Angiotensin II-induced Ca2+ mobilization and prolactin release in normal and hyperplastic pituitary cells

Graciela Díaz-Torga, Arturo González Iglesias, Rita Achával-Zaia, Carlos Libertun, and Damasia Becú-Villalobos

Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas V, Obligado 2490, (1428) Buenos Aires, Argentina

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We evaluated the effects of angiotensin II (ANG II) and its antagonists on prolactin release, intracellular calcium ([Ca2+]i) mobilization, and [3H]thymidine uptake in cells from normal rat pituitaries and from estrogen-induced pituitary tumors. ANG II (10-7 to 10-9 M) increased prolactin release significantly in control and not in tumoral cells. In control cells, ANG II (10-6 to 10-9 M) produced an immediate spike of [Ca2+]i followed by a plateau. Spike levels rose significantly between 10-10 and 10-8 M ANG II, whereas the onset of the spike was retarded with decreasing concentrations. In tumoral cells, ANG II did not produce a spike phase even at 10-6 M. ANG II-induced prolactin release and calcium mobilization were blocked by losartan (AT1 receptor antagonist) and not by PD-123319 (AT2 antagonist). Finally, [3H]thymidine uptake was not modified by ANG II (10-7 to 10-10 M) or its antagonists in either group. Our results suggest that chronic in vivo estrogenic treatment alters in vitro pituitary response to ANG II. Alterations might function to limit excessive prolactin secretion of hypersecreting tumors. Besides, ANG II does not modify DNA synthesis in vitro of cells from normal or tumor-derived hypophyses.

calcium; estrogen

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IT HAS BEEN DESCRIBED that all the components of the renin-angiotensin system (RAS) are present in the pituitary and that angiotensin II (ANG II) is produced locally (7). ANG II releases prolactin (PRL) both in vivo and in vitro (8), and specific ANG II receptors, belonging to the AT1 subtype, have been identified mainly in lactotrophs (18). In the anterior pituitary AT1 receptor, stimulation is coupled to phospholipase C-mediated hydrolysis of membrane phosphoinosites, leading to the formation of diacylglycerol and inositol phosphates and subsequently to the activation of protein kinase C (PKC) and to an increase in intracellular calcium ([Ca2+]i) levels (4, 21).

On the other hand, ANG II has been proposed to act as a growth factor in several tissues on the basis of its ability to stimulate protein synthesis and cell growth (29, 31). In fact, increases in both PKC and [Ca2+]i induced by ANG II have been shown to promote the expression of the growth-related immediate early genes such as c-fos, c-jun, and c-myc in smooth muscle cells (24). The role of ANG II on growth is complex, and the peptide has been postulated to modulate growth in concert with other promoters (17, 31). This feature would be of great importance in the regulation of the proliferation of malignant cells and in the progression of tumor growth, keeping in mind the potential ability of ANG II to accelerate angiogenesis (16). Although both subtypes of ANG II receptors (AT1 and AT2) have been shown to be expressed in tumoral and in developing tissues, there is increasing evidence that shows that distinct growth-modulating actions (proliferative or antiproliferative) of the octapeptide are coupled to different receptor subtypes (29).

Chronic administration of estrogens to rats induces enlargement of the anterior pituitary and increased synthesis and secretion of PRL. Histologically, tumors are composed of hyperplastic and hypertrophied lactotrophs, with involution of somatotrophs and gonadotropin-producing cells (6). Damage to hypothalamic dopaminergic neurons in response to estrogen has been described (26), and a direct action of estrogen at the pituitary level has also been suggested. For example, a high rate of DNA polymerase activity (19) and a description of an estrogen-responsive element in the 5'-flanking region of the PRL gene in the tumors (9) are consistent with direct stimulation of lactotroph proliferation by estrogens. In addition, estrogen-induced hyperplasia of the anterior pituitary is associated with the development of a direct arterial blood supply (10), and the mechanism of arterial growth might be analogous to the process of angiogenesis observed coincident with tumor formation.

Therefore, in view of the PRL-releasing action of ANG II and of its participation in cellular proliferation and in angiogenesis in several tissues, we attempted to elucidate if the octapeptide was involved in the regulation of prolactinomas induced by estradiol in rats. In this context we examined comparatively in dispersed adenohypophyseal cells from normal pituitaries and from estrogen-induced prolactinomas (tumors) the effects of ANG II and of antagonists of ANG II receptor subtypes on 1) PRL release as a measure of the secretory capacity of the adenohypophysis, 2) [Ca2+]i mobilization as this cation has been shown to participate both in hormonal secretion and in the trophic actions of ANG II, and 3) [3H]thymidine uptake to evaluate DNA synthesis.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Female 60-day-old Sprague-Dawley rats were housed in an air-conditioned room with lights on at 0700 and off at 1900. They had free access to laboratory chow and tap water. Pituitary tumors were induced by subcutaneous administration of estradiol-valerate (0.5 mg · kg body wt-1 · wk-1) for 6-7 wk. Rats in diestrus were used as controls. Pituitary weights at euthanasia were 7.8 ± 2.3 and 32.5 ± 7.2 mg for control and tumor group, respectively.

Drugs. ANG II was from Sigma (St. Louis, MO), losartan (DUP-753) was a gift from DuPont Merck (Wilmington, DE), and PD-123319-121b was a gift from Parke-Davis (Ann Arbor, MI). PD-123319 is similar to AT2 ligands PD-123177 and PD-121981 (27). Epidermal growth factor (EGF) was from Sigma.

Cell dispersion. Rats were killed by decapitation at 0900, and normal or tumoral pituitaries were removed on ice, separated from the neurointermediate lobe, and placed in chambers containing freshly prepared Krebs-Ringer bicarbonate buffer (KRBGA) without Ca2+ or Mg2+. Buffer containing 14 mM glucose, 1% bovine serum albumin (BSA, Sigma), 2% Eagle's minimum essential medium (MEM) amino acids (GIBCO, Buenos Aires, Argentina), and 0.025% phenol red was previously gassed for 15 min with 95% O2 and 5% CO2 and adjusted to pH 7.35-7.40. Buffer was filtered through a membrane (Nalgene) with a pore diameter of 0.22 µm. Hypophyses were washed three times with KRBGA and then cut in 1-mm pieces. Obtained fragments were washed and incubated in the same buffer containing 0.2% trypsin for 30 min at 37°C, 95% O2, and 5% CO2. They were then treated for two additional minutes with deoxyribonuclease I (Sigma, 1 mg/ml), and digestion was ended by adding 0.2% of newborn calf serum (GIBCO). Fragments were dispersed in individual cells by gentle trituration through siliconized Pasteur pipettes. Resulting suspension was filtered through a nylon gauze (160 µm) and centrifuged 10 min at 1,200 g. Before centrifugation, an aliquot of cellular suspension was taken to quantify hypophyseal cell yield, using a Neubauer chamber. Viability of cells determined by trypan blue was always >95%.

Cell cultures. The cell pellet from tumoral or control rats was resuspended in a Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% horse serum, 2.5% fetal calf serum, 1% MEM nonessential amino acids, 25,000 U/l of micostatin and 25 ng/l gentamicin. Cells were plated in sterile tissue culture plates (Corning, Cluster 96; 60,000 cells/well) and incubated with 300 µl DMEM (GIBCO) in a metabolic incubator at 37°C with 5% CO2 and 95% O2. After long-term incubation (96 h), cells were washed twice with DMEM [with addition of F-12 nutrient mixture (GIBCO), 2.2 g/l CO3HNa, and 0.1% BSA] to remove all traces of serum. Experimental incubations were performed in 300 µl DMEM alone (controls) or with different combinations of the pharmacological agents (ANG II 1 × 10-6 to 1 × 10-10 M, losartan 1 × 10-6 M, PD-123319 1 × 10-6 M, in quadruplicate). For analysis of PRL secretion, samples were taken at 30 min of incubation period. They were subsequently stored at -20°C until analyzed by radioimmunoassay after appropriate dilution with 0.01 M phosphate-buffered saline with 1% egg albumin. Experiments were repeated five times. Time and concentrations were chosen according to our previous experience (3)

Radioimmunoassays. PRL was measured by radioimmunoassay using kits provided by the National Institute of Diabetes and Digestive and Kidney Diseases. Results are expressed in terms of PRL reference preparation. Intra- and interassay coefficients of variation were 7.2 and 12.8%, respectively.

[Ca2+]i measurements. Fura 2-acetoxymethyl ester (fura 2-AM, Sigma) was used as a fluorescent indicator. In preliminary experiments, studies on calcium mobilization were carried out in cells that had been cultured in identical conditions as those in experiments of secretion and [3H]thymidine incorporation. Because no differences in calcium response both in control and tumoral cells were observed using freshly isolated cells, this last methodology was chosen. The pellet of adenohypophyseal cells of each experimental group was redispersed and incubated in a buffered saline solution [BSS (in mM): 127 NaCl, 5 KCl, 0.5 KH2PO4, 5 NaHCO3, 1.8 CaCl2, 2 MgCl2, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; pH 7.5] in the presence of 1.5 µM fura 2-AM, 10 mM glucose, and 0.1% BSA. Cells were incubated for 30 min at 37°C in an atmosphere of 5% CO2, the time during which fura 2-AM was incorporated into the cells and converted by endogenous esterases to the fluorescent indicator fura 2 in the cellular cytoplasm. Cells were then washed twice in BSS without fura 2-AM and prepared at a density of 1.7-2 × 106 cells/ml. Fluorescence was measured in a spectrofluorometer (Jasco, Tokyo, Japan) provided with the accessory CA-261 to measure Ca2+ with continuous stirring, thermostat adjusted to 37°C, and injection chamber. [Ca2+]i levels were registered every second by exposure to alternating 340- and 380-nm light beams, and the intensity of light emission at 505 nm was measured. In this way, light intensities and their ratio (F340/F380) were followed. Drugs (5 µl) were injected into the chamber as a 100-fold concentrated solution. ANG II (concentration in chamber 10-5 to 10-10 M) was administered at minute 2. When the effect of ANG II antagonists was tested, losartan (AT1 specific) or PD-123319 (AT2 specific) was added (final concentration: 10-7 M) 1 min before the ANG II stimulus (final concentration: 10-9 M). The preparation was calibrated, determining maximal fluorescence induced by Triton X-100 0.1% and minimal fluorescence in the presence of 5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH adjusted to >8.3). [Ca2+]i was calculated according to Grynkiewicz et al. (13). Basal values were considered as those measured during the 1st min of the experiment. Values were corrected for dye leakage and autofluorescence. Resulting graphs were scanned, processed, and quantified using software Ungraph 2.0, and Excel 5.0. Results were normalized with respect to average basal levels (between 90 and 120 s).

DNA synthesis. Culture procedure was the same as described in Cell cultures. [3H]thymidine (0.2 µCi/well; DuPont NEN, 87.7 Ci/mmol) was added to cultures after addition of stimuli (ANG II 10-7 to 10-10 M and EGF 20 ng/ml). After 24 h of incubation, medium was discarded and the cells were removed and lysed by treatment with 0.05% trypsin and 0.02% EDTA in deionized water. Twenty minutes later the reaction was stopped by filtering under vacuum through GF/C Whatman filters using the Nunc Cell Harvester 8. After five washes with deionized water, the filters were placed in plastic vials with 3 ml of scintillation solution and radioactivity was counted in a Beckman counter. Each experiment was repeated four times.

Statistical analyses. Hormone secretion and [3H]thymidine uptake results were analyzed by two-way analysis of variance (ANOVA) for the effects of group (control or tumor) and drug. If F of interaction was found to be significant (P < 0.05), individual means were compared by Scheffé's test; if it was not significant, groups of means were analyzed by the same test.

For calcium experiments means of peak values (maximum levels achieved in the first 40 s after application of stimulus) and latency to the peak (seconds elapsed from stimulus to peak values) were analyzed by one-way ANOVA.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of ANG II on PRL secretion. At 30 min of incubation, basal PRL release was greater in tumoral than in control cells (143.0 ± 43.3 vs. 60.6 ± 15.1 ng/well, P < 0.05).

The effect of ANG II on PRL secretion differed between groups. ANG II released PRL significantly at the concentrations 1 × 10-7 to 1 × 10-9 M in control cells, whereas with the use of two-way ANOVA no significant effect was evident in tumoral cells at any concentration used (Fig. 1).


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Fig. 1.   Percentage of prolactin release induced by angiotensin II (ANG II) at different concentrations in control and tumoral cells. * P < 0.05 vs. respective control without ANG II (basal levels: 143.0 ± 43.3 and 60.6 ± 15.1 ng/well in control and tumoral cells, respectively, P < 0.05).

When we tested the effect of 1 × 10-8 and 10-9 M thyrotropin-releasing hormone (TRH), we found that the percent response was similar in both groups (control cells: 144.8 ± 9.8 and 140.2 ± 12.6%; tumoral cells: 142.5 ± 7.6 and 137 ± 6.7% for the concentrations of 10-8 and 10-9 M, respectively). This indicated that, even though basal levels in tumoral cells were high, they could respond to specific releasing stimuli.

ANG II (1 × 10-8 M)-induced PRL release in control cells was inhibited by losartan 1 × 10-6 M and not by PD-123319 1 × 10-6 M (Fig. 2). In control and tumoral cells neither losartan nor PD-123319 modified basal release of PRL, suggesting that ANG II does not participate in basal hormone release in control or tumoral cells.


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Fig. 2.   Effect of ANG II antagonists on basal and ANG II (10-8 M)-induced prolactin release in control and tumoral cells. Los, losartan (10-6 M), AT1 receptor antagonist; PD, PD-123319 (10-6 M), AT2 receptor antagonist. *P < 0.05 vs. respective control.

Effect of ANG II on [Ca2+]i mobilization. Changes in [Ca2+]i induced by ANG II (1 × 10-6 M and 1 × 10-8 to 1 × 10-10 M) were monitored in a suspension of dispersed pituitary control or tumoral cells. We used freshly dispersed adenohypophyseal cells, considering that ANG II receptors are mainly located in lactotrophs and secondarily in corticotrophs, which constitute <5% of hypophyseal cell population. Basal [Ca2+]i was 180.10 ± 8.67 and 217.13 ± 18.30 nM, respectively. The response evoked in control cells was significantly greater and markedly different from that in tumoral cells.

As illustrated in Fig. 3, ANG II-induced rise of [Ca2+]i was biphasic in control cells. At all concentrations except 10-10 M, an immediate and transient spike was observed during the 1st min, followed by a prolonged plateau of elevated [Ca2+]i, which was still above basal values 4 min after addition of the stimulus. The effect of ANG II on Ca2+ mobilization was concentration dependent. The spike phase of [Ca2+]i response rose with increasing concentrations of ANG II (Fig. 4, left), and response at 10 nM was already maximal. Latency to [Ca2+]i peak was also concentration dependent; the onset of the calcium spike was retarded with decreasing concentrations of ANG II (Fig. 4, right).


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Fig. 3.   Intracellular calcium ([Ca2+]i) mobilization induced by ANG II at different concentrations in control and tumoral cells. Basal [Ca2+]i was 180.10 ± 8.67 and 217.13 ± 18.30 nM, respectively (P > 0.05). Curves represent the average of 5-6 experiments (percent increase vs. basal values), and lines on top represent SE for each point. ANG II was administered at minute 2.


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Fig. 4.   Left: maximum spike values (in %) after administration of different concentrations of ANG II in control cells. Right: latency to [Ca2+]i peak (in seconds) in relation to increasing concentrations of ANG II in control cells.

On the other hand, the pattern of ANG II-induced [Ca2+]i mobilization in tumoral cells was different (Fig. 3). ANG II was unable to produce the spike phase even at concentrations of 10-6 and 10-5 M (last concentration not shown). The cells exhibited an initial [Ca2+]i rise that reached a sustained plateau, which remained invariable for the period tested. The ANG II calcium mobilizing ability was also concentration dependent in tumoral cells with regard to the delay in onset of plateau levels (At 0.1, 1, 10, 1,000, and 10,000 nM latency of plateau was 54.2 ± 3.7, 35.4 ± 2.7, 30.1 ± 3.4; 22.7 ± 0.8 and 27.1 ± 2.1 s). Plateau levels were not significantly different among concentrations.

Despite clear differences in ANG II-induced [Ca2+]i mobilization patterns between control and tumoral cells, responses in both groups were blocked by previous addition of losartan (10-7 M), the AT1 receptor specific antagonist, and not by the AT2 receptor specific antagonist PD-123319 (10-7 M) (Fig. 5), indicating both responses were mediated by the same receptor subtype. Neither losartan nor PD-123319 modified per se [Ca2+]i during minute 1-2.


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Fig. 5.   Effect of ANG II antagonists on [Ca2+]i mobilization induced by ANG II (10-9 M) in control and tumoral cells.

Effect of ANG II on cell DNA synthesis. No significant effect of ANG II (at the doses of 10-7 to 10-10 M) or of the antagonists (10-7 M) on cell DNA synthesis was observed in either group (Fig. 6). Combinations of ANG II and both antagonists were equally ineffective (data not shown). EGF used as positive control increased [3H]thymidine uptake, and the effect was significantly greater in tumoral cells.


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Fig. 6.   Effect of ANG II (10-8 to 10-10 M) and losartan 10-6 M (L-6M) and PD-123319 10-6 M (P-6M) on percent [3H]thymidine uptake by control and tumoral cells. Epidermal growth factor (EGF) was used as a positive control of DNA synthesis. * P < 0.05 vs. respective control without drug (100%). Basal levels (counts · min-1 · 60,000 cells-1) were 1,584.4 ± 359.4 (n = 7) and 2,087.7 ± 220.0 (n = 7; P > 0.05)

    DISCUSSION
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Materials & Methods
Results
Discussion
References

It is well documented that ANG II releases PRL by interaction with pituitary AT1 receptor through a calcium-dependent process. The initial spike response of Ca2+ mobilization induced by ANG II is independent of Ca2+ influx to the cell and reflects Ca2+ release from the endoplasmic reticulum induced by inositol trisphosphate (IP3), whereas the plateau phase requires Ca2+ entry to the cells mainly through voltage-sensitive calcium channels (VSCC). In the present experiments we first studied in detail the concentration-related response of [Ca2+]i to ANG II in adenohypophyseal control cells and describe an increase in spike magnitude and a decrease in latency to spike with increasing concentrations of ANG II. It has been described that ANG II increased IP3 formation already at 10 s (4), in good correlation with our results in which [Ca2+]i spikes were demonstrated at 14-41 s, depending on the concentration used. Latency between arrival of the agonist and the onset of the calcium response was particularly long at low agonist concentrations and may be explained by the time required to generate sufficient IP3 to release [Ca2+]i. This phenomenon has also been described for TRH acting on lactotrophs (1).

In cells from tumor-bearing rats the pattern of response was altered. ANG II did not evoke clear spike elevations of [Ca2+]i even at high concentrations. In correlation, the sensitivity of PRL release in response to different concentrations of ANG II was lower in this group, even though ANG II produced a plateau rise in [Ca2+]i levels. These results enhance the importance of the transient spike phase of Ca2+ mobilization in relation to hormone release. PRL secretion can be achieved by increasing Ca2+ entry through VSCC, but it has been shown that only the initial burst in [Ca2+]i correlated with TRH-stimulated PRL production (25) and that, in gonadotropes, inhibition of this burst by gonadotropin-releasing hormone (GnRH) pretreatment prevented luteinizing hormone release in response to a subsequent pulse of GnRH (30). On the other hand, altered sensitivity of the associated PRL-releasing effect of ANG II was specific and did not merely reflect an incapacity of the hypersecreting cell to increase secretion even further. This was evidenced using TRH, which evoked a marked PRL release in tumoral as well as in control cells. It has been described that acute in vitro treatment with estrogen did not alter PRL response to ANG II (5). Therefore, it is probable that, in pituitaries that have been administered estrogen for 6 wk, there are marked alterations that cannot be detected after only 48 h of treatment.

Estrogens can modulate many aspects of the peripheral and pituitary RAS. They increase plasma angiotensinogen levels, enhance angiotensinogen mRNA in the liver, brain, and pituitary (15), and, what is more important, ANG II receptor number in the pituitary fluctuates during the estrous cycle, with highest binding in diestrus and lowest in estrus (27). This feature is in accordance with the finding that estrogen treatment reduces ANG II receptor number in the hypophysis, as measured by binding studies and by autoradiography (5, 18, 27). Nevertheless, changes in receptor number described range from two- to ninefold, and the decrease in sensitivity of ANG II-induced PRL release and Ca2+ mobilization is greater. Therefore, we cannot conclude that differences found are simply due to changes in receptor number. Reduced efficiency of Ca2+ mobilization by D-myo-inositol 1,4,5-trisphosphate, as described in homologous desensitization to GnRH (22), or another mechanism could also participate. The decrease in ANG II receptors coupled to altered [Ca2+]i mobilization and hormone release, which we describe, could represent a safety mechanism to control excessive PRL secretion when the effect of the major inhibitory neurohormone, dopamine, is reduced.

On the other hand, the modification in [Ca2+]i mobilization could reflect an overall altered Ca2+ metabolism in tumoral cells. Elevation of cytosolic free calcium plays an important role in the regulation of growth, participating in gene expression and cell division. For example, ANG II evoked alterations in [Ca2+]i in human lung adenocarcinoma cell lines and not in a normal lung cell line (2). In contrast, in our cells from pituitary tumors ANG II-induced [Ca2+]i mobilization was reduced, or at least markedly altered. In several studies, tumoral or clonal cell lines are used to evaluate the effects of neurohormones on calcium metabolism (11, 23). The present results indicate that tumor cell lines are not always adequate models to study calcium dynamics in pituitary cells. On the other hand, alterations in calcium metabolism in tumoral cells were not associated with changes in DNA synthesis in the present experiments, as revealed by the studies of [3H]thymidine uptake, but were probably associated with alterations in hormone secretion.

It has been well documented that estrogen treatment produces not only hyperprolactinemia, but also cell proliferation of lactotrophs and arteriogenesis. ANG II increases protein synthesis and promotes cell growth in a number of cells, including fibroblasts, vascular smooth muscle cells, adrenocortical cells, and myocardial cells, by a mechanism involving PKC activation, increase in [Ca2+]i levels, and induction of c-fos (12, 31). It also participates in angiogenesis (16). These findings inspired us to investigate the effects of ANG II on DNA synthesis in control and tumoral pituitary cells. Results indicate that in this experimental model ANG II is not a mitogenic agent in the pituitary. In contrast, it has been described in estrogen-induced pituitary hyperplasia that ANG II at the concentration of 10-10 M and not at higher or lower concentrations induced pituitary cell proliferation, which was not inhibited either by losartan or by PD-123177 (20). Differences in the experimental model (rat strain, estrogenic dose, or time of treatment) might account for discrepancies encountered. Besides, it has been described that ANG II-induced hypertrophy in renal proximal tubular cells depends on previous ANG II induction of TGF-beta (31) and that, in vascular smooth muscle cells, ANG II requires the presence of a competence growth factor, such as platelet-derived growth factor, or of EGF and fibroblast growth factor to exert its mitogenic effect (17). Therefore, we cannot rule that in the hypophysis ANG II may induce proliferation acting in conjunction with other growth factors or at longer periods of time.

Nevertheless, from the present results the participation of ANG II in transformed adenohypophyseal cells cannot be excluded, because it has been described that certain genes are expressed only in malignant phenotype in rat pituitary tumors (28).

The growth modulatory actions (proliferative or antiproliferative) of ANG II depend on the type of ANG II receptor present on a given cell under physiological conditions (29). For example, in coronary endothelial cells the growth-promoting effects mediated by the AT1 receptor were offset by the antiproliferative actions of the AT2 receptor and could therefore be unmasked only blocking AT2 receptors with PD-123177 (29). In the anterior pituitary the receptor subtype described is the AT1 (18), but, as in certain tissues that undergo tumorigenesis, there is induction of different ANG II receptor subtypes (14). We evaluated the effect of ANG II antagonists of receptor subtypes alone and in combination with ANG II on basal DNA synthesis, PRL release, and [Ca2+]i mobilization. Neither antagonist per se had any effect on DNA synthesis, hormone release, or basal [Ca2+]i, and the positive actions of ANG II were blocked only by losartan and not by PD-123319. From these results it can be implied that in tumoral pituitaries the receptor subtype present is the AT1 as in the normal pituitary.

In conclusion, we have characterized the concentration-related response of [Ca2+]i to ANG II in control adenohypophyseal cells in correlation with PRL secretion induced by the secretagogue. We also describe that chronic in vivo treatment with estrogens alters the response of pituitary cells to ANG II in vitro. There is a decrease in [Ca2+]i mobilization induced by the octapeptide in correlation with a decreased sensitivity to the PRL-releasing effect of the same. These alterations might function to limit the magnitude of PRL secretion of the hypersecreting tumors. On the other hand, there is no modification in the subtype of receptor to ANG II (AT1) involved in such responses. Finally, ANG II does not modify in vitro DNA synthesis of cells from normal or tumor-derived hypophyses.

    ACKNOWLEDGEMENTS

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina; Fundación Antorchas and Universidad de Buenos Aires.

    FOOTNOTES

Address for reprint requests: C. Libertun, Instituto de Biología y Medicina Experimental, CONICET V, Obligado 2490, (1428) Buenos Aires, Argentina.

Received 10 July 1997; accepted in final form 6 November 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Endocrinol Metab 274(3):E534-E540
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