Wynick, David; (1994) Purification of functional lactotrophs and somatotrophs. Doctoral thesis (M.D), UCL (University College London).

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Abstract

Data is presented in this thesis demonstrating that using flow cytometry, dispersed, anterior pituitary cells can be analysed and enriched on the basis of their size, granularity and cell surface fluorescence. Two distinct populations of cells, differing in granularity were defined. 26 ± 2.2% were more granular and 74 ± 3.5% less granular. Acutely dispersed anterior pituitary cells were labelled with antibodies against four of the anterior pituitary hormones, and cell size and granularity were compared amongst the different hormonal cell types. Somatotrophs were the most granular cell type, gonadotrophs were the largest and corticotrophs the smallest whilst lactotrophs were of intermediate size. Labelling was demonstrated to be dependant upon the secretory state of the cell. Hypothalamic stimulating factors increased cell surface labelling, whilst dopamine and somatostatin decrease labelling. These changes compare favourably with published data obtained by immunocytochemistry. Fluorescence-activated cell sorting (FACS) was applied to labelled pituitary cells and percentage purity and depletion of other cell types assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 ± 1.7% and 98 ± 1.0% respectively by immunocytochemistry and to 95.8 ± 1.1% and 97 ± 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 ± 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Having established the technique of FACS enrichment this was then compared to magnetic bead separation. Whilst magnetic bead separation is inferior to FACS enrichment it allows the study of newly released hormone and an hypothesis to be advanced as to how the prolactin secretory granule is packaged and exocytosed from the lactotroph. The secretory characteristics of FACS enriched lactotrophs have been studied using the reverse haemolytic plaque assay and compared to unenriched populations, with particular reference to regulation of prolactin secretion by the 29 amino acid peptide galanin. Using a novel cell blot assay to detect galanin secretion from single cells, a sub-population of lactotrophs were identified (9% ± 1%) which secrete galanin. These galanin secreting cells were enriched by FACS and their autocrine regulation of the non-galanin secreting lactotrophs studied. Galanin would appear to be cruicial to the regulation of basal and vasoactive intestinal polypeptide stimulated prolactin release. Hyperoestrogenisation increases the number of galanin secreting cells to 39 ± 2% of all lactotrophs and the resulting increase in basal prolactin release is completely abolished by treatment with galanin antiserum. These findings represent direct evidence for paracrine and autocrine regulation of lactotroph function and demonstrate that the effect of oestrogen on prolactin release would appear to be mediated by locally secreted galanin. Lastly, having demonstrated that a specific galanin antiserum inhibits prolactin release from FACS enriched lactotrophs I was able to show that the same cells do not respond to the galanin antagonist, galantide, allowing the characterisation of a single high affinity anterior pituitary galanin receptor with a Kd of 4.4 ± .34 nM and a Bmax of 79 ± 8.3 fmol/mg of protein. These data suggest the presence of a novel pituitary galanin receptor, designated GAL-R2, in which the region 3-10 and amino acid 25 are crucial for membrane binding and biological activity, in contrast to the known gut/brain galanin receptor (designated GAL-R1). A number of tissues known to bind galanin were screened. GAL-R2 would only appear to be expressed in the anterior pituitary and hypothalamus.

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