Effects of endotoxin on neutrophil-mediated I/R injury in isolated perfused rat hearts

With the use of a syngeneic model, we demonstrate that rat polymorphonuclear neutrophils (PMNs) exacerbate ischemia-reperfusion injury in the isolated rat heart. However, PMNs (19 x 10(6) cells) from lipopolysaccharide (LPS)-treated rats (LPS-PMNs; 100 mg/kg administered 7 h before exsanguination) induce less reperfusion injury in the isolated heart. Average recovery of left ventricular developed pressure after 20 min of ischemia and 60 min of reperfusion was 51 +/- 4% in hearts receiving PMNs from saline-treated control rats (saline-PMNs) versus 78 +/- 2% in hearts receiving LPS-PMNs. Ischemic hearts reperfused with LPS-PMNs recovered to the same extent as did hearts reperfused with Krebs buffer only. LPS-PMNs and saline-PMNs showed no difference in basal or phorbol ester-induced superoxide production. Whereas twice the number of LPS-PMNs was positive for nitroblue tetrazolium, the percent positive for L-selectin, a receptor integral in PMN-adhesion to endothelium, was 50% less in LPS-PMNs than in controls. After reperfusion, three-fourths of the saline-PMNs remained within the hearts, whereas only one-fourth of LPS-PMNs were trapped. These data suggest that PMNs from LPS-treated rats do not exacerbate ischemia-reperfusion injury as do control PMNs, possibly, due to impaired PMN adhesion to endothelium as a result of decreased L-selectin receptors.     

Restoration of myocardial contractile function during gradual reperfusion after total ischemia

Isolated guinea pig hearts subjected to 25-min total normothermic ischemia and 30-min reperfusion with the initial rate exhibited a great rise in isovolumic diastolic pressure while the contractile function recovered to 34 +/- 4% of initial value. Reperfusion with gradually increased rate from 13% of initial rate to 100% resulted in better recovery of the contractile function--to 54 +/- 3% and markedly less rise in the diastolic pressure. This coincided with 28% less inosine loss. More better recovery of the myocardial contractile function (to 80 +/- 5%) was observed in experiments in which gradual reperfusion was combined with recirculation. In both gradual reperfusion series, the recovery of the heart rate and the contractile function were much delayed during first 5 min reperfusion and were associated with a rise in coronary resistance.     

Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improves myocardial function and coronary perfusion in senescent hearts.

Cardiovascular ageing is associated with an increase in cardiac susceptibility to ischaemia and reperfusion and production of reactive oxygen species has been suspected to be responsible for this age-associated particular vulnerability. To determine whether administration of antioxidant treatment could afford some protection against ischaemia and reperfusion during aging, isolated perfused hearts from adult and senescent rats were submitted to normoxia (180 min), prolonged low-flow ischaemia (15% of initial coronary flow;180 min) or low-flow ischaemia/reperfusion (45 min/30 min), without or with antioxidant enzymes (superoxide dismutase+catalase; 50IU/ml). Contractile function and coronary perfusion were measured and protein oxidation was quantitated in left ventricle after normoxia, ischaemia and ischaemia/reperfusion. Protein oxidation was higher in senescent than in adult hearts after ischaemia-reperfusion, in contrast to prolonged ischaemia. During prolonged ischaemia, antioxidant treatment prevented coronary vasoconstriction at both ages and delayed contractile dysfunction in senescent hearts but did not limit protein oxidation. During reperfusion, antioxidant treatment prevented coronary vasoconstriction and protein oxidation at both ages and considerably improved recovery of contractile function in senescent hearts. In conclusion, antioxidant treatment fully protects the senescent heart against ischaemia/reperfusion but not against prolonged ischaemia injury, indicating that oxidative stress plays a central role in the age-associated vulnerability to ischaemia-reperfusion.     

Dipyridamole inhibits hydroxylamine augmented nitric oxide (NO) production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism

Polymorphonuclear neutrophils (PMN) are thought to play a role in reperfusion injury and ischemia. These effects are partly mediated by toxic oxygen species (superoxide anion, hydrogen peroxide and hydroxyl radical) acting at the level of the endothelium. It was demonstrated recently that the superoxide anion reacts with nitric oxide (NO) and that interaction leads to the generation of highly toxic peroxynitrite. Several drugs were tested so far in order to affect PMN function. It was demonstrated that dipyridamole (2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d)-pyrimidine) can influence neutrophil function by inhibiting adenosine uptake. However, this action can not fully explain all of the observed effects of dipyridamole action on PMN metabolism. The aim of our study was to evaluate the influence of dipyridamole on nitric oxide production by activated polymorphonuclear neutrophils. Incubation of PMNs with hydroxylamine (HA) and phorbol myristate acetate (PMA) generated nitrite (36.4+/-4.2 nmol/h 2x10(6) PMN), dipyridamole at 100 micromol/l, 50 micromol/l and 10 micromol/l caused a considerable drop in nitrite production (11.8+/-1.8, 19.7+/-2.7 and 27.4+/-3.2 nmol/h, respectively). Neither adenosine nor the adenosine analogue could mimic the dipyridamole effect. Moreover theophylline, an adenosine inhibitor could not reverse the dipirydamole action on PMN metabolism. We also found that dipyridamole inhibited hydrogen peroxide release from neutrophils. Catalase that scavenges hydrogen peroxide also largely abolished nitric oxide release from PMN. It is evident that dipyridamole inhibits hydroxylamine-augmented nitric oxide production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism.     

Subcellular distribution of energy metabolites in the pre-ischaemic and post-ischaemic perfused working rat heart

Isolated working rat hearts were subjected to 20 min of global ischaemia and either 5 min or 15 min of reperfusion. The subcellular distribution of ATP, ADP, AMP, phosphocreatine and Pi were determined before and after ischaemia by the method of non-aqueous tissue fractionation. Ventricular function and the cytosolic, mitochondrial and ATPase-associated compartmentation of metabolites were measured. After 5 min of reperfusion, only 13 +/- 9% of the pre-ischaemic contractile function was restored compared to 67 +/- 8% after 15 min reperfusion. ATP was reduced in all cellular compartments after 5 min of reperfusion but was only decreased from pre-ischaemic values in the cytosolic compartment after 15 min of reperfusion (17.1 +/- 3.9 nmol/mg vs. 4.3 +/- 1.5 nmol/mg total protein; P less than 0.05). The mitochondrial [ATP]/[ADP] was reduced from a normal value of 4.36 to 1.79 after 5 min but recovered to 4.62 after 15 min of reperfusion. Most of the Pi was located in the mitochondria or with the ATPase fraction of the cell, with only 16% of the total Pi free in the cytosol. This study indicates that the capacity of the heart to recover function may be compromised during early reperfusion by a 59% increase in mitochondrial phosphate content and during late reperfusion by a reduced cytosolic/mitochondrial concentration ratio of both ATP (from 0.85 to 0.19) and phosphocreatine (from 3.9 to 1.24).     

Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.     

Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance.

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.     

Protein kinase C-zeta inhibition exerts cardioprotective effects in ischemia-reperfusion injury

Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in marked cardiac contractile dysfunction. A cell-permeable PKC-zeta peptide inhibitor was used to test the hypothesis that PKC-zeta inhibition could attenuate PMN-induced cardiac contractile dysfunction by suppression of superoxide production from PMNs and increase nitric oxide (NO) release from vascular endothelium. The effects of the PKC-zeta peptide inhibitor were examined in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. The PKC-zeta inhibitor (2.5 or 5 microM, n = 6) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n = 6) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dt(max)) cardiac function indexes (P < 0.01), and these cardioprotective effects were blocked by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (50 microM). Furthermore, the PKC-zeta inhibitor significantly increased endothelial NO release 47 +/- 2% (2.5 microM, P < 0.05) and 54 +/- 5% (5 microM, P < 0.01) over basal values from the rat aorta and significantly inhibited superoxide release from phorbol-12-myristate-13-acetate-stimulated rat PMNs by 33 +/- 12% (2.5 microM) and 40 +/- 8% (5 microM) (P < 0.01). The PKC-zeta inhibitor significantly attenuated PMN infiltration into the myocardium by 46-48 +/- 4% (P < 0.01) at 2.5 and 5 microM, respectively. In conclusion, these results suggest that the PKC-zeta peptide inhibitor attenuates PMN-induced post-I/R cardiac contractile dysfunction by increasing endothelial NO release and by inhibiting superoxide release from PMNs thereby attenuating PMN infiltration into I/R myocardium.     

L-selectin is required for fMLP- but not C5a-induced margination of neutrophils in pulmonary circulation

To study the role of L-selectin in neutrophil (PMN) margination and sequestration in the pulmonary microcirculation, maximally active concentrations of C5a (900 pmol/g) and N-formylmethionyl-leucyl-phenylalanine (fMLP; 0.34 pmol/g) were injected into the jugular vein of wild-type or L-selectin-deficient C57BL/6 mice. In wild-type mice administered C5a or fMLP, 92 +/- 1% and 34 +/- 9%, respectively, of peripheral blood PMN were trapped mostly in the pulmonary circulation as determined by immunohistochemistry and myeloperoxidase activity. In wild-type mice treated with F(ab')(2) fragments of the L-selectin monoclonal antibody MEL-14 or in L-selectin-deficient mice, C5a-induced neutropenia was not significantly reduced, but the decrease in peripheral PMN in response to fMLP was completely abolished, indicating that L-selectin is necessary for fMLP- but not C5a-induced pulmonary margination. Immunostained lung sections of fMLP- or C5a-treated mice showed sequestered neutrophils in alveolar capillaries with no evidence of neutrophil aggregates. We conclude that chemoattractant-induced PMN margination in the pulmonary circulation can occur by two separate mechanisms, one of which requires L-selectin.     

Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these interactions. Whether platelets also promote PMN transmigration across the endothelium is less clear. We tested the hypothesis that platelets enhance PMN transmigration across the inflamed endothelium and that PSGL-1 is involved. We studied the effects of platelets on PMN transmigration in vivo and in vitro using a well-characterized corneal injury model in C57BL/6 mice and IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) under static and dynamic conditions. In vivo, platelet depletion altered PMN emigration from limbal microvessels after injury, with decreased emigration 6 and 12 h after injury. Both PSGL-1-/- and P-selectin-/- mice, but not Mac-1-/- mice, also had reduced PMN emigration at 12 h after injury relative to wild-type control mice. In the in vitro HUVEC model, platelets enhanced PMN transendothelial migration under static and dynamic conditions independent of firm adhesion. Anti-PSGL-1 antibodies markedly inhibited platelet-PMN aggregates, as assessed by flow cytometry, and attenuated the effect of platelets on PMN transmigration under static conditions without affecting firm adhesion. These data support the notion that platelets enhance neutrophil transmigration across the inflamed endothelium both in vivo and in vitro, via a PSGL-1-dependent mechanism.     

Role for tumor necrosis factor as mediator of lung injury following lower torso ischemia

Ischemia and reperfusion of the ischemic lower torso lead to a neutrophil- (PMN) dependent lung injury characterized by PMN sequestration and permeability edema. This mimics the injury seen after infusion of tumor necrosis factor alpha (TNF), a potent activator of PMN and endothelium. This study tests whether TNF is a mediator of the lung injury after lower torso ischemia. Anesthetized rats underwent 4 h of bilateral hindlimb tourniquet ischemia, followed by reperfusion for 10 min, 30 min, 1, 2, 3, and 4 h (n = 6 for each time point). Quantitative lung histology indicated progressive sequestration of PMN in the lungs, 25 +/- 3 (SE) PMN/10 high-power fields (HPF) 10 min after reperfusion vs. 20 +/- 2 PMN/10 HPF in sham animals (NS), increasing to 53 +/- 5 PMN/10 HPF after 4 h vs. 23 +/- 3 PMN/10 HPF in sham animals (P less than 0.01). There was lung permeability, shown by increasing protein accumulation in bronchoalveolar lavage (BAL) fluid, which 4 h after reperfusion was 599 +/- 91 vs. 214 +/- 35 micrograms/ml in sham animals (P less than 0.01). Similarly, there was edema, shown by the lung wet-to-dry weight ratio, which increased by 4 h to 4.70 +/- 0.12 vs. 4.02 +/- 0.17 in sham animals (P less than 0.01). There was generation of leukotriene B4 in BAL fluid (720 +/- 140 vs. 240 +/- 40 pg/ml, P less than 0.01), and in three of six rats tested at this time TNF was detected in plasma, with a mean value of 167 pg/ml. TNF was not detectable in any sham animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of endotoxin on neutrophil-mediated ischemia/reperfusion injury in the rat heart in vivo

We have previously shown that a nonlethal dose of lipopolysaccharide (LPS) decreases L-selectin expression of neutrophils (PMNs), thereby preventing PMN-mediated reperfusion injury in the isolated heart. In the present study we determined whether or not that dose of LPS would protect hearts during in vivo ischemia and reperfusion by preventing PMN-induced reperfusion injury. Rats receiving saline vehicle showed marked myocardial injury (necrotic area/area at risk = 82%+/-2%) and significant depression in left ventricular function as assessed in the isolated isovolumic heart preparation at constant flow rates of 5, 10, 15, and 20 ml/min. The administration of LPS (100 microg/kg body wt) 7 hr prior to ischemia resulted in a reduction in myocardial damage (necrotic area/area at risk = 42%+/-3%) and preservation of function. Myocardial function was similar to that of sham ischemic saline- and LPS-treated rats. Moreover, PMN infiltration as determined by histology was quantitatively more severe in hearts of saline-treated rats than in hearts of LPS-treated rats. Isolated hearts from vehicle- and LPS-treated animals undergoing sham ischemia in vivo recovered to the same extent after in vitro ischemia/reperfusion, suggesting that LPS did not induce protection by altering intrinsic properties of the heart. Our results indicate that LPS-induced protection of the heart from in vivo PMN-mediated ischemia/reperfusion injury may be due to decreased L-selectin expression of PMNs in LPS-treated animals.     

Importance of extracellular divalent cations to polarisation of polymorphonuclear leukocytes induced by plasma

The roles of extracellular calcium and magnesium ions in the polarisation of human peripheral blood polymorphonuclear leukocytes (PMN) induced by autologous fresh heparinised plasma were investigated by studying the effects of 5 mM chelators of divalent cations [ethylenediamine tetra-acetic acid (EDTA), ethylenebis-(oxyethylene-nitrilo)-tetra-acetic acid (EGTA) or disodium hydrogen citrate]. In addition, the effects of a blocker of membrane calcium channels (verapamil) were studied. Polarisation of PMN suspended in plasma (84.1 +/- 11.9%) was reduced by each chelating agent over 30 min (to 20.0 +/- 15.6% by EDTA, to 42.5 +/- 19.3% by EGTA and to 29.4 +/- 22.9% by citrate). Polarisation of PMN suspended in plasma treated with EDTA or EGTA was restored by inclusion of equimolar additional Ca2+ ions, and in plasma treated with EDTA, EGTA or citrate, by equimolar additional Mg2+ ions. Additional Mg2+ had no effect on the spherical shape of PMN in Hanks' solution and additional cations had no effects on the polarisation of PMN induced by fMLP. Cells rendered spherical by each chelating agent in plasma for 30 min retained their ability to polarise on addition of fMLP to the plasma-chelator medium. Verapamil (10(-4) M) markedly reduced polarisation in plasma (to 52 +/- 11.3%) but the same drug (10(-5) M) had no such effect. In contrast to the polarisation of cells in plasma, the polarisation response of PMN to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) in buffered Hanks' solution was not affected by any of the chelating agents or by verapamil, even in high concentration. These results indicate that extracellular divalent cations are necessary for the polarisation of PMN suspended in autologous plasma and that the mechanism of polarisation of PMN in plasma may be different to that of polarisation induced by fMLP.     

Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.     

A new method to evaluate extravascular albumin and blood cell accumulation in the lung

A method was developed to evaluate blood volume, accumulation of extravascular albumin (ALBev), and platelet (PL) or polymorphonuclear neutrophil (PMN) sequestration in lungs after challenge with inflammatory agents. Erythrocytes (RBC), albumin, and PL or PMN, labeled with 99mTc, 131I, and 111In,-respectively, were injected intravenously into anesthetized and ventilated guinea pigs. The different parameters were calculated from in vivo lung and blood radioactivity values. When N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was injected intravenously at 10 micrograms.kg-1, lung RBC content dropped by 14.7 +/- 1.8% (SE; n = 10), indicating a reduced lung blood volume, ALBev rose to 15.0 +/- 3.2% of the initial albumin vascular content, and the circulating PMN were sequestered by 9.2 +/- 1.7%. A transient PL sequestration was also observed 1 min after the injection of fMLP (13.1 +/- 2.0%, n = 7). During the infusion of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, the lung PL content rose dose dependently from 10.1 +/- 2.2% of the circulating pool with 3 ng.kg-1.min-1 to 54.9 +/- 20.1% with 44 ng.kg-1.min-1, the lung RBC content decreased by greater than 10%, and the ALBev increased beyond 16%. Our method allows the study of the correlations between cell entrapment and the variations of the albumin exchanges in the lung and may lead to a better understanding of the correlations between cell activation and edema.     

Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning

Ischemic preconditioning (Pre-con) is an adaptive response triggered by a brief ischemia applied before a prolonged coronary occlusion. We tested the hypothesis that repetitive ischemia applied during early reperfusion, i.e., postconditioning (Post-con), is cardio-protective by attenuating reperfusion injury. In anesthetized open-chest dogs, the left anterior descending artery (LAD) was occluded for 60 min and reperfused for 3 h. In controls (n = 10), there was no intervention. In Pre-con (n = 9), the LAD was occluded for 5 min and reperfused for 10 min before the prolonged occlusion. In Post-con (n = 10), at the start of reperfusion, three cycles of 30-s reperfusion and 30-s LAD reocclusion preceded the 3 h of reperfusion. Infarct size was significantly less in the Pre-con (15 +/- 2%, P < 0.05) and Post-con (14 +/- 2%, P < 0.05) groups compared with controls (25 +/- 3%). Tissue edema (% water content) in the area at risk was comparably reduced in Pre-con (78.3 +/- 1.2, P < 0.05) and Post-con (79.7 +/- 0.6, P < 0.05) versus controls (81.5 +/- 0.4). Polymorphonuclear neutrophil (PMN) accumulation (myeloperoxidase activity, Deltaabsorbance.min-1.g tissue-1) in the area at risk myocardium was comparably reduced in Post-con (10.8 +/- 5.5, P < 0.05) and Pre-con (13.4 +/- 3.4, P < 0.05) versus controls (47.4 +/- 15.3). Basal endothelial function measured by PMN adherence to postischemic LAD endothelium (PMNs/mm2) was comparably attenuated by Post-con and Pre-con (15 +/- 0.6 and 12 +/- 0.6, P < 0.05) versus controls (37 +/- 1.5), consistent with reduced expression of P-selectin on coronary vascular endothelium in Post-con and Pre-con. Endothelial function assessed by the maximal vasodilator response of postischemic LAD to acetylcholine was significantly greater in Post-con (104 +/- 6%, P < 0.05) and Pre-con (109 +/- 5%, P < 0.05) versus controls (71 +/- 8%). Plasma malondialdehyde (microM/ml), a product of lipid peroxidation, was significantly less at 1 h of reperfusion in Post-con (2.2 +/- 0.2, P < 0.05) versus controls (3.2 +/- 0.3) associated with a decrease in superoxide levels revealed by dihydroethidium staining in the myocardial area at risk. These data suggest that Post-con is as effective as Pre-con in reducing infarct size and preserving endothelial function. Post-con may be clinically applicable in coronary interventions, coronary artery bypass surgery, organ transplantation, and peripheral revascularization where reperfusion injury is expressed.     

Effects of 5-hydroxydecanoate and ischemic preconditioning on the ischemic-reperfused heart of fed and fasted rats

This investigation aimed to assess whether the mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) could abolish the protection conferred by fasting and ischemic preconditioning (IPC) and to ascertain whether these effects are associated with glycogen breakdown and glycolytic activity. Langendorff perfused hearts of fed and 24-h fasted rats were exposed to 25 min ischemia plus 30 min reperfusion. IPC was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. 5-HD (100 microM) perfusion begun 5 min before IPC or 13 min before sustained ischemia in the non preconditioned groups. Fasting improved the reperfusion recovery of contraction, decreased the contracture and the lactate production, increased glycogenolysis and did not affect the percentage of viable tissue. 5-HD abolished the effects of fasting on the contractile recovery but did not affect the contracture. 5-HD decreased the lactate production in the fed group, increased the preischemic glycogen content in both nutritional groups and did not affect the ischemic glycogen fall. IPC improved the contractile function but prevented the contracture only in the fed group, reduced lactate accumulation and glycogenolysis and evoked an increase of the viable tissue. 5-HD abolished the effects of IPC on the contractile recovery and did not affect its effect on the contracture, lactate production, glycogenolysis and viable tissue. These data suggest that the mitocondrial ATP-sensitive potassium channel is involved in the effects of fasting and IPC on the contractile function but the other cardioprotective and metabolic effects appear evoked through other mechanisms. Also suggest that besides the inhibition of the mitochondrial potassium channel, other mechanisms mediate the effects of 5-HD.     

Neutrophil adhesion to xenogeneic endothelium via iC3b

Neutrophils are thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. We asked whether the interactions of human polymorphonuclear leucocytes (PMN) with xenogeneic endothelium might be promoted by the binding of natural anti-endothelial antibodies and complement by using porcine aortic endothelial cells (PAEC), human serum, and human PMN in an in vitro model of hyperacute rejection. Pretreatment of PAEC with 10% human serum followed by washing markedly increased PMN adhesion from 15.7 +/- 1.8% to 62.5 +/- 3.6% (p less than 0.001). Complement and anti-endothelial antibodies were necessary for the increase, because heat-inactivated serum or serum depleted of IgM did not significantly increase PMN adhesion to treated endothelium. The induction of increased PMN adhesion to PAEC by human serum was observed within 1 min. The essential role of complement was defined using complement-depleted serum. Ten percent C2-deficient serum did not increase PMN adhesion whereas 10% C5-depleted or 10% C8-depleted serum caused the same increase in PMN adhesion as observed with normal human serum. These results suggested that C3 might play a critical role in enhanced neutrophil adhesion. In fact, PAEC treated with 10% human serum for 15 min and incubated with an F(ab')2 antihuman C3 for 10 min completely abolished the enhanced adhesion. PAEC treated with 10% human serum or C5-depleted serum displayed fluorescence of iC3b whereas monolayers treated with heat-inactivated serum or C2-deficient serum were non-reactive. The enhanced PMN adhesion to serum-treated PAEC was mediated through neutrophil receptors binding iC3b because mAb directed against CD11b/CD18 inhibited the serum-enhanced adhesion of PMN. We conclude that PMN adhesion to endothelium can be significantly enhanced by the endothelial deposition of iC3b.     

Mitochondrial and cytoplasmic aspartate aminotransferase enzyme release in the calcium paradox

The release of mitochondrial and cytoplasmic aspartate aminotransferase (AST) enzymes from the myocardium was studied in the isolated rabbit heart under conditions of the calcium paradox. Four different periods of calcium-free perfusion for 10, 15, 20, and 25 min were selected to produce different degrees of the calcium paradox and the associated myocardial damage which was indicated by impairment in the left ventricular contractile function. Calcium-free perfusion periods of less than 20 min were associated with partial recovery of ventricular function, while periods of 20 min or greater were associated with little or no recovery of contraction after reperfusion with calcium. Mitochondrial (ASTm) and cytoplasmic (ASTc) aspartate aminotransferase were released from the heart beginning within 1 min of reintroduction of Ca2+. The cumulative amount of ASTm release was about one-tenth the amount of ASTc release. The cumulative amount of ASTm and ASTc released were significantly (p less than 0.05) related to the duration of calcium-free perfusion. The time to 90% of maximum AST release was slightly longer for ASTm compared with ASTc (6.8 +/- 0.6 vs. 5.7 +/- 0.5 min, 0.10 greater than p greater than 0.05). ASTc but not ASTm correlated significantly (p less than 0.05) with total protein release from the myocardium, while ASTm was not as consistently related to protein loss. The cumulative amount of ASTm and ASTc were inversely related to the extent of recovery of left ventricular contractile function. Disparities did occur as the longest duration of the calcium-free period, which did not produce any further damage to left ventricular function, was nonetheless associated with more enzyme release from the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.