| Research Abstract |
Both angiotensin II and adrenocorticotropic hon-none(ACTH) are well known to play a crucial role on the regulation of aldosterone production in adrenal glomerulosa cells. Recent observations suggest that the steroidogenic action of ACTH is mediated via the cAMP messenger system, whereas angiotensin II acts mainly through the phosphoinositide pathway. However, there have been no reports conceming the interaction between the cAMP messenger system activated by ACTH and the Ca^<2+> messenger system induced by angiotensin II. Both ACTH and angiotensin II simultaneously act on adrenal cells for regulating steroidogenesis under physiological conditions. Thus the present experiment s were performed to examine the effect of ACTH on the action of angiotensin II by measuring angiotensin II receptor activity, cytosolic Ca^<2+> movement, and aldosterone production. The major findings of the present study are that short-term exposure to a high dose of ACTH(10-^7 M) inhibited ^<125>I-angiotensin II b
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inding to bovine adrenal glomerulosa cells, decreased the initial spike phase of[Ca^<2+>]_i induced by angiotensin II, and inhibition of angiotensin II-induced aldosterone production. Low dose of ACTH(10-^<10> M), which did not increase cAMP formation, did not affect angiotensin II receptor activity. These studies have shown that angiotensin II receptors of bovine adrenal glomerulosa cells can be downregulated by 1 mM dibutyryl cyclic AMP, as well as by effectors which are able to activate cAMP formation (10^<-7> M ACTH) and 10^<-5> M forskolin). The rapid decrease in angiotensin II receptors induced by 10^<-7>M ACTH was associated with a decreased steroidogenic responsiveness and a decreased rise in the [Ca^<2+>]_i response induced by angiotensin II. These studies show that the cAMP-dependent processes activated by ACTH have the capacity to interfere with signal transduction mechanisms initiated by receptors for angiotensin II.
[1-^<14>C] arachidonic acid was incubated with isolated bovine adrenal fasciculate cells for 15 min at 37ﾟC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6, 8, 11, 14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE), leukotriene B_4 and 11, 14, 15-trihydroxy-5, 8, 12-eicosatrienoic acid (11, 14, 15-THET). Addition of 15-hydroperoxy-5, 8, 11, 13- eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculate cells resulted in the formation of 1 1, 14, 15-THET. The formation of 11, 14, 15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome PA50 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculate cells and that 15-HPETE is further metabolized to 11, 14, 15-THETbyadrenalmicrosomalcytochromeP-450. Less
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