The H2O2 induced effects on purine metabolism in human endothelial cells.

The effects of hydrogen peroxide (H2O2) on the purine metabolism of human endothelial cells were investigated. An incubation with 0.01 mM H2O2 over 60 min led to an increase in the intracellular adenosine-5-triphosphate (ATP) and creatine phosphate (CP) levels by 51.3% and 18.2%, respectively. A 60 min incubation with 0.1 mM H2O2 showed no effect. The uptake and salvage of 14C-adenine (14C-AD) and 14C-adenosine (14C-ADO) was significantly (p < 0.005) increased using 0.01 mM H2O2. Only an increase of 14C-ADO incorporation was observed using 0.1 mM H2O2. A concentration of 0.01 mM H2O2 reduced 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP-S) activity by 60% and at the same time increased the activity of purine nucleoside phosphorylase, which converts inosine to hypoxanthine (PNP I), by 24%. Adenosine kinase (AK) activity was reduced by H2O2, whereas adenine phosphoribosyltransferase (APRT) activity was found to be elevated. In conclusion, the observed elevation of cellular ATP and CP levels could be partially caused by an increased purine salvage resulting from changes in purine enzyme activities.

Endothelial cells' responses to hypoxia and reperfusion.

Our results suggest that longer periods of hypoxia lead to a deficiency of high energy phosphates. Reoxygenation leads to the formation of ROS, irrespectively of the duration of hypoxia. It might be concluded that a diminished level of intracellular high energy phosphates upon hypoxia followed by oxidative stress plays a key role in the reperfusion associated cellular dysfunctions.

In vitro effects of hypoxia and reoxygenation on human umbilical endothelial cells.

We investigated metabolic changes in human umbilical venous endothelial cells, when these were incubated under hypoxic followed by hyperoxic conditions, thus simulating hypoxia and reoxygenation. The human umbilical venous endothelial cells were incubated with a degassed buffer (oxygen content: 0-0.5%) for either 3 h or 24 h, followed by a 60 min incubation with oxygen-perfused buffer (oxygen content: 100%). Three hours of hypoxia led to a slight decrease in the ATP and creatine phosphate content (-16% +/- 5%), while a pronounced decrease of high energy phosphates (-54% +/- 4%) was observed after 24 h of hypoxia. Reoxygenating the cells after 3 h of hypoxia led to restoration of the content of high energy phosphates, while reoxygenation after 24 h resulted in a strong decrease (-66% +/- 4%). The prostaglandin I2 release during the first 3 h of hypoxia exceeded the release in the following 21 h. In all cases, reoxygenation increased the prostaglandin I2 release. Under normoxic conditions the ratio between oxidised glutathione and reduced glutathione shifted from 1:100 to 1:4.5 after 3 h of hypoxia. The content of lipid peroxidation products was almost unaffected during hypoxia, whereas reoxygenation resulted in a pronounced increase (+380% +/- 60%). The results of this in vitro study suggest that relatively long periods of hypoxia lead to a deficiency of high energy phosphates in the cell. Reoxygenation leads to the formation of oxygen-derived radicals, irrespectively of a prior hypoxia.

Myocardial protection with Bretschneider cardioplegic solution--an evaluation of full oxygenation.

In the present study the effect of oxygenated Bretschneider cardioplegia on high-energy phosphates [adenosine triphosphate (ATP), adenosine diphosphate (ADP) and creatine phosphate (CP)] and hemodynamics was evaluated in the isolated working rabbit heart. Hearts were obtained from 37 adult white Elco rabbits (3,100 +/- 110 g). After a 20-min working period 14 hearts were arrested with Bretschneider cardioplegia (8 degrees C) oxygenated with 98% oxygen (O2) and 2% carbon dioxide in comparison to 14 hearts receiving Bretschneider solution saturated with 98% nitrogen (N2) and 2% carbon dioxide as a control group for either 60 or 90 min (O(2)60, O(2)90, N(2)60, N(2)90 groups, n = 7). Seven hearts were used to determine preischemic baseline values of ATP, ADP and CP, 2 were excluded. The results showed a significantly poorer preservation of high-energy phosphates in hearts receiving oxygenated Bretschneider cardioplegia as compared to hearts receiving nitrogenated cardioplegia (p < 0.05). Postischemic recovery of hemodynamics did not demonstrate any statistically significant differences between the groups. However, the intragroup analysis showed a tendency towards weaker hemodynamic recovery in hearts treated with oxygenated cardioplegia. in contrast to the beneficial effect of oxygenated St. Thomas solution. In conclusion our findings suggest that oxygenated Bretschneider cardioplegia leads to significantly poorer preservation of high-energy phosphates and depressed hemodynamic recovery.