ABSTRACT
To investigate the influence of neuropeptide Y (NPY) on heart function in a relatively simple model system, the effects of eel NPY (eNPY) on the eel atrium were examined. Eel NPY enhanced the contractile force of the isolated atrium in a dose-dependent manner, without altering the rate of contraction. Although adrenaline also exerts a positive inotropic effect, the effect of eNPY was not blocked by the β1-adrenoceptor antagonist betaxolol, indicating that eNPY does not act via adrenaline release. When eNPY and adrenaline were applied simultaneously, their effects were additive at lower concentrations but not at higher concentrations. The plateau reached at high concentrations suggests that these two regulators act through a common signal transduction process. One candidate for this is an elevation of the concentration of intracellular free Ca2+ ([Ca2+]i), since treatment with eNPY or adrenaline enhanced [Ca2+]i, as assessed by fluorescence of Calcium Green-1. The increase in [Ca2+]I after eNPY and adrenaline treatment is presumably due to Ca2+ influx from the external medium, since the effect was greatly reduced in Ca2+-free Ringer’s solution and after treatment with verapamil, a Ca2+ channel blocker. Although both eNPY and adrenaline enhanced the atrial contractile force, the time courses were different, with the effect of eNPY being gradual and long-lasting, and that of adrenaline being immediate and transient. It is plausible, therefore, that eNPY and catecholamine(s) act synergistically to cause a long-lasting enhancement of contraction force if the two regulators arrive at the atrium simultaneously. The present study also demonstrates that the eel heart contains eNPY.
Introduction
Since the discovery of neuropeptide Y (NPY) in porcine brain (Tatemoto, 1982), NPY-like immunoreactivity has been detected in both the central and peripheral nervous system of mammals (reviewed by Dumont et al. 1992). High levels of NPY-like immunoreactivity have also been detected in the mammalian heart, especially in neurones innervating the atrial and ventricular myocardium (Gu et al. 1984; Dalsgaard et al. 1986; Corr et al. 1990). In some mammalian cardiac sympathetic nerve endings, NPY has been shown to co-exist with noradrenaline (Gu et al. 1984; Allen et al. 1985; Sternini and Brecha, 1985; Dalsgaard et al. 1986; Corr et al. 1990).
However, the effects of NPY on the mammalian heart are controversial. In the right atrium isolated from guinea pig, positive inotropic and chronotropic effects of NPY have been reported by Lundberg et al. (1984), while no effects of NPY were seen by Allen et al. (1986). NPY was also reported to have no effect on the papillary muscle isolated from cat, guinea pig and rat (Allen et al. 1986), on the auricles of human hearts (Franco-Cereceda et al. 1987; Michel et al. 1989) and on the atria and ventricles of the rat (Scoot et al. 1990). In contrast, in perfused whole hearts isolated from rabbit and guinea pig, NPY exerts a negative inotropic effect (Allen et al. 1983b; Rioux et al. 1986). Treatment with NPY produced a similar negative inotropic effect in atrial and ventricular strips isolated from rat (Balasubramaniam et al. 1988; Piper et al. 1989) and dog (Rigel et al. 1989). These inconsistent effects of NPY may be attributed to the complexity of the mammalian heart, which is influenced by a blood supply from the coronary artery. To analyze the direct effect of NPY on the heart, a simpler model is required.
In this study, an isolated eel atrium was used as such a model. The eel atrium can be easily isolated from the heart and beats spontaneously for more than 10 h in normal Ringer’s solution (Yasuda et al. 1996). Using this preparation, the effects of eel NPY (eNPY) were examined and compared with those of adrenaline. Furthermore, this study also demonstrated the existence of eNPY in eel heart.
Materials and methods
Biological activity in the eel atrium
Japanese eels Anguilla japonica, from farmed stock and weighing approximately 220 g, were kept in sea water (20 °C) for more than 1 week. After decapitation, the heart was rapidly excised and the atrium isolated on ice. It was then tied with two cotton threads, one being connected to the bottom of an experimental chamber and the other to a force transducer (type 45196A, Sanei, Tokyo, Japan). The atrium was bathed in Krebs bicarbonate Ringer’s solution consisting of (in mmol l–1): 118.5 NaCl, 4.7 KCl, 3.0 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 24.9 NaHCO3 and 10 lactic acid. In Ca2+-free Ringer’s solution, 3 mmol l–1 CaCl2 was replaced with 4.5 mmol l–1 NaCl. The bathing solution (2.0 ml) was bubbled with a 95 % O2:5 % CO2 gas mixture (pH 7.4) at room temperature (24–27 °C). After preloading by 5.9 mN, spontaneous isometric contractions were converted into electrical signals by a transducer connected to a strain amplifier (6M82, Sanei) and these were recorded using an electronic polyrecorder (EPR-10B, Toa, Tokyo, Japan). The rate of contraction was measured simultaneously using a tachometer (type 1321, Sanei).
Measurements of intracellular Ca2+ levels
The isolated eel atrium was cut into pieces in Ringer’s solution and treated with collagenase (1 mg ml–1) in the same solution for 1 h at room temperature. After rinsing with Ringer’s solution, the partially dispersed tissue was incubated with Calcium Green-1 acetoxymethyl ester (AM) (5 μmol l–1) and with Pluronic F-127 (0.05 %) as a surfactant for 40 min at room temperature. The atrial cells were stuck onto a glass slide, and this was mounted in a small chamber on the stage of an inverted microscope (Axiovert 135MTV, Zeiss, Tokyo, Japan) and superfused with normal Ringer’s solution bubbled with 95 % O2:5 % CO2 at room temperature.
The fluorescent signal from Calcium Green-1 was recorded with a laser scanning confocal imaging system (MRC-600, Bio-Rad, Tokyo, Japan; dichroic reflector 510LP; emission filter 515LP) equipped with a Kr–Ar laser (5470K, Ion Laser Technology, Salt Lake City, UT, USA; 488 nm for excitation) and with an inverted microscope (objectives, Zeiss Plan-Neofluar 20×/NA 0.50). The intensity of the fluorescence was expressed on an arbitrary scale of units ranging from 0 to 255.
Isolation of eNPY from heart
The entire heart was removed from 60 eels and the hearts were washed in ice-cold Ringer’s solution and stored at -40 °C. A boiled-water extract of eel hearts (91 g wet mass) was prepared following a method described previously (Uesaka et al. 1994). The extract was applied to Sep-Pak C18 cartridges (Millipore, Milford, MA). The retained material was eluted with 80 % 2-propanol containing 0.1 % trifluoroacetic acid (TFA), and the eluate was evaporated. The concentrated material was separated by HPLC (LC-6AD system, Shimadzu, Kyoto, Japan) using a C8 reverse-phase column (C8P-50, Asahi Chemical, Kanagawa, Japan). Retained material was eluted with a 90 min linear gradient of 0 % to 90 % acetonitrile containing 10 % 2-propanol and 0.1 % TFA. Fractions with a similar retention time to that of the synthesized eNPY were pooled and further purified by cation-exchange and reverse-phase HPLC. The cation-exchange column (CM-5PW, Tosoh, Tokyo, Japan) was equilibrated with 10 % ethanol and 20 mmol l–1 phosphate buffer (pH 6.7). The concentration of NaCl in the eluting solvent was raised to 0.3 mol l–1 for the initial 30 min and thereafter to 1.0 mol l–1 over 35 min with a linear gradient. In the reverse-phase step (ODS-120T, Tosoh), elution was performed with a linear gradient of 22 % to 32 % acetonitrile containing 10 % 2-propanol and 0.1 % TFA for 50 min. The primary structure of the purified peptide was determined by automated Edman degradation using a gas-phase sequencer (PPSQ-10, Shimadzu).
Reagents
EelNPY (Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Gln-Ala-Lys-Tyr-Tyr-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2), which had been isolated from pancreas and intestine of eel (Conlon et al. 1991; Uesaka et al. 1996), was synthesized automatically using an Nα-9-fluorenylmethoxycarbonyl (Fmoc) protection strategy (PSSM-8, Shimadzu) and purified by HPLC on a C18 column (ODS-120T). Drugs used were (-)-adrenaline (Sigma Chemical, St Louis, MO, USA), Calcium Green-1 AM, Pluronic F-127 (Molecular Probes, Eugene, OR, USA), (±)-betaxolol HCl (Mitsubishi Kasei, Tokyo, Japan), collagenase and verapamil HCl (Wako Pure Chemical, Osaka, Japan).
Statistical analyses
Data are reported as mean ± S.E.M.; N represents the number of preparations. The statistical significance of differences between means was examined using a Mann–Whitney U-test. The null hypothesis was rejected for P<0.05.
Results
Effects of eNPY on atrial beating
The isolated atrium from the eel heart continues to beat spontaneously at a constant rate for more than 10 h in normal Ringer’s solution. The basal atrial beating force was 1.6±0.1 mN and the rate was 55.7±2.0 beats min–1 (N=34). when eNPY (10–7 mol l–1) was added to the bathing fluid, the contractile force was enhanced without a significant change in the rate of beating (Fig. 1A). The increase in contractile force was dependent on the concentration of eNPY, with a threshold at 3×10–9 mol l–1 and a maximal effect at 10–6 mol l–1 (Fig. 1B).
Table 1 shows the effect of eNPY after blocking adrenaline action with betaxolol, a β1-adrenoceptor antagonist. In the presence of betaxolol (10–5 mol l–1), the force and rate of spontaneous beating tended to be reduced, although the effects were not significant. The effects of adrenaline were completely blocked after treatment with betaxolol (Table 1, upper half). Even after blocking adrenaline with betaxolol, eNPY (3×10–7 mol l–1) enhanced the contractile force significantly (P<0.05).
Interaction between adrenaline and eNPY
The maximal effects of eNPY and adrenaline were then compared (Table 2, upper half). Even at 10–6 mol l–1, eNPY had no effect on the beating rate, whereas adrenaline (3×10–7 mol l–1) increased it significantly (P<0.05). The contractile force was enhanced both by eNPY and by adrenaline. Although the enhancement caused by adrenaline tended to be greater than that produced by eNPY, the difference was not significant. When eNPY and adrenaline were applied simultaneously, the combined effect was not significantly different from that of adrenaline alone. When 10–6 mol l–1 eNPY was applied after pretreatment with 3×10–7 mol l–1 adrenaline, further enhancement was not observed (data not shown). These results indicate that there is an upper limit to the effects produced by the combined actions of eNPY and adrenaline. In contrast, when lower concentrations of eNPY (10–8 mol l–1) and adrenaline (10–8 mol l–1) were applied simultaneously, the increase in contractile force was significantly greater (P<0.05) than that induced by adrenaline alone (Table 2, lower half).
Effect of eNPY and adrenaline on the intracellular concentration of free Ca2+
As shown in Fig. 2, both eNPY and adrenaline enhanced the peak concentrations of intracellular free Ca2+ ([Ca2+]i) in the eel atrium. After eNPY administration, the peak levels of [Ca2+]i were enhanced gradually, while they were enhanced transiently after adrenaline application.
To determine whether this Ca2+ originates from the extracellular medium or from intracellular stores, Ca2+ in the bathing medium was omitted. As shown in Fig. 3A, both the contractile force and rate of beating were reduced in the absence of external Ca2+. After reintroducing Ca2+ into the bathing solution, both parameters recovered (Fig. 3Ai). In Ca2+-free Ringer’s solution, the effect of eNPY was completely abolished (Fig. 3Aii) and that of adrenaline was markedly reduced (Fig. 3Aiii). Similar reductions in the effects of eNPY and adrenaline were also observed in the presence of verapamil (10–5 mol l–1), a Ca2+ channel blocker (Fig. 3B). After washing out verapamil, however, spontaneous beating did not recover.
Time courses of the effects of eNPY and adrenaline
Using submaximal dosages of eNPY (10–7 mol l–1) and adrenaline (3×10–8 mol l–1), the time courses of the effects of eNPY and adrenaline on atrial beating were compared (Fig. 4). When adrenaline was added to the bathing fluid, both contractile force and rate of beating increased immediately and maximal effects were obtained after 4–5 min. The contractile force then decreased gradually, i.e. the effect of adrenaline on atrial contractile force was transient. In contrast, eNPY started to enhance the contractile force after 2 min and the effect reached a maximum after 16 min with no change in the rate of beating. When eNPY and adrenaline were applied simultaneously, both the contractile force and the rate of beating increased initially in the same way as in response to adrenaline alone, but the maximal force was maintained for more than 16 min.
Isolation of eNPY from an extract of eel heart
To prove that eNPY was present in eel heart, I attempted to isolate eNPY from the heart. Using synthesized eNPY as a marker, a peptide was purified using HPLC. The retention time of the isolated peptide (native) was exactly the same as that of the synthesized eNPY on both reverse-phase and cation-exchange HPLC (Fig. 5Ai,ii, Bi,ii). When a mixture of the native and the synthesized peptides was applied, only a single peak was obtained on both types of HPLC (Fig. 5Aiii, Biii).
The amino acid sequence of the isolated peptide was determined as follows: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Gln-Ala-Lys-Tyr-Tyr-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. This sequence was identical to that of the synthesized eNPY. The isolated peptide increased atrial contractile force without changing the rate of beating (data not shown).
Discussion
Although the role of NPY in the mammalian heart is still obscure, the present study clearly demonstrates that a homologous NPY (eNPY) increases the contractile force of the isolated eel atrium without altering the rate at which the atrium beats. The effect of eNPY was not inhibited by treatment with betaxolol, a β1-adrenoceptor antagonist (Table 1), indicating that eNPY does not act via adrenaline release.
Adrenaline also enhanced atrial contractile force, but the enhancement was not additive to that of a supramaximal dose of eNPY (Table 2). This suggests that the two regulators enhance contractile force through a common process. Enhancement of [Ca2+]i can be considered as a possible common factor, since eNPY and adrenaline both increase [Ca2+]i in the eel myocardium (Fig. 2). Similarly, enhancement of [Ca2+]i by NPY has been reported in many mammalian cells, including vascular smooth muscle cells (Mihara et al. 1989; Wahlestedt et al. 1992b), dorsal root ganglion (Perney and Miller, 1989), erythroleukaemia cells (Motulsky and Michel, 1988), neuroblastoma cells (Aakerlund et al. 1990), adrenal chromaffin cells (Wahlestedt et al. 1992a) and cells in the ovary (Selbie et al. 1995).
Contraction of the eel atrium was dependent on the presence of extracellular Ca2+, and the effects of eNPY and adrenaline were both reduced in Ca2+-free Ringer’s solution or by verapamil, a Ca2+ channel blocker (Fig. 3). This suggests that the elevation in [Ca2+]i after eNPY and adrenaline administration is caused by an influx of Ca2+ from the extracellular fluid. Extracellular Ca2+ has also been implicated in ventricle contraction in frogs (Morad et al. 1983; Bers, 1985) and teleosts (Driedzic and Gesser, 1988; Hove-Madsen and Gesser, 1989), whereas intracellular stores of Ca2+ are involved in contraction of the mammalian ventricle (see Wier, 1990).
Although both eNPY and adrenaline enhanced the contractility of eel atrium, the time courses of the effects of these regulators were different; the effect of eNPY being gradual and long-lasting, while the effect of adrenaline was immediate and transient (Fig. 4). Since eel heart contains eNPY (as shown in the present study) and catecholamines (Pennec and Le Bras, 1984; Yasuda et al. 1996), it is plausible that eNPY and catecholamine(s) act synergistically in the eel atrium. Although at present there is no evidence that eNPY and catecholamine(s) co-exist in the same cell and are co-released in eel heart, there is considerable evidence for the co-existence or co-release of these regulators in mammalian sympathetic nerves (Gu et al. 1984; Dalsgaard et al. 1986; Corr et al. 1990), adrenal gland (Allen et al. 1983a; Varndell et al. 1984; Kuramoto et al. 1986) and pancreas (Ahren et al. 1989).
ACKNOWLEDGEMENTS
I wish to thank Professor Masaaki Ando, Faculty of Integrated Arts and Sciences, Hiroshima University, for his valuable advice and for reading the manuscript and Mr H. Teranishi for the amino acid sequence analysis. This research was supported in part by Grants-in-Aid no. 02804062 from the Ministry of Education, Sciences and Culture, Japan, and no. 9233 from the Salt Science Research Foundation, Japan.