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Endocrinology Vol. 145, No. 4 2064-2070
Copyright © 2004 by The Endocrine Society

Long-Term Treatment of Anterior Pituitary Cells with Nitric Oxide Induces Programmed Cell Death

Miguel Omar Velardez, Ariel Hernán Poliandri, Jimena Paula Cabilla, Cristian Carlos Armando Bodo, Leticia Inés Machiavelli and Beatriz Haydeé Duvilanski

Centro de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires C1121ABG, Argentina

Address all correspondence and requests for reprints to: Beatriz H. Duvilanski, Ph.D., Centro de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155, Piso 10, Buenos Aires C1121ABG, Argentina. E-mail: neuroend{at}fmed.uba.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) plays a complex role in modulating programmed cell death. It can either protect the cell from apoptotic death or mediate apoptosis, depending on its concentration and the cell type and/or status. In this study, we demonstrate that long-term exposition to NO induces cell death of anterior pituitary cells from Wistar female rats. DETA NONOate (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, 1 mM], a NO donor that releases NO for an extended period of time, decreased cellular viability and prolactin release from primary cultures of anterior pituitary cells. Morphological studies showed an increase in the number of cells with chromatin condensation and nuclear fragmentation at 24 and 48 h after DETA/NO exposure. DNA internucleosomal fragmentation was also observed at the same time. Reversibility of the NO effect on cellular viability and prolactin release was observed only when the cells were incubated with DETA/NO for less than 6 h. Most apoptotic cells were immunopositive for prolactin, suggesting a high susceptibility of lactotrophs to the effect of NO. The cytotoxic effect of NO is dependent of caspase-9 and caspase-3, but seems to be independent of oxidative stress or nitrosative stress. Our results show that the exposition of anterior pituitary cells to NO for long periods induces programmed cell death of anterior pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ANTERIOR PITUITARY, the different cell types behave as dynamic populations. Under normal conditions, the gland maintains a continuous renewal of cells that is a consequence of the equilibrium between the processes of cell division, differentiation, cell cycle arrest, and apoptosis (1, 2, 3). The term apoptosis refers to a constellation of morphological events that are associated with the activation of the machinery for programmed cell death (4, 5). These morphological and ultrastructural changes include plasma membrane blebbing, cell rounding and shrinkage, chromatin condensation, and nuclear fragmentation. On average, each anterior pituitary cell undergoes either mitosis or apoptosis once every 63 d, indicating a high incidence of cell death process in the gland (2). Moreover, anterior pituitary cell turnover varies depending on the hormonal status of the animal (2, 7, 8, 9).

Very little is known about the factors that regulate programmed cell death at the pituitary level; these factors need to be elucidated to establish the physiological mechanisms that regulate changes in anterior pituitary cell populations. It has been demonstrated that bromocriptine induces apoptosis in the GH3 somatomammotroph-derived cell line by a mechanism involving p38 MAPK and down-regulation of Bcl-2 protein (10, 11). A dominant negative estrogen receptor also promotes apoptosis in GH4 cells, another pituitary-derived cell line (12).

Despite the fact that cell death is a frequent event in the anterior pituitary gland, there is little evidence of possible apoptosis-inducing factors synthesized by anterior pituitary cells. It has been demonstrated that both TNF-{alpha} and the transcription factor Zac-1 cause apoptosis (1, 3). TNF-{alpha} could indeed participate in the renewal of anterior pituitary cells during the estrous cycle of the rat (1).

Nitric oxide (NO) is a free radical synthesized by NO synthases (NOS) that are expressed in anterior pituitary cells (13, 14, 15, 16). We have previously shown that NO plays a role as a regulator of prolactin release in the anterior pituitary (17, 18, 19). In addition to its function as a neurotransmitter and second messenger, NO behaves as a regulator of apoptosis in a series of cell types including thymocytes, pancreatic cells, hepatocytes, and neurons (20, 21, 22, 23). Cytokine actions on hormonal secretion in the anterior pituitary can be mediated by an increase in NOS expression and activity (15, 24). Remarkably, both NOS1 expression and the apoptotic effect of TNF-{alpha} on lactotrophs are maximal during proestrus (1, 25), suggesting the participation of NO in the proapoptotic effect of this cytokine. Moreover, the highest increase in inducible NOS mRNA after lipopolysaccharide injection occurs in the anterior pituitary gland, which may make secretory cells from the pituitary particularly vulnerable during the normal infection process (26, 27). These findings prompted us to test the hypothesis that anterior pituitary cells might undergo apoptosis in response to NO. In the present work, we found that long-term treatment with NO induces apoptosis in anterior pituitary cells. The proapoptotic effect of NO impacted mainly lactotrophs and resulted in partial dependence of caspase-9 and caspase-3 activation, but does not seem to be related to nitrosative or oxidative stress generation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs and reagents
DETA NONOate [(Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, a NO donor], Ac-DEVD-CHO (N-acetyl-DEVD-aldehyde), and Ac-LEHD-CHO (N-acetyl-LEHD-aldehyde), caspase inhibitors, N-acetyl-cysteine and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were purchased from Alexis (San Diego, CA). All other drugs were obtained from Sigma Chemical Co. (St. Louis, MO). All drugs were freshly prepared in DMEM supplemented with 10% fetal bovine serum (FBS). DETA NONOate unable to release NO (DETA) was obtained by incubating a solution of 1 mM DETA NONOate for 48 h at 37 C to allow the complete release of NO. Stock solutions (10-2 M) of Ac-LEHD-CHO and Ac-DEVD-CHO were prepared in dimethylsulfoxide and then diluted in DMEM-10% FBS.

Cell culture
Adult female Wistar rats (200–250 g), kept on 12-h light, 12-h dark cycles with controlled temperature (20–25 C), were used. Food and water were supplied ad libitum. The animals were maintained in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. At least nine animals per experiment were killed by decapitation, and the anterior pituitary glands were removed.

The cells were obtained from the glands by enzymatic (trypsin/deoxyribonuclease I) and mechanical dispersion (extrusion through a Pasteur pipette) as described previously (19). In all cases, the cells were cultured for 3 d (37 C, 5% CO2 in air) in DMEM supplemented with 10% FBS, 10 µl/ml MEM amino acids, 2 mM glutamine, 5.6 µg/ml amphotericin B, and 25 µg/ml gentamicin (DMEM-S-10% FBS). For cell activity and hormone release experiments, cells were seeded onto 96-well tissue culture plates (0.2 x 106 cells/well). For immunostaining, cells were seeded on glass coverslips onto 24-well tissue culture plates (0.1 x 106 cells/well). For DNA ladder experiments, cells were seeded onto six-well tissue culture plates (2.5 x 106 cells/well), and the content of two wells was combined for each treatment.

Cell activity assay
MTT assay was used to determine cell activity (28). In brief, cells were washed twice with Krebs Ringer bicarbonate buffer and then incubated with 110 µl of a MTT solution (500 µg/ml) for 4 h at 37 C. After this incubation period, 90 µl of the solution was removed, 100 µl of 0.04 N HCl in isopropanol was added to each well, and the plate was gently shaken for 3 min. OD was determined at 600 nm on an ELISA plate reader.

Immunostaining and nuclear morphology analysis
Cells were fixed in methanol/acetic acid (1:3), permeabilized with 0.1% Triton X-100 in PBS for 10 min at 4 C, and incubated in blocking solution (5% normal serum, 2% BSA in 0.1% Triton X-100) for 1 h at room temperature. Polyclonal antibodies against the different hormones (1:2000) (Dr. A. F. Parlow, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA) and monoclonal antibody against S100 protein (1:50) (Sigma) were used. The cells were incubated with primary antibodies for 1 h at room temperature, and after three washes, secondary antibodies conjugated to fluorescein isothiocyanate (1:200) were added. Nuclear morphology was visualized by propidium iodide. To distinguish between necrotic and apoptotic nuclei, cells were stained with a solution of acridine orange/ethidium bromide (10 µg/ml each). Cells were observed and quantified in an Axiophot D-7082 (Carl Zeiss MicroImaging, Inc., Thornwood, NY) microscope. Data of at least 500 nuclei obtained from random fields were expressed as number of apoptotic nuclei x 100/number of total nuclei, or number of immunostained cells with apoptotic nuclei x 100/number of total apoptotic cells.

Determination of internucleosomal DNA fragmentation
Cells were scratched with a rubber policeman in calcium- and magnesium-free buffer and centrifuged for 5 min at 1500 x g. The cell pellets were resuspended and incubated at 55 C for 150 min in 100 mM NaCl, 50 mM Tris-HCl (pH 8.0), 4 mM EDTA (pH 8.0), 0.5% SDS, and 0.1 mg/ml proteinase K. The DNA fraction from the digested cells was extracted using 1 M potassium acetate and chloroform. The DNA was precipitated and treated for 1 h at 37 C in 1 µg/ml ribonuclease solution. After that, equal amounts of DNA (10 µg) were separated by electrophoresis on a 1.8% agarose gel. The DNA was stained with ethidium bromide, and the DNA pattern was examined by UV transillumination.

Hormone determination
Hormones were measured by a double antibody RIA (29, 30) using reagents gently provided by Dr. A. F. Parlow. Prolactin and LH RP-3 were used as reference preparations and NIDDK-anti-rPRL-S-9 and anti-rLH-11 as antiserum, respectively. The intra- and interassay coefficients of variation were lower than 10%.

Statistical analysis
The results were expressed as mean ± SEM and evaluated by Student’s t test or one- or two-way ANOVA followed either by the Scheffé multiple comparison test for unequal replicates or Dunnett’s test, depending on the experimental design. Differences between groups were considered significant if P < 0.05. Results were confirmed by at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Long-term incubation with DETA/NO decreases anterior pituitary cell activity
Treatment of anterior pituitary cells with NO had a biphasic effect on cellular activity. The NO donor DETA/NO (1 mM) increased cellular activity when cells were exposed for 3 h and remained high until 12 h of incubation (Fig. 1AGo); at 24 h of incubation, the cellular activity of NO-treated cells matched the control levels whereas longer incubation times resulted in a steep decrease in cell viability (Fig. 1AGo, data for 72 h; control, 0.149 ± 0.005; DETA/NO, 0.008 ± 0.003; P < 0.001, Student’s t test). The effect of DETA/NO was dose dependent. When evaluated at 48 h, 0.5–1.0 mM DETA/NO decreased, whereas 0.1 mM failed to modify cellular activity (Fig. 2AGo).



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  		FIG. 1. NO effect on cellular activity (CA) (A) and prolactin release (B). Anterior pituitary cells were incubated with or without 1 mM DETA/NO for the indicated times. At the end of the experiment, CA and prolactin release cumulated during the different times were measured. Bars represent mean ± SEM; n = 8 replicates of a representative experiment from four independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. respective control (Student’s t test).

 


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  		FIG. 2. NO decreases cellular activity (CA) in a concentration-dependent manner (A). Anterior pituitary cells were incubated with increasing concentrations of DETA/NO for 48 h. Neither CA (B) nor prolactin release (C) were modified by 1 mM DETA (without the ability to release NO) treatment for 48 h. Bars represent mean ± SEM; n = 8 replicates of a representative experiment from four independent experiments. ***, P < 0.001 vs. respective control (Dunnett’s test).

 
In the same experiments, we determined prolactin release as an indicator of NO action on anterior pituitary hormone secretion. DETA/NO decreased basal prolactin release in cells incubated for 1 h (data not shown). This inhibitory effect was not observed in incubations of 3 h with the NO donor (Fig. 1BGo). However, a significant decrease in basal prolactin release was observed for periods equal and higher to 6 h of exposure to DETA/NO (Fig. 1BGo).

The effect of DETA/NO on anterior pituitary cells resulted from the specific action of NO because DETA NONOate unable to release NO (DETA) failed to modify cellular activity and prolactin release (Fig. 2Go, B and C).

Because DETA/NO releases NO for several hours, we also investigated the minimum time of exposure capable of inducing cell death, as well as the reversibility of this process. Thus, we exposed anterior pituitary cells to 1 mM DETA/NO during different incubation times, after which the medium was changed by medium without NO donor and cellular activity was assessed at 48 h. In these conditions, DETA/NO produced a decrease in cell viability starting from 6 h of incubation, the same time of exposure needed to reduce prolactin release (Fig. 3Go).



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  		FIG. 3. Determination of the minimum time of exposure to NO capable of producing a decrease on cellular activity (CA) or prolactin release. Anterior pituitary cells were incubated with or without 1 mM DETA/NO for the indicated times and then incubated with medium without NO donor to complete 48 h. CA (A) and cumulative prolactin release (B) were measured. Bars represent mean ± SEM, n = 8 replicates of a representative experiment from three independent experiments. *, P < 0.05; ***, P < 0.001 vs. respective control (Student’s t test).

 
NO induces apoptosis of lactotrophs
Cells incubated with DETA/NO showed condensation and marginization of chromatin characteristic of an apoptotic process (Fig. 4Go, A and B). These morphological changes were observed after 12 h of incubation with 1 mM DETA/NO, but they became clearly evident when cells were incubated for 24 or 48 h. The percentage of apoptotic nuclei was similar in cells treated during 24 or 48 h with DETA/NO (Table 1Go). Necrotic cells were occasionally seen without differences between control and treated groups (data not shown). Cells treated for 48 h with 1 mM DETA/NO also showed typical internucleosomal DNA fragmentation (Fig. 4CGo), which constitutes a direct evidence of apoptosis induction by NO.



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  		FIG. 4. DETA/NO induces apoptosis of anterior pituitary cells. Cells were incubated with medium alone (A) or with 1 mM DETA/NO (B) for 48 h. DETA/NO induced the appearance of chromatin condensation (B, arrow) and internucleosomal DNA fragmentation (C). Cells were stained with acridine orange/ethidium bromide. Bar, 10 µm.

 

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  		TABLE 1. DETA/NO induces nuclear morphological changes in anterior pituitary cells

 
Once we established the proapoptotic action of NO in pituitary cultures, the specific cell types affected were determined by immunostaining followed by propidium iodide nuclei staining (Fig. 5Go). As shown in Table 2Go, 50% of the cells exhibiting apoptotic nuclei were prolactin positive, whereas the second cell type affected by NO was gonadotrophs. We observed that the intensity of LH staining in apoptotic gonadotrophs was much weaker than in gonadotrophs of the control group (Fig. 5CGo; data not shown), which agrees with the decrease of basal LH release produced by the treatment with the NO donor (Velardez, M. O., and B. H. Duvilanski, unpublished results). Thus, the number of gonadotrophs undergoing apoptosis may be indeed underestimated.



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  		FIG. 5. DETA/NO induces apoptosis of lactotrophs and gonadotrophs. Cells were incubated with 1 mM DETA/NO for 48 h. NO donor induced the appearance of chromatin condensation (B and D, arrows) both in prolactin- and LH-secreting cells (A and C, respectively, arrows). Arrowhead, Prolactin-negative apoptotic cell. Bars, 10 µm.

 

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  		TABLE 2. Identification of cell type affected by NO

 
Mechanisms of NO action on anterior pituitary cell death
To determine whether caspases were involved in the NO effect on anterior pituitary cell viability, the cells were incubated with 1 mM DETA/NO for 24 h in the presence of inhibitors of caspase-9 or caspase-3, and cellular activity was determined after 48 h. Both caspase inhibitors partially reversed the decrease of cellular activity induced by DETA/NO (Fig. 6Go).



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  		FIG. 6. Cytotoxic action of NO is partially mediated by caspase-9 and caspase-3 activation. Changes in cellular activity (CA) of anterior pituitary cells incubated with 1 mM DETA/NO in the presence of caspase-9 (A) or caspase-3 (B) inhibitors for 24 h and with incubation medium alone for another 24 h. Bars represent mean ± SEM; n = 8 replicates of a representative experiment from three independent experiments. *, P < 0.05; **, P < 0.01 vs. respective control without caspase inhibitor; {Delta}{Delta}{Delta}, P < 0.001 vs. respective control without DETA/NO (Scheffé test).

 
NO has been shown to catalyze S-nitrosylation of diverse enzymes involved in apoptotic processes. NO is also known as an oxidative stressor capable of damaging mitochondria and DNA leading to apoptosis. The involvement of nitrosylation events and oxidative stress action in the NO proapoptotic effect on anterior pituitary cells was evaluated by using N-acetyl-cysteine as a nitrosylation blocker and the hydrosoluble derivative of vitamin E, Trolox, as an antioxidant protector of lipidic membranes. Neither N-acetyl-cysteine (10 mM) nor Trolox (0.1 mM) was able to reverse the cytotoxic action of NO on anterior pituitary cells (Fig. 7Go).



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  		FIG. 7. Involvement of oxidative stress in the cytotoxic action of NO. Changes in cellular activity (CA) of anterior pituitary cells incubated with 1 mM DETA/NO in the presence of N-acetyl-cysteine (NAC) (A) or Trolox (B) for 24 h and with incubation medium alone for another 24 h. Bars represent mean ± SEM; n = 8 replicates of a representative experiment from four independent experiments. **, P < 0.01; ***, P < 0.001 vs. respective control (Scheffé test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The anterior pituitary gland can be considered as an expansive cell population (31). Under normal conditions, the homeostasis of anterior pituitary cell population reflects the balance between proliferation and programmed cell death (2, 3).

Programmed cell death of anterior pituitary cells has not been extensively studied yet, but a number of factors synthesized by the pituitary have been proposed to trigger this process. In the present study, we show evidence that NO decreases cellular activity in a concentration- and time-dependent manner. The cytotoxic effect of NO became evident after 48 h incubation with DETA/NO, and it was not observed in cells treated with DETA (unable to release NO), proving the specificity of the NO action.

Short-term treatments with DETA/NO resulted in an increase in cellular activity whereas long-term treatments decreased it. However, the same incubation times that initially resulted in an augmented cellular activity appear to commit the cells to a program that ultimately leads to their death after 48 h. The minimum incubation time required to produce a decrease on cellular activity (evaluated after 48 h) was 6 h of treatment. Thus, it is reasonable to assume that after 6 h of exposure to NO, irreversible changes within the cells are triggered, leading to apoptosis. Remarkably, a minimum of 6 h was also required to steadily inhibit prolactin release. These results suggest that the inhibition of prolactin release may be one of the earlier manifestations of cytotoxic action of NO on lactotrophs. Moreover, NO inhibited LH release (data not shown) in the same periods of exposure. This result, along with the result of immunostaining, supports the idea that NO also has a cytotoxic effect on gonadotrophs.

Morphological analysis showed that NO induces changes in chromatin and nuclear fragmentation along with a reduction of cytoplasmic volume that are typical of the programmed cell death process. Although the percentage of apoptotic nuclei observed in cells treated for 24 and 48 h was similar, in preparations treated for 48 h the number of attached cells was lower, which suggests that NO might be interfering in the ability of anterior pituitary cells to attach to substrate, and this, in turn, could have led us to underestimate the real percentage of apoptosis in 48-h treatments.

As shown by immunostaining, NO seems to affect mainly lactotrophs (a high percentage of cells immunopositive for prolactin showed apoptotic nuclei). Gonadotrophs were the second most affected cell type. These results suggest a greater sensitivity of lactotrophs to NO.

Caspase activation is the main pathway that triggers apoptosis (32), and NO frequently induces apoptosis by activating these proteases (33, 34). Both caspase-9 and caspase-3 inhibitors partially reversed the decrease in cellular activity caused by NO, which suggests the involvement of these enzymes in the proapoptotic action of NO on anterior pituitary cells. The fact that the cytotoxic action of NO was only partially reversed by caspase inhibitors led us to hypothesize that NO may exert its proapoptotic effect not only by activating caspases but also through a caspase-independent pathway. It has been demonstrated that p38 MAPK activation by NO promotes Bax translocation to the mitochondria, ultimately causing cell death, and this effect cannot be blocked by caspase inhibitors (35). In addition, we cannot rule out the possibility that higher concentrations of the inhibitors may be required for a complete inhibition of caspase activity. In our experimental conditions, we were unable to test concentrations above 1 µM due to the cytotoxicity of the solvent.

Many effects of NO, both pro- and antiapoptotic, can be triggered by the ability of NO to nitrosylate proteins (33, 36, 37, 38). In our system, N-acetyl-cysteine, a nitrosylation blocker, was unable to prevent the cytotoxic action of NO on anterior pituitary cells, suggesting that this effect is independent of the nitrosylating capability of NO.

NO can also induce apoptosis by reacting with superoxide anion to produce peroxynitrite, which, in turn, decomposes to generate highly reactive molecules such as hydroxyl radical (OH·) that can attack lipidic membranes (33, 34, 39, 40, 41). In Jurkat cells, it was shown that Trolox, a soluble derivative of vitamin E that protects membranes from lipidic peroxidation, reduces the cytotoxic effect of NO (42). Because Trolox did not reverse the proapoptotic action of NO in anterior pituitary cells, the mechanism of action of NO in our system does not seem to involve a disruption of membrane integrity.

When the cells were incubated for short periods of time, NO induced an increase in cellular activity. MTT assay measures the level of mitochondrial dehydrogenase activity. NO, in the concentration range used on this study (<1 µM), inhibits cytochrome c oxidase (43) thus facilitating the delivery of electrons by reduced nicotinamide adenine dinucleotide oxidase to MTT. This would result in an apparent increase in cellular activity. Therefore, our results suggest that the mitochondria could be a site of cytotoxic action of NO.

Little is known about the intrapituitary factors that regulate apoptosis of the anterior pituitary cells. In the rat, changes in cell number during different physiological statuses are extremely important for the processes of estrous cycle, pregnancy, and lactation (2, 7, 8, 9, 44). It is known that NO acts as mediator of TNF-{alpha} and inteferon-{gamma} actions on hormonal secretion at the anterior pituitary level (15, 24). Moreover, TNF-{alpha} has a specific proapoptotic action mainly on lactotrophs during the proestrous phase (1). Both expression and enzymatic activity of NOS1 are also up-regulated during proestrus (25). More investigations are necessary to find out whether NO is the molecule that mediates the apoptotic process induced by TNF-{alpha} in anterior pituitary cells. The present results demonstrate that it is capable of performing such a role in vitro, making it an interesting hypothesis to test in the future.

Finally, it is worth noting that during inflammatory processes, expression of NOS2 is incremented 50 times in the anterior pituitary, resulting in the production of NO in high concentrations and during long periods (26, 27). Although a higher rate of apoptosis during inflammatory processes in the anterior pituitary has not been demonstrated yet, the available evidence points to NO as an ideal candidate to mediate such an effect.

Our findings demonstrate a higher susceptibility of lactotrophs to undergo apoptosis in response to NO exposition, which opens the possibility to develop new strategies for the treatment of pituitary tumors such as prolactinomas and to control bromocriptine-refractory hyperprolactinemia.

To summarize, the present study shows for the first time that NO induces programmed cell death in rat anterior pituitary cells. NO produced morphological and biochemical changes characteristic of an apoptotic process, and lactotrophs seem to be the cell type preferentially affected by NO. The cytotoxic effect of NO involves the activation of caspase-9 and caspase-3, but not nitrosative or oxidative stress. Our results allow us to postulate NO as a key regulatory factor of programmed cell death in the anterior pituitary gland.


   Footnotes
 
This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Ramón Carrillo-Arturo Oñativia Grant from Ministerio Nacional de Salud, and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT).

Abbreviations: Ac-DEVD-CHO, N-Acetyl-DEVD-aldehyde; Ac-LEHD-CHO, N-acetyl-LEHD-aldehyde; CA, cellular activity; DETA NONOate, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; FBS, fetal bovine serum; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NO, nitric oxide; NOS, NO synthase.

Received September 22, 2003.

Accepted for publication December 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Candolfi M, Zaldivar V, De Laurentiis A, Jaita G, Pisera D, Seilicovich A 2002 Tumor necrosis factor-{alpha} induces apoptosis of lactotropes from female rats. Endocrinology 143:3611–3617[Abstract/Free Full Text]
  2. 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]
  3. Pagotto U, Arzberger T, Ciani E, Lezoualc’h F, Pilon C, Journot L, Spengler D, Stalla GK 1999 Inhibition of Zac1, a new gene differentially expressed in the anterior pituitary, increases cell proliferation. Endocrinology 140:987–996[Abstract/Free Full Text]
  4. Brenner C, Kroemer G 2000 Apoptosis. Mitochondria—the death signal integrators. Science 289:1150–1151[Free Full Text]
  5. Chai J, Du C, Wu JW, Kyin S, Wang X, Shi Y 2000 Structural and biochemical basis of apoptotic activation by Smac/DIABLO. Nature 406:855–862[CrossRef][Medline]
  6. Deleted in proof
  7. Haggi ES, Torres AI, Maldonado CA, Aoki A 1986 Regression of redundant lactotrophs in rat pituitary gland after cessation of lactation. J Endocrinol 111:367–373[Abstract]
  8. Oishi Y, Okuda M, Takahashi H, Fujii T, Morii S 1993 Cellular proliferation in the anterior pituitary gland of normal adult rats: influences of sex, estrous cycle, and circadian change. Anat Rec 235:111–120[CrossRef][Medline]
  9. Yin P, Arita J 2000 Differential regulation of prolactin release and lactotrope proliferation during pregnancy, lactation and the estrous cycle. Neuroendocrinology 72:72–79[CrossRef][Medline]
  10. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E 2000 Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells. Biol Reprod 62:1486–1494[Abstract/Free Full Text]
  11. Yin D, Tamaki N, Kokunai T, Yasuo K, Yonezawa K 1999 Bromocriptine-induced apoptosis in pituitary adenoma cells: relationship to p53 and bcl-2 expression. J Clin Neurosci 6:326–331[CrossRef][Medline]
  12. Lee EJ, Duan WR, Jakacka M, Gehm BD, Jameson JL 2001 Dominant negative ER induces apoptosis in GH(4) pituitary lactotrope cells and inhibits tumor growth in nude mice. Endocrinology 142:3756–3763[Abstract/Free Full Text]
  13. 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]
  14. Okere CO, Murata E, Higuchi T 1998 Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction. Brain Res 784:337–340[CrossRef][Medline]
  15. Vankelecom H, Matthys P, Denef C 1997 Involvement of nitric oxide in the interferon-{gamma}-induced inhibition of growth hormone and prolactin secretion in anterior pituitary cell cultures. Mol Cell Endocrinol 129:157–167[CrossRef][Medline]
  16. Vincent SR, Kimura H 1992 Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46:755–784[CrossRef][Medline]
  17. Duvilanski BH, Zambruno C, Seilicovich A, Pisera D, Lasaga M, Díaz MC, Belova N, Rettori V, McCann SM 1995 Role of nitric oxide in control of prolactin release by the adenohypophysis. Proc Natl Acad Sci USA 92:170–174[Abstract/Free Full Text]
  18. Velardez MO, Ogando D, Franchi AM, Duvilanski BH 2001 Role of nitric oxide in the metabolism of arachidonic acid in the rat anterior pituitary gland. Mol Cell Endocrinol 172:7–12[CrossRef][Medline]
  19. Velardez MO, Benitez AH, Cabilla JP, Bodo CC, Duvilanski BH 2003 Nitric oxide decreases the production of inositol phosphates stimulated by angiotensin II and thyrotropin-releasing hormone in anterior pituitary cells. Eur J Endocrinol 148:89–97[Abstract]
  20. Fehsel K, Kroncke KD, Meyer KL, Huber H, Wahn V, Kolb-Bachofen V 1995 Nitric oxide induces apoptosis in mouse thymocytes. J Immunol 155:2858–2865[Abstract]
  21. Heneka MT, Loschmann PA, Gleichmann M, Weller M, Schulz JB, Wullner U, Klockgether T 1998 Induction of nitric oxide synthase and nitric oxide-mediated apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor-{alpha}/lipopolysaccharide. J Neurochem 71:88–94[Medline]
  22. Kim YM, De Vera ME, Watkins SC, Billiar TR 1997 Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-{alpha}-induced apoptosis inducing heat shock protein 70 expression. J Biol Chem 272:1402–1411[Abstract/Free Full Text]
  23. McDaniel ML, Corbett JA, Kwon G, Hill JR 1997 A role for nitric oxide and other inflammatory mediators in cytokine-induced pancreatic ß-cell dysfunction and destruction. Adv Exp Med Biol 426:313–319[Medline]
  24. Theas S, Pisera D, Duvilanski B, De Laurentiis A, Pampillo M, Lasaga M, Seilicovich A 2000 Estrogens modulate the inhibitory effect of tumor necrosis factor-{alpha} on anterior pituitary cell proliferation and prolactin release. Endocrine 12:249–255[CrossRef][Medline]
  25. Lozach A, Garrel G, Lerrant Y, Berault A, Counis R 1998 GnRH-dependent up-regulation of nitric oxide synthase I level in pituitary gonadotrophs mediates cGMP elevation during rat proestrus. Mol Cell Endocrinol 143:43–51[CrossRef][Medline]
  26. McCann SM, Licinio J, Wong ML, Yu WH, Karanth S, Rettori V 1998 The nitric oxide hypothesis of aging. Exp Gerontol 33:813–826[CrossRef][Medline]
  27. Wong ML, Rettori V, al-Shekhlee A, Bongiorno PB, Canteros G, McCann SM, Gold PW, Licinio J 1996 Inducible nitric oxide synthase gene expression in the brain during systemic inflammation. Nat Med 2:581–584[CrossRef][Medline]
  28. Abdallah MA, Biellmann JF 1980 Activity determination of 3-iodopyridineadenine dinucleotide and its phosphate as hydride acceptors in the presence of dehydrogenases using a coupled redox system. Eur J Biochem 112:331–333[Medline]
  29. Niswender GD, Chen CL, Midgley Jr AR, Meites J, Ellis S 1969 Radioimmunoassay for rat prolactin. Proc Soc Exp Biol Med 130:793–797[Medline]
  30. Niswender GD, Midgley Jr AR, Monroe SE, Reichert Jr LE 1968 Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH-131I. Proc Soc Exp Biol Med 128:807–811[Medline]
  31. Mastro A, Shelton E, Hymer WC 1969 DNA synthesis in the rat anterior pituitary: an electron microscope radioautographic study. J Cell Biol 43:626–629[Free Full Text]
  32. Thornberry NA, Lazebnik Y 1998 Caspases: enemies within. Science 281:1312–1316[Abstract/Free Full Text]
  33. Borutaite V, Morkuniene R, Brown GC 2000 Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms. FEBS Lett 467:155–159[CrossRef][Medline]
  34. Moriya R, Uehara T, Nomura Y 2000 Mechanism of nitric oxide-induced apoptosis in human neuroblastoma SH-SY5Y cells. FEBS Lett 484:253–260[CrossRef][Medline]
  35. Ghatan S, Larner S, Kinoshita Y, Hetman M, Patel L, Xia Z, Youle RJ, Morrison RS 2000 p38 MAP kinase mediates Bax translocation in nitric oxide-induced apoptosis in neurons. J Cell Biol 150:335–347[Abstract/Free Full Text]
  36. Brüne B, Mohr S, Messmer UK 1996 Protein thiol modification and apoptotic cell death as cGMP-independent nitric oxide (NO) signaling pathways. Rev Physiol Biochem Pharmacol 127:1–30[Medline]
  37. Haendeler J, Weiland U, Zeiher AM, Dimmeler S 1997 Effects of redox-related congeners on apoptosis and caspase-3 activity. Nitric Oxide 1:282–293[CrossRef][Medline]
  38. Li J, Billiar TR, Talanian RV, Kim YM 1997 Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem Biophys Res Commun 240:419–424[CrossRef][Medline]
  39. Bosca L, Hortelano S 1999 Mechanisms of nitric oxide-dependent apoptosis: involvement of mitochondrial mediators. Cell Signal 11:239–244[CrossRef][Medline]
  40. Burney S, Caulfield JL, Niles JC, Wishnok JS, Tannenbaum SR 1998 The chemistry of DNA damage from nitric oxide and peroxynitrite. Mutat Res 424:37–49
  41. Yabuli M, Kariya S, Ishisaka R, Yasuda T, Yoshioka T, Horton AA, Utsumi K 1999 Resistance to nitric oxide-mediated apoptosis in HL-60 variant cells is associated with increased activities of Cu, Zn-superoxide dismutase and catalase. Free Radic Biol Med 26:325–332[CrossRef][Medline]
  42. Ushmorov A, Ratter F, Lehmann V, Droge W, Schirrmacher V, Umansky V 1999 Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release. Blood 93:2342–2352[Abstract/Free Full Text]
  43. Poderoso JJ, Carreras MC, Lisdero C, Riobo N, Schopfer F, Boveris A 1996 Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys 328:85–92[CrossRef][Medline]
  44. Ahlbom E, Grandison L, Zhivotovsky B, Ceccatelli S 1998 Termination of lactation induces apoptosis and alters the expression of the bcl-2 family members in the rat anterior pituitary. Endocrinology 139:2465–2471[Abstract/Free Full Text]



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