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 O₂ and CO₂ /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 Ca²⁺ (Δ[Ca²⁺ ]_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-[Ca²⁺ ]_i -Panx-1 signalling pathway. What is the main finding and its importance? Src family kinase and protein kinase A, acting downstream from Δ[Ca²⁺ ]_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-HT₂ receptors, leading to intracellular Ca²⁺ elevation (Δ[Ca²⁺ ]_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 μm), but not by its inactive analogue, PP3 (1 μm). Moreover, I_5-HT was reversibly inhibited (>90%) by H89 (1 μm), a protein kinase A blocker, whereas the protein kinase C blocker GF109203X (2 μm) was largely ineffective. In contrast, the P2Y2R agonist UTP (100 μm) activated Panx-1-like currents that were reversibly inhibited (∼60%) by either H89 or GF109203X. Using fura-2 spectrofluorimetry, the 5-HT-induced Δ[Ca²⁺ ]_i was unaffected by PP2, H89 and GF109293X, suggesting that the kinases acted downstream of the Ca²⁺ rise. Given that intracellular Ca²⁺ 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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