Angiotensinogen Secretion by Single Rat Pituitary Cells: Detection by a Reverse Haemolytic Plaque Assay and Cell Identification by Immunocytochemistry

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Abstract
A reverse haemolytic plaque assay (RHPA) for angiotensinogen was developed in rat hepatoma H4 cells and applied to investigate the possible secretion of angiotensinogen from rat pituitary cells in primary culture. Over a 24-hour incubation period in Cunningham chambers plaques with a mean area of 2,800 ± 430 and 590 ± 220 µmV plaque (SD, n = 6) formed around all viable H4 cells and 2.8 ± 0.59% of viable pituitary cells respectively. As a positive control PRL secretion from lactotrophs was routinely checked by the RHPA and shown to form plaques with a mean area of 4,050 ± 1,850 µm2/ plaque after a 4-hour incubation. By comparing plaque size in H4 cells with angiotensinogen release in cell culture, as quantified by radioimmunoassay, the secretion rate of angiotensinogen from pituitary cells was calculated as 22 ± 8 ng/106 cells/24 h. Plaque-forming cells consisted of two morphologically distinct populations; 78% being small cells (9 µm diameter) cells with an abundant cytoplasm. Immunocytochemical staining of pituitary cells after formation of plaques with anti-angiotensinogen, anti-LH and anti-PRL antiserum showed that the large plaque-forming cells were gonadotrophs and none were lactotrophs. All plaque-forming cells stained for angiotensinogen but only 44% of the viable cells which stained for angiotensinogen actually formed plaques. The possibility that cellular angiotensinogen was imported from extracellular sources was investigated by incubation of pituitary cells with pure 125I-angiotensinogen for periods up to 24 h. No uptake of the radiolabelled protein was found. It was concluded that gonadotrophs and a second, probably perisinusoidal, pituitary cell type synthesize and secrete angiotensinogen which is then used in the intracellular (gonadotroph) or extracellular (sinusoidal spaces) generation of angiotensin II.

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