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Effect of eledoisin, physalaemin and some eledoisin- or physalaemin-like peptides on water intake and arterial blood pressure in conscious pigeons
Pharmacological Research Communications, Vol. 11, No. 10, 1979
891
EFFECT OF ELEDOISIN, PHYSALAEMIN AND SOME ELEDOISIN- OR PHYSALAEMIN-LIKE PEPTIDES" ON WATER INTAKE AND ARTERIAL BLOOD PRESSURE IN CONSCIOUS PIGEONS G. de Caro, M. Hassi and L,G, Hicossi
I n s t i t u t e of Pharmacology and Pharmacognosy, F a c u l t y of Pharmacy, U n i v e r s i t y of Camerino, 52052 Camerino ( I t a l y ) .
SUMMARY
The hypotensive and dipsogenic effects of eledoisin, physalaemin and some
synthetic
scious
eledoisin- or physalaemin-like peptides were studied in con-
pigeons,
respectively after intravenous or intracerebroventricular
administration. The effects of thepeptides
were compared to the hypotensi-
ve or dipsogenic activities of eledoisin considered equal to I00. i) Shortening
of eledoisin molecule by means of progressive elimination of
up to four N-terminal aminoacid residues left practically unmodified the dipsogenic effect, while it progressively reduced the hypotensive effect. 2) Physalaemin
was
a dipsogen as potent as eledoisin, but its hypotensive
activity was approximately double the one of eledoisin. 3) Modifying of
physalaeminmolecule
produced different alterations in the
dipsogen~c and hypotensive effect. Apparently
there was no relationship between =he dipsogen~c and the hy-
potensive effect of the substances tested.
INTRODUCTION
Intracerebroventricular (i.c.v.) injection of the tachykin i n s e l e d o i s i n and p h y s a l a e m i n e l i c i t s in the r a t a p o t e n t antidipsogenic effect (de Caro e t a l . , 1977; de Caro e_~t a_~l., 1978). Conversely, i.c.v, a d m i n i s t r a t i o n of t h e s e p e p t i d e s to p i g e o n s p o t e n t l y s t i m u l a t e s w a t e r i n t a k e ( E v e r e d e t a l . , 1977; de Caro e t a l . , 1 9 7 8 a ) . The mechanism by w h i c h t a c h y k i n i n s m o d i f y d r i n k i n g b e h a v i o u r has n o t y e t b e e n c l a r i f i e d . 0031-6989/791100891 --11/$02.00/0
©1979 The Italian Pharmacoloo.ical Society
Pharmacological Research Communications, VoL 11, No. 10, 1979
892
In the rat, a neurogenic mechanism for the antidipsogenic ef fect of eledoisin has been suggested (de Caro et al., 1977}. In fact, eledoisin inhibits water intake at doses which do not modify arterial blood pressure [de Caro et al., 1978b); moreover, the
undecapeptide
!~ viv_.__@_o induces in the rat behavioural and
electrocortical alterations (de Caro e__tta~l., 1974) and i__n_nvitro , exercises a direct neurotropic effect on isolated neurons of punctata and Helix pomatia
(Brancati, personal communi-
cation). In
the pigeon, instead, the similar time course and dose-re
sponse relationship of drinking following i.c.v, administration of angiotensin II or eledoisin and the same distribution of sen sitive brain sites suggest for the undecapeptide a mechanism si milar to that of angiotensin If, namely a vascular one (Nicola~ dis & Fitzsimons, 1975). Similar
considerations
and
hypotheses have
been
made to
explain the mechanism by which physalaemin affects drinking behaviour in rats and pigeons. Data at present available allow us neither to reach any conclusion on the mechanism(s) of the effects of eledoisin and ph Z salaemin on water intake nor to explain the opposing effects elicited by the peptides in rats and pigeons. The experiments we report in this paper were carried out
to
compare
the effect on drinking behaviour and on arterial blood
pressure
of eledoisin, physalaemin and some eledoisin- or phy-
salaemin-like peptides in an attempt to investigate whether th£ re is or not any relationship between the two effects. The aim was to contribute to the elucidation of the mechanism of the tachykinins dipsogenic effect, by obtaining evidence in support or against the vascular hypothesis suggested by Nicolaidis et al. (1977).
MATERIAL AND METHODS Animals. Pigeons (Columba livia) of both sexes, weighing
350 -
-400 g, were housed individually in a room in which temperature averaged water
were
21°C
with little variation. Food (Papp, Martini) and
freely a v a i l a b l e .
Pharrnaco/ogica/ Research Communications, VoL 11, No. 10, 1979
o_f£
Implantation
intraventricular cannulae.
cannula (o.d. 600 ~) was stereotaxically thesin anaesthesia,
into
stainless-steel
implanted, under equi-
the third ventricle, according to the
technique described by Evered & Fitzsimons Animals
A
893
(1977).
were tested at least IO days after they were prepa-
red. Intracranial in~eqtions.
The peptides were dissolved in Tyrode
solution (pH 7.4) and were administered in a constant volume of 1 ~I at doses of i-i000 ng/pigeon. Injections were made in conscious birds through a stainless-steel
injector
(o.d. 3OO ~) inserted
into the guide cannula
about 50 sec before the injection. Water intake determination.
Animals were treated in their home
cages. During the experiments the pigeons had free access to wa ter from graduated drinking tubes and received food ad libitum. Water intake was determined at 5 min interv~s for ]5 min to the nearest 0.i ml. Groups of 6-8
animals
were employed and the results were
expressed as means ± s.e. of the mean. Blood r e s ~
determination.
The brachial artery and vein were
cannulated under local anaesthesia, and blood corded bed
pressure was re-
in conscious pigeons according to the technique descri-
by
Evered & Fitzsimons (1977). The arterial cannula (o.d.
0.7 mm) was filled with heparinized saline and connected to a pressure transducer. Arterial blood pressure was recorded by means of a Gemini polygraph (Basile, Milan). During the experiment, foam
rubber
was wrapped lightly around the ventral half
of the body and taped over the birds' back to prevent wing mov£ ments. of ZO ~I
Drugs
were injected intravenously in a constant volume
and
after the injection the vein cannula was flushed
with fresh saline, i00 ~i. The effect on arterial blood pressure induced by the polype~ tides
was compared in the same animal by measuring the maximum
decrease of diastolic blood pressure after rapid injections, a! ternatively,
of
the
substances
which was employed as reference
to be tested or of eledoisin substance.
Four doses of each
substance were employed, ranging between 12.5 and iOO ng per P!
Pharmaco/ogical Research Communications, Vol. 11, iVo, 10, 1979
894
geon for eledoisin, and between 12.5 and 50 ng per pigeon for physalaemin. Data obtained on 6-8 animals were pooled and reco~ ded as means ± s.e. of the mean. The hypotensive effect of the peptides tested was expressed as per cent of eledoisin activity taken as 1OO. Substances. Synthetic eledoisin, physalaemin and nine synthetic eledoisin- or physalaemin-like peptides were employed. Their structure is reported in table 1. Table I, Structure of the peptides tested. COMPOUND I ELEDOISIN II
STRUCTURE PYR-PRO-SER-LYS-ASP(OH)-ALA-PHE-ILE-GLY-LEU-MET(NH
2)
PRO-SER-LYS-ASP (OH)-ALA-PHE-ILE-GLY-LEU-MET(NH 2)
III
SER-LYS-ASP (OH) -ALA-PHE-ILE-GLY-LEU-MET(NH 2)
IV
LYS-ASP (OH) -ALA-PHE-[LE-GLY-LEU-MET(NH 2)
V
ASP (OH)-ALA-PHE-ILE-GLY-LEU-MET(NH 2)
VI
ALA-PHE-ILE-GLY-LEU-ETI(NH 2)
VII PHYSALAEMIN
pYR-ALA-ASP-PRO-ASP(NH2)-LYS-PHE-TYR-GLY-LEU-MET(NH 2)
VIII
PYR-ALA-ASP-PRO-ASP(NH2)-LYS-PHE-TYR-GLY-LEU-ETI(NH 2)
IX
ASP(NH2)-LYS-PHE-ILE-GLY-LEU-MET(NH 2)
X
LYS-PHE-TYR-GLY-LEU-ETI(NH 2)
XI
LYS-PHE-ILE-GLY-LEU-ETI(NH 2)
Compounds II-V
are
partial
sequences,
a n d c o m p o u n d VI i s
an
analogue of eledoisin. Compound VII is physalaemin, an undecapeptide found in the skin of the South American amphibian Physalaemus fuscumaculatus (Erspamer et al., 1962). It is closely related to eledoisin
Pharmacological Research Communications, Vol. ! 1, No. 10, 1979
895
,oo1
2001 +
+ I I
I ~
I
50 ~
o"
I
I
|1
Ill
IV
V
Vl
VII
VIII COMPOUND
IX
X ,
Fig. i. Hypotensive effect of i.v. injection of eledoisin, physalaomin and related peptides. Ordinate: hypotensive activity of the peptides reported as per cent of that of eledoisin considered equal to leO. from a chemical point of view and shares with this peptide pra~ tically the same activity spectrum (Lembek & Fischer, 1967; Erspamer, 1971). Compounds VIII-XI are analogues of physalaemin. Besides these substances, in some experiments angiotensin II (synthetic 5-isoleucin-angiotensin If, Calbiochem) was employed. Eledoisin and related peptides were synthetized at the Farm! talia Laboratories, Milan. RESULTS Effects o n.n arterial blood pressure. In conscious pigeons e l e d o ~ sin, given by rapid intravenous injection, produced a fall in blood pressure which was proportional, both in intensity and du ration, to the dose. In the interval of doses between 12.5 and 100 ng per pigeon the fall in diastolic blood pressure ranged between 5 ± 1.3 and 40 ± 2.5 mmHg.
Pharmacological Research Communications, Vol. 11, No. 10, 1979
896
150"
+ ~100o
-
+
+
+
u $ 3
50-
F
0I
II
Ill
IV
v
Vl
V,
v,,, ,x
x
~"
COMPOUND
Fig.
2. D i p s o g e n i c e f f e c t o f i . c . v , i n j e c t i o n o f e l e d o i s i n , ph Z s a l a e m i n and r e l a t e d p e p t i d e s . Ordinate: dipsogenic activity of the substances report e d as p e r c e n t o f t h a t o f e l e d o i s i n c o n s i d e r e d e q u a l to
I00.
The effect was reproducible,
complete sign
recovery
always
tachyphylaxis never occurred. A
followed the hypotensive effect. No
of discomfort was ever observed, even at the maximum dose
we employed -effect
(I00 ng/pigeon). The similar time course and dose-
relationship of hypotension induced by eledoisin or by
the other peptides tested allowed us to compare the relative po tencies of these substances. The eledoisin-like compound II and the physalaemin-like compound X were slightly more active than the reference substance, while physalaemin (compound VII) and the physalaemin-like compound VIII appeared to be respectively twice and three
times
as
active as eledoisin itself. All the
other peptides were less effective than eledoisin itself. The activity of these compounds was compared to that of eledoisin
considered equal to I00. Their per cent activity is re-
ported in fig. I. ~ffects on water intake. ~ledoisin injected into the third ventricle caused the pigeons to drink. Controls which received in the third ventricle I ~I of Tyrode solution did not drink at all. ~ledoisin at doses of I, iO, I00 and IO00 ng per pigeon caused the birds to drink 2.06 ±
Pharmacological Research Communications, Vol. .11, No. 10, 1979
25"
,o.,°° ,,.,,i ,,,°°, ,°°°°, ,.J°. 20,,.,,e ,°.°°°, ,,.°t° °,e,°, ,°~,oo, °,°°,~
Z 0 tu ~5, (.2
a,°°° o,,D° oo,~°,
r
o°.°. ,°•°°, °°.o°, .°o°o, .°o°o ,°.o°, .°.,°, °~,°° o°o°o, °°.°°, °°~°°, °,°o°, °°,,°, °.°.°,
E u;lO"
°°.°~, °°°°o, °°°.°, .°°°,,
_z
°°o°°, °,,°°, °°,o°, °,°°°. °°°°°,
°°o.P, °...°... .° . . ° °~°,, °°°o°, °°,o°, °.o°°° .°°.,,
iiHi
°°°.,o °°°°o, °.,°o, °°,o°°
°°,°,,
co
T~
Spontmnwout drinking
Lco A~
i.duced
dvinklr,9
Fig. 3. Drinking inhibitory effect of i.c.v, injection of compound [XI), IOO ng/pigeon. Open bars: spontaneous drinking. Water intake (ml/pigeo~) in controls (CO) and treated animals (TR). Dotted bars: angiotensin II [iOO ng/pigeon) induced drinking. Water intake (ml/pigeon) in controls (CO) and treated animals (TR). Each bar is the mean of 6-8 experiments ± s.e. of mean. ± 1.56, 7.5 ± 1.68, 20.0 ± 2.93 and 36.8 ± 4.37 ml of water per pigeon. Latency to the onset of drinking was extremely short, usually ranging between 50 and 180 sec. Drinking was copious but absolutely normal. After eledoisin administration, even at the ma ximum
dose,
the birds were not excited nor depressed, did not
eat or engage in any other behaviour. All the peptides but one (compound Xl) stimulated water int~ ke.
Drinking response was identical to that which was observed
following the administration of eledoisin. Thus, it was possible to compare their potency to that of the reference peptide considered equal to IOO. Compounds II, III, IV, VII (physalaemin) and IX were practi-
cally as active as eledoisin, while the effect elicited by compounds V, VI, VIII and X
was clearly inferior to that produced
897
Pharmacological Research Communications, Vol. ; 1, No. 10, 1979
898
by the reference peptide. These data are reported in fig~ 2. Surprisingly, compound XI did not both spontaneous and angiotensin-induced ministration of the peptide, IO0 ng per inhibition of the water intake elicited
stimulate but inhibited drinking. In fact, adpigeon, produced a 54% by angiotensin II, iO0
ng per pigeon. These data are reported in fig. 3. DISCUSSION Our experiments confirmed that in conscious pigeons eledoisin elicits drinking when administered into the third ventricle and demonstrated that, injected intravenously, in this animal species it produces a potent hypotensive effect which is reproducible and dose-dependent. Moreover, from present experiments it emerged that the dipso genic and not
the
hypotensive activity of the peptides tested did
vary in a parallel fashion.
In fact, physalaemin
(compound
VII) proved to be a dipsogen as potent as eledoisin, confirming exactly what w a s observed in a previous paper (de Caro et al., 1978a). Conversely, its activity on arterial blood pressure was double that of eledoisin. Shortening of eledoisin molecule by means of progressive eli mination of N-terminal aminoacid residues practically left unmodified the dipsogenic effect, while it progressively reduced the hypotensive activity. Compound VIII, which differs from physalaemin because of the presence of a C-terminal ethionin¢ residue instead of a methionine residue, was less effective than eledoisin and physalaemin itself in stimulating water intake but more effective than the mother s u b s t a n c e in lowering arterial blood pressure. On the contrary, the physalaemin-like heptapeptide compound IX was more active than physalaemin or eledoisin itself in eliciting drinking, but was about one third as active as eledoisin and about six times less potent than physalaemin in lowering arterial blood pressure. Compound XI, a physalaemin-like hexapeptide bearing and ethionine instead of a methionine C-terminal residue, was a poor hypotensive agent (its activity was 15% of
Pharmaco/ogica/ Research Communications, Vol. 11, No. 10, 1979 that of eledoisin) but showed a potent inhibitory effect on both spontaneous and angiotensin II induced drinking. Nicola[dis et al. (1977) suggested that the effect of intracranial angiotensin II on water intake may be due, in pigeons as well as in rats, to the ischaemic action of the peptide on the higly vascular circumventricular organs. It might be possible that the peptides tested produce in the brain the same haemodynamic alterations induced by angiotensin II and therefore the same alterations on drinking behaviour. Indeed, it is possible that eledoisin and related substances have in cerebral vessels effects different from those produced in other vascular beds, namely that they produce cerebral vasoconstriction instead of vasodilation. However, in all the animal species so far tested, including man, and in all the vascular beds studied up to now, eledoisin and physalaemin always proved to possess a va'soplegic effect fBertaccini et al., 1965; de Care et al., 1966; Fregnan & GI~sset, 1968; Erspamer et al., 1972; Bertaccini, 1976). From the cited papers it is evident that physalaemin is 2-100 times more effective than eledoisin, according to the animal species or the vascular district considered. Moreover, these peptides produce vasodilation by a direct action on the vascular muscle receptors [Fregnan & Gl~sser, 1968). On the basis of these considerations, it is possible to hyp£ thesize that eledoisin, physalaemin and related peptides have in the br~in the same vasoplegic effect as in other districts, and that in the brain vessels the potency ratio between eledoisin and the other peptides is similar to that in other vascular beds. If so) it can be hardly admitted that substances (e.g. compound IV~ which elicit poor vascular effects, potently stimu late drinking) while other substances {e.g. compound VIII) which evoke potent vascular effects, are poor dipsogens. Of particular interest is the puzzling behaviour of compound XI. In previous papeTs [Bernardi et al., 1965; de Care, 1966; de Care et al., 1966; de Care et al., 1966a) it has been demonstrafed that this peptide shares with eledoisin,physalaemin and related peptides the same activity spectrum. In the present experiments compound XI is different from the above peptides,
899
900
Pharmacological Research Communications, Vol. 11, No. I0, 1979
in that it proved to posses, instead of a stimulating activity, a potent inhibitory effect on water intake. Since in the present as well as in the previous papers compound XI proved to elicit the same haemodynamic alterations as eledoisin and related substances, it is reasonable to think that it induces in the brain of pigeons the same vascular modifications produced by the above peptides. If this statement is correct, clearly the effect of compound Xl on drinking behaviour cannot be explained in terms of vascular alterations. In conclusion, on the basis of the above considerations, the results of the present experiments suggest that the alterations induced by eledoisin, physalaemin and related peptides on drinking behaviour of pigeons cannot be explained in terms of effects on brain vessels. However, the problem of the mechanism(s) of the effect of the above peptides on drinking behaviour, as well as the interpretation of the puzzling behaviour of compound XI, remains open and additional clarificatory experiments are needed.
REFERENCES BERNARDI L , , BOSISIO G., CHILLE~I F . , DE CARD G., DE CASTIGLIONE R . , ERSPAMER V., GLOSSER A. & GOFFREDO O. ( 1 9 6 S ) . Experientia, 21, 695-696. BERTACCINI G~-~, CEI J.M. & ERSPA~ER V. ( 1 9 6 5 ) . Br. J . P h a r m a c . , 25, 380-391. BERT'~CINI G. (1976}. Pharmac. R e v . , 28, 127-177. DE ~J~D G. [1966). Arch. int. Pharmaco-~yn., 162, 437-446. DE ~ARO G,, FARRUGGIA L., MINARBI E. & NOVART-NT A. (1966). Naunyn-Schmiedeberg,s Arch. Pharmac., 2S4, 194-198. DE CARO G.j FARRUGGIA L. (1966a). Arch. int. Pharmacodyn., 160,
44-54. DE CARO G.p NICOSSI L.G., VENTUR! F . , BRANCATI A. & SCARNATI E. ( 1 9 7 4 ) . P s y c h o p h a r m a c o l o g i a ( B e r l i n ) ) 38, 211-218. DE CARe G., NICOSSI L.G. & PICCININ G. - ' [ 1 9 7 7 ) . Pharmac. Res. Commun., 9, 489-500. DE CARe G., ~ASSI H. & NICOSSI L.G. ( 1 9 7 8 ) . Neuropharmacology, 17p 925-929. DE ~¢~RO G., HASSI M. & MICOSSI L.G. {1978a). Pharmac. Res. Commun., 9, 861-866. DE CARe G?, HASSI M. & MICOSSI L.G. (1978b). Pharmac. Res. Commun., lOp 633-642° ERSPAMER~T?p BERTACCINI G. & CEI J.M. (1962). Experientia) 18,
562-563.
Pharmacological Research Communications, Vol. 11, No. 10, 1979 ERSPAMER V. ( 1 9 7 1 ) . Ann. Rev. P h a r m a c o l . , 11, 5 2 7 - 3 5 0 . ERSPAMER V . , MELCHIORRI P. & SOPRANZI N . - - ( 1 9 7 2 ) . Br. J . P h a r mac., 45, 4 4 Z - 4 5 0 . EVERED M.~., FITZSIMONSJ.T. & DE CARO G. (1977). Nature, 268, 332-333.
EVERED ~.D. & FITZSIMONS J.T. (1977). J. Physiol., 271, AP~. FREGNAN G.B. & GLOSSER A.H. C1968). Arch. int. ~ r m a c o d y n . , 171, 435-448. LEM~K F. & FISCHER G. ( 1 9 6 7 ) . A r c h . P h a r m a c . Exp. Path., 258, 452-56. NICOLAYDIS S. & FITZSIMONS J.T. (1975). C. r. hebd S~anc. Acad. Sci., Paris, 281, 1417-1420. NICOLAYDIS S., E~h~ED M.D. & FITZSIMONS J.T. {1977). 6th International Conference on the Physiology of Food and Fluid Int£ ke, Paris (France).
901
891
EFFECT OF ELEDOISIN, PHYSALAEMIN AND SOME ELEDOISIN- OR PHYSALAEMIN-LIKE PEPTIDES" ON WATER INTAKE AND ARTERIAL BLOOD PRESSURE IN CONSCIOUS PIGEONS G. de Caro, M. Hassi and L,G, Hicossi
I n s t i t u t e of Pharmacology and Pharmacognosy, F a c u l t y of Pharmacy, U n i v e r s i t y of Camerino, 52052 Camerino ( I t a l y ) .
SUMMARY
The hypotensive and dipsogenic effects of eledoisin, physalaemin and some
synthetic
scious
eledoisin- or physalaemin-like peptides were studied in con-
pigeons,
respectively after intravenous or intracerebroventricular
administration. The effects of thepeptides
were compared to the hypotensi-
ve or dipsogenic activities of eledoisin considered equal to I00. i) Shortening
of eledoisin molecule by means of progressive elimination of
up to four N-terminal aminoacid residues left practically unmodified the dipsogenic effect, while it progressively reduced the hypotensive effect. 2) Physalaemin
was
a dipsogen as potent as eledoisin, but its hypotensive
activity was approximately double the one of eledoisin. 3) Modifying of
physalaeminmolecule
produced different alterations in the
dipsogen~c and hypotensive effect. Apparently
there was no relationship between =he dipsogen~c and the hy-
potensive effect of the substances tested.
INTRODUCTION
Intracerebroventricular (i.c.v.) injection of the tachykin i n s e l e d o i s i n and p h y s a l a e m i n e l i c i t s in the r a t a p o t e n t antidipsogenic effect (de Caro e t a l . , 1977; de Caro e_~t a_~l., 1978). Conversely, i.c.v, a d m i n i s t r a t i o n of t h e s e p e p t i d e s to p i g e o n s p o t e n t l y s t i m u l a t e s w a t e r i n t a k e ( E v e r e d e t a l . , 1977; de Caro e t a l . , 1 9 7 8 a ) . The mechanism by w h i c h t a c h y k i n i n s m o d i f y d r i n k i n g b e h a v i o u r has n o t y e t b e e n c l a r i f i e d . 0031-6989/791100891 --11/$02.00/0
©1979 The Italian Pharmacoloo.ical Society
Pharmacological Research Communications, VoL 11, No. 10, 1979
892
In the rat, a neurogenic mechanism for the antidipsogenic ef fect of eledoisin has been suggested (de Caro et al., 1977}. In fact, eledoisin inhibits water intake at doses which do not modify arterial blood pressure [de Caro et al., 1978b); moreover, the
undecapeptide
!~ viv_.__@_o induces in the rat behavioural and
electrocortical alterations (de Caro e__tta~l., 1974) and i__n_nvitro , exercises a direct neurotropic effect on isolated neurons of punctata and Helix pomatia
(Brancati, personal communi-
cation). In
the pigeon, instead, the similar time course and dose-re
sponse relationship of drinking following i.c.v, administration of angiotensin II or eledoisin and the same distribution of sen sitive brain sites suggest for the undecapeptide a mechanism si milar to that of angiotensin If, namely a vascular one (Nicola~ dis & Fitzsimons, 1975). Similar
considerations
and
hypotheses have
been
made to
explain the mechanism by which physalaemin affects drinking behaviour in rats and pigeons. Data at present available allow us neither to reach any conclusion on the mechanism(s) of the effects of eledoisin and ph Z salaemin on water intake nor to explain the opposing effects elicited by the peptides in rats and pigeons. The experiments we report in this paper were carried out
to
compare
the effect on drinking behaviour and on arterial blood
pressure
of eledoisin, physalaemin and some eledoisin- or phy-
salaemin-like peptides in an attempt to investigate whether th£ re is or not any relationship between the two effects. The aim was to contribute to the elucidation of the mechanism of the tachykinins dipsogenic effect, by obtaining evidence in support or against the vascular hypothesis suggested by Nicolaidis et al. (1977).
MATERIAL AND METHODS Animals. Pigeons (Columba livia) of both sexes, weighing
350 -
-400 g, were housed individually in a room in which temperature averaged water
were
21°C
with little variation. Food (Papp, Martini) and
freely a v a i l a b l e .
Pharrnaco/ogica/ Research Communications, VoL 11, No. 10, 1979
o_f£
Implantation
intraventricular cannulae.
cannula (o.d. 600 ~) was stereotaxically thesin anaesthesia,
into
stainless-steel
implanted, under equi-
the third ventricle, according to the
technique described by Evered & Fitzsimons Animals
A
893
(1977).
were tested at least IO days after they were prepa-
red. Intracranial in~eqtions.
The peptides were dissolved in Tyrode
solution (pH 7.4) and were administered in a constant volume of 1 ~I at doses of i-i000 ng/pigeon. Injections were made in conscious birds through a stainless-steel
injector
(o.d. 3OO ~) inserted
into the guide cannula
about 50 sec before the injection. Water intake determination.
Animals were treated in their home
cages. During the experiments the pigeons had free access to wa ter from graduated drinking tubes and received food ad libitum. Water intake was determined at 5 min interv~s for ]5 min to the nearest 0.i ml. Groups of 6-8
animals
were employed and the results were
expressed as means ± s.e. of the mean. Blood r e s ~
determination.
The brachial artery and vein were
cannulated under local anaesthesia, and blood corded bed
pressure was re-
in conscious pigeons according to the technique descri-
by
Evered & Fitzsimons (1977). The arterial cannula (o.d.
0.7 mm) was filled with heparinized saline and connected to a pressure transducer. Arterial blood pressure was recorded by means of a Gemini polygraph (Basile, Milan). During the experiment, foam
rubber
was wrapped lightly around the ventral half
of the body and taped over the birds' back to prevent wing mov£ ments. of ZO ~I
Drugs
were injected intravenously in a constant volume
and
after the injection the vein cannula was flushed
with fresh saline, i00 ~i. The effect on arterial blood pressure induced by the polype~ tides
was compared in the same animal by measuring the maximum
decrease of diastolic blood pressure after rapid injections, a! ternatively,
of
the
substances
which was employed as reference
to be tested or of eledoisin substance.
Four doses of each
substance were employed, ranging between 12.5 and iOO ng per P!
Pharmaco/ogical Research Communications, Vol. 11, iVo, 10, 1979
894
geon for eledoisin, and between 12.5 and 50 ng per pigeon for physalaemin. Data obtained on 6-8 animals were pooled and reco~ ded as means ± s.e. of the mean. The hypotensive effect of the peptides tested was expressed as per cent of eledoisin activity taken as 1OO. Substances. Synthetic eledoisin, physalaemin and nine synthetic eledoisin- or physalaemin-like peptides were employed. Their structure is reported in table 1. Table I, Structure of the peptides tested. COMPOUND I ELEDOISIN II
STRUCTURE PYR-PRO-SER-LYS-ASP(OH)-ALA-PHE-ILE-GLY-LEU-MET(NH
2)
PRO-SER-LYS-ASP (OH)-ALA-PHE-ILE-GLY-LEU-MET(NH 2)
III
SER-LYS-ASP (OH) -ALA-PHE-ILE-GLY-LEU-MET(NH 2)
IV
LYS-ASP (OH) -ALA-PHE-[LE-GLY-LEU-MET(NH 2)
V
ASP (OH)-ALA-PHE-ILE-GLY-LEU-MET(NH 2)
VI
ALA-PHE-ILE-GLY-LEU-ETI(NH 2)
VII PHYSALAEMIN
pYR-ALA-ASP-PRO-ASP(NH2)-LYS-PHE-TYR-GLY-LEU-MET(NH 2)
VIII
PYR-ALA-ASP-PRO-ASP(NH2)-LYS-PHE-TYR-GLY-LEU-ETI(NH 2)
IX
ASP(NH2)-LYS-PHE-ILE-GLY-LEU-MET(NH 2)
X
LYS-PHE-TYR-GLY-LEU-ETI(NH 2)
XI
LYS-PHE-ILE-GLY-LEU-ETI(NH 2)
Compounds II-V
are
partial
sequences,
a n d c o m p o u n d VI i s
an
analogue of eledoisin. Compound VII is physalaemin, an undecapeptide found in the skin of the South American amphibian Physalaemus fuscumaculatus (Erspamer et al., 1962). It is closely related to eledoisin
Pharmacological Research Communications, Vol. ! 1, No. 10, 1979
895
,oo1
2001 +
+ I I
I ~
I
50 ~
o"
I
I
|1
Ill
IV
V
Vl
VII
VIII COMPOUND
IX
X ,
Fig. i. Hypotensive effect of i.v. injection of eledoisin, physalaomin and related peptides. Ordinate: hypotensive activity of the peptides reported as per cent of that of eledoisin considered equal to leO. from a chemical point of view and shares with this peptide pra~ tically the same activity spectrum (Lembek & Fischer, 1967; Erspamer, 1971). Compounds VIII-XI are analogues of physalaemin. Besides these substances, in some experiments angiotensin II (synthetic 5-isoleucin-angiotensin If, Calbiochem) was employed. Eledoisin and related peptides were synthetized at the Farm! talia Laboratories, Milan. RESULTS Effects o n.n arterial blood pressure. In conscious pigeons e l e d o ~ sin, given by rapid intravenous injection, produced a fall in blood pressure which was proportional, both in intensity and du ration, to the dose. In the interval of doses between 12.5 and 100 ng per pigeon the fall in diastolic blood pressure ranged between 5 ± 1.3 and 40 ± 2.5 mmHg.
Pharmacological Research Communications, Vol. 11, No. 10, 1979
896
150"
+ ~100o
-
+
+
+
u $ 3
50-
F
0I
II
Ill
IV
v
Vl
V,
v,,, ,x
x
~"
COMPOUND
Fig.
2. D i p s o g e n i c e f f e c t o f i . c . v , i n j e c t i o n o f e l e d o i s i n , ph Z s a l a e m i n and r e l a t e d p e p t i d e s . Ordinate: dipsogenic activity of the substances report e d as p e r c e n t o f t h a t o f e l e d o i s i n c o n s i d e r e d e q u a l to
I00.
The effect was reproducible,
complete sign
recovery
always
tachyphylaxis never occurred. A
followed the hypotensive effect. No
of discomfort was ever observed, even at the maximum dose
we employed -effect
(I00 ng/pigeon). The similar time course and dose-
relationship of hypotension induced by eledoisin or by
the other peptides tested allowed us to compare the relative po tencies of these substances. The eledoisin-like compound II and the physalaemin-like compound X were slightly more active than the reference substance, while physalaemin (compound VII) and the physalaemin-like compound VIII appeared to be respectively twice and three
times
as
active as eledoisin itself. All the
other peptides were less effective than eledoisin itself. The activity of these compounds was compared to that of eledoisin
considered equal to I00. Their per cent activity is re-
ported in fig. I. ~ffects on water intake. ~ledoisin injected into the third ventricle caused the pigeons to drink. Controls which received in the third ventricle I ~I of Tyrode solution did not drink at all. ~ledoisin at doses of I, iO, I00 and IO00 ng per pigeon caused the birds to drink 2.06 ±
Pharmacological Research Communications, Vol. .11, No. 10, 1979
25"
,o.,°° ,,.,,i ,,,°°, ,°°°°, ,.J°. 20,,.,,e ,°.°°°, ,,.°t° °,e,°, ,°~,oo, °,°°,~
Z 0 tu ~5, (.2
a,°°° o,,D° oo,~°,
r
o°.°. ,°•°°, °°.o°, .°o°o, .°o°o ,°.o°, .°.,°, °~,°° o°o°o, °°.°°, °°~°°, °,°o°, °°,,°, °.°.°,
E u;lO"
°°.°~, °°°°o, °°°.°, .°°°,,
_z
°°o°°, °,,°°, °°,o°, °,°°°. °°°°°,
°°o.P, °...°... .° . . ° °~°,, °°°o°, °°,o°, °.o°°° .°°.,,
iiHi
°°°.,o °°°°o, °.,°o, °°,o°°
°°,°,,
co
T~
Spontmnwout drinking
Lco A~
i.duced
dvinklr,9
Fig. 3. Drinking inhibitory effect of i.c.v, injection of compound [XI), IOO ng/pigeon. Open bars: spontaneous drinking. Water intake (ml/pigeo~) in controls (CO) and treated animals (TR). Dotted bars: angiotensin II [iOO ng/pigeon) induced drinking. Water intake (ml/pigeon) in controls (CO) and treated animals (TR). Each bar is the mean of 6-8 experiments ± s.e. of mean. ± 1.56, 7.5 ± 1.68, 20.0 ± 2.93 and 36.8 ± 4.37 ml of water per pigeon. Latency to the onset of drinking was extremely short, usually ranging between 50 and 180 sec. Drinking was copious but absolutely normal. After eledoisin administration, even at the ma ximum
dose,
the birds were not excited nor depressed, did not
eat or engage in any other behaviour. All the peptides but one (compound Xl) stimulated water int~ ke.
Drinking response was identical to that which was observed
following the administration of eledoisin. Thus, it was possible to compare their potency to that of the reference peptide considered equal to IOO. Compounds II, III, IV, VII (physalaemin) and IX were practi-
cally as active as eledoisin, while the effect elicited by compounds V, VI, VIII and X
was clearly inferior to that produced
897
Pharmacological Research Communications, Vol. ; 1, No. 10, 1979
898
by the reference peptide. These data are reported in fig~ 2. Surprisingly, compound XI did not both spontaneous and angiotensin-induced ministration of the peptide, IO0 ng per inhibition of the water intake elicited
stimulate but inhibited drinking. In fact, adpigeon, produced a 54% by angiotensin II, iO0
ng per pigeon. These data are reported in fig. 3. DISCUSSION Our experiments confirmed that in conscious pigeons eledoisin elicits drinking when administered into the third ventricle and demonstrated that, injected intravenously, in this animal species it produces a potent hypotensive effect which is reproducible and dose-dependent. Moreover, from present experiments it emerged that the dipso genic and not
the
hypotensive activity of the peptides tested did
vary in a parallel fashion.
In fact, physalaemin
(compound
VII) proved to be a dipsogen as potent as eledoisin, confirming exactly what w a s observed in a previous paper (de Caro et al., 1978a). Conversely, its activity on arterial blood pressure was double that of eledoisin. Shortening of eledoisin molecule by means of progressive eli mination of N-terminal aminoacid residues practically left unmodified the dipsogenic effect, while it progressively reduced the hypotensive activity. Compound VIII, which differs from physalaemin because of the presence of a C-terminal ethionin¢ residue instead of a methionine residue, was less effective than eledoisin and physalaemin itself in stimulating water intake but more effective than the mother s u b s t a n c e in lowering arterial blood pressure. On the contrary, the physalaemin-like heptapeptide compound IX was more active than physalaemin or eledoisin itself in eliciting drinking, but was about one third as active as eledoisin and about six times less potent than physalaemin in lowering arterial blood pressure. Compound XI, a physalaemin-like hexapeptide bearing and ethionine instead of a methionine C-terminal residue, was a poor hypotensive agent (its activity was 15% of
Pharmaco/ogica/ Research Communications, Vol. 11, No. 10, 1979 that of eledoisin) but showed a potent inhibitory effect on both spontaneous and angiotensin II induced drinking. Nicola[dis et al. (1977) suggested that the effect of intracranial angiotensin II on water intake may be due, in pigeons as well as in rats, to the ischaemic action of the peptide on the higly vascular circumventricular organs. It might be possible that the peptides tested produce in the brain the same haemodynamic alterations induced by angiotensin II and therefore the same alterations on drinking behaviour. Indeed, it is possible that eledoisin and related substances have in cerebral vessels effects different from those produced in other vascular beds, namely that they produce cerebral vasoconstriction instead of vasodilation. However, in all the animal species so far tested, including man, and in all the vascular beds studied up to now, eledoisin and physalaemin always proved to possess a va'soplegic effect fBertaccini et al., 1965; de Care et al., 1966; Fregnan & GI~sset, 1968; Erspamer et al., 1972; Bertaccini, 1976). From the cited papers it is evident that physalaemin is 2-100 times more effective than eledoisin, according to the animal species or the vascular district considered. Moreover, these peptides produce vasodilation by a direct action on the vascular muscle receptors [Fregnan & Gl~sser, 1968). On the basis of these considerations, it is possible to hyp£ thesize that eledoisin, physalaemin and related peptides have in the br~in the same vasoplegic effect as in other districts, and that in the brain vessels the potency ratio between eledoisin and the other peptides is similar to that in other vascular beds. If so) it can be hardly admitted that substances (e.g. compound IV~ which elicit poor vascular effects, potently stimu late drinking) while other substances {e.g. compound VIII) which evoke potent vascular effects, are poor dipsogens. Of particular interest is the puzzling behaviour of compound XI. In previous papeTs [Bernardi et al., 1965; de Care, 1966; de Care et al., 1966; de Care et al., 1966a) it has been demonstrafed that this peptide shares with eledoisin,physalaemin and related peptides the same activity spectrum. In the present experiments compound XI is different from the above peptides,
899
900
Pharmacological Research Communications, Vol. 11, No. I0, 1979
in that it proved to posses, instead of a stimulating activity, a potent inhibitory effect on water intake. Since in the present as well as in the previous papers compound XI proved to elicit the same haemodynamic alterations as eledoisin and related substances, it is reasonable to think that it induces in the brain of pigeons the same vascular modifications produced by the above peptides. If this statement is correct, clearly the effect of compound Xl on drinking behaviour cannot be explained in terms of vascular alterations. In conclusion, on the basis of the above considerations, the results of the present experiments suggest that the alterations induced by eledoisin, physalaemin and related peptides on drinking behaviour of pigeons cannot be explained in terms of effects on brain vessels. However, the problem of the mechanism(s) of the effect of the above peptides on drinking behaviour, as well as the interpretation of the puzzling behaviour of compound XI, remains open and additional clarificatory experiments are needed.
REFERENCES BERNARDI L , , BOSISIO G., CHILLE~I F . , DE CARD G., DE CASTIGLIONE R . , ERSPAMER V., GLOSSER A. & GOFFREDO O. ( 1 9 6 S ) . Experientia, 21, 695-696. BERTACCINI G~-~, CEI J.M. & ERSPA~ER V. ( 1 9 6 5 ) . Br. J . P h a r m a c . , 25, 380-391. BERT'~CINI G. (1976}. Pharmac. R e v . , 28, 127-177. DE ~J~D G. [1966). Arch. int. Pharmaco-~yn., 162, 437-446. DE ~ARO G,, FARRUGGIA L., MINARBI E. & NOVART-NT A. (1966). Naunyn-Schmiedeberg,s Arch. Pharmac., 2S4, 194-198. DE CARO G.j FARRUGGIA L. (1966a). Arch. int. Pharmacodyn., 160,
44-54. DE CARO G.p NICOSSI L.G., VENTUR! F . , BRANCATI A. & SCARNATI E. ( 1 9 7 4 ) . P s y c h o p h a r m a c o l o g i a ( B e r l i n ) ) 38, 211-218. DE CARe G., NICOSSI L.G. & PICCININ G. - ' [ 1 9 7 7 ) . Pharmac. Res. Commun., 9, 489-500. DE CARe G., ~ASSI H. & NICOSSI L.G. ( 1 9 7 8 ) . Neuropharmacology, 17p 925-929. DE ~¢~RO G., HASSI M. & MICOSSI L.G. {1978a). Pharmac. Res. Commun., 9, 861-866. DE CARe G?, HASSI M. & MICOSSI L.G. (1978b). Pharmac. Res. Commun., lOp 633-642° ERSPAMER~T?p BERTACCINI G. & CEI J.M. (1962). Experientia) 18,
562-563.
Pharmacological Research Communications, Vol. 11, No. 10, 1979 ERSPAMER V. ( 1 9 7 1 ) . Ann. Rev. P h a r m a c o l . , 11, 5 2 7 - 3 5 0 . ERSPAMER V . , MELCHIORRI P. & SOPRANZI N . - - ( 1 9 7 2 ) . Br. J . P h a r mac., 45, 4 4 Z - 4 5 0 . EVERED M.~., FITZSIMONSJ.T. & DE CARO G. (1977). Nature, 268, 332-333.
EVERED ~.D. & FITZSIMONS J.T. (1977). J. Physiol., 271, AP~. FREGNAN G.B. & GLOSSER A.H. C1968). Arch. int. ~ r m a c o d y n . , 171, 435-448. LEM~K F. & FISCHER G. ( 1 9 6 7 ) . A r c h . P h a r m a c . Exp. Path., 258, 452-56. NICOLAYDIS S. & FITZSIMONS J.T. (1975). C. r. hebd S~anc. Acad. Sci., Paris, 281, 1417-1420. NICOLAYDIS S., E~h~ED M.D. & FITZSIMONS J.T. {1977). 6th International Conference on the Physiology of Food and Fluid Int£ ke, Paris (France).
901