Segmental microvascular permeability in ischemia-reperfusion injury in rat lung.

Segmental microvascular permeabilities were measured using pre- and postalveolar vessel capillary filtration coefficient (Kfc) values (ml. min-1. cmH2O-1. 100 g-1) in isolated rat lungs subjected to ischemia-reperfusion (I/R). Total Kfc values measured in flowing and nonflowing lungs were highly correlated (r = 0.98, P < 0.0001). Kfc values were then measured in another group of lungs under no-flow conditions when airway pressure was increased to 20 cmH2O and either the arterial or venous pressure was elevated to 7-8 cmH2O to measure the prealveolar and postalveolar Kfc values. Control total and postalveolar Kfc values were 0.0225 +/- 0.001 and 0.0219 +/- 0.001 ml. min-1. cmH2O-1. 100 g-1, respectively, and the prealveolar permeability was extremely small (0.00003 +/- 0.00005 ml. min-1. cmH2O-1. 100 g-1). Kfc values were again made in nonflowing lungs that had been subjected to 45 min of ischemia followed by 30 min of reperfusion. After I/R, the total membrane Kfc increased 10-fold to 0.2597 +/- 0.006 ml. min-1. cmH2O-1. 100 g-1, the prealveolar Kfc increased to 0.0677 +/- 0.003 ml. min-1. cmH2O-1. 100 g-1, and the postalveolar Kfc increased to 0.1354 +/- 0.008 ml. min-1. cmH2O-1. 100 g-1 (P < 0.05 for all I/R values). These data indicate that normal solvent microvascular permeability was predominantly postalveolar, and after I/R damage, the postalveolar (venular) permeability comprised 52% of the total, whereas the prealveolar and alveolar vessels comprised only 27 and 23%, respectively, of the total Kfc.

Tumor necrosis factor-alpha in ischemia and reperfusion injury in rat lungs.

The effects of both recombinant rat tumor necrosis factor-alpha (TNF-alpha) and an anti-TNF-alpha antibody were studied in isolated buffer-perfused rat lungs subjected to either 45 min of nonventilated [ischemia-reperfusion (I/R)] or air-ventilated (V/R) ischemia followed by 90 min of reperfusion and ventilation. In the I/R group, the vascular permeability, as measured by the filtration coefficient (Kfc), increased three- and fivefold above baseline after 30 and 90 min of reperfusion, respectively (P < 0.001). Over the same time intervals, the Kfc for the V/R group increased five- and tenfold above baseline values, respectively (P < 0.001). TNF-alpha measured in the perfusates of both ischemic models significantly increased after 30 min of reperfusion. Recombinant rat TNF-alpha (50,000 U), placed into perfusate after baseline measurements, produced no measurable change in microvascular permeability in control lungs perfused over the same time period (135 min), but I/R injury was significantly enhanced in the presence of TNF-alpha. An anti-TNF-alpha antibody (10 mg/rat) injected intraperitoneally into rats 2 h before the lung was isolated prevented the microvascular damage in lungs exposed to both I/R and V/R (P < 0.001). These results indicate that TNF-alpha is an essential component at the cascade of events that cause lung endothelial injury in short-term I/R and V/R models of lung ischemia.

Vasoconstriction increases pulmonary nitric oxide synthesis and circulating cyclic GMP.

Vascular shear stress increases when blood flow or blood viscosity increases or when vessel diameter decreases. In the systemic circulation, shear stress is a potent stimulus for endothelial nitric oxide synthesis. We studied isolated rat lungs to determine whether increasing shear stress increases nitric oxide synthesis in the pulmonary circulation. Lungs were given the vasoconstrictor, U46619 (a thromboxane analogue), and perfused at constant flow rates or at constant pressure, since constant pressure perfusion minimizes changes in shear stress with vasoconstriction. The subsequent effect of the NOS inhibitor, N omega-methyl-L-arginine (LMA), or the soluble guanylyl cyclase inhibitor, 6-anilino-5,8-quinolinodione (LY83583) was assessed. Changes in pulmonary vascular resistance (PVR), pulmonary vascular compliance, and perfusate cyclic GMP concentration were measured as indicators of nitric oxide synthesis. The effect of the cyclic GMP-specific (type V) phosphodiesterase inhibitor, zaprinast, on perfusate cyclic GMP concentrations was also examined. An infusion of U46619 consistently increased PVR and decreased compliance. LMA and LY83583 also increased PVR in U46619-treated lungs perfused at constant flow rates, primarily by increasing precapillary resistance. LMA had no effect in U46619-treated lungs perfused at constant pressure. Perfusate cyclic GMP concentrations increased significantly after U46619 in lungs perfused at constant flow rates, but cyclic GMP levels did not change after U46619 in lungs perfused at constant pressure. Zaprinast also increased perfusate cyclic GMP, demonstrating that increases in intracellular cyclic GMP are reflected in circulating cyclic GMP concentrations. We conclude that vasoconstriction with U46619 increases nitric oxide synthesis in isolated rat lungs. Lungs perfused at constant pressure respond differently to NOS inhibitors compared to those perfused at constant flow, suggesting that shear stress may increase nitric oxide synthesis in the lung. Perfusate concentrations of cyclic GMP reflect activation of soluble guanylyl cyclase in this model.

Role of nitric oxide in lung ischemia and reperfusion injury.

We studied the effects of nitric oxide synthase (NOS) inhibitors and nitric oxide (NO.) donors on ischemia-reperfusion (I/R)-induced microvascular permeability increase in isolated buffer-perfused rat lungs. Microvascular permeability (Kf,c) was significantly increased in lungs subjected to 45 min of ischemia followed by 30 min of reperfusion. Lungs that were pretreated with 300 and 600 microM N omega-nitro-L-arginine methyl ester (L-NAME), 1, 300, and 600 microM NG-monomethyl-L-arginine (L-NMMA), or 600 microM L-N6-(1-iminoethyl) ornithine (L-NIO) still showed significant increases in Kf,c after I/R. Lungs that were pretreated with 5 mM L-NAME or 5 mM N omega-nitro-D-arginine methyl ester showed no increase in Kf,c after I/R. However, both compounds at these concentrations produced significant decreases in perfusate pH. The decreased pH was responsible for the protective effects, since lungs pretreated with 5 mM L-NAME and supplemented with NaHCO3 to prevent the perfusate pH decrease still showed a significant elevation in Kf,c after I/R. In additional experiments, NO.donors were administered to isolated lungs at the onset of reperfusion. Spermine-NO (100 microM) and S-nitroso-N-acetylpenacillamine (300 microM) both prevented the increase in Kf,c associated with I/R. We conclude from these studies that peroxynitrite does not mediate microvascular permeability increase after lung I/R injury in this model, and exogenous NO. does not exacerbate injury; rather, it prevents microvascular damage.

Role of calmodulin and myosin light-chain kinase in lung ischemia-reperfusion injury.

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.

Blocked ETA receptors prevent ischemia and reperfusion injury in rat lungs.

The effects of endothelin (ET)-A (ETA)- and ETB-receptor agonist and antagonists were studied in isolated buffer-perfused rat lungs subjected to 45 min of ischemia followed by 105 min of reperfusion (I/R). For the I/R group after 30 and 90 min of reperfusion, the Kfc had increased three- and fivefold above control values, respectively (P < 0.01), and the number of circulating neutrophils in the perfusate decreased by 65 +/- 7.65%. Both an ETA-receptor antagonist (BQ-610) and an ETAB-receptor antagonist (PD-156707-0015) given before the ischemic period protected the lung endothelial barrier from injury associated with I/R. Also, these compounds attenuated the I/R-induced neutrophil accumulation in the lung (31.94 +/- 4.16 and 34.38 +/- 1.05%, respectively; P < 0.01 compared with I/R). Neither an ETB-receptor agonist (IRL-1620) nor an ETB-receptor antagonist (IRL-1038) affected the I/R-induced endothelial injury. In addition, they did not alter the number of circulating polymorphonuclear cells during I/R. ET-1 administration alone caused a dose-dependent increase in pulmonary arterial pressure, but no measurable increase in microvascular permeability occurred. We conclude that ET-1 is involved in I/R-induced lung endothelial injury and speculate that it acts in concert with some other coactivator(s), most likely platelet-activating factor, through ETA receptors. This mechanism requires polymorphonuclear leukocyte activation with subsequent release of oxygen radicals and/or expression of adhesive molecules on the neutrophil surface.

Endothelial damage caused by ischemia and reperfusion and different ventilatory strategies in the lung.

Not all possible mediators of lung I/R injury that have been studied, such as cyclooxygenase and lipoxygenase products, have been presented in this review, but it is very clear that oxygen free radicals are the primary mediators of the damage, regardless of their origin. Oxygen radicals are generated by neutrophils, which are sequestered and activated in the ischemic-reperfused pulmonary tissue, and by xanthine oxidase, which is upregulated by ischemia and/or activated neutrophils. The contributions to lung injury by different species of oxygen radicals may very depending upon the lung model used to study I/R. Also, nitric oxide may be injurious or protective in lung I/R injury, depending upon some critical alveolar PO2 level present either during ischemia or at reperfusion. I/R-induced lung microvascular injury ultimately depends upon some balance between lung metabolic stress, the extent of the I/R-induced inflammatory response, endogenous antioxidant levels, and the timing, magnitude, and duration of oxygen free radical generation during both periods of ischemia and reperfusion. The final common pathway causing microvascular permeability to increase after lung I/R is the activation of the endothelial cell's contractile machinery. Particularly, endothelial contraction may occur in a MLCK-dependent fashion. Endothelial contraction may also be related to an intracellular Ca++ increase and subsequent calmodulin activation. The initiating event causing increased intracellular Ca++ is not known, but may be due to endothelial cell/leukocyte interactions, oxygen radical-mediated Ca++ transients, mobilization of intracellular Ca++ pools by various second messengers, or stimulation of Ca++ influx secondarily to changes in the activity of membrane ion pumps such as the Na+/H+ antiport. Increasing cAMP levels in the postischemic lung can prevent and actually reverse I/R-induced microvascular injury, by affecting MLCK, the endothelial cell cytoskeleton, and/or the function of sequestered leukocytes. Also, cAMP elevation aids the resolution of pulmonary edema by facilitating capillary fluid reabsorption. Whatever the mechanism, elevation of cAMP in the setting of lung I/R injury represents a potentially useful therapy for improving early lung function following lung transplantation. Finally, additional studies are necessary to elucidate the complete mechanisms responsible for producing microvascular injury during lung I/R. Specifically, a better understanding of the relationships between the many factors required to produce lung damage is needed. Many interventions into the lung I/R process provide protection against microvascular injury, suggesting that regulation of the endothelial barrier permeability to fluid, protein, and leukocytes is accomplished by several redundant systems. This situation may be similar to mechanisms reported to regulate the immune response mediated by T cells (62a), where T cell activation depends upon multiple signal inputs for the full immune response to occur. Thus, multiple signals in a correct sequence delivered to the endothelium may be necessary to produce the microvascular injury associated with lung ischemia and reperfusion.

Restoration of normal pH triggers ischemia-reperfusion injury in lung by Na+/H+ exchange activation.

The effects of acidotic extracellular pH and Na+/H+ exchange inhibition on ischemia-reperfusion (I/R)-induced microvascular injury were studied in the isolated, buffer-perfused rat lung. When lungs were subjected to 45 min of ischemia followed by 30 min of reperfusion, the capillary filtration coefficient (Kfc) increased significantly, resulting in a change in Kfc (delta Kfc) of 0.360 +/- 0.09 ml.min-1.cmH2O-1.100 g-1. Addition of hydrochloric acid to the perfusate before ischemia at a concentration sufficient to reduce perfusate pH from 7.38 +/- 0.03 to 7.09 +/- 0.04 completely prevented the increase in Kfc associated with I/R (delta Kfc = 0.014 +/- 0.034 ml.min-1.cmH2O-1.100 g-1). Addition of a Na+/H+ exchange inhibitor, 5-(N,N-dimethyl)-amiloride, to the perfusate either before ischemia or at reperfusion also prevented the I/R-induced permeability increase (delta Kfc = 0.01 +/- 0.02 and -0.001 +/- 0.02 ml.min-1.cmH2O-1.100 g-1, respectively). We conclude that restoration of flow at physiological pH to the postischemic lung activates the Na+/H+ exchange system, which may represent the "triggering mechanism" responsible for initiating reperfusion-induced microvascular injury.

ATP-sensitive K+ channels are not involved in ischemia-reperfusion lung endothelial injury.

The role of ATP-sensitive K+ channels (KATP) in ischemia and reperfusion (I/R) was studied in isolated rat lungs. I/R produced a sixfold increase in endothelial permeability as measured by the capillary filtration coefficient. Cromakalim (10 microM) given at 46 min after reperfusion reversed the filtration coefficient increase. This effect was not blocked by either a protein kinase A inhibitor (adenosine-3',5'-cyclic monophosphothioate; 100 microM) or an adenosine antagonist [8-(p-sulfophenyl)-theophylline; 20 microM]. Cromakalim given before ischemia or at the beginning of reperfusion protected the endothelial barrier from injury. Glibenclamide (500 microM) given before the ischemic period, at the beginning of reperfusion, or 46 min after reperfusion did not alter the changes in microvascular permeability produced by I/R. Glibenclamide blocked the ability of cromakalim to reverse endothelial damage but not the ability of either isoproterenol (10 microM) or an adenosine A2-receptor agonist, CGS-21680 (300 nM). We conclude that opening of KATP channels does not produce endothelial injury in I/R. The activation of KATP channels can both protect against and reverse the endothelial damage associated with I/R. This novel mechanism(s) is independent from known pathways that employ cAMP-protein kinase system and adenosine.

Platelet-activating factor increases vascular resistance in rat hindquarters by thromboxane A2.

The effects of platelet-activating factor (PAF) on vascular resistance and capillary permeability were studied in the isolated rat hindquarter. Six groups were studied (n = 30): control; PAF alone (1.4 microM); and PAF (1.4 microM) pretreated with ibuprofen (30 mg/kg), thromboxane A2 (TxA2)-receptor antagonist (BM-13505, 2 mg/kg), PAF-receptor antagonist (WEB-2086, 5 mg/kg), or dexamethasone (5 mg/kg). The vascular resistance was calculated, and the reflection coefficient (sigma) was determined as an index of capillary permeability. Exogenous PAF caused a threefold increase in vascular resistance peaking at 5 min and a 2.5-fold increase in capillary permeability. The increased vascular resistance caused by PAF alone was significantly attenuated by ibuprofen, BM-13505, and dexamethasone. The PAF-induced permeability was neither attenuated by ibuprofen nor BM-13505. However, both the increased vascular resistance and permeability were blocked and attenuated by WEB-2086 and dexamethasone, respectively. We conclude that TxA2 mediates the PAF-induced increased vascular resistance; however, the increased vascular permeability is independent of the formation of TxA2 in the isolated hindquarter.

Muscarinic agonists and antagonists cause vasodilation in isolated rat lung.

The present study investigated the ability of atropine and different muscarinic receptor subtypes to affect acetylcholine (ACh)-induced bronchoconstriction and vasodilation in the isolated rat lung model. ACh (10(-7) M) given after U-46619 decreased total (RT), precapillary, and postcapillary vascular resistances and increased peak airway pressure. Atropine (20 microM) decreased RT and precapillary and postcapillary vascular resistances and blocked ACh-induced increases in peak airway pressure. The M1-selective agonist McN-A-343 (1.3 x 10(-5) M) decreased RT from 40.27 +/- 2.98 to 29.20 +/- 2.81 cmH2O.l-1.min-100 g lung wt (P = 0.01), and ACh caused no further dilation. The M1-selective antagonist pirenzepine (1.6 x 10(-6) M) blocked ACh-induced vasodilation. The M2-selective antagonist gallamine (7.5 x 10(-7) M) decreased RT from 45.50 +/- 3.19 to 34.86 +/- 1.25 cmH2O.l-1.min.100 g lung wt (P < 0.05), and after gallamine, ACh further decreased RT to 28.59 +/- 1.75 cmH2O.l-1.min.100 g lung wt (P < 0.01). Neither the selective muscarinic agonists nor antagonists affected peak airway pressures. We conclude that ACh-induced vasodilation in isolated rat lungs preconstricted with U-46619 is mediated by M1 receptors. Atropine-induced vasodilation in this model is mediated through the inhibition of the M2 receptor. We postulate that this represents either a blockade of postganglionic receptors, permitting release of vasodilator substances from local nerve terminals, or a direct vasodilatory effect on the vascular smooth muscle.

Adenosine A2 receptors reverse ischemia-reperfusion lung injury independent of beta-receptors.

To evaluate the adenosine systems ability to reverse the endothelial damage produced by ischemia and reperfusion (I/R), we studied several different selective adenosine-receptor agonists and antagonists, a protein kinase A inhibitor, and a beta-adrenoreceptor antagonist in isolated buffer-perfused rat lungs. I/R (45 min/105 min) produced a sixfold increase in endothelial permeability as measured by the capillary filtration coefficient. Both a selective A2-receptor agonist (CGS-21680, 300 nM) and a beta-receptor agonist (isoproterenol, 10 microM) reversed the increased microvascular permeability. A nonselective adenosine-receptor antagonist (SPT, 20 microM) and a selective A1-receptor antagonist (DPCPX, 10 nM) had no effect on increased microvascular permeability. Also, isoproterenol and CGS-21680 reversed the damage being introduced after a selective A1-receptor agonist (CCPA, 100 nM). The nonspecific adenosine A1- and A2-receptor agonist NECA (12 nM) appeared to desensitize the A2 receptors and a protein kinase A inhibitor, adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS, 100 microM), blocked the reversal of endothelial damage by isoproterenol or A2-receptor agonist. Propranolol (100 microM) blocked the effect of isoproterenol but not the effect of CGS-21680. From this study we conclude that A2-receptor activation reverses endothelial damage associated with I/R by a mechanism independent of beta-receptors or Gi protein. However, a protein kinase A-3',5',-cyclic adenosine monophosphate pathway is activated by both the adenosine systems and beta-receptor activation.

Vascular permeability and epithelial transport effects on lung edema formation in ischemia and reperfusion.

To determine the role of various Na+ transport systems in the edema fluid accumulation after ischemia and reperfusion in the lung, we evaluated the effect of amiloride (a Na+ channel blocker), ouabain (a Na(+)-K(+)-adenosinetriphosphatase blocker), and phloridzin (a Na(+)-glucose cotransport blocker) in isolated rat lungs. Ischemia and reperfusion (I/R) significantly increased the edema accumulation, with the wet-to-dry weight ratios increasing to 10.14 +/- 0.58 from 6.03 +/- 0.05 in control lungs (P < 0.04). Amiloride significantly augmented the amount of edema fluid (wet-to-dry weight ratio 12.26 +/- 0.77), and ouabain further increased the amount of edema (wet-to-dry weight ratio 18.58 +/- 1.00). Phloridzin did not significantly affect edema formation associated with I/R. Isoproterenol decreased the amount of edema formation in the presence and absence of amiloride. This occurred because the endothelial permeability as assessed by filtration coefficient was restored to normal values and less edema formed. The present study indicates that Na+ channels and Na(+)-K(+)-adenosinetriphosphatase, components of the active Na+ absorption transport system, are very important in opposing edema fluid accumulation in rat lungs subjected to I/R injury and operate as an edema safety factor. However, if the endothelial damage associated with I/R is allowed to persist, then the transport processes, even if operative, are insufficient to prevent continuous edema accumulation.

[Effect of adrenergic beta blockers on hemodynamics, diastolic relaxation and distensibility of left-ventricular myocardium in patients with ischemic heart disease]. / Vliianie adrenoblokatorov na gemodinamiku, diastolicheskoe rasslablenie i rastiazhimost' miokarda levogo zheludochka u bol'nykh ishemicheskoi bolezn'iu serdtsa.

Altogether 32 patients were investigated by catheterization of the left ventricular cavity with volume loading of 76% solution of verographin++ administered at 0.65 ml per kg body mass. beta-blockade with preliminary i.v. administration of 5 mg obsidan or 10 mg cordanum was shown to improve myocardial diastolic function as a result of improved myocardial distensibility in CHD patients.

[Effect of the complex treatment of stable stenocardia on the status of the prostacyclin-thromboxane system]. / Vliianie kompleksnogo lecheniia stabil'noi stenokardii na sostoianie prostatsiklin-tromboksanovoi sistemy.

Raised function of platelets with an increase in their aggregation activity in vitro and an increase in spontaneous intravascular activation as well as imbalance of the prostacyclin-thromboxane system with a shift to the predominance of proaggregation agents were detected in CHD patients after myocardial infarction suffering from stable angina. An inhibiting effect of beta-adrenergic receptor blocking agents and acetylsalicylic acid on the synthesis of thromboxane A2 was observed. No marked effect of dipyridamole on the prostacyclin-thromboxane system was noted.