Intravenous administration of eel angiotensin II (eANG II), histamine (HA), serotonin (5-HT), acetylcholine (ACh) or carbachol (CCh), mammalian substance P (mSP) and isoproterenol (β-adrenoceptor agonist) enhanced drinking rate in the seawater eels. The dipsogenic effects of HA and 5-HT seem to be due to ANG II synthesis, because these effects were completely blocked by captopril, an inhibitor of angiotensin converting enzyme (ACE). Captopril blocked eANG I effect, but not eANG II effect, suggesting existence of ACE in seawater eels. 800 μl Hemorrhage also enhanced water intake, and this effect was completely blocked by captopril. Therefore, it is likely that blood withdrawal stimulates renin-angiotensin system (RAS) in seawater eels. Effects of ACh, CCh and mSP were not inhibited by captopril, suggesting separate action of these regulators from ANG II synthesis. Isoproterenol action was partially inhibited by captopril, suggesting existence of some β-adrenoceptors other than the RAS. On the other hand, intravenous eel atrial natriuretic peptide (eANP), arginine vasotocin (AVT), human vasoactive intestinal peptide (hVIP), mammalian bradykinin (mBK), eel intestinal pentapeptide (EIPP), cholecystokinin (CCK-8), and phenylephrine (α-adrenoceptor agonist) depressed the drinking rate. In the presence of mBK, HA and 5-HT enhanced water intake similarly as in the absence of mBK. Plasma hyperosmolarity also reduced drinking. Although the in vivo system is so complicated and many regulators are involved in the drinking behavior, a possible regulatory mechanisms are proposed. Compared to mammalian results, eels seem to be a suitable model for anlayzing drinking mechanisms in vertebrates.
Maintenance of blood homeostasis is essential for life of vertebrates. Especially, water intake is most important for terrestrial vertebrates or seawater teleosts. However, the mechanisms how the drinking is controlled are complicated and not clear yet even in mammals (Bourque et al., 1994; Fitzsimons, 1998). Looking from another viewpoint, the obscurity in mammals may be due to complexity in their drinking behavior. After perception of thirst, they must seek for water, ingest water into their mouth, and finally swallow. For analyzing drinking mechanisms, more simple model may be needed. As a candidate for such model, fishes will be suitable, because they hold water constantly in their mouth for respiration. They can swallow immediately after perceiving thirst, and may skip seeking and ingestion.
Among fishes, eels are a suitable subject for study, because their drinking rate can be measured continuously using esophagus cannulations as developed by Hirano (1974). However, his system is for fresh-water eels and not for sea-water eels. Seawater eels must absorb water from the diluted sea water in the intestine. Without water absorption across the intestine, the seawater eels die (Takei et al., 1998). Therefore, a new system was developed by Takei et al. (1998). Simplifying their method, effects of various regulators on the drinking behavior were examined in this study. The present study was aimed to classify various regulators into dipsogens and antidipsogens. Most dipsogens in mammals also enhanced the drinking rate in eels. Furthermore, in the present study, interactions between these dipsogens were examined pharmacologically.
MATERIALS AND METHODS
Cultured Japanese eels, Anguilla japonica, weighing about 200 g were obtained from a commercial source. They were kept in sea-water aquaria at 20°C for more than 1 week before use without food. For measurement of water intake, the esophagus was cannulated with a vinyl tube (o.d. 2.0 mm) as described previously (Ando and Nagashima, 1996). The cannula was connected to a drop counter (PG-602, Keyence, Osaka, Japan) for continuous recording of the drinking rate. Each drop was recorded as a spike on a chart recorder (EPR-121A, TOA, Tokyo, Japan). One drop was 27.8 μl. Another tube (o.d. 1.1 mm) was inserted into the stomach to apply 0.2 mol l−1 NaCl solution, which was determined from the NaCl concentration at the esophagogastric junction in seawater eels (Ando and Nagashima, 1996). The perfusion through the cannula was made with a peristaltic pump (MS-1 Reglo 160, Ismatic, Zurich, Switzerland) controlled by an electric stimulator (SEN-3201, Nihon Koden, Tokyo), which was connected to the drop counter. Through this system, the swallowed sea water can be reintroduced into the stomach.
A third cannula (SP-10, Natume, Tokyo) filled with heparinized saline (100 IU/ml) was inserted into the posterior cardinal vein. Through the cannula, various regulators were administered into the blood. All reagents were dissolved in 0.9% NaCl solution (vehicle), and 100 μl of their appropriate concentrations was injected slowly (100 μl/ min) into the vein, followed by an injection of 50 μl vehicle to push out whole reagent remaining in the cannula. In case of administration of hyperosmotic NaCl and sucrose solutions, 200 μl was injected for 2 min. 200 μl injection of vehicle at the same rate did not alter the drinking rate significantly. 800 μl blood was withdrawn from the posterior caudal vein with microinjector (IM-1, Narishige, Tokyo) for 30 min.
After operation, the incision was closed using silk suture and all cannulae were fixed to the body with threads, then the eels was transferred to a plastic trough of the same size as the eel. Well-aerated sea water was circulated continuously through the trough at room temperature (20–23°C).
Eel angiotensin II ([Asp1, Val5]ANG II, [Asn1, Val5]ANG II), [Sar1, Ile5, Ala8]ANG II, [Sar1, Ile5,8]ANG II, non-sulfated cholecystokinin-related peptide (CCK-8), human vasoactive intestinal peptide (hVIP), mammalian substance P (mSP), and mammalian bradykinin (mBK) were obtained from Peptide Institute Inc., Osaka, Japan. Tricain methanesulfonate (MS222), Captopril, cimetidine, cyproheptadine, 5-hydroxytriptamine creatine sulfate (5-HT), phenylephrine hydro-chloride, carbamylcholine chloride (CCh), acetylcholine (ACh), and sheep prolactin (sPRL) were purchased from Sigma, St. Louis, USA. Histamine dihydrochloride, thioperamide maleate, PD 123319 ditrifluoroacetate, CGP 42112 were obtained from Research Biochemicals International, Natick, USA. Eel angiotensin I ([Asn1, Val5, Gly9]ANG I) and angiotensin III ([Val4]ANG III) were purchased from Peninsula Laboratories, Belmont, USA. Heparin sodium (Katayama Chemical, Osaka), isoproterenol hydrochloride (Nakarai Tesque, Kyoto, Japan) and Atropine sulfate (Merck, Darmstadt, Germany) were purchased commercially. Losartan potassium (Banyu, Tokyo, Japan), Exp 3174 (Dupont, Wilmington, USA) and CV11974 (Takeda Chemical Industries, Tokyo) were kindly supplied. Eel atrial natriuretic peptide (eANP) was kindly gifted by Prof. Y. Takei, Ocean Research Institute, University of Tokyo.
Statistical analyses of results were performed using Mann-Whitney U-test. Results are given as mean±S.E.M. and considered significant at P<0.05.
Effect of various eel angiotensins
The operated eels started drinking sea water immediately after recovery from anesthesia. The drinking rate was high initially but reached a relatively steady level after 20 hr. Thus all experiments were started 20 hr after operation. When 0.9% NaCl solution was injected into the vein, the drinking rate was not significantly altered (0.21±0.08 ml/15 min vs 0.19±0.05 ml/15 min, n=5).
Although eels have two kinds of angiotensin II (eANG II), [Asp1]eANG II and [Asn1]eANG II (Hasegawa et al., 1983), both peptides had qualitatively similar effects. After administration of eANG II, the drinking rate was raised initially for 15–30 min, and then inhibited for more than 1 hr. Similar biphasic effect of ANG II has been reported in freshwater and 1/3 seawater eels (Takei et al., 1979; 1988). Fig. 1 shows comparison between the dipsogenic effects of these 2 peptides. [Asn1]eANG II was nearly 10 times potent than [Asp1]eANG II. Eel ANG III (Arg-Val-Tyr-Val-His-Pro-Phe) also enhanced water intake initially, but the potency was 1/100 compared to [Asn1]eANG II (data not shown). The dipsogenic effect of [Asn1]eANG II was not inhibited by cimetidine nor atropine.
Eel ANG I (Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Gly-Leu) also enhanced drinking rate similarly as [Asn1]eANG II. However, this effect of eANG I was completely blocked by captopril (10−6 mol), an inhibitor of angiotensin converting enzyme (ACE)(Fig. 2).
To determine receptor type bound with eANG II, various ANG II antagonists were pretreated before application of [Asn1]eANG II. However, neither AT1 receptor antagonists (saralasin, losartan, Exp 3174, CV 11974) nor AT2 receptor antagonists (PD 123319, CGP42112) blocked the [Asn1]ANG II effect (data not shown). These results confirm previous observations in fishes (Nishimura et al., 1978; Tierney et al., 1997).
Effect of other dipsogens
Beside angiotensins, histamine (HA), serotonin (5-HT), isoproterenol (β-adrenoceptor agonist), acetylcholine (ACh), carbachol (CCh, a cholinergic agonist), and mammalian substance P (mSP) also enhanced drinking rate (Table 1). Similar dipsogenic effect was observed after injection of sheep prolactin (sPRL, 10−9 mol) (data not shown). The dipsogenic effect of HA was dose-dependent from 10−12 to 10−7 mol, and was completely blocked by cimetidine, a H2 receptor antagonist (Fig. 3b). Similar blockage was also observed after treatment with thioperamide, another H2 receptor antagonist, but not with cyproheptadine, a H1 receptor antagonist. These results indicate existence of H2-type receptor in the eel. After treatment with captopril, the effect of HA was also blocked completely (Fig. 3c). Even in the presence of cimetidine, dipsogenic effect of [Asn1]eANG II still remained, suggesting that ANG II does not act through HA release.
Effects of various dipsogens on water intake in seawater eels. Water intake was measured for 15 min before and after administration of dipsogens
After administration of 5-HT (10−8–10−7 mol), the drinking rate was enhanced initially, followed by an inhibition (Fig. 4a). This dipsogenic effect of 5-HT was also completely blocked by captopril (Fig. 4b), suggesting that 5-HT acts through ANG II synthesis. The 5-HT effect was not inhibited by cimetidine (Fig.4c).
The effect of CCh (10−9–10−8 mol) was not inhibited by captopril (10−6 mol), but inhibited by atropine (10−6 mol) (data not shown), suggesting existence of muscarinic ACh receptor in the eel. The dipsogenic effect of mSP (10−9–10−8 mol) was not inhibited by captopril (10-6 mol), and the effect of isoproterenol (10−9–10−8 mol) was only partially inhibited by captopril (10−6 mol) (data not shown).
Effect of blood volume and osmolarity
When 800 μl blood was withdrawn from the posterior cardinal vein, drinking rate was enhanced. However, the effect of blood withdrawal was inhibited by a pretreatment with captopril (10−6 mol). Figure 5 shows effect of hemorrhage in the absence or presence of captopril. In the presence of captopril, hemorrhage did not enhance the water intake at all.
When concentrated NaCl or sucrose solution was injected into the vein, the water intake decreased. The decrease was dose-dependent (Fig. 6).
Effect of antidipsogens
Eel atrial natriuretic peptide (eANP, 10−10–10−8 mol), argi-nine vasotocin (AVT, 10−10–10−8 mol), and eel intestinal pentapeptide (EIPP, Gly-Phe-Trp-Asn-Lys, 10−9–10−8 mol) isolated from eel intestine (Uesaka et al., 1991) inhibited the drinking rate (Fig. 7). Similar antidipsogenic effect was observed after cholecystokinin (CCK-8, 10−9–10−8 mol), phenylephrine (α-adrenoceptor agonist, 10−9–10−8 mol), human vasoactive intestinal peptide (hVIP, 10−10–10−8 mol), and mammalian bradykinin (mBK, 10−10–10−8 mol), Even after pretreatment with 10−8 mol mBK, HA and 5-HT enhanced the drinking rate similarly as in the absence of mBK (data not shown).
The drinking rate in seawater eels was enhanced by eANG II, HA, 5-HT, ACh (CCh), mSP, sPRL and isoproterenol, while inhibited by eANP, AVT, hVIP, EIPP, mBK, CCK-8 and phenylephrine (Table 2). Since these dipsogens, except for SP, are also known to increase water intake in mammals (Fitzsimons, 1998), the drinking behavior in the seawater eels may be controlled similarly as in mammals. ANP is also known to inhibit water intake induced by dehydration or ANG II in the rat (Antunes-Rodrigues et al., 1985). However, the effect of ACE inhibitor (captopril) is reverse: inhibitory in eels (present study) and stimulatory in mammals (Fitzsimons, 1998).
Regulators affecting drinking behavior in seawater eels
Among dipsogens examined in the present study, HA and 5-HT seem to act through ANG II synthesis, since the effects of HA and 5-HT were completely blocked by a pretreatment with captopril, an ACE inhibitor. Although captopril is also known to inhibit kininase thus increase kinin levels (Campbell, 1987, Olson, 1992), high concentration of mBK did not inhibit HA and 5-HT actions in the eels. The existence of ACE in the seawater eels is supported by a result that eANG I effect is blocked by captopril but eANG II effect not. Renin-angiotensin system (RAS) in teleosts has been demonstrated previously (see Olson, 1992). Since RAS exists in eels (Sokabe and Ogawa, 1974; Henderson et al., 1976; Tierney et al., 1995), the hemorrhage may activate the RAS, thus activate ANG II synthesis, and the synthesized ANG II may enhance drinking (Fig. 8). The blockage of hemorrhage-induced drinking by captopril supports this explanation. Increased plasma ANG II level after hemorrhage is already observed in eels (Takei, 1988). ACh and SP seem to act separately from RAS, since the effects of these regulators are not inhibited by captopril. β-Adrenoceptors may exist in RAS and in others, since the effect of isoproterenol is only partially inhibited by captopril. Although the present results appear to suggest that captopril inhibits RAS, other sites for captopril action can not be ruled out.
ANG II receptor in eels seems to be distinct from mammalian types, since mammalian AT1 and AT2-receptor antagonists did not inhibit the eANG II action. Similar no antagonistic action of salarasin or losartan has been observed in eels (Nishimura et al., 1978) or elasmobranchs (Tierney et al., 1997). N-terminal Asn in the eel ANG II seems to be important for the dipsogenic action of the peptide, since replacement of Asn with Asp([Asp1]eANG II) or deletion of Asn (eANG III) lowers dipsogenic potency.
The effects of eANG II were always biphasic, initial dipsogenic and secondary antidipsogenic. Because in vivo system is so complicated, many factors may be involved in the biphasic phenomena. ANG II may stimulate catechola-mine release and make hypertension as in other teleosts (Platzack et al., 1993; Perry et al., 1999), and the hypertension may inhibit the drinking as observed by Hirano and Hasegawa (1984). However, it can be explained simply by a secretion of antidipsogens. Antidipsogenic effect of ANP has been reported in eels previously (Takei and Balment, 1993; Tsuchida and Takei, 1998). In addition, eel plasma ANP level is elevated after infusion of ANG II (Tsuchida and Takei, 1999).
In contrast to mammalian (Fitzsimons, 1979), avian (Kaufman and Peters, 1980; Kobayashi and Takei, 1982) and reptilian (Fitzsimons and Kaufman, 1977) cases, plasma hyperosmolarity reduces water intake in seawater eels. Similar inhibition by hyperosmolarity has been reported in freshwater and 1/3 seawater eels (Takei et al., 1979; 1988). The mechanisms how the plasma hyperosmolarity depresses drinking rate in the eel are not known yet. Administration of hypertonic solution into eels increases plasma ANG II level (Takei et al., 1988). However, enhanced secretion of antidipsogens, such as ANP, AVT, VIP, BK, catecholamines or EIPP, might explain this phenomenon simply. In fact, plasma hyperosmolarity increases plasma ANP level in eels (Kaiya and Takei, 1996). Although the mechanisms are not clear yet, this phenomenon seems to be significant physiologically, especially in seawater eels, because the lowered drinking rate accelerates desalination of the ingested sea water through their esophagus, more diluted sea water enters into the gastrointestinal tract (Ando and Nagashima, 1996), which enhances water absorption across the intestine (Skadhauge, 1969).
Previously, we demonstrated that intestinal Cl− reduced drinking rate in seawater eels and suggested involvement of humoral mediator(s) released from the intestine and acting on the brain (Ando and Nagashima, 1996). EIPP can be a candidate for such mediators, because the peptide is isolated from the eel intestine (Uesaka et al., 1991) and reduces drinking rate (present study).
This research was supported by Grants-in-Aid no. 08640864 from the Ministry of Education, Sciences and Culture, Japan, and also by the Fisheries Agency of Japan.