Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca2+ concentration in rat carotid body glomus cells

https://doi.org/10.1016/j.resp.2014.01.020Get rights and content

Highlights


  • Activators of AMPK had no effect on TASK channel activity.


  • Activators of AMPK had no effect on resting membrane potential and [Ca2+]i.


  • Hypoxia inhibited TASK activity in the presence of Compound C.


  • Activators of AMPK phosphorylated AMPKα in PC12 cells.


  • AMPK may not mediate the hypoxic-induced excitation of isolated glomus cells.

Abstract

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that AMP-activated protein kinase (AMPK) mediates these effects of hypoxia by inhibiting the background K+ channels such as TASK. Here we studied the effects of modulators of AMPK on TASK activity in cell-attached patches. Activators of AMPK (1 mM AICAR and 0.1–0.5 mM A769662) did not inhibit TASK activity or cause depolarization during acute (10 min) or prolonged (2–3 h) exposure. Hypoxia inhibited TASK activity by ∼70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15–40 min) failed to increase [Ca2+]i in glomus cells. Compound C (40 μM), an inhibitor of AMPK, showed no effect on hypoxia-induced inhibition of TASK. AICAR and A769662 phosphorylated AMPKα in PC12 cells, and Compound C blocked the phosphorylation. Our results suggest that AMPK does not affect TASK activity and is not involved in hypoxia-induced elevation of intracellular [Ca2+] in isolated rat carotid body glomus cells.

Introduction

In carotid body glomus cells, hypoxia inhibits the outward K+ current, and thereby causes cell depolarization, Ca2+ influx via voltage-dependent Ca2+ channels and secretion of transmitters (Ortega-Saenz et al., 2007, Peers et al., 2010, Prabhakar, 2006). The hypoxia-sensitive K+ current in glomus cells is believed to consist mainly of Kv, BK and TASK (TASK-1, TASK-3 and TASK-1/3) channels, but the signaling pathways by which hypoxia inhibits each of these K+ channels are not well defined. Several mechanisms for hypoxia-induced inhibition of K+ current have been proposed, including inhibition of heme-oxygenase-2 (Williams et al., 2004), inhibition of mitochondrial oxidative phosphorylation (Buckler and Vaughan-Jones, 1998, Wyatt and Buckler, 2004), and an undefined rotenone-sensitive pathway (Ortega-Saenz et al., 2003). It may be that different O2 sensors and signals are involved in the inhibition of specific K+ channels, but this remains to be determined. The inhibition of mitochondrial oxidative phosphorylation is probably responsible for the hypoxia-induced reduction of TASK, as mitochondrial inhibitors and uncouplers reversibly inhibit these two-pore domain background K+ channels (Buckler, 2007, Buckler, 2012, Kim, 2013).

Inhibition of mitochondrial oxidative phosphorylation results in the reduction of ATP production and rise in [ADP]/[ATP] ratio. Adenylate kinase converts ADP to AMP and ATP, which causes an increase in cell [AMP]/[ATP] ratio (Oakhill et al., 2011). Increases in both [ADP]/[ATP] and [AMP]/[ATP] ratios have been shown to stimulate AMP-activated protein kinase (AMPK) to regulate cell energy consumption (Hardie and Carling, 1997, Steinberg and Kemp, 2009). In glomus cells, AICAR, a well-known activator of AMPK, was found to inhibit the outward whole-cell K+ current sensitive to iberiotoxin, suggesting that BK was a target of AMPK (Wyatt et al., 2007). In the same study, AICAR caused cell membrane depolarization, elevated intracellular calcium concentration ([Ca2+]i) in glomus cells and increased the carotid sinus nerve discharge in carotid body-sinus nerve preparations. AICAR also inhibited a Ba2+-sensitive, voltage-independent K+ current, suggesting that a background K+ current was also targeted by AICAR (Wyatt et al., 2007). In support of this finding, AICAR inhibited TASK-3 expressed in HEK293 cells by ∼50%, and this inhibition was blocked by Compound C, an inhibitor of AMPK (Dallas et al., 2009). These findings have led to the hypothesis that AMPK mediates the hypoxia-induced excitation of glomus cells by inhibition of K+ channels such as BK and TASK that are both well expressed in glomus cells (Peers et al., 2010, Wyatt et al., 2007).

In the course of our studies to identify the effect of phosphorylation by AMPK on TASK single channel behavior and potential amino acid residues involved, we tested the effect of AICAR on TASK single channel kinetics to confirm its inhibitory action. Our preliminary tests using cell-attached patches showed no effect of AICAR on TASK function in glomus cells or in COS-7 cells expressing TASK-3. As our findings are in direct contradiction to the proposal that AMPK inhibits TASK and mediates the hypoxia-induced excitation of glomus cells, we further investigated the effects of AMPK on hypoxia-induced inhibition of TASK and intracellular [Ca2+]i in glomus cells. TASK channel activity in cell-attached patches and intracellular [Ca2+]i were recorded in response to modulators of AMPK. Consistent with our preliminary observation, AMPK activators failed to inhibit TASK, and hypoxia still inhibited TASK and produced strong depolarization even after blockade of AMPK with Compound C. Furthermore, AMPK activators failed to produce depolarization or an increase in [Ca2+]i in glomus cells. Our results show that AMPK is unlikely to be a signal for hypoxia-induced inhibition of TASK and depolarization in isolated rat glomus cells.

Section snippets

Cell isolation

Rats (postnatal 14–18 day; Sprague-Dawley) were anesthetized with isoflurane and used according to the animal protocols approved by the Animal Care and Use Committees of Rosalind Franklin University and University of Arkansas for Medical Sciences. The carotid bodies were removed and placed in ice-cold low-Ca2+, low-Mg2+ phosphate buffered saline solution (low Ca2+/Mg2+-PBS: 137 mM NaCl, 2.8 mM KCl, 2 mM KH2PO4, 0.07 mM CaCl2, 0.05 mM MgCl2, pH 7.4). Each carotid body was cut into 3–4 pieces and

AICAR and A769662 do not affect TASK activity in cell-attached patches

In our recent study, biophysical and pharmacological studies showed that TASK was the primary K+ channels that was open at rest in cell-attached patches of glomus cells (Kim et al., 2009). In this study, TASK was clearly identified by the single channel conductance level (∼34-pS) and short mean open time (∼1 ms), and was recorded without applying any potential to the pipette (i.e., a pipette potential of 0 mV). Under this condition, single channel openings of TASK current can be recorded, because

Discussion

One of the important questions regarding the mechanism of O2 sensing by chemoreceptors relates to how hypoxia inhibits K+ channels to cause excitation of glomus cells. Various signals such as reactive oxygen species, carbon monoxide and hydrogen sulfide have been proposed, but the pathway involving AMPK has been gaining attention. The reason for this is that AICAR was found to inhibit the outward K+ current and also inhibit the hypoxia-induced elevation of [Ca2+]i. Furthermore, Compound C was

Acknowledgments

This work was funded by grant awards to D. Kim (NIH-HL111497), J.L. Carroll (NIH-HL054621) and D. Kang (Korean Research Foundation #2010-0024258).

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