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Heterogeneity of growth hormone production in human pituitary tumour cells

In the past few years, a number of techniques have been developed which allow the study of hormone production and secretion in individual cells. Most of our previous understanding of cellular physiology was based on studies of large populations of cells, but it is now becoming apparent that endocrine tissues are heterogeneous, and that sub-populations of cells may behave differently from the tissue as a whole. The paper by Hofland et al. in this issue of Clinical Endocrinology has addressed the question of cellular heterogeneity in the adenomatous somatotrophic cells obtained from the pituitary tumours of acromegalic patients. Two techniques were used, namely the reverse haemolytic plaque assay, to study growth hormone (GH) secretion from individual cells, and quantitative in-situ hybridization, to study G H mRNA levels. Both techniques were studied in cells from each of four somatotroph adenomas, to provide comparative data about G H production in single cells. The results show that the cells in these tumours are heterogeneous with respect to both G H secretion and G H mRNA, and imply that a small number of cells contribute most of the overall G H production within each tumour. The reverse haemolytic plaque assay (RHPA) was first applied to demonstrate the characteristics of prolactin release from individual pituitary cells by Neil1 and Frawley (1983). A lawn of ovine red blood cells coated with staphylococcal Protein-A is layered over a monolayer culture of pituitary cells. The cells are incubated in the presence of specific antiserum against the hormone of interest, and hormone-antibody complexes become bound to the ProteinA attached to the red blood cells. In the presence of added complement (usually in the form of guinea-pig serum), complement-mediated cytolysis results in a plaque of haemolysed red cells surrounding an active hormone-secreting pituitary cell. Plaque diameter is proportional to the amount of hormone secreted. This assay was used by Frawley and Neil1 (1984) to demonstrate that G H production was heterogeneous in normal pituitary cells. This first suggested that a population of a given pituitary cell type might contain variable sub-populations of low secretors and high secretors, and that overall hormone production was therefore a function not only of secretion rate, but alsozof the number of cells recruited into a high-secretor pool. This hypothesis was confirmed in the GH3 pituitary cell line, where the RHPA demonstrated sub-groups of G H secretors and prolactin secretors; the 2: 1 proportion of G H secretors to prolactin secretors was reversed by treatment with TRH or oestradiol, and increased by cortisol (Boockfor ei ol., 1985). A recent study of bovine pituitary cells combined RHPA with immunocytochemistry to show that only 53% of the cells that contained G H actually secreted it, but this proportion rose to 86% on exposure to GH-releasing factor suggesting recruitment of 'quiescent' somatrotrophs (Kineman et al., 1990). The same principles may apply to G H secretion in the human anterior pituitary gland. In a study of human fetal somatotrophs, somatostatin reduced the percentage of plaque-forming cells and also the average area of the plaques; G R F accelerated plaque formation and increased average plaque area but had no eventual effect on the percentage of plaque-forming cells (Mulcahey et al., 1988). These data indicated that G R F transiently increased the rate of G H secretion but (in contrast to the animal data) there was no recruitment of additional non-secreting cells into the secretory pool. On the other hand, somatostatin reduced the amount of G H secreted per cell and also reduced the total number of secreting cells. Turning to human pituitary adenomas, Hofland et 01. (1989) have previously found in a series of eight tumours that somatotrophic cells secreted very variable amounts of GH. Plaque areas were not normally distributed, and there was a preponderance of small plaques: 24-38% of somatotroph cells formed larger than average plaques, and this minority of cells accounted for most (63-80%) of the total G H released. Exposure to the somatostatin analogue SMS 20 1-995 preferentially inhibited formation of the larger plaques (i.e. the high secretors), with less effect on the smaller plaques surrounding low-secretor cells. G R F and T R H had no preferential effect on any sub-population of cells. In summary therefore, only a small sub-population of cells secrete a a lot of G H and respond to somatostatin, whereas most cells secrete little GH and are less responsive to inhibition by somatostatin. Somatotrophic cells also appear to be heterogeneous in terms of G H mRNAcontent. In-situ hybridization data have shown, by uneven patterns of probe binding to tumour sections (Levy & Lightman, 1988; Pixley et al., 1987; Lloyd ef al., 1989), that G H mRNA content is not homogeneously distributed across pituitary adenomas. The data in the present paper by Hotland ef 01. (1991) show that the