Evidence for protein kinase involvement in the 5‐HT–[Ca²⁺]_i–pannexin‐1 signalling pathway in type II glial cells of the rat carotid body

New Findings What is the central question of this study? The mammalian carotid body (CB) is a peripheral chemoreceptor organ involved in O 2 and CO 2 /H + homeostasis. Recent studies suggest that 5‐HT, released from CB receptor cells, can stimulate adjacent glial‐like type II cells, leading to an increase in intracellular Ca 2+ (Δ[Ca 2+ ] i ) and activation of ATP‐permeable pannexin‐1 (Panx‐1) channels. The aim of this study was to elucidate the role of protein kinases in the 5‐HT–[Ca 2+ ] i –Panx‐1 signalling pathway. What is the main finding and its importance? Src family kinase and protein kinase A, acting downstream from Δ[Ca 2+ ] i , played central roles in 5‐HT‐mediated Panx‐1 channel activation. This provides new insight into mechanisms regulating CB excitation, especially in pathophysiological conditions. Abstract Chemoreceptor (type I) cells of the rodent carotid body (CB) synthesize and release several neurotransmitters/neuromodulators, including 5‐hydroxytryptamine (5‐HT), implicated in enhanced CB excitation after exposure to chronic intermittent hypoxia, e.g. sleep apnoea. However, recent studies suggest that 5‐HT can robustly stimulate adjacent glial‐like type II cells via ketanserin‐sensitive 5‐HT 2 receptors, leading to intracellular Ca 2+ elevation (Δ[Ca 2+ ] i ) and activation of ATP‐permeable pannexin‐1 (Panx‐1) channels. Using dissociated rat CB cultures, we investigated the role of protein kinases in the intracellular signalling pathways in type II cells. In isolated type II cells, 5‐HT activated a Panx‐1‐like inward current ( I 5‐HT ) that was reversibly inhibited by the Src family kinase inhibitor PP2 (1 μ m ), but not by its inactive analogue, PP3 (1 μ m ). Moreover, I 5‐HT was reversibly inhibited (>90%) by H89 (1 μ m ), a protein kinase A blocker, whereas the protein kinase C blocker GF109203X (2 μ m ) was largely ineffective. In contrast, the P2Y2R agonist UTP (100 μ m ) activated Panx‐1‐like currents that were reversibly inhibited (∼60%) by either H89 or GF109203X. Using fura‐2 spectrofluorimetry, the 5‐HT‐induced Δ[Ca 2+ ] i was unaffected by PP2, H89 and GF109293X, suggesting that the kinases acted downstream of the Ca 2+ rise. Given that intracellular Ca 2+ chelation was previously shown to block receptor‐mediated Panx‐1 current activation in type II cells, these data suggest that CB neuromodulators use overlapping, but not necessarily identical, signalling pathways to activate Panx‐1 channels and release ATP, a CB excitatory neurotransmitter. In conclusion, these studies provide new mechanistic insight into 5‐HT signalling in the CB that has pathophysiological relevance.

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