Jack Ham

Adenosine-induced IL-6 expression in pituitary folliculostellate cells is mediated via A2b adenosine receptors coupled to PKC and p38 MAPK

Activation of adenosine receptors in folliculostellate (FS) cells of the pituitary gland leads to the secretion of IL‐6 and vascular endothelial growth factor (VEGF). We investigated the action of adenosine A2 receptor agonists on IL‐6 and VEGF secretion in two murine FS cell lines (TtT/GF and Tpit/F1), and demonstrated a rank order of potency, 5′‐N‐ethylcarboxamidoadenosine (NECA)>2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine>adenosine, suggesting mediation via the A2b receptor. NECA‐mediated IL‐6 release was inhibited by the PLC inhibitor 1‐[6‐((17β‐3‐methoxyestra‐1,3,5(10)‐tiene‐17‐yl)amino)hexyl]‐1H‐pyrrole‐2,5‐dione, the PI3 kinase inhibitor wortmannin and the PKC inhibitors bisindolylmaleimide 1 and bisindolymaleimide X1 HCl (Ro‐32‐0432). NECA‐mediated IL‐6 release was attenuated (<50%) by the extracellular signal‐regulated kinase MAPK inhibitor 2′‐amino‐3′‐methoxyflavone, and completely (>95%) inhibited by the p38 MAPK inhibitor 4‐(4‐fluorophenyl)‐2‐(4‐methylsulphinylphenyl)‐5‐(4‐pyridyl)1H‐imidazole. NECA stimulates p38 MAPK phosphorylation that is inhibited by Ro‐32‐0432 but not by wortmannin. Dexamethasone inhibits NECA‐stimulated IL‐6 and VEGF secretion. These findings indicate that adenosine can stimulate IL‐6 secretion in FS cells via the A2b receptor coupled principally to PLC/PKC and p38 MAPK; such an action may be important in the modulation of inflammatory response processes in the pituitary gland.

GH3 cells expressing constitutively active Gsα (Q227L) show enhanced hormone secretion and proliferation

cAMP levels in GH3gsp cells (Q227L mutation of Gs alpha), in comparison with uninfected GH3 and GH3vt (with vector alone) cells, were two to three fold (P< 0.01) higher (basal), and 10-20 fold (P<0.001) higher (in the presence of isobutyl methylxanthine, (IBMX)). Proliferation of GH3gsp cells after 7 days in culture, as determined by cell number and [(3)H]thymidine incorporation, were up to 25% (respectively P <0.001 and P < 0.02) higher. After chronic (4 days) but not acute (15 min) exposure to forskolin (10 microM) or dibutyryl cAMP (50 microM) all cell types showed a greater than 200% (P < 0.001) increase in [(3)H]thymidine incorporation. Secretion of prolactin and growth hormone by GH3gsp cells were two to four fold (P <0.001) higher than GH3 and GH3vt cells after 4 h and 10-12 fold (P <0.001) higher after 8 h. In conclusion GH3 cells possessing Q227L have a higher proliferation rate and secrete higher levels of prolactin and growth hormone which are associated with higher levels of cAMP.

Adenosine-Regulated Cell Proliferation in Pituitary Folliculostellate and Endocrine Cells: Differential Roles for the A1 and A2B Adenosine Receptors

A(1) and A(2) adenosine receptors have been identified in the pituitary gland, but the cell type(s) on which they are located and their effects on pituitary cell growth are not known. Therefore, we analyzed the expression of A(1) and A(2) receptors in primary rat anterior pituitary cells, two pituitary folliculostellate (TtT/GF and Tpit/F1) and two pituitary endocrine (GH(3) and AtT20) cell lines, and compared their effects on cell proliferation. In anterior pituitary and folliculostellate cells, adenosine and adenosine receptor agonists (5-N-ethylcarboxamidoadenosine, a universal agonist, and CGS 21680, an A(2A) receptor agonist) stimulated cAMP levels with a rank order of potency that indicates the presence of functional A(2B) receptors. This stimulation, however, was not observed in either GH(3) or AtT20 cells, where adenosine and the A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine inhibited VIP/forskolin-stimulated cAMP production. Expression of A(2B) and A(1) receptors in the folliculostellate cells and that of the A(1) receptor in the endocrine cells were confirmed by RT-PCR, immunocytochemistry, and ligand binding. Adenosine and 5-N-ethylcarboxamidoadenosine dose-dependently (10 nM to 10 microM) stimulated growth in the folliculostellate, but not in the endocrine, cells, whereas in the latter, 100 microM adenosine and 2-chloro-N(6)-cyclopentyladenosine inhibited cell proliferation by slowing cell cycle progression. These data highlight the differential expression of A(1) and A(2B) adenosine receptors in pituitary cells and provide evidence for opposing effects of adenosine on pituitary folliculostellate and endocrine cell growth.

Novel insights into how purines regulate pituitary cell function.

Purine nucleosides and nucleotides are widely distributed substances that exhibit a diverse range of effects in a number of tissues, acting as important extracellular signalling molecules in addition to their more established roles in cellular metabolism. They mediate their effects via activation of distinct cell surface receptors, termed adenosine (or P1) and P2 purinergic receptors. Although roles for adenosine and adenine nucleotides have been described previously in the pituitary gland, the distribution of the receptor subtypes and the effects of their activation on pituitary function are not well defined. Recent evidence, however, has emerged to describe a complex signalling system for purines in the pituitary gland. Data from a variety of studies have shown that the expression pattern, number and affinity of adenosine and/or P2 receptors may be cell-type specific and that non-endocrine in addition to endocrine cells elaborate these receptors. These variations, along with the diverse range of signalling pathways activated, dictate the response of individual cell types to extracellular purines, with roles now emerging for these substances in the regulation of hormone release, pituitary cell proliferation and cytokine/growth factor expression. In this review, we discuss these advances and examine some implications for pituitary growth control and the response of the hypothalamic-pituitary-adrenal axis to stress and inflammation.

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence

The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5 end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5 truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5 truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5 truncated POMC gene occurring in pituitary cells.

Growth factors and pituitary tumors.

Growth within the anterior pituitary gland is probably controlled by several interacting extracellular messenger molecules, including hypothalamic peptides, target gland hormones, and several growth factors acting in autocrine or paracrine fashion. Adenoma formation may result from abnormal production of such factors or their specific cellular receptors, loss of local inhibitory influences, activation of the intracellular secondary message pathways conveying the mitogenic signal to the nucleus, or deregulation of the nuclear processes controlling mitosis.

Mitogen-Activated Protein Kinase Mediates Epidermal Growth Factor-Induced Morphogenesis in Pituitary GH3 Cells

Epidermal growth factor (EGF) causes pituitary GH3 cells to change from their normal predominantly rounded morphology to much more elongated cells with extensive filopodia, and this effect is accompanied by a parallel increase in cell volume. In view of this, and because EGF receptor expression is increased in some pituitary tumours, we examined the mechanism of this EGF‐induced morphological effect as it may play a role in tumour invasiveness. The effect of treatment of the cells with EGF (1u2003nm, 4 days) was determined visually (expressed as percent non round cells) and by measuring the cell volume by Coulter Counter analysis. EGF treatment caused the cells to change their morphology with percent non round cells increasing from 37% in control cells to 74% in EGF‐treated cultures; this was accompanied by a parallel increase in cell volume. Treatment of the cells with EGF in the presence of the MEK1 inhibitor (PD98059) completely blocked the EGF‐induced morphological changes, showing that activation of the mitogen‐activated protein kinase (MAPK) pathway is necessary to mediate this effect. Transfection of the cells with a constitutively activated mutant of MEK1 produced a similar morphological change to that produced by EGF treatment, with the proportion of non round cells increasing to 62% with a parallel increase in cell volume compared to cells transfected with the empty vector, demonstrating that direct activation of MAPK pathway is sufficient to mediate the observed morphological effects. The effects produced by activated MEK1 transfection could be blocked by PD98059. EGF had opposing effects on prolactin and growth hormone (GH) secretion by the cells, increasing prolactin release and inhibiting GH release. Transfection of the cells with activated MEK1 produced similar effects on hormone release as EGF treatment. In conclusion, the morphological effects of EGF on GH3 cells are mediated by activation of the MAPK pathway as blockade of this pathway abolished the observed effect, and direct activation of this pathway by transfection with an activated mutant of MEK1 was able to duplicate these effects. This mechanism may contribute to the growth and possibly local invasiveness of some pituitary tumours that express the EGF receptor.

Differential responses of rat cerebral somatostatinergic and cholinergic cells to glutamate agonists

Reductions in cortical somatostatin (SRIH) and choline acetyl-transferase (ChAT) are major biochemical deficits in Alzheimer disease (AD). SRIH and ChAT were measured in fetal rat cerebral neurons after exposure to the glutamate agonists N-methyl-D-aspartate (NMDA), kainate (KA), and quisqualate (Q). NMDA (96 h incubation) stimulated SRIH release and content in a dose-dependent manner with a Bmax of 10(-5)M and EC50 of 2-3 x 10(-6)M. KA showed a small stimulation in SRIH levels at 10(-5)M, but produced marked inhibition at 10(-4)M. Q decreased both intracellular and secreted SRIH. KA (51-76% of basal) and Q (27-56% of basal) but not NMDA (91-114% of basal) also inhibited the incorporation of [35S]methionine into proteins. In similar experiments 10(-4)M Q (23 +/- 9% of basal) and KA (20 +/- 3% of basal) but not NMDA (80 +/- 16% of basal) reduced ChAT levels in hypothalamic/septal cultures. These inhibitory actions on ChAT activity by KA and Q were reversed by gamma-glutamyltaurine (GT) but not by 2-amino-5-phosphonopentanoic acid (AP5). Chronic NMDA exposure partially inhibited muscarinic acetylcholine receptor (mAChR) mediated inositol phospholipid (PI) turnover, whereas it was abolished after KA and Q pretreatment. These findings suggest that in cerebral cell cultures, NMDA has a stimulatory action on somatostatinergic neurons and non-NMDA receptor agonism could play an important role in EAA-mediated neural damage.

Prolonged exposure to N-methyl-D-aspartate increases intracellular and secreted somatostatin in rat cortical cells.

Somatostatin (SRIF) release from fetal rat cortical cells was stimulated by exposure to 10(-5) M N-methyl-D-aspartate (NMDA) (250 +/- 20% of basal at 96 h). A similar but much less potent effect was seen with kainate (KA) but not with quisqualate (Q) which inhibited SRIF release (KA 150 +/- 13%, Q 65 +/- 18% of basal at 96 h). Similar data were obtained for intracellular levels of SRIF. Dose-dependent experiments showed that the EC50 for the stimulatory action of NMDA was 2-3 x 10(-6) M with a Bmax of around 10(-5) M. At 10(-4) M KA and Q but not NMDA reduced tissue content and release of SRIF (KA: 47 +/- 14, 67 +/- 17%; Q: 36 +/- 13, 42 +/- 6% of basal for content and release, respectively). These findings indicate that cortical SRIF content and release is enhanced by exposure to NMDA but not by KA or Q. We suggest that SRIF-containing neurones are sensitive to glutamate damage through the activation of non-NMDA rather than NMDA receptors.

The glutamate inhibition of carbachol stimulated inositol phosphate production in rat cortical cells is mediated through an ionotropic NMDA receptor

Carbachol (0.1 mM) stimulated accumulation of inositol monophosphate (IP1) (3-4 fold of basal, P < 0.001) in fetal rat cortical cells is attenuated by glutamate (at 0.1 mM, 40-70% of carbachol alone, P < 0.001). This inhibition by glutamate was reduced by 2-amino-5-phosphonopentanoic acid (AP5), but not by gamma-D-glutamylaminomethyl sulphonic acid (GAMS) [corrected] or 2-amino-3-phosphonopropionic acid (AP3). The metabotropic receptor agonist (1S,3R)-1-aminocyclopentane-1-3-dicarboxylic acid [(1S,3R)-ACPD] (up to 0.1 mM) had no effect upon carbachol stimulated IP1. Staurosporine and quinacrine were unable to prevent the inhibition of carbachol stimulated IP1 by glutamate. These data suggest that the inhibition of carbachol-stimulated IP1, by glutamate in rat cortical cells is mediated through an NMDA ionotropic receptor.