Short Communications cAMP Production by GHRH in GH-Producing Pituitary Adenoma Cells K.Nakagawa1,2, T. Ishizuka2 , C.Shimizu2, Y. Matsuura2 , N. Wada2 , H. Kijima2 , Y.Ito2, 71 Aida3 andH. Abe3 1HealthAdministration Center, Hokkaido University of Education, Sapporo Second Department of Medicine and 3Department of Neurosurgery, Hokkaido University School of Medicine, Sapporo, Japan

Introduction

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It has been well established that GHRH increases cAMP production in pituitary cells {Bilezikjian and Vale 1983). In 1987 Vallar, Spada and Giannattasio reported high basal cAMP levels and very low GHRH-responsiveness in a group (20/68 = 29%) of human GH-secreting pituitary adenomas and they postulated the alteration of Gs protein in this group. Landis, Masters, Spada, Pace, Bourne and Vallar (1989) demonstrated the point mutations at codons 201 and 227 of alpha-subunit of Gs protein. Lyons, Landis, Harsh, Vallar, Griinewald, Feichtinger, Duh, Clark, Kawasaki, Bourne and McCormick (1990) screened various human endocrine tumors and found these mutations in 18 out of 42 (43 %) of GH-secreting pituitary adenomas. As the responsiveness to GHRH seems to be critical to differentiate 2 groups, we examined the relation between cAMP production by GHRH in vitro and GH release by GHRH in vivo in the cases of acromegaly treated surgically in our hospital. Materials and Methods Pituitary GH-secreting adenoma tissues, obtained at transsphenoidal pituitary adenomectomy for acromegaly during the period between January 1988 and July 1991, were minced and incubated at 37 °C in 0.15 M phosphate buffer, pH 7.4, containing 2.5 mg/ml trypsin, 0.2 mg/ml collagenase and 0.5 mg/ml glucose. The dispersed cells, approximately 8 x 10 /dish, were incubated in 3.5 ml Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum at 37 °C for 4 days under humidified 95 % air and 5 % CO2. Then the dishes were divided into two groups; the medium of one group was replaced with 2 ml DMEM containing 0.2% bovine serum albumin (DMEM-BSA), the other with 2 ml DMEM-BSA containing 10 nM GHRH. The dishes were incubated for 2 hours in the same condition, then the medium was removed and stored at -30 °C for the determination of extracellular hGH. After washing with 1 ml of DMEM-BSA, 1 ml DMEM-BSA and 0.1 ml of 1 N HC1 were added. The monolayer cells were removed with a rubber policeman and the suspension was storedat — 30 C for the determination of intracellular cAMP. Before surgery each acromegalic patient received GHRH test with 100 ug of GHRH injected intravenously. Blood samples for hGH measurement were obtained — 30, 0, 15, 30, 45, 60, 90,120,150 and 180 minutes after the injection. cAMP was assayed with the RIA kit purchased from Yamasa Shoyu Co., Choshi, Japan. Human GH was determined with our in-house RIA.

Fig. 1 Intracellular cAMP (upper panel) and extracellular hGH (middle panel) in basal (open bars) and GHRH (10 nM)stimulated (stippled bars) states in in vitro incubation of 10 pituitary GH-secreting adenomas. In the lower panel, basal (open bars) and maximal (stippled bars) plasma hGH levels in in vivo GHRH test of corresponding cases are shown. Horizontal bars: one SE, * * * p < 0.001, * * p < 0 . 0 1 , *p

cAMP production by GHRH in GH-producing pituitary adenoma cells.

Short Communications cAMP Production by GHRH in GH-Producing Pituitary Adenoma Cells K.Nakagawa1,2, T. Ishizuka2 , C.Shimizu2, Y. Matsuura2 , N. Wada2...
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