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Endocrinology, doi:10.1210/en.2003-1446
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Endocrinology Vol. 145, No. 4 2006-2013
Copyright © 2004 by The Endocrine Society

Prolactin Prevents Acute Stress-Induced Hypocalcemia and Ulcerogenesis by Acting in the Brain of Rat

Takahiko Fujikawa, Hideaki Soya, Kellie L. K. Tamashiro, Randall R. Sakai, Bruce S. McEwen, Naoya Nakai, Masato Ogata, Ikukatsu Suzuki and Kunio Nakashima

Department of Biochemistry, Faculty of Medicine, Mie University (T.F., N.N., M.O.), Tsu, Mie 514-8507, Japan; Department of Exercise Biochemistry, Institute of Health and Sports Science, Tsukuba University (H.S.), Tsukuba, Ibaraki 305-8574, Japan; Department of Psychiatry, University of Cincinnati Medical Center (T.F., K.L.K.T., R.R.S.), Cincinnati, Ohio 45267-0559; Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University (B.S.M.), New York, New York 10021-6399; Department of Clinical Nutrition, Faculty of Health and Hygiene, Suzuka University of Medical Science (I.S.), Suzuka, Mie 510-0293, Japan; and Faculty of Human Health Science, Tokai Gakuen University (K.N.), Tenpaku, Nagoya, Aichi 468-8514, Japan

Address all correspondence and requests for reprints to: Dr. Takahiko Fujikawa, Department of Biochemistry, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. E-mail: t-fuji{at}doc.medic.mie-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stress causes hypocalcemia and ulcerogenesis in rats. In rats under stressful conditions, a rapid and transient increase in circulating prolactin (PRL) is observed, and this enhanced PRL induces PRL receptors (PRLR) in the choroid plexus of rat brain. In this study we used restraint stress in water to elucidate the mechanism by which PRLR in the rat brain mediate the protective effect of PRL against stress-induced hypocalcemia and ulcerogenesis. We show that rat PRL acts through the long form of PRLR in the hypothalamus. This is followed by an increase in the long form of PRLR mRNA expression in the choroid plexus of the brain, which provides protection against restraint stress in water-induced hypocalcemia and gastric erosions. We also show that PRL induces the expression of PRLR protein and corticotropin-releasing factor mRNA in the paraventricular nucleus. These results suggest that the PRL levels increase in response to stress, and it moves from the circulation to the cerebrospinal fluid to act on the central nervous system and thereby plays an important role in helping to protect against acute stress-induced hypocalcemia and gastric erosions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCIUM (Ca2+) PLAYS a major role in fundamental biological processes, such as intracellular signal transduction, bone mineralization, and muscle contraction. Extracellular circulating levels of Ca2+ are about 10,000 times higher than intracellular levels (1, 2, 3, 4). PTH (5, 6), calcitonin (7), 1,25-dihydroxyvitamin D, and a blood calcium-lowering peptide (8) from the acid-producing part of the stomach control blood Ca2+ levels in the rat. In rats, stress causes a decrease in circulating Ca2+ levels (1, 4), and the homeostatic imbalance of Ca2+ produces the chance for psychological and physical disorders, such as anxiety, depression, and gastric ulcers (1, 9, 10). The hypothalamus, which is a pivotal target for both calciotropic hormones and stress hormones, regulates circulating Ca2+ homeostasis via the vagus nerve innervating the stomach and the parathyroid glands (6). In particular, the hypothalamic paraventricular (PVN) and ventromedial nuclei are thought to regulate the development of stress-induced hypocalcemia and ulcerogenesis (1).

The hypothalamus is also a target site of prolactin (PRL), and it contains abundant PRL receptor (PRLR) mRNA (11) and protein (12, 13). PRL is closely associated with the stress response, and a striking increase in serum PRL has been observed during defensive, but not offensive, fighting in rat colonies (14). We have previously reported that restraint stress in water (RSW) causes up-regulation of the long form of PRLR [PRLR(L)] in the rat brain. Specifically, the enhanced expression of PRLR(L) in the choroid plexus (CP) occurs 1.5 h after a rapid and transient increase in circulating PRL at 0.5 h of RSW (15); this suggests that circulating PRL is one of the stress hormones, and that PRLR(L) is related to the control of brain function during stress. Although the lowering of PRL levels occurs immediately after the elevation at 0.5 h of RSW, it is not clear whether it is a reason why this leads to hypocalcemia and ulcerogenesis.

In situ hybridization histochemical (ISHH) (12) and immunohistochemical (IHC) (11) analyses have revealed that PRLR is expressed widely in the brain, i.e. in the CP, PVN, medial preoptic nucleus, ventromedial nucleus, arcuate nucleus, hippocampus, and cortex. Although PRL’s exact function in the stress response is not totally clear, it appears to induce an internal gastro-cytoprotective action, because intracisternally administered PRL (16) suppresses the generation of gastric ulcers. Results from recent reports suggest that PRL is a novel neuromodulator of emotionality and hypothalamo-pituitary-adrenal (HPA) axis reactivity in the rat (17, 18). However, little is known about how or where PRL acts in the central nervous system (CNS) to cause these effects. Thus, we hypothesized that an increase in circulating PRL acts on PRLRs in the hypothalamus to attenuate RSW-induced hypocalcemia and ulcerogenesis.

We tested this hypothesis using an animal model of acute stress, RSW. We used ISHH and IHC to examine the effects of pretreating rats with ip or intracerebroventricular (icv) injection of rat PRL (rPRL) before RSW. We further tested our hypothesis by inhibiting PRL or its receptor, PRLR.

First, to examine whether rPRL prevents RSW-induced hypocalcemia and gastric erosions via activation of PRLR in the CNS, we used rats treated ip or icv with rPRL before exposing them to RSW for 7 h. In a second set of experiments, animals were PRL- or PRLR-neutralized by the addition of antiserum. These results, ISHH of PRLR(L) mRNA in the CP and Western blots of PRLR(L) in the hypothalamus, showed that the preventive effect of rPRL on RSW-induced hypocalcemia and ulcerogenesis was largely regulated through activation of PRLR(L) in the brain. The prevention afforded by administering rPRL was almost perfectly blocked by anti-PRL serum and anti-PRLR serum.

Finally, we carried out icv injections of rPRL to examine whether rPRL induces PRLR protein and corticotropin-releasing factor (CRF) mRNA expression in the PVN. We found that PRL in the brain increases PRLR protein and CRF mRNA expression in the PVN. These data demonstrate that circulating PRL acts on PRLR in the PVN to prevent RSW-induced hypocalcemia and ulcerogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male adult (8 wk old) rats (Sprague Dawley) were purchased from SLC, Inc. (Shizuoka, Japan) and group-housed (three or four per cage), with food (CE-2 rat chow, CLEA, Tokyo, Japan) and drinking water available ad libitum. All animals were handled daily for 2 wk before the start of the experiment. The animals were housed in a temperature- and humidity-controlled room maintained at 23–25 C and 50–60% humidity with a 12-h light, 12-h dark cycle, with lights on at 0710 h.

RSW
Adult male rats were placed into individual wire-mesh restraint cages and immersed tail first in water up to chest level. The water temperature was maintained at 23 ± 0.5 C. After 7 h of RSW, the animals were removed from the cages and killed. As dietary restriction was not carried out before the start of RSW at midnight, there was food in the stomach of all animals. For ISHH and IHC, animals were decapitated, and their brains were quickly removed, immediately frozen on powdered dry ice, and stored at -80 C until sectioning.

The RSW experiments were initiated at midnight (0000 h) when rats were highly active, as previously described (15, 19, 20). The animal facilities and protocols were approved by the institutional animal care and use committees at Mie University Faculty of Medicine and University of Cincinnati. All procedures were performed in accordance with the NIH’s guidelines regarding the principles of animal care (1996).

RSW-induced hypocalcemia and gastric erosions
Ca2+ concentrations and pH in whole blood were measured using ion-selective electrodes (643 Ca2+ /pH analyzer, Ciba Corning, Medfield, MA) with balanced heparin (20 IU/ml total blood; Ciba Corning) (2, 4). Measurements were made in duplicate, and the circulating level of Ca2+ was adjusted to pH 7.4 to evaluate pH-independent changes in the circulating Ca2+, using an equation previously described (21). At death, stomachs were removed and inflated by injection of 1% formalin (~10 ml), then immersed in 1% formalin solution for 30 min. The stomachs were then opened along the greater curvature and examined for gastric lesions. The gastric erosions obtained from this stress model were in the shape of a point or a line, and no wide areas appeared. A point was set at 1 mm, regardless of the size of the bleeding point, and the gastric lesion index was calculated as the cumulative length (millimeters) of gastric lesions (19). For example, when there were 35 hemorrhage points and a hemorrhage line 15 mm long in the gastric corpus, the gastric lesion index (millimeters) was set at 50.

Peripheral pretreatment with rPRL
Rats were housed and handled for 2 wk as described above, then pretreated with either rPRL (1, 5, 25, or 50 µg/rat in 200 µl saline, ip) or saline (200 µl/rat, ip) 30 min before the start of RSW. Animals were then subjected to 7-h RSW.

Central pretreatment with rPRL
In the icv administration group, rats were fitted with a 22-gauge stainless steel guide cannula in the right lateral cerebroventricle under sodium pentobarbital anesthesia (40 mg/kg body weight, ip), according to a procedure described previously (22). The animals were allowed to recover for at least 7 d before RSW experiments. Rats were pretreated with rPRL (50 or 500 ng/rat in 3 µl saline, icv) administered through the guide cannula 30 min before the start of the RSW experiments. Control rats underwent the same treatment with heat-denatured rPRL (DrPRL: 500 ng/rat in 3 µl saline, icv). Animals were then subjected to 7-h RSW. Whole blood Ca2+ levels and the index of gastric erosions were then determined. The rPRL used in this experiment was provided by Dr. A. F. Parlow (NIDDK).

Treatment with antiserum for PRL or PRLR before icv injection of rPRL
Rats were fitted with a 22-gauge stainless steel guide cannula in the right lateral cerebroventricle and were allowed to recover for at least 7 d. Antisera for PRL (6 µl, icv) and PRLR (6 µl, icv) were icv injected into rats 5 min before icv injection of rPRL. Thirty minutes after icv administration of rPRL, the rats were exposed to 7 h of RSW. Control animals underwent the same treatment with DrPRL (500 ng/rat in 1 or 3 µl saline, icv) or normal rabbit serum (NRS: 6 µl, icv, or 100 µl, ip). After RSW, whole blood Ca2+ levels and the index of gastric mucosal erosions were determined (2, 13).

ISHH for PRLR(L) mRNA and CRF mRNA
ISHH was carried out on 16-µm-thick frozen sections of brain. Briefly, sections were thaw-mounted on glass slides and fixed in 4% paraformaldehyde. 35S-labeled antisense and sense (control) cRNA probes for PRLR(L) (15) and CRF (23) (1.2 kb CRF cDNA, provided by T. Imaki) were used under conditions described in detail previously (15, 23). The general hybridization buffer contained 50% deionized formamide, with final washes in 0.1x standard saline citrate containing 1 mM dithiothreitol at 60 C for 30 min. For emulsion autoradiography, slides were dipped in Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY). After counterstaining with 0.3% thionine, densitometric units per square micrometer area were determined using image analysis software (NIH Image, 1.61).

IHC for PRLR immunoreactivity
IHC was performed on 4% paraformaldehyde-fixed, 16-µm-thick frozen sections of the brain from adult male rats of the Sprague Dawley strain, and mouse antirat monoclonal antibody U5 (MA1-610, Affinity Bioreagents, Inc., Golden, CO) was prepared using a procedure that has been fully characterized (24). Sections were sequentially incubated in the following solutions: primary antibody U5 diluted in PBS containing 2% normal horse serum, 7.5 mg/ml biotinylated horse antimouse IgG diluted in PBS containing 2% normal horse serum and 2% normal rat serum, PBS containing 0.5% hydrogen peroxide to quench endogenous peroxidase activity avidin-biotinylated horseradish peroxidase complex (ABC, Vector Laboratories, Inc., Burlingame, CA), and 3,3'-diaminobenzidine (Sigma Fast DAB, Sigma-Aldrich Corp., St. Louis, MO) in the presence of hydrogen peroxide to produce light-brown immunostaining. Densitometric units square micrometers of area and positive cell numbers were measured using image analysis software (NIH Image, 1.61).

Membrane preparation after dissection of hypothalamus
Each microsomal membrane was prepared from the pool of hypothalamus dissected from the brain of two or three rats as described previously (25). Briefly, tissues were homogenized on ice, in volumes (wt/vol) of homogenization buffer (300 mM sucrose and 50 mM HEPES with protease inhibitors, pH 8.0) using a homogenizer. The homogenates were centrifuged at 20,000 x g for 30 min. The resulting supernatant was centrifuged at 10,000 x g for 1 h to pellet microsomal membrane fractions. The pellets were then washed in a buffer containing 50 mM HEPES, 10 mM EDTA, and protease inhibitors (pH 7.5) and centrifuged again. Membrane proteins were solubilized by vigorous agitation of the pellet in solubilization buffer (10 mM EDTA, 150 mM NaCl, and 2% Triton X-100, pH 7.5). An aliquot of each membrane preparation was assayed for total protein using a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Samples were stored at -80 C for later use in Western blotting.

Western blots for PRLR(L)
Solubilized membrane proteins from the rat hypothalamus (50 mg protein/lane for each hypothalamus dissection sample) were separated on a 10% SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Prestained standards (Bio-Rad Laboratories, Hercules, CA) were used as molecular weight markers. The membranes were incubated with 1 µg/ml mouse antirat monoclonal antibody U5 (MA1-610, Affinity Bioreagents, Inc.) diluted in PBS containing 2% normal horse serum for 2 h, followed by 30-min incubation with horse antimouse IgG horseradish peroxidase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted (1:50,000) in PBS containing 2% normal horse serum. The PRLR bands were detected using Amersham ECL Plus chemiluminescence reagents and ECL Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ) exposure. Densitometric units per square micrometer of area were measured using image analysis software (NIH Image, 1.61).

Antisera
In our studies rat anti-rPRL serum was used to neutralize endogenous or exogenous rPRL in the rat brain, whereas rat anti-rPRLR serum was used as a receptor antagonist to rPRL. Rat anti-PRL serum was provided by Shikibo Co. Ltd. (Shiga, Japan). The neutralization effect of antisera to exogenous rPRL was evaluated using Nb2 cells after modifying the method described by Krishnan et al. (26). PRL-dependent Nb2 cells were maintained at 37 C in Fischer’s medium including 10% fetal bovine serum as a source of lactogens, 10% horse serum, 2-mercaptoethanol (100 µM), penicillin (50 U/ml), and streptomycin (50 µg/ml). Nb2 cells were rendered quiescent by incubation for 20 h in lactogen-free medium [Fischer’s medium, 2-mercaptoethanol, antibiotics, and 10% nonmitogenic gelding serum (ICN Biochemicals, Irvine, CA)], then cultured in 96-well plates in the presence of various dilutions of rPRL and 1 µl antiserum. After 48 h, cells were harvested onto glass-fiber filters using a PHD cell harvester (Cambridge Technology, Watertown, MA). The radioactivity of trichloroacetic acid-insoluble material was determined after a 4-h pulse of [3H]thymidine (48 h; Amersham Pharmacia Biotech, Arlington Heights, IL; specific activity, 90 Ci/mmol; 0.5 µCi/well). The relative neutralization concentrations of antisera were determined from a standard curve generated using rPRL (NIDDK). One microliter of the undiluted antiserum was able to bind to approximately 109.2 ng rPRL using the Nb2 cell. Rat anti-rPRLR serum (NO.3136-2) was produced by the peptide, which was complementary to 16 amino acid residues (83–97 amino acids; ATNQMGSSSSDPL-YVC; molecular weight, 1658.8, hydrophobicity, 0.26; charge, 0) of the extracellular domain of rPRLR. By using the Nb2 cell, 1 µl undiluted antiserum was made to approximately 83.3 ng rPRL.

Statistical analysis
Macintosh SuperANOVA was used in the data analysis. Data are shown as the mean ± SEM. The significance of differences between the values was determined by Scheffé’s F/Fisher’s protected least significant difference (PLSD) post hoc procedure test after evaluating differences among treatment groups by one-way ANOVA; P < 0.05 was taken as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increase in circulating PRL levels by RSW
When rats were exposed to RSW, circulating PRL levels were rapidly and transiently increased (Table 1Go). The maximal circulating PRL level was obtained after 30 min of RSW, and the elevated level decreased to the initial level after 7 h of RSW.


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  		TABLE 1. Changes in circulating PRL in rats exposed to RSW

 
Effect of rPRL on RSW-induced hypocalcemia and gastric erosions
To determine whether PRLR in the rat brain is involved in the mediation of RSW-induced hypocalcemia and ulcerogenesis, we exposed rats to 7 h of RSW with or without prior ip or icv administration of rPRL. As previously observed, RSW elicited hypocalcemia and gastric ulceration, and both were prevented by ip or icv administration of rPRL (Fig. 1Go). The preventive effect of peripherally or centrally administered rPRL against hypocalcemia and gastric erosions was dose dependent (Fig. 1Go).



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  		FIG. 1. Protective effect of rPRL on hypocalcemia (A and C) and gastric erosions (B and D) during RSW. A and B, Rats were subjected to 7 h of RSW after ip administration of saline (Sal; 200 µl) or rPRL. C and D, Animals were subjected to 7 h of RSW after icv administration of DrPRL (D-rP) or rPRL. Numbers at the top of the column indicate the percent inhibition of gastric erosions compared with Sal/RSW+ or D-rP/RSW+ in B and D. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001 (vs. Sal/RSW+ or D-rP/RSW+). n = 5–11.

 
Induction of PRLR(L) mRNA expression in the CP by ip or icv administration of rPRL
Intraperitoneal administration of rPRL (50 µg/200 µl saline) significantly increased PRLR(L) mRNA in the CP at 7 h under non-RSW conditions (Fig. 2Go). Similarly, icv administration of rPRL increased PRLR(L) mRNA expression in the CP after 7 h of RSW (Fig. 2Go).



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  		FIG. 2. Up-regulation of PRLR(L) mRNA expression in CP in rats treated with rPRL. A, ISHH signals of PRLR(L) mRNA in the CP (magnification, x40). B, Rats were treated with saline (200 µl, ip) or rPRL (50 µg/200 µl saline, ip) under non-RSW and with DrPRL (500 ng/3 µl saline, icv) or rPRL (500 ng/3 µl saline, icv) under 7-h RSW. ***, P < 0.0005; ****, P < 0.0001 (vs. DrPRL, ip/RSW-). ####, P < 0.0001 (vs. DrPRL, icv/RSW+). n = 4–5.

 
Induction of PRLR(L) protein expression in the hypothalamus dissected from the brain by ip or icv administration of rPRL
Intraperitoneal administration of rPRL (50 µg/200 µl saline) significantly produced 1.8- and 1.7-fold increases, respectively, in PRLR(L) protein levels in the hypothalamus at 7 h under non-RSW conditions (Fig. 3AGo). Similarly, icv administration of rPRL (500 ng/3 µl saline) also resulted in a 1.8-fold increase in PRLR(L) protein levels in the hypothalamus at 7 h (Fig. 3BGo).



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  		FIG. 3. Increased hypothalamic PRLR(L) protein expression in rats treated with rPRL. A, Rats were treated with DrPRL (200 µl saline) and rPRL (50 µg/200 µl saline). B, Animals were treated with DrPRL (500 ng/3 µl saline) and RSW or rPRL (500 ng/3 µl saline). A: ****, P < 0.0001 (vs. DrPRL, ip/RSW-). B: ****, P < 0.0001 (vs. DrPRL, icv/RSW+). n = 7.

 
Central action, followed by change in circulating PRL and prevention of stress hypocalcemia and gastric erosions
Prior icv administration of antiserum for PRL or PRLR facilitated the exacerbation of hypocalcemia and gastric erosions in the rats after 7 h of RSW, and these antisera completely abolished the preventive effects caused by icv-administered rPRL (Table 2Go). The exacerbation of hypocalcemia and ulcerogenesis that occurred after 7 h of RSW was significantly promoted by prior ip administration of anti-rPRL serum compared with NRS (Table 2Go).


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  		TABLE 2. Effect of antiserum for PRL or PRLR on rPRL-induced prevention of 7-h stress hypocalcemia and ulcer

 
Induction of PRLR protein and CRF mRNA expressions in the PVN by rPRL
Thirty minutes after icv injection of rPRL (500 ng/3 µl saline) into normal rats, the expression levels of PRLR protein and CRF mRNA in the PVN were up-regulated by 42% and 44%, respectively, over control levels (Fig. 4Go). In contrast, icv injection of DrPRL (500 ng/3 µl saline) caused only 17% and 21% increases, respectively, in the expression levels for PRLR protein and CRF mRNA in the PVN (Fig. 4Go). The icv administration of rPRL elicited a slight decrease in PRLR protein and CRF mRNA levels in the PVN of rats for at least 4 h compared with the icv administered DrPRL (Fig. 4Go). The generalization is described in Fig. 5Go.



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  		FIG. 4. Effect of icv administered PRL on the expression of PRLR protein (A) and CRF mRNA (B) in the PVN without RSW. A, IHC signals of PRLR protein in the PVN (magnification, x50). B, ISHH signals of CRF mRNA expression in the PVN (magnification, x50). **, P < 0.001; ****, P < 0.0001 (vs. the value at each time point of DrPRL). #, P < 0.01; ###, P < 0.0005; ####, P < 0.0001 (vs. the value at time zero of DrPRL or rPRL). n = 4.

 


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  		FIG. 5. Proposed mechanism by which the protective effect of circulating PRL acts against stress-induced hypocalcemia and ulcerogenesis. PRL released rapidly and transiently into general circulation from the anterior pituitary stimulates PRLR(L) located on the choroid plexus and increases stress-induced and choroid plexus PRLR(L)-mediated transport of circulating PRL into cerebrospinal fluid. PRL transported to the brain increases PRLR(L) expression in the PVN and thus stimulates CRF neurons to produce the antistress effect. After ip injection, exogenous rPRL bind to PRLR(L) in the CP to exert the antistress effect by the same mechanism. Similarly, after icv injection, exogenous rPRL bind to PRLR(L) in the PVN to exert the antistress effect through vagus nerves (47 48 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PRL knockout mice are very useful for examining the physiological functions of PRL at birth (26, 27). However, it is unclear whether alternative compensatory mechanisms are activated as the animal matures. There is evidence of some compensation for the lack of PRL in the adult knockout mouse, although systematic studies have not been conducted (26, 27, 28). To avoid endocrinological effects of, for example, placental lactogen (29, 30) associated with compensation in PRL knockout mice, we used a rat model of stress that had been previously shown to produce hypocalcemia and ulcerogenesis (15, 16, 20). In this study we report that circulating PRL acting on PRLR(L) in the brain protects rats from RSW-induced hypocalcemia as well as gastric erosions, two conditions that may be caused by PRL insufficiency (the lowering of PRL to initial levels) after a rapid and transient increase in PRL during stress. In addition, CRF mRNA expression increases in the PVN in a similar fashion as PRLR expression.

As observed previously (1, 2), RSW elicited hypocalcemia with gastric ulceration, and gastric erosions were prevented by prior administration of rPRL (16, 20). The occurrence of gastric erosions is also inhibited by icv treatment with CRF (31, 32, 33), vasopressin (34), IL-1 (22), neurotensin (35), or oxytocin (36), whereas mortality and the severity of gastric erosions caused by cysteamine are augmented by sc treatment with 22K human GH (37, 38, 39). We found that ip or icv administration of rPRL suppressed RSW-induced hypocalcemia and gastric erosions. The ip administered rPRL remarkably increased PRLR(L) mRNA levels in the CP and its protein levels in the hypothalamus in the absence of RSW. In rats during RSW, icv administered rPRL markedly enhanced the expression level of PRLR(L) mRNA in the CP and of its protein in the hypothalamus.

Furthermore, it has been reported that the increase in serum PRL levels in rats results in increased PRL-binding sites at the CP and enhanced transport of PRL from blood to the cerebrospinal fluid (40). Our results from rats icv administered antiserum for PRL and PRLR show that an insufficiency of PRL or PRLR in the brain causes RSW-induced accentuation of hypocalcemia and ulcerogenesis, and results from rats icv treated with rPRL and antiserum for PRL and PRLR also show that PRL largely acts at the PRLRs in the brain to induce antistress effects. Another experiment indicates that ip administration of antiserum for rPRL (0.1 ml/rat) 30 min before RSW accentuates hypocalcemia and ulcerogenesis compared with rats that were given ip administration of NRS (0.1 ml/rat). These findings strongly suggest that the antistress effects of PRL on hypocalcemia and ulcerogenesis may not depend on the change in PRL produced from brain parenchymal cells but, rather, on changes in circulating PRL acting on the CNS during RSW. CRF administered icv is known to inhibit gastric acid secretion (29, 31) and to protect against stress-induced ulcers (32) by acting through an autonomic nervous system- and adrenal-dependent mechanism or the ß-endorphin pathway (41). A recent study indicates a close interaction between CRF and PRL during stress, as the centrally administered, nonpeptide CRF1 receptor antagonist, R121919, attenuates the stress-induced release of corticosterone, PRL, and oxytocin (42). Our results from rats treated icv with rPRL show a significant elevation in PRLR immunoreactivity and CRF mRNA in the PVN at 0.5 h, and these elevations are maintained for an additional 3.5 h. This result is supported by the facts that hyperprolactinemia increases CRF levels in hypophysial portal blood and ACTH levels in peripheral blood (43), and that rPRL stimulates hypothalamic CRF and pituitary ACTH secretion in vitro (44). The stress-induced increase in PRL has been shown to be closely related to HPA axis activity. The bed nucleus of the stria terminals receives input from the hippocampus, amygdala, and limbic cortex and sends heavy axonal projections to the PVN. Ibotenic acid lesions in the bed nucleus of the stria terminal not only attenuate the increase in HPA activity, but also increase circulating PRL levels under stress (45). In contrast, the stress-induced increase in ACTH secretion is decreased by chronic icv infusion of ovine PRL and is increased by antisense targeting of brain PRLR (18). These results suggest that PRL has an opposing effect at the receptor level in the brain on the response of the HPA axis to acute and chronic stress.

During acute stress, several studies indicate that intracisternally administered CRF inhibits TRH-induced gastric acid secretion (41). Similarly, corticosterone released during stress exerts a gastroprotective action (46). Therefore, activation of the HPA axis by PRL acting in the PVN during acute stress may partially contribute to the protective effect against RSW-induced ulcerogenesis, and this may occur in addition to preventing hypocalcemia caused by acute stress. In summary, our studies demonstrate that rPRL have similar effects in preventing hypocalcemia and on the formation of gastric ulcers in our RSW model. Additionally, PRLR(L) mRNA expression in the CP and its protein expression in the hypothalamus are enhanced with rPRL administration. The icv administration of rPRL causes an increase in CRF mRNA and PRLR mRNA expressions in the PVN in the absence of RSW. Our results also indicate that circulatory PRL may act at the PRLR(L) in the PVN to stimulate CRF neurons to produce the antistress effects. These results suggest that an increase in circulating PRL during acute stress is necessary and that PRLR-driven actions in the PVN are important for coping with acute stress. It is unclear why PRL from the periphery is the primary source of PRL acting on the CNS, rather than PRL synthesized centrally. This is an intriguing and important question that remains to be answered.


   Acknowledgments
 
We thank Dr. T. Imaki for advice and for providing CRF plasmid DNA, A. F. Parlow (NIDDK) for providing rPRL, and Dr. Shingami (Shikibo Co. Ltd.) for providing antiserum for rat PRL.


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan.

Abbreviations: CNS, Central nervous system; CP, choroid plexus; CRF, corticotropin-releasing factor; DrPRL, heat-denatured rat prolactin; HPA, hypothalamo-pituitary-adrenal; icv, intracerebroventricular; IHC, immunohistochemical analysis; ISHH, in situ hybridization histochemical analysis; NRS, normal rabbit serum; PRL, prolactin; PRLR, prolactin receptor; PRLR(L), long form of prolactin receptor; PVN, paraventricular nucleus; rPRL, rat prolactin; RSW, restraint stress in water.

Received October 27, 2003.

Accepted for publication December 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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T. Fujikawa, K. Tamura, T. Kawase, Y. Mori, R. R. Sakai, K. Sakuma, A. Yamaguch, M. Ogata, H. Soya, and K. Nakashima
Prolactin Receptor Knockdown in the Rat Paraventricular Nucleus by a Morpholino-Antisense Oligonucleotide Causes Hypocalcemia and Stress Gastric Erosion
Endocrinology, August 1, 2005; 146(8): 3471 - 3480.
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