Illuminating chromaffin granules: compartmentalization of endocannabinoids in bovine adrenal glands supports role in hormone modulation

The endocannabinoid system (ECS) plays a major role in the central stress response. However, its role in the sympathetic nervous system remains poorly understood. Knowledge about the spatial distribution and function of endocannabinoids (eCBs) in adrenals is lacking. Here, we analysed lipid eCBs and alpha-hemoglobin-derived peptide endocannabinoids (pepcans) across distinct zones of the bovine adrenal gland and investigated the effects of RVD-hemopressin (pepcan-12) and anandamide (AEA) on steroidogenesis. To challenge the hypothesis that pepcans are produced in the adrenal medulla, bovine chromaffin granules (CGs) were isolated and pepcans, eCBs, amino acids and neurotransmitters quantified by targeted metabolomics. To label CGs for flow cytometry, we synthesized the novel vesicular monoamine transporter 2 (VMAT2)-targeting probe CV-28 which specifically stained large dense-core vesicles. While the classical eCBs were present in CGs, pepcans were absent, indicating that these peptides are not released via CGs from chromaffin cells. Interestingly, besides neurotransmitters also N-linoleoylethanolamine (LEA) and L-leucine were markedly enriched in CGs. Overall, N-acylethanolamines including AEA, RVD-hemopressin and its precursor pepcan-23 were predominant in the cortex while 2-arachidonoylglycerol (2-AG) was found in the medulla. This intriguing distribution was supported by the differential expression of serine hydrolases visualized by activity-based protein profiling (ABPP). In NCI-H295R cells, AEA and RVD-hemopressin increase progesterone and reduce testosterone secretion. In basal state, the effect of AEA on increased progesterone secretion was potentiated by RVD-hemopressin. Overall, this study illuminates the CG metabolome and gives impetus to the further investigation of ECS-mediated regulation of adrenal hormone production and adrenal physiology.