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

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 (Delta[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 (I5-HT) that was reversibly inhibited by the Src family kinase inhibitor PP2 (1 mu m), but not by its inactive analogue, PP3 (1 mu m). Moreover, I5-HT was reversibly inhibited (>90%) by H89 (1 mu m), a protein kinase A blocker, whereas the protein kinase C blocker GF109203X (2 mu m) was largely ineffective. In contrast, the P2Y2R agonist UTP (100 mu m) activated Panx-1-like currents that were reversibly inhibited (similar to 60%) by either H89 or GF109203X. Using fura-2 spectrofluorimetry, the 5-HT-induced Delta[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.