Vitamin B6 (pyridoxal 5’ phosphate) antagonizes carotid body P2X3 receptors in hypertension
ABSTRACT Background: ATP acting on P2X3 receptors (P2X3R) within carotid bodies (CBs) underpins chemoreflex-mediated sympathetic overactivity in Spontaneously Hypertensive rats (SHR). Given that pyridoxal 5’phosphate (PLP), the active form of vitamin B6 (pyridoxine hydrochloride; PHC), is a non-selective P2X receptor blocker, we hypothesized that PLP antagonism in the CB of SHR would lower blood pressure in hypertension. Methods: Herein, we employed in silico, in vitro, in situ and in vivo models to investigate the capability of PLP to bind to P2X3R to attenuate CB hyperexcitability in SHR. We performed a double-blind randomized clinical trial to investigate the capability of PLP to attenuate CB hyperexcitability in hypertensive patients. Results: First, PLP inhibited Ca2+ responses evoked by α,β-methylene ATP in cells lines expressing human (h) P2X2/3R with IC50 of 8.7µM (N=5). It also significantly increased the EC50 of α,β-methylene ATP from 433 nM to 1706 nM (P<0.01) and reduced its maximal evoked response by 63% (P<0.01), suggesting an allosteric mode of antagonism. Next, in silico, we demonstrated a similar predicted binding for PLP to that observed for MK-7264 (a P2X3R blocker, Gefapixant) in the crystal structure of its complex with hP2X3R, supporting an allosteric antagonistic effect. On the other hand, molecular dynamic simulations suggest PLP binds weakly to the same site of MK-7264. Using an in vitro isolated perfused carotid artery bifurcation-CB preparation, arterial infusion of PLP (50 µM ;15 min) attenuated CBs sensory firing in SHR (P=0.022). Using our in situ working-heart brainstem preparation, close arterial injections of PLP (1-5 mM) attenuated chemoreflex-evoked sympathetic (P=0.023) but not phrenic (P=0.62) responses; the CB was stimulated with potassium cyanide (KCN, 50 µL; 0.04%). In awake telemetered adult SHR (n=5), intravenous infusion of PLP (48 mg/Kg/h; 30 min) attenuated KCN-evoked chemoreflex responses and prompted falls in systolic, diastolic, and mean blood pressures (ΔMBP = -16 mmHg; P=0.005). In recruited volunteers with hypertension (n=14), acute oral supplementation with PHC (600 mg) attenuated CB activity (measured via hypoxia ventilatory response) only in the patients with high peripheral chemoreflex sensitivity but had no effect on MBP or the hypoxic pressor response. Conclusion: Our findings suggest that PLP binds to and antagonizes P2X3R and is a viable candidate for larger clinical trials to treat CB dysregulation in cardiovascular disease.