Anion exchanger and chloride channel in cat carotid body chemotransduction

Anion exchanger and chloride channel in cat carotid body chemotransduction

Journal of the Autonomic Nervous System 70 Ž1998. 23–31 Anion exchanger and chloride channel in cat carotid body chemotransduction Rodrigo Iturriaga ...

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Journal of the Autonomic Nervous System 70 Ž1998. 23–31

Anion exchanger and chloride channel in cat carotid body chemotransduction Rodrigo Iturriaga a

a,b

, Anil Mokashi a , Sukhamay Lahiri

a,)

Department of Physiology, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104-6085, USA b Laboratory of Neurobiology, Catholic UniÕersity of Chile, Santiago 1, Chile Received 30 June 1997; revised 12 January 1998; accepted 13 January 1998

Abstract y In order to test the hypothesis that carotid body ŽCB. chemoreception depends on the functions of anion channels and HCOy 3 rCl exchangers, we studied the effects of the anion channel blocker anthracene-9-carboxylic acid Ž9-ANC., the carbonic anhydrase inhibitor y methazolamide, and the HCOy exchanger blocker 4,4 diisothiocyanatostilbene-2-2X disulfonic acid ŽDIDS. on the chemosensory 3 rCl discharges of cat CB, perfused–superfused in vitro at 36.5 " 0.58C, with a modified Tyrode solution. The chemosensory responses to hypoxia ŽPO 2 f 50 Torr., hypercapnia ŽPCO 2 f 60 Torr, pH s 7.10., nicotine Ž2–4 nmol. and NaCN Ž20–40 nmol. were recorded. 9-ANC Ž2 mM. and DIDS Ž10 m M. decreased the chemosensory baseline activity, and eliminated the initial peak responses to hypercapnia and hypoxia and increased the time to achieve it. Methazolamide Ž0.13 mM. did not alter the effect of 9-ANC. The steady state responses to hypoxia and hypercapnia were not diminished after 9-ANC but DIDS lowered the responses. Responses to NaCN effects were all diminished but those to nicotine were not affected. The results suggest that the functions of anion channels and y exchangers are important for the resting dischargers and for the fast responses to hypoxia and hypercapnia. q 1998 Elsevier HCOy 3 rCl Science B.V. All rights reserved.

Keywords: Acid; 9-ANC; Cyanide; Chemosensory discharge; DIDS; Hypercapnia; Hypoxia; Methazolamide; Nicotine

1. Introduction The glomus Žtype I. cells in the carotid body ŽCB. presumably act as sensors of the chemical changes in their environment, initiating the chemoreception process by secreting one or more neurotransmitters that in turn generate the chemosensory discharge in the nerve terminals of the petrosal chemosensory neurons w7,8x. The chemosensory responses are well characterized w6,19x, but the underlying mechanisms of chemotransduction are not well understood and remain controversial, partly because of the difficulty in making recordings from the nerve endings and synaptic sites w7,10x. However, the chemosensory transmission in the CB is still the most consistent expression of chemoreception, and it can be used to study possible links between chemosensory discharge and cellular events in the CB. )

Corresponding author. Department of Physiology, B-400, Richard Bldg., School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085, USA. Tel.: q1 215 8989480; fax: q1 215 5735851. 0165-1838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 1 8 3 8 Ž 9 8 . 0 0 0 1 9 - 8

Several hypotheses of chemotransduction have been advanced w7,8,22,26x. According to the most prevalent membrane hypothesis, hypoxia and hypercapnia suppress the Kq current by binding with the membrane component, which depolarize the cells, leading to the opening of the Ca2q channel, and allowing Ca2q entry, thus enhancing the neurotransmitter release and neural discharge w8x. In this scenario, anion channels have not been directly implicated in the transduction of hypoxia and hypercapnia. However, these channels are involved in pH i Žintracellular pH. regulation, and hence, in the regulation of chemosensory activity. We w12,13x and others w28x have previously shown that perfusion of cat CB without CO 2 –HCOy 3 nearly lost sensitivity to hypoxia. Buckler et al. w5x, Stea and Nurse w30x and Wilding et al. w32x showed that rat glomus cell pH i increased on switching from CO 2 – Ž HCOy 3 -containing medium to one without at the pH o extracellular pH. of 7.4. Notwithstanding the species difference, the pH i regulation in glomus cells and intact carotid bodies indicate that the processes are similar.

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Buckler et al. w5x also showed that the rate of intracellular acidification in buffer containing CO 2 –HCOy 3 was considerably reduced by the membrane permanent carbonic anhydrase inhibitor. They also showed that adding DIDS Ž150 m M., an inhibitor of transmembrane bicarbonate movement, slowed down recovery of pH i of CB cells from acidity due to an NH 4 Cl prepulse. NH 4 Cl prepulse causes cellular-acidity increase after alkalinization. In the presence of CO 2 –HCOy 3 , Cl-free medium caused cellular alkalosis w5,32x, but the effect was abolished by DIDS Ž150 m M., promoting acidity rise. But DIDS often caused alkalosis inside CB cells in the presence of CO 2 –HCOy 3, inhibiting a background acid influx. This background acid y influx presumably worked via HCOy anion exchang3 rCl ers w5x. Thus, it was left uncertain as to which of the two effects of DIDS would predominate in the intact CB cells without wClyx o alteration. The anion channels in the cell membrane are insensitive to voltage changes, have a large conductance Ž296 pS., show selectivity for HCOy 3 and y . w29x. Cly ŽHCOy rCl s 0.7 , and are blocked by 9-ANC 3 y The blockade of the HCOy rCl exchanger with DIDS or 3 by the removal of external Cly made the rat glomus cell w x alkaline even in the presence of CO 2 –HCOy 3 5 , suggesting that, normally, there is a net efflux of HCOy 3 through y the HCOy rCl exchanger or anion channels. In the cat 3 CB preparation in vitro, a decrease of external wClyx from 110 to 10 mM by replacing it with gluconate decreased the baseline discharge, delayed the response to hypoxia and eliminated the initial peak response to acid hypercapnia w14,20x. The above observations suggest that anion channels and anion exchangers, increasing acidity in the glomus cell, may modulate the speed and amplitude of the chemosensory responses, although they may not be essential for chemoreception. We tested this hypothesis using y agents expected to block the function of the HCOy 3 rCl exchangers and anion channels in the whole CB in vitro with sensory discharge. We also studied the dependence of the responses on carbonic anhydrase because the CB pH i is also dependent of the enzyme function in the glomus cell w5x. We showed that functions of the anion channels y and HCOy exchangers are particularly critical for the 3 rCl resting discharge and for the fast responses to hypoxia and hypercapnia. Some of the results have been briefly reported w14,20x. 2. Methods 2.1. Carotid body preparations and protocols The experiments were performed on 19 CBs from 12 cats of either sex Ž2.5–3.8 kg., anesthetized with sodium pentobarbitone Ž35 mgrkg, i.p... The carotid bifurcation, including the CB, was exposed and the carotid sinus nerve ŽCSN. was sectioned at its junction with the glossopharyngeal nerve. The CBs were perfused and superfused as previously described w16x. The arteries originating from the

carotid bifurcation, except the ascending pharyngeal artery, were ligated and cut. The CB veins were identified and cut to allow perfusate outflow. The common carotid artery was cannulated and the preparation was perfused and superfused simultaneously with a modified Tyrode solution ŽPCO 2 s 29.8 " 0.7 Torr at pH s containing CO 2 –HCOy 3 7.39 " 0.01, equilibrated with PO 2 of 124.7 " 3.4 Torr for perfusate and PO 2 of - 25 Torr for superfusate.. The composition of the modified Tyrode solution was Žin mM. NaCl 114; KCl 4.7; CaCl 2 2.2; MgCl 2 1.1; Na-glutamate 22.0; NaHCO 3 21.4; HEPES 5.0; and glucose 5.0 and dextran 5 grl. The CBs were perfused by gravity at a constant hydrostatic pressure of about 80 Torr, and superfused at a flow of about 1.5 mlrmin. The temperature of the fluid in the chamber was maintained at 36.5 " 0.58C with a heated water jacket. The chemosensory discharge was recorded from the whole desheathed CSN. The CSN was placed on a pair of platinum electrodes and lifted into paraffin oil. The effluent was maintained by a vacuum system at a constant level sufficient to cover the CB. The electroneurogram was fed to a differential AC-preamplifier and amplifier system, provided with a band pass Ž10 Hz – 1 kHz. and a notch filter Ž60 Hz.. The amplified signals were displayed on an oscilloscope, and stored in a digital recording VCR system for later analysis. The electroneurogram was displayed later in an electrostatic recorder, ES 1000 ŽGould Instruments, OH, USA.. Actions potentials above the baseline noise were selected with an amplitude window discriminator ŽFrederick Haer, USA.. The resulting standardized pulses were counted by a frequency meter and printed. The carotid chemosensory responses were studied during perfusion with Tyrode solution and during the following experimental conditions: Ž1. perfusion of Tyrode containing 2 mM 9-ANC for 30 min to block anion channels w9,29x in 7 CBs; Ž2. perfusion of Tyrode containing 2 mM 9-ANC plus 0.13 mM methazolamide for 30 min to block carbonic anhydrase w15,21x in 4 CBs; Ž3. perfusion with Tyrode containing 10 m M DIDS for 30–90 min to block y HCOy exchanger w2,5x in 5 CBs. 3 rCl The carotid chemosensory responses were assessed during perfusion with Tyrode solution Žcontrol., during the experimental conditions, and then 30–60 min after washout with Tyrode solution Žrecovery.. The protocols were the following: Ž1. perfusion with hypoxic ŽPO 2 f 50 Torr. and normocapnic Tyrode solution for 1–2 min; Ž2. perfusion with acid hypercapnic ŽPCO 2 f 60 Torr at pH s 7.10. and normoxic Tyrode solution for about 2 min; Ž3. bolus injections into the perfusate line of saline solution ŽNaCl 9 grl, 0.2 ml. with and without NaCN Ž20–40 nmol. or nicotine bitartrate Ž2–4 nmol.. 2.2. Statistical analysis The results are expressed as mean " S.E.M. The baseline chemosensory discharges, and the half-time and ampli-

R. Iturriaga et al.r Journal of the Autonomic NerÕous System 70 (1998) 23–31

tude of the chemosensory responses were statistically analyzed. Half-time is defined as the time required for halfmaximal response from the stimulus application. A part of this time included the transit time of the stimulus in the tubing system. The statistical difference for two groups of paired data was assessed by the nonparametric tests of Wilcoxon. The Friedman test Žnonparametric equivalent of a Two-way ANOVA. was used for comparison of several related samples and was followed by the Conover test for

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multiple comparisons between groups w31x. Differences were considered significant at 95% confidence level Ž P 0.05..

3. Results The effects of 9-ANC are illustrated in Fig. 1, and then the mean results are given in Tables 1 and 2. Fig. 2

Fig. 1. Effects of 9-ANC Ž2 mM. and methazolamide Ž0.13 mM. on the chemosensory response to hypoxia ŽPO 2 f 50 Torr, left-hand panel. and acid hypercapnia ŽPCO 2 f 55 Torr at pH 7.1, right-hand panel.. ŽA. Perfusion of Tyrode. ŽB. Perfusion of Tyrode containing 9-ANC. ŽC. Perfusion of Tyrode containing 9-ANC and methazolamide. ŽD. Recovery. Bars indicate duration of hypoxic and hypercapnic perfusate.

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Table 1 Effects of 9-ANC Ž2 mM. on the carotid chemosensory responses to hypoxia and hypercapnia Condition

Control 9-ANC Recovery

Hypoxia

Hypercapnia

Bas fx Žimprs.

Max fx Žimprs.

ss fx Žimprs.

Half-time Žs.

Max fx Žimprs.

ss fx Žimprs.

Half-time Žs.

45.2 " 9.0 3.8 " 1.2 ) 45.6 " 1.7

361.2 " 34.7 270.4 " 36.9 ) 397.0 " 35.7

322.7 " 33.2 270.4 " 36.5 330.6 " 30.0

7.9 " 2.0 36.5 " 8.4 ) 9.3 " 1.1

312.4 " 29.2 212.0 " 44.5 ) 346.2 " 39.5

217.4 " 30.4 207.8 " 42.5 243.9 " 39.7

7.0 " 1.1 25.3 " 5.4 ) 6.6 " 1.2

Values are the mean " S.E.M. of 7 CBs. Bas fx: baseline chemosensory activity. Max fx: maximal chemosensory activity. ss fx: semi steady-state response measured at the end of the maneuvers. During perfusion without 9-ANC, PO 2 was 131.2 " 4.0 Torr, PCO 2 29.6 " 3.9 Torr, and pH s 7.39 " 0.01 and during perfusion with Tyrode with 9-ANC, PO 2 was 128.4 " 3.9 Torr, PCO 2 29.4 " 0.5 Torr, and pH s 7.38 " 0.01. Hypoxia was PO 2 s 50.9 " 1.3 Torr, PCO 2 s 29.5 " 0.8 and pH s 7.39 " 0.01. Hypercapnia was PCO 2 s 53.5 " 1.5 Torr, pH s 7.09 " 0.02 and PO 2 s 122.3 " 2.5 Torr. Significant differences from the control were assessed by Conover test for multiple comparison after the Friedman test Ž ) P - 0.01..

illustrates the effects of DIDS, and Table 3 summarizes the effects of DIDS. The mean effects of nicotine and NaCN injections with and without 9-ANC and DIDS are described, respectively, in Figs. 3 and 4. 3.1. Effects of 9-ANC on the baseline carotid chemosensory actiÕity and responses to hypoxia and hypercapnia Fig. 1 shows the effects of 9-ANC Ž2 mM. on carotid chemosensory responses to hypoxia ŽPO 2 f 50 Torr. and to acid hypercapnia ŽPCO 2 f 55 Torr at pH 7.15.. In the control, the chemosensory discharge increased rapidly to maximal levels, which showed a small adaptation to hypoxia and a large to hypercapnia ŽFig. 1A.. Perfusion with Tyrode solution containing 9-ANC significantly reduced the chemosensory baseline activity immediately, eliminated and slowed down the initial peak responses to both

hypoxia and hypercapnia, and delayed the onset of the responses. However, the late steady-state responses to hypoxia and hypercapnia were hardly affected ŽFig. 1B.. The effect of 9-ANC was reversible within 30 min of washout Žnot shown.. Table 1 summarizes the chemosensory responses from 7 CBs. Administration of 9-ANC reduced the chemosensory baseline from 45.2 " 9.0 imprs to 3.8 " 1.2 imprs Ž P 0.01., eliminated the initial peak responses Ž P - 0.01. but did not affect the late responses Ž P ) 0.05., and delayed the responses to hypoxia and hypercapnia Ž P - 0.01.. 3.2. Effect of 9-ANC and methazolamide on the carotid chemosensory actiÕity and responses to hypoxia and hypercapnia In separate experiments Ž n s 4 CBs. 9-ANC effects were recorded first, and then methazolamide Ž0.13 mM.

Table 2 Effects of 9-ANC Ž2 mM. and methazolamide on the carotid chemosensory responses to hypoxia and hypercapnia Condition

Control 9-ANC q MET Recovery

Hypoxia

Hypercapnia

Bas fx Žimprs.

Max fx Žimprs.

ss fx Žimprs.

Half-time Žs.

Max fx Žimprs.

ss fx Žimprs.

Half-time Žs.

50.1 " 8.5 4.0 " 2.5 ) 3.0 " 1.9 ) 70.0 " 15.0

391.9 " 22.3 336.5 " 40.3 ) 335.2 " 43.8 ) 428.3 " 19.3

345.1 " 30.1 323.1 " 48.9 332.9 " 44.9 327.2 " 50.0

6.8 " 1.5 22.8 " 3.0 ) 31.5 " 5.1) 5.8 " 1.1

350.6 " 29.7 288.9 " 48.3 ) 276.8 " 48.4 ) 387.6 " 30.6

277.4 " 32.7 279.6 " 44.8 266.7 " 49.5 318.3."36.9

6.3 " 1.3 26.0 " 3.0 ) 30.5 " 3.3 ) 4.3 " 1.6

Values are the mean " S.E.M. of 4 CBs. Bas fx: baseline chemosensory activity. Max fx: maximal chemosensory activity. ss fx: semi steady-state chemosensory response, measured at the end of the maneuvers. During perfusion without 9-ANC, PO 2 was 133.0 " 2.3 Torr, PCO 2 30.5 " 0.9 Torr, and pH 7.38 " 0.01. During perfusion with Tyrode containing 9-ANC, PO 2 was 130.7 " 5.0 Torr, PCO 2 30.0 " 0.4 Torr and pH 7.37 " 0.01. During perfusion with Tyrode containing 9-ANC and methazolamide, PO 2 was 134.0 " 54.9 Torr, PCO 2 29.8 " 0.8 Torr, and pH 7.36 " 0.01. Hypoxic perfusion was PO 2 s 50.5 " 1.1 Torr, PCO 2 s 29.1 " 1.1 and pH 7.37 " 0.01. Hypercapnic perfusion was PCO 2 s 53.3 " 3.2 Torr, pH s 7.10 " 0.05 and PO 2 s 122.5 " 5.8 Torr. Significant differences from the control were assessed by Conover test for multiple comparison after the Friedman test Ž ) P - 0.01..

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Fig. 2. Effects of DIDS Ž10 m M. on the carotid chemosensory response to acidic hypercapnia ŽPCO 2 f 55 Torr at pH 7.1, left-hand panel. and to hypoxia ŽPO 2 f 50 Torr, right-hand panel.. ŽA. Control Tyrode. ŽB. Tyrode containing DIDS. Bars indicate duration of hypercapnic and hypoxic perfusates.

was administered in addition to 9-ANC Ž2 mM. in the perfusate. Methazolamide slightly delayed the onset of the responses to both stimuli, the effect being more prominent in hypercapnic response. The late peak responses were not significantly affected ŽFig. 1C.. Upon removal of 9-ANC and methazolamide Ž30 min. both the responses to hypoxia and hypercapnia were exaggerated ŽFig. 1D.. These exaggerated responses were similar to those seen in increased wCa2q x i response to anoxia w1x after returning the glomus cells from zero wCa2q x o to 2 mM wCa2q x o . Table 2 summarizes the characteristics of the combined effects of 9-ANC and methazolamide. The half-time of the hypoxic response increased further with methazolamide from 22.8 " 3.0 to 31.5 " 5.1 s Ž P - 0.05. and the half-time of the hypercap-

nic response increased from 26 " 3.3 to 30.5 " 3.3 s Ž P 0.05.. The late responses to hypoxia and hypercapnia were not further reduced by methazolamide. 3.3. Effects of DIDS on the carotid chemosensory responses to hypoxia and hypercapnia Fig. 2 shows the effects of DIDS Ž10 m M. on the chemosensory responses to acid hypercapnia and hypoxia. Application of DIDS reduced the chemosensory baseline activity within 30 s. The overall response to acid hypercapnia was reduced, initial burst of activity was slow, but the pattern of the response Žpeak followed by adaptation. remained the same ŽFig. 2B, left-hand panel.. The response

Table 3 Effects of DIDS Ž10 m M. on the carotid chemosensory responses to hypoxia and hypercapnia Condition

Control DIDS

Hypoxia

Hypercapnia

Bas fx Žimprs.

Max fx Žimprs.

ss fx Žimprs.

Half-time Žs.

Max fx Žimprs.

ss fx Žimprs.

Half-time Žs.

42.9 " 6.7 6.1 " 2.2 )

320.6 " 23.5 128.6 " 26.6.3 )

249.9 " 26.3 93.2 " 22.3 )

8.8 " 1.8 23.6 " 9.4 )

294.6 " 46.9 195.4 " 11.4 )

185.1 " 25.8 128.8 " 14.9 )

4.8 " 1.0 13.8 " 3.0 )

Values are the mean " S.E.M. of 5 CBs. Bas fx: baseline chemosensory activity. Max fx: maximal chemosensory activity. ss fx: semi steady-state chemosensory response measured at the end of the maneuvers. During perfusion without DIDS, PO 2 was 120.2 " 9.5 Torr, PCO 2 s 29.5 " 1.1 Torr, and pH 7.39 " 0.01. During perfusion with Tyrode containing DIDS, PO 2 was 110.8 " 7.6 Torr, PCO 2 s 30.2 " 1.0 Torr, and pH 7.39 " 0.01. Hypoxic perfusion was PO 2 s 53.0 " 1.2 Torr, PCO 2 s 29.6 " 1.3, and pH 7.37 " 0.01. Hypercapnia was PCO 2 s 57.8 " 1.9 Torr, pH s 7.11 " 0.03 and PO 2 s 121.6 " 2.1 Torr. Significant differences from the control were assessed by the Wilcoxon test Ž ) P - 0.01..

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chemosensory baseline activity ŽFig. 3A. was drastically reduced by 9-ANC, but the minimal activity could not be reduced further with methazolamide Ž0.13 mM.. The maximal response ŽFig. 3B. to nicotine Ž2–4 nmol. was reduced but not delayed. The response to NaCN Ž20–40 nmol. was reduced and delayed by 9-ANC. Application of methazolamide did not further reduce or delay the response to nicotine but further delayed the chemosensory response to NaCN ŽFig. 3C.. Following Tyrode perfusate, free of 9-ANC and methazolamide for 20 min, both responses were recovered Žnot shown..

3.5. Effects of DIDS on the carotid chemosensory responses to nicotine and NaCN Fig. 4 summarizes the effects of DIDS Ž10 m M.. The chemosensory baseline responses were reduced, and the maximal chemosensory response to NaCN were reduced and delayed, but not to nicotine.

Fig. 3. Summary of the effects of 2 mM 9-ANC Ž ns6 CBs. and 2 mM 9-ANC q0.13 mM methazolamide Ž ns 4 CBs., on the chemosensory responses to 2–4 nmol of nicotine boluses injection Žleft-hand panels. and to 20–40 nmol NaCN Žright-hand panels.. ŽA. Baseline carotid chemosensory activity. ŽB. Maximal carotid chemosensory activity. ŽC. Half-time of the response. Control, open bars; 9-ANC, hatched bars; 9-ANC plus methazolamide, cross hatched bars; recovery, solid bars. Ž ) P - 0.01, Conover test after Friedman test..

to hypoxia, however, was strikingly reduced, and the initial peak response was eliminated ŽFig. 2B, right-hand panel.. Note that DIDS also abolished the undershoot in the response after the stimulus withdrawal. The effects of DIDS at low concentration Ž10 m M. for 10–15 min were reversible, but prolonged perfusion, even at this low concentration, finally suppressed the chemosensory responses to hypoxia and hypercapnia. Perfusion of Tyrode containing DIDS at higher concentrations Ž0.1–1 mM. further reduced or eliminated all the responses Ž n s 3, not shown.. Table 3 summarizes the effects of DIDS Ž10 m M. for 20 min on the carotid response to hypoxia and acid hypercapnia. The baseline activity was significantly reduced. The mean maximal responses to hypoxia and hypercapnia were reduced significantly. The half-time of the hypoxic response increased from 8.8 " 1.8 s to 23.6 " 9.4 s Ž P - 0.01. and that of the hypercapnic response increased from 4.8 " 1.1 s to 13.8 " 3.0 Ž P - 0.01, n s 5.. 3.4. Effects of 9-ANC on the carotid chemosensory responses to nicotine and NaCN Fig. 3 shows the effects of 9-ANC and methazolamide on responses to nicotine and cyanide Ž n s 6 CBs.. The

Fig. 4. Summary of the effects of DIDS Ž10 m M, ns 4. on the chemosensory responses to 2–4 nmol of nicotine Žleft-hand panels. and to 20–40 nmol NaCN Žright-hand panels.. ŽA. Baseline carotid chemosensory activity. ŽB. Maximal carotid chemosensory activity. ŽC. Half-time of the response. Control, open bars; DIDS, cross-hatched bars Ž ) P - 0.01, Wilcoxon test..

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4. Discussion The essential results are summarized as follows, with Žy. indicating a significant decrease, Žq. a significant increase, and Ž0. no change: 9-ANC Ž2 mM. Baseline Max Žimprs. discharge rate Žflux. Žimprs. Hypoxia y y Hypercapnia y y NaCN y y Nicotine y y DIDS (10 m M) Hypoxia y Hypercapnia y NaCN y Nicotine y

y y y 0

Steady-state discharge rate Žflux. Žimprs. 0 0

Half-time of max discharge Žs. q q q 0

y y

q q q 0

4.1. Effects of 9-ANC and DIDS on baseline actiÕity, and hypoxic and hypercapnic responses Baseline discharges were significantly inhibited by 9ANC and DIDS. Maximal peak responses to hypoxia, hypercapnia, cyanide and nicotine were all Žexcept for DIDS on nicotine. inhibited. Half-time of responses to all the stimuli except for nicotine increased. That is, 9-ANC and DIDS caused a decrease of acid influx, making the cells alkaline, which in turn reduced the activity. The effects of DIDS promoting acidity increase was not evident in these results. That is, inhibition of background acid influx by DIDS dominated the response. Permeable inhibitors of carbonic anhydrase produced alkalinization of rat glomus cells w5x, and delayed the CB responses to hypercapnia and hypoxia in vitro w15,21x. Thus, the results of this study is consistent with those results suggesting that CO 2 –HCOy 3 , at pH o of 7.4, maintains a relative acid pH i by CO 2 hydration and HCOy 3 y extrusion through HCOy exchangers and anion 3 rCl y channels. Blockade of HCOy exchanger and anion 3 rCl channels is equivalent to removal of CO 2 –HCOy 3 buffer in vitro, which makes the cells alkaline w5x. On the basis of a reduced baseline activity, the responses to the excitatory stimuli were expected to be diminished. However, this was not the case for all instances and there were differences in the response to different stimuli. For instance, 9-ANC Ž2 mM. eliminated the initial rapid responses to hypoxia and hypercapnia, but not the steady-state responses. Presuming a membrane potential for glomus cell in the range of y50 to y55 mV, the anion channels would allow a net efflux of HCOy 3 and could be a major pathway for acid-equivalent entry into the cell. Also, Cly conduc-

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tance does contribute to the resting membrane potential and hence to the process w24x. The resulting alkalinization following anion channel blocker may neutralize the initial rise of Hq due to hypercapnia. Methazolamide delayed the onset and reduced the rate of rise of the chemosensory responses only slightly more to hypercapnia and hypoxia than with 9-ANC alone. The lack of effect of methazolamide on the late responses is expected because, in the presence of CO 2 –HCOy 3 , methazolamide does not affect the equilibrium reaction; only the rate of reaction is affected w21x. In the absence of CO 2 –HCOy 3 , methazolamide did not reduce the baseline chemosensory activity in the cat CB in vitro preparation w15x. DIDS did not eliminate, although slowed down the rate of response to hypercapnia, but the response to hypoxia was strikingly reduced. The reason for the differential response is presumably due to the fact that their mechanisms of responses are different. The irreversibility of the effects of DIDS agrees with the known effects of the disulfonic stilbene derivates Žsuch as DIDS., which covalently bind the extracellular domain of the protein and irreversibly blocked the anion transport through the membrane w18x. Panisello and Donnelly w25x also briefly reported that the chemosensory discharge to transient hypoxia was diminished by 9-ANC Ž5 mM. and DIDS Ž100 m M., agreeing with our observation on maximal response. But they used much higher concentrations of the pharmacological agents, which could explain more intense effects. 4.2. Effects of cellular H q during hypoxia The cellular alkalinity produced by the blockade of the y HCOy exchanger and the anion channel made the 3 rCl chemoreception less responsive, presumably by generating lesser amount of a second messenger, like Ca2q. Recently, Buckler and Vaughan-Jones w3,4x reported that both hypoxia and hypercapnia increased wCa2q x i in isolated rat glomus cells. This is consistent with the observations of Sato w27x. Hypercapnia simultaneously increased CB tissue acidity and chemosensory discharge while hypoxia only increased the chemosensory discharge without pH i change w17x. Moreover, Mokashi et al. w23x and He et al. w11x showed that hypercapnia reduced the pH i in the cat and rat glomus cells, respectively, whereas hypoxia either did not show a pH i change w23x, or caused inconsistent changes w11x. Accordingly, these results do not agree with the hypothesis that hypoxic chemotransduction is initiated by chemoreceptor cell acidosis. 4.3. Effects on the nerÕe endings Part of the effects of anion channels blocker 9-ANC, y and HCOy exchangers blocker DIDS on CB 3 rCl chemosensory discharge can be attributed to a likely pH i increase in the chemoreceptor nerve endings. We measured

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only the neural discharges, which included the effects of the blockers on the nerve endings also. The blockers could have impaired the pH i regulation not only in the glomus cells, but also in the sensory nerve terminals. We cannot rule out that part of the effects reported here which could be attributed to changes in the nerve endings in the same direction.

w5x

w6x

w7x

4.4. Effects of 9-ANC and DIDS on NaCN and nicotine responses The effects of these agents on NaCN responses are similar to those on hypoxia and hypercapnia, and are explained by alkalinity produced by these agents. Nicotine effects on chemosensory discharge were not affected by DIDS. Also, nicotine effects were not delayed by 9-ANC. These are distinct from the effects of cyanide, indicating different mechanisms of effects.

5. Conclusion The present results support the hypothesis that the y exchanger and the Cly channel in the glomus HCOy 3 rCl cells contributed to the pH i regulation by extruding bicarbonate. The cellular alkalinization caused by blocking the Cly and HCOy 3 transport through channels and exchangers delayed the responses to hypoxia and hypercapnia but did not prevent chemotransduction. The rapidity of the initial responses are dependent on the normal movement of these ions across the plasma membrane. Suppressions of Kq currents by hypoxia w22x and hypercapnia w26x may mediate the same membrane response but hypoxia does not cause cellular acidosis w23x, whereas hypercapnia does w5,23,32x. Thus, the delayed and depression of responses to hypoxia and hypercapnia by 9-ANC and DIDS cannot be explained by suppression of Kq currents.

w8x

w9x w10x

w11x

w12x

w13x w14x

w15x

w16x

w17x

w18x w19x

Acknowledgements w20x

We are grateful to Ms Mary Pili for her secretarial assistance. This work was supported in part by NIH grants HL-50180-3, HL-43413-8, and T-32-HL-07027-23.

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