[CANCER RESEARCH 40, 3768-3771. October 1980] 0008-5472/80/0040-OOOOS02.00 Multiple Hormone Receptors in the Adenylate Cyclase of Human Adrenocortical Tumors1 Shigeru Matsukura,2 Teruo Kakita,3 Satoru Sueoka, Hiroki Yoshimi, Yukio Mirata, Makoto Yokota, and Takuo Fujita Third Division. Department of Medicine. Kobe University School of Medicine. Ikuta-ku, Kobe 650. Japan ABSTRACT relate the presence of altered hormone receptors to the pa thology of the human adrenal gland. Adenylate cyclase responses to pituitary hormones including adrenocorticotropic hormone (ACTH), biogenetic amines, pros- MATERIALS AND METHODS taglandin E, (PGE,), angiotensin II, and glucagon were evalu ated in adrenocortical tumors and hyperplastic adrenal tissues, obtained from patients with Cushing's syndrome at surgery, Normal adrenal tissues were obtained at therapeutic total adrenalectomy from 2 patients with breast carcinoma. Two and in normal adrenals. The adenylate cyclase of two normal bilaterally hyperplastic adrenocortical tissues due to excess adrenals was activated only by ACTH and PGEi among the ACTH secretion from the pituitary, 5 clinically ACTH-responsive hormones tested, while that of two hyperplastic adrenal tissues adrenal adenomas, an ACTH-unresponsive carcinoma, and an due to excessive pituitary ACTH secretion was stimulated only ACTH-responsive adrenocortical nodular hyperplastic tissue by ACTH. Of five ACTH-responsive adrenocortical adenomas, were obtained at surgery from patients presenting with clinical in contrast, three were stimulated by norepinephrine, two by and biochemical evidence of Cushing's syndrome (1). The epinephrine, one by thyroid-stimulating hormone, and one by tissue specimens were processed, and the whole homogenates luteinizing hormone in addition to ACTH, indicating the pres were mainly used in duplicate or triplicate for the adenylate ence of multiple receptors for hormones other than ACTH and cyclase assay described in the previous paper (11). In brief, PGEi in these four tumors. The cyclase of an ACTH-unrespon- the adenylate cyclase response was expressed as cyclic AMP sive adrenocortical carcinoma was activated only by PGE, and (pmol/mg protein) produced during 20-min incubation at 37° not by other hormones including ACTH, whereas that of an with enzyme fractions and hormones, based on the conversion ACTH-responsive adrenocortical nodular hyperplasia was stim of [14C]ATP to [14C]cyclic AMP. To obtain the fractions, the ulated by ACTH and glucagon but not by other hormones homogenate of an adrenal adenoma (Case 1) was centrifuged including PGE,. at 600 x g for 10 min. The sediment was collected, and the These results indicate the presence of multiple receptors for supernatant was centrifuged at 10,000 x g for 10 min. The hormones other than ACTH and PGE,, the normal adrenocor sedimented particles (600 x g and 10,000 x g) were resus- tical stimulants, in human adrenocortical tumors, particularly in pended in the medium, utilizing volumes adjusted to yield adrenal adenomas, but not in normal and hyperplastic (of approximately equal protein concentrations and used for the whichever an etiology) adrenocortical tissues, suggesting a adenylate cyclase assay. functional alteration of the cellular membrane receptors in ACTH (Cortrosyn) was supplied by Daiichi, Tokyo, Japan; human adrenocortical tumors. PGE, was supplied by Ono, Osaka, Japan; and glucagon was furnished by Novo Industrials, Copenhagen, Denmark. Angio INTRODUCTION tensin II aspartic-/6-amide 5-valine was supplied by Ciba-Geigy, Basel, Switzerland. Ovine or bovine TSH, LH, follicle-stimulat The altered cell surface characteristics distinguishing cancer ing hormone, and prolactin were kindly provided by the Na cells from normal ones (8, 14) are well known. In view of the tional Institute of Arthritis, Metabolism, and Digestive Diseases, initial binding of many hormones to their specific cell surface NIH, Bethesda, Md. Dopamine and serotonin were purchased receptors on their target (6), prior to the action, qualitative from Nakarai, Kyoto, Japan; norepinephrine was supplied by changes of hormone responsiveness may be expected in the Fluka AG, B, Switzerland; and epinephrine was purchased tumors of several endocrine glands (5, 18, 19). The presence from Merck, Darmstadt, West Germany. [8-14C]ATP or [U- of multiple hormone receptors for hormones other than ACTH4 14C]ATP and [3H]cyclic AMP were purchased from New Eng has been demonstrated in the adrenocortical tumors of rats land Nuclear, Boston, Mass. (20, 21) and humans (10). The present investigation was de Statistical analysis was performed by Student's f test. Con signed to investigate further the adenylate cyclase responses sidered significant were p values less than 0.05. of normal, hyperplastic, and tumorous adrenocortical tissues of humans to hormones and other active substances and to RESULTS 1This study was supported by grants from the Ministry of Health and Welfare and the Ministry of Education, Science, and Culture, Japan. '' To whom requests for reprints should be addressed. Adenylate Cyclase of Normal Adrenals. The adenylate cy ' Present address: Endocrine Division. Department of Medicine. Kanazawa clase responses of 2 normal adrenals are shown in Table I. The Medical College, Uchinada-cho. Ishikawa 920-20. Japan. adrenal homogenates possess a cyclase system responsive to " The abbreviations used are: ACTH, adrenocorticotropic hormone; cyclic ACTH, PGE,, and NaF. Other polypeptide hormones and bio- AMP, cyclic adenosine 3':5'-monophosphate; PGE,, prostaglandin E,: TSH, thyroid-stimulating hormone; LH, luteinizing hormone. genetic amines tested were without effect in activating the Received March 6, 1980; accepted June 19, 1980. cyclase. 3768 OCTOBER 1980 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1980 American Association for Cancer Research. Receptors of Human Adrenocortical Tumors Table 1 Table 2 Adenylate cyclase responses of 2 normal adrenal glands Adenylate cyclase responses of 2 bilateral hyperplastic adrenocortical tissues Whole tissue homogenates were incubated with or without the addition of Conditions of the experiment are similar to those described in Table 1. hormones as described in the text. Adenylate cyclase activity is expressed as pmol of cyclic AMP produced per mg of protein per 20 min at 37°. Adenylate cyclase activity (cyclic AMP (pmol/mg protein/20 min)| Adenylate cyclase activity Concentra pro-AdditionControlACTHTSHProlactinLHFollicle-stimulatinghormoneEpinephrineNorepinephrineAngiotensin[cyclic AMP (pmol/mg Addition tion (M) Case 1 Case 2 mm)]Case Control 2a117± auun 4677 1333*46 ACTHTSHEpinephrineNorepinephrineGlucagonAngiotensin"10 (M)10101010101010102.8 1110a197111929314696119639Case21965042102231742071901962154392071691296±±±±±±±i±±±+26t>13C101371622122457C3814± 510 590 250 5io-510 573 253 568 457 "10s2.8 373 956 IIPGE, 2100 ± 249 ± X 10 5 ± 14 ±4 99 ±2b NaF10 10 282 112± 4b52±3a Mean ±S.E. of triplicate determinations. 1p < 0.01 (stimulated versus control values). IIPGE,GlucagonSerotoninNaF^UM^enir x101.2b-410'5-2tein/20 Table 3 x10-«-5-5-5-6-5-S-a10 102C Adenylate cyclase responses of whole homogenates and fractionated particles from an adrenocortical adenoma (Case 1) Mean of duplicate determinations. 0 Mean ±S.E. of triplicate determinations Conditions of the experiment are similar to those described in Table 1 except c p < 0.01 (stimulated versus control values). for use of the particulate fractions for the assay. Adenylate cyclase activity [cyclic AMP (pmol/ min)]AdditionControlACTHEpinephrineNorepinephrineAngiotensinCon- mg protein/20 Adenylate Cyclase of Bilateral Hyperplastic Adrenocorti cal Tissues Due to Excess ACTH Secretion. The adenylate Wholehomog-(M) x g parti gparticle66x cyclase responses to 2 bilaterally hyperplastic adrenal tissues enate2391010101037639236320710cle99 18a+ 5a176+ due to excess ACTH secretion obtained from the patients with +237 37"± +279 16Ö192+ Cushing's disease are shown in Table 2. The adenylate cyclase 25°± 156265± ±215 14°± 14°78± of the hyperplastic tissues was stimulated by ACTH and NaF ±115 IINaFtration 24±1506600±1 10327± but not by other hormones. This finding is quite similar to that ±43o 1450+ 191 ±19a37"Bb10"1210,000 of normal adrenal tissues except for the failure of activation of Mean ±S.E. of triplicate determinations. the cyclase of the hyperplastic tissues by PGE,. 1p < 0.05 (stimulated versus control values). Adenylate Cyclase of ACTH-responsive Adrenocortical Ad enomas. The activity of the adenylate cyclase in the whole tissue homogenates and in the 600 x g and 10,000 x g Table 4 particulate fractions from an adrenal adenoma (Case 1) is Adenylate cyclase responses of 3 adrenocortical adenomas shown in Table 3. The cyclase of the homogenate and of each Conditions of the experiment are similar to those described in Table 1. fraction was stimulated by ACTH, epinephrine, norepinephrine, AMPAdditionControlACTHTSHLHEpinephrineNorepinephrineGlucagonAngiotensinAdenylate cyclase activity [cyclic and NaF despite a decrease of the control cyclase levels in the min)]Case protein/20 600 x g and 10,000 x g particulate fractions relative to that 2161 3134b29212410814460108807Case317 in the whole homogenate, thus making the percentage of 12a243+ +533 20C240± stimulation over the control greater. The whole homogenate, 'io-5IO'510 ±330 45C234+ however, was used for the adenylate cyclase assay to ensure +519 ±2802623248508232654 a rapid and simple processing