Role of endogenous nitric oxide in TNF-alpha and IL-1beta generation in hepatic ischemia-repefusion.

In the present study, we examined the role of nitric oxide (NO) in early-response cytokine production by using a rat model of hepatic ischemia-reperfusion (HI/R). The left and median lobes of the liver were subjected to 30 min of ischemia, followed by 4 h of reperfusion. Group I and II rats were sham-operated controls that received saline (vehicle) or N(W)-nitro-L-arginine methylester (L-NAME) (10 mg/kg, iv); group III and IV rats were subjected to HI/R and received vehicle or L-NAME (10 mg/kg, iv, 10 min before reperfusion), respectively. Administration of L-NAME to rats subjected to I/R resulted in a fourfold decrease in plasma NO levels, accompanied by a marked increase of plasma alanine aminotransferase (ALT) activity relative to group III. These changes in group IV were associated with elevation of superoxide generation in ischemic liver lobes by 2.1-fold and circulating leukocyte number by 1.42-fold, compared with group III. Normalized for expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger ribonucleic acid (mRNA), expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) mRNA in ischemic liver of group IV was augmented by 207% and 175% compared with Group III. The expression of (iNOS) mRNA was also increased (223%) relative to group III. Moreover, in group IV, plasma TNF-alpha levels at 4 h of reperfusion and IL-1beta levels at 90 min and 4 h of reperfusion were significantly increased compared with group III. No statistically significant changes were observed between groups I and II in plasma ALT activity, plasma NO levels, circulating leukocyte counts, superoxide generation in the ischemic lobes of liver, and plasma TNF-a and IL-1beta concentrations. The observed enhancement of I/R injury by L-NAME is consistent with the hypothesis that endogenous NO down-regulates TNF-alpha and IL1beta generation, thereby decreasing HI/R injury.

Peroxynitrite induces apoptosis of HL-60 cells by activation of a caspase-3 family protease.

Apoptosis is an active process critical for the homeostasis of organisms. Enzymes of the caspase family are responsible for executing this process. We have previously shown that peroxynitrite (ONOO-), a biological product generated from the interaction of nitric oxide and superoxide, induces apoptosis of HL-60 cells. The aim of this study was to elucidate the mechanisms involved in the execution process of peroxynitrite-induced apoptosis. Proteolytic cleavage of poly(ADP-ribose) polymerase, an indication of caspase-3 family protease activation and an early biochemical event accompanying apoptosis, was observed in a time-dependent manner during peroxynitrite-induced apoptosis of HL-60 cells. Activation of caspase-3 during peroxynitrite-induced apoptosis was substantiated by monitoring proteolysis of the caspase-3 proenzyme and by measuring caspase-3 activity with a fluorogenic substrate. Furthermore, pretreatment of HL-60 cells with N-acetyl-Asp-Glu-Val-Asp-aldehyde, a specific inhibitor of caspase-3, but not N-acetyl-Tyr-Val-Ala-Asp-aldehyde, a specific inhibitor of caspase-1, decreased peroxynitrite-induced apoptosis. These results suggest that the activation of a caspase-3 family protease is essential for initiating the execution process of peroxynitrite-induced apoptosis of HL-60 cells.

Venous thrombosis produced in the vena cava of rabbits by vascular damage and stasis.

The main objective of these experiments was to develop and characterize a new experimental model of venous thrombosis, and determine whether a combination of vascular wall damage (crushing with hemostat clamps) and prolonged stasis produced more reproducible clots than prolonged stasis per se. Rabbits were laparotomized, and a segment of the vena cava was dissected free and looped with two silk ligatures, 2.5 cm apart. The proximal tie was ligated 5 min after release of the clamps; the distal tie applied shortly thereafter, trapping a volume of blood in the isolated segment. At 2 hr after ligation, the isolated venous sac was excised and examined for the presence of a clot. Large, well-formed clots, which could be readily transferred and weighed, were invariably observed in the "clamp" and "no clamp" groups, the latter being a sham control. Mean clot weights did not differ in the two groups (23.1 +/- 1.6 versus 30.8 +/- 5.4 mg dry weight, clamped versus no clamp, respectively, p greater than 0.05). However, the precision of the method was improved significantly (p less than 0.005) by clamping as determined by homogeneity of variance testing. Time-course studies showed that a considerable lag period (about 60 min) preceded development of a detectable clot, and that the thrombus evolved rapidly during the interval of 60-90 min postligation. The location of the small clots at 60 min in clamped segments, as well as the failure of prolonged (120 min) stasis without caval isolation to cause substantial thrombi, strongly suggests that clot formation attributed to "stasis per se" is in fact due to focal vascular lesions created at the tie-down points. The present study is also the first report of blockade of localized venous thrombosis by recombinant tissue factor pathway inhibitor (rTFPI). When given as an i.v. bolus 20 min prior to ligation, rTFPI at 400 and 800 micrograms/kg completely blocked formation of the thrombus or greatly reduced its size in five of the six animals tested.

Recombinant lipoprotein-associated coagulation inhibitor inhibits tissue thromboplastin-induced intravascular coagulation in the rabbit.

Lipoprotein-associated coagulation inhibitor produces feed-back inhibition of tissue factor (tissue thromboplastin)-induced coagulation in the presence of factor Xa Recombinant lipoprotein-associated coagulation inhibitor (rLACI) was tested for its ability to modify thromboplastin-induced intravascular coagulation in a rabbit model that allows monitoring of iodine-125 fibrin accumulation/disappearance in the lung and sampling of blood for the measurement of coagulation parameters. Infusion of thromboplastin into the rabbit caused a rapid increase of radioactivity over the lungs, possibly due to the accumulation of 125I fibrin in the lungs, followed by a rapid decline of radioactivity, suggestive of removal of fibrin from the lungs. Thromboplastin also caused a rapid decrease of systemic fibrinogen that was accompanied by a lengthening of the activated partial thromboplastin time and prothrombin time. The effect of coinfusion of rLACI with thromboplastin or bolus injection of rLACI before thromboplastin infusion was studied. At a high dose of rLACI (800 micrograms/kg body weight), the thromboplastin-induced radioactivity increase in the lungs and the systemic fibrinogen decrease were completely suppressed. The activated partial thromboplastin time and prothrombin time of the plasma samples lengthened, possibly due to the presence of thromboplastin in circulation. The thromboplastin-induced radioactivity increase over the lungs was not completely suppressed by lower doses of rLACI (135 to 270 micrograms/kg body weight), but these doses of rLACI prevented systemic fibrinogen decrease. At a bolus dose of 23 micrograms/kg body weight, rLACI provided 50% protection of the fibrinogen consumption (fibrinogen decreased to 82% compared with 65% in rabbits treated with thromboplastin alone). These results show that rLACI is effective in the inhibition of thromboplastin-induced coagulation in vivo.

Cardiovascular effects of chronic high-dose atriopeptin III infusion in normotensive rats.

Seventy-eight Sprague-Dawley rats received continuous intravenous infusions of either atriopeptin III (APIII), 60 micrograms/kg/hr, or distilled water vehicle for a period of 7 days by means of osmotic minipumps. On Day 7 approximately one-half of the animals (20 vehicle-treated rats and 21 APIII-treated rats) were instrumented for evaluation of cardiac function and terminated for measurement of heart weight. The minipumps remained in place during the evaluation of cardiac function. Also on Day 7, the osmotic pumps were removed from the remaining animals and an additional 7 days were allowed to elapse before heart weight and cardiac function were evaluated. Mean arterial blood pressure (MAP) of rats receiving APIII for 7 days was significantly lower (-9%, p less than 0.05) than that of rats receiving vehicle for 7 days. In addition, reductions (p less than 0.05) of total ventricular weightdry (-7%), left ventricular weightdry (-8%), and right ventricular weightdry (-9%) were observed in the APIII-treated rats (all ventricular weights are normalized for body weight). Hematocrit (HCT) was significantly higher (13%, p less than 0.05) in the APIII-treated group. Chronic APIII infusion did not influence ventricular performance nor did it affect regional vascular resistances. Seven days after termination of the APIII infusion the differences in MAP and HCT between vehicle-treated and APIII-treated animals were no longer evident. Partial recovery of the effect on heart weights was apparent, with total ventricular weightdry and left ventricular weightdry remaining slightly reduced (-4 and -5%, respectively; p less than 0.05). No differences were found between the two recovery groups for any index of cardiac function. In separate experiments, it was demonstrated that APIII, 60 micrograms/kg/hr iv, caused a significant increase in urine volume (p less than 0.05 relative to vehicle) during the initial 24 hr of infusion. The results indicate that chronic infusion of a large diuretic dose of APIII exerts relatively little influence on overall cardiovascular function in normotensive rats.

Hepatic transformation of prostaglandin D2 to a new prostanoid, 9 alpha,11 beta-prostaglandin F2, that inhibits platelet aggregation and constricts blood vessels.

The metabolic transformation of tritium-labeled prostaglandin D2 ([3H]PGD2) was investigated in the isolated Tyrode's-perfused rabbit liver. One major product was isolated and identified in the perfusate as a new prostanoid. The structure of this metabolite was further confirmed by gas chromatography-mass spectrometry and chemical methods to be 9 alpha,11 beta,15-L-trihydroxyprosta-5-cis, 13-trans-dienoic acid, namely (9 alpha,11 beta-PGF2). This new prostanoid was found to be an inhibitor of platelet aggregation and to cause constriction of canine coronary artery strips. These results suggested that on passage through the hepatic circulation exogenous PGD2 is converted to 9 alpha,11 beta-PGF2, the latter having a biological profile which differs from that of PGD2 and PGF2 alpha.

Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta.

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis.

Intima-related vasodilatation of the perfused rat caudal artery.

We examined the endothelial dependence of responses to ACh and some vasodilator drugs by using the central tail artery of the rat perfused with Krebs buffer. Perfusion with ACh (100 nM-100 microM) produced dose-dependent vasodilatation of arteries preconstricted with norepinephrine and antagonized pressor responses to periarterial electrical stimulation. Endothelium was removed by introducing a fine catheter through the lumen or a stream of gas (O2 95%-CO2 5%) intraluminally. Both procedures prevented the vasodilator effect of ACh. Gassing also abolished the vasodilatation in response to hydralazine 334 nM but not to equidilator amounts of papaverine 13 microM, or nitroglycerin 50 nM. These results indicating endothelial dependence of hydralazine and ACh responses are in accord with our previous studies on vascular rings.

Endothelial mechanism in the vascular action of hydralazine.

Relaxation of precontracted isolated chains of aortic rings with intact endothelium and in those with the endothelium removed was studied in response to various antihypertensive vasodilator drugs. Of the drugs tested--nitroprusside, nitroglycerin, prazosin, minoxidil, diazoxide and hydralazine--only the vascular relaxant effects of hydralazine were found to be dependent, in part, on the presence of intact endothelium. The endothelial component of the hydralazine response represented a major contribution to the net relaxant effect on the vascular smooth muscle, particularly at lower concentrations, 90 nM to 1 microM, which are also clinically relevant.

Metabolism of prostaglandin E2 in the isolated perfused kidney of the rabbit.

In the Krebs-perfused rabbit isolated kidney, [3H]PGE2 (5 microCi, 165 Ci/mmole) was infused intra-artially for 5 min; venous and urinary effluents were collected every 2 min for 20 min. Efflux of radioactive material peaked at 8 min and declined thereafter. The kidney retained 35% of the infused 3H. Samples were extracted for acidic lipids; PGE2, PGF2 alpha and metabolites were separated by TLC and quantified by a radiometric method. Efflux of [3H]PGF2 alpha into urinary and venous outflows increased progressively over the first 12 min and then plateaued for the remaining 4 min. By 12 min, conversion of [3H]PGE2 to [3H]PGF2 alpha was 70 and 80% as determined by radiolabeled products recovered in the urinary and venous effluents respectively. Estimates of total conversion of [3H]PGE2 to [3H]PGE2 alpha were 62 and 52% of the radiolabeled material exiting in the urinary and venous effluents respectively. The 15-keto and 13,14-dihydro-15-keto metabolites of [3H]PGF2 alpha appeared in the urine but were not found in the venous outflow. We conclude that PGE-9-ketoreductase (PGE-9KRD) activity is high in the rabbit isolated perfused kidney. Further, the extent of conversion of PGE2 to PGF2 alpha and metabolism of newly formed PGF2 alpha may differ within the vascular and tubular compartments of the kidney. PGE-9KRD activity may be important in the regulation of renal vascular tone, compliance of veins, and salt and water balance.

Prostaglandin-related renin release from rabbit renal cortical slices.

The possibility that 6-keto-prostaglandin E1 (6-keto-PGE1) affects renin release was studied using rabbit renal cortical slices, a preparation that eliminates hemodynamic, neural, and blood-borne factors that might influence renin release. The medium used for incubating the slices was collected for renin assay at the end of each of four successive 20-minute periods. Test agents were added only once, at the beginning of Period 3 (experimental period). Between Periods 3 an 4 (recovery period), the medium was aspirated and the slices rinsed with Krebs solution before replacing the medium. Renin release did not change in vehicle-treated slices. Unlike the PGI2-induced changes, the effects of 6-keto-PGE1 on renin release were sustained in Period 4. Indomethacin potentiated renin stimulation induced by 10 microM concentrations of PGI2 and 6-keto-PGE1 in Period 3 and by 6-keto-PGE1 in Period 4. Using platelet antiaggregatory activity as an index of stability, we found that PGI2 was largely inactivated within 10 minutes under the conditions used for incubating the slices (pH 7.4, 37 degrees C), while 6-keto-PGE1 was stable. The results lend further support to the concept that 6-keto PgE1 is capable of releasing renin through a direct action.

Stimulation of renin release by 6-oxo-prostaglandin E1 and prostacyclin.

1 Renin release induced by 6-oxo-prostaglandin E1 (6-oxo-PGE1) was compared to release in response to prostacyclin (PGI2) and 6-oxo-PGF1 alpha in slices of rabbit renal cortex. 2 Krebs-Ringer medium bathing slices of renal cortex was collected for renin assay after four successive 20 min intervals (periods I-IV). Renin release did not increase during periods I to IV in untreated slices. Agonists were added, only once, at the beginning of period III. Between periods III and IV, the incubation solution was aspirated and replaced with fresh medium. 3 PGI2 increased renin release during period III while 6-oxo-PGE1 stimulated release during periods III and IV. 6-oxo-PGE1 stimulated renin release (24%-74%) in concentrations ranging from 1-33 microM while PGI2 stimulated release at 10 microM (60%) but not at 5 microM. 6-oxo-PGF1 alpha, 10 microM, did not release renin during period III (period III, 9%), but caused a small rise in period IV (29%). 4 6-oxo-PGE1, unlike PGI2, was stable under the incubation conditions (pH 7.4, 37 degrees C) as indicated by recovery of undiminished platelet anti-aggregatory material after 20 min. 5 In the rabbit kidney, activity of 9-hydroxyprostaglandin dehydrogenase was greatest in the cortex and negligible in the papilla, corresponding to the zonal distribution of renin. 6 The prominent and sustained in vitro renin releasing effect of 6-oxo-PGE1, as well as the cortical localization of enzyme activity capable of generating this stable prostacyclin metabolite, suggest that formation of 6-oxo-PGE1 may contribute to PGI2-induced renin release and may explain the delayed stimulation caused by 6-oxo-PGF1 alpha.