- Email: [email protected]
Effects of biologically active peptides on the excitability of identifiable giant neurones of an African giant snail (Achatina fulica Férussac)
Neurrrpharmoroloy
I..
1977. 16. 593-601.
Pergamon
Press
Prmted m Great Bream
EFFECTS OF BIOLOGICALLY ACTIVE PEPTIDES ON THE EXCITABILITY OF IDENTIFIABLE’ GIANT NEURONES OF AN AFRICAN GIANT SNAIL (ACK4TINA FULIC.4 FlQxm4C) HIROSHI TAKEUCHI. MICHIKO MATSUMOTOand AKINORI SAKAI Institute
[or Neurobiology.
Okayama University Okayama. Japan
Medical
School.
(Accepted 18 February 1977) Summary-Effects of biologically active peptides, including vaso-active peptides. hypothalamic hormonal peptides and peptides analogous to neurohypophyseal hormones, were examined on the excitability of two giant neurones (the TAN, tonically autoactive neurone and the PON. periodically oscillating neurone) of Acltatinu jiuu[icu Fdrussuc. In a 10m4kg/l bath application. an excitatory effect was produced on the TAN by physalaemin only, and on the PON by deamino-dicarba-(o-D-)oxytocin and D-D-Arg-vasotocin (not by D-D-kg-VaSOpreSSin). Microdrop application of physalaemin produced a biphasic effect (hyperpolarizing-depolarizing. dominant depolarizing effect) on the TAN, but only depolarization with the application of D-D-oxytocin and D-D-kg-VaSOtOCin to the PON. The excitatory effect of physalaemin on the TAN was lost after trypsin treatment. Contrary to expectation, inhibition was produced by chymotrypsin-treated physalaemin. D-D-Arg-vasotocin and D-D-oxytocin retained their effect on the PON after chymotrypsin treatment for 6 hr. D-D-kg-VdSOtOCin. however, lost tts effect after trypsin treatment. The current-voltage relationship (the I-V curve) of neurones was measured by application of a transmembrane triangular current of long duration. It was concluded that physalaein the same concenmm at 2 x IO-“keil does not affect the TAN I-V curve, and that o-o-oxytocin tration also does not affect the PON 1-V curve.
Some biologically active peptides, such as substance P. are thought to be putative synaptic transmitters of mammalian central nervous system neurones (Zetler. 1956; Phillis and Limacher, 1974a. b). The aim of the present study was to investigate the effects of vaso-active peptides, hypothalamic hormonal peptides and peptides analogous to neurohypophyseal hormones, and to attempt to clarify their structureactivity relationships on the excitability of two spontaneously firing giant neurones (the TAN, tonically autoactive neurone: and the PON, periodically oscillating neurone) identified in the subesophageal ganglia of an African giant snail (Achatina jdicu Fe’russac). Some pharmacological properties of the two identifiable neurones have already been reported (Takeuchi. Mori and Kohsaka. 1973a. 1974b, c: Takeuchi. Mori, Kohsaka and Ohmori, 1974d: Takeuchi, Yokoi, Mori and Kohsaka, 1975a. b; Takeuchi. Yokoi, Mori and Ohmori. 1976; Takeuchi, Yokoi and Hiramatsu, 1977). Modification of the effects of these peptides on the two neurones by treatment with trypsin and chymotrypsin was also examined. Observation of changes in membrane resistance of the identifiable neurones caused by the application of certain active peptides was performed. As is well known some molluscan neurones show membrane rectification (Kandel and Taut, 1966; Arvanitaki, Romey and Takeuchi, 1967) so, in the present study, as the indicator of the membrane resistance, the current-voltage relationship (I-V curve) of the neuro593
membrane was measured by injection of a transmembrane triangular current of long duration. MATERIALS AND
METHODS
Two spontaneously firing giant neurones (the TAN, tonically autoactive neurone. and the PON, periodically oscillating neurone) were identified in the subesophageal ganglia of an African giant snail, Achatina The TAN is excited by fiulica FPrtrssac. 5-hydroxytryptamine and histamine, inhibited by dopamine. GABA and acetylcholine. and not affected by L-glutamic acid. The PON is excited by 5-hydroxytryptamine and dopamine, inhibited by L-homocysteic acid and erythro-P-hydroxy-L-glutamic acid, and not affected by acetylcholine, GABA, histamine and L-glutamic acid. Arrangements for the electrophysiological experiments and localizations of the neurones in the ganglia have been described in previous papers (Takeuchi rt al., 1975b, 1976, 1977). A micropipette was implanted into the soma of the identifiable neurones, and their intracellular biopotentials were recorded by a penwriting galvanometer. The number of spike discharges per minute was automatically counted by a spike counter (Takeuchi, Kondoh and Misaki. 1974a). The following peptides, obtained in an analytically pure state, were tested in the present study: substance P. physalaemin. eledoisin-related peptide. neurotensin, bradykinin. Lys-bradykinin. Met-Lys-bradykinin. angiotensin I, II, III, luteinizing hormone-releasing
horlone (LH-RH ). tllyrt~tropin-releasing hormone (TRH), .~matost~~tin, MSH-release inhibiting factor (MRIH). (Nair, Kastin and Schally. 1971I. deaminodic;~rh3-(r,-u-)oxytocin ~Yamana~a. Hase. Sakakibara, Schwa&.. Dubois and Walter, 1970), I>-rt-Arg-vasoprcssin, I>-I>-Arg-vasotocin (Hase Sakakibara. Wahrcnburg, Kirchbergcr. Schwartz and Walter, 1972), liver-cell growth fitctor (Pickart. Thaycr. L. and Thqcr, M. 1473). tuftsin (Nishioku, Satoh. Constantopouios and Naj.jar, 1973) (products of Protein Research Fout~d~~t~on, Osaka). xenopsin (Araki. Tachihana. Uchiyama, Nakajima and Yasuhara, 1973) (donated by Eisai Co.). hypertensin (donated by CIBA-GEIGY Co.) and caerulein (donated by Falmitalia Co.). These peptides were applied to the experimental material by both bath application and microdrop appiic~~ti~~tl,In the bath appljcation, the peptidc to be examined, dissolved in the snail’s physiological solution (Takeuchi. Morimasa. Kohsaka, Kobayashi and Morii, 1973b). was applied directly to the dissected ganglia. Peptides demonstrated as cffectivr in the bath application were further tested by ioca1 application (the microdrop application). A microdrop (about IS0 ~lrn in diameter) of the peptide solution was made in air at the tip of a micropipette containing the peptide solution. and the microdrop c~cli~liy p&cd on the surface of an idcnti~~~ll~lencurone (diameters of the ncuroncs wcrc about I50 jrm 200 inni.
FO
r-Chymotrypsin (product of Wortliingt~~n Biochemical C~~rporation, 55 u,/mg). TPCK(r.- I-tosylamide-I!-phenylethyl-chloromrthyl ketone)-treated tryp‘hh sin ~Wortbiil~ton Riochemical Corporation. u/m@ and pancreatic trypsin-chym~trypsili inhi~~it~~l (Trasylol. Bayer A.G.) wet-c used for the enzyme trcatment of peptides. All substances used wt‘re dissolved in a physiological solution for the Afric:rn giant sn;iii, the pH value of which was around 7.5. One ml of a peptide solution (2.5 x Ii)-’ kg,‘!) was incubated at 37°C with 0.1 mi of an enzyme solution (5 x IO ’ kg/l) for either 30 min or G hr. After incubation, 0.17 ml of Trasylol f2 x IO-’ kg:l) W;LSaddcd to ;Irrc
in
the
physiological
st:tto
snd
the
TAN
60
2X 10~4neurotensin Fig. 1. Effects of vase-active pcptides on the TAN (tonically autoactive neurone) excitability (bath application) Ordinate. the number of spike discharges per minute. Abscissa. the time cowsc. each histogram is I min 2 x IO- ’ kgjl substance P. bradykinin, phy~~l~emin, 4 x lWi ph~~ll~lernin. 2 x if)-’ cledoisill-rci~lted substance, ~~n~iotensin I. angiotensin II and neurotensin were applied respectively.
otltc~
Peptide effects on molluscan giant neurones
Fig. 2. Comparison of effects of three peptides analogous to neurohypophyseal hormones on the PON (periodical11 oscillating neurone) excitability (bath application). Ordinate. the number of spike discharges per minute. Abscissa. the time course. each histogram is 1 min 2 x 10W4kg/l deamino-dicarha(i)-r)-tArg-vasopressin. I,-D-oxytocin, D-D-Arg-vasotocin, IO-’ D-D-oxytocin and o-o-Arg-vasotocin were applied respectively. measured in the presence of an effective peptide) were superimposed, the recordings of the applied current intensities were erased after confirming their fairly triangular shapes (no rectification of the current-applying pipette). The temperature of the experimental room was maintained at 23 + 1 C. The pH value of the examined peptide solutions was close to 7.5 in all cases. RESC’LTS
Vase-active peptides (substance P. physalaemin. elcdoisin-related peptide. neurotensin. xenopsin. Lys-bradykinin, bradykinin. Met-Lys-bradykinin. angiotensin 1. II. III and hypertensin). hypothalamic hormonal peptides (LH-RH. TRH, somatostatin and MRIH), peptides analogous to neurobypophyseal hormones (D-D-oxytocin, D-D-Arg-vasopressin and D-DArg-vasotocin) and others (caerulein, liver-cell growth factor and tuftsin) were tested by bath application. Of the peptides examined, only physalaemin, a vaso-active peptide from an amphibian skin, showed an excitatory effect on the TAN. The critical concentration to produce the effect was about 10-4kg/l. The other peptides examined had no effect on this neurone at 2 x IO--” kg/l. As shown in Figure 1. application
of phy~laemin at 2 x 10e4 kg/l resulted in an increase in the number of spike discharges of the neurone to more than twice that of the physiological state. This effect disappeared after washing the ganglia with the physiological solution. An effect of physalaemin at 4 x lo- ’ kg/l was no longer demonstrable. Substance P, bradykinin, eledoisin-related peptide, angiotensin I and II and neurotensin were without effect at 2 x 10m4 kg/l. The sensitivity of another identifiable neurone, the PON, to biologically active peptides was completely different from that of the TAN. Of the peptides examined, two peptides analogous to neurol~ypophyseal D-D-oxytocin and D-D-Arg-vasot~in, hormones. showed an excitatory effect on the PON. The critical concentration of these two peptides was about lo-” kg/l. The other peptides examined at 2 x 10s4 kg/l. including D-D-Arg-vasopressin, produced no effect. In Figure 2. the effect of D-D-OXytOCin, D-D-Arg-vasototin and D-D-Arg-vasopressin on the PON excitability is compared. D-D-OXytOCin and D-D-Arg-vasotocin at 10-j kg/l clearly prodbced an increase in the number of the PON spike discharges, although D-D-Arg-vasopressin, even at 2 x 10 -4 kg/l, did not affect the PON. Eficts of‘ local ~l~~licat~uiz~t~~j~~od~~~ a~p~jcatioi~)qf p~~t~d~~on f/w hvo id~~lti~abl~rwuronrs To distinguish between the direct effect of peptides
596
Hmosrn
TAKEUCHI
er cd.
TAN
f Physofaemine
2X IO-’
~O.tSmm 30 set
(3.5ng) XImV I
20mV I
Fig. 3. Effects of l~hysalaemin on the TAN biopotential (microdrop application). The upper trace is a full spike recording of the TAN biopotential by a pen-writing galvanometer. The lower trace is a high amplification recording of the upper trace (spike peaks have been cut by an electronic voltage clipper). A microdrop (150 jirn in diameter) of 2 x 1W3kgJ physalaemil~ (the total ~~lnount of applied phfsaldemin estimated to he about 3.5 ng) was applied on the TAN surface (arrow). Left vertical bar, calibration for the upper trace (50 mV). Right vertical bar. calibration for the lower trace (20 mV). Horizontal bar. the time course (30 set). After local application of physalacmin. the TAN biopotentiul showed a slight hyperpolarization followed by a marked depolarization.
PON
A
Fig. 4. Comparison of effects of three peptides analogous to neurohypophyseal hormones on the PON excitability (microdrop application). The upper traces of A. B and C are full-spike recordings of the PON biopotential by a pen-writing galvanometer. The lower traces of A. B and C are high amplification recordings of the upper traces (spike peaks have been cut by an electronic voltage clipper). In A. a microdrop (IS0 pm in diameter) of 4 x 10m4 kg,1 deamino-di~arba(~-I.~-)-ox~toc~n (the total amount of o-o-oxytocin estimated to be about 700 pg) was applied on the PON surface (arrow). In B, a microdrop (the same diameter) of 4 x IO-’ u-D-Arg-vasotocin (the total amount estimated to be about 700 pg) was applied (arrow). In C. a microdrop (the same diameter) of 4 x 10m4 rl-o-Arg-vasopressin (the total amount estimated to be about 700 pg) was applied (arrow). Left vertical bar. calibration for the upper traces (50 mV). Right vertical bar, calibration for the lower traces (20 mV). Horizontal bar, the time course (30 set). After local application of D-D-oxytocin and u-u-Arg-vasotocin. the PON biopotenti~il showed a depolarization. Local application of ~I~-Arg-vasopressin had no ell’ect.
Peptide
etl’ects on molluscan
giant
597
neurones
Table I. Effects of enLyme-treated peptides. compared with non-treated ones, on the two identifiable (the TAN. tonically autoactive neurone; and the PON. periodically oscillating neurone) of Achatina All peptides were examined at a concentration of 2 x 10V4 kg/l (bath application)
Substance Substance P Substance P Substance P Substance P Substance P Physalaemin Physalaemin Physataemin Physalaemin Physataemin Etedoisin-related substance Eiedoisin-related substance Eiedoisin-related substance Eledoisin-related substance Eledoisin-related substance Deaminodicarbaoxytocin
Deaminodicarbaoxytocin Dcaminodicarbaoxytocin Deaminodicarba-Argvasotocin Deaminodicarba-Agvasotocin Deaminodicarhn-Argvasotocin Dermino‘ dicarba-iirgvasotocm Deaminodicarba-Argvasotocin Deaminodicarba-Argvasopressin Deaminodicarba-Argvasopressm Deaminodicurba-Agvasopressin Deaminodicarba-Argvasoprcssin Dcaminodicarba-Arg-
vasopressin
Enzyme treatment None T T CT CT None ; CT CT
Incubation time
Effects on TAN
Amino
Effects on PON
(--I
i-1 (- 1
(-) (--I
(-) (6) (6) C-1 (6)
30 min hhr 30 min 6 hr
c-i
30 min 6 hr 30 min 6 hr
(;J* (1)
None
(;’
I
(4
(-)
(4
T
30 min
(--I
I-1
T
6hr
(-)
(4
CT
30 min
(--)
t-1
CT
6hr
(-)
f-1
(-)
E
None CT
30 min
(-)
E
CT
6 hr
(-)
E
(-)
E
None
T
30 min
(-)
t-1
T
6hr
(-f
(-)
CT
30 min
(-)
E
CT
6 hr
(-)
E
(-)
i-4
T
30 min
(-)
T
6hr
(-
CT
30 min
t-1
(-)
CT
6 hr
t-1
k-)
acid sequence and main sites cleaved by enzymes
CT
T
cr
CT
CT
Pyr-Ala-Asp-Pro-Asn-Ly~~Plle!Tqr!Gly-Leu-Met-NH2
: Lys-Phe~;le-Gly-Leu-Met-Nap
,+Tyr-Ile-Gin-Am-Asu-Pro-Leu-Gly-NH2
+
~Tyr!Ile-Gin-Asn-ALju-Pro-Arg~GIy-NHI
L‘T’
)
neurones Ferussac.
Arg~Pro-Lys-Pro-Gtn-Gin-Ph~~Phe!Gi~-L~,,-Met-N~~
CT*
None
giant fulica
CT“
f ) rtTyi-Phe!Cln-Asn-ASu-Pro-Arg~Gty-NHz
E---excitatory effect; (-+no effect: (-)*-no effect, but sometimes a slight excitatory effect: T-trypsin: CT--chymotrypsin; T--main sites of cleavage by trypsin; ‘T-main sites of cleavage by chymotrypsin; Pyr-r--pyroglutamic acid; Asu-L-a-amino suberic acid; +-cleavage was demonstrated (Walter and Hoffman, 1974): ++---cleavage is deduced from peptide structure.
Fig. 5. EtTecta ill’ on7~m+trcatcd physnluemin on the TAN excitability (bath application). Ordin;ttc. the numhcr of spike d~schargas par minute. Abscissa. the time course, each histogram is I min 7 x lVJ kg,1 ph~solaemrn. trcalctl hj trypsin (T) for 6 hr. b> T for 30 min. by chymotrypsin (CT) for 30 miu and by CT for 6 hr, were applied respectively.
ng ( I50 izrn in diamctcr. at 2 x f 0” 3 kg ‘I) was applied to the TAN surface (Fig. 3). Several seconds after the
application, the TAN biopotentinl showed a slight hyperpolarization followed by a marked dcpolarization. The TAN biopotential changes thus caused were clearly biphasic. the excitatory phase being dominant. These biopotentiai changes disappeared even if the examined ganglia remained unwashed. Presumably.
oxytocin and t~~~-Ar~-vi~~otuc~l~ caused ~~~~~~~ri~~~tion of &he PON neuromembrane with frequency augmentation of the slow oscillation of biopotential? while n-rz-Arg-vasapressin applied in the samc u;t) did not affect the hiopotential (Fig. 4). The depolnrization of the ncuromembrane occurred almost immediately after the microdrop application, and disapfared after severed minutes. even without ~shing
PUN
Fig. 6. Effects of enzyme-trcntcd phvsahemm on the PON the number of spike discharges per mjnute. Abscissa, the time physalaemin. treated by cbymotrypsin (CT) for 30min, by and hy T for 6 hr. were applied
excitability (bath application). Ordinate. course, each histogram is I min 2 x IO-” CT for 6 hr, by trppsin (T) for 30mit1 respectively.
Peptide effects on molluscan giant neurones
?t:10-‘4 d-ddrg-vasotocln ! T-6h )
2x10”)
599
y-;_$r;-)vascpressin
Fig. 7. Administration or trypsin (T)-treotcd peptides a&logous to neurohypophyseal hormones with respect to the PON cucttnhlhty (hath application). Ordinate, the number of spike discharges per minute. Abscissa. the time course. cuch histogram is 1mm 2 x lo-.” kg/l de~mino-dic~rb~ (u-u)-Arg-vasopressin (treated hg T l’or 30 min). i~-l)-Ar~-v~lsotocin (T for 30 mint, ~-~Arg-~sotoci~l (T for 6 hrl and [~-I~-Ark-v~~sopressin (T for 6 hr) were applied respectively.
the ganglia. Microdrop application also indicated a difference in effect between the two peptides, D-D-oxytocin and D-D-Arg-vasotocin, and D-D-Arg-vasopressin.
The results of representative peptides are summarized in Table I. Figure 5 shows the modi~cation of the effect of phy~~laemin on the TAN caused by enzyme treatment. After trypsin treatment for 30 min phy~~emin still exhibited a marked excitatory effect on the TAN. When treated with the silme enzyme for 6 hr, however. this peptidc either lost its effect entirely, or. as on a few occasions, demonstrated it only weakly. After chymotrvpsin treatment for both 30 min and 6 hr. this peptide showed a marked inhibitory effect, in contrast to its effect before enzyme treatment. The TAN inhibition occurred within 1 to 1 min of the application of the chymotrypsin-treated peptide. Two other vase-active peptides (substance P and eledoisinrelated peptide) and three synthetic peptides, analogous to neurohypophys~ll hormonal peptide, showed no effect on the TAN excitability, even after the enzvme trcttment. ‘ Although non-treated physalaemin does not affect the PON excitability. this peptide produced a marked inhibitory effect when treated by chymotrypsin for 30 min (Fig. 6). This inhibitory affect disappeared. however. after physalaemin was treated by chymotrypsin for 6 hr. Trypsin-treated physalaemin had no effect on the PON at a concentration of 2 x lo-’ kg&. The
vaso-active peptides other than physalaemin, listed in Table 1. had no effect on the neurone either before or after enzyme treatment. As shown in Figure 7, D-D-Arg-vasotocin lost its excitatory effect on the PON after trypsin treatment. D-D-Oxytocin and D-D-Arg-vasotocin, however, were still excitatory after incubation with chymotrypsin for 6 hr under the above conditions. D-D-Arg-vasopressin had no effect on the PON either before or after treatment by two enzymes. Besides the cxperimen~l results shown in Table 1. Lys-bradykinin, bradykinin. Met-Lys-bradykinin, angiotensin I, II. III and caerulein, after the ei~zyme treatment, were examined. All were, however, without effect on the excitability of the two neurones.
Figure 8 shows superimposed I-V curves (a. measured in the physiological state: b, measured 3 min after the bath application of physalaemin at 2 x 10-j kg/l) of the same TAN neuromembrane. Three minutes after the application of 2 x IO-’ kg/l physalaemin, the TAN neuromembrane was markedly depolarized with augmentation of the number of its spike discharges. In Figure 8A, the two I-V curves were superimposed using the initial polarization level (just before the transmembrane current application) as the common standard. In this Figure, it appears that the membrane resistance was reduced by physalaemin application. However, superimposition of the two I- V curves using the firing level of the neurone
HIROSHITAKEUCHIet al
600 TAN
superimposed using the firing level of the neuronc as the common standard (Fig. 9B), a concordance of the two curves in a wide range of the polarization level was apparent. The conclusion. therefore. is that the 1-V curve of the PON is not affected by D-foxytocin in this concentration. DlSClJSSlORi (~1) Physlol (b)
3mfn 2xIO-4physalaem~n
Flung
IOnA 30sec
Fig. 8. Changes of the current voltage relationship (I-V curve) of the TAN neuromembrane, caused by physalaemin (bath application). To measure the I-V curve, a triangular current (hyperpokirizing, depolarizing and hyperpolarizing (I.2 x IO’)-“Hz). has been applied into the TAN soma. (a), the TAN 1-V curve measured in the physiological state. (b), the TAN 1-V curve measured 3 min after the application of 2 x 10m4 kg/l physalaemin. In A, the two I-V curves have been superimposed using the initial polarization level (just before the triangular current application) as the common standard. Ordinate (V), the voltage of biopotential shift (mV). Abscissa (I), the intensity of applied current (nA). Horizontal bar, the time course (30 set). In B. the two I-V curves have been superimposed
using the firing level as the common standard. Vertical bar, calibration of biopotential shift (40 mV). Upper horizontal bar. intensity of applied current (IO nA). Lower horizontal bar, the time course (30 set). Note that the two I-V curves are concordant in the wide range of membrane polarization level, when the two curves have been superimposed using the firing level as the common standard.
as the common standard (Fig. 8B) showed their concordance from the hyperpolarized state to above the firing level of the neurone. It was concluded, therefore. that the TAN 1-V curve was not affected by physalaemin in this concentration. As is apparent in this Figure. the TAN 1-V curve is not linear, although the degree of its non-linearity is less than that of the PON. Concordance was achieved, therefore, by compensating in the graph for the depolarization of the neuromembrane caused by physalaemin when superimposing the two I V curves. In Figure 9, two 1-V curves of the same PON measured in different conditions (a, in the physiological state; b. 3 min after the application of D-D-OXytOtin at 2 x 10m4 kg/l) are superimposed. The application of o-D-oxytocin produced a marked excitatory effect on the PON. The manner of superimposing this Figure was the same as in Figure 8. The membrane rectification of the PON was stronger than that of the TAN. When the two 1-V curves of the PON were
It has been reported previously (Takeuchi c’f uI.. 1975a) that all amino acids making up the peptides examined in the present study were without effect on the two identifiable giant neuroncs. so the effects 01 these peptides, therefore, cannot be due to their rcspective amino acids. Physalaemin. a hypotensive peptidc extracted from the skin of a South American amphibian (Phpr/uen~us~irscurt~a~ulu~u.s) (Erspamer, Bertaccini and Cci. 1962; Erspamer, Anastasi, Bertaccini and Cei. 1964: Anastasi, Erspamer and Cei, 1964). clearly showed :I biphasic effect (inhibitory-excitatory) on the TAN biopotential by microdrop application. The excitatory phase was much more dominant than the inhibitory one. so only excitation of the TAN was ohserlcd with
1V(mV)
PON
A
(a) (b)
Physiol 3mln 2~lO-~d-d-oxytocin
30 5et
Fig. 9. Changes of the PON I V CIII’~Cca~~scd ha I)-I)-osvtocin (bath apphcation). (a), the PON I V curve measured in the physiological state. (b), the PON 1 V curve measured 3 min after the application of 3 x IO m4 kg/l r,-I,-oxytocln. In A, the two 1-V curves have been superimposed usmg the initial polarization level (just before the triangular current application) as the common standard. Ordinate (V). the voltage of biopotential shift (mV). Abscissa (I). the intensity of applied current (nA). Horizontal bar. the time course (30 set). In B. the two 1-V curves have been superimposed using the firing level as the common standard. Vertical bar, calibration of biopotential shift (40 mV). Upper horizontal bar. intensity of applied current (I 0 nA). Lower horizontal bar, the time course (30 set). Note that the two 1-V curves are concordant in the wide range of membrane polarization level. when the two curves hate been superimposed using the firing level as the common standard.
Peptide effects on molluscan giant neurones bath application of physalaemin. Two explanations of this phenomenon are possible: either two kinds of receptors for physalaemin (inhibitory and excitatory) are present in the TAN neuromembrane, or there are two different groups of active sites in the physalaemin structure. vertebrate neurones. Phillis and Concerning Limacher (1974a, b) reported the effects of vaso-active peptides on Betz neurones and spontaneously active unidentified neurones in the rat cerebral cortex. They detected an excitatory effect of substance P, bradykinin and physalaemin on the majority of the neurones examined. Konishi and Otsuka (1974) reported the effects of vaso-active peptides on the ventral root potential (a mass response) of the frog spinal cord (Rmu cutrshrimu). Negative deflection of the ventral root potential occurred after application of substance P. physalaemin and eledoisin. They postulated that the change in this potential was due to the depolarization of the spinal motoneurones caused by the three peptides having a common C-terminal sequence (-Phe-X-Gly-Leu-Met-NHJ. The present study, on the other hand. demonstrated that the TAN neuromembrane was selectively sensitive to physalaemin. Treatment by trypsin to cleave the peptide bond between “Lys-Phe” resulted in the loss of the effect of physalaemin on the TAN. The subcomponcnts produced by this trypsin treatment, most probably “Pyr-Ala-Asp-Pro-Asn-Lys” and “PheTyr-Gly-Leu-Met-NH,“, should, therefore, be similarly devoid of effect. The results of the present experiments could not confirm the hypothesis of Konishi and Otsuka. When treated by chymotrypsin for either 30 min or 6 hr, physalaemin showed a marked inhibitory effect on the TAN. This unexpected phenomenon must be due to some inhibitory peptide (possibly an oligopeptide) produced by the treatment. The present study did not elucidate the amino acid sequence of the inhibitory peptide. but was able to demonstrate that the C-terminal sequence of physalaemin, “Glywas not inhibitory. since substance Leu-Met-NH?“. P and eledoisin-related substance, did not affect the TAN after chymotrypsin treatment although the same sequence must be produced in these cases. Another identifiable neurone, the PON, was excited by deamino-dicarba-(D-D-)oxytocin and D-D-Argvasotocin, but not by D-D-Arg vasopressin. Barker, Ifshin and Gainer (1975) reported that neurohypophyseal hormones and their analogues, including Argvasopressin, Arg-vasotocin and oxytocin. caused depolarization and augmentation of bursting pacemaker potentials (BPP’s) of two molluscan slowlyoscillating giant neurones. cell 11 of Otala lactea (Gainer. 1972) and R 15 of Aplysia californica (Frazier, Kandel. Kupfetmann, Waziri and Coggeshall. 1967). Their experimental results, however. do not completely agree with those of the present study of the PON. The concentration of neurohypophyseal peptides producing the depolarization reported by Barker
601
et ul. (1975) was much lower than that of deaminodicarba peptides analogous to neurohypophyseal hormones exciting the PON. Further, Barker et (II. (1975) reported that not only oxytocin and Arg-vasotocin but,also Arg-vdsopressin were effective on the neurones examined; however. the. present ‘study has not demonstrated any effect of D-D-Arg-vasopressin on the PON. Concerning mammalian central neurones, Moss, Dyball and Cross (1972) observed that the majority of neurosecretory neurones in the paraventricular nucleus of rabbits and rats were excited by iontophoretically applied oxytocin, but not by vasopressin. Deamino-dicarba-(D-D-)Arg-vasotocin lost its excitatory effect on the PON after trypsin treatment. Since trypsin cleaves the peptide bond at “Arg-Gly”, the “Gly-NH2” in the C-terminal of this peptide is apparently indispensable to the production of this effect on the PON. As mentioned above, D-D-oxytocin and D-D-Arg-vasotocin (but not D-D-Arg-vasopressin) have an excitatory effect on the same neurone. This means that the “Ile” at the second position in the amino acid sequence of the two effective peptides must also be necessary for the production of this effect. Almost the entire structure of the two effective peptides may, therefore, be necessary to produce excitation of the PON. According to Walter and Hoffman (1974) and Sakakibara (personal communication), oxytocin and Lysvasopressin (not deamino-dicarba form) were resistant to the cc-chymotrypsin treatment, but the deaminodicarba analogues of these peptides were much less resistant to the same treatment. In the present experiments with the PON, the activity of D-D-oxytocin and D-D-Arg-vasotocin still existed after a-chymotrypsin treatment for 6 hr. Without analysis of the structural change caused by cc-chymotrypsin in these peptides. however, the relationship between these chemical modifications and their effects is not clear. It was not anticipated that the TAN. a molluscan giant neurone, would be selectively sensitive to physalaemin. a peptide extracted from an amphibian skin. There is no evidence that physalaemin is physiologically active as. for example, a neurotransmitter of the TAN. However, there is the possibility that unknown peptides having active sites similar to those of physalaemin (excitatory or even inhibitory) might play a physiological role in the excitability change of the identifiable neurone. Although substance P has been assumed to be a neurotransmitter of several kinds of neurones, the two molluscan giant neurones examined were not sensitive to this substance. The possibility also exists that the effect of D-D-OXytOCin or D-DArg-vasotocin on the PON is physiological. perhaps acting as an analogous substance of a neurotransmitter. although there is as yet insufficient evidence for this view. It would appear that the structure-activity relationships of such .peptides as those examined in the present study must be selective and regular at the
neuronal level. as in the case of alnines and amino acids, but further experiments are necessary to confirm this.
Phillis. J. W. and Limacher. J. J. (19741). Substance P cwtation of cerebral cortical Bet7 cells. Rrtrh RLJ.~. 69: 158 163.
.-lc~rtoi~irtt~n~l~,/~rs-The authors wish to thank Associate Professor Sadnaki iwanaga of Osaka University and DF Atsuo Inotte of Daiichi Pharmaceutical Co. for their helpful advice. and Miss Hiroko Tamura for her ‘technical assistance.
REFERENCES Anastasi. A.. Erspamer. V. and Cci. J. M. (1964). Isolation and amino acid sequence of physalaemin, the main active polypcptidc of the skin of Physalaemus fuscumaculatus. At&i ~i~j~~~~~fl.Biopitys. 108: 341&34X. Araki, K.. Tachibana, S.. Uchiyama. M.. Nakajima. T. and Yasuhara, T. (1973). lsofation and structure of u new active peptide “xenopsin” on the smooth muscle. especially on a strip of ftmdus from a rat stomach. from the skm of Xenopus Inevis. Cl~cr~. pllclrrt~. Blrll.. 7i&,lto 21: 2801-1804. Arvanitaki. A., Romey. G. et Takeuchi. H. (1967). Variations de la relation caracthistique V(i).do la ncuromemhrane en fonction de la ~0:. Implications fonc~ioni~clles. C.r. Siur~c. Sot. Bioi. 161: 1629.-1633. Barker. J. L., Ifshin. M. S. and Gainer. H. (1975). Studies on bursting pacemaker potential activity in molluscan neurons. III. Effects of hormones. Brairl Rev. 84: 501&51?. Erspamer. V., Rertaccim, G. and Cei. J. M. (1963). Occurrence of an eledoisin-like polypeptide (phy~laemin) in skin extracts of Physalaemus fuscumoculatus. E.uprricwtiu 18: 56’- 563. Erspamer. V.. Anastasi. A.. Bertaccini. G. and Cei. J. M. (I 964). Structure and pharmacological actions of Physalaemin, the main active polypeptide of the skin of physalaemus fuscumaculatus. E.xpcriottici 20: 489-440. Frazier. W. T., Kandel, E. R.; Kupferm~~nn. I., Waziri. R. and Coogeshall. R. E. (1967). Mt~rnholo~ical and functional properties of identified nct&ns in-the abdominal ganglion of Aplysia californica. .I. lYewop/~~sio/. 30: 12x8~1351. Gainer. H. (1971). Patterns of protein synthesis in individual, identified molluscan neurons. Bruin RLJS. 39: 369-3X5. Hase. S., Saknkibara. S., Wahrenbtlr8, M.. Kirchberger, M.. Schwartz. 1. L. and Walter, R. (1972). 1.6-Amino-suberic acid analogs of lysine- and arginine-vasoprcssiri and -vasotocin. Synthesis and biological properties. J. -i/n. &/?I. Sot. 94 : 3590- 3600. Kandel. E. R. and Taut. L. (1966). Anomalous rectification in the m&cerebral giant cells and its conseauences for synaptic transmission. J. fI1ysiof.. Lo&. 18% 2X7- 304. Konishi. S. and Otsuka. M. (1974). The effects of substance P and other peptides on spinal neurons of the frog. Brcrirr Res. 65: 397110. Moss. R. L.. Dyball. R. E. J. and Cross. B. A. (1972). Escitation of antidromically identified neurosecretory cells of the pamventricular nucleus by oxytocin applied iontophoretically. Espl Neud 34: 95.- IO?. Nair, R. M. G.. Kastin. A. J. and Schally, A. V. (1971). Isolation and structure of ~lypoth~~lamic MSH relrasrinhibiting hormone. 5io&m. hioph~x Res. Cormwr~ 43: Ii7h~l3XI. Ntshioka. K.. S; ttoh. P. S.. Constantopoulos, A. and Najjar. V. A. 11973). ’ rhe chemical synthesis of the phagocytosisstimulating t etrapeptide tuftsin (Thr-Lys-Pro-Arg) and its biological properties. Ulf>c~lri~tt. hifl$j~.s. &‘to 310: 23s 2.77.
Tukeuchi. H.. Mortmasa. T.. Kohsaka. M.. et Moril. F. (1973bl. Concentrations des qucs dans I’ht?molymphe tic I’Escargot / .~c~/I~J~~w fdim Fc~,ussrcc) selon l’i-tat de S&12I.. SO<,. Biol. 167: 59X-607.
Kobayashi, J. ions morgani_cknl if&iii1 nutrition. (‘.I,.
Effects of glutamic acid relatives on the electrical usti\lt! of an identified molluscan giant nt’urone ( 1~4trr1mr fuliw ftkwrc,). Mtuin Rcz. 103: 2hI 17-1.
Yamanaka. 1.. Hasc. S.. Sakakihara. S.. Schuarti. I. L,. Dub&. B. M. and Walter. R. ( 1970).Crystalline ticamine-dicarbo oxptocin. Prcpar’otion and bomc pharmacological propzrttes. ,llolcc,. Phmvuwl. 6: 371 JXO. Zetler. G. ( 19%). Substone P. ein Polypeptid ati? Darm tmd Gehirn mit deprcssivcn. hyperalgctischen und Morphinanta@onistischetl Wirkungen auf dab Zcntntl-ncr\cns\stem. N~(i(l7!.ll-Si.l7ii2ii’ll~l)i’t211. Irt,ii.
I..
1977. 16. 593-601.
Pergamon
Press
Prmted m Great Bream
EFFECTS OF BIOLOGICALLY ACTIVE PEPTIDES ON THE EXCITABILITY OF IDENTIFIABLE’ GIANT NEURONES OF AN AFRICAN GIANT SNAIL (ACK4TINA FULIC.4 FlQxm4C) HIROSHI TAKEUCHI. MICHIKO MATSUMOTOand AKINORI SAKAI Institute
[or Neurobiology.
Okayama University Okayama. Japan
Medical
School.
(Accepted 18 February 1977) Summary-Effects of biologically active peptides, including vaso-active peptides. hypothalamic hormonal peptides and peptides analogous to neurohypophyseal hormones, were examined on the excitability of two giant neurones (the TAN, tonically autoactive neurone and the PON. periodically oscillating neurone) of Acltatinu jiuu[icu Fdrussuc. In a 10m4kg/l bath application. an excitatory effect was produced on the TAN by physalaemin only, and on the PON by deamino-dicarba-(o-D-)oxytocin and D-D-Arg-vasotocin (not by D-D-kg-VaSOpreSSin). Microdrop application of physalaemin produced a biphasic effect (hyperpolarizing-depolarizing. dominant depolarizing effect) on the TAN, but only depolarization with the application of D-D-oxytocin and D-D-kg-VaSOtOCin to the PON. The excitatory effect of physalaemin on the TAN was lost after trypsin treatment. Contrary to expectation, inhibition was produced by chymotrypsin-treated physalaemin. D-D-Arg-vasotocin and D-D-oxytocin retained their effect on the PON after chymotrypsin treatment for 6 hr. D-D-kg-VdSOtOCin. however, lost tts effect after trypsin treatment. The current-voltage relationship (the I-V curve) of neurones was measured by application of a transmembrane triangular current of long duration. It was concluded that physalaein the same concenmm at 2 x IO-“keil does not affect the TAN I-V curve, and that o-o-oxytocin tration also does not affect the PON 1-V curve.
Some biologically active peptides, such as substance P. are thought to be putative synaptic transmitters of mammalian central nervous system neurones (Zetler. 1956; Phillis and Limacher, 1974a. b). The aim of the present study was to investigate the effects of vaso-active peptides, hypothalamic hormonal peptides and peptides analogous to neurohypophyseal hormones, and to attempt to clarify their structureactivity relationships on the excitability of two spontaneously firing giant neurones (the TAN, tonically autoactive neurone: and the PON, periodically oscillating neurone) identified in the subesophageal ganglia of an African giant snail (Achatina jdicu Fe’russac). Some pharmacological properties of the two identifiable neurones have already been reported (Takeuchi. Mori and Kohsaka. 1973a. 1974b, c: Takeuchi. Mori, Kohsaka and Ohmori, 1974d: Takeuchi, Yokoi, Mori and Kohsaka, 1975a. b; Takeuchi. Yokoi, Mori and Ohmori. 1976; Takeuchi, Yokoi and Hiramatsu, 1977). Modification of the effects of these peptides on the two neurones by treatment with trypsin and chymotrypsin was also examined. Observation of changes in membrane resistance of the identifiable neurones caused by the application of certain active peptides was performed. As is well known some molluscan neurones show membrane rectification (Kandel and Taut, 1966; Arvanitaki, Romey and Takeuchi, 1967) so, in the present study, as the indicator of the membrane resistance, the current-voltage relationship (I-V curve) of the neuro593
membrane was measured by injection of a transmembrane triangular current of long duration. MATERIALS AND
METHODS
Two spontaneously firing giant neurones (the TAN, tonically autoactive neurone. and the PON, periodically oscillating neurone) were identified in the subesophageal ganglia of an African giant snail, Achatina The TAN is excited by fiulica FPrtrssac. 5-hydroxytryptamine and histamine, inhibited by dopamine. GABA and acetylcholine. and not affected by L-glutamic acid. The PON is excited by 5-hydroxytryptamine and dopamine, inhibited by L-homocysteic acid and erythro-P-hydroxy-L-glutamic acid, and not affected by acetylcholine, GABA, histamine and L-glutamic acid. Arrangements for the electrophysiological experiments and localizations of the neurones in the ganglia have been described in previous papers (Takeuchi rt al., 1975b, 1976, 1977). A micropipette was implanted into the soma of the identifiable neurones, and their intracellular biopotentials were recorded by a penwriting galvanometer. The number of spike discharges per minute was automatically counted by a spike counter (Takeuchi, Kondoh and Misaki. 1974a). The following peptides, obtained in an analytically pure state, were tested in the present study: substance P. physalaemin. eledoisin-related peptide. neurotensin, bradykinin. Lys-bradykinin. Met-Lys-bradykinin. angiotensin I, II, III, luteinizing hormone-releasing
horlone (LH-RH ). tllyrt~tropin-releasing hormone (TRH), .~matost~~tin, MSH-release inhibiting factor (MRIH). (Nair, Kastin and Schally. 1971I. deaminodic;~rh3-(r,-u-)oxytocin ~Yamana~a. Hase. Sakakibara, Schwa&.. Dubois and Walter, 1970), I>-rt-Arg-vasoprcssin, I>-I>-Arg-vasotocin (Hase Sakakibara. Wahrcnburg, Kirchbergcr. Schwartz and Walter, 1972), liver-cell growth fitctor (Pickart. Thaycr. L. and Thqcr, M. 1473). tuftsin (Nishioku, Satoh. Constantopouios and Naj.jar, 1973) (products of Protein Research Fout~d~~t~on, Osaka). xenopsin (Araki. Tachihana. Uchiyama, Nakajima and Yasuhara, 1973) (donated by Eisai Co.). hypertensin (donated by CIBA-GEIGY Co.) and caerulein (donated by Falmitalia Co.). These peptides were applied to the experimental material by both bath application and microdrop appiic~~ti~~tl,In the bath appljcation, the peptidc to be examined, dissolved in the snail’s physiological solution (Takeuchi. Morimasa. Kohsaka, Kobayashi and Morii, 1973b). was applied directly to the dissected ganglia. Peptides demonstrated as cffectivr in the bath application were further tested by ioca1 application (the microdrop application). A microdrop (about IS0 ~lrn in diameter) of the peptide solution was made in air at the tip of a micropipette containing the peptide solution. and the microdrop c~cli~liy p&cd on the surface of an idcnti~~~ll~lencurone (diameters of the ncuroncs wcrc about I50 jrm 200 inni.
FO
r-Chymotrypsin (product of Wortliingt~~n Biochemical C~~rporation, 55 u,/mg). TPCK(r.- I-tosylamide-I!-phenylethyl-chloromrthyl ketone)-treated tryp‘hh sin ~Wortbiil~ton Riochemical Corporation. u/m@ and pancreatic trypsin-chym~trypsili inhi~~it~~l (Trasylol. Bayer A.G.) wet-c used for the enzyme trcatment of peptides. All substances used wt‘re dissolved in a physiological solution for the Afric:rn giant sn;iii, the pH value of which was around 7.5. One ml of a peptide solution (2.5 x Ii)-’ kg,‘!) was incubated at 37°C with 0.1 mi of an enzyme solution (5 x IO ’ kg/l) for either 30 min or G hr. After incubation, 0.17 ml of Trasylol f2 x IO-’ kg:l) W;LSaddcd to ;Irrc
in
the
physiological
st:tto
snd
the
TAN
60
2X 10~4neurotensin Fig. 1. Effects of vase-active pcptides on the TAN (tonically autoactive neurone) excitability (bath application) Ordinate. the number of spike discharges per minute. Abscissa. the time cowsc. each histogram is I min 2 x IO- ’ kgjl substance P. bradykinin, phy~~l~emin, 4 x lWi ph~~ll~lernin. 2 x if)-’ cledoisill-rci~lted substance, ~~n~iotensin I. angiotensin II and neurotensin were applied respectively.
otltc~
Peptide effects on molluscan giant neurones
Fig. 2. Comparison of effects of three peptides analogous to neurohypophyseal hormones on the PON (periodical11 oscillating neurone) excitability (bath application). Ordinate. the number of spike discharges per minute. Abscissa. the time course. each histogram is 1 min 2 x 10W4kg/l deamino-dicarha(i)-r)-tArg-vasopressin. I,-D-oxytocin, D-D-Arg-vasotocin, IO-’ D-D-oxytocin and o-o-Arg-vasotocin were applied respectively. measured in the presence of an effective peptide) were superimposed, the recordings of the applied current intensities were erased after confirming their fairly triangular shapes (no rectification of the current-applying pipette). The temperature of the experimental room was maintained at 23 + 1 C. The pH value of the examined peptide solutions was close to 7.5 in all cases. RESC’LTS
Vase-active peptides (substance P. physalaemin. elcdoisin-related peptide. neurotensin. xenopsin. Lys-bradykinin, bradykinin. Met-Lys-bradykinin. angiotensin 1. II. III and hypertensin). hypothalamic hormonal peptides (LH-RH. TRH, somatostatin and MRIH), peptides analogous to neurobypophyseal hormones (D-D-oxytocin, D-D-Arg-vasopressin and D-DArg-vasotocin) and others (caerulein, liver-cell growth factor and tuftsin) were tested by bath application. Of the peptides examined, only physalaemin, a vaso-active peptide from an amphibian skin, showed an excitatory effect on the TAN. The critical concentration to produce the effect was about 10-4kg/l. The other peptides examined had no effect on this neurone at 2 x IO--” kg/l. As shown in Figure 1. application
of phy~laemin at 2 x 10e4 kg/l resulted in an increase in the number of spike discharges of the neurone to more than twice that of the physiological state. This effect disappeared after washing the ganglia with the physiological solution. An effect of physalaemin at 4 x lo- ’ kg/l was no longer demonstrable. Substance P, bradykinin, eledoisin-related peptide, angiotensin I and II and neurotensin were without effect at 2 x 10m4 kg/l. The sensitivity of another identifiable neurone, the PON, to biologically active peptides was completely different from that of the TAN. Of the peptides examined, two peptides analogous to neurol~ypophyseal D-D-oxytocin and D-D-Arg-vasot~in, hormones. showed an excitatory effect on the PON. The critical concentration of these two peptides was about lo-” kg/l. The other peptides examined at 2 x 10s4 kg/l. including D-D-Arg-vasopressin, produced no effect. In Figure 2. the effect of D-D-OXytOCin, D-D-Arg-vasototin and D-D-Arg-vasopressin on the PON excitability is compared. D-D-OXytOCin and D-D-Arg-vasotocin at 10-j kg/l clearly prodbced an increase in the number of the PON spike discharges, although D-D-Arg-vasopressin, even at 2 x 10 -4 kg/l, did not affect the PON. Eficts of‘ local ~l~~licat~uiz~t~~j~~od~~~ a~p~jcatioi~)qf p~~t~d~~on f/w hvo id~~lti~abl~rwuronrs To distinguish between the direct effect of peptides
596
Hmosrn
TAKEUCHI
er cd.
TAN
f Physofaemine
2X IO-’
~O.tSmm 30 set
(3.5ng) XImV I
20mV I
Fig. 3. Effects of l~hysalaemin on the TAN biopotential (microdrop application). The upper trace is a full spike recording of the TAN biopotential by a pen-writing galvanometer. The lower trace is a high amplification recording of the upper trace (spike peaks have been cut by an electronic voltage clipper). A microdrop (150 jirn in diameter) of 2 x 1W3kgJ physalaemil~ (the total ~~lnount of applied phfsaldemin estimated to he about 3.5 ng) was applied on the TAN surface (arrow). Left vertical bar, calibration for the upper trace (50 mV). Right vertical bar. calibration for the lower trace (20 mV). Horizontal bar. the time course (30 set). After local application of physalacmin. the TAN biopotentiul showed a slight hyperpolarization followed by a marked depolarization.
PON
A
Fig. 4. Comparison of effects of three peptides analogous to neurohypophyseal hormones on the PON excitability (microdrop application). The upper traces of A. B and C are full-spike recordings of the PON biopotential by a pen-writing galvanometer. The lower traces of A. B and C are high amplification recordings of the upper traces (spike peaks have been cut by an electronic voltage clipper). In A. a microdrop (IS0 pm in diameter) of 4 x 10m4 kg,1 deamino-di~arba(~-I.~-)-ox~toc~n (the total amount of o-o-oxytocin estimated to be about 700 pg) was applied on the PON surface (arrow). In B, a microdrop (the same diameter) of 4 x IO-’ u-D-Arg-vasotocin (the total amount estimated to be about 700 pg) was applied (arrow). In C. a microdrop (the same diameter) of 4 x 10m4 rl-o-Arg-vasopressin (the total amount estimated to be about 700 pg) was applied (arrow). Left vertical bar. calibration for the upper traces (50 mV). Right vertical bar, calibration for the lower traces (20 mV). Horizontal bar, the time course (30 set). After local application of D-D-oxytocin and u-u-Arg-vasotocin. the PON biopotenti~il showed a depolarization. Local application of ~I~-Arg-vasopressin had no ell’ect.
Peptide
etl’ects on molluscan
giant
597
neurones
Table I. Effects of enLyme-treated peptides. compared with non-treated ones, on the two identifiable (the TAN. tonically autoactive neurone; and the PON. periodically oscillating neurone) of Achatina All peptides were examined at a concentration of 2 x 10V4 kg/l (bath application)
Substance Substance P Substance P Substance P Substance P Substance P Physalaemin Physalaemin Physataemin Physalaemin Physataemin Etedoisin-related substance Eiedoisin-related substance Eiedoisin-related substance Eledoisin-related substance Eledoisin-related substance Deaminodicarbaoxytocin
Deaminodicarbaoxytocin Dcaminodicarbaoxytocin Deaminodicarba-Argvasotocin Deaminodicarba-Agvasotocin Deaminodicarhn-Argvasotocin Dermino‘ dicarba-iirgvasotocm Deaminodicarba-Argvasotocin Deaminodicarba-Argvasopressin Deaminodicarba-Argvasopressm Deaminodicurba-Agvasopressin Deaminodicarba-Argvasoprcssin Dcaminodicarba-Arg-
vasopressin
Enzyme treatment None T T CT CT None ; CT CT
Incubation time
Effects on TAN
Amino
Effects on PON
(--I
i-1 (- 1
(-) (--I
(-) (6) (6) C-1 (6)
30 min hhr 30 min 6 hr
c-i
30 min 6 hr 30 min 6 hr
(;J* (1)
None
(;’
I
(4
(-)
(4
T
30 min
(--I
I-1
T
6hr
(-)
(4
CT
30 min
(--)
t-1
CT
6hr
(-)
f-1
(-)
E
None CT
30 min
(-)
E
CT
6 hr
(-)
E
(-)
E
None
T
30 min
(-)
t-1
T
6hr
(-f
(-)
CT
30 min
(-)
E
CT
6 hr
(-)
E
(-)
i-4
T
30 min
(-)
T
6hr
(-
CT
30 min
t-1
(-)
CT
6 hr
t-1
k-)
acid sequence and main sites cleaved by enzymes
CT
T
cr
CT
CT
Pyr-Ala-Asp-Pro-Asn-Ly~~Plle!Tqr!Gly-Leu-Met-NH2
: Lys-Phe~;le-Gly-Leu-Met-Nap
,+Tyr-Ile-Gin-Am-Asu-Pro-Leu-Gly-NH2
+
~Tyr!Ile-Gin-Asn-ALju-Pro-Arg~GIy-NHI
L‘T’
)
neurones Ferussac.
Arg~Pro-Lys-Pro-Gtn-Gin-Ph~~Phe!Gi~-L~,,-Met-N~~
CT*
None
giant fulica
CT“
f ) rtTyi-Phe!Cln-Asn-ASu-Pro-Arg~Gty-NHz
E---excitatory effect; (-+no effect: (-)*-no effect, but sometimes a slight excitatory effect: T-trypsin: CT--chymotrypsin; T--main sites of cleavage by trypsin; ‘T-main sites of cleavage by chymotrypsin; Pyr-r--pyroglutamic acid; Asu-L-a-amino suberic acid; +-cleavage was demonstrated (Walter and Hoffman, 1974): ++---cleavage is deduced from peptide structure.
Fig. 5. EtTecta ill’ on7~m+trcatcd physnluemin on the TAN excitability (bath application). Ordin;ttc. the numhcr of spike d~schargas par minute. Abscissa. the time course, each histogram is I min 7 x lVJ kg,1 ph~solaemrn. trcalctl hj trypsin (T) for 6 hr. b> T for 30 min. by chymotrypsin (CT) for 30 miu and by CT for 6 hr, were applied respectively.
ng ( I50 izrn in diamctcr. at 2 x f 0” 3 kg ‘I) was applied to the TAN surface (Fig. 3). Several seconds after the
application, the TAN biopotentinl showed a slight hyperpolarization followed by a marked dcpolarization. The TAN biopotential changes thus caused were clearly biphasic. the excitatory phase being dominant. These biopotentiai changes disappeared even if the examined ganglia remained unwashed. Presumably.
oxytocin and t~~~-Ar~-vi~~otuc~l~ caused ~~~~~~~ri~~~tion of &he PON neuromembrane with frequency augmentation of the slow oscillation of biopotential? while n-rz-Arg-vasapressin applied in the samc u;t) did not affect the hiopotential (Fig. 4). The depolnrization of the ncuromembrane occurred almost immediately after the microdrop application, and disapfared after severed minutes. even without ~shing
PUN
Fig. 6. Effects of enzyme-trcntcd phvsahemm on the PON the number of spike discharges per mjnute. Abscissa, the time physalaemin. treated by cbymotrypsin (CT) for 30min, by and hy T for 6 hr. were applied
excitability (bath application). Ordinate. course, each histogram is I min 2 x IO-” CT for 6 hr, by trppsin (T) for 30mit1 respectively.
Peptide effects on molluscan giant neurones
?t:10-‘4 d-ddrg-vasotocln ! T-6h )
2x10”)
599
y-;_$r;-)vascpressin
Fig. 7. Administration or trypsin (T)-treotcd peptides a&logous to neurohypophyseal hormones with respect to the PON cucttnhlhty (hath application). Ordinate, the number of spike discharges per minute. Abscissa. the time course. cuch histogram is 1mm 2 x lo-.” kg/l de~mino-dic~rb~ (u-u)-Arg-vasopressin (treated hg T l’or 30 min). i~-l)-Ar~-v~lsotocin (T for 30 mint, ~-~Arg-~sotoci~l (T for 6 hrl and [~-I~-Ark-v~~sopressin (T for 6 hr) were applied respectively.
the ganglia. Microdrop application also indicated a difference in effect between the two peptides, D-D-oxytocin and D-D-Arg-vasotocin, and D-D-Arg-vasopressin.
The results of representative peptides are summarized in Table I. Figure 5 shows the modi~cation of the effect of phy~~laemin on the TAN caused by enzyme treatment. After trypsin treatment for 30 min phy~~emin still exhibited a marked excitatory effect on the TAN. When treated with the silme enzyme for 6 hr, however. this peptidc either lost its effect entirely, or. as on a few occasions, demonstrated it only weakly. After chymotrvpsin treatment for both 30 min and 6 hr. this peptide showed a marked inhibitory effect, in contrast to its effect before enzyme treatment. The TAN inhibition occurred within 1 to 1 min of the application of the chymotrypsin-treated peptide. Two other vase-active peptides (substance P and eledoisinrelated peptide) and three synthetic peptides, analogous to neurohypophys~ll hormonal peptide, showed no effect on the TAN excitability, even after the enzvme trcttment. ‘ Although non-treated physalaemin does not affect the PON excitability. this peptide produced a marked inhibitory effect when treated by chymotrypsin for 30 min (Fig. 6). This inhibitory affect disappeared. however. after physalaemin was treated by chymotrypsin for 6 hr. Trypsin-treated physalaemin had no effect on the PON at a concentration of 2 x lo-’ kg&. The
vaso-active peptides other than physalaemin, listed in Table 1. had no effect on the neurone either before or after enzyme treatment. As shown in Figure 7, D-D-Arg-vasotocin lost its excitatory effect on the PON after trypsin treatment. D-D-Oxytocin and D-D-Arg-vasotocin, however, were still excitatory after incubation with chymotrypsin for 6 hr under the above conditions. D-D-Arg-vasopressin had no effect on the PON either before or after treatment by two enzymes. Besides the cxperimen~l results shown in Table 1. Lys-bradykinin, bradykinin. Met-Lys-bradykinin, angiotensin I, II. III and caerulein, after the ei~zyme treatment, were examined. All were, however, without effect on the excitability of the two neurones.
Figure 8 shows superimposed I-V curves (a. measured in the physiological state: b, measured 3 min after the bath application of physalaemin at 2 x 10-j kg/l) of the same TAN neuromembrane. Three minutes after the application of 2 x IO-’ kg/l physalaemin, the TAN neuromembrane was markedly depolarized with augmentation of the number of its spike discharges. In Figure 8A, the two I-V curves were superimposed using the initial polarization level (just before the transmembrane current application) as the common standard. In this Figure, it appears that the membrane resistance was reduced by physalaemin application. However, superimposition of the two I- V curves using the firing level of the neurone
HIROSHITAKEUCHIet al
600 TAN
superimposed using the firing level of the neuronc as the common standard (Fig. 9B), a concordance of the two curves in a wide range of the polarization level was apparent. The conclusion. therefore. is that the 1-V curve of the PON is not affected by D-foxytocin in this concentration. DlSClJSSlORi (~1) Physlol (b)
3mfn 2xIO-4physalaem~n
Flung
IOnA 30sec
Fig. 8. Changes of the current voltage relationship (I-V curve) of the TAN neuromembrane, caused by physalaemin (bath application). To measure the I-V curve, a triangular current (hyperpokirizing, depolarizing and hyperpolarizing (I.2 x IO’)-“Hz). has been applied into the TAN soma. (a), the TAN 1-V curve measured in the physiological state. (b), the TAN 1-V curve measured 3 min after the application of 2 x 10m4 kg/l physalaemin. In A, the two I-V curves have been superimposed using the initial polarization level (just before the triangular current application) as the common standard. Ordinate (V), the voltage of biopotential shift (mV). Abscissa (I), the intensity of applied current (nA). Horizontal bar, the time course (30 set). In B. the two I-V curves have been superimposed
using the firing level as the common standard. Vertical bar, calibration of biopotential shift (40 mV). Upper horizontal bar. intensity of applied current (IO nA). Lower horizontal bar, the time course (30 set). Note that the two I-V curves are concordant in the wide range of membrane polarization level, when the two curves have been superimposed using the firing level as the common standard.
as the common standard (Fig. 8B) showed their concordance from the hyperpolarized state to above the firing level of the neurone. It was concluded, therefore. that the TAN 1-V curve was not affected by physalaemin in this concentration. As is apparent in this Figure. the TAN 1-V curve is not linear, although the degree of its non-linearity is less than that of the PON. Concordance was achieved, therefore, by compensating in the graph for the depolarization of the neuromembrane caused by physalaemin when superimposing the two I V curves. In Figure 9, two 1-V curves of the same PON measured in different conditions (a, in the physiological state; b. 3 min after the application of D-D-OXytOtin at 2 x 10m4 kg/l) are superimposed. The application of o-D-oxytocin produced a marked excitatory effect on the PON. The manner of superimposing this Figure was the same as in Figure 8. The membrane rectification of the PON was stronger than that of the TAN. When the two 1-V curves of the PON were
It has been reported previously (Takeuchi c’f uI.. 1975a) that all amino acids making up the peptides examined in the present study were without effect on the two identifiable giant neuroncs. so the effects 01 these peptides, therefore, cannot be due to their rcspective amino acids. Physalaemin. a hypotensive peptidc extracted from the skin of a South American amphibian (Phpr/uen~us~irscurt~a~ulu~u.s) (Erspamer, Bertaccini and Cci. 1962; Erspamer, Anastasi, Bertaccini and Cei. 1964: Anastasi, Erspamer and Cei, 1964). clearly showed :I biphasic effect (inhibitory-excitatory) on the TAN biopotential by microdrop application. The excitatory phase was much more dominant than the inhibitory one. so only excitation of the TAN was ohserlcd with
1V(mV)
PON
A
(a) (b)
Physiol 3mln 2~lO-~d-d-oxytocin
30 5et
Fig. 9. Changes of the PON I V CIII’~Cca~~scd ha I)-I)-osvtocin (bath apphcation). (a), the PON I V curve measured in the physiological state. (b), the PON 1 V curve measured 3 min after the application of 3 x IO m4 kg/l r,-I,-oxytocln. In A, the two 1-V curves have been superimposed usmg the initial polarization level (just before the triangular current application) as the common standard. Ordinate (V). the voltage of biopotential shift (mV). Abscissa (I). the intensity of applied current (nA). Horizontal bar. the time course (30 set). In B. the two 1-V curves have been superimposed using the firing level as the common standard. Vertical bar, calibration of biopotential shift (40 mV). Upper horizontal bar. intensity of applied current (I 0 nA). Lower horizontal bar, the time course (30 set). Note that the two 1-V curves are concordant in the wide range of membrane polarization level. when the two curves hate been superimposed using the firing level as the common standard.
Peptide effects on molluscan giant neurones bath application of physalaemin. Two explanations of this phenomenon are possible: either two kinds of receptors for physalaemin (inhibitory and excitatory) are present in the TAN neuromembrane, or there are two different groups of active sites in the physalaemin structure. vertebrate neurones. Phillis and Concerning Limacher (1974a, b) reported the effects of vaso-active peptides on Betz neurones and spontaneously active unidentified neurones in the rat cerebral cortex. They detected an excitatory effect of substance P, bradykinin and physalaemin on the majority of the neurones examined. Konishi and Otsuka (1974) reported the effects of vaso-active peptides on the ventral root potential (a mass response) of the frog spinal cord (Rmu cutrshrimu). Negative deflection of the ventral root potential occurred after application of substance P. physalaemin and eledoisin. They postulated that the change in this potential was due to the depolarization of the spinal motoneurones caused by the three peptides having a common C-terminal sequence (-Phe-X-Gly-Leu-Met-NHJ. The present study, on the other hand. demonstrated that the TAN neuromembrane was selectively sensitive to physalaemin. Treatment by trypsin to cleave the peptide bond between “Lys-Phe” resulted in the loss of the effect of physalaemin on the TAN. The subcomponcnts produced by this trypsin treatment, most probably “Pyr-Ala-Asp-Pro-Asn-Lys” and “PheTyr-Gly-Leu-Met-NH,“, should, therefore, be similarly devoid of effect. The results of the present experiments could not confirm the hypothesis of Konishi and Otsuka. When treated by chymotrypsin for either 30 min or 6 hr, physalaemin showed a marked inhibitory effect on the TAN. This unexpected phenomenon must be due to some inhibitory peptide (possibly an oligopeptide) produced by the treatment. The present study did not elucidate the amino acid sequence of the inhibitory peptide. but was able to demonstrate that the C-terminal sequence of physalaemin, “Glywas not inhibitory. since substance Leu-Met-NH?“. P and eledoisin-related substance, did not affect the TAN after chymotrypsin treatment although the same sequence must be produced in these cases. Another identifiable neurone, the PON, was excited by deamino-dicarba-(D-D-)oxytocin and D-D-Argvasotocin, but not by D-D-Arg vasopressin. Barker, Ifshin and Gainer (1975) reported that neurohypophyseal hormones and their analogues, including Argvasopressin, Arg-vasotocin and oxytocin. caused depolarization and augmentation of bursting pacemaker potentials (BPP’s) of two molluscan slowlyoscillating giant neurones. cell 11 of Otala lactea (Gainer. 1972) and R 15 of Aplysia californica (Frazier, Kandel. Kupfetmann, Waziri and Coggeshall. 1967). Their experimental results, however. do not completely agree with those of the present study of the PON. The concentration of neurohypophyseal peptides producing the depolarization reported by Barker
601
et ul. (1975) was much lower than that of deaminodicarba peptides analogous to neurohypophyseal hormones exciting the PON. Further, Barker et (II. (1975) reported that not only oxytocin and Arg-vasotocin but,also Arg-vdsopressin were effective on the neurones examined; however. the. present ‘study has not demonstrated any effect of D-D-Arg-vasopressin on the PON. Concerning mammalian central neurones, Moss, Dyball and Cross (1972) observed that the majority of neurosecretory neurones in the paraventricular nucleus of rabbits and rats were excited by iontophoretically applied oxytocin, but not by vasopressin. Deamino-dicarba-(D-D-)Arg-vasotocin lost its excitatory effect on the PON after trypsin treatment. Since trypsin cleaves the peptide bond at “Arg-Gly”, the “Gly-NH2” in the C-terminal of this peptide is apparently indispensable to the production of this effect on the PON. As mentioned above, D-D-oxytocin and D-D-Arg-vasotocin (but not D-D-Arg-vasopressin) have an excitatory effect on the same neurone. This means that the “Ile” at the second position in the amino acid sequence of the two effective peptides must also be necessary for the production of this effect. Almost the entire structure of the two effective peptides may, therefore, be necessary to produce excitation of the PON. According to Walter and Hoffman (1974) and Sakakibara (personal communication), oxytocin and Lysvasopressin (not deamino-dicarba form) were resistant to the cc-chymotrypsin treatment, but the deaminodicarba analogues of these peptides were much less resistant to the same treatment. In the present experiments with the PON, the activity of D-D-oxytocin and D-D-Arg-vasotocin still existed after a-chymotrypsin treatment for 6 hr. Without analysis of the structural change caused by cc-chymotrypsin in these peptides. however, the relationship between these chemical modifications and their effects is not clear. It was not anticipated that the TAN. a molluscan giant neurone, would be selectively sensitive to physalaemin. a peptide extracted from an amphibian skin. There is no evidence that physalaemin is physiologically active as. for example, a neurotransmitter of the TAN. However, there is the possibility that unknown peptides having active sites similar to those of physalaemin (excitatory or even inhibitory) might play a physiological role in the excitability change of the identifiable neurone. Although substance P has been assumed to be a neurotransmitter of several kinds of neurones, the two molluscan giant neurones examined were not sensitive to this substance. The possibility also exists that the effect of D-D-OXytOCin or D-DArg-vasotocin on the PON is physiological. perhaps acting as an analogous substance of a neurotransmitter. although there is as yet insufficient evidence for this view. It would appear that the structure-activity relationships of such .peptides as those examined in the present study must be selective and regular at the
neuronal level. as in the case of alnines and amino acids, but further experiments are necessary to confirm this.
Phillis. J. W. and Limacher. J. J. (19741). Substance P cwtation of cerebral cortical Bet7 cells. Rrtrh RLJ.~. 69: 158 163.
.-lc~rtoi~irtt~n~l~,/~rs-The authors wish to thank Associate Professor Sadnaki iwanaga of Osaka University and DF Atsuo Inotte of Daiichi Pharmaceutical Co. for their helpful advice. and Miss Hiroko Tamura for her ‘technical assistance.
REFERENCES Anastasi. A.. Erspamer. V. and Cci. J. M. (1964). Isolation and amino acid sequence of physalaemin, the main active polypcptidc of the skin of Physalaemus fuscumaculatus. At&i ~i~j~~~~~fl.Biopitys. 108: 341&34X. Araki, K.. Tachibana, S.. Uchiyama. M.. Nakajima. T. and Yasuhara, T. (1973). lsofation and structure of u new active peptide “xenopsin” on the smooth muscle. especially on a strip of ftmdus from a rat stomach. from the skm of Xenopus Inevis. Cl~cr~. pllclrrt~. Blrll.. 7i&,lto 21: 2801-1804. Arvanitaki. A., Romey. G. et Takeuchi. H. (1967). Variations de la relation caracthistique V(i).do la ncuromemhrane en fonction de la ~0:. Implications fonc~ioni~clles. C.r. Siur~c. Sot. Bioi. 161: 1629.-1633. Barker. J. L., Ifshin. M. S. and Gainer. H. (1975). Studies on bursting pacemaker potential activity in molluscan neurons. III. Effects of hormones. Brairl Rev. 84: 501&51?. Erspamer. V., Rertaccim, G. and Cei. J. M. (1963). Occurrence of an eledoisin-like polypeptide (phy~laemin) in skin extracts of Physalaemus fuscumoculatus. E.uprricwtiu 18: 56’- 563. Erspamer. V.. Anastasi. A.. Bertaccini. G. and Cei. J. M. (I 964). Structure and pharmacological actions of Physalaemin, the main active polypeptide of the skin of physalaemus fuscumaculatus. E.xpcriottici 20: 489-440. Frazier. W. T., Kandel, E. R.; Kupferm~~nn. I., Waziri. R. and Coogeshall. R. E. (1967). Mt~rnholo~ical and functional properties of identified nct&ns in-the abdominal ganglion of Aplysia californica. .I. lYewop/~~sio/. 30: 12x8~1351. Gainer. H. (1971). Patterns of protein synthesis in individual, identified molluscan neurons. Bruin RLJS. 39: 369-3X5. Hase. S., Saknkibara. S., Wahrenbtlr8, M.. Kirchberger, M.. Schwartz. 1. L. and Walter, R. (1972). 1.6-Amino-suberic acid analogs of lysine- and arginine-vasoprcssiri and -vasotocin. Synthesis and biological properties. J. -i/n. &/?I. Sot. 94 : 3590- 3600. Kandel. E. R. and Taut. L. (1966). Anomalous rectification in the m&cerebral giant cells and its conseauences for synaptic transmission. J. fI1ysiof.. Lo&. 18% 2X7- 304. Konishi. S. and Otsuka. M. (1974). The effects of substance P and other peptides on spinal neurons of the frog. Brcrirr Res. 65: 397110. Moss. R. L.. Dyball. R. E. J. and Cross. B. A. (1972). Escitation of antidromically identified neurosecretory cells of the pamventricular nucleus by oxytocin applied iontophoretically. Espl Neud 34: 95.- IO?. Nair, R. M. G.. Kastin. A. J. and Schally, A. V. (1971). Isolation and structure of ~lypoth~~lamic MSH relrasrinhibiting hormone. 5io&m. hioph~x Res. Cormwr~ 43: Ii7h~l3XI. Ntshioka. K.. S; ttoh. P. S.. Constantopoulos, A. and Najjar. V. A. 11973). ’ rhe chemical synthesis of the phagocytosisstimulating t etrapeptide tuftsin (Thr-Lys-Pro-Arg) and its biological properties. Ulf>c~lri~tt. hifl$j~.s. &‘to 310: 23s 2.77.
Tukeuchi. H.. Mortmasa. T.. Kohsaka. M.. et Moril. F. (1973bl. Concentrations des qucs dans I’ht?molymphe tic I’Escargot / .~c~/I~J~~w fdim Fc~,ussrcc) selon l’i-tat de S&12I.. SO<,. Biol. 167: 59X-607.
Kobayashi, J. ions morgani_cknl if&iii1 nutrition. (‘.I,.
Effects of glutamic acid relatives on the electrical usti\lt! of an identified molluscan giant nt’urone ( 1~4trr1mr fuliw ftkwrc,). Mtuin Rcz. 103: 2hI 17-1.
Yamanaka. 1.. Hasc. S.. Sakakihara. S.. Schuarti. I. L,. Dub&. B. M. and Walter. R. ( 1970).Crystalline ticamine-dicarbo oxptocin. Prcpar’otion and bomc pharmacological propzrttes. ,llolcc,. Phmvuwl. 6: 371 JXO. Zetler. G. ( 19%). Substone P. ein Polypeptid ati? Darm tmd Gehirn mit deprcssivcn. hyperalgctischen und Morphinanta@onistischetl Wirkungen auf dab Zcntntl-ncr\cns\stem. N~(i(l7!.ll-Si.l7ii2ii’ll~l)i’t211. Irt,ii.