Effects of modulators of AMP-activated protein kinase on TASK-1/3 and intracellular Ca(2+) concentration in rat carotid body glomus cells

Abstract

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca(2+) concentration ([Ca(2+)]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 (1mM AICAR and 0.1-0.5mM A769662) did not inhibit TASK activity or cause depolarization during acute (10min) or prolonged (2-3h) exposure. Hypoxia inhibited TASK activity by ∼70% in cells pretreated with AICAR or A769662. Both AICAR and A769662 (15-40min) failed to increase [Ca(2+)]i in glomus cells. Compound C (40μM), 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 [Ca(2+)] in isolated rat carotid body glomus cells.

Keywords: AMP kinase; Background K(+) channels; Carotid body; Chemoreceptors; Hypoxia.

Figure 1. No effect of AICAR and A769662 on TASK activity in glomus cells

A. Cell-attached patches were perfused with solution 1 mM AICAR or 0.1 mM A769662 until patches broke or up to 12 min. Pipette potential was set at 0 mV. Representative single-channel currents at ~1 min and ~10 min following application of the drugs are shown. B. TASK activity (NPo) was determined at various time points and plotted as a function of perfusion time. TASK activity was calculated and normalized to that obtained at time zero (1.0). Each point is the mean±SD of 12 determinations from 6 separate cell preparations. C. TASK activity in cell-attached patches was determined from cells incubated with 0.1% DMSO, 1 mM AICAR or 0.1 mM A769662 for 2–3 h. Each bar is the mean±SD of 12 patches.

Figure 2. AMPK modulators do not affect the hypoxia-induced inhibition of TASK activity

A. Cell-attached patches were perfused with normoxic solution, then with hypoxia solution for ~30 sec, and then back to normoxic solution. Glomus cells were incubated for 2–3 h with 0.1% DMSO. B-C. Same experiment as in A except that glomus cells were incubated with 1 mM AICAR (B) or A760662 (C) for 2–3 h before recording TASK in cell-attached patches. Perfusion solutions contained either AICAR or A769662. D. Glomus cells were incubated with 40 µM Compound C for 2–3 hr. Cell-attached patches were then formed and perfused with normoxic and hypoxic solution containing Compound C. For all bar graphs (A-D), each bar is the mean±SD of 6–8 patches. Asterisk (*) indicates a significant reduction from control (normoxia) at p < 0.05. For all experiments in this figure, data were gathered from 4 separate cell preparations.

Figure 3. Phosphorylation of AMPKα by AICAR and A769662

PC12 cells were treated with 1 mM AICAR or 100 µM A769662 for 60 min. Compound C (40 µM) was pretreated for 30 min to block AMPK activation. Activated AMPK (p-AMPKα) was detected in PC12 cells using a specific antibody for the phosphorylated form of AMPKα. Whole-cell lysate was used as total protein. Equal amounts (30 µg) of total protein were loaded in each lane. The plus and minus signs (+ and −) represent treatment conditions with and without chemical, respectively. The bands obtained from Western blot were quantified by Quantity One software (version 4.6.3) attached to GS-800 Calibrated densitometer (Bio-Rad). The numbers below the pAMPKα band represent the ratio of the expression levels of p-AMPKα to those of AMPKα for each lane. For example, in lanes 1and 4 (control), the level of phosphorylated p-AMPKα compared to that of AMPKα is 0.6 and 0.8, respectively. AICAR or A769662 increased the fractional levels of AMPKα phosphorylation (lanes 2 and 5) and Compound C blocked this effect (lanes 3 and 6). The α-tubulin was used as a loading control. The size of molecules: p-AMPKα (62 kDa), AMPKα (62 kDa), and α-tubulin (50 kDa). The experiment was done three times and an image from one experiment is shown.

Figure 4. AICAR and A769662 do not elevate intracellular [Ca²⁺]

A. Representative averaged trace of changes in [Ca²⁺]_i in glomus cells in response to 20 mM KCl, hypoxia, AICAR (1 mM), and AICAR (1 mM) + Compound C (40 µM) are shown. B. Representative averaged trace of changes in [Ca²⁺]_i in glomus cells in response to 20 mM KCl, hypoxia, A769662 (0.1 mM), and A769662 (0.1 mM) + 0% O₂ are shown. C. Averaged [Ca²⁺]_i increase above the basal level in response to 20 mM KCl (n=15), 0% O₂ (n=15), 1 mM AICAR (n=15), AICAR (1 mM) + Compound C (40 µM) (n=15), A769662 (0.1 mM) (n=25), and A769662 (0.1 mM) + 0% O₂ (n=8). Each bar represents the mean±SE. Asterisk(*) indicates significant difference at p < 0.001. NS= not significant. D-F. Glomus cells were incubated in medium containing 1 mM AICAR (D), 0.5 mM A769662 (E) or 0.5% DMSO (F) for 30 min at 37°C before recording [Ca²⁺]. Representative averaged traces of changes in [Ca²⁺]_i in response to hypoxia and 20 mM KCl are shown. For these experiments, cells from 3 separate preparations were used.