Abstract-Adrenal zona glomerulosa (ZG) cells do not contain nitric oxide (NO) synthase (NOS).Key Words: nitric oxide Ⅲ aldosterone Ⅲ adrenal glands Ⅲ adenovirus Ⅲ nitric oxide synthase Ⅲ zona glomerulosa N itric oxide (NO) derived from NO donors inhibits aldosterone synthesis in adrenal zona glomerulosa (ZG) cells from bovine, rat, and human adrenal glands. 1-3 NOmediated inhibition occurs in ZG cells stimulated with angiotensin II (Ang II), adrenocorticotropic hormone, and potassium ion, as well as with the steroidogenic substrates 25-hydroxycholesterol and progesterone. 2,3 NO stimulates the accumulation of cGMP in adrenal ZG cells; however, this appears to be unrelated to NO-mediated inhibition of aldosterone release. 3 The mechanism of inhibition appears to occur primarily via direct interaction of NO with cytochrome P450 enzymes of the steroidogenic pathway. 3 The results of studies of the effects of NO on other steroidogenic cell types indicate that the inhibitory effects are not limited to aldosterone. NO inhibits the synthesis of testosterone in rat Leydig cells and the synthesis of corticosterone in rat adrenal zona fasciculata cells. 4 -8 Thus, it appears likely that the inhibitory effects of NO may be seen in a variety of cells containing steroidogenic cytochrome P450 enzymes. Studies of the biochemical interactions between NO and various cytochrome P450 enzymes have clearly demonstrated the ability of NO to rapidly bind the heme portion of the cytochrome P450 enzyme. 9,10 This binding process inactivates the enzyme by preventing binding to oxygen. 11 Raman spectrophotometric analysis of the cytochrome P450/NO interaction suggests that there may be 2 phases to the inhibition by NO. The first phase involves a rapid occupation of the heme group by NO and results in a reversible inhibition of enzymatic activity. After a 2-hour exposure to NO, a second phase of inhibition appears, which likely involves the nitrosylation of a tyrosine of the cytochrome P450 enzyme. 9,10 This second phase results in irreversible inhibition of enzymatic activity.Determination of the physiological role of NO in aldosterone synthesis is complicated by the interactions of vasodilatory effects of NO with the regulatory pathways that stimulate aldosterone synthesis. The administration of NO donors such as sodium nitroprusside and nitroglycerin has long been recognized to dramatically lower blood pressure. Conversely, the inhibition of endogenous NO production through the use of NO synthase (NOS) inhibitors increases blood pressure. 12 The effects of these manipulations on plasma aldosterone concentrations have produced conflicting results. Usui et al 13 demonstrated an increase in plasma aldosterone concentrations and ZG cell angiotensin II type 1 (AT 1 ) receptor expression in rats after NOS inhibition

The regulation of aldosterone synthesis by endogenous nitric oxide (NO) was examined in cultured cells of the adrenal cortex. Endothelial NO synthase (eNOS) was detected by Western blot in cultured adrenal endothelial cells (ECs) but not in zona glomerulosa (ZG) cells or adrenal fibroblasts. Neither inducible (iNOS) nor neuronal NOS (nNOS) isoforms were detected in the cells. Only ECs had NOS activity and converted [(3)H]L-arginine to [(3)H]L-citrulline. Angiotensin II (ANG II, 100 nM) increased EC production of nitrate/nitrite by 2.4-fold. Coincubation with ECs or treatment with DETA nonoate increased the fluorescence of ZG cells loaded with an NO-sensitive dye, diaminofluorescein 2 diacetate (DAF-2 DA). DETA nonoate inhibited ANG II (1 nM) and potassium (10 mM) -stimulated aldosterone release in a concentration-related manner. This inhibitory effect of NO was enhanced >10-fold by decreasing the oxygen concentration from 21 to 8%. Coincubation of EC and ZG cells in 8% oxygen inhibited ANG II-induced aldosterone release, and inhibition was reversed by blockade of NOS. These findings indicate that adrenal EC-derived NO inhibits aldosterone release by cultured ZG cells and that the sensitivity to NO inhibition is increased at low oxygen concentrations.

Abstract-Prostaglandin E 2 (PGE 2 ) is an endogenous hormone of adrenal zona glomerulosa cells and is released in response to stimulation by agonists such as angiotensin II (Ang II). It stimulates the release of aldosterone from cultured bovine adrenal zona glomerulosa cells. These studies were designed to determine whether this steroidogenic effect of PGE 2 was mediated by an EP 1 , EP 2 , or EP 3 receptor. Prostaglandin E 2 and 11-deoxy PGE 1 , an EP 2 -selective agonist, stimulated aldosterone release in a concentration-related manner with an ED 50 of 300 nmol/L for PGE 2 and 2 mol/L for 11-deoxy PGE 1 . The maximal effect of PGE 2 was less than that of angiotensin II. 17-Phenyl trinor PGE 2 , an EP 1 -selective agonist, required concentrations of 100 mol/L to stimulate aldosterone release and sulprostone, an EP 3 /EP 1 -selective agonist, failed to alter aldosterone release. The EP 1 -selective antagonist SC19220 failed to alter basal or PGE 2 -stimulated aldosterone release over a range of concentrations. PGE 2 and 11-deoxy PGE 1 also stimulated an increase in both intracellular and extracellular cAMP. This increase was time-and concentration-related. The ED 50 for PGE 2 was 9.8 mol/L. 17-Phenyl trinor PGE 2 and sulprostone were without effect. Using fura-2 loaded cells, PGE 2 (2 mol/L), dibutyryl cAMP (2 mmol/L), and Ang II (2 mol/L) increased intracellular calcium over basal concentrations by 5.5-fold, 3-fold, and 6.2-fold, respectively. Like PGE 2 , dibutyryl cAMP also stimulated aldosterone release. PGE 2 -and dibutyryl cAMP-induced aldosterone release were blocked by the calcium channel inhibitor diltiazem. These studies indicate that PGE 2 is a potent stimulus for aldosterone release and that the effect is mediated by EP 2 receptors. Both cAMP and calcium appear to mediate the steroidogenic effect of PGE 2 and calcium seems to be distal to cAMP. (Hypertension. 1998;31:575-581.)Key Words: zona glomerulosa Ⅲ cyclic AMP Ⅲ calcium Ⅲ receptors, prostanoid Ⅲ angiotensin II T here are five classes of prostanoid (P) receptors designated as EP, FP, DP, IP, and TP, corresponding to their naturally occurring agonists, prostaglandin E 2 , prostaglandin F 2␣ , prostaglandin D 2 , prostaglandin I 2 , and thromboxane A 2 , respectively.1 Synthetic and natural analogues of these prostaglandins also exist that possess selectivity at these five classes of receptors. 2,3The EP receptor has been subclassified into three subtypes: EP 1 , EP 2 , and EP 3 . [4][5][6][7][8] Based on the analysis of Coleman et al, 1,7 Eglin and Whiting, 3 and Muallem et al, 9 we know that prostanoid receptors differ in the second messengers that mediate their biological effects. Agonists acting on the EP 1 , FP, and TP receptors stimulate the IP 3 /DAG pathway and exert their effects through an increase in intracellular calcium. The EP 2 , DP, or IP receptor agonists stimulate adenylyl cyclase and the accumulation of cAMP. Finally, EP 3 receptor activation may increase IP 3 /DAG formation or inhibit adenylyl cyclase.There is considerable evid...

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