Effects of ATP and other purine derivatives on steroidogenesis in pri mary cultured bovine adrenocortical fasciculata cells were examined. At concentra tions higher than 1 uM, ATP showed a potent stimulative effect on the cortisol pro duction of the cells. The potency order of the steroidogenic effect of the tested purine derivatives was ATP > ADP >> adenosine > AMP. a ,f'-Methylene ATP had no stimulative effect on the steroidogenesis at concentrations as high as l mM. Theophylline did not antagonize the steroidogenic effect of ATP. These results sug gest that bovine adrenocortical fasciculata cells possess the Pty purinoceptors that are linked to steroidogenesis.Recently, many researchers have proposed that extracellular ATP is involved in the regu lation of many biological functions; and the plasma concentration of ATP is elevated above 10 mM under stressful conditions (1). Because glucocorticoid from the adrenal cor tex plays an important role in adapting the body to stressors, the effect of extracellular ATP on steroidogenesis in bovine adrenocor tical fasciculata cells was investigated.The isolated bovine adrenocortical fascicu lata cells were prepared aseptically by the use of collagenase (Cooper Biomedicals) and de oxyribonuclease (Sigma Chemical Co.). The isolated cells were cultured as previously de scribed in Ham's F-10 medium supplemented with sera and antibiotics (2). The 3-day pri mary cultured monolayer cells were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) containing 3 mg/ml bovine serum albumin and 1 mg/ml glucose for 1 hr at 37TC in a CO-, incubator (5% CO-, in air) in the presence or absence of the test reagents (2). Glucocorti coid in the incubation medium was determined fluorometrically using cortisol as the standard (3). Extracellular ATP had a stimulative effect on cortisol production at concentrations higher than 1 uM, and the maximum response was obtained at 100,uM (Fig. 1). The result indi cated that bovine adrenocortical cells have purinoceptors that are linked to steroidogene sis, and the physiological concentration of ATP influences adrenocortical function. To estimate the receptor subtype, the effects of other purine derivatives on steroidogenesis were examined. At concentrations tested, up to 1 mM, ADP, AMP, and adenosine also stimulated steroidogenesis. However, the potency order of the effect was ATP > ADP >> adenosine >> AMP. a ,,3-Methylene ATP did not stimulate steroidogenesis at concentra

ABSTRACT-The effect of extracellular ATP on steroidogenesis in primary cultured bovine adreno cortical fasciculata cells was investigated. I observed that in the presence of extracellular Ca t+, ATP caused a dose-dependent elevation of intracellular Ca 2+ ([Ca2+];) and induced steroidogenesis concen tration and time-dependently. However, in the absence of extracellular Ca t+, ATP had no effect on steroidogenesis. In the presence of extracellular Ca `+, calmodulin inhibitors inhibited the ATP-induced steroidogenesis, but dihydropyridine calcium channel blockers did not. Furthermore, ATP did not cause an elevation of cyclic AMP in bovine adrenocortical fasciculata cells even if extracellular Ca 2+ existed. These results suggest that extracellular ATP might have an influence on bovine adrenocortical cells via the purinoceptor (P2Y) in connection with calcium mobilization, open the non-selective calcium chan nel and induce steroidogenesis by means of an elevation of [Ca 2+]i via the calcium-calmodulin system.

ABSTRACT-We examined the effect of extracellular adenosine 5'-triphosphate (ATP) on adrenocorticotropic hormone (ACTH)-and angiotensin II-induced steroidogenesis in bovine adrenocortical fasciculata cells. The low concentration of ATP (5 mM) potentiated ACTH-induced steroidogenesis synergistically. However, the purine derivative did not affect angiotensin II-induced steroidogenesis. Although adenosine (100 mM) (a metabolite of ATP) showed a weak steroidogenic effect, it did not potentiate ACTH-induced steroidogenesis. ATP also enhanced the steroidogenesis by NaF synergistically in bovine adrenocortical cells, but did not potentiate forskolin-and dibutyryl cyclic AMP-induced steroidogenesis. The stimulating effect of ACTH on cyclic AMP production was synergistically accelerated by ATP (5 mM), which has no effect by itself on cyclic AMP formation. These results suggest that extracellular ATP affected the ACTH receptor-adenylyl cyclase coupling processes, and potentiation of steroidogenesis by ACTH ensued in bovine adrenocortical fasciculata cells.Keywords: Adrenal cortex, Adrenocorticotropic hormone, Steroidogenesis, ATP, P2Y receptor Extracellular adenosine 5'-triphosphate (ATP) plays many biological roles in diverse cell functions via its cell membrane receptors (1 -4). Receptors for ATP are classified into two groups: a ligand-gated ion channel P2X receptor family and a G protein-coupled P2Y receptor family (5). We previously reported that extracellular ATP stimulated glucocorticoid production (steroidogenesis) in bovine adrenocortical fasciculata cells (BAFC) via P2Y receptors at micromolar concentrations (6).Recently, the importance of cross-talk between two biological active substances on the functions in several kinds of cells and organs has been mentioned. In the case of extracellular ATP, the nucleotide potentiates the insulin secretion stimulated by acetylcholine in isolated perfused rat pancreas (7), and the cytosolic Ca 2+ response to parathyroid hormone in rat osteoblastic cells (8). In steroidogenic organs, the cross-talk between serotonin and angiotensin II (9), interleukin-6 and adrenocorticotropic hormone (ACTH) (10) in rat adrenal steroidogenesis and angiotensin II and ACTH on cyclic AMP formation in bovine adrenal glomerulosa cells (11, 12) had been reported. From these observations, there might be possible cross-talk between extracellular ATP and ACTH (and/ or another steroidogenic substances) on glucocorticoid production in BAFC. Therefore, we investigated the effect of ATP on ACTH-and angiotensin II-induced steroidogenesis in BAFC. MATERIALS AND METHODS Primary culture of BAFCIsolated bovine adrenocortical fasciculata cells were prepared aseptically using collagenase and deoxyribonuclease I as previously described (13). The isolated cells were cultured in Ham's F-10 medium supplemented with 5% fetal calf serum, 10% newborn calf serum, 2.5% horse serum and antibiotics in a 24-well-type dish (10 -15 10 5 cells / well) in a CO2 incubator (humidified atmosphere of 5% CO 2 in air) at 37°C as repor...

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