Cardiovascular actions of adenosine. Granulocyte and blood platelet adhesion in the reperfused myocardium.
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Adenosine↗
Biomedical subjects
C Kupatt
Publications and source records attributed to C Kupatt.
The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method.
Most comparative studies on neutrophil (PMN) isolation techniques have shown either activation or functional impairment of the cells due to the different separation processes. We have established a preparation method for PMN from human whole blood employing iron tagged, magnetizable antibodies against the cell surface antigen CD15. The aim of our study was to test whether this magnetic separation (MACS) alters cellular functions of PMN in comparison to a conventional density gradient technique (Percoll). The purity, cell yield, and pre-activation of the cells were evaluated. The latter was assessed by quantifying the expression of the integrin CD11b using flow cytometry. Furthermore, as functional tests, cell morphology and the oxidative burst reaction were investigated. We have shown that the use of 'magnetic' antibodies leads to highly purified PMN (> 99% of isolated leukocytes), while there is still contamination by eosinophils (about 6%) after Percoll separation. Platelet contamination was about the same in both procedures (approx. one platelet per two PMN). The basal expression of CD11b and, hence, neutrophil activation, was significantly lower and the upregulation of CD11b in response to FMLP was more pronounced after magnetic separation, as compared to density gradient centrifugation. The MACS technique did not lead to polarisation of PMN, nor did it affect the oxidative burst. This study suggests that magnetic separation is a simple, time-saving technique, yielding highly purified and functionally intact PMN.
Centrifugation, Density Gradient↗
Cardioprotection by cyclosporine A in experimental ischemia and reperfusion--evidence for a nitric oxide-dependent mechanism mediated by endothelin.
The acute effect of cyclosporine A (CSA) on myocardial function after ischemia and reperfusion and the mechanism of action was investigated in isolated working guinea-pig hearts. Myocardial function was experimentally infringed by imposing short-term global ischemia and reperfusion (15 min each). External heart work (EHW), determined before and after ischemia, served as the criterion for quantitation of recovery. Control hearts were perfused with modified Krebs-Henseleit buffer, other hearts received buffer supplemented with CsA +/- an endothelin receptor antagonist or exogenous endothelin +/- an inhibitor of nitric oxide (NO) synthesis. To assess the importance of endothelial production of mediators directly, NO release in coronary effluent (continuously measured with an amperometric sensor) and release of 6-keto-prostaglandin F1, (6-keto-PGFb), a stable metabolite of prostacyclin (PGI2), were determined in non-working. Langendorff hearts. Oxidative stress during reperfusion was assessed by measuring glutathione release in coronary venous effluent. Cyclosporine A (0.8 microM) improved post-ischemic function significantly (59% recovery of EHW nu 31% for controls). At 0.08 microM. CsA was without beneficial effect (30% recovery). The endothelin (ET)A- and ETB-receptor antagonist bosentan inhibited the protective action of 0.8 microM CsA (32% recovery). Exogenous ET-1 (80 pM) improved recovery to 53%, an effect which was blocked by the inhibitor of NO-synthase, NG-nitro-L-arginine (NOLAG. 1 microM. 31% recovery. In the control group, post-ischemic NO release in coronary effluent recovered from zero to about 100% of the pre-ischemic value by 10 min. but then decreased rapidly during the subsequent 15 min of reperfusion. In hearts treated with 0.8 microM CsA, NO release stayed at 100% of the pre-ischemic value throughout reperfusion, the difference between controls and CsA-treated hearts being significant after 20 min of reperfusion. On the other hand, coronary venous release of 6-keto-PGF1a was not different between the groups. Release of glutathione during early reperfusion first 5 min) was significantly lowered (P < 0.05) to about 50% in CsA (0.8 microMI- and ET-I-treated compared with controls (8.8 nmol/min). Cyclosporine A acts as a cardioprotective agent in our model of ischemia and reperfusion, presumably by elevating the level of endogenous nitric oxide and thereby reducing oxidative stress.
6-Ketoprostaglandin F1 alpha↗
Nitric oxide attenuates reoxygenation-induced ICAM-1 expression in coronary microvascular endothelium: role of NFkappaB.
Enhanced leukocyte adhesion has been shown to occur in post-ischemic reperfused hearts due to the upregulation of specific cell-surface adhesion molecules. Therefore, we investigated the influence of 4 h of reoxygenation after 20 h of hypoxia on ICAM-1 induction in primary cultures of rat coronary microvascular endothelial cells (CMEC). ICAM-1 surface expression as well as oxygen free radical formation were measured by flow cytometry. Changes in ICAM-1 mRNA levels were assessed by Northern blot and activation of NFkappaB and AP-1 signalling were analysed by electrophoretic mobility shift assays (EMSA) in CMEC lysates. Although hypoxia alone did not affect cell-surface ICAM-1 expression, 4 h of reoxygenation induced a significant upregulation of ICAM-1. ICAM-1 mRNA could not be found after hypoxia alone, but could be detected as early as 1 h following reoxygenation. Unlike AP-1, the activation of which could be detected in CMEC lysates following hypoxia alone, NFkappaB binding activity was induced only following reoxygenation, concurrent with an increase in the formation of reactive oxygen species (ROS). A proteasome inhibitor, nor-Leu (25 microM) inhibited NFkappaB activation by reoxygenation and ICAM-1 expression. Blockade of endogenous nitric oxide (NO) synthesis in CMEC with L-nitroarginine (10 microM) accentuated post-reoxygenation ICAM-1 expression. Finally, an exogenous NO donor, S-nitrosoacetyl-penicillamine (SNAP, 100 microM), suppressed the generation of ROS upon reoxygenation, and blocked the activation of NFkappaB and the upregulation of ICAM-1. Thus, ICAM-1 upregulation in CMEC primary cultures is not induced by hypoxia alone, but appears shortly after reoxygenation in the absence of exogenous cytokines or inflammatory cells. Because upregulation of AP-1 through hypoxia alone did not affect ICAM-1 expression, we conclude that redox-sensitive NFkappaB activation triggers ICAM-1 upregulation. NO inhibits reoxygenation-specific ICAM-1 upregulation, most likely by diminishing oxidative stress that leads to NFkappaB activation.
Animals↗
Effects of ACE-inhibition on redox status and expression of P-selectin of endothelial cells subjected to oxidative stress.
Redox stress during post-ischemic reperfusion may be the prime signal for processes leading to myocardial remodelling and hypertrophy. Nitric oxide (NO) is antioxidative, antiadhesive for neutrophils (PMN) and antiproliferative. Thus, enhancing endothelial production of NO, e.g. by inhibiting breakdown of endogenous bradykinin via angiotensin converting enzyme (ACE), could be beneficial. The effect of cilazaprilat (CILA, 10 micro M), an ACE inhibitor, on redox status, expression of the adhesion molecule P-selectin, and PMN adhesion under conditions of oxidative stress was investigated in cultured human umbilical vein endothelial cells (HUVECs). Incubation of the cells with H2O2 (0.1 and 1 mm) for 15 min served as oxidative stimulus. The intra- and extracellular concentrations of reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC as indicators of endothelial redox status. Expression of P-selectin was measured by flow cytometry. Furthermore, firm leukocyte adhesion to HUVECs was assessed. In controls, the intracellular ratio GSH/GSSG averaged 47 and dropped to 30 after incubation with 0.1 mm H2O2. The ratio declined to 6.5 with 1 mm H2O2. CILA blocked the effects of 0.1 mm H2O2, but was ineffective against 1 mm peroxide. The extracellular ratio did not discriminate between 0.1 and 1 mm H2O2, falling from 18 to 1 in both situations. P-selectin expression rose from 100% (control) to 146% after 1 mm H2O2 without CILA, but only to 114% in the presence of CILA. PMN adhesion was enhanced from about 1600 PMN per microwell (control) to 4300/well by 1 mm H2O2. CILA had no significant effect on adhesion (3900 PMN/well). Exposure of HUVECs to 0.1 mm H2O2 affected neither P-selectin expression nor PMN adhesion. Consequently, ACE inhibition can mitigate mild (0.1 mm H2O2) but not more severe redox stress in HUVECs. Irrespectively, CILA reduced the upregulation of P-selectin at the higher H2O2 concentration, indicating that this process is regulated independently of the cellular redox status. The firm adhesion of PMN to HUVECs was independent of P-selectin expression.
Angiotensin-Converting Enzyme Inhibitors↗
Retention of leucocytes in reperfused, isolated hearts does not cause haemodynamically relevant permanent capillary plugging.
Effects of microspheres (5 microns or 10 microns diameter) and polymorphonuclear leucocytes (PMN) on coronary resistance were compared in beating, non-working isolated guinea-pig hearts (Langendorff preparation). The hearts were buffer perfused (5 ml/min, constant flow) and particles or cells were infused into the coronary system as a bolus (1 ml, 1 min). Coronary perfusion pressure, coronary flow and formation of epicardial transudate were measured before and after bolus administration. Coronary resistance was estimated from these parameters. Retention of particles or cells was monitored by quantifying the numbers emerging in the coronary effluent in relation to the number administered. The effects of PMN were also studied after 15 min of global ischemia. Coronary resistance correlated with the number of 10-micron particles infused, which were almost quantitatively retained. In contrast, 5-micron beads had no such effect and were not retained in the coronary system. Though considerable numbers of PMN were retained in the hearts (about 21% under control conditions and 35% after ischaemia), coronary resistance was not increased in either case. Blockage of the CD18 adhesion complex by monoclonal antibodies lowered basal retention to 11% and completely prevented the elevation of retention by ischaemia. We conclude that, in this experimental model, PMN, permanently retained in the hearts under normal flow conditions and especially after brief ischaemia, do not cause acute, haemodynamically relevant capillary plugging, but adhere to postcapillary venules via CD18.
Animals↗
ACE-inhibition prevents postischemic coronary leukocyte adhesion and leukocyte-dependent reperfusion injury.
OBJECTIVE: Polymorphonuclear leukocytes (PMN), retained in the microvascular bed, can contribute to postischemic myocardial reperfusion injury. Since a beneficial effect of ACE-inhibition on reperfusion injury has been reported, we investigated the impact of cilazaprilat on PMN dependent reperfusion injury in isolated guinea pig hearts. METHODS: Hearts (n = 5 per group) were subjected to 15 min of ischemia. Immediately thereafter, a bolus of PMN was injected into the coronary system. External heart work (EHW) and total cardiac nitric oxide release were measured. For microscopic evaluation, hearts received rhodamine 6G labelled PMN after ischemia, were arrested 5 min later and further perfused with FITC dextran (0.1%). Localization of retained PMN was assessed by fluorescence microscopy. Leukocyte activation was studied by FACS analysis of the adhesion molecule CD11b before and after coronary passage of the PMN. The ACE-inhibitor cilazaprilat (Cila, 2 microM) and the NO-synthase inhibitor nitro-L-arginine (NOLAG, 10 microM) were used to modulate nitric oxide formation of the heart. RESULTS: Postischemic EHW recovered to 67 +/- 5% (controls) and 64 +/- 6% (Cila) of the preischemic value. Addition of PMN severely depressed recovery of EHW (39 +/- 2%) and NO release (39 +/- 6% of the preischemic value). Simultaneously, ischemia led to a substantial increase in postcapillary PMN adhesion (from 21 +/- 5 to 172 +/- 27 PMN/mm2 surface) and CD11b-expression of the recovered PMN (3-fold). Cila attenuated postischemic PMN adhesion (83 +/- 52 PMN/mm2) and activation of PMN, whereas it improved recovery of work performance (64 +/- 4%) and NO release (65 +/- 4%) in the presence of PMN. Conversely, NOLAG increased PMN adhesion (284 +/- 40 PMN/mm2) and myocardial injury. We conclude that ACE-inhibition prevents leukocyte dependent reperfusion injury mainly by inhibition of postcapillary leukocyte adhesion. The effect may be mediated by NO, given the proadhesive effect of NOLAG.
Analysis of Variance↗
Nitric oxide mitigates leukocyte adhesion and vascular leak after myocardial ischemia.
Tissue edema is a facet of ischemia/reperfusion injury in many organs, polymorphonuclear leukocytes (PMN) presumably playing a contributory role. We studied the intracoronary adhesion of PMN and its effect on vascular permeability during reperfusion in isolated guinea-pig hearts. After a global ischemia of 15 min duration. PMN (10(7)) were infused into the coronary system during the first minute of reperfusion. PMN adhesion was measured as difference of applied PMN and those recovered in the effluent perfusate. Coronary permeability was assessed by measuring the rate of transudate formation (TF) on the epicardial surface, before as well as 5, 15 and 30 min after ischemia. Experiments were also performed in the presence of the NO-synthase inhibitor nitro-L-arginine (10 microM) and the ACE-inhibitor ramiprilat (2 microM), the latter known to enhance endogenous nitric oxide formation. Furthermore, the radical scavenger uric acid (0.5 mM) was applied either before and during ischemia or starting after PMN application. Ischemia/reperfusion increased coronary PMN adherence from 23 +/- 1% (basal) to 33 +/- 2%. Whereas ischemia alone did not influence TF (about 100 microliters/min during reperfusion), postischemic PMN infusion led to progressive TF. With nitro-L-arginine, PMN adhesion rose to 45 +/- 3%; TF increased to 212 +/- 30 microliters/min. In contrast, ramiprilat caused post-ischemic adhesion and TF to decline to basal values. In the presence of uric acid (UA) PMN adhesion declined to 26 +/- 2%, however, the subsequent increase in TF after withdrawal of UA was not markedly attenuated. On the other hand, infusion of UA after application of PMN caused a significant decrease of TF. The extracellular antioxidants SOD/catalase were without effect. As shown using luminol enhanced chemiluminescence. No was able to scavenge oxygen free radicals released by activated PMN. These findings indicate that enhanced PMN adhesion in reperfusion leads to an increase in coronary permeability. Scavenging of oxygen free radicals with NO or UA appears to mitigate both, postischemic PMN adhesion and PMN-induced vascular injury, even after adhesion.
Angiotensin-Converting Enzyme Inhibitors↗
[Interaction of adenosine with leukocytes and thrombocytes].
Platelets and polymorphonuclear granulocytes (PMN) contribute to post-ischemic myocardial reperfusion damage. However, to elicit any deleterious actions, they first need to become adherent to the vascular endothelium. Numerous studies have documented an A2-receptor mediated platelet-stabilizing action of adenosine and an A2-dependent antiinflammatory effect on PMN themselves. Intriguingly, an A1-receptor mediated chemotactic action of adenosine on isolated PMN has also been reported. A1-receptors are more sensitive towards adenosine than A2-receptors, and interactions between platelets and leukocytes could alter the net-adhesive potential. Furthermore, the endothelial cells also express adenosine A1- and A2-receptors. In the situation of ischemia and reperfusion both, the intracoronary concentration of adenosine and the shear forces, vary with time. We have, therefore, investigated the influence of adenosine on intracoronary adhesion of PMN and platelets, applied to isolated heart preparations (guinea pig), both separately and in combination, and determined the resultant effect on postischemic myocardial pump function. At submicromolar adenosine concentrations, as found after brief ischemia (15 min stopped-flow or 30 min low-flow), adenosine enhanced intracoronary PMN retention by preferentially stimulating endothelial A1-receptors. The effect required the intermediate formation of platelet activating factor (PAF) and occurred via CD11/CD18 adhesion molecules on the PMN. Higher, i.e., micromolar levels of adenosine, in contrast, inhibited PMN adhesion via an A2-receptor dependent mechanism. Thrombin-induced platelet adhesion was inhibited by adenosine at high shear rates by both A1- and A2-receptor dependent mechanisms. However, adenosine was not protective at low shear rates, or at high flow in the presence of PMN. Pertinently, adhesion of either PMN or platelets, alone or in combination, regularly caused deterioration of post-ischemic myocardial function. Thus, depending on its concentration and on the phase of ischemia/reperfusion, adenosine may elicit cardioprotective or detrimental effects in the reperfused myocardium, which makes general prognosis of its role in such situations difficult. However, in the course of every reperfusion, the adenosine levels will inevitably fall into the proadhesive range. Thus, prophylactic inhibition of A1-receptor effects may be beneficial.
Adenosine↗
[Organ transplantation and human dignity. Editorial].
Modern medicine has succeeded in achieving enormous technical developments. One recent highlight has been the introduction of postmortem organ transplantation. At the same time, serious objections have been raised concerning the radical changes in the cultural conception of the inviolable body. One major objection arises from the conflict of considering a brain-dead person as dead. The presence of brain death is a prerequisite for post-mortem organ donation, because only during this phase of dying does the individual quality as dead while the organs, other than the brain, remain viable. The objection implies scepticism as to the physician's ability to distinguish a dead from a living person. On the other hand, even the critics must rely on the physician's ability to discriminate, e.g., when to discontinue resuscitation. The medical community has not found reasons to restrict the definition of irreversible coma 25 years after its first formulation. It must be clearly recognised that reasons other than medical ones can be decisive for refusing organ donation. One ethical problem is the therapeutic benefit of organ transplantation. The beneficiary of the treatment is not the donor, but another person, the recipient. The concept of human dignity does not allow the use of a person for purposes other than the ones he/she consents to, as Immanual Kant stated. Although the human corpse is not a person in the full sense, even if it is protected by the thought of respect for the former person, the life-interest of the organ recipient had to be considered legitimate.(ABSTRACT TRUNCATED AT 250 WORDS)
Brain Death↗