1. Volume 127, Issue 3, Pages 914-923 (September 2004)
Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning 
Rita Carini, Maria Grazia De Cesaris, Roberta Splendore, Gianluca Baldanzi, Maria Paola Nitti, Elisa Alchera, Nicoletta Filigheddu, Cinzia Domenicotti, Maria Adelaide Pronzato, Andrea Graziani, Emanuele Albano 
Gastroenterology 
Volume 127, Issue 3, Pages (September 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
Activation of phosphatidylinositole-3-phosphate kinase (PI3K) by hepatocyte preconditioning and its role in improving cell tolerance to hypoxia. A shows the incorporation of (γ-32P)ATP into phosphatidylinositole-3,4,5-triphosphate (PIP3) catalyzed by phosphorylated proteins immunoprecipitated with antiphosphotyrosine antibodies from isolated rat hepatocytes preconditioned by exposure to transient hypoxia/reoxygenation (PC) (see Materials and Method section) or by the addition of adenosine A2A receptor agonist CGS21680 (CGS; 1 μmol/L). The effect on PIP3 formation of tyrosine phosphatase inhibition with sodium orthovanadate (1 mmol/L) is shown as a positive control. B shows the phosphorylation of PKB/Akt in preconditioned hepatocytes incubated 15 minutes under hypoxic conditions in the presence or in the absence of the adenosine A2A receptor antagonist 3,7-dimethylpropargylxantine (DMPX; 100 μmol/L), adenosine A1 receptor antagonist 8-cyclopenthyl-1,3-dipropylxanthine (DPCPX; 100 μmol/L) or with PI3K inhibitors wortmannin (WM; 250 nmol/L) and LY (LY; 75 μmol/L). The relative intensity of phosphorylated and nonphosphorylated PKB/Akt bands is expressed as arbitrary units after normalization at 1 for the control sample. One experiment representative of 3. C and D show the effects of PI3K inhibition by wortmannin (WM) or LY on the capacity of preconditioning (PC) or CGS21680 to preserve hepatocyte viability during 90-minute incubation under hypoxic conditions. The results are means of 4 or 5 different experiments ± SD. Statistical significance: *P < vs. nonpreconditioned hepatocytes or cells receiving PI3K inhibitors.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

3. Figure 2
Effects of the modulation of cAMP/PKA-mediated signals on PI3K activation (A) and hepatocyte protection against hypoxic injury (B) induced by adenosine A2A receptor agonist CGS PKB/Akt phosphorylation was measured as an index of PI3K activation 15 minutes after the addition of CGS21680 (CGS; 1 μmol/L) in combination with adenylate cyclase inhibitor 2,5-dideoxyadenosine (2,5-DDA, 1 μmol/L) or PKA inhibitor H89 (0.1 μmol/L). As a reference, the effects of cAMP analogue 8Br-cAMP (500 μmol/L) and PKA activator forskolin (50 μmol/L) are shown. The relative intensity of phosphorylated and nonphosphorylated PKB/Akt bands is expressed as arbitrary units after normalization at 1 for the control sample. One experiment representative of 3. Hepatocyte viability was assessed following 60-minute incubation under hypoxic conditions. The results are means of 3 or 4 different experiments ± SD. Statistical significance: *P < 0.01 vs. untreated hepatocytes or cells receiving cAMP/PKA agonists and antagonists.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

4. Figure 3
Role of etherotrimeric Gi protein and Scr tyrosine kinase on PI3K activation (A), hepatocyte protection against hypoxic injury (B), and Scr phosphorylation on the regulatory Tyr418 (C) induced by hypoxic preconditioning (PC) or adenosine A2A receptor agonist CGS21680 (CGS; 1 μmol/L). PKB/Akt phosphorylation was measured in the presence or in the absence of pertussis toxin (PX; 200 ng/mL) or Src inhibitor 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2; 5 μmol/L) 15 minutes after preconditioning or the addition of CGS The relative intensity of phosphorylated and nonphosphorylated PKB/Akt bands is expressed as arbitrary units after normalization at 1 for the control sample. One experiment representative of 3. Hepatocyte viability was assessed following 60-minute incubation under hypoxic conditions. The results are means of 3 or 4 different experiments ± SD. Statistical significance: *P < 0.01 vs. untreated hepatocytes or cells receiving pertussis toxin or PP2. The relative intensity of the bands corresponding to the phosphorylated and nonphosphorylated forms of Scr were measured by videodensitometry in the protein fraction obtained from preconditioned or CGS21680-treated hepatocytes incubated 15 minutes with or without LY (LY; 250 nmol/L). The values are expressed as arbitrary units after normalization at 1 for the control sample. One experiment representative of 3.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

5. Figure 4
Role of PKA in promoting adenosine A2A receptor phosphorylation upon the addition of adenosine A2A receptor agonist CGS21680 (CGS; 1 μmol/L). The relative intensity of the bands corresponding to the phosphorylated and nonphosphorylated forms of adenosine A2A receptor were measured by videodensitometry in the membrane fractions obtained from isolated hepatocytes incubated 5 minutes with CGS21680 (1 μmol/L) with or without adenylate cyclase inhibitor 2,5-dideoxyadenosine (2,5-DDA, 1 μmol/L) or PKA inhibitor H89 (0.1 μmol/L). The effect of direct activation of PKA by forskolin (50 μmol/L) in control cell or cell receiving CGS21680 plus 2,5-DDA is also shown. The values are expressed as arbitrary units after normalization at 1 for the control sample. One experiment representative of 3.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

6. Figure 5
Effect of PI3K inhibitors on the activity of protein kinase C (PKC) δ and ϵ isoforms in the cytosol and in the membrane fractions of isolated rat hepatocytes exposed to hypoxic preconditioning (PC), adenosine A2A receptor agonist CGS21680 (CGS; 1 μmol/L), or the diacylglycerol analogues dioctanoylglycerole (DOG; 20 μmol/L). The effects of wortmannin (WM; 250 nmol/L) or LY (75 μmol/L) on PKC activity were measured in the immunoprecipitated fractions obtained from hepatocytes incubated 15 minutes after preconditioning or the supplementation with CGS21680 or DOG. The results are expressed as percentage of the values in the controls and are means of 3 different experiments ± SD. Statistical significance: *P < 0.05 vs. cells not receiving PI3K inhibitors.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

7. Figure 6
Role of PI3K in promoting the activation and the plasma membrane translocation of phospholypase Cγ (PLCγ). Isolated rat hepatocytes were exposed to hypoxic preconditioning (PC) or adenosine A2A receptor agonist CGS21680 (CGS; 1 μmol/L) alone or in combination with wortmannin (WM; 250 nmol/L). The presence of PLCγ in hepatocyte membrane fraction was assessed by Western blotting (A). The detection of the α1 subunit of the Na+/K+ ATPase was used as a marker of plasma membrane proteins. The activity of PLCγ was evaluated by the production of inositol-3-phosphate (IP3). The results are expressed as arbitrary units and are means of 3 different experiments ± SD. Statistical significance: *P < vs. controls and cells receiving the PI3K inhibitor.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

8. Figure 7
Proposed mechanisms leading to the activation of phosphatidylinositol 3-kinase (PI3K) as a result of hepatocyte hypoxic preconditioning and possible role of PI3K in the development of tolerance against ischemia/reperfusion injury. The stimulation by adenosine of adenosine A2A receptors (A2AR) induces the sequential activation of adenylate cyclase and protein kinase A (PKA). The latter, by phosphorylating A2AR, allows the receptor interaction with heterotrimeric Gαi-proteins (Gαi) and the activation of PI3K through the stimulation of Src tyrosine kinase. In its turn, PI3K-mediated activation of phospholypase Cγ (PLCγ) and phosphoinositide-dependent kinase 1 (PDK1) mediates the sequential stimulation of protein kinase C (PKC-δ and ϵ) and p38 mitogen-activated protein kinase (p38 MAPK). The action of p38 MAPK is then responsible for preventing hepatocyte death by necrosis. It is also possible that PI3K-mediated activation of serine-threonine kinase PKB/Akt might interfere with the processes leading to liver cell apoptosis.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions