Generation, breakdown and responses of ATP in the carotid body.

Purinergic signaling involving adenosine triphosphate (ATP) acting on P2X2/3 receptors modulates physiological carotid body (CB) afferent discharge and chemoreflex activation however, the upregulation of P2X3 receptors on petrosal sensory neurons is partly responsible for aberrant CB tonicity and hyperreflexia, and the increase in blood pressure in the Spontaneously Hypertensive rat (SHR). The enhanced chemoreflex occurs prior to the onset of hypertension and elevated CB activity is thought to contribute to the underlying pathophysiology of the disease but the exact mechanisms remain unclear. We hypothesize that there is greater release of ATP and/or decreased ATP breakdown in the pre-hypertensive SHR CB compared with that in the Wistar rat, and this evokes an increase in the chemoreflex response and subsequent activation of the sympathetic nervous system. We performed three studies. (i) Using digital droplet PCR (ddPCR), we measured mRNA expression levels of enzymes involved in the breakdown of ATP in CBs extracted from male 4-week-old prehypertensive SHR and Wistar rats; these included: ectonucleotide pyrophosphatase/phosphodiesterase 1-3 (Enpp1-3), ectonucleoside triphosphate diphosphohydrolase 2-3 (Entpd2-3), and ecto-5'-nucleotidase (Nt5e). (ii) We quantified ATP release from CBs in vitro during baseline conditions and during cyanide-evoked ATP release using a colorimetric assay (213-579-1 - Millipore), comparing between male 4-week-old pre-hypertensive SHR and Wistar rats. (iii) In an in situ working heart-brainstem preparation of 4-week-old Wistar rats, simultaneous recordings were made from phrenic (PN), hypoglossal (HN), recurrent laryngeal (RLN), abdominal (ABN), and thoracic sympathetic nerves (tSNA). The peripheral chemoreceptors were stimulated with either potassium cyanide (KCN 20-100 μL, 0.04%) or ATP at varying concentrations (10-100 µL, 50 µM - 2 mM) delivered via the internal carotid artery. In whole CB extracts, ddPCR revealed increased Enpp2-3 and Nt5e expression (p<0.05) in pre-hypertensive SHRs relative to Wistar rats suggesting greater capacity to break down ATP in the pre-hypertensive SHR CB. There were no differences in the expressions of Enpp1 or Entpd2-3 between rat strains. However, the amount of ATP released from the CB in both baseline and during cyanide stimulation was two-fold greater in the pre-hypertensive SHR (P<0.05). Stimulation of the CB with KCN evoked hyperpnoea, bradycardia and sympathoexcitation at all doses. In contrast, CB injection of low dose ATP increased PN rate with little effect on tSNA, whereas higher doses induced apnea accompanied by repetitive burst discharge of post-inspiratory activity in the RLN with tonic elevations in the discharge of both ABN and tSNA. In sum, despite the upregulation of several ATP degrading enzymes in the CBs of the SHR, CB release of ATP is higher in this species. We hypothesize that the increase in ATP release in the SHR CB drives the CB hyperexcitability and that the shift towards an upregulation of ATP degrading enzymes even prior to the development of hypertension is compensatory to oppose the increase in ATP signaling. The dose-dependent effect of exogenous ATP at supraphysiological levels evoking greater CB-mediated expiratory and sympathetic outflow in the Wistar rat mimics aspects of pathophysiological CB chemoreflex signaling in the SHR.