The paradoxical increase in cortisol secretion induced by dexamethasone in primary pigmented nodular adrenocortical disease involves a glucocorticoid receptor-mediated effect of dexamethasone on protein kinase A catalytic subunits

Abstract

Context: Primary pigmented nodular adrenocortical disease (PPNAD) results in most cases from mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene. Patients with PPNAD exhibit a paradoxical increase in cortisol secretion in response to dexamethasone.

Objective: The aim was to investigate the mechanism of the action of dexamethasone on adrenocortical cells removed from patients with PPNAD and a transgenic model of PPNAD [Tg(tTA/X2AS) mice].

Design and setting: We performed an in vitro study in an academic research laboratory.

Patients: Eleven patients with histologically proven PPNAD were included in the study.

Intervention: Cultured PPNAD cells were incubated with dexamethasone in the presence of various modulators of the cAMP/PKA pathway and the glucocorticoid receptor antagonist RU486.

Main outcome measure: Cortisol and corticosterone were measured by radioimmunological assays in cell culture supernatants.

Results: Dexamethasone stimulated in vitro cortisol secretion from PPNAD tissues in six patients. The stimulatory effect of dexamethasone on cortisol release was not reduced by the adenylyl cyclase inhibitor SQ22536 or potentiated by the phosphodiesterase inhibitor IMBX and the cAMP analog 8Br-cAMP. Conversely, the PKA inhibitor H89 and RU486 inhibited the cortisol response to dexamethasone. Dexamethasone had no effect on cortisol production from normal human adrenocortical cells but stimulated corticosteroidogenesis in the presence of RU486. Similarly, dexamethasone failed to influence corticosterone release by adrenocortical cells removed from Tg(tTA/X2AS) mice but stimulated corticosteroidogenesis in the presence of RU 486.

Conclusions: These results indicate that, in human PPNAD tissues, dexamethasone paradoxically stimulates cortisol release through a glucocorticoid receptor-mediated effect on PKA catalytic subunits.

Figure 1

Typical profiles illustrating the action of modulators of the cAMP/PKA pathway on dexamethasone-induced cortisol secretion by PPNAD cells. A, Effect of the adenylyl cyclase inhibitor SQ22536 on basal and dexamethasone-induced cortisol secretion by cultured P2 cells. Dexamethasone (10⁻⁶ m, 24 h) stimulated by 40 ± 4% and 31 ± 8% cortisol secretion (P = 0.37) in the absence and presence of SQ22536 (5 × 10⁻⁴ m; 24 h), respectively. B, Effect of the PDE inhibitor IBMX on basal and dexamethasone-induced cortisol secretion from cultured P3 cells. Dexamethasone (10⁻⁶ m, 24 h) stimulated by 26 ± 6% and 39 ± 4% cortisol secretion (P = 0.12) in the absence and presence of IBMX (10⁻⁴ m; 24 h), respectively. C, Effect of the cAMP analog 8Br-cAMP on basal and dexamethasone-induced cortisol secretion from cultured P4 cells. Dexamethasone (10⁻⁶ m; 24 h) stimulated by 297 ± 14% and 264 ± 154% cortisol secretion (P = 0.84) in the absence and presence of 8Br-cAMP (10⁻⁴ m; 24 h), respectively. Data values represent mean ± sem from four experiments. NS, Not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2

Typical profiles illustrating the action of the PKA inhibitor H89 on the cortisol response to dexamethasone by PPNAD cells. Effect of graded concentrations of dexamethasone (10⁻⁹ to 10⁻⁵ m) on cortisol secretion by cultured cells derived from P1 (A) and P11 (B) cells in the absence (▪) and presence (□) of the PKA inhibitor H89 (10⁻⁵ m). Data values represent mean ± sem from four experiments. For evaluation of the effect of dexamethasone in the presence of H89, basal level was calculated as the mean cortisol production by cultured cells incubated with H89 alone.

Figure 3

Effect of the GR antagonist RU486 on the cortisol response to dexamethasone by PPNAD cells. A, Effect of graded concentrations of dexamethasone (10⁻⁹ to 10⁻⁶ m) on cortisol secretion by cultured cells derived from P11 cells in the absence (▪) and presence (□) of RU486 (10⁻⁶ m). B, Effect of RU486 (10⁻⁶ m) on the cortisol responses to dexamethasone (10⁻⁶ m) by PPNAD cells derived from patients P2–5 and P11. Data values represent mean ± sem from four experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4

Action of dexamethasone on glucocorticoid production from human and mouse adrenocortical cells. A, Effect of dexamethasone (10⁻⁶ m, 24 h) on cortisol secretion by cultured normal human adrenocortical cells obtained from four different subjects in the absence and presence of RU486 (10⁻⁶ m). Dexamethasone did not affect corticosteroidogenesis but stimulated cortisol secretion by 39 ± 15% (P = 0.03) in the presence of RU486. B and C, Effect of graded concentrations of dexamethasone (10⁻⁹ to 10⁻⁶ m) on corticosterone secretion by cultured wild-type (B) and transgenic Tg(tTA/X2AS) (C) mice adrenocortical cells in the absence (▪) and presence (□) of RU486 (10⁻⁶ m). Data values represent mean ± sem from four experiments. NS, Not significant. *, P < 0.05; ***, P < 0.001.