Modulation by APGW-Amide, an Achatina Endogenous Inhibitory Tetrapeptide, of Currents Induced by Neuroactive Compounds on Achatina Neurons: Peptides

Gen. Pharmac. Vol. 29, No. 4, pp. 531–538, 1997 Copyright  1997 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/97 $17.00 1 .00 PII S0306-3623(96)00579-4 All rights reserved

Modulation by APGW-Amide, an Achatina Endogenous Inhibitory Tetrapeptide, of Currents Induced by Neuroactive Compounds on Achatina Neurons: Peptides Xiao Yan Han,1 Thucydides L. Salunga,1 Wei Zhang,2 Hiroshi Takeuchi1* and Ken-ichi Matsunami1 1 Department of Physiology, Institute of Equilibrium Research, Gifu University School of Medicine, Tsukasa-machi 40, Gifu 500, Japan [Fax: 181-58-265-9004] and 2Department of Physiology, Norman Bethune University of Medical Sciences, Jilin, Changchun, China

ABSTRACT. 1. Modulatory effects of APGW-amide (Ala-Pro-Gly-Trp-NH2), proposed as an inhibitory neurotransmitter of Achatina neurons, perfused at 331026 M on the currents induced by neuroactive peptides, ejected by brief pressure, were examined by using Achatina giant neuron types, vRCDN (ventral-right cerebral distinct neuron) and PON (periodically oscillating neuron), under voltage clamp. 2. Outward current (Iout) caused by FMRFamide (Phe-Met-Arg-Phe-NH2) on v-RCDN, which was probably K1 dependent, was inhibited with membrane conductance (g) increase by APGW-amide. From the dose (pressure duration)-response curves of FMRFamide and a Lineweaver-Burk plot of these data, the inhibition caused by APGW-amide was mainly in an uncompetitive manner. 3. Iout caused by APGW-amide on v-RCDN, which was probably K1 dependent, was inhibited with g increase by APGW-amide. The inhibition caused by APGW-amide was partly in a competitive manner and partly in a noncompetitive manner. 4. Iout caused by [Ser2]-Mytilus inhibitory peptide, [Ser2]-MIP (Gly-Ser-Pro-Met-Phe-Val-NH2) on v-RCDN, which was probably K1 dependent, was inhibited with g increase by APGW-amide. Because the modulation of this current was not so marked, a dose-response study of this compound was not carried out. Iin induced by oxytocin on PON was not affected by APGW-amide. 5. From the dose-response curves of APGW-amide, perfused consecutively, the inhibitory effects of APGW-amide on the Iout caused by APGW-amide were stronger than those on the Iout caused by FMRFamide. 6. The inhibition of the APGW-amide–induced Iout on v-RCDN by APGW-amide was partly due to the competition in the receptor sites and partly to the g increase. The inhibition by APGW-amide on the Iout induced by FMRFamide and [Ser2]-MIP would be partly due to the g increase. In addition, we consider that APGW-amide affects intracellular signal transduction systems or ionic channels, thus modulating these currents. 7. The currents modulated by APGW-amide were different from those modulated by achatin-I, another Achatina endogenous neuroexcitatory peptide. We consider that the mechanisms underlying the modulatory effects of APGW-amide are different from those of achatin-I. gen pharmac 29;4:531– 538, 1997.  1997 Elsevier Science Inc. KEY WORDS. Neuroactive peptides, APGW-amide, achatin-I, neuromodulatory effects, neurotransmitters, giant neuron, snail (Achatina fulica Fe´russac) INTRODUCTION APGW-amide (Ala-Pro-Gly-Trp-NH2), isolated from the ganglia of a prosobranch (Fusinus ferrugineus) and an African giant snail (Achatina fulica Fe´russac), was proposed as an inhibitory neurotransmitter of Achatina neurons. ED50 (confidence limit at 95%) of APGW-amide, applied by bath consecutively at different concentrations, for producing the outward current (Iout) on an Achatina neuron type, RAPN (right anterior pallial nerve neuron), under voltage clamp was 6.231026 M (5.0–7.83102 6 M) (Liu et al., 1991b). It was demonstrated that APGW-amide at 33102 6 M modulated the currents *To whom all correspondence should be addressed. Received 17 October 1996; accepted 14 November 1996.

induced by small-molecule putative neurotransmitters dopamine, 5-hydroxytryptamine (5-HT) and erythro-b-hydroxy-l-glutamic acid (erythro-l-BHGA), applied by brief pneumatic pressure ejection, on Achatina neuron types (Han et al., 1997). Among these compounds, l-BHGA, in either erythro or threo type, was proposed as a putative inhibitory neurotransmitter of neuron type v-RCDN (ventral-right cerebral distinct neurone) (Zhang et al., 1996a, 1996b). Achatin-I (Gly-d-Phe-Ala-Asp), also isolated from the Achatina ganglia, excited (depolarized) Achatina neuron types including v-RCDN and PON (periodically oscillating neuron) (Araki et al., 1995; Kamatani et al.,1989; Kim et al., 1991a, 1991b). Several neuroactive peptides including APGW-amide at 331026 M modulated

532 the inward current (Iin ) induced by achatin-I on Achatina neuron types, such as v-RCDN (Liu and Takeuchi, 1995). Following these previous reports, the present study aimed to examine the modulatory effects of APGW-amide on the currents induced by other neuroactive peptides isolated from molluscan nervous tisues, FMRFamide (Phe-Met-Arg-Phe-NH2), APGW-amide, [Ser2]-Mytilus inhibitory peptide, [Ser2]-MIP (Gly-Ser-Pro-Met-PheVal-NH2) and oxytocin, ejected locally to the neuron by brief pressure, by using two Achatina giant neuron types, v-RCDN and PON, under voltage clamp. Of the neuroactive peptides tested, FMRFamide was originally isolated from the ganglia of a clam (Macrocallista nimbosa) (Price and Greenberg, 1977), and [Ser2]-MIP was isolated from the ganglia of a mussel (Mytilus edulis) (Hirata et al., 1988). A peptide structurally related to oxytocin was found in the ganglia of a fresh water snail (Lymnaea stagnalis) (Ebberink and Joosse, 1985). It was found that FMRFamide, APGW-amide and [Ser2]-MIP inhibited (hyperpolarized) Achatina neuron types including v-RCDN under current clamp but did not affect PON (Liu et al., 1991a, 1991b; Yongsiri et al., 1989). In contrast, oxytocin excited (depolarized) PON but did not affect v-RCDN (Liu et al., 1991a). In addition, achatin-I at 331026 M modulated the currents induced by small-molecule putative neurotransmitters and neuroactive peptides. With these findings, we proposed that achatin-I is acting as an excitatory neurotransmitter and a neuromodulator in the Achatina nervous system (Liu and Takeuchi, 1993a, 1993b). In the present study, the modulatory effects of APGW-amide on the currents tested are compared with those of achatin-I.

MATERIALS AND METHODS The materials and methods adopted in the present study were essentially similar to those of the previous report (Han et al., 1997). Briefly, among the giant neuron types identified in the ganglia of the African giant snail Achatina, v-RCDN and PON were used in the present experiments. Characteristics of these neuron types were described in the following reports: Takeuchi et al. (1985a, 1985b, 1987, 1991), Goto et al. (1986), Liu et al. (1991a, 1991b), Araki et al. (1995). The ganglia were dissected from the animal and incubated with trypsin (type III, Sigma, USA) to soften the connective tissue covering the ganglia. The connective tissue was removed with fine forceps to expose the neuron to be tested. With the use of two microelectrodes implanted into a neuron soma (Okamoto et al., 1976), the neuromembrane was set (holding voltage, Vh) at260 mV, near the resting potential of the Achatina neurons. The membrane conductance was measured by superimposing repetitive hyperpolarizing pulses (5 mV, 1 sec in duration and 0.5 Hz) on Vh. APGW-amide, FMRFamide and oxytocin were obtained commercially from Sigma. [Ser2]-MIP was donated by Dr. K. Nomoto of Suntory Institute for Bioorganic Research, Osaka, Japan. The Achatina physiological solution was formulated according to the amounts of main inorganic ions in the Achatina hemolymph (Takeuchi et al., 1973). The application methods of the compounds were described in detail in the previous report (Han et al., 1997). Briefly, to prevent the transsynaptic effects as much as possible, neuroactive peptide was applied by brief pneumatic pressure ejection. The peptide was dissolved at 1023 M in physiological solution, pipetted into a glass micropipette together with Fast Green, and ejected by the opening of an electromagnetic valve (mainly 23105 Pa and 400 msec) under the perfusion of physiological solution in the experimental chamber at a constant rate (about 2 ml/min). In the screening trials of

X. Y. Han et al. APGW-amide for its modulatory effects, this peptide at 331026 M was perfused in the chamber at the same rate. Statistical calculations were similar to those of the previous report (Han et al., 1997). Briefly, the data were expressed as mean values6 SEM in n observations. Two data obtained from one neuron were compared by the two-tailed Student’s t-test for paired data. Multiple data obtained repeatedly from one neuron were compared by analysis of variance (ANOVA) for repeated measurements and Bonferroni’s t-test (Glantz, 1987). The dose-response curve was analyzed by the probit method (Litchfield and Wilcoxon, 1949) to get ED50 (confidence limit at 95%), ideal sigmoidal curve (r value), slope (i.e., tangent at ED50), and Hill coefficient (r value) of the curve. A Line-weaver-Burk plot was performed from the mean of the reciprocal of each datum of the dose-response curve.

RESULTS

Effects of APGW-amide on the Iout produced by FMRFamide The outward current (Iout) caused by FMRFamide, applied by pneumatic brief pressure ejection (23105 Pa, 400 msec, 102 3 M and 5-min intervals), on v-RCDN in physiological solution was stable for at least 70 min. The relation between the time course and the Iout (n54), obtained by the linear regression, was Y (nA)51.42551 0.002700X (min) (Fig. 1A). Membrane conductance (g) values, measured before the Iout caused by FMRFamide and at the peak of the Iout, in physiological solution also were stable for the same period; the relation between the time course and the g value was Y (mS)5 0.1588310.0011571X (min) for g before the Iout and Y (mS)5 0.2711710.0011286X (min) for g at the peak of the Iout. The Iout caused by FMRFamide, measured in the same manner, on v-RCDN was inhibited by APGW-amide, perfused at 33102 6 M. This inhibition occurred rapidly and recovered after wash out; the Iout (mean6SEM; n56) was 3.2460.39 nA for the mean of the values obtained before APGW-amide perfusion (control), 1.7460.16 nA 10 min after the perfusion (**, P,0.01; compared with the mean of the control by ANOVA for repeated measurements and Bonferroni’s t-test) and 3.2060.59 nA 30 min after wash out (NS, not significantly different; compared with the mean of the control) (Figs. 1B and 1D). The g value, measured before the Iout caused by FMRFamide, was increased under APGW-amide at 331026 M and recovered after wash out; the g (n56) was 0.1760.02 mS for the mean of the control, 0.2260.02 mS 10 min after APGW-amide perfusion (***, P,0.001) and 0.1960.02 mS 20 min after wash out (NS). On the other hand, the g value at the peak of Iout was not changed by APGW-amide at this concentration. The difference between the g value measured before the Iout and that at the peak of Iout (g delta) was decreased significantly 10 min after APGW-amide perfusion (*, P,0.05) (Figs. 1C and 1D). The dose (pressure duration)-response curves of FMRFamide, ejected by brief pressure, on v-RCDN (n55) were measured by varying the pressure durations from 30 to 600 msec in physiological solution (control curve) and under APGW-amide at 331026 M (drug curve) from one neuron. The measurement of the drug curve started 10 min after APGW-amide perfusion. The two curves were analyzed by the probit method as follows: ED50 (confidence limit at 95%), slope (i.e., tangent at ED50), Hill coefficient (r value) and Emax were 38.5 msec (16.6–58.8 msec), 0.92235, 1.70854 (0.956449) and 1.7360.31 nA, respectively, for the control curve and 29.1 msec (0.0–69.4 msec), 1.00258, 1.93966 (0.925582) and 1.2160.33 nA (*, P,0.05; compared with Emax of the control curve by Student’s t-test for paired data), respectively, for the drug curve. ED50 of the drug curve was a bit smaller than that of the control curve, and Emax

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FIGURE 1. Inhibitory effects of APGW-amide, perfused at 33102 6 M, on the outward currents (Iout) produced by FMRFamide, applied by brief pressure ejection (23105 Pa, mostly 400 msec, 102 3 M and 5–10-min intervals), on v-RCDN. Number of observations is indicated in parentheses; small bar represents SEM. (A) Iout caused by FMRFamide in physiological solution. (B) FMRFamide-induced Iout under APGW-amide. (C) Changes in g caused by FMRFamide under APGW-amide. (D) Recordings of the FMRFamide-induced Iout. (E) Dose (pressure duration)-response curves of FMRFamide measured by varying pressure duration. (F) Lineweaver-Burk plot of the data shown in E. In A, the line was drawn by the linear regression. In B and C, the horizontal bar represents APGW-amide perfusion. In C, open circles represent g before the Iout caused by FMRFamide; solid circles represent g at the peak of the Iout; triangles represent the difference between g values measured at the peak of the Iout and before the Iout (g delta). In B and C, values under APGW-amide and after wash out were compared with the mean of the values obtained before the drug perfusion (control) by ANOVA for repeated measurements and Bonferroni’s t-test (*, P,0.05; **, P,0.01; ***, P,0.001). Traces are in D (a) control, (b) 30 min after APGW-amide perfusion and (c) 20 min after wash out. Arrows indicate the brief pressure ejection of FMRFamide. In E, the two dose-response curves of FMRFamide were measured from one neuron in (open circles) physiological solution (control curve) and under (solid circles) APGW-amide (drug curve). Values of the drug curve were compared with those of the control curve in the corresponding pressure duration by Student’s t-test for the paired data. The curves were drawn by fitting the ideal sigmoidal curves calculated by the computer program (r50.9745 for the control curve and 0.9421 for the drug curve). In F, the two lines were drawn by the linear regression [r50.9970 for the control line (open circles) and 0.9349 for the drug line (solid circles)]. Membrane potential was kept (holding voltage, Vh) at260 mV.

of the drug curve also was smaller than that of the control curve. From a Lineweaver-Burk plot of these data, the relation between the reciprocal of the pressure duration (abscissa in 1/sec) and the reciprocal of the Iout (ordinate in 1/nA), obtained by the linear regression, was Y50.6353610.029515X for the control (control line) and Y51.2447010.026883X under the drug (drug line). The two lines were almost parallel, indicating that the inhibition was caused in an uncompetitive manner (Figs. 1E and 1F).

Effects of APGW-amide on the Iout produced by APGW-amide Modulatory effects of APGW-amide on the Iout caused by the same peptide on v-RCDN were examined. The Iout caused by APGW-amide, ejected by brief pressure, on v-RCDN in physiological solution was stable for at least 70 min; the relation between the time course and the Iout (n54) was Y (nA)50.886520.000700X (min) (Fig. 2A). The g also was stable for the same period; the relation between the time course and the g value was Y (mS)50.1390010.00037143X (min) for

g before the Iout induced by APGW-amide and Y (mS)50.178921 0.00062143X (min) for g at the peak of the Iout. APGW-amide at 331026 M inhibited the Iout induced by APGW-amide, applied by brief pressure, on v-RCDN. The inhibition was produced rapidly and recovered after wash out; the Iout (n55) was 0.8360.14 nA for the mean of the control, 0.4360.05 nA 10 min after APGW-amide perfusion (*) and 0.7260.14 nA 30 min after wash out (NS). The g value measured before the Iout caused by APGW-amide showed a tendency to be increased under APGWamide (NS), and the g measured at the peak of the Iout was almost unchanged (NS) (Figs. 2B–2D). The dose (pressure duration)-response curves of APGW-amide on v-RCDN (n54) were measured in physiological solution (control curve) and under APGW-amide at 331026 M (drug curve) from one neuron. The measurement of the drug curve started 10 min after APGW-amide perfusion. ED50 (confidence limit at 95%), slope, Hill coefficient (r value) and Emax of the curves were 42.8 msec (7.8–78.3 msec), 0.729958, 1.33524 (0.879512) and 1.7360.11 nA, respectively,

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FIGURE 2. Inhibitory effects of APGW-amide, perfused at 331026 M, on the outward currents (Iout) produced by APGW-amide, ejected by brief pressure (23105 Pa, mostly 400 msec, 1023 M and 5–10-min intervals), on v-RCDN. Number of observations is indicated in parentheses; small bar represents SEM. (A) Iout caused by APGW-amide in physiological solution. (B) APGW-amide-induced Iout under APGWamide. (C) Changes in g caused by APGW-amide under APGW-amide. (D) Recordings of the APGW-amide–induced Iout. (E) Dose (pressure duration)-response curves of APGW-amide measured by varying pressure duration. (F) Lineweaver-Burk plot of the data shown in E. In A, the line was drawn by the linear regression. In B and C, the horizontal bar represents APGW-amide perfusion. In C, open circles represent g before the Iout caused by APGW-amide; solid circles represent g at the peak of the Iout; triangles represent the difference between g values measured at the peak of the Iout and before the Iout (g delta). In B and C, values under APGW-amide and after wash out were compared with the mean of the control by ANOVA for repeated measurements and Bonferroni’s t-test. Traces in D are (a) control, (b) 30 min after APGW-amide perfusion and (c) 20 min after wash out. Arrows indicate the brief pressure ejection of APGW-amide. In E, the two dose-response curves of APGW-amide were measured from one neuron in (open circles) physiological solution (control curve) and under (solid circles) APGW-amide (drug curve). Values of drug curve were compared with those of control curve in the corresponding pressure duration by Student’s t-test for paired data. The curves were drawn by fitting the ideal sigmoidal curves calculated by the computer program (r50.9119 for control curve and 0.9420 for drug curve). In F, the two lines were drawn by the linear regression [r50.9889 for control line (open circles) and 0.9920 for drug line (solid circles)]. Vh5260 mV.

for the control curve and 71.5 msec (41.5–107.5 msec), 0.831554, 1.48283 (0.916223) and 0.7860.09 nA (*, compared with that of the control curve), respectively, for the drug curve. ED50 of the drug curve was larger than that of the control curve, and Emax of the drug curve was smaller than that of the control curve. From a Lineweaver-Burk plot of these data, the relation between the reciprocal of the pressure duration and the reciprocal of the Iout was Y (1/nA)50.58711 0.025224X (1/sec) for the control line and Y (1/nA)51.31191 0.113000X (1/sec) for the drug line. The cross point of the two lines was X528.26 and Y50.38, which was between the two axes. This suggested that the inhibition was caused partly in a noncompetitive manner and partly in a competitive manner (Figs. 2E and 2F).

Effects on currents produced by [Ser 2]-MIP and oxytocin The Iout caused by [Ser2]-MIP, ejected by brief pressure, on v-RCDN was stable in physiological solution for at least 70 min; the relation between the time course and the Iout (n54) was Y (nA)51.95291 0.0006786X (min) (Fig. 3A). The g values measured before the Iout

caused by [Ser2]-MIP and at the peak of the Iout also were stable in physiological solution; the relation between the time course and the g was Y (mS)50.1153310.0004000X (min) for the g before the Iout and Y (mS)50.2145010.0007286X (min) for the g at the peak of the Iout. The Iout induced by [Ser2]-MIP on v-RCDN was inhibited significantly under APGW-amide at 331026 M, and the inhibition recovered after wash out; the Iout (n55) was 1.0660.11 nA for the mean of the control, 0.8060.10 nA 30 min after APGW-amide perfusion (*, compared with the mean of the control), and 1.2460.11 nA 20 min after wash out (NS). The g value before the Iout caused by [Ser2]MIP was increased under APGW-amide at this concentration on the same neuron type; the g (n55) was 0.1460.02 mS for the mean of the control and 0.1760.03 mS 20 min after APGW-amide perfusion (*). On the other hand, the g at the peak of the Iout was almost not changed under APGW-amide (Figs. 3B–3D). Because the inhibition caused by APGW-amide on the [Ser2]MIP–induced Iout was not so marked, the dose-response study of [Ser2]-MIP was not performed.

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FIGURE 3. Effects of APGW-amide, perfused at 331026 M, on the currents induced by [Ser2]-MIP and oxytocin, ejected by brief pressure (23105 Pa, 400 msec, 102 3 M and 5–10-min intervals). Number of observations is indicated in parentheses; small bar represents SEM. (A) Iout caused by [Ser2]-MIP on v-RCDN in physiological solution. (B) [Ser2]-MIP–induced Iout on v-RCDN under APGW-amide. (C) Change in g caused by [Ser2]-MIP on v-RCDN under APGW-amide. (D) Recordings of the [Ser2]-MIP–induced Iout. (E) Iin caused by oxytocin on PON under APGW-amide. (F) Change in g caused by oxytocin on PON under APGW-amide. In B, C, E and F, the horizontal bar represents APGW-amide perfusion. In C and F, open circles represent g before the currents caused by [Ser2]-MIP (C) and oxytocin (F); solid circles represent g at the peak of the currents; triangles represent the difference between g values measured at the peak of the currents and before the currents (g delta). In B, C, E and F, values under APGW-amide and after wash out were compared with the mean of the control by ANOVA for repeated measurements and Bonferroni’s t-test. Traces in D are (a) control, (b) 30 min after APGW-amide perfusion and (c) 20 min after wash out. Arrows indicate the brief pressure ejection of [Ser2]-MIP. Vh5260 mV.

The Iin caused by oxytocin, ejected by brief pressure, on PON was stable for 60 min in physiological solution. This Iin was not affected under APGW-amide at 33102 6 M. The g values measured before the Iin and at the peak of the Iin on PON were almost not changed under APGW-amide at this concentration (Figs. 3E and 3F).

Comparison of APGW-amide potencies for suppressing currents produced by FMRFamide and APGW-amide The potencies of APGW-amide, perfused consecutively from 1027 to 33102 6 M, for inhibiting the Iout induced by FMRFamide and APGW-amide, ejected by brief pressure, were compared. APGW-amide at concentrations from 1027 M dose-dependently inhibited the Iout caused by FMRFamide on v-RCDN; the Iout (n54) was 1.5060.04 nA for the mean of the control, 1.3060.11 nA 10 min after APGW-amide perfusion at 102 7 M (*, compared with the mean of the control), 1.1360.07 nA 10 min after the perfusion at 331027 M (*), 1.0060.06 nA 10 min after the perfusion at 1026 M (**) and 0.9160.06 nA 10 min after the perfusion of 33102 6 M (***, P,0.001). The g value measured before the Iout caused by FMRFamide showed a tendency to be increased under APGWamide (NS), but the g measured at the peak of the Iout was not changed (Figs. 4A and 4B).

The dose-resposne curve of APGW-amide for inhibiting the Iout caused by FMRFamide was measured by the relative values (to the mean of the control) of the currents obtained 20 min after APGWamide perfusion at each concentration. ED50 (confidence limit at 95%), slope and Hill coefficient (r value) of the curve (n54) were 3.98931026 M (2.224–11.688310 26 M), 0.285406 and 0.482958 (0.990547), respectively (Fig. 4C). APGW-amide at concentrations from 331027 M showed a tendency to dose-dependently inhibit the Iout caused by APGW-amide on v-RCDN; the Iout (n54) was 1.9860.19 nA for the mean of the control, 1.8560.35 nA 16 min after APGW-amide perfusion at 1027 M (NS), 1.3360.20 nA 16 min after the perfusion at 331027 M (NS), 0.8660.07 nA 16 min after the perfusion at 1026 M (NS), and 0.5360.02 nA 16 min after the perfusion at 331026 M (*). The g value measured before the Iin caused by APGW-amide was increased under APGW-amide, whereas the g at the peak of the Iout was almost not changed (Figs. 4D and 4E). From the dose-response curve of APGW-amide, perfused consecutively, for inhibiting the Iout caused by APGW-amide, ejected by brief pressure, ED50 (confidence limit at 95%), slope and Hill coefficient (r value) of the curve (n54) were 0.857310 26 M (0.355– 1.82931026 M), 0.549591 and 0.926349 (0.98057), respectively (Fig. 4F). The comparison of the two ED50 values of APGW-amide indicated that the inhibitory effects of this peptide on the Iout caused

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FIGURE 4. Dose-response studies of APGW-amide, perfused consecutively from 1027 to 331026 M, for suppressing the currents produced by FMRFamide and APGW-amide, ejected by brief pressure (23105 Pa, 400 msec and 102 3 M). Number of observations is indicated in parentheses; small bar represents SEM. (A) Iout caused by FMRFamide on v-RCDN under APGW-amide. (B) Changes in g caused by FMRFamide on v-RCDN under APGW-amide. (C) Dose-response curve of APGW-amide for suppressing the Iout caused by FMRFamide on v-RCDN. (D) Iout caused by APGW-amide on v-RCDN under APGW-amide. (E) Changes in g caused by APGW-amide on v-RCDN under APGW-amide. (F) Dose-response curve of APGW-amide for suppressing the Iout caused by APGW-amide on v-RCDN. In A, B, D and E, the horizontal bar represents APGW-amide perfusion at varied concentrations. In B and E, open circles represent g value before the current induced by FMRFamide (B) and APGW-amide (E); solid circles represent g at the peak of the currents; triangles represent the difference between g values measured at the peak of the currents and before the currents (g delta). In A, B, D and E, values under APGW-amide and after wash out were compared with the mean of control by ANOVA for repetitive measurements and Bonferroni’s t-test. The ordinate in C and F is relative value (to the mean of control) of the current measured 10 min (C) or 16 min (F) after APGW-amide perfusion at each concentration. The curves were drawn by fitting the ideal sigmoidal curves calculated by the computer program (r50.9920 for C and 0.9848 for F). Vh5260 mV.

by APGW-amide were significantly stronger than those on the Iout caused by FMRFamide. DISCUSSION It was demonstrated that APGW-amide, an Achatina endogenous neuroinhibitory peptide, at 331026 M modulated the currents induced by small-molecule putative neurotransmitters erythro-L-BHGA, dopamine and 5-HT (fast component), ejected by brief pressure, on Achatina neuron types (Han et al., 1997). It was also found that several neuroactive peptides, including APGW-amide, modulated the inward current (Iin ) caused by achatin-I, an Achatina endogenous neuroexcitatory peptide, ejected by brief pressure (Liu and Takeuchi, 1995). It was shown in the present study that APGW-amide at 331026 M also modulated (inhibited) the outward current (Iout ) caused by neuroactive peptides originally isolated from molluscan nervous tissues, FMRFamide, APGW-amide and [Ser2]-MIP, ejected by brief pressure, on v-RCDN. However, APGW-amide did not affect the inward current (Iin) induced by oxytocin on PON.

Regarding the ionic dependencies of the currents mentioned, the Iout caused by APGW-amide on RAPN was K1 dependent (Liu et al., 1991b). We consider that the Iout induced by APGW-amide on v-RCDN, which was tested in the present study, is also K1 dependent; the membrane conductance (g) was increased during the Iout, and the duration of the Iout was longer than that of the Cl2 -dependent Iout induced by g-aminobutyric acid (GABA) on TAN (tonically autoactive neurone) (Kim and Takeuchi, 1990). The Iout caused by FMRFamide and [Ser2]-MIP on v-RCDN would also be due to K1 for the same reasons. Besides, it was found that the Iout caused by [Ser2]-MIP on RAPN was K1 dependent (Yongsiri et al., 1989). The Iin caused by oxytocin on PON was probably Na1 dependent; the g was virtually unaltered during the Iin. In addition, the Iin caused by achatin-I on v-RCDN, which was inhibited by APGWamide (Liu and Takeuchi, 1995), was only partly Na1 dependent (Emaduddin and Takeuchi, unpublished data), whereas the same currents of PON and TAN were mainly Na1 dependent (Kamatani et al., 1989; Kim et al., 1991a). Dose (pressure duration)-response curves of FMRFamide and a Lineweaver-Burk plot of these data indicated that the inhibition of

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TABLE 1. Characteristics of modulation by APGW-amide, perfused at 331026 M, on currents induced by neuroactive compounds, ejected by brief pressure, on Achatina neuron types Dose-response curve of the compound ED50 (msec) Compound

Neuron

I

Erythro-BHGA 1 Dopamine1 5-HT (fast)1 Achatin-I2 FMRFamide3 APGW-amide 3 [Ser2]-MIP3

v-RCDN v-RCDN TAN v-RCDN v-RCDN v-RCDN v-RCDN

Iout Iout Iin Iin Iout Iout Iout

Emax (nA)

Modulation Cont APGW enhanced inhibited inhibited inhibited inhibited inhibited inhibited

83.1 32.2 20.3 68.2 38.5 42.8

76.9 29.5 18.5 63.4 29.1 71.5

Cont 3.41 6 0.35 0.75 6 0.08 1.29 6 0.24 1.73 6 0.23 1.73 6 0.31 1.73 6 0.11 (not tested)

D-R curve of APGW

APGW 5.54 0.38 0.72 0.58 1.21 0.78

6 6 6 6 6 6

0.69* 0.03* 0.14* 0.13** 0.33* 0.09*

Competition

ED50 (mM)

on abscissa noncomp/uncomp noncomp noncomp uncomp comp/noncomp

Hill

(not tested) 3.49 0.522 1.29 0.586 0.14 0.344 3.98 0.483 0.86 0.926 (not tested)

Data were obtained from dose (pressure duration)-response curves of neuroactive compounds in physiological solution (control) and under APGW-amide and from the dose-response curves of APGW-amide to inhibit the currents. 1 Han et al. (1997). 2 Liu and Takeuchi (1995). 3 Present study. Abbreviations: D-R, dose-response; cont, control; Hill, Hill coefficient; fast, fast component; on abscissa, by Lineweaver-Burk plot, the cross point of control line and drug line is on abscissa; noncomp, noncompetitive; uncomp, uncompetitive; comp, competitive; *, P , 0.05, compared with the control by Student’s t-test for paired data.

the FMRFamide-induced Iout by APGW-amide was in an uncompetitive manner. We consider that the inhibition of this Iout is not a receptor event but partly due to the g increase caused by APGWamide. In contrast, APGW-amide, perfused at 33102 6 M, inhibited the Iout caused by APGW-amide, ejected by brief pressure, partly in a competitive and partly in a noncompetitive manner. The inhibition of this Iout by APGW-amide is also partly due to the g increase. Because the inhibitory effects of APGW-amide on the Iout induced by [Ser2]-MIP were not so marked, the dose (pressure duration)-response curves of this peptide were not measured. However, the inhibition of this current by APGW-amide would be at least partly due to the g increase caused by APGW-amide. In addition, we consider that APGW-amide alters the intracellular signal transduction systems or ionic channels, thus modulating the currents—for example, the Iout induced by FMRFamide.

Dose-response curves of APGW-amide, perfused consecutively, indicated that APGW-amide dose-dependently inhibited the currents induced by FMRFamide and APGW-amide. We summarize in Table 1 the features of the modulatory effects of APGW-amide on the currents induced by the small-molecule neurotransmitters and neuroactive peptides studied in the previous (Liu and Takeuchi, 1995; Han et al., 1997) and present experiments. Among the seven currents modified by APGW-amide, only the Iout induced by erythro-l-BHGA was enhanced by APGW-amide, whereas the others were inhibited. According to the dose-response curves of APGW-amide, perfused consecutively, the order of the sensitivities of the currents mentioned to APGW-amide is: the Iin caused by achatin-I.the Iout induced by APGW-amide.the Iin (fast component) induced by 5-HT$the Iout induced by dopamine and FMRFamide. It was demonstrated that achatin-I also acts as a neuromodulator

TABLE 2. Comparison of modulatory effects of APGW-amide and achatin-I, perfused at 3 3 1026 M, on the currents induced by neuroactive compounds, ejected by brief pressure, on Achatina neuron types Compound

Neuron

I

Erythro-l-BHGA Dopamine 5-HT (fast) 5-HT (slow) Acetylcholine GABA GABA l-Glu Achatin-I FMRFamide APGW-amide [Ser2]-MIP Oxytocin

v-RCDN v-RCDN TAN TAN v-RCDN v-RCDN TAN RAPN v-RCDN v-RCDN v-RCDN v-RCDN PON

Iout Iout Iin Iin Iin Iin Iout Iin Iin Iout Iout Iout Iin

1

Ionic depend K1! K1 Na1! Na1! (NT) Cl2 Cl2 (NT) Na1# K1! K1! K1! Na1!

g value at 3 3 1026 M APGW-amide increased increased no change no change increased increased no change no change increased increased increased increased no change

Achatin-I no change no change no change no change no change no change no change (not measured) no change no change no change no change (not measured)

Liu and Takeuchi (1993a). 2 Liu and Takeuchi (1993b). 3 Liu and Takeuchi (1995). 4 Han et al. (1997). 5 Present study. Abbreviations: !, probably with this ionic dependency; NT, not tested; #, partly with this ionic dependency.

Modulation at 3 3 1026 M APGW-amide enhanced4 inhibited4 inhibited4 no effect4 no effect4 no effect4 (not tested) no effect4 inhibited3 inhibited5 inhibited5 no effect5 no effect5

Achatin-I no effect2 no effect2 enhanced1 no effect1 no effect2 (not tested) no effect2 (not tested) (not tested) enhanced2 inhibited2 no effect2 inhibited2

538 of Achatina neurons (Liu and Takeuchi, 1993a, 1993b). We compare in Table 2 the features of the modulatory effects of APGWamide on the currents mentioned with those of achatin-I. Although the g increase caused by APGW-amide at 331026 M was a cause of the modulatory effects of this peptide, we found in the present study that achatin-I at the same concentration did not affect the g of v-RCDN and TAN for at least 60 min (data are not shown). As shown in Table 2, the currents modified by APGW-amide were different from those modified by achatin-I. For example, the Iout induced by erythro-l-BHGA on v-RCDN was enhanced by APGW-amide but was not affected by achatin-I. The Iin (fast component) induced by 5-HT on TAN was inhibited by the former peptide but enhanced by the latter. The g change was ruled out as the cause of modification of the two currents by the previous (Han et al., 1997) and current experiments. Then, we consider that the mechanisms underlying the modulatory effects of APGW-amide are different from those of achatin-I. For example, the two peptides would affect different intracellular signal transduction systems or ionic channels. It should be noted that achatin-I showed excitatory effects on Achatina neurons, whereas APGW-amide showed inhibitory effects. For the next study, we plan to elucidate the involvement of the intracellular signal transduction systems in both the currents and the modulations caused by APGW-amide and achatin-I by using inhibitors of these systems. The authors wish to express their thanks to Dr. K. Nomoto of Suntory Institute for Bioorganic Research, for his donation of [Ser2]-Mytilus inhibitory peptide, and to Mrs. M. Matsuo, for her excellent secretarial assistance. This work was partly supported by Grants-in-Aid for the International Scientific Research Program, Joint Research No. 04044075 in 1992–94 and No. 10033333 in 1995, and by those for General Scientific Research No. 06680758 in 1994–95, from the Ministry of Education and Culture of Japan.

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