Vasoactive intestinal peptide (VIP) stimulates cortisol secretion from the H295 human adrenocortical tumour cell line via VPAC1 receptors.

Vasoactive intestinal peptide (VIP) shows a wide tissue distribution and exerts numerous physiological actions. VIP was shown in a dose-dependent manner to increase cortisol secretion in the NCI-H295R human adrenocortical carcinoma (H295) cell line (threshold dose 3.3x10(-10) M, maximal dose 10(-7) M), coupled with a parallel increase in cAMP accumulation. Receptor-specific agonists were employed to determine which of the two known VIP receptor subtypes was involved in cortisol secretion. Treatment with the VPAC1 receptor agonist, [K(15), R(16), L(27)]VIP(1-7)/GRF(8-27), produced a dose-dependent increase in H295 cell cortisol secretion (threshold dose 10(-11) M, maximal dose 10(-7) M) similar to that seen with VIP. Meanwhile, the high-affinity VPAC2 receptor agonist, RO-25-1553, failed to stimulate significantly cortisol or cAMP production from H295 cells. Inhibition of VIP-mediated H295 cell cortisol secretion by PG97-269, a competitive VPAC1-specific antagonist, produced parallel shifts of the dose-response curve and a Schild regression slope of 0.99, indicating competitive inhibition at a single receptor subtype. VIP is known also to interact with the PAC1 receptor, albeit with lower affinity (EC(50) of approximately 200 nM) than the homologous ligand, PACAP (EC(50) of approximately 0.5 nM). PACAP stimulated cortisol secretion from H295 cells (EC(50) of 0.3 nM), suggesting the presence of functional PAC1 receptors. However, stimulation of cortisol secretion by nanomolar concentrations of VIP (EC(50) of 5 nM), coupled with real-time PCR estimation that VPAC1 receptor transcripts appear 1000-fold more abundant than PAC1 transcripts in H295 cells, makes it unlikely that VIP signals via PAC1 receptors. Together, these data suggest that VIP directly stimulates cortisol secretion from H295 cells via activation of the VPAC1 receptor subtype.

Direct stimulation of cortisol secretion from the human NCI H295 adrenocortical cell line by vasoactive intestinal polypeptide.

OBJECTIVE: To investigate a possible direct action of vasoactive intestinal polypeptide (VIP) on adrenal cortisol secretion and to define its mechanism of action. DESIGN: The human adrenocortical carcinoma cell line NCI H295, which is not contaminated by medullary chromaffin cells, was used to aid distinction between a direct action of VIP on adrenocortical cells and an indirect mechanism involving VIP-stimulated release of catecholamines. METHODS: NCI H295 cells were challenged with 10(-11)-10(-7) mol/l VIP for 4 h, with or without prior exposure for 72 h to 10 micromol/l forskolin. Cortisol and cyclic AMP contents of the overlying media were measured using in-house radioimmunoassays. Cells were treated with 10(-8)-10(-6) mol/l adrenaline or 3.3 x 10(-8) mol/l VIP with and without 10(-8)-10(-6) mol/l propranolol to exclude the possibility that an indirect mechanism of action involving beta-adrenoceptors was operating. RESULTS: VIP treatment produced an increase in cortisol secretion without pre-incubation, but this was markedly enhanced by prior exposure of cells to forskolin. VIP was potent, with a threshold of 10(-11) mol/l (n = 4), reaching a maximum 3.9+/-0.9-fold increase in effect on cells pre-exposed to forskolin (n = 4) by 3.3 x 10(-8) mol/l. This increase matched the 4 h response to 10 micromol/l forskolin. Cortisol secretion was accompanied by a parallel, dose-dependent increase in accumulation of cAMP. CONCLUSIONS: VIP potently and directly stimulates secretion of cortisol from these adrenocortical cells of human origin via an adenylate cyclase-coupled VIP receptor. These findings raise the possibility of a significant and direct effect of VIP in the control of steroid secretion from the adrenal cortex in humans.

Forskolin treatment directs steroid production towards the androgen pathway in the NCI-H295R adrenocortical tumour cell line.

The human adrenocortical tumour cell line, NCI-H295, secretes steroids on the mineralocorticoid, glucocorticoid and adrenal androgen pathways. We have investigated the effects of 96 h treatment of cells in monolayer culture with either forskolin (10 microM) (a direct activator of adenylate cyclase), angiotensin II (10 nM) or no agonist ('control') on the steroidogenic phenotype of this cell line. Androstenedione, cortisol and corticosterone secreted into the medium in response to a subsequent 4 hour treatment with angiotensin II (10nM) indicated that the steroidogenic phenotype of NCI-H295 cells changes away from 17-deoxysteroid biosynthesis towards adrenal androgen production in response to forskolin. The NCI-H295R cell line therefore serves as a useful model for investigation of the differential regulation of the steroidogenic pathways in the human adrenal cortex.

Endothelins and rat carotid body: autoradiographic and functional pharmacological studies.

The effects of ET-1 and ET-3 on ventilation and carotid chemosensory discharge have been studied in rats anaesthetised with pentotarbitone. Autoradiographic studies were also performed in vitro to investigate the binding of [125I]ET-1 to rat carotid body, nodose ganglion and brain stem. ET-1 caused a dose-related hyperventilation that was abolished by cutting both carotid sinus nerves. Recordings of chemosensory discharge from the carotid sinus nerve confirmed that ET-1 caused chemoexcitation. ET-3 had only slight effects. The hyperventilation evoked by ET-1 was antagonised by the ETA receptor antagonist FR139317, but responses to hypoxia (10% oxygen) and to cyanide were unaffected. [125I]ET-1 bound to the carotid body, the nodose ganglion and to the brain stem, particularly in the region of the nucleus tractus solitarii. ET-1 binding in the carotid body was displaceable by FR139317, which is consistent with the functional evidence for ETA receptors in the carotid body. The effects of ET-1 on ventilation, coupled with the presence of ET binding sites in areas involved in respiratory and cardiovascular regulation, is consistent with a physiological role for ET in the control of respiration, but our evidence suggests that ET is not crucial for chemotransduction in acute hypoxia.