Contribution of TASK like potassium channels to the enhanced rat carotid body responsiveness to hypoxia

Texto completo

(1)Chapter 49. Contribution of TASK-Like Potassium Channels to the Enhanced Rat Carotid Body Responsiveness to Hypoxia Fernando C. Ortiz, Rodrigo Del Rio, Rodrigo Varas, and Rodrigo Iturriaga. Abstract A major hallmark of obstructive sleep apnea is the potentiation of the carotid body (CB) chemosensory response to acute hypoxia, as result of the chronic intermittent hypoxia (CIH) exposition. Several mechanisms have been involved in this CB chemosensory potentiation, but the primary target of CIH remains elusive. In physiological conditions, hypoxia depolarized CB chemoreceptor cells, trigger an increase of intracellular Ca2+, and the subsequent transmitter’s release. Since the depolarization is initiated by the inhibition of a TASK-like K+ channel, we studied if CIH may increase the amplitude of the hypoxic-induced depolarization in the chemoreceptor cells, due to an enhanced inhibition of the TASK-like current. CBs obtained from adult rats exposed to CIH (5% O2, 12 times/hr for 8 hr/day) for 7 days were acute dissociated, and the membrane potential and TASK-like current were recorded from isolated chemoreceptor cells. Resting membrane properties were not modified by CIH, but the amplitude of the hypoxic-evoked depolarization increases ~2-fold. The same result was obtained when all the voltage-dependent K+ currents were pharmacologically blocked. Accordingly, the inhibition of the TASK-like current induced by acute hypoxia (PO2 ~5 torr) increased from ~62% in control cells to ~96% in the CIH cells. Present results show that acute hypoxic inhibition of TASK-like K+ channel is potentiated by CIH exposure, suggesting that the enhancing effect of CIH on CB chemosensory responsiveness to hypoxia occurs at the initial step of the oxygen transduction in the CB chemoreceptor cells. Keywords Carotid body • Chemosensory discharge • Glomus cells • TASK channels • Acute hypoxia • Single channel recording • Intermittent hypoxia • Obstructive sleep apnea. 49.1. Introduction. Chronic intermittent hypoxia (CIH) is the main characteristic of the obstructive sleep apnea (OSA) syndrome, a growing health problem that affects 5% of the worldwide population (Somers et al. 2008). The OSA syndrome is recognized as an independent risk factor for sympathetic overactivation. F.C. Ortiz • R. Del Rio • R. Varas • R. Iturriaga (*) Laboratorio de Neurobiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile e-mail: [email protected]; [email protected] C.A. Nurse et al. (eds.), Arterial Chemoreception: From Molecules to Systems, Advances in Experimental Medicine and Biology 758, DOI 10.1007/978-94-007-4584-1_49, © Springer Science+Business Media Dordrecht 2012. 365.

(2) 366. F.C. Ortiz et al.. and hypertension (Somers et al. 2008; Garvey et al. 2009). A major contributing mechanism to the cardiovascular alterations induced by CIH is the potentiation of the carotid body chemosensory responses to acute hypoxia (Peng et al. 2003; Rey et al. 2004; Del Rio et al. 2010). Oxidative stress, endothelin-1 and pro-inflammatory molecules have been involved in the carotid chemosensory potentiation, but the primary target for CIH in the carotid body remains elusive (Iturriaga et al. 2009). The most accepted model for carotid body chemoreception states that in response to acute hypoxia the CB chemoreceptors (type I) cells depolarize, triggering an intracellular calcium increase and the subsequent release of one or more excitatory neurotransmitters (Iturriaga et al. 2007). In neonatal rats it has been shown that the depolarization is initiated by the hypoxic inhibition of a background TASK-like potassium channel, probably a TASK-1/TASK-3 heterodimer (Buckler 1999, 2010; Kim et al. 2009). We hypothesized that the enhanced carotid chemosensory responses to hypoxia is partially explained by the potentiation of the hypoxia-induced depolarization, due to a greater inhibition of the TASK-like current by acute hypoxia. Therefore, we evaluated if CIH may increase the amplitude of the hypoxic-induced depolarization by enhancing the inhibition of the TASK-like current in chemoreceptor cells from rats exposed to CIH for 7 days.. 49.2 49.2.1. Methods Animals and Exposure to Intermittent Hypoxia. Experiments were performed on adult male Sprague-Dawley rats (200–250 g), fed with standard chow diet ad libitum, and kept on a 12-h light/dark schedule (8:00 a.m.–8:00 p.m.). Animals were randomly assigned to CIH or to Control (sham) conditions. The experimental procedures were approved by the Bio-Ethical Committee of the Biological Sciences Faculty, P. Universidad Católica de Chile, and were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were housed in individual chambers and exposed to either hypoxic cycles of 5% inspired O2 for 20 s, followed by room air for 280 s, applied 12 times/h; 8 h/day or control (sham) group exposed to air/air cycles for 7 days (Del Rio et al. 2010). The O2 level in the chambers was continuously monitored with an oxygen analyzer and the CO2 was maintained low by continuous air extraction. The room temperature was kept at 23–25°C.. 49.2.2. Recordings of Ventilatory Reflex and Carotid Chemosensory Responses to Acute Hypoxia. Rats were anesthetized with sodium pentobarbitone (40 mg/kg, i.p.). The trachea was cannulated for recording the airflow signal with appropriate pneumotachographs. Minute inspiratory volume (VI), tidal volume and respiratory frequency were digitally obtained from the airflow signal. The physiological data was analyzed using the LabChart 7.2 Pro software (AD Instruments, Australia). To assess the effects of CIH on the sensitivity and reactivity of peripheral chemoreceptor reflexes, we studied the ventilatory responses elicited by several isocapnic levels of PO2, maintained during 30 s. At the end of the ventilatory physiological recordings, one carotid sinus nerve was dissected and placed on a pair of platinum electrodes, and covered with warm mineral oil. The neural signal was pre-amplified (Grass P511, USA), filtered (30 Hz to 1 kHz) and fed to an electronic spike-amplitude discriminator, allowing the selection of action potentials of given amplitude above the noise to be counted with a.

(3) 49. Contribution of TASK-Like Potassium Channels to the Enhanced Rat Carotid…. 367. frequency meter to measure the frequency of carotid chemosensory discharge (ƒx), expressed in Hz. Carotid sinus barosensory fibers were eliminated by crushing the common carotid artery wall between the carotid sinus and the carotid body. The contralateral carotid sinus nerve was cut to prevent vascular and ventilatory reflexes evoked by the activation of the CB. The chemosensory discharge was measured at different (isocapnic) levels of PO2.. 49.2.3. Electrophysiological Recordings. The carotid bodies extracted from anesthetized rats were enzymatic (trypsin/collagenase) and mechanically dissociated as previously described (Buckler 1999). Cellular suspension was plated into poly-D-lysine coated glass coverslip. The acute dissociated cells were studied upon either cellattached or whole-cell configuration of the voltage clamp technique. Cells were bathed with standard HCO3-buffered saline contained (in mM) 117 NaCl, 4.5 KCl, 23 NaHCO3, 1.0 MgCl2, 2.5 CaCl2 and 11 glucose; bubbled with either 5% CO2 and 95% air (normoxia, PO2 ~140 mmHg) or 5% CO2 and 95% N2 (during hypoxic stimuli, PO2 ~5 mmHg by 30 s). Pipette solution for cell attached recordings contained (in mM) 140 KCl, 4 MgCl2, 1 EGTA, 10 HEPES, 10 tetraethylammonium (TEA)-Cl and 5 4-aminopyridine (4-AP), equilibrated to pH = 7.43, and for whole-cell recordings (in mM) 140 KCl, 1.5 MgCl2, 10 EGTA, 2 CaCl2, 10 HEPES (pH 7.21). Channel activity was reported as the open probability times the number of active channels in a given patch (NPo). Membrane potential recordings were performed under whole-cell current clamp conditions with no imposed current. Mean values were expressed as MEAN ± SEM.. 49.3 49.3.1. Results Effect of CIH on Ventilatory and Carotid Chemosensory Responses to Acute Hypoxia. Rats exposed to CIH for 7 days showed potentiated reflex ventilatory responses to hypoxia when compared with control rats (Fig. 49.1a, p < 0.001, two-way ANOVA). The carotid chemosensory responses to acute hypoxia were also enhanced by the exposure to CIH (Fig. 49.1b). The two-way ANOVA analysis showed that the overall carotid chemosensory curve for PO2 was different in CIH rats (p < 0.001) compared with the control group. The posthoc test showed that carotid chemosensory discharges were higher (p < 0.01) not only in the hypoxic range, but also in normoxia (Fig. 49.1b).. 49.3.2. Effects of CIH on the Resting Membrane Potential of Isolated Chemoreceptor Cells. Exposure of rats to CIH for 7 days did not modify the resting membrane potential of chemoreceptor cells. The resting membrane potential measured in chemoreceptor cells of the CIH group was −54.3 ± 2.4 mV (n = 30), while in the control cells was −55.1 ± 1.4 mV (n = 28) (p > 0.05, Student T test)..

(4) 368. F.C. Ortiz et al.. Fig. 49.1 Effects of intermittent hypoxia exposure for 7 days on ventilatory and carotid chemosensory responses to acute hypoxia. a Ventilatory response (V, minute volume) to acute hypoxia in rats exposed to CIH for 7 days (CIH, n = 10) and control rats (Control, n = 10). b Carotid chemosensory responses to hypoxia (two way ANOVA, followed by Bonferroni posthoc test. *** p < 0.001, ** p < 0.01 and * p < 0.05). ƒx, frequency of carotid chemosensory discharge expressed in Hz. 49.3.3. Effects of CIH on the Depolarization Induced by Acute Hypoxia in Chemoreceptor Cells. The depolarization (DVmHx) induced by acute hypoxia (PO2 ~5 Torr) was enhanced by the CIH exposure (Fig. 49.2). Indeed, we recorded a DVmHx of 27.5 ± 2.8 mV in 16 chemoreceptor cells from CIH-treated rats versus DVmHx of 15.7 ± 1.5 mV in 20 control cells (p < 0.05, unpaired T-test). The same result was obtained when the voltage-dependent K+ currents were pharmacologically blocked with tetraethyl ammonium and 4-aminopiridine (not shown)..

(5) 49. Contribution of TASK-Like Potassium Channels to the Enhanced Rat Carotid…. 369. Fig. 49.2 Depolarization induced by acute hypoxia (DVmHx) in chemoreceptor cells was enhanced by CIH exposition. (CIH, n = 16) as compared to control cells (control, n = 20). * p < 0.05, Unpaired T-test. Fig. 49.3 TASK-like current inhibition by acute hypoxia is enhanced by CIH exposition. Upper panel, representative cell-attached record of TASK-like channel activity from cells of control and CIH groups during acute hypoxia stimulation (pipette potential 0 mV). Scale: 1 pA, 5 s Bar, hypoxic estimulation. Bottom panel summarized the effects of hypoxia inhibition in 16 control cells and 12 cells from rats exposed to CIH, respectively. * p < 0.05, Mann-Whitney Test. 49.3.4. Effect of CIH on TASK-Channel Inhibition Induced by Acute Hypoxia. In cell-attached recordings under resting conditions (pipette potential of 0 mV) we observed robust TASK-like channel activity in patches from cells of control and CIH groups. Interestingly, we found that TASK-like current inhibition by acute hypoxia was enhanced by CIH exposition. Fig. 49.3 shows representative traces of TASK-like channel activity recorded from cell-attached patches of chemoreceptor cells in response to acute hypoxia (PO2 ~ 5 Torr). The hypoxic inhibition, measured as percentage of NPo in normoxic conditions was 62.5 ± 2.4% (n = 16) in the control group while in the cells from rats exposed to CIH the inhibition was 96.4 ± 1.5% (n = 12). Thus, present results showed that the hypoxic.

(6) 370. F.C. Ortiz et al.. inhibition of TASK-like K+ channel is potentiated by the CIH exposure, suggesting that the enhancing effects of CIH on carotid chemosensory responsiveness to hypoxia occurs at the initial step of the oxygen transduction in the chemoreceptor cells.. 49.4. Discussion. Exposure to CIH induces a potentiation of the rat carotid body chemosensory and ventilatory responses to hypoxia (Del Rio et al. 2010). In the present report the enhanced carotid chemosensory response to hypoxia was associated with a large inhibition of the TASK-like current in chemoreceptor cells in response to acute hypoxia. It is worth noting that the present study was performed in adult rats, which functionally expresses a TASK-like current in the carotid body chemoreceptor cells. This current was reversibly inhibited by acute hypoxia, and therefore, could be involved in the adult carotid hypoxic response in this animal model. The resting TASK-like channel activity in normoxia was not modified by the CIH treatment. This result agrees with the data showing no effects of CIH in the resting membrane potential. However, the TASK-like current inhibition by acute hypoxia clearly increased, ~96% in CIH group versus ~62% of inhibition in control group. In order to establish a relationship between the enhanced TASK-like current inhibition and the augmented depolarization observed in chemoreceptor cells from CIH-treated rats, it was studied the hypoxia-induced depolarization when the main voltage-gated K+ conductances were blocked with TEA and 4-AP. In this condition the depolarization induced by acute hypoxia in both control and CIH group were roughly the same, suggesting that the enhanced depolarization observed in CIH cells is explained at least in part by a potentiation in the inhibition of TASKlike current. It is interesting to note that while the normoxic TASK-like current remains unaffected by the CIH treatment, its sensitivity to hypoxia enhances. One explanation for these observations is that CIH treatment changes the CB type-I cell metabolic state, triggering modifications on TASK-like channel regulatory factors involved in the hypoxic response (Varas et al. 2007) rather than a direct effect on channel structure or expression. In conclusion, present results showed that the chemoreceptor cells depolarization to acute hypoxia was enhanced by the CIH treatment, due to the increase of the acute hypoxia inhibitory effect on TASKlike potassium channel. This raises a new mechanism to explain the effects of chronic intermittent hypoxia on carotid body acclimatization. Acknowledgements This work was supported by grant 1100405 from the National Fund for Scientific and Technological Development of Chile (FONDECYT).. References Buckler KJ (1999) Background leak K+-currents and oxygen sensing in carotid body type 1 cells. Respir Physiol 115:179–187 Buckler KJ (2010) Two-pore domain K+ channels and their role in chemoreception. Adv Exp Med Biol 66:15–30 Del Rio R, Moya EA, Iturriaga R (2010) Carotid body and cardiorespiratory alterations in intermittent hypoxia: the oxidative link. Eur Resp J 36:143–150 Garvey JF, Taylor CT, McNicholas WT (2009) Cardiovascular disease in obstructive sleep apnoea syndrome: the role of intermittent hypoxia and inflammation. Eur Resp J 33:1195–1205 Iturriaga R, Varas R, Alcayaga J (2007) Electrical and pharmacological properties of petrosal ganglion neurons that innervate the carotid body. Resp Physiol Neurobiol 157:130–139 Iturriaga R, Moya EA, Del Rio R (2009) Carotid body potentiation induced by intermittent hypoxia: implications for cardiorespiratory changes induced by sleep apnoea. Clin Exp Pharmacol Physiol 36:1197–1204.

(7) 49. Contribution of TASK-Like Potassium Channels to the Enhanced Rat Carotid…. 371. Kim D, Cavanaugh EJ, Kim I, Carroll JL (2009) Heteromeric TASK-1/TASK-3 is the major oxygen-sensitive background K+ channel in rat carotid body glomus cells. J Physiol 587:2963–2975 Peng YJ, Overholt JL, Kline D, Kumar GK, Prabhakar NR (2003) Induction of sensory long-term facilitation in the carotid body by intermittent hypoxia: implications for recurrent apneas. Proc Natl Acad Sci U S A 100:10073–10078 Rey S, Del Rio R, Alcayaga J, Iturriaga R (2004) Chronic intermittent hypoxia enhances cat chemosensory and ventilatory responses to hypoxia. J Physiol 560:577–586 Somers VK, White DP, Amin R, Abraham WT, Costa F, Culebras A, Daniels S, Floras JS, Hunt CE, Olson LJ, Pickering TG, Russell R, Woo M, Young T (2008) Sleep apnea and cardiovascular disease. J Am Coll Cardiol 52:686–717 Varas R, Wyatt CN, Buckler KJ (2007) Modulation of TASK-like background potassium channels in rat arterial chemoreceptor cells by intracellular ATP and other nucleotides. J Physiol 583:521–536.

(8)