A neuropeptide Y (eNPY) was isolated from the intestinal extract of eels. This peptide enhanced significantly the serosa-negative transepithelial potential difference (PD) and short-circuit current (lsc) across the intestine of the seawater eel after pretreatment with isobutylmethylxanthine, serotonin and methacholine. The effects of eNPY on the lsc were concentration-dependent with a threshold concentration of 3×10−9 M and a maximal effect at 3×10−7 M. Similar concentration-responce curve was obtained by porcine peptide YY (pPYY). Since 9 amino acid residues are replaced in the pPYY, this result indicates that these substitutions do not change the potency and the efficacy. These stimulatory actions of eNPY were not blocked by tetrodotoxin, an inhibitor of neural firing, or yohimbine, an α2-adrenoceptor antagonist, indicating that eNPY acts without enteric neural firing or catecholamine release. When eNPY and adrenaline (AD) were applied simultaneously, the effects were additive only at lower dosage (3×10−8 M for eNPY, 3×10−8 M for AD), but not at high dosage (10−6 M eNPY, 10−7 M AD). The ceiling effect at high dosage suggests that these two regulators act through common signal transduction systems and affect the Na -K -Cl− cotransport system, since both effects were completely blocked by bumetanide, a specific inhibitor of Na -K -Cl− cotransporter.
INTRODUCTION
Regulation of ion and water transport across the intestine is essential for survival in sea water and fresh water of euryhaline teleosts such as eel and flounder. However, only a few regulators have been proposed in the intestinal ion transport of these teleosts. As an inhibitor, vasoactive intestinal peptide (O'Grady and Wolters, 1990; Uesaka et al., 1995) and atrial natriuretic peptide (Ando et al., 1992; O'Grady et al., 1985) have been proposed in both eel and flounder. In the eel intestine, acetylcholine and serotonin (5-HT) (Mori and Ando, 1991) have also been reported to reduce NaCI absorption. In contrast, as an enhancer, only catecholamines have been reported to enhance the short-circuit current (lsc) from serosa to mucosa across the intestine of the seawater eel (Ando and Kondo, 1993; Ando and Omura, 1993). The lsc is due to active Cl− transport from mucosa to serosa across the intestine of these fish (Ando et al., 1975; Huang and Chen, 1971).
To find out another endogenous regulators in the eel intestine, we tried to isolate peptides from the intestinal extract, and found previously that various somatostatin-related peptides enhanced the lsc, accompanied by an enhancement in NaCI and water absorptions (Uesaka et al., 1994a, b). During the isolation process, other bioactive fractions which enhance the lsc were also obtained.
The present study was aimed to characterize one of such bioactive peptides. A neuropeptide Y-related peptide (eNPY) was isolated as such a regulatory peptide. In the present study, the effects of eNPY on intestinal ion transport were also examined, in relation to the adrenaline (AD) actions.
MATERIALS AND METHODS
Intestinal extraction and peptide purification
A boiled-water extraction of 100 eel intestine (199 g wet weight), from which pancreas was removed, was prepared by the method described previously (Uesaka et al., 1994b). The extract enhanced the transepithelial potential difference (PD) across the intestine of the seawater eel. The bioactive material was isolated from the extract with Sep-Pak C18 cartridges (Millipore, Milford, MA). The retained material was further purified by reverse-phase (C8P-50, Asahi Chemical, Kanagawa) and cation-exchange (CM-5PW, Tosoh, Tokyo) HPLC. In all reverse-phase steps, elution was performed with a linear gradient of 0-70% acetonitrile containing 5% 2-propanol and 0.1% trifluoroacetic acid (TFA) for 70 min. In cation-exchange step, elution was performed with a linear gradient of 0-0.5 M NaCI in 10% ethanol and 20 mM phosphate buffer (pH 6.7) for 50 min. The final purification was performed with HPLC on C18 column (ODS-120T, Tosoh), eluted isocratically using 26% acetonitrile containing 5% 2-propano! and 0.1% TFA.
Peptide structural analysis
For amino acid analysis, a small portion of the purified peptide was dried down in tube. The tube was placed into a hydrolysis chamber with 6 N HCl containing 1% phenol and heated to 110°C for 20 hr. After cooling, the amino acids were analyzed on PICO-TAG amino acid analyzer (Millipore). Amino acid sequence of the purified peptide was determined by automated Edman degradation with a gas-phase sequencer (PPSQ-10, Shimadzu, Kyoto).
Measurement of electrical parameters
The cultured Japanese eels, Anguilla japonica, weighing about 210 g, were kept in sea water (20°C) for more than 1 week. After decapitation, the posterior part of the intestine was excised rapidly and the outer muscle layers were stripped off according to previous methods (Ando and Kobayashi, 1978). The mucosal segment was opened and mounted on an Ussing chamber with an exposed area of 0.5 cm2. The intestinal sheet was bathed in Krebs bicarbonate Ringer's solutions consisting of (mM): 118.5 NaCI, 4.7 KCl, 3.0 CaCl2, 1.2 MgS04, 1.2 KH2P04, 24.9 NaHC03, 5 glucose and 5 alanine. The bathing solution (3 ml each) was bubbled with 95% 02-5% C02 gas mixture (pH 7.4) at 20°C. Details are described elsewhere (Uesaka et al., 1994b)
The PD was recorded through a pair of calomel electrodes with a polyrecorder (EPR-151A, Toa, Tokyo) as a serosal potential with respect to the mucosa. Rectangular pulses, 30 μA for 500 msec, were applied across the tissue every 5 min. The tissue resistance (Rt) was calculated from the deflection of the PD (Rt = ΔPD / 30 μA). The short-circuit current (lsc) was obtained from the ratio of PD to Rt (lsc = PD / Rt). intestinal extract and synthesized peptides were applied to the serosal bathing medium after pretreatment with isobutylmethylxanthine (IBMX), serotonin (5-HT) and methacholine (MCh).
Reagents
Eel neuropeptide Y (eNPY) for this study was synthesized automatically using a Nα-9-fluorenylmethoxycarbonyl protection strategy (PSSM-8, Shimadzu, Kyoto) and purified by HPLC on C18 column (TSKgel ODS-120T). Acetyl-β-methylcholine bromide (MCh), (−)adrenaline (AD), 5-hydroxytryptamine creatine sulfate (5-HT), 3-isobutyl-1-methylxanthine (IBMX), porcine peptide YY (pPYY), tetrodotoxin (TTX) and yohimbine HCl were purchased from Sigma Chemical (St. Louis, MO). Bumetanide was kindly gifted from Sankyo Co. (Tokyo).
RESULTS
Isolation and characterization of eel neuropeptide Y
A bioactive material was isolated from the intestinal extract. The purified substance enhanced the serosa-negative PD across the seawater eel intestine after pretreatment with IBMX, 5-HT and MCh. Without such pretreatment, its effects varied among preparations (data not shown).
By sequence analysis, the bioactive substance was found to be a peptide with the following amino acid sequence; TyrPro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Ser-Pro-GluGlu-Gln-Ala-Lys-Tyr-Tyr-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-LeuIle-Thr-Arg-Gln-Arg-Tyr. The quantitative amino acid analysis gave the following amino acid composition in this peptide (residues/mol peptide); Asx 3.0; Glx 5.5; Ser 1.3; Gly 1.4; His 1.3; Arg 2.9; Thr 2.6; Ala 3.2; Pro 5.4; Tyr 4.7; lle 2.0; Leu 2.0; Lys 1.9. This confirms the primary structure obtained by the sequence analysis. On the basis of these results, the isolation yield from 199 g of guts was 15.5 μg (3.6 nmol). However, the same sequence had already been reported as eel NPY-related peptide (eNPY) by Conlon et al. (1991). They isolated it from the pancreas of American eels. Since the pancreas eNPY is a-amidated, we synthesized C-terminally amidated eNPY. The synthesized peptide was eluted at the same retention time of the native eNPY both in a reverse-phase and a cation-exchange HPLC (Fig. 1). Since C-terminally free eNPY elutes more fast than the amidated eNPY on the cation-exchange HPLC, this indicates that the native eNPY is amidated C-terminally. Therefore, the following experiments were all performed by using the synthesized C-terminally amidated eNPY.
Fig. 1.
Comparison of chemical characteristics between native (N) and synthetic (S) eel neuropeptide Y (eNPY). (A) Reverse-phase chromatograms of N, S and N+S. Each peptide and thier mixture were eluted with a 30-min linear gradient of 22%-28% acetonitrile in 5% 2-propanol and 0.1% TFA at a flow rate of 0.5 ml/min. (B) Cation-exchange chromatograms with CM-5PW column. N, S and N+S were eluted with a 20-min linear gradient of 0.3-0.7 M NaCl in 10% 2-propanol and 20 mM phosphate buffer (pH 6.7). Flow rate was 0.5 ml/min.
Effects on the eel intestine
Figure 2A shows the effects of eNPY in the presence of TTX. Even after blocking neural firing with TTX (10−6 M), eNPY (3×10−7 M) enhanced the serosa-negative PD and lsc significantly (ΔPD=4.6±0.6 mV, Δlsc=25.5±5.9 μA/cm2, n=5, P<0.05). But the effect on Rt was not significant (ΔRt=11.7±4.8 Ω cm2). As similar enhancements in the PD and lsc were observed after AD in this preparation (Ando and Omura, 1993), we examined a possibility that eNPY acts through AD release. However, as shown in Fig. 2B, even after blocking AD (10−8 M) actions completely with yohimbine (5×10−6 M), an α2-adrenoceptor antagonist, eNPY (3×10−7 M) enhanced the PD and lsc significantly (ΔPD=6.1±1.1 mV, Δlsc=38.9±7.6 μA/cm2, n=5, P<0.05) but not significantly in case of Rt (ΔRt=36.7±14.9 Ω cm2). Table 1 summarizes the effects of eNPY on these three electrical parameters after pretreatment with IBMX, 5-HT and MCh.
Fig. 2.
Independent action of eNPY from neural firing and catecholamine release. (A) Effects of eNPY on the PD (•), lsc (▪) and Rt (▴) in the presence of tetrodotoxin (TTX). At the first arrows, IBMX (10−6 M), 5-HT (10−6 M) and MCh (10−6 M) were added to the serosal fluid. After pretreatment with TTX (10−6 M, horizontal bar), eNPY (3×10−7 M) was added to the serosal bathing medium (second arrows). (B) Effects of eNPY on the PD (•), lsc (▪) and Rt (▴) after pretreatment with yohimbine. In the presence of yohimbine (5×10−6 M, horizontal bar), AD (10−8 M) was added to the serosal medium (second arrows), and eNPY (3×10−7 M) at the third arrows. Tracing is representative of four experiments.
Table 1.
Effects of eel neuropeptide Y (eNPY) on the transepithelial potential difference (PD), short-circuit current (lsc) and tisssue resistance (Rt) after pretreatment with IBMX, 5-HT and MCh
The stimulatory effects of eNPY on lsc were concentration-dependent, with a threshold concentration of 3×10−9 M and a maximal effect at 3×10−7 M (Fig. 3). In the next experiment, effect of porcine PYY (pPYY), 75% identity to eNPY, was compared with that of eNPY. As shown in Fig. 3, the concentration-response curve of pPYY were almost identical to that of eNPY.
Fig. 3.
Concentration-response curve for the effect of eNPY on the lsc across the seawater eel intestine. The increase in the lsc (Δlsc) after addition of eNPY (•) was plotted against concentrations (on a logarithmic scale). For comparison, effect of porcine peptide YY (pPYY) was also plotted (▴). Each point and vertical bar indicate the mean value and S.E.M. (n=5). Primary structures of these peptides are shown above with a single letter code.
When bumetanide (10−6 M), a specific inhibitor of Na+-K+Cl− cotransporter, was added to the mucosal fluid after pretreatment with IBMX, 5-HT and MCh, the serosa-negative PD and lsc were further diminished (−4.8±0.3 to −0.9±0.2 mV for PD (n=5), P<0.05, −53.9±7.8 to −11.2±2.7 μA/cm2 for lsc (n=5), P<0.05). In the presence of bumetanide (10−6 M), the effects of eNPY were completely blocked (Table 2). AD actions were also blocked by mucosal bumetanide (data not shown).
Table 2.
Effects of eNPY after pretreatment with bumetanide. In the absence of bumetanide (control), eNPY enhanced the serosa-negative PD and lsc (denoted by an increase in negativity in ΔPD and Δlsc), but bumetanide blocked these eNPY actions completely.
Interaction between eNPY and AD
As shown in Table 3, eNPY and AD enhanced the lsc similarly. When the supramaximal doses of eNPY and AD were applied simultaneously, the combined effect was not significantly different from that of eNPY alone or AD alone. On the other hand, when lower concentrations of eNPY (3×10−8 M) and AD (3×10−9 M) were applied simultaneously, the enhancement in lsc was significantly greater (P<0.05) than that induced by eNPY alone or AD alone, i.e. both effects were additive.
Table 3.
Comparison of the effects of eNPY and AD on the lsc across the seawater eel intestine. All values are expressed as an increase in negativity of the lsc after eNPY, AD or their mixture.
DISCUSSION
The present study is the first report to show the effect of eNPY in an homologous system. Eel NPY enhanced the lsc across the intestine in a concentration-depended manner (Fig. 3). Since the lsc is due to active Cl− transport from mucosa to serosa (Ando and Utida, 1986; Ando et al., 1975) and water follows this transport (Ando, 1975; Ando et al., 1986, 1992; Mori and Ando, 1991; Uesaka et al., 1994b), Cl− and water absorption will be enhanced by eNPY. The present study also demonstrates that replacement of Pro3, Asn7, Glu17, Ala18, Lys19, Thr22, Ala23, IIe28, IIe31 in the eNPY with Ala3, Ala7, Leu17, Ser18, Arg19, Ala22, Ser23, Leu28, Val31, respectively, does not change the potency and the efficacy, since eNPY and pPYY had similar effect on the lsc (Fig. 3).
Similar enhancement in lsc is also induced by AD (Table 3) (Ando and Omura, 1993). However, the effect of eNPY is not due to AD release, because the eNPY actions were not blocked by yohimbine (Fig. 2), a potent antagonist for AD action in the seawater eel intestine (Ando and Omura, 1993). In addition, the effects of eNPY were not blocked by TTX (Fig. 2), suggesting that the peptide does not act on neurons. Together, eNPY might act directly on the intestinal epithelium.
Although eNPY and AD had similar effect on the lsc, the dose-response curve was more steep in case of AD; threshold concentration of 10−9 M and a maximal effect at 3×10−8 M (Ando and Omura, 1993). When eNPY and AD were applied simultaneously, the effects were additive only at low dosage, but not at high dosage (Table 3). If eNPY and AD act through a common signal transduction pathway such as cAMP, these results can be explained. Although details of such signal transduction pathways are not clear yet in case of eNPY and AD, both regulators may enhance the Na+-K+-Cl− cotransport finally, since the effects of eNPY and AD were completely blocked by bumetanide, a specific inhibitor of the Na+-K+-Cl− Chcotransporter. In mammals, coexistence of NPY and catecholamines have been observed in some sympathetic neurons (Wahlestedt et al., 1986; Wiley and Owyang, 1987). If eNPY coexists with AD in the enteric nervous system of the eel, it will accelerate AD action and stimulate the Cl− absorption at lower concentrations, though a possibility that circulating AD regulates the Cl− absorption hormonally is still remained.
The experimental condition used in the present study was determined after several trials to obtain a constant response (Ando and Omura, 1993). Under such condition, eNPY (present study), somatostatins (Uesaka et al., 1994a, b; Uesaka et al., 1995) and catecholamines (Ando and Kondo, 1993; Ando and Omura, 1993) enhance the lsc in a concentration-dependent manner without exceptions. Probably these regulators may antagonize the inhibitory effects of 5-HT or ACh.
Although eNPY enhances Cl− transport across the seawater eel intestine, mammalian NPY inhibits Cl− transport across the rat intestine (Cox et al., 1988). The discrepancy of the effect of NPY may be due to different characteristics between these preparations. Both tissues contain the loopdiuretic sensitive Na+-K+-Cl− cotransporter. However, the localization of the transporter is opposite; on the brushborder membrane of the epithelium in the seawater eel (Ando and Utida, 1986) vs on the basolateral membrane in mammals (see Ref. Anderson et al., 1992). Recently, it is clarified from cDNA cloning of various Na+-K+-Cl− cotransporters that molecular structure of these cotransporters varies considerably depending on their localization, apical or basolateral (Soybel et al., 1995).
Acknowledgments
This research was supported in part by Grants in Aid no. 02804062 from the Ministry of Education, Scinece and Culture, Japan, and no. 9233 from Salt Science Research Foundation, Japan.
