Regulation of AMPK activity by type 10 adenylyl cyclase: contribution to the mitochondrial biology, cellular redox and energy homeostasis

The downregulation of AMP-activated protein kinase (AMPK) activity contributes to numerous pathologies. Recent reports suggest that the elevation of cellular cAMP promotes AMPK activity. However, the source of the cAMP pool that controls AMPK activity remains unknown. Mammalian cells possess two cAMP sources: membrane-bound adenylyl cyclase (tmAC) and intracellularly localized, type 10 soluble adenylyl cyclase (sAC). Due to the localization of sAC and AMPK in similar intracellular compartments, we hypothesized that sAC may control AMPK activity. In this study, sAC expression and activity were manipulated in H9C2 cells, adult rat cardiomyocytes or endothelial cells. sAC knockdown depleted the cellular cAMP content and decreased AMPK activity in an EPAC-dependent manner. Functionally, sAC knockdown reduced cellular ATP content, increased mitochondrial ROS formation and led to mitochondrial depolarization. Furthermore, sAC downregulation led to EPAC-dependent mitophagy disturbance, indicated by an increased mitochondrial mass and unaffected mitochondrial biogenesis. Consistently, sAC overexpression or stimulation with bicarbonate significantly increased AMPK activity and cellular ATP content. In contrast, tmAC inhibition or stimulation produced no effect on AMPK activity. Therefore, the sAC-EPAC axis may regulate basal and induced AMPK activity and support mitophagy, cellular energy and redox homeostasis. The study argues for sAC as a potential target in treating pathologies associated with AMPK downregulation

Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture

BACKGROUND: Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury by provoking hypercontracture of cardiomyocytes (acute reperfusion injury). Previous studies indicated that halothane can protect ischemic-reperfused myocardium. The aim of the present study was to analyze on the cellular level the mechanism by which halothane may protect against reoxygenation-induced hypercontracture. METHODS AND RESULTS: To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in the presence or absence of 0.4 mmol/L halothane. Reoxygenation was started when intracellular Ca2+ (measured with fura 2) had increased to > or = 10(-5) mol/L and pHi (BCECF) had decreased to 6.5. Development of hypercontracture was determined microscopically. In the control group, reoxygenation provoked oscillations of cytosolic Ca2+ (72+/-9 per minute at fourth minute of reoxygenation) accompanied by development of hypercontracture (to 65+/-3% of end-ischemic cell length). When halothane was added on reoxygenation, Ca2+ oscillations were markedly reduced (4+/-2 per minute, P <.001) and hypercontracture was virtually abolished (90+/-4% of end-ischemic cell length, P <.001). Halothane did not influence the recovery of pHi during reoxygenation. Similar effects on Ca2+ oscillations and hypercontracture were observed when ryanodine (3 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ release, or cyclopiazonic acid (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, were applied instead of halothane. CONCLUSIONS: Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca2+ during the early phase of reoxygenatio