B M Lewis’s research while affiliated with Keele University and other places

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.

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 (1 nm, 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.

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.

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have in- fected normal human, postmortem pituitary cells in monolayer cul- ture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, des- ignated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of se- rum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for hu- man a-subunit and focal staining for TSHb. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cy- togenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing a-subunit and TSHb .( J Clin Endocrinol Metab 83: 1598 -1603, 1998)

We investigated the role of hypothalamic glutamate receptors in mediating the stimulatory effect of low glucose (< 5 mM) on somatostatin release. We also studied whether alteration in glutamate release might contribute to the reduced hypothalamic somatostatin response to low glucose observed in diabetic (Goto-Kakizaki) rat hypothalami. Hypothalamic somatostatin release in response to incubation with 1 mM D-glucose was inhibited by the ionotropic glutamate receptor antagonists MK801, D-AP5 and DNQX but not by the metabotropic antagonists L-AP3 or MCPG. The release of somatostatin was increased by the ionotropic agonists NMDA, AMPA and kainate but not by metabotropic agonists t-ACPD or L-AP4. Basal and peak glutamate release in response to incubation with 1 mM glucose, were significantly lower from GK hypothalami There were no significant differences in the basal or stimulated release of serine and GABA. These data indicate that ionotropic NMDA/AMPA/kainate receptors and not metabotropic receptors mediate the effects of glucose on rat hypothalamic somatostatin release. Reduced hypothalamic somatostatin release in response to low glucose in diabetic (Goto-Kakizaki) rats may well be secondary, at least in part, to reduced glutamate release.