AMP-activated protein kinase mobilises calcium from a thapsigargin-sensitive, cyclopiazonic acid-insensitive sarcoplasmic reticulum calcium store via ryanodine receptors but not via IP3 receptors

E. A. Blanco, C. N. Wyatt, A. M. Evans, C. Peers. Division of Biomedical sciences, School of Biology, Bute Building, University of St.Andrews, Fife KY16 9TS.

We have previously suggested that AMP-activated protein kinase (AMPK), a metabolic sensor, may underpin sarcoplasmic reticulum (SR) Ca2+ signalling in response to hypoxia in isolated pulmonary arterial smooth muscle cells (PASMCs) and thereby hypoxic pulmonary vasoconstriction (Evans et al., 2005). However, we have yet to characterise the SR store from which AMPK mobilises Ca2+. We have previously shown that hypoxia mobilises Ca2+ in a cADPR-dependent manner and from a cyclopiazonic acid (CPA)-insensitive SR Ca2+ store via ryanodine receptors (RyRs; Dipp & Evans, 2001). In this study we sought to characterise the SR store from which AMPK mobilizes Ca2+.

Male Wistar rats (150-300 g) were killed by cervical dislocation, PASMCs from 2nd and 3rd order branches of the pulmonary arterial tree isolated and changes in intracellular Ca2+ concentration monitored using Fura-2 as described previously (Evans et al., 2005). Activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR, 1 mM) increased the Fura-2 fluorescence ratio (F340/F380) by 0.154 ± 0.07 (mean ± S.E.M. for all experiments; n = 77). Pre-incubation (30 min) of PASMCs with either the RyR antagonist ryanodine (40 µM; n = 18) or the cADPR antagonist 8-bromo-cADPR (100 µM; n = 23) abolished the increase in the Fura-2 fluorescence ratio induced by AICAR respectively. In marked contrast, the IP3 receptor (IP3R) antagonist xestospongin C (0.1 µM, 30 min) had no inhibitory effect on the response to AICAR (0.33 ± 0.04; n = 12) . Furthermore, after pre-incubation (30 min) of cells with the SR Ca2+ ATPase (SERCA) antagonist cyclopiazonic acid (CPA; 10 µM) the increase in the Fura-2 fluorescence ratio induced by AICAR remained unaffected ( 0.144 ± 0.016; n = 32). In marked contrast, a 2nd SERCA antagonist, thapsigargin (1µM, 30 min), abolished the increase in Fura-2 fluorescence ratio induced by AICAR ( n = 8).

Most significantly, using small vessel myography as described previously (Dipp & Evans, 2001), we found that AICAR-induced constriction (1.44 ± 0.57 mN/mm; n = 4) of pulmonary artery rings (3rd order branches, 250-400 µm i.d.) remained unaffected after pre-incubation (30 min) of arteries with CPA (10µM; 1.45 ± 0.35 mN/mm; n = 6), whilst being abolished by pre-incubation (30 min) with thapsigargin (1µM; n = 4).

We conclude that AMPK activation, like hypoxia, induces cADPR-dependent Ca2+ mobilisation rom a thapsigargin-sensitive, CPA-insensitive SR Ca2+ store via RyRs, but not via IP3Rs.

Dipp and Evans (2001) Circ. Res.89, 77-83.
Evans et al., (2005) J. Biol. Chem. 280, 41504

Supported by The British Heart Foundation.