No abstract

Just as the recognition of the role of the phosphoinositides and phosphoinositols as a cellular signalling pathway has seen a dramatic advance in the last 10 years, so parallel investigations in adrenocortical cells have led to an equally dramatic increase in our understanding of the mechanisms involved in the control of adrenal steroidogenesis. In rat and bovine adrenocortical cells, the non-cAMP stimulatory agonists AII, acetylcholine and vasopressin have been shown to promote receptor/G-protein-mediated activation of a polyphosphoinositide-specific phospholipase C. In turn, studies in rat ZG and bovine ZG and ZFR cells have provided strong evidence for a causal relationship between the rapid and sustained formation of inositol 1,4,5-trisphosphate and DG by phospholipase C, and the subsequent increase in steroidogenesis in these cell types. In addition to describing the stimulatory effects of the various agonists on phospholipase C activity, this review has considered whether agonists may act through stimulation of phospholipase A2. No agonist can be said to act exclusively through phospholipase A2, and only AII can be said not to act through phospholipase A2 in adrenocortical cells. It seems unlikely that many studies will focus on this question in future unless an alternative physiological role for phospholipase A2 becomes apparent.

Bovine adrenal cortex tissue expresses high levels of glutathione S-transferase (GST) from each of the alpha, mu and pi gene families. We describe the purification and characterization of an abundant alpha-class GST from this tissue that has not been identified previously because of its failure to bind to S-hexylglutathione-Sepharose 6B (S-hexG-Ag). This enzyme has been affinity purified on glutathione-Sepharose 6B (GSH-Ag) and was obtained in a highly purified form by employing S-hexG-Ag to remove the bulk of GST before chromatography on GSH-Ag. The purified GST eluted from GSH-Ag was found to exhibit marked peroxidase and delta 5-ketosteroid isomerase activities (19.2 and 1.67 U/mg respectively). The bovine enzyme also showed high GST activity towards 4-hydroxynonenal (5.09 U/mg). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the bovine GST contains two distinct polypeptides, one with an Mr of 25,900 and the other with an Mr of 26,500. An abundant alpha-class GST was also purified from human adrenal cortex that possessed properties which were similar to the bovine alpha-class GST described above; however, unlike the bovine enzyme, the corresponding human alpha-class GST bound to S-hexG-Ag. As with the bovine enzyme, the purified human GST displayed marked peroxidase and isomerase activities (27 and 4.02 U/mg respectively). Further analysis on SDS-PAGE (Mr 25,800) and reverse-phase high-performance liquid chromatography established that this abundant alpha-class GST in human adrenal cortex is equivalent to the human liver GST B1B1 enzyme. As both human and bovine adrenal cortex contain high levels of alpha-class GST with similar catalytic properties, we discuss the possible functions of these enzymes in this tissue.

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 3·3×10 −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 ∼200 nM) than the homologous ligand, PACAP (EC 50 of ∼ 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.

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.