Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP

Abstract

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K ϩ , ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca 2ϩ ) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca 2ϩ content are controversial and far from being completely resolved. Here, we found that Ca 2ϩ /calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCIcBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2Ј-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca 2ϩ or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion. angiotension II; adenosine 3Ј,5Ј-cyclic monophosphate; adenosine 3Ј,5Ј-cyclic monophosphate-dependent protein kinase; Ca 2ϩ /calmodulin-dependent kinase ALL STEROID HORMONES, INCLUDING ALDOSTERONE, are synthesized from a common precursor, cholesterol. Stimulation of steroidproducing cells results in prompt mobilization of cholesterol esters from intracellular lipid droplets and their enzymatic hydrolysis to free cholesterol by cholesterol ester hydrolase (CEH) (8). Free cholesterol is then transported to the outer mitochondrial membrane (28,29,50,57), where it is transferred to the side-chain cleavage enzyme system that is localized at the inner mitochondrial membranes. The latter step is the rate-limiting step in steroidogenesis and depends on the activity and phosphorylation of the steroidogenic acute regulatory protein (StAR) (49).The steroid hormone aldosterone produced in adrenal zona glomeruloza (ZG) cells is a major regulator of intravascular volume and blood pressure. Many hormonal and paracrine factors are involved in the regulation of aldosterone production; however, under physiological conditions, the most important ones are adrenocorticotropin (ACTH), angiotensin II (ANG II), and extracellular K ϩ (19, 46). The main action of ACTH in ZG cells is connected with the activation of transmembrane G s ␣-coupled ACTH receptors and the generation of cAMP as a second messenger (11,37). Until the recent discovery of a new family of cAMP-binding proteins [exchange proteins directly activated by cAMP (Epacs)] that can directly activate small GTPases Rap1 and Rap2 (12), cAMP signaling was mainly linked to the activation of cAMP-de...

The Orphan Nuclear Receptor NUR77 Regulates Hormone-Induced StAR Transcription in Leydig Cells through Cooperation with Ca2+/Calmodulin-Dependent Protein Kinase I

“…The exact mechanism responsible for the cross talk between the cAMP and CaMK signaling pathways remains to be elucidated. An intriguing possibility that has recently been proposed (71) is that perhaps a cAMP mediator/ adaptor molecule, such as exchange protein directly activated by cAMP, responsible for linking cAMP to the MAPK pathway (72,73), could also be responsible for the cross talk between cAMP and CaMK. Such an adaptor molecule, however, has yet to be identified.…”

“…The role of the CaMK signaling pathway has mainly been studied in adrenal steroidogenic cells where it was found to be involved in the stimulation of aldosterone production in response to angiotensin II and K ϩ (71,(74)(75)(76)(77)(78). Much less is known regarding the CaMK pathway in Leydig cell steroidogenesis, although Ca 2ϩ has been shown to be important for LH-stimulated testosterone production (13,(79)(80)(81).…”

Short term regulation of aldosterone secretion after stimulation and suppression experiments in mice

“…Increased intracellular calcium levels are followed by activation of the calcium binding protein calmodulin (CaM) by CaM kinase (CaMK) I and/or CAMK IV (Condon et al 2002). Furthermore, several lines of evidence suggest additional activation of CaMK directly through cAMP dependent effects (Gambaryan et al 2006). CaMK induced phosphorylation of ATF-1 and CREB and transcriptional activation of NGF1-B and Nurr1 in turn induces promotor activation of Cyp11b2 (Bassett et al 2004a).…”

N6-Substituted cAMP Analogs Inhibit bTREK-1 K+Channels and Stimulate Cortisol Secretion by a Protein Kinase A-Independent Mechanism

“…cAMP has been reported to activate other kinases in adrenocortical cells and cell lines. cAMP activates CaMK in bovine adrenal zona glomerulosa cells by a PKA-independent mechanism (Gambaryan et al, 2006). The addition of the specific calmodulin antagonist KN-93 (5 M) to the pipette solution in addition to the PKA inhibitors H-89 and PKI (6-22) amide did not blunt bTREK-1 inhibition by 6-Bnz-cAMP (Fig.…”

“…However, they do not exclude the possibility that bTREK-1 inhibition by cAMP could be mediated by pathways independent of both PKA and Epac2. In bovine adrenal glomerulosa cells, cAMP has been reported to activate calmodulin-dependent protein kinase (CaMK) and stimulate aldosterone secretion through a PKA-and Epac-independent mechanism (Gambaryan et al, 2006). In these same cells, aldosterone secretion is coupled to membrane depolarization by bTREK-1 inhibition (Enyeart et al, 2004).…”