Primary aldosteronism is the most common cause of secondary hypertension, most frequently due to an aldosterone-producing adenoma or idiopathic hyperaldosteronism. Somatic mutations of the potassium channel KCNJ5 in the region of the selectivity filter have been found in a significant number of aldosterone-producing adenomas. There are also familial forms of primary aldosteronism, one of which, familial hyperaldosteronism type 3 which to date has been found in one family who presented with a severe abnormality in aldosterone and 18-oxocortisol production and hypertrophy and hyperplasia of the transitional zone of the adrenal cortex. In familial hyperaldosteronism type 3, there is a genomic mutation causing a T158A change of amino acids within the selectivity filter region of the KCNJ5 gene. We are reporting our studies demonstrating that lentiviral-mediated expression of a gene carrying the T158A mutation of the KCNJ5 in the HAC15 adrenal cortical carcinoma cell line causes a 5.3-fold increase in aldosterone secretion in unstimulated HAC15-KCNJ5 cells and that forskolin-stimulated aldosterone secretion was greater than that of angiotensin II. Expression of the mutated KCNJ5 gene decreases plasma membrane polarization, allowing sodium and calcium influx into the cells. The calcium channel antagonist nifedipine and the calmodulin inhibitor W-7 variably inhibited the effect. Overexpression of the mutated KCNJ5 channel resulted in a modest decrease in HAC15 cell proliferation. These studies demonstrate that the T158A mutation of the KCNJ5 gene produces a marked stimulation in aldosterone biosynthesis that is dependent on membrane depolarization and sodium and calcium influx into the HAC15 adrenal cortical carcinoma cells.

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4.

Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex under primary regulation by the renin-angiotensin system. Angiotensin II (A-II) acts through the angiotensin types 1 and 2 receptors (AT1R and AT2R). A-II is metabolized in different tissues by various enzymes to generate two heptapeptides A-III and angiotensin 1-7, which can then be catabolized into smaller peptides. A-II was more potent than A-III in stimulating aldosterone secretion in the adrenocortical cell line HAC15, and A-II, but not A-III, stimulated cortisol secretion. A-II stimulated mRNA expression of steroidogenic acute regulatory protein, 3β-hydroxysteroid dehydrogenase, CYP11B1, and CYP11B2, whereas A-III stimulated 3β-hydroxysteroid dehydrogenase, CYP11B1, and CYP11B2 but decreased the expression of CYP17A1 required for cortisol synthesis. The stimulation of aldosterone secretion by A-II and A-III was blocked by the AT1R receptor blocker, losartan, but not by an AT2R blocker. A-II was rapidly metabolized by the HAC15 cells to mainly to angiotensin 1-7, but not to A-III, and disappeared from the supernatant within 6 h. A-III was metabolized rapidly and disappeared within 1 h. In conclusion, A-II was not converted to A-III in the HAC15 cell and is the more potent stimulator of aldosterone secretion and cortisol of the two. A-III stimulated aldosterone secretion but not cortisol secretion.

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