Identifying ATP evoked responses from the carotid body in spontaneously hypertensive rats
In spontaneously hypertensive rats (SHR), the carotid body (CB) is enlarged. Functionally, ATP acting on P2X3 receptors expressed in petrosal chemoreceptive afferent neurones underpins chemoreflex sympathetic overactivation (i.e., hyperreflexia). As the exact mechanisms remain elusive, the following experiments were performed to investigate the generation, breakdown and physiological responses of adenosine 5’-triphosphate (ATP) in the CB of SHRs: First, we quantified the amount of ATP released from CBs of Wistar and SHRs (3-5 weeks old) in vitro; second, we carried out quantitative analysis of gene expression of six enzymes, which are all involved with the extracellular metabolism of ATP Ectonucleotide pyrophosphatase/phosphodiesterase, Enpp1-3; Ectonucleoside triphosphate diphosphohydrolase, Entpd2-3; and ecto-5′-nucleotidase, Nt5e). Second, we quantified the gene expression for tyrosine hydroxylase (Th), Pannexin 1 channel (Panx 1), and the gene encoding, hypoxia-inducible factor-2α (HIF-2α; Epas1). Third, using live cell confocal imaging of ATP depletion, we quantified the amount of ATP released from dissociated glomus cells in both strains. Lastly, we used the working heart-brainstem preparation for functional analysis of CB-evoked responses in both strains. We found, that the CBs of SHRs release a greater amount of ATP relative to normotensive rats (P<0.05). This was associated with the upregulation of enzyme transcripts, Enpp2-3, Nt5e, and Th (p<0.05). Live cell confocal imaging did not show any difference in ATP depletion between both strains, thus indicating that the latter is mediated by a greater number of glomus cells in the hyperplasic CB. The latter was accompanied by upregulation of Epas1, which is known to mediate glomus cell lineage expansion in models of chronic sustained hypoxia. focal application to the CB of either ATP or α, β-methylene ATP produced hyperpnoea and sympathetic activation. In SHRs, α, β-methylene ATP microperfusion produced an enhanced sympathoexcitation relative to Wistars rats. In addition, P2X3 receptor antagonism produced a greater reduction in CB sensory firing evoked by cyanide in SHR. Thus, P2X3 receptor antagonism remains an attractive option for lower sympathetic activity in CB-mediated cardiovascular disease.