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Title: The characterisation of vasoactive intestinal peptide as a secretagogue in human H295R adrenocortical cells
Author: Cobb, Vanessa Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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The aims of this thesis were to investigate the direct action of VIP on adrenocortical steroid production, by using the human H295R adrenocortical carcinoma cell-line, and to determine the second messenger systems activated and the receptor types involved. The steroid responses to AII and forskolin were first characterised in the H295R cell-line, including the effect of pretreatment with these agonists on the steroid phenotype of the cells. Forskolin-pretreatment directed steroidogenesis towards adrenal androgen production and away from 17-deoxysteroid production compared with AII-pretreated cells and control cells, demonstrating that the steroid phenotype of the cells could be modified by different agonists which utilise different second messenger systems. VIP increased steroid secretion from H295R cells in a time-dependent manner. The pattern of steroid secretion was altered depending on the pretreatment for the cell-line. VIP increased cortisol, corticosterone and androstenedione secretion from forskolin-pretreated cells but had no effect on androstenedione production from control cells or AII-pretreated cells. Although VIP increased cortisol and corticosterone from control and AII-pretreated cells, forskolin pretreatment enhanced the cortisol and corticosterone responses to VIP. The effect of VIP on cortisol production from forskolin-pretreated H295R cells was studied further. VIP dose-dependently increased cortisol secretion (threshold dose ≈ 10-11 M, maximal dose ≈ 3.3 x 10-8 M, pEC50 = 9.2 (±0.4) (n=4). This response was accompanied by a parallel, dose-dependent increase in cAMP accumulation (threshold dose ≈ 10-11 M, maximal dose ≈ 3.3 x 10-8 M, pEC50 = 8.6(±0.5) (n=4). Changes in total phosphoinositide turnover and cGMP production were not detected in response to VIP treatment of H295R cells. A β-adrenoceptor-mediated mechanism for VIP-induced cortisol production was excluded. Comparison of dose-response curves demonstrated a similar potency of VIP and PACAP (a highly homologous peptide which shares some receptors with VIP) for cortisol secretion and cAMP production (pEC50s for cortisol response to VIP and PACAP were 9.4 (±0.4) and 9.8 (±0.1) respectively (n=3) and the pEC50s for cAMP response to VIP and PACAP were 8.7 (±0.5) and 9.3 (±0.1) respectively (n=3), suggesting that these responses were mediated primarily by activation of VPAC receptors, not PAC1 receptors. The VPAC2 receptor superagonist, RO-25-1553, failed to stimulate cortisol or cAMP production from H295R cells at doses of 10-9 M and 10-8 M (P≥0.05)) whilst the VPAC1 receptor antagonists, C1-phe6, Leu17-VIP and acetyl-tyr-GRF-amide, had no effect on VIP-stimulated increases in cortisol and cAMP production from H295R cells (P≥0.05). Therefore, the VPAC receptor subtype involved in VIP-mediated cortisol and cAMP responses was not clearly defined using these pharmacological tools.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available