Dopamine inhibits in vitro release of VIP and proliferation of VIP-immunoreactive pituitary cells

Neuropeptides(1996) 30 (1), 61-86 © PearsonProfessionalLtd 1996

Dopamine inhibits in vitro release of VIP and proliferation of VI P-immunoreactive pituitary cells J. Carretero ~, R. J. Vazquez ~, M. Santos 1, L. Cacicedo a, M. Rubio ~, Fo S a n c h e z - F r a n c o 3, R. Vazquez 1 1Department of Human Anatomy and Histology, Facultad de Medicina, Universidad de Salamanca. aService of Endocrinology, Hospital Ram6n y Cajal. aService of Endocrinology, CNIC Carlos III, Madrid, Spain.

Summary A double immunohistochemical study for VIP and proliferating ceil nuclear antigen (PCNA) was carried out on monolayer cultures from adult male rats pituitary glands treated with dopamine (ranging from 10`9 to 10-SM), in order to establish whether or not dopamine is involved in the regulation of the proliferation rate of pituitary VIP-immunoreactive cells. For all doses of dopamine assayed, the release of VIP to the culture medium, the numerical density of VIP-immunoreactive cells and the percentages of VIP- and PCNA-immunoreactive cells decreased significantly after dopamine treatment. These results suggest that dopamine could be a physiological inhibitor involved in the modulation of pituitary VIP proliferation rate.

INTRODUCTION

Vasoactive intestinal peptide (VIP) is synthesized at hypothalamic level '-2 and released into the pituitary portal system 3-4 with evident effects on pituitary mammotroph cells? Pituitary synthesis of V I P 6-z and autocrine or paracrine effects on prolactin release have been reported, s The pituitary content of VIP is modulated by several peripheral hormones such as thyroid hormones z,9-1° or estradio111-13 and hypothalamic factor as dopamine2 4-]6 Modulation of proliferation rate of pituitary VIP cells is not clear. Only some VIPomas induced by chronic

estradiol treatment have been reported 17but if dopamine is involved in this, regulation is unknown. In order to determine whether or not dopamine is involved in the regulation of the proliferation of VIPimmunoreactive cells, an immunocytochemical study for VIP and PCNA, jointly, was carried out on pituitary monolayer cultures after dopamine treatment, because PCNA immunohistochemical labelling has proved to be a good approach to evaluate the proliferation rate in different tissues, including the pituitary gland.18-2° MATERIALS AND METHODS Pituitary cultures

Received 18 July 1995 Accepted 12 September 1995 Correspondence to: J. Carretero, Dpto. de Anatomfa e Histologia Humanas, Facultad de Medicina, Avda. Campo Charro, s/n, E-37007 Salamanca, Spain. Fax: 34-23-294559.

Following anaesthesia with isoflurane, male Wistar rats (175-200 g) were killed by decapitation and the anterior pituitary glands were removed and washed in Earle's balanced salt solution. Enzymatic dispersion was carried out by incubation for 15 rain at 37°C in Hank's solution to 81

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which 0.15% MgC12, 0.1% papain, 0.01% DNase and 0.1% of neutral protease had been added. Mechanical dispersion was achieved by passing the pituitaries through Pasteur pipettes and 20-22 gauge needles. After centrifugation, the supernatant was removed and the cells were resuspended in an appropriate volume of Dulbecco's modified Eagle's medium, supplemented with 10% calf serum, 2.5% fetal calf serum, 2% L-glutamine, 1000 IU/ml of penicillin and 1000 IU/ml of streptomycin. 15,19-21The cells were seeded on 40 culture dishes (30 x 15 mm) at a final concentration of 5 x 105 cells/dish and incubated at 37°C in a 5% CO2/95% air atmosphere for 7 days. On the 4th day of incubation the medium was changed by fresh additioned medium. On the 7th day of incubation, the medium was replaced by fresh medium in the control dishes and fresh medium plus 10-9, 10 -8, 10z, 10-6 or 10.5 M dopamine, in the treated dishes, and incubated for 1, 3, 6, 12 or 24 h. Five dishes by treatment and time were employed. To avoid the oxidation of dopamine, the medium was additioned with 60 gM ascorbic acid. After incubation the culture medium was removed, the dishes were carefully washed with Dulbecco's sterile PBS, and the cells were fixed in Somogyi solution for 1 h, followed by careful rinsing in PBS. Radioimmunoassay of VIP

The levels of VIP in the culture media were determined from duplicate aliquots of media by RI& as described by Lorenzo et al.~5,~6Vip was quantified by radioimmunoassay using an antiserum raised in rabbits against porcine VIP. VIP was conjugated to BSA with carbodiimide hydrochloride. The initial dilution of the antiserum was 1:250 000. Assay sensitivity was 9 pg/ml, the within and between assay variations being 4-6 and 10-15% respectively.

immunolabelling, the peroxidase-antiperoxidase {PAP) reaction was performed for the detection of VIE using as primary serum anti-VIP rabbit serum at a dilution of 1:800, swine anti-rabbit serum (Dako, diluted 1:100) and rabbit-PAP complex (Dako, diluted 1:100). The characterization and specificity of the primary serum had been checked in a previous study (Carretero et al, 1992). Preabsorption tests with VIP and tests substituting the specific serum by normal rabbit serum abolished the reaction. By EUSA,specificity of swine anti-rabbit IgG was lower than 1% for rat and mouse IgG and 100% for rabbit IgG. For the washes and dilutions of the sera, Tris buffer (0.05 M, pH7.4) containing 0.8% NaC1 was used. The reaction was developed in freshly prepared 4-chloro1-naphthol (1.7x 10.3M in 3% absolute ethanol and TRISbuffer containing 0.3% H202). Quantitation of PCNA, VIP and PCNA-ViP immunoreactive cells

Four thousand cells per dish were evaluated using an Axioplan Zeiss microscope equipped with an ocular grid at a final magnification of 400x. The cells were randomly selected from different areas of the dishes. Dishes with high degree of agglomerations of overlapping cells were rejected and only non-overlapping cells were considered. Because the PCNA protein is known to have a 20 h halflife in vitro system '8 and is detected in cells that have recently completed mitosis, 19,2°only nuclear PCNA expression was considered, except in metaphasic-mitotic cells. The following parameters were determined: (1) The total number of cells, (2) PCNA-positive cells, (3) VIPpositive cells and (4) PCNA-positive cells/VIP-positive cells; 2, 3 and 4 were calculated as the percentages from the total number of cells analysed. The percentage of proliferating VIP cells from the VIP-positive cells, labelled jointly for PCNA and VIE was calculated.

Immunocytochemistry

To study PCNA-positive cells and to determine the PCNA-VIP labelling index, a double labelling immunohistochemical method for PCNA and VIP was developed. Endogenous peroxidase was blocked with H202 in methanol and non-specific reactions of the secondary antibody by incubation in normal goat serum (Dako, diluted 1:30). Cultures were incubated overnight at 4°C with the mouse P C 10 mAb (Dako, lot. 121 diluted 1:2000 in TBS). Biotinylated goat anti-mouse IgG (Dako, lot. 061 diluted 1:100) and Avidin-Biotinylated horseradish peroxidase complex (ABC kit, DAKO lot. 081 diluted 1:100) were successively applied at room temperature for 40 min and 30 min, respectively. The reaction was developed in freshly prepared 3-3'DAB (0.025% in TRIS buffer containing 0.03% of H=O=. Following the PCNA Neuropeptides (1996) 30(1), 81-86

Statistical analysis

The results obtained were processed statistically and the differences observed were compared using analysis of variance, accepting as significant values of P<0.05 for the Fisher-PLSD and Scheff6 F tests jointly. Two pituitary cultures under similar experimental conditions were carried out, in order to verify the reproducibility of the results (within assay error always was less than 1.850/0). RESULTS

In the control experiments, from 0 to 24 h, the VIP ranged between 60 to 90 pg/ml. Except for 3, 6, 12 and 24 h of incubation (P <0.05 in relation to 0-1 h), nonsignificant variations were observed among the different © Pearson Professional Ltd 1996

Dopamine inhibits in vitro release of VIP and profiferation of VIP-immunoreactive pituitary cells 83

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periods of incubation (Fig. 1). Dopamine (see Fig. 1) induced a significant decrease (P<0.05, in relation to control dishes) for all doses and times studied and a signif-icant dose-dependent inhibitory effect on the release of VIP was observed. .As result of double immunostaining, negative, PCNA-, VIP- and PCNA-VIP-immunoreactive cells were founded. The immunoreaction for PCNA was present in the nuclei, except for 2.34% of the cells that showed cytoplasmic reaction. The immunoreaction for VIP was always restricted to cytoplasm. Control dishes showed a slow and progressive increase of the percentage of VIP-immunoreactive cells from 0 to 24 h of incubation. As result of lineal increase, in 24th h the percentage of VIP-immunoreactive cells was twice more numerous than those observed on 0 h (P<0.01)

(Fig. 2). However, the percentage of VIP-immunoreactive cells was decreased in the dishes treated with dopamine in relation to control dishes (see Fig. 2). After 1 h of treatment a significant decrease (P<0.05) was observed for all doses assayed. Following 3 h of treatment a dose-dependent effect was evidenced: the higher doses of dopamine decrease the percentage of VIPimmunoreactive cells more than lower doses. After 6 h of treatment discrepancies in response to different doses of © Pearson Professional Ltd 1996

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dopamine were found, however, always significant lower percentages of VIP-immunoreactive cells than in control dishes were observed (P<0.01) (Fig. 2). Control dishes showed a sustained proliferation rate of VIP-immunoreactive cells from 0 to 24 h of incubation. Modifications of proliferation were not induced by the increase of the percentage of VIP-immunoreactive cells. After 3 h of incubation significant increases (P<0.05) of the proliferation rate of VIP-immunoreactive cells in relation to 0 h were observed, but no differences among 1, 3, 6, 12 or 24 h of incubation were founded (Fig. 3). Proliferation rate of VIP-immunoreactive cells decreased after treatment with dopamine in relation to control dishes. Dopamine always decreases the proliferation rate of VIP-immunoreactive cells in relation to control dishes: following 1 h of treatment, 10.7 and 10-6M dopamine were more effective than the other doses and after 3 h a significant dose-dependent effect was found. In a similar way that was described for the percentage of VIPimmunoreactive cells, discrepancies among the doses and periods of incubation were found following 6 h of treatment with dopamine, but the values observed always were lower than in control dishes. Mirdmum values were found for 10.8 and 10-gM dopamine on 12th and 24th h after the treatment (Fig. 3). Neuropeptides (1996) 30(1), 81-86

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DISCUSSION PCNA is an auxiliary protein of DNA polymerase ~, necessary for DNA replication. It is expressed in the G1 phase, reaches a maximum in the S phase, and declines again during the G2 and M phases of the cell cycle.22-24 Although some discrepancies have been reported between PCNA immunoreactivity and data obtained with other cell proliferation assessing methods, various studies have shown that the PCNA labelling index represents a valuable approach for the evaluation of cellular proliferation in human and animal tissuesY -29 The 'in vivo' kinetics of anterior pituitary cells have been studied by the PCNA labelling index; this has been reported to be more sensitive than other methods such as the BrDU labelling index) s Moreover, PCNA was revealed as a good in vitro marker for proliferation of pituitary prolactin- and VIP-immunoreactive cells.ig,20 VIP is a neuropeptide involved in the regulation of the synthesis and release of prolactin 6,s,3°,31 that is synthesized in pituitary gland 6,7 and is immunohistochemically expressed, '3,32 Dopaminergic system is involved in VIPergic regulation of prolactin secretion, because dopamine blocks the VIP-induced prolactin response in Neuropeptides (1996) 30(1), 81-86

women 2s and, when the dopaminergic inhibitor tone is blocked, exogenous VIP is unable to increase prolactin secretionY Moreover, the dopamine agonist bromocryptine significantly reduces the in vivo pituitary VIP levels of female rats 35 and in a previous work 15 we demonstrated an inhibitory role for dopamine in the modulation of pituitary VIP cells. However, to date, no studies about dopaminergic effects on the proliferation of pituitary VIP cells have been reported. The present study demonstrated decreases in the percentage of VIP- and PCNA-VIP-immunoreactive cells after dopamine treatment, associated to decreases of the release of VIP to the culture medium. It could be speculated that VIP-immunoreactive cells are lactotroph cells as was described by other authors 36 and because dopamine and its agonist inhibits the proliferation of lactotroph cells37-41 this could explain the decrease of the proliferation rate of VIP-immunoreactive cells observed in our study. However, there are discrepancies in relation to the identity of VIP-immunoreactive pituitary population 7'9'32'36'42 and, without rejecting the possibility that some VIP-immunoreactive cells can be lactotroph cells, the resuks observed in the present study after dopamine treatment point to a decrease in the in vitro proliferation rate of pituitary VIP cells. The dose-time dependent discrepancies observed in our study suggest a faster inhibition of proliferation rate on VIP-immunoreactive cells induced by higher doses than those observed for lower doses of dopamine. Globally considered, our study suggests that dopamine, in addition to its inhibitory effect on in vitro pituitary VIP expression, could be a physiological factor involvedin the regulation of proliferation of pituitary VIP-immunoreactive cells.

ACKNOWLEDGEMENTS This work was supported by the Spanish National Research Program (DGICYT PM 91-0105).

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Dopamine inhibits in vitro release of VIP and proliferation of VIP-immunoreactive pituitary cells

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40. Kovacs, K., Stefaneanu, L., Horvath, E., Lloyd, R. V., Lancrajan, I., Buchelder, M., and Fahlbusch, R. Effect of dopamine agonist medication on prolactin producing pituitary adenomas. A morphological study including immunocytochemistry, electron microscopy and in situ hybridization. Virchows Arch. A Pathol. Anat. Histopathol. 1991; 418: 439-446. 41. Drewett, N., Jacobi, J. M., Willgoss, D. and Lloyd, H. M. Apoptosis in the anterior pituitary gland of the rat: Studies with estrogen and bromocriptine. Neuroendocrinology 1993; 57: 89-95.

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