Differential regulation and action of estrogen receptors alpha and beta in GH3 cells.

The pituitary lactotroph, a well established target for estrogens, expresses estrogen receptor-alpha (ER alpha) and -beta (ER beta). A truncated isoform of ER alpha, named TERP, is expressed in the pituitary, but not in the uterus. In this study we used the somatolactotroph cell line, GH3 cells, to examine 1) the expression of ER alpha, TERP, or ER beta and their regulation by estradiol; 2) the presence of receptor proteins; and 3) the effects of overexpressing ER beta or TERP on estrogen induction of the PRL gene and activation of the estrogen response element (ERE). Incubation of GH3 cells with estradiol (0.1-10 nM) produced dose-dependent increases in messenger RNA levels of ER beta and TERP, but not ER alpha, as determined by quantitative RT-PCR. Cell incubation with 1 nM estradiol resulted in a time-dependent biphasic increase in TERP and a delayed rise in ER beta, suggesting activation by both direct and indirect mechanisms. A polyclonal ER beta antibody directed against an N-terminal synthetic peptide was generated. This antibody detected ER beta-positive cells in ovarian granulosa cells and in many cells throughout the pituitary; its specificity was demonstrated by preabsorption with the synthetic peptide. The antibody detected a 58- to 60-kDa protein by Western blotting of ovarian, pituitary, and GH3 cell extracts. Cotransfection of ER beta and reporter genes (PRL promoter/luciferase or ERE/luciferase) into GH3 cells resulted in a dose-dependent increase in estrogen-induced PRL gene expression, with a lesser activation of the ERE. A 20-kDa TERP protein was undetectable in untreated GH3 cells and was weakly induced by estradiol. Overexpression of TERP had no effect on estrogen induction of either PRL or ERE. We conclude that 1) both ER beta and TERP messenger RNAs in GH3 cells are increased by estradiol in a dose- and time-dependent manner, whereas ER alpha is not altered; 2) a 58-kDa ER beta protein is expressed in both the pituitary and GH3 cells; and 3) overexpression of ER beta increases estrogen-induced PRL gene expression.     

Activin-A, inhibin and transforming growth factor-beta modulate growth of two gonadal cell lines

The proliferative and differentiating effects of the gonadal hormones inhibin and activin-A were examined on cell lines derived from the ovary and testis. Activin-A was found to inhibit the growth of CHO-K1 (Chinese hamster ovary) cells in culture, with an IC50 of 3 ng/ml. The maximal response (50% inhibition) required 3 days of incubation in the presence of 40 ng/ml activin-A, and the inhibitory effect was accompanied by morphological changes. Inhibin (10 ng/ml) partially blocked the inhibition of growth by activin. Transforming growth factor-beta (TGF beta), which is structurally related to activin and inhibin, was a very potent inhibitor of the proliferation of CHO-K1 cells, with an IC50 of 0.2 ng/ml and a maximal effect (70% inhibition) at 2 ng/ml. The combination of high concentrations of both TGF beta and activin-A did not result in a greater inhibitory effect than that observed with TGF beta alone, suggesting an overlapping step in the mechanism of action for both factors. In contrast to the results with CHO-K1 cells, differential effects of activin-A and TGF beta were observed in R2C (rat Leydig cell testicular tumor) cells. Activin-A had only a slight effect on proliferation over a 4-day incubation, but inhibited progesterone accumulation ina concentration-dependent fashion within 12 h. TGF beta, on the other hand, was a potent inhibitor of both growth and steroidogenesis in R2C cells. These studies suggest that activin-A and inhibin may regulate proliferation as well as functions of gonadal cells.     

Possible role of transforming growth factor-beta as a mediator of luteotropic action of prolactin in rat luteal cell cultures

PRL is known to be a hormone carrying luteotropic action in rats and enhances progesterone secretion by suppressing 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD) activity in the corpus luteum. In this study, we investigated the effects of PRL and transforming growth factor-beta (TGF beta) on the 20 alpha HSD activity of rat luteal cells in vitro and examined whether TGF beta is involved in the luteotropic action of PRL. 20 alpha HSD activity of luteal cells (from midpseudopregnant rats), which had been suppressed by PRL in vivo, increased when the cells were cultured for 48 h without PRL addition. TGF beta (0.01, 0.1, 1.0, and 10.0 ng/ml) as well as PRL (2, 20, 200, and 2000 ng/ml) suppressed this increase in a dose-dependent manner. Furthermore, the suppressive effect of PRL on 20 alpha HSD activity was significantly attenuated by anti-TGF beta antibody. Activin, having homology with TGF beta in its chemical structure, also suppressed the increase in enzyme activity, although the effect was much less marked than that of TGF beta. TGF beta or PRL did not affect total progestin (progesterone plus 20 alpha-dihydroprogesterone) secretion, but induced reduction in 20 alpha-dihydroprogesterone secretion during a 48-h culture period, without any alteration of DNA or protein content per culture dish. These results indicate that TGF beta, like PRL, can suppress luteal 20 alpha HSD activity without producing nonspecific cell damage, and that the luteotropic action of PRL is at least in part mediated by TGF beta or an immunoreactive TGF beta-like substance(s).     

Effects of the gp130 cytokines ciliary neurotropic factor (CNTF) and interleukin-11 on pituitary cells: CNTF receptors on human pituitary adenomas and stimulation of prolactin and GH secretion in normal rat anterior pituitary aggregate cultures

Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor (LIF) and interleukin (IL)-6, which belong to the cytokine family using the common gp130 signal transducer. Recently, the expression and action of two other members of this family, IL-11 and ciliary neurotrophic factor (CNTF), on different cell lines has also been demonstrated. We studied the expression of the specific receptor subunits for CNTF in mammotropic, non-functioning and somatotropic tumors and the action of CNTF and IL-11 in the regulation of hormone secretion in these and normal pituitary cells. The mRNA for the alpha chain specific for the CNTF receptor was detected by Northern blot in tumors secreting prolactin (PRL) and GH and in non-functioning tumors. We found that both IL-11 and CNTF exerted a similar stimulatory effect on GH mRNA expression in somatotropic monolayer cell cultures from acromegalic tumors, but these cytokines had no significant influence on GH secretion. CNTF stimulates prolactin secretion in lactotropic monolayer cell cultures from patients with prolactinoma. In monolayer cell cultures from normal rat anterior pituitary, IL-11 and CNTF had no significant effect on the release of either GH or PRL, or on GH mRNA. However, when the cells were cultured in aggregate cultures, in which the three-dimensional structure of the cells is reconstituted, both cytokines, in doses at which they had no effect on monolayer cultures, significantly stimulated both PRL and GH secretion. These data show that IL-11 and CNTF may act as regulatory factors in anterior pituitary cells, in which the three-dimensional structure of the gland is of critical importance.     

Bihormonal cells producing gonadotropins and prolactin in a rat pituitary tumor cell line (RC-4B/C)

Using single hormone-staining immunocytochemical methods, we have recently characterized a novel cell line, RC-4B/C, established from an aged male rat pituitary adenoma. This cell line contained all known anterior pituitary cell types including gonadotropes. Gonadotropin-releasing hormone receptors were also present. A recent re-examination of the cell types using single hormone stains showed that the cell line underwent an alteration in the percentage of different cell types as compared to the data obtained 2 years ago. The proportion of follicle-stimulating hormone-beta (FSH beta), luteinizing hormone-beta (LH beta), prolactin (PRL), and adrenocorticotropic hormone cells increased significantly (p less than or equal to 0.001), while the proportion of growth hormone (GH), and thyrotropin-beta cells did not change. Dual staining of the monolayers showed that the cell line contained many bihormonal cells producing FSH beta + LH beta, FSH beta + PRL and LH beta + PRL. The proportion of bihormonal FSH beta + LH beta and FSH beta + PRL cells was preponderant over monohormonal cells, while the proportion of bihormonal LH beta + PRL cells was in the same range as that of monohormonal cells. Preliminary data with dual labeling also revealed the presence of GH and PRL in the same cell, but complete absence of a combination such as FSH beta + GH. No search for the presence of other bihormonal or multihormonal cells was performed. In short, our data show that the majority of the cells in the cell line RC-4B/C contain FSH beta, LH beta and PRL and that among these cells many bihormonal cells are present.     

The gp130 cytokines interleukin-11 and ciliary neurotropic factor regulate through specific receptors the function and growth of lactosomatotropic and folliculostellate pituitary cell lines

Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor and interleukin-6 (IL-6), which belong to the cytokine receptor family using the common gp130 signal transducer. We studied the actions of two other members of this family, IL-11 and ciliary neurotropic factor (CNTF), on folliculostellate (FS) cells (TtT/GF cell line) and lactosomatotropic cells (GH3 cell line). The messenger RNA (mRNA) for the alpha-chain specific for the IL-11 receptor (1.7 kb) and CNTF receptor (2 kb) are expressed on both cell types. In addition, we detected CNTF receptor mRNA in normal rat anterior pituitary cells. IL-11 (1.25-5 nM) dose dependently stimulated the proliferation of FS cells. CNTF, at doses from 0.4-2 nM, also significantly stimulated the growth of these cells. In addition, both cytokines significantly stimulated proliferation of lactosomatotropic GH3 cells, and CNTF stimulated hormone production (GH and PRL) at 24 h by these cells. At 16-72 h, IL-11 stimulates the secretion of the angiogenic factor vascular endothelial growth factor by FS cells. In addition, both GH3 and FS cells express CNTF mRNA. These data suggest that IL-11 and CNTF may act as growth and regulatory factors in anterior pituitary cells.     

Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture

Bombesin (BBS) has been previously shown to stimulate the secretion of PRL and GH in steroid-primed rats. To determine whether these effects were mediated by the central nervous system or were due to direct action on the pituitary gland, we studied the interaction of BBS with GH4C1 cells, a clonal strain of rat pituitary cells which synthesizes and secretes PRL and GH. The addition of 100 nM BBS to GH4C1 cells for 60 min increased PRL release to 140 +/- 3% of the control value (mean +/- SE) and GH release to 133 +/- 5% of the control value. Stimulation of hormone secretion was observed within 15 min of treatment with 100 nM BBS and continued for at least 2 h. Half-maximal stimulation of PRL release occurred with 0.5 nM BBS, and a maximal effect was observed with 10 nM peptide. The BBS analogs ranatensin, litorin, and [Tyr4]BBS, each at a concentration of 100 nM, caused the same stimulation of PRL release as maximal concentrations of BBS itself. BBS stimulated hormone release selectively in two of five different clonal pituitary cell strains examined. Pretreatment of GH4C1 cells with 1 nM estradiol and/or 100 nM insulin resulted in more powerful stimulation of PRL release by both TRH and BBS. When epidermal growth factor and vasoactive intestinal peptide were added simultaneously with BBS, PRL release was greater than in the presence of either peptide alone. In contrast, the stimulatory effects of TRH and BBS were not additive. Somatostatin inhibited both basal and stimulated PRL release. Thus, low concentrations of BBS can directly stimulate PRL and GH release by a clonal pituitary cell strain in culture. These results suggest that BBS may stimulate PRL and GH secretion in vivo by direct action on the pituitary gland.     

Dopamine receptor activation inhibits estrogen-stimulated transforming growth factor-alpha gene expression and growth in anterior pituitary, but not in uterus.

Transforming growth factor-alpha (TGF alpha) has been localized to the anterior pituitary, specifically to the lactotroph and somatotroph cell populations, by our previous studies. Since pituitary lactotrophs are known to undergo growth in response to estrogens, we have used an estradiol-induced pituitary hyperplasia/adenoma model. Estradiol treatment resulted in induction of TGF alpha mRNA in anterior pituitary, evident by 48 h, preceding actual macroscopic growth, which attained a maximum greater than 500% by 12 weeks. This rapid effect of estradiol also enhanced PRL mRNA, but did not affect other species of mRNA encoding for proenkephalin, D2 receptor mRNA, or hexosaminidase-A. TGF alpha mRNA remained elevated for the duration of rapid pituitary growth. D2 receptor activation by its agonist bromocriptine resulted in marked attenuation of TGF alpha mRNA preceding regression of growth. Coadministration of bromocriptine with estradiol resulted in an involution of pituitary size, indicating the overriding influence of dopamine in spite of a continued estrogenic stimulus. Epidermal growth factor receptor mRNA was not affected by any of these manipulations, suggesting that the receptor was not coregulated in this tissue similarly to TGF alpha. Estradiol also induced uterine TGF alpha mRNA and marked growth of the organ, but TGF alpha in this location was not regulated by dopamine. These results indicate that TGF alpha in the anterior pituitary is rapidly induced by estrogen in a time course preceding the growth of the gland. Estrogen-induced TGF alpha is rapidly attenuated by D2 dopamine receptor activation and is accompanied by a regression of pituitary growth. Interaction between these opposing hormonal/transmitter responses will determine the growth potential of the anterior pituitary.     

Transforming growth factor-beta blocks protein kinase-A-mediated iodide transport and protein kinase-C-mediated DNA synthesis in FRTL-5 rat thyroid cells.

Recent studies have shown that transforming growth factor-beta (TGF beta) alters DNA synthesis and iodide metabolism in human, porcine, and rat thyroid cells. In the present work we studied the mechanism of TGF beta action in FRTL-5 rat thyroid cells. The cells were treated with TGF beta in the presence of TSH, growth factors, and cellular modulators for various periods of time; then, [3H]thymidine incorporation and DNA content were measured as indicators of DNA synthesis, and [125I]iodide uptake was measured to assess cell function. TGF beta (10 ng/ml) inhibited TSH-induced DNA synthesis and iodide uptake. TGF beta also inhibited DNA synthesis induced by insulin-like growth factor-I, fibroblast growth factor, and endothelial cell growth factor. The protein kinase-A (PKA) activator 8-bromo-cAMP increased both iodide uptake and DNA synthesis; TGF beta inhibited 8-bromo-cAMP-induced [125I]iodide uptake, but not [3H]thymidine incorporation. The protein kinase-C (PKC) activator phorbol 12-myristate 13-acetate increased [3H]thymidine incorporation, and TGF beta inhibited this action of phorbol 12-myristate 13-acetate. The results show that activation of PKA or PKC increases DNA synthesis. TGF beta inhibited PKC-mediated, but not PKA-mediated, DNA synthesis in these cells. The results also show that TGF beta selectively inhibits PKA-mediated iodide uptake, but not PKA-mediated DNA synthesis. These findings suggest that TGF beta is a strong inhibitor of the proliferation and function of thyroid cells.     

Early human thymocyte proliferation is regulated by an externally controlled autocrine transforming growth factor-beta 1 mechanism

Early thymocytes undergo extensive proliferation after their entry into the thymus, but cellular interactions and cytokines regulating this intrathymic step remain to be determined. We analyzed the effects of various T-cell growth factors and cellular interactions on in vitro proliferation of early CD2+CD3/TCR-CD4-CD8- (triple negative [TN]) human thymocytes. Freshly isolated TN cells were then assayed for their growth capacity after incubation with CD2I+III-monoclonal antibody (MoAb), recombinant human interleukin-2 (IL-2), IL-7, and/or IL-4. These cells displayed significant proliferative responses with IL-4, IL- 7, or CD2-MoAb+IL-2. The addition of recombinant transforming growth factor beta (TGF beta) or autologous irradiated CD3+CD8+CD4- cells to TN cell cultures dramatically decreased their growth responses to IL-2 and IL-7, whereas IL-4-induced proliferation was less sensitive to growth inhibition. We thus asked whether the CD8+ cell-derived inhibitory effect was due to TGF beta. The addition of neutralizing anti-TGF beta MoAb completely abolished CD8+ cell-derived inhibition of TN cell growth. Analysis of CD8+ cell-derived supernatants indicated that these cells had low TGF beta 1 production capacity, whereas TN cells secrete significantly high levels of TGF beta 1. Cell fixation studies showed that TN cells were the source of the TGF beta. TGF beta 1 released from TN cells was in the latent form that became the active inhibitory form through interaction of TN cells with CD8+ cells. Together, these data suggest a role for TGF beta 1 as an externally controlled, autocrine inhibitory factor for human early thymocytes, with a regulatory role in thymic T-cell output.     

Inhibitory effects of serum and stimulatory effects of estrogen on prolactin mRNA levels in GH3 rat pituitary tumor cells.

We investigated the effects of serum and estrogen on prolactin (PRL) mRNA accumulation in GH3 cells under different cell culture conditions. Hybridization analysis of GH3 cellular RNA indicated that PRL mRNA levels decreased more than 20-fold in cells cultured for 1 week in medium containing dextran-charcoal-treated serum (stripped serum). No effects on actin mRNA levels were observed under these conditions. Furthermore, this inhibition of PRL mRNA accumulation depended on the concentration of stripped serum in the medium. Although incubation in stripped-serum medium inhibited cell growth, these culture conditions did not appear to irreversibly affect the GH3 cell population. These data indicate that a potent inhibitor of PRL mRNA accumulation is present in stripped serum. GH3 cells grown in stripped-serum medium were shown to be responsive to estrogen. Treatment of these cells with 10(-9) M estradiol resulted in a 6.6-fold stimulation of PRL gene expression. However, estrogen had no effect on cell growth under these conditions, suggesting that estrogen stimulates PRL gene expression and cell proliferation by independent mechanisms.     

Transient dopaminergic inhibition of prolactin release from hybrid cells derived by fusion of normal rat pituitary and GH4C1 tumor cells

The clonal rat pituitary tumor cell line GH4C1 secretes PRL but does not respond to dopamine, a physiological inhibitor of PRL. In an attempt to generate a dopamine-responsive cell line, GH4C1 cells, which lack the enzyme hypoxanthine-guanine phosphoribosyltransferase, were fused with cells from the pituitary glands of lactating rats to generate cell hybrids. The GH4C1 cells were fused with dispersed normal pituitary cells by either chemical fusion in 40% polyethylene glycol or electrofusion. The fused cells were grown in medium with hypoxanthine, aminopterin, and thymidine (HAT) for 4 weeks to select for hybrid cells. Control fusions between GH4C1 cells only or normal cells only did not produce viable colonies. Of 36 HAT-selected colonies, 3 responded to 10 nM bromocryptine (BCR) with inhibition of TRH-stimulated PRL release. These hybrid colonies had an inhibitory response similar to that of normal pituitary cells in culture. Both TRH- and vasoactive intestinal peptide-stimulated PRL release were inhibited to basal levels by 10 nM BCR, with an IC50 for BCR of approximately 0.25 nM. Basal hormone release was not inhibited by BCR. The BCR-sensitive hybrid cells grew more slowly than the parental GH4C1 line both in culture and when passaged in female Wistar-Furth rats. The response of the hybrid cells to the dopamine agonist and the characteristic of slow growth were lost after 9 months of continuous culture and after freezing cells. The parental GH4C1 cells were grown in female Wistar-Furth rats, the resulting tumors were dissociated, and the cells were grown in culture. This resulted in a brief establishment of the dopamine response. Stimulated PRL and GH release from freshly dispersed GH4C1 tumor cells was inhibited by BCR at concentrations from 0.1-10 nM, and spiroperidol reversed the inhibition. The inhibitory response to the dopaminergic agonist was lost quickly as the cells were carried in culture. These results demonstrate that GH4C1 cells may have the genetic information necessary for dopaminergic inhibition of PRL synthesis, but that the dopamine response is not observed under standard tissue culture conditions.     

Differential effects of estrogen receptor antagonists on pituitary lactotroph proliferation and prolactin release

Anti-estrogens act by inhibiting estrogen receptor (ER) function. Unlike raloxifene and tamoxifen which exhibit both antagonist and agonist properties, ICI 182,780 (ICI) is considered a "pure" anti-estrogen devoid of any agonistic activities. Whereas there is ample information on the effects of anti-estrogens on the breast and uterus, little is known about their action on the pituitary, the estrogen-sensitive master endocrine gland. Our objectives were to: (1) compare the effects of ICI, tamoxifen and raloxifene on lactotroph proliferation in the absence of estrogen, (2) determine whether their action is mediated through the ER, and (3) compare their effects on prolactin (PRL) release. We are reporting that ICI is a potent inhibitor of lactotroph proliferation (both GH3 and MMQ cells) with maximal inhibition of 45-50% seen with 1nM. ICI is several orders of magnitude more potent than raloxifene while tamoxifen has no effect. Neither anti-estrogen affects T47D breast cancer cell proliferation. GH3 cell incubation with ICI for 1h only causes maximal suppression of cell proliferation, an effect which is reversed by co-incubation with estrogen. Such a short exposure to ICI is sufficient to cause rapid and persistent downregulation of ERalpha protein, whereas downregulation of ERbeta is significantly delayed; tamoxifen and raloxifene have no appreciable effects on ER(s) levels. The ability of ICI to inhibit GH3 cell proliferation is dependent upon ERalpha, since an ERalpha, but not ERbeta, specific agonist reverses the effect of ICI. PRL release is differentially regulated by the anti-estrogens. ICI at 0.1nM suppresses PRL release from GH3 cells by 80%, with a similar strong suppression also seen with 10nM raloxifene. However, tamoxifen at 0.01nM inhibits PRL release but has no effect at 10nM. Cell co-incubation with ICI and estradiol results in a four-fold increase in PRL release. Taken together, our study shows that ICI, in the absence of exogenous estrogens, inhibits lactotroph proliferation and PRL release by downregulating or inactivating ERalpha. The dissimilar responses of cell proliferation and PRL release to the anti-estrogens suggest that both processes are regulated by different mechanisms. These data highlight the importance of studying the effects of anti-estrogens in multiple systems.     

Comparison of the pituitary effects of the mammalian endothelins: vasoactive intestinal contractor (endothelin-beta, rat endothelin-2) is a potent inhibitor of prolactin secretion.

Direct pituitary effects of vasoactive intestinal contractor (VIC), which has been described recently to be the rat form of endothelin-2 (ET-2), were compared to those previously reported for rat ET-1, rat ET-3, and human ET-2. In static incubations of cultured dispersed anterior pituitary cells, the minimum effective dose of VIC necessary to inhibit PRL release after 1-h incubation was 1 pM, and the maximum effective dose was 1 nM. Similar inhibition was observed with human ET-2. The minimum effective inhibitory dose of ET-1 was also 1 pM; however, that of ET-3 was 0.1 nM. PRL release inhibition by VIC was not mediated via the D2-dopamine receptor and was not prevented by calcium channel blockade with 100 nM nifedipine. The inhibitory effect of VIC was not present in cells treated with 100 nM staurosporine, a dose that inhibits protein kinase-C activity. Time-course studies revealed a transient stimulation of PRL release with higher doses of VIC (10 and 100 nM), which occurred within the first 15 min of incubation and was unaffected by calcium channel blockade or inhibition of protein kinase-C activity. No stimulation of PRL release was observed with doses of VIC lower than 10 nM. Instead, we observed the maintenance of the inhibitory effect for 4 h of incubation. GH release was not significantly affected by doses of VIC ranging from 10(-13)-10(-7) M; however, the release of LH was slightly, yet significantly, stimulated by 10 and 100 nM VIC. This release was prevented by pretreatment with nifedipine, but unaffected by protein kinase-C inactivation. A physiological role for VIC (rat ET-2) in the control of lactotroph function is suggested by its effectiveness at picomolar doses and its long-lasting action.     

Interferon-alpha-2a is a potent inhibitor of hormone secretion by cultured human pituitary adenomas.

Interferon-alpha (IFN alpha) may exert direct inhibitory effects on cell proliferation and on the production of different peptide hormones. We investigated the effect of IFN alpha on hormone production by 15 GH-secreting pituitary adenomas, 4 clinically nonfunctioning or gonadotroph pituitary adenomas, and 4 prolactinomas in vitro. In the GH-secreting pituitary adenoma cultures, a short term (72-h) incubation with IFN alpha (50-100 U/mL) significantly inhibited GH secretion in 3 of 7 cases and PRL secretion in 6 of 7 cultures. During prolonged incubation (14 days) with IFN alpha, GH and/or PRL secretion was significantly inhibited in 7 of 8 cultures (GH, 17-78% inhibition; PRL, 39-88% inhibition). In the clinically nonfunctioning or gonadotroph cultures, incubation with IFN alpha resulted in inhibition of the secretion of gonadotropins and/or alpha-subunit in all cases (27-62%), whereas in the prolactinoma cultures PRL secretion was inhibited by IFN alpha in all cases (37-76%). The effect of IFN alpha was additive to the inhibitory effects of the dopamine agonist bromocriptine (10 nmol/L) or the somatostatin analog octreotide (10 nmol/L). The inhibition of hormone secretion by IFN alpha was accompanied by inhibition of the intracellular hormone concentrations. The effect of IFN alpha was dose dependent, with an IC50 for inhibition of hormone secretion of 2.3 +/- 0.3 U/mL (n = 5), which is relatively low compared with the concentrations that are reached in patients treated with IFN alpha for various malignancies. In conclusion, the potent antihormonal effect of IFN alpha on cultured pituitary adenomas suggests that this drug might be of benefit in the treatment of selected patients with secreting pituitary adenomas. As treatment with IFN alpha is associated with considerable adverse reactions, studies with this drug should only be considered in inoperable, invasive aggressive, and dopamine agonist- and/or somatostatin analog-resistant functioning pituitary macroadenomas.     

Differential effects of ketoconazole on prolactin and growth hormone release by normal and tumoral rat anterior pituitary cells in vitro

The imidazole derivative ketoconazole (1-100 microM) was shown to stimulate the release of prolactin (PRL) from rat anterior pituitary cells in vitro. In contrast, this drug did not affect growth hormone (GH) release from the same cells. In addition, ketoconazole was found to have no effect on PRL or GH release from a tumoral pituitary cell clone (GH3). Treatment of normal pituitary cells with ketoconazole (10 microM) for more than 20 min abolished TRH-induced hormone release. TRH-stimulated release was both attenuated and delayed in the ketoconazole-treated tumoral cells. Ketoconazole (10 microM) did not affect the basal electrophysiological properties of GH3 cell membranes, although it did affect the TRH-induced response. The action of ketoconazole of the spontaneous release of PRL by normal cells and the TRH-stimulated release of PRL and GH is consistent with an interference with arachidonic acid metabolism.     

Implication of pituitary vasoactive intestinal peptide in dopaminergic inhibition of estrogen-induced pituitary hyperplasia and vascular endothelial growth factor expression

We have shown that pituitary vasoactive intestinal peptide (VIP) mediates the effects of estrogen on lactotrope hyperplasia, angiogenesis and hyperprolactinemia, and reduces the pituitary content of transforming growth factor beta beta1 (TGF-beta1, an inhibitor of lactotrope proliferation). Dopamine agonists reverse lactotrope hyperplasia and hyperprolactinemia and also reduce the pituitary VIP content in hyperestrogenized rats. To elucidate the interaction of bromocriptine (BC) and pituitary VIP, a VIP receptor antagonist (VA), BC, or both drugs were administered for 5 days to F344 rats treated with diethylstilbestrol (DES). Both BC and VA similarly blocked the effects of DES on pituitary weight and pituitary content of prolactin (PRL), proliferating cell nuclear antigen, and vascular endothelial growth factor, without evidence of synergism. The estrogen effect on pituitary TGF-beta1 was completely inhibited by VA, but only partially by BC. On the contrary, serum PRL was close to the normal levels in the BC group 2 h after the first dose, while VA only reduced serum PRL after 5 days. DES increased VIP and VIP mRNA levels specifically at the pituitary, this effect being partially blocked by BC. These data suggest that the dopamine agonists inhibit lactotrope proliferation and angiogenesis by blocking the autocrine/paracrine action of VIP. On the other hand, the dopamine agonists inhibit the estrogen-induced hyperprolactinemia by acting through different pathways than those implicated in the proliferative process.