Protein Kinase D restrains angiotensin II-induced aldosterone secretion in primary bovine adrenal glomerulosa cells

Abstract

Misregulation of the renin-angiotensin II (Angll)-aldosterone (Aldo) system is a key feature of cardiovascular disease. A focus of study in this system is the Angll-elicited secretion of Aldo from the adrenocortical zona glomerulosa. An excellent model in which to study this phenomenon is primary cultures of bovine adrenal glomerulosa (AG) cells. These cells secrete detectable quantities of Aldo in response to secretagogues, such as Angll, elevated potassium (K+), adrenocorticotrophic hormone (ACTH) and phorbol 12-myristate 13-acetate (PMA), within 30 minutes. The serine (Ser)/threonine kinase protein kinase D (PKD) is reported to be activated by Angll in several systems, including the adrenocortical carcinoma cell line NCI H295R, and is thought to have a positive role in chronic (24 hours) Angll-evoked Aldo secretion. Because the role ofPKD in acute Angll-elicited Aldo secretion has never been examined in a primary culture system, we undertook to study the role of PKD in acute (minutes to one hour) Aldo secretion. Thus, Angll (10 nM) and PMA (100 nM), but not elevated K+ (15 mM) and ACTH (10 nM), induced phosphorylation of PKD on Ser9l 0, a marker of PKD activation, in primary bovine AG cells. This finding was confirmed by an in vitro kinase activity assay. Angll and PMA were also able to induce PKD activation in H295R cells. Furthermore, this activation was concentration dependent, and was rapidly induced (by 5 min). PKD activation was dependent on Angll type 1 (AT-I), but not AT-2 receptor, signaling, and was independent of tyrosine kinase signaling. Finally, we introduced, via adenovirus transduction, wild-type PKDw' imd dominant negative PKDs738n4 zA constructs into primary AG cells and monitored Angll-evoked Aldo secretion. PKDw' -transduced AG cells exhibited decreased Angll-stimulated Aldo secretion, while in the PKDs738n42 Ainfected AG cells Angll-stimulated Aldo was enhanced. Thus, we hypothesize that PKD has an anti-secretory role in Angll-induced acute Aldo secretion.