GH Release from Anterior Pituitary Cells

The pituitaries of male Sprague-Dawley rats weighing about 100 g are quickly removed after decapitation. The posterior lobe is discarded and the anterior lobe is divided into two halves by a midsagittal cut. Five bisected hemipituitaries are incubated in plastic vials containing 4 ml TCM 199 with 0.1 % BSA, 15 pg/ml penicillin and 25 fig/ml streptomycin. The vials are gassed with 95 % O2 and 5 % C02. After 30 min of control incubation, the medium is changed and various doses of standard and test substances are added for an incubation of 90 min. GH content in the medium and in the pituitary tissue after incubation is determined by a specific radioimmunoassay (Schalch and Reichlin 1966). Other of hormones may be tested in the same procedure, for example prolactin. 

Dose-response curves are established for standard and test compounds measuring GH release into the medium and GH depletion from the pituitaries. Potencies ratios with confidence limits are calculated from dose-concentration curves. 

GH release was determined using cultured rat pituitary cells (Brazeau et al. 1982 Perkins et al. 1983 Scheikl-Lenz et al. 1985). Pituitary cells were prepared by enzyme dispersion with collagenase, DNAase and pancreatin. The cells were cultured for 3 days in microbiological Petri dishes in Dulbecco s modified essential medium with 20 mM HEPES, 15 % fetal calf serum, 100 mU/ml penicillin-G and 100 (ig/ml streptomycin at 37 °C and 10 % C02. 

Serum growth hormone levels were dose-dependently increased after oral and intravenous administration. Moreover, an increase of insulin-like growth factor and serum cortisol was found. Clearly, the pituitary incubation method can only identify compounds which are suitable for further in vivo characterization, because the responses found in test animals at the target organ level are vast different from those found at the pituitary cell level. 

The advantage of pituitary cell incubation with test compounds of suspected endocrine activity is that useful initial information can be provided rapidly, especially for compounds that show neuroendocrine modulating activity, e.g. neuroleptics and dopamine receptor agonists. The response at a clearly defined cellular level needs than to be compared with the response in intact animals, where multiple feedback loops are operating, and may markedly changed their response profile when the test result is compared in vitro and in vivo. The possibility of using pituitary cells ex-vivo from treated animals should always be kept in mind. 

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