Angiotensin II-activated protein kinase D mediates acute aldosterone secretion

Abstract

Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24h) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion.

Figure 1

AngII and PMA, but not elevated K⁺ or ACTH, induced PKD activation. Cultured adrenal glomerulosa cells were treated with _eqKRB⁺ with or without 10 nM AngII, 15 mM K⁺ (NaCl in the KRB⁺ was replaced isoosmotically with KCl), 100 nM PMA or 10 nM ACTH for 30 minutes. Panel (A) illustrates a representative experiment and (B) shows the quantitation of multiple experiments in which pPKD^S910 levels were normalized using actin. (C) Aldosterone levels were quantified in the supernatants. Values represent the means ± SEM from at least 3 experiments; *p<0.05 vs. control.

Figure 2

AngII and PMA, but not elevated K+, stimulated PKD activity. Cultured adrenal glomerulosa cells were treated with _eqKRB⁺ with or without 10 nM AngII, 15 mM K⁺, 100 nM PMA or 10 nM ACTH for 30 minutes. Equal amounts of cellular protein were immunoprecipitated and PKD activity was determined using an in vitro kinase assay as described in the Methods section. Data represent means ± SEM from 5 experiments performed in duplicate; *p<0.05 vs. control.

Figure 3

AngII and PMA induced PKD activation in a time- and a dose-dependent manner. (A) Cultured glomerulosa cells were treated with or without _eqKRB⁺ containing various concentrations of AngII, as indicated, for 30 minutes, and immunoblot analysis was performed using antibody directed towards pPKD^S910. Panel (A) illustrates a representative western blot and panel (B) shows the quantitation of multiple experiments in which pPKD^S910 levels were normalized using actin. Values represent means ± SEM from 4 experiments performed in duplicate; * p<0.05, **p<0.01 vs. control. (C and D) Cultured glomerulosa cells were treated with or without _eqKRB⁺ containing (C) 10 nM AngII or (D) 100 nM PMA for 5, 15 or 30 minutes, and immunoblot analysis was performed as above. Values represent means ± SEM from 4 experiments performed in duplicate; *p<0.05, **p<0.01 vs. control.

Figure 4

Candesartan, but not PD123,319, inhibited AngII-induced PKD activation. (A) Cultured glomerulosa cells were pretreated with _eqKRB⁺ containing no addition, 10 M candesartan (Cand) or 10 M PD123,319 (PD) for 30 minutes. Cells were then treated with or without 10 nM AngII in the presence or absence of the agents as indicated for another 30 minutes, and aldosterone assays were performed on the supernatants. Data represent means ± SEM from 3 experiments performed in duplicate; *p<0.05 vs. control, †p<0.05 vs. AngII alone or AngII plus PD123,319. (B and C) Immunoblot analysis was performed on the cell lysates. Panel (B) illustrates a representative experiment and (C) shows the quantitation of multiple experiments in which pPKD^S910 levels were normalized using actin. Data represent means ± SEM from at least 3 experiments performed in duplicate; *p<0.05 vs. control, †p<0.05 vs. AngII alone or AngII plus PD123,319.

Figure 5

Adenovirus-induced overexpression of PKD^S738/742E enhanced AngII-induced aldosterone secretion from adrenal glomerulosa cells. Cultured glomerulosa cells were incubated for 4 hours with adenovirus expressing pAdtrackCMV (vector) or PKD^S738/742E constructs on the second day of culture. The supernatant was removed, and was replaced with serum-free medium for 20 hours. pAdtrackCMV- (vector-) or PKD^S738/742E-infected primary cells were then treated with or without 10 nM AngII for one hour in _eqKRB⁺, and aldosterone secretion was assayed. Panel (A) illustrates a representative experiment showing PKD^S738/742E and GFP overexpression in primary adenovirus infected cells 20–24 hours post-infection. Results are representative of a minimum of 3 separate experiments. The samples were separated on the same blot but intervening lanes were removed for clarity. Panel (B) illustrates aldosterone secretion data representing the means ± SEM from 3 experiments performed in duplicate; the results are expressed relative to the maximal aldosterone secretory response for each experiment. The maximal aldosterone secretory values for the 3 experiments were 1,123 ± 21; 1,298 ± 88 and 2,248 ± 6 (means ± SEM) pg aldosterone/mL/60 min; *p<0.05 vs. control (vector-infected cells without AngII), †p<0.05 versus the vector-infected control with AngII.

Figure 6

Adenovirus-induced overexpression of PKD^S738/742A inhibits AngII-induced aldosterone secretion from adrenal glomerulosa cells. Cultured glomerulosa cells were incubated for 4 hours with adenovirus expressing pAdtrackCMV (vector) or PKD^S738/742A constructs on the second day of culture. The supernatant was removed, and was replaced with serum-free medium for 20 hours. pAdtrackCMV- (vector-) or PKD^S738/742A-infected primary cells were then treated with or without 10 nM AngII for one hour in _eqKRB⁺, and aldosterone secretion was assayed. Panel (A) illustrates a representative experiment showing PKD^S738/742A and GFP overexpression in primary adenovirus infected cells 20–24 hours post-infection. Results are representative of a minimum of 4 separate experiments. The samples were separated on the same blot but intervening lanes were removed for clarity. Panel (B) illustrates aldosterone secretion data representing the means ± SEM from 4 experiments performed in duplicate; the results are expressed relative to the maximal aldosterone secretory response for each experiment. The maximal aldosterone secretory values for the 4 experiments were 284 ± 20; 984 ± 66; 1,695 ± 116 and 3,964 ± 442 (means ± SEM) pg aldosterone/mL/60 min; *p<0.05 vs. control (vector-infected cells without AngII), †p<0.05 versus the vector-infected control with AngII.