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Endocrinology Vol. 140, No. 5 2334-2342
Copyright © 1999 by The Endocrine Society

ARTICLES

Centrally Administered Adrenomedullin Increases Plasma Oxytocin Level with Induction of c-fos Messenger Ribonucleic Acid in the Paraventricular and Supraoptic Nuclei of the Rat1

Ryota Serino, Yoichi Ueta, Yuko Hara, Masayoshi Nomura, Yukiyo Yamamoto, Izumi Shibuya, Yukio Hattori, Kazuo Kitamura, Kenji Kangawa, John A. Russell and Hiroshi Yamashita

Department of Physiology, School of Medicine, University of Occupational and Environmental Health (R.S., Y.U., M.N., Y.Y., I.S., H.Y.), Kitakyushu 807-8555; the Department of Foods and Human Nutrition, Faculty of Human Life Sciences, Notre Dame Seishin University (Y.Har., Y.Hat.), Okayama 700-8516; the First Department of Internal Medicine, Miyazaki Medical College (K.Ki.), Kihara Kiyotake, Miyazaki 889-1601; and the National Cardiovascular Center Research Institute (K.Ka.), Fujishirodai, Suita, Osaka 565-0873, Japan; and the Department of Physiology, University Medical School (J.A.R.), Edinburgh, United Kingdom EH8 9AG

Address all correspondence and requests for reprints to: Hiroshi Yamashita, M.D., Ph.D., Department of Physiology, University of Occupational and Environmental Health School of Medicine, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. E-mail: yama{at}med uoeh.-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of intracerebroventricular (icv) administration of adrenomedullin (AM) on plasma oxytocin (OXT), c-Fos protein (Fos), and c-fos messenger RNA (mRNA) in the paraventricular (PVN) and supraoptic nuclei (SON) of the rat were investigated using RIA for OXT, immunohistochemistry for Fos, and in situ hybridization histochemistry for c-Fos mRNA. Central administration of AM caused a significant increase in the plasma OXT level. Intracerebroventricular administration of AM caused a marked induction of Fos-like immunoreactivity (LI) in the PVN and in the dorsal parts of the SON. In the PVN and SON, OXT-LI cells predominantly exhibited nuclear Fos-LI in comparison with arginine vasopressin-LI cells. In situ hybridization histochemistry revealed that the induction of c-fos mRNA in the PVN and SON was increased in a dose-related manner 30 min after icv administration of AM. This induction was reduced by pretreatment with the AM receptor antagonist, human AM-(22–52)-NH2. These results suggest that central AM is responsible for activating the neurosecretory cells in the PVN and SON via selective AM receptors, and that AM stimulates the secretion of OXT by activating hypothalamic OXT-producing cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A POTENT hypotensive peptide, adrenomedullin (AM), was originally isolated from human pheochromocytoma (1). AM immunoreactivity and AM gene expression are found not only in the peripheral organs but also in the central nervous system (CNS) (2, 3, 4, 5). There are abundant and specific binding sites for AM in the rat brain,, including in the hypothalamus (6). Although iv administration of AM causes potent hypotension, central administration of AM induces hypertension, tachycardia, and activation of sympathetic outflow in anesthetized and conscious rats (7, 8). Central administration of AM suppresses water intake (9) and salt appetite (10). This evidence indicates that central AM may play an important role in body fluid homeostasis and central regulation of the cardiovascular system.

Yokoi et al. demonstrated that intracerebroventricular (icv) administration of AM had no significant effect on the basal plasma arginine vasopressin (AVP) level (11). However, icv administration of AM attenuated the plasma AVP increase induced by hyperosmolality and hypovolemia (11). The hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) are well known to synthesize AVP and oxytocin (OXT) and then release them into the general circulation through terminal axons located in the posterior pituitary. In addition, AM-like immunoreactive (LI) cells exist in the PVN and SON in the rat (12) and in humans (13).

There has been no evidence about whether central AM may have some effects on OXT release. In the present study, first we examined the effects of icv administration of AM on the plasma oxytocin level, using RIA for OXT. Second, we examined the effects of icv administration of AM on c-Fos protein (Fos) and c-Fos messenger RNA (mRNA) in the PVN and SON, using immunohistochemistry and in situ hybridization histochemistry. Finally, we examined the effects of pretreatment with AM receptor antagonists on the expression of c-fos mRNA in the PVN and SON induced by central administration of AM. The expression of the c-fos gene has recently been widely used to detect neuronal activity in the CNS (14). The expression of Fos in the dorsal brain stem, including the area postrema (AP) was also examined using immunohistochemistry, as the AP is thought to be one of the primary sites in the CNS activated by AM (15, 16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Wistar rats, weighing between 210–250 g, were used in all experiments. They were housed individually in a plastic cage in an air-conditioned room (23–25 C) under a 12-h light (0700–1900 h), 12-h dark (1900–0700 h) cycle.

Surgical procedures
For icv administration of AM, AM antagonist, or vehicle, the animals were anesthetized (sodium pentobarbital, 50 mg/kg BW, ip injection) and then placed in a stereotaxic frame in a prone position. A stainless steel guide cannula (550-µm od; 10.5-mm length) was implanted stereotaxically, following coordinates given in the atlas of Paxinos and Watson (17). These coordinates were 0.8 mm posterior to bregma, 1.4 mm lateral to midline, and 2.0 mm below the surface of the left cortex, such that the tip of the cannula was 1.5 mm above the left cerebral ventricle. Two stainless steel anchoring screws were fixed on the skull, and the cannula was secured in place by dental acrylic cement. The animals were then returned to their cages and allowed to recover for at least 5 days. The animals were handled and housed individually before the start of the experiments.

Central administration of the AM, AM antagonist, or vehicle
For icv administration of AM, a stainless steel injector (300-µm od) was introduced through the cannula to a depth of 1.5 mm beyond the end of the guide. The total volume of solution injected into the lateral ventricle was 10 µl. Rat AM and human AM-(22–52)-NH2 were purchased from the Peptide Institute (Minoh, Japan). Both were dissolved in sterile 0.9% saline solution.

Experimental procedure
In the first experiment, icv administration of AM (1, 10 µg/rat) or vehicle was performed (n = 5–8 in each group). Five, 10, and 15 min after the icv injection, the animals were decapitated. Trunk blood was collected for measurement of plasma OXT level by RIA.

In the second experiment, icv administration of AM (1, 5, and 10 µg/rat) or vehicle was performed (n = 2 or 3 in each group). Ninety minutes after icv injection, the animals were anesthetized deeply with an ip injection of sodium pentobarbital (75 mg/kg BW) and then used for immunohistochemistry for Fos, OXT, and AVP.

In the third experiment, icv administration of AM (1 and 10 µg/rat) or vehicle was also performed (n = 6 in each group). Thirty minutes after injection, the animals were decapitated, and the brains were removed and then placed on powdered dry ice for in situ hybridization histochemistry for c-fos mRNA. Other animals were decapitated 0, 30, 60, or 180 min after icv administration of AM (5 µg/rat; n = 6 in each group).

In the final experiment, the effects of pretreatment with the AM receptor antagonist, human AM-(22–52)-NH2 (100 µg/5 µl saline, icv) on the induction of c-fos gene expression by icv administration of AM (1 µg/5 µl saline) were examined.

All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.

RIA for OXT
Plasma OXT levels were determined using a RIA with a specific OXT antiserum and [125I]OXT tracer as previously described (18), using antiserum provided by Prof. T. Higuchi. The sensitivity of the assay was 3–5 pg/ml. The inter- and intraassay variations were 12% and 15%, respectively.

Dual detection of Fos and OXT/AVP immunoreactivities
Deeply anesthetized animals were perfused transcardially with 0.1 M phosphate buffer (PB; pH 7.4) containing heparin (1000 U/liter) followed by 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB. The brains were then removed and divided into blocks, which included the hypothalamus and the brain stem. The blocks were postfixed with 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB for 48 h at 4 C. The tissues were then cryoprotected in 20% sucrose in 0.1 M PB for 24 h at 4 C. Serial sections of either 30 µm for dual staining for Fos and OXT/AVP or 40 µm for immunostaining for Fos were cut using a microtome. The sections were rinsed twice with 0.1 M PBS containing 0.3% Triton X-100 and then incubated in 0.1 M PBS containing 0.3% Triton X-100 with 1% hydrogen peroxidase for 60 min. The sections were rinsed twice with 0.1 M PBS containing 0.3% Triton X-100. The floating sections were incubated with a primary Fos antibody (sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then diluted at 1:100 in 0.1 M PBS containing 0.3% Triton X-100 at 4 C for 3 days. After washing for 20 min in 0.1 M PBS solution containing 0.3% Triton X-100, the sections were further incubated for 120 min with a biotinylated secondary antibody solution (1:250) and finally with an avidin-biotin peroxidase complex (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA) for 120 min. The peroxidase in the sections was visualized with 0.02% diaminobenzidine in a Tris buffer containing 0.05% hydrogen peroxidase for 10–15 min. In the dual staining for OXT and AVP, they were sequentially incubated in OXT antibody (INCSTAR Corp., Stillwater, MN; diluted 1:2000) or AVP antibody (INCSTAR Corp.; diluted 1:6000) for 5 days each at 4 C. The avidin-biotin peroxidase complex was visualized with simainobenzidine using nickel sulfate enhancement. Both OXT- and AVP-like immunoreactivity (LI) were revealed to be a violet cytoplasmic and axonal precipitate. Fos-LI was revealed as dark brown-labeled nuclei. Details of the imunohistochemistry have been published previously (19, 20). The sections were mounted onto slides coated with gelatin, air-dried, dehydrated in 100% ethanol, cleared with xylene, and then finally coverslipped. To count the double labeled cells, four serial sections, including the PVN and SON, per animal were chosen and observed by microscope.

In situ hybridization histochemistry for c-fos mRNA
In situ hybridization histochemistry was performed on frozen 12-µm thick coronal brain sections cut by cryostat at -20 C, thawed, and mounted onto gelatin/chrome alum-coated slides that had been kept at -80 C until needed. The locations of the PVN and SON were determined according to coordinates given by Paxinos and Watson (17). Eight sites from four sections in the PVN and SON per rat were used to measure the density of the autoradiographs. The slides were warmed to room temperature and allowed to dry for 10 min, then fixed in 4% formaldehyde in PBS for 5 min. They were then washed twice in PBS and incubated in 0.9% NaCl containing 0.25% acetic anhydride (vol/vol) and 0.1 M triethanolamine at room temperature for 10 min. The sections were then dehydrated using a series of 70% (1 min), 80% (1 min), 95% (2 min), and 100% (1 min) ethanol solutions in a stepwise manner and delipidated in 100% chloroform for 5 min. The slides were then partially rehydrated in first 100% (1 min) and then 95% (1 min) ethanol and allowed to dry briefly in air. Hybridization was performed at 37 C overnight in 45 µl buffer solution consisting of 50% formamide and 4 x SSC (1 x SSC = 150 mM NaCl and 15 mM sodium citrate), which contains 500 µg/ml sheared salmon sperm DNA (Sigma Chemical Co., St. Louis, MO), 250 µg/ml baker’s yeast total RNA (Boehringer Mannheim, Mannheim, Germany), 1 x Denhardt’s solution, and 10% dextran sulfate (500,000 mol wt; Sigma Chemical Co.). The hybridization was performed under a Nescofilm (Bando Chemical IMD Ltd., Osaka, Japan) coverslip. A 35S 3'-end-labeled deoxyoligonucleotide complementary to transcripts coding for c-fos (complementary to bases 138–185 of the rat c-fos gene) and OXT (complementary to bases 912–941 of rat OXT nucleotides) was used. The specificity of the probes has been described previously (21, 22). A total of 1 x 106 cpm/slide for c-fos transcripts was used. A total of 3 x 105 cpm/slide for OXT transcripts was used. After hybridization, the sections were washed for 1 h in four separate 1 x SSC rinses at 55 C and for a further 1 h in two changes of 1 x SSC at room temperature. All independent experimental sections were treated simultaneously to minimize the variable effects of hybridization and wash stringency. Hybridized sections of the PVN and SON were apposed using autoradiography film (Hyperfilm, Amersham, Aylesbury, UK) for 7–14 days for c-fos transcripts and 8 h for OXT transcripts. The resulting images were analyzed by computerized densitometry using an MCID imaging analyzer (Imaging Research, Inc., Ontario, Canada). The mean optical density of autoradiographs was measured by comparing it with simultaneously exposed 14C microscale samples (Amersham). Slides hybridized with the c-fos probe were dipped in a nuclear emulsion (K-5, Ilford, Cheshire, UK) and further exposed for 28 days.

Statistical analysis
Data for plasma oxytocin levels are expressed as the mean ± SEM. Each group within an experiment was compared with the control group. The data were analyzed using a one-way fractional ANOVA followed by a Bonferroni-type adjustment for multiple comparison. The statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of icv administration of AM on plasma OXT level
Plasma OXT levels were measured 5, 10, and 15 min after icv administration of AM (1, 10 µg/rat) or vehicle. AM increased plasma oxytocin secretion dose dependently. The concentrations of plasma OXT were 55.4 ± 6.1 pg/ml (n = 6), 56.8 ± 11.7 pg/ml (n = 7), and 12.2 ± 2.8 pg/ml (n = 6) 5, 10, and 15 min after icv administration of AM (1 µg/rat), respectively. The concentrations of plasma OXT were 192.8 ± 56.8 pg/ml (n = 5), 240.9 ± 65.5 pg/ml (n = 8), and 24.8 ± 4.5 pg/ml (n = 6) 5, 10, and 15 min after icv administration of AM (10 µg/rat), respectively. The concentrations of plasma OXT were 36.6 ± 6.1 pg/ml (n = 7), 17.0 ± 1.0 pg/ml (n = 7), and 8.2 ± 1.2 pg/ml (n = 6) 5, 10, and 15 min after icv administration of vehicle, respectively. Figure 1Go shows the changes in plasma OXT level after central administration of AM or vehicle.



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  		Figure 1. Effects of intracerebroventricular administration of AM on the plasma oxytocin level in conscious rats. Data for plasma oxytocin are expressed as the mean ± SEM. *, P < 0.05 compared with vehicle-treated. rats. n, Number of rats.

 
Effects of icv administration of AM on Fos-LI in the PVN, SON, and AP
Immunohistochemical staining revealed that icv administration of AM (1, 10 µg/rat) caused a marked induction of Fos-LI in the PVN and the dorsal parts of the SON (Fig. 2Go). Figure 2CGo showed that parvocellular cells predominantly expressed Fos-LI, and few magnocellular cells expressed Fos-LI in the PVN. The Fos-LI in the magnocellular cells was particularly within the ventromedial zone, where there are OXT cells that are likely to be projecting to the medulla. A few Fos-LI cells were observed in the PVN and SON after icv administration of vehicle (saline; Fig. 2Go, A and D). The pattern of distribution of the Fos-LI in the PVN and SON after administration of 1 µg AM/rat icv was not different from that after administration of 10 µg/rat (Fig. 2Go, B and C, E, and F). The 5 µg/rat dose also caused a similar induction of Fos-LI in the PVN and SON. Fos-LI cells were seen throughout the ependymal cells of the cerebral ventricles (Fig. 2Go, A, B, and C). In the brain stem, Fos-LI cells were observed in the AP and the nucleus of the tractus solitarius (NTS) after icv administration of AM (10 µg/rat; Fig. 3Go, C and F). Fos-LI cells in the AP after AM administration were distributed in the peripheral regions of the rostral and ventral AP. Although Fos-LI cells were observed in the AP and NTS after the administration of 1 and 5 µg AM/rat, these were fewer than after 10 µg/rat (Fig. 3Go).



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  		Figure 2. Photomicrographs showing changes in Fos-like immunoreactivity in the paraventricular nucleus (A–C) and supraoptic nucleus (D–F). A and D are sections from control rats (icv, vehicle). B and E are sections from AM-treated rats (1 µg/rat AM, icv). C and F are sections from AM-treated rats (10 µg/rat AM, icv). Animals were decapitated at 90 min after icv administration of AM. 3V, Third ventricle; OC, optic chiasma. Bars indicate 50 µm.

 


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  		Figure 3. Photomicrographs showing changes in Fos-like immunoreactivity in the dorsal brain stem, including the AP and NTS. A and D are sections from control rats (vehicle, icv). B and E are sections from AM-treated rats (1 µg/rat AM, icv). C and F are sections from AM-treated rats (10 µg/rat AM, icv). Animals were decapitated at 90 min after icv administration of AM. The bar indicates 50 µm.

 
Dual detection of Fos-LI and OXT-/AVP-LI in the PVN and SON
Fos-LI cells in the magnocellular parts of the PVN after icv administration of AM (10 µg/rat) were exhibited by OXT-LI rather than AVP-LI cells (Fig. 4Go, B and E). The numerous Fos-LI cells in the parvocellular parts of the PVN were reactive for neither OXT nor AVP (Fig. 4Go, C and F). In the SON, OXT-LI cells predominated in the nuclear Fos-LI region compared with AVP-LI cells (Fig. 5Go).



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  		Figure 4. Coexistence of Fos-LI and oxytocin-/vasopressin-LI in the paraventricular nucleus after central administration of AM (10 µg/rat, icv). A shows coexistence of Fos-LI (brown) and oxytocin-LI (violet). B and C are enlargements from the boxed areas in A, indicated by b and c, respectively. D shows Fos-LI (brown) and vasopressin-LI (violet). E and F are enlargements from the boxed areas in D, indicated by e and f, respectively. D, E, and F do not show the coexistence of Fos-LI (brown) and vasopressin-LI (violet). Animals were decapitated at 90 min after icv administration of AM. Bars indicate 50 µm.

 


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  		Figure 5. Coexistence of Fos-LI and oxytocin-/vasopressin-LI in the supraoptic nucleus after central administration of AM (10 µg/rat, icv). A shows the coexistence of Fos-LI (brown) and oxytocin-LI (violet). B is the enlargement from the boxed area in A. C shows the coexistence of Fos-LI (brown) and vasopressin-LI (violet). D is the enlargement from the boxed area in C. D does not show the coexistence of Fos-LI (brown) and vasopressin-LI (violet). Animals were decapitated at 90 min after icv administration of AM. Bars indicate 50 µm.

 
Taking the total OXT-LI-positive cells counted in each nucleus as 100%, the percentage of Fos-LI-positive cells (n = 3) was 79.0 ± 3.0% in the PVN and 92.0 ± 0.7% in the SON after icv AM (10 µg/rat). Similarly, the percentage of AVP-LI-positive cells that were also Fos-LI positive (n = 3) was 13 ± 2.0% in the PVN and 12 ± 1.0% in the SON after icv AM (10 µg/rat).

Effects of icv administration of AM on c-fos gene induction in the hypothalamus
In situ hybridization histochemistry revealed that c-fos gene expression in the PVN and SON increased in a dose-related manner 30 min after icv administration of AM (1 and 10 µg/rat; Fig. 6AGo). Thirty minutes after administration of AM (1 µg/rat), c-fos gene expression increased significantly in the PVN (3995 ± 873.8 vs. 1012 ± 170.4 arbitrary units) and SON (2203 ± 166.1 vs. 639.7 ± 35.0 arbitrary units; n = 6 in each case; P < 0.01; Fig. 6AGo). Thirty minutes after icv administration of AM (10 µg/rat), c-fos gene expression had also increased significantly in the PVN (12740 ± 2575 vs. 1012 ± 170.4 arbitrary units) and SON (5794 ± 746.0 vs. 639.7 ± 35.0 arbitrary units; n = 6 in each case; P < 0.01; Fig. 6AGo). The icv administration of AM at 0, 30, 60, and 180 min (5 µg/rat) induced expression of the c-fos gene to the greatest degree at 30 min in the PVN (5339 ± 757.4 arbitrary units) and SON (2427 ± 68.7 arbitrary units; Figs. 6BGo and 7Go). The expression of the c-fos gene in the PVN began to decrease by 60 min and eventually reached basal levels 180 min after icv administration of AM (5 µg/rat). The expression of the c-fos gene in the SON also decreased after 30 min, but remained significantly above basal levels even 60 and 180 min after icv administration of AM (5 µg/rat). The expression of the c-fos gene in the PVN and SON at 0, 30, 60, and 180 min after icv administration of vehicle did not change significantly (Fig. 6BGo). Strong expression of the c-fos gene was seen throughout the ependymal cells of the cerebral ventricle 30 min after icv administration of vehicle (Fig. 7Go, F and N).



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  		Figure 6. Effects of central administration of vehicle and AM on c-fos transmittance values in the paraventricular (PVN) and supraoptic nuclei (SON). A, Expression of the c-fos gene in the PVN and SON was increased in a dose-related manner 30 min after central administration of AM. B, The peak of c-fos gene expression in the PVN and SON was 30 min after central administration of AM (5 µg/rat). Values represent the mean ± SEM (n = 6). *, P < 0.01 compared with vehicle-treated rats. The transmittance of autoradiographs (arbitrary units) was measured by comparing it with simultaneously exposed 14C-labeled microscale samples.

 


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  		Figure 7. Representative autoradiographs of slides hybridized to a 35S-labeled oligodeoxynucleotide probe to c-fos mRNA in the paraventricular (A–H) and supraoptic nuclei (I–P) after central administration of AM (5 µg/rat; A–D and I–L) and vehicle (E–H and M–P). A and I, and E and M are sections from 0 min after AM and vehicle administration, respectively. B and J, and F and N are sections from 30 min after AM and vehicle administration, respectively. C and K, and G and O are sections from 60 min after AM and vehicle administration, respectively. D and L, and H and P are sections from 180 min after AM and vehicle administration, respectively. The bar indicates 1 mm. The transmittance of autoradiographs was measured by comparing it with simultaneously exposed 14C-labeled microscale samples (kilobecquerels per g).

 
Effects of icv administration of AM on OXT mRNA in the hypothalamus
The expression of OXT mRNA in the PVN and SON was examined by in situ hybridization histochemistry 0, 30, 60, and 180 min after icv administration of AM (5 µg/rat). The expression of OXT mRNA did not change significantly after icv administration of AM. The mean measurements of autoradiograph density, expressed as a percentage of the control value, in the PVN were 97.9 ± 4.3%, 104.5 ± 3.7%, and 110.2 ± 2.9% 30, 60, and 180 min after icv administration of AM, respectively. The mean percentages for the SON were 102.5 ± 1.8%, 96.8 ± 4.0%, and 102.5 ± 3.2% 30, 60, and 180 min after icv administration of AM, respectively.

Effects of icv pretreatment with an AM antagonist on AM-induced c-fos gene expression
Thirty minutes after icv administration of AM (1 µg/5 µl), the expression of the c-fos gene had significantly increased in the PVN (18,680 ± 868.5 vs. 5981 ± 364.0 arbitrary units in vehicle controls) and SON (10,600 ± 460.0 vs. 3948 ± 233.6 arbitrary units in controls; n = 6 in each case; P < 0.01; Fig. 8Go). Thirty minutes after icv administration of the AM receptor antagonist, human AM-(22–52)-NH2 (100 µg/5 µl), and AM (1 µg/5 µl), the expression of the c-fos gene had increased in the PVN (10,440 ± 813.1 arbitrary units) and SON (5,808 ± 203.8 arbitrary units; n = 6 in each case; P < 0.01). However, the induction of c-fos gene expression as caused by icv administration of AM was significantly reduced by pretreatment with the AM receptor antagonist, human AM-(22–52)-NH2 (Figs. 8Go and 9Go; P < 0.01).



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  		Figure 8. The effects of pretreatment with AM antagonist on AM-induced c-fos gene expression in the PVN and SON. The induction of c-fos transmittance values in the PVN and SON after central administration of AM (1 µg/rat) was partially blocked by pretreatment with AM antagonist (100 µg/rat). Values represent the mean ± SEM (n = 6). *, P < 0.01 compared with vehicle-treated rats.

 


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  		Figure 9. Representative autoradiographs of sections hybridized with a 35S-labeled oligodeoxynucleotide probe to c-fos mRNA in the PVN (A–D) and SON (E-H) after central administration of AM (1 µg/rat) and pretreatment with AM antagonist (100 µg/rat, icv). A and E are sections taken after central administration of vehicle and pretreatment with vehicle. B and F are sections taken after central administration of AM (1 µg/rat) and pretreatment with vehicle. C and G are sections taken after central administration of AM (1 µg/rat) and pretreatment with AM antagonist (100 µg/rat). D and H are sections taken after central administration of vehicle and pretreatment with AM antagonist (100 µg/rat). The bar indicates 1 mm. The transmittance of autoradiographs (arbitrary units) was measured and compared with simultaneously exposed 14C-labeled microscale samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has provided the first evidence that centrally administered AM causes an increase in the plasma OXT level. Centrally administered AM induced c-fos gene expression in the PVN and SON, and in particular, Fos was predominantly induced in immunocytochemically identified OXT cells. The induction of c-fos gene expression caused by central administration of AM in the PVN and SON was significantly reduced by pretreatment with the AM antagonist, human AM-(22–52)-NH2. The results of the present study also demonstrated that centrally administered AM induced the expression of Fos in the AP and NTS. Allen and Ferguson reported in an in vitro study that AM excites AP neurons directly and modulates the activity of NTS neurons indirectly (15). Allen et al. also reported that AM acts on the AP directly and produces a significant cardiovascular response (16). Based on these findings, they suggested that the AP is one of the most potent sites of action for centrally administered AM. Thus, icv administration of AM was expected to induce the expression of Fos in both the AP and the NTS. This was confirmed by the present finding that Fos-LI in the AP and the NTS was induced by central administration of AM.

The finding from dual immunohistochemical staining for Fos and OXT/AVP that Fos was predominantly in OXT-LI cells in the PVN and SON indicates that central AM preferentially activates OXT-producing magnocellular neurosecretory cells in the PVN and SON. We did not detect a significant change in the expression of the OXT gene after AM injection. It may be difficult to determine whether transcription of the OXT gene is rapidly activated by icv administration of AM because of the large cytoplasmic pool of OXT mRNA.

Physiological stimuli such as hyperosmolality and hypovolemia stimulate the release of both AVP and OXT (23). However, systemic cholecystokinin is known to stimulate the release of OXT, but not AVP, in rats (24). It has been demonstrated in previous studies that peripheral administration of cholecystokinin activates OXT-secreting cells in the PVN and SON via noradrenergic neurons in the caudal NTS in the region of the brain stem receiving visceral vagal afferents (25, 26). Verbalis et al. reported that central OXT may inhibit salt appetite and feeding behavior (27). They suggested that release of OXT into the systemic circulation may correlate with the activation of central OXTergic pathways. The dual immunostaining study indicates that activation of both centrally OXT projecting neurons and magnocellular OXT neurons projecting to the posterior pituitary may be elicited by central administration of AM. Central administration of AM also inhibited salt appetite, which had been induced by peripheral administration of polyethylene glycol (10), and feeding induced by fasting (28). It is thus hypothesized that the inhibitory effects of central AM on salt appetite and feeding may involve central OXTergic pathways. It has been further shown that icv administration of AM inhibited water drinking, which had been induced by icv administration of angiotensin II, dehydration, and hyperosmotic challenge (9). However, it is unclear whether a central OXTergic pathway is associated with the inhibitory effects of AM on water-drinking behavior.

In the present study only a few AVP-LI cells in the PVN and SON exhibited nuclear Fos-LI after icv administration of AM. Recent studies have demonstrated that icv administration of AM had no significant effect on the basal level of plasma AVP in the rat (11) or sheep (29) and, further, that it inhibited the release of AVP that had been evoked by hypertonic and hypovolemic stimulation in the rat (11). Thus, central AM may not stimulate the release of AVP under basal conditions.

The present study showed that expression of the c-fos gene in the PVN and SON was increased transiently after icv administration of AM in a dose-related manner. However, it remains unclear whether centrally administered AM induces the expression of the c-fos gene and Fos in the PVN and SON directly or indirectly. The distribution of Fos-LI cells in the PVN and SON 90 min after the icv administration of AM coincided with that in c-fos mRNA-expressing cells. As we did not perform a time-course study of the expression of Fos-LI and c-fos mRNA in the PVN and SON after icv administration of AM, we could not exclude the possibility that there might be a discrepancy between the expression of Fos-LI and c-fos mRNA in the PVN and SON after central administration of AM. The rapid and transient increase in plasma OXT levels is quite dramatic. The elevation of plasma OXT reached a peak 10 min after the icv administration of AM and returned to normal 5 min later. On the other hand, the marked increase in c-fos mRNA in the PVN and SON was observed 30 min after icv administration of AM. The number of Fos-LI cells in the PVN and SON was also increased 90 min after icv administration of AM. The effects of the centrally administrated AM on plasma OXT is much faster than the induction of c-fos mRNA and Fos in the PVN and SON when plasma OXT levels returned to normal. Therefore, it should be determined whether both events might be significantly related or independent of each other. At the least, induction of Fos and c-fos mRNA in the PVN and SON should reflect the neural activation either directly or indirectly. Given that previous studies have demonstrated that AM-LI cells exist in the PVN and SON in the rat (12) as well as in humans (13), the possibility exists, then, that the neuronal activity of neurons in the PVN and SON is modulated by the release of AM in an autocrine and/or paracrine manner.

Central administration of AM causes vasopressor and sympathetic responses in anesthetized rats (7) and conscious rats (8). As the PVN is known to be an integrative site for the coordination of neuroendocrine and autonomic functions (30, 31), centrally administered AM may activate the PVN neurons that project to the regions of the brain stem and the spinal cord related to the sympathetic outflow. In the present study we demonstrated that icv AM induced the expression of Fos and the c-fos gene in the subdivision of the PVN that may project to the brain stem and spinal cord. Central administration of AM causes hemodynamic changes that may be responsible for the expression of the c-fos gene within autonomic-related nuclei in the PVN. Central administration of AM provokes an alteration in blood pressure, which consequently activates the regions involved in the central cardiovascular system. The high doses of AM (5 and 10 µg/rat) used in the present study caused strong Fos expression in the PVN; this induction of Fos and c-fos gene expression after administration of icv administration of AM (5 and 10 µg/rat) may depend on hemodynamic changes. However, the lowest dose of AM (1 µg/rat) used here caused Fos expression in the PVN and SON. As this dose of centrally administered AM (1 µg/rat) does not cause any corresponding change in blood pressure (9, 11), this induction of Fos and c-fos gene expression after icv administration of AM (1 µg/rat) may be independent of changes in blood pressure.

The presence of Fos-LI after icv administration of AM was also observed in the parvocellular parts of the PVN. These parts of the PVN comprise cells containing hypophysiotropic factors, such as CRH- and TRH-producing cells. As the possibility that AM may play an important role in the regulation of the hypothalamo-pituitary-adrenal axis has been suggested (32, 33), the central effects of AM may include actions on the hypothalamo-adenohypophysial system as well as on the hypothalamo-neurohypophysial system.

In the present study it was demonstrated that pretreatment with a selective AM receptor antagonist partially blocked AM-induced c-fos mRNA induction in the PVN and SON. Centrally administered AM-induced suppression of feeding and vasopressor and sympathetic responses are partially blocked by pretreatment with a calcitonin gene-related peptide (CGRP) antagonist (8, 28). The possibility exists that AM-induced c-fos mRNA induction in the PVN and SON may be mediated not only by AM-selective receptors but also by CGRP receptors. A recent study demonstrated that a combination of calcitonin receptor-like receptor and receptor activity-modifying proteins, which are members of a new family of single transmembrane domain proteins, play an important role in AM and CGRP receptors (34). However, the regional expression of calcitonin receptor-like receptor and receptor activity-modifying proteins and their distribution in the CNS have not been identified.

In conclusion, centrally administered AM may activate OXTergic cells in the PVN and SON in part via an AM-selective receptor.


   Acknowledgments
 
The technical assistance of Dr. P. M. Bull is gratefully acknowledged.


   Footnotes
 
1 This work was supported in part by Grant-in-Aid for Scientific Research 10470019 and 10218210 (to H.Y.); Grant-in-Aid for Encouragement of Young Scientists 09770046 (to Y.U.) from the Ministry of Education, Science, Sports, and Culture, Japan; a special grant from the Ministry of Labor for Occupational Health Studies; a grant provided by the Ichiro Kanehara Foundation; the Uehara Memorial Foundation; a research grant from the Ministry of Health and Welfare; and the Salt Science Research Foundation. Back

Received June 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T 1993 Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 192:553–560[CrossRef][Medline]
  2. Ichiki Y, Kitamura K, Kangawa K, Kawamoto M, Matsuo H, Eto T 1994 Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett 338:6–10[CrossRef][Medline]
  3. Kitamura K, Sakata J, Kangawa K, Kojima M, Matsuo H, Eto T 1993 Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem Biophys Res Commun 194:720–725[CrossRef][Medline]
  4. Sakata J, Shimokubo T, Kitamura K, Nakamura S, Kangawa K, Matsuo H, Eto T 1993 Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide. Biochem Biophys Res Commun 195:921–927[CrossRef][Medline]
  5. Sakata J, Shimokubo T, Kitamura K, Nishizono M, Ichiki Y, Kangawa K, Matsuo H, Eto T 1994 Distribution and characterization of immunoreactive rat adrenomedullin in tissue and plasma. FEBS Lett 352:105–108[CrossRef][Medline]
  6. Owji AA, Smith DM, Coppock HA, Morgan DGA, Bhogal R, Ghatei MA, Bloom SR 1995 An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat. Endocrinology 136:2127–2134[Abstract]
  7. Takahashi H, Watanabe TX, Nishimura M, Nakanishi T, Sakamoto M, Yoshimura M, Komiyama Y, Masuda M, Murakami T 1994 Centrally induced vasopressor and sympathetic responses to a novel endogenous peptide, adrenomedullin, in anesthetized rats. Am J Hypertens 7:478–482[Medline]
  8. Saita M, Shimokawa A, Kunitake T, Kato K, Hanamori T, Kitamura K, Eto T, Kannan H Central actions of adrenomedullin on cardiovascular parameters, and sympathetic outflow in conscious rats. Am J Physiol 274:R979–R984
  9. Murphy TC, Samson WK 1995 The novel vasoactive hormone, adrenomedullin, inhibits water drinking in the rat. Endocrinology 136:2459–2463[Abstract]
  10. Samson WK, Murphy TC 1997 Adrenomedullin inhibits salt appetite. Endocrinology 138:613–616[Abstract/Free Full Text]
  11. Yokoi H, Arima H, Murase T, Kondo K, Iwasaki Y, Oiso Y 1996 Intracerebroventricular injection of adrenomedullin inhibits vasopressin release in conscious rats. Neurosci Lett 216:65–67[CrossRef][Medline]
  12. Ueta Y, Kitamura K, Isse T, Shibuya I, Kabashima N, Yamamoto S, Kangawa K, Matsuo H, Eto T, Yamashita H 1995 Adrenomedullin-immunoreactive neurons in the paraventricular and supraoptic nuclei of the rat. Neurosci Lett 202:37–40[CrossRef][Medline]
  13. Satoh F, Takahashi K, Murakami O, Totsune K, Sone M, Ohneda M, Sasano H, Mouri T 1996 Immunocytochemical localization of adrenomedullin-like immunoreactivity in the human hypothalamus and the adrenal gland. Neurosci Lett 203:207–210[CrossRef][Medline]
  14. Sagar SM, Sharp FR, Curran T 1988 Expression of c-Fos protein in brain: metabolic mapping at the cellular level. Science 240:1328–1331[Abstract/Free Full Text]
  15. Allen MA, Ferguson AV 1996 In vitro recordings from area postrema neurons demonstrate responsiveness to adrenomedullin. Am J Physiol 270:R920–R925
  16. Allen MA, Smith PM, Ferguson AV 1997 Adrenomedullin microinjection into the area postrema increases blood pressure. Am J Physiol 272:R1698–R1703
  17. Paxinos G, Watson C 1982 The Rat Brain in Stereotaxic Coordinates. Academic Press, Sydney
  18. Leng G, Mansfield S, Bicknell RJ, Brown D, Chapman C, Dyer RG, Hollingsworth S, Shibuki K, Yates JO, Way SA 1988a Endogenous opioid actions and effects of environmental disturbance on parturition and oxytocin secretion in rats. J Reprod Fertil 84:345–356
  19. Ison A, Yuri K, Ueta Y, Leng G, Koizumi K, Yamashita H, Kawata M 1993 Vasopressin- and oxytocin-immunoreactive hypothalamic neurones of inbred polydipsic mice. Brain Res Bull 31:405–414[CrossRef][Medline]
  20. Onaka T, Luckman SM, Guevara-Guzman R, Ueta Y, Kendrick K, Leng G 1995 Presynaptic actions of morphine: blockade of cholecystokinin-induced noradrenaline release in the rat supraoptic nucleus. J Physiol 482.1:69–79
  21. Harbuz MS, Chalmers J, De Souza L, Lightman SL 1993 Stress-induced activation of CRF and c-fos mRNAs in the paraventricular nucleus are not affected by serotonin depletion. Brain Res 609:167–173[CrossRef][Medline]
  22. Kawata M, McCabe JT, Pfaff DW 1988 In situ hybridization histochemistry with oxytocin synthetic oligonucleotide: strategy for making the probe and its application. Brain Res Bull 20:693–697[CrossRef][Medline]
  23. Stricker EM, Verbalis JG 1986 Interaction of osmotic and volume stimuli in regulation of neurohypophyseal secretion in rats. Am J Physiol 250:R267–R275
  24. Verbalis JG, McCann MJ, McHale CM, Stricker EM 1986 Oxytocin secretion in response to chelecystokinin and food: differentiation of nausea from satiety. Science 232:1417–1419[Abstract/Free Full Text]
  25. Luckman SM 1992 Fos-like immunoreactivity in the brainstem of the rat following peripheral administration of cholecystokinin. J Neuroendocrinol 4:149–152
  26. Verbalis JG, Stricker EM, Robinson AG, Hoffman GE 1991 Cholecystokinin activates c-fos expression in hypothalamic oxytocin and corticotropin-releasing hormone neurons. J Neuroendocrinol 3:205–213
  27. Verbalis JG, Blackburn RE, Hoffman GE, Stricker EM 1995 Establishing behavioral and physiological functions of central oxytocin: insights from studies of oxytocin and ingestive behaviors. In: Ivell R, Russel J (eds) Oxytocin. Plenum Press, New York, pp 209–225
  28. Taylor GM, Meeran K, O’Shea D, Smith DM, Ghatei MA, Bloom SR 1996 Adrenomedullin inhibits feeding in the rat by a mechanism involving calcitonin gene-related peptide receptors. Endocrinology 137:3260–3264[Abstract]
  29. Parkes DG, May CN 1995 ACTH-suppressive and vasodilator actions of adrenomedullin in conscious sheep. J Neuroendocrinol 7:923–929[CrossRef][Medline]
  30. Swanson LW, Sawchenko PE 1983 Hypothalamic integration: organization of the paraventricular and supraoptic nuclei. Annu Rev Neurosci 6:269–324[CrossRef][Medline]
  31. Yamashita H, Kannan H, Ueta Y 1989 Involvement of caudal ventrolateral medulla neurons in mediating visceroreceptive information to the hypothalamic paraventricular nucleus. Prog Brain Res 81:293–302[Medline]
  32. Nussdorfer GG, Rossi GP, Mazzocchi G 1997 Role of adrenomedullin and related peptides in the regulation of the hypothalamo-pituitary-adrenal axis. Peptides 18:1079–1089[CrossRef][Medline]
  33. Samson WK 1998 Proadrenomedullin-derived peptides. Front Neuroendocrinol 19:100–127[CrossRef][Medline]
  34. McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG, Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333–339[CrossRef][Medline]




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