The effect of amp-kinase activators on oxygen-sensing type 1 cells of the rat carotid body

C. N.Wyatt1, C. Peers2, P. Kumar3, D. G. Hardie4, and A. M. Evans1. 1Division of Biomedical Sciences, School of Biology, Bute Building, Westburn Lane, St Andrews, Fife, KY16 9TS. 2Faculty of Medicine, Worsley Building, University of Leeds, W. Yorkshire, LS2 9JT. 3Department of Physiology, The Medical School, University of Birmingham, B15 2TT. 4Division of Molecular Physiology, School of Life Sciences, University of Dundee, DD1 5EH.

The carotid bodies play a critical role in initiating compensatory ventilatory responses to hypoxia. However, the complete mechanism by which hypoxia excites the oxygen-sensing carotid body type 1 cells has not been fully defined. We have previously proposed that the enzyme adenosine monophosphate-activated protein kinase (AMPK) may couple hypoxic inhibition of oxidative phosphorylation to carotid body type 1 cell excitation (Evans et al, 2005). Here we present evidence that activating AMPK by 2 distinct mechanisms mimics the effect of hypoxia on the oxygen-sensing carotid body type 1 cells.

Wistar rats (age 10-25 days) were killed by schedule 1 methods, their carotid bodies removed and enzymically dispersed (Wyatt et al, 2004). Isolated type 1 cells were fixed in methanol for immunocytochemical studies or maintained in tissue culture for subsequent electrophysiological and Ca2+ imaging studies. Cells were recorded from using the perforated-patch configuration of the patch-clamp technique and intracellular Ca2+ ([Ca2+] i) was monitored using the dye FURA-2 (ex; 340/380 nm, em; 510 nm, HEPES buffer, 37 oC). All data is presented as mean ± S.E.M and statistical analysis was carried out using the Student’s paired t test.

Immunocytochemistry demonstrated that AMPKα1 subunits were targeted to the plasma membrane of type 1 cells. Application (10 min) of 2 commonly used AMPK activators, the AMP-mimetic 5-aminoimidazole-4-carboxamide riboside (AICAR, 1mM, see Evans et al, 2005) or the mitochondrial complex 1 inhibitor phenformin (10mM) caused type 1 cells to depolarise by 12.6 ± 0.9 mV (n=6, P<0.05) and 24.8 ± 6.2 mV (n=6, P<0.04) respectively. AICAR (1mM) and phenformin (10mM) inhibited macroscopic currents at +30 mV by 40.0 ± 12.2 % (n=5, P<0.05) and 49.6 ± 12.7 % (n=5, P<0.03) respectively. The inhibited currents were found to be the oxygen-regulated BKCa current and a Ba2+-sensitive leak current. Additionally, AICAR and phenformin caused a rise in FURA-2 fluorescence ratio from a baseline of 0.62 ± 0.01 to 1.71 ± 0.23 (n=7, P<0.001) and 0.60 ± 0.01 to 2.02 ± 0.30 (n=7, P<0.006) respectively. The voltage-gated calcium channel blocker nickel (5mM) reversed these increases in [Ca2+]i.

Thus AMPK activation mimics excitation by hypoxia in carotid body type 1 cells by triggering membrane depolarisation via inhibition of the oxygen–sensitive BKCa currents and oxygen regulated, Ba2+-sensitive leak currents. This results in voltage-gated Ca2+ entry to the type 1 cell.

Evans A. M. et al (2005) J. Biol. Chem. 41504 - 41511
Wyatt C. N. et al (2004) J. Physiol (Lond) 556.1, 175-191.

This work was supported by the Wellcome Trust