SAT-306 Muscarinic and Adrenergic Receptor Cooperativity in a Human Adrenocortical Carcinoma Cell Line

The role of autonomic receptors in the regulation of the adrenal cortex is poorly understood. We recently showed that activation of M(3) muscarinic receptors stimulates intracellular calcium oscillations, aldosterone production, and expression of CYP11B2 (1). The present study explores the relationship between muscarinic and adrenergic receptors in corticosteroid production. Using live-cell fluorescence imaging of HAC15 adrenocortical cells with the calcium-sensitive probe Fluo-4, we have shown that stimulation of adrenergic receptors with the endogenous, non-selective adrenergic agonist norepinephrine (10μM) enhances intracellular Ca(2+) oscillations caused by the cholinergic agonist carbachol (1μM). However, Ca(2+) is not affected by norepinephrine alone. Adrenergic enhancement of carbachol-induced Ca(2+) oscillations is blocked by the ⍺ adrenergic receptor antagonist phentolamine, but not by the β adrenergic receptor antagonist propanolol. Specifically, ⍺2 and β2 antagonists (such as yohimbine and butoxamine, respectively) significantly suppressed the norepinephrine effect, but ⍺1 and β1 antagonists (such as tamsulosin and metoprolol, respectively) had no effect. RT qPCR identified ⍺2A receptors as the most abundant adrenergic receptor in HAC15 cells. Saturation experiments using (3)H-NMS and (3)H-Rauwolscine confirmed the presence of muscarinic M(2) and M(3) receptors as well as ⍺2A receptors. Using competition radiolabeled binding assays we explored the cooperation between M(2)/M(3) and ⍺2A adrenergic receptors. Our results suggest that autonomic regulation of intracellular Ca(2+) depends on an interplay of M(3) and ⍺2A receptors. Additional experiments will use ELISA methods to determine the functional impact of autonomic receptor cooperativity on steroid synthesis and secretion. References: (1) Malaiyandi et al., Mol Cell Endocrinol. 2018 478: 1-9.