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Endocrinology Vol. 144, No. 10 4403-4409
Copyright © 2003 by The Endocrine Society

Autocrine/Paracrine Action of Pituitary Vasoactive Intestinal Peptide on Lactotroph Hyperplasia Induced by Estrogen

Oscar Gómez and José Antonio Balsa

Servicio de Endocrinología, Hospital Ramón y Cajal, 28034 Madrid, Spain

Address all correspondence and requests for reprints to: José A. Balsa, M.D., Ph.D., Servicio de Endocrinología, Hospital Ramón y Cajal, Carretera de Colmenar Km 9, 28034 Madrid, Spain. E-mail: jbalsa.hrc{at}salud.madrid.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vasoactive intestinal polypeptide (VIP) content is increased in the hyperplastic pituitaries of estrogen (E)-treated rats, thus suggesting that this neuropeptide could mediate the E effect on lactotrophs. E also decreases pituitary TGF-ß1 content, an autocrine/paracrine inhibitor of lactotroph proliferation, and induces pituitary angiogenesis. To elucidate the role of VIP in this context, lactotroph hyperplasia was induced in female Fisher 344 rats by implanting sc pellets of diethylstilbestrol (DES). Twenty-five days later, the rats were treated with three different increasing doses of a VIP receptor antagonist or the vehicle for 5 d. DES treatment resulted in a marked increase of serum prolactin (PRL), pituitary PRL content, PRL mRNA expression, pituitary weight, and pituitary proliferating cell nuclear antigen. DES treatment also increased pituitary VIP content and VIP mRNA levels, but not in the hypothalamus and cerebral cortex. Simultaneously, DES treatment decreased the pituitary TGF-ß1 content and increased the pituitary content of vascular endothelial growth factor. VIP receptor antagonist partially reverted the effect of DES on serum PRL and pituitary PRL, proliferating cell nuclear antigen, TGF-ß1, and vascular endothelial growth factor contents, as well as on pituitary weight, in a dose-dependent relation. These data suggest that pituitary VIP mediates the effect of E on lactotroph hyperplasia, pituitary TGF-ß1, and angiogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS LONG been recognized that estrogen (E) induces lactotroph hyperplasia and simultaneous hyperprolactinemia in rats in a time- and dose-response relationship and finally results in induction of prolactinomas (1, 2, 3). The most widely used strain for studying the mitogenic action of E at the pituitary is the Fisher 344 (F344) as its lactotrophs are specially sensitive to this action. The mechanisms of induction at which E acts to promote lactotroph hyperplasia and tumorigenesis are not fully understood. Several growth factors and neuropeptides expressed at the pituitary such as TGF-ß1 (4), TGF-ß3 (5), basic fibroblast growth factor (6), or Galanin (7, 8), as well as disruption of the dopaminergic axis (9, 10) have been demonstrated to be involved in the mediation of the E proliferative effect on lactotrophs. Of these factors, only TGF-ß1 has an antiproliferative action exerted through an autocrine and/or paracrine way.

Vasoactive intestinal polypeptide (VIP), a neuropeptide that activates the adenylate cyclase-cAMP pathway (11), is synthesized in the cerebral cortex, hypothalamus and anterior pituitary, as well as in other tissues. It has been shown that hypothalamic VIP may act as a PRL-releasing factor during lactation (12). On the other hand, both we and other authors have demonstrated the role of pituitary VIP as an autocrine/paracrine modulator of both basal and stimulated PRL release in vitro (13, 14, 15, 16, 17, 18). Chronic E treatment has also been reported to increase pituitary VIP content and VIP mRNA expression without modifying these parameters in the hypothalamus and cerebral cortex (19, 20). However, a previous study had found that chronic E treatment for 2 wk increased VIP content both in the pituitary and median eminence, although the effect on the pituitary was clearly larger than on the median eminence (21).

It has been shown that after chronic E treatment there is an overall increase in the amount of blood flowing through the hyperplastic gland that may be explained by newly formed supplying vessels (22). The process of angiogenesis is essential for the growth of experimentally E induced pituitary hyperplasia (23). Vascular endothelial growth factor (VEGF) is one of the several regulators of angiogenesis that have been identified in different tumoral and hyperplastic tissues (24). Increased VEGF expression has been also described in the pituitary after chronic E treatment (25).

For a better understanding of the VIP involvement in the development of lactotroph hyperplasia, angiogenesis and hyperprolactinemia induced by long-term treatment with E, as well as the VIP and TGF-ß1 interaction, we performed the following study.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal preparation
Female 5-wk-old F344 rats were housed in a room equipped with a regulated 12-h light, 12-h dark photoperiod and a temperature maintained at 23 ± 2 C. The rats were routinely housed, five per cage, and allowed water and Purina Rat Chow (Purina Rat Chow 5001, Purina Mills, St. Louis, MO) ad libitum. The rats were ovariectomized under anesthesia (90 mg/kg ketamine, administered ip) to reduce any variability due to changing serum E levels during the estrous cycle. A sc pellet containing diethylstilbestrol (DES; 5 mg, 60 d release) (Innovative Research of America, Sarasota, FL) was simultaneously implanted in all but the control group. Twenty-five days thereafter, when serum PRL reached 200 ng/ml, the DES-implanted rats were randomized (n = 5) into four groups, and treated with increasing doses of a VIP-receptor antagonist (VA) (p-chloro-D-phe6, Leu17-VIP): 0 (vehicle); 0.015; 0.15 and 1.5 µg/g weight/d (Bachem, Basel, Switzerland). The control group (n = 7) was also injected with the vehicle. We chose this selective antagonist for VIP receptors (26) based on our previous experience with it in cultures of normal pituitary cells (16, 17, 18). To our knowledge, its selectivity for the two VIP receptors VPAC1 and VPAC2 (previously referred as PVR2 and PVR3) has not been completely characterized. The VA was administrated by twice daily ip injection for 5 d. Along this period, and 2 h after the early injection, the animals, while moving free in metacrylate box, were anesthetized with halotane (2%) and blood samples to assay sera PRL concentration were collected. This anesthesia was chosen after testing three anesthetic drugs—ether, chloroform, and halotane—in normal rats due to the absence of any stressing effect that could interfere with the serum PRL measurement. Mean serum PRL levels in control rats anesthetized with ether or chloroform were higher than 40 ng/ml, but they were lower than 10 ng/ml with halotane. Two hours after the last VA dose, the animals were decapitated under the same anesthesia and trunk blood, anterior pituitary, hypothalamus, and cerebral cortex were taken. Blood samples were conserved at -20 C. Pituitaries were weighed after freezing and finally stored at -80 C with the rest of tissues till their processing.

Western blot analysis
To evaluate the changes in the pituitary proliferating cell nuclear antigen (PCNA), TGF-ß1, and VEGF content, cellular proteins were isolated by sonication in 0.4 ml of buffer [0.01 M Tris-HCl (pH 7.0), 0.001 M EDTA (pH 8.0), 0.1 M NaCl, 1 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonyl fluoride], added from a stock solution just before the suspension buffer was used. Protein concentration was quantified by the Bradford method (27). Equal amounts of protein (25 µg per sample) were added to equal volumes of 2x sodium dodecyl sulfate gel loading buffer [0.125 M Tris-Cl (pH 6.8); 4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol] and 4 µl of 1% coomassie blue R-250 was used as stain. The samples were then placed in a boiling water bath for 5 min. Prestained SDS-PAGE Standard, Broad Range (Bio-Rad, Hercules, CA) range was used as molecular weight standard. The total volume sample per lane were passed through a 10% polyacrylamide gel at 100 V for 2 h at 22 C. Resolved proteins were transferred electrophoretically to Hybond P transfer membranes (Amersham Pharmacia Biotech, Amersham Biosciences, Amersham, Buckinghamshire, UK) at 4 C overnight at 24 V in a buffer containing 25 mmol/liter glycine, 192 mmol/liter Tris, and 20% methanol. Nonspecific binding of the antibodies to the membranes was diminished by preincubation of the blots in PBS 0.01 M in the presence of 5% skim milk and 1% Tween 20 for 1 h at room temperature. Blots were incubated with monoclonal mouse anti-PCNA (Dako A/S, Glostrup, Denmark) dilution 1:10,000 in PBS containing 0.5% skim milk and 0.1 Tween 20 at room temperature for 1 h and then with peroxidase conjugated goat antimouse IgGs (Dako) dilution 1:10,000 in the same buffer at room temperature for 1 h. After each incubation, blots were washed twice with PBS containing 0.1% Tween 20 for 10 min. To evaluate TGF-ß1 pituitary content polyclonal rabbit anti-TGF-ß1 (TGF-ß1 V; sc-146, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), dilution 1:2,000, and antirabbit IgG peroxidase conjugated (Pierce, Rockford, IL), dilution 1:10,000, were employed. Polyclonal goat anti-VEGF (VEGF-20, sc-183, Santa Cruz Biotechnology), dilution 1:2,000, and antigoat IgG peroxidase conjugated (sc2020, Santa Cruz Biotechnology), dilution 1:10,000, were used to evaluate pituitary VEGF content. Immunoreactive bands were visualized with the use of an enhancing chemiluminescent solution (ECL) plus Western blotting detection system (Amersham Pharmacia Biotech), films were then scanned and the optical density was determined by the National Institutes of Health Image 1.52 program.

RIAs
Pituitary, hypothalamus, and cortex samples were sonicated in 1 M acetic acid and incubated at 5 C for 2 h. After removing aliquots of 25 µl for protein determination, the samples were placed in boiling water for 10 min followed by centrifugation at 14,000 rpm for 15 min at 4 C. The supernatant fluids were stored at -20 C for subsequent VIP and PRL determination. Heparined blood samples were centrifuged and plasma stored at -20 C for subsequent PRL determination.

The VIP content in the pituitary, hypothalamus, and cortex samples was assayed as previously described (15) using an antiserum against porcine VIP, generously provided by Dr. L. Cacicedo (Ph.D., Servicio de Endocrinología, Hospital Ramón y Cajal). The pituitary PRL content as well as serum PRL were measured by RIA performed with materials generously provided by the National Pituitary Hormone Distribution Program (National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, MD).

RNA extraction and Northern hybridization
The total RNA extraction protocol used was that described by Chomczynski and Sacchi (28). Briefly, pituitary samples were homogenized in a buffer containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7), 0.5% N-laurylsarkosine sodium salt, and 0.7% ß-mercaptoethanol. Total RNA was isolated by repeated phenol chloroform extractions, and isopropanol precipitation. The quality of the RNA was monitored by the ratio of UV absorbance at 260 and 280 nm and samples were kept at -80 C until assayed. A260/A280 ratios were consistently 1.8–2.0, indicating that the samples were essentially free of contaminating protein.

Northern blot analysis and hybridization were performed with material and methods as previously described (16). To ensure that equal amounts of RNA were loaded and the transfer was quantitative, ribosomal 28S RNA was stained with ethidium bromide after transfer from agarose/formaldehyde gels to nylon membrane. PRL and VIP mRNA levels were quantitated by densitometric analysis of exposed x-ray film using a Color Image Scanner (Seiko, Nagano-Ken, Japan) and Adobe Photoshop 4.0 (Adobe Systems, Inc., San Jose, CA) and National Institutes of Health Image 1.52 programs.

Statistical analysis
Results are presented as mean ± SEM. Statistical analysis was made by two way ANOVA followed by Student-Newman-Keuls test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pituitary weight and PCNA content
Mean weight of pituitary glands in the DES-treated group was 2.5-fold greater than that of control rats at the end of the experiment: 8.7 ± 1.6 mg in the control group vs. 22.1 ± 3.3 mg in the DES-treated group (P < 0.05). VA treatment for 5 d partially inhibited the DES effect in a dose-response relationship and was statistically significant at 1.5 µg/g weight/d. At this dose, VA blocked the DES effect by 25.3% (Fig. 1Go).



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  		FIG. 1. Upper panel, Effect of VA on anterior pituitary weight in DES-treated F344 rats. Female 5-wk-old F344 rats were ovariectomized and sc DES pellet (5 mg; 60 d release) was simultaneously implanted in all but control group. Twenty-five days thereafter, the DES-treated rats were treated with increasing doses of VA (0, 0.015, 0.15, and 1.5 µg/g weight/d). Bottom panel, Effect of VA on anterior pituitary PCNA levels in DES-treated rats. Twenty-five micrograms of total protein were loaded to evaluate the pituitary PCNA protein expression by Western blot. Only the blots in which the load was homogeneous were estimated. Each load represents the pooled samples from three different rats. The blots were made using different pools. Each point represents the mean ± SEM from three independent blots. a, P < 0.05 compared with the control group. b, P < 0.05 compared with the VA 0 group after 5 d of VA treatment.

 
E increased pituitary PCNA levels by 250% (P < 0.05) and VA inhibited this effect in a dose-dependent response relationship. At 1.5 µg/g weight/d, VA blocked the DES effect by 90% (Fig. 1Go).

Serum PRL
Basal serum PRL levels were 6.9 ± 3.1 ng/ml and increased progressively in the rats bearing the DES pellet in a time-dependent relation. At the end of the experiment, serum PRL was approximately 100-fold greater than that of control rats (637 ± 126 ng/ml) (P < 0.05). VA at 1.5 µg/g weight/d inhibited the DES effect on serum PRL by 54.8% (288 ± 92) (P < 0.05), whereas no differences were observed when smaller doses of VA were injected (Fig. 2Go).



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  		FIG. 2. Effect of VA on serum PRL in DES-treated F344 rats. Rats were treated as detailed in Fig. 1Go. a, P < 0.05 compared with the VA 0 group after 5 d of VA treatment. The thick arrows indicate the day when the DES pellets were implanted and when the VA treatment was started. [{square}, Control; , DES + 0 µg VA/g weight/d; {diamond}, 0.015 µg VA/g weight/d; {Delta}, DES + 0.15 µg VA/g weight/d; {blacktriangledown}, DES + 1.5 µg VA/g weight·d.

 
Pituitary PRL content and PRL mRNA expression
DES treatment increased the pituitary PRL content from 12.7 ± 1.5 µg/mg total protein in the control group to 297 ± 39 µg/mg total protein. VA inhibited this effect in a dose-dependent response reaching statistical significant at a dose of 1.5 µg/g weight/d (206 ± 22; P < 0.05) (Fig. 3Go).



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  		FIG. 3. Upper panel, Effect of VA administration on anterior pituitary PRL content in DES-treated F344 rats. Bottom panel, Effect of VA administration on anterior pituitary VIP content in DES-treated F344 rats. Rats were treated as detailed in Fig. 1Go. a, P < 0.05 compared with the control group. b, P < 0.05 compared with the VA 0 group after 5 d of VA treatment.

 
Chronic DES treatment increased PRL mRNA expression by 237% (P < 0.05). VA did not change this effect although VA inhibited the DES effect by 18% at 1.5 µg/g weight/d dose (Fig. 4Go).



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  		FIG. 4. Northern blot analysis of the effect of VA administration on pituitary PRL mRNA levels in DES-treated F344 rats. Rats were treated as detailed in Fig. 1Go. Ten micrograms of total RNA were loaded per lane. Each sample represents the pooled total RNA of tissues from three rats. The blots were made using different pools. Autoradiographs were quantified by scanning densitometry and the values were normalized to the levels of 28S rRNA in each sample. a, P < 0.05 compared with the control group.

 
VIP content and VIP mRNA expression in anterior pituitary, hypothalamus, and cerebral cortex
DES treatment increased pituitary VIP content from 0.72 ± 0.24 ng/mg of total protein in the control group to 22.76 ± 3.06 ng/mg of total protein (P < 0.05). VIP content did not change in VA-treated rats (Fig. 3Go).

Pituitary VIP mRNA signal was almost undetectable by Northern blot in the control group. DES markedly increased (P < 0.05) the pituitary VIP mRNA expression, and this effect did not change after VA treatment (Fig. 5Go).



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  		FIG. 5. Northern blot analysis of the effect of VA administration on pituitary VIP mRNA levels in DES-treated F344 rats. Rats were treated as detailed in Fig. 1Go. The blot shown in the Fig. 4Go was rehybridized with the VIP probe. For details see Fig. 4Go. a, P < 0.05 compared with the control group.

 
Neither DES nor VA modified the VIP content and VIP mRNA levels in the hypothalamus and cerebral cortex (Figs. 6Go and 7Go).



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  		FIG. 6. Upper panel, Effect of VA treatment on hypothalamic VIP content in DES-treated F344 rats. Rats were treated as detailed in Fig. 1Go. Bottom panel, Effect of VA on hypothalamic VIP mRNA levels in DES-treated rats. 10 µg of total RNA were loaded per lane. Each sample represents the pooled total RNA of tissues from three rats. The blots were made using different pools. Each point represents the mean and SEM from two independent blots. Autoradiographs were quantified by scanning densitometry and the values were normalized to the levels of 28S rRNA in each sample.

 


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  		FIG. 7. Upper panel, Effect of VA treatment on cerebral cortex VIP content in DES-treated F344 rats. Rats were treated as detailed in Fig. 1Go. Bottom panel, Effect of VA on cerebral cortex VIP mRNA levels in DES-treated rats. Ten micrograms of total RNA were loaded per lane. Each sample represents the pooled total RNA of tissues from three rats. The blots were made using different pools. Each point represents the mean and SEM from three independent blots. Autoradiographs were quantified by scanning densitometry and the values were normalized to the levels of 28S rRNA in each sample.

 
Pituitary TGF-ß1 content
DES treatment completely blocked the pituitary TGF-ß1 signal assayed by Western blot by 93%. VA overcame this effect in a dose-dependent relationship. At 1.5 µg/g weight/d dose, TGF-ß1 signal was similar the control group (Fig. 8Go).



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  		FIG. 8. Upper panel, Western blot analysis of the effect of VA administration on anterior pituitary TGF-ß1 protein expression. Bottom panel, Western blot analysis of the effect of VA administration on anterior pituitary VEGF protein expression. Rats were treated as detailed in Fig. 1Go. Only the blots in which the load was homogeneous were estimated. Each load represents the pooled samples from three different rats. The blots were made using different pools. Each point represents the mean ± SEM from three independent blots. a, P < 0.05 compared with the control group. b, P < 0.05 compared with the VA 0 group after 5 d of VA treatment.

 
Pituitary VEGF content
Pituitary VEGF signal was almost undetectable by Western blot in the control group. DES markedly increased (P < 0.05) the pituitary VEGF expression and VA overcame this effect in a dose-dependent relationship. At 1.5 µg/g weight/d dose, VEGF signal was equivalent to the control group (P < 0.05) (Fig. 8Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we show that VA partially inhibits the lactotroph hyperplasia, evaluated by pituitary weight and pituitary PCNA levels, completely blocks the pituitary VEGF increase and overcomes the inhibition of pituitary TGF-ß1 induced by chronic E treatment. As shown by morphologic studies, the increase of pituitary growth of F344 rats treated with E is almost exclusively due to lactotroph hyperplasia (2, 3). On the other hand, PCNA, a nuclear protein that functions as cofactor of DNA polymerase {delta} and that is synthesized at a greater rate in the S phase of growing cells (29), represents a valuable approach for the evaluation of cellular proliferation in different tissues including the pituitary (30, 31, 32, 33, 34). Furthermore, the content of this nuclear antigen is increased in proliferating lactotrophs under E treatment (35). In this study, and in agreement with previous published papers (19, 20), we have found that E induces a tissue-specific VIP synthesis in the pituitary. Although different cells such as folliculo-stellate or thyrotrophs cells have been proposed as source of pituitary VIP (36, 37), both this peptide and its mRNA signals are increased in lactotrophs of rats under E treatment (38, 39). Although the in vivo study does not completely permit the exclusion of a possible block of VA on hypothalamic VIP, collectively, these data clearly support that the VA administrated ip specifically blocked the autocrine/paracrine VIP action on lactotroph proliferation, whereas hypothalamic VIP did not act as endocrine proliferating factor mediating the E effect.

VIP has been shown to regulate cell proliferation and differentiation in many cell types and, on the contrary, different VIP receptor antagonists inhibit the proliferation of several tumoral cell lines (40); therefore, blocking VIP receptors has been suggested as possible antitumoral treatment. In pituitary cultures, both the activation of the adenylate cyclase-cAMP pathway with forskolin or dibutyryl-AMP (41) and VIP (42), as recently demonstrated, induce lactotroph proliferation. These data support the role of VIP as a lactotroph proliferative factor and its mediator role of lactotroph proliferation induced by E as we are now reporting.

The inhibitory effect of VA on serum PRL does not necessarily mean an inhibition of PRL secretion because it might be secondary to the proliferation block. However, we believe that both the inhibition of lactotroph proliferation and PRL secretion account for the whole inhibitory effect on serum PRL because VIP antiserum inhibits the PRL release in vitro in pituitaries obtained from F344 rats treated with DES (43) and this antagonist also inhibits PRL release in vitro under different stimuli (15, 16, 17, 18). The fact that VA also decreased the pituitary PRL content when expressed per mg of total protein supports the double inhibition both on lactotroph proliferation and PRL secretion.

TGF-ß1 is known to inhibit cell growth and proliferation of many E-responsive normal and transformed cells (44, 45, 46). In the pituitary, E treatment reduces the expression and action of TGF-ß1 on lactotrophs (47), but on the contrary, this growth factor inhibits lactotroph proliferation and PRL secretion induced by E both in vivo and in vitro (48, 49). In agreement with these data, we found that pituitary TGF-ß1 content was decreased by chronic E treatment, overcoming its antiproliferative and antisecretory effect on lactotrophs, and that VA partially reverted the effect of E on TGF-ß1. These data clearly support the fact that the autocrine/paracrine action of VIP is an early mediator of the E-proliferative effect on lactotrophs and that at least another implicated autocrine factor such as TGF-ß1 is secondarily inhibited. Probably, both events in this chain, i.e. the increase of pituitary VIP and the inhibition of pituitary TGF-ß1, are necessary to trigger the lactotroph proliferation. Thus, as has been expounded above, VIP activates the proliferation of normal and tumoral cells through the adenylate cyclase-cAMP pathway increasing gene expression of c-fos, c-jun, and c-myc (40). On the other hand, the inhibition of pituitary TGF-ß1 is also necessary because intrapituitary administration of TGF-ß1 inhibits the lactotroph proliferation in hyperestrogenized rats (48). As previously commented, different neuropeptides and growth factors synthesized in the anterior pituitary have been propounded of being implicated in the E-induced lactotroph hyperplasia by acting in a autocrine or paracrine way. Moreover, paracrine modulation links between two of these factors have been previously demonstrated in the case of TGF-ß3 and fibroblast growth factor, which display a highly complex paracrine/autocrine regulatory mechanism in the effect of E on lactotroph (50).

In agreement with previous studies, we found that E increases VEGF content in the pituitary (25), and our data show that this effect is blocked by VA treatment. Earlier studies have also found that VEGF expression is increased through the cAMP pathway in MCF-7 breast carcinoma cells (51) and that cAMP can enhance the transcription of E-regulated genes in Chinese hamster ovary cells (52). All of these data plus the result now presented suggest that the E effect on the pituitary VEGF is mediated through pituitary VIP. In agreement with this hypothesis, it has been reported that the addition of VIP increases the VEGF mRNA expression in lung cancer cells in vitro (53). As E specifically increase VIP (38, 39) and decrease TGF-ß1 (54) in lactotrophs and VEGF has been only detected in folliculo-stellate cells in the normal pituitary (55), our data suggest that the VIP synthesized by the lactotrophs mediates the E-induced hyperplasia, and that both autocrine and paracrine actions are simultaneously triggered to induce lactotroph proliferation and angiogenesis, respectively.

Previous in vitro studies from our laboratory have shown that pituitary VIP mediates the PRL release induced by TRH, hypothyroidism, dopamine withdrawal, serotonin, and IGF-I (15, 16, 17, 18). Collectively, all these data plus the results now presented support a major role of VIP as autocrine or paracrine modulator of lactotroph physiology.

In summary, our data suggest that: 1) VIP mediates, at least partially, the lactotroph hyperplasia and hyperprolactinemia induced by E through an autocrine/paracrine action. 2) TGF-ß1, an autocrine/paracrine inhibitor factor of lactotroph response to E, is simultaneously down-regulated by pituitary VIP, displaying a multi-step sequence of mediation in the E-induced lactotroph proliferation at pituitary level. 3) Pituitary VIP mediates the E effect on pituitary VEGF expression and, consequently, on the E-induced angiogenesis in the hyperplastic pituitary. Further studies are necessary to elucidate whether other mediators of E proliferative and angiogenic action are also linked in a single chain of local regulation or, on the contrary, are simultaneously but independently activated.


   Footnotes
 
This work was supported by Grant 98/1342 from Fondo de Investigaciones Sanitarias.

Abbreviations: DES, Diethylstilbestrol; E, estrogen; PCNA, proliferating cell nuclear antigen; PRL, prolactin; VA, VIP receptor antagonist; VEGF, vascular endothelial growth factor; VIP, vasoactive intestinal polypeptide.

Received February 27, 2003.

Accepted for publication June 16, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. A. Blake, L. M. Brown, M. W. Duncan, S. W. Hunsucker, and S. M. Helmke
Estrogen Regulation of the Rat Anterior Pituitary Gland Proteome
Experimental Biology and Medicine, December 1, 2005; 230(11): 800 - 807.
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