calcimycin and Reperfusion-Injury

Intestinal ischemia-reperfusion (IIR) injury is associated with inflammation and oxidative stress, yet its precise mechanisms remain not fully understood. IIR injury is closely linked to the gut microbiota and its metabolites. The anti-inflammatory and antioxidant effects of Lactiplantibacillus plantarum are specific to IIR. In our study, we conducted a 30-day pre-treatment of SD rats with both a standard strain of Lactiplantibacillus plantarum and Lactiplantibacillus plantarum GL001. After a 7-day cessation of treatment, we induced an IIR injury model to investigate the mechanisms by which Lactiplantibacillus plantarum alleviates IIR damage. The results demonstrate that Lactiplantibacillus plantarum effectively mitigates the inflammatory and oxidative stress damage induced by IIR. Lactiplantibacillus plantarum GL001 can improve the gut microbiota by reducing the abundance of harmful bacteria and increasing the abundance of beneficial bacteria. In IIR intestinal tissue, the levels of secondary bile acids are elevated. The content of the bacterial metabolite Calcimycin increases. Annotations of metabolic pathways suggest that Lactiplantibacillus plantarum GL001 can alleviate IIR damage by modulating calcium-phosphorus homeostasis through the regulation of parathyroid hormone synthesis, secretion, and action. Microbiota-metabolite correlation analysis reveals a significant negative correlation between calcimycin and Lactonacillus and a significant positive correlation between calcimycin and Shigella. There is also a significant positive correlation between calcimycin and secondary bile acids. Lactiplantibacillus plantarum GL001 can alleviate oxidative damage induced by IIR through improvements in gut microbiota and intestinal tissue metabolism.

Topics: Animals; Bacteria; Bile Acids and Salts; Calcimycin; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury

The role of SK(Ca) and IK(Ca) channels in myogenic tone and endothelium-derived hyperpolarizing factor (EDHF) responsiveness was investigated under control conditions and after ischemia and reperfusion in parenchymal arterioles (PA) versus middle cerebral arteries (MCA).. MCA and PA were dissected from male Wistar rats that were ischemic for 1 hour with 24 hours of reperfusion (n=12) or sham controls (n=12). Basal tone and reactivity to apamin (300 nmol/L), TRAM-34 (1.0 micromol/L), and nitro-L-arginine (0.1 mmol/L) were compared in PA and MCA pressurized to 40 mm Hg and 75 mm Hg, respectively. SK(Ca) and IK(Ca) channel mRNA expression was measured using real-time PCR.. PA developed greater basal tone than MCA (42+/-4% versus 19+/-3%; P<0.01). Addition of apamin and TRAM-34 increased tone of PA by 25+/-3% and 16+/-2%, respectively, whereas MCA had no response to either inhibitor. After ischemia and reperfusion, the response to nitric oxide synthase inhibition (NOS) was diminished in PA, whereas EDHF responsiveness was preserved. In addition, stimulated EDHF dilation was partially reversed by apamin and completely reversed by TRAM-34 in both control and ischemic PA. SK(Ca) and IK(Ca) channel mRNA expression was similar in PA and MCA and not altered by ischemia and reperfusion. However, IK(Ca) channel mRNA expression was 4- to 5-fold greater than SK(Ca) channels.. It appears that SK(Ca) and IK(Ca) channel activity diminishes basal tone of PA, but not MCA. The preservation of EDHF responsiveness of PA after ischemia and reperfusion suggests an important role for this vasodilator under conditions when NOS is inhibited.

Topics: Animals; Apamin; Arterioles; Biological Factors; Brain Ischemia; Calcimycin; Disease Models, Animal; Enzyme Inhibitors; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionophores; Male; Middle Cerebral Artery; Nitric Oxide; Nitroarginine; Pyrazoles; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Small-Conductance Calcium-Activated Potassium Channels; Vasodilation

Renal ischemic/reperfusion injury in vivo results in a significant increase of acute renal failure (ARF) and death. Nevertheless, there are many limitations in using in vivo models of renal ischemic injury to elucidate the detailed mechanisms of renal injury. Adenosine triphosphate (ATP), an extracellular signal, has been shown to be an important factor in regulation of epithelial cell function. Thus, the present study was performed to establish in vitro ischemic model using primary cultured rabbit renal proximal tubule cells (PTCs) and to examine the effect of ATP in this model. We established an in vitro model of ischemic injury, causing severe depletion of intracellular ATP by using the combination of a mitochondrial respiration inhibitor (antimycin A), non-metabolizable glucose analog (2-deoxyglucose), and calcium ionophore (A23187) in PTCs. Indeed, this ischemic injury significantly increased LDH release, a marker of structural damage, and ATP blocked ischemic injury-induced LDH release. 2-Methylthio-ATP and ATP-gamma-S (P2Y purinoceptor agonists) also blocked ischemic injury-induced LDH release, whereas AMP-CPP (P2X purinoceptor agonist) did not block it. In experiments to examine the relationship between ischemic injury and NF-kappaB activation, ischemic injury increased NF-kappaB translocation, DNA binding activity, and CAT activity. On the other hand, ATP, ATP-gamma-S, or 2-methylthio-ATP protected ischemic injury-induced NF-kappaB activation. These results suggest that the protective effect of ATP on ischemic injury is, in part, related to inhibition of NF-kappaB activation via P2Y receptor in PTCs.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Cell Survival; Cells, Cultured; I-kappa B Kinase; Kidney; Lactate Dehydrogenases; Male; NF-kappa B; Phosphorylation; Protein Serine-Threonine Kinases; Rabbits; Receptors, Purinergic P2; Reperfusion Injury

The role of poly (adenosine diphosphate-ribose) synthetase (PARS) in the contractile and relaxant responses of pulmonary arteries injured by ischemia and reperfusion (IR) of splanchnic artery has not been evaluated. We examined these responses by using 3-aminobenzamide, a pharmacological inhibitor of PARS. IR models in rats were induced by clamping the superior mesenteric artery for 60 min, followed by release of the clamp for 60 min. In the 2 treated groups, 5 or 10 mg/kg of 3-aminobenzamide was administered as an IV bolus at 10 min before reperfusion, followed by infusion rates of 5 and 10 mg.kg(-1).h(-1), respectively, during the period of reperfusion (IR + PARS inhibitor 5 and 10 groups). In the vehicle-treated group, 3-aminobenzamide was not given, but IV saline was administered (IR group). In the control group, surgery was performed, but the superior mesenteric artery was not occluded (sham group). The pulmonary arteries were isolated, and effects of drugs were evaluated in vitro. The IR group showed no attenuation of the contractile responses of the pulmonary artery to phenylephrine. The relaxant responses to endothelium-dependent vasodilators, acetylcholine, and A23187 in the IR group were significantly inhibited when compared with the sham group. The reduction in the relaxant response to endothelium-dependent vasodilators was improved in the IR + PARS inhibitor 5 and 10 groups when compared with the IR group. We concluded that IR attenuated the relaxant responses of the pulmonary artery to endothelium-dependent vasodilators and that PARS inhibitors ameliorate the reduction in the relaxant response.

Topics: Acetylcholine; Animals; Benzamides; Calcimycin; Endothelium, Vascular; Enzyme Inhibitors; Male; Mesenteric Artery, Superior; Nitroprusside; Poly(ADP-ribose) Polymerase Inhibitors; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Vasodilation

The function of the vascular endothelium after storage at room temperature (24 degrees Celsius) for four, eight, and twenty-four hours was investigated with use of an ex vivo canine tibial perfusion model. Function was assessed in terms of changes in perfusion pressure and changes in the concentration of endothelin-1 in the venous effluent of the perfused tibiae. Endothelin-1 is a potent vasoconstrictor that is produced in low concentrations by normal endothelial cells and in increased concentrations by injured vascular endothelial cells. The mean perfusion pressures at flow rates of 1.0 and 1.5 milliliters per minute were significantly higher in the tibiae that had been stored for eight hours than in the tibiae that had been stored for four hours (p < 0.05), and they were significantly higher in the tibiae that had been stored for twenty-four hours than in the tibiae that had been stored for four or eight hours (p < 0.05). The increase in perfusion pressure with increasing duration of storage was associated with an increase in production of endothelin-1. The production of endothelin-1 in the tibiae that had been stored for eight hours (10.6 +/- 0.46 picograms per milliliter) was approximately ten times greater than that in the tibiae that had been stored for four hours (1.1 +/- 0.29 picograms per milliliter). The tibiae that had been stored for twenty-four hours had 19.1 +/- 1.5 picograms of endothelin-1 per milliliter, nearly twice that produced in the tibiae that had been stored for eight hours. Injection of acetylcholine demonstrated muscarinic receptor-mediated vasodilation in the tibiae that had been stored for four hours. In contrast, the tibiae that had been stored for eight and twenty-four hours had no evidence of acetylcholine-induced vasodilation of baseline perfusion vascular smooth-muscle tone. However, there was some preservation of endothelium-dependent vascular smooth-muscle relaxation in the tibiae that had been stored for eight and twenty-four hours, as norepinephrine-induced vascular smooth-muscle contraction was significantly greater in the presence of N(G)-monomethyl-L-arginine acetate (p < 0.05). Moreover, in the second phase of the study, a bolus injection of calcium ionophore A23187 in tibiae that had been stored for twenty-four hours relaxed vascular smooth muscle. Adrenomedullin, a novel peptide with known vasodilator properties, relaxed vascular smooth muscle in all three groups and also attenuated the pressor response to norepi

Topics: Acetylcholine; Adrenomedullin; Animals; Calcimycin; Dogs; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; Female; In Vitro Techniques; Ionophores; Ischemia; Male; Muscle, Smooth, Vascular; Nitric Oxide; Oligopeptides; Peptides; Pressoreceptors; Reperfusion Injury; Temperature; Tibia; Vasodilator Agents

Chronic angiotensin-converting enzyme (ACE) inhibition initiated days to weeks after acute myocardial infarction can reduce ventricular dilatation and improve patient survival. However, the effects on coronary vascular and myocardial function of very early ACE inhibitor therapy for acute myocardial infarction remain unresolved.. Hemodynamics, segmental shortening, coronary blood flow, and in vitro coronary microvascular relaxation responses were studied in noninstrumented control pigs (n = 8) and pigs subjected to 30 minutes of left anterior descending ischemia followed by administration of 30 mL IV normal saline (IR-saline, n = 8), 5 mg/kg IV captopril (IR-captopril, n = 6), or 1.5 mg/kg IV enalaprilat (IR-enalaprilat, n = 6) before 1 hour of reperfusion. Hemodynamics were similar at baseline, end of ischemia, and end of reperfusion. However, coronary blood flow immediately on reperfusion was significantly enhanced in the IR-enalaprilat cohort (59 +/- 10 mL/min) compared with the IR-saline group (32 +/- 3 mL/min, P < .05). Segmental shortening in the dyskinetic ischemic region improved only minimally at the end of reperfusion to 1 +/- 2%, -7 +/- 3%, and -2 +/- 6% for the IR-saline, IR-captopril, and IR-enalaprilat groups, respectively (P < .05, IR-captopril versus IR-saline). Arteriolar microvascular endothelium-dependent responses to ADP (P < .01) and calcium ionophore A23187 (P < .01) were impaired after ischemia-reperfusion, whereas bradykinin responses were preserved (P = .95). Endothelium-dependent venular responses to ADP and serotonin were maintained despite ischemia-reperfusion. Endothelium-independent responses to sodium nitroprusside were unaltered in arterioles and venules. Either captopril or enalaprilat restored ADP and A23187 arteriolar responses to control levels and increased bradykinin responses above control levels.. Brief ischemia followed by reperfusion induces arteriolar microvascular endothelial dysfunction, while venular endothelial function is preserved in this porcine model. ACE inhibition enhances coronary blood flow at the time of reperfusion and can prevent impairment of endothelium-dependent arteriolar responses. However, ACE inhibition does not enhance ventricular segmental shortening acutely despite improved microvascular endothelial function and augmented postischemic coronary blood flow in this model of ischemia-reperfusion.

Topics: Adenosine Diphosphate; Angiotensin-Converting Enzyme Inhibitors; Animals; Calcimycin; Captopril; Coronary Vessels; Enalaprilat; Endothelium, Vascular; Female; Hemodynamics; Ionophores; Male; Microcirculation; Reperfusion Injury; Swine

It is generally accepted that microvascular permeability is controlled by intercellular endothelial cell gap size. This process is controlled in endothelial cell monolayers and peripheral blood vessels by calmodulin (CaM)-dependent myosin light-chain kinase (MLCK), which phosphorylates MLC20 with subsequent actin-myosin interaction. In the present study both CaM and MLCK blockers were studied during ischemia-reperfusion (I/R)-induced injury in isolated buffer-perfused rat lungs. The effects of a calcium ionophore (CaI) were tested in isolated intact rat lungs to compare the effects of increasing intracellular Ca2+ to I/R-induced damage. Because protein kinase C (PKC) could also be a mediator of I/R injury, a PKC inhibitor was studied in lungs subjected to either I/R or CaI. In lungs subjected to I/R alone, a fivefold increase in microvascular permeability occurred after 30 min of reperfusion (P < 0.001), and a tenfold increase was present after an additional 60 min of reperfusion (P < 0.01). Pretreatment of the I/R lungs with a CaM inhibitor (trifluoperazine, 100 microM) or with a MLCK inhibitor (ML-7,500 nM) blocked the microvascular damage at both 30 and 90 min of reperfusion. When the CaM inhibitor was introduced into the venous reservoir after 46 min of reperfusion, after the microvascular damage was present, no further increase in microvascular permeability occurred. Pretreatment of the lungs with a PKC inhibitor (staurosporine, 100 nM) did not alter the magnitude of the increased microvascular permeability produced by I/R or the time course of the damage. The calcium ionophore A23187 (7.5 microM) caused increases in Kfc values similar to those produced by I/R. Pretreatment of A23187-treated lungs with a CaM inhibitor produced no protective effect on the microvascular injury at 30 min after administration. Pretreatment of the CaI-challenged lungs with staurosporine significantly increased the microvascular barrier injury at 30 min compared with that occurring with I/R. When a beta-adrenergic receptor agonist (isoproterenol, 10 microM) was introduced to the lung after CaI-induced damage had occurred, no further increase in microvascular permeability was observed, and a trend toward reversal of injury occurred. We conclude from these studies that CaM/MLCK/MLC20 system is involved in our model of I/R-induced rat lung injury but is not involved in lung injury associated with Ca2+ entering the cell.

Topics: Adrenergic beta-Agonists; Animals; Calcimycin; Calcium; Calmodulin; In Vitro Techniques; Ischemia; Isoproterenol; Male; Microcirculation; Myosin-Light-Chain Kinase; Protein Kinase C; Pulmonary Circulation; Rats; Rats, Inbred Strains; Reperfusion Injury; Trifluoperazine

The goal of these experiments was to determine whether the perturbation of ischemia-reperfusion has an age-dependent effect on subsequent endothelial cell production of nitric oxide. Three- and 35-d-old swine in the experimental group were exposed to 1-h partial ischemia (90% flow reduction) and 2-h reperfusion in vivo by creation and then removal of a mesenteric artery coarctation. Control subjects underwent exposure of the mesenteric artery only. After reperfusion, gut vascular resistance had increased 44 +/- 6% in 3-d-old, but had decreased 41 +/- 4% in 35-d-old subjects. At the completion of the in vivo portion of the protocol mesenteric artery was removed, and nitric oxide production was estimated in vitro, by measuring cGMP production by vessel segments or by measuring relaxation of phenylephrine-precontracted rings, both after stimulation of nitric oxide production by substance P or the calcium ionophore A23187. Compared with control, mesenteric artery segments from 3-d-old subjects demonstrated reductions in basal, substance P-stimulated (10(-8) M) and A23187-stimulated (10(-7) M) cGMP accumulation of 50 +/- 7%, 66 +/- 6% and 78 +/- 7%. Mesenteric artery segments from 35-d-old subjects demonstrated increases in basal, substance P-stimulated, or A23187-stimulated cGMP accumulations of 114 +/- 14%, 92 +/- 8%, or 78 +/- 9%. Compared with control, I/R rings from 3-d-old subjects demonstrated reductions in substance P-induced (10(-8) M) or A23187-induced (10(-7) M) relaxations of 56 +/- 7% or 30 +/- 7%. In contrast, 35-d-old ischemia-reperfusion rings demonstrated increases in substance P- or A23187-induced relaxation of 36 +/- 8% or 98 +/- 11%. It is concluded that ischemia-reperfusion has an age-dependent effect on endothelial production of NO within in vitro postnatal mesenteric artery and that these changes mirror the effects of ischemia-reperfusion on gut vascular resistance in vivo.

Topics: Age Factors; Animals; Calcimycin; Cyclic GMP; Endothelium; Hemodynamics; Intestinal Mucosa; Ionophores; Mesenteric Arteries; Nitric Oxide; Reperfusion Injury; Substance P; Swine

A major hemodynamic feature of acute lung injury is pulmonary hypertension caused by pulmonary vasoconstriction. Impairment of the mechanisms of pulmonary vasorelaxation may contribute to this pulmonary vasoconstriction. This study examined the effect of mesenteric ischemia/reperfusion (I/R) on lung neutrophil accumulation and endothelial-dependent and -independent cyclic 3'-5' guanosine monophosphate-mediated pulmonary vasorelaxation in rats.. Rats were studied after 1 hour of superior mesenteric artery occlusion and 2 hours of reperfusion. Lung neutrophil accumulation was determined by myeloperoxidase assay (MPO). The following mechanisms of pulmonary vasorelaxation were studied in isolated pulmonary artery rings by generating dose response curves (10(-9) to 10(-6)mol/L): (1) receptor-dependent, endothelial-dependent relaxation (response to acetylcholine), (2) receptor-independent, endothelial-dependent relaxation (response to the calcium ionophore, A23187), and (3) endothelial-independent relaxation (response to sodium nitroprusside [SNP]).. Lung MPO activity was significantly increased from 2.4 +/- 0.2 units/gm lung weight in controls to 10.3 +/- 0.4 after mesenteric I/R (p < 0.05). The vasorelaxation response to SNP was not different after mesenteric I/R, but vasorelaxation by both acetylcholine and A23187 were significantly impaired.. Endothelial-dependent pulmonary vasorelaxation is significantly impaired after mesenteric I/R. Such impairment of pulmonary vasorelaxation may help tip the net balance of pulmonary vasomotor tone toward vasoconstriction and contribute to the pulmonary hypertension seen in acute lung injury.

Topics: Acetylcholine; Animals; Calcimycin; Cyclic GMP; Endothelium, Vascular; Hypertension, Pulmonary; In Vitro Techniques; Lung; Lung Injury; Male; Neutrophils; Nitroprusside; Peroxidase; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Vasodilation; Vasodilator Agents

The severity of "reperfusion injury" is dependent on the extent to which the involved pathways are activated and on the degree of tissue susceptibility to them. This study was undertaken to ascertain whether preexistent ischemic proximal tubular damage (ischemic "pre-conditioning") significantly alters the expression of two purported mediators of reperfusion damage: oxidant stress and cytosolic Ca2+ loading.. Male Sprague-Dawley rats underwent 35 minutes of bilateral renal arterial occlusion. Fifteen minutes or 24 hours later, the kidneys were removed, proximal tubular segments (PTS) were isolated, and their susceptibility to oxidant stress (H2O2 or FeSO4) and to cytosolic Ca2+ loading (Ca2+ ionophore, A23187) was determined. Results were contrasted to those obtained with normal PTS. Cell injury was quantified by percentage of cellular lactate dehydrogenase released. Lipid peroxidation was gauged by PTS malondialdehyde (MDA) concentrations. As an index of endogenous antioxidant defenses, PTS catalase and superoxide dismutase activities were determined. Vulnerability to lipid peroxidation is highly dependent on phospholipid unsaturated fatty content, so PTS fatty acid concentrations also were assessed.. Although PTS harvested at 15 minutes postischemia manifested sublethal injury (increased lactate dehydrogenase release under control conditions), no increased vulnerability to the oxidant insults or to the Ca2+ ionophore was noted. By 24 hours of reflow, cytoresistance to each of the insults had developed. Postischemic PTS demonstrated no increase in basal MDA concentrations (indicating a lack of in vivo lipid peroxidation), and when challenged with H2O2 or FeSO4, significantly less MDA generation developed (vs. the normal PTS). This resistance to lipid peroxidation was not associated with increased superoxide dismutase/catalase levels or altered PTS fatty acid content.. Sublethal ischemic proximal tubular injury does not directly predispose to oxidant stress or cytosolic Ca2+ loading, and by 24 hours postischemia, increased resistance to these insults develops. Decreased membrane susceptibility to lipid peroxidation may contribute to this result.

Topics: Animals; Calcimycin; Calcium; Ferrous Compounds; Hydrogen Peroxide; Ischemia; Kidney Tubules, Proximal; Lipid Peroxidation; Male; Oxidants; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury

To examine whether endocardial microvascular function is preferentially impaired by ischemia and reperfusion, we studied endothelium-dependent responses of epicardial and endocardial coronary microvessels (130-220 microns) from control pigs and from pigs subjected to 1-h regional myocardial ischemia (circumflex occlusion) followed by 1-h reperfusion (n = 8) in vitro using videomicroscopy. In control animals (n = 8), no significant transmural differences were apparent in microvascular responses to the endothelium-dependent agents bradykinin or the calcium ionophore A23187, to the endothelium-independent agent sodium nitroprusside (SNP), or to adenosine. Serotonin caused a slight but statistically insignificant greater relaxation of endocardial than of epicardial microvessels. After ischemia-reperfusion, relaxations to all endothelium-dependent agents (serotonin, bradykinin, A23187) and to adenosine were significantly reduced (p < 0.05 for all agents) as compared with the respective control responses. There were no significant differences between epicardial and endocardial responses in the ischemia-reperfusion group for any of the vasoactive agents. Endothelium-independent responses to SNP were not affected by ischemia-reperfusion, indicating no alteration in the ability of vascular smooth muscle to relax through guanylate cyclase-mediated mechanisms. Control epicardial microvascular responses were examined after endothelial denudation and after pretreatment with NG-monomethyl-L-arginine (L-NMMA), indomethacin, or glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Animals; Arginine; Bradykinin; Calcimycin; Coronary Circulation; Endocardium; Endothelium, Vascular; Female; Glyburide; Guanylate Cyclase; Indomethacin; Male; Microcirculation; Muscle, Smooth, Vascular; Nitroprusside; omega-N-Methylarginine; Reperfusion Injury; Serotonin; Swine

Activated neutrophils cause conversion of xanthine dehydrogenase to its oxidase form (xanthine oxidase) in endothelial cells, the mechanism of which may be related to the cytotoxic effect of activated neutrophils. The elastase inhibitors, elastatinal, alpha 1-antitrypsin, and MeO-Suc-(Ala)2-Pro-Val-CH2Cl, significantly inhibited xanthine dehydrogenase to oxidase conversion by phorbol myristate acetate-stimulated neutrophils without inhibition of neutrophil adherence to the endothelial cell monolayer. The role of elastase in this enzyme conversion process was confirmed by the ability of purified elastase to cause conversion of xanthine dehydrogenase to xanthine oxidase in intact endothelial cells (or cell extracts) without causing cytotoxicity. In contrast, cathepsin G failed to cause conversion. The kinetics of conversion induced by elastase was relatively rapid, being essentially completed by 30 min. Upon removal of elastase, the effect was slowly (greater than 12 h) reversible and could be inhibited by cycloheximide treatment. Exposure of endothelial cells to hypoxia failed to enhance the elastase-induced conversion. Treatment of endothelial cells with Ca2+ ionophores failed to cause conversion of xanthine dehydrogenase to oxidase, suggesting that intracellular Ca(2+)-activated proteases are not sufficient to induce this process. Neutrophil-induced xanthine dehydrogenase to oxidase conversion was inhibited by concomitant treatment with antibodies to CD11b. The results suggest that activated neutrophils induce conversion of xanthine dehydrogenase to oxidase by secretion of elastase in close proximity to the endothelial cells and that this intimate contact between the two cell types enables high local concentrations of elastase to be attained, which are sufficient to cause xanthine dehydrogenase to xanthine oxidase conversion.

Topics: Animals; Calcimycin; Cell Cycle; Cycloheximide; Endothelium, Vascular; In Vitro Techniques; Ionomycin; Neutrophils; Oxygen; Pancreas; Pancreatic Elastase; Rats; Reperfusion Injury; Tetradecanoylphorbol Acetate; Xanthine Dehydrogenase; Xanthine Oxidase

Although the specific cause(s) of inflammatory bowel diseases (IBD) has not been identified, one theory suggests ischemia as the early event that occurs in IBD and reperfusion causes sustained release of oxyradicals, leading to inflammation and ulceration. In this study, we have confirmed that H2O2 in the concentration seen during ischemia/reperfusion is primarily responsible for cellular membrane damage in the rat colonic fragments in vitro. Hydrogen peroxide caused a time and dose-dependent increase in 6-keto-PGF1 alpha and TXB2 release. Hydrogen peroxide-stimulated 6-keto-PGF1 alpha release was blocked (50%) by phospholipase A2 (PLA2) inhibitors quinacrine and dimethyleicosadienoic acid at 5 min. Hydrogen peroxide-stimulated 6-keto-PGF1 alpha release was completely blocked by indomethacin, significantly blocked (69%) by nordihydroguiaretic acid, and completely blocked by catalase. Superoxide dismutase and uric acid failed to inhibit H2O2-stimulated 6-keto-PGF1 alpha release. Endogenous catalase inhibitors 3-aminotriazole and sodium azide further enhanced the release of 6-keto-PGF1 alpha stimulated by H2O2 by 29% and 73%, respectively. Xanthine-xanthine oxidase also increased 6-keto-PGF1 alpha release from the fragments by 110%. This release was not inhibited by superoxide dismutase and uric acid, but was completely inhibited by catalase. These studies suggest a direct effect of H2O2 on colonic fragments leading to submicroscopic cellular membrane damage and excess prostanoid production utilizing a PLA2/cyclooxygenase and catalase-sensitive pathway without the formation of toxic hydroxyl ions. The quick release of 6-keto-PGF1 alpha also suggests an early manifestation of H2O2-induced damage in rat colonic fragments.

Topics: 6-Ketoprostaglandin F1 alpha; Adrenal Cortex Hormones; Animals; Antioxidants; Calcimycin; Colon; Epithelium; Hydrogen Peroxide; Indomethacin; Intestinal Mucosa; Male; Organ Culture Techniques; Oxidation-Reduction; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Reperfusion Injury; Thromboxane B2; Uric Acid