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Penile erection and yawning induced by oxytocin and related peptides: Structure-activity relationship
Peptides, Vol. 10, pp. 559-563. e Pergamon Press plc, 1989. Printed in the U.S.A.
0196-9781/89 $3.00 + .00
Penile Erection and Yawning Induced by Oxytocin and Related Peptides: Structure-Activity Relationship A. A R G I O L A S , M. R. M E L I S , R. S T A N C A M P I A N O
A N D G. L. G E S S A
Department of Neurosciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy R e c e i v e d 18 July 1988
ARGIOLAS, A., M. R. MELIS, R. STANCAMPIANO AND G. L. GESSA. Penileerection and yawning inducedby oxytocin and relatedpeptides: Structure-activity relationship. PEPTIDES 10(3) 559-563, 1989.--The potency of several oxytocin-related peptides in inducing penile erection and yawning after injection into a lateral ventricle of male rats was compared. Substitution of two amino acids in the oxytocin molecule or deletion of the C-terminal glycinamide as in des-GlyNH2-oxytocin [oxytocin(1-8)] reduced oxytocin potency in inducing both effects, the rank order being: oxytocin > [Thr4,GlyT]-oxytocin = isotocin ([Ser4,Ileg]-oxytocin) > vasopressin ([Phe3,ArgS]-oxytocin) > des-GlyNH2-oxytocin. Oxytocin's ability to induce penile erection and yawning was abolished by permanent opening of the disulfide bridge by reduction and carboxymethylation. Oxytocin(1-6) and oxytocin(7-9) were also inactive. Penile erection and yawning induced by oxytocin-related peptides were antagonized in a dose-dependent manner by nonapeptide antagonists with a rank order of potency that follows their antioxytocic activity (d[(CH2)sTyr(Me)OrnS]-vasotocin -~ [Pent,Phe(Me)2,Thr4,Orns]-oxytocin > d[(CH2)sTyr(Me)ArgSl-vasopressin).Carboxymethylated oxytocin, oxytocin(1-6), and oxytocin(79) were devoid of antagonistic activity. The present results suggest that central oxytocin receptors mediating the expression of penile erection and yawning are structurally related to those present in the uterus and in the mammary gland. Oxytocin
Oxytocin-vasopressin analogs
Oxytocin antagonists
OXYTOCIN injected into the lateral ventricles (ICV) or into the paraventricular nucleus of the hypothalamus (PVN) or the CA1 field of the hippocampus induces repeated episodes of penile erection and yawning in male rats (1-3, 23). While the importance of penile erection in reproduction does not need to be stressed, it is pertinent to recall that yawning is considered an arousal mechanism to antagonize sleep when it is pressing under dangerous conditions or, as in civilized society, under improper circumstances [see (6)]. Oxytocin-induced penile erection and yawning seem to be mediated by the stimulation of central oxytocinergic receptors, since they are prevented by the ICV injection of the potent nonapeptide antagonist d[(CH2)sTyr(Me)OrnS]-vasotocin (2) and by electrolytic lesion of the PVN (3), that causes an almost complete depletion of brain oxytocin content (17,20). The present study was aimed at the screening of oxytocin-related peptides for their ability to induce penile erection and yawning in male rats and to compare the structure-activity relationship with those well characterized for their effects on the uterus and mammary gland (28), and on memory processes (12-14).
Penile erection
Yawning
humidity 60%, with water and standard laboratory food ad lib.
ICV Injections Stainless steel guide cannulas (22 gauge) aimed at one lateral ventricle were stereotaxically implanted (David Kopf Instruments, USA) under chloral hydrate anaesthesia 5 days before the experiments (coordinates: 1 mm anterior to bregma, 1.5 lateral to midline and 2 mm ventral to dura) (27). Substances dissolved in saline or saline alone were injected into a lateral ventricle (ICV) via an internal cannula (28 gauge), which extended 2 mm below the tip of the guide cannula and was connected by polyethylene tubing to a 10 lxl Hamilton syringe driven by a micrometric screw. Volumes injected ICV were 10 txl in 2 min.
Behavioral Studies Soon after injection, the animals were placed individually into Plexiglas cages (30 x 30 x 30 cm) and observed for 60 min during which penile erection and yawning episodes were counted. At the end of the experiments, the animals were killed by decapitation, the brains were removed and visually inspected to ascertain the correct position of the cannula tip into the lateral ventricle. Only those animals that were found to have the cannula tip positioned correctly were considered for statistical analysis of the results
METHOD
Animals Male Sprague-Dawley rats (250-300 g) were used in all the experiments. The animals were caged in groups of 4-6 at 24°C,
559
560
ARGIOLAS, MELIS, STANCAMPIANO AND GESSA TABLE 1 AMINO ACID SEQUENCE OF OXYTOCIN AND RELATED PEPTIDES
Peptide
1
2
3
Oxytocin [Thr4, Gly 7 ]-Oxy
Cys Cys
Tyr Tyr
Ile Ile
Isotocin Vasotocin Vasopressin Oxytocin(1-8) Oxytocin(1-6)
Cys Cys Cys Cys Cys
Tyr Tyr Tyr Tyr Tyr
lie lie
Phe lie lie
4 Gin
Thr Ser Gin Gin Gin Gin
Residue 5
6
7
8
Asn Asn
C)'s C)'s
Pro
Leu Leu
GlyNH2 GlyNH 2
Asn Ash Asn Ash Asn
C)'s C),s C)'s C)'s C)'s
Pro Pro Pro Pro
lle Arg Arg
GIyNH2 GlyNH2 GlyNH2
Pro
Leu
GlyNH 2
Oxytocin(7-9) CM-oxytocin d [(C H 2)5 Tyr(Me) Orn8 ]_vasotocin [Pen 1, Phe(Me) 2, Thr4 Orn 8 ]-oxytocin d[(Cn 2)5 Tyr(Me) ArgS]-vasopressin
Gly
9
Leu
CMCys
Tyr
Ile
Gin
Asn
CMCys
Pro
Leu
GlyNH2
Mpr
Tyr(Me)
lie
Gin
Asn
C,vs
Pro
Orn
GlyNH 2
Pen
Phe(Me) Ile
Thr
Asn
C)'s
Pro
Orn
GlyNH 2
Mpr
Tyr(Me)
Gin
Asn
C),s
Pro
Arg
GlyNH2
Ile
Different residues are italicized. Mpr=-l(13-mercapto-13, I]-cyclopentamethylene)-propionic acid. Pen--Penicillamine.
(Duncan's new multiple range test).
Peptides The following peptides were purchased from Peninsula Laboratories (San Carlos, CA): oxytocin, oxytocin(1-6) (=tocinoic acid), oxytocin(7-9) (= Pro-Leu-GlyNH2), [Thr4,GlyT]-oxytocin, vasotocin, vasopressin, d[(CH2)sTyr(Me)OmS]-vasotocin, d[(CH2)5 Tyr(Me)ArgS]-vasopressin. Isotocin was purchased from Sigma (St. Louis, MO). Oxytocin(1-8) (=des-9-glycinamide-oxytocin) (Organon, The Netherlands) was kindly provided by Prof. David De Wied (Rudolph Magnus Institute, Utrecht, The Netherlands). [Pen 1,Phe(Me) 2,Thr4,Orns]-OxytOcin was kindly provided by Prof. V. J. Hruby (University of Arizona, Tucson, AZ). Carboxymethylated oxytocin was prepared from oxytocin after reduction of the disulfide bridge with dithiothreitol and carboxymethylation with iodoacetic acid and purified by high pressure liquid chromatography on a 0.39 × 30 cm I~Bondapak C18 column (Waters Associates). The chromatographic conditions were reported elsewhere (1). The amino acid sequence of the above peptides is shown in Table 1. The antioxytocic, antivasopressor and antidiuretic activity of nonapeptide antagonists is summarized in Table 2. RESULTS Figure 1 shows the dose-response curve of different oxytocinlike peptides for inducing penile erection and yawning in male rats. As previously reported, oxytocin showed a bell-shaped dose-response curve, the maximal effect being induced by doses between 10 and 60 ng, while doses higher than 100 ng were almost
ineffective. A similar bell-shaped dose-response curve was also found with [Thra,GlyT]-oxytocin, a synthetic agonist of peripheral and central oxytocin receptors (15, 21, 22), and with the bony fish isotocin ([Ser4,IleS]-oxytocin), although they were about 6 times less potent than oxytocin, the minimal and the maximal effective dose being 30 ng and between 100 and 500 ng, respectively. Oxytocin(1-8) also induced penile erection and yawning but it was 100 times less potent than oxytocin. In contrast, carboxymethylated oxytocin, oxytocin(1--6) and oxytocin(7-9) were ineffective at doses up to 1 p,g. Vasotocin ([ArgS]-oxytocin), and vasopressin ([Phe3,ArgS]-oxytocin) were also ineffective. However, the study TABLE 2 BIOLOGICALACTIVITIESOF THE NONAPEPTIDEANTAGONISTS Activity (In Vivo) Antioxytocic AntivasopressorAntidiuretic Compound
(pA2)
(pA2)
U/mg
d[(CH2)sTyr(Me)OmS]-VT 7.88* [PenI,Phe(Me)2,Thr4,OmS]-Oxy 7.30f d[CH2)sTyr(Me)ArgS]-VP 6.62:~
7.96* 6~7t 8.62:~
0.009* n.a. 0.31:~
pA2= Negative logarithm of the molar concentration that reduces by 50% the response to agonist. For the definition of U/mg see (19). *(5). ¢(11). $(19). n.a. = Not available.
PENILE ERECTION AND YAWNING BY OXYTOCIN
2o ,~;.,o~.z~ VSae,,a-'oxx 15- Aoxy 1-8
g N
/
*~.~-~6~"~ /r x "~i~F..~ /~
*
~ ~,,d
561
/'l
.
•1~
20
-II TI
4("
1,
g N n
0
10
100
1000
ng i.c.v,
FIG. 1. Penile erection and yawning induced by the ICV injection of oxytocin and related peptides: dose-response curves. Peptides were dissolved in saline and injected ICV in a volume of 10 ~1 through chronically implanted guide cannulas as described in the Method section. After injection, rats were placed individually into Plexiglas cages and observed for 60 min during which penile erection and yawning episodes were counted. Values are means---S.E.M, of 3 experiments (15 rats per group). *p<0.001 with respect to saline-treated rats.
of the effect of the latter two peptides was complicated by the fact that both peptides, at doses higher than 5 ng, induced barrel rotation and motor disturbances, which lasted for 15-30 min. After this time, the animals looked normal, but no yawning or penile erection episodes were observed during the remaining observation period. Penile erection and yawning induced by 30 ng of ICV oxytocin were prevented in a dose-dependent manner by the ICV pretreatment with the nonapeptide antagonists d[(CH2)~Tyr(Me)OrnS]vasotocin (5) and [Penl,Phe(Me)2,Thra,oruS]-oxytocin (11) at doses between 10 and 100 ng. d[(CH2)sTyr(Me)ArgS]-vasopressin (19,32) also antagonized the oxytocin effect, but it was 10-20 times less potent (Fig. 2). In contrast, penile erection and yawning induced by oxytocin were not prevented by the ICV pretreatment with doses up to 1 txg of either carboxymethylated oxytocin or oxytocin(1-6) or oxytocin(7-9) (Fig. 2). Like the oxytocin effect, penile erection and yawning induced by 100 ng of [Thra,GlyT]oxytocin or 1 p,g of oxytocin(1-8) were also prevented by pretreatment with the above nonapeptide antagonists (Fig. 3). DISCUSSION
The present results show that penile erection and yawning are induced not only by the ICV injection of oxytocin but also by its analogs isotocin and [Thr4,Gly7]-oxytocin. Interestingly, the three peptides showed a bell-shaped dose-response curve, suggesting that they act on the same receptor to induce the above effects. In contrast, ICV vasotocin ([Arga]-oxytocin) and vasopressin ([Phe3,Arga]-oxytocin) were found to be ineffective. However, such ineffectiveness may be due to the fact that both peptides induced barrel rotations and motor disturbances, which might have
letP~I,Ph~(M,.RThr~O.#]--O~ o 0
I
10
i x
. ~ 100
*
\* : 1000
peptide ontogonist (ng i.c.v.)
FIG. 2. Penile erection and yawning induced by oxytocin: effect of nonapeptide antagonists, CM-oxytocin, oxytocin(1-6) and oxytocin(7-9). Peptides were dissolved in saline and injected ICV in a volume of 5 ~1, as described in the Method section. All rats received 30 ng of oxytocin. Nonapeptide antagonists, CM-oxytocin, oxytocin(l~), oxytocin(7-9) or saline were injected 10 min before the administration of oxytocin. After oxytocin injection, the animals were placed individually into Plexiglas cages and observed for 60 min, during which penile erection and yawning episodes were counted. Values are means---S.E.M, of 3 experiments (15 rats per group). *p<0.001 with respect to rats pretreated with saline (nonapeptide antagonist = 0).
precluded the appearance of penile erection and yawning. This possibility is supported by the observation that vasopressin has been found to be able to induce both penile erection and yawning when microinjected into the PVN or the CA1 region of the hippocampus, although being 10-20 times less potent than oxytocin (23). Moreover, while deletion of the glycinamide in position 9, as in oxytocin(1-8), resulted in a 100 times decrease in oxytocin potency for inducing penile erection and yawning, deletion of the C-terminal tripeptide, as in oxytocin(1-6), completely destroyed the activity. Similarly, the C-terminal tripeptide oxytocin(7-9) was ineffective in inducing yawning and penile erection. Finally, a complete loss of activity was also induced by permanent opening of the disulfide bridge by reduction and carboxymethylation of the oxytocin molecule, suggesting that the cyclic structure conferred by the disulfide bridge plays an important role in the interaction of oxytocin with its binding site. As to the prevention of penile erection and yawning induced by oxytocin and related peptides by nonapeptide antagonists, our results confirm and extend previous findings showing that oxytocininduced penile erection and yawning were prevented in a dosedependent manner by an oxytocin analog which is a potent antagonist of peripheral and central oxytocin effects (2,5). Interestingly, the prevention of the effect of oxytocin, [Thr4,GlyT]oxytocin and oxytocin(1-8) by nonapeptide antagonists is correlated with the antioxytocic activity of these peptides, rather than with their antivasopressor or antidiuretic activity (Table 2). Indeed d[(CHz)sTyr(Me)ArgS]-vasopressin, which is the less potent as antioxytocic of the three analogs, and used as a selective ligand of peripheral and central vasopressin receptors of the V1 type (13,
562
ARGIOLAS, MELIS, STANCAMPIANO AND GESSA I--'l None
TABLE 3 C O M P A R I S O N OF THE A C T I V I T Y O F O X Y T O C I N A N D RELATED PEPTIDES O N THE I N D U C T I O N O F PENILE ERECTION A N D Y A W N I N G , UTERUS C O N T R A C T I O N , M I L K EJECTION A N D ON PASSIVE A V O I D A N C E
Activity (%)*
Peptide Oxytocin [Thr4,Gly7]-oxy Isotocin Vasotocin Vasopressin Oxytocin(1-8) Oxytocin(l~5) Oxytocin(7-9) CM-oxytocin
Penile Erection Yawning 100 17 17 n.a. 107 1 0 0 0
Passive Avoidance 100 n.a. n.a. 100§ 0:~'§ 100§ n.a. O~:'?t n.a.
Uterus 100 33# 25¶ 26¶ 5¶ 1¶'** OH OH OH
Milk Ejection 100 n.a.
70¶ 42¶ 15¶ n.a. 0H 0H 0¶
*Activity is expressed as percent of that of oxytocin. t(23). $Opposite effect. §(12,14). ¶(28). #(21,22). **(34). ?t(33). n.a. = Not available.
15, 21, 22, 32) was found to be also the less potent in antagonizing penile erection and yawning induced by oxytocin and related peptides. Such selective reversal of the effect of oxytocin and related peptides by nonapeptide antagonists provides further support to the hypothesis that oxytocin effect is mediated by the stimulation of central oxytocinergic receptors. It is likely that these receptors are located in the hypothalamic PVN, the brain area most sensitive to oxytocin for the induction of penile erection and yawning (23) and whose destruction prevents oxytocin effect (3). Although the mechanism by which oxytocin and related peptides induce penile erection and yawning by acting in the PVN is unknown at present, our results provide further evidence for a neurotransmitter role of oxytocin in the central nervous system. Accordingly, it is noteworthy that 1) the PVN contains not only the cell bodies of magnocellular oxytocinergic neurons projecting mainly to the neurohypophysis, but also the cell bodies of parvocellular oxytocinergic neurons which project to several extrahypothalamic brain areas (see [8,29]), 2) oxytocin receptors have been found in this hypothalamic nuclei (7), 3) exogenous oxytocin has been shown to enhance in vivo the electrical activity of oxytocinergic neurons (16) and to stimulate in vitro the release of endogenous oxytocin (24), and 4) oxytocin-immunoreactive synapses have been found to impinge on oxytocinergic neurons in hypothalamic nuclei (30). Furthermore, oxytocin has been implied in the modulation of memory and learning processes (12-14) and maternal behavior (26). On the other hand, the failure of carboxymethylated oxytocin, oxytocin(1-6) and oxytocin(7-9) either to induce or to prevent oxytocin effect suggests that these peptides do not interact with oxytocin receptors. Taken all together, the above findings indicate that the structureactivity relationship of oxytocin and related peptides for their ability to induce penile erection and yawning resembles that found for their oxytocic and milk ejection activities (21, 22, 28), but not
d(CH2)sTyr(Me)-OrnB-VT 100 ng
I ~ Pen T,Phe(Me)2,Thr~Orr~-oxY 100 ng mm d(CH2)5Tyr(Me),-Ar~-VP 1000 ng
20 ~15 z~
~ Z
1o
4
o
3
i,i ° o¢"
2
"'
1
Z
"' 0..
,, I
0
Thr ,GI); -Oxy 100 ng
Oxy '1-8 1 ,ug
FIG. 3. Prevention of penile erection and yawning induced by [Thr4,Gly7]oxytocin and oxytocin(1-8) by nonapeptide antagonists. Peptides were dissolved in saline and injected ICV in a volume of 5 ILl as described in the Method section. Saline or one nonapeptide antagonist was injected 10 min before 100 ng of [Thr4,GlyT]-oxytocinor 1 I~g of oxytocin(1-8). After injection, the rats were placed individually into Plexiglas cages and observed for 60 min during which penile erection and yawning episodes were counted. Values are means_ S.E.M. of 3 experiments (15 rats per group), p<0.001 with respect to rats pretreated with saline (no nonapeptide antagonist).
that reported for the effect of oxytocin and its fragments on avoidance behavior (12,14) (Table 3). Such resemblance and the prevention of oxytocin effect by nonapeptide antagonists with a potency that is parallel to their antioxytocic activity led us to infer that the central oxytocinergic receptors, whose activation is responsible for the induction of penile erection and yawning, are structurally related to the oxytocinergic receptors present in the uterus and mammary gland. Interestingly, 1) a uterine-type oxytocinergic receptor has been already characterized in the hippocampus (15,25), one of the brain areas most sensitive for the induction of penile erection and yawning by oxytocin (23); 2) the oxytocic activity of oxytocin and its fragments has been correlated with their potency in inducing grooming behavior (9) and 3) the antioxytocic activity of nonapeptide antagonists correlates with their potency in antagonizing oxytocin-induced analgesia (10). On the other hand, the different structure-activity relationships of oxytocin-related peptides for the effect on yawning and penile erection and avoidance behavior (Table 3) indicate that the two responses are mediated by different receptors. This is also suggested by the opposite effects of vasopressin- and oxytocin-related peptides on memory processes, the first peptides having a protective effect against retrograde amnesia, and the second ones attenuating memory processes (12,14) and by the finding that other extrahypothalamic brain areas have been found to mediate the effect of neurohypophyseal peptides on memory processes (18). The presence of structurally-related oxytocinergic receptors in brain, uterus and mammary gland suggests the existence of a closely related oxytocinergic function in these tissues. In this regard, it is noteworthy that the above tissues play a primary role in physiological functions related to the reproduction and survival of the species, i.e., penile erection, parturition and lactation. Thus, the involvement of oxytocin in the above functions might be seen as one of Mother Nature's ways for the achievement of
PENILE ERECTION AND YAWNING BY OXYTOCIN
563
the survival of the species. Indeed oxytocin-vasopressin nonapeptides are among the most ancient and highly conserved hormones from a phylogenetic point of view, since they are present in fishes, amphibians, reptiles, birds and mammals with one or two amino acid substitutions due often to single base changes in the genetic code (4,31). In conclusion, substitution of one or two amino acids in the oxytocin molecule causes marked changes in its potency to induce
penile erection and yawning after central administration in rats. As for uterine and mammary oxytocinergic receptors, the integrity of the oxytocin molecule and its cyclic structure conferred by the disulfide bridge as well, other than the amino acid sequence, appear to be necessary for the interaction of oxytocin with the central oxytocinergic receptors that mediate the expression of penile erection and yawning.
REFERENCES
1. Argiolas, A.; Melis, M. R.; Gessa, G. L. Oxytocin: a potent inducer of penile erection and yawning in male rats. Eur. J. Pharmacol. 130:265-272; 1986. 2. Argiolas, A.; Melis, M. R.; Gessa, G. L. d(CH2)sTyr(Me)-Orn 8vasotocin, a potent oxytocin antagonist, antagonizes penile erection and yawning induced by oxytocin and apomorphine, but not by ACTH (1-24). Eur. J. Pharmacol. 134:221-224; 1987. 3. Argiolas, A.; Melis, M. R.; Mauri, A.; Gessa, G. L. Paraventricular nucleus lesion prevents yawning and penile erection induced by apomorphine and oxytocin, but not by ACTH 1-24. Brain Res. 421:349-352; 1987. 4. Ascher, R. Chemistry of the neurohypophyseal hormones: an example of molecular evolution. In: Greep, R. O., et al., eds. Handbook of physiology, section 7: Endocrinology vol. IV. Washington, DC: American Physiological Society; 1974:119-130. 5. Bankowski, K.; Manning, M.; Seto, J.; Halder, J.; Sawyer, W. H. Design and synthesis of potent in vivo antagonists of oxytocin. Int. J. Pept. Prot. Res. 16:382-391; 1980. 6. Bertolini, A.; Gessa, G. L. Behavioural effects of ACTH and MSH peptides. J. Endocrinol. Invest. 4:241-251; 1981. 7. Brinton, R. E.; Wamsley, J. K.; Gee, K. W.; Wan Yeih, P.; Yamamura, H. I. 3H-Oxytocin binding sites in the rat brain demonstrated by quantitative light microscopic autoradiography. Eur. J. Pharmacol. 102:365-367; 1984. 8. Bujis, R. M. Vasopressin and oxytocin--their role in neurotransmission. Pharmacol. Ther. 22:127-141; 1983. 9. Caldwell, J. D.; Prange, A. J., Jr.; Pedersen, C. A. Is oxytocin induced grooming mediated by uterine-like receptors? Neuropeptides 8:77-86; 1986. 10. Caldwell, J. D.; Mason, J. A.; Stanley, D. A.; Gerdak, G.; Hrnby, V. J.; Hill, P.; Prange, A. J., Jr.; Pedersen, C. A. Effects of nonapeptides antagonists on oxytocin- and arginine-vasopressin-induced analgesia in mice. Regul. Pept. 18:233-241; 1987. 11. Chan, W. Y.; Hruby, W. J.; Rockway, T. W.; Hlavacek, J. Design of oxytocin antagonists with prolonged action: Potential tocolytic agents for the treatment of preterm labor. J. Pharmacol. Exp. Ther. 239: 84-87; 1986. 12. De Wied, D.; Bohus, B. The modulation of memory processes by vasotocin, the evolutionarily oldest neurosecretory principle. Prog. Brain Res. 48:327-334; 1978. 13. De Wied, D.; Gaffori, O.; Van Ree, J. M.; De Jong, W. Vasopressin antagonists block peripheral as well as central vasopressin receptors. Pharmacol. Biochem. Behav. 21:393-400; 1984. 14. De Wied, D.; Gaffori, O.; Burbach, J. P. H.; Kovacs, L.; Van Ree, J. M. Structure-activity relationship studies with C-terminal fragments of vasopressin and oxytocin on avoidance behaviors of rats. J. Phannacol. Exp. Ther. 241:268-274; 1987. 15. Elands, J.; Barberis, C.; Jard, S. [3H]-[Thra,GlyT]OT: a highly selective ligand for central and peripheral OT receptors. Am. J. Physiol. 254:E31-E38; 1988. 16. Freund-Mercier, M. J.; Richard, P. Excitatory effectss of intraventricular injections of oxytocin on the milk ejection reflex in the rat. Neurosci. Lett. 23:193-198; 1981. 17. Hawthorn, J.; Ang, V. T.; Jenkins, J. S. Effects of lesions in the hypothalamic paraventricular, supraoptic and suprachiasmatic nuclei on vasopressin and oxytocin in rat brain and spinal cord. Brain Res. 346:51-57; 1985. 18. Kovacs, G. L.; Bohus, B.; Versteeg, D. H. G.; De Kloet, E. R.; De
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0196-9781/89 $3.00 + .00
Penile Erection and Yawning Induced by Oxytocin and Related Peptides: Structure-Activity Relationship A. A R G I O L A S , M. R. M E L I S , R. S T A N C A M P I A N O
A N D G. L. G E S S A
Department of Neurosciences, University of Cagliari, Via Porcell 4, 09124 Cagliari, Italy R e c e i v e d 18 July 1988
ARGIOLAS, A., M. R. MELIS, R. STANCAMPIANO AND G. L. GESSA. Penileerection and yawning inducedby oxytocin and relatedpeptides: Structure-activity relationship. PEPTIDES 10(3) 559-563, 1989.--The potency of several oxytocin-related peptides in inducing penile erection and yawning after injection into a lateral ventricle of male rats was compared. Substitution of two amino acids in the oxytocin molecule or deletion of the C-terminal glycinamide as in des-GlyNH2-oxytocin [oxytocin(1-8)] reduced oxytocin potency in inducing both effects, the rank order being: oxytocin > [Thr4,GlyT]-oxytocin = isotocin ([Ser4,Ileg]-oxytocin) > vasopressin ([Phe3,ArgS]-oxytocin) > des-GlyNH2-oxytocin. Oxytocin's ability to induce penile erection and yawning was abolished by permanent opening of the disulfide bridge by reduction and carboxymethylation. Oxytocin(1-6) and oxytocin(7-9) were also inactive. Penile erection and yawning induced by oxytocin-related peptides were antagonized in a dose-dependent manner by nonapeptide antagonists with a rank order of potency that follows their antioxytocic activity (d[(CH2)sTyr(Me)OrnS]-vasotocin -~ [Pent,Phe(Me)2,Thr4,Orns]-oxytocin > d[(CH2)sTyr(Me)ArgSl-vasopressin).Carboxymethylated oxytocin, oxytocin(1-6), and oxytocin(79) were devoid of antagonistic activity. The present results suggest that central oxytocin receptors mediating the expression of penile erection and yawning are structurally related to those present in the uterus and in the mammary gland. Oxytocin
Oxytocin-vasopressin analogs
Oxytocin antagonists
OXYTOCIN injected into the lateral ventricles (ICV) or into the paraventricular nucleus of the hypothalamus (PVN) or the CA1 field of the hippocampus induces repeated episodes of penile erection and yawning in male rats (1-3, 23). While the importance of penile erection in reproduction does not need to be stressed, it is pertinent to recall that yawning is considered an arousal mechanism to antagonize sleep when it is pressing under dangerous conditions or, as in civilized society, under improper circumstances [see (6)]. Oxytocin-induced penile erection and yawning seem to be mediated by the stimulation of central oxytocinergic receptors, since they are prevented by the ICV injection of the potent nonapeptide antagonist d[(CH2)sTyr(Me)OrnS]-vasotocin (2) and by electrolytic lesion of the PVN (3), that causes an almost complete depletion of brain oxytocin content (17,20). The present study was aimed at the screening of oxytocin-related peptides for their ability to induce penile erection and yawning in male rats and to compare the structure-activity relationship with those well characterized for their effects on the uterus and mammary gland (28), and on memory processes (12-14).
Penile erection
Yawning
humidity 60%, with water and standard laboratory food ad lib.
ICV Injections Stainless steel guide cannulas (22 gauge) aimed at one lateral ventricle were stereotaxically implanted (David Kopf Instruments, USA) under chloral hydrate anaesthesia 5 days before the experiments (coordinates: 1 mm anterior to bregma, 1.5 lateral to midline and 2 mm ventral to dura) (27). Substances dissolved in saline or saline alone were injected into a lateral ventricle (ICV) via an internal cannula (28 gauge), which extended 2 mm below the tip of the guide cannula and was connected by polyethylene tubing to a 10 lxl Hamilton syringe driven by a micrometric screw. Volumes injected ICV were 10 txl in 2 min.
Behavioral Studies Soon after injection, the animals were placed individually into Plexiglas cages (30 x 30 x 30 cm) and observed for 60 min during which penile erection and yawning episodes were counted. At the end of the experiments, the animals were killed by decapitation, the brains were removed and visually inspected to ascertain the correct position of the cannula tip into the lateral ventricle. Only those animals that were found to have the cannula tip positioned correctly were considered for statistical analysis of the results
METHOD
Animals Male Sprague-Dawley rats (250-300 g) were used in all the experiments. The animals were caged in groups of 4-6 at 24°C,
559
560
ARGIOLAS, MELIS, STANCAMPIANO AND GESSA TABLE 1 AMINO ACID SEQUENCE OF OXYTOCIN AND RELATED PEPTIDES
Peptide
1
2
3
Oxytocin [Thr4, Gly 7 ]-Oxy
Cys Cys
Tyr Tyr
Ile Ile
Isotocin Vasotocin Vasopressin Oxytocin(1-8) Oxytocin(1-6)
Cys Cys Cys Cys Cys
Tyr Tyr Tyr Tyr Tyr
lie lie
Phe lie lie
4 Gin
Thr Ser Gin Gin Gin Gin
Residue 5
6
7
8
Asn Asn
C)'s C)'s
Pro
Leu Leu
GlyNH2 GlyNH 2
Asn Ash Asn Ash Asn
C)'s C),s C)'s C)'s C)'s
Pro Pro Pro Pro
lle Arg Arg
GIyNH2 GlyNH2 GlyNH2
Pro
Leu
GlyNH 2
Oxytocin(7-9) CM-oxytocin d [(C H 2)5 Tyr(Me) Orn8 ]_vasotocin [Pen 1, Phe(Me) 2, Thr4 Orn 8 ]-oxytocin d[(Cn 2)5 Tyr(Me) ArgS]-vasopressin
Gly
9
Leu
CMCys
Tyr
Ile
Gin
Asn
CMCys
Pro
Leu
GlyNH2
Mpr
Tyr(Me)
lie
Gin
Asn
C,vs
Pro
Orn
GlyNH 2
Pen
Phe(Me) Ile
Thr
Asn
C)'s
Pro
Orn
GlyNH 2
Mpr
Tyr(Me)
Gin
Asn
C),s
Pro
Arg
GlyNH2
Ile
Different residues are italicized. Mpr=-l(13-mercapto-13, I]-cyclopentamethylene)-propionic acid. Pen--Penicillamine.
(Duncan's new multiple range test).
Peptides The following peptides were purchased from Peninsula Laboratories (San Carlos, CA): oxytocin, oxytocin(1-6) (=tocinoic acid), oxytocin(7-9) (= Pro-Leu-GlyNH2), [Thr4,GlyT]-oxytocin, vasotocin, vasopressin, d[(CH2)sTyr(Me)OmS]-vasotocin, d[(CH2)5 Tyr(Me)ArgS]-vasopressin. Isotocin was purchased from Sigma (St. Louis, MO). Oxytocin(1-8) (=des-9-glycinamide-oxytocin) (Organon, The Netherlands) was kindly provided by Prof. David De Wied (Rudolph Magnus Institute, Utrecht, The Netherlands). [Pen 1,Phe(Me) 2,Thr4,Orns]-OxytOcin was kindly provided by Prof. V. J. Hruby (University of Arizona, Tucson, AZ). Carboxymethylated oxytocin was prepared from oxytocin after reduction of the disulfide bridge with dithiothreitol and carboxymethylation with iodoacetic acid and purified by high pressure liquid chromatography on a 0.39 × 30 cm I~Bondapak C18 column (Waters Associates). The chromatographic conditions were reported elsewhere (1). The amino acid sequence of the above peptides is shown in Table 1. The antioxytocic, antivasopressor and antidiuretic activity of nonapeptide antagonists is summarized in Table 2. RESULTS Figure 1 shows the dose-response curve of different oxytocinlike peptides for inducing penile erection and yawning in male rats. As previously reported, oxytocin showed a bell-shaped dose-response curve, the maximal effect being induced by doses between 10 and 60 ng, while doses higher than 100 ng were almost
ineffective. A similar bell-shaped dose-response curve was also found with [Thra,GlyT]-oxytocin, a synthetic agonist of peripheral and central oxytocin receptors (15, 21, 22), and with the bony fish isotocin ([Ser4,IleS]-oxytocin), although they were about 6 times less potent than oxytocin, the minimal and the maximal effective dose being 30 ng and between 100 and 500 ng, respectively. Oxytocin(1-8) also induced penile erection and yawning but it was 100 times less potent than oxytocin. In contrast, carboxymethylated oxytocin, oxytocin(1--6) and oxytocin(7-9) were ineffective at doses up to 1 p,g. Vasotocin ([ArgS]-oxytocin), and vasopressin ([Phe3,ArgS]-oxytocin) were also ineffective. However, the study TABLE 2 BIOLOGICALACTIVITIESOF THE NONAPEPTIDEANTAGONISTS Activity (In Vivo) Antioxytocic AntivasopressorAntidiuretic Compound
(pA2)
(pA2)
U/mg
d[(CH2)sTyr(Me)OmS]-VT 7.88* [PenI,Phe(Me)2,Thr4,OmS]-Oxy 7.30f d[CH2)sTyr(Me)ArgS]-VP 6.62:~
7.96* 6~7t 8.62:~
0.009* n.a. 0.31:~
pA2= Negative logarithm of the molar concentration that reduces by 50% the response to agonist. For the definition of U/mg see (19). *(5). ¢(11). $(19). n.a. = Not available.
PENILE ERECTION AND YAWNING BY OXYTOCIN
2o ,~;.,o~.z~ VSae,,a-'oxx 15- Aoxy 1-8
g N
/
*~.~-~6~"~ /r x "~i~F..~ /~
*
~ ~,,d
561
/'l
.
•1~
20
-II TI
4("
1,
g N n
0
10
100
1000
ng i.c.v,
FIG. 1. Penile erection and yawning induced by the ICV injection of oxytocin and related peptides: dose-response curves. Peptides were dissolved in saline and injected ICV in a volume of 10 ~1 through chronically implanted guide cannulas as described in the Method section. After injection, rats were placed individually into Plexiglas cages and observed for 60 min during which penile erection and yawning episodes were counted. Values are means---S.E.M, of 3 experiments (15 rats per group). *p<0.001 with respect to saline-treated rats.
of the effect of the latter two peptides was complicated by the fact that both peptides, at doses higher than 5 ng, induced barrel rotation and motor disturbances, which lasted for 15-30 min. After this time, the animals looked normal, but no yawning or penile erection episodes were observed during the remaining observation period. Penile erection and yawning induced by 30 ng of ICV oxytocin were prevented in a dose-dependent manner by the ICV pretreatment with the nonapeptide antagonists d[(CH2)~Tyr(Me)OrnS]vasotocin (5) and [Penl,Phe(Me)2,Thra,oruS]-oxytocin (11) at doses between 10 and 100 ng. d[(CH2)sTyr(Me)ArgS]-vasopressin (19,32) also antagonized the oxytocin effect, but it was 10-20 times less potent (Fig. 2). In contrast, penile erection and yawning induced by oxytocin were not prevented by the ICV pretreatment with doses up to 1 txg of either carboxymethylated oxytocin or oxytocin(1-6) or oxytocin(7-9) (Fig. 2). Like the oxytocin effect, penile erection and yawning induced by 100 ng of [Thra,GlyT]oxytocin or 1 p,g of oxytocin(1-8) were also prevented by pretreatment with the above nonapeptide antagonists (Fig. 3). DISCUSSION
The present results show that penile erection and yawning are induced not only by the ICV injection of oxytocin but also by its analogs isotocin and [Thr4,Gly7]-oxytocin. Interestingly, the three peptides showed a bell-shaped dose-response curve, suggesting that they act on the same receptor to induce the above effects. In contrast, ICV vasotocin ([Arga]-oxytocin) and vasopressin ([Phe3,Arga]-oxytocin) were found to be ineffective. However, such ineffectiveness may be due to the fact that both peptides induced barrel rotations and motor disturbances, which might have
letP~I,Ph~(M,.RThr~O.#]--O~ o 0
I
10
i x
. ~ 100
*
\* : 1000
peptide ontogonist (ng i.c.v.)
FIG. 2. Penile erection and yawning induced by oxytocin: effect of nonapeptide antagonists, CM-oxytocin, oxytocin(1-6) and oxytocin(7-9). Peptides were dissolved in saline and injected ICV in a volume of 5 ~1, as described in the Method section. All rats received 30 ng of oxytocin. Nonapeptide antagonists, CM-oxytocin, oxytocin(l~), oxytocin(7-9) or saline were injected 10 min before the administration of oxytocin. After oxytocin injection, the animals were placed individually into Plexiglas cages and observed for 60 min, during which penile erection and yawning episodes were counted. Values are means---S.E.M, of 3 experiments (15 rats per group). *p<0.001 with respect to rats pretreated with saline (nonapeptide antagonist = 0).
precluded the appearance of penile erection and yawning. This possibility is supported by the observation that vasopressin has been found to be able to induce both penile erection and yawning when microinjected into the PVN or the CA1 region of the hippocampus, although being 10-20 times less potent than oxytocin (23). Moreover, while deletion of the glycinamide in position 9, as in oxytocin(1-8), resulted in a 100 times decrease in oxytocin potency for inducing penile erection and yawning, deletion of the C-terminal tripeptide, as in oxytocin(1-6), completely destroyed the activity. Similarly, the C-terminal tripeptide oxytocin(7-9) was ineffective in inducing yawning and penile erection. Finally, a complete loss of activity was also induced by permanent opening of the disulfide bridge by reduction and carboxymethylation of the oxytocin molecule, suggesting that the cyclic structure conferred by the disulfide bridge plays an important role in the interaction of oxytocin with its binding site. As to the prevention of penile erection and yawning induced by oxytocin and related peptides by nonapeptide antagonists, our results confirm and extend previous findings showing that oxytocininduced penile erection and yawning were prevented in a dosedependent manner by an oxytocin analog which is a potent antagonist of peripheral and central oxytocin effects (2,5). Interestingly, the prevention of the effect of oxytocin, [Thr4,GlyT]oxytocin and oxytocin(1-8) by nonapeptide antagonists is correlated with the antioxytocic activity of these peptides, rather than with their antivasopressor or antidiuretic activity (Table 2). Indeed d[(CHz)sTyr(Me)ArgS]-vasopressin, which is the less potent as antioxytocic of the three analogs, and used as a selective ligand of peripheral and central vasopressin receptors of the V1 type (13,
562
ARGIOLAS, MELIS, STANCAMPIANO AND GESSA I--'l None
TABLE 3 C O M P A R I S O N OF THE A C T I V I T Y O F O X Y T O C I N A N D RELATED PEPTIDES O N THE I N D U C T I O N O F PENILE ERECTION A N D Y A W N I N G , UTERUS C O N T R A C T I O N , M I L K EJECTION A N D ON PASSIVE A V O I D A N C E
Activity (%)*
Peptide Oxytocin [Thr4,Gly7]-oxy Isotocin Vasotocin Vasopressin Oxytocin(1-8) Oxytocin(l~5) Oxytocin(7-9) CM-oxytocin
Penile Erection Yawning 100 17 17 n.a. 107 1 0 0 0
Passive Avoidance 100 n.a. n.a. 100§ 0:~'§ 100§ n.a. O~:'?t n.a.
Uterus 100 33# 25¶ 26¶ 5¶ 1¶'** OH OH OH
Milk Ejection 100 n.a.
70¶ 42¶ 15¶ n.a. 0H 0H 0¶
*Activity is expressed as percent of that of oxytocin. t(23). $Opposite effect. §(12,14). ¶(28). #(21,22). **(34). ?t(33). n.a. = Not available.
15, 21, 22, 32) was found to be also the less potent in antagonizing penile erection and yawning induced by oxytocin and related peptides. Such selective reversal of the effect of oxytocin and related peptides by nonapeptide antagonists provides further support to the hypothesis that oxytocin effect is mediated by the stimulation of central oxytocinergic receptors. It is likely that these receptors are located in the hypothalamic PVN, the brain area most sensitive to oxytocin for the induction of penile erection and yawning (23) and whose destruction prevents oxytocin effect (3). Although the mechanism by which oxytocin and related peptides induce penile erection and yawning by acting in the PVN is unknown at present, our results provide further evidence for a neurotransmitter role of oxytocin in the central nervous system. Accordingly, it is noteworthy that 1) the PVN contains not only the cell bodies of magnocellular oxytocinergic neurons projecting mainly to the neurohypophysis, but also the cell bodies of parvocellular oxytocinergic neurons which project to several extrahypothalamic brain areas (see [8,29]), 2) oxytocin receptors have been found in this hypothalamic nuclei (7), 3) exogenous oxytocin has been shown to enhance in vivo the electrical activity of oxytocinergic neurons (16) and to stimulate in vitro the release of endogenous oxytocin (24), and 4) oxytocin-immunoreactive synapses have been found to impinge on oxytocinergic neurons in hypothalamic nuclei (30). Furthermore, oxytocin has been implied in the modulation of memory and learning processes (12-14) and maternal behavior (26). On the other hand, the failure of carboxymethylated oxytocin, oxytocin(1-6) and oxytocin(7-9) either to induce or to prevent oxytocin effect suggests that these peptides do not interact with oxytocin receptors. Taken all together, the above findings indicate that the structureactivity relationship of oxytocin and related peptides for their ability to induce penile erection and yawning resembles that found for their oxytocic and milk ejection activities (21, 22, 28), but not
d(CH2)sTyr(Me)-OrnB-VT 100 ng
I ~ Pen T,Phe(Me)2,Thr~Orr~-oxY 100 ng mm d(CH2)5Tyr(Me),-Ar~-VP 1000 ng
20 ~15 z~
~ Z
1o
4
o
3
i,i ° o¢"
2
"'
1
Z
"' 0..
,, I
0
Thr ,GI); -Oxy 100 ng
Oxy '1-8 1 ,ug
FIG. 3. Prevention of penile erection and yawning induced by [Thr4,Gly7]oxytocin and oxytocin(1-8) by nonapeptide antagonists. Peptides were dissolved in saline and injected ICV in a volume of 5 ILl as described in the Method section. Saline or one nonapeptide antagonist was injected 10 min before 100 ng of [Thr4,GlyT]-oxytocinor 1 I~g of oxytocin(1-8). After injection, the rats were placed individually into Plexiglas cages and observed for 60 min during which penile erection and yawning episodes were counted. Values are means_ S.E.M. of 3 experiments (15 rats per group), p<0.001 with respect to rats pretreated with saline (no nonapeptide antagonist).
that reported for the effect of oxytocin and its fragments on avoidance behavior (12,14) (Table 3). Such resemblance and the prevention of oxytocin effect by nonapeptide antagonists with a potency that is parallel to their antioxytocic activity led us to infer that the central oxytocinergic receptors, whose activation is responsible for the induction of penile erection and yawning, are structurally related to the oxytocinergic receptors present in the uterus and mammary gland. Interestingly, 1) a uterine-type oxytocinergic receptor has been already characterized in the hippocampus (15,25), one of the brain areas most sensitive for the induction of penile erection and yawning by oxytocin (23); 2) the oxytocic activity of oxytocin and its fragments has been correlated with their potency in inducing grooming behavior (9) and 3) the antioxytocic activity of nonapeptide antagonists correlates with their potency in antagonizing oxytocin-induced analgesia (10). On the other hand, the different structure-activity relationships of oxytocin-related peptides for the effect on yawning and penile erection and avoidance behavior (Table 3) indicate that the two responses are mediated by different receptors. This is also suggested by the opposite effects of vasopressin- and oxytocin-related peptides on memory processes, the first peptides having a protective effect against retrograde amnesia, and the second ones attenuating memory processes (12,14) and by the finding that other extrahypothalamic brain areas have been found to mediate the effect of neurohypophyseal peptides on memory processes (18). The presence of structurally-related oxytocinergic receptors in brain, uterus and mammary gland suggests the existence of a closely related oxytocinergic function in these tissues. In this regard, it is noteworthy that the above tissues play a primary role in physiological functions related to the reproduction and survival of the species, i.e., penile erection, parturition and lactation. Thus, the involvement of oxytocin in the above functions might be seen as one of Mother Nature's ways for the achievement of
PENILE ERECTION AND YAWNING BY OXYTOCIN
563
the survival of the species. Indeed oxytocin-vasopressin nonapeptides are among the most ancient and highly conserved hormones from a phylogenetic point of view, since they are present in fishes, amphibians, reptiles, birds and mammals with one or two amino acid substitutions due often to single base changes in the genetic code (4,31). In conclusion, substitution of one or two amino acids in the oxytocin molecule causes marked changes in its potency to induce
penile erection and yawning after central administration in rats. As for uterine and mammary oxytocinergic receptors, the integrity of the oxytocin molecule and its cyclic structure conferred by the disulfide bridge as well, other than the amino acid sequence, appear to be necessary for the interaction of oxytocin with the central oxytocinergic receptors that mediate the expression of penile erection and yawning.
REFERENCES
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