Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation.

AIMS: Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion. METHODS AND RESULTS: Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development. CONCLUSION: sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Cytosolic Ca2+ oscillations in human cerebrovascular endothelial cells after subarachnoid hemorrhage.

Molecular mechanisms of cerebral vasospasm after subarachnoid hemorrhage (SAH) include specific modes of cell signaling like activation of nuclear factor (NF)-kappaB and vascular cell adhesion molecules (VCAM)-1 expression. The study's hypothesis is that cisternal cerebral spinal fluid (CSF) from patients after SAH may cause Ca(2+) oscillations which induce these modes of vascular inflammation in an in vitro model of human cerebral endothelial cells (HCECs). HCECs were incubated with cisternal CSF from 10 SAH patients with confirmed cerebral vasospasm. The CSF was collected on days 5 and 6 after hemorrhage. Cytosolic Ca(2+) concentrations and cell contraction as an indicator of endothelial barrier function were examined by fura-2 microflurometry. Activation of NF-kappaB and VCAM-1 expression were measured by immunocytochemistry. Incubation of HCEC with SAH-CSF provoked cytosolic Ca(2+) oscillations (0.31+/-0.09 per min), cell contraction, NF-kappaB activation, and VCAM-1 expression, whereas exposure to native CSF had no significant effect. When endoplasmic reticulum (ER) Ca(2+)-ATPase and ER inositol trisphosphate (IP3)-sensitive Ca(2+) channels were blocked by thapsigargin or xestospongin, the frequency of the Ca(2+) oscillations was reduced significantly. In analogy to the reduction of Ca(2+) oscillation frequency, the blockers impaired HCEC contraction, NF-kappaB activation, and VCAM-1 expression. Cisternal SAH-CSF induces cytosolic Ca(2+) oscillations in HCEC that results in cellular constriction, NF-kappaB activation, and VCAM-1 expression. The Ca(2+) oscillations depend on the function of ER Ca(2+)-ATPase and IP3-sensitive Ca(2+) channels.

The role of poly(ADP-ribose) polymerase (PARP) in the autonomous proliferative response of endothelial cells to hypoxia.

OBJECTIVE: The autonomous proliferative response of endothelial cells to hypoxia has been shown to be dependent on activation of NAD(P)H oxidase, on the cytosolic Ca2+ load, and, consequently, on nuclear translocation of extracellular signal-regulated kinase (ERK)1/2 during transient hypoxia. The aim of the present study was to investigate whether poly(ADP-ribose) polymerase (PARP) is a downstream signal of NAD(P)H oxidase, mediating cytosolic Ca2+ load and hence nuclear translocation of ERK1/2 and endothelial cell proliferation. METHODS: Porcine aortic endothelial cells were incubated under hypoxic conditions for 40 min. Cytosolic [Ca2+] and reactive oxygen species (ROS) formation were measured in fura-2- and DCF-loaded cells, respectively. PARP activation was detected by immunocytochemistry, and endothelial cell proliferation was determined 24 h after 60 min of transient hypoxia. RESULTS: Inhibition of NAD(P)H oxidase with antisense oligonucleotide against the p22(phox) subunit, MEK/ERK signalling with UO 126 (30 microM), or PARP with PJ 34 (10 microM) leads to a marked reduction in hypoxia-induced cytosolic Ca2+ load and activation of PARP. Hypoxia-induced translocation of ERK1/2 and endothelial cell proliferation were also prevented when NAD(P)H oxidase or PARP were inhibited; however, hypoxic ROS formation was not affected in the presence of PARP inhibitor. CONCLUSION: PARP represents a downstream effector of NADP(H) oxidase and acts as a necessary intermediate step for the hypoxic proliferative response of endothelial cells.

Insulin protects cardiomyocytes against reoxygenation-induced hypercontracture by a survival pathway targeting SR Ca2+ storage.

OBJECTIVE: Experimental and clinical studies have shown that administration of insulin during reperfusion is cardioprotective, but the underlying mechanisms are still unknown. In this study, we investigated in isolated rat cardiomyocytes subjected to hypoxia and reoxygenation whether administration of insulin during reoxygenation reduces reoxygenation-induced hypercontracture, a hallmark of acute reperfusion injury. The effects of insulin on potential pro-survival kinases, i.e., PI 3-kinase, NO synthase (eNOS), and cGMP-dependent protein kinase (PKG), and on cytosolic Ca2+ control in reoxygenated cardiomyocytes were investigated. RESULTS: Administration of insulin (10 mU/L) during reoxygenation protected cardiomyocytes against hypercontracture development (cell length as % of end-hypoxic length: control 61.6+/-3.2; insulin 76.3+/-2.9; n=26; p<0.05 vs. control). Cytosolic [Ca2+] recovery during the first 2 min of reoxygenation was accelerated (fura-2 ratio after 2 min of reoxygenation; control 1.01+/-0.05; insulin 0.79+/-0.04; n=26; p<0.05 vs. control). The beneficial effects of insulin on cytosolic [Ca2+] recovery and hypercontracture were suppressed in the presence of inhibitors of PI 3-kinase (LY294002, 1 microM), eNOS (L-NMMA, 100 microM), PKG (KT 5823, 1 microM), or sarcoplasmic reticulum Ca2+ pump (SERCA) (thapsigargin, 150 nM). Insulin increased phosphorylation and activity of eNOS and augmented phospholamban phosphorylation in reoxygenated cardiomyocytes. Correlated with phospholamban phosphorylation, insulin also augmented SR Ca2+ load. CONCLUSIONS: Insulin protects cardiomyocytes against reoxygenation-induced hypercontracture. This is due to acceleration of cytosolic [Ca2+] recovery by enhanced Ca2+ sequestration into the sarcoplasmic reticulum via SERCA activation. This protective mechanism is activated through the survival pathway consisting of PI 3-kinase, eNOS, and PKG.