Zonal Expression of Endothelial Nitric Oxide Synthase (eNOS) in Normal Human Adrenal Cortex and Functional Insights from Comparison with Adrenals in Cushing's Disease

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 41-52-HPA Axis & Disease States
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-43
Subarna Mani Dhital*1, Theresa M Duello2, Yoram Shenker3 and Ian Michael Bird4
1Patan Academy of Health Sciences, Kathmandu, Nepal, 2Univ Wisconsin-Madison Sch Med, Madison, WI, 3University of Wisconsin, Madison, WI, 4Univ of Wisconsin-Madison, Madison, WI
Background: Nitric oxide (NO) is known to modulate adrenal steroidogenesis (1). Because of short half-life of NO (2), presence of catalyzing enzyme in proximity to target tissue is important. The main isozyme of NOS expressed in adrenal cortex is eNOS with marked interspecies variation in zonal expression (3). In humans, expression of eNOS and functional modulation of steroidogenesis by NO has been demonstrated in zona glomerulosa (ZG) cells but has not been evaluated in zona fasciculata (ZF) and zona reticularis (ZR).   

Sample collection: Histologically normal-looking adrenal glands were collected from adrenalectomy specimens in 5 patients. Two adrenal glands were collected from Cushing’s disease patient. Tissues were processed immediately for immunohistochemistry (IHC) and western blot.

Methodology: IHC was performed for eNOS in paraffin-embedded sections of adrenal tissues. P450c17 IHC served as zonal marker for ZR and ZF and Cytochome b5 (b5) as ZR marker. In addition, western blot for eNOS was performed in dissected tissue blocks from different adrenal zones. Digital grayscale densitometry was performed to compare staining intensity and results analyzed with one-way ANOVA using Kruskall-Wallis test. P <0.05 was considered significant.

Results: Capsule-subtracted value of eNOS staining on IHC was significantly higher than medullary eNOS expression in ZG, ZF and ZR (p= 0.012) in normal adrenal glands.  These results also corresponded to eNOS expression pattern on western blot. Zonal differentiation was confirmed by appropriate expression patterns of P450c17 and b5 in both IHC and western blot. In adrenals from Cushing’s disease patient, ZF expression of eNOS was significantly lower than in ZG or ZR (p = 0.001).

Discussion: eNOS expression was demonstrated in ZG, ZF and ZR of histologically normal-looking human adrenals using IHC and western blot. The presence of eNOS in adrenal cortex points to the potential role for NO in local modulation and fine-tuning of adrenal steroidogenesis. Inhibitory role of NO has been reported in human ZG cells (4). But these results point to its relevance in ZF and ZR as well. Low expression of eNOS in adrenals from Cushing’s disease patient further support its functional relevance because lowered inhibition of steroidogenic enzymes by decreased NO production fits the profile of increased cortisol output from ZF.

(1) Ducsay CA, Myers DA. eNOS activation and NO function: differential control of steroidogenesis by nitric oxide and its adaptation with hypoxia. J Endocrinol. 2011;210(3):259-69. (2) Ignarro LJ. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol. 1990;30:535-60. (3) Peterson JK, Moran F, Conley AJ, Bird IM. Zonal expression of endothelial nitric oxide synthase in sheep and rhesus adrenal cortex. Endocrinology. 2001;142(12):5351-63. (4) Natarajan R, Lanting L, Bai W, Bravo EL, Nadler J. The role of nitric oxide in the regulation of aldosterone synthesis by adrenal glomerulosa cells. J Steroid Biochem Mol Biol. 1997;61(1]2):47]53.

Nothing to Disclose: SMD, TMD, YS, IMB

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: Supported by internal grant from Division of Endocrinology, Department of Medicine, University of Wisconsin School of Medicine and Public Health.