J. Clin. Biochem. Nutr., 2, 171-177, 1987 Phospholipid/Calcium-Dependent Protein Kinase Activity in Human Cortisol-Hypersecreting Adrenocortical Adenoma Tatsuo ISHIZUKA,1,* Hiroshi MURASE,1 Midori YASUE,1 Kiyoshi MIURA,1 Seiji NAGAO,2 and Yoshinori NOZAWA2 1The Third Department of Internal Medicine, 2 Department of Biochemistry, School of Medicine, Gifu University, Gifu 500, Japan (Received September 13, 1986) Summary We examined resected adrenocortical adenoma and adjacent atrophic adrenal tissue from a patient with Cushing's syndrome for phospholipid/calcium-dependent protein kinase (C-kinase) activity. Cytosolic C-kinase activity was higher in adrenocortical adenoma than in adjacent adrenal tissue, while C-kinase activity in the particulate fraction was lower in adrenocortical adenoma than in adjacent adrenal tissue. Upon stimulation of isolated adrenocortical adenoma cells with Ĉ 1-24 ACTH (10-7M), C-kinase activity in the cytosolic fraction was increased from 7 pmol/ min/mg protein (basal) to 107 pmol/min/mg protein by 120 min after stimulation. On the other hand, C-kinase activity in the particular fraction of the isolated cells was considerably decreased from 884 pmol/min/ mg protein (basal) to 179 pmol/min/mg protein overr the same period. These results suggest that C-kinase activity of adrenocortical adenoma cells was translocated from the particulate to the cytosolic fraction by ACTHstimulation. Key Words: C-kinase, translocation, ACTH, adrenocortical adenoma, Cushing's syndrome It is generally documented that C-kinase plays an important role in mediating functional responses in various cell types such as pancreatic acinar cells [1], platelets [2], pituitary cells [3], and neutrophils [4]. Recently, vilgrain et al. [5] presented evidence showing that cytosolic C-kinase activity in isolated bovine adrenocortical cells was increased by stimulation with,l 1-24 ACTH. Also, Anderson et al. [6] *To whom correspondence should be addressed. 171
172 T. ISHIZUKA et a1. have demonstrated an increase in C-kinase activity in the particulate fraction in accordance with malignant transformation. Thus, we examined the distribution of C-kinase activity in human cortisol-hypersecreting adrenocortical adenoma. MATERIALS AND METHODS Case history. A 38-year-old female presented with a 5-year history of petechien at extremities, moon face, central obesity and hypertension (systolic, 172-200 mmhg; diastolic, 100-120 mmhg). Plasma cortisol level was high, 26.4 gig/dl, and the diurnal rhythm of the plasma cortisol level disappeared. One milligram, 2 mg, and 8 mg of a single dose dexamethasone suppression test showed nonsuppressive high plasma cortisol levels. Abdominal computerized tomography and ultrasonography revealed a left adrenal tumor (Fig. 1). Based on these findings she was diagnosed as. having Cushing's syndrome due to a left adrenal cortisol hypersecreting tumor. Six months later, left adrenectomy was performed. Size of this adrenal tumor was 20 x 21 x 32 mm (Fig. 2(a)). Histological examination revealed that the tumor consisted of cells in various sizes with eosinophilic cytoplasm and large nuclei, and this tumor was classified pathologically as a adenoma (Fig. 2(b)). Materials. [7-32P]ATP was purchased from New England Nuclear, Boston, MA. DEAF Sephacel was obtained from Pharmacia, Uppsala. Phosphatidylserine, 1,2-diolein, and histone (type III-s) were from Sigma, St. Louis, MO. E64 [(N-(N-(L-3-trans-carboxiran-2-carbonyl)-L-leucyl)-agamatine)] was donated by Taisho Pharmaceutical Co., Tokyo. Preparation o f adrenal tumor and adjacent adrenal gland. Resected adenoma was divided into two portions. The first portion was immediately cut with scissors into small pieces in Krebs-Ringer bicarbonate (KRB) buffer. These fragments of adrenocortical tumors were then incubated at 37 C in 20 ml of KRB buffer containing 2 mg/m1 collagenase (Worthington Type IV) for 20 min. Following incubation the isolated adrenocortical adenoma cells were immediately washed with K.RB buffer. The cells were then resuspended (900 pl, 4.5 x 106 cells) in KRB buffer containing 0.5% albumin and exposed to 100 pl of 106 M 1S 1-24 ACTH at 37 C, and the reaction was terminated by cooling in an ice bath. Samples taken at different times were washed and homogenized in 3 ml of ice cold buffer I (20 mm Tris/HCI, ph 7.5, 2 mm EGTA, 0.25 M sucrose, 2 mm 2-mercaptoethanol, 50 ~cm E64). The homogenate was centrifuged at 105,000 x g for 60 min, and the resultant supernatant (cytosolic fraction) was used for the C-kinase assay without prior purification through ion exchange column. The pellet was rehomogenized in buffer I containing 0.2/ NP-40 and centrifuged at 105,000 x g for 60 min to obtain the supernatant (solubilized particulate fraction). The second portion of adrenocortical tumor and adjacent atrophic adrenal tissue were homogenized in buffer I, and the cytosolic and solubilized particulate fractions were prepared by the same procedures described above. J. Gin. Biochem. Nutr.
C-KINASE Fig. 1. the Abdominal left adrenal computerized IN ADRENAL tomography 173 ADENOMA showing left adrenal tumor. Arrows show tumor. DEAE-sephacel chromatography. One milliliter of cytosolic or solubilized particulate fraction was applied onto a DEAE-Sephacel column (bed volume: 1 ml) [5] and eluted with 3 ml of 80 mm in buffer 11 (20 mm Tris/HCI, ph 7.5, 2 mm 2-mercaptoethanol, 2 mm EGTA, 50 UME64). The column was further eluted with 300 mm NaCI to obtain solubilized particulate fraction. Protein kinase assay. C-kinase activity was assayed by measuring the phosphorylation of histone. The standard assay mixture (250 al) contained 20 mm Tris/HCI, ph 7.5, 5 mm magnesium acetate, 0.02% histone, 0.2 pg of 1,2-diolein, 10 pg of phosphatidylserine, 0.5 mm CaC12, 2.5 nmol of [1-32P]ATP (1.5 x 105 cpm/nmol), and sample protein. After incubation for 5 min at 30 C, the reaction was stopped addition of 25 trichloroacetic acid, and the precipitate was collected and washed on nitrocellulose membrane filters of 0.45 um pore size (Millipore Corp.), and the radioactivity was measured (Beckman, LS-7500). Vol. 2, No. 2, 1987
174 T. ISHIZUKA et a!. (a) (b) Fig. 2. (a) Resected adrenal tumor. The size of this tumor, 20 x 21 x 32 cm; its weight 10.5 g. (b) Histological examination of the resected adrenal tumor. RESULTS C-kinase activity in resected adjacent adrenal tissue and in adrenocortical tumor C-kinase activities in the cytosolic and particulate fractions from adjacent adrenal tissue were 31 pmol/min/mg protein and 880 pmol/min/mg protein, respectively (Fig. 3). On the other hand, C-kinase activity in the cytosolic fraction from adrenocortical adenoma was 205 pmol/min/mg protein and in the particulate fraction 155 pmol/min/mg protein (sum of C-kinase activities eluted with 80 mm and 300 mm NaC1) (Fig. 3). There was significant difference in C-kinase activity between adjacent adrenal tissue and adrenocortical adenoma. C-kinase activity in the J. Clin. Biochem. Nutr.
C-KINASE IN ADRENAL ADENOMA 175 Fig. 3. C-kinase activity in adjacent adrenal tissue and adrenocortical adenoma. Adjacent adrenal tissue and adrenocortical adenoma were homogenized in buffer I as described under "Materials and Methods." Cytosolic and particulate fractions were assayed either in the absence (open bar) or presence (closed bar) of a phosphatidylserine/diolein/ca2+ mixture. particulate fraction of adjacent adrenal tissue was highest among the cytosolic and particulate fractions from adjacent adrenal tissue and adenoma. The ratio of C-kinase activity in the particulate to that in the cytosolic fraction was higher in adjacent adrenal tissue than in adrenocortical adenoma. Changes in C-kinase activity in isolated adrenocortical adenoma cells stimulated by p 1-24 ACTH Cytosolic C-kinase activity without DEAE-Sephacel column was increased from 7 pmol/min/mg protein to 107 pmol/min/mg protein after stimulation with p 1-24 ACTH (l0~? M) for 120 min (Fig. 4). On the other hand, C-kinase activity in the particulate fraction was decreased from 884 pmol/min/mg protein to 179 pmol/.min/mg protein after stimulation with p 1-24 ACTH for the same period of time (Fig. 4). These changes were well reflected in variations of total C-kinase activity. DISCUSSION In this study adrenocortical adenoma and adjacent atrophic adrenal tissue were examined for C-kinase activity. Cytosolic C-kinase activity of adjacent adrenal tissue, in which secretion of cortisol was suppressed by the adrenocortical adenoma, was significantly lower than that of adrenocortical adenoma which was hypersecreting cortisol, whereas C-kinase activity in the particulate fraction was significantly higher in control adrenal tissue than in adrenocortical adenoma. Anderson et al. [6] demonstrated an increase in C-kinase activity in the particulate frac- Vol. 2, No. 2, 1987
176 T. ISHIZUKA et al. Fig. 4. Changes in C-kinase activity in isolated adrenocortical adenoma cells stimulated by S 1-24 ACTH. Isolated adrenocortical cells were incubated with S 1-24 ACTH (107 M). C-kinase activity in crude cytosolic (open circle) and in particulate fraction (closed circle) obtained by DEAF-Sephacel chromatography was assayed as described in "Materials and Methods." tion of NRK cells in accordance with malignant transformation. Considering that the adrenocortical tumor used in this study was an adenoma but not a cancer, it may be reasonable that C-kinase activity is relatively low in the particulate fraction. Our data also have demonstrated that C-kinase activity of isolated adrenocortical adenoma cells was increased in the cytosolic fraction, whereas the activity was decreased in the particulate fraction upon stimulation with p 1-24 ACTH. Vilgrain et al. [5] also showed an increase in C-kinase activity in the cytosolic fraction, with an accompanying decrease in that in the particulate fraction in p 1-24 ACTH-stimulated bovine adrenocortical cells. These observations are in contrast with those for other cell types such as pancreatic acinar cells [1], platelets [2], pituitary cells [3] and neutrophils [4], in which C-kinase activity was translocated from the cytosol to the particulate fraction by the different types of agonists. However, Averdunk and Gunther [7] recently reported that upon stimulation of rat thymocytes with concanavalin A C-kinase activity was increased in the cytosolic fraction and decreased in the particulate fraction. These findings are consisted with those obtained in the present study, which demonstrates that C-kinase activity in adrenocortical cortisol-hypersecreting adenoma cells is translocated from the particulate to the cytosol fraction after stimulation with ACTH. Further investigations on redistribution of C-kinase activity in other hormone-producing adenomas will be required for clarification of the relationship between C-kinase activity and hormone secretion in the receptor-activated cells upon stimulation..1. Clin. Biochem. Nutr.
C-KINASE IN ADRENAL ADENOMA 177 REFERENCES 1. Noguchi, M., Adachi, H., Gardner, J.D., and Jensen, R.T. (1985): Calcium-activated, phospholipid-dependent protein kinase in pancreatic acinar cells. Am. J. Physiol., 248, G692-701. 2. Uratsuji, Y., Nakanishi, H., Takeyama, Y., Kishimoto, A., and Nishizuka, Y. (1985) : Activation of cellular protein kinase C and mode of inhibitory action of phospholipid-interacting compounds. Biochem. Biophys. Res. Commun., 130, 654-661. 3. Fearon, C.W., and Tashjian, A.H., Jr. (1985): Thyrotropin-releasing hormone induces redistribution of protein kinase C in GH4Cl rat pituitary cells. J. Biol. Chem., 260, 8366-8371. 4. Nishihara, J., McPhail, L.C., and O'Flaherty, J.T. (1986): Stimulus-dependent mobilization of protein kinase C. Biochem. Biophys. Res. Commun., 134, 587-594. 5. Vilgrain, I., Cochet, C., and Chambaz, E.M. (1984): Hormonal regulation of a calciumactivated, phospholipid-dependent protein kinase in bovine adrenal cortex. J. Biol. Chem., 259, 3403-3406. 6. Anderson, W.B., Estival, A., Tapiovaara, H., and Gopalakrishna, R. (1985): Altered subcellular distribution of protein kinase C (a phorbol ester receptor). Possible role in tumor promotion and the regulation of cell growth : relationship to changes in adenylate cyclase activity, in Advances in Cyclic Nucleotide and Protein Phosphorylation Research, ed. by Cooper, D.M.F., and Seamon, K.B., Raven Press, New York, Vol. 19, pp. 287-303. 7. Averdunk, R., and Gunther, T. (1986) : Protein kinase C in cytosol and cell membranes of concanavalin A stimulated rat thymocytes. FEBS Lett., 195, 357-361. Vol. 2, No. 2, 1987