Endocrinology, Vol 135, 1553-1560, Copyright © 1994 by Endocrine Society

ARTICLES

Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine 5'-triphosphate, adenosine 5'- diphosphate, and uridine 5'-triphosphate via a nucleotide receptor that may be coupled to two signal generation systems [corrected and republished with original paging, article originally printed in Endocrinology 1994 Mar;134(3):1553-60]

DE Hoey, M Nicol, BC Williams and SW Walker
Department of Clinical Biochemistry, Royal Infirmary, Edinburgh, United Kingdom.

Cultured inner zone cells isolated from bovine adrenal cortex secreted cortisol in a dose-dependent fashion in response to ATP and ADP. The threshold response was at 10(-6) M ATP, reaching a maximum by 10(-4) M ATP, at which concentration the n-fold increase relative to basal was 43.8 +/- 22.3 (mean +/- SD; n = 3). Cells were also responsive to the pyrimidine nucleotide UTP. EC50 values for ATP, ADP, and UTP were 5.83 +/- 3.98 x 10(-6), 13.7 +/- 5.67 x 10(-6), and 7.33 +/- 4.52 x 10(-7) M, respectively (mean +/- SD; n = 3). The response to 10(-4) M ATP was linear for at least 60 min, and the cells appeared morphologically normal after removal of the stimulus. The purinergic antagonist suramin was relatively ineffective. The potency order of a range of purines was as follows: ATP = UTP > ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Stimulation of cortisol secretion by ATP was evident after 24 h in primary culture and reached a maximum after 48-72 h, thereafter declining. No response was detected in static incubations of freshly isolated cells. The possibility that added ATP was degraded over the course of the incubation was investigated by separating ATP, ADP, AMP, and adenosine by high resolution anion exchange chromatography after different times of exposure to the cells. Although there was degradation, largely to ADP, about 50% of the ATP remained at 1 h. Cells grown in the presence of [3H]inositol (10 microCi/ml) for 48 h (to prelabel the membrane phosphoinositide pool to isotopic equilibrium) showed a time- and dose- dependent increase in [3H]inositol-labeled total phosphoinositols to ATP or ADP; the response was linear for at least 20 min. Cells labeled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium to 10(-4) M ATP on days 3 and 4 of culture. The basal intracellular Ca2+ concentration was 57.3 +/- 39.3 nmol/liter (mean +/- SD; n = 12 cell suspensions), rising to 171 +/- 84 nmol/liter (mean +/- SD; n = 12 cell suspensions) after the addition of ATP (10(-4) M). Bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min of stimulation with ATP. It was not possible to account for the cAMP response on the basis of conversion of ATP to adenosine, which then acted at an A2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)