help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0103
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3102    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subburaju, S.
Right arrow Articles by Aguilera, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subburaju, S.
Right arrow Articles by Aguilera, G.
Endocrinology Vol. 148, No. 7 3102-3110
Copyright © 2007 by The Endocrine Society

Vasopressin Mediates Mitogenic Responses to Adrenalectomy in the Rat Anterior Pituitary

Sivan Subburaju and Greti Aguilera

Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Greti Aguilera, M.D., Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10 Room 10N262, Bethesda, Maryland 20892. E-mail: Greti_Aguilera{at}nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether increased vasopressinergic activity during chronic stress or adrenalectomy mediates trophic changes in the corticotroph, we examined the effect of peripheral V1 receptor blockade in rats, using the antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]vasopressin (VP), on the number of pituitary cells taking up bromodeoxyuridine (BrdU) and cells containing immunoreactive ACTH (irACTH). Adrenalectomy significantly increased the number of BrdU- and ACTH-labeled cells at 3 and 6 d, and a much larger increase was observed at 28 d. Minipump infusion of V1 antagonist for 28 d, at doses blocking the increases in ACTH and corticosterone induced by exogenous VP, prevented the increases in BrdU incorporation, but not irACTH cells observed 28 d after adrenalectomy. Unexpectedly, colocalization of BrdU with ACTH-positive cells was minor (about three cells per pituitary section), and this was unaffected by adrenalectomy or V1 antagonist infusion. In contrast, adrenalectomy for 6 or 14 d failed to increase BrdU incorporation or irACTH cells in V1b receptor knockout mice while inducing the expected increase in wild-type mice. The data show that VP is required for pituitary mitogenesis after adrenalectomy but, at least in rats, not for increasing the number of corticotrophs. The lack of colocalization of ACTH in mitotic cells suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A LARGE BODY of evidence indicates that vasopressin (VP) has a role in regulating pituitary corticotroph function (1, 2, 3). Unlike VP secreted into the peripheral circulation from magnocellular neurons, VP responsible for corticotroph regulation is secreted into the pituitary portal circulation from parvocellular axon terminals in the external zone of the median eminence (2). In humans and rodents, VP alone is a weak stimulant of ACTH secretion, but it markedly potentiates the stimulatory effect of CRH (2, 3, 4). The expression of VP in parvocellular neurons increases during physiological states or experimental conditions involving activation of the hypothalamic-pituitary-adrenal (HPA) axis, such as stress or adrenalectomy (3, 5). Studies in the rat have shown that about 50% of CRH-containing neurons in the parvocellular hypothalamic paraventricular nucleus (PVN) coexpress VP, and this proportion increases substantially during stress, especially chronic and prolonged adrenalectomy (5, 6).

The actions of VP are mediated by plasma membrane receptors belonging to the guanyl nucleotide binding protein (G protein) family (7, 8). The receptor present in pituitary corticotrophs is the V1b receptor subtype, which is coupled to Gq/11 and phospholipase C, leading to activation of protein kinase C and increases in cytosolic calcium (9, 10, 11, 12). It has been shown that there is a good general correlation between V1b receptor content in the pituitary and ACTH responses by the pituitary corticotroph (3, 13). The up-regulation of pituitary V1b receptors, in conjunction with the increase in VP expression in parvocellular neurons during activation of the HPA axis, has suggested that regulation of the V1b receptor plays an important role in the sensitivity of ACTH responses, maintaining corticotroph responsiveness even in the presence of elevated glucocorticoid levels (3). Unlike the parvocellular vasopressinergic system, the expression of hypothalamic CRH and pituitary type 1 CRH receptors can be unchanged or reduced in conditions of high HPA axis activity (3).

The above observations have led to the hypothesis that in conditions such as chronic stress or adrenalectomy there is a switch from CRH to vasopressinergic regulation of ACTH secretion (3, 14). Although studies using Brattleboro rats or V1b receptor knockout mice appear to indicate an unaltered ACTH regulation in the absence of VP (15, 16, 17), it is generally accepted that VP plays a role mediating full ACTH responses to acute stress (18, 19, 20). On the other hand, the role of the marked increases in parvocellular VP and pituitary V1b receptor expression during chronic stress and adrenalectomy is less understood. Recent studies using minipump infusion of a V1 VP antagonist have shown that VP is required for full ACTH responses to acute stress (ip saline injection), but it does not mediate the hypersensitivity to a novel stress in rats subjected to repeated restraint (Chen, Y., and G. Aguilera, unpublished). This suggests that the marked increase in parvocellular vasopressinergic activity during chronic HPA activation mediates pituitary effects other than purely stimulating ACTH secretion. The aim of this study is to test the hypothesis that the increases in VP expression and pituitary V1b receptors during chronic stimulation of the HPA axis regulate corticotroph proliferation and/or differentiation rather than stimulation of ACTH secretion. Because adrenalectomy is associated with marked increases in parvocellular VP expression and increases in the number of corticotrophs (5, 21, 22, 23, 24), we used this model to examine the effect of VP V1 receptor blockade on proliferative responses in the pituitary.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and in vivo procedures
Adult male Sprague Dawley rats (200–250 g) (Harlan, Indianapolis, IN) were housed four per cage for at least 1 wk before experiments with a 14-h light, 10-h dark cycle. V1b receptor knockout mice were provided by Drs. W. S. Young III and S. J. Lolait, National Institute of Mental Health, National Institutes of Health (16). Male V1b receptor knockout mice and their wild-type littermates were derived from heterozygous breeding. All animal procedures were performed according to National Institutes of Health guidelines, and experimental protocols were approved by the National Institute of Child Health and Human Development Animal Care and Use Committee.

To induce corticotroph mitogenesis (23, 24, 25), bilateral adrenalectomy was carried out via a dorsal approach under ketamine/xylazine anesthesia (26). Sham-operated rats/mice were subjected to the same surgical procedure without removing the adrenals. Adrenalectomized rats/mice had access to normal saline as drinking fluid in addition to tap water. Because of problems with survival of mice after adrenalectomy, all adrenalectomized mice were administered a single ip injection of 0.025 µg of the mineralocorticoid flurocortisone on the day of surgery and subsequently the same daily amount in the drinking water for the duration of the experiment. This treatment, performed to avoid sodium loss after adrenalectomy-induced mineralocorticoid withdrawal, prevented mortality in subsequent experiments. The number of cells undergoing mitosis was examined by immunohistochemistry after injection of 100 mg/kg of bromodeoxyuridine (BrdU) as described below. To determine the optimal time after adrenalectomy, groups of rats were adrenalectomized and killed 3–28 d later after receiving daily injections of BrdU for 3 d. Because the highest number of BrdU-stained cells was found at 28 d, this time point was used to evaluate the effect of VP antagonism. In a second preliminary experiment, 28-d sham-operated or adrenalectomized rats were killed either 2 h after a single BrdU injection (100 mg/kg body weight) or after daily injections for 4 or 7 d. Because injection for 7 d yielded the highest number of BrdU-stained cells, this number of injections was used in subsequent experiments with rats.

To study the role of VP on adrenalectomy-induced pituitary mitogenesis, we administered the peptide VP V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP (Bachem, Torrance, CA), which displays about equal binding affinities to V1a and V1b receptors. The antagonist was delivered sc using Alzet osmotic minipumps (model 2004; Durect Corp., Cupertino, CA), with a delivery rate of 230 ng/h, for 28 d. In preliminary experiments, we tested the effectiveness of the antagonist blocking pituitary VP receptors by examining the ability of the minipump infusion to prevent the increases in plasma ACTH and corticosterone levels in response to a single iv injection of VP in intact rats. Control rats received sc implants of empty silastic tubing of dimensions similar to the minipump. One day before VP testing, a jugular catheter (Braintree Scientific Inc., Braintree, MA) was implanted under ketamine/xylazine anesthesia. The catheter was exteriorized in the neck of the animal, filled with heparinized saline (50 IU/ml), protected by a metal spring, and connected to a swivel fixed to the top of the cage using a counterbalanced beam. After collecting a basal blood sample at 0800 h, rats received an injection of 100 ng arginine VP through the jugular vein catheter in 100 µl saline. Additional blood samples (0.3 ml) were collected at 30 and 60 min after the VP injection from VP V1 antagonist-infused as well as control animals. Blood samples, 300 µl, were collected in ice-cold 1.5-ml microfuge tubes containing 300 µg EDTA and 30 kIU aprotinin. After centrifugation at 1500 x g for 20 min, plasma fractions were stored at –80 F until assayed for ACTH and corticosterone. ACTH and corticosterone were measured by RIA, using kit reagents from Diagnostic Products Co. (Los Angeles, CA) for steroid hormones and DiaSorin (Stillwater, MN) for ACTH, respectively.

To study the effect of VP on pituitary mitogenesis, rats were divided into four experimental groups: sham-operated controls (n = 4), sham plus VP V1 antagonist (n = 4), 28-d adrenalectomy (n = 4), and adrenalectomy plus VP V1 antagonist (n = 4). Infusion of dGly[Phaa1,D-tyr(et), Lys, Arg]VP, was performed for 28 d as described above. BrdU injections were carried out from d 21–27. At d 28, rats were killed by decapitation, and pituitary glands were rapidly removed, immersion-fixed in ice-cold Bouin’s fixative (Accustain, HT10-32; Sigma-Aldrich, St. Louis, MO), and postfixed overnight at 4 C. Five-micrometer pituitary sections were cut and used for immunohistochemical detection.

To study the effect of adrenalectomy on pituitary cell proliferation in V1b receptor knockout mice, matched males from the same litters, wild-type or V1b receptor knockout mice, were divided into two groups each: sham (n = 6) and adrenalectomy (n = 6). Six or 14 d after adrenalectomy, mice were killed by decapitation and the pituitaries quickly removed, immersion fixed, and processed as described above for immunohistochemical detections. All mice received BrdU injections (30 mg/kg, ip) for the last 6 d before being killed.

Double-staining immunohistochemistry
BrdU was detected using kit regents (Roche Applied Sciences, Indianapolis, IN) as follows. The sections were blocked against nonspecific reactions (PBS, 5% BSA, and 30% normal goat serum for 1 h), incubated with a monoclonal anti-BrdU antibody (1:10) for 1 h at room temperature, washed three times with PBS containing 0.1% Triton X-100, and then incubated with antimouse IgG conjugated with alkaline phosphatase. Immunoreactive cells were visualized by addition of alkaline phosphatase substrate containing levamisole to block endogenous alkaline phosphatase (Dako, Carpinteria, CA). For double labeling, before the second immunostaining, sections were treated with 0.2 M glycine-HCl buffer (pH 2.2) for 30 min to remove residual antibodies, incubated with 30% normal goat serum for 1 h to block nonspecific antibody binding, and incubated with one of following rabbit polyclonal antisera: anti-ACTH (1:2000), anti-GH (1:2000), anti-prolactin (1:2000), anti-FSH/LH (1:1000), anti-TSHß (1:1000) (A. F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases). The rabbit polyclonal antibodies, anti-nestin (R&D Systems, Minneapolis, MN) and anti-S100 (folliculo stellate cell) (Dako), were used at dilutions of 1:200 and 1:300, respectively. A polyclonal anti-Tpit antibody provided by Prof. J. Drouin, Institute de Recherche de Montreal, Canada, was used at 1:200. After washing with PBS containing 0.1% Triton X-100, sections were incubated for 1 h with alkaline phosphatase-conjugated antirabbit IgG at room temperature and developed with the Fast Red substrate system (Dako). Negative control sections incubated in the absence of either primary or secondary antibody showed no staining. Immunopositive cells were counted using a grid; five random sections per pituitary were evaluated and averaged to obtain the number of cells per square millimeter (approximately 2500 µm2/section). All slides were coded and counted without the observer being aware of the treatment.

Statistical analysis
Data were expressed as the mean ± SEM. Differences between groups were evaluated by Student’s t test or one- or two-way ANOVA followed by Student-Newman-Keuls post hoc test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of VP V1 receptor antagonism on VP-induced release of ACTH and corticosterone
To determine the effectiveness of the minipump infusion of the VP V1 receptor antagonist to block VP receptors in the pituitary, we measured plasma ACTH and corticosterone responses to a single injection of VP (100 ng, iv) in controls and V1 antagonist-infused rats. In control rats (without V1 antagonist infusion), plasma ACTH (Fig. 1AGo) and corticosterone (Fig. 1BGo) levels increased significantly 30 and 60 min after VP injection compared with basal values (P < 0.001). In contrast, VP injection failed to increase plasma ACTH or corticosterone in rats receiving chronic V1 antagonist infusion (Fig. 1Go), indicating that the mode of administration effectively blocked pituitary VP receptors.


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 1. Effectiveness of chronic minipump infusion of V1 receptor antagonist blocking pituitary VP receptors. A and B, Plasma ACTH (A) and corticosterone (B) responses to a single injection of 100 ng of VP, iv, in control rats or rats receiving mini-pumps infusion of the V1 receptor antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP, for 28 d. Data points represent mean and SE of values obtained in eight rats per experimental point. *, P < 0.01, VP injection vs. basal; #, P < 0.01, V1 antagonist infusion vs. respective control.

 
Effect of adrenalectomy on pituitary cell proliferation and ACTH-stained cells
The time course of the effect of adrenalectomy on the number of corticotrophs in the anterior pituitary is shown in Fig. 2AGo. Adrenalectomy caused a small but significant increase in the number of ACTH-stained cells by 3, 6, and 14 d (1.8-fold, P < 0.05; 1.6-fold, P < 0.05; and 2.3-fold, P < 0.001) and a much larger increase of 4.5-fold by 28 d (P < 0.001) vs. sham. The time course of the effect of adrenalectomy on BrdU incorporation is shown in Fig. 2BGo. Because in sham-operated rats there was no significant difference in BrdU incorporation at the different time points, fold differences in adrenalectomized rats were compared with the pooled sham-operated values. Adrenalectomy significantly increased BrdU-labeled cells compared with sham-operated controls at 3, 6, and 28 d but not at 14 d. Although increases of 2.0- and 1.8-fold (P < 0.05) over sham-operated controls were observed at 3 and 6 d, a much larger increase of 3.5-fold (P < 0.001) was found at 28 d. Therefore, all consecutive experiments were performed in 28-d adrenalectomized rats. The effect of the number of BrdU injections in 28-d adrenalectomized rats is shown in Fig. 2CGo. Increasing the number of BrdU injections resulted in a slightly higher number of BrdU-stained cells in sham-operated rats. Irrespective of the number of injections, there was a significant (P < 0.001) increase in the number of BrdU-stained nuclei in pituitaries from adrenalectomized rats compared with sham-operated controls, but values were significantly higher in pituitaries from rats receiving injections for 7 d (d 21–27) compared with a single injection or 4 d injections (Fig. 2CGo).


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 2. Effect of adrenalectomy (ADX) on pituitary cell proliferation and number of corticotrophs (ir ACTH). A and B, Time course of the changes in the number of irACTH cells (A) and BRdU-stained nuclei (B) after adrenalectomy. Adrenalectomized rats received BrdU injections for 3 d before being killed at 3, 6, or 28 d after adrenalectomy. C, Effect of the number of BrdU injections in 28-d adrenalectomized or sham-operated rats. Bars represent the mean and SE of the number of immunopositive cells counted in 10–12.5 mm2 per pituitary (n = 4 rats per group). *, P < 0.05; ***, P < 0.001 vs. sham. Time 0 represents the pooled values of sham-operated rats after 3, 14, and 28 d.

 
Effect of V1 antagonism on adrenalectomy-induced increases in BrdU incorporation and ACTH-stained cells in the anterior pituitary
To determine the role of VP in mediating adrenalectomy-induced pituitary cell proliferation, groups of sham-operated and adrenalectomized rats underwent minipump infusion of the V1 antagonist, dGly[Phaa1,D-tyr(et), Lys, Arg]VP, for 28 d starting at the time of surgery. Consistent with the data in Fig. 2Go, without V1 receptor antagonism, the number of BrdU-labeled nuclei increased from 30.14 ± 12.61/mm2 in sham-operated controls to 104.67 ± 23.74/mm2 in 28-d adrenalectomized rats (P < 0.001). Infusion of the V1 receptor antagonist for 28 d had no significant effect on BrdU incorporation in sham-operated rats (30.14 ± 12.61/mm2 and 42.76 ± 11.51/mm2 in controls and V1 antagonist infusion, respectively), but it prevented the increase in BrdU incorporation after adrenalectomy (P < 0.05, lower than adrenalectomy control) (Fig. 3Go). The number of ACTH-immunopositive cells also increased from 39.19 ± 8.28 to 213.07 ± 13.90/mm2 (P < 0.002, sham vs. adrenalectomy) after 4 wk adrenalectomy, but in contrast to BrdU incorporation, the number of ACTH-labeled cells was not affected by VP V1 antagonist (Fig. 3Go).


Figure 3
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 3. Effect of V1 receptor antagonist infusion on adrenalectomy (ADX)-induced changes in the number of cells immunostained for ACTH or BrdU 28-d adrenalectomized rats that received minipump infusion of V1 receptor antagonist or vehicle starting at the time of surgery and BrdU injections for the last 7 d before being killed. Bars represent the mean and SE of immunopositive cells counted in pituitary sections of four rats per experimental group. *, P < 0.05 vs. sham; **, P < 0.002 vs. sham; ***, P < 0.001 vs. sham; $, P < 0.05 vs. ADX.

 
BrdU incorporation and pituitary cell types
To determine the phenotype of proliferating cells, we performed double immunocytochemistry with BrdU and antibodies against pituitary hormones (Fig. 4Go). Analysis of double staining for BrdU and ACTH in pituitary sections of sham-operated and adrenalectomized rats revealed minor colocalization of BrdU-labeled nuclei in immunoreactive (ir)ACTH cells (Figs. 4Go and 5AGo). The absolute number of cells colocalizing BrdU and ACTH tended to increase after 28 d adrenalectomy, but the differences were not statistically significant (1.4 ± 0.2/mm2 and 2.0 ± 0.64/mm2 for sham-operated and adrenalectomized rats, respectively; Fig. 5BGo). On the other hand, when expressed as percentage of BrdU-labeled cells coexpressing ACTH, values decreased from 5.1 ± 0.8% in sham-operated rats to 2.1 ± 0.4% in adrenalectomized rats (P < 0.001), reflecting the higher increase in BrdU-labeled cells compared with double-stained cells (not shown). The tendency to increase was not evident in the adrenalectomy group treated with V1 antagonist, but the differences between groups were not statistically significant because of the large individual variation shown by the SE (Fig. 5BGo). Similar minor or lack of colocalization was observed with GH, TSH, prolactin, or FSH and LH (gonadotrophs) (Fig. 4AGo).


Figure 4
View larger version (116K):
[in this window]
[in a new window]

  		FIG. 4. Double staining of BrdU and markers for specific pituitary cell types in 28-d adrenalectomized rats. The 28-d adrenalectomized rats were injected with BrdU for 7 d before collecting pituitaries for immunohistochemistry. A, Nuclei incorporating BrdU were stained with alkaline phosphatase (shown in dark purple), and hormonal staining for ACTH, GH, TSH, prolactin (PRL), FSH, and LH are shown by the FastRed cytoplasmic staining; B, high magnification for Brdu/ACTH costaining; C, double labeling of BrdU (FastRed staining) and markers for folliculostellate (S100) and stem cells (nestin) stained in purple with alkaline phosphatase. Figures are representative of observations in pituitaries from four rats. Magnification is shown at the bottom right of the images.

 

Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 5. Effect of adrenalectomy (ADX) and V1 receptor blockade on the colocalization of BrdU with ACTH or Tpit. A, Time course of the effect of adrenalectomy on the number of BrdU nuclei colocalizing irACTH; B and C, 28-d adrenalectomized rats received minipump infusion of V1 receptor antagonist or vehicle starting at the time of surgery and BrdU injections for the last 7 d before being killed. Colocalization of BrdU and ACTH is shown in B and BrdU and Tpit in C. Bars represent the mean and SE of immunopositive cells counted in pituitary sections of four rats per experimental group. *, P < 0.05 vs. sham. The differences between adrenalectomized and sham-operated rats for BrdU/Tpit colocalization were significant only when compared using the Student’s t test: a, *, P < 0.05.

 
To determine whether BrdU-labeled nuclei corresponded to folliculostellate cells or stem cells, sections were double stained for BrdU and the glial cell marker S100 or the stem cell marker nestin (Fig. 4Go, B and C). Again, microscopic analysis of the sections showed an insignificant proportion of double-labeled cells.

The lack of colocalization of BrdU-stained nuclei with markers for specific pituitary cell types raised the question of whether the cells undergoing mitosis during late adrenalectomy correspond to still undifferentiated corticotroph precursors. To examine this possibility, we carried out immunostaining for Tpit, a transcription factor expressed exclusively in proopiomelanocortin (POMC)-expressing cells and involved in the differentiation of the corticotroph lineage (27). As expected, Tpit-labeled nuclei were observed in the anterior pituitary and intermediate lobe, and Tpit-expressing nuclei largely corresponded to ACTH-expressing cells (Fig. 6Go). In sham-operated rats, there was more than 80% colocalization of ACTH with Tpit nuclei, whereas the percent colocalization decreased to 60% in adrenalectomized rats. Only a minor proportion of Tpit-expressing nuclei, 20 of 2334 cells counted, showed colocalization with BrdU, and the majority of BrdU-stained nuclei did not show Tpit staining. In contrast to the decrease in Tpit-ACTH colocalization, the number of cells expressing both Tpit and BrdU staining increased by about 2-fold in pituitaries from adrenalectomized rats (4.78 ± 2.0/mm2 vs. 2.20 ± 0.38/mm2 in sham-operated controls, P < 0.05; Fig. 5CGo). The effect of adrenalectomy on the number of cells colocalizing Tpit and BrdU was completely abolished by infusion of the V1 antagonist (1.33 ± 0.15/mm2, P < 0.05 vs. adrenalectomy).


Figure 6
View larger version (129K):
[in this window]
[in a new window]

  		FIG. 6. Representative photomicrographs showing double staining for BrdU and ACTH or Tpit in 28-d adrenalectomized rats. Cells showing colocalization of Tpit and ACTH or Tpit and Brdu are shown by the arrows. AP, Anterior pituitary lobe; IL, intermediate pituitary lobe. Magnification is shown at the bottom right.

 
Effect of adrenalectomy on pituitary cell proliferation in V1b receptor knockout mice
To further study the role of VP on cell proliferation, we examined the effect of adrenalectomy on BrdU incorporation and number of ACTH-stained cells in V1b receptor-deficient mice. In wild-type mice, there was a significant (P < 0.05) increase in incorporation of BrdU after adrenalectomy for 6 and 14 d, compared with sham controls (Fig. 7Go, A and B). In contrast, the number of BrdU-positive cells was almost identical in sham-operated and adrenalectomized V1b receptor-deficient mice at 6 or 14 d. In wild-type mice, the number of ACTH-immunopositive cells also increased significantly after 6 d (P < 0.001) and 14 d (P < 0.05) adrenalectomy compared with sham-operated controls. However, similar to BrdU incorporation, in V1b receptor knockout mice, adrenalectomy did not increase the number of ACTH cells either at 6 or 14 d.


Figure 7
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 7. Effect of adrenalectomy (ADX) on pituitary cell proliferation (BrdU staining) and number of ACTH-positive cells (irACTH) in V1b receptor-deficient wild-type mice (WT) and V1b receptor-deficient mice (KO). Mice were adrenalectomized for 6 or 14 d and subjected to daily ip BrdU injections for 6 d before collecting pituitaries for analysis. Bars represent the mean and SE of the number of immunopositive cells counted in three mice per group. *, P < 0.05 vs. sham; ***, P < 0.001 vs. sham.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The lack of conclusive evidence for a role of the marked increased parvocellular vasopressinergic activity (15, 28, 29) controlling ACTH secretion during chronic stimulation of the HPA axis prompted us to examine the effect of VP on pituitary mitogenesis. The present study demonstrates that VP is essential for the pituitary mitogenic responses during prolonged adrenalectomy, suggesting new functions of VP on HPA axis regulation. It is well known that adrenalectomy results in marked and progressive increases of VP expression in parvocellular neurons of the PVN (30, 31, 32) as well as hypertrophy and hyperplasia of the corticotrophs (21, 22, 24, 31, 33, 34, 35). However, the exact mechanisms regulating proliferative responses of pituitary corticotrophs during chronic stress and adrenalectomy are not fully understood. The increase in the number of corticotrophs after adrenalectomy is associated with increases in [3H]thymidine or BrdU incorporation in the anterior pituitary, indicative of mitogenic activity (36, 37, 38). Consistent with previous reports, the present study shows early increases in the number of BrdU-positive cells (3 and 6 d) followed by a decline at 14 d (38, 39). It is likely that the decline is due to apoptotic cell death, because it has been shown that cells undergoing recent mitosis are more susceptible to apoptosis (40). However, in contrast to the slight and not significant recovery in mitogenic activity observed at 4 wk in the latter study (40), in the present experimental conditions, the number of both BrdU- and ACTH-stained cells increased again by 4 wk. The reason for this discrepancy could relate to the strain of rats (Wistar vs. Sprague Dawley) or the procedure to evaluate mitogenesis (BrdU incorporation vs. computerized counting of mitotic figures). The second proliferative phase detected 4 wk after adrenalectomy in this study suggests that a mitogenic stimulus can override apoptotic cell death.

Because VP expression in parvocellular neurons increases markedly during long-term adrenalectomy, and VP can act as a mitogen, we sought to examine the role of VP on pituitary cell proliferation. It has been shown that VP stimulates mitogenesis in a number of systems, including mouse Swiss 3T3 cells (41), rat bone marrow cells after hemorrhage (42), rat liver cells (43), mesangial cells (44), human osteoblast-like cells (45), and the murine corticotroph tumor cell line AtT20 (46). VP also has been shown to increase the number of cells incorporating BrdU in primary cultures of rat anterior pituitary cells (47). The ability of the V1 antagonist to prevent the increase in the number of cells incorporating BrdU during long-term adrenalectomy, shown in the present study, supports the hypothesis that VP mediates the mitogenic activity in the pituitary after glucocorticoid withdrawal. The lack of mitogenic responses to adrenalectomy in V1b receptor knockout mice is also in agreement with this hypothesis. Although the main VP receptor subtype found in the pituitary is the V1b receptor, the antagonist used in these experiments has similar binding affinity for V1a and V1b receptors (48). Thus, it is possible that blockade of V1a receptors located in pituitary cells other than corticotrophs or in the periphery could contribute to the effects of the antagonist. In contrast with the present data supporting a critical role for VP on pituitary mitogenesis, previous studies have shown that PVN lesions are unable to prevent the increase in pituitary cell proliferation induced by adrenalectomy (49). The latter findings suggest that pituitary mitogenesis is more likely to result from direct pituitary effects of glucocorticoid withdrawal rather than to the increases in CRH and/or VP. However, because the supraoptic nuclei remained intact in these rats, it is not possible to rule out that VP released to the pituitary portal circulation from these nuclei was sufficient to promote mitogenesis. In this regard, there is anatomical (50) and functional (51, 52) evidence that VP can access the pituitary portal circulation from magnocellular nerve fibers in the internal zone of the median eminence. It is also possible that the lack of effect of PVN lesions observed by Nolan et al. (49) reflect the shorter time of glucocorticoid withdrawal, 7 vs. 28 d in the present study. Thus, it is conceivable that VP becomes critical as a pituitary mitogenic agent during longer-term adrenalectomy.

It should be noted that the effects of the V1 antagonist on the number of BrdU-labeled cells did not parallel changes in the number of ACTH-labeled cells. The preliminary experiments showed that using the same dose and mode of administration, the V1 receptor antagonist prevented the rise in plasma ACTH and corticosterone elicited by VP (Fig. 7Go, A and B), indicating that V1b receptors in the corticotroph were effectively blocked. This suggests that in the rat, VP is required for pituitary cell proliferation but not for corticotroph differentiation, at least for the duration of the experiment, 28 d. On the other hand, the inability of V1b receptor knockout mice to increase the number of ACTH-stained cells after adrenalectomy suggests that lifetime deficient pituitary vasopressinergic activity has a more profound impact on the corticotroph population. Alternatively or in addition, it is possible that regulation of corticotroph differentiation varies between rat and mouse. It was recently reported that plasma ACTH responses to repeated restraint for 14 d, but not to acute stress or basal levels, are reduced in V1b receptor knockout mice compared with wild-type controls. This is consistent with the present data and raises the possibility that the lack of VP action in the pituitary also impairs stress-induced corticotroph proliferation (53). Although the mechanism for the difference between the effects of the V1 antagonist and genetic V1b receptor deficiency remains to be elucidated, the data strongly suggest that VP influences pituitary mitogenesis as well as the number of corticotrophs. Previous reports showing a reduced number of corticotrophs in the VP-deficient Brattleboro rat compared with control Long Evans rats (54, 55) also support this view.

Because adrenalectomy increases the number of corticotrophs, it was quite unexpected to find only minor colocalization of BrdU and irACTH. This raises questions about the origin of the newly formed corticotrophs as well as the identity of the BrdU-labeled cells. Although corticotrophs derive from embryonic precursors displaying corticotroph-specific markers, some reports using primary pituitary cell cultures, showing colocalization of ACTH in pituitary cell types other than corticotrophs, have suggested that mature pituitary cells can cross-differentiate (56). However, the lack of colocalization of BrdU-stained nuclei in lactotrophs, thyrotrophs, somatotrophs, or gonadotrophs shown in this study is against this possibility. The minor increase in corticotrophs undergoing mitosis in adrenalectomized rats prompted us to examine the presence of BrdU-stained nuclei in early corticotroph precursors or undifferentiated stem cells. It has been shown that the T-box transcription factor Tpit is exclusively found in POMC-expressing cells in the pituitary (27). Adrenalectomy increased the number of Tpit-positive cells in the pituitary as well as the number of cells expressing both Tpit and ACTH (Fig. 6Go); however, the increase of Tpit was not statistically significant (sham vs. adrenalectomy). Again, the minor colocalization of BrdU-stained nuclei with Tpit or ACTH cells renders it unlikely that the increase in corticotrophs originates from the division of existing corticotrophs or already differentiated corticotroph precursors but suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells. Previous studies have also shown lack of colocalization of BrdU in corticotrophs after adrenalectomy (34, 39, 57). A recent study in 3- and 6-d adrenalectomized or gonadectomized rats also reports minor incidence of mitogenesis in corticotrophs or gonadotrophs (38). In addition, the lack of additivity in mitogenic responses to adrenalectomy and gonadectomy in the latter study suggested to the authors that mitogenesis in response to both stimuli occurs in an undifferentiated progenitor population (38).

The present study examined mitogenic activity of two populations of potential progenitor cells, folliculostellate and nestin-labeled stem cells. Folliculostellate cells are non-hormone-producing pituitary cells with glial cell characteristics, which influence the local hormone secretion by producing basic fibroblast growth factor (58), vascular endothelial growth factor (59), IL-6 (60), follostatin (61), NO (62), and lipocortin1 (63). It has been postulated that folliculostellate and hormone-secreting cells are derived from the same progenitor cells, based on the common presence of the homeobox 1 gene in these cells (64) as well as in muscle cells derived from transplanted pituitary cells (65). Others have suggested that folliculostellate cells are formed by retro-differentiation and that they act as adult stem cells in the human pituitary (66). The lack of BrdU incorporation in cells stained with the glial/folliculostellate cell marker, S100, in this study suggest that folliculostellate cells do not act as precursor for the newly formed corticotroph after adrenalectomy. Recently, Chen et al. (67) identified a population of cells in adult pituitaries, which express transcription factors mostly expressed during embryonic development, such as the 220-kDa filament protein nestin, and suggested that these cells represent adult pituitary stem/progenitor cells. An antibody against nestin, used as a marker for pituitary stem cells (68), also failed to show significant colocalization with any pituitary hormone, indicating that nestin-expressing stem cells are unlikely to be corticotroph precursors. Studies in human pituitaries, obtained from autopsy (69), have questioned the validity of nestin as an endocrine pituitary stem cell marker (at least in humans). Thus, it is possible that non-nestin-expressing stem cells are responsible for the mitogenic responses to long-term adrenalectomy. Taken as a whole, BrdU colocalized only with a small number of ACTH- and Tpit-positive cells, but this accounts for a small proportion of the overall proliferative response to adrenalectomy. Considering that the colocalization studies were performed within 7 d of the first BrdU injection, the lack of major colocalization of BrdU in corticotrophs suggests that differentiation of cells undergoing mitogenesis to corticotrophs is a delay process, requiring a time period longer than 7 d. Additional studies in rats receiving BrdU injections early during long-term adrenalectomy will be required to test this hypothesis. An additional unanswered question is whether pituitary cell types other than corticotrophs express VP receptors. In situ hybridization studies have shown the expected colocalization of V1b receptor mRNA and POMC mRNA (10). However, it is evident from the image shown in the latter study (10) that some clusters of V1b receptor mRNA grains do not overlay POMC-stained cells, suggesting V1b receptor expression in additional cell types. Whether these cells correspond to pituitary progenitor cells remains to be elucidated.

In conclusion, this study provides evidence indicating that VP can mediate proliferative responses to prolonged adrenalectomy in the rat. The mechanisms mediating corticotroph differentiation appear to be more complex and at least in the rat are likely to involve factors other than VP. The minor colocalization of BrdU-stained nuclei in ACTH- or Tpit-stained cells (or other pituitary cell type) renders it unlikely that the increase in corticotrophs originates from division of existing differentiated cells and suggests that recruitment of corticotrophs during adrenalectomy occurs from undifferentiated cells. The data suggest that a major role for the marked increases in parvocellular vasopressinergic activity during adrenalectomy (and probably chronic stress) is regulating cell proliferation and remodeling of the pituitary tissue.


   Acknowledgments
 
We thank Drs. W. S. Young III and S. J. Lolait (Section on Neural Gene Expression, National Institute of Mental Health) for the V1b receptor knockout mice, Prof. J. Drouin (Institut des Recherches Cliniques de Montréal, Canada) for the Tpit antibody, and Mr. Jafri Syed for genotyping V1b receptor knockout mice.


   Footnotes
 
This work was supported by the Intramural Research Program of the National Institutes of Health (National Institute of Child Health and Human Development).

Disclosure: The authors have nothing to disclose.

First Published Online April 5, 2007

Abbreviations: BrdU, Bromodeoxyuridine; HPA, hypothalamic-pituitary-adrenal; ir, immunoreactive; POMC, proopiomelanocortin; PVN, paraventricular nucleus; VP, vasopressin.

Received January 24, 2007.

Accepted for publication March 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McCann SM, Brobeck JR 1954 Evidence for a role of the supraopticohypophyseal system in regulation of adrenocorticotrophin secretion. Proc Soc Exp Biol Med 87:318–324[Medline]
  2. Antoni FA 1993 Vasopressinergic control of pituitary adrenocorticotropin secretion comes of age. Front Neuroendocrinol 14:76–122[CrossRef][Medline]
  3. Aguilera G 1994 Regulation of pituitary ACTH secretion during chronic stress. Front Neuroendocrinol 15:321–350[CrossRef][Medline]
  4. Gillies GE, Linton EA, Lowry PJ 1982 Corticotropin releasing activity of the new CRF is potentiated several times by vasopressin. Nature 299:355–357[CrossRef][Medline]
  5. Whitnall MH 1993 Regulation of the hypothalamic corticotropin-releasing hormone neurosecretory system. Prog Neurobiol 40:573–629[Medline]
  6. de Goeij DC, Kvetnansky R, Whitnall MH, Jezova D, Berkenbosch F, Tilders FJ 1991 Repeated stress-induced activation of corticotropin-releasing factor neurons enhances vasopressin stores and colocalization with corticotropin-releasing factor in the median eminence of rats. Neuroendocrinology 53:150–159[Medline]
  7. Jard S, Barberis C, Audigier S, Tribollet E 1987 Neurohypophyseal hormone receptor systems in brain and periphery. Prog Brain Res 72:173–187[Medline]
  8. Peter J, Burbach H, Adan RA, Lolait SJ, van Leeuwen FW, Mezey E, Palkovits M, Barberis C 1995 Molecular neurobiology and pharmacology of the vasopressin/oxytocin receptor family. Cell Mol Neurobiol 15:573–595[CrossRef][Medline]
  9. Seibold A, Brabet P, Rosenthal W, Birnbaumer M 1992 Structure and chromosomal localization of the human antidiuretic hormone receptor gene. Am J Hum Genet 51:1078–1083[Medline]
  10. Lolait SJ, O’Carroll A, Mahan LC, Felder CC, Button DC, Young III WS, Mezey E, Brownstein MJ 1995 Extrapituitary expression of the rat V1b vasopressin receptor gene. Proc Natl Acad Sci USA 92:6783–6787[Abstract/Free Full Text]
  11. Sugimoto T, Saito M, Mochizuki S, Watanabe Y, Hashimoto S, Kawashima H 1994 Molecular cloning and functional expression of a cDNA encoding the human V1b vasopressin receptor. J Biol Chem 269:27088–27092[Abstract/Free Full Text]
  12. Lolait SJ, O’Carroll AM, Brownstein MJ 1995 Molecular biology of vasopressin receptors. Ann NY Acad Sci 771:273–292[Medline]
  13. Volpi S, Rabadan-Diehl C, Aguilera G 2004 Regulation of vasopressin V1b receptors and stress adaptation. Ann NY Acad Sci 1018:293–301[Abstract/Free Full Text]
  14. Ma XM, Levy A, Lightman SL 1997 Emergence of an isolated arginine vasopressin (AVP) response to stress after repeated restraint: a study of both AVP and corticotropin-releasing hormone messenger ribonucleic acid (RNA) and heteronuclear RNA. Endocrinology 138:4351–4357[Abstract/Free Full Text]
  15. Makara GB, Mergl Z, Zelena D 2004 The role of vasopressin in hypothalamo-pituitary-adrenal axis activation during stress: an assessment of the evidence. Ann NY Acad Sci 1018:151–161[Abstract/Free Full Text]
  16. Wersinger SR, Ginns EI, O’Carroll AM, Lolait SJ, Young III WS 2002 Vasopressin V1b receptor knockout reduces aggressive behavior in male mice. Mol Psychiatry 7:975–984[CrossRef][Medline]
  17. Tanoue A, Ito S, Honda K, Oshikawa S, Kitagawa Y, Koshimizu TA, Mori T, Tsujimoto G 2004 The vasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity under both stress and resting conditions. J Clin Invest 113:302–309[CrossRef][Medline]
  18. Olsen L, Knigge U, Warberg J 1992 Gastrin-releasing peptide stimulation of corticotropin secretion in male rats. Endocrinology 130:2710–2716[Abstract]
  19. Suda T, Nakano Y, Tozawa F, Sumitomo T, Sato Y, Yamada M, Demura H 1992 The role of corticotropin-releasing factor and vasopressin in hypoglycemia-induced proopiomelanocortin gene expression in the rat anterior pituitary gland. Brain Res 579:303–308[CrossRef][Medline]
  20. Rivier J, Rivier C, Vale W 1984 Synthetic competitive antagonists of corticotropin-releasing factor: effect on ACTH secretion in the rat. Science 224:889–891[Abstract/Free Full Text]
  21. Crane WA, Loomes RS 1967 Effect of age, sex, and hormonal state on tritiated thymidine uptake by rat pituitary. Br J Cancer 21:787–792[Medline]
  22. Childs GV, Yamauchi K, Unabia G 1989 Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes. Am J Anat 185:223–235[CrossRef][Medline]
  23. Childs GV, Lloyd J, Unabia G, Rougeau D 1989 Growth and secretory responses of enriched populations of corticotropes. Endocrinology 125:2540–2549[Abstract]
  24. Rappay G, Makara GB 1981 A quantitative approach to trace the corticotrophs in culture after adrenalectomy. Histochemistry 73:131–136[CrossRef][Medline]
  25. Nolan LA, Kavanagh E, Lightman SL, Levy A 1998 Anterior pituitary cell population control: basal cell turnover and the effects of adrenalectomy and dexamethasone treatment. J Neuroendocrinol 10:207–215[CrossRef][Medline]
  26. Ma XM, Aguilera G 1999 Differential regulation of corticotropin-releasing hormone and vasopressin transcription by glucocorticoids. Endocrinology 140:5642–5650[Abstract/Free Full Text]
  27. Lamolet B, Pulichino AM, Lamonerie T, Gauthier Y, Brue T, Enjalbert A, Drouin J 2001 A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 104:849–859[CrossRef][Medline]
  28. Zelena D, Foldes A, Mergl Z, Barna I, Kovacs KJ, Makara GB 2004 Effects of repeated restraint stress on hypothalamo-pituitary-adrenocortical function in vasopressin deficient Brattleboro rats. Brain Res Bull 63:521–530[CrossRef][Medline]
  29. Juaneda C, Lafon-Dubourg P, Ciofi P, Sarrieau A, Corio M, Tramu G 1999 Immune challenge-stimulated hypophysiotropic corticotropin-releasing hormone messenger RNA expression is associated with an induction of neurotensin messenger RNAs without alteration of vasopressin messenger RNAs. Neuroscience 93:393–400[CrossRef][Medline]
  30. Whitnall MH, Smyth D, Gainer H 1987 Vasopressin coexists in half of the corticotropin-releasing factor axons present in the external zone of the median eminence in normal rats. Neuroendocrinology 45:420–424[Medline]
  31. Sawchenko PE, Swanson LW, Vale WW 1984 Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat. Proc Natl Acad Sci USA 81:1883–1887[Abstract/Free Full Text]
  32. Plotsky PM, Sawchenko PE 1987 Hypophysial-portal plasma levels, median eminence content, and immunohistochemical staining of corticotropin-releasing factor, arginine vasopressin, and oxytocin after pharmacological adrenalectomy. Endocrinology 120:1361–1369[Abstract]
  33. Kiss JZ, Mezey E, Skirboll L 1984 Corticotropin-releasing factor-immunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy. Proc Natl Acad Sci USA 81:1854–1858[Abstract/Free Full Text]
  34. Gulyas M, Pusztai L, Rappay G, Makara GB 1991 Pituitary corticotrophs proliferate temporarily after adrenalectomy. Histochemistry 96:185–189[CrossRef][Medline]
  35. McNicol AM, Carbajo-Perez E 1999 Aspects of anterior pituitary growth, with special reference to corticotrophs. Pituitary 1:257–268[CrossRef][Medline]
  36. Stepien H, Karasek E, Pawlikowski M 1981 Effects of adrenalectomy and hydrocortisone on DNA synthesis in the rat anterior pituitary gland. Biochem Biophys Res Commun 100:1531–1536[CrossRef][Medline]
  37. Nolan LA, Thomas CK, Levy A 2003 Enhanced anterior pituitary mitotic response to adrenalectomy after multiple glucocorticoid exposures. Eur J Endocrinol 149:153–160[Abstract]
  38. Nolan LA, Levy A 2006 A population of non-luteinising hormone/non-adrenocorticotrophic hormone-positive cells in the male rat anterior pituitary responds mitotically to both gonadectomy and adrenalectomy. J Neuroendocrinol 18:655–661[CrossRef][Medline]
  39. Taniguchi Y, Tamatani R, Yasutaka S, Kawarai Y 1995 Proliferation of pituitary corticotrophs following adrenalectomy as revealed by immunohistochemistry combined with bromodeoxyuridine-labeling. Histochem Cell Biol 103:127–130[CrossRef][Medline]
  40. Nolan LA, Levy A 2003 Temporally sensitive trophic responsiveness of the adrenalectomized rat anterior pituitary to dexamethasone challenge: relationship between mitotic activity and apoptotic sensitivity. Endocrinology 144:212–219[Abstract/Free Full Text]
  41. Rozengurt E, Legg A, Pettican P 1979 Vasopressin stimulation of mouse 3T3 cell growth. Proc Natl Acad Sci USA 76:1284–1287[Abstract/Free Full Text]
  42. Hunt NH, Perris AD, Sandford PA 1977 Role of vasopressin in the mitotic response of rat bone marrow cells to haemorrhage. J Endocrinol 72:5–16[Abstract]
  43. Russell WE, Bucher NL 1983 Vasopressin modulates liver regeneration in the Brattleboro rat. Am J Physiol 245:G321–G324
  44. Ghosh PM, Mikhailova M, Bedolla R, Kreisberg JI 2001 Arginine vasopressin stimulates mesangial cell proliferation by activating the epidermal growth factor receptor. Am J Physiol Renal Physiol 280:F972–F979
  45. Lagumdzija A, Bucht E, Stark A, Hulting AL, Petersson M 2004 Arg-vasopressin increases proliferation of human osteoblast-like cells and decreases production of interleukin-6 and macrophage colony-stimulating factor. Regul Pept 121:41–48[CrossRef][Medline]
  46. van Wijk PA, van Neck JW, Rijnberk A, Croughs RJ, Mol JA 1995 Proliferation of the murine corticotropic tumour cell line AtT20 is affected by hypophysiotrophic hormones, growth factors and glucocorticoids. Mol Cell Endocrinol 111:13–19[CrossRef][Medline]
  47. McNicol AM, Murray JE, McMeekin W 1990 Vasopressin stimulation of cell proliferation in the rat pituitary gland in vitro. J Endocrinol 126:255–259[Abstract]
  48. Thibonnier M, Preston JA, Dulin N, Wilkins PL, Berti-Mattera LN, Mattera R 1997 The human V3 pituitary vasopressin receptor: ligand binding profile and density-dependent signaling pathways. Endocrinology 138:4109–4122[Abstract/Free Full Text]
  49. Nolan LA, Thomas CK, Levy A 2004 Pituitary mitosis and apoptotic responsiveness following adrenalectomy are independent of hypothalamic paraventricular nucleus CRH input. J Endocrinol 181:521–529[Abstract]
  50. Buma P, Nieuwenhuys R 1988 Ultrastructural characterization of exocytotic release sites in different layers of the median eminence of the rat. Cell Tissue Res 252:107–114[Medline]
  51. Holmes MC, Antoni FA, Aguilera G, Catt KJ 1986 Magnocellular axons in passage through the median eminence release vasopressin. Nature 319:326–329[CrossRef][Medline]
  52. Antoni FA, Fink G, Sheward WJ 1990 Corticotrophin-releasing peptides in rat hypophysial portal blood after paraventricular lesions: a marked reduction in the concentration of corticotrophin-releasing factor-41, but no change in vasopressin. J Endocrinol 125:175–183[Abstract]
  53. Lolait SJ, Stewart LQ, Jessop DS, Young III WS, O’Carroll AM 2007 The hypothalamic-pituitary-adrenal axis response to stress in mice lacking functional vasopressin V1b receptors. Endocrinology 148:849–856[Abstract/Free Full Text]
  54. Tankosic P, Burlet A, Jegou S, Chateau M, Vaudry H, Burlet C, Boulange M 1982 Fetal and postnatal maturation of corticotrope function in the vasopressin-deficient rat (Brattleboro strain): a radioimmunological, immunocytochemical, and morphometric study. Ann NY Acad Sci 394:560–573[CrossRef][Medline]
  55. Schmale H, Richter D 1984 Single base deletion in the vasopressin gene is the cause of diabetes insipidus in Brattleboro rats. Nature 308:705–709[CrossRef][Medline]
  56. Denef C, Pals K, Hauspie A, Vankelecom H, Seuntjens E 2005 Combinatorial expression of phenotypes of different cell lineages in the rat and mouse pituitary. Ann NY Acad Sci 1040:84–88[Abstract/Free Full Text]
  57. Nolan LA, Levy A 2001 Anterior pituitary trophic responses to dexamethasone withdrawal and repeated dexamethasone exposures. J Endocrinol 169:263–270[Abstract]
  58. Ferrara N, Schweigerer L, Neufeld G, Mitchell R, Gospodarowicz D 1987 Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5773–5777[Abstract/Free Full Text]
  59. Ferrara N, Henzel WJ 1989 Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161:851–858[CrossRef][Medline]
  60. Vankelecom H, Matthys P, Van Damme J, Heremans H, Billiau A, Denef C 1993 Immunocytochemical evidence that S-100-positive cells of the mouse anterior pituitary contain interleukin-6 immunoreactivity. J Histochem Cytochem 41:151–156[Abstract]
  61. Gospodarowicz D, Lau K 1989 Pituitary follicular cells secrete both vascular endothelial growth factor and follistatin. Biochem Biophys Res Commun 165:292–298[CrossRef][Medline]
  62. Ceccatelli S, Hulting AL, Zhang X, Gustafsson L, Villar M, Hokfelt T 1993 Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc Natl Acad Sci USA 90:11292–11296[Abstract/Free Full Text]
  63. Traverso V, Christian HC, Morris JF, Buckingham JC 1999 Lipocortin 1 (annexin 1): a candidate paracrine agent localized in pituitary folliculo-stellate cells. Endocrinology 140:4311–4319[Abstract/Free Full Text]
  64. Kurotani R, Tahara S, Sanno N, Teramoto A, Mellon PL, Inoue K, Yoshimura S, Osamura RY 1999 Expression of Ptx1 in the adult rat pituitary glands and pituitary cell lines: hormone-secreting cells and folliculo-stellate cells. Cell Tissue Res 298:55–61[CrossRef][Medline]
  65. Inoue K, Mogi C, Ogawa S, Tomida M, Miyai S 2002 Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells? Arch Physiol Biochem 110:50–53[CrossRef][Medline]
  66. Horvath E, Kovacs K 2002 Folliculo-stellate cells of the human pituitary: a type of adult stem cell? Ultrastruct Pathol 26:219–228[CrossRef][Medline]
  67. Chen J, Hersmus N, Van D, V, Caesens P, Denef C, Vankelecom H 2005 The adult pituitary contains a cell population displaying stem/progenitor cell and early embryonic characteristics. Endocrinology 146:3985–3998[Abstract/Free Full Text]
  68. Krylyshkina O, Chen J, Mebis L, Denef C, Vankelecom H 2005 Nestin-immunoreactive cells in rat pituitary are neither hormonal nor typical folliculo-stellate cells. Endocrinology 146:2376–2387[Abstract/Free Full Text]
  69. Rotondo F, Kovacs K, Horvath E, Bell CD, Lloyd RV, Scheithauer BW 2006 Immunohistochemical expression of nestin in the nontumorous hypophysis and in pituitary neoplasms. Acta Neuropathol (Berl) 111:272–277[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
A. H. Veenema, S. O. Reber, S. Selch, F. Obermeier, and I. D. Neumann
Early Life Stress Enhances the Vulnerability to Chronic Psychosocial Stress and Experimental Colitis in Adult Mice
Endocrinology, June 1, 2008; 149(6): 2727 - 2736.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3102    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Subburaju, S.
Right arrow Articles by Aguilera, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Subburaju, S.
Right arrow Articles by Aguilera, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.