Among the many changes in pregnancy that are
controlled by central mechanisms (1) are major adaptations in the control of
blood volume and osmolality in women and other species, including the rat. By
the end of pregnancy blood volume is increased by ca. 55% and plasma volume
by ca. 40% (2, 3) while plasma sodium concentration ([Na+
and osmolarity, are decreased by ca. 4% (2). Clearly, homeostats regulating
these variables are reset, and the blood volume expansion involves substantial
retention of water and electrolytes, especially NaCl. These changes ensure adequate
blood supply to the gravid uterus and other organs in pregnancy. The question
addressed here is what are the mechanisms that lead to or maintain this state
of physiological hyponatraemic hypervolaemia in pregnancy? Multiple mechanisms
regulate blood volume and [Na+] (and osmolarity); roles of aldosterone, angiotensin
II (AII), arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) are
well-established (4). In some species, a role for oxytocin in promoting natriuresis
has been shown (e.g.
dog, rat) (5-7). Post-prandial natriuresis may involve
oxytocin, ANP and satiety peptides, such as leptin (8). Here we focus on the
adaptations in the responsiveness of the vasopressin and oxytocin neurohypophysial
systems in late pregnancy, and the roles of the high levels of oestrogens and
progesterone and of the pregnancy hormone relaxin in these changes.
Vasopressin is secreted from the posterior pituitary gland by the axon terminals
of magnocellular neurosecretory neurones when action potentials arrive from
the cell bodies in the supraoptic (SON) and paraventricular nuclei (PVN) (9).
The major stimuli that excite these vasopressin neurones are an increase in
extracellular osmolality, generally a result of increased [Na+] (with Cl-),
and decreased blood volume and pressure (9, 10). The peripheral actions of vasopressin
counteract such changes through stimulating renal water reabsorption (via
receptors) and vasoconstriction (via
receptors) (11). Consequently, decreased
extracellular fluid osmolality and increased blood volume or pressure normally
inhibit vasopressin neurones and secretion (10, 12). However, the hyponatraemic
hypervolaemia of pregnancy is associated with a reduced threshold (without a
change in slope) for hyperosmotic stimulation of vasopressin secretion (13).
Accordingly, this shift in threshold indicates that the hyponatraemic hypervolaemia
of pregnancy is a simple result of increased water retention caused by vasopressin.
Also, the augmentation of vasopressin secretory responses to hyperosmolarity
by reduced blood volume persists in pregnancy (3). The role of vasopressin in
the hyponatraemic hypervolaemia of pregnancy is complemented by increased water
Oxytocin, like vasopressin, is also secreted from the posterior pituitary gland. Its peripheral roles are primarily to promote uterine contractions during parturition and to effect milk ejection during lactation (14). However, like vasopressin, oxytocin secretion is also stimulated by hyperosmolarity of extracellular fluid, at least in the rat (15-17), and it has peripheral natriuretic actions. These natriuretic actions involve direct effects in the kidney (4) and actions in the right atrium to stimulate ANP secretion (7). Oxytocin receptors are expressed in the heart, and so also is oxytocin (18). Oxytocin is secreted post-prandially (7), and may contribute to post-prandial ANP secretion (19), and hence post-prandial natriuresis.
Salt appetite is increased in pregnancy (2) and total circulating sodium is
clearly increased, although water retention reduces plasma [Na+
while increasing volume. In these circumstances a natriuretic response can be
expected, through stimulation of ANP secretion by right atrial distension (7),
but clearly any such response is either ineffective or suppressed in pregnancy.
Indeed, circulating ANP levels decrease in late pregnancy (19-21). Reduced oxytocin
content in the atria in pregnancy, and reduced oxytocin receptor mRNA expression
near term (19) may contribute to this reduced ANP secretion. Also, in late pregnancy
the kidney is refractory to the natriuretic actions of ANP (22), through increased
phosphodiesterase-5 activity (23).
The maintenance of the hypervolaemia of pregnancy might thus involve Na+ retention resulting from reduced stimulation of oxytocin secretion due to the hyponatraemia caused by vasopressin action and by increased drinking. Moreover, in contrast with the stimulation of vasopressin secretion by hypovolaemia, oxytocin neurones and secretion are stimulated by hypervolaemia; hence consequent natriuresis will contribute to normalisation of expanded blood volume (4, 24, 25). Thus, it may be that reduced oxytocin responses to the hypervolaemia of pregnancy also contribute to the expanded blood volume of pregnancy. Alternatively, stimulation of oxytocin secretion by further volume increase in late pregnancy may be preserved to prevent volume overload. Hence, it becomes important to consider whether adaptations in the control of oxytocin neurones are involved in maintaining the hyponatraemic hypervolaemia of late pregnancy.
Additional actions of oxytocin that are important in this context are that centrally-released oxytocin, potentially from the dendrites of magnocellular neurones (26) as well as from centrally-projecting parvocellular neurones (27), has salt-appetite suppressing (27, 28, 29) and anorectic actions (30). Hence, reduced central release of oxytocin in pregnancy would favour the increased food and salt intake that is seen especially in late pregnancy (2, 30). Overall, increased vasopressin secretion, and reduced oxytocin secretion (and hence reduced ANP secretion) in pregnancy could underlie the hypervolaemia and hyponatraemia in late pregnancy.
MECHANISMS OF HYPEROSMOTIC STIMULATION
OF VASOPRESSIN AND OXYTOCIN NEURONES
Both magnocellular vasopressin and oxytocin neurones are directly osmosensitive;
they respond to increases in extracellular osmolarity by depolarising and increasing
their firing-rate (10, 17). Since these neurones control body water and [Na+
they are also osmoreceptors. Depolarisation is the result of shrinkage of the
cell-bodies of the neurones, consequent on net water flux out of the cells into
the hyperosmotic environment, which activates a stretch-inactivated cationic
conductance channel (the osmoreceptor transduction channel, possibly transient
receptor potential vanilloid [TRPV]) (10). The firing-rate and secretory responses
of vasopressin neurones to graded systemic hyperosmotic challenge are correlated
with changes in osmolarity (9, 31). This provides effective negative feedback
control of extracellular osmolarity through graded antidiuretic actions of vasopressin
(17). The firing-rate and secretory responses of oxytocin neurones show a similar
relationship after an intraperitoneal hyperosmotic saline challenge (31, 32),
and during intravenous hypertonic saline infusion (a hyperosmotic/ hypervolaemic
stimulus) the responses are closely correlated with the amount of salt infused
INPUTS FROM THE LAMINA TERMINALIS AND BRAINSTEM
Although the magnocellular vasopressin and oxytocin neurones are directly osmosensitive, in vivo
they rely upon input from lamina terminalis structures to express this property as changes in firing rate. Acute lesion of the ventral lamina terminalis, in the region anterior and ventral to the third ventricle (AV3V: including the organum vasculosum of the lamina terminalis [OVLT] and median preoptic nucleus [nucleus medianus, MnPO], and projections from the subfornical organ [SFO]) induces adipsia, profoundly impairs osmoregulation, and prevents activation of electrical or secretory activity of magnocellular oxytocin and vasopressin neurones (33- 36).
The lamina terminalis input to magnocellular neurones has two roles; firstly, it provides tonic glutamatergic excitatory drive (35), and secondly, the OVLT, SFO and the MnPO contain osmosensitive neurones, which transmit information to the magnocellular neurones (10). The inhibitory transmitter GABA is also important in this pathway. During systemic hyperosmotic stimulation the release in the SON of both glutamate and GABA is increased, and GABA has an important modulatory role to limit the excitation of the magnocellular neurones, although the net effect of electrically stimulating the AV3V region is to excite oxytocin neurone firing rate and oxytocin secretion (16, 35). There is also evidence for roles of both AII and ANP as excitatory transmitters in the lamina terminalis input to the magnocellular neurones (4, 37, 38).
The OVLT and SFO are circumventricular organs, providing access for circulating peptides which act via
receptors expressed in these structures to regulate oxytocin and vasopressin neurone activity and thirst. These peptides include AII, which registers low blood volume and pressure, and the pregnancy hormone relaxin (4, 39, 40). AII and relaxin actions via
the OVLT or SFO, respectively, regulate magnocellular neurones and thirst (36, 39, 40). In humans, intravenous hypertonic saline infusion induces thirst and activates the lamina terminalis, as visualised with fMRI/BOLD imaging (41). The lamina terminalis has an important role in the regulation of natriuresis, as well as in the control of water balance through the renal actions of vasopressin and through thirst mechanisms; thus an AV3V lesion strongly attenuates the excretion of a systemically infused isotonic or hypertonic Na+ and volume load (7, 42). This has been interpreted as a result of reduced release of ANP by the brain, or perhaps more likely to result from suppressed oxytocin secretion as a result of loss of an ANP input from the lamina terminalis (7). Oxytocin neurones do express ANP, and intriguingly, ANP mRNA expression in the SON is increased near term, while expression in the preoptic area is decreased (43). Since central ANP contributes to mediation of osmotic stimulation of oxytocin neurones (38), reduced central ANP level in late pregnancy might contribute to their reduced response to a hyperosmotic stimulus.
A wide spectrum of information from the periphery is conveyed by projections
from the brainstem to the magnocellular oxytocin and vasopressin neurones in
the hypothalamus. These are predominantly from the noradrenergic neurones in
the A1 group in the ventrolateral medulla and the A2 group in the nucleus tractus
solitarii (9). The inputs include neural signals from arterial baroreceptors
and atrial volume receptors, though these project predominantly to vasopressin
neurones (44), so the route for cardiovascular inputs to oxytocin neurones (25)
is unclear. Peptide hormones from the gastro-intestinal tract signal information
about food intake and digestion (e.g.
cholecystokinin, CCK) (45, 46)
and cytokines signalling infection (47) also act via
relays to the hypothalamus
in the brainstem. Since food intake involves a Na+
load activation of magnocellular oxytocin neurones, and hence oxytocin secretion,
by CCK after a meal would contribute to Na+
OXYTOCIN NEURONE RESPONSES TO HYPEROSMOTIC STIMULATION IN PREGNANCY
Intraperitoneal injection of hypertonic saline (1.5M, 4 ml/kg) reliably increases
circulating oxytocin concentrations in urethane-anaesthetised virgin female
rats, but in late pregnant rats the increase in oxytocin secretion is reduced
), despite a similar increase in plasma [Na+
(48). The first question from this finding was whether oxytocin neurones are
excited less by activation of the lamina terminalis input. However, this was
found not to be the case as the substantial oxytocin secretory response to electrical
stimulation of the AV3V region is not different between late pregnant and virgin
rats (48). This result indicated that either the hyperosmotic stimulus used
in late pregnancy was insufficient if the threshold for stimulation was not
reduced pari passu
with the physiological hyponatraemia of pregnancy,
or that the oxytocin neurones or the lamina terminalis input, is actively inhibited
in late pregnancy.
1. Oxytocin responses to different stimuli in late pregnancy. Female
rats were given naloxone (2-5 mg/kg i.v.) to antagonise actions
of endogenous opioids before exposure to the stimuli. Changes in plasma
oxytocin concentration were measured in blood samples collected from urethane-anaesthetised
virgin and pregnant (day 21) rats after (i) electrical stimulation of
the AV3V region (0.5 mA, 1 ms pulses, 10s/10s off at 25 Hz for 2 min),
(ii) hypertonic saline (1.5 M NaCl i.p.), (iii) cholecystokinin (CCK;
20 µg/kg i.v.); or from conscious rats following (iv) interleukin-1ß
(IL-1ß; 500 ng/kg i.v.). After naloxone both AV3V stimulation and
i.p. hypertonic saline less effectively stimulate oxytocin secretion
in late pregnant rats compared to virgin rats, indicating an opioid-independent
reduction in the drive by lamina terminalis inputs in late pregnancy.
Conversely, oxytocin secretory responses to CCK and IL-1ß are greater
in late pregnant rats than in virgin rats after naloxone, indicating that
without endogenous opioid inhibition the brainstem input to the oxytocin
neurones is more effective in late pregnancy. *p<0.05 vs within
treatment (One-way ANOVA). See (48, 51, 52, 53).
In studies of hyponatraemia induced by V2
(DDAVP) treatment and liquid diet feeding, the threshold for hyperosmotic stimulation
of oxytocin secretion does not shift to the left (31). Consequently, the curve
describing the relationship between plasma [Na+
and oxytocin secretion is non-linear, rising sharply for increases just above
the normal plasma [Na+
] (31). Hence, the decrease
in plasma [Na+
] in late pregnancy can be predicted
to mean that a larger increase in plasma [Na+
is needed to produce a measurable oxytocin secretory response. Indeed, increasing
the hypertonic saline stimulus by giving late pregnant rats an i.p. injection
of 5 ml/kg 2M NaCl produces a similarly large stimulation of oxytocin secretion
to that produced by 4 ml/kg 1.5M NaCl in virgin rats at 40-50 minutes post-injection
(15). Furthermore, the oxytocin secretory response to combined hypovolaemia
and hyperosmotic stimulation in pregnancy, induced by subcutaneous polyethylene
glycol injection, is retained (49).
Local mechanisms inhibiting oxytocin neurones
Although the explanation for the reduced oxytocin responses to a hyperosmotic stimulus in late pregnancy seems simple, there are several mechanisms that inhibit oxytocin neurones which might be involved. These include nitric oxide (NO), endogenous opioids, endocannabinoids and allopregnanolone.
Oxytocin neurones strongly express neuronal nitric oxide synthase (nNOS), which generates (NO) (15). However, the expression of nNOS mRNA in the supraoptic nucleus is decreased towards the end of pregnancy, and whilst a nNOS inhibitor (L-NNA) enhances the oxytocin secretory response to an i.p. hypertonic saline stimulus in virgin rats, it is without effect near the end of pregnancy (15). Hence inhibitory (NO) mechanisms in oxytocin neurones are evidently inactive by the end of pregnancy.
In virgin rats endogenous opioids inhibit oxytocin secretion only at the level
of the posterior pituitary (50). In vitro
studies show that this inhibitory
mechanism is down-regulated as pregnancy nears term (50). Indeed, treatment
of non-pregnant and late pregnant female rats with the opioid antagonist naloxone
before challenge with an i.p.
hypertonic saline stimulus is much less
effective in enhancing the oxytocin secretory response in the pregnant rats
) (48, 51). This finding confirms the reduced response of oxytocin
neurones to the hyperosmotic stimulus. Moreover, electrical stimulation of the
AV3V region after naloxone is also less effective in stimulating oxytocin secretion
in late pregnant compared with non-pregnant rats (Fig. 1
In striking contrast, the oxytocin response to systemic CCK is greatly potentiated
after naloxone in late pregnant rats compared with non-pregnant rats (Fig.
) (52). CCK acts via
the brainstem, and this effect of naloxone
in late pregnancy is typical of other stimuli that act via
including systemic interleukin-1ß (Fig. 1
) (53, 54) and intravenous
hypertonic saline infusion, which acts as a hypernatraemic and hypervolemic
stimulus (42, 51). The brainstem input to the oxytocin neurones (specifically
the A2 noradrenergic projection mediating IL-1ß signalling) is less effective
in late pregnancy due to preterminal opioid inhibition of noradrenaline release
(47). The source of this opioid is likely to be the A2 neurones themselves,
from up-regulated pro-enkephalin-A gene expression (47). Hypersecretion of oxytocin
in response to interleukin-1ß after naloxone pre-treatment in late pregnancy
compared with non-pregnant rats is evidently due to increased efficiency of
stimulus-secretion coupling in the posterior pituitary, probably consequent
on the enlarged oxytocin store (55). Similar analysis has not been performed
for hypervolaemic stimulation of oxytocin neurones in late pregnancy, but it
would seem advantageous for effectiveness of this input to be restrained to
maintain the physiological hypervolaemia of pregnancy.
Stimulated oxytocin neurones generate endocannabinoids, which act presynaptically to retrogradely inhibit synaptic inputs (56). For example, endocannabinoids inhibit the activation of oxytocin neurones by electrical stimulation of the OVLT (57). Whether any altered production or actions of endocannabinoids in pregnancy is involved in the attenuation of oxytocin neurone responses to osmotic stimulation has not been investigated.
Allopregnanolone is a neuroactive steroid metabolite of progesterone which allosterically
receptors, including those on
oxytocin neurones. When the receptors are occupied by GABA, allopregnanolone
prolongs the opening time of the receptor channel, and consequently the neurones
are more potently inhibited (58). Whether the high levels of allopregnanolone
in pregnancy, consequent on metabolism of high progesterone levels (59), are
involved in the reduced oxytocin neurone responses to a hyperosmotic stimulus
has not been investigated. However, allopregnanolone does seem to be responsible
for upregulating expression of pro-enkephalin A in the NTS in pregnancy (54),
hence inhibiting the effectiveness of excitatory brainstem inputs to oxytocin
neurones. So, in the absence of evidence for up-regulated inhibition of oxytocin
neurone responses to hyperosmotic stimulation in pregnancy, the question of
whether responsiveness is reduced because of the hyponatraemia of pregnancy
HORMONES OF PREGNANCY AND OSMORESPONSIVENESS
OF OXYTOCIN NEURONES
Sex steroids: oestrogen and progesterone
Circulating levels of 17ß-oestradiol and, to a larger extent, progesterone,
are greatly increased in late pregnancy (1). There are several sites where oestrogen
and progesterone might act to modulate central osmoreceptor mechanisms, though
resulting actions on oxytocin and vasopressin secretion in this context are
not clear. Classical oestrogen receptor (ER)-
is expressed in neurones in the SFO, OVLT and MnPO, whereas only ER-ß
is expressed in the magnocellular vasopressin and oxytocin neurones, predominantly
in the former (60-62). The SFO, OVLT and MnPO neurones that express ER-
respond to hyperosmotic stimulation (63) and project to the magnocellular neurones
(60). Magnocellular PVN and SON neurones strongly express G protein-coupled
receptor 30 (GPR 30), regarded as the non-genomic ER, mediating rapid membrane
actions (64), such as on electrical activity of oxytocin neurones (65). Progesterone
receptor is not expressed in the magnocellular neurones, and few neurones in
the lamina terminalis express progesterone receptor (66). However, allopregnanolone,
the neuroactive steroid metabolite of progesterone which is abundant in late
pregnancy, modulates oxytocin neurones via
allosteric enhancement of
receptor function (58).
We manipulated sex steroid levels in late pregnant and virgin rats, aiming to
reverse or mimic, respectively, the reduced oxytocin response to hyperosmotic
stimulation (67). Firstly, oxytocin responses to i.p.
were measured in pregnant rats on day 21, after bilateral ovariectomy 24 h previously,
which reduced plasma 17ß-oestradiol and progesterone levels by ca. 67%,
and increased [Na+] by ca 2 nmol/l. However, ovariectomy did not alter the suppressed
oxytocin secretory response to hypertonic saline (67). Secondly, virgin rats
were exposed to pregnancy levels of 17ß-oestradiol and progesterone for
17 days, via
s.c. implanted capsules [as in (68)]. In these rats, the
oxytocin response to the i.p.
hypertonic saline stimulus was not different
from that in control virgin rats, even after i.v.
naloxone (5 mg/kg)
(67). Hence ovarian steroids alone seem not to be responsible for the reduced
oxytocin responses to the hyperosmotic stimulus in pregnancy. Similarly, chronic
17ß-oestradiol and progesterone treatment does not reduce the threshold
for hyperosmotic stimulation of vasopressin secretion (69).
Ovariectomy removes the source of relaxin as well as 17ß-oestradiol and
progesterone, so the experiment described above indicated that relaxin does
not seem important in maintaining suppressed oxytocin responses to hyperosmotic
stimulation in just the last day of pregnancy. We next evaluated a role for
more prolonged actions of relaxin.
Relaxin is an ovarian insulin-like peptide hormone, produced by the corpora lutea in pregnancy (70). In the rat, relaxin is detectable in blood around day 10 of gestation and levels increase throughout pregnancy (71). Relaxin relaxes the interpubic ligament, inhibits uterine contractions, contributes to softening of the uterine cervix and promotes nipple development (72). There is strong evidence that relaxin is also the driver for the major changes in blood volume and osmolality changes in pregnancy. In late pregnancy passive immunization with relaxin antibody reduces water intake (73, 74), and relaxin knockout mice do not have reduced plasma osmolality near the end of pregnancy, unlike wild-type mice (72).
Systemic relaxin infusion activates neurones in the SFO, OVLT, MnPO, and magnocellular
neurones in the SON and PVN, as shown by Fos immunocytochemistry (40). Direct
actions have been shown in the SFO by electrophysiological recording, and most
relaxin-responsive neurones also respond to AII (40). AII is also a powerful
dipsogen and stimulates vasopressin secretion (39), and may partly mediate the
central actions of relaxin (40, 75). Relaxin stimulates drinking via
the SFO since actions on drinking are abolished by SFO lesion, while activation
of the OVLT, and SON and PVN magnocellular neurones is unaffected (40, 76).
OVLT lesion reduces relaxin-induced Fos expression in the SON and magnocellular
PVN without affecting the drinking response to relaxin (40), indicating that
stimulation of vasopressin and oxytocin secretion by relaxin is mediated by
the OVLT. Given acutely, by i.v.
injection, relaxin excites both vasopressin
and oxytocin neurones in non-pregnant rats (77), but importantly, the stimulatory
action on oxytocin (but not vasopressin) secretion is lost in late pregnancy
(78). Hence, in late pregnancy central actions of relaxin will favour water
retention through sustained stimulation
of vasopressin secretion without stimulation of oxytocin secretion (Fig.2
2. Neuropeptide mechanisms in the hyponatraemic hypervolaemia of late
pregnancy. Hypernatraemia (high [Na+])
stimulates magnocellular oxytocin and vasopressin neurones in the paraventricular
(PVN) and supraoptic (SON) nuclei directly, and via osmoreceptive
neurones in the organum vasculosum of the lamina terminalis (OVLT), subfornical
organ (SFO) and nucleus medianus (NM; median preoptic nucleus). The projection
from the lamina terminalis involves glutamate (GLU; excitatory) and -aminobutyric
acid (GABA; inhibitory) transmission, atrial natriuretic peptide (ANP)
and angiotensin II (AII). The SFO also mediates drinking responses to
high [Na+] and to relaxin. The high level
of relaxin from the corpora lutea in late pregnancy stimulates magnocellular
vasopressin neurones via relaxin receptors (RXFP1) in the OVLT
and SFO. Outwith pregnancy and in early but not late pregnancy, relaxin
similarly stimulates oxytocin neurones. AII in the pathway from the lamina
terminalis partly mediates relaxin actions on magnocellular neurones.
In late pregnancy relaxin-stimulated vasopressin secretion, consequent
renal water reabsorption, and drinking lead to the hyponatraemic hypervolaemia
of pregnancy. Vasopressin neurone responsiveness to a hyperosmotic stimulus
is maintained, despite the increased blood volume and [Na+].
In contrast the threshold for osmotic stimulation of oxytocin secretion
is not decreased, and together with reduced ANP secretion and action,
Na+ retention is favoured. These effects
of relaxin may require interaction with actions of oestrogen on the SFO,
NM and OVLT, and progesterone actions, perhaps through its neuroactive
steroid metabolite, allopregnanolone (AP) at GABA receptors.
The action of relaxin via
the OVLT on drinking (79) and vasopressin secretion (which is increased 3.5-fold) (40, 80) is maintained over 3 weeks. Consequently, chronic central relaxin treatment, or relaxin expression in brain induced via
a viral vector, reduces plasma osmolality by ca. 3% (80). This effect of relaxin is a result of a reduced threshold for hyperosmotic stimulation of vasopressin secretion, and increased drinking (81).
Radiolabelled relaxin binds strongly in the SFO, and there is weaker binding in the OVLT, PVN and SON (82). Of the family of four G-protein coupled relaxin receptors, LGR7 (or RXFP1) (70) is the primary receptor for relaxin; this receptor activates adenylate cyclase, and interacts with nitric oxide signalling mechanisms (70). RXFP1 (LGR7) mRNA and protein are similarly strongly expressed in the SFO, the magnocellular PVN and SON, but weakly in the OVLT (83).
We have tested the hypothesis that chronic relaxin exposure is responsible for
the reduced responses of oxytocin neurones to hyperosmotic stimulus in late
pregnancy. Contrary to expectation, removing the source of endogenous relaxin
in pregnancy by bilateral ovariectomy on day 15 (with 17b-oestradiol and progesterone
replacement to maintain pregnancy) did not alter the reduced oxytocin response
to the hyperosmotic stimulus tested 5 days later. The oxytocin response was
indistinguishable from that in sham-operated pregnant rats, and significantly
less than the response in virgin rats, even after naloxone administration (Brunton
PJ, Bull PM, Russell JA, unpubl.). While this experiment did not support a continuing
role for relaxin on oxytocin neurone responsiveness in the last few days of
pregnancy, we next tested the action of relaxin infused centrally for 7 days
in virgin rats with or without 17ß-oestradiol and progesterone implants
to simulate pregnancy levels. As expected, i.c.v.
relaxin infusion for
seven days increased water intake (Fig. 3a
), and reduced plasma [Na+
). Importantly, the combination of i.c.v.
and systemic pregnancy levels of 17ß-oestradiol and progesterone significantly
reduced the oxytocin secretory response to the acute i.p.
stimulus, provided that opioid inhibition, presumably at the posterior pituitary
gland (50) was antagonised by naloxone treatment. Hence, these results indicate
that a chronic action of relaxin with 17ß-oestradiol and progesterone
in late pregnancy may cause the reduced oxytocin response to hyperosmotic stimulation.
The mechanisms and sites at which relaxin and the female sex steroids interact
to modulate oxytocin neurone responsiveness need further study.
OTHER ADAPTATIONS IN PREGNANCY:
3. Oxytocin response to acute hyperosmotic stimulus in virgin rats
after chronic central relaxin infusion. Virgin rats were fitted, under
halothane anaesthesia, with an intracerebroventricular (i.c.v.)
cannula aimed at the right lateral ventricle by a dorsal approach, secured
to the skull with dental acrylic and connected to a s.c. osmotic minipump
(Alzet 2001, infusion rate 1ml/h). The minipump infused for 7 days either
artificial cerebrospinal fluid (aCSF; n=5) or synthetic human recombinant
relaxin (RLX) at a rate of 250ng/h. Rats were also implanted with either
empty capsules (RLX; n=5) or oestradiol-17ß (E) and progesterone
(P) filled silastic capsules implanted s.c. (RLX+E/P; n=5) see (68). An
additional group were untreated controls (n=6). Post-surgery the rats
were housed singly, and daily water intake was monitored by weighing the
drinking bottle. After 7 days the rats were anaesthetised with urethane
(1.25 g/kg i.p.), and the femoral artery and vein were cannulated
for blood sampling and drug administration, respectively. Two basal blood
samples were collected 10 min apart before i.p. injection of 1.5M NaCl
(4ml/kg). Further samples were withdrawn 35 and 45 minutes later, then
naloxone was injected (5 mg/kg i.v.) to antagonise opioid inhibition
of oxytocin secretion, and a final sample was drawn 10 minutes later.
Plasma oxytocin concentration was determined by radioimmunoassay [as in
(68)], and plasma [Na+] by flame photometry.
Data are presented as group means ± sem. Data were analysed by ANOVA (water
intake), or RM ANOVA (oxytocin data), with significance level at p<0.05.
Post-hoc tests were by the Newman-Keuls method. Relaxin infusion (a) increased
mean daily water intake (across days 3-7; *p<0.004 vs non-relaxin-infused
groups) and (b) significantly reduced plasma [Na+]
(*p<0.05 vs basal levels in same group; +p<0.01 vs RLX+E/P
group; ++p<0.02 vs relaxin-infused groups); hence effectiveness
of the i.c.v. relaxin infusion was confirmed (see 79, 80). Hypertonic
saline given i.p. (c) increased plasma oxytocin concentration similarly
across the 4 groups (*p<0.01) at 35 min (and at 45 min, data not shown).
Naloxone further increased oxytocin secretion in each group (#p<0.001),
however the increase from basal was significantly attenuated in the RLX+E/P
group versus the other groups (15-fold increase in the RLX+E/P
group, vs 62-fold, 53-fold and 50-fold increases in the control,
aCSF and RLX only groups, respectively; **p<0.05). Basal plasma oxytocin
concentration was greater in the RLX+E/P group than in the other groups
experiments were conducted in accordance with the UK Animals (Scientific
Procedures) Act 1986.
APPETITE PEPTIDES AND OXYTOCIN NEURONES
CCK and neuropeptide Y (NPY) are peptides that regulate appetite but also stimulate oxytocin neurones (45, 84, 85), indicating additional roles in regulating post-prandial natriuresis. Magnocellular oxytocin neurones release oxytocin from their dendrites when stimulated, which may contribute to suppression of salt and food appetite (30), complementing the natriuretic actions of oxytocin. Changes in pregnancy in release of central oxytocin in response to CCK, NPY, or indeed hyperosmotic stimulation, have not been reported. In late pregnant rats, systemic CCK is less effective in stimulating oxytocin secretion compared with virgin rats (86).
Moreover, in late pregnancy the oxytocin response to exogenous NPY, given centrally
(NPY is produced by arcuate nucleus neurones and oxytocin neurones express NPY
receptors) (87), is lost (85). In contrast, the eating response to NPY is maintained
in late pregnancy (85). This lack of a stimulatory effect of NPY on oxytocin
neurones in pregnancy is expected to contribute to both increased food and Na+
intake and the retention of Na+
, supporting the
hypervolaemia of pregnancy. We have sought similar changes in oxytocin neurone
responses to leptin, an adipocyte cytokine-like peptide that signals fat deposition
and acts centrally to suppress appetite and regulate metabolism, predominantly
the arcuate nucleus, but also via
the nucleus tractus solitarii
(88). Furthermore, leptin can enter the brain via
the choroid plexus
and oxytocin neurones express leptin receptors (89). Leptin has natriuretic
actions on the kidney, particularly in conditions of mild sodium and volume
expansion (8), so its central actions on oxytocin neurones and any changes in
pregnancy become of interest. The paradoxical increase in plasma leptin levels
during the latter half of pregnancy, when food consumption is markedly increased
(90) indicates that pregnancy is a physiological state of leptin resistance
(91). In a preliminary study, we have investigated acute actions of intravenous
leptin administration in virgin and late pregnant rats by recording extracellularly
the firing of individual identified SON oxytocin neurones in urethane-anaesthetised
rats (Arunachalam S, Leng G, Russell JA, unpubl.). We found that rat recombinant
leptin (i.v. 100 µg/rat) excited oxytocin neurones similarly in ad libitum
fed virgin and late pregnant rats within a few minutes, indicating oxytocin
neurones do not become leptin resistant in late pregnancy. We also studied oxytocin
neurone responses to leptin after overnight fasting, predicting that the decreased
circulating leptin level (92) might increase oxytocin neurone responsiveness
to exogenous leptin in late pregnant rats. Instead, leptin did not excite oxytocin
neurones in fasted pregnant rats but still excited oxytocin neurones in fasted
virgin rats. This loss of leptin action on oxytocin neurones with fasting in
late pregnancy is unexplained, but might contribute to maintenance of hyponatraemic
hypervolaemia, as well as favouring increased food intake.
Overall, the reduced oxytocin response in late pregnancy to the hypertonic saline
challenge used in our studies seems not to be a result of up-regulation of any
of the several inhibitory mechanisms investigated. Rather, the reduced response
can be interpreted as a result of the stimulation by relaxin of vasopressin
secretion and drinking, with a consequent reduction in extracellular [Na+
that simply shifts the operational range on the osmotic stimulus-oxytocin response
curve to the left. Since oxytocin is natriuretic, this shift will support the
hypervolaemia of pregnancy. Other adaptations that restrain oxytocin neurone
responsiveness in pregnancy will contribute to maintaining this state.
PJB and JAR are supported by the BBSRC. SA is supported by a PhD Studentship
from the College of Medicine and Veterinary Medicine, University of Edinburgh
and an ORS award. Dr PM Bull provided technical assistance. Human recombinant
relaxin was kindly donated by Professor GW Tregear, Howard Florey Institute,
University of Melbourne, Australia.
Conflicts of interest statement: None declared.
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