The role of calcium influx pathways in phospholipase D activation in bovine adrenal glomerulosa cells.

The steroid hormone aldosterone maintains sodium homeostasis and is therefore important in the control of blood volume and pressure. Angiotensin II (AngII) and elevated extracellular potassium concentrations ([K(+)](e)), the prime physiologic regulators of aldosterone secretion from adrenal glomerulosa cells, activate phospholipase D (PLD) in these cells. The role of Ca(2+) in the activation by these agents is unknown, although nitrendipine, a voltage-dependent Ca(2+) channel antagonist, does not inhibit AngII-elicited PLD activation, despite the fact that this compound blocked elevated [K(+)](e)-stimulated PLD activity. PLD activation triggered by AngII was also unaffected by the T-type calcium channel inhibitor nickel. Nevertheless, Ca(2+) influx was required for AngII-induced PLD activation in both primary cultures of bovine adrenal glomerulosa cells and a glomerulosa cell model, the NCI H295R adrenocortical carcinoma cell line. The involvement of store-operated Ca(2+) (SOC) influx and Ca(2+) release-activated Ca(2+) (CRAC) influx pathways in PLD activation was investigated using thapsigargin, an endoplasmic reticulum Ca(2+) pump inhibitor that empties the store to induce SOC influx, and the SOC inhibitor YM-58483 (BTP2), as well as a CRAC inhibitor, tyrphostin A9. In bovine glomerulosa cells, tyrphostin A9 inhibited AngII-induced PLD activation without affecting elevated [K(+)](e)-stimulated enzyme activity. On the other hand, differences were observed between the bovine adrenal glomerulosa and H295R cells in the involvement of Ca(2+) influx pathways in PLD activation, with the involvement of the SOC pathway suggested in the H295R cells. In summary, our results indicate that Ca(2+) entry only through certain Ca(2+) influx pathways is linked to PLD activation.

Phorbol ester increases mitochondrial cholesterol content in NCI H295R cells.

The first step in steroidogenesis is cholesterol mobilization from cytosolic lipid droplets to the initiating rate-limiting enzyme complex located on the inner mitochondrial membrane. Angiotensin II (AngII), the primary agonist of aldosterone secretion from adrenal glomerulosa cells, is known to induce cholesterol mobilization to mitochondria. However, the role of the protein kinase C (PKC) pathway in mediating cholesterol mobilization is unknown. To determine PKC's involvement, human adrenocortical carcinoma cells were incubated with or without PKC-activating phorbol 12-myristate 13-acetate (PMA) and mitochondrial cholesterol content assayed. Like AngII, PMA significantly elevated mitochondrial cholesterol content as well as aldosterone secretion. Thus, PKC may play a role in cholesterol mobilization to mitochondria and hence steroid production. Atrial natriuretic peptide (ANP) inhibited both AngII- and PMA-stimulated mitochondrial cholesterol content. These findings suggest that the ability of ANP to inhibit steroidogenesis induced by multiple agents may be related to its capacity to reduce cholesterol mobilization.