply is limited and stirring may be insufficient, since one compartment of the Ussing chamber must be closed completely.

In the present study, by improving the perfusion method, the rate of net water flow is directly calculated from the difference between the rates of perfusate and effluent flow in the perfusion system, where both media are continuously supplied with sufficient oxygen. Using this apparatus, it is clearly demonstrated that water transport across the seawater eel intestine is stimulated by L-alanine but not by D-glucose.

Japanese cultured eels, Anguilla japonica, weighing about 200 g, were obtained from a commercial supplier and kept in seawater aquaria at 20 °C for more than 1 week before use. They were decapitated. The intestine was excised and the outer muscle layers were stripped off following our previous method (Ando & Kobayashi, 1978). A cylindrical polyester mesh (NBC Kogyo, T.N08OT) 6cm long was inserted into the middle part of the intestinal tube and both ends of the intestine were tied to polyethylene tubes (4 mm o.d.) with silk thread. After filling the inside of the intestinal tube with Ringer solution (approx. 1-1 ml), the preparation was set horizontally in a trough (30 ml) which was placed in a water jacket maintained at 20 °C.

Ringer solution was pumped into the tube via a damper (to eliminate pulsations) using a pump (FMI, RP-G6-0, usually; Iwaki, MM-1, otherwise) led from a constant-level reservoir. The damper was made from a three-way tube with one end closed as an air cell. The reservoir was filled by a peristaltic pump (EYELA, MP-3C). All connections were made with Teflon tubing (1mm i.d.). The temperature of the perfusate was adjusted to 20°C by placing the reservoir and the damper in water jackets maintained at 20°C.

Effluent was collected with fraction collector (EYELA, DC-20) with droplets of about 7 μl The collected water volume was measured gravimetrically by using a balance (Sartorius, 1601MP8) connected to a computer (NEC, PC-8801 mkll), and was printed out automatically (NEC, PC-8826) at intervals. After measuring the rate of effluent flow (V_e) through the intestine, the perfusion rate (V_i) of the pump itself was measured by collecting fluid directly from the damper. Net water flux was calculated by subtracting the perfusion rate from the rate of effluent flow; ⁠, where is the mean of each V, value. Perfusion could be maintained at a constant rate for more than 4h (Fig. 2). Therefore, changes in the rate of effluent flow can be regarded as changes in the net water flux across the intestine.

According to the previous perfusion method, net water flux was also calculated simultaneously by using [¹⁴C]PEG (New England Nuclear) which was counted with a liquid scintillation counter (Aloka, LSC-701). Collection of the effluent was started after a 90-min equilibration period.

Measurements of the transepithelial potential difference (PD) were made using 2% Ringer-agar bridges, calomel electrodes and a preamplifier (Nihon Kohden, MEZ-7101) connected to a polyrecorder (Toadempa, ERP-200A).

The standard Ringer solution contained (mmol l⁻¹): 118·5 NaCl; 4·7 KC1; 3·0 CaCl₂; 1·2 MgSO₄; 1·2 KH₂PO₄; 24·9 NaHCO₃; 5·0 glucose (pH 7·3). Glucose-free Ringer was identical to the standard Ringer solution with the omission of glucose. Both sides of the intestine were bathed with identical Ringer solutions and bubbled continuously with 95% O₂–5% CO₂ gas mixture throughout the experiments. Most experiments were started when the PD had almost reached a steady level. Ouabain (Merck), L-alanine, L-valine or L-glycine (Katayama Chemical Co., Osaka, Japan) was added to either side as indicated. At the end of the experiments, the intestine was cut longitudinally and spread on graph paper; the surface area of the intestine was measured using a planimeter (Ushikata, 220L). All data are expressed as mean ± S.E.

When the lumen of the non-everted intestine was perfused, the rate of effluent flow was less than the perfusion rate by 20 μl/10 min (negative water gain in Fig. 3), indicating that this amount of water was absorbed through the seawater eel intestine (mucosa to serosa). After everting the intestine and perfusing the serosal side, on the other hand, the net water flux from mucosa to serosa was 70 μl/10min. This difference in rate was also observed when measurements were made on the everted intestine before the non-everted intestine. The serosa-negative PD was nearly equal in both preparations. Since mucus secreted from the mucosa frequently stuck in the outlet tube in the non-everted intestine, the following experiments were all done only in the everted intestine.

The values of net water flux appear reliable for two reasons. First, net water flux and PD were abolished by the addition of 10⁻³ mol l⁻¹ ouabain to the serosal fluid for Ih, confirming previous results obtained in the sac preparation (Ando, 1981). Secondly, the net water flux was independent of perfusion rate (Fig. 4).

Even when perfusion was stopped completely by switching off the main pump and clamping the inlet tube into the intestine, effluent from the everted intestine was collected at a nearly constant rate (68·2 ± 7·7 μl cm⁻²h⁻¹; N = 7), corresponding to net water flux from mucosa to serosa. Values in Fig. 4 are expressed as a percentage of this level (initial standard flux). Even when perfusion rate was increased stepwise, the mean net water flux obtained at each perfusion rate, ⁠, was almost identical with the initial standard water flux if the perfusion rate was below 500 μl min⁻¹. However, when the perfusion rate was increased above 500 μl min⁻¹, the net water flux was decreased drastically, presumably due to a rise in hydraulic pressure of intestinal fluid. After these experiments, water flux was measured again under zero perfusion (final standard water flux), and those preparations whose final standard water flux was less than 70% of the initial one were discarded. Less than 20% of all preparations were adopted in this way. In most cases, net water flux reduced gradually with time, accompanied by a slight drop in the PD.

To compare this new method with the previous perfusion method, which uses a marker substance such as PEG, simultaneous measurements of flow rates and PEG concentration in each flow were made (Table 1). The concentration of PEG in the effluent was lower by 333·3 c.p.m./50 μl than that in the perfusate, and the total amount of PEG (the product of the concentration of PEG and the rate of effluent flow) was almost equal to that in the perfusate flow, i.e. V_eC_c = V_iC_i, if mean values obtained from 10 successive measurements during 100 min were used. Therefore, the mean water flux obtained by the present new method (202·9 μl/10 min) was close to that calculated from the ratio of PEG concentrations (210·7 μl/10 min), according to the previous perfusion method. When net water fluxes were calculated every 10 min, however, the values of net water fluxes obtained from the previous method were erratic, while relatively constant net water fluxes were obtained by the present new method (Fig. 5).

Net water flux and PD continued to decrease during perfusion, and were not increased after addition of adrenaline (10⁻⁸–10⁻⁴mol l⁻¹), noradrenaline (10⁻⁸–10⁻⁴ mol l⁻¹), phenylephrine (10⁻⁶–10⁻⁵mol l⁻¹), naphazoline (10⁻⁶–10⁻⁵mol l⁻¹), dopamine (10⁻⁶–10⁻⁴mol l⁻¹), cortisol (50 μgml⁻¹) or insulin (1 μgml⁻¹) into the serosal fluid (data not shown).

Addition of 5 mmol l⁻¹ L-alanine into the mucosal fluid increased both the net water flux from mucosa to serosa and the serosa-negative PD, after latent periods of 20–30 min for net water flux and about 10min for the PD (Fig. 6). Further addition of L-alanine (10 mmol l⁻¹) did not add to these increases, and 1 mmol l⁻¹ alanine did not induce any enhancements in these parameters (data not shown), implying an all- or-nothing type of response with a threshold concentration of about 5 mmol l⁻¹. Although an effect of serosal L-alanine (5 mmol l⁻¹) is not demonstrated in Fig. 6, a slight enhancement of the net water flux and the PD was induced by serosal L-alanine (5 mmol l⁻¹) in other preparations.

Since mucosal alanine might stimulate Na⁺ transport from mucosa to serosa and thus stimulate water transport, the effects of another stimulant of Na⁺ transport were examined and compared with those of L-alanine (Fig. 7). After the PD had reached a steady level in glucose-free Ringer solution, 5 and 10 mmol l⁻¹ glucose was added into the mucosal fluid, but no enhancement of net water flux or PD was observed.

When 5 mmol l⁻¹ was added into the mucosal fluid, however, the net water flux and the PD increased gradually, with latent periods as cited above.

To examine the specificity of the effect of L-alanine, other neutral amino acids, L-valine and L-glycine, were applied. These amino acids are known to be absorbed across the fish intestine (for a review, see Ferraris & Ahearn, 1984). As shown in Fig. 8, mucosal addition of 5 mmol l⁻¹ L-valine or 10 mmol l⁻¹ L-glycine did not enhance the net water flux significantly, and tended to increase the PD only negligibly. Responses to L-alanine were subsequently observed in these preparations.

Table 2 summarizes the effects of D-glucose (5 mmol l⁻¹) and L-alanine (5 mmol l⁻¹) on the PD and the net water flux. When both sides of the seawater eel intestine were bathed with nutrient-free Ringer solution, serosa-negative PD (−3·2 ± 0·3 mV) and the net water flux (58·1 ±3·5 μl cm⁻²h⁻¹) from mucosa to serosa were observed, although they tended to decrease with time. Similar results were obtained even in the presence of 5 mmol l⁻¹ D-glucose. On the other hand, when the Ringer solution contained 5 mmol l⁻¹ L-alanine, higher serosa-negative PD (−6·8 ± 0·6 mV) and net water flux (84·0 ± 6·0 μl cm⁻² h⁻¹) were obtained, and they stayed at a nearly steady level for more than 4h. Further addition of 5mmol l⁻¹ D-glucose to the alanine-containing bathing solutions did not significantly enhance these two parameters.

The present study reports a new technique for measuring net water flux across the intestine in vitro, which overcomes the defects of previous methods (see Introduction). In this new technique, net water flux was calculated directly from the difference between the rates of effluent and perfusate flow (⁠⁠) without measuring the concentration of a marker substance. The net water flux obtained in this way appears to be reliable : its direction is always from mucosa to serosa in both everted and non-everted intestines; ouabain, an inhibitor of ions and water transport across the seawater eel intestine (Ando, 1981, 1985), reduces it to zero (Fig. 3); it is almost identical with the standard water flux obtained under zero perfusion (Fig. 4) ; and it is identical with the net water flux obtained by the previous perfusion method, if mean values for 100 min are compared (Table 1). However, the values of net water flux obtained by the previous perfusion method were erratic, if they were calculated every 10 min (Fig. 5), presumably due to insufficient mixing of PEG as reported by Worning & Amdrup (1965). In contrast, the net water fluxes calculated every 10 min by this new method were relatively steady (Fig. 5). Therefore, it may be reasonable to conclude that the present simple method is more accurate than the previous complicated perfusion method. The new method is also convenient for controlling the ionic condition and oxygen supply to both sides of the intestine.

The net water flux was always much lower in the non-everted intestine than in the everted one (Fig. 3). With the non-everted intestine, the polyester mesh in the lumen may prevent solutes and water from arriving at the mucosal membrane of the epithelium; in the everted intestine, the boundary layer on the mucosal membrane is thinner, allowing transporting substances to reach the mucosal membrane more readily. The experiments with the non-everted intestine were also complicated by the accumulation of mucus in the outlet tube. (This intestine, compared with that in mammals, has numerous goblet cells: Ando & Kobayashi, 1978.)

The net water flux and the serosa-negative PD (explained by an active Cl⁻ transport from mucosa to serosa: Ando, Utida & Nagahama, 1975) were found to be insensitive to adrenergic agents or cortisol, although these substances are known to stimulate NaCl and fluid absorption across the mammalian intestine (reviewed by Berridge, 1983; Tapper, 1983; Turnberg, 1983). Instead, the PD and the water flux were stimulated by L-alanine. It is possible that the gradual decline in these parameters observed in standard saline may be due to the loss of L-alanine from the tissue.

L-Alanine had a greater effect on net water flux when it was applied on the mucosal rather than the serosal side. This suggests that Na⁺ transport stimulated by mucosal L-alanine may enhance water transport since transport of L-alanine and D-glucose are coupled with Na⁺ transport in the marine fish intestine (Eveloff, Field, Kinne & Mure, 1980; Bogé & Rigal, 1981) as in the mammalian intestine (see Schultz, 1979). However, D-glucose did not enhance the net water flux in the seawater eel intestine (Fig. 7; Table 2), although D-glucose is transported by the intestine of many fish (for a review, see Ferraris & Ahearn, 1984) including moray eel intestine (Ferraris & Ahearn, 1983). Moreover, other neutral amino acids such as L-valine and L-glycine, which are transported in the intestine of goldfish (Kitchin & Morris, 1971) and rainbow trout (Bogé, Rigal & Pérès, 1979), respectively, do not enhance the net water flux across the seawater eel intestine (Fig. 8). In addition, the enhanced serosanegativity in the PD after application of L-alanine cannot simply be explained by the stimulation of Na⁺ transport from mucosa to serosa. Therefore, the enhancement of the net water flux induced by L-alanine does not appear to be due to a stimulation of Na⁺ transport, but seems to be due to a specific action of L-alanine.

We are pleased to acknowledge the considerable assistance of Dr Akira Kawahara, Faculty of Integrated Arts and Sciences, Hiroshima University. This research was supported in part by Grant-in-Aid no. 587004 from the Ministry of Education, Sciences and Culture, Japan, and by funds from the Cooperative Program (nos 83119 and 84117) provided by The Ocean Research Institute, University of Tokyo.