Cytosolic and mitochondrial Ca2+ handling in pulmonary arterial smooth muscle and endothelial cells

Cytosolic Ca2+ elevated by receptor-operated (ROCE) or store operated Ca2+ entry (SOCE) and buffered by mitochondrial Ca2+ uptake plays an essential role in physiological processes of lung cells. Disturbed Ca2+ homeostasis in precapillary pulmonary arterial smooth muscle cells (PPASMC) induces pulmonary arterial hypertension (PAH) or disrupts barrier function of murine pulmonary microvascular endothelial cells (MPMVEC) resulting in vascular leakage and lung edema. While SOCE is mediated by stromal interaction molecule (STIM1 and 2) dimers sensing Ca2+ in Ca2+ stores and plasma membrane Orai channels, ROCE is mediated by classical transient receptor potential (TRPC) channels via diacylglycerol (DAG) and receptor-activated phospholipase C (PLC). To investigate if TRPCs are also involved in SOCE, Ca2+ entry was analyzed in PPASMCs isolated from STIM1/2flox/flox/TRPC1/3/6-/- mice as well as control mice and infected with Cre-recombinase expressing lentiviruses to delete STIM1/2 protein expression. Deficiency of STIM1/2 did only impair the long-lasting receptor-operated Ca2+ entry (ROCE), but TRPC1, TRPC3 and TRPC6 channels may be activated indirectly by store-operated Ca2+ entry (SOCE). Lack of STIM1/2 or Orai1 in MPMVECs did not impair acetylcholine-induced vasodilatation of isolated aortic rings but pulmonary microvascular endothelial cells (MPMVECS) isolated from endothelial cell-specific STIM1/2 or Orai1 knockout mice (STIM1/2ΔEC and Orai1ΔEC) were protected from Ca2+-dependent nuclear factor of activated T-cells c3 (NFATc3) nuclear translocation which is involved in expression of proinflammatory mediators highlighting their important role in inflammation processes. Migration and proliferation of MPMVECs was reduced in STIM1/2ΔEC and Orai1ΔEC. Disturbed mitochondrial Ca2+ uptake results in endothelial dysfunction and may affect energy production. The influence of mitochondrial Ca2+ uniporter (MCU) and mitochondrial Ca2+ uniporter regulator 1 (MCUR1) on mitochondrial bioenergetics was investigated in MPMVECs from respective endothelial cell-specific knockout mice. Mitochondrial membrane potential and oxygen consumption rate was not impaired in MCUR1ΔEC or MCUΔEC. ATP production was reduced in MCUR1ΔEC and MCUΔEC. Probably resulting from reduced ATP levels, uncoupling protein 2 (UCP2) was upregulated in MCUR1ΔEC. The upregulated UCP2 might flux protons to prevent mitochondrial membrane potential (ΔΨm) hyperpolarization, induce a metabolic shift from oxidative phosphorylation to glycolysis or stimulate compensatory mechanisms to rescue [Ca2+]m-uptake. In accordance with lower superoxide production in MCUR1ΔEC and MCUΔEC, we observed a reduced proliferation and migration in these cells. Low mitochondrial Ca2+ uptake and low cellular ATP levels in MCUR1ΔEC and MCUΔEC activated AMPK and turned on autophagy. Therefore, cytosolic and mitochondrial Ca2+ handling is of foremost importance for lung cell function and proteins involved in these processes may serve as pharmacological targets for therapeutic approaches in patients with pulmonary hypertension or lung edema.