A Reverse Hemolytic Plaque Assay for Microscopic Visualization of Growth Hormone Release from Individual Cells: Evidence for Somatotrope Heterogeneity

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Abstract
Growth hormone (GH) secreting cells direct complement-mediated plaque formation (clear zones of hemoly-sis surrounding the somatotropes) in mixed pituitary cell cultures incubated as a monolayer with protein-A coupled ovine erythrocytes (oRBC) in the presence of GH antiserum. Plaques were maximal in number after 4 h; growth hormone-releasing hormone (GHRH) and somatostatin increased and decreased, respectively, the rate of formation of plaques and their final sizes. Approximately 30% of all pituitary cells formed GH plaques and a similar fraction stained for GH using peroxidase-antiperoxidase immunocytochemistry (ICC). The plaque areas of individual somatotropes (reflecting the amount of GH released) covered a 20-fold range from the smallest to the largest in the 3 treatment groups. Somatostatin-treated and untreated cells formed frequency distributions of plaque sizes that were unimodal. In contrast, GHRH produced a bimodal frequency distribution suggestive of a sub-population of somatotropes preferentially responsive to this secretagogue. This new assay coupled with other morphological and biochemical techniques that can be applied to single cells will permit further analysis of these sub-populations of somatotropes.

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