Intracellular calcium release activates carotid body afferents by fentanyl

We recently reported [Peng YJ, Nanduri J, Wang N, Xie Z, Fox AP, Prabhakar NR. Function (Oxf) 6: zqaf020, 2025] that fentanyl activates carotid body (CB) afferents via kappa opioid receptors (KORs), while CB denervation exacerbates, coadministration of fentanyl with a KOR agonist attenuates opioid-induced respiratory depression (OIRD). These findings indicated that CB chemoreflex activation by fentanyl may counteract OIRD. The present study investigated the cellular mechanisms underlying CB afferent activation by fentanyl. We hypothesized that Ca²⁺ signaling in glomus cells mediates CB activation by fentanyl. Using Fura-2 calcium imaging in rat glomus cells, we observed that fentanyl increased intracellular Ca²⁺ even in the absence of extracellular calcium. Pretreatment with thapsigargin, which depletes internal Ca²⁺ stores, abolished Ca²⁺ response, suggesting that fentanyl releases Ca²⁺ from intracellular stores. In human embryonic kidney cells expressing KOR and G protein alpha q subunit (Gαq), fentanyl promoted KOR-Gαq complex formation and stimulated phospholipase C (PLC), elevating inositol trisphosphate (IP₃) levels in the CB. Pharmacological blockade of KOR, Gαq, PLC, or IP₃ receptors prevented both the rise in [Ca²⁺]_i and CB afferent activation. Collectively, these results identify a previously uncharacterized KOR-Gαq-PLC-IP₃R-Ca²⁺ signaling pathway in glomus cells that mediates CB afferent activation by fentanyl, providing new mechanistic insight into how CB chemoreflex activation by fentanyl may mitigate OIRD.