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CO-induced K+ currents in rat glomus cells are insensitive to light unlike carotid body neural discharge and Vo O2
Respiratory Physiology & Neurobiology 131 (2002) 285– 290 www.elsevier.com/locate/resphysiol
Short communication
CO-induced K+ currents in rat glomus cells are insensitive to light unlike carotid body neural discharge and V, O2 Jinqing Li, Arijit Roy, Anil Mokashi, Sukhamay Lahiri * Department of Physiology, Uni6ersity Pennsyl6ania School of Medicine, B400 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104 -6085, USA Received 12 March 2002; accepted 25 March 2002
Abstract The hypothesis that the light sensitive properties of CO-induced chemosensory nerve (CSN) discharge and oxygen consumption of the carotid body (CB) were shared by the pre-synaptic glomus cells was tested. The light effect on K+ currents were measured before and during perfusion of the isolated rat glomus cells with high PCO of 550 Torr during nomoxia (PO2 $100 Torr) at extra-cellular pH 7.0 and intracellular pH 6.8 with HEPES buffer. CO increased the K+ currents with a left ward shift of the reversal potential, which showed no light effect. Thus the K+ permeability of the glomus cell membrane were not shared by the light-sensitive CSN discharge of the CB and oxygen consumption in the presence of high PCO. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Carotid body, CO-induced chemosensing, O2 consumption; CO, chemosensing, carotid body; Glomus, cell, pre-synaptic; Mammals, rat; Nerve, carotid sinus
1. Introduction Type I cells (glomus cells) are synaptically connected with carotid sinus nerve (CSN). It is thought that type I cells are the chemoreceptors that sense changes in blood PO2, pH and PCO2. The consensus model is that stimulation by hypoxia is followed by K+ Channel inhibition with membrane depolarization that induces Ca2 + entry and subsequent release of neurotransmitters that activate the afferent fibers of the CSN (Gon* Corresponding author. Tel.: +1-215-898-9125; fax: + 1215-573-5851. E-mail address: [email protected] (S. Lahiri).
zalez et al., 1994). The alternative hypothesis is that the nerve endings themselves are directly excited by hypoxia, and type I cells which are also excited are secretory in nature (Lahiri et al., 1999, 2001). One distinguishing feature of carotid body chemoreception is that the CSN discharge is intensely and promptly stimulated by high PCO of \ 300 Torr during normoxia in the absence of light. Exposure to light instantly silenced the CSN discharge (Joels and Neil, 1962; Lahiri et al., 1993) and increased oxygen consumption of the carotid body (Lahiri et al., 1995; Warburg and Negelein, 1928). The CO-induced light effects are shared by mitochondrial respiratory chain (Warburg and Negelein, 1928; Wilson et al., 1994).
0034-5687/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 4 8 ( 0 2 ) 0 0 0 2 9 - 0
286
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
The outward K+ currents of type I cells, which are suppressed by hypoxia (Lopez-Barneo et al., 1988), are opposed by high PCO (Lopez-Lopez and Gonzalez, 1992; Riesco-Fagundo et al., 2001). But their light sensitivity has not been reported. If the light sensitivity does not exist, then the CO effects on type I cells would be dissociated from those on oxygen consumption and CSN responses. The objective of the present investigation was to determine whether there exists a CO-induced light effects on K+ currents of type I cells of rat carotid body.
2. Methods The glomus cells were separated enzymatically using collegenase IV (Sigma Co). The dissociated cells transferred to growth medium, plated on glass cover slips in Petri dishes and maintained at 37 °C in a CO2 –air incubator for up to 48 h for electrophysiological recording. Before the recording, the cells along with the cover slip were transferred into the patch clamp recording chamber and were perfused at the rate of 1.5 ml/min by gravity with desired bath solution. The temperature was maintained at 36– 37 °C and monitored with TC344B dual automatic temperature controller (Warner Instrument CO) during the entire procedure. The pipette filling solution contained (in mM): 110 K-glutamate, 30 KCl, 2.0 MgCl2, 5 CaCl2, 10 HEPES, with 100–200 mg/ml nystatin at pH 6.8, adjusted with KOH. Extracellular solution consisted of (in mM) 140 NaCl, 5 KCl, 2.0 MgCl2, 5 CaCl2, 10 HEPES, with 50 mM of ATP at pH 7.0, adjusted with NaOH. These pH values with HEPES buffer were chosen because the sensory discharge gave the optimal activity at this pH (Osanai et al., 1997a,b). High PCO (550 Torr) with PO2 (120 Torr) solution was obtained by bubbling above extracellular solution with appropriate CO and O2. The pipettes had resistances of 2– 6 MV when filled with above solution. The glomus cells were identified based on the presence of granular birefringence appearance and the response of the K+ currents to pH. The cells we considered as the glomus cells if their K+
currents were lower at [pH]e 7.0 than at [pH]e 7.4. To avoid the cytosolic constituents washout effect, following patch excision, perforated whole cell configuration was used to measure the K+ currents, using nystatin (Horn and Marty, 1988). Whole cell recordings were performed using an Axopatch 200B patch clamp amplifier (Axon Instruments, CA). The voltage clamp commands were generated using DigiData 1200 interface (Axon Instruments, CA) connected to a PC (Gateway E-4200). The 90 ms depolarizing voltage steps from − 80 to + 60 mV with 10 mV increments were used to elicit the outward currents. Signals were filtered at 1 kHz and acquired at 10 kHz. Pclamp 8.0 programs (Axon Instruments) were used for data acquisition and analysis. All experiments were conducted in pairs (in the absence and presence of high PCO with lighton and light-off conditions). Currents were recorded only when stable membrane potentials and currents were reached after the bath solution or the illumination had been changed. The data analyses were performed from the average currents over the range 78–88 ms of the voltage steps. Mean current-voltage (I–V) relationships were calculated from 4 cells. Membrane conductance was determined by fitting a linear regression to the current-voltage relationship over the range − 10 to +40 mV. Illumination was controlled using the microscope light source. The strength of bright light was 100 W/12 V.Results were presented as means9S.E.M. (n, is the number of observations). Statistical significance was assessed using Student’s two-tailed paired t-test. P was considered significant at B 0.05.
3. Results The K+ currents were increased significantly on exposure to 550 Torr PCO, following the depolarizing voltage steps regardless of the presence of bright light. The representative recordings and I–V relationships in the presence of the bright light are presented in Fig. 1A–C. Fig. 1A shows the currents recorded in the absence of high PCO, and Fig. 1B shows that in the presence of high PCO. The I–V relationships were plotted in Fig.
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
1C. The curves with filled square are for the control and those with the open circle for those in the presence of high PCO. With the light off, the representative recordings are shown in Fig. 1D, E in the absence and presence of high PCO respec-
287
tively. Fig. 1F shows the I–V relationships in both absence (filled squares) and presence (open circles) of 550 Torr PCO. The light did not change the CO-induced increases of K+ currents. The 550 Torr PCO also increased the K+ conductances
Fig. 1. Representative recordings of the High PCO effects on the whole cell outward K+ currents in the presence and the absence of bright light. Temperature was 36.8 °C. The membrane was held at − 85 mV and voltage steps were from −80 to +60 mV in 10 mV increment. All recordings were from the same patch. (A) It shows the whole cell outward K+ currents in the absence of high PCO with the light on. (B) It shows the currents in the presence of high PCO. 550 Torr PCO significantly increased the whole cell outward K+ currents when the glomus cell exposed to the bright light. (C) It is the I – V curves from the (A) (·, without CO) and B (, with CO). (D and E) These were recorded respectively in the absence and the presence of 550 Torr PCO with the light off. (F) It is the I – V curves from D and E. High PCO resulted in a considerable increase of the outward K+ currents and left ward shift of the reversal potential as those in the presence of bright light.
288
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
Fig. 2. Summary of high PCO-induced light independent increases of the whole cell conductance and of the left ward shift of the reversal potential. 550 Torr PCO significantly increased the whole cell K+ conductance regardless of light was on (A) or off (B), (P B 0.05 for both). However, CO-induced increases of the conductances were insensitive to the light (P \0.05). 2C and D show the high PCO induced the reverse potential shifts from − 33.09 8.6 to − 50.8 910.8 mV with the light on (C) and from 35.8 9 10.7 to 50.09 11.3 mV (D), (P B 0.05 for both). This CO-induced reversal potential shift was not affected by high PCO (P\ 0.05).
from 3.7791.56 to 7.22 9 2.20 nS with the light on (Fig. 2A, PB 0.05) and from 3.369 1.59 to 5.75 91.68 nS with the light off condition (Fig. 2B, PB 0.05). High PCO increased conductances respectively by 140.43942.76% with light on and by 115.3097.93% with light off conditions. The increases of the conductances in the presence and
absence of light were not statistically different (P\ 0.05). The high PCO resulted in left ward shifts of the reversal potentials in both presence and absence of the bright light (PB 0.05). However, CO-induced reversal potential shifts were not light sensitive (P\ 0.05). The 550 Torr PCO induced 17.7593.71 mV left ward shift (Fig. 2C)
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
and 14.2593.47 mV (Fig. 2D) of the reversal potentials in the presence and absence of light respectively. These results suggest no photolabile effect on the CO-induced K+ permeability in rat glomus cells. As control, we also examined the possibility of light effect on the K+ permeability in the absence of CO. No light effect was observed on both K+ conductances (Fig. 2A, B, unfilled bars) and the reversal potentials (Fig. 2C, D, unfilled bars), P\ 0.05. The results indicated that the K+ channels were not sensitive to light.
4. Discussion It is well known that CO stimulates CSN discharges and concomitantly decreases oxygen uptake (Lahiri et al., 1995). The dogma is that glomus (type I) cells are the initial sites of transduction and they release neurotransmitters in response to hypoxia and high PCO, which in turn depolarize the nearby afferent nerve endings, leading to an increase in CSN discharge (Gonzalez et al., 1994). One hypothesis suggests that K+ channel proteins of glomus cells are associated with the oxygen sensing and that high PCO inhibits this channels, ensuing depolarization of glomus cell and CO-mediated CSN excitation (Gonzalez et al., 1994; Lahiri et al., 2001). Hypoxia inhibits glomus cell K+ currents, which is reversed by CO. (Lopez-Lopez and Gonzalez, 1992; Riesco-Fagundo et al., 2001), as if CO hyperpolarized the cells during moderate hypoxia. CO effects on CSN discharge also were suppressed during moderate hypoxia and the light further inhibited the response (Lahiri et al., 1993). But the light effects were reversed during extreme hypoxia (Lahiri et al., 1999). It was also reported that CO hyperpolarized single smooth muscle cells and that the whole-cell outward K+ currents were enhanced by CO (Wang et al., 1997). However, no light effects were reported in these studies. The CSN discharge is immediately stimulated by high PCO in the absence of light and is accompanied by a decreased oxygen uptake, which are immediately reversed by bright light (Lahiri et al., 1995). These characteristics match that of cy-
289
tochrome a3 (Warburg and Negelein, 1928). Furthermore, the action spectrum characteristics of the CO compound match those of mitochondria cytochrome a3 (Wilson et al., 1994). CO no doubt acted on the glomus cells, and perhaps neurotransmitters were secreted but the light effects were not there. On the other hand, if CO might have acted on the nerve endings which are well equipped with mitochondria and other organelles to elicit a neural response, there is a possible disconnection. This disconnection between K+ permeability of the glomus cells and CSN discharge is consistent with the lack of the effects of the K+ channel blockers, charybdotoxin (Osanai et al., 1997a,b), TEA and 4 AP (Lahiri et al., 1998; Roy et al., 1998) on CSN discharge unlike the response of glomus cells (Lopez-Barneo et al., 1988) during hypoxia.
Acknowledgements This work is supported in part by the grants of R37-HL43413-12, RO1-HL 50180-7 and N001401-0948.
References Gonzalez, C., Almaraz, L., Obeso, A., Rigual, R., 1994. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol. Rev. 74, 829 – 898. Horn, R., Marty, A., 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145 – 159. Joels, N., Neil, E., 1962. The action of high tension of carbon monoxide on the carotid chemoreceptor. Arch. Int. Pharmacol. Ther. 139, 528 – 534. Lahiri, S., Iturriaga, R., Mokashi, A., Ray, D.K., Chugh, D., 1993. CO reveals dual mechanisms of O2 chemoreception in the cat carotid body. Respir. Physiol. 94, 227 – 240. Lahiri, S., Buerk, D.G., Chugh, D., Osanai, S., Mokashi, A., 1995. Reciprocal photolabile O2 consumption and chemoreceptor excitation by carbon monoxide in the cat carotid body: evidence for cytochrome a3 as the primary O2 sensor. Brain Res. 684, 194 – 200. Lahiri, S., Roy, A., Rozanov, C., Mokashi, A., 1998. K+-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor. Brain Res. 794, 162 –165. Lahiri, S., Ehleben, W., Acker, H., 1999. Chemoreceptor discharges and cytochrome redox changes of the rat
290
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
carotid body: role of heme ligands. Proc. Natl. Acad. Sci. USA 96, 9427 – 9432. Lahiri, S., Rozanov, C., Roy, A., Storey, B., Buerk, D.G., 2001. Regulation of oxygen sensing in peripheral arterial chemoreceptors. Int. J. Biochem. Cell Biol. 33, 755 –774. Lopez-Barneo, J., Lopez-Lopez, J.R., Urena, J., Gonzalez, C., 1988. Chemotransduction in the carotid body: K + current modulated by PCO2 in type I cells. Science 241, 580 –582. Lopez-Lopez, J.R., Gonzalez, C., 1992. Time course of K+ current inhibition by low oxygen in chemoreceptor cells of adult rabbit carotid body. Effects of carbon monoxide. FEBS Lett. 299, 251 –254. Osanai, S., Buerk, D.G., Mokashi, A., Chugh, D.K., Lahiri, S., 1997a. Cat carotid body chemosensory discharge (in vitro) is insensitive to charybdotoxin. Brain Res. 747, 324 – 327. Osanai, S., Rozanov, C., Mokashi, A., Buerk, D.G., Lahiri, S., 1997b. CO interact with intracellular [H+] with and without CO2 – HCO− in the cat carotid chemosensory dis3 charge. Brain Res. 764, 221 –224.
Riesco-Fagundo, A.M., Perez-Garcia, M.T., Gonzalez, C., Lopez-Lopez, J.R., 2001. O2 modulates large-conductance Ca2 + -dependent K + channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism. Circ. Res. 89, 430 – 436. Roy, A., Rozanov, C., Buerk, D.G., Mokashi, A., Lahiri, S., 1998. Suppression of glomus cell K+ conductance by 4-aminopyridine is not related to [Ca2 + ]i, dopamine release and chemosensory discharge from carotid body. Brain Res. 785, 228 – 235. Wang, R., Wu, L., Wang, Z., 1997. The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells. Pflu¨ gers Arch. 434, 285 – 291. Warburg, O., Negelein, E., 1928. ber die photochemische Dissoziation bei intermittierender Bleichuterung und das absolute Absorp-sionsspektrum des Atmungsferments. Biochem. Z. 2, 202 – 208. Wilson, D.F., Mokashi, A., Chugh, D., Vinogradov, S., Osanai, S., Lahiri, S., 1994. The primary oxygen sensor of the cat carotid body is cytochrome a3 of the mitochondrial respiratory chain. FEBS Lett. 351, 370 – 374.
Short communication
CO-induced K+ currents in rat glomus cells are insensitive to light unlike carotid body neural discharge and V, O2 Jinqing Li, Arijit Roy, Anil Mokashi, Sukhamay Lahiri * Department of Physiology, Uni6ersity Pennsyl6ania School of Medicine, B400 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104 -6085, USA Received 12 March 2002; accepted 25 March 2002
Abstract The hypothesis that the light sensitive properties of CO-induced chemosensory nerve (CSN) discharge and oxygen consumption of the carotid body (CB) were shared by the pre-synaptic glomus cells was tested. The light effect on K+ currents were measured before and during perfusion of the isolated rat glomus cells with high PCO of 550 Torr during nomoxia (PO2 $100 Torr) at extra-cellular pH 7.0 and intracellular pH 6.8 with HEPES buffer. CO increased the K+ currents with a left ward shift of the reversal potential, which showed no light effect. Thus the K+ permeability of the glomus cell membrane were not shared by the light-sensitive CSN discharge of the CB and oxygen consumption in the presence of high PCO. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Carotid body, CO-induced chemosensing, O2 consumption; CO, chemosensing, carotid body; Glomus, cell, pre-synaptic; Mammals, rat; Nerve, carotid sinus
1. Introduction Type I cells (glomus cells) are synaptically connected with carotid sinus nerve (CSN). It is thought that type I cells are the chemoreceptors that sense changes in blood PO2, pH and PCO2. The consensus model is that stimulation by hypoxia is followed by K+ Channel inhibition with membrane depolarization that induces Ca2 + entry and subsequent release of neurotransmitters that activate the afferent fibers of the CSN (Gon* Corresponding author. Tel.: +1-215-898-9125; fax: + 1215-573-5851. E-mail address: [email protected] (S. Lahiri).
zalez et al., 1994). The alternative hypothesis is that the nerve endings themselves are directly excited by hypoxia, and type I cells which are also excited are secretory in nature (Lahiri et al., 1999, 2001). One distinguishing feature of carotid body chemoreception is that the CSN discharge is intensely and promptly stimulated by high PCO of \ 300 Torr during normoxia in the absence of light. Exposure to light instantly silenced the CSN discharge (Joels and Neil, 1962; Lahiri et al., 1993) and increased oxygen consumption of the carotid body (Lahiri et al., 1995; Warburg and Negelein, 1928). The CO-induced light effects are shared by mitochondrial respiratory chain (Warburg and Negelein, 1928; Wilson et al., 1994).
0034-5687/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 4 8 ( 0 2 ) 0 0 0 2 9 - 0
286
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
The outward K+ currents of type I cells, which are suppressed by hypoxia (Lopez-Barneo et al., 1988), are opposed by high PCO (Lopez-Lopez and Gonzalez, 1992; Riesco-Fagundo et al., 2001). But their light sensitivity has not been reported. If the light sensitivity does not exist, then the CO effects on type I cells would be dissociated from those on oxygen consumption and CSN responses. The objective of the present investigation was to determine whether there exists a CO-induced light effects on K+ currents of type I cells of rat carotid body.
2. Methods The glomus cells were separated enzymatically using collegenase IV (Sigma Co). The dissociated cells transferred to growth medium, plated on glass cover slips in Petri dishes and maintained at 37 °C in a CO2 –air incubator for up to 48 h for electrophysiological recording. Before the recording, the cells along with the cover slip were transferred into the patch clamp recording chamber and were perfused at the rate of 1.5 ml/min by gravity with desired bath solution. The temperature was maintained at 36– 37 °C and monitored with TC344B dual automatic temperature controller (Warner Instrument CO) during the entire procedure. The pipette filling solution contained (in mM): 110 K-glutamate, 30 KCl, 2.0 MgCl2, 5 CaCl2, 10 HEPES, with 100–200 mg/ml nystatin at pH 6.8, adjusted with KOH. Extracellular solution consisted of (in mM) 140 NaCl, 5 KCl, 2.0 MgCl2, 5 CaCl2, 10 HEPES, with 50 mM of ATP at pH 7.0, adjusted with NaOH. These pH values with HEPES buffer were chosen because the sensory discharge gave the optimal activity at this pH (Osanai et al., 1997a,b). High PCO (550 Torr) with PO2 (120 Torr) solution was obtained by bubbling above extracellular solution with appropriate CO and O2. The pipettes had resistances of 2– 6 MV when filled with above solution. The glomus cells were identified based on the presence of granular birefringence appearance and the response of the K+ currents to pH. The cells we considered as the glomus cells if their K+
currents were lower at [pH]e 7.0 than at [pH]e 7.4. To avoid the cytosolic constituents washout effect, following patch excision, perforated whole cell configuration was used to measure the K+ currents, using nystatin (Horn and Marty, 1988). Whole cell recordings were performed using an Axopatch 200B patch clamp amplifier (Axon Instruments, CA). The voltage clamp commands were generated using DigiData 1200 interface (Axon Instruments, CA) connected to a PC (Gateway E-4200). The 90 ms depolarizing voltage steps from − 80 to + 60 mV with 10 mV increments were used to elicit the outward currents. Signals were filtered at 1 kHz and acquired at 10 kHz. Pclamp 8.0 programs (Axon Instruments) were used for data acquisition and analysis. All experiments were conducted in pairs (in the absence and presence of high PCO with lighton and light-off conditions). Currents were recorded only when stable membrane potentials and currents were reached after the bath solution or the illumination had been changed. The data analyses were performed from the average currents over the range 78–88 ms of the voltage steps. Mean current-voltage (I–V) relationships were calculated from 4 cells. Membrane conductance was determined by fitting a linear regression to the current-voltage relationship over the range − 10 to +40 mV. Illumination was controlled using the microscope light source. The strength of bright light was 100 W/12 V.Results were presented as means9S.E.M. (n, is the number of observations). Statistical significance was assessed using Student’s two-tailed paired t-test. P was considered significant at B 0.05.
3. Results The K+ currents were increased significantly on exposure to 550 Torr PCO, following the depolarizing voltage steps regardless of the presence of bright light. The representative recordings and I–V relationships in the presence of the bright light are presented in Fig. 1A–C. Fig. 1A shows the currents recorded in the absence of high PCO, and Fig. 1B shows that in the presence of high PCO. The I–V relationships were plotted in Fig.
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
1C. The curves with filled square are for the control and those with the open circle for those in the presence of high PCO. With the light off, the representative recordings are shown in Fig. 1D, E in the absence and presence of high PCO respec-
287
tively. Fig. 1F shows the I–V relationships in both absence (filled squares) and presence (open circles) of 550 Torr PCO. The light did not change the CO-induced increases of K+ currents. The 550 Torr PCO also increased the K+ conductances
Fig. 1. Representative recordings of the High PCO effects on the whole cell outward K+ currents in the presence and the absence of bright light. Temperature was 36.8 °C. The membrane was held at − 85 mV and voltage steps were from −80 to +60 mV in 10 mV increment. All recordings were from the same patch. (A) It shows the whole cell outward K+ currents in the absence of high PCO with the light on. (B) It shows the currents in the presence of high PCO. 550 Torr PCO significantly increased the whole cell outward K+ currents when the glomus cell exposed to the bright light. (C) It is the I – V curves from the (A) (·, without CO) and B (, with CO). (D and E) These were recorded respectively in the absence and the presence of 550 Torr PCO with the light off. (F) It is the I – V curves from D and E. High PCO resulted in a considerable increase of the outward K+ currents and left ward shift of the reversal potential as those in the presence of bright light.
288
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
Fig. 2. Summary of high PCO-induced light independent increases of the whole cell conductance and of the left ward shift of the reversal potential. 550 Torr PCO significantly increased the whole cell K+ conductance regardless of light was on (A) or off (B), (P B 0.05 for both). However, CO-induced increases of the conductances were insensitive to the light (P \0.05). 2C and D show the high PCO induced the reverse potential shifts from − 33.09 8.6 to − 50.8 910.8 mV with the light on (C) and from 35.8 9 10.7 to 50.09 11.3 mV (D), (P B 0.05 for both). This CO-induced reversal potential shift was not affected by high PCO (P\ 0.05).
from 3.7791.56 to 7.22 9 2.20 nS with the light on (Fig. 2A, PB 0.05) and from 3.369 1.59 to 5.75 91.68 nS with the light off condition (Fig. 2B, PB 0.05). High PCO increased conductances respectively by 140.43942.76% with light on and by 115.3097.93% with light off conditions. The increases of the conductances in the presence and
absence of light were not statistically different (P\ 0.05). The high PCO resulted in left ward shifts of the reversal potentials in both presence and absence of the bright light (PB 0.05). However, CO-induced reversal potential shifts were not light sensitive (P\ 0.05). The 550 Torr PCO induced 17.7593.71 mV left ward shift (Fig. 2C)
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
and 14.2593.47 mV (Fig. 2D) of the reversal potentials in the presence and absence of light respectively. These results suggest no photolabile effect on the CO-induced K+ permeability in rat glomus cells. As control, we also examined the possibility of light effect on the K+ permeability in the absence of CO. No light effect was observed on both K+ conductances (Fig. 2A, B, unfilled bars) and the reversal potentials (Fig. 2C, D, unfilled bars), P\ 0.05. The results indicated that the K+ channels were not sensitive to light.
4. Discussion It is well known that CO stimulates CSN discharges and concomitantly decreases oxygen uptake (Lahiri et al., 1995). The dogma is that glomus (type I) cells are the initial sites of transduction and they release neurotransmitters in response to hypoxia and high PCO, which in turn depolarize the nearby afferent nerve endings, leading to an increase in CSN discharge (Gonzalez et al., 1994). One hypothesis suggests that K+ channel proteins of glomus cells are associated with the oxygen sensing and that high PCO inhibits this channels, ensuing depolarization of glomus cell and CO-mediated CSN excitation (Gonzalez et al., 1994; Lahiri et al., 2001). Hypoxia inhibits glomus cell K+ currents, which is reversed by CO. (Lopez-Lopez and Gonzalez, 1992; Riesco-Fagundo et al., 2001), as if CO hyperpolarized the cells during moderate hypoxia. CO effects on CSN discharge also were suppressed during moderate hypoxia and the light further inhibited the response (Lahiri et al., 1993). But the light effects were reversed during extreme hypoxia (Lahiri et al., 1999). It was also reported that CO hyperpolarized single smooth muscle cells and that the whole-cell outward K+ currents were enhanced by CO (Wang et al., 1997). However, no light effects were reported in these studies. The CSN discharge is immediately stimulated by high PCO in the absence of light and is accompanied by a decreased oxygen uptake, which are immediately reversed by bright light (Lahiri et al., 1995). These characteristics match that of cy-
289
tochrome a3 (Warburg and Negelein, 1928). Furthermore, the action spectrum characteristics of the CO compound match those of mitochondria cytochrome a3 (Wilson et al., 1994). CO no doubt acted on the glomus cells, and perhaps neurotransmitters were secreted but the light effects were not there. On the other hand, if CO might have acted on the nerve endings which are well equipped with mitochondria and other organelles to elicit a neural response, there is a possible disconnection. This disconnection between K+ permeability of the glomus cells and CSN discharge is consistent with the lack of the effects of the K+ channel blockers, charybdotoxin (Osanai et al., 1997a,b), TEA and 4 AP (Lahiri et al., 1998; Roy et al., 1998) on CSN discharge unlike the response of glomus cells (Lopez-Barneo et al., 1988) during hypoxia.
Acknowledgements This work is supported in part by the grants of R37-HL43413-12, RO1-HL 50180-7 and N001401-0948.
References Gonzalez, C., Almaraz, L., Obeso, A., Rigual, R., 1994. Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiol. Rev. 74, 829 – 898. Horn, R., Marty, A., 1988. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145 – 159. Joels, N., Neil, E., 1962. The action of high tension of carbon monoxide on the carotid chemoreceptor. Arch. Int. Pharmacol. Ther. 139, 528 – 534. Lahiri, S., Iturriaga, R., Mokashi, A., Ray, D.K., Chugh, D., 1993. CO reveals dual mechanisms of O2 chemoreception in the cat carotid body. Respir. Physiol. 94, 227 – 240. Lahiri, S., Buerk, D.G., Chugh, D., Osanai, S., Mokashi, A., 1995. Reciprocal photolabile O2 consumption and chemoreceptor excitation by carbon monoxide in the cat carotid body: evidence for cytochrome a3 as the primary O2 sensor. Brain Res. 684, 194 – 200. Lahiri, S., Roy, A., Rozanov, C., Mokashi, A., 1998. K+-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor. Brain Res. 794, 162 –165. Lahiri, S., Ehleben, W., Acker, H., 1999. Chemoreceptor discharges and cytochrome redox changes of the rat
290
J. Li et al. / Respiratory Physiology & Neurobiology 131 (2002) 285–290
carotid body: role of heme ligands. Proc. Natl. Acad. Sci. USA 96, 9427 – 9432. Lahiri, S., Rozanov, C., Roy, A., Storey, B., Buerk, D.G., 2001. Regulation of oxygen sensing in peripheral arterial chemoreceptors. Int. J. Biochem. Cell Biol. 33, 755 –774. Lopez-Barneo, J., Lopez-Lopez, J.R., Urena, J., Gonzalez, C., 1988. Chemotransduction in the carotid body: K + current modulated by PCO2 in type I cells. Science 241, 580 –582. Lopez-Lopez, J.R., Gonzalez, C., 1992. Time course of K+ current inhibition by low oxygen in chemoreceptor cells of adult rabbit carotid body. Effects of carbon monoxide. FEBS Lett. 299, 251 –254. Osanai, S., Buerk, D.G., Mokashi, A., Chugh, D.K., Lahiri, S., 1997a. Cat carotid body chemosensory discharge (in vitro) is insensitive to charybdotoxin. Brain Res. 747, 324 – 327. Osanai, S., Rozanov, C., Mokashi, A., Buerk, D.G., Lahiri, S., 1997b. CO interact with intracellular [H+] with and without CO2 – HCO− in the cat carotid chemosensory dis3 charge. Brain Res. 764, 221 –224.
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