G. Gaja

Adriamycin: Energy metabolism and mitochondrial oxidations in the heart of treated rabbits

Abstract Heart mitochondria isolated from rabbits subjected to intermittent treatment with adriamycin show a reduced respiratory control, resulting from increase in state 4b oxidation. Continuous daily treatment causes an impairment of respiratory control which is more severe, is due both to increase of state 4b and decrease of state 3 oxygen uptake and occurs after a total amount of drug which does not produce appreciable effects with the intermittent schedule of treatment: however, these changes disappear within 2 weeks from the interruption of the treatment. Mitochondria isolated from adriamycin-treated rabbits show constantly increased permeability to the addition of NADH. On the contrary the ADP/O ratio measured in vitro is essentially unchanged: the same happens with the tissue contents of ATP, ADP, AMP and the metabolites chosen to estimate cytoplasmic and mitochondrial redox states and measured in quick-frozen hearts in vivo . The results are discussed in relation to the possible role of mitochondrial functional defects in the onset of adriamycin cardiomyopathy.

Phosphorylation and redox states in ischemic liver

Abstract Metabolite concentrations, redox and phosphorylation states were studied in ischemic and postischemic rat livers; oxidative phosphorylation and related functional parameters also were investigated with mitochondria derived from these tissues. Time periods were chosen to assess the effects of short-duration (15 min), long-duration reversible (60 min), and necrogenic ischemia (120 min). The energy-charge of liver cells drops promptly and deeply within the first 15 min of ischemia, with a concurrent profound decrease of the [NAD + ] [NADH] ratio both in cytoplasm and mitochondria. Once affected by this initial change, redox and phosphorylation states do not vary appreciably by prolonging blood deprivation for 120 min. The functional capacity of isolated mitochondria declines steadily with increasing duration of ischemia and severe reductions—especially of the ADP O ratios—are attained only at the end of 120 min of ischemia. The reestablishment of the blood supply promotes a good recovery of the energy-charge and redox states (at a slightly different pace) only if previous ischemia did not last more than 60 min. Results with isolated mitochondria correlate well with chemical determinations carried out on postischemic tissue, but respiratory control index does not recover completely over the investigated period following reversible ischemia, thus indicating a less tight coupling of phosphorylation to oxidation during the repair of reversible cellular damage. The relationships between impaired mitochondrial functions, phosphorylation and redox states, and the relevance of their changes to the fate of ischemic cells are discussed.

The effect of somatostatin on experimental inflammation in rats

The aim of the present study was to investigate the effect of somatostatin administration on experimentally induced inflammation in rats.Inflammation was induced by the intraplantar injection of carrageenan (50 micro L) into the hind paw of the rat. Animals were treated intraplantarly with somatostatin in a volume of 50 micro L at different doses (2.5, 25, and 250 ng, 10 micro g). The inflammatory response was studied 120, 180, and 240 min after drug administration. The antinociceptive effect of somatostatin was determined by using the Randall and Selitto test and by local production of beta-endorphin from lymphocytes obtained from popliteal lymph nodes. Data show that small doses of somatostatin were the most effective in reducing hyperalgesia. Moreover, our results show that somatostatin treatment significantly increased beta-endorphin in lymphocytes from popliteal lymph nodes. The secretion of opioid peptides, which enhance analgesia, could be stimulated by locally administered somatostatin. Implications: Acute pain because of intraplantar inflammation induced in rats by carrageenan injection was significantly reduced by small-dose, local administration of somatostatin, which possibly favors beta-endorphin release as a mechanism. These results may have implications regarding treatment of pain conditions associated with an inflammatory response. (Anesth Analg 1997;85:1112-5)

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion

1 Leukocyte‐endothelial cell interactions play an important role during ischaemia‐reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2 Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia‐reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3 In basal conditions, defibrotide (1000 μg ml−1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% ± 3.6 (P < 0.05), and after endothelial cell stimulation (TNF‐α, 500 u ml−1) or after leukocyte stimulation (fMLP, 10−7 m), it inhibited leukocyte adhesion by 26.5% ± 3.4 and 32.4% ± 1.8, respectively (P < 0.05). 4 In adhesion blockage experiments, the use of the monoclonal anntibody anti‐CD31 (5 μg ml−1) did not demonstrate a significant inhibitory effect whereas use of the monoclonal antibody anti‐LFA‐1 (5 μg ml−1) significantly interfered with the effect of defibrotide. 5 This result was confirmed in NIH/3T3‐ICAM‐1 transfected cells. 6 We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM‐1/LFA‐1 adhesion system is involved in the defibrotide mechanism of action.

Protection by L-2-oxothiazolidine-4-carboxylic acid of hydrogen peroxide-induced CD3ζ and CD16ζ chain down-regulation in human peripheral blood lymphocytes and lymphokine-activated killer cells

We investigated whether L-2-oxothiazolidine-4-carboxylic acid (OTC) [in the form of Procysteine, kindly donated by Transcend Therapeutics] could protect peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells from CD3zeta and CD16zeta chain down-regulation induced by H2O2 produced by lipopolysaccharide (LPS)-activated autologous monocytes. OTC is known to enhance glutathione production in cells in which glutathione was depleted by reactive oxygen species. Our data showed that OTC induced a significant increase in CD3zeta and CD16zeta chain expression in peripheral blood lymphocytes and LAK cells, respectively, pretreated for 12 hr at 37 degrees. Moreover, OTC significantly protected peripheral blood lymphocytes and LAK against decreased zeta chain expression induced by lipopolysaccharide-activated monocytes or the addition of H2O2 to the culture medium. Our experiments thus suggested that alterations in signal-transducing molecules, such as decreased CD3zeta and CD16zeta expression observed in cytotoxic T lymphocytes and LAK cells in response to oxidative stress, could be prevented by the use of OTC.

METABOLIC FUNCTION OF GRAFTED LIVER IN RATS

We studied the metabolic variations in grafted livers at different times after transplant by measuring the hepatic energy and redox states. Five groups of rats were studied: control ungrafted Wistar (RT1y) rats (group 1), ungrafted Wistar rats with ligature of the hepatic artery (group 2), isografted Wistar rats (group 3), allografted Wistar rats with livers from ACI (RT1a) donors (group 4, long-term surviving rat strain combination), and allografted Wistar rats with livers from BN (RT1n) rats (group 5, rejector rats). The metabolism of grafted livers was studied for 7 days in groups 2 and 3, for 2 months in group 4, and at the time of rejection in group 5. Adenine nucleotide levels (ATP, ADP, AMP) were significantly impaired at 24 hr and at 48 hr from grafting in isografted and in allografted livers, and the reestablishment of normal values began at the 7th day from grafting. Cytoplasmic NAD+/NADH ratios were lowered at 24 hr from grafting in isografted and in allografted livers. Mitochondrial NAD+/NADH ratios were lowered at 24 hr in isografted livers and at 24 hr and 48 hr from grafting in allografted livers. The metabolic studies performed for 2 months revealed a significant correlation between well-maintained metabolic functions and transplant survival. On the contrary, an important energy loss was evidenced in livers of group 5, at the time of rejection.

Protection of rat heart from damage due to ischemia-reperfusion during procurement and grafting by defibrotide

We studied the efficacy of defibrotide, a prostacyclin-stimulating agent, in preventing ischemia reperfusion injury in Wistar rat heart by using three experimental models: (1) hearts from donors were perfused with the drug (32 mg/kg/hr) during 15, 30, 45, and 60 min of cold ischemia following 5, 10, and 15 min of warm ischemia; (2) hearts from donors treated with the drug were cold-stored for 12 or 24 hr; and (3) procured hearts perfused with the drug were isografted, after 30 or 60 min of warm ischemia, in recipient rats treated daily with defibrotide. Hearts perfused with saline and/or vehicle of the drug were used as controls. At the end of established ischemia times, and after 30 min, and 2, 4, 7 and 14 days from transplantation, hearts were rapidly cooled in liquid nitrogen. ATP, ADP, AMP, cAMP contents, and NAD+/NADH ratios were evaluated in prepared tissue extracts. Cardiac ATP and ADP levels and NAD+/NADH ratios were significantly higher in defi-brotide-treated organs than in controls. Isografted defibrotide-treated hearts were also significantly preserved, with respect to controls, from the loss of ATP levels until rejection occurred. Our results demonstrate the protective activity of the drug against the myocardial metabolic damage due to ischemia-reperfusion.

Effect of somatostatin on β-endorphin release in rat experimental chronic inflammation

The aim of the present study was to investigate the effect of somatostatin administration in arthritic rats. Inflammation was induced by daily interplantar injection of 100 microl of Freunds complete adjuvant into the left hind paw of the rat. Arthritis developed 20 days following the first injection and was stable in the inoculate paw. Arthritic rats were treated interplantarly with somatostatin (5 or 10 microg) or with indomethacin (100 microg) daily for 14 days. Inflammatory response was studied at 12 h, 7 and 14 days following drug administration. The effect of somatostatin was determined by local (into popliteal lymph nodes) and systemic production of beta-endorphin. Our results showed that somatostatin treatment significantly increased beta-endorphin levels in the blood and lymphocytes from popliteal lymph nodes. Greater efficiency was seen when 5 microg instead of 10 microg of somatostatin was used. A significant decrease of absolute leukocytosis was observed at the 14th day following somatostatin administration. Moreover, a significant reduction of plasmatic beta-globulins at 12 h and the 7th day and of plasmatic alpha2-globulins at the 14th day was observed after the beginning of somatostatin treatment.

Effect of cyclosporine and aminophylline on streptozotocin-induced diabetes in rats

Diabetes induced in rats by multiple low doses of streptozotocin is thought to mimic type 1 disease in man. We tested the effect of concomitant treatment with immunomodulator drugs in this diabetic experimental model. Administration of cyclosporine resulted in a rapid appearance of hyperglycemia, perhaps by a potentiation of the direct cytotoxic action of streptozotocin on beta cells. By contrast, aminophylline administration protected the animals from the diabetogenic action of streptozotocin. Concomitant treatment with aminophylline and cyclosporine failed to protect the rats from the hyperglycemia induced by streptozotocin.

Prevention of impaired liver metabolism due to ischemia in rats: Efficacy of defibrotide administration

The effect of defibrotide treatment in protecting liver metabolism from ischemic damage was studied. The drug was administered to male Wistar rats as a bolus (30 mg/kg body weight) at the beginning of 60 min ischemia and then continuously during 60 min of postischemic reperfusion at a dose of 30 mg/kg body weight. This dose was previously identified as useful to protect against myocardial ischemia induced in the cat. ATP and ADP intrahepatic levels were significantly higher in drug-treated rats than in untreated animals. The liver cytoplasmic NAD+/NADH ratio in defibrotide-treated rats was no different from that observed in sham-operated rats. The mitochondrial NAD+/NADH ratio in the liver was also improved by defibrotide treatment. Our data suggest that defibrotide may exert protective activity on hepatocytes useful for inducing a rapid restoration of their metabolism. Such a restoration is possibly related to improvement of microcirculation through an increase in prostaglandin I2 production or oxygen delivery due to drug administration.