Purinergic Receptors in the Carotid Body As a New Drug Target For

© 2016 Nature America, Inc. All rights reserved. California, California, USA. should Correspondence be addressed to J.F.R.P. ( 5 Bristol, UK. Ribeirao Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil. 1 anastomosis baroreceptors carotid denervation renal including studies, interventional device-based of series a to led has agents antihypertensive cological its question into relationship benefit–harm call long-term effects adverse associated and questionable, is blockers receptor and inhibitors enzyme angiotensin–aldosterone for hypertension in 13 years, yet its beyond efficacy the existing renin- renin inhibitor released in 2007. This was the first new drug to emerge pressure. blood control to ways for drug resistance with people worldwide million hypertension 900 estimated an of 8% least at Furthermore, disease cardiovascular from death of risk the doubles mmHg) (80 pressure (DBP) above blood levels in normal 10-mmHg rise diastolic mmHg <140/90 to reduced be could pressure blood individual’s an if result would costs in savings healthcare Substantial of medications. current effects side tolerated poorly the and hypertension of nature tomatic asymp the to owing adherence, drug with problem serious a reflect controlled pressure is blood medication such taking those of million) (~41 53% only in tion, medica antihypertensive use that 75% of the fact despite individuals USA, the In hypertension. has population human the of One-third the control of human hypertension. bodies in individuals with These hypertension. data support the of identification the P2X3 receptor as a potential new target for in rats without Wehypertension. verified P2X3 receptor expression in human carotid bodies and observed of hyperactivity carotid basal sympathetic activity and normalizes carotid body in hyperreflexia conscious rats with no hypertension; effect was observed phenomena are normalized by the blockade of P2X3 receptors. Antagonism of P2X3 receptors also reduces arterial pressure and These hypertension. neurons generate both tonic drive and in hyperreflexia hypertensive (but not rats, normotensive) and both also known as aberrant signaling that contributes to high blood pressure in Wehypertension. discovered that purinergic receptor P2X3 ( of this medication, new drugs to treat hypertension are urgently needed. Here we show that peripheral generate chemoreceptors In view of the high proportion of individuals with resistance to medication and/or antihypertensive poor compliance or tolerance H Benedito Machado Wioletta Pijacka target for controlling hypertension Purinergic receptors in the carotid body as a new drug nature medicine nature Received 2 May; accepted 27 July; published online 5 September 2016; Department Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand. School of Physiology, Pharmacology & Biomedical Neuroscience, Sciences, University of Bristol, Bristol, UK. The most recently introduced antihypertensive drug is aliskiren, a is aliskiren, drug antihypertensive introduced recently most The 2 . In adults over 40 years of age, there is clear evidence that a that evidence clear is there age, of years 40 over adults In . 4 CardioNomics Group, Department of Cardiology, Bristol Heart Institute, University Hospitals Bristol National Health Service Foundation Trust, Bristol, UK. 11 , 1 5 2 P2x3 , 6 . . Here we introduce a new mechanistic approach that . . Categorically, there remains an unmet clinical need

1 advance online publication online advance , Davi J A Moraes ) ) mRNA expression is upregulated in substantially petrosal chemoreceptive sensory neurons in rats with 9 , deep brain stimulation brain deep , 4 have developed and currently experience experience currently and developed have 1 2 . This low rate of control might, in part, part, in might, control of rate low This . , , Fiona D McBryde 7 . The paucity of new pharma new of paucity The . 2 , Laura E K Ratcliffe 1 0 and arterial venous venous arterial and 1 8 ,

5 , stimulation of of stimulation , , , Ana P Abdala [email protected] doi:10.1038/nm.417 3 CardioNomics CardioNomics Research Group, Clinical Research and Imaging Centre, University of Bristol, 3 3 - - - . , Angus K Nightingale approaches, as we propose here. propose we as approaches, antihypertensive the pharmacological new refining for pivotal be Although could it unknown, hypertension. is tone aberrant body to carotid the for basis mechanistic contributing as carotid discharge aberrant of body presence the support data human and animal substantially pressure arterial reduce can resection activity sympathetic lowers hyperoxia tension, strain rat are normotensive but a arterial rats, in SH in brain, unaffected reduced the are from activity body sympathetic and carotid pressure the disconnect to nerve or section hyperoxia through off switched is tone body carotid this pressure blood activity high causes sympathetic up and drives that tone excitatory aberrant erate gen chemoreceptors body carotid the hypertension, of conditions the in pressure rat blood SH high both of control to maintenance way and effective development an the evidently is body carotid the of hypertensive hypertension with humans in rat—and spontaneously (SH) hypertension—the of model pre a both clinical in potentiated is response chemoreflex- body sympathoexcitatory carotid evoked The disease. cardiovascular for target hypertension. for trial clinical future a in use its justify to provide data clinical we and and preclinical antagonist, small-molecule selective highly a uses 1 , , Anthony P Ford Currently, the carotid body is under consideration as a therapeutic as Currently, a is consideration therapeutic body the under carotid 19 3 , 2 ). 0 . On the basis of these findings, we concluded that in in that concluded we findings, these of basis the On . 2 Department Department of Physiology, School of Medicine of 6 4 & Julian F R Paton , Emma C Hart 6 Afferent Pharmaceuticals, Afferent San Pharmaceuticals, Mateo, 14 , 2 1 . Indeed, we found that when when that found we Indeed, . 1 4 1 . In humans with hyper with humans In . , Melina P da Silva 13– 1 1 1 8 8 , and carotid body body carotid and , s e l c i t r a , and denervation denervation and , 2 2 . Thus, these these Thus, . P2rx3 , 2 , - - -

© 2016 Nature America, Inc. All rights reserved. Neither the membrane input resistance of these chemoreceptive chemoreceptive these of resistance input membrane the Neither ( rats Wistar in spikes 25 evoked pulse current nA 1 a example, for pulses; current injected to excitability enhanced ited recorded from SH rats, as compared to from those Wistar chemoreceptive rats, exhib of neurons the that We found rats. Wistar excitability and SH in neurons petrosal electrical the compared next We Chemoreceptive petrosal neuron excitability in SH rats hyperreflexia. and tone the carotid of body an SH rat is generating both aberrant hyperactive, rat, normotensive the to compared when In sum, rats. normotensive 123 (22.5 NaCN of dose equivalent an to response firing flex-evoked (1.5 P (−51.5 depolarized more were rats, sive hyperten from recorded neurons, Such neurons. ganglion afferent petrosal primary chemoreceptive as them characterizing stimulus, the NaCN to the in as responding characterized were neurons preparation, recordings animals in intracellular tonicity During body hypertension. carotid with of presence the suggests which in Wistar rats not ( an observed effect respiration, depressed dopamine infusion (10 of to discharge dopamine body carotid inhibit respectively; spikes/s, respectively, spikes/s, 24.9 basal, rats: Wistar versus (SH hypertension were elevated in rats with hypertension, as compared to those without (15–30 volleys chemoreflex-evoked hours of course a over repeatable is that treatment stimulus this controlled temporally and because quantifiable specific, a provides response chemoreflex peripheral stimu the to used late was hypoxia, simulating (NaCN), cyanide Sodium bypass). cardiopulmonary on rat decerebrated the unanesthetized of (an nerve sinus carotid the from afferent discharge body carotid recorded We pressure. con blood in high active of more ditions was body carotid the whether tested first We Hyperactivity of carotid bodies in hypertensive rats RESULTS hypertension. with humans to findings preclinical of these applicability the and have demonstrated in on antagonists rats hypertension P2X3-receptor of selective highly effects the we Totested have this, effect. address an antihypertensive and of that in receptor would this blockade cause body hypertension, and aberrant chemo-hyperreflexia discharge peripheral of the carotid types mice reduces the ventilatory response to hypoxia, as compared to wild rats normotensive of neurons ganglia petrosal and bodies carotid both body carotid the in ing signal hypoxic in involved transmitters of number a of one is ATP organs of variety a in states disease hyperreflexic to contribute subtypes, P2X3-receptor which are commonly with associated afferent and sensitization might C-fiber-localized, the specifically tors), triphosphate (ATP)-gated ion P2X purinergic channels recep (called aden of role the studied have we investigation, current our In A p

e < 0.001, A plethora of mechanisms governs carotid body signaling body carotid governs mechanisms of plethora A trosal trosal neurons (119 s e l c i t r 3 ± ± 6 3 . Moreover, combined P2X2- and P2X3-subunit deletion in in deletion P2X3-subunit and P2X2- combined Moreover, . 5 versus 52 52 versus 5 0.8 versus 0 Hz; 0 Hz; versus 0.8 7 . We hypothesized that the P2X3 receptor contributes to both to both contributes receptor P2X3 the that . We hypothesized Fig. Fig. 2a , c and ± 3 spikes; spikes; 3 P ± P Fig. 2 Fig. µ Supplementary Fig. 2a Fig. Supplementary < 0.001; chemoreflex, 62.3 62.3 < chemoreflex, 0.001; 3 versus 117 30– Supplementary Fig. 1 Supplementary < 0.001, < 0.001, g/kg/min g/kg/min intravenously (i.v.), as described 3 5 . P2X3-receptor expression is present in in present is expression P2X3-receptor . a ) and displayed an enhanced chemore an enhanced displayed ) and P ± < 0.001, 0.001, < 1.1 spikes in SH rats versus 15 15 versus rats SH in spikes 1.1 Fig. 1a Fig. ± µ 9 M g NaCN, intra-arterially (i.a.)) (i.a.)) intra-arterially NaCN, g – c ± . Fig. 2a Fig. Ω ). ). Next, we a used low dose Both basal discharge and and discharge basal Both 0.9 versus −55.7 −55.7 versus 0.9 ; ; Supplementary Supplementary Fig. 1c ), ), exhibited tonic firing 38 , , 3 b , 9 n situ in ± d , , . . In SH rats ± 2 versus 1.4 1.4 versus 2 ), as compared to to compared as ), n 3 28.3 versus = 6; 6; = preparation preparation P < 0.05). 0.05). < Fig. Fig. 1 ± in in vivo 1 mV; 1 n situ in o 23– ± ± 28 sine sine 0.3 0.3 1.7 1.7 µ ± , d 3 2 2 g, g, 9 2 7 9 ), ), ------) ) . . ,

stimulation ( stimulation ( spikes/s ( (22.5 cyanide sodium with bodies carotid the of stimulation through reflexively evoked situ in and (CSN nerve sinus carotid ( rats. (SH) hypertensive 1 Figure reduced in SH rat neurons (for example, at 1 nA, 25 25 nA, 1 at example, (for neurons rat SH in reduced was pulses current depolarizing injected to response firing intrinsic ( drug the of washout upon body SH in rats reversible was antagonism of P2X3 effect this (n.s.)); cant carotid signifi not spikes; 32 Wistar, versus 34 (SH, antagonism, strains rat the between neurons petrosal in responses firing P2X3-receptor comparable yielded ( activation rats during Wistar were in spiking than Notably, rats SH chemoreflex-evoked in in greater reduction the and zation P 52 (from volley chemoreflex-evoked (−4.4 animals sive (from 123 neurons chemoreceptive petrosal of sensitivity chemoreflex ( activity ongoing of abolition (−9.9 polarization 20 hyper nl, caused body carotid noncompetitive ipsilateral into the 15 inserted micropipette selective, (AF-353; highly antagonist a of P2X3-receptor delivery focal rats, SH In rats SH in dysfunction body carotid in involved is receptor organs other in numerous sitization of body carotid the in rats normotensive expressed is receptor P2X3 the that Given neuron excitability in SH rats P2X3-receptor antagonism normalizes chemoreceptive petrosal ( strains. rat the between different capacitance whole-cell their nor means are data All baseline. own group’s the to compared are group rat each for test. post Dunnett’s ANOVA One-way activity. body carotid endogenous silence to indicated as infused i.v.) was (10 Dopamine rats. SH of groups separate in performed (CBR) resection body carotid selective without or with rats SH and Wistar *** b d spikes/s a < 0.001; 0.001; < , ∫ CSN c CSN

Respiratory rate (bpm) 10 P ) Quantitation of the results from the experiment in in experiment the from results the of ) Quantitation –20 –10 ± < 0.001). ( < 0.001). 10 20 0 5 to 34 34 to 5 perfused preparation, showing both basal discharge and discharge discharge and discharge basal both showing preparation, perfused ± –6 ±

s.e.m. 1.8 mV; mV; 1.8 b Overactive peripheral chemoreceptors in spontaneously spontaneously in chemoreceptors peripheral Overactive Baseline –4 Wistar ) and the change in the number of spikes/s after NaCN NaCN after spikes/s of number the in change the ) and c NaCN i. 2b Fig. –2 ) (one-way ANOVA Bonferroni post test; test; post Bonferroni ANOVA ) (one-way in situ in µ ± g NaCN i.a., arrows) in SH rats (SHR) and Wistar rats. rats. Wistar and (SHR) rats SH in arrows) i.a., g NaCN 2 spikes; spikes; 2 d 8 6 4 2 0 advance online publication online advance ) Respiration rate in conscious radiotelemetered radiotelemetered conscious in rate ) Respiration Dopamine 3 Time (min) n 6 ± , AF-353 delivery also caused hyperpolarization hyperpolarization caused also delivery AF-353 , , and is associated with pathological afferent sen afferent pathological with and is associated d = 10, 10, = 2.1 mV; 2.1 . h mgiue o bt te hyperpolari the both of magnitudes The ). a ) Original recordings of raw and integrated integrated and raw of recordings ) Original * n = 12, 12, = * SHR ∫ NaCN CSN, respectively) activity from the the from activity respectively) CSN, P 10 Fig. 2 Fig. * < 0.001; 0.001; < n Fig. 2 Fig. Recovery = 12, 12, = 12 P < 0.001, 0.001, < n 14 2 b = 5 per group; * group; = 5 per 28 s ). After AF-353 treatment, the the treatment, AF-353 After ). b upeetr Fg 1d Fig. Supplementary 16 P , ), as well as a reduction in the the in reduction a as well as ), 29 ± < 0.001; 0.001; < , 3 to 32 32 to 3 3 i. 2 Fig. SHR &CBR SHR Wistar Increased Increased sensitivity 3 tonicity , we assessed whether this this whether , we assessed Fig. 2b Fig. reflex P < 0.001; 0.001; < c

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© 2016 Nature America, Inc. All rights reserved. body body produced dose-dependent increases in the firing and membrane application (15 nl) of ATP or The rat strains. the between to ATP neurons differed of these sitivity chemoreceptive petrosal neurons of SH rats, we tested whether the of sen upregulation the of basis the On SH rat chemoreceptive petrosal cells are sensitized to ATP ( body ( cells glomus positive 2f Fig. ( solitarii tractus nucleus the in located neurons tive chemoreceptive neurons ( petrosal either in mRNA expression cells) glomus of marker (a ylase the rat strains in their expression of between differences no were there Moreover, neurons. chemoreflex ( strains in nonchemoreceptive neuronspetrosal was between the observed rat ( blot western by confirmed was bodies carotid in level protein the at n of lation is from SH rats recorded neurons receptors ganglion petrosal P2X3 chemoreceptive of characterized of expression analysis PCR Single-cell neurons. the petrosal rat SH in upregulated whether tested next We P2X3 receptors are upregulated in petrosal neurons of SH rats (15 neurons spikes; in Data post-test. Two-way Bonferroni ANOVA neurons). 22.5 (NaCN, stimulus a chemoreceptor with activation their by assessed as chemoreceptive, as defined cells those on only performed was RT–qPCR modalities, sensory the in neurons chemoreceptive petrosal in means are ( responses firing chemoreflex-evoked ( washout. AF-353 after rat SH an from neuron same (20 AF-353 with blockade 22.5 cyanide, sodium (NaCN, stimulation preparation the from recorded right) (SHR, rat SH and (left) of upregulation the with 2 Figure nature medicine nature the in neurons Notably, petrosal neurons. petrosal of depolarization Fig. 3 Fig. = 6, 6, = Supplementary Fig. 3a Fig. Supplementary P a . 23 eetr wr osre i tyrosine-hydroxylase- in observed were receptors P2X3 ).

P ). Notably, no difference in in difference Notably,no ). < 0.001). A comparable upregulation of the P2X3 receptor receptor P2X3 the of upregulation comparable A 0.001). < Supplementary Fig. 2e Fig. Supplementary P2X3-receptor-mediated hyperreflexia and tonicity of chemoreceptive petrosal neurons in spontaneously hypertensive (SH) rats are associated associated are rats (SH) hypertensive spontaneously in neurons petrosal chemoreceptive of tonicity and hyperreflexia P2X3-receptor-mediated P2x3 < 0.05; 0.05; < b a ± –60 mV s.e.m. One-way ANOVA Bonferroni post-test ( post-test Bonferroni ANOVA One-way s.e.m. –55 mV Firing (Hz) freq ± in situ Firing 25 receptor mRNA relative to that in Wistar rats ( rats Wistar in that to mRNA relative receptor (Hz) freq 2 to 12 12 to 2 0 25 0 Supplementary Fig. 2a Fig. Supplementary

( 20 mV 5 Fig. Fig. 2 WISTAR advance online publication online advance µ s 205 mV NaCN g). Shown are a recording from one such chemoreceptive neuron ( neuron chemoreceptive such one from a recording are Shown g). Fig. 3 Fig. P2x3 ± s NaCN 2, n.s.). 2, WISTAR µ Supplementary Fig. 2c Supplementary e M, 20 nl), which was delivered focally into the ipsilateral carotid body by picoinjection. Also shown is a recording from the the from a recording is shown Also picoinjection. by body carotid ipsilateral the into focally delivered was which nl), 20 M, ) ) revealed a greater than four-fold upregu receptor mRNA. ( mRNA. receptor –61 mV α b ), as well as in axons within the carotid carotid the within axons in as well as ), - , β b ), which is indicative of specificity to of specificity indicative is which ), -methylene-ATP focally to the carotid ). NaCN –51 mV SHR d P2x2 ) with and without P2X3-receptor antagonism in Wistar and SH rats. Data in ( in Data rats. SH and Wistar in antagonism P2X3-receptor without and ) with P2x3 P2X3 –52 mV µ in situ in ( g i.a., arrows) were compared between rat strains. ( strains. rat between compared were arrows) g i.a., or or Fig. Fig. 2 receptor expression in the the in expression receptor , b a P2x2 ), but not in Wistar rat rat Wistar in not but ), ) Representative whole-cell patch clamp recordings from chemoreceptive petrosal neurons from a Wistar a Wistar from neurons petrosal chemoreceptive from recordings clamp patch whole-cell ) Representative arterially perfused preparation. Given that the petrosal ganglion contains neurons with different different with neurons contains ganglion petrosal the that Given preparation. perfused arterially NaCN SHR–washout NaCN f SHR ) ) or tyrosine hydrox c ) ) or in chemorecep mRNA expression expression mRNA , d f ) Grouped mean data quantifying membrane potential ( potential membrane quantifying data mean ) Grouped are means means are Supplementary Supplementary

n = 12 SH, SH, = 12 in situ in . Ongoing discharge, membrane potential and reflex-evoked responses to carotid body body carotid to responses reflex-evoked and potential membrane discharge, . Ongoing Fig. 2 Fig. ± d c s.e.m. *** s.e.m. n

= 10 Wistar rats). ( rats). Wistar = 10 Chemoreflex evoked spikes Membrane potential (mV) 100 150 –70 –40 –60 –50 50 f - - - - 0 ;

WISTAR WISTAR AF-219 (8 mg/kg/h) still caused a residual fall of −17 −17 of fall residual a caused still mg/kg/h) (8 AF-219 of in SH infusion a rats, Notably, 30-min resection body carotid after breaths/min, respectively; respectively; breaths/min, −38 (by decreased also frequency tory 3/−26 6 Fig. ( rats Wistar in observed was response no ( pressure arterial in drop dose-dependent a produced 219 151 was pressure arterial basal ( (SBP) pressure blood concentra in systolic falls with tions, drug plasma between correlations found subsequently concentra plasma assess ( rats SH to in AF-219 of tions analysis pharmacokinetic a formed humans in AF-353 AF-219 for because has used clinically been other indications for use preferred was compound i.v.This mg/kg/h, 8 and 4 1, at made were AF-219 called antagonist P2X3-receptor noncompetitive highly selective, a of infusions 1-h rats. SH in pressure blood arterial lowers receptors P2X3 body carotid of antagonism whether tested we Next, Systemic blockade of P2X3 receptors is antihypertensive ( Wistar rats in SH than in greater was neurons petrosal chemoreceptive ATPin by evoked current inward sensitive 4a Fig. ( respectively rats, Wistar versus SH in 2.4 and for 17.2 8.8 were values concentration) effective (half-maximal SH rat were more sensitive than from those Wistar rats; for ATP, EC P *** < 0.001. *** ± ). At a dose of 8 mg/kg/h, SBP/DBP in SH rats fell by −28 −28 by fell rats SH in SBP/DBP mg/kg/h, 8 of dose a At ). 1.3 in SH versus Wistar ( rats, respectively SHR SHR *** ± in vivo α , 4 mmHg ( mmHg 4 b *** - . osset ih hs fnig, h P2X3-receptor- the findings, these with Consistent ). β e 2 WISTAR WISTAR -methylene-ATP, EC AF-353 e 9 AF-353 ) and quantitation of of quantitation ) and *** , and does not cross the blood–brain barrier. We per We barrier. blood–brain the cross not does and f over the related, structurally more lipophilic antagonist *** ) Single-cell RT–qPCR for for RT–qPCR ) Single-cell b ) Recordings of neurons as in in as neurons of ) Recordings AF-353 AF-353 Fig. 4 Fig. SHR SHR a –55 mV ; ; f e Supplementary Fig. 6 Fig. Supplementary n

Fig. 4 Fig. Relative mRNA = 7, 7, = expression (arbitrsry units) c 0 1 2 3 4 5 6 ; see also also ; see P2x3 ±

50 20 mV 1s 3/109 3/109 P a P values were 9.4 values and and < 0.001). Heart rate and respira and rate Heart 0.001). < c P2 < 0.001 ) are means means ) are and and × NaCN P2x3 3 Supplementary Fig. 5 Fig. Supplementary ± Supplementary Fig. 1a Fig. Supplementary ± P2x2 3 (SBP/DBP) mmHg. AF- mmHg. (SBP/DBP) 3 8 and −12 −12 and 8 P and and Fig. 4 Fig. a < 0.05; 0.05; < after P2X3-receptor P2X3-receptor after mRNA ( mRNA SHR WISTAR P ± P2x2 P < 0.0001; < 0.0001; s.d., and those in ( in those and s.d., Fig. 4 Fig. P < 0.001; b s e l c i t r a ± 2 ; ; ( ; ; n × 2.1 versus 16.2 16.2 versus 2.1 n =5) 2 Supplementary Supplementary Supplementary Supplementary was performed performed was = 7, 7, = ( n n ± a = 5 or 6 = 5 or ± =6) 3 beats and and beats 3 ). ). In SH rats, 3 mmHg in in mmHg 3 ± 2.2 versus versus 2.2 Fig. Fig. 3c P Fig. 3e Fig. < 0.01). 0.01). < Fig. 4 Fig. , b ), and and ), ) and ) and , , d a f d 50 ); ); ). ), ), ± ± - - - - )

© 2016 Nature America, Inc. All rights reserved. by AF-219 (bolus doses given i.v. >1 mg/kg; i.v. mg/kg; >1 given doses (bolus AF-219 by receptors P2X3 of blockade systemic after reduced also was rats SH conscious in pressure arterial in increase chemoreflex-evoked The origin. body non-carotid of activity afferent P2X3-receptor-driven SBP ( A aberrant reduces antagonism P2X3-receptor that suggest data These unpaired unpaired an using made were comparisons and preparation, perfused arterially the from were data Neurophysiological rats. SH and Wistar in ( ATP, before body carotid indicated. as ipsilateral the to applied were 20 traces (bottom rats SH traces (top Wistar in body carotid ATPof ipsilateral the to application the by evoked neurons chemoreceptive petrosal identified ( curve. the under area AUC, rats. SH and Wistar in body carotid the to applied ATPof concentrations the increasing by evoked neurons chemoreceptive ( indicated. ( ( Wistar in neurons chemoreceptive petrosal 25 and left) 100 bars, Scale antibody. P2X3-receptor the of specificity the on See rat. a SH in receptors P2X3 and TH expressing cells glomus body carotid of photomicrographs power high- three and left) (top a low show images immunofluorescence The (TH; (red) hydroxylase tyrosine for staining by identified cells glomus on labeling (green) immunofluorescence receptor ( lane. per loaded was rat each from right) and (left bodies carotid of pair One strains. rat between difference the to refers protein of (Mann–Whitney rats SH and ( rats. SH ATPto in neurons petrosal chemoreceptive of sensitization and rats SH 3 Figure f n e –58 mV c a

) Quantification of the AF-353-sensitive, ATP-evoked inward current current inward ATP-evoked AF-353-sensitive, the of ) Quantification Firin = 13). ATP was micro-infused into the ipsilateral carotid body, as carotid ipsilateral the into ATP = 13). micro-infused was (Hz) freq s e l c i t r µ

25 Relative P2X3 protein M) or the nonselective P2X-receptor antagonist suramin (100 (100 suramin antagonist P2X-receptor nonselective the or M) 0 g Fig. 4 Fig. expression (arbitrary units)

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2 20 mV P2X3 s s Upregulation of P2X3-receptor protein in the carotid body of body carotid the in protein P2X3-receptor of Upregulation t a -test. All data are means means are data All -test. s ATP (10 ATP (10 ) P2X3-receptor protein levels in the carotid body of Wistar Wistar of body carotid the in levels protein ) P2X3-receptor d Wistar b µ ) The levels of membrane depolarization from petrosal petrosal from depolarization membrane of levels ) The ; ; m (all others). ( others). m (all SHR WISTAR µ n M) = 7, µ µ –52 mV ( M) M) n =5) P ( n ; ; n = < 0.05; < 0.05; =4) ATP (10 Suramin (100 AF-353 (20 ATP 12). The P2X3-receptor antagonist (AF-353, (AF-353, antagonist P2X3-receptor The 12). SH t * c -test; -test; Suramin (100 AF-353 (20 ATP µ R ) Whole-cell recording (current clamp) from from clamp) (current recording ) Whole-cell M) Supplementary Fig. 7 Fig. Supplementary Supplementary Figure 10 Figure Supplementary µ M) 66 kDa µ n ± M) = 4 or 5). The relative expression expression relative The 5). = 4 or s.e.m. * s.e.m. µ d M) µ b

M) AUC (mV.s) 100 150 50 n 0 = 10) and SH rats rats SH and = 10) P n < 0.05; *** < 0.05; = 3 Wistar, 0.5 n f * Concentration ofATP SHR WISTAR = 4, 4, = AF-353-sensitive

(pA × ms) 2.0 *** 1,000 1,200 ( n 200 400 600 800 P =13) ), which suggests suggests ), which 10.0 < 0.05; 0.05; < ; ; n = 0 ( *** n for control data data control for b =10) P ) ) P2X3- n < 0.001. 20.0 = 3 SH rats). rats). = 3 SH Wistar n in situ in P *** 12) and and 12) =12 <0.0001

40.0 Fig. 4 Fig. µ *** µ n m (top m (top ( SHR =12 µ

M) M) M) 100.0 *** c ). ). SH rats. ( rats. SH 4 Figure were reduced from 20.8 20.8 from reduced were recorded (SNA) activity nerve sympathetic basal lumbar of Ongoing levels activity. autonomic cardiovascular on antagonism of P2X3-receptor effect the in we SH evaluated rats, pressure arterial lowers blockade P2X3-receptor which by mechanisms the Tostudy P2X3 receptor blockade reduces sympathetic tone in SH rats sensitivity. reflex its normalizing and afferent drive from the carotid body, thereby pressure lowering blood * post-test. Dunnett’s mg/kg/h). (0.5–8.0 AF-219 of doses indicated the or vehicle of infusion the during rats SH radiotelemetered conscious in SBP in responses pressor evoked ( cases. three in rats separate in and cases four in rat same the in CBR after and before performed were measurements n ( without rats SH in ( pharmacokinetic. 8 mg/kg/h; 4 and (1, AF-219 of doses also see lines; (dotted sampling plasma of basis the on predicted concentration adenosine-5 reversible. were discharge ing chemoreflex-evoked lumbar activity chain sympathetic were ( reversed reflex-evoked) and basal the SNAon (both AF-353 of min, effects depressant 20–30 After strains. rat between different not were levels respectively ( 76 to reduced were significantly, levels these bodies, carotid to delivery AF-353 After 209 were SNA expiratory-modulated in increases Chemoreflex-evoked n.s.). 2.6 (12.3 before either rats normotensive in those to similar were ery in ies SH bilaterally rats. The SNAafter AF-353 deliv observed levels P b = 7) and in intact Wistar rats ( rats Wistar intact in and = 7) a < 0.001) after focal delivery of AF-353 (15 nl, 20

e elctd hs dt uig 2 using data these replicated We ∆SBP (mmHg) ∆SBP (mmHg) µ –3 –2 –2 –1 –1 –5 –4 –3 –2 –1 –5 10 10 20 V) or after AF-353 application (11.7 (11.7 application AF-353 after or V) 0 5 0 5 0 5 0 0 0 0 0 0 0

Supplementary Fig. 5 Fig. Supplementary –3 Experimental P2X3-receptor antagonism lowers arterial pressure in conscious conscious in pressure arterial lowers antagonism P2X3-receptor Modeled PK a n SBP data ± ) Changes in SBP (solid lines) and the AF-219 plasma plasma AF-219 the and lines) (solid SBP in ) Changes 0 = 7 per group. Data are means means are Data group. = 7 per 12% and 131 131 and 12% Baseline data 8 mg/kg/h 4 mg/kg/h 1 mg/kg/h ′ SH ∆ -triphosphate tetra(triethylammonium) salt or TNP-ATP Fig. Fig. 5a SBP S R Fig. Fig. 5 advance online publication online advance b * n ) Changes in SBP induced with 8 mg/kg/h AF-219 i.v. AF-219 8 mg/kg/h with induced SBP in ) Changes 0 = 7) and with carotid body resection (SHR & CBR; & CBR; (SHR resection body carotid with and = 7) P – HR &CB ± b < 0.05, ** < 0.05, e 11% and 69 69 and 11% ) indicating ) that indicating of the effects AF-353 on ongo *** ; ; AF-219 lnfusion n = 12, ± ± W R 11% for SH and Wistar rats, respectively. respectively. rats, Wistar and SH for 11% 2.2 to 13.7 13.7 to 2.2 ** ) after intravenous infusion of the indicated indicated the of infusion intravenous ) after 30 n P = 4). For CBR in SHR rats, rats, SHR in CBR For = 4). Time (min) P < 0.001); after AF-353 delivery, these istar < 0.01, *** < 0.01, n ± = 7 SH rats per drug dose). PK, PK, dose). drug per rats = 7 SH 7.8% in SH and Wistar rats, rats, Wistar and SH in 7.8% 60 ′ ± ± c ,3 2.5 2.5 s.e.m. One-way ANOVA One-way s.e.m. ∆SBP (mmHg) ′ - 100 120 ± 20 40 60 80 O c P 0 2.3 2.3 ) Peripheral chemoreflex- ) Peripheral Recovery -(2,4,6-trinitrophenyl) µ Vehicle < 0.001. V ( V

0.5 mg/kg 90 µ µ nature medicine nature Fig. 5a Fig. V; M) M) to carotid bod 1 mg/kg Fig. 5 Fig.

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c 4 mg/kg c ; ; ; ; 0 1,000 2,000 3,000 n n * in situ in 8 mg/kg = 12, 12, = = 12, 12, =

) (ng/ml conentration plasma AF-219 ± - - -

© 2016 Nature America, Inc. All rights reserved. baroreflex gain was enhanced after AF-219 (0.14 (0.14 AF-219 after enhanced was gain baroreflex sympathetic renal Spontaneous response. nerve sympathetic renal chemoreflex-evoked the in reduction drug-evoked the of reversal a ( activity thetic ( SBP 5 Fig. (−37 activity sympathetic receptor antagonism using AF-219. This reduced both the renal basal in conscious SH telemetry rats receptors. other and/or conductances on ionic actions nonselective to than rather receptors, due to the blockade of P2X3 homomeric and P2X2/P2X3 heteromeric are AF-353 of effects antihypertensive the that concept the supports evidence This receptors. P2X7 and P2X4 P2X2, homomeric over ity selectiv a with 1,000-fold P2X2/P2X3, and heteromeric P2X3 P2X1, inhibits that and AF-353 from distinct structurally is that antagonist ( ( ( ISN modulated respiratory basal ( ( Wistar in nl) 15 22.5 NaCN, by (evoked stimulation chemoreceptor ( 5 Figure nature medicine nature ± measures one-way ANOVA, with Holm–Sidek Holm–Sidek with ANOVA, one-way measures from data mean of 22.5 (NaCN ( infusion). drug of duration and timing indicates arrow horizontal and RSNA recorded means are data post-test; Bonferroni ANOVA One-way n ∫ a Supplementary Fig. 8 Fig. Supplementary PN) is shown. Also shown is a sympathetic nerve recording from the same preparation of SH rat after AF-353 washout. ( washout. AF-353 after rat SH of preparation same the from recording nerve a sympathetic is shown Also shown. is PN) 0.02 %/mmHg; %/mmHg; 0.02 , = 12 per group). Inspiratory and expiratory modulation of lSN was determined by using averaged triggering from the integrated phrenic nerve ( nerve phrenic integrated the from triggering averaged using by determined was lSN of modulation expiratory and Inspiratory group). per = 12 Recordings were made from renal sympathetic nerves using radio were nerves made from Recordings renal sympathetic b ) Ongoing lumbar chain sympathetic activity (arrow, raw: lSN; integrated: integrated: lSN; raw: (arrow, activity sympathetic chain lumbar ) Ongoing Fig. 4 Fig. f ; ;

P in vivo in The antihypertensive action of P2X3-receptor antagonism is associated with a reduction in sympathetic activity in SH rats rats SH in activity sympathetic in a reduction with associated is antagonism P2X3-receptor of action antihypertensive The < 0.05), which followed a similar time course to the fall in in fall the to course time similar a followed which 0.05), < ∫ b a ∫ ISN ISN ∫ ISN ISN ∫ PN a PN µ ), and the chemoreflex-evoked increase in renal sympa renal in increase chemoreflex-evoked the and ), g i.a.) in the RSNA in a conscious radiotelemetered SH rat before (control) and after AF-219 infusion in the same animal. ( animal. same the in infusion AF-219 after and (control) before rat SH radiotelemetered a conscious in RSNA the in g i.a.) W from conscious radiotelemetered SH rats ( rats SH radiotelemetered conscious from Fig. 5g Fig. a ashout ) and SH rats ( rats SH ) and g NaCN NaCN P . In . In

< 0.05), but not vehicle, infusion. Although no no Although infusion. vehicle, not but 0.05), < advance online publication online advance C , h C ∫ h ISN ontr , washout refers to the chemoreflex-evoked RSNA in SH rats the day after they had been infused with AF-219. Repeated Repeated AF-219. with infused been had they after day the rats SH in RSNA chemoreflex-evoked the to refers , washout ontr ), a high-affinity, nonselective P2X-receptor P2X-receptor nonselective high-affinity, a ), ; ; P ol ol ± < 0.05). Washout < 0.05). ( data 13 normalized units relative to baseline; baseline; to relative units normalized 13 NaCN b in in vivo c ) ) ( ), and chemoreflex-evoked changes in both inspiratory- ( inspiratory- both in changes chemoreflex-evoked and ), n = 12 per group). Activity from the phrenic nerve (PN) (neural inspiration) was recorded, and its integrated form form integrated its and recorded, was inspiration) (neural (PN) nerve phrenic the from Activity group). per = 12 during chronic P2X3- systemic NaCN NaCN post hoc post AF AF -353 -353 µ

g i.a.) without (control) and with bilateral local application of AF-353 to the carotid bodies (20 (20 bodies carotid the to AF-353 of application local bilateral with and (control) without g i.a.)

comparison. * comparison. µ V ± 10 ± Fig. 5 Fig. s.e.m. from from s.e.m. 1 0.07 versus 0.01 0.01 versus 0.07 s

µ µ V 10 V = 5) during the infusion of AF-219 (8 mg/kg/h i.v.; vertical arrows indicate samples of raw raw of samples indicate arrows i.v.; vertical mg/kg/h (8 AF-219 of infusion the during = 5) 10 1 1 h s s ) indicated ) indicated e d c ∆ Expiratory ISN (%) ∆ Inspiratory ISN (%) Respiratory related 200 250 15 10 10 in situ in P 25 50 75 50 ISN burst (µV) < 0.05, *** < 0.05, 0 0 0 0 0 20 25 15 10 0 5 WI WI WI - - - ST ST rat preparations. ( preparations. rat ST AR AR g *** AR ∫ ) A representative recording of the peripheral chemoreflex-evoked response response chemoreflex-evoked peripheral the of recording ) A representative sine hydroxylase ( hydroxylase sine Fig. 11a ( bodies carotid these in prevalent was ( peptide ing by was using determined a block specificity antibody P2X3-receptor deter was minedexpression by both immunocytochemistry P2X3 ( and hypertension, of bodies. a history with carotid medical individuals of human cadavers in from obtained were presence bodies their Carotid of step demonstration first a essential an is then humans, in target as considered antihypertensive be an to are body carotid the within receptors P2X3 If P2X3-receptor expression and tonic drive in human carotid bodies a of use potential hypertension. human for antagonist and P2X3-receptor data rat SH the of translatability the tested ms/mmHg; 0.05 (0.13 increased was baroreflex cardiac spontaneous ( infusion AF-219 con in after rats detected SH were scious heart the to activity autonomic in changes *** lSN) and the sympathetic nerve reflex response (arrowhead) to peripheral peripheral to (arrowhead) response reflex nerve sympathetic the and lSN) *** SHR *** SHR SHR *** P < 0.001. WI WI *** AF WI AF *** AF ) ) and showed co-expression with the glomus cell marker tyro *** ST ST -353 ST -353 -353 AR AR AR d AF ) and expiratory- ( expiratory- ) and AF AF f SHR upeetr Fg 10 Fig. Supplementary SHR SHR ) Changes in renal sympathetic nerve activity (RSNA) (RSNA) activity nerve sympathetic renal in ) Changes -353 -353 -353 Fig. 6 Fig. upeetr Fg 9c Fig. Supplementary f g RSNA (µV) RSNA (µV) ∆RSNA (norm. units) –60 –40 –20 20 30 40 50 60 10 10 20 a 0 5 0 0 ). Axons in the carotid body also expressed expressed also body carotid the in Axons ). C + e NaCN ontr AF ) modulated lSN in Wistar and SH rats rats SH and Wistar in lSN ) modulated 0 -21 ol NaCN 9 T RSNA AF ime (min) 30 h * -21 Fig. 6a Fig. n RSNA ). P2X3-receptor expression expression P2X3-receptor ). ∆ c = 5) and western blot ( 9 Supplementary Fig. 9a Fig. Supplementary * – (% baseline) 1,000 e 200 400 600 800 ) Effects of AF-353 on AF-353 of ) Effects 60 ; ; * 0 1 ,

P C b ontr S < 0.001). We next next We 0.001). < and and ol 20 2 AF in situ in s e l c i t r a 90 S -21 µ

W V 9 * Supplementary Supplementary ashout ± 0.04 to 0.27 0.27 to 0.04 h and and ) Quantitation ) Quantitation in vivo in ∫ PN). PN). n = 4). µ , b M, M, . ), ), ± - - - -

© 2016 Nature America, Inc. All rights reserved. of humans with hypertension, we next tested whether tonicity in the the in tonicity whether tested we next hypertension, with of humans ( rats SH in seen that to similar was the P2X3 receptor ( A using assessed as exists, hypertension with humans of body carotid (mean of 5 min); 5 min); of (mean ** depression; peak versus vehicle * post-test. Bonferroni ANOVA One-way terminated. was infusion dopamine after response hyperventilatory a rebound of appearance the Note infusion. dopamine the during reached response ventilatory lowest the was response Peak line. dotted black the by indicated is response ( mean The zero. time at reference and a control as used was infusion vehicle A dextrose individual). one to corresponds line solid (each position supine the in hypertension with individuals awake six (2 infusion dopamine low-dose during ventilation ( kDa). (44 receptor P2X3 human the of weight ( body carotid human ( labeling. axonal for and experiments control antibody P2X3-receptor See bodies. carotid human five for those bar, 25 (scale red) (TH, hydroxylase tyrosine and (green) receptor P2X3 of co-localization showing images bar, 100 scale blue; (DAPI; phenylindole 4 with staining nuclear and (green) immunofluorescence P2X3-receptor showing cadaver a human from body a carotid of a section ( hypertension. with individuals from bodies carotid in generation tone aberrant and hypertension, of history a medical with individuals 6 Figure

Having verified the presence of P2X3 receptors in carotid bodies bodies carotid in receptors P2X3 of presence the verified Having s e l c i t r

c b a P2X3-receptor expression in carotid bodies from cadavers of cadavers from bodies carotid in expression P2X3-receptor P2X3

∆ Minute ventilation (L/min) / D 52 kD 38 kD –4 –2 API 0 2 4 † P < 0.05, vehicle versus rebound. versus vehicle < 0.05, a a P b Supplementary Supplementary Fig. 11b n eak r ) Western blot of the P2X3-receptor protein in the the in protein P2X3-receptor the of blot ) Western = 4). Arrow indicates bands at the molecular molecular the at bands indicates Arrow = 4). Dopamine (2 esponse P

µ < 0.01, depression versus rebound rebound versus depression < 0.01, µ g/k * m). Images are representative of representative are Images m). Fig. 3 Fig. g/h i. R Supplementary Figure 10 Figure Supplementary µ ebound m). Right, high-power confocal confocal high-power Right, m). v. 1 mi ) P2X3 TH P2X3 b n ). c ). ). This expression pattern 44 kD ) Changes in minute minute in ) Changes P2X3 Supplementary Figure 11 Figure Supplementary / µ TH g/kg/min i.v.) in g/kg/min / a D API ** † ′ ,6-diamidino-2- ± s.e.m.) s.e.m.) P a < 0.05 < 0.05 ) Left, ) Left, for for

units were both found to be present in the rat carotid body rat carotid the present in found to be were both units Moreover, sub question. an open remains and P2X3-receptor P2X2- ATP release by these cells and any of the aforementioned mechanisms and catthe rat the in hypoxia to response in cells glomus ATPforfrom release flow blood sulfide channels ion acid-sensing and channels potassium pore-domain two involving mechanisms including states, disease in in described the carotid body that could contribute to its sensitization Many mechanisms that mediate reflex responses to hypoxia have been DISCUSSION discharge. aberrant this of identification the enables infusion dopamine that and drive, that the carotid body of humans with hypertension can generate tonic (1.28 of infusion dopamine termination the after infusion) vehicle to (relative overshoot an (−1.28 infusion the during sion minute in ventilation response to dopamine infusion ( biphasic a observed we hypertension, with als previously,described to inactivate the carotid body 2 min; (5 infusion dopamine low-dose rats, which could contribute to the hyperactive state of the petrosal petrosal the of state hyperactive the to contribute could which rats, larger receptive field in the carotid body of SH, as compared to Wistar, a have could neurons petrosal chemoreceptive that We acknowledge system. nervous sympathetic the of hyperactivity downstream and neurons petrosal of of chemoreceptive ogy the tonicity, hyperreflexia expressionP2X3-receptor in the of carotid body seems, in part, upregulation to be causal forthe the etiol in rats, not SH but In SH, rats. in normotensive, sensitivity reflex its controlling in role crucial a receptors. heteromeric and homomeric P2X2/P2X3 P2X3 of involvement probable the and neurons petrosal oreceptive to current nondesensitizing a of presence the indicate experiments voltage-clamp from Data nit. shift toward the expression of channels dominated by the P2X3 subu than mRNA rather nucleotide tor the to ies displays substantially lower EC ATP sensitivity, because this form of the channel in recombinant stud receptor upregulation in the carotid body could account for increased receptors P2X2/P2X3 heteromeric than homomeric P2X3 for affinity higher a have used P2X2/ we that antagonists the heteromeric Although responses. such in versus receptors P2X3 P2X3 homomeric of contribution rela tive the establish fully cannot we that is study our to limitation A neurons. petrosal on receptors P2X3-subunit-containing on act can exogenous both and ATPendogenous from the released carotid body reflexes. of body carotid sensitization pathological Thus, changes in P2X3-subunit expression seem to be with associated hyporeflexia show ventilatory mice, butsingle-knockout not P2X3 mice, double-knockout P2X3 and P2X2 and single-knockout P2X2 to response given that hypoxemia, both ventilatory mal physiological nor the dominate probably homomeric) P2X3 and/or homomeric (P2X2 channels P2X2-containing antagonist. P2X nonselective and rosal neurons. was on based This evidence the use of a suramin, weak leagues of using co-cultures glomus cells and petrosal Our data indicate that P2X3 receptors in the carotid body have have body carotid the in receptors P2X3 that indicate data Our that found we antagonists, selective using study,by present the In 23 3 6 , demonstrated that ATP via acts P2X receptors to excite pet 2 4 2 3 cro monoxide carbon , 7 1 . However, . studies , , as well as in humans advance online publication online advance P2x2 4 0 2 . In addition, the marked upregulation of upregulation marked the In. addition, 8 , it seems reasonable that P2X3 homomeric homomeric P2X3 that reasonable seems it , would probably precipitate a stoichiometric probably precipitate stoichiometric a would ± 0.41 l/min; l/min; 0.41 α - 2 β in vitro in 4 ± -methyl-ATP recorded from chem from recorded -methyl-ATP nti oxide nitric , 50 Fig. 6 0.52 l/min; l/min; 0.52 3 values than the homomeric recep 5 . Whether there is a link between . is there a between Whether link have also shown a major role role major a shown also have P c ), ), which comprised a depres < 0.05). These data indicate indicate data These < 0.05). µ g/kg/min i.v.), which was was which i.v.), g/kg/min in vitro P 2

< 0.05), followed by followed 0.05), < 4 and carotid body body carotid and nature medicine nature 3 8 . . In six individu , , Nurse and col 14 , 2 5 , hydrogen hydrogen , 3 6 , , and, by P2x3 3 3 7 0 ------ .

© 2016 Nature America, Inc. All rights reserved. glomus tissue in the thorax and abdomen, but this remains to be be to remains this but abdomen, and thorax the in tissue glomus include could afferents sensory Such active. become have receptors P2X3 which in afferents, sensory other from a contribution suggests ablation body carotid after remains that AF-219 of presence the in body carotid the into 353 activity sympathetic in by a reduction arte accompanied was which in pressure, rial fall the that is body carotid the on AF-219 of action direct a for support Further rats. intact to compared ablation body carotid pressure in was substantially, arterial reduced but not after abolished, fall body,the carotid within that given the antagonist-induced acting exclusively, not but primarily, is antagonism P2X3-receptor of effect SH to Wethe rat. the that is not restricted propose antagonism P2X3 with rats in of effect that indicates data), which unpublished the antihypertensive pressure Paton, J.F.R. & blood McBryde F.M. in (W.Pijacka, fall hypertension a Goldblatt to AF-219 led rats. also SH to administration AF-219 of administration systemic after sure rapid less show channels desensitization these that given receptors, heteromeric P2X2/P2X3 of involvement possible a out rule not do we although body, carotid the in receptor P2X3 of the control the under be might hyperalgesic of priming condition a to leading as C-fibers nociceptor sory somatosen in described as agent, excitatory other any by activation for threshold the lower that terminals afferent in transients calcium activation might P2X3-receptor lead to generator local and potentials desensitization of SH rats any (or body body carotid the carotid in condition postulated the a pH, Notably, low organ). from drive tonic maintain to receptors current tivating translation of P2X3 as a new drug target into the clinical arena. and body other organs now anbecomes important issue to enable the at both the transcriptional and cytosolic trafficking levels in the carotid Elucidation of the mechanisms that control P2X3-receptor expression skeletal muscle der sitization of sensing visceral mechanisms from, for example, the blad signaling has been previously observed rats SH of body carotid the release be hypoxic and hypercapnic, a condition that is known to induce ATP drive sympathetic high receive body carotid rat SH the in is hypertrophied body of carotid the microvasculature the that given in carotid signaling the body SH pathological rat is unclear. However, for sufficient is ATP clearance, reduced or ATP release increased for need the without alone, upregulation P2X3-receptor Whether rats. Wistar from recorded neurons in activity of dearth the explain also to temper carotid body discharge are which known conditions, were hyperoxic under made recordings underestimates carotid body tonicity in conscious SH rats because the from preparations report we that tonicity body carotid The cells). petrosal oreceptive nonchem in upregulated not was it (i.e., neurons petrosal receptive Notably, neurons. upregulated nature medicine nature induced activity sympathetic in decrease the Mechanistically, tested. e on a out oedpnet oeig f reil pres arterial of lowering dose-dependent robust a found We inac rapidly a evokes activation P2X3-receptor that finding The The association of P2X3-receptor upregulation and aberrant afferent 44 , 4 5 4 , urinary tract 2 54 in vivo in . . Additionally, inflammation, which is known to be present in 4 , 1 5 , can potentiate P2X3-receptor inward currents and reduce 5 . Such threshold attenuation and chemoceptive priming priming chemoceptive and attenuation threshold Such . 4 1 5 , and given the possibility that arterioles feeding the the feeding arterioles that possibility the given and , 5 5 , was also observed after the direct injection of AF- of injection direct the after observed also was , 0 2 3 in vitro in in numerous disease states, including chronic pain , which is consistent with our voltage-clamp data. voltage-clamp our with consistent is which , that developed has been concept the . Furthermore, in in situ

4 advance online publication online advance 6 , larynx 5 2 (1.5 Hz per petrosal neuron) most probably (1.5 Hz per petrosal seems inconsistent with an ability of P2X3 P2X3 of ability an with inconsistent seems 4 in situ in 1 , can also drive ATP release drive also can , 2 9 , lungs P2x3 14 . The residual depressor response response depressor residual The . , 2 0 mRNA was exclusive to chemo to exclusive mRNA was . The hyperoxic conditions might 4 2 7 8 , gastro-intestinal tract and is consistent with the sen 4 1 , the tissue is likely to to likely is tissue the ,

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trial to treat chronic pathological cough, in which coughing was was coughing seem not did reflexes which cough protective and in physiological but abated cough, pathological chronic treat to trial These animals. results are entirely control consistent with the to results of a recent akin human stimulation, to responsive physiologically remained system the yet rats, hypertensive in antagonism receptor cept; carotid body tonic drive and were hyperreflexia abated by P2X3- con this support signaling data Our function. aberrant physiological affecting without attenuate to able be might antagonist receptor to neurons petrosal of a P2X3- and Administration not underestimated. be should pathology body carotid the in expression receptor rat. SH the in antagonism P2X3-receptor contribute to the and ofsympatho-inhibition effects antihypertensive individuals with hypertension sinus baroreceptor reflex, as reported previously in both SH rats of removal of the influence carotid on body the inhibitory the carotid the reflects possibly which reflex, baroreceptor cardiac spontaneous ance in hypertension imbal autonomic of driver major a as generator respiratory central hypertension of models numerous in upregulated is that component this is It tion. respira of component expiratory the by particularly respiration, by by antagonism a included in P2X3-receptor reduction its modulation University of Bristol’s Wolfson Bioimaging Facility BBSRC 13 Alert capital grant Project (B.H.M.) 2013/06077-5 and research grant (D.J.A.M.).2013/10484-5 The ‘Fundação de Amparo à Pesquisa do Estado de São Paulo’ FAPESP Thematic of Bristol (A.K.N. & J.F.R.P.). Studies Hospitals Bristol National FoundationHealth Service Trust and the University (NIHR) Biomedical Research Unit in atCardiovascular Disease the University This research was supported by the National Institute for Health Research (A.P.F. & J.F.R.P.) and the James Tudor Foundation (J.F.R.P.) are acknowledged. British Heart Foundation RG/12/6/29670 (J.F.R.P.), Afferent Pharmaceuticals and D. Carr (electronic workshop) is appreciated. The research support of the cardiovascular data. Technical support from P. Chappell (mechanical workshop) for on his softwareexpertise used for analyzing ofaspects some of the We wish to thank J.-C. Isner of (School UniversityBiological Sciences, of Bristol) online version of the pape Note: Any Supplementary Information and Source Data files are available in the the v in available are references associated any and Methods M antagonism. P2X3 of effects antihypertensive the test to trial clinical a of undertaking the support discharge body of dopamine infusion to select those individuals with aberrant carotid ATP from human glomus cells in response to hypoxia of release the humans, P2X3 hypertensive of of body carotid the in presence receptors The system. nervous sympathetic overactive an trolled hypertension, demonstrable aberrant carotid body activity and in of a might subpopulation humans be most efficacious with uncon therapeutics tensive this. negate to able be should dosing appropriate of on P2X3 antagonism effects known adverse perception taste the be might antagonists P2X3 of use clinical the for issue An tion. func body carotid normal preserving while signaling abolish can pathological it whether is AF-219 as such antagonist P2X3 a of use functions reflex antagonism receptor by P2X3 altered be to A e ckn ethods r The potential clinical importance of the upregulation of P2X3- of upregulation the of importance clinical potential The Over the past 20 years, there has been a dearth of antihyper novel a dearth been has there 20 years, past the Over protective- and regulatory both has chemoreception Peripheral s i o o n wled

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p men a 5 56 p 9 . Thus, an important consideration for the clinical clinical the for consideration important an Thus, . , e 5 r 7 t . 7 , a finding that supports the importance of the the of importance the supports that finding a , r 5 s . . We speculate that P2X3-receptor antagonism antagonism P2X3-receptor that We speculate . 7 . . P2X3-receptor antagonism also improved the 5 in situ 8 . . This improved baroreflex gain might were supported by grants from 2 9 . s e l c i t r a 3 5 and the ability in vivo o 2 2

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© 2016 Nature America, Inc. All rights reserved. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprint at online available is information permissions and Reprints version of The authors declare competing interests:financial details are available in the wrote the manuscript and it,revised and raised the funding. the design of the project, provided with supervision data acquisition and analysis, in design anddrug trial manuscript preparation and revision. J.F.R.P. orchestrated P2X3-receptor antagonists, outcarried the pharmacokinetic analysis and assisted of the first onimmunohistochemistry human carotid A.P.F.bodies. provided the this also included data analysis and figure preparation. A.P.A. performed some the design, data experimental analysis and manuscript preparation. F.D.M. conducted the dopamine-infusion study. B.H.M. supported all with hypertension, and with L.E.K.R. E.C.H. performed and analyzed data from study. with L.E.K.R. A.K.N. outcarried the diagnosis and recruitment of humans data analysis and figure preparation. M.P.d.S. performed the single-neuron PCR rat and nerve neuronpetrosal whole-cell recording studies; this also included analysis and figure and manuscript preparation. D.J.A.M. performed all humanand western immunocytochemistry blotting; this also included data W.P. conducted all the National the HealthNIHR or Service, the Department of Health. (NIHR). The expressed views are ofthose the author(s) and not ofnecessarily those presents independent research funded by the National Institute for Health Research Community Framework Program under grant agreement no. 612280. This article Curie International Research Staff Exchange Scheme within the 7th European is BB/L014181/1 acknowledged. This project also received funding from the Marie A C Aut

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MPE in vivo Sin´M. ski, ofthe sensitivity J.Increased &Przybylski, M. Zoltowski, M., Tafil, A., Trzebski, nerve sympathetic of Potentiation F.M. Abboud, of & pathogenesis A.L. the Mark, in V.K., role Somers, a play chemoreceptors arterial Do J. Przybylski, Z.Y. Tan, K. Narkiewicz, M.D. Lobo, Arteriovenous J.F. Paton, & P.A. Sobotka, N., Sulke, M.D., Lobo, A.E., Burchell, N.K. Patel, K. Heusser, H. Krum, Aliskiren. M.J. Brown, R&D. cardiovascular of pulse the Accelerating A. Plump, hypertension. Resistant R.M. Carey, P.M. Kearney, pressure blood of usual relevance Age-specific Collaboration. Studies Prospective uncontrolled of costs health and Financial N. Marchant, Schmieder,& A., C. Lloyd, A.S. Go, nrae smahtc rv i esnil hypertension. essential in (2012). 487–491 drive sympathetic increased 16 hypertension. mild with men young in drive chemoreceptor arterial subjects. hypertensive borderline in (1988). 608–612 hypoxia to responses hypertension? in hypertension of onset the SHR. before channels TASK and ASIC of overexpression apnea. sleep obstructive 97 with patients in pressure blood and activity randomised a study): HTN trial. CONTROL controlled ROX (the hypertension uncontrolled with hypertension? control to way (2014). the this is anastomosis: 76 (2010). patients. hypertensive in pressure blood and function, (2009). 1275–1281 study.cohort proof-of-principle and safety multicentre a hypertension: (2010). 823–824 Lancet in adults studies. million prospective one 61 for data individual of meta-analysis a mortality: vascular to (2003). Kingdom. United the in pressure blood (2014). Association. Heart American the from report a update: h , 163–172 (1982). 163–172 , (1998). 943–945 , (2011). 405–407 , s/index.htm o TI r Circ. Res. Circ. the pape

radiotelemetry study radiotelemetry for recording renal sympathetic activity; nerve Co 365 N G t al. et t al. et et al et n et al. et , 217–223 (2005). 217–223 , F t al. et et al. et et al. et t I Med. Hypotheses Med. NANC l t al. et r . in vivo t al. et r xctv smay hat ies ad toe statistics—2014 stroke and disease heart summary: Executive

. Tonic activity of carotid body chemoreceptors contributes to the to contributes chemoreceptors body carotid of activity Tonic . Lancet . Deep brain stimulation relieves refractory hypertension. refractory relieves stimulation brain Deep 106 i hmrcpo hprestvt, yptei ectto, and excitation, sympathetic hypersensitivity, Chemoreceptor Carotid baroreceptor stimulation, sympathetic activity,baroreflex sympathetic stimulation, baroreceptor Carotid Central arteriovenous anastomosis for the treatment of patients of treatment the for anastomosis arteriovenous Central b ahtrbsd ea smahtc eevto fr resistant for denervation sympathetic renal Catheter-based utio lbl udn f yetnin aayi o wrdie data. worldwide of analysis hypertension: of burden Global , 536–545 (2010). 536–545 , Contribution of tonic chemoreflex activation to sympathetic activation chemoreflex tonic of Contribution I radiotelemetry blood pressure blood radiotelemetry studies and the rat and

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© 2016 Nature America, Inc. All rights reserved. 0.5, 2, 10, 20, 40, 100 mM), response to sodium cyanide injected into the aorta (0.03%, 50 chemosensitive andpetrosal NTS neurons, as characterized by their excitatory stimulation of the carotid sinus nerve (the axons of petrosal neurons) to find the We ganglia. electrical petrosal used the of visualization permitting while tion, along its lateral aspect. A mesh grid was lowered onto the ganglion for stabiliza suction. To enable stable whole-cell recordings, the petrosal ganglion wasGigaseals (>1 G opened (Molecular Devices). Molecular amplifier 10.0, (version software acquisition pClamp and Devices) integrating Axopatch-200B an with performed were of resistance a had pipettes 3–8 M filled, when and mOsmol, ~300 an of had osmolarity solution This 7.3. pH Na-GTP; 0.3 Na-ATP; 4 EGTA; 5 HEPES; 10 MgCl 4.5 K-gluconate, 130 mM): (in following the containing solution a with filled electrodes with solitarii tractus nucleus the patch clamp recordings of chemoreceptive petrosal ganglionic or neurons from right side. By using recently described techniques carotid sinus nerve and petrosal ganglion complex was isolated on the neurons. animal’s chemoreceptive from recordings Whole-cell The head of the preparation was fixed by ear bars. signal. integrated the from subtracted was level this chain; sympathetic the to each of experiment, the end noise the level Atwas measured kHz. after 2–5 the applicationat sampled of lidocaine and (2%) integrated rectified, kHz), 5 to electrode. All nerve signals were AC-amplified, band-pass filtered (from 0.5 Hz suction glass bipolar a using by chain sympathetic lumbar the from recorded also was (SNA) activity nerve Sympathetic mechanically.preparation the lize bromide, 40 (vecuronium blocker neuromuscular a commenced, movements continuous physiological index of preparation viability. After respiratory-related bipolar electrode held in a 3Dsuction micromanipulator.glass a using by end central Rhythmic cut the ramping from (PNA) activity PNAits recorded gave a to used was monitor pressure. catheter perfusion aortic was The phrenicisolated,left nerve and we the of lumen second The circulation. arterial the into directly drugs of administration the enabled catheter this to connector side A 25- size: (pore screen nylon a using filtered and carbogenation gassed with carbogen (95% O Sigma-UK), 1.5%; glycol, (polyethylene agent oncotic an containing 10) cose mM: NaCl 120, NaHCO in (containingsolution Ringer isosmotic an was perfusate The Marlow505S). One lumen was used to deliver perfusate pumped using a roller pump (Watson chamber, and a double-lumen catheter was intoinserted the descending aorta. recording a to transferred and preparationskinned aspiration. gentleThe was by pre-collicularly decerebrated were they and solution Ringer ice-cooled in rats were below transected the diaphragm, their upper werebodies submerged by a failure to respond to a noxious pinch of either a paw or thewere anesthetized deeply using tail. isoflurane (5%). Anesthetic depth Anesthetizedwas assessed 4–5 weeks, weighing 70–100 g were prepared perfused as Arterially originally described them. blind to possible not long was it studies, the the of duration and nature technical highly the Given experiment. of type each of rate success technical expected the and dosages, drug and tested be to drugs into the taking number papers consideration and oflished pilot experiments), pub experience, previous (our variance data experiments), pilot or reports previous of basis the on or (arbitrarily magnitude response determining on: or old, weeks for 14–16 radiotelemetry for g), (70–100 old weeks 4–5 either were that rats used to a 12 h cycle and day–night-shift had access unlimited to food and water. We perature (21 tem controlled with housed and litters different from selected were Animals Bristol. of University the of facility animal the within bred were which rats, (SH) Weor committee. Wistar hypertensive review spontaneously male, used ethical- of Bristol University by approved the were and Act 1986 Procedures) studies. Animal ONLINE METHODS nature medicine nature µ g/ml, Norcuron Organon Teknika) was added to the perfusate to stabi to perfusate the to added was Teknika) Organon Norcuron g/ml, Ω when tested in bath solution. Current and voltage-clamp experiments ± 2 °C) and humidity (55 Ω All procedures conformed to the UK Animals (Scientific (Scientific Animals UK the to conformed procedures All ) ) were formed, and whole-cell configuration was obtained by in situ in 3 24, KCl 5, CaCl α - juvenile rat preparation. rat juvenile β -methylene ATP (15 nl of 0.5, 2, 10, 20, 40, 100 mM) 2 and 5% CO ± 10%). Wistar and SH rats were exposed in in vivo 2 2.5, MgSO 2 ) ) and warmed to 32 °C, pH 7.3 after . Power calculations were based were . based Power calculations 57 , 6 1 2 , we performed whole-cell ; 14 trisphosphocreatine, trisphosphocreatine, 14 ; 4 Male Wistar or SH rats, WistarSH orMale 1.25, KH in situ in h crtd body, carotid The µ l). ATP (15 nl of 2 µ preparations, preparations, 6 PO m diameter). diameter). m 0 . In brief, rats 4 1.25, glu - - - - -

rons from SH rats relative to the Wistar group was determined by 2 C ( genes housekeeping the of average the to normalized and the by determined was quantitation relative The data. the normalize to gene trol control. negative a as cDNA, of instead used, was (Life Technologies), according to the manufacturer’s recommendations. Water kitTaqMan Mix the Master and UniversalPCR above described probes same the using Biosystems), Applied System, (StepOnePlus triplicate in performed RT–qPCR single-cell respectively.wereforthe min, reactions 14 The and s 15 hold temperature at 95 °C during 10 min and 14 cycles of 95 °C and 60a pre-amplificationof protocol consisted The gene). (reference °C NM_031144.2 during TH: Rn00562500_m1, post-synaptic density-95: Rn00571479_m1 and ( Rn04219592_g1 probes: following the with was performed using the TaqMan PreAmp Master Mix Kit (Life Technologies) mocycler (Mastercycler Gradient, Eppendorf). A pre-amplification of the cDNA ther a in transcription subsequent forwater Technologies)nuclease-free and tube containing High Capacity cDNA TranscriptionReverse Kit reagents (Life pressure, recently as described petrosal and NTS neurons was pulled into RT–qPCR. a patch neuron pipette with a petrosal slightly negative Single attached to a picopump (Picospritzer II, Parker Instrumentation). 2- a of use the through body carotid the 20 of nl (15 AF-353 or were carried out in rats housed singularly. Animals were acclimated to the the to acclimated were Animals singularly. housed rats in out (i.v.) carried infusions were Drug independently. working were who researchers two by studies rat for protocols Experimental dose). d per 3 for mg/kg 0.01 treatment (buprenorphine, analgesic with postoperatively, d 7 rats least and at for closed, was recovered incision The mesh. cellulose and adhesive tissue with place in secured was complex elastomer nerve–electrode the and Eu), wire WPI, silicone (Kiwk-Sil, silver biocompatible a bipolar with over isolated and lifted electrodes then recording were nerve(s) renal The underneath. freed was placed of piece and parafilm a gently small tissue, connective were from surrounding nerves renal the approach, retroperitoneal a via exposed was artery renal right the recording, nerve renal For above. given are dures proce pressure blood for Details Texas, USA). Houston, Millar, (TRM56SP; U other heparin) dayevery to maintain their patency. solution tion. saline saline/100 Catheters (0.9% were with heparinized flushed protocol catheter placed in the right jugular vein was perforated, according to a previous sampling respectively.femoral infusions, for vein andleft and blood drug The jugular right (TDMAC, the into placed heparinized catheters Germany) with Eppelheim, fitted Plolysciences, were rats Some Germany). Boehringer Metacam, Ingelheim, of g mg/100 (0.006 postoperatively d 3–5 for istered A nonsteroidal period. analgesic was admin antiinflammatory a recovery 7-d The transmitter body was placed in the abdominal cavity. Animals were allowed held with a in place tissue adhesive (VetBond, matrix. and USA) 3M, cellulose inserted until the tip rested just below the left renal artery branch point and was was (PA-C40;transmitter USA) the DSI, of cannula The bifurcation. iliac the the abdomen, and the abdominal aorta was andexposed cannulated just above Elanco Animal Health, Hampshire, UK), injected intramuscularly. Vetalar, Zoetis, London, UK)/medetomidine hydrochloride (1 mg/ml Domitor, were described previously Blood pressure and renal nerve monitoring tive cells from SH rats. in six petrosal chemoreceptive cells from Wistar and five petrosal chemorecep petrosal chemoreceptive cells from SH rats; nine in PSD-95 and TH Wistarrats; from cells chemoreceptive petrosal 11 in Wistar PSD-95 the probinggroup.ofandthat to mRNATH of Dual included ∆ ∆ treferencegene For renal nerve recording, rats were implanted with telemetric devices devices telemetric with implanted were rats recording, nerve renal For of midline the in made was incision only,an recording pressure blood For C ∆ t = ∆ C ∆ 6 t C method. For each sample, the threshold cycle (C cycle threshold the sample, each For method. 2 . Animals were allowed a 5-d recovery period after . vein wereAnimals catheteriza allowed a period 5-d recovery tUnknown ). ). The fold change of mRNA content in the sample of petrosal neu − ∆ C tControl µ 2 M; a P2X3-receptor antagonist) P2X3-receptor a M; 0 . Rats were anesthetized with ketamine (100 mg/ml; . Data are presented as mRNA expression relative 6 1 . . The cytoplasm was then placed into a micro The cytoplasm of the chemosensitive chemosensitive the of cytoplasm The µ in vivo in m tip diameter glass microelectrode microelectrode glass diameter tip m P2x2 in vivo P2x2 . and These studies were repeated repeated were studies These ), Rn00579301_m1 ( Rn00579301_m1 ), β . -actin was used as a con a as used was -actin Most surgical techniques P2x3 doi: 4 receptor expression 0 t was injected into injected was ∆ ) was determined determined was ) 10.1038/nm.4173 C t = = − C ∆ ∆ tUnknown C β t , where -actin: -actin: P2x3 − ), ), ------

© 2016 Nature America, Inc. All rights reserved. ous cardiac and renal sympathetic baroreflex gain (sBRG). The sBRG for the the for sBRG The (sBRG). gain baroreflex sympathetic renal and cardiac ous with GraphPad Prism 6 (GraphPad Software, USA). We analyzed the spontane analyzed were treatment and baseline between differences mean The vehicle. to relative AF-219 with SNA renal in change the We report level. normalized 100%- the as taken infusion before immediately min 30 the with scaled, then the baseline noise level and removed from the signal. The renal SNA signal was instruments, Cambridge, UK). The interburst interval in renal SNA was taken as Power spectral analysis was performed on heart rate using Spike2 software (CED arterial pressure, we derived pulse pressure, heart rate and respiratory frequency. in Spike2 software (Cambridge Electronic Designs, Cambridge, UK). From the sampled at 1k Hz using acquisition a 1401 scripts system and purpose-written low-pass filter with a 20-ms time constant. Arterial pressure and renal SNA were was amplified, filtered (50–5,000 Hz), full-wave rectified and integrated using a Arterial pressure was measured during resting conditions.period. infusion The vehicle renal or SNA drug signal the during continuously collected were nals analysis. Statistical details were similar to given those below for the human studies. experimental other All mixed. gently and °C 4 at overnight serum goat 2% in Alomone, Israel). Incubation with anti-P2X3 and TH antibody was carried out (APR026AN0202, antibody anti-P2X3-receptor and body carotid rat mount immunohistochemistry. Immunohistochemistry was carried out on the whole- right together) for western blotting or fixed in and ice-cold(left nitrogen liquid in snap-frozen and methanol/DMSOthese from dissected were bodies (4:1) for ( nohistochemistry. of receptorP2X3 the The specificity antibody was validated immu for used wereWistar rats six and SH Six lane. per loaded was rat each and ratfrom right) (left strains. of One pair between difference carotid bodies relative expressionproteinofThe the blotting. refers to forwestern wereused ratsWistar four and (TedSH Five Microscope Pella). Stereo Series K Motic a under dissected were bodies Carotid saline. ice-cold into transferred diately bifurcations were removed surgically from deeply anesthetized rats and imme Western blotting and immunocytochemical studies. i.v.) on separate days. (10 hydrochloride dopamine i.v.)and mg/kg/h (8 AF-219 vehicle, with infused were rats confirmed, was ablation body carotid selective Once and respiratory response to NaCN (i.v.). Animals were allowed 6 d for recovery. Effective carotid body ablation was confirmed by an absence of a cardiovascular tive, the carotid body was visualized and ablated using fine watchmakers forceps. neck, and by using microscope. a Fromdissecting binocular a perspec medial bifurcation via a midline incision through the skin on the ventral surface of the We bilateral resection. carotid body selective underwent carotid the identified heart rate and respiratory frequency. carotid bodies reversibly and assess their tonicity by measuring arterial pressure, chloride was (10 infused hydro Dopamine USA). MA, apparatus, (Harvard pump syringe standard a out using wereInfusionscarried dose. drug each and drug or vehicle between to check for chemoreflex sensitivity. period recovery were 24-h Rats a allowed was given 45 min before, and 60 and 90 ml) min after, 0.1 in the beginning (0.04% of the cyanide infusion sodium of injection bolus A ml)). (0.1 saline 0.9% bolus ml (0.1 i.v. cyanide sodium NaCN;0.04% BDH, Poole, with flushed UK, labeled AF-219 as internal standard. The peripheral chemoreflex was tested with infusion period by using high-pressure liquid chromatography assay and60-min the stable- after concentrations plasma AF-219 quantifying by validated was a pharmacokinetic profile similar to that achievable for humans. The modeling These doses were selected by modeling pharmacokinetic data to obtain for rats period. The administration order of the drug doses and vehicle was randomized. for at min into60 femoral 5 ml/kg/h their vein, followed by washout a 60-min AF-219 mg/kg/h 8 and 4 1, and vehicle of infusion an received Rats infusion. the jugular catheter at two time points: 1 h from the start of infusion andpharmacokinetic 1 analysish afterof AF-219 was a performed measured, by was pressurewithdrawing blood blood fromarterial which in animals some In (vehicle). HCl mM 10 containing saline 0.9% in dissolved was AF-219 stable. remained pressure blood point, time this At h. 2 least at for env doi: Supplementary Fig. 10 Rats that had been tested with NaCN and dopamine hydrochloride hydrochloride dopamine and NaCN with tested been had that Rats 10.1038/nm.4173 Digitally transmitted arterial pressureSNA renal and sig arterial transmitted Digitally ). Carotid artery bifurcations were removed, and carotid µ g/kg/min; Sigma-Aldrich, Dorset, UK) Sigma-Aldrich, g/kg/min; to inactivate Rat left and right carotid µ g/kg/min g/kg/min ------

barrier, whereas AF-353 does. The chemical formula for AF-219 is (amino- is AF-219 for formula chemical The does. AF-353 whereas barrier, in vivo this time AF-353 was the only antagonist available to us. For subsequent studies used in all studies contribution of homomeric, as compared to heteromeric, receptors. AF-353 was heteromeric P2X2/P2X3 receptors the for than receptors homomeric P2X3 for affinity higher have used we that AF-353ies: and AF-219 (Afferent antagonistsAlthough the Pharmaceuticals). stud these in used were antagonists receptor (heteromeric) P2X2/P2X3 and Drugs. data for the Supplementary Figures is provided in means as were compared statistically. Unless stated in werethe normally figure distributed legends, and variance data was comparable were betweenexpressed the groups that renal SNA sBRG vehiclebetween and AF-219 infusions. (pre-infusion) level set at 100%. A paired student baseline the with normalized SNA was renal above, described As analysis. for renal SNA the changeopposed in the corresponding DBP ramp were included and rate heart both which in ramps All UK). Cambridge, instruments, (CED at least five consecutive beats were identified automatically using Spike2 scripts over andDBP or beats, smoothed positive five was negative pressure ramps of before, and the final 10 min of the 60-min infusion period, of AF-219 or vehicle. arterial pressure–renal SNA relationship was calculated for 10 min immediately carotid bodies were obtained from twelve cadavers with a medical history of history medical a with cadavers twelve from obtained were bodies carotid studies. immunocytochemical and Western blotting The data was analyzed by GraphPad Prism 5 (GraphPad Software, USA). tion using Spike2 (Cambridge ventila Electronic Design) minute and MATLAB and volume (MathWorks). tidal rate, respiratory calculate to analyzed and mask. face a to attached Data were valve acquired using non-rebreathing PowerLab and a LabChart7 software of (AD Instruments) port expiratory the to connected flow-head respiratory a via measured were volumes Respiratory metabolized. be to dopamine the for time air, allowing room breathingmin while 5 of period recovery a by followed was This infusions. the of order the to blinded were Participants administered. were dopamine and dextrose of air.breathingroom volumes while Equivalent min for5 started was dextrose) 100 concentration, (infusion dopamine of infusion intravenous the and purged was line the infusion, vehicle the After control. (5%) for 5 min via peripheral venous access while breathing room air as a vehicle cines were selected. Participants were given an intravenous infusion of dextrose >139/91 mmHg) and prescribed a mean of 2.83 different antihypertensivepressure, blood (ambulatory medimmHg >140/90 pressureof blood office an with Six humans with hypertension (age 48.8 (2 infusion brainstem the into input afferent ( concentrations dopamine humans. hypertensive in tone body Carotid Committee (REC numbers: and14/SW/0054 12/SW/0277). Ethics Research Bristol Central the by approved were studies human All ers. studies, and consent was also obtained for removing carotid bodies from cadav studies. Human subunit containing receptors has previously notedbeen non-P2X3 any on AF-219 or AF-353 either of effect inhibitory no that Note A.P.Ford). data; (unpublished respectively AF-219, and AF-353 for nM 250 and nM 100 were values example, for receptors; heteromeric P2X2/P2X3 for nM (unpublished data; A.P. Ford), respectively. Higher IC receptors, the IC data half) by reduced is response the 481 pdfaiw.uspto.gov/ 5yl)ether. The structure of AF-219 is contained in a published patent (see (4-isopropyl-2-methoxyphenyl)sulfone)-5-yl-(2,4-diaminopyrimidine- The type of statisticaloftype The testperformed indicatedfigureis legends.the in Data & ). Doses used are based on IC , we used AF-219 because this drug is known not to cross the blood–brain Two highly potent and selective noncompetitive P2X3 (homomeric) (homomeric) P2X3 noncompetitive selective and potent highly Two ± s.e.m. and were assumed to be significantwhen be toassumed were ands.e.m. µ g/kg/min) was used to test for any tonicity from carotid bodies. bodies. carotid from tonicity any for test to used was g/kg/min) 50 All participants gave informed consent to participate in the the in participate to consent informed gave participants All .aiw?PageNum=0&docid in situ values for AF-353 (ref. , which were performed before the ≤ 2 2 µ g/kg) can decrease peripheral chemoreceptor peripheral decrease can g/kg) 28 50 6 3 , (the concentration of the antagonist where 40 . Thus, low-dose intravenous dopamine dopamine intravenous low-dose Thus, . 40 , 4 , 4 4 , they cannot fully establish the relative 4 ± . For recombinant P2X3 homomeric recombinant ForP2X3 . 4 =20150057299&IDKey=DA73323D4 3 years; 28.6 BMI 0 ) ) and AF-219 were ~8 nM and ~30 Small increases in circulating circulating in increases Small t Supplementary Data Set 1 -test was used to compare the µ g/ml of dopamine in 5% 5% in dopamine of g/ml 40 Human left and right right and left Human nature medicine nature , in vivo 4 50 4 . values were found ± P 4, three female) 4, three < 0.05. Source 0.05. < studies, and at http . :// - - - -

© 2016 Nature America, Inc. All rights reserved. peptide peptide (APR026AG0140, Alomone; the anti-P2X3 antibody was confirmed by membrane incubation with blocking were quantified using ImageQuant software (GE Healthcare). The specificity of (Typhoon, scanner ProteinHealthcare). expressionsGE laser fluorescent a on brane was thoroughly washed. bywas Signal detected scanning the membrane 488 was carried out in the dark at room temperature. Before imaging, the mem 10 min at for room temperature. Incubation antibody with Alexa times Fluorsecondary three PBS/0.1%Tween × 1 with washed subsequently were branes with anti-P2X3 (APR026AN0202; 1:200 dilution) overnight at 4 °C. The mem with 2% Advance blocking agent (GE Healthcare). Membranes were incubated After protein transfer, the membrane was blocked for 1 h at room temperature linearity. high and range dynamic broad a with quantification and detection diluted according to manufacturer’s instructions) were used. This system enablesand Alexa Fluor 488 secondary antibody (Life Technologies, UK, cat. #R37116; UK) Buckinghamshire, Healthcare, (GE membrane PVDF low-fluorescent a Gel, Life Technologies). The Amersham ECL Plex western blotting system using used for protein electrophoresis on 4–12% Bis-Tris gel (NuPage 4–12% Bis-Tris Technologies),(NuPage, Life 4×, Sample Buffer LDS 20 and protein assay kit (Bio-Rad Laboratories). Samples were mixed with loading dye DC- the using method Lowry the by determined was supernatant the of tion at 10,000 centrifuged was Homogenate Biotechnology). Cruz (Santa RIPABuffer Lysis with homogenized subsequently were Samples pestle. and mortar a with gen used for immunohistochemistry. and the right carotid body was immediately fixed in 4% paraformaldehyde and blotting, western for used and snap-frozen directly was body carotid left The out, cut were placed into cold ×1 PBS, bifurcations and both carotid bodies were carotid dissected and processed. Briefly, mmHg). (>140/90 hypertension nature medicine nature Westernblotting. g for 20 min at 4 °C, and the pellet was discarded. Protein discarded. was for at concentra min pellet and 20 °C, the 4 The left human carotid body was crushed in liquid nitro liquid in crushed was body carotid human left The Supplementary Supplementary Fig. 10 µ ). g ofprotein g was - - - -

61. 60. Adobe in processed were Illustrator CS3. Images sensitivity. greater for detectors GaAsP inverted epifluorescence microscope, and imaging was performed using ‘hybrid’ I6000 DM Leica a to attached microscope laser-scanning AOBSconfocal SP8 Leica a under examined were bodies Carotid Laboratories). Vector H-1000, and the samples were mounted on slides using mounting medium (Vectashield, above, described as bywashing, followed was incubation Secondary-antibody hydroxylase. tyrosine visualize to used was 594 Fluor Alexa anti-mouse and tem perature. Anti-rabbit receptor, P2X3 the Fluor toAlexa visualize was 488 used room at h 1 for applied was antibody secondary the and temperature, room at min 10 for times four PBS × 1 in washed then were Samples °C. 4 at according to the manufacturer’s recommendations, in 2% goat serum overnight (TH, F-11, sc-25269, Santa Cruz dilutionBiotechnology; 1:25) was carried out, (APR026AN0202, Alomone, Israel; 1:25 dilution) and anti–tyrosine hydroxylase gently agitated overnight. and Primary temperature antibody room incubationat h with 1 anti-P2X3for receptor UK) (Sigma-Aldrich, Saponin serum/0.1% goat 10% with incubated were slices body Carotid cryostat. a on slices thick 63. 62. Immunofluorescence.

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65 , 63–68 (1996). 63–68 , t al. et t al. et lcrpyilgcl rpris f ota vnrltrl medulla ventrolateral rostral of properties Electrophysiological ua rgt aoi bde wr ct no 20- into cut were bodies carotid right Human Carotid chemoreceptor modulation of blood flow during flow blood of modulation chemoreceptor Carotid J. Physiol. (Lond.) Physiol. J. xeietl n sria tcnqe i te rat the in techniques surgical and Experimental

589 , 6219–6230 (2011). 6219–6230 , doi: 10.1038/nm.4173 . Neurosci. J. J. Neurosci. J. µ m - .