Purinergic receptors in the carotid body as a new drug target for controlling hypertension

Journal name:
Nature Medicine
Volume:
22,
Pages:
1151–1159
Year published:
DOI:
doi:10.1038/nm.4173
Received
Accepted
Published online

Abstract

In view of the high proportion of individuals with resistance to antihypertensive medication and/or poor compliance or tolerance of this medication, new drugs to treat hypertension are urgently needed. Here we show that peripheral chemoreceptors generate aberrant signaling that contributes to high blood pressure in hypertension. We discovered that purinergic receptor P2X3 (P2rx3, also known as P2x3) mRNA expression is upregulated substantially in chemoreceptive petrosal sensory neurons in rats with hypertension. These neurons generate both tonic drive and hyperreflexia in hypertensive (but not normotensive) rats, and both phenomena are normalized by the blockade of P2X3 receptors. Antagonism of P2X3 receptors also reduces arterial pressure and basal sympathetic activity and normalizes carotid body hyperreflexia in conscious rats with hypertension; no effect was observed in rats without hypertension. We verified P2X3 receptor expression in human carotid bodies and observed hyperactivity of carotid bodies in individuals with hypertension. These data support the identification of the P2X3 receptor as a potential new target for the control of human hypertension.

At a glance

Figures

  1. Overactive peripheral chemoreceptors in spontaneously hypertensive (SH) rats.
    Figure 1: Overactive peripheral chemoreceptors in spontaneously hypertensive (SH) rats.

    (a) Original recordings of raw and integrated carotid sinus nerve (CSN and ∫CSN, respectively) activity from the in situ perfused preparation, showing both basal discharge and discharge evoked reflexively through stimulation of the carotid bodies with sodium cyanide (22.5 μg NaCN i.a., arrows) in SH rats (SHR) and Wistar rats. (b,c) Quantitation of the results from the experiment in a, showing spikes/s (b) and the change in the number of spikes/s after NaCN stimulation (c) (one-way ANOVA Bonferroni post test; n = 10 or 11; ***P < 0.001). (d) Respiration rate in conscious radiotelemetered Wistar and SH rats with or without selective carotid body resection (CBR) performed in separate groups of SH rats. Dopamine (10 μg/kg/min i.v.) was infused as indicated to silence endogenous carotid body activity. One-way ANOVA Dunnett's post test. n = 5 per group; *P < 0.05. Data for each rat group are compared to the group's own baseline. All data are means ± s.e.m.

  2. P2X3-receptor-mediated hyperreflexia and tonicity of chemoreceptive petrosal neurons in spontaneously hypertensive (SH) rats are associated with the upregulation of P2x3 receptor mRNA.
    Figure 2: P2X3-receptor-mediated hyperreflexia and tonicity of chemoreceptive petrosal neurons in spontaneously hypertensive (SH) rats are associated with the upregulation of P2x3 receptor mRNA.

    (a) Representative whole-cell patch clamp recordings from chemoreceptive petrosal neurons from a Wistar (left) and SH rat (SHR, right) recorded from the preparation in situ. Ongoing discharge, membrane potential and reflex-evoked responses to carotid body stimulation (NaCN, sodium cyanide, 22.5 μg i.a., arrows) were compared between rat strains. (b) Recordings of neurons as in a after P2X3-receptor blockade with AF-353 (20 μM, 20 nl), which was delivered focally into the ipsilateral carotid body by picoinjection. Also shown is a recording from the same neuron from an SH rat after AF-353 washout. (c,d) Grouped mean data quantifying membrane potential (c; see also Supplementary Fig. 1a,b) and chemoreflex-evoked firing responses (d) with and without P2X3-receptor antagonism in Wistar and SH rats. Data in (c) are means ± s.d., and those in (d) are means ± s.e.m. One-way ANOVA Bonferroni post-test (n = 12 SH, n = 10 Wistar rats). (e,f) Single-cell RT–qPCR for P2x3 and P2x2 was performed in petrosal chemoreceptive neurons in the in situ arterially perfused preparation. Given that the petrosal ganglion contains neurons with different sensory modalities, RT–qPCR was performed only on those cells defined as chemoreceptive, as assessed by their activation with a chemoreceptor stimulus (NaCN, 22.5 μg). Shown are a recording from one such chemoreceptive neuron (e) and quantitation of P2x3 and P2x2 mRNA (n = 5 or 6 neurons). Two-way ANOVA Bonferroni post-test. Data in f are means ± s.e.m. ***P < 0.001.

  3. Upregulation of P2X3-receptor protein in the carotid body of SH rats and sensitization of chemoreceptive petrosal neurons to ATP in SH rats.
    Figure 3: Upregulation of P2X3-receptor protein in the carotid body of SH rats and sensitization of chemoreceptive petrosal neurons to ATP in SH rats.

    (a) P2X3-receptor protein levels in the carotid body of Wistar and SH rats (Mann–Whitney t-test; n = 4 or 5). The relative expression of protein refers to the difference between rat strains. One pair of carotid bodies (left and right) from each rat was loaded per lane. (b) P2X3-receptor immunofluorescence (green) labeling on glomus cells identified by staining for tyrosine hydroxylase (red) (TH; n = 3 Wistar, n = 3 SH rats). The immunofluorescence images show a low (top left) and three high-power photomicrographs of carotid body glomus cells expressing TH and P2X3 receptors in a SH rat. See Supplementary Figure 10 for control data on the specificity of the P2X3-receptor antibody. Scale bars, 100 μm (top left) and 25 μm (all others). (c) Whole-cell recording (current clamp) from petrosal chemoreceptive neurons in Wistar (n = 10) and SH rats (n = 13). ATP was micro-infused into the ipsilateral carotid body, as indicated. (d) The levels of membrane depolarization from petrosal chemoreceptive neurons evoked by increasing the concentrations of ATP applied to the carotid body in Wistar and SH rats. AUC, area under the curve. (e) Voltage-clamp recordings showing the inward current from identified petrosal chemoreceptive neurons evoked by the application of ATP to the ipsilateral carotid body in Wistar (top traces; n = 12) and SH rats (bottom traces; n = 12). The P2X3-receptor antagonist (AF-353, 20 μM) or the nonselective P2X-receptor antagonist suramin (100 μM) were applied to the ipsilateral carotid body before ATP, as indicated. (f) Quantification of the AF-353-sensitive, ATP-evoked inward current in Wistar and SH rats. Neurophysiological data were from the in situ arterially perfused preparation, and comparisons were made using an unpaired t-test. All data are means ± s.e.m. *P < 0.05; ***P < 0.001.

  4. P2X3-receptor antagonism lowers arterial pressure in conscious SH rats.
    Figure 4: P2X3-receptor antagonism lowers arterial pressure in conscious SH rats.

    (a) Changes in SBP (solid lines) and the AF-219 plasma concentration predicted on the basis of plasma sampling (dotted lines; see also Supplementary Fig. 5) after intravenous infusion of the indicated doses of AF-219 (1, 4 and 8 mg/kg/h; n = 7 SH rats per drug dose). PK, pharmacokinetic. (b) Changes in SBP induced with 8 mg/kg/h AF-219 i.v. in SH rats without (n = 7) and with carotid body resection (SHR & CBR; n = 7) and in intact Wistar rats (n = 4). For CBR in SHR rats, measurements were performed before and after CBR in the same rat in four cases and in separate rats in three cases. (c) Peripheral chemoreflex-evoked pressor responses in SBP in conscious radiotelemetered SH rats during the infusion of vehicle or the indicated doses of AF-219 (0.5–8.0 mg/kg/h). n = 7 per group. Data are means ± s.e.m. One-way ANOVA Dunnett's post-test. *P < 0.05, **P < 0.01, ***P < 0.001.

  5. The antihypertensive action of P2X3-receptor antagonism is associated with a reduction in sympathetic activity in SH rats in situ and in vivo.
    Figure 5: The antihypertensive action of P2X3-receptor antagonism is associated with a reduction in sympathetic activity in SH rats in situ and in vivo.

    (a,b) Ongoing lumbar chain sympathetic activity (arrow, raw: lSN; integrated: ∫lSN) and the sympathetic nerve reflex response (arrowhead) to peripheral chemoreceptor stimulation (evoked by NaCN, 22.5 μg i.a.) without (control) and with bilateral local application of AF-353 to the carotid bodies (20 μM, 15 nl) in Wistar (a) and SH rats (b) (n = 12 per group). Activity from the phrenic nerve (PN) (neural inspiration) was recorded, and its integrated form (∫PN) is shown. Also shown is a sympathetic nerve recording from the same preparation of SH rat after AF-353 washout. (ce) Effects of AF-353 on basal respiratory modulated ISN (c), and chemoreflex-evoked changes in both inspiratory- (d) and expiratory- (e) modulated lSN in Wistar and SH rats (n = 12 per group). Inspiratory and expiratory modulation of lSN was determined by using averaged triggering from the integrated phrenic nerve (∫PN). One-way ANOVA Bonferroni post-test; data are means ± s.e.m. from in situ rat preparations. (f) Changes in renal sympathetic nerve activity (RSNA) recorded in vivo from conscious radiotelemetered SH rats (n = 5) during the infusion of AF-219 (8 mg/kg/h i.v.; vertical arrows indicate samples of raw RSNA and horizontal arrow indicates timing and duration of drug infusion). (g) A representative recording of the peripheral chemoreflex-evoked response (NaCN 22.5 μg i.a.) in the RSNA in a conscious radiotelemetered SH rat before (control) and after AF-219 infusion in the same animal. (h) Quantitation of mean data from g. In h, washout refers to the chemoreflex-evoked RSNA in SH rats the day after they had been infused with AF-219. Repeated measures one-way ANOVA, with Holm–Sidek post hoc comparison. *P < 0.05, ***P < 0.001.

  6. P2X3-receptor expression in carotid bodies from cadavers of individuals with a medical history of hypertension, and aberrant tone generation in carotid bodies from individuals with hypertension.
    Figure 6: P2X3-receptor expression in carotid bodies from cadavers of individuals with a medical history of hypertension, and aberrant tone generation in carotid bodies from individuals with hypertension.

    (a) Left, a section of a carotid body from a human cadaver showing P2X3-receptor immunofluorescence (green) and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI; blue; scale bar, 100 μm). Right, high-power confocal images showing co-localization of P2X3 receptor (green) and tyrosine hydroxylase (TH, red) (scale bar, 25 μm). Images are representative of those for five human carotid bodies. See Supplementary Figure 10 for P2X3-receptor antibody control experiments and Supplementary Figure 11 for axonal labeling. (b) Western blot of the P2X3-receptor protein in the human carotid body (n = 4). Arrow indicates bands at the molecular weight of the human P2X3 receptor (44 kDa). (c) Changes in minute ventilation during low-dose dopamine infusion (2 μg/kg/min i.v.) in six awake individuals with hypertension in the supine position (each solid line corresponds to one individual). A dextrose vehicle infusion was used as a control and reference at time zero. The mean (± s.e.m.) response is indicated by the black dotted line. Peak response was the lowest ventilatory response reached during the dopamine infusion. Note the appearance of a rebound hyperventilatory response after dopamine infusion was terminated. One-way ANOVA Bonferroni post-test. *P < 0.05 vehicle versus peak depression; **P < 0.01, depression versus rebound (mean of 5 min); P < 0.05, vehicle versus rebound.

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Author information

Affiliations

  1. School of Physiology, Pharmacology & Neuroscience, Biomedical Sciences, University of Bristol, Bristol, UK.

    • Wioletta Pijacka,
    • Emma C Hart,
    • Fiona D McBryde,
    • Ana P Abdala &
    • Julian F R Paton
  2. Department of Physiology, School of Medicine of Ribeirao Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil.

    • Davi J A Moraes,
    • Melina P da Silva &
    • Benedito H Machado
  3. CardioNomics Research Group, Clinical Research and Imaging Centre, University of Bristol, Bristol, UK.

    • Laura E K Ratcliffe
  4. CardioNomics Group, Department of Cardiology, Bristol Heart Institute, University Hospitals Bristol National Health Service Foundation Trust, Bristol, UK.

    • Angus K Nightingale
  5. Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.

    • Fiona D McBryde
  6. Afferent Pharmaceuticals, San Mateo, California, USA.

    • Anthony P Ford

Contributions

W.P. conducted all in vivo radiotelemetry blood pressure studies and the rat and human immunocytochemistry and western blotting; this also included data analysis and figure and manuscript preparation. D.J.A.M. performed all in situ rat nerve and petrosal neuron whole-cell recording studies; this also included data analysis and figure preparation. M.P.d.S. performed the single-neuron PCR study. L.E.K.R. with A.K.N. carried out the diagnosis and recruitment of humans with hypertension, and L.E.K.R. with E.C.H. performed and analyzed data from the dopamine-infusion study. B.H.M. supported all in situ studies and assisted in experimental design, data analysis and manuscript preparation. F.D.M. conducted the in vivo radiotelemetry study for recording renal sympathetic nerve activity; this also included data analysis and figure preparation. A.P.A. performed some of the first immunohistochemistry on human carotid bodies. A.P.F. provided the P2X3-receptor antagonists, carried out the pharmacokinetic analysis and assisted in drug trial design and manuscript preparation and revision. J.F.R.P. orchestrated the design of the project, provided supervision with data acquisition and analysis, wrote the manuscript and revised it, and raised the funding.

Competing financial interests

A.P.F. is Chief Scientific Officer for Afferent Pharmaceuticals. The other authors declare no competing financial interests.

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