Excitatory effect of ATP on rat area postrema neurons ATP-induced inward currents and increases in the cytosolic Ca2 + concentration -LRB- -LSB- Ca -RSB- in -RRB- were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry , respectively . The ATP-induced current -LRB- IATP -RRB- and -LSB- Ca -RSB- in increases were mimicked by 2-methylthio-ATP and ATP-γS , and were inhibited by P2X receptor -LRB- P2XR -RRB- antagonists . The current -- voltage relationship of the IATP exhibited a strong inward rectification , and the amplitude of the IATP was concentration-dependent . The IATP was markedly reduced in the absence of external Na + , and the addition of Ca2 + to Na + - free saline increased the IATP . ATP did not increase -LSB- Ca -RSB- in in the absence of external Ca2 + , and Ca2 + channel antagonists partially inhibited the ATP-induced -LSB- Ca -RSB- in increase , indicating that ATP increases -LSB- Ca -RSB- in by Ca2 + influx through both P2XR channels and voltage-dependent Ca2 + channels . There was a negative interaction between P2XR - and nicotinic ACh receptor -LRB- nAChR -RRB- - channels , which depended on the amplitude and direction of current flow through either channel . Current occlusion was observed at Vhs between − 70 and − 10 mV when the IATP and ACh-induced current -LRB- IACh -RRB- were inward , but no occlusion was observed when these currents were outward at a Vh of +40 mV . The IATP was not inhibited by co-application of ACh when the IACh was markedly decreased either by removal of permeant cations , by setting Vh close to the equilibrium potential of IACh , or by the addition of d-tubocurarine or serotonin . These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR - and nAChR-channels . Introduction The rat area postrema -LRB- AP -RRB- is a medullary circum-ventricular organ located in the hindbrain at the level of the obex , with a dense vascular supply devoid of a blood-brain barrier . The lack of a blood-brain barrier makes the AP ideally placed to act as a chemosensitive trigger zone involved in the control of vomiting in response to circulating emetic substances -LRB- Borison -LSB- 1 , 2 -RSB- -RRB- . Neurons in the AP are also responsive to changes in osmolarity or sodium concentration -LRB- Franchini et al. -LSB- 3 -RSB- -RRB- , and can be activated by circulating vasoactive peptides such as angiotensin II -LRB- Fink et al. -LSB- 4 -RSB- -RRB- , and arg-vasopressin -LRB- Undesser et al. -LSB- 5 -RSB- -RRB- . Anatomical studies have revealed that the AP sends dense efferent projections to the nucleus tractus solitarius , parabrachial nucleus , nucleus ambiguus , and the dorsal motor nucleus of the vagus , and receives afferent inputs from the hypothalamic paraventricular and dorsomedial nucleii , and from the caudal nucleus tractus solitarius -LRB- Morest -LSB- 6 -RSB- ; Kooy and Koda -LSB- 7 -RSB- ; Shapiro and Miselis -LSB- 8 -RSB- -RRB- . Thus , the AP is not only capable of responding to circulating hormones , but is also anatomically well situated to regulate a range of other central neurons , including those important in cardiovascular control -LRB- Sun and Spyer -LSB- 9 -RSB- -RRB- . The low intrinsic firing rates of AP neurons in vivo -LRB- Papas et al. -LSB- 10 -RSB- -RRB- suggests that understanding and modulating excitatory inputs to AP could be particularly important in the functional output of AP neurons . A number of transmitters can evoke excitatory currents in the AP . Inward currents and increases in cytosolic Ca2 + -LRB- -LSB- Ca -RSB- in -RRB- via non-NMDA-glutamate receptors have been reported in rabbit and rat AP neurons , respectively -LRB- Jahn et al. -LSB- 11 -RSB- ; Hay and Lindsley -LSB- 12 -RSB- -RRB- . Our preliminary reports indicated that ATP also induces inward currents and -LSB- Ca -RSB- in increases via the activation of P2X receptor -LRB- P2XR -RRB- in isolated rat AP neurons -LRB- Sorimachi et al. -LSB- 13 , 14 -RSB- -RRB- . In addition , pre - and post-synaptic nicotinic ACh receptors -LRB- nAChR -RRB- have been demonstrated in the AP in rat brain slices -LRB- Funahashi et al. -LSB- 15 -RSB- -RRB- , and we have recently reported the presence of nAChRs in dissociated rat AP neurons -LRB- Sorimachi and Wakamori -LSB- 16 -RSB- -RRB- . During that study , we also noticed that many of these AP neurons also responded to ATP , which has prompted us to further investigate ATP responses in AP neurons , and potential interactions between nAChR and P2XR responses . In a variety of different peripheral neurons , including sympathetic neurons of bullfrog -LRB- Akasu and Koketsu -LSB- 17 -RSB- -RRB- , rat -LRB- Nakazawa et al. -LSB- 18 -RSB- -RRB- , and guinea-pig -LRB- Searl et al. -LSB- 19 -RSB- -RRB- , cultured guinea-pig enteric and submucosal neurons -LRB- Zhou and Galligan -LSB- 20 -RSB- Barajas-Lopez et al. -LSB- 21 -RSB- -RRB- , a negative interaction between P2XR - and nAChR responses has been reported . Such an interaction has also been observed for recombinant P2X2 and α3β4 nAChR channels in Xenopus oocytes and HEK cells -LRB- Khakh et al. -LSB- 22 , 23 -RSB- ; Boue-Grabot et al. -LSB- 24 -RSB- -RRB- , where it has been recently suggested that this results from direct physical interactions between co-localized receptors -LRB- Khakh et al. -LSB- 23 -RSB- -RRB- . In this study we more fully describe P2XR responses in AP neurons , demonstrate cross-inhibition between P2XRs and nAChRs and characterize some of the features of this cross-inhibition . Experimental procedures Preparation of AP neurons The study was approved by the Committee on Animal Experimentation , Kagoshima University . Wistar rats -LRB- 13 -- 18 days-old -RRB- were anaesthetized with ether and decapitated . The brain was quickly removed from the skull and placed in ice-cold HEPES-buffered saline containing 150 mM NaCl , 5 mM KCl , 2 mM CaCl2 , 1 mM MgCl2 , 10 mM HEPES and 5.5 mM glucose . The pH of the saline solution was adjusted to 7.4 by adding tris -LRB- hydroxymethyl -RRB- aminomethane . The brain was sliced at a thickness of 400 µm with a microslicer -LRB- DTK-1000 , Dosaka , Kyoto , Japan -RRB- , and the slices were kept in bicarbonate-buffered saline bubbled continuously with 95 % O2 -- 5 % CO2 at room temperature -LRB- 21 -- 26 °C -RRB- . The bicarbonate-buffered saline contained 120 mM NaCl , 5 mM KCl , 2 mM CaCl2 , 1 mM MgCl2 , 20 mM NaHCO3 , 2 mM KH2PO4 and 5.5 mM glucose . Following 30 -- 60 min incubation , the slices were treated with pronase -LRB- 0.2 mg/ml -RRB- in bicarbonate-buffered saline for 20 min at 33 °C , followed by incubation for 30 min with bicarbonate-buffered saline containing thermolysin -LRB- 0.12 -- 0.15 mg/ml -RRB- . The bilateral AP regions were identified with a binocular microscope -LRB- Zeiss , Germany -RRB- , and were cut out using the tip of an injection needle , and subsequently mechanically triturated with fire-polished glass pipettes of decreasing diameters . Dissociated neurons were placed on to the bottom of 35 mm culture dishes -LRB- Falcon , USA -RRB- for electrophysiological recordings , or on to glass coverslips -LRB- Matsunami , Japan -RRB- coated with poly-l-lysine for -LSB- Ca -RSB- in measurements . Electrophysiological recordings Electrical measurements were done using the whole-cell patch-clamp recording configuration under voltage-clamp conditions . Patch pipettes were fabricated from borosilicate glass tubes in five or six stages using a pipette puller -LRB- Model P-97 , Sutter Instrument , San Rafael , CA , USA -RRB- . The resistance between the recording electrode filled with the internal solution , and the reference electrode in the external solution , was 3 -- 6 MΩ . Ionic currents were measured , and voltages controlled , using a patch-clamp amplifier -LRB- EPC-9 , Heka , Darmstadt , Germany -RRB- . All experiments were carried out at 24 -- 26 °C . Culture dishes were placed on an inverted microscope -LRB- TE200 , Nikon , Japan -RRB- , and drugs were rapidly applied to single cells using a Y-tube perfusion device . The internal solution -LRB- patch pipette solution -RRB- contained 70 mM K-gluconate , 50 mM KCl , 10 mM NaCl , 0.5 mM CaCl2 , 3 mM MgCl2 , 10 mM HEPES , 10 mM EGTA , and 2 mM ATP . The pH was adjusted to 7.2 with KOH . For measuring current-voltage -LRB- I -- V -RRB- relationships , a Cs-based internal solution was employed , which contained 98 mM CsOH , 40 mM CsCl , 98 mM aspartate , 2 mM MgCl2 , 5 mM HEPES , 5 mM EGTA , and 2 mM ATP . pH was adjusted to 7.2 with CsOH . The external solution was the HEPES-buffered saline , pH7 .4 , described above . When N-methyl-d-glucamine -LRB- NMDG + -RRB- or sucrose was substituted for external Na + or NaCl , respectively , the osmolarity of the solution was kept constant as measured using an osmometer -LRB- Vogel OM801 , Germany -RRB- . To obtain the Ca2 + / Na + permeability ratio -LRB- PCa/PNa -RRB- , we measured the reversal potential of the ATP-activated current -LRB- EATP -RRB- in the presence of 1 or 110 mM of external Ca2 + by stepping the holding potential -LRB- Vh -RRB- between 10 and 30 mV , using increments of 10 mV . The 1 and 110 mM Ca2 + solutions contained 155 or 0 mM NaCl , respectively , in addition to 10 mM HEPES with pH adjusted to 7.4 with NaOH or Ca -LRB- OH -RRB- 2 , respectively . The values of PCa/PCs and PNa/PCs were calculated using the constant field equation as described by Lewis -LRB- -LSB- 25 -RSB- -RRB- , taking the activity coefficients of Na + , Cs + , and Ca2 + as 0.75 , 0.75 , and 0.3 , respectively . Measurement of -LSB- Ca -RSB- in Dissociated AP neurons on a glass coverslip were incubated with 1 -- 2 µM fura-2 acetoxymethyl ester -LRB- fura-2 / AM -RRB- , 0.1 % dimethyl sulfoxide , and 1 % bovine serum albumin for 45 min at 37 °C . The coverslips were then mounted in a superfusion chamber and placed on the stage of an inverted microscope -LRB- Diaphot-TMD , Nikon , Japan -RRB- . Cells were continuously superfused at a rate of 1 ml/min with HEPES-buffered saline at 24 -- 26 °C via a polyethylene tube placed 1 -- 2 mm away from the cells . Cells were viewed with a 40 × Fluor objective lens -LRB- Nikon -RRB- , and a single cell -LRB- 10 -- 12 µm diameter -RRB- was fixed in a window positioned between the photomultiplier and the microscope . The changes in fluorescence ratios at 340 and 380 nm excitation wavelengths were measured using a CAM-200 spectrometer -LRB- Jasco , Japan -RRB- . The absolute value of -LSB- Ca -RSB- in was calculated using the formulae as given by Grynkiewicz et al. -LSB- 26 -RSB- : . Calibration constants were determined in separate experiments with the same experimental set-up , as described previously -LRB- Sorimachi et al. -LSB- 27 -RSB- -RRB- . Student 's paired t test was used to evaluate differences between mean values obtained from the same cells , and Student 's unpaired t test was used for data obtained from different groups of cells . Reagents The following reagents were used : Fura-2 / AM -LSB- Dojindo , Kumamoto , Japan -RSB- , ACh -LSB- Horai Chem . Co. , Japan -RSB- , PPADS -LRB- pyridoxal-phosphate-6-azophenyl-2 ′ , 4 ′ - disulphonic acid -RRB- -LSB- RBI , USA . -RSB- , pronase -LSB- Calbiochem. , USA -RSB- , thermolysin , ATP , 2-methylthio-ATP , ATPγS , α,β-methylene-ATP , β,γ-methylene-ATP , ADP , nitrendipine , nicardipine , suramin -LSB- all from Sigma , Aldrich , Tokyo , Japan -RSB- , ω-conotoxin-MVIIA , ω-conotoxin-MVIIC and ω-agatoxin-IVA -LSB- all from Peptide Institute , Osaka , Japan -RSB- . Results ATP-induced current Rapid application of ATP -LRB- 100 µM -RRB- to isolated AP neurons voltage clamped at a Vh of − 70 mV induced an inward current , which desensitized slowly -LRB- Fig. 1A -RRB- . The ATP-induced current -LRB- IATP -RRB- was recorded at various Vhs between − 70 and +50 mV -LRB- Fig. 1 : N = 6 -RRB- , and the relative current amplitude was plotted against Vh exhibiting a strong inward rectification . The EATP , estimated from the intersection of the current response and the zero voltage-axis -LRB- Fig. 1B -RRB- , was 22.7 ± 0.9 mV -LRB- N = 18 -RRB- . Fig. 1Current-voltage relationship for ATP-induced currents . A -RRB- Representative currents in response to 200 µM ATP recorded at Vhs between − 70 and +50 mV . B -RRB- Current-voltage relationship for the IATP . All responses are normalized to the peak current amplitude obtained at a Vh of − 70 mV , and each point is the mean ± S.E.M. of five neurons . C -RRB- Averaged concentration-current relationships for ATP in the presence of 0.1 , 2 , and 10 mM external Ca2 + . All responses were normalized to the mean of two control responses induced by 100 µM ATP in saline containing 2 mM external Ca2 + before and after the test response . Each point is the average ± S.E.M of responses from four to six neurons . To investigate the ATP concentration-response relationship , care was taken to adjust the pH of the solutions containing higher concentrations of ATP to 7.3 , since addition of ATP reduced pH and a small decrease in pH to 7.1 increased the IATP to 189 ± 8 % -LRB- N = 6 -RRB- of control -LRB- pH7 .3 -RRB- , as previously reported for recombinant P2X2 receptors -LRB- King et al. -LSB- 28 -RSB- -RRB- . The responses to different ATP concentrations were flanked by responses to the control concentration of 100 µM , and normalized responses were expressed relative to the average of these control responses -LRB- Fig. 1C ; Ca2mM -RRB- . We also examined the effects of 0.1 and 10 mM Ca2 + on IATP . As summarized in Fig. 1C , an increase in the external concentration of Ca2 + shifted the concentration-response curve for ATP to the right , with the half-maximum effective concentration -LRB- EC50 -RRB- values at 0.1 , 2 and 10 mM Ca2 + being 30 , 70 , and 190 µM , respectively . The purinergic agonists , 2-methylthio-ATP -LRB- 50 µM -RRB- and ATPγS -LRB- 50 µM -RRB- induced currents that were 41 ± 3 % -LRB- N = 4 -RRB- , and 41 ± 4 % -LRB- N = 5 -RRB- , respectively , of the IATP in response to 100 µM ATP . ADP -LRB- 0.5 mM -RRB- did not produce any response . The IATP in response to 50 µM ATP was inhibited by the P2 antagonists ; suramin -LRB- 10 µM and 20 µM -RRB- and PPADS -LRB- 50 µM -RRB- , with the response being 35 ± 2 % -LRB- N = 5 -RRB- , 25 ± 2 % -LRB- N = 4 -RRB- , and 56 ± 5 % -LRB- N = 5 -RRB- , of the control IATP , respectively . ATP-induced -LSB- Ca -RSB- in increase ATP also increased -LSB- Ca -RSB- in in a dose-dependent manner -LRB- Fig. 2A -RRB- . These responses were all recorded in the presence of 2 mM external Ca2 + , and the -LSB- Ca -RSB- in increases by various concentrations of ATP were normalized to that induced by 100 µM ATP . The normalized and averaged responses to ATP , 2-methylthio-ATP and ATPγS are shown in Fig. 2B . There was no ATP-induced -LSB- Ca -RSB- in increase in the absence of external Ca2 + -LRB- N = 6 ; data not shown -RRB- . Furthermore , neither ADP , α,β-methylene-ATP nor β,γ-methylene-ATP increased -LSB- Ca -RSB- in when tested at concentrations of 200 µM -LRB- data not shown -RRB- . The ATP-induced -LSB- Ca -RSB- in increases were inhibited by suramin and PPADS -LRB- Table 1 -RRB- . Fig. 2Concentration-response relationships for the -LSB- Ca -RSB- in increases in response to ATP and ATP analogues . A -RRB- Representative -LSB- Ca -RSB- in increases induced by various concentrations of ATP in the presence of 2 mM external Ca2 + . B -RRB- Averaged concentration-response relationships for ATP and ATP analogues . All responses were normalized to the mean of two control responses induced by 100 µM ATP before and after the test response . Each point is the mean ± S.E.M. of data from five to nine neurons . MeS-ATP : 2-methylthio-ATP . Table 1Effects of P2 receptor antagonists and Ca2 + channel blockers on the -LSB- Ca -RSB- in increases induced by ATP and high K + - salineStimulusBlockersPercent of controlATP100 MSuramin 10 M55 ± 7 -LRB- N = 5 -RRB- 20 M30 ± 5 -LRB- N = 6 -RRB- 50 M13 ± 3 -LRB- N = 5 -RRB- PPADS 10 M96 ± 6 -LRB- N = 3 -RRB- 20 M70 ± 8 -LRB- N = 9 -RRB- 50 M25 ± 5 -LRB- N = 6 -RRB- 100 M11 ± 5 -LRB- N = 4 -RRB- 110 mMKClNitrendipine 2 M44 ± 4 -LRB- N = 16 -RRB- − CT. M C 2 M61 ± 4 -LRB- N = 16 -RRB- − CT. M A 2 M62 ± 6 -LRB- N = 16 -RRB- − CT. M A 2 M + − CT. M C 2 M58 ± 5 -LRB- N = 10 -RRB- ATP100 MNitrendipine 2 M56 ± 4 -LRB- N = 21 -RRB- Nicardipine 2 M47 ± 9 -LRB- N = 10 -RRB- Cd2 + 50 M27 ± 3 -LRB- N = 14 -RRB- − CT. M A 2 M + − CT. M C 2 M77 ± 9 -LRB- N = 4 -RRB- The mean -LSB- Ca -RSB- in increase induced by the first and third applications of ATP -LRB- 100 μM -RRB- or 110 μM KCl were averaged and referred to as 100 % , and the response in the presence of the P2 antagonist or Ca2 + channel blocker was expressed as a percentage of this control value . The cell was pre-treated for 30 s with the indicated agent before the second stimulation with 100 μM ATP or 110 mM KCl . Number of experiments is shown in parentheses . ω-CT ω-conotoxin . Effects of Ca2 + channel antagonists on ATP-induced -LSB- Ca -RSB- in increase Previous results , using cultured rabbit AP neurons , demonstrated the presence of a ω-conotoxin-M Csensitive Ca2 + response , but that did not involve L - or N-type Ca2 + channels -LRB- Hay et al. -LSB- 29 -RSB- -RRB- . We also investigated the effects of various Ca2 + channel antagonists on -LSB- Ca -RSB- in increases induced by high KCl -LRB- 110 mM -RRB- and ATP -LRB- 100 µM -RRB- . Antagonist for the L-type -LRB- nitrendipine , nicardipine -RRB- , N-type -LRB- ω-conotoxin-M A -RRB- , or P/Q-type -LRB- ω-conotoxin-M C -RRB- Ca2 + channels each substantially inhibited these -LSB- Ca -RSB- in increases -LRB- Table 1 -RRB- . In contrast , the selective P-type Ca2 + channel antagonist , ω-agatoxin IVA -LRB- 1 µM -RRB- , did not have any inhibitory effect on the -LSB- Ca -RSB- in increases -LRB- 98 ± 5 % of control , N = 4 -RRB- . ATP-induced current in the absence of external Na + When external Na + was completely replaced by NMDG + , and in the absence of external Ca2 + -LRB- 0 Ca2 + plus 0.5 mM EGTA -RRB- , the IATP was markedly reduced to 8 ± 1 % of the control IATP recorded in the presence of external Ca2 + and 150 mM Na + -LRB- N = 9 , Fig. 3A -RRB- . This current was further reduced to 4 ± 1 % -LRB- N = 3 -RRB- in the presence of 20 µM suramin . The small suramin-sensitive , Na + - independent current supports the previous suggestion that the P2XR channel is permeable to glucosamine -LRB- Nakazawa -LSB- 30 -RSB- -RRB- . To confirm this , ATP did not induce a current at all when sucrose -LRB- 0.25 M -RRB- was substituted for NaCl -LRB- again in the absence of external Ca2 + , N = 5 -RRB- . Addition of Ca2 + to the NMDG + - substituted saline further increased IATP . The currents induced by 500 µM ATP in the presence of 2 and 10 mM Ca2 + , but in the absence of external Na + -LRB- replaced by NMDG + -RRB- were 18 ± 2 % -LRB- N = 11 -RRB- and 29 ± 2 % -LRB- N = 14 , Fig. 3B -RRB- of the control IATP , respectively . Similarly , the currents induced by 500 µM ATP in the presence of 2 and 10 mM Ca2 + added to the sucrose-substituted saline were 6 ± 1 % -LRB- N = 6 -RRB- , and 13 ± 2 % -LRB- N = 16 ; Fig. 3C -RRB- of the control IATP , respectively . Fig. 3ATP-induced current in Na + - and NaCl-free saline containing 0 and 10 mM Ca2 + . A -RRB- A representative IATP recorded in Na + - free -LRB- Na + replaced by NMDG + -RRB- and Ca2 + - free saline -LRB- containing 0.5 mM EGTA -RRB- at a Vh of − 70 mV . B -RRB- A representative IATP in Na + - free -LRB- NMDG + -RRB- saline containing 10 mM Ca2 + . C -RRB- A representative IATP in NaCl-free -LRB- NaCl replaced by sucrose -RRB- saline containing 10 mM Ca2 + . We also measured the EATP of IATP in the presence of 1 mM external Ca2 + and 150 mM NaCl or 110 mM external Ca2 + and 0 mM NaCl . The EATP at 110 mM Ca2 + was 21.1 ± 1.0 mV -LRB- N = 18 -RRB- , from which we calculated a PCa/PCs of 6.3 . The EATP at 1 mM Ca2 + was 20.2 ± 1.0 mV -LRB- N = 13 -RRB- , from which we calculated a PNa/PCs ratio of 2.1 . From these , we obtained a PCa/PNa ratio of 3.0 , confirming the substantial Ca2 + permeability of P2XRs . Negative interaction between P2XR and nAChR channels Requirement of actual current flow through receptor channels for cross-inhibition It has previously been reported that there is mutual occlusion between P2XR and nAChR in some neurons -LRB- Nakazawa et al. -LSB- 18 -RSB- ; Nakazawa -LSB- 30 -RSB- ; Searl et al. -LSB- 19 -RSB- ; Zhou and Galligan -LSB- 20 -RSB- ; Barajas-Lopez et al. -LSB- 21 -RSB- ; Khakh et al. -LSB- 22 , 23 -RSB- ; Boue-Grabot et al. -LSB- 24 -RSB- -RRB- . To examine whether there were negative interactions between the ACh-activated current -LRB- IACh -RRB- and IATP , one receptor agonist was added in the presence of the other . As shown in Fig. 4A , the IACh -LRB- 100 µM -RRB- was markedly reduced when activated in the presence of ATP -LRB- 50 µM -RRB- . We next examined IACh -LRB- 100 µM -RRB- in the presence of various concentrations of ATP . With 2 , 10 , 20 , and 100 µM ATP , IACh was reduced to 95 ± 1 % -LRB- N = 20 -RRB- , 84 ± 2 % -LRB- N = 24 -RRB- , 60 ± 4 % -LRB- N = 15 -RRB- , and 46 ± 6 % -LRB- N = 7 -RRB- , respectively , of control -LRB- P < 0.01 except at 2 µM ATP -RRB- . Thus , the IACh inhibition became stronger as the concentration of ATP was increased and with a higher agonist-receptor occupancy . In fact , when IATP at 100 µM ATP was markedly inhibited in the presence of 200 mM PPADS -LRB- 8 ± 2 % of control , N = 6 -RRB- , IACh was not occluded -LRB- 97 ± 1 % of control ; N = 6 -RRB- . When the peak amplitude of IATP , evoked by various concentrations of ATP , was plotted against the ratio of IACh in the presence and absence of ATP , there was an inverse correlation between the amplitudes of these responses -LRB- Fig. 4B -RRB- . Conversely , when ATP -LRB- 50 µM -RRB- was applied in the presence of ACh -LRB- 100 µM -RRB- , IATP was also occluded -LRB- Fig. 4C -RRB- . Again , when IACh at 200 mM ACh was nullified in the presence of 1 mM hexamethonium , a nAChR antagonist -LRB- 0.2 ± 0.2 % of control , N = 6 -RRB- , IATP was 96 ± 2 % -LRB- N = 6 -RRB- of control . IACh at 10 , 20 , 50 , and 200 µM ACh were 6 ± 2 % -LRB- N = 5 -RRB- , 20 ± 4 % -LRB- N = 5 -RRB- , 61 ± 8 % -LRB- N = 6 -RRB- , and 139 ± 8 % -LRB- N = 6 -RRB- of IACh at 100 µM . There was also an inverse correlation between the peak amplitude of IACh , evoked by various concentrations of ACh , and the ratio of IATP in the presence and absence of ACh -LRB- Fig. 4D -RRB- . However , the inhibition of IATP by nAChR activation was weaker than that of IACh by P2XR activation . As the IACh desensitized faster than the IATP , the weaker inhibition of IATP by ACh could be due to the reduced amplitude of the IACh at the time of ATP application . Fig. 4Inhibition of IACh and IATP , respectively , in the presence of ATP and ACh . A -RRB- Sample currents in response to ACh -LRB- 100 µM -RRB- in the presence of ATP -LRB- 50 µM -RRB- . The responses to ACh applied in the presence of ATP were reduced . The extent of occlusion was normalized to the control ACh responses recorded before and after ATP application . This sequence was routinely repeated twice or three times , and the averaged amount of occlusion was plotted in B. B -RRB- Relative amplitude of the ACh current amplitude in the presence of ATP -LRB- ordinate -RRB- plotted against the peak amplitude of IATP obtained in the same neuron -LRB- abscissae -RRB- . Note the negative correlation between these two variables . The line was drawn using KaleidaGraph -LRB- Synergy Software -RRB- . Vh : − 70 mV . C -RRB- The IATP was recorded in the presence of 100 µM ACh and this response was flanked by control ATP alone responses . This sequence was routinely obtained twice or three times in the same cell , and the averaged amount of occlusion was plotted in D. D -RRB- The ratio of the ATP current amplitude in the presence and absence of ACh -LRB- ordinate -RRB- is negatively correlated with the peak amplitude of IACh -LRB- abscissae -RRB- . Vh : − 70 mV . To examine this possibility , IATP was measured in the presence of both ACh and d-tubocurarine -LRB- dTc , 10 µM -RRB- or serotonin -LRB- 5HT , 50 µM -RRB- , two experimental conditions which change the time course of the IACh response . The former agent , dTc , has been shown to be both an open channel blocker and a competitive antagonist of nAChR -LRB- Manalis -LSB- 31 -RSB- -RRB- , and , as expected , a low concentration -LRB- 10 µM -RRB- slightly reduced the peak amplitude of IACh -LRB- 79 ± 4 % of control , N = 11 -RRB- and caused the IACh to be terminated within 0.5 s -LRB- Fig. 5A , insert -RRB- . Once the IACh response had returned to baseline -LRB- in the presence of ACh and 10 µM dTc -RRB- and ATP was applied , there was no inhibition of IATP -LRB- 95 ± 1 % of control , N = 5 ; Fig. 5A -RRB- . The IATP in the presence of ACh alone was 77 ± 4 % of control -LRB- N = 5 , P < 0.01 ; Fig. 5A -RRB- . When we measured the current -LRB- IACh + ATP + dTc -RRB- induced by a combination of ACh , ATP and dTc , the peak amplitude of IACh + ATP + dTc was occluded under these conditions , being 74 ± 2 % -LRB- N = 10 -RRB- of the predicted sum of IATP and IACh + dTc -LRB- Fig. 5B -RRB- . The level of IACh + ATP + dTc at 0.5 s following ligand application , which corresponds to a time when the nAChR channels are completely blocked -LRB- Fig. 5A , insert -RRB- , was 96 ± 3 % -LRB- N = 8 -RRB- of the control IATP , measured immediately before and after -LRB- Fig. 5C -RRB- . These results suggest that the inhibition of IATP by IACh disappears immediately after the IACh is abolished , although ACh is still present and bound to the nAChRs . 5HT has been shown to also accelerate the decay of IACh -LRB- Grassi et al. -LSB- 32 -RSB- ; Garcia-Colunga and Miledi -LSB- 33 -RSB- ; Sorimachi and Wakamori -LSB- 34 -RSB- -RRB- , but not to the same extent as dTc . 5HT at 50 µM decreased the peak amplitude of IACh slightly , to 88 ± 4 % -LRB- N = 6 -RRB- of control , and reduced the time constant -LRB- tau -RRB- of IACh decay from 2.02 ± 0.18 to 1.06 ± 0.19 s -LRB- N = 6 -RRB- . As shown in Fig. 5D , the extent of inhibition of IATP by ACh was smaller -LRB- to 92 ± 2 % of control , N = 6 -RRB- in the presence of ACh and 5HT compared to that in the presence of ACh alone -LRB- to 73 ± 3 % of control , N = 6 ; P < 0.01 -RRB- . Fig. 5Loss of the inhibition of IATP by ACh in the presence of dTc and 5HT . A -RRB- Representative IATP in control conditions -LRB- applied alone -RRB- , in the presence of ACh and in the presence of ACh and dTc . This sequence was routinely repeated twice , and the averaged response was obtained . Note the very transient nature of IACh in the presence of dTc , with the current returning to baseline levels within 0.5 s -LRB- insert -RRB- . Note also the lack of occlusion under these conditions . Vh : − 70 mV . B -RRB- Effect of dTc on IACh + ATP . The sequence of applications was repeated multiple times in each neuron . The peak amplitude of IACh + ATP + dTc was smaller than the predicted sum of IATP and IACh + dTc . C -RRB- Rapid recovery of IATP from inhibition by ACh in the presence of dTc . The amplitude of IACh + ATP + dTc 0.5 s following application was equal to the mean of two control IATP at an equivalent time . Inset shows the rapid decline of IACh + ATP + dTc on an expanded time scale . D -RRB- Representative IATP in control conditions , in the presence of ACh , and in the presence of both ACh and 5HT . In this example , the tau of IACh in response to the first application was 2.7 s , while the tau of IACh +5 HT was 0.9 s , showing the acceleration of the IACh decay by 5HT . Note the reduced occlusion under these conditions . Vh : − 70 mV . Next , we measured the current induced by the concomitant applications of ACh and ATP -LRB- IACh + ATP -RRB- at a Vh of − 70 mV . As shown in Fig. 6A , IACh + ATP was 75 ± 1 % -LRB- N = 44 ; P < 0.01 -RRB- of the predicted sum of IACh and IATP . The decay phase of IACh , IATP and IACh + ATP were fit to a single exponential function , giving tau of 3.0 ± 0.2 , 7.5 ± 0.6 and 3.7 ± 0.2 s , respectively . When the decay of IACh was accelerated by the presence of 100 µM 5HT , the tau of IACh , IATP , and IACh + ATP were 0.8 ± 0.3 s -LRB- N = 7 -RRB- , 7.4 ± 1.4 s -LRB- N = 7 -RRB- and 2.6 ± 0.8 s -LRB- N = 7 -RRB- , respectively -LRB- the amplitude of IACh + ATP under these conditions was 76 ± 2 % of the predicted sum of IACh and IATP -RRB- . Thus , IACh + ATP desensitization occurs with kinetics that can not be explained by the desensitization kinetics of IACh or IATP alone , suggesting that IACh + ATP is mediated via both receptors . Fig. 6Voltage dependence of IACh and of IATP occlusion . A -RRB- At a Vh of − 70 mV , co-application of ACh -LRB- 200 µM -RRB- and ATP -LRB- 100 µM -RRB- induced an inward current that was smaller in amplitude than the predicted sum of the individual IACh and IATP . B -RRB- At a Vh of +40 mV , the amplitude of the outward current induced by the co-application of ACh -LRB- 500 µM -RRB- and ATP -LRB- 200 µM -RRB- was equal to the predicted sum of the individual IACh and IATP . C -RRB- Co-application of ATP and ACh does not markedly change EACh or EATP . Representative currents induced by ACh , ATP , and by co-application of ACh and ATP at a Vh of +10 mV . Note that IACh + ATP is composed of early outward and delayed inward current , corresponding to the ACh component and the ATP component , respectively . When Ca2 + was omitted from extracellular solution , the negative interaction between the two receptor channels was still obtained ; with IACh + ATP being 77 ± 1 % -LRB- N = 5 ; P < 0.01 -RRB- of the predicted sum of IACh and IATP . Hence negative interaction is not mediated by a Ca2 + influx-dependent mechanism , although both nAChR and P2XR are Ca2 + - permeable cation channels -LRB- Fieber and Adams -LSB- 35 -RSB- ; Rogers and Dani -LSB- 36 -RSB- -RRB- . This result , however , does not necessarily rule out the possible involvement of -LSB- Ca -RSB- in in negative interaction . Occlusion was not only observed at a Vh of − 70 mV ; IACh + ATP at Vhs of − 20 , and − 10 mV were 80 ± 2 % -LRB- N = 12 ; P < 0.01 -RRB- , and 76 ± 2 % -LRB- N = 8 ; P < 0.01 -RRB- , respectively , of the predicted sum of IACh and IATP . In sharp contrast , such occlusion was not observed at a positive potential ; the outward current caused by coapplication of ACh and ATP at a Vh of +40 mV was 100 ± 1 % -LRB- N = 10 -RRB- of the predicted sum of individual current -LRB- Fig. 6B -RRB- . We next investigated the possibility that the combined addition of ACh and ATP alters the driving force for Na + , by attempting to measure EACh + ATP . In these studies , currents in response to ACh , ATP and both ligands were measured at various fixed potentials close to the reversal potential for IACh and IATP . The EACh and EATP were 8.7 ± 1.5 mV -LRB- N = 5 -RRB- and 21.0 ± 1.6 mV -LRB- N = 7 -RRB- , respectively , and IACh + ATP measured at a potential between these two reversal potentials -LRB- e.g. 10 mV , Fig. 6C -RRB- showed a combination of both the ACh-induced outward current and the ATP-induced inward current . These results suggest that the driving force for permeant ions during combined ACh and ATP is similar to that during the application of each ligand separately . The question arises as to whether occlusion is specific just for Na + ions or whether inward currents carried by cations other than Na + can also contribute to occlusion during co-activation of nAChRs and P2XRs . To address this question , we measured IACh , IATP , and IACh + ATP in the presence of Na + - free -LRB- NMDG + -RRB- saline containing 10 mM Ca2 + -LRB- Fig. 7 -RRB- . NMDG + does not permeate through nAChR -LRB- Sorimachi and Wakamori -LSB- 16 -RSB- -RRB- , and hence IACh would be mediated by only Ca2 + influx , whereas IATP would be mediated via both NMDG + and Ca2 + influx -LRB- Fig. 3 -RRB- . Under these conditions , IACh + ATP was again occluded , being 70 ± 3 % of the predicted sum of IACh and IATP -LRB- N = 11 ; P < 0.01 ; Fig. 7 -RRB- . Fig. 7Occlusion of IACh + ATP in the presence of Na + - free saline containing 10 mM Ca2 + . IACh , IATP or IACh + ATP was recorded 20 s after switching to Na + - free -LRB- replaced by NMDG + -RRB- saline containing 10 mM Ca2 + . The sequence of applications was repeated multiple times . Following each ligand application , the external solution was changed back to normal saline containing 150 mM Na + before the next ligand application . Vh : − 70 mV . In a further attempt to distinguish whether channel activation or ion permeation was primarily responsible for occlusion of one channel by the other , we compared IATP with IACh + ATP under two experimental conditions , in which a higher concentration of ACh should activate nAChR but produce very little current . In the absence of Na + and Ca2 + -LRB- replaced by NMDG + -RRB- application of ACh induces a negligible current -LRB- Sorimachi and Wakamori -LSB- 16 -RSB- -RRB- , while ATP induces a substantial current under the same conditions -LRB- Fig. 3A -RRB- . As shown in Fig. 8B , there was no occlusion in these conditions ; the amplitude of IATP+AC h was 105 ± 1 % -LRB- N = 10 -RRB- of the predicted sum of IATP and IACh . We also investigated currents in response to the ligands at a potential close to the EACh -LRB- 7 ± 2 mV in this experiment , N = 13 -RRB- , taking advantage of the more positive EATP -LRB- 22.7 ± 0.9 mV -RRB- , so that at this potential only the IATP was observed . The amplitude of IATP+AC h was 105 ± 3 % -LRB- N = 13 -RRB- of that of IATP , and IACh was close to zero -LRB- Fig. 8D -RRB- . These results are , however , in contrast to that obtained in sympathetic neurons -LRB- Nakazawa -LSB- 30 -RSB- -RRB- , in which IATP in the absence of both Na + -LRB- replaced by glucosamine -RRB- and Ca2 + was inhibited in the presence of ACh , which caused no current -LRB- Nakazawa -LSB- 30 -RSB- -RRB- . In these experiments , we only included data in which IATP was large enough to be clearly resolved , greater than 50 pA -LRB- corresponding to a control IATP at a Vh of − 70 mV larger than 1.5 nA -RRB- . These results suggest that actual current flow through both nAChR and P2XR is responsible for occlusion . Fig. 8Lack of inhibition in response to co-application of ACh and ATP under two experimental conditions designed to virtually eliminate IACh . A -RRB- Control IACh recorded at a Vh of − 70 mV in standard saline . B -RRB- Current responses recorded at − 70 mV from 20 s after exchanging the standard saline for a Na + - free -LRB- replaced by NMDG + -RRB- , Ca2 + - free saline . This sequence was repeated twice or three times . Note the small amplitude of IACh , and the lack of occlusion of IACh + ATP . C -RRB- Control IACh recorded at a Vh of − 70 mV in another neuron . D -RRB- Representative traces of IACh , IATP , and IACh + ATP recorded at a Vh of +7 mV , which is very close to EACh . Note the lack of occlusion at this potential , when current flow through the AChR is negligible . Discussion In this study , we found that ATP induced an inward current and increased -LSB- Ca -RSB- in in isolated rat AP neurons . The ATP-induced current and -LSB- Ca -RSB- in increase were mimicked by ATPγS and 2-methylthio-ATP , but not by α,β-methylene-ATP , β , γ-methylene-ATP nor ADP , and was inhibited by suramin and PPADS , suggesting that it was mediated by P2XRs . These results are in good agreement with previous histochemical findings , which demonstrated the presence of P2X2 , P2X4 , and P2X6 receptor mRNAs -LRB- Collo et al. -LSB- 37 -RSB- -RRB- , and of P2X2 receptor immunoreactivity in AP neurons -LRB- Kanjhan et al. -LSB- 38 -RSB- ; Atkinson et al. -LSB- 39 -RSB- -RRB- . The IATP showed strong inward rectification and the EATP was 22.7 ± 0.9 mV -LRB- Fig. 1B -RRB- . The amplitude of IATP varied inversely with the extracellular Ca2 + concentrations -LRB- Fig. 1C -RRB- . The inhibitory effect of increasing extracellular Ca2 + has also been reported in rat sensory neurons -LRB- Krishtal et al. -LSB- 40 -RSB- -RRB- , PC-12 cells -LRB- Nakazawa et al. -LSB- 18 -RSB- -RRB- , ventromedial hypothalamic neurons -LRB- Sorimachi et al. -LSB- 41 -RSB- -RRB- , and in cells expressing recombinant P2X2 receptors -LRB- Evans et al. -LSB- 42 -RSB- ; Virginio et al. -LSB- 43 -RSB- -RRB- , where an allosteric alteration of the ATP binding sites has been suggested to be the underlying mechanism . The IATP was markedly reduced , but still persisted when NMDG + was substituted for external Na + even in the absence of Ca2 + -LRB- Fig. 3A -RRB- . Since ATP did not induce a current when sucrose was substituted for external NaCl , our result suggests that NMDG + could permeate through P2XR . A substantial IATP has been similarly reported in PC-12 cells and sympathetic neurons when glucosamine was substituted for Na + -LRB- Nakazawa et al. -LSB- 18 -RSB- ; Nakazawa -LSB- 30 -RSB- -RRB- . The addition of Ca2 + to NMDG-Cl - and sucrose-substituted saline increased the IATP -LRB- Fig. 3B and C , respectively -RRB- , indicating that Ca2 + also permeates through P2XR channel . We quantified the relatively high Ca2 + permeability , obtaining a PCa/PNa ratio of 3.0 . A relatively high permeability of P2XR to Ca2 + has been reported in previous studies -LRB- Nakazawa et al. -LSB- 18 -RSB- ; Sorimachi et al. -LSB- 41 -RSB- ; Evans et al. -LSB- 42 -RSB- ; Virginio et al. -LSB- 43 -RSB- -RRB- . Although direct influx of extracellular Ca2 + through P2XR channel may thus contribute to the ATP-induced -LSB- Ca -RSB- in increase , membrane depolarization and the secondary activation of voltage-dependent Ca2 + channels could also make a significant contribution to the -LSB- Ca -RSB- in increase . In fact , the high K + - and ATP-induced -LSB- Ca -RSB- in increases were substantially inhibited by a range of Ca2 + channel antagonists , including those which block L - and N-type Ca2 + channels -LRB- Table 1 -RRB- . This is in contrast to a previous study , which reported an absence of the L - and N-type Ca2 + channels in rabbit AP neurons -LRB- Hay et al. -LSB- 12 -RSB- -RRB- . The discrepancies between their results and ours could be , at least in part , accounted for by the use of different AP preparations -LRB- cultured rabbit neurons vs. acutely dissociated rat neurons -RRB- . The present results , in combination with our previous demonstration of nAChR on AP neurons -LRB- Sorimachi and Wakamori -LSB- 16 -RSB- -RRB- , suggest that both ATP and ACh may act as excitatory neurotransmitters in AP neurons , although their release from presynaptic nerve terminals has not yet been reported . We also report a negative functional interaction between P2XRs and nAChRs in AP neurons , as has been observed in a variety of peripheral neurons and in recombinant expression systems -LRB- Nakazawa et al. -LSB- 18 -RSB- ; Nakazawa -LSB- 30 -RSB- ; Zhou and Galligan -LSB- 20 -RSB- ; Barajas-Lopez et al. -LSB- 21 -RSB- ; Searl et al. -LSB- 19 -RSB- ; Khakh et al. -LSB- 22 , 23 -RSB- ; Boue-Grabot et al. -LSB- 24 -RSB- -RRB- . This is the first report , that we are aware of , of such interactions occurring in central neurons . When ACh was applied in the presence of ATP , there was a positive correlation between the peak amplitude of inward IATP and the amount of occlusion of inward IACh -LRB- Fig. 4A and B -RRB- . The converse was also true when ATP was applied in the presence of ACh -LRB- Fig. 4C and D -RRB- . Non-additivity of the IACh + ATP was observed even when the inward current was carried by NMDG + and/or Ca2 + -LRB- Fig. 7 -RRB- . Co-application of two agonists did not seem to change the driving force for Na + , because at a Vh between EACh and EATP an outward current due to the activation of nAChR , followed by an inward current due to the activation of P2XR , was observed -LRB- Fig. 6C -RRB- . Occlusion was also observed at a Vh of − 10 mV , which is closer to EACh or EATP . Furthermore , the removal of external Ca2 + did not alter the occlusion , and thus a Ca2 + - mediated mechanism does not contribute to the current occlusion . The IACh + ATP occlusion was observed at all negative holding potentials when the current was inward but was not observed for outward currents at a Vh of +40 mV . Here the outward IACh + ATP was not different from the predicted sum of IACh and IATP -LRB- Fig. 6B -RRB- . The same dependence on current direction has been reported in guinea-pig enteric and submucosal neurons -LRB- Zhou and Galligan -LSB- 20 -RSB- ; Barajas-Lopez et al. -LSB- 21 -RSB- -RRB- , suggesting that the current occlusion was triggered by the inward movement of cations through two channels . Some investigators have shown that the amplitude of IACh + ATP is equal to or even smaller than that of either IACh or IATP . For instance , the concentration of one agonist causing little or no inward current produced dramatic occlusion of the inward current generated by the other agonist -LRB- Searl et al. -LSB- 19 -RSB- -RRB- . Khakh et al. -LSB- 22 -RSB- using Xenopus oocytes co-expressing P2X2 and α3β4 nAChR channels , provided several lines of evidence which indicated that occlusion was mostly mediated by the inhibition of the nAChR channel by activation of the P2XR . However , we found that concomitant application of two agonists always caused a larger current than either agonist -LRB- Fig. 6A -RRB- , and that the amount of occlusion of one channel current in the presence of the other channel agonist was correlated with the amplitude of current through the other channel -LRB- Fig. 4 -RRB- . Furthermore , we found that IACh + ATP desensitizes faster than IATP , but more slowly than IACh , and IACh + ATP at a Vh between EACh and EATP showed a combination pattern of early outward IACh and delayed inward IATP -LRB- Fig. 6C -RRB- . These results both strongly suggest that in AP neurons , IACh + ATP are carried through both nAChR and P2XR channels , and that inhibition between these channels is reciprocal . The IATP was not inhibited by co-application of ACh when the inward IACh was markedly reduced in the absence of permeant cations -LRB- Fig. 8B -RRB- or at a Vh very close to EACh -LRB- Fig. 8D -RRB- , and the inhibited IATP in the presence of ACh -LRB- Fig. 5B -RRB- recovered as soon as the nAChR channel closed in the presence of dTc -LRB- Fig. 5C -RRB- . These results thus suggest that the inhibitory interaction not only requires the activation of both receptor channels , but also requires a substantial current to flow through these channels . These results are in contrast to that obtained in sympathetic neurons , in which the ATP-induced glucosamine influx was inhibited in the presence of ACh -LRB- Nakazawa -LSB- 30 -RSB- -RRB- . The cause of this inconsistency remained unknown , but may reflect a real difference in the underlying mechanisms of occlusion in these two types of neurons . Altogether , our results have characterized the P2XR responses in AP neurons and the cross-inhibition between P2XRs and nAChRs in AP neurons . We show that the current flow through one receptor channel hinders the current flow through the other channel . These interactions support the notion that these channels are positioned very close to each other , as has been previously considered -LRB- Zhou and Galligan -LSB- 20 -RSB- ; Barajas-Lopez et al. -LSB- 21 -RSB- ; Khakh et al. -LSB- 22 -RSB- ; Boue-Grabot et al. -LSB- 24 -RSB- -RRB- and more recently demonstrated for recombinant channels -LRB- Khakh et al. -LSB- 23 -RSB- -RRB- . These results will be important to consider when designing ligands to modify excitability of ATP neurons and may have some physiological function during co-activation of P2XRs and nAChRs by synaptically released transmitters .

O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O B O B O O O O O B O B O O O O O O O O O O O B O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O

negative interaction between nicotinic and p2x channels