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Hypothermia induced by centrally administered vasopressin in rats
Neuropharmacology Vol. 23, No. 10, pp. 1195-1200, Printed in Great Britain. All rights reserved
1984 Copyright
0
0028-3908184 $3.00 + 0.00 1984 Pergamon Press Ltd
HYPOTHERMIA INDUCED BY CENTRALLY ADMINISTERED VASOPRESSIN IN RATS A STRUCTURE-ACTIVITY
STUDY
G. MEISENBERG* and W. H. SIMMONS Department
of Biochemistry
and Biophysics, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, U.S.A.
(Accepted 16 February 1984) Summary-Vasopressin and related peptides cause short-lasting hypothermia when injected into the lateral ventricle of the rat. In the present study, the structure-activity relationships for the induction of this effect were examined. For the agonist peptides studied, the structural requirements were found to be similar to those required to cause peripheral vasoconstriction and to induce behavioral excitation in mice. However, an antagonist of the pressor and behavioral effects of vasopressin was ineffective in antagonizing the hypothermic response. Moreover, this analog and another pressor antagonist themselves caused hypothermia. Comparison with the structure-activity relationships for other effects on the central nervous
system strongly suggests that the hypothermic response is unrelated to the effects of vasopressin on consolidation of memory, development of tolerance to drugs, and mechanisms of reinforcement. Key words: vasopressin, oxytocin, body temperature,
In addition to its peripheral hormonal actions (Beck, Hassid and Dunn, 1980; Valiquette, 1980; de Wulf, Keppens, Vandenheede, Haustraete, Proost and Carton, 1980), vasopressin has been found to induce a variety of effects on the central nervous system (for a review, see Meisenberg and Simmons, 1983), including modification of spontaneous behavior (Abood, Knapp, Mitchell, Booth and Schwab, 1980; Delanoy, Dunn and Tintner, 1978; Delanoy, Dunn and Walter, 1979; Kasting, Veale and Cooper, 1980; Meisenberg, 1981; Meisenberg and Simmons, 1982), jearning and memory (Krejci, Kupkova, Metys, Barth and Jost, 1979; Walter, Hoffman, Flexner and Flexner, 1975; Walter, van Ree and de Wied, 1978; de Wied, 1976; de Wied and Versteeg, 1979), development and maintenance of tolerance to drugs and dependence (Hoffman and Tabakoff, 1981; Krivoy, Zimmermann and Lande, 1974; Walter, Flexner, Ritzmann, Bhargava and Hoffman, 1980) and reinforcement mechanisms (Dorsa and van Ree, 1979; van Ree and de Wied, 1977; Schwarzbcrg, Betschen and Unger, 1980). Vasopressin has also been shown to produce centrally-mediated alterations of body temperature. There is evidence that exogenous vasopressin suppresses fever, induced experimentally, and that endogenous vasopressin, released from peptidergic terminals in the septal area, is involved physiologically in the suppression of fever (Cooper, Kasting, Lederis and Veale, 1979; Kasting, Cooper and Veale, 1979; Kasting, Veale, Cooper and Lederis,
*Address correspondence of Biochemistry, Ross Portsmouth, Commonwealth
to: G. Meisenberg, Department University Medical School, of Dominica, West Indies.
structure-activity
relationships.
1981; Veale, Kasting and Cooper, 1981). In addition to this antipyretic effect, alterations of resting body temperature have been observed in different species
(Kasting, Veale and Cooper, 1980; Lipton and Glyn, 1980). In the rat, vasopressin induces short-lasting hypothermia after intracerebroventricular injection (Kasting et al., 1980). In the study reported here, the active doses, the time-course, and the structureactivity relationships for the induction of hypothermia in rats have been determined. The results are compared with the known structure-activity relationships for the induction of other biological effects of vasopressin. METHODS
Animals and procedures Male Long-Evans rats, weighing 180-220 g, were housed in groups of three per cage in a 12 x 12 hr light-dark cycle at a constant temperature of 24°C with food and water available ad libitum. Unilateral injections into the lateral ventricle were performed under light ether anesthesia using a Hamilton microsyringe and an injection volume of 20 ~1. The injection site was 2.0mm lateral of the midline. Control injections with a 1% solution of methylene blue showed that this procedure resulted in a distribution of the injected material throughout the ventricular system. The vehicle for the injections was artificial cerebrospinal fluid containing 128 mM NaCl, 2.55 mM KCl, 1.25 mM CaCl,, 1.0 mM MgCl, and 1.0 mM glucose. Body temperature was recorded using a rectal probe and a telethermometer. The rectal probe was inserted while the rat was lightly
1195
1196
G.
MEISENBERG
restrained manually and was kept in place until a stable temperature was reached. In experiments involving repeated measurements, the rat was kept in its home cage in between measurements. All experiments were performed at an ambient temperature of 23°C. Peptides 1 - Deamino - [ 1,6 - dicarba, 8 - ar~ninelvasopressin
( = [ 1,6-aminosuberic acid, 8-arginine]vasop~ssin, = Asu-AVP), [l-/?-mercapto-&fl-cyclopentamethylenepropionic acid, 2-(~-methyl)tyrosine, &arginine] vasopressin (dPM-AVP) and desglycinamide9[8-lysinelvasopressin (DG-LVP) were obtained from Peninsula Labs. [%Arginine]Vasotocin (AVT) and isotocin (Iso) were purchased from Sigma Chemical Company. ~-a~tyl-[2-(~-methyl)tyrosine,8-arginine] vasopressin (AC-AVP) was the generous gift of Dr David A. Jones, Searle Research and Development, Chicago, Illinois and Dr Wilbur H. Sawyer, Columbia University, New York. [8_Arginine]Vasopressin (AVP),oxytocin(~xy), l-deamino-~4-(ff-~inobuty~c &arginine]vasopressin (dAbu-AVP) and acid, 1-deamino-[2-phenylalanine, 7-(3,4_dehydro)proline, 8-arginine]vasopressin (dPdP-AVP) were available at the University of Illinois at the Medical Center, Chicago, Illinois and were synthesized in the laboratory of the late Dr R. Walter. The biological activities in the assay of behavior in the mouse of the samples of oxytocin and ~8-ar~nine]vasopressi~ used in the present experiments were 1.5 and 140 U/mg, respectively. statistical analysis
Data are expressed in terms of the change in body temperature from IO min before injection to the indicated time after injection in order to eliminate variability due to individual differences in resting body temperatures. In most experiments, changes in body temperature in rats injected with peptide were compared with those in the controls injected with CSF, by means of Mann-Whitney’s U-test. In experiments on the time-course of hypothermia induced by vasopressin, changes in body temperature in the treated and control rats were compared at each time period by Student’s t-test.
and W. H. SIMMONS hypothermia observed tion of CSF is probably sia (Clark and Clark, later on in these rats handling.
5min after the control injecinduced by the ether anesthe1981). The hyperthermia seen may be induced by repeated
Effects of analogs of vasopressin
Table 1 shows the results of an experiment in which body taco was determined 10 and 20min after the injection of various analogs of vasopressin. Both vasopressin and AVT were much more potent than oxytocin, dAbu-AVP, dPdP-AVP and DG-LVP in inducing hypothermia. Comparison of these results with the known structure-activity relationships for other biological effects (Meisenberg and Simmons, 1982) suggests a good correlation between the potency of these analogs in inducing hypothermia and their ability to elevate blood pressure in the rat after peripheral administration or to induce behavioral excitation in mice after intracerebroventricular injection [blood pressure in rats and behavioral effects in mice are thought to be mediated by a closely-related type of receptor (Meisenberg and Simmons, 1982)]. There seemed to be no correlation between the observed hypothermic potencies and the uterotonic or depressor potencies of the analogs in avians. The correlation with the antidiuretic potency was weak, since dAbu-AVP and dPdP-AVP, two highly potent and selective anti~uretic analogs, were only moderately active in the induction of hypothermia. The inactivity of desglycinamide-LVP (DG-LVP) shows that the hypothermia induced by vasopressin was mechanistically unrelated to vasopressin-induced al-
1.2
r
T
I
T
0.0
0.4 v e
00
2
-0.4 -08 c) CSF
RESULTS Time-course of hypother~iu
induced hy v~opress~~
Figure 1 shows the body temperature of rats at different time intervals after the intracerebroventricular (i.c.v.) injection of either 1 pg ~ginine-vasopressin (AVP) or artificial CSF. Arginine-v~opressin caused a statistically”si~ificant decrease in body temperature compared to control (CSF) at 10 and 15 min after injection. The effect was short-lasting, with complete recovery within 30 min. These results are essentially in agreement with those reported previously (Kasting et al., 1980). The slight
I
q
-1.2 -1.6
I 5
if 10
, 15
(n=8)
AVP,ljtg
t 20
I 30
fn=8) I 45
I 60
Time (min i Fig. 1. Time-course of hypothermia induced by vasopressin. Rats were iniected with 1 tig (i.c.v.) AVP in 20 ul artificial CSF or wit& CSF alone. kzy temperature (rekal probe) was determined before injection and at various times after injection. The change in body temperature from the preinjection value was determined at each time period for each rat. Data is expressed as mean change zf:standard error of the mean. *P < 0.05, Student’s t-test between rats treated with vasopressin and controls given CSF at the indicated
time after injection.
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Vasopressin and hypothermia Table 1. Hypothermic N*
Peptidet
16 8
CSF AVP 10 ng 100 ng
8 6 8
effects of various analogs of vasopressin
1% AVT 10 ng IOOng
1Pi
OXY1Pg 1OM 2ON Is0 20 /lg dAbu-AVP 300 ng 9 6 11 12 7 8
1Pk? 3/c&! dPdP-AVP 300 ng DG-L++-.lO pg 2Opg
AT,,,,,l - 0.29 f 0.19 - 0.28 + 0.3 1 -0.99*0.13$ - 1.63 k 0.2811 - 0.28 * 0.34 - 0.55 * 0.20 - 1.46 k 0.2411 - 0.27 f 0.34 - 0.84 f 0.29 - 1.15 f 0.1% + 0.60 + 0.49 -0.13&0.34 - 0.68 f 0.34 - 0.70 k 0.18 - 0.58 If: 0.30 - 0.80 + 0.27 - 0.06 : 0.28 - 0.39 + 0.30
ATx,m,nf +0.28*0.19 + 0.35 k 0.32 - 0.20 * 0.09 - 1.06 k 0.40/l + 0.33 + 0.25 + 0.11 * 0.22 - 1.15 kO.3811 + 0.53 f 0.34 - 0.23 + 0.36 - 0.70 * 0.285 + 1.26 k 0.44 + 0.60 5 0.45 - 0.01 * 0.32 - 0.47 + 0.26 - 0.23 k 0.23 - 0.48 + 0.236 + 0.57 + 0.19” - 0.05 + 0.27
‘Number of rats per group. tSee Methods for IMean k SEM. $P < 0.05; 11~c 0.01; Mann-Whitney compared with controls given CSF.
details. U-test
terations of learning and memory (Krejci et al., 1979; Walter et al., 1975; Walter et al., 1978; de Wied, 1976; de Wied and Versteeg, 1979), formation of tolerance (Hoffman and Tabakoff, 1981; Krivoy et al., 1974; Walter et al., 1980) and mechanisms of reinforcement (van Ree and de Wied, 1977) where DG-LVP was highly active. The properties of antagonists
Two putative antagonists were tested for their ability to block hypothermia induced by vasopressin. The peptide dPM-AVP is a potent antagonist of the pressor response to vasopressin (Kruszynski, Lammek, Manning, Seto, Haldar and Sawyer, 1980), while Asu-AVP is a pressor agonist (Hase, Morikawa and Sakakibara, 1969), but may be an antagonist of the antipyretic effect of vasopressin in sheep (Veale et al., 1981). Table 2 shows that both of these peptides did not suppress, but seemed to enhance, the hypothermic effect of vasopressin. despite the fact that the ratio of dPM-AVP to vasopressin used in this experiment was sufficient to prevent AVP-induced behavioral excitation in mice induced by vasopressin (Simmons and Meisenberg, 1983). Table 3 shows that, surprisingly, dPM-AVP and Asu-AVP, as well as another pressor antagonist, AC-AVP (Jones and Sawyer, 1980), were able to induce hypothermia themselves. The pressor antagonists also acted as agonists in inducing motor disturbances in rats. In the experiment shown in Table 2, an intracerebroventricular injection of 5 pg of the analogs along with 250 ng of vasopressin resulted in ataxia and occasionally “barrel rotation” (Abood et al., 1980; Kruse, van Wimersma Greidanus and de Wied, 1977). In other experiments, medium or large hypothermic doses of vasopressin, analogs, or pressor antagonists by themselves frequently induced slight to moderate motor abnormalities including hindlimb weakness and ataxia, but not barrel rotation. No attempt was made to quantitate these effects.
These results are in contrast to the effects of the pressor antagonists on behavioral excitation in mice induced by vasopressin, where the pressor antagonist, AC-AVP, was reported to act as an antagonist (Meisenberg and Simmons, 1982). The same also has been found for dPM-AVP. Both peptides were essentially devoid of agonist activity in this behavior assay in the mouse (Simmons and Meisenberg, 1983). DISCUSSION
The peripheral hormonal actions of vasopressin are thought to be mediated by specific receptors, and the structure-activity relationships for most of these effects are well studied (Beck et al., 1980; Butlen, Guillon, Rajerison, Jard, Sawyer and Manning, 1978; Guillon, Courand, Butlen, Cantau and Jard, 1980; Walter, Rudinger and Schwarz, 1967; de Wulf et al., 1980). More recently, similar structure-activity relationships have also been determined for some of the effects on the central nervous system (Abood et al., 1980; Delanoy et al., 1979; van Dijk, Lodewijks, van Ree and van Wimersma Greidanus, 1981; Walter et al., 1975; Walter et al., 1978; Walter et al., 1980; de Wied, 1976; Meisenberg and Simmons, 1982). As discussed elsewhere (Meisenberg and Simmons, 1983), the various actions of vasopressin which are mediated centrally appear to involve several types of receptors since the active doses, durations of action, and the structural requirements for these effects are different. The data presented here suggest that the putative receptor which mediates the hypothermic effect of vasopressin in the rat after intracerebroventricular injection has structural requirements similar to those of the peripheral pressor receptor (Botos, Smith, Chan and Walter, 1979; Chan and du Vigneaud, 1962; van Dyke, Sawyer and Overweg, 1963; Gillessen and du Vigneaud, 1970; Guttmann, Table 2. Hypothermic N*
effects of vasopressin combined with two putative antagonists Peptidet
AT,,,,“t: -0.38+0.10 - 0.79 * 0.14$ - 1.33 i 0.2311 - 1.04i 0.q
.._ CSF AVP 250 ng AVP 250 ng + Asu-AVP 5 pg AVP 250 ng + dPM-AVP 5 pg
16 16 16 16
*Number of rats per group. tSee Methods for details of peptides. fMean 5 SEM. IF’ < 0.05; 11~< 0.01; Mann-Whitney U-test compared with controls given CSF.
Table 3. Test of putative antagonists for agonist activity in inducing hypothermia N*
Peptidet
11 9 11 10 11 8 I1 9
CSF AVP 150 ng AC-AVP 2 pg AC-AVP 20 /~g dPM-AVP 150 ng dPM-AVP 1.5 pg Asu-AVP 200 ng Asu-AVP 2pg
-
AT,o,,.S 0.54 f 0.21 1.38 f 0.215 1.20 + 0.26 1.70 * 0.21 I/ 0.98 + 0.30 1.31 f 0.275 1.15 f 0.20 2.09 + 0.3711
*Number of rats per group. tSee Methods for details of peptides. fMean + SEM. §P c 0.05; 11~< 0.01; Mann-Whitney I/-test compared with controls injected with CSF.
1198
G. MEISENBERG and W. H. SIMMONS
1962; Hase et al., 1969; Jones and Sawyer, 1980; Kruszynski et al., 1980; Meienhofer, Trzeciak, Havran and Walter, 1970; Wafter et al., 1967) as well as the receptor mediating behavioral excitation in mice (Delanoy et al., 1978; Delanoy et al., 1979; Meisenberg, 1981; Meisenberg and Simmons, 1982; Meisenberg and Simmons, 1983). The receptor is clearly different from that mediating the peripheral antidiuretic or oxytocic activities of neurohypophyseal hormones. However, the two pressor antagonists, AC-AVP and dPM-AVP, which also antagonize behavioral excitation in mice induced by vasopressin (Meisenberg and Simmons, 1983), showed agonist activity in the induction of hypothermia. These results suggest that the “hypothermia receptor” may represent a new vasopressin-r~eptor subtype. There appears to be no relationship between the hypothermic effect of vasopressin and other actions of neurohypophyseal hormones on the central nervous system described in the rat. The hypothermic effect required considerably larger doses (100 ng AVP, i.c.v) than those required to improve learning and memory and to suppress intracranial selfstimulation ( < 1 ng, i.c.v) (de Wied, 1976; de Wied and Versteeg, 1979; Schwartzberg et al., 1980). Furthermore, the structure-activity relationships are quite different (Moal, Koob, Koda, Bloom, Manning, Sawyer and Rivier, 1981; Walter et al., 1975; Walter et al., 1978; de Wied, 1976; de Wied and Versteeg, 1979). In particular, oxytocin acts in an opposite manner to vasopressin in these paradigms, while exhibiting weak agonist activity in the induction of hypothermia. The analog DG-LVP, which is very potent in improving the consolidation of memory (de Wied and Versteeg, 19791, did not produce hypothermia. Vasopressin and oxytocin can induce maternal behavior in estrogen-primed virgin female rats (Pedersen and Prange, 1979; Pedersen, Ascher, Monroe and Prange, 1982). Although the active doses for this effect are similar to those required for the induction of h~othermia, the structural requirements are different since oxytocin is clearly more potent than vasopressin in stimulating maternal behavior. The structure-activity relationships for the induction of convulsant activity and “barrel rotations” in the rat (Abood et al., 1980; Kruse et al,, 1977) appear to be similar to those for the hypothetic effect. However, the present authors’ observation that “barrel rotation” and overt convulsions occurred very rarely after doses which caused hypothermia suggests that these effects are not casually related. Rather, they may be mediated by structurally-related receptor types, possibly in different areas of the brain. The relationship between the hypothermic and the antipyretic effects of vasopressin is not clear. Using push-pull perfusion of the septal region in sheep, the doses of vasopressin reported to be antipyretic were 0.8-4pg/ml, which is comparable to the doses used in the present experiments (Cooper et ai., 1979;
Kasting et al., 1979). By the same route of administration, vasotocin was found to be partially active while oxytocin was inactive (Veale et al., 1981). Ad~tional detailed structur~activity data on the antipyretic effect are currently not available. The peptide Asu-AVP ( = deamino-dicarba-AVP) was reported to act in an opposite manner to vasopressin in febrile sheep and a competitive antagonism by this peptide was suggested (Veale et al., 1981). However, it was not shown whether Asu-AVP actually blocked the antipyretic effect when administered along with vasopressin. In the rat, the sites of action for the antipyretic and the hypothermic effects seem to be different, as shown by different effects after intracerebroventricular injection and microinjection into the septal region (Eagan, Veale and Cooper, 1980; Kasting et al., 1980). The physiological role of vasopressin-induced hypothermia is unclear. Several vasopressin-containing fiber tracts have been described in the brain (Buijs, Velis and Swaab, 1980; Dogterom and Buijs, 1980), and vasopressin-containing synapses have been identified in structures of the brain stem and the limbic system (Buijs and Swaab, 1979; Sterba, Naumann and Hoheisel, 1980). In the septal region of the sheep, vasopressin was found in push-pull perfusates (Cooper et al., 1979), showing that significant quantities of vasopressin are released physiologically in this region of the brain. This suggests that large local concentrations of vasopressin can temporarily be reached following release from peptidergic synapses. The finding of the present study that, among various related peptides, the naturally occurring vasopressin was most active lends support to the suggestion that the hypothermic effect is not “non-specific” but may be of physiological significance. Tonic release of vasopressin, however, seems less likely than the activation of a vasopressinergic system in specific physiological situations. acknowledgement-This work was supported by USPHS grants AM30970 and HL28710. REFERENCES Abood L. G., Knapp R., Mitchell T., Booth H. and Schwab L. (1980) Chemical requirements of vasopressins for barrel rotation convulsions and reversal by oxytocin. J. .?&ri~osci.Res. 5: 191-199. Beck Th. R., Ha&d A. and Dunn M. J. (1980) The effect of arginine vasopressin and its analogs on the synthesis of prostaglandin E, by rat renal medullary interstitial cells in culture. J. Pharmac. exp. Ther. 215: E-29. Botes C. R., Smith C. W., Chan Y.-L. and Walter R. (1979) Synthesis and biological activities of arginine-vasopressin analogues designed from a confo~ation-activity approach. J. med. C&em. 22: 926931. Buijs R. M. and Swaab D. F. (1979) Immuno-electron microscopical demonstration of vasopressin and oxytocin synapses in the limbic system of the rat. Cell Tiss. Res. 204~ 355-365. Buijs R. M., Velis D. N. and Swaab D. F. (1980) Extrahypothalamic vasopressin and oxytocin innervation of fetal and adult rat brain. Prag. Brain Res. 53: 159-167.
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Fedn Proc. Fedn Am. Sacs exp.
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Waiter R., Rudinger J. and Schwan I. L. (1967) foists and structur~activity relations of the antidiuretic hormones. Am. J. Med. 42: 653-677. Walter R., Hoffman P. L., Flexner .I. B. and Flexner L. B. (1975) Neurohypophyseal hormones, analogs and fragments: Their effect on puromycin induced amnesia. Proc. natn. Acad. Sci. U.S.A. 72: 41804184.
Walter R., Ree J. M. van and Wied D. de (1978) Modification of conditioned behavior of rats by neurohypophyseal hormones and analogues. Proc. natn. Acad. Sci. U.S.A. 75: 2493-2496.
Walter R., Flexner L. B., Ritzmann R. F., Bhargava H. N. and Hoffman P. L. (1980) Central nervous system effects of posterior pituitary hormones, fragments, and their
derivatives on drug to~eran~/dependence and behavior. In: Poiypeptide Hormones (Beers R. F. Jr and Bassett E. G., E&t), pp. 321-335. Raven Press, New York. Wied D. de (1976) Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments. Life Sci. 19: 685-690. Wied D. de and Versteeg D. H. G. (1979) Neurohypophyseal principles and memory. Fedn Proc. Fedn Am. Sots exp. Biol. 38: 2348-2354.
Wulf H. de, Keppens S., Vandenheede J. R., Haustraete F., Proost C. and Carton H. (1980) Cyclic AMP-independent regulation of liver glycogenolysis.. In: Hormones and Cell Regulation, Vol. 4 (Dumont J. and Nunez J., Eds), pp. 47-71. Eise~er/No~h_Hoiland, New York.
1984 Copyright
0
0028-3908184 $3.00 + 0.00 1984 Pergamon Press Ltd
HYPOTHERMIA INDUCED BY CENTRALLY ADMINISTERED VASOPRESSIN IN RATS A STRUCTURE-ACTIVITY
STUDY
G. MEISENBERG* and W. H. SIMMONS Department
of Biochemistry
and Biophysics, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, U.S.A.
(Accepted 16 February 1984) Summary-Vasopressin and related peptides cause short-lasting hypothermia when injected into the lateral ventricle of the rat. In the present study, the structure-activity relationships for the induction of this effect were examined. For the agonist peptides studied, the structural requirements were found to be similar to those required to cause peripheral vasoconstriction and to induce behavioral excitation in mice. However, an antagonist of the pressor and behavioral effects of vasopressin was ineffective in antagonizing the hypothermic response. Moreover, this analog and another pressor antagonist themselves caused hypothermia. Comparison with the structure-activity relationships for other effects on the central nervous
system strongly suggests that the hypothermic response is unrelated to the effects of vasopressin on consolidation of memory, development of tolerance to drugs, and mechanisms of reinforcement. Key words: vasopressin, oxytocin, body temperature,
In addition to its peripheral hormonal actions (Beck, Hassid and Dunn, 1980; Valiquette, 1980; de Wulf, Keppens, Vandenheede, Haustraete, Proost and Carton, 1980), vasopressin has been found to induce a variety of effects on the central nervous system (for a review, see Meisenberg and Simmons, 1983), including modification of spontaneous behavior (Abood, Knapp, Mitchell, Booth and Schwab, 1980; Delanoy, Dunn and Tintner, 1978; Delanoy, Dunn and Walter, 1979; Kasting, Veale and Cooper, 1980; Meisenberg, 1981; Meisenberg and Simmons, 1982), jearning and memory (Krejci, Kupkova, Metys, Barth and Jost, 1979; Walter, Hoffman, Flexner and Flexner, 1975; Walter, van Ree and de Wied, 1978; de Wied, 1976; de Wied and Versteeg, 1979), development and maintenance of tolerance to drugs and dependence (Hoffman and Tabakoff, 1981; Krivoy, Zimmermann and Lande, 1974; Walter, Flexner, Ritzmann, Bhargava and Hoffman, 1980) and reinforcement mechanisms (Dorsa and van Ree, 1979; van Ree and de Wied, 1977; Schwarzbcrg, Betschen and Unger, 1980). Vasopressin has also been shown to produce centrally-mediated alterations of body temperature. There is evidence that exogenous vasopressin suppresses fever, induced experimentally, and that endogenous vasopressin, released from peptidergic terminals in the septal area, is involved physiologically in the suppression of fever (Cooper, Kasting, Lederis and Veale, 1979; Kasting, Cooper and Veale, 1979; Kasting, Veale, Cooper and Lederis,
*Address correspondence of Biochemistry, Ross Portsmouth, Commonwealth
to: G. Meisenberg, Department University Medical School, of Dominica, West Indies.
structure-activity
relationships.
1981; Veale, Kasting and Cooper, 1981). In addition to this antipyretic effect, alterations of resting body temperature have been observed in different species
(Kasting, Veale and Cooper, 1980; Lipton and Glyn, 1980). In the rat, vasopressin induces short-lasting hypothermia after intracerebroventricular injection (Kasting et al., 1980). In the study reported here, the active doses, the time-course, and the structureactivity relationships for the induction of hypothermia in rats have been determined. The results are compared with the known structure-activity relationships for the induction of other biological effects of vasopressin. METHODS
Animals and procedures Male Long-Evans rats, weighing 180-220 g, were housed in groups of three per cage in a 12 x 12 hr light-dark cycle at a constant temperature of 24°C with food and water available ad libitum. Unilateral injections into the lateral ventricle were performed under light ether anesthesia using a Hamilton microsyringe and an injection volume of 20 ~1. The injection site was 2.0mm lateral of the midline. Control injections with a 1% solution of methylene blue showed that this procedure resulted in a distribution of the injected material throughout the ventricular system. The vehicle for the injections was artificial cerebrospinal fluid containing 128 mM NaCl, 2.55 mM KCl, 1.25 mM CaCl,, 1.0 mM MgCl, and 1.0 mM glucose. Body temperature was recorded using a rectal probe and a telethermometer. The rectal probe was inserted while the rat was lightly
1195
1196
G.
MEISENBERG
restrained manually and was kept in place until a stable temperature was reached. In experiments involving repeated measurements, the rat was kept in its home cage in between measurements. All experiments were performed at an ambient temperature of 23°C. Peptides 1 - Deamino - [ 1,6 - dicarba, 8 - ar~ninelvasopressin
( = [ 1,6-aminosuberic acid, 8-arginine]vasop~ssin, = Asu-AVP), [l-/?-mercapto-&fl-cyclopentamethylenepropionic acid, 2-(~-methyl)tyrosine, &arginine] vasopressin (dPM-AVP) and desglycinamide9[8-lysinelvasopressin (DG-LVP) were obtained from Peninsula Labs. [%Arginine]Vasotocin (AVT) and isotocin (Iso) were purchased from Sigma Chemical Company. ~-a~tyl-[2-(~-methyl)tyrosine,8-arginine] vasopressin (AC-AVP) was the generous gift of Dr David A. Jones, Searle Research and Development, Chicago, Illinois and Dr Wilbur H. Sawyer, Columbia University, New York. [8_Arginine]Vasopressin (AVP),oxytocin(~xy), l-deamino-~4-(ff-~inobuty~c &arginine]vasopressin (dAbu-AVP) and acid, 1-deamino-[2-phenylalanine, 7-(3,4_dehydro)proline, 8-arginine]vasopressin (dPdP-AVP) were available at the University of Illinois at the Medical Center, Chicago, Illinois and were synthesized in the laboratory of the late Dr R. Walter. The biological activities in the assay of behavior in the mouse of the samples of oxytocin and ~8-ar~nine]vasopressi~ used in the present experiments were 1.5 and 140 U/mg, respectively. statistical analysis
Data are expressed in terms of the change in body temperature from IO min before injection to the indicated time after injection in order to eliminate variability due to individual differences in resting body temperatures. In most experiments, changes in body temperature in rats injected with peptide were compared with those in the controls injected with CSF, by means of Mann-Whitney’s U-test. In experiments on the time-course of hypothermia induced by vasopressin, changes in body temperature in the treated and control rats were compared at each time period by Student’s t-test.
and W. H. SIMMONS hypothermia observed tion of CSF is probably sia (Clark and Clark, later on in these rats handling.
5min after the control injecinduced by the ether anesthe1981). The hyperthermia seen may be induced by repeated
Effects of analogs of vasopressin
Table 1 shows the results of an experiment in which body taco was determined 10 and 20min after the injection of various analogs of vasopressin. Both vasopressin and AVT were much more potent than oxytocin, dAbu-AVP, dPdP-AVP and DG-LVP in inducing hypothermia. Comparison of these results with the known structure-activity relationships for other biological effects (Meisenberg and Simmons, 1982) suggests a good correlation between the potency of these analogs in inducing hypothermia and their ability to elevate blood pressure in the rat after peripheral administration or to induce behavioral excitation in mice after intracerebroventricular injection [blood pressure in rats and behavioral effects in mice are thought to be mediated by a closely-related type of receptor (Meisenberg and Simmons, 1982)]. There seemed to be no correlation between the observed hypothermic potencies and the uterotonic or depressor potencies of the analogs in avians. The correlation with the antidiuretic potency was weak, since dAbu-AVP and dPdP-AVP, two highly potent and selective anti~uretic analogs, were only moderately active in the induction of hypothermia. The inactivity of desglycinamide-LVP (DG-LVP) shows that the hypothermia induced by vasopressin was mechanistically unrelated to vasopressin-induced al-
1.2
r
T
I
T
0.0
0.4 v e
00
2
-0.4 -08 c) CSF
RESULTS Time-course of hypother~iu
induced hy v~opress~~
Figure 1 shows the body temperature of rats at different time intervals after the intracerebroventricular (i.c.v.) injection of either 1 pg ~ginine-vasopressin (AVP) or artificial CSF. Arginine-v~opressin caused a statistically”si~ificant decrease in body temperature compared to control (CSF) at 10 and 15 min after injection. The effect was short-lasting, with complete recovery within 30 min. These results are essentially in agreement with those reported previously (Kasting et al., 1980). The slight
I
q
-1.2 -1.6
I 5
if 10
, 15
(n=8)
AVP,ljtg
t 20
I 30
fn=8) I 45
I 60
Time (min i Fig. 1. Time-course of hypothermia induced by vasopressin. Rats were iniected with 1 tig (i.c.v.) AVP in 20 ul artificial CSF or wit& CSF alone. kzy temperature (rekal probe) was determined before injection and at various times after injection. The change in body temperature from the preinjection value was determined at each time period for each rat. Data is expressed as mean change zf:standard error of the mean. *P < 0.05, Student’s t-test between rats treated with vasopressin and controls given CSF at the indicated
time after injection.
1197
Vasopressin and hypothermia Table 1. Hypothermic N*
Peptidet
16 8
CSF AVP 10 ng 100 ng
8 6 8
effects of various analogs of vasopressin
1% AVT 10 ng IOOng
1Pi
OXY1Pg 1OM 2ON Is0 20 /lg dAbu-AVP 300 ng 9 6 11 12 7 8
1Pk? 3/c&! dPdP-AVP 300 ng DG-L++-.lO pg 2Opg
AT,,,,,l - 0.29 f 0.19 - 0.28 + 0.3 1 -0.99*0.13$ - 1.63 k 0.2811 - 0.28 * 0.34 - 0.55 * 0.20 - 1.46 k 0.2411 - 0.27 f 0.34 - 0.84 f 0.29 - 1.15 f 0.1% + 0.60 + 0.49 -0.13&0.34 - 0.68 f 0.34 - 0.70 k 0.18 - 0.58 If: 0.30 - 0.80 + 0.27 - 0.06 : 0.28 - 0.39 + 0.30
ATx,m,nf +0.28*0.19 + 0.35 k 0.32 - 0.20 * 0.09 - 1.06 k 0.40/l + 0.33 + 0.25 + 0.11 * 0.22 - 1.15 kO.3811 + 0.53 f 0.34 - 0.23 + 0.36 - 0.70 * 0.285 + 1.26 k 0.44 + 0.60 5 0.45 - 0.01 * 0.32 - 0.47 + 0.26 - 0.23 k 0.23 - 0.48 + 0.236 + 0.57 + 0.19” - 0.05 + 0.27
‘Number of rats per group. tSee Methods for IMean k SEM. $P < 0.05; 11~c 0.01; Mann-Whitney compared with controls given CSF.
details. U-test
terations of learning and memory (Krejci et al., 1979; Walter et al., 1975; Walter et al., 1978; de Wied, 1976; de Wied and Versteeg, 1979), formation of tolerance (Hoffman and Tabakoff, 1981; Krivoy et al., 1974; Walter et al., 1980) and mechanisms of reinforcement (van Ree and de Wied, 1977) where DG-LVP was highly active. The properties of antagonists
Two putative antagonists were tested for their ability to block hypothermia induced by vasopressin. The peptide dPM-AVP is a potent antagonist of the pressor response to vasopressin (Kruszynski, Lammek, Manning, Seto, Haldar and Sawyer, 1980), while Asu-AVP is a pressor agonist (Hase, Morikawa and Sakakibara, 1969), but may be an antagonist of the antipyretic effect of vasopressin in sheep (Veale et al., 1981). Table 2 shows that both of these peptides did not suppress, but seemed to enhance, the hypothermic effect of vasopressin. despite the fact that the ratio of dPM-AVP to vasopressin used in this experiment was sufficient to prevent AVP-induced behavioral excitation in mice induced by vasopressin (Simmons and Meisenberg, 1983). Table 3 shows that, surprisingly, dPM-AVP and Asu-AVP, as well as another pressor antagonist, AC-AVP (Jones and Sawyer, 1980), were able to induce hypothermia themselves. The pressor antagonists also acted as agonists in inducing motor disturbances in rats. In the experiment shown in Table 2, an intracerebroventricular injection of 5 pg of the analogs along with 250 ng of vasopressin resulted in ataxia and occasionally “barrel rotation” (Abood et al., 1980; Kruse, van Wimersma Greidanus and de Wied, 1977). In other experiments, medium or large hypothermic doses of vasopressin, analogs, or pressor antagonists by themselves frequently induced slight to moderate motor abnormalities including hindlimb weakness and ataxia, but not barrel rotation. No attempt was made to quantitate these effects.
These results are in contrast to the effects of the pressor antagonists on behavioral excitation in mice induced by vasopressin, where the pressor antagonist, AC-AVP, was reported to act as an antagonist (Meisenberg and Simmons, 1982). The same also has been found for dPM-AVP. Both peptides were essentially devoid of agonist activity in this behavior assay in the mouse (Simmons and Meisenberg, 1983). DISCUSSION
The peripheral hormonal actions of vasopressin are thought to be mediated by specific receptors, and the structure-activity relationships for most of these effects are well studied (Beck et al., 1980; Butlen, Guillon, Rajerison, Jard, Sawyer and Manning, 1978; Guillon, Courand, Butlen, Cantau and Jard, 1980; Walter, Rudinger and Schwarz, 1967; de Wulf et al., 1980). More recently, similar structure-activity relationships have also been determined for some of the effects on the central nervous system (Abood et al., 1980; Delanoy et al., 1979; van Dijk, Lodewijks, van Ree and van Wimersma Greidanus, 1981; Walter et al., 1975; Walter et al., 1978; Walter et al., 1980; de Wied, 1976; Meisenberg and Simmons, 1982). As discussed elsewhere (Meisenberg and Simmons, 1983), the various actions of vasopressin which are mediated centrally appear to involve several types of receptors since the active doses, durations of action, and the structural requirements for these effects are different. The data presented here suggest that the putative receptor which mediates the hypothermic effect of vasopressin in the rat after intracerebroventricular injection has structural requirements similar to those of the peripheral pressor receptor (Botos, Smith, Chan and Walter, 1979; Chan and du Vigneaud, 1962; van Dyke, Sawyer and Overweg, 1963; Gillessen and du Vigneaud, 1970; Guttmann, Table 2. Hypothermic N*
effects of vasopressin combined with two putative antagonists Peptidet
AT,,,,“t: -0.38+0.10 - 0.79 * 0.14$ - 1.33 i 0.2311 - 1.04i 0.q
.._ CSF AVP 250 ng AVP 250 ng + Asu-AVP 5 pg AVP 250 ng + dPM-AVP 5 pg
16 16 16 16
*Number of rats per group. tSee Methods for details of peptides. fMean 5 SEM. IF’ < 0.05; 11~< 0.01; Mann-Whitney U-test compared with controls given CSF.
Table 3. Test of putative antagonists for agonist activity in inducing hypothermia N*
Peptidet
11 9 11 10 11 8 I1 9
CSF AVP 150 ng AC-AVP 2 pg AC-AVP 20 /~g dPM-AVP 150 ng dPM-AVP 1.5 pg Asu-AVP 200 ng Asu-AVP 2pg
-
AT,o,,.S 0.54 f 0.21 1.38 f 0.215 1.20 + 0.26 1.70 * 0.21 I/ 0.98 + 0.30 1.31 f 0.275 1.15 f 0.20 2.09 + 0.3711
*Number of rats per group. tSee Methods for details of peptides. fMean + SEM. §P c 0.05; 11~< 0.01; Mann-Whitney I/-test compared with controls injected with CSF.
1198
G. MEISENBERG and W. H. SIMMONS
1962; Hase et al., 1969; Jones and Sawyer, 1980; Kruszynski et al., 1980; Meienhofer, Trzeciak, Havran and Walter, 1970; Wafter et al., 1967) as well as the receptor mediating behavioral excitation in mice (Delanoy et al., 1978; Delanoy et al., 1979; Meisenberg, 1981; Meisenberg and Simmons, 1982; Meisenberg and Simmons, 1983). The receptor is clearly different from that mediating the peripheral antidiuretic or oxytocic activities of neurohypophyseal hormones. However, the two pressor antagonists, AC-AVP and dPM-AVP, which also antagonize behavioral excitation in mice induced by vasopressin (Meisenberg and Simmons, 1983), showed agonist activity in the induction of hypothermia. These results suggest that the “hypothermia receptor” may represent a new vasopressin-r~eptor subtype. There appears to be no relationship between the hypothermic effect of vasopressin and other actions of neurohypophyseal hormones on the central nervous system described in the rat. The hypothermic effect required considerably larger doses (100 ng AVP, i.c.v) than those required to improve learning and memory and to suppress intracranial selfstimulation ( < 1 ng, i.c.v) (de Wied, 1976; de Wied and Versteeg, 1979; Schwartzberg et al., 1980). Furthermore, the structure-activity relationships are quite different (Moal, Koob, Koda, Bloom, Manning, Sawyer and Rivier, 1981; Walter et al., 1975; Walter et al., 1978; de Wied, 1976; de Wied and Versteeg, 1979). In particular, oxytocin acts in an opposite manner to vasopressin in these paradigms, while exhibiting weak agonist activity in the induction of hypothermia. The analog DG-LVP, which is very potent in improving the consolidation of memory (de Wied and Versteeg, 19791, did not produce hypothermia. Vasopressin and oxytocin can induce maternal behavior in estrogen-primed virgin female rats (Pedersen and Prange, 1979; Pedersen, Ascher, Monroe and Prange, 1982). Although the active doses for this effect are similar to those required for the induction of h~othermia, the structural requirements are different since oxytocin is clearly more potent than vasopressin in stimulating maternal behavior. The structure-activity relationships for the induction of convulsant activity and “barrel rotations” in the rat (Abood et al., 1980; Kruse et al,, 1977) appear to be similar to those for the hypothetic effect. However, the present authors’ observation that “barrel rotation” and overt convulsions occurred very rarely after doses which caused hypothermia suggests that these effects are not casually related. Rather, they may be mediated by structurally-related receptor types, possibly in different areas of the brain. The relationship between the hypothermic and the antipyretic effects of vasopressin is not clear. Using push-pull perfusion of the septal region in sheep, the doses of vasopressin reported to be antipyretic were 0.8-4pg/ml, which is comparable to the doses used in the present experiments (Cooper et ai., 1979;
Kasting et al., 1979). By the same route of administration, vasotocin was found to be partially active while oxytocin was inactive (Veale et al., 1981). Ad~tional detailed structur~activity data on the antipyretic effect are currently not available. The peptide Asu-AVP ( = deamino-dicarba-AVP) was reported to act in an opposite manner to vasopressin in febrile sheep and a competitive antagonism by this peptide was suggested (Veale et al., 1981). However, it was not shown whether Asu-AVP actually blocked the antipyretic effect when administered along with vasopressin. In the rat, the sites of action for the antipyretic and the hypothermic effects seem to be different, as shown by different effects after intracerebroventricular injection and microinjection into the septal region (Eagan, Veale and Cooper, 1980; Kasting et al., 1980). The physiological role of vasopressin-induced hypothermia is unclear. Several vasopressin-containing fiber tracts have been described in the brain (Buijs, Velis and Swaab, 1980; Dogterom and Buijs, 1980), and vasopressin-containing synapses have been identified in structures of the brain stem and the limbic system (Buijs and Swaab, 1979; Sterba, Naumann and Hoheisel, 1980). In the septal region of the sheep, vasopressin was found in push-pull perfusates (Cooper et al., 1979), showing that significant quantities of vasopressin are released physiologically in this region of the brain. This suggests that large local concentrations of vasopressin can temporarily be reached following release from peptidergic synapses. The finding of the present study that, among various related peptides, the naturally occurring vasopressin was most active lends support to the suggestion that the hypothermic effect is not “non-specific” but may be of physiological significance. Tonic release of vasopressin, however, seems less likely than the activation of a vasopressinergic system in specific physiological situations. acknowledgement-This work was supported by USPHS grants AM30970 and HL28710. REFERENCES Abood L. G., Knapp R., Mitchell T., Booth H. and Schwab L. (1980) Chemical requirements of vasopressins for barrel rotation convulsions and reversal by oxytocin. J. .?&ri~osci.Res. 5: 191-199. Beck Th. R., Ha&d A. and Dunn M. J. (1980) The effect of arginine vasopressin and its analogs on the synthesis of prostaglandin E, by rat renal medullary interstitial cells in culture. J. Pharmac. exp. Ther. 215: E-29. Botes C. R., Smith C. W., Chan Y.-L. and Walter R. (1979) Synthesis and biological activities of arginine-vasopressin analogues designed from a confo~ation-activity approach. J. med. C&em. 22: 926931. Buijs R. M. and Swaab D. F. (1979) Immuno-electron microscopical demonstration of vasopressin and oxytocin synapses in the limbic system of the rat. Cell Tiss. Res. 204~ 355-365. Buijs R. M., Velis D. N. and Swaab D. F. (1980) Extrahypothalamic vasopressin and oxytocin innervation of fetal and adult rat brain. Prag. Brain Res. 53: 159-167.
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1199
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Kruse H., Wimersma Greidanus Tj. B. van and Wied D. de (1977) Barrel rotation induced by vasopressin and related peptides in rats. Pharmac. Biochem. Behav. 7: 31 t-313. Kruszynski M., Lammek B., Manning M., Seto J., Haldar J. and Sawyer W. H. (1980) [l-(fi-Mercapto-fl,fl-cyclopentamethylenepropionic acid), 2-(O-methyl)’ iyrosine] arginine-vasopressin and [I-(fi-Mercapto$,B-cyclopentamethylenepropionic acid)] arginine-vasopressin; two highly potent antagonists of the vasopressor response to arginine-vasopressin. J. med. Chem. 23: 364-368. Lipton J. M. and Glyn J. R. (1980) Central administration of peptides alters thermoregulation in the rabbit. Peptides 1: 15-18. Meienhofer J., Trzeciak A., Havran R. T. and Walter R. (1970) A solid phase synthesis of 8arginine vasopressin through a crystalline protected nonapeptide inte~ediate and biological properties of the hormone. J. Am. them, Sot. 92: 7199-7202.
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