Primary cultures of bovine inner zone adrenocortical cells secrete cortisol in response to adenosine triphosphate, adenosine diphosphate, and uridine triphosphate via a nucleotide receptor which may be coupled to two signal generation systems

Abstract

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 relative to basal was 43.8 +/- 22.3 (mean +/- SD, n = 3). The response to 10(-4) M ATP remained linear for up to 2 h, and the cells appeared morphologically normal after removal of the stimulus. 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 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. The potency order of a range of purines was as follows: ATP = ADP > 2-methyl-S-ATP > alpha, beta-methylene ATP = beta, alpha-methylene ATP = AMP. Cells were also responsive to the pyrimidine nucleotide uridine 5'-triphosphate, which was equipotent with ATP. The purinergic antagonist suramin was relatively ineffective. 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 in response to ATP or ADP; the response was linear for at least 60 min. Cells labelled with the Ca2+ indicator fura-2 showed an increase in intracellular calcium in response to 10(-4) M ATP on days 3 and 4 of culture. Basal intracellular Ca2+ was found to be 57.3 +/- 39.3 nmol/liter (mean +/- SD, n = 12 cell suspensions) rising to 171 +/- 84 nmol/liter (mean +/- SD, n = 12 cell suspensions) in response to ATP (10(-4) M). In response to ATP, bovine inner zone cells also demonstrated a dose-dependent increase in intracellular cAMP measured after 1 min stimulation. 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)