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Suppression of glomus cell K+ conductance by 4-aminopyridine is not related to [Ca2+]i, dopamine release and chemosensory discharge from carotid body
Brain Research 785 Ž1998. 228–235
Research report
Suppression of glomus cell Kq conductance by 4-aminopyridine is not related to wCa2qx i , dopamine release and chemosensory discharge from carotid body Arijit Roy a , Charmaine Rozanov a , Donald G. Buerk
b,c
, Anil Mokashi a , Sukhamay Lahiri
a, )
a Department of Physiology, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104, USA Department of Bioengineering, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104, USA Institute for EnÕironmental Medicine, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104, USA b
c
Accepted 14 October 1997
Abstract The hypothesis that suppression of O 2-sensitive Kq current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine Ž4-AP., a known suppressant of Kq current, on intracellular wCa2q x i , dopamine secretion and chemosensory discharge in cat carotid body ŽCB.. In vitro experiments were performed with superfused–perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP Ž0.2, 1.0 and 2.0 mM in CO 2-HCO 3- buffer. and recording wCa2q x i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in wCa2q x i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered wCa2q x i by 4-AP are not consistent with the implied meaning of the suppressant effect on Kq current of glomus cells. q 1998 Elsevier Science B.V. Keywords: Carotid body; Intracellular calcium; Dopamine; Perfusion; Hypoxia; Kq–O 2 current
1. Introduction Glomus cells Žtype I. of the carotid body ŽCB. are excitable, and the cell membrane contains voltage-dependent Naq, Kq and Ca2q channels w19,32x. Of these channels, the conductance of Kq channels can be inhibited by low PO 2 when glomus cells are artificially depolarized in voltage clamp experiments, positive to resting membrane potential w22,27,30x. This event is supposed to result in opening of voltage-gated Ca2q channels and Ca2q entry, release of neurotransmitters and excitation of the afferent carotid sinus nerves ŽCSN. which synaptically connect with the glomus cells, forming a chemoreceptor unit. But Kq current properties are not the same in different species. In adult rabbits, Kq current inhibition by hypoxia is )
Correspondence author. Department of Physiology, B-400 Richards Bldg., University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, USA. Fax: q1-215-573-5851; E-mail: [email protected]. upenn.edu 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 2 7 6 - 6
insensitive to calcium w12,22x; in rats, it is calcium dependent w27,33x, and in cats, these cells show both calcium sensitive and insensitive Kq currents w7x. Despite all these differences, the suppression of O 2-sensitive Kq currents is postulated to be the key event in cellular excitation w4,7,12,14,19,21,25,30,32,33 x. In a detailed study Lopez-Lopez et al. w22x showed that 4-aminopyridine Ž4-AP. suppressed the PO 2-sensitive Kq currents in rabbit carotid body glomus cells up to 1 mM in a dose dependent manner. In neonatal rat carotid body, Peers and O’Donnell w26x have shown a reduction in Kq current with 2 mM 4-AP. In a recent study, Chou and Shirahata w7x showed that O 2 sensitive Kq current in cat glomus cells was also inhibited by 4-AP Ž10 mM.. Therefore, if 4-AP suppressed Kq current in different species, then it is expected to excite the glomus cells leading to wCa2q x i rise, neurosecretion and sensory excitation. We tested the hypothesis on perfused–superfused cat carotid body by measuring simultaneously dopamine release and chemosensory discharge without and with 4-AP Ž0.2, 1.0
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and 2 mM.. We found that 4-AP inhibited systematically the chemosensory discharge during normoxia, and did not alter the effects of hypoxia and perfusate flow interruption. On the other hand, 4-AP Ž1 mM. decreased the tissue dopamine release with normoxia and hypoxia. Also, 4-AP Ž1 mM. did not evoke any rise of wCa2q x i in isolated cat and rat glomus cells with and without hypoxia. Thus, contrary to expectation, 4-AP did not increase wCa2q x i and dopamine release nor did it increase sensory discharge.
2. Materials and methods 2.1. Carotid body isolation Experiments were performed in 16 carotid bodies from eight cats of either sex Ž2.5–3.9 kg., anaesthetized with sodium pentobarbital Ž35 mgrkg, i.p., initially.. Carotid bodies were sequentially removed for the study in a perfusion–superfusion system, as described previously w16x, and given here briefly. Carotid bodies from two cats were used for cell separation for calcium studies. Also four rats of either sex Ž300–350 g., anaesthetized with sodium pentobarbital Ž90–100 mgrkg., were used for carotid body cell separation for calcium studies. 2.2. Solutions A modified Tyrode buffer containing ŽmM. 112 NaCl, 4.7 KCl, 2.2 CaCl 2 , 1.1 MgCl 2 , 21.4 NaHCO 3 , 5.0 N-2hydroxyethyl-piperazine-N X -2 ethanesulfonic acid ŽHEPES., 5.0 glucose and 22.0 sodium glutamate and 4 grl of dextran Žmol wt. 74,200. were used to perfuse the carotid body. The normoxic and hypoxic solutions were equilibrated by bubbling with compressed gases containing 5% of CO 2 in 21% of O 2 ŽPCO 2 f 35 Torr, PO 2 110–120 Torr. and 5% of CO 2 in 5% of O 2 ŽPCO 2 f 35 Torr, PO 2 39–43 Torr.. The pH was adjusted to f 7.4. A stock solution of 4-AP was prepared in Tyrode buffer, and diluted to 0.2, 1.0 and 2 mM. 2.3. Carotid chemosensory actiÕity measurement A section of the carotid artery bifurcation with the intact carotid body attached to the carotid sinus nerve was cannulated, removed and mounted in a perfusion chamber. The isolated carotid body was perfused and superfused from both at a constant pressure Žabout 80 Torr., temperature of which, along with the perfusion chamber, was maintained by circulating warm water at 37 " 0.58C. The perfusion could be switched from normoxic to hypoxic solutions and vice versa. The effluent from the chamber was removed by continuous suction, thus maintaining a constant fluid level. The whole carotid sinus nerve was desheathed, placed on a platinum electrode and lifted up into paraffin oil layer for electrical isolation. The neural
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discharge signals were passed through a differential amplifier and a notch filter, and an electronic amplitude discriminator counted the impulses with a frequency meter and printed every 10 s. An analog neural discharge signal from the frequency meter was also tape recorded. 2.4. Measurement of dopamine (DA) Dopamine was measured by DA microsensors positioned into carotid body tissue by a motorized, microdriven, piezoelectric head polarized at q150 mV relative to a grounded Ag–AgCl reference, in contact with the carotid body preparation w5x. There were minor interferences from epinephrine and norepinephrine Ž- 10% compared to dopamine. at this potential w5x. Microelectrode current was amplified with a sensitive picoammeter and recorded in a VCR tape. Calibrations were made before and after each experiment. Tissue dopamine levels were measured before and during application of 4-AP at normoxia, hypoxia and momentary flow interruption Ž n s number of observations from 7 CBs.. 2.5. Measurement of intracellular calcium, [Ca 2 q ]i wCa2q x i in isolated glomus cells were studied separately in two cats and four rats. The glomus cells were separated enzymatically as described before w23x. Briefly, the carotid bodies were identified, surgically removed and kept in an ice-cold growth medium. After the removal of excessive connective tissue, the carotid bodies were transferred to 1 ml of Ca2q- and Mg 2q-free phosphate buffer ŽpH 7.4.,containing 0.1% collagenase Žtype IV, Sigma Chemicals. and was digested for 20 min for rat CB and 30 min for cat CB in a water bath at 378C with continuous bubbling of humidified 100% O 2 . The digested tissue was then transferred to the growth medium that contained 90 parts Ham F-12 Žcontaining NaHCO 3 . and 10 parts of fetal bovine serum fortified with 10 mg of streptomycin, 10,000 U of penicillin G and 80 U of insulin, and was triturated with a fire polished Pasteur pipette. The cells were plated on a 18 mm coverslip, and kept undisturbed for a period of 36 h in a Petri dish in a humidified incubator in which 5% CO 2 and air were circulated at 378C. The cells were then loaded with a fluorescent probe, Indo-1-AM Ž10 m M, Molecular Probes Inc.., in HEPES buffer ŽpH s 7.2. for a period of 30 min at room temperature Žabout 258C. prior to the start of an experiment. The coverslip with cells attached formed the bottom part of the chamber Žvolume capacity 125 m l., through which the buffer flowed. The chamber was attached to an inverted fluorescent microscope ŽFluovert– Leitz.. The cells in a selected field were excited at 340 nm with xenon-arc light source Ž75 W. that was fitted with a neutral density filter Ž10%.. The output emission signals were recorded with a ratio fluorometer equipped with two photomultiplier tubes that were attached near the eye-piece ŽBiomedical Instrumentation Group, University of Penn-
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sylvania.. The fluorescent analog signals at 405 nm and 495 nm and the ratio Ž405:495. were simultaneously recorded on a multichannel recorder ŽLinear Instruments..
The peak digital ratio signals of fluorescence change were also noted manually. The cells were superfused during hypoxia by a syringe-infusion pump ŽSage Instruments.
Fig. 1. Effects of 4-AP on carotid sinus nerve discharge during perfusion with normoxia ŽNx. and hypoxia ŽHx.. ŽA. Effect of 0.2 mM 4-AP. CSN activities during normoxia wPO 2 f 113 Torrx followed by hypoxia wPO 2 f 43 Torrx and back to normoxia. Perfusion with 0.2 mM 4-AP caused suppression of activity during normoxia but had no effect during hypoxia. ŽB. Effect of 1.0 mM 4-AP. CSN activities during normoxia wPO 2 f 130 Torrx followed by hypoxia wPO 2 f 40 Torrx and momentary normoxia. Peak hypoxic response with 1 mM 4-AP was less than by hypoxia alone. Normoxic activity was inhibited with 1 mM 4-AP, and the activity was restored without 4-AP. ŽC. Effect of 2.0 mM 4-AP. CNS activities during normoxia was wPO 2 f 121 Torrx followed by hypoxia wPO 2 f 40 Torrx and back to normoxia are shown first. Perfusion with 2 mM 4-AP caused inhibition of hypoxic as well as normoxic responses.
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Fig. 1. Žcontinued.
which were kept at 378C with a warm water bath. The chamber containing the cells was heated with Peltier heating device to maintain 378C. The hypoxic solutions ŽPO 2 f 12–18 Torr. without and with 4-AP were prepared from 100% O 2 , N2 and CO 2 saturated solution made in Tyrode buffer containing bicarbonate and mixed in proportion to get the required hypoxic PO 2 and PCO 2 levels. The hypoxic PO 2 was kept low compared to the sensory activity protocol because the cell PO 2 was expected to be lower than the perfusate PO 2 in the whole CB w17x. Test solutions Žnormoxia and hypoxia. were analysed on blood gas machine ŽPHM-73 Radiometer. for pH, PCO 2 and PO 2 prior to wCa2q x i measurements. The syringe infusion pump was set at a flow rate of 1 mlrmin. Response to various test solutions were studied in 3–5 cells in the field. Intracellular calcium concentration was measured by Indo-1 fluorescence ratio at 405 nm and 495 nm with excitation at 340 nm w8x. The conversion of fluorescence ratio into wCa2q x i was accomplished by in situ calibration of separate groups of cells, essentially as described by Thomas and Delaville w31x. Briefly, cells loaded with Indo-1-AM were incubated in Ca2q-free HEPES-Tyrode solution, containing 5 mM EGTA, for 20–30 min at room temperature Ž; 258C.. The cover slips with the cells were then superfused with same buffer at 378C with 140 mM Kq plus 10 m M ionomycin. The fluorescence measured with this solution gave R min Ž0 Ca2q .. The cells were then superfused with the same Tyrode solution containing 2
mM Ca2q with 10 m M ionomycin, and the measured ratio gave R max Ž2 mM Ca2q .. wCa2q x i was calculated according to the equation: wCa2q x i s K d P Ž SfrS b . P Ž R y R min .rŽ R max y R ., where R s the experimental fluorescence ratio at 405:495 nm, SfrS b s fluorescence ratio free form at 495 nm:bound form at 495 nm, and K d is the dissociation constant and was assumed to be 250 nM w15x. No detectable change of cell fluorescence in the absence of probe Žautofluorescence. was observed in this procedure. 2.6. Experimental protocol Carotid sinus nerve activities Žimprs. were measured during normoxia ŽPO 2 f 110–120 Torr. and hypoxia ŽPO 2 f 39–43 Torr. and interruption of perfusate flow was done to measure the maximum response of carotid chemosensory discharge. All these were performed with and without 4-AP in solutions. The nerve discharge rates and dopamine release were observed for 2 to 4 min. In a separate study with isolated cat and rat glomus cells, wCa2q x i levels ŽnM. were measured during normoxia ŽPO 2 f 125–135 Torr. and hypoxia ŽPO 2 f 12–18 Torr. in presence and absence of 1 mM 4-AP. 2.7. Statistical analysis Comparison between carotid chemosensory activities in different experimental groups were analyzed using one-way
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Fig. 2. Quantitative analysis of carotid chemosensory responses Žpercent of maximum response to flow interruption. during normoxia Ž'., hypoxia ŽB. and flow interruption Žv . without and with 0.2, 1.0 and 2.0 mM 4-AP, respectively. During normoxia all levels of 4-AP showed a significant decrease in chemosensory responses compared to normoxia alone Žwith 0.2 mM 4-AP, F s 7.08 and P s 0.016; 1.0 mM 4-AP, F s 7.47, P s 0.012 and 2.0 mM 4-AP, F s 15.07, P s 0.001.. Peak hypoxic response remained unaltered with 0.2 mM 4-AP, but with 1 mM Ž F s 5.68, P s 0.026. and 2 mM Ž F s 13.32, P s 0.001. 4-AP CSN responses were reduced. With flow interruption no significant alterations of CSN activities were observed with all levels of 4-AP. All values were represented as mean " S.E.M. % of maximum response and P - 0.05 was considered significant. Normoxia PO 2 was f 123 Torr and hypoxia PO 2 was f 39 Torr.
Fig. 3. Flow interruption Žnear anoxic condition. with and without 4-AP increased DA release significantly. With normoxia and 1 mM 4-AP, DA release decreased Ž ) P - 0.0001.. DA release during hypoxia ŽPO 2 39–43 Torr. was suppressed by 0.2 mM 4-AP Ž ) P - 0.0001.. Increasing the dose of 4-AP Ž1.0 and 2.0 mM. did not have any further effects Ž ) P - 0.0001..
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ANOVA Ž F-test.. wCa2q x i and dopamine levels were analyzed using t-test. Data were expressed as mean " S.E.M., and P - 0.05 was considered significant.
3. Results 3.1. Effects of 4-AP on carotid chemosensory response The details of time course of effects are given in the legends to Fig. 1A–C. Fig. 1A illustrates the effects of 0.2 mM 4-AP on CSN activities. The basal normoxic activity decreased considerably with 0.2 mM 4-AP compared to normoxia alone but had no effect during hypoxia, which alone stimulated the activity. Fig. 1B shows equal suppression of CSN activities with 1.0 mM 4-AP during hypoxia and normoxia. In Fig. 1C, the decrease in CSN activities with 2 mM 4-AP during normoxia and hypoxia were of the same magnitude. Fig. 2 shows a comparative analysis of the quantitative chemosensory responses Žpercent of maximum response with flow interruption. at 0.2, 1.0 and 2.0 mM 4-AP. During normoxia Žsolid triangle., 4-AP treatment showed a significant decrease in basal chemosensory responses from 33 " 4% Ž n s 14. to 13 " 3% Ž n s 5., 18 " 3% Ž n s 11. and 13 " 3% Ž n s 11. respectively with 0.2 mM, 1.0 mM and 2.0 mM 4-AP. Peak hypoxic response Žsolid square. remained unaltered with 0.2 mM 4-AP Ž n s 5., but with 1.0 mM and 2.0 mM 4-AP the sensory response reduced from 62 " 4% Ž n s 14. to 46 " 4% Ž n s 11. and 39 " 3% Ž n s 11. respectively. The suppression with 4-AP during hypoxia was no more than what was seen during normoxia. With flow interruption Žsolid circle., CSN activities were all stimulated with all 4-AP levels. 3.2. Effects of 4-AP on dopamine leÕel Dopamine release Ždifference, D ., as shown in Fig. 3, decreased significantly with 1 mM 4-AP Žy2.0 " 0.5 m M. during normoxia. Peak dopamine release during hypoxia was reduced from 11.4 " 0.9 m M Ž n s 79. to 3.7 " 0.8 m M Ž n s 21., 3.1 " 0.8 m M Ž n s 19. and 3.1 " 0.9 m M Ž n s 13. respectively with 0.2, 1.0 and 2.0 mM 4-AP. No significant alteration to dopamine level was observed during flow interruption with 1 mM 4-AP. 3.3. Effect of 4-AP on [CA 2 q ]i leÕel Intracellular calcium level, measured in isolated cat and rat glomus cells ŽFig. 4., showed no increase in presence of 4-AP Ž1 mM. Žcat: 91 " 3 nM, n s 7, rat: 118 " 7.5 nM, n s 6. during normoxia as compared to basal normoxic value of wCa2q x i Žcat: 88 " 3 nM, n s 7; rat: 114 " 3 nM, n s 7.. During hypoxic stimulation, wCa2q x i levels in
Fig. 4. Effects of 4-AP on wCa2q x i level in rat and cat glomus cells. There were no alterations in intracellular calcium levels in presence of 1 mM 4-AP during normoxia ŽPO 2 f125 Torr.. During hypoxic stimulation ŽPO 2 f12–18 Torr. there were significant increase of wCa2q x i Ž ) P - 0.05, hypoxia vs. normoxia. but 4-AP did not further stimulate the hypoxic wCa2q x i level Ž ) ) n.s., hypoxia vs. hypoxia 4-AP..
cat and rat increased to 347 " 15 nM Ž n s 7. and 211 " 21 nM Ž n s 8. respectively, but further increase was not observed with 4-AP Ž1 mM. when applied in addition to hypoxia Žcat: 313 " 10 nM, n s 7; rat: 223 " 12 nM, n s 8..
4. Discussion 4.1. K q–O2 currents of glomus cells Õs. chemosensory discharge The focus of this study was on the relation between Kq-current of glomus cell membrane and wCa2q x i , neurosecretion and neural discharge. The oxygen sensitive Kq-current is suppressed in glomus cells of rat, rabbit and cat by the pharmacological agent, 4-AP w7,22,26x, and accordingly it is expected to increase wCa2q x i , neurosecretion and neural discharge during normoxia and hypoxia. During normoxia in the cat, 4-AP did not influence wCa2q x i , diminished DA release and decreased the neural discharge. During hypoxia, 4-AP did not influence the wCa2q x i and neural discharge and decreased the DA release. In no cases, 4-AP stimulated any of the foregoing parameters that were measured. The properties of the oxygen-sensitive Kq currents are not the same among different species, like rabbit, cat and rat as explained in the introduction. It has been suggested that inhibition of these channels by hypoxia is responsible for membrane depolarization of glomus cells w7,14,19,21,26–28x. But the sensitivity of these Kq currents to PO 2 differs among authors. Ganfornina and Lopez-Barneo w12x and Montoro et al. w24x have two ranges of PO 2 s: 150–80 Torr and 160–10 Torr which gave a
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linear relationship, while others have used one level of PO 2 w27,30x. However, the relationship between microvascular PO 2 in intact carotid bodies is curvilinear below 40 Torr w17,18x. The intracellular wCa2q x i also shows a relationship to PO 2 w4,11,20,24x similar to the whole carotid body w17,18x. Thus, Kq current shows a different PO 2 profile. Recently, Lopez-Lopez et al. w21x stated that the electrical properties of glomus cells from adult rat and rabbit carotid bodies are strikingly different, yet the chemosensory responses to hypoxia are the same w17x. These raises difficulty in our understanding to the mechanisms of Kq–O 2 current involved in the O 2 chemotransduction process. Previously, Biscoe and Duchen w1,2x suggested that the electrophysiological response is a consequence of the change in wCa2q x i and not its cause. More recently, Donnelley w9x and Osanai et al. w25x expressed doubt as to the role of Kq–O 2 current of glomus cells in the chemosensory response to hypoxia. The pharmacological compounds, 4-AP w9,10x, TEA w6,9x, charybdotoxin w25x and GSH ŽA. Roy, C. Rozanov and S. Lahiri; unpublished observations., which also inhibit the O 2-sensitive Kq-current, failed to excite the chemosensory discharge in carotid bodies, implying that electrophysiological events seen in isolated glomus cells have little to do with O 2 transduction in intact organs. More recently, abandoning the role of these large voltage-gated Kq channels, Buckler w3x has produced evidence of a new type of small conductance Kq channel which is insensitive to 4-AP, TEA and voltage but is largely responsible for mediating the hypoxic membrane depolarization in resting glomus cells. It is postulated that this small resting Kq current Žleak current. mediates the initial electrical activity which will then be controlled by the modulation of large voltage-gated Kq channels. But it is not clear how the large voltage-gated Kq current which is insensitive to charybdotoxin w25x, 4-AP Žpresent communication. and TEA w9x could contribute. Our results extend beyond those by Cheng and Donnelley w6x, Donnelley w9x, and Doyle and Donnelly w10x, who found no effect of 4-AP Ž4 mM. on baseline chemosensory discharge Žsingle afferent. but the peak activity to transient anoxia was diminished w10x in rat carotid body. In contrast recently, Donnelley w9x has reported that 1 mM 4-AP reduced total outward current to 66% of control, with no alteration in basal nerve activity and the nerve response to hypoxia remained intact. These findings are different from ours with cat carotid body because we observed that basal CSN responses were significantly diminished with 4-AP Ž0.2 mM. during normoxia whereas hypoxia or flow interruption showed no inhibitory effect. They have further shown w10x that 4-AP significantly increased baseline catecholamine levels but the peak level significantly decreased during severe transient hypoxia. We found that the dopamine levels in the cat carotid body always decreased with 4-AP during normoxia. The decrease in dopamine release by 4-AP during hypoxia was independent of wCa2q x i
because glomus cell wCa2q x i did not change with 4-AP ww3x, present observationx. Thus, the inhibition of DA release by 4-AP could be due to other effects of 4-AP. 4.2. Comparison with other O2-sensitiÕe cells It has been reported that carotid body type I cells and pulmonary artery smooth muscle cells ŽPASMC. w29x appear to share a similar response to PO 2 . Hypoxic pulmonary vasoconstriction is mediated through inhibition of Kq current, thereby causing depolarization of the resting membrane potential and Ca2q entry through voltage dependent Ca2q channels w34x. In these studies, only one level of hypoxia has been used. Moreover, Kq channel blockers, 4-AP has been shown to mimic the hypoxic response causing an increase in wCa2q x i and membrane depolarization of PASMC w34x. Thus, these results are different from those of glomus cells. Also, PC-12 cells have been reported to be O 2 sensitive but their responses are linear over wide range of PO 2 , 160–0 Torr w35x unlike the chemosensory response w17,18x. On the other hand, erythropoietin ŽEPO. production in human hepatoma cells in chronic hypoxia are stimulated by PO 2 below 70 Torr w13x like carotid bodies w17,18x in acute hypoxia. This expression in EPO cells is time dependent. And also, they are not excitable and are not known to possess O 2 sensitive Kq channels ŽN.R. Prabhakar, personal communication.. In summary, the present study demonstrate that the effect of 4-AP is not excitatory and did not increase glomus cell wCa2q x i . The suppression of dopamine release by 4-AP is contrary to the model of O 2-sensitive Kq channels conductance w22,26x. We feel that these results do not provide any evidence for a link between O 2-sensitive Kq currents and chemosensory excitation.
Acknowledgements This study was supported by grants HL-43413-7, HL50180-3 and T32-HL-07027-22. Charmaine Rozanov is a recipient of NRSA.
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A. Roy et al.r Brain Research 785 (1998) 228–235 w6x P.M. Cheng, D.F. Donnelley, Relationship between changes of glomus cell current and neural response of rat carotid body, J. Neurophysiol. 74 Ž1995. 2077–2086. w7x C. Chou, M. Shirahata, Two types of voltage-gated K channels in carotid body cells of adult cats, Brain Res. 742 Ž1996. 34–42. w8x L.L.T. Dasso, K.J. Buckler, R.D. Vaughan-Jones, Muscarinic and nicotinic receptors raise intracellular Ca2q levels in rat carotid body type I cells, J. Physiol. 498 Ž1997. 327–338. w9x D.F. Donnelley, Modulation of glomus cell membrane currents of intact rat carotid body, J. Physiol. 489 Ž1995. 677–688. w10x T.P. Doyle, D.F. Donnelley, Effect of Naq and Kq channel blockade on baseline and anoxia-induced catecholamine release from rat carotid body, J. Appl. Physiol. 77 Ž1994. 2606–2611. w11x M.R. Duchen, T.J. Biscoe, Relative mitochondrial membrane potential and wCa2q x i in type I cells isolated from the rabbit carotid body, J. Physiol. 450 Ž1992. 33–62. w12x M.D. Ganfornina, J. Lopez-Barneo, Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen, J. Gen. Physiol. 100 Ž1992. 401–426. w13x M.A. Goldberg, G.A. Glass, J.M. Cunningham, F.H. Bunn, The regulated expression of erythropoietin by two human hepatoma cell lines, Proc. Natl. Acad. Sci. U.S.A. 84 Ž1987. 7972–7976. w14x C. Gonzalez, L. Almaraz, A. Obeso, R. Rigual, Oxygen and acid chemoreception in the carotid body chemoreceptors, Trends Neurosci. 15 Ž1992. 146–153. w15x G. Grynkiewicz, R. Jacob, J.E. Merrit, A new generation of Ca2q indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 Ž1985. 3440–3450. w16x R. Iturriaga, W.L. Rumsey, A. Mokashi, D. Spergel, D.F. Wilson, S. Lahiri, In vitro perfused–superfused cat carotid body for physiological and pharmacological studies, J. Appl. Physiol. 70 Ž1991. 1393–1400. w17x S. Lahiri, Chromophores in O 2 chemoreception: the carotid body model, NIPS 9 Ž1994. 161–165. w18x S. Lahiri, W.L. Rumsey, D.F. Wilson, R. Iturriaga, Contribution of in vivo microvascular PO 2 in the cat carotid body chemotransductiuon, J. Appl. Physiol. 75 Ž1993. 1035–1043. w19x J. Lopez-Barneo, J.R. Lopez-Lopez, J. Urena, C. Gonzalez, Chemotransduction in the carotid body: Kq current modulated by PO 2 in type I chemoreceptor cells, Science 241 Ž1988. 580–582. w20x J. Lopez-Barneo, Oxygen-sensing by ion channels and the regulation of cellular functions, TINS 19 Ž1996. 435–440. w21x J.R. Lopez-Lopez, C. Gonzalez, M.T. Perez-Garcia, Properties of ionic currents from isolated adult rat carotid body chemoreceptor cells: Effect of hypoxia, J. Physiol. 499 Ž1997. 429–441.
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w22x J.R. Lopez-Lopez, D.A. DeLuis, C. Gonzalez, Properties of a transient Kq current in chemoreceptor cells of rabbit carotid body, J. Physiol. 460 Ž1993. 15–32. w23x A. Mokashi, D. Ray, F. Botre, M. Katayama, S. Osanai, S. Lahiri, Effect of hypoxia on intracellular pH of glomus cells cultured from cat and rat carotid bodies, J. Appl. Physiol. 78 Ž1995. 1875–1881. w24x R.J. Montoro, J. Urena, R. Fernandez-Chacon, G. Alvarez de Toledo, J. Lopez-Barneo, Oxygen-sensing by ion channels and chemotransduction in single glomus cells, J. Gen. Physiol. 107 Ž1996. 133–143. w25x S. Osanai, D.G. Buerk, A. Mokashi, D.K. Chugh, S. Lahiri, Cat carotid body chemosensory discharge Žin vitro. is insensitive to charybdotoxin, Brain Res. 747 Ž1997. 324–327. w26x C. Peers, O’Donnell, Potassium currents recorded in type I carotid body cells from the neonatal rat and their modulation by chemoexcitatory agents, Brain Res. 522 Ž1990. 259–266. w27x C. Peers, Hypoxic suppression of Kq currents in type I carotid body cells: selective effect on the Ca2q activated Kq current, Neurosci. Lett. 119 Ž1990. 253–256. w28x C. Peers, Actions of doxapram on Kq currents in isolated type I cells of the neonatal rat carotid body, Adv. Exp. Med. Biol. 337 Ž1993. 421–427. w29x J.M. Post, J.R. Hume, S.L. Archer, E.K. Weir, Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction, Am. J. Physiol. 262 Ž1992. C882–C890. w30x A. Stea, C.A. Nurse, Whole-cell and perforated-patch recordings from O 2 -sensitive rat carotid body cells grown in short- and long-term culture, Eur. J. Physiol. 418 Ž1991. 93–101. w31x A.P. Thomas, F. Delaville, The use of fluorescent indicators for measurements of cytosolic calcium concentration in cell populations and single cells, in: J.G. McCormack, P.H. Cobbold ŽEds.., Cellular Calcium: A Practical Approach, Oxford Univ. Press, London, 1991, pp. 1–54. w32x J. Urena, J. Lopez-Lopez, C. Gonzalez, J. Lopez-Barneo, Ionic currents in dispersed chemoreceptor cells of the mammalian carotid body, J. Gen. Physiol. 93 Ž1989. 979–999. w33x C.N. Wyatt, C. Peers, Ca2q-activated Kq channels in isolated type I cells of the neonatal rat carotid body, J. Physiol. 438 Ž1995. 559–565. w34x X.J. Yuan, M.L. Tod, L.J. Rubin, M.P. Blanstein, Hypoxia and metabolic regulation of voltage-gated Kq channels in rat pulmonary artery smooth muscle cells, Exp. Physiol. 80 Ž1995. 803–813. w35x W.H. Zhu, L. Conforti, M.F. Czyzyk-Krzcska, D.E. Millhorn, Membrane depolarization in PC-12 cells during hypoxia is regulated by an O 2 -sensitive Kq current, Am. J. Physiol. 271 Ž1996. C658–C665.
Research report
Suppression of glomus cell Kq conductance by 4-aminopyridine is not related to wCa2qx i , dopamine release and chemosensory discharge from carotid body Arijit Roy a , Charmaine Rozanov a , Donald G. Buerk
b,c
, Anil Mokashi a , Sukhamay Lahiri
a, )
a Department of Physiology, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104, USA Department of Bioengineering, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104, USA Institute for EnÕironmental Medicine, UniÕersity of PennsylÕania School of Medicine, Philadelphia, PA 19104, USA b
c
Accepted 14 October 1997
Abstract The hypothesis that suppression of O 2-sensitive Kq current is the initial event in hypoxic chemotransduction in the carotid body glomus cells was tested by using 4-aminopyridine Ž4-AP., a known suppressant of Kq current, on intracellular wCa2q x i , dopamine secretion and chemosensory discharge in cat carotid body ŽCB.. In vitro experiments were performed with superfused–perfused cat CBs, measuring chemosensory discharge, monitoring dopamine release by microsensors without and with 4-AP Ž0.2, 1.0 and 2.0 mM in CO 2-HCO 3- buffer. and recording wCa2q x i by ratio fluorometry in isolated cat and rat glomus cells. 4-AP decreased the chemosensory activities in normoxia but remained the same in hypoxia and in flow interruption. It decreased the tissue dopamine release in normoxia, and showed an additional inhibition with hypoxia. Also, 4-AP did not evoke any rise in wCa2q x i in glomus cells either during normoxia and hypoxia, although hypoxia stimulated it. Thus, the lack of stimulatory effect on chemosensory discharge, inhibition of dopamine release and unaltered wCa2q x i by 4-AP are not consistent with the implied meaning of the suppressant effect on Kq current of glomus cells. q 1998 Elsevier Science B.V. Keywords: Carotid body; Intracellular calcium; Dopamine; Perfusion; Hypoxia; Kq–O 2 current
1. Introduction Glomus cells Žtype I. of the carotid body ŽCB. are excitable, and the cell membrane contains voltage-dependent Naq, Kq and Ca2q channels w19,32x. Of these channels, the conductance of Kq channels can be inhibited by low PO 2 when glomus cells are artificially depolarized in voltage clamp experiments, positive to resting membrane potential w22,27,30x. This event is supposed to result in opening of voltage-gated Ca2q channels and Ca2q entry, release of neurotransmitters and excitation of the afferent carotid sinus nerves ŽCSN. which synaptically connect with the glomus cells, forming a chemoreceptor unit. But Kq current properties are not the same in different species. In adult rabbits, Kq current inhibition by hypoxia is )
Correspondence author. Department of Physiology, B-400 Richards Bldg., University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085, USA. Fax: q1-215-573-5851; E-mail: [email protected]. upenn.edu 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 2 7 6 - 6
insensitive to calcium w12,22x; in rats, it is calcium dependent w27,33x, and in cats, these cells show both calcium sensitive and insensitive Kq currents w7x. Despite all these differences, the suppression of O 2-sensitive Kq currents is postulated to be the key event in cellular excitation w4,7,12,14,19,21,25,30,32,33 x. In a detailed study Lopez-Lopez et al. w22x showed that 4-aminopyridine Ž4-AP. suppressed the PO 2-sensitive Kq currents in rabbit carotid body glomus cells up to 1 mM in a dose dependent manner. In neonatal rat carotid body, Peers and O’Donnell w26x have shown a reduction in Kq current with 2 mM 4-AP. In a recent study, Chou and Shirahata w7x showed that O 2 sensitive Kq current in cat glomus cells was also inhibited by 4-AP Ž10 mM.. Therefore, if 4-AP suppressed Kq current in different species, then it is expected to excite the glomus cells leading to wCa2q x i rise, neurosecretion and sensory excitation. We tested the hypothesis on perfused–superfused cat carotid body by measuring simultaneously dopamine release and chemosensory discharge without and with 4-AP Ž0.2, 1.0
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and 2 mM.. We found that 4-AP inhibited systematically the chemosensory discharge during normoxia, and did not alter the effects of hypoxia and perfusate flow interruption. On the other hand, 4-AP Ž1 mM. decreased the tissue dopamine release with normoxia and hypoxia. Also, 4-AP Ž1 mM. did not evoke any rise of wCa2q x i in isolated cat and rat glomus cells with and without hypoxia. Thus, contrary to expectation, 4-AP did not increase wCa2q x i and dopamine release nor did it increase sensory discharge.
2. Materials and methods 2.1. Carotid body isolation Experiments were performed in 16 carotid bodies from eight cats of either sex Ž2.5–3.9 kg., anaesthetized with sodium pentobarbital Ž35 mgrkg, i.p., initially.. Carotid bodies were sequentially removed for the study in a perfusion–superfusion system, as described previously w16x, and given here briefly. Carotid bodies from two cats were used for cell separation for calcium studies. Also four rats of either sex Ž300–350 g., anaesthetized with sodium pentobarbital Ž90–100 mgrkg., were used for carotid body cell separation for calcium studies. 2.2. Solutions A modified Tyrode buffer containing ŽmM. 112 NaCl, 4.7 KCl, 2.2 CaCl 2 , 1.1 MgCl 2 , 21.4 NaHCO 3 , 5.0 N-2hydroxyethyl-piperazine-N X -2 ethanesulfonic acid ŽHEPES., 5.0 glucose and 22.0 sodium glutamate and 4 grl of dextran Žmol wt. 74,200. were used to perfuse the carotid body. The normoxic and hypoxic solutions were equilibrated by bubbling with compressed gases containing 5% of CO 2 in 21% of O 2 ŽPCO 2 f 35 Torr, PO 2 110–120 Torr. and 5% of CO 2 in 5% of O 2 ŽPCO 2 f 35 Torr, PO 2 39–43 Torr.. The pH was adjusted to f 7.4. A stock solution of 4-AP was prepared in Tyrode buffer, and diluted to 0.2, 1.0 and 2 mM. 2.3. Carotid chemosensory actiÕity measurement A section of the carotid artery bifurcation with the intact carotid body attached to the carotid sinus nerve was cannulated, removed and mounted in a perfusion chamber. The isolated carotid body was perfused and superfused from both at a constant pressure Žabout 80 Torr., temperature of which, along with the perfusion chamber, was maintained by circulating warm water at 37 " 0.58C. The perfusion could be switched from normoxic to hypoxic solutions and vice versa. The effluent from the chamber was removed by continuous suction, thus maintaining a constant fluid level. The whole carotid sinus nerve was desheathed, placed on a platinum electrode and lifted up into paraffin oil layer for electrical isolation. The neural
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discharge signals were passed through a differential amplifier and a notch filter, and an electronic amplitude discriminator counted the impulses with a frequency meter and printed every 10 s. An analog neural discharge signal from the frequency meter was also tape recorded. 2.4. Measurement of dopamine (DA) Dopamine was measured by DA microsensors positioned into carotid body tissue by a motorized, microdriven, piezoelectric head polarized at q150 mV relative to a grounded Ag–AgCl reference, in contact with the carotid body preparation w5x. There were minor interferences from epinephrine and norepinephrine Ž- 10% compared to dopamine. at this potential w5x. Microelectrode current was amplified with a sensitive picoammeter and recorded in a VCR tape. Calibrations were made before and after each experiment. Tissue dopamine levels were measured before and during application of 4-AP at normoxia, hypoxia and momentary flow interruption Ž n s number of observations from 7 CBs.. 2.5. Measurement of intracellular calcium, [Ca 2 q ]i wCa2q x i in isolated glomus cells were studied separately in two cats and four rats. The glomus cells were separated enzymatically as described before w23x. Briefly, the carotid bodies were identified, surgically removed and kept in an ice-cold growth medium. After the removal of excessive connective tissue, the carotid bodies were transferred to 1 ml of Ca2q- and Mg 2q-free phosphate buffer ŽpH 7.4.,containing 0.1% collagenase Žtype IV, Sigma Chemicals. and was digested for 20 min for rat CB and 30 min for cat CB in a water bath at 378C with continuous bubbling of humidified 100% O 2 . The digested tissue was then transferred to the growth medium that contained 90 parts Ham F-12 Žcontaining NaHCO 3 . and 10 parts of fetal bovine serum fortified with 10 mg of streptomycin, 10,000 U of penicillin G and 80 U of insulin, and was triturated with a fire polished Pasteur pipette. The cells were plated on a 18 mm coverslip, and kept undisturbed for a period of 36 h in a Petri dish in a humidified incubator in which 5% CO 2 and air were circulated at 378C. The cells were then loaded with a fluorescent probe, Indo-1-AM Ž10 m M, Molecular Probes Inc.., in HEPES buffer ŽpH s 7.2. for a period of 30 min at room temperature Žabout 258C. prior to the start of an experiment. The coverslip with cells attached formed the bottom part of the chamber Žvolume capacity 125 m l., through which the buffer flowed. The chamber was attached to an inverted fluorescent microscope ŽFluovert– Leitz.. The cells in a selected field were excited at 340 nm with xenon-arc light source Ž75 W. that was fitted with a neutral density filter Ž10%.. The output emission signals were recorded with a ratio fluorometer equipped with two photomultiplier tubes that were attached near the eye-piece ŽBiomedical Instrumentation Group, University of Penn-
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sylvania.. The fluorescent analog signals at 405 nm and 495 nm and the ratio Ž405:495. were simultaneously recorded on a multichannel recorder ŽLinear Instruments..
The peak digital ratio signals of fluorescence change were also noted manually. The cells were superfused during hypoxia by a syringe-infusion pump ŽSage Instruments.
Fig. 1. Effects of 4-AP on carotid sinus nerve discharge during perfusion with normoxia ŽNx. and hypoxia ŽHx.. ŽA. Effect of 0.2 mM 4-AP. CSN activities during normoxia wPO 2 f 113 Torrx followed by hypoxia wPO 2 f 43 Torrx and back to normoxia. Perfusion with 0.2 mM 4-AP caused suppression of activity during normoxia but had no effect during hypoxia. ŽB. Effect of 1.0 mM 4-AP. CSN activities during normoxia wPO 2 f 130 Torrx followed by hypoxia wPO 2 f 40 Torrx and momentary normoxia. Peak hypoxic response with 1 mM 4-AP was less than by hypoxia alone. Normoxic activity was inhibited with 1 mM 4-AP, and the activity was restored without 4-AP. ŽC. Effect of 2.0 mM 4-AP. CNS activities during normoxia was wPO 2 f 121 Torrx followed by hypoxia wPO 2 f 40 Torrx and back to normoxia are shown first. Perfusion with 2 mM 4-AP caused inhibition of hypoxic as well as normoxic responses.
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Fig. 1. Žcontinued.
which were kept at 378C with a warm water bath. The chamber containing the cells was heated with Peltier heating device to maintain 378C. The hypoxic solutions ŽPO 2 f 12–18 Torr. without and with 4-AP were prepared from 100% O 2 , N2 and CO 2 saturated solution made in Tyrode buffer containing bicarbonate and mixed in proportion to get the required hypoxic PO 2 and PCO 2 levels. The hypoxic PO 2 was kept low compared to the sensory activity protocol because the cell PO 2 was expected to be lower than the perfusate PO 2 in the whole CB w17x. Test solutions Žnormoxia and hypoxia. were analysed on blood gas machine ŽPHM-73 Radiometer. for pH, PCO 2 and PO 2 prior to wCa2q x i measurements. The syringe infusion pump was set at a flow rate of 1 mlrmin. Response to various test solutions were studied in 3–5 cells in the field. Intracellular calcium concentration was measured by Indo-1 fluorescence ratio at 405 nm and 495 nm with excitation at 340 nm w8x. The conversion of fluorescence ratio into wCa2q x i was accomplished by in situ calibration of separate groups of cells, essentially as described by Thomas and Delaville w31x. Briefly, cells loaded with Indo-1-AM were incubated in Ca2q-free HEPES-Tyrode solution, containing 5 mM EGTA, for 20–30 min at room temperature Ž; 258C.. The cover slips with the cells were then superfused with same buffer at 378C with 140 mM Kq plus 10 m M ionomycin. The fluorescence measured with this solution gave R min Ž0 Ca2q .. The cells were then superfused with the same Tyrode solution containing 2
mM Ca2q with 10 m M ionomycin, and the measured ratio gave R max Ž2 mM Ca2q .. wCa2q x i was calculated according to the equation: wCa2q x i s K d P Ž SfrS b . P Ž R y R min .rŽ R max y R ., where R s the experimental fluorescence ratio at 405:495 nm, SfrS b s fluorescence ratio free form at 495 nm:bound form at 495 nm, and K d is the dissociation constant and was assumed to be 250 nM w15x. No detectable change of cell fluorescence in the absence of probe Žautofluorescence. was observed in this procedure. 2.6. Experimental protocol Carotid sinus nerve activities Žimprs. were measured during normoxia ŽPO 2 f 110–120 Torr. and hypoxia ŽPO 2 f 39–43 Torr. and interruption of perfusate flow was done to measure the maximum response of carotid chemosensory discharge. All these were performed with and without 4-AP in solutions. The nerve discharge rates and dopamine release were observed for 2 to 4 min. In a separate study with isolated cat and rat glomus cells, wCa2q x i levels ŽnM. were measured during normoxia ŽPO 2 f 125–135 Torr. and hypoxia ŽPO 2 f 12–18 Torr. in presence and absence of 1 mM 4-AP. 2.7. Statistical analysis Comparison between carotid chemosensory activities in different experimental groups were analyzed using one-way
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Fig. 2. Quantitative analysis of carotid chemosensory responses Žpercent of maximum response to flow interruption. during normoxia Ž'., hypoxia ŽB. and flow interruption Žv . without and with 0.2, 1.0 and 2.0 mM 4-AP, respectively. During normoxia all levels of 4-AP showed a significant decrease in chemosensory responses compared to normoxia alone Žwith 0.2 mM 4-AP, F s 7.08 and P s 0.016; 1.0 mM 4-AP, F s 7.47, P s 0.012 and 2.0 mM 4-AP, F s 15.07, P s 0.001.. Peak hypoxic response remained unaltered with 0.2 mM 4-AP, but with 1 mM Ž F s 5.68, P s 0.026. and 2 mM Ž F s 13.32, P s 0.001. 4-AP CSN responses were reduced. With flow interruption no significant alterations of CSN activities were observed with all levels of 4-AP. All values were represented as mean " S.E.M. % of maximum response and P - 0.05 was considered significant. Normoxia PO 2 was f 123 Torr and hypoxia PO 2 was f 39 Torr.
Fig. 3. Flow interruption Žnear anoxic condition. with and without 4-AP increased DA release significantly. With normoxia and 1 mM 4-AP, DA release decreased Ž ) P - 0.0001.. DA release during hypoxia ŽPO 2 39–43 Torr. was suppressed by 0.2 mM 4-AP Ž ) P - 0.0001.. Increasing the dose of 4-AP Ž1.0 and 2.0 mM. did not have any further effects Ž ) P - 0.0001..
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ANOVA Ž F-test.. wCa2q x i and dopamine levels were analyzed using t-test. Data were expressed as mean " S.E.M., and P - 0.05 was considered significant.
3. Results 3.1. Effects of 4-AP on carotid chemosensory response The details of time course of effects are given in the legends to Fig. 1A–C. Fig. 1A illustrates the effects of 0.2 mM 4-AP on CSN activities. The basal normoxic activity decreased considerably with 0.2 mM 4-AP compared to normoxia alone but had no effect during hypoxia, which alone stimulated the activity. Fig. 1B shows equal suppression of CSN activities with 1.0 mM 4-AP during hypoxia and normoxia. In Fig. 1C, the decrease in CSN activities with 2 mM 4-AP during normoxia and hypoxia were of the same magnitude. Fig. 2 shows a comparative analysis of the quantitative chemosensory responses Žpercent of maximum response with flow interruption. at 0.2, 1.0 and 2.0 mM 4-AP. During normoxia Žsolid triangle., 4-AP treatment showed a significant decrease in basal chemosensory responses from 33 " 4% Ž n s 14. to 13 " 3% Ž n s 5., 18 " 3% Ž n s 11. and 13 " 3% Ž n s 11. respectively with 0.2 mM, 1.0 mM and 2.0 mM 4-AP. Peak hypoxic response Žsolid square. remained unaltered with 0.2 mM 4-AP Ž n s 5., but with 1.0 mM and 2.0 mM 4-AP the sensory response reduced from 62 " 4% Ž n s 14. to 46 " 4% Ž n s 11. and 39 " 3% Ž n s 11. respectively. The suppression with 4-AP during hypoxia was no more than what was seen during normoxia. With flow interruption Žsolid circle., CSN activities were all stimulated with all 4-AP levels. 3.2. Effects of 4-AP on dopamine leÕel Dopamine release Ždifference, D ., as shown in Fig. 3, decreased significantly with 1 mM 4-AP Žy2.0 " 0.5 m M. during normoxia. Peak dopamine release during hypoxia was reduced from 11.4 " 0.9 m M Ž n s 79. to 3.7 " 0.8 m M Ž n s 21., 3.1 " 0.8 m M Ž n s 19. and 3.1 " 0.9 m M Ž n s 13. respectively with 0.2, 1.0 and 2.0 mM 4-AP. No significant alteration to dopamine level was observed during flow interruption with 1 mM 4-AP. 3.3. Effect of 4-AP on [CA 2 q ]i leÕel Intracellular calcium level, measured in isolated cat and rat glomus cells ŽFig. 4., showed no increase in presence of 4-AP Ž1 mM. Žcat: 91 " 3 nM, n s 7, rat: 118 " 7.5 nM, n s 6. during normoxia as compared to basal normoxic value of wCa2q x i Žcat: 88 " 3 nM, n s 7; rat: 114 " 3 nM, n s 7.. During hypoxic stimulation, wCa2q x i levels in
Fig. 4. Effects of 4-AP on wCa2q x i level in rat and cat glomus cells. There were no alterations in intracellular calcium levels in presence of 1 mM 4-AP during normoxia ŽPO 2 f125 Torr.. During hypoxic stimulation ŽPO 2 f12–18 Torr. there were significant increase of wCa2q x i Ž ) P - 0.05, hypoxia vs. normoxia. but 4-AP did not further stimulate the hypoxic wCa2q x i level Ž ) ) n.s., hypoxia vs. hypoxia 4-AP..
cat and rat increased to 347 " 15 nM Ž n s 7. and 211 " 21 nM Ž n s 8. respectively, but further increase was not observed with 4-AP Ž1 mM. when applied in addition to hypoxia Žcat: 313 " 10 nM, n s 7; rat: 223 " 12 nM, n s 8..
4. Discussion 4.1. K q–O2 currents of glomus cells Õs. chemosensory discharge The focus of this study was on the relation between Kq-current of glomus cell membrane and wCa2q x i , neurosecretion and neural discharge. The oxygen sensitive Kq-current is suppressed in glomus cells of rat, rabbit and cat by the pharmacological agent, 4-AP w7,22,26x, and accordingly it is expected to increase wCa2q x i , neurosecretion and neural discharge during normoxia and hypoxia. During normoxia in the cat, 4-AP did not influence wCa2q x i , diminished DA release and decreased the neural discharge. During hypoxia, 4-AP did not influence the wCa2q x i and neural discharge and decreased the DA release. In no cases, 4-AP stimulated any of the foregoing parameters that were measured. The properties of the oxygen-sensitive Kq currents are not the same among different species, like rabbit, cat and rat as explained in the introduction. It has been suggested that inhibition of these channels by hypoxia is responsible for membrane depolarization of glomus cells w7,14,19,21,26–28x. But the sensitivity of these Kq currents to PO 2 differs among authors. Ganfornina and Lopez-Barneo w12x and Montoro et al. w24x have two ranges of PO 2 s: 150–80 Torr and 160–10 Torr which gave a
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linear relationship, while others have used one level of PO 2 w27,30x. However, the relationship between microvascular PO 2 in intact carotid bodies is curvilinear below 40 Torr w17,18x. The intracellular wCa2q x i also shows a relationship to PO 2 w4,11,20,24x similar to the whole carotid body w17,18x. Thus, Kq current shows a different PO 2 profile. Recently, Lopez-Lopez et al. w21x stated that the electrical properties of glomus cells from adult rat and rabbit carotid bodies are strikingly different, yet the chemosensory responses to hypoxia are the same w17x. These raises difficulty in our understanding to the mechanisms of Kq–O 2 current involved in the O 2 chemotransduction process. Previously, Biscoe and Duchen w1,2x suggested that the electrophysiological response is a consequence of the change in wCa2q x i and not its cause. More recently, Donnelley w9x and Osanai et al. w25x expressed doubt as to the role of Kq–O 2 current of glomus cells in the chemosensory response to hypoxia. The pharmacological compounds, 4-AP w9,10x, TEA w6,9x, charybdotoxin w25x and GSH ŽA. Roy, C. Rozanov and S. Lahiri; unpublished observations., which also inhibit the O 2-sensitive Kq-current, failed to excite the chemosensory discharge in carotid bodies, implying that electrophysiological events seen in isolated glomus cells have little to do with O 2 transduction in intact organs. More recently, abandoning the role of these large voltage-gated Kq channels, Buckler w3x has produced evidence of a new type of small conductance Kq channel which is insensitive to 4-AP, TEA and voltage but is largely responsible for mediating the hypoxic membrane depolarization in resting glomus cells. It is postulated that this small resting Kq current Žleak current. mediates the initial electrical activity which will then be controlled by the modulation of large voltage-gated Kq channels. But it is not clear how the large voltage-gated Kq current which is insensitive to charybdotoxin w25x, 4-AP Žpresent communication. and TEA w9x could contribute. Our results extend beyond those by Cheng and Donnelley w6x, Donnelley w9x, and Doyle and Donnelly w10x, who found no effect of 4-AP Ž4 mM. on baseline chemosensory discharge Žsingle afferent. but the peak activity to transient anoxia was diminished w10x in rat carotid body. In contrast recently, Donnelley w9x has reported that 1 mM 4-AP reduced total outward current to 66% of control, with no alteration in basal nerve activity and the nerve response to hypoxia remained intact. These findings are different from ours with cat carotid body because we observed that basal CSN responses were significantly diminished with 4-AP Ž0.2 mM. during normoxia whereas hypoxia or flow interruption showed no inhibitory effect. They have further shown w10x that 4-AP significantly increased baseline catecholamine levels but the peak level significantly decreased during severe transient hypoxia. We found that the dopamine levels in the cat carotid body always decreased with 4-AP during normoxia. The decrease in dopamine release by 4-AP during hypoxia was independent of wCa2q x i
because glomus cell wCa2q x i did not change with 4-AP ww3x, present observationx. Thus, the inhibition of DA release by 4-AP could be due to other effects of 4-AP. 4.2. Comparison with other O2-sensitiÕe cells It has been reported that carotid body type I cells and pulmonary artery smooth muscle cells ŽPASMC. w29x appear to share a similar response to PO 2 . Hypoxic pulmonary vasoconstriction is mediated through inhibition of Kq current, thereby causing depolarization of the resting membrane potential and Ca2q entry through voltage dependent Ca2q channels w34x. In these studies, only one level of hypoxia has been used. Moreover, Kq channel blockers, 4-AP has been shown to mimic the hypoxic response causing an increase in wCa2q x i and membrane depolarization of PASMC w34x. Thus, these results are different from those of glomus cells. Also, PC-12 cells have been reported to be O 2 sensitive but their responses are linear over wide range of PO 2 , 160–0 Torr w35x unlike the chemosensory response w17,18x. On the other hand, erythropoietin ŽEPO. production in human hepatoma cells in chronic hypoxia are stimulated by PO 2 below 70 Torr w13x like carotid bodies w17,18x in acute hypoxia. This expression in EPO cells is time dependent. And also, they are not excitable and are not known to possess O 2 sensitive Kq channels ŽN.R. Prabhakar, personal communication.. In summary, the present study demonstrate that the effect of 4-AP is not excitatory and did not increase glomus cell wCa2q x i . The suppression of dopamine release by 4-AP is contrary to the model of O 2-sensitive Kq channels conductance w22,26x. We feel that these results do not provide any evidence for a link between O 2-sensitive Kq currents and chemosensory excitation.
Acknowledgements This study was supported by grants HL-43413-7, HL50180-3 and T32-HL-07027-22. Charmaine Rozanov is a recipient of NRSA.
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