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Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria 3052, Australia
Address all correspondence and requests for reprints to: Dr. M. J. McKinley, Howard Florey Institute, University of Melbourne, Parkville, Victoria, 3052 Australia. E-mail: mmck{at}hfi.unimelb.edu.au
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Abstract |
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The water drinking induced by iv infusion of relaxin in the rat suggests that blood-borne relaxin may be a dipsogenic hormone. Potentiation of this relaxin-induced drinking by moderate levels of circulating angiotensin II is additional evidence in support of this view. The results also indicate that a central angiotensinergic neural pathway, utilizing AT1 receptors, subserves relaxin-induced drinking.
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Introduction |
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The physiological control of drinking involves the interaction of many factors (e.g. osmotic, hormonal, cardiovascular, and social), which are integrated within the brain to produce the behavior (11, 12, 13), and little is known as to how other dipsogenic stimuli influence drinking responses to relaxin. Increased plasma tonicity (resulting from dehydration or feeding) or angiotensin II levels (resulting from extracellular fluid losses) are well-characterized dipsogenic stimuli (11, 12, 13), which may occur in the pregnant animal, together with increased plasma relaxin concentration. Therefore, a further aim of this study was to determine whether the dipsogenic potency of blood-borne relaxin is influenced by other dipsogens, such as hypertonicity and angiotensin II. In addition, we tested the effect of a centrally administered angiotensin AT1 receptor antagonist on drinking induced by iv administered relaxin, because of evidence that central angiotensinergic pathways may be involved in relaxin-induced drinking (4).
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Materials and Methods |
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-irradiation (Steritech Pty Ltd; Dandenong,
Australia) and was inserted aseptically using instruments that had been
sterilized in 70% alcohol. Three days were allowed to elapse before
experiments involving iv infusions were commenced. For central injection of losartan (Merck Laboratories, Rahway, NJ), rats were anesthetized with equithesin (3 ml/kg), and a stainless steel (23 g) needle was implanted surgically into the lateral ventricle of the brain while the animal’s head was held in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). The ventricular cannula was held in place with dental acrylic moulded around three jeweler’s screws inserted into the skull. Rats were allowed at least 1 week recovery before a polyethylene cannula was inserted into the femoral vein, as described above. Animals were handled regularly before experiments commenced. All experiments had received prior approval from the Animal Ethics Committee of the Howard Florey Institute and adhered to the Australian National Health and Medical Research Council’s Code of Practice for the Care and Use of Animals for Scientific Purposes.
Experimental protocols
Exp 1. Water intake caused by iv infusion of relaxin. A
graduated glass pipette with an attached drinking spout was fixed to
the cage throughout the experiments, which were performed during
daylight hours (1100–1500 h). The femoral vein cannula was connected
to an infusion pump (Perfusor VIl; B. Braun, Melsungen, Germany) via
1 m polyethylene tubing (outside diameter, 0.96 mm; inside
diameter, 0.58 mm; Dural Plastics) filled with synthetic human
gene 2 relaxin (15) dissolved at various concentrations in isotonic
0.15 M NaCl. Iv infusions were then made at 1 ml/h, so that
relaxin was infused at 0 (vehicle), 25, 40, 55, or 80 µg/kg·h for
1 h, and water intake was measured at 15-min intervals for 2
h after commencement of infusion. The order of infusion was random, and
at least 3 days were allowed to elapse between successive experimental
trials. At the end of each experiment, the cannula was flushed with
heparinized saline (50 U/ml), and an obturator was inserted in the free
end. Both male (n = 6) and female rats (n = 4) were used in
these trials.
Exp 2. Water intake resulting from iv infusion of relaxin combined with hypertonic saline. Relaxin (human) was infused into the femoral vein of female rats (n = 10) at 40 µg/kg·h in combination with 1 M NaCl at 1 ml/h for 1 h, and water intake was measured at 15-min intervals during the hour of infusion and during the following hour. In another trial (n = 8, control experiment), 1 M NaCl was infused at 1 ml/h for 1 h, and water intake was measured at 15-min intervals. The results were compared with the water intake in response to iv infusion of relaxin (40 µg/kg·h, n = 6). The dose of relaxin used in this and the subsequent experiment was chosen on the basis that the response to this dose was considerably less than maximal and would allow a potentiation to be observed if it occurred. Because female rats are more responsive than males to some dipsogenic stimuli, e.g. angiotensin II (11), and relaxin probably circulates only in female rats (1), it was considered more appropriate to test females, for potentiation of relaxin responses by other stimuli, rather than males.
Exp 3. Water intake resulting from iv infusion of relaxin in combination with angiotensin II. Relaxin (40 µg/kg·h) was infused into the femoral vein of female rats together with angiotensin II at 0.5 µg/h for 1 h (n = 7). Water intake was measured at 15-min intervals for 2 h. The water drinking response to iv infusion of angiotensin II (0.5 µg/h) was measured in another trial (n = 4). The results were compared with the water intake in response to iv infused relaxin (40 µg/kg·h) obtained in Exp 2.
Exp 4. Effect of icv losartan on water intake resulting from iv relaxin. An iv infusion of relaxin, at 40 µg/kg·h for 1 h, was preceded by an injection of either artificial cerebrospinal fluid (CSF, 2 µl, n = 4) or losartan (10 µg in 2 µl artificial CSF, n = 6) into the lateral ventricle of female rats. Water drinking during the following hour was measured.
Statistical analysis
Results are expressed as mean ± SEM.
Statistical analysis of data involved a two-way ANOVA with repeated
measures on one variable (dose of relaxin, Fig. 1
; time, Figs. 2-4) and independent measures on one
variable (sex, Fig. 1; treatment,
Figs. 2–4). The subsequent multiple
comparison test used was Fisher’s least significant difference
test (Statistica, Statsoft).
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Results |
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, and the threshold required to initiate drinking was less than 25
µg/kg·h. Water intake was positively correlated with dose of
relaxin (intake in ml/100 g BW = 0.02 dose + 0.14, r = 0.86,
P < 0.001). Drinking typically began 15–30 min after
the commencement of the iv infusion and continued during the remainder
of the infusion and in the hour after its termination.
Exp 2. Effect of iv infusion of relaxin, combined with hypertonic
saline, on water intake
iv infusion of relaxin at 40 µg/kg·h, in combination with 1
M NaCl, caused about 2 ml water/100 g BW to be drunk during
the 2 h of observation, whereas iv infusion of 1 M
NaCl alone caused water intake only slightly less than that during this
time (Fig. 2). The water intake resulting from iv relaxin at 40
µg/kg·min was approximately 1.5 ml water/100 g BW. Thus, the water
drinking resulting from the combined iv infusion of relaxin and
hypertonic NaCl was not significantly different from that resulting
from either of these dipsogenic stimuli infused individually (Fig. 2).
Exp 3. Effect of iv infusion of relaxin, combined with angiotensin
II, on water intake
The iv infusion of angiotensin II, at 0.5 µg/h, by itself did
not cause increased water drinking; and iv relaxin, at 40 µg/kg·h,
alone caused a moderate drinking response. However, iv infusion of
relaxin at 40 µg/kg·min, combined with iv angiotensin II at 0.5
µg/h, resulted in a much greater dipsogenic response, with a total of
approximately 3.5 ml/100 g BW being drunk during the observation
period. Thus, a subthreshold dose of angiotensin II caused a
potentiation of relaxin-induced water intake (Fig. 3).
Exp 4. Effect of icv losartan on water intake resulting from iv
infusion of relaxin
Significantly less drinking occurred in response to iv infusion of
relaxin (40 µg/kg·h), when it was preceded by a prior icv injection
of losartan, than when icv artificial CSF preceded the iv relaxin
infusion (Fig. 4).
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Discussion |
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In contrast to the rapid drinking response that has been reported to occur with icv relaxin (latencies of 1 min, Refs. 4, 5), we observed that systemically infused relaxin had a longer latency for the induction of drinking (15–30 min). Whether this reflects the time needed for the blood levels to reach the threshold level after the commencement of the iv infusion or whether the dipsogenic response to circulating relaxin is intrinsically more delayed than that to icv relaxin was not determined. It should be noted that Thornton and Fitzsimons (3) observed a comparatively slow dipsogenic response to injections of porcine relaxin into the third ventricle of rats, and the site of the cannulae within the ventricle may be a factor in this regard. Recently, it was reported that centrally injected relaxin gave a stronger dipsogenic response if it was infused during the circadian dark period, although this factor did not affect the latency to drink (5). We did not test whether iv infusion of relaxin was a more effective dipsogen if administered at night, and the possibility remains that circulating relaxin may be a more potent dipsogen if administered during the circadian dark period.
In an earlier study, human relaxin (10 µg/h) was infused iv for 6 days to ovariectomized rats receiving estrogen replacement, and increased water intake was not observed (9). Although we have found such a rate of infusion of relaxin to be dipsogenic, the lack of increased water intake in the earlier study is not necessarily inconsistent with the present work, because water intake continued in the face of an induced hyposmolality (9). It seems likely that relaxin may have had a role in maintaining fluid intake in those animals. Our results are also in accord with conclusions obtained from studies of pregnant rats that had been ovariectomized and administered progesterone, estrogen, and relaxin replacement (7), and also from studies of the effects of relaxin-neutralizing antibody administration during pregnancy (8), that circulating relaxin may make a modest contribution to water drinking during the last few days of pregnancy in the rat.
In the present study, both male and female rats exhibited similar dose-dependent dipsogenic responses to iv relaxin, and it was shown previously that icv relaxin also stimulates both male and female rats to drink (3). These results show that relaxin-induced drinking is not dependent on the contemporaneous secretion of other ovarian hormones, such as progesterone and estrogen, unlike some other responses to relaxin that are influenced by ovarian steroids (1). We are unable to determine whether estrous behavior affects relaxin-induced drinking in the present experiments, because the stage of estrous in the female rats studied was not controlled and therefore was probably variable between animals.
Although we infused synthetic human relaxin into rats in these experiments, it has been shown previously that human and rat relaxin have similar potencies in causing a number of responses in the rat (17). We have also observed similar patterns and amounts of stimulation of c-fos expression in rat brain with iv (18, 19) or icv infusions (20) of relaxin, regardless of whether rat or human relaxin was infused, suggesting that conclusions drawn from these results with infusions of human relaxin may be physiologically relevant.
The pronounced potentiation of the dipsogenic response to circulating relaxin caused by iv infusion of angiotensin II in female rats raises the possibility that blood-borne relaxin and angiotensin II may act in concert in pregnant animals, to increase water intake in hypovolemic conditions. The rate of infusion of angiotensin II used has been reported to yield blood levels of angiotensin II of approximately 300 fmol/ml plasma (21), which may occur physiologically under hypovolemic conditions (21). Such conditions could arise as a result of extracellular volume losses from the gastrointestinal tract or by hemorrhage, impairing the circulatory function of both mother and fetus.
Plasma hypertonicity is a major physiological stimulus for water drinking (12, 13). Surprisingly, there was no potentiation or even additivity of osmotically-induced drinking by circulating relaxin. Therefore, not all dipsogenic stimuli potentiate relaxin-induced drinking as angiotensin II does.
In regard to the site(s) in the central nervous system, where blood-borne relaxin may act to stimulate drinking, specific binding sites for relaxin occur in two of the forebrain circumventricular organs, the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (22). Being a peptide molecule of 55 amino acid residues, it is unlikely that relaxin would cross rapidly from the circulation into the brain interstitium except in regions that lack the blood-brain barrier. Both the subfornical organ and OVLT are sites in the brain lacking a blood-brain barrier (23). They are also part of the neural circuitry subserving drinking behavior in response to dipsogenic stimuli, such as angiotensin II and hypertonicity (12, 23, 24, 25). In addition, ablation of the subfornical organ has been shown to attenuate the pressor response (26) and milk ejection (27) in response to icv injection of relaxin, and we have shown recently that iv infusion of relaxin (rat or human) activates neurons in the periphery of the subfornical organ and also in the dorsal part of OVLT of the rat (18, 20). Therefore it seems likely that blood-borne relaxin acts at one or both of these circumventricular organs, to stimulate neural pathways subserving thirst. Because angiotensin II acts at the subfornical organ, to stimulate drinking in the rat (24), it is feasible that its potentiation of relaxin-induced drinking could be attributable to both peptides acting on the same neurons in the subfornical organ, an effect we have observed with electrophysiological recordings from isolated rat subfornical organ in vitro (Rauch, M., H. Schmid, M. J. McKinley; unpublished observations). Such an explanation awaits further investigation, as does the actual central neural pathways subserving relaxin-induced drinking.
It has been found previously that prior central administration of a peptidic angiotensin II receptor antagonist, saralasin (which blocks both AT1 and AT2 receptors) inhibited drinking (4), as well as oxytocin and VP secretion, and increased arterial pressure in response to icv injection of porcine relaxin (28, 29). icv administration of losartan has been shown previously to block thirst and salt appetite, in response to centrally administered angiotensin II (30), by blockade of AT1 receptors. In the present study, icv treatment with losartan significantly attenuated water intake induced by iv administration of relaxin. This is consistent with the earlier finding of Summerlee and Robertson (4), and it shows that the dipsogenic effect of blood-borne relaxin, like a number of its other centrally mediated responses, is probably mediated through a neural pathway that, at least in part, involves an action of centrally derived angiotensin II acting through AT1 rather than AT2 receptors in the brain. It is unlikely that the effect of icv losartan on relaxin-induced drinking is attributable to a nonspecific depressive effect on behavior, because icv losartan does not inhibit water intake after dehydration, or salt intake after sodium depletion in rats (31). In addition, we have observed that an icv injection of 10 µg losartan in rats did not reduce drinking to another dipsogenic agent, icv injection of carbachol (P. Sinnayah and M. J. McKinley, unpublished observations).
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Acknowledgments |
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Footnotes |
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Received March 23, 1999.
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