Bovine pituitary cells exhibit a unique form of somatotrope secretory heterogeneity

Use of reverse hemolytic plaque assays revealed that, as in the rat, bovine somatotropes are functionally heterogeneous with respect to regulated GH release. Uniquely, this heterogeneity is manifested by the appearance of a distinct subpopulation of GH cells that release hormone only in the presence of the hypothalamic secretagogues GRF and/or TRH. Although these cells did not release GH in the absence of stimulatory agents, immunocytochemical detection of GH demonstrated these cells were capable of hormone storage. The relative abundance of silent somatotropes varied according to the hypothalamic secretagogue applied to the culture (GRF and/or TRH) and the physiologic state of the tissue donor. In cell cultures obtained from castrated males, 4.2 +/- 1.2%, 3.0 +/- 0.7%, and 6.8 +/- 1.2% (mean +/- SEM; n = 14) of all pituitary cells were induced to release detectable amounts of GH by GRF, TRH, and GRF/TRH, respectively. When pituitary cells obtained from gonad-intact males (n = 4) and females (n = 4) were tested, only incubation with GRF (14.1 +/- 2.5%; 6.9 +/- 2.3%, respectively) and GRF/TRH (15.2 +/- 2.4%; 9.2 +/- 2.6%, respectively) stimulated a significant population of these silent cells. A combined analysis of the proportion of GH secretors and the relative amount of hormone released per cell revealed that a substantial fraction of the GH secreted in vitro after stimulation is attributable to the recruitment of silent somatotropes into the secretory pool. Taken together, these results reveal the existence of a unique form of GH cell heterogeneity--the silent somatotrope. Moreover, our findings demonstrate that this somatotrope population could provide a cellular basis for the readily releasable pool of GH in the bovine pituitary.     

Different regulation of growth hormone-releasing factor-sensitive adenylate cyclase in the anterior pituitary of young and aged rats

Low basal GH secretion and reduced GH responsiveness to different GH secretagogues, including GHRF, have been reported in aged animals and humans. Parallel to the in vivo findings, an impaired GH responsiveness to GHRF is evident in somatotropes from old rats of either sex. We report here that in anterior pituitaries (APs) from aged male and female rats GHRF-induced stimulation of adenylate cyclase (AC) activity was strikingly reduced (male rats, change from baseline 700% in young and 100% in old rats) or lacking (female rats, change from baseline 430% in young and 13% in old rats) when compared to that evoked by GHRF in the APs from young counterparts. Pretreatment with GHRH (5 micrograms/rat iv for 3 days) decreased the high basal AC activity of old male rats [from 33.38 +/- 3.60 to 15.99 +/- 5.75 (SEM) pmol cAMP/min.mg protein], did not alter the GHRF-stimulated rise in AC activity in old male rats, and induced a small but unequivocal rise in AC activity in old female rats (change from baseline 35% vs. 13%, respectively). Pretreatment with GHRF markedly reduced the acute effect of GHRF in the APs from young rats of both sexes (male rats, change from baseline 360% and 700%; female rats, change from baseline 230% and 430% in GHRF-pretreated and control rats, respectively). In parallel studies performed in female rats, it was shown that in vivo pretreatment with GHRF at the same schedule markedly reduced the effect of acute GHRF stimulation on GH secretion from cultured pituitary cells of young rats but left unchanged GHRF-induced stimulation of GH secretion from pituitary cells of old rats. In all, these data suggest that deficiency of endogenous GHRF synthesis and/or release may underlie defective GH secretion in old rats and that a GHRF replacement regimen that reduces the sensitivity of the young somatotrope cells does not alter the sensitivity of (male rats) or exerts a priming effect (female rats) on the old somatotrope cell.     

Subpopulations of somatotropes with differing intracellular calcium concentration responses to secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca2+]i). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Somatotropes were identified and distinguished from other adenohypophyseal cells, by their unique ability to respond to GH-releasing hormone with increased [Ca2+]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca2+]i (222 +/- 16 nM) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca2+]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca2+]i in only 21% of somatotropes. Perfusion with gonadotropin-releasing hormone elevated [Ca2+]i, but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogues.     

GH in the dwarf dopaminergic D2 receptor knockout mouse: somatotrope population, GH release, and responsiveness to GH-releasing factors and somatostatin

Recently, the importance of the dopaminergic D2 receptor (D2R) subtype in normal body growth and neonatal GH secretion has been highlighted. Disruption of D2R alters the GHRH-GH-IGF-I axis and impairs body growth in adult male mice. The D2R knockout (KO) dwarf mouse has not been well characterized; we therefore sought to determine somatotrope function in the adult pituitary. Using immunohistochemistry and confocal microscopy, we found a significant decrease in the somatotrope population in pituitaries from KO mice (P=0.043), which was paralleled by a decreased GH output from pituitary cells cultured in vitro. In cells from adult mice the response amplitude to GHRH differed between genotypes (lower in KO), but this difference was less dramatic after taking into account the lower basal release and hormone content in the KO cells. Furthermore, there were no significant differences in cAMP generation in response to GHRH between genotypes. By Western blot, GHRH-receptor in pituitary membranes from KO mice was reduced to 46% of the level found in wildtype (WT) mice (P=0.016). Somatostatin induced a concentration-dependent decrease in GH and prolactin (PRL) secretion in both genotypes, and 1x10(-7) M ghrelin released GH in cells from both genotypes (P=0.017) in a proportionate manner to basal levels. These results suggest that KO somatotropes maintain a regulated secretory function. Finally, we tested the direct effect of dopamine on GH and PRL secretion in cells from both genotypes at 20 days and 6 months of life. As expected, we found that dopamine could reduce PRL levels at both ages in WT mice but not in KO mice, but there was no consistent effect of the neurotransmitter on GH release in either genotype at the ages studied. The present study demonstrates that in the adult male D2R KO mouse, there is a reduction in pituitary GH content and secretory activity. Our results point to an involvement of D2R signaling at the hypothalamic level as dopamine did not release GH acting at the pituitary level either in 1-month-old or adult mice. The similarity of the pituitary defect in the D2R KO mouse to that of GHRH-deficient models suggests a probable mechanism. A loss of dopamine signaling via hypothalamic D2Rs at a critical age causes the reduced release of GHRH from hypophyseotropic neurons leading to inadequate clonal expansion of the somatotrope population. Our data also reveal that somatotrope cell number is much more sensitive to changes in neonatal GHRH input than their capacity to develop properly regulated GH-secretory function.     

Sexually dimorphic effects of aging on rat somatotropes and lactotropes

During the aging process modified functions of hypothalamic factors may cause sexually dimorphic changes in pituitary somatotropes and lactotropes. To test this hypothesis, pituitary tissue from young adult (4 months) and old (20-22 months) male and female rats was labeled immunocytochemically for growth hormone (GH) and prolactin (PRL). The total amount of immunoreactive material as well as the total area and number of immunoreactive structures were evaluated. With increasing age the intracellular GH content was moderately increased in male and decreased in female rats. An age-dependent PRL increase, due both to increased cell number and intracellular hormone content, was present only in female rats. The amount of GH- and PRL-immunoreactive material, distributed into classes of increasing density, differed both between sex and age groups. Our results indicate that the aging process of the somatotrope and lactotrope cell populations in rats appears to be different in the two sexes.     

Capacity of individual somatotropes to release growth hormone varies according to sex: analysis by reverse hemolytic plaque assay

A reverse hemolytic plaque assay was used to investigate the mechanism(s) underlying sexual differences in GH release which evolve at puberty in rats. The percentages of GH-secreting cells in 24-h pituitary cultures from each sex were similar for pituitary donors up to 30 days of age (range = 38.9% to 41.7% of all cells in culture, n = 3 separate experiments) but decreased by day 50. The decrease was more striking for females (to 24.1 +/- 0.3% mean +/- SE) than for males (to 33.2 +/- 1.1%). However, owing to the greater increase in total pituitary cell number exhibited by female rats at this time, the absolute numbers of somatotropes recovered from male and female pituitaries were almost identical on 50 and 100 days of age. To assess the secretory capacities of individual somatotropes, we measured the sizes of plaques formed. In prepubertal rats (days 10-30), the plaque areas under basal conditions were comparable for males and females at each age studied, and treatment with GH-releasing factor increased plaque sizes to approximately the same degree (10-fold) for both sexes at each age. However, by day 100, plaques that formed under both basal and stimulated conditions were consistently larger (P less than 0.01) for male than for female donors. Taken together, our results demonstrate that sexual differences in GH release are attributable to the secretory capacities of individual somatotropes rather than to differences in the numbers of GH cells in pituitaries of male and female rats.     

The influence of dietary restriction on the process of age-related disorders in male Donryu rats

Life span, which is mainly influenced by pathologic lesions, was compared between specific pathogen-free male Donryu rats fed ad libitum (AL group) and those with dietary restriction (DR group, restricted to 60% of the ad libitum intake). The major age-related lesions observed were pituitary tumor, chronic nephropathy, cardiomyopathy, and myopathy (anterior tibial and masseter muscle). Dietary restriction was effective in slowing the progression of pituitary tumor, chronic nephropathy and myopathy in anterior tibial muscle. Although cardiomyopathy worsened with age, no difference was seen between the AL and DR group. In conclusion, 1) dietary restriction acts to suppress or delay the development of pathologic lesions that occur with age, 2) the onset phase of a pathologic lesion differs with the lesion and organ involved, 3) inhibiting the development of pituitary adenoma and chronic nephropathy can help prolong the life span of rats, 4) for muscle lesions, depending on their anatomical location, the physiological condition of exercise, as well as the relationship with other organs can be involved.     

Endogenous hypothalamic somatostatins differentially regulate growth hormone secretion from goldfish pituitary somatotropes in vitro

Using Southern blot analysis of RT-PCR products, mRNA for three different somatostatin (SS) precursors (PSS-I, -II, and -III), which encode for SS(14), goldfish brain (gb)SS(28), and [Pro(2)]SS(14), respectively, were detected in goldfish hypothalamus. PSS-I and -II mRNA, but not PSS-III mRNA, were also detected in cultured pituitary cells. We subsequently examined the effects of the mature peptides, SS(14), gbSS(28), and [Pro(2)]SS(14), on somatotrope signaling and GH secretion. The gbSS(28) was more potent than either SS(14) or [Pro(2)]SS(14) in reducing basal GH release but was the least effective in reducing basal cellular cAMP. The ability of SS(14), [Pro(2)]SS(14), and gbSS(28) to attenuate GH responses to GnRH were comparable. However, gbSS(28) was less effective than SS(14) and [Pro(2)]SS(14) in diminishing dopamine- and pituitary adenylate cyclase-activating polypeptide-stimulated GH release, as well as GH release resulting from the activation of their underlying signaling cascades. In contrast, the actions of a different 28-amino-acid SS, mammalian SS(28), were more similar to those of SS(14) and [Pro(2)]SS(14). We conclude that, in goldfish, SSs differentially couple to the intracellular cascades regulating GH secretion from pituitary somatotropes. This raises the possibility that such differences may allow for the selective regulation of various aspects of somatotrope function by different SS peptides.     

Reporter expression, induced by a growth hormone promoter-driven Cre recombinase (rGHp-Cre) transgene, questions the developmental relationship between somatotropes and lactotropes in the adult mouse pituitary gland

This report describes the development and validation of the rGHp-Cre transgenic mouse that allows for selective Cre-mediated recombination of loxP-modified alleles in the GH-producing cells of the anterior pituitary. Initial screening of the rGHp-Cre parental line showed Cre mRNA was specifically expressed in the anterior pituitary gland of adult Cre+/- mice and cephalic extracts of e17 Cre+/- fetuses. Heterozygote rGHp-Cre transgenic mice were crossbred with Z/AP reporter mice to generate Cre+/-,Z/AP+/- offspring. In this model system, the GH promoter-driven, Cre-mediated recombination of the Z/AP reporter leads to human placental alkaline phosphatase (hPLAP) expression that serves to mark cells that currently produce GH, in addition to cells that would have differentiated from GH cells but currently do not express the GH gene. Double immunocytochemistry of adult male and female Cre+/-,Z/AP+/- pituitary cells revealed the majority (approximately 99%) of GH-producing cells of the anterior pituitary also expressed hPLAP, whereas ACTH-, TSH-, and LH-producing cells were negative for hPLAP, confirming previous reports that corticotropes, thyrotropes, and gonadotropes develop independently of the somatotrope lineage. A small subset (approximately 10%) of the prolactin-producing cells was positive for hPLAP, consistent with previous reports showing lactotropes can arise from somatotropes during pituitary development. However, the fact that 90% of prolactin-producing cells were negative for hPLAP suggests that the majority of lactotropes in the adult mouse pituitary gland develop independently of the somatotrope lineage. In addition to developmental studies, the rGHp-Cre transgenic mouse will provide a versatile tool to study the role of a variety of genes in somatotrope function and neoplastic transformation.     

Differential expression of gonadotropin and prolactin antigens by GHRH target cells from male and female rats.

There is a 2- to 3-fold increase in luteinizing hormone-beta (LHbeta) or follicle-stimulating hormone-beta (FSHbeta) antigen-bearing gonadotropes during diestrus in preparation for the peak LH or FSH secretory activity. This coincides with an increase in cells bearing LHbeta or FSHbeta mRNA. Similarly, there is a 3- to 4-fold increase in the percentage of cells that bind GnRH. In 1994, we reported that this augmentation in gonadotropes may come partially from subsets of somatotropes that transitionally express LHbeta or FSHbeta mRNA and GnRH-binding sites. The next phase of the study focused on questions relating to the somatotropes themselves. Do these putative somatogonadotropes retain a somatotrope phenotype? As a part of ongoing studies that address this question, a biotinylated analog of GHRH was produced, separated by HPLC and characterized for its ability to elicit the release of GH as well as bind to pituitary target cells. The biotinylated analog (Bio-GHRH) was detected cytochemically by the avidin-peroxidase complex technique. It could be displaced by competition with 100-1000 nM GHRH but not corticotropin-releasing hormone or GnRH. In cells from male rats exposed to 1 nM Bio-GHRH, 28+/-6% (mean+/-s.d) of pituitary cells exhibited label for Bio-GHRH (compared with 0.8+/-0.6% in the controls). There were no differences in percentages of GHRH target cells in populations from proestrous (28+/-5%) and estrous (25+/-5%) rats. Maximal percentages of labeled cells were seen following addition of 1 nM analog for 10 min. In dual-labeled fields, GHRH target cells contained all major pituitary hormones, but their expression of ACTH and TRH was very low (less than 3% of the pituitary cell population) and the expression of prolactin (PRL) and gonadotropins varied with the sex and stage of the animal. In all experimental groups, 78-80% of Bio-GHRH-reactive cells contained GH (80-91% of GH cells). In male rats, 33+/-6% of GHRH target cells contained PRL (37+/-9% of PRL cells) and less than 20% of these GHRH-receptive cells contained gonadotropins (23+/-1% of LH and 31+/-9% of FSH cells). In contrast, expression of PRL and gonadotropins was found in over half of the GHRH target cells from proestrous female rats (55+/-10% contained PRL; 56+/-8% contained FSHbeta; and 66+/-1% contained LHbeta). This reflected GHRH binding by 71+/-2% PRL cells, 85+/-5% of LH cells and 83+/-9% of FSH cells. In estrous female rats, the hormonal storage patterns in GHRH target cells were similar to those in the male rat. Because the overall percentages of cells with Bio-GHRH or GH label do not vary among the three groups, the differences seen in the proestrous group reflect internal changes within a single group of somatotropes that retain their GHRH receptor phenotype. Hence, these data correlate with earlier findings that showed that somatotropes may be converted to transitional gonadotropes just before proestrus secretory activity. The LH and FSH antigen content of the GHRH target cells from proestrous rats demonstrates that the LHbeta and FSHbeta mRNAs are indeed translated. Furthermore, the increased expression of PRL antigens by these cells signifies that these convertible somatotropes may also be somatomammotropes.     

Mutation in the neural cell adhesion molecule interferes with the differentiation of anterior pituitary secretory cells.

The neural cell adhesion molecule (NCAM) and its polysialylated isoform (PSA-NCAM) have been shown to influence the proliferation, differentiation and survival of different cell types. Here, we report the pattern of expression of NCAM and PSA-NCAM in the anterior lobe (AL) of the pituitary gland of the adult mouse. We demonstrate that the majority of cells express NCAM, while PSA-NCAM is retained mostly on corticotropes. Analysis of bromodeoxyuridine (BrdU) incorporation shows that the presence of PSA-NCAM on corticotropes is not related to proliferation but most likely to their functional properties. We subsequently analyzed defects induced by NCAM deficiency in adult NCAM knockout mice. In these mice, all secretory cell types in the AL are present and their distribution within the gland is similar to that in wild-type mice. However, proliferation of AL cells is significantly increased. In particular, more BrdU-positive cells are detected among somatotropes and mammotropes in NCAM-deficient mice. In addition, the percentages of secretory cells are changed: somatotropes are more numerous while the number of corticotropes is reduced. These data demonstrate the involvement of NCAM in the proper generation and/or maintenance of the different cell populations in the AL and suggest the importance of PSA in corticotrope functioning.     

Identification of the somatostatin receptor subtypes (sst) mediating the divergent, stimulatory/inhibitory actions of somatostatin on growth hormone secretion

It is well established that somatostatin acts through G protein-coupled receptors, termed sst, to inhibit GH release. However in pigs somatostatin can stimulate or inhibit in vitro GH secretion in a dose- and somatotrope subpopulation-dependent manner. We report herein that somatostatin-stimulated GH release is blocked by pretreatment with GTPgamma-S, suggesting an involvement of G protein-coupled receptors. Consistent with this, an sst5 selective agonist stimulated spontaneous GH secretion at doses ranging 10(-13) to 10(-9) m, without influencing GHRH-induced GH release. Conversely, sst1-, sst2-, sst3-, and sst4-specific agonists inhibited GHRH-evoked GH release but not basal GH secretion. Examination of the effects of sst-specific agonists on two subpopulations of somatotrope cells separated by density gradient centrifugation [low- (LD) and high-density (HD) cells] showed that only a low dose of the sst5 agonist stimulated GH release in LD somatotropes, whereas both low and high doses of this agonist stimulated GH release in HD cells. In marked contrast, sst1 and sst2 agonists blocked GHRH-stimulated GH release in LD cells at all doses tested, whereas only a high dose of the sst2 agonist inhibited GHRH-induced GH release in HD somatotropes. Interestingly, sst expression pattern in these subpopulations correlates with the distinct actions of sst-selective agonists; specifically, sst5 is more abundant in HD somatotropes, whereas sst1 and sst2 mRNA predominate in LD cells. These results indicate that in the pig, sst1 and sst2 are the primary mediators of the inhibitory effects of somatostatin, whereas sst5 or an sst5-related mechanism mediates the stimulatory action of somatostatin on GH release.     

Differentiation of fetal rat somatotropes in vitro: effects of cortisol, 3,5,3'-triiodothyronine, and glucagon, a light microscopic and radioimmunological study

Cortisol stimulates somatotrope differentiation in vitro. T3 and/or glucagon may also be involved. Fetal rat pituitary primordia were explanted at 14 days gestation and cultured for 7 days in medium supplemented with cortisol (50-500 nM), and either T3 (0.67 nM) or glucagon (0.5 nM). Also, to determine the time of first appearance of the somatotropes, explants were cultured 4, 5, or 6 days with cortisol alone. Immunoreactive somatotropes were detected by immunohistochemistry, and their size and number were estimated for each medium. GH was measured by RIA in explants and media. Immunoreactive somatotropes first appear at 18-19 days gestation. Their size and number depend on cortisol concentration: no cells at 50 nM, a few small ones at 100 nM, and many large ones at 250-500 nM. This progression was reflected by RIA of GH in explants and media, although small quantities were detected with 50 nM. The effect of T3 was only visible with a low dose of cortisol. With 100 nM cortisol, it increased the size and number of cells. Differentiation was also triggered with 50 nM cortisol plus T3. RIA detected significantly higher GH content and secretion after T3 stimulation. The decreases in number, size, and GH secretion and content elicited by glucagon were not significant, probably due to the high variability. Both techniques used provide similar information on somatotrope differentiation: stimulation by cortisol alone, or alternatively by a synergistic action between cortisol and T3.     

Pituitary alterations involved in the decline of growth hormone gene expression in the pituitary of aging rats

Growth hormone (GH) declines during aging. This study investigates whether pituitary constitutive alterations may be involved in the GH decline. Two groups of male Wistar rats were studied (young: 3-month-old; old: 24-month-old). The old rats showed lower pituitary GH messenger RNA (mRNA) levels, immunoreactive rat (IR)-GH content, and GH secretion with no difference in pituitary Pit-1 and cAMP-response element-binding protein (CREB) expression. Pituitary GH releasing hormone receptor (GHRH-R), GH secretagogue receptor (GHS-R), sstr2, and sstr5 mRNA levels were significantly reduced in old rats. The percentage of GH immunoreactive cells was similar in both groups. In vitro, pituitary IR-GH response to GHRH, forskolin (FK), ghrelin, and insulin-like growth factor I (IGF-I) was similar when compared with respective basal secretion and somatostatin-diminished GHRH- and ghrelin-induced IR-GH release in both groups. These results indicate that, as somatotrope function is maintained in aging, the changes observed in GH gene expression and secretion could be reversed by GHS.     

Growth hormone (GH) secretion in the pituitary-grafted male rat: in vivo effects of GH-releasing hormone and isoproterenol and in vitro release by individual somatotropes

Although the pituitary-grafted rat is a classic model of chronic PRL excess, the presence of somatotropes in grafted pituitary tissue indicates a potential for GH secretion. The current study was designed to investigate GH-releasing hormone (GRH)-induced GH secretion and beta-adrenergic inhibition of GH release in animals bearing ectopic pituitary tissue free from hypothalamic control. Positive findings with regard to these in vivo experiments led us to an initial determination of GH secretion by individual somatotropes from transplanted pituitary tissue. In litters of 10 30-day-old Fisher rats, 2 male animals received subcapsular renal grafts of 3 littermate pituitary glands each. Thirty-five days after grafting, 1 group received saline (SAL) followed by GRH, and the other received the beta-adrenergic agonist isoproterenol (ISO) followed by GRH. Blood samples were taken before and after SAL or ISO treatment, GRH was then infused, and sampling was continued. Plasma was assayed for GH and PRL, and the reverse hemolytic plaque assay was used to determine GH release by individual somatotropes from transplanted pituitary tissue. Plasma PRL was clearly elevated in pituitary-grafted compared to muscle-grafted animals, but there was no difference in either body weight gain or basal GH levels between the groups. As shown previously, ISO itself induced a brief release of GH due to its direct effect on the pituitary gland. The GH response to GRH was greater in pituitary-grafted animals than in muscle-grafted controls after both SAL and ISO. GRH-induced GH release was suppressed by ISO pretreatment in muscle-grafted animals, but not in pituitary-grafted animals. The reverse hemolytic plaque assay unequivocally showed that transplanted pituitary tissue was capable of tonic as well as GRH-stimulated GH release. These results demonstrate that despite similar basal GH levels, animals bearing pituitary grafts release significantly greater amounts of GH in response to GRH. The evidence for GH secretion by individual somatotropes from transplanted pituitary tissue directly shows the grafted tissue to be a source of GRH-stimulated GH. The lack of beta-adrenergic inhibition of GRH-induced GH release in pituitary-grafted animals is consistent with the hypothesis that beta-adrenergic inhibition of GRH-induced GH secretion is mediated by an effect on the hypothalamus.     

Long-term culture of rat mammotrope and somatotrope subpopulations separated on continuous Percoll density gradients: effects of dopamine, TRH, GHRH and somatostatin

Normal adult female rat mammotrope and somatotrope subpopulations were separated on continuous Percoll density gradients according to differences in their density. Viable cells were recovered in 16 fractions. The cells from each fraction were cultured during 7 days after which period 4-h incubations were performed. rPRL secretion per cell increased towards the higher density fractions. No major difference in TRH, dopamine and somatostatin responsiveness was observed between mammotropes that were recovered in the different gradient fractions. In addition, no differences in somatostatin responsiveness between the somatotrope cells in the different gradient fractions were observed. However, somatotropes that were recovered in the highest density region of the gradient appeared to be more responsive to GHRH than the lower density somatotropes. In the various gradient fractions there were no paradoxical effects of TRH and dopamine on rGH release and of GHRH on rPRL release. Conclusions: 1. In long-term cultures there is no evidence for functionally different subpopulations of mammotropes and somatotropes, separated according to differences in their density, with regard to dopamine and TRH responsiveness and with regard to somatostatin responsiveness, respectively. 2. There is no evidence for a (mammosomatotrope?) subpopulation of cells showing paradoxical responses of PRL or GH release to GHRH and dopamine or TRH, respectively.     

Vascular endothelial growth factor production and regulation in rodent and human pituitary tumor cells in vitro

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope alpha T3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-alpha (TGF-alpha), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and alpha T3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-alpha, PACAP-38, and 17 beta-estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas.     

Progesterone antagonizes the permissive action of estradiol on tumor necrosis factor-alpha-induced apoptosis of anterior pituitary cells

We previously reported that TNF-alpha-induced apoptosis of lactotropes is estrogen dependent and predominant at proestrus. Here we observed that TNF-alpha (50 ng/ml) failed to induce apoptosis of anterior pituitary cells from ovariectomized rats cultured in the presence of progesterone (10(-6) m). However, progesterone blocked the apoptotic effect of TNF-alpha in anterior pituitary cells and lactotropes cultured with 17beta-estradiol (10(-9) m). In addition, 17beta-estradiol induced apoptosis of somatotropes and triggered the proapoptotic action of TNF-alpha in these cells, effects completely blocked by ICI 182 780 (10(-6) m), an estrogen receptor antagonist. Progesterone reverted the permissive effect of 17beta-estradiol on TNF-alpha-induced apoptosis of somatotropes. TNF-alpha induced apoptosis of somatotropes from rats killed at proestrus but not at diestrus. The antiprogestine ZK 98,299 (10(-6) m) completely inhibited the protective action of progesterone on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Although progesterone can interact with glucocorticoid receptors, dexamethasone (10(-6) m) had no effect on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Our results show that progesterone, by interacting with progesterone receptors, antagonizes the permissive action of estrogens on TNF-alpha-induced apoptosis of lactotropes and somatotropes. These observations suggest that the steroid milieu may modulate the apoptotic response of anterior pituitary cells during the estrous cycle.