Phorbol ester increases mitochondrial cholesterol content in NCI H295R cells

Abstract

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.

Figure 1. PMA stimulated aldosterone secretion from and mitochondrial cholesterol content in the human adrenocortical carcinoma cell line, NCI H295R

H295R cells were incubated for 24 hours with serum-free medium containing 500 µM aminoglutethimide in the presence and absence of 10 nM PMA. Mitochondria were isolated and assayed for cholesterol content (light bars) as described in Methods. Values represent the means ± S.E.M. of 12 separate experiments; ***p<0.002 versus a control of 1.0 by a one-sample t-test. The mean relative enrichment of citrate synthase activity was 6.7 ± 1.7-fold. (Note that one value of relative enrichment in this set was estimated, because the actual citrate synthase activity in the homogenate could not be exactly calculated due to the loss of the homogenate protein aliquot prior to assay.) In additional, parallel experiments, H295R cells were incubated for 24 hours with medium containing no additions or 10 nM PMA. Supernatants were collected and assayed for aldosterone secretion (dark bars) by radioimmunoassay. Values represent the means ± S.E.M. of 14 separate experiments; ***p<0.002 versus a control of 1.0 by a one-sample t-test.

Figure 2. Atrial natriuretic peptide (ANP) inhibited AngII-stimulated aldosterone secretion and mitochondrial cholesterol content

(A) H295R cells were incubated for 24 hours with serum-free medium containing no additions, 10 nM AngII or 10 nM AngII plus 100 nM ANP. Supernatants were collected and assayed for aldosterone secretion by radioimmunoassay. Values represent the means ± S.E.M. of 5 separate experiments. (B) H295R cells were incubated for 24 hours with serum-free medium containing no additions, 10 nM AngII, or 10 nM AngII plus 100 nM ANP. Mitochondria were isolated and cholesterol measured as described in Methods. Values represent the means ± S.E.M. of 4 separate experiments; *p<0.05, **p<0.01 versus control and †p<0.01 versus Ang II by ANOVA and a Student-Neuman-Keul's post-hoc test. The mean relative enrichment of citrate synthase activity was 7.8 ± 2.9-fold.

Figure 3. Atrial natriuretic peptide (ANP) inhibited PMA-stimulated aldosterone secretion and mitochondrial cholesterol content

(A) H295R cells were incubated for 24 hours with serum-free medium containing no addition, 10 nM PMA or 10 nM PMA plus 100 nM ANP. Supernatants were collected and assayed for aldosterone secretion by radioimmunoassay. Values represent the means ± S.E.M. of 8 separate experiments. (B) H295R cells were incubated for 24 hours with serum-free medium containing 500 µM aminoglutethimide and no other additions, 10 nM PMA or 10 PMA plus 100 nM ANP. Mitochondria were isolated and cholesterol measured as described in Methods. Values represent the means ± S.E.M. of 8 separate experiments; *p<0.05 versus control by ANOVA and a Student-Neuman-Keul's post-hoc test. The mean relative enrichment of citrate synthase activity was 6.7 ± 1.7-fold. (Note that one value of relative enrichment in this set was estimated, because the actual citrate synthase activity in the homogenate could not be exactly calculated due to the loss of the homogenate protein aliquot prior to assay.)