Cadmium exposure modifies lactotrophs activity associated to genomic and morphological changes in rat pituitary anterior lobe

Cadmium (Cd) is widely used in industrial applications and is an important contaminant of agricultural products. As an endocrine disruptor, Cd modifies the hormone release of pituitary anterior lobe (PAL). This work was undertaken to evaluate a possible association between phospholipase D (PLD) and prolactin mRNA expressions and the activity of lactotrophs and folliculostellate cells (FSC) in PAL of Cd exposed adult male Wistar rats (Cd, 0.133 mM per liter for 2 months). The PALs were submitted to immunohistochemical and morphometric analysis to determine the percentage of lactotrophs (PRL-ir) and FSC (S-100-ir). Cultured PAL cells were stained with Hoechst 33258 to determine the presence of alterations in nuclear morphology consistent with apoptosis. The expressions of PLD and prolactin mRNA were assessed by RT-PCR. Cd treated rats showed a decrease of PLD mRNA levels that can be associated to both high number of apoptotic cells and increase of S-100 protein expression in FSC. Cd decreased prolactin mRNA expression, number of lactotrophs and percentage of PRL-ir suggesting a low availability of prolactin to be secreted from PAL. Cd modifies the lactotrophs activity of pituitary gland through biochemical, genomic and morphological changes and contributes directly or indirectly to the levels of serum prolactin.     

Multifactorial Modulation of TRH Metabolism

1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through medianeminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary.2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell.3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure.4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times.TRH mRNA are thus controlled by transsynaptic and hormonal influences.5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglu-tamyl peptidase II (PPII).6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity.7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.     

3,3',5'-triiodo-L-thyronine reduces efficiency of mRNA knockdown by antisense oligodeoxynucleotides: a study with pyroglutamyl aminopeptidase II in adenohypophysis

The impact of hormones on the efficacy of antisense oligodeoxynucleotides (ASOs) is a poorly analyzed subject. We designed, based on the identification of potentially favorable local elements of mRNA secondary structure, eight phosphorothioate ASOs to knock down the expression of an ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in primary cultures of adenohypophysis. Two of the PPII ASOs were very efficient, sequence-specific, and target-specific. Because the expression of PPII is upregulated by 3,3',5'-triiodo-L-thyronine (T3), we studied the impact of varying the protocol of PPII induction on the knockdown efficacy. Hormone removal at transfection increased markedly the ability of (1) PPII ASOs to reduce PPII mRNA levels or PPII activity in adenohypophyseal cells or in C6 rat glioma cells and (2) a thyrotropin-releasing hormone (TRH) receptor-1 (TRH-R1) ASO to reduce TRH-R1 mRNA levels in adenohypophyseal cells. There was no effect of hormone removal on transfection efficacy and no correlation between target mRNA levels and ASO efficacy. These data demonstrated that ASO efficacy could depend on T3 levels; this might be due to regulation of a step generally critical for ASO efficiency.     

Various proteinase inhibitors decrease prolactin and growth hormone release by anterior pituitary cells

Proteinase inhibitors were tested for their ability to inhibit prolactin (PRL) and growth hormone (GH) release by cultured anterior pituitary cells of the rat. Inhibitors of microbial origin (chymostatin, elastatinal, leupeptin) had either no or a moderate effect on hormone release while some tripeptide aldehydes, especially those with lysine at their C terminus, inhibited markedly PRL and to a lesser extent GH release. Boc-DPhe-Phe-lysinal was the most effective on lactotrophs inhibiting PRL release more than 50% at 10(-4) M. The site(s) of action of tripeptide aldehydes remain to be elucidated.     

Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells

In vivo and in vitro effects of elevated androgens on agonist-induced gonadotropin secretion have been addressed previously. Here we investigated the effects of testosterone on hormonal content and basal (in the absence of agonists) hormone release in pituitary lactotrophs, somatotrophs and gonadotrophs from female rats. Furthermore we tested the hypothesis that testosterone action is dependent on the pattern of spontaneous and Bay K 8644 (a L-type calcium channel agonist) -induced calcium signalling. Mixed anterior pituitary cells were cultured in steroid containing or depleted media, and testosterone (1pM to 10nM) was added for 48h. Cells were studied for their spontaneous and Bay K 8644-induced calcium signalling pattern and total hormone levels (release and hormonal content). In lactotrophs, somatotrophs and gonadotrophs testosterone did not affect the pattern of spontaneous calcium signalling. Bay K 8644-induced calcium signalling and hormone release were not affected by testosterone. In both steroid-depleted and -containing medium, testosterone inhibited prolactin (PRL), luteinizing hormone (LH) and growth hormone (GH) cellular content and release in a dose-dependent manner, with IC(50)s in a sub-nanomolar concentration range. These results indicate that testosterone inhibits basal hormone release from lactotrophs, somatotrophs and gonadotrophs without affecting intracellular calcium signalling. This action of testosterone is not dependent on the presence of other steroid hormones.     

In vitro stimulation of prolactin release by vasoactive intestinal peptide

The effect of vasoactive intestinal peptide (VIP) on anterior pituitary hormone release was examined in a variety of in vitro preparations. Synthetic VIP was capable of stimulating increased prolactin (PRL) release from male rat hemipituitaries in doses as low as 10⁻⁹ M only when the enzyme inhibitor bacitracin was present in the incubation medium. Natural porcine VIP was similarly capable of stimulating PRL release, but only at higher doses (10⁻⁶ M). Additionally, synthetic VIP was capable of stimulating PRL release from dispersed anterior pituitary cells harvested from adult male and lactating female rats and from an enriched population of lactotrophs obtained by unit gravity sedimentation of similar dispersed cells from infantile female rats. No effect of VIP on luteinizing hormone, growth hormone or thyroid stimulating hormone release was seen. These findings taken in concert with the presence of VIP in the hypothalamus, pituitary and hypophyseal portal plasma of the rat suggest a physiological role for VIP in the control of PRL secretion.     

In vitro stimulation of prolactin release by vasoactive intestinal peptide

The effect of vasoactive intestinal peptide (VIP) on anterior pituitary hormone release was examined in a variety of in vitro preparations. Synthetic VIP was capable of stimulating increased prolactin (PRL) release from male rat hemipituitaries in doses as low as 10⁻⁹ M only when the enzyme inhibitor bacitracin was present in the incubation medium. Natural porcine VIP was similarly capable of stimulating PRL release, but only at higher doses (10⁻⁶ M). Additionally, synthetic VIP was capable of stimulating PRL release from dispersed anterior pituitary cells harvested from adult male and lactating female rats and from an enriched population of lactotrophs obtained by unit gravity sedimentation of similar dispersed cells from infantile female rats. No effect of VIP on luteinizing hormone, growth hormone or thyroid stimulating hormone release was seen. These findings taken in concert with the presence of VIP in the hypothalamus, pituitary and hypophyseal portal plasma of the rat suggest a physiological role for VIP in the control of PRL secretion.     

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.     

Endogenous thyrotropin-releasing hormone in the anterior pituitary: sites of activity as identified by immunocytochemical staining.

Thyrotropin-releasing hormone (TRH) immunoreactivity was localized in the rat anterior pituitary with rabbit anti-TRH sera and the unlabeled antibody peroxidase-antiperoxidase complex (PAP) technique. Stain was present in secretory granules of cells possessing morphological characteristics of thyrotropes, gonadotropes and lactotropes. Antibody absorption studies with anti-TRH sera absorbed with TRH, 3 diastereoisomeric analogues of TRH, gonadotropin-releasing hormone (GnRH), bovine serum albumin, thyrotropin, prolactin, adrenocorticotropin, luteinizing hormone, follicle stimulating hormone were performed to determine the specificity of the staining reaction. Only absorption with TRH resulted in a significant reduction in staining intensity. In vitro experiments were then begun with hemipituitaries to ascertain if intrapituitary TRH might originate by sequestration of exogenous, plasma membrane bound TRH or by de novo synthesis. The results suggest that anterior pituitary TRH is of endogenous origin.     

PMA-sensitive protein kinase C is not necessary in TRH-stimulated prolactin release from female rat primary pituitary cells.

In GH3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRH-stimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10(-6) M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary cells with TRH (10(-7) M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin. sn-1,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent response to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C.     

Different Behavior of Lactotroph Cell Subpopulations in Response to Angiotensin II and Thyrotrophin-Releasing Hormone

1. In the present investigation we have extended the study of lactotroph subpopulations in primary pituitary cell cultures. Male rats with or without previous estrogenization followed by A-II or TRH treatments were selected as experimental models.2. The TRH increased up to 50% the PRL released in both whole and ORQX + EB rats (P < 0.05). In contrast, A-II treatment introduced no changes in PRL secretion from cell cultures derived from whole male rats but attained a significant augmentation (about 75%) of PRL secreted by ORQX + EB pituitary cells.3. The addition of TRH and A-II to cultures of ORQX + EB-derived lactotrophs induced cytological changes compatible with a high secretory activity. In estrogen-treated rats the prevailing lactotroph subpopulation is type I. In cell cultures from control and A-II treated whole male pituitaries, the majority of lactotrophs consists of atypical subpopulations of II and III cells, with smaller secretory granules (between 150 and 300 nm in diameter).4. Morphometry of immunostained lactotrophs performed on light microscopic preparations revealed that about 30–36% of the total cell count were lactotrophs. This percentage was fixed and did not change significantly after TRH and A-II treatments.5. The present results confirm the presence of morphological and functional subtypes of lactotroph cells in rat pituitary. Typical PRL cell population shows the highest responsiveness to angiotensin II and TRH action. This functional heterogeneity of lactotroph subtypes may reflect an important and scarcely explored factor in the regulatory process of prolactin secretion.     

Impaired calcium mobilisation in the 7315a prolactin-secreting pituitary tumour

The 7315a tumour secretes prolactin, but is refractory to enhancement of prolactin release by thyrotrophin-releasing hormone (TRH). In order to investigate further this refractoriness of the 7315a tumour cell, we compared cells from the tumour and from the normal pituitary with regard to TRH-enhanced fractional 45Ca2+ efflux and inositol phosphate production. TRH caused a large efflux of calcium from normal pituitary cells, but only mildly enhanced calcium efflux from the tumour cells. In contrast, TRH enhanced total inositol phosphate generation in both groups of cells to a similar degree. We therefore conclude that prolactin release from 7315a tumour cells is refractory to TRH due, at least in part, to impaired mobilisation of intracellular calcium by inositol phosphates.     

Thyrotropin releasing hormone

Summary Thyrotropin releasing hormone (TRH) acutely stimulates release of thyrotropin (TSH) and prolactin from anterior pituitary cells. A considerable number of studies have been performed with neoplastic and nonneoplastic pituitary cells in culture to elucidate the sequence of intracellular events involved in this action. Although cyclic AMP was suggested as an intracellular messenger, it has been demonstrated that TRH stimulation of hormone release can be dissociated from changes in cyclic AMP concentration, thereby supporting the contention that cyclic AMP is not a required mediator. In contrast, stimulation of hormone release by TRH requires Ca²⁺ and it seems likely that Ca²⁺ is the intracellular coupling factor between TRH stimulation and hormone secretion. TRH has been shown to stimulate ⁴⁵Ca²⁺ efflux from preloaded pituitary cells. Enhanced ⁴⁵Ca²⁺ efflux is thought to reflect an increase in the free intracellular Ca²⁺ concentration which leads to hormone release; however, the source of this Ca^2– is uncertain. Results are reviewed from a series of experiments in pituitary cells which attempt to determine the pool (or pools) of Ca²⁺ that is affected by TRH. These include the following: the effects of decreasing the extracellular Ca^2– concentration on hormone release stimulated by TRH; the effect of TRH on cellular Ca²⁺ as monitored by chlortetracycline; the effects of TRH on Ca²⁺ influx; the effects of the organic Ca²⁺ channel blocking agents, verapamil and methoxyverapamil, on TRH-stimulated hormone release; and the effects of TRH on plasma membrane potential difference and on Ca²⁺-dependent action potentials. Based on these data, separate hypotheses of the early events in TRH stimulation of hormone release in mammotropes and thyrotropes are proposed. In mammotropes, TRH is thought to stimulate prolactin release optimally by elevating the free intracellular Cat+ concentration by mobilizing cellular Ca^2– only. In contrast, in thyrotropes under normal physiological conditions, TRH is thought to stimulate TSH release by mobilizing Ca² from a cellular pool (or pools) and to augment this effect by also inducing influx of extracellular Ca²⁺ through voltage-dependent channels in the plasma membrane.     

17β-Estradiol modulates the prolactin secretion induced by TRH through membrane estrogen receptors via PI3K/Akt in female rat anterior pituitary cell culture

Considering that estradiol is a major modulator of prolactin (PRL) secretion, the aim of the present study was to analyze the role of membrane estradiol receptor-α (mERα) in the regulatory effect of this hormone on the PRL secretion induced by thyrotropin-releasing hormone (TRH) by focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway activation. Anterior pituitary cell cultures from female rats were treated with 17β-estradiol (E(2), 10 nM) and its membrane-impermeable conjugated estradiol (E(2)-BSA, 10 nM) alone or coincubated with TRH (10 nM) for 30 min, with PRL levels being determined by RIA. Although E(2), E(2)-BSA, TRH, and E(2)/TRH differentially increased the PRL secretion, the highest levels were achieved with E(2)-BSA/TRH. ICI-182,780 did not modify the TRH-induced PRL release but significantly inhibited the PRL secretion promoted by E(2) or E(2)-BSA alone or in coincubation with TRH. The PI3K inhibitors LY-294002 and wortmannin partially inhibited the PRL release induced by E(2)-BSA, TRH, and E(2)/TRH and totally inhibited the PRL levels stimulated by E(2)-BSA/TRH, suggesting that the mER mediated the cooperative effect of E(2) on TRH-induced PRL release through the PI3K pathway. Also, the involvement of this kinase was supported by the translocation of its regulatory subunit p85α from the cytoplasm to the plasma membrane in the lactotroph cells treated with E(2)-BSA and TRH alone or in coincubation. A significant increase of phosphorylated Akt was induced by E(2)-BSA/TRH. Finally, the changes of ERα expression in the plasmalemma of pituitary cells were examined by confocal microscopy and flow cytometry, which revealed that the mobilization of intracellular ERα to the plasma membrane of lactotroph cells was only induced by E(2). These finding showed that E(2) may act as a modulator of the secretory response of lactotrophs induced by TRH through mER, with the contribution by PI3K/Akt pathway activation providing a new insight into the mechanisms underlying the nongenomic action of E(2) in the pituitary.     

Regulation of hormonal secretion and DNA synthesis in the lactotrophs of the rat adenohypophysis in vitro in primary cell cultures

Changes in DNA synthesis in lactotrophs of primary monolayer cultures of the rat pituitary cells were studied, using immunoperoxidase staining in combination with autoradiography. Pituitary cell cultures were treated for 3 days with thyroliberin (TRH), bromocriptine (CB154) or somatostatin (SRIF). The proportion of lactotrophs labelled with 3H-thymidine in the total pool of labelled cells served as a criterion for the estimation of DNA synthesis in prolactin-secreting cells. Prolactin secretion by the same cultures was measured by homologous radioimmunoassay. TRH (10 ng/ml) stimulated DNA synthesis in the total population of pituitary cells, but not in lactotrophs. SRIF decreased selectively the proliferation of lactotrophs, but failed to depress or even stimulated DNA synthesis in some cell types of the rat pituitary gland in the cultures. The quantitative method of studying DNA synthesis in anterior pituitary may be used to evaluate the effects of a number of biologically active compounds on various cell systems.     

Effect of [D-Trp6]LHRH infusion on prolactin secretion by perifused rat pituitary cells

The effect of a superactive agonistic analog of luteinizing hormone-releasing hormone (LHRH), [D-Trp6]LHRH on prolactin (PRL) secretion by perifused rat pituitary cells was investigated. Constant infusion of [D-Trp6]LHRH (0.5 ng/min) for 2-3 h elicited a significant decrease in PRL secretion by these cells. This decrease in PRL release started ca. 30 min after the beginning of the infusion with the LHRH analog and lasted up to 1.5-2 h. [D-Trp6]LHRH significantly stimulated luteinizing hormone (LH) secretion during the first 30 min of peptide infusion; thereafter, LH levels began to return to control values. In animals pretreated in vivo with 50 micrograms of [D-Trp6]LHRH (s.c.) 1 h before sacrifice, PRL secretion by the rat pituitary cell perifusion system was significantly lower than vehicle-injected controls throughout the entire [D-Trp6]LHRH infusion period. On the other hand, thyrotropin-releasing hormone (TRH)-stimulated PRL secretion was slightly, but significantly imparied by [D-Trp6]LHRH infusion, while dopamine (DA) inhibition of PRL release was unaffected by this same treatment. These results reinforce previous observations of a modulatory effect of [D-Trp6]LHRH, probably mediated by pituitary gonadotrophs, on PRL secretion by the anterior pituitary. In addition, our findings suggest that basal PRL secretion by the lactotroph may be dependent on a normal function of the gonadotroph. The collected data from this and previous reports support the existence of a functional link between gonadotrophs and lactotrophs in the rat pituitary gland.     

DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures. Effects of thyroliberin, bromocriptine and somatostatin

Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154). The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells. TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs. TRH only weakly counteracted the noticeable inhibitory effect of CB 154 (0.75 microM/l) on thymidine incorporation into lactotrophs. SRIF (20 ng/ml) inhibited DNA synthesis in lactotrophs to a lesser extent than CB 154. The combination of methods used in this paper may be useful for studying the selective effects of regulators on the proliferative activity of various pituitary cell types in vivo and in culture.