Profiling Endogenous Opioid Peptide Release from Adrenal Chromaffin Cells

Despite longstanding recognition that the adrenal medulla is a major source of both catecholamines and opioid peptides, direct measurements of opioid peptide release kinetics─and characterization of the full complexity of the peptidergic forms released─remain under studied. Here, this gap is addressed by introducing a workflow that integrates real-time electrochemical detection of catecholamine (CA) and enkephalin (ENK) release kinetics with tandem mass spectrometry to profile opioid peptide signaling from adrenal chromaffin cells. Fast-scan cyclic voltammetry was used to quantitatively monitor the exocytotic release of ENK peptides and CA from large dense-core vesicles in single chromaffin cells. Interestingly, ENK release events were evident on two separate time scales. The first phase of release was consistent with the codetected CA signal, suggesting release from the same population of vesicles. A slower signal was also present, suggestive of a strong association with the dense-core matrix and hindered mass transport. Liquid chromatography was coupled with mass spectrometry (LC-MS) to profile the activity-dependent opioid peptide release from these cells. The MS data show that proenkephalin-derived peptides dominate the adrenal peptidome, and that extended variants of M-ENK that contain the YGGFM motif plus additional amino acids, including YGGFM-RF, YGGFM-RGL, peptide E, BAM-18 and BAM-22, are actively released in response to both chemical and electrical stimulation. Overall, the results of this study inform on the temporal complexity and chemical diversity of peptide participants in opioid signaling while highlighting a versatile and powerful workflow for further exploration of these molecules in physiological systems.