icatibant and Hypoxia

Our previous study has shown that human tissue kallikrein protected against ischemia/reperfusion-induced myocardial injury. In the present study, we investigated the protective role of local kallikrein gene delivery in ischemia/reperfusion-induced cardiomyocyte apoptosis and its signaling mechanisms in promoting cardiomyocyte survival. Adenovirus carrying the human tissue kallikrein gene was delivered locally into the heart using a catheter-based technique. Expression and localization of recombinant human kallikrein in rat myocardium after gene transfer were determined immunohistochemically. Kallikrein gene delivery markedly reduced reperfusion-induced cardiomyocyte apoptosis identified by both in situ nick end-labeling and DNA fragmentation. Delivery of the kallikrein gene increased phosphorylation of Src, Akt, glycogen synthase kinase (GSK)-3beta, and Bad(Ser-136) but reduced caspase-3 activation in rat myocardium after reperfusion. The protective effect of kallikrein on apoptosis and its signaling mediators was blocked by icatibant and dominant-negative Akt, indicating a kinin B2 receptor-Akt-mediated event. Similarly, kinin or transduction of kallikrein in cultured cardiomyocytes promoted cell viability and attenuated apoptosis induced by hypoxia/reoxygenation. The effect of kallikrein on cardiomyocyte survival was blocked by dominant-negative Akt and a constitutively active mutant of GSK-3beta, but it was facilitated by constitutively active Akt, catalytically inactive GSK-3beta, lithium, and caspase-3 inhibitor. Moreover, kallikrein promoted Bad.14-3-3 complex formation and inhibited Akt-GSK-3beta-dependent activation of caspase-3, whereas caspase-3 administration caused reduction of the Bad.14-3-3 complex, indicating an interaction between Akt-GSK-caspase-3 and Akt-Bad.14-3-3 signaling pathways. In conclusion, kallikrein/kinin protects against cardiomyocyte apoptosis in vivo and in vitro via Akt-Bad.14-3-3 and Akt-GSK-3beta-caspase-3 signaling pathways.

Topics: 14-3-3 Proteins; Adenoviridae; Animals; Apoptosis; bcl-Associated Death Protein; Blotting, Western; Bradykinin; Carrier Proteins; Caspase 3; Caspases; Cell Survival; DNA; DNA Fragmentation; DNA, Complementary; Gene Transfer Techniques; Genes, Dominant; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Hypoxia; Immunohistochemistry; Immunoprecipitation; In Situ Nick-End Labeling; Ischemia; Kallikreins; Kinins; Lithium; Male; Myocardium; Myocytes, Cardiac; Oxygen; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Recombinant Proteins; Reperfusion Injury; Signal Transduction

Although prior studies suggest that hypoxia may increase pulmonary vascular permeability, the mechanisms responsible for that effect remain uncertain. Neprilysin (neutral endopeptidase) is a cell surface metallopeptidase that degrades several vasoactive peptides including substance P and bradykinin. We hypothesized that hypoxia could reduce lung neprilysin expression, leading to increased vascular leak. Weanling rats were exposed to normobaric hypoxia (inspired O(2) fraction = 0.1). Lung neprilysin activity was significantly decreased after 24 and 48 h of hypoxia (P < 0.006). The decrease in enzyme activity was associated with decreased lung neprilysin protein content and decreased lung neprilysin mRNA expression. Immunohistochemistry showed a predominantly perivascular distribution of neprilysin, with clear reductions in neprilysin immunoreactivity after exposure to hypoxia. Exposure to hypoxia for 24 h also caused marked increases in vascular leak (P = 0.008), which were reversed by the administration of recombinant neprilysin. The hypoxia-induced increase in leak was also reversed by substance P and bradykinin receptor antagonists. We conclude that in young rats hypoxia decreases lung neprilysin expression, which contributes to increased pulmonary vascular leak via substance P and bradykinin receptors.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bradykinin; Gene Expression Regulation, Enzymologic; Hypoxia; Lung; Male; Methionine; Microcirculation; Neprilysin; Neurokinin-1 Receptor Antagonists; Oxygen; Piperidines; Protease Inhibitors; Pulmonary Edema; Quinuclidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; RNA, Messenger; Specific Pathogen-Free Organisms; Substance P; Water

Cyclooxygenase (COX) products play an important role in modulating sepsis and subsequent endothelial injury. We hypothesized that COX inhibitors may attenuate endothelial dysfunction during sepsis, as measured by receptor-mediated bradykinin (BK)-induced vasoconstriction and/or receptor-independent hypoxic pulmonary vasoconstriction (HPV). Rats were administered intraperitoneally a nonselective COX inhibitor (indomethacin, 5 or 10 mg/kg) or a selective COX-2 inhibitor (NS-398, 4 or 8 mg/kg) 1 h before lipopolysaccharide (LPS, 15 mg/kg), or saline (control). Three hours later, the rats were anesthetized, the lungs were isolated, and pulmonary vasoreactivity was assessed with BK (0.3, 1.0, and 3.0 microg) and HPV (3% O(2)). Perfusion pressure was monitored as an index of vasoconstriction. To investigate what receptor-subtype is mediating BK responses, the BK(1)-receptor antagonist des-Arg(9)-[Leu(8)]-BK, the BK(2)-receptor antagonist HOE-140, or the thromboxane A(2)-receptor antagonist SQ 29548 (all at 1 microM) were added to the perfusate. BK-induced vasoconstriction was significantly increased in LPS lungs (1.4-5.2 mm Hg) compared with control (0.1-1.1 mm Hg). In LPS lungs, indomethacin 10 mg/kg significantly decreased BK vasoconstriction by 78% +/- 9%, whereas 5 mg/kg did not. NS-398, 4 mg/kg, significantly attenuated BK vasoconstriction at 0.3 microg (71% +/- 7%) and 1.0 microg (56% +/- 12%), whereas 8 mg/kg attenuated 0.3 microg BK (57% +/- 14%), compared with LPS lungs. HPV was increased in LPS lungs (21.5 +/- 2 mm Hg) compared with control lungs (9.8 +/- 0.6 mm Hg). Indomethacin 5 mg/kg increased HPV in LPS lungs; otherwise, HPV was not altered by COX inhibition. BK-induced vasoconstriction was prevented by BK(2), but not BK(1) or thromboxane A(2)-receptor antagonism. This study suggests that nonselective COX inhibition, and possibly inhibition of the inducible isoform COX-2, may attenuate sepsis-induced, receptor-mediated vasoconstriction in rats.. This study demonstrated that, in an isolated rat lung model, nonselective inhibition of the cyclooxygenase pathway, and possibly selective inhibition of the inducible cyclooxygenase-2 isoform, may attenuate sepsis-induced endothelial dysfunction.

Topics: Animals; Bradykinin; Bridged Bicyclo Compounds, Heterocyclic; Cyclooxygenase Inhibitors; Fatty Acids, Unsaturated; Hydrazines; Hypoxia; Lipopolysaccharides; Lung; Male; Nitric Oxide; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Receptors, Bradykinin; Sepsis; Vasoconstriction

We previously reported that hypoxic coronary vasodilatation (HCVD) is initiated by endothelial NO and sustained by adenosine. Prolonged ischemia/reperfusion impairs endothelium-dependent coronary vasodilatation, whereas transient ischemia (ie, preconditioning) protects the myocardium from subsequent ischemic events. Accordingly, we assessed whether prolonged ischemia/reperfusion impairs HCVD and whether preconditioning prevents this dysfunction. HCVD, elicited in isolated guinea pig hearts by a 1-minute exposure to 100% N2, consisted of an approximately 70% increase in coronary flow associated with enhanced nitrite/nitrate and adenosine overflow (+40% and 5-fold, respectively). After 30-minute global ischemia and 20-minute reperfusion, HCVD was decreased by approximately 60%, and the increases in nitrite/nitrate and adenosine overflow were abolished. Preconditioning (ie, three cycles of 5-minute global ischemia+5-minute reperfusion) prevented the impairment of HCVD and fully restored the increase in nitrite/nitrate overflow, but not that of adenosine. The protective effect of preconditioning was mimicked by perfusion with the adenosine A1 receptor agonist N6-cyclopentyladenosine and prevented by the A1 receptor antagonist N-0861. In addition, the A3 receptor agonist N6-(3-iodobenzyl)adenosine-5'-N-methyl-carboxamide had a similar protective effect. The bradykinin B2 receptor antagonist HOE 140 abolished the protective effect of preconditioning, whereas the NO synthase inhibitor N(omega)-methyl-L-arginine and the cycloxygenase inhibitor indomethacin did not. Our data indicate that preconditioning restores HCVD by a process that is triggered by activation of adenosine A1/A3 and bradykinin B2 receptors. The action of bradykinin is independent of NO and prostacyclin production. Once restored by preconditioning, HCVD is mediated by NO but no longer sustained by adenosine.

Topics: Adenine; Adenosine; Animals; Bradykinin; Bradykinin Receptor Antagonists; Coronary Vessels; Guinea Pigs; Hypoxia; In Vitro Techniques; Ischemic Preconditioning, Myocardial; Male; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase; Norbornanes; Receptor, Adenosine A3; Receptor, Bradykinin B2; Receptors, Bradykinin; Receptors, Purinergic P1; Vasodilation