Local Renin-Angiotensin System Is Involved in K ⁺ -Induced Aldosterone Secretion from Human Adrenocortical NCI-H295 Cells

Abstract

Abstract-NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K ϩ stimulated the formation of both angiotensin I and angiotensin II 1.9-and 2.5-fold, respectively, and increased aldosterone release 3.0-fold. The K ϩ -induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT 1 )-receptor antagonist losartan (28%). Key Words: NCI-H295 cell line Ⅲ renin-angiotensin system Ⅲ angiotensin II Ⅲ aldosterone Ⅲ potassium T he renin-angiotensin-system (RAS) plays an important role in the regulation of blood pressure as well as sodium and volume homeostasis. 1 In addition to the circulating RAS, local RAS have been documented in a number of animal and human tissues including the adrenal gland (for review, see Reference 2 ). In the adrenal cortex, the role of the local RAS has not been fully characterized. [3][4][5] The human adrenal zona glomerulosa expresses all the components of the RAS, including angiotensinogen (AOG), renin, and angiotensinconverting enzyme (ACE) 2,5 as well as the prohormone convertase PC5 mRNA. 6 Locally produced angiotensin II (Ang II) has been proposed to exercise an autocrine/paracrine control of aldosterone secretion. 2 In many tissues, the various components of the RAS are found in different cells, 7 and it is not clear whether the components of the adrenocortical RAS are produced by different cells or if one cell contains all components. Precise data on the localization and regulation of the RAS components are a requirement for the investigation and interpretation of this system. The examination of an isolated adrenal RAS is especially important to differentiate between the effects caused by circulating and local adrenal systems.The human adrenocortical carcinoma cell line NCI-H295 is a widely accepted model for human adrenocortical studies. This cell line, originally cultured from a human adrenocortical tumor in 1980, represents the first cell line to maintain the ability to produce all adrenocortical steroids, expressing the 3 major pathways of adrenal steroidogenesis including the main steroidogenic enzymes. 8 As in normal human adrenocortical cells, synthesis of aldosterone is regulated by Ang II through AT 1 receptors 9,10 and potassium. 11 These cells therefore may be a suitable system for the characterization of a local RAS.

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3β-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

“…By contrast, steroidogenesis of R cells is reported to be regulated by AngII , potassium , Pezzi et al 1996, Hilbers et al 1999, and ACTH , Staels et al 1993, Mountjoy et al 1994. These investigators observed that the responsiveness of R cells is highly dependent on growth conditions .…”

StAR expression and the long-term aldosterone response to high-potassium diet in Wistar-Kyoto and spontaneously hypertensive rats

“…This idea is supported by evidence that adrenal renin expression increases in response to small and transient increases of serum potassium levels; this increase has been shown to contribute to the rise in aldosterone production (10,26). Furthermore, also in human NCI-H295 cells, a local renin-angiotensin system is involved in potassium-induced aldosterone secretion (14).…”

Renin knockout rat: control of adrenal aldosterone and corticosterone synthesis in vitro and adrenal gene expression

“…The second is that the local expression of adrenal renin is required for normal ZG function (7,9,10,14,16,26,28,29,31). One of the theories is that local generation of adrenal ANG II mediates the ZG response to increases in potassium and that this is mediated by AT 1 -type angiotensin receptors (14,26,29).…”

“…However, it has been known for decades that the adrenal cortex also expresses a local RAAS that operates independently of the renal RAAS (2,7,9,14,16,26,28). One theory is that adrenal renin-generated ANG II mediates the ZG response to increases in plasma potassium, and this is mediated by expression of the angiotensin AT 1 receptor (5,9,14,26,29).The development of the renin knockout (KO) rat provides a unique opportunity to begin to evaluate these phenomena (13).In the current study, we have begun this process by evaluating the aldosterone and corticosterone response to cAMP in vitro by the measurement of adrenal steroidogenic gene expression and by morphological examination of adrenal zonation by immunohistochemical analysis of the two major late-pathway steroidogenic enzymes: P450aldo (CYP11B2) that mediates the conversion of corticosterone to aldosterone in the ZG and P45011␤ (CYP11B1) that mediates corticosterone production in the zona fasciculata/reticularis (ZFR) (8). …”

“…The classic renal renin-angiotensin-aldosterone system (RAAS) is well known as a major regulator of sodium and potassium balance. However, it has been known for decades that the adrenal cortex also expresses a local RAAS that operates independently of the renal RAAS (2,7,9,14,16,26,28). One theory is that adrenal renin-generated ANG II mediates the ZG response to increases in plasma potassium, and this is mediated by expression of the angiotensin AT 1 receptor (5,9,14,26,29).…”