Diabetes abolishes sildenafil-induced cGMP-dependent protein kinase-I expression and cardioprotection.

The selective phosphodiesterase type 5 inhibitor sildenafil has been demonstrated to produce cardioprotection; however, diabetes is known to abolish cardioprotective signaling. We tested the hypothesis that sildenafil-induced cGMP-dependent protein kinase-I (PKG-I) expression and cardioprotection are attenuated by diabetes. Barbiturate-anesthetized dogs (n = 38) were instrumented for measurement of hemodynamics and subjected to 60-minute occlusion of the left anterior descending coronary artery and 3-hour reperfusion. Dogs were randomly assigned to receive 0.9% saline (control) or intravenous sildenafil (0.7 or 1.4 mg/kg) in the absence or presence of diabetes (3 weeks after administration of alloxan and streptozotocin). No differences in hemodynamics or coronary collateral blood flow (radioactive microspheres) were observed between groups before and during ischemia and reperfusion, except that infusion of sildenafil produced transient decreases in left ventricle systolic pressure. Sildenafil significantly (P < 0.05) reduced infarct size (16 +/- 2% of the left ventricular area at risk; triphenyltetrazolium staining) as compared to control (31 +/- 39%). Diabetes alone did not alter infarct size (31 +/- 2%) but abolished the protective effect of sildenafil (0.7 mg/kg: 26 +/- 3%; 1.4 mg/kg: 26 +/- 3%). Sildenafil increased PKG-I expression (immunohistochemistry and Western blotting) in the absence but not the presence of diabetes. The results indicate that diabetes abolishes cardioprotection by sildenafil and implicates PKG-I in the signal transduction pathway activated by this drug.

Inflammatory response of tracheobronchial epithelial cells to endotoxin.

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.

Isoflurane inhibits cardiac myocyte apoptosis during oxidative and inflammatory stress by activating Akt and enhancing Bcl-2 expression.

BACKGROUND: Volatile anesthetics attenuate apoptosis. The underlying mechanisms remain undefined. The authors tested whether isoflurane reduces apoptosis in cardiomyocytes subjected to oxidative or inflammatory stress by enhancing Akt and B-cell lymphoma-2 (Bcl-2). METHODS: Adult and neonatal rat ventricular myocytes and atrial HL-1 myocytes were exposed to hypoxia, hydrogen peroxide, or neutrophils with or without isoflurane pretreatment. The authors assessed cell damage and investigated apoptosis using mitochondrial cytochrome c release, caspase activity, and TUNEL assay. They determined expression of phospho-Akt and Bcl-2 and tested their involvement by blocking phospho-Akt with wortmannin and Bcl-2 with HA14-1. RESULTS: Isoflurane significantly reduced the cell damage and apoptosis induced by hypoxia, H2O2, and neutrophils. Isoflurane reduced hypoxia-induced mitochondrial cytochrome c release in HL-1 cells by 45 +/- 12% and caspase activity by 28 +/- 4%; in neonatal cells, it reduced caspase activity by 43 +/- 5% and TUNEL-positive cells by 50 +/- 2%. Isoflurane attenuated H2O2-induced caspase activity in HL-1 cells by 48 +/- 16% and TUNEL-positive cells by 78 +/- 3%; in neonatal cells, it reduced caspase activity by 30 +/- 3% and TUNEL-positive cells by 32 +/- 7%. In adult cardiomyocytes exposed to neutrophils, isoflurane decreased both mitochondrial cytochrome c and caspase activity by 47 +/- 3% and TUNEL-positive cells by 25 +/- 4%. Isoflurane enhanced phospho-Akt and Bcl-2 expression. Wortmannin and HA14-1 prevented the action of isoflurane (53 +/- 8% and 54 +/- 7% apoptotic cells vs. 18 +/- 1% without blockers). CONCLUSIONS: Isoflurane protects cardiomyocytes against apoptosis induced by hypoxia, H2O2, or activated neutrophils through Akt activation and increased Bcl-2 expression. This suggests that a reduction in apoptosis contributes to the cardioprotective effects of isoflurane.