In ischemic-reperfused myocardium, necrosis of cardiomyocytes may develop not only due to the ischemic conditions but also the specific circumstances of reperfusion. The existence of reperfusion ...injury becomes apparent when modifications of the conditions of reperfusion can prevent cell death otherwise occurring. Three prime causes of rapidly developing reperfusion injury are here discussed, ie, reenergization of cells at increased cytosolic Ca2+ contents, rapid normalization of tissue pH, and rapid normalization of tissue osmolality. All three causes lead to severe mechanical stress of cardiomyocytes which can cause their rapid deterioration. Propagation of cell injury among adjacent cells can cause a spreading of necrosis throughout myocardial tissue. The understanding of these initial causes of rapidly developing lethal reperfusion injury leads to new concepts for specific protection of reperfused myocardium.
ABSTRACT
Release of nitric oxide (NO) during inflammation can induce apoptosis in the heart. Here we analyzed the involvement of members of the mitogen‐activated protein kinase (MAPK) family and ...their downstream target, the transcription factor AP‐1, in induction of apoptosis by NO in isolated adult cardiomyocytes of rat. The NO‐donor (±)‐S‐nitroso‐N‐acetylpenicillamine (100 μM SNAP)‐induced apoptosis in 10.5 ± 0.7% of cardiomyocytes and activated the transcription activator protein AP‐1 by 333.6 ± 122.3%. Intracellular scavenging of AP‐1 with decoy‐oligonucleotides blocked NO‐induced apoptosis to control levels (3.8 ± 0.5% apoptotic cells). Activation of AP‐1 with a c‐Jun amino‐terminal kinase (JNK) activator (Ro318220, 10 μM) provoked apoptosis in 18.7 ± 1.2% cardiomyocytes, which was again blocked by intracellular scavenging of AP‐1. NO activated JNK by 87.0 ± 27.3% and extracellular signal‐regulated kinase (ERK) by 35 ± 3%. Inhibition of ERK by the mitogen‐activated protein kinase kinase (MEK1) inhibitor PD98059 (10 μM) abolished AP‐1 activation and apoptosis induction with SNAP. Evidence that p38 MAPK plays a role in NO‐induced apoptosis was not found. These results clearly demonstrate the involvement of the transcription factor AP‐1 in NO‐induced apoptosis in cardiomyocytes. The activation of AP‐1 is mediated by the two MAP kinases JNK and ERK.
Aims
Intermedin (IMD) is a novel member of the calcitonin gene-related peptide family, which acts via calcitonin receptor-like receptors (CLRs), mediating activation of cAMP signalling. The main ...objective of the present study was to analyse the molecular mechanisms of the differential effects of IMD on the macromolecule permeability of endothelial cells of different vascular beds.
Methods and results
Here we demonstrate that IMD increases permeability of rat coronary microvascular endothelial cells (RCECs) and reduces permeability of human umbilical vein endothelial cells (HUVECs) and rat aortic endothelial cells via CLRs and cAMP. Intermedin causes a derangement of the actin cytoskeleton accompanied by loss of vascular endothelial cadherin (VE-cadherin) in RCECs, while it causes a rearrangement of the actin cytoskeleton and VE-cadherin at cell-cell junctions in HUVECs. Intermedin inactivates the RhoA/Rho-kinase (Rock) pathway in both cell types; however, it inactivates Rac1 in RCECs but not in HUVECs. Inhibition and rescue experiments demonstrate that both RhoA and Rac1 are required for the RCEC barrier stability, while in HUVECs the inhibition of RhoA/Rock signalling does not interfere with basal permeability.
Conclusion
The opposite effects of IMD on permeability of RCECs and HUVECs are due to differential regulation of actin cytoskeleton dynamics via RhoA and Rac1. Moreover, Rac1 activity is regulated by the RhoA/Rock pathway in RCECs but not in HUVECs.
Abstract ATP can differentially affect the micro- and macrovascular endothelial barrier. It has been shown that it can both increase and/or decrease macromolecule permeability of microvascular ...endothelial cells and microvessels, in vivo . We hypothesised that the barrier stabilising effect is mediated by ATP itself via P2 receptors, while barrier-disrupting effect is mediated by its metabolite adenosine via adenosine receptors. The effects of ATP, ADP, AMP and adenosine on barrier function were studied in cultured rat coronary microvascular endothelial monolayers (RCEC) in vitro , as well as in rat mesentery vessels, and in rat hearts in vivo . ATP and ADP showed a biphasic effect on permeability of RCEC monolayers with a reduction followed by a later increase in albumin permeability. The permeability decreasing effect of ATP was enhanced by ecto-nucleotidase inhibitor ARL67156 while permeability increasing effect was enhanced by apyrase, an extracellular ecto-nucleotidase. Moreover, the permeability increasing effect was abrogated by adenosine receptor antagonists, 8-phenyltheophylline (8-PT) and DMPX. Adenosine and adenosine receptor agonists 5′-(N-ethylcarboxamido)-adenosine (NECA), CGS21680, and R-PIA enhanced albumin permeability which was antagonised by 8-PT, A1 , and A2 but not by A3 receptor antagonists. Likewise, immunofluorescence microscopy of VE-cadherin and actin showed that NECA induces a disturbance of intercellular junctions. Pre-incubation of ATP antagonised the effects of NECA on permeability, actin cytoskeleton and intercellular junctions. Similar effects of the applied substances were observed in rat mesentery artery by determining the vascular leakage using intravital microscopy as well as in rat hearts by assessing myocardial water contents in vivo . In conclusion, the study demonstrates that in RCEC, ATP, ADP, and its metabolite adenosine play opposing roles on endothelial barrier function.
Adult ventricular cardiomyocytes show an unusual structure‐function relationship for cyclic AMP‐dependent effects of PTHrP. We investigated whether PTHrP(1 – 16), void of biological activity on ...classical PTHrP target cells, is able to mimic the positive contractile effect of PTHrP(1 – 34), a fully biological agonist on cardiomyocytes.
Adult ventricular cardiomyocytes were paced at a constant frequency of 0.5 Hz and cell contraction was monitored using a cell‐edge‐detection system. Twitch amplitudes, expressed as per cent cell shortening of the diastolic cell length, and rate constants for maximal contraction and relaxation velocity were analysed.
PTHrP(1 – 16) (1 μmol l
−1
) mimicked the contractile effects of PTHrP(1 – 34) (1 μmol l
−1
). It increased the twitch amplitude from 5.33±0.72 to 8.95±1.10 (% dl l
−1
) without changing the kinetic of contraction.
PTH(1 – 34) (10 μmol l
−1
) affected the positive contractile effect of PTHrP(1 – 34), but not that of PTHrP(1 – 16).
RpcAMPS (10 μmol l
−1
) inhibited the positive contractile effect of PTHrP(1 – 34), but not that of PTHrP(1 – 16).
The positive contractile effect of PTHrP(1 – 16) was antagonized by the ET
A
receptor antagonist BQ123.
Sarafotoxin 6b and PTHrP(1 – 16), but not PTHrP(1 – 34), replaced
3
H‐BQ123 from cardiac binding sites.
We conclude that N‐terminal PTHrP peptides void of a PTH/PTHrP‐receptor binding domain are able to bind to, and activate cardiac ET
A
receptors.
British Journal of Pharmacology
(2001)
132
, 427–432; doi:
10.1038/sj.bjp.0703830
Ischemia-reperfusion provokes barrier failure of the coronary microvasculature, leading to myocardial edema development that jeopardizes functional recovery of the heart during reperfusion. Here, we ...tested whether adenosine 5'-triphosphate (ATP), either exogenously applied or spontaneously released during reperfusion, protects the endothelial barrier against an imminent reperfusion injury and whether interventions preventing ATP breakdown augment this protective ATP effect.
Cultured microvascular coronary endothelial monolayers and isolated-perfused hearts of rat were used.
After ischemic conditions were induced, reperfusion of endothelial monolayers activated the endothelial contractile machinery and caused intercellular gap formation. It also led to the release of ATP. When its breakdown was inhibited by 6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP (ARL 67156; 100 microM), a selective ectonucleotidase inhibitor, contractile activation and gap formation were significantly reduced. Reperfusion in the presence of exogenously added ATP (10 microM) plus ARL caused an additional reduction of both aforementioned effects. In contrast, elevation of ATP degradation by apyrase (1 U/ml), a soluble ectonucleotidase, or addition of adenosine (10 microM) provoked an increase in gap formation during reperfusion that could be completely inhibited by 8-phenyltheophylline (8-PT; 10 microM), an adenosine receptor antagonist. In Langendorff-perfused rat hearts, the reperfusion-induced increase in water content was significantly reduced by ARL plus ATP. Under conditions favouring ATP degradation, an increase in myocardial edema was observed that could be blocked by 8-PT.
ATP, either released from cells or exogenously applied, protects against reperfusion-induced failure of the coronary endothelial barrier. Inhibition of ATP degradation enhances the stabilizing effect of ATP on barrier function.
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