Involvement of α2- and ß2-adrenoceptors on breast cancer cell proliferation and tumour growth regulation.

BACKGROUND AND PURPOSE: ß-Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours. EXPERIMENTAL APPROACH: ß-Adrenoceptor expression was analysed by immunofluorescence and RT-PCR. Cell proliferation was assessed by [(3) H]-thymidine incorporation, tumour growth by measuring with a calliper and ERK 1/2 phosphorylation by Western blotting. KEY RESULTS: ß(2) -Adrenoceptor expression was confirmed in the mouse and human cells tested. Cell proliferation was increased by adrenaline (by α(2) -adrenoceptor action) and decreased in every tested cell line by the ß-adrenoceptor agonist isoprenaline and the ß(2) -adrenoceptor agonist salbutamol. Isoprenaline and salbutamol reduced tumour growth in every tumour tested (mouse C4-HD and CC4-3-HI and human IBH-4, IBH-6 and MDA-MB-231 cell lines growing as xenografts in nude mice). These effects were reversed by the ß-adrenoceptor antagonist propranolol. The α(2) -adrenoceptor antagonist rauwolscine and the ß(2) -adrenoceptor agonist salbutamol were equally effective in diminishing tumour growth. ERK 1/2 activation analysed in IBH-4 tumours correlated with tumour growth, with the ß-adrenoceptor agonists decreasing its activation. Inhibition of ERK 1/2 phosphorylation in vitro was mainly mediated by the PKA pathway. CONCLUSIONS AND IMPLICATIONS: In our experimental models, the ß-adrenoceptor agonists inhibited breast cancer cell proliferation and tumour growth, probably mediated by inhibition of ERK 1/2 phosphorylation. The ß-adrenoceptor agonists were as effective as the α(2) -adrenoceptor antagonist rauwolscine, providing possible novel adjuvant treatments for breast cancer.

Adrenoceptors: non conventional target for breast cancer?

Epinephrine and Norepinephrine, typically released during stress bind to nine different adrenoceptors (AR) which classically control the cardiovascular and respiratory systems. New targets were described for the many agonists and antagonists developed for these AR, as the central nervous system. During the last three decades, AR expression and action on the mammary gland/breast were extensively investigated. In the cow mammary gland, good milkability was associated with low density of beta(2)-AR and high density of alpha(2)-AR. In the rat normal mammary gland, beta-AR are expressed in the epithelial cells, alveoli, ducts, and adipocytes showing an exquisite regulation by steroid hormones and prolactin. In rat dimethylbenz(a)anthracene (DMBA) tumors, a close correlation was observed between tumor growth and beta-AR concentration. beta(2)-AR were described in numerous human cell lines and breast tumors. The action of beta-adrenergic compounds on cell proliferation is contradictory. While some authors found that beta-agonists significantly inhibit cancer cell proliferation and tumor growth in mice, others described a significant reduction in DNA synthesis by beta-blockers. Also, positive effects of beta-AR on human carcinoma cell migration have been described. alpha(2)-AR are expressed in human breast cancer and non-cancer cell lines, their stimulation being associated with increased cell proliferation. In vivo clonidine increased tumor growth and alpha (2)-adrenergic antagonists completely reversed this effect. When administered alone, rauwolscine inhibited tumor growth behaving as an inverse agonist. Therefore, the numerous adrenergic beta- and alpha-AR agonists or antagonists could prove to be unexpected therapeutic options for mammary gland/ breast and mainly breast cancer.

Alpha2-adrenoceptor action on cell proliferation and mammary tumour growth in mice.

BACKGROUND AND PURPOSE: Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described alpha(2)-adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on alpha(2)-adrenoceptors in a reliable model of breast cancer. EXPERIMENTAL APPROACH: The expression of alpha(2)-adrenoceptors was confirmed by immunocytochemistry, immunofluorescence and reverse transcription-PCR in the mouse mammary tumour cell line MC4-L5. Proliferation was assessed by [(3)H]thymidine incorporation and tumours were measured daily. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling. KEY RESULTS: Incubation for 2 days with alpha(2)-adrenoceptor agonists (clonidine and dexmedetomidine) significantly enhanced proliferation of the mouse mammary tumour cell line MC4-L5. These agonists also significantly stimulated tumour growth of the progestin-dependent tumour C4-HD even in the presence of medroxyprogesterone acetate (MPA). In every tumour tested (C4-HD, CC4-2-HD and CC4-3-HI), regardless of MPA sensitivity, clonidine significantly enhanced tumour growth in the absence of MPA. The alpha(2)-adrenoceptor antagonists, yohimbine and rauwolscine, completely reversed the effects of clonidine. However, the group receiving yohimbine alone showed a nonsignificant but constant increase in tumour growth, whereas rauwolscine alone diminished tumour growth significantly, behaving as a reverse agonist. In CC4-3-HI tumours, rauwolscine treatment enhanced apoptosis and diminished the mitotic index, whereas clonidine had the inverse effect. CONCLUSIONS AND IMPLICATIONS: Alpha(2)-adrenoceptor agonists enhanced tumour growth and rauwolscine behaved in vivo as a reverse agonist, suggesting that it may be tested for adjuvant treatment.

Androgen receptors in human melanoma cell lines IIB-MEL-LES and IIB-MEL-IAN and in human melanoma metastases.

The presence and characteristics of androgen receptors (ARs) have been described by our group in one human melanoma cell line. We have now investigated their presence in two other human melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, as well as in biopsies from human metastatic melanoma. Scatchard analysis revealed a single binding component for both cell lines, the apparent dissociation constant obtained being 15 nM, with a binding capacity of 280 fmol/mg total cell protein, for IIB-MEL-LES cells and 14 nM, with a binding capacity of 206 fmol/mg total cell protein for IIB-MEL-IAN cells. When specificity was assessed, not only androgen and anti-androgen but also non-androgenic compounds were able to compete for [3H]R1881 binding, as seen before. When immunocytochemistry of IIB-MEL-LES and IIB-MEL-IAN cells was performed for ARs, both cell lines were deeply stained in the nucleus, whereas no staining was found for oestrogen or progesterone receptors. Every specimen of melanoma metastases tested for the presence of ARs was deeply stained, and in the majority the intensity of the staining was high. Several hormones and anti-hormones were tested for their ability to affect cell proliferation. In both cell lines, testosterone, dihydrotesterone, oestradiol and progesterone significantly stimulated cell proliferation, and this was reversed by hydroxyflutamide, bicalutamide or tamoxifen.

Alpha2-adrenergic effect on human breast cancer MCF-7 cells.

(-)Epinephrine (Epi) and (-)Norepinephrine (NEpi) significantly stimulated tritiated Thymidine incorporation in MCF-7 cells at concentrations 10-30pM to 10nM, with an EC50 of 10pM for Epi and 14.2pM for NEpi. To characterize this action, cells were incubated in the presence of NEpi or Epi and different antagonists. The beta-adrenergic antagonist Propanolol showed no effect on the agonist's stimulation, whereas the alpha-adrenergic antagonist Phentolamine, reverted it completely at high concentrations (100 microM). The alpha1-adrenergic antagonist Prazosin (Pra) acted only at high concentrations, while the alpha2-adrenergic antagonist Yohimbine (Yo) reverted the stimulation at an EC50 of 0.11 microM. Likewise, when the cells were incubated in the presence of the specific alpha2-adrenergic agonist Clonidine (Clo), Thymidine incorporation was significantly stimulated at an EC50 of 0.298 pM. Again, the incubation of the cells in the presence of the alpha1-adrenergic antagonist Pra exerted its action at high concentrations, whereas the alpha2-adrenergic antagonist Yo showed a clear reversal of the agonist's enhancement at an EC50 of 0.136 microM. Moreover, Clo caused a clear and significant inhibition of stimulated cAMP levels both in the intracellular and the extracellular fractions. Yo showed a complete reversion of cAMP levels to control values in the presence of Clo, while Pra had the opposite effect. These data suggest that the stimulation provoked in Thymidine incorporation by the agonists Epi, NEpi, and Clo is, at least in part, due to an alpha2-adrenergic mechanism directly on tumoral cells, and that the effect is coupled with inhibition of cAMP levels, as described for this kind of receptors.

Progesterone receptor involvement in independent tumor growth in MPA-induced murine mammary adenocarcinomas.

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.

Binding of 125I-prolactin to spermatozoa from normospermic and asthenospermic men.

Scatchard analysis of prolactin binding sites (PRL-BS) from ejaculated spermatozoa showed a single population of binding sites (apparent association constant: 2.51+/-0.186 nmol/l[-1]) with 0.317+/-0.0743 fmol/10(6) sperm binding sites. Different pools of spermatozoa were incubated with increasing concentrations of several hormones. There was a decrease in [125I]-oPRL binding with purified ovine prolactin (oPRL) and human growth hormone (hGH) which was not observed in the presence of synthetic ACTH and recombinant FSH, suggesting that binding was hormone specific. When the patient's samples were analyzed using the single point assay at saturation concentration, asthenospermic patients showed a significantly higher concentration of binding sites compared to normospermic ones. Both groups of patients displayed similar PRL levels in seminal plasma measured by DELFIA. Moreover, individual values of PRL levels in seminal plasma did not correlate with PRL-BS concentrations. We thus conclude that [125I]-oPRL binding to ejaculated spermatozoa was hormone specific and with similar parameters as seen in other target tissues. PRL-BS concentration in asthenospermic patients was significantly higher than in normospermic but this was not due to different levels of PRL in seminal plasma.

Prazosin and stress effect on tumoral growth of 7,12-dimethylbenz[A]anthracene-induced rat mammary tumors.

Repeated isolation stress and prazosin effect were evaluated in 7,12-dimetylbenz[A]anthracene (DMBA) mammary tumors. Tumor volume was significantly lower in stressed than in control animals from 10 to 52 days considering day 1 the moment when tumors became palpable and treatment began. Control Prazosin (0.5 mg/kg) rats showed diminished tumor volume after 40 days. Stress Prazosin curve was similar to stress alone. The proportion of progressing tumors in control was significantly higher than in stressed groups, regardless of Prazosin administration. Body weight gain was similar in every group throughout the experiment. Behavioral studies were performed when stress effect was no longer evident. Grooming and the number of fecal boli were similar in all groups, as well as prolactin serum levels, suggesting that habituation took place. No significant differences were observed between groups for estrogen receptors. However, a greater concentration of progesterone receptors was found in Stressed rats, compared to all other groups. We conclude that the decrease of tumor volume provoked by stress could not be reversed by the alpha 1-adrenergic antagonist prazosin. Then, it appears that the main effect of stress is not mediated by the alpha 1-adrenergic receptors. Higher progesterone receptors in stressed rats could explain the differences observed.

Effect of medroxyprogesterone acetate (MPA) and serum factors on cell proliferation in primary cultures of an MPA-induced mammary adenocarcinoma.

The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on the in vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblast-enriched cultures using 3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10(-11) to 10(-7) M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10(-11) M. At nM concentrations, neither DEXA nor DHT stimulated 3H-thymidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10(-7)-10(-9) M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 +/- 115 fmoles/10(5) cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10(5) cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of 3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals. It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.

Atypical androgen receptor in the human melanoma cell line IIB-MEL-J.

To evaluate the presence of androgen receptors in the human melanoma cell line IIB-MEL-J, a Scatchard plot analysis was performed. Cells in culture revealed a single binding component with an apparent dissociation constant (KD) at 37 degrees C of 11 nM and a binding capacity of 326 fmol/mg protein when measured with [3H]-R1881. Competition analysis revealed an atypical relaxation of specificity, since not only androgen (testosterone, dihydrotestosterone [DHT], R1881) and antiandrogen (hydroxy-flutamide [OH-FLU]) competed for [3H]-R1881 binding, but also estradiol, progesterone, and cortisol at 500-fold excess concentration. Binding of [3H]-estradiol and [3H]-R5020 in the absence of unlabeled DHT were completely suppressed in its presence. Immunohistochemistry of androgen receptor with a monoclonal antibody showed that nuclei were vigorously stained. Different doses of flutamide (FLU) and OH-FLU tested on cultured IIB-MEL-J cells in the presence of serum inhibited significantly cell proliferation in a dose-dependent manner. When cells were incubated with 10 nM DHT and 1% charcoal-adsorbed serum, a significant stimulation of growth that was observed was inhibited by 4 microM OH-FLU. DHT stimulation was completely reversed by the antiestrogen tamoxifen. In addition, male nude mice transplanted with IIB-MEL-J tumor were treated with FLU when tumors were palpable. FLU was effective in diminishing tumor growth and increasing survival rate of the animals. As a conclusion, the presence of functional androgen receptors in these cells has been demonstrated by growth inhibition in vitro and in vivo with antiandrogens, and their atypical nature is suggested by binding cross-reactivity and competition studies.

Effects of hyperprolactinemia on ornithine decarboxylase activity and polyamine levels in seminal vesicles of genetically prolactin-deficient adult dwarf mice.

Prolactin (PRL) has been shown to exert many different actions in various biological systems. Polyamines are known to influence the growth and function of the seminal vesicles (SV). Furthermore, ornithine decarboxylase (ODC) is considered a key enzyme in the biosynthesis of polyamines and is regulated by PRL in certain target tissues. Adult Ames dwarf mice (df/df), genetically deficient in PRL, were used for this study. The experimental groups were as follows: Group 1, pituitary-grafted; Group 2, sham-operated; Group 3, castrated + testosterone propionate (TP)-treated (25 micrograms/mouse, 3 times/wk, s.c.) + grafted; and Group 4, castrated + TP as above. The animals were killed 40 days later, and polyamines and ODC activity in SV and liver were determined. Serum PRL, FSH, and testosterone (T) were also measured. In the grafted groups, there were significant elevations in serum PRL and FSH levels. In the gonad-intact, pituitary-grafted group, animals exhibited an elevation in plasma T levels, and similar levels were achieved in the castrated, androgen-replaced groups. In hyperprolactinemic mice, the weights of SV were significantly greater than in the corresponding control groups. The relative weights of the SV showed a similar pattern. An increase in ODC activity was observed in both SV and liver in hyperprolactinemic groups. In those animals in which serum T levels were held constant, an increase in the enzyme activity in SV was detected in hyperprolactinemic group whereas in liver, no significant difference was observed. Concentrations of polyamines in the SV were increased in hyperprolactinemic, castrated, TP-treated mice. The present results indicate that PRL can exert a direct stimulatory effect on the growth, ODC activity, and polyamine levels in the SV.

Androgenic activity of synthetic progestins and spironolactone in androgen-sensitive mouse mammary carcinoma (Shionogi) cells in culture.

A series of compounds designed to block the action of androgens in target tissues, and called antiandrogens, have been developed for the treatment of androgen-sensitive diseases, especially prostate cancer, hirsutism, precocious puberty and deviant sexual behavior. In order to further assess the androgenic activity of these compounds, we have studied their effect on the growth of an androgen-sensitive clone of the mouse mammary carcinoma Shionogi SC-115 cells in culture. Hydroxy-flutamide did not affect the doubling time (7.40 +/- 0.09 vs 7.20 +/- 0.12 days) characteristic of these cells. However, all of the other compounds tested stimulated cell growth. Thus, in the presence of cyproterone acetate, cells had an accelerated growth rate and shorter generation time of 6.28 +/- 0.06 days (P less than 0.01). In the presence of 1 microM spironolactone, the generation time was 4.96 +/- 0.04 days (P less than 0.01). With chlormadinone acetate, the doubling time was reduced to 3.79 +/- 0.08 days while for megestrol acetate, the doubling time was 3.63 +/- 0.04 days (P less than 0.01). The synthetic progestin Medroxyprogesterone acetate had the most potent androgenic effect reducing the doubling time to 1.85 +/- 0.05 days (P less than 0.01). For comparison, dihydrotestosterone gave a doubling time of 1.76 +/- 0.07 days. When hydroxy-flutamide (5 microM) was added simultaneously with each "progestin", the ED50 value of action of all the compounds was increased in a competitive manner, thus indicating that the mitogenic effect on cell growth of all compounds is mediated by the androgen receptor. Of all the compounds used, only hydroxy-Flutamide was devoid of any androgenic activity and thus meets the criteria of a pure antiandrogen.

Mediation by the androgen receptor of the stimulatory and antiandrogenic actions of 17 beta-estradiol on the growth of androgen-sensitive Shionogi mammary carcinoma cells in culture.

Increasing concentrations of 17 beta-estradiol (E2) led to a maximal 7-fold stimulation of growth of the highly androgen-sensitive clone (SEM-1) of the mammary carcinoma Shionogi cell line. Half-maximal stimulation by the estrogen was observed at 100 nM E2. Diethylstilbestrol (DES), on the other hand, a synthetic estrogen with no affinity for the androgen receptor, had no significant stimulatory effect on cell growth but caused growth inhibition at concentrations above 1 microM. Mediation of the action of E2 by the androgen receptor is indicated by the absence of interference of E2 action by the antiestrogen LY156758 while the antiandrogen hydroxyflutamide (3 microM) caused a 50% inhibition of E2 action. While increasing concentrations of E2 led to a progressive increase in cell growth, a progressive shift in the ED50 value of action of dihydrotestosterone (DHT) was observed at intermediate (10-100 nM) concentrations of E2 while 10 microM E2 completely inhibited DHT action. At those high E2 concentrations, however, E2 itself led to a stimulation of cell growth equivalent to approximately 50% of the maximal value achieved by DHT. E2 competed with the specific uptake of [3H]testosterone in intact cells at an inhibition constant (Ki) value of 15 nM, thus indicating direct interaction of E2 with the androgen receptor. Preincubation with E2 had no influence on the apparent affinity of testosterone for the androgen receptor nor on the number of androgen binding sites. The present data demonstrate that both the stimulatory and antiandrogenic action of E2 on the growth of the androgen-sensitive mammary carcinoma cell line SEM-1 are mediated through direct interaction of the estrogen with the androgen receptor. Such data may offer an explanation for the subjective improvements reported in prostate cancer patients receiving a high dose of E2 when relapsing after castration.

Adrenal precursor C19 steroids are potent stimulators of growth of androgen-sensitive mouse mammary carcinoma Shionogi cells in vitro.

Since there is convincing evidence for a role of adrenal steroids as precursors of active sex steroids in peripheral tissues, especially prostate cancer, we have studied the effect of the four main adrenal steroids, namely dehydroepiandrosterone sulfate (DHEA-S), DHEA, 5-androstene-3 beta,17 beta-diol (delta 5-diol) and 4-androstene-3,17-dione (delta 4-dione) on the growth of an androgen-sensitive clone (SEM-1) of the mouse mammary carcinoma Shionogi. From a control doubling time of 6.69 +/- 0.03 days, 0.1 microM DHT, 1.0 microM delta 4-dione, 10 microM delta 5-diol, 10 microM DHEA-S and 10 microM DHEA decreased generation time to 1.60 +/- 0.01, 1.69 +/- 0.01, 1.95 +/- 0.01, 4.37 +/- 0.02 and 5.66 +/- 0.03 days, respectively (P less than 0.01 vs. control). The same compounds exerted their stimulatory effects on cell growth at the following ED50 values: 0.06 nM, 16 nM, 90 nM, 150 nM and 16 microM for DHT, delta 4-dione, DHEA, delta 5-diol and DHEA-S, respectively. The stimulatory effect of all compounds was inhibited in a competitive manner by the pure antiandrogen hydroxyflutamide. Further evidence for an action of the adrenal steroids through the androgen receptor is indicated by competition of [3H]testosterone uptake in the tumor cells at the following IC50 values: 0.21 nM, 0.63 nM, 50 nM, 75 nM and 680 nM for DHT, testosterone, delta 4-dione, delta 5-diol and DHEA, respectively. The present data show that the four main adrenal steroids present in the serum of adult men can exert potent stimulatory effects on the growth of an androgen-sensitive cancer cell line through an androgen receptor-mediated mechanism.

Bromocriptine and sulpiride competitively inhibit estrogen binding to its receptor in the adrenal gland.

Bromocriptine and sulpiride incubated simultaneously with [3H]-estradiol in the cytosol from adrenal glands of adult male rats, yielded curves typical of competitive inhibition as analyzed by Lineweaver-Burk plots. The inhibition constant for both drugs was approximately 10(8) M-1, only 10 times lower than the association constant for estradiol.

Effect of median eminence lesions and hormonal replacement on the prolactin receptors in the adrenal gland and Langerhans islets from ovariectomized adult rats.

The effect of hyperprolactinemia induced by median eminence lesions (MEL) and ACTH and glucocorticoid replacement on prolactin (Prl) receptors was studied in the adrenal, isolated Langerhans islets and the liver. Adult rats were ovariectomized 15 days before MEL and they were divided in the following groups: 1) SHAM: injected with saline solution 3 times in alternate days; 2) MEL: saline solution; 3) MEL + ACTH: 50 micrograms: 10 IU/rat, s.c. (Synacthen) and 4) MEL + DEXA: 10 micrograms/rat (dexamethasone). For measuring total lactogenic binding sites an in vitro treatment of the membrane fraction with 4M MgCl2 was used. MEL originated a significant increase in Prl serum levels, which was not altered by injections of ACTH or dexamethasone. In contrast, serum corticosterone (B) levels in MEL rats were significantly lowered, and it was restored by ACTH. Unexpectedly, B levels increased when dexamethasone was administered to MEL rats. Prl receptors were diminished in the adrenal gland and Langerhans islets from MEL animals, as compared with the SHAM group. ACTH and glucocorticoid administration did not affect the pancreatic Prl receptors, while the adrenal gland exhibited a further lowering of Prl binding sites during ACTH treatment. Since no effect was found when dexamethasone was injected, a possible direct action of ACTH is suggested. On the other hand, Prl receptors were induced in the liver by MEL, and this action was abolished by dexamethasone and ACTH. Binding affinity in every tissue studied remained unchanged. Our data suggest that endogenous Prl is able to regulate its own receptors not only in the liver, but also in the adrenal gland and pancreatic islets.

Prolactin binding sites in the brain and kidneys of the toad, Bufo arenarum Hensel.

(125I)-ovine prolactin (oPRL) binding was found in several brain areas of the toad, Bufo arenarum Hensel. The olfactory bulb, cerebral hemispheres, and both dorsal and ventral mesencephalic regions showed saturable, high affinity, (125I)-oPRL binding, ranging between 5.6 to 29.9 fmol/mg protein, while the association constant (Ka) by Scatchard analysis was between 4.0 to 8.7 x 10(9) M-1. This binding was compared with the Scatchard plot of the kidney, which has been already described by other groups, and gave 41.7 fmol/mg protein and Ka 2.5 x 10(9) M-1. Liver showed no binding and in the cerebral hemispheres (125I)-oPRL was not displaced by non-lactogenic hormones, indicating that binding was hormone and tissue specific.

Ontogeny of the sex steroid and prolactin receptors in the male rat adrenal gland.

Cytosolic estrogen and androgen receptors and membrane prolactin-binding sites in the male adrenal glands showed a definite pattern during sexual development. The level of sexual steroid receptors paralleled adrenal growth, whereas prolactin binding reached its maximum value in mature rats.

Effects of prolactin, bromocriptine and sulpiride on estrogen and lactogenic receptors in the rat adrenal gland.

In prepubertal male rats, the injection of bromocriptine (Br) for 10 days caused an increase in adrenal weight (Br 0.75 mg/kg BW (Br I): 2.83%; Br 1.5 mg/kg BW (Br II): 12.1% and Br 3 mg/kg BW (Br III): 24.7%), and this effect was only significant at the highest dose. Sulpiride (S, 30 mg/kg BW/day) for 10 days produced a significant decrease in adrenal weight (18.6%), whereas ovine prolactin (oPRL) administered at doses of 0.5 or 5 mg/kg BW/day for 10 had no effect on this parameter. The action of these drugs on corticosterone serum levels was for Br III a 50.6% increase and for S a 29.2% decrease. Bromocriptine caused a significant increment of cytosolic available estrogen receptors C: 7.65 +/- 0.36 (SE); Br I: 10.2 +/- 0.36; Br II: 11.0 +/- 0.23 and Br III: 13.3 +/- 0.35) and total lactogenic receptors in the adrenal gland (C: 125.2 +/- 2.84; Br I: 203.8 +/- 4.43; Br II: 213.1 +/- 7.58 and Br III: 251.3 +/- 10.4), and this effect was dose-related. oPRL diminished adrenal estrogen receptors only at the highest dose used (C: 11.1 +/- 1.73; PRL 5: 8.2 +/- 0.75) as did S (C: 11.2 +/- 1.84 and S: 5.2 +/- 1.07); while the former originated a marked decrease in lactogenic adrenal binding sites at both doses (C: 198.7 +/- 12.2; PRL 0.5: 52.9 +/- 5.00 and PRL 5: 38.8 +/- 4.76), S also had a highly significant diminution over these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)