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First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan
Address all correspondence and requests for reprints to: Dr. Hiroshi Arima, First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan. E-mail: harima-ngy{at}umin.u-tokyo.ac.jp
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Abstract |
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Introduction |
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The area postrema (AP) is one of circumventricular organs (CVOs) located adjacent to the NTS on the dorsal surface of the medulla in rats. The AP has an extremely rich capillary plexus that lacks a blood brain barrier (9). High densities of receptors for various intrinsic substances such as angiotensin II are reported to be within the AP (10, 11), and these substances activate AP neurons (12, 13). It is also shown that AP projects to various brain regions (14), and thus AP has been postulated to be a "window" through which circulating peripheral substances may influence the central nervous system. Previous studies have revealed that AP lesion (APX) attenuates stress-induced ACTH and corticosterone secretion in rats (15, 16), suggesting the possibility that AP might be involved in the neuroendocrine regulatory system. Along with other CVOs such as the organum vasculosum of the lamina terminalis and the subfornical organ (4), AP has been indicated to play some role in the water and sodium balance (17). Although APX is reported to cause polyuria in rats (18, 19), the role of AP in the hypothalamo-neurohypophyseal system is still uncertain.
In this study, we examined the effects of APX on basal plasma AVP level and on AVP release induced by either hyperosmolality or hypovolemia in unanesthetized rats to see whether AP is involved in the regulation of AVP release. We also examined AVP gene expression in the hypothalamus using in situ hybridization, to clarify the role of AP in AVP synthesis as well.
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Materials and Methods |
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AP lesion
The rats were anesthetized with ip injection of pentobarbital
(50 mg/kg) and placed in a stereotaxic instrument with incisor bar 10
mm below the center of the aural bars. A midline incision was made from
the skull to the second cervical vertebra, and the underlying muscle
was retracted laterally. The meninges were incised to expose the dorsal
medulla, and the AP was aspirated through a blunted 25-gauge needle
with a surgical microscope. Sham operation was performed in the same
fashion except that the AP was not aspirated. After completion of the
studies, the brain stem was cut in 25-µm sections coronally. The
sections were stained with cresyl violet and examined microscopically
to determine the completeness of AP lesion. All experiments were
performed in unanesthetized rats 6 weeks after the operation.
Stimuli for AVP release
To examine the effect of APX on hyperosmolality-induced AVP
release, hypertonic saline (2% BW, 600 mosmol/kg) or isotonic saline
(2% BW) was injected ip 30 min before decapitation in both APX and
Sham groups. Polyethylene glycol (PEG) is known to reduce plasma volume
without altering plasma osmolality (20). To examine the effect of APX
on hypovolemia-induced AVP release, PEG (2% BW, MW 3,000; Wako Pure
Chemical Industries Ltd., Osaka, Japan) dissolved in isotonic saline
(20% wt/vol) or isotonic saline (2% BW) was injected ip 90 min before
decapitation in both APX and Sham groups. The time points used in the
analyses were selected in consideration of previous studies (21, 22).
Plasma AVP, Na, total protein (TP), pituitary AVP content, and
blood pressure
After decapitation, trunk blood was collected into chilled tubes
containing EDTA (potassium salt) for plasma AVP, Na, and TP assay.
After immediate separation at 4 C, AVP was extracted from 1 ml volume
of plasma sample through a Sep-Pak C18 Cartridge (Waters Associates
Inc., Milford, MA) and measured using a RIA kit (AVP-RIA kit, kindly
provided by Mitsubishi Chemical Co., Ltd., Tokyo, Japan). The
sensitivity of the assay for AVP was 0.063 pg/tube with less than
0.01% cross-reactivity with oxytocin, and plasma AVP values were
log-transformed before statistical analyses (20). AVP concentrations in
plasma were corrected for recovery, which averaged 83.0–90.8%. Plasma
Na and TP were measured using an autoanalyzer (Hitachi Ltd., Tokyo,
Japan) for estimation of plasma osmolality and change in plasma volume,
respectively (23, 24). Immediately after decapitation, pituitary glands
were removed and kept in acetone at -20 C. The glands were homogenized
and stirred in 0.1 N HCl at 4 C for 24 h. After
centrifugation, the supernates were diluted in buffer and assayed.
Systolic blood pressure was measured by a tail cuff method using a
programmable sphygmomanometer (Model PS-100; Rikenkaihatsu Co., Tokyo,
Japan).
In situ hybridization
The levels of AVP messenger RNA (mRNA) in PVN, SON, and
suprachiasmatic nucleus (SCN) were examined in both basal and
dehydrated states. For dehydration, rats in both APX and Sham groups
were deprived of water for 3 days while food was available ad
libitum. After decapitation, brains were removed as soon as
possible and kept at -80 C. Coronal brain sections were cut in 12 µm
by a cryostat, thaw-mounted onto gelatin/chrome alum-coated slides, and
kept at -80 C until hybridization. They were warmed to room
temperature, allowed to dry, fixed in 4% formaldehyde in PBS for 5
min, washed twice in PBS, placed in 0.9% NaCl containing 0.25% acetic
anhydride and 0.1 M triethanolamine for 10 min, then passed
through 70, 80, 95, and 100% ethanol, 100% chloroform, and 100 and
95% ethanol before air drying. The AVP probe, directed against bases
encoding the last sixteen amino acids of the glycopeptide sequence
(25), was labeled using 35S-deoxy ATP and terminal
deoxynucleotidyl transferase, and applied to each section (2 x
105 cpm). Hybridization was performed overnight at 37 C.
The sections were then washed in 1 x SSC (0.15 M NaCl
and 0.015 M sodium citrate) for 4 x 15 min at 55 C
and 2 x 30 min at room temperature. After two water dips to
remove salts, the sections were exposed to hyperfilm MP (Amersham
International plc, Buckinghamshire, UK) together with the
[14 C] microscale (Amersham) (26). The relationship
between the signal intensities and exposure time was examined by
exposing sections to the x-ray films for various periods, and then the
exposure times yielding appropriate signal intensities within the
linear range of the detection system were determined (24 h for PVN and
SON, and 5 days for SCN). The autoradiographic images were quantified
using a Macintosh IIci computer equipped with an image capture board
(Data Translation, Inc., Marlboro, MA) running the program Image (Image
1.41; Wayne Rasband, NIMH, Bethesda, MD), and the data representing the
hybridization signals were expressed as percentages from control level.
No attempt was made to differentiate between signals present in
parvocellular and magnocellular divisions of PVN.
Statistics
Results were expressed as mean ± SE.
Comparison between groups was performed by Student’s t
test, except where multiple comparisons were made, when Fisher PLSD was
employed. Differences were considered statistically significant at
P < 0.05. The group size was six in all
experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), when the BW in the
APX group was still less than in the Sham group (APX, 357 ±
13 g; Sham, 416 ± 8 g; P < 0.01). The
urine output in the APX group became higher than in the Sham group by 2
weeks after the operation, and the polyuria continued (Table 1). Six
weeks after the operation, the plasma AVP level in the APX group was
significantly lower than in the Sham group (APX, 1.03 ± 0.06
pg/ml; Sham, 1.65 ± 0.04 pg/ml; P < 0.01; Fig. 2 and Table 2). There was no significant difference
between the groups in the levels of plasma Na, TP, AVP content in
pituitary (Tables 2 and 3) or blood
pressure (APX, 117.5 ± 1.9 mmHg; Sham, 117.8 ± 2.3
mmHg).
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), or AVP (APX, 1.12 ± 0.10 pg/ml; Sham,
1.61 ± 0.09 pg/ml; P < 0.01; Fig. 2) was not
significantly different from each basal level in both APX and Sham
groups. Following ip injection of hypertonic saline, the plasma Na
increased similarly in both APX and Sham groups but the plasma TP was
not affected (Table 3). The plasma AVP level in the APX group was
significantly lower than in the Sham group (APX, 4.88 ± 0.42
pg/ml; Sham, 9.09 ± 0.68 pg/ml; P < 0.01; Fig. 2), whereas the plasma AVP levels were significantly increased from
control levels in both groups (APX, 335%; Sham, 465%; increase of
each control level; Fig. 2).
Effect of APX on AVP release induced by hypovolemia
Ninety minutes after ip injection of isotonic saline, the level of
plasma Na, TP (Table 3), or AVP (APX, 1.05 ± 0.11 pg/ml; Sham,
1.60 ± 0.10 pg/ml; P < 0.01) was not
significantly different from each basal level in both APX and Sham
groups. Following ip injection of PEG, the plasma TP increased
similarly in both APX and Sham groups, but the plasma Na was not
affected (Table 3), and the estimated decrease in plasma volume was
about 11%. The plasma AVP level in the APX group was significantly
lower than in the Sham group (APX, 3.54 ± 0.55 pg/ml; Sham,
9.37 ± 0.99 pg/ml; P < 0.01; Fig. 2), whereas
the plasma AVP levels were significantly increased from control levels
in both groups (APX, 237%; Sham, 486%; increase of each control
level; Fig. 2).
Effect of APX on AVP gene expression in PVN, SON, and SCN
The levels of AVP mRNA in the APX group were significantly lower
than in the Sham group in the basal state in both PVN (100 ± 11
vs. 76 ± 6%; P < 0.05) and SON
(100 ± 8 vs. 82 ± 3%; P <
0.05) (Figs. 3 and 4). After water deprivation for 3 days,
plasma Na and TP were increased in both groups, and there was no
significant difference in the levels between the groups (Table 3). The
plasma AVP level in the APX group after dehydration was significantly
lower than in the Sham group, indicating impaired AVP release in the
APX group during dehydration (APX, 6.25 ± 1.77 pg/ml; Sham,
12.88 ± 2.43 pg/ml; P < 0.05; Table 2). As shown
in Fig. 4, the AVP mRNA levels in PVN and SON after dehydration were
significantly lower in the APX group than in the Sham group, whereas
the AVP mRNA levels were significantly increased from the basal levels
in both groups (APX, 45% and 30%; Sham, 46% and 38%; increase of
each basal level in PVN and SON, respectively). There was no
significant difference in the levels of pituitary AVP content between
the groups after dehydration (Table 2). The level of AVP mRNA in SCN
was not affected by dehydration in both groups, and there was no
significant difference in the levels between APX and Sham groups in
both basal and dehydrated states (Figs. 3 and 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We showed that the level of basal plasma AVP was decreased by APX and that the decrease of plasma AVP level was not associated with the changes of variables such as plasma Na, TP, and blood pressure. These data indicate that AVP release is tonically stimulated in the basal state by the neuronal input from the AP to the hypothalamo-neurohypophyseal system. This tonic stimulation is supported by a previous study which showed that APX reduced the firing rate of AVP cells in the SON in the basal state of rats (27). While we first elucidated the tonic stimulation of AVP release by AP, this result is not in accordance with previous studies which showed no significant effects of APX on the level of basal plasma AVP (28, 29). Considering the transient hypophagia by APX, our study was performed 6 weeks after the operation. As the previous studies examined the effects 5–12 days after the APX, this discrepancy might be attributed to their relatively short recovery period. Alternatively, it might be due to a difference in the sensitivity of the assay as the detection limit of the AVP RIA used in this study is very low.
Although the role of AP in the osmoregulation of AVP release has been controversial (27, 29), we clearly showed that AVP release induced by hyperosmolality was attenuated by APX. It is reported that AP neurons are responsive to sodium ion (30), and that ip injection of hypertonic saline induced immediate early gene c-fos expression in AP (31). Thus, it is likely that AP is sensitive to plasma osmolality and that AVP release induced by hyperosmolality is regulated, at least partially, by AP. It is well known that the AV3V are critically involved in the osmoregulation of AVP release, and AVP release induced by hyperosmolality is reported to be impaired in AV3V lesioned rats (4, 5, 6). The reciprocal connections between CVOs have been shown, and AP is supposed to be embedded within the network (14). Collectively, it might be appropriate to consider that AP coordinates with AV3V in the osmoregulation.
The ip injection of hypertonic saline is supposed to be not only an osmotic stimulus but also a painful stress (31). In contrast to the critical role of plasma osmolality in the regulation of AVP release, the effect of stress such as pain on plasma AVP level is ambiguous (2). Although stressors are generally not considered to stimulate AVP release (32, 33, 34), there is a study suggesting that stimuli of ip structures might induce AVP release (35). We showed that ip injection of isotonic saline had no significant effect on plasma AVP level. Nevertheless, one cannot exclude the possibility that the pain accompanying hypertonic saline injection had an effect on the plasma AVP level.
It is widely accepted that AVP release induced by hypovolemia occurs through reduction in tonic inhibitory input from the baroreceptor to the NTS (1, 2, 3), even though some studies disagree as to the tonic inhibition (7, 8). Schreihofer et al. (8) showed that NTS lesion did not prevent AVP release induced by hypovolemia, suggesting that baroreceptor input to the NTS might not be necessary for hypovolemia-induced AVP release. It should be noted that AP and the commissural portion of NTS, which receives dense projections from AP (36), were spared in their lesions. It is also reported that humoral factors such as angiotensin II, which could act at CVOs, might be involved in the baroregulation, and that glucose utilization was increased in AP after hypovolemia (37). Our study does not address the issue whether the tonic inhibitory input from the baroreceptor to the NTS is involved in the baroregulation. Instead, we first showed that the inputs for hypovolemia-induced AVP release are mediated, at least partially, via the AP.
In the present study, we showed the levels of AVP mRNA in PVN and SON in the basal state were decreased by APX, indicating that the AVP synthesis is tonically stimulated by the neuronal mechanism involving AP. We also showed that the levels of AVP mRNA in PVN and SON in the APX group after dehydration were significantly lower than in the Sham group. This could not be attributed to a difference in the degree of dehydration because the levels of plasma Na and TP in the APX group were not significantly different from those in the Sham group after dehydration. Thus, it is indicated that AP regulates, at least partially, the AVP synthesis in PVN and SON reflecting plasma osmolality and/or plasma volume. The similar levels of pituitary AVP content between APX and Sham groups in both basal and dehydrated states might be attributed to the fact that both synthesis and release of AVP were decreased by APX. We also showed that the level of AVP mRNA in SCN, which is not directly involved in water balance (38), was not affected by APX, indicating that AP stimulates AVP synthesis exclusively in PVN and SON.
Whereas the plasma AVP level in the hypertonic or hypovolemic state and the AVP mRNA levels in PVN and SON in the dehydrated state were lower in the APX group than in the Sham group, both AVP release and synthesis in the APX group were increased by stimuli and the responses were still robust, which was shown by the percentage changes relative to the control values. This suggests that some other than AP-related mechanisms are involved in the regulation of AVP release and synthesis and that AP might play a critical role, especially in the regulation of the set point for AVP release and synthesis.
AP is exposed to the substances borne in the peripheral circulation as it lacks a blood brain barrier (9). In addition to humoral inputs, AP receives neuronal inputs from not only the vagus (39) but also the central nervous system, such as NTS, parabrachial nucleus, and PVN (14). On the other hand, heavy inputs are directed from the AP to the catecholaminergic neurons in the caudal NTS (36), which in turn give rise to substantial inputs to the hypothalamus (40). The A1 noradrenergic neurons in the ventral medulla receive the projection from the NTS (41) as well as from the AP (14) and are well known to project to AVP cells in the hypothalamus (40, 42). Moreover, the existence of a direct projection from AP to SON is also suggested (29). Thus, it is likely that AP receives neuronal as well as humoral inputs, transmits the influence to the hypothalamo-neurohypophyseal system directly or via other brain stem regions such as the NTS and the A1 neurons, and stimulates both synthesis and release of AVP.
In conclusion, AVP synthesis and release are stimulated by the input from the AP to the hypothalamo-neurohypo-physeal system.
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Footnotes |
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Received July 21, 1997.
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-receptor agonist U50488H on the secretion
of arginine vasopressin. Neuroendocrinology 48:658–662[Medline]
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