HUMAN NCI-H295 ADRENOCORTICAL CARCINOMA-CELLS - A MODEL FOR ANGIOTENSIN-II-RESPONSIVE ALDOSTERONE SECRETION

Excessive secretion of aldosterone from the adrenal results in the mos t common form of endocrine hypertension. An understanding of the regul atory processes involved in aldosterone synthesis and release is neede d to define the biomolecular mechanisms controlling excessive producti on of aldosterone. However, in vitro studies regarding the regulatory mechanisms of human aldosterone production have been limited because o f difficulties in obtaining tissue and the subsequent isolation of ald osterone-secreting glomerulosa cells. Herein we describe an adrenocort ical carcinoma cell line, NCI-H295, which provides a suitable angioten sin-II (AII)-responsive model system to investigate the acute and chro nic regulation of aldosterone synthesis. The cells were characterized with regard to the effects of AII on second messenger systems, aldoste rone release, and levels of aldosterone synthase (P450c18) mRNA. In th e presence of lithium, AII caused a rapid, but transient, increase in the production of inositol tris- and bisphosphates, whereas a prolonge d gradual accumulation of inositol monophosphate occurred. Treatment w ith AII resulted in a 4.5-fold increase in total inositol phosphates i n a concentration-dependent manner and increase in intracellular cytop lasmic free Ca2+. Significant increases an in aldosterone (3.5-fold) w ere detected within 1 h of AII addition. Aldosterone release occurred in a concentration- and time-dependent manner. The type 1 AII (ATI) re ceptor was shown to be responsible for activation of phosphoinositidas e-C, increased intracellular free Ca2+, and aldosterone production, as determined by use of the AT1 receptor antagonist DuP753. In addition, AII treatment resulted in a time-dependent increase in levels of P450 c18 mRNA, as detected by RNAse protection assay. In summary, NCI-H295 cells provide a valuable model system to define mechanisms regulating human aldosterone production.