Oxygen metabolites stimulate thromboxane production and vasoconstriction in isolated saline-perfused rabbit lungs.

Generation of reactive oxygen metabolites, thromboxane increases, and vasoconstriction have been implicated in the pathogenesis of acute edematous lung injury, such as that seen in patients with the Adult Respiratory Distress Syndrome (ARDS), but their interactions are unknown. We hypothesized that reactive O2 products would stimulate arachidonic acid metabolism in lungs and that vasoactive products of arachidonate, such as the potent vasoconstrictor thromboxane A2, might then mediate O2-metabolite-induced pulmonary vasoconstriction. We found that O2 metabolites generated by injection of purine plus xanthine oxidase caused increases in mean pulmonary artery perfusion pressures (27 +/- 4 mmHg) in isolated perfused lungs. In addition, purine plus xanthine oxidase also caused 30-fold increases in perfusate levels of thromboxane B2 (the stable metabolite of thromboxane A2) compared with only twofold increases in 6-keto-PGF1a (the stable metabolite of prostacyclin). Moreover, prior addition of catalase inhibited both vasoconstriction and the thromboxane B2 production seen in isolated lungs following injection of purine plus xanthine oxidase. Similarly, pretreatment with cyclooxygenase inhibitors, either aspirin or indomethacin, also completely blocked thromboxane generation and markedly attenuated pressor responses usually seen after purine plus xanthine oxidase (increase in mean pulmonary artery perfusion pressures, 4.4 +/- 1.5 mmHg). Furthermore, imidazole, a thromboxane synthetase inhibitor, also decreased O2-metabolite-induced thromboxane generation and vasoconstriction. These results suggested that thromboxane generation might participate in O2-metabolite-induced vasoconstriction. However, since a significant correlation between thromboxane levels and the degree of vasoconstriction could not be demonstrated, and since addition of superoxide dismutase reduced thromboxane generation but did not affect the intensity of vasoconstriction, it is possible that thromboxane is not the only vasoactive mediator in this model. We conclude that exposing lungs to O2 metabolites results in thromboxane generation and that thromboxane is a major mediator of oxidant-induced vasoconstriction.     

Human platelets modulate edema formation in isolated rabbit lungs.

The role of platelet glucose-6-phosphate dehydrogenase (G-6-PD) in mediating the effects of human platelets on oxidant-induced edema in the isolated perfused rabbit lung was investigated using dehydroepiandrosterone, a specific steroidal inhibitor of G-6-PD. Xanthine oxidase (0.003 and 0.012 U/ml) caused lung edema that was attenuated by coinfusion of washed human platelets. Platelets that were incubated with DEA to inhibit G-6-PD activity augmented xanthine oxidase-induced lung edema and pulmonary hypertension at both doses of xanthine oxidase. Infusion of papaverine to maintain stable pulmonary artery (PA) pressures, incubation of G-6-PD-inhibited platelets with acetylsalicylate, or infusion of a thromboxane-prostaglandin endoperoxide receptor site antagonist, SQ 29548, into the lung perfusate prevented augmentation of lung edema and the PA pressor response by G-6-PD-inhibited platelets. It was concluded that antioxidant-intact platelets attenuate oxidant-induced lung edema by preventing increased membrane permeability, and that G-6-PD-inhibited platelets augment lung edema through hydrostatic mechanisms mediated by release of platelet cyclooxygenase products.     

Acetyl glyceryl ether phosphorylcholine-stimulated human platelets cause pulmonary hypertension and edema in isolated rabbit lungs. Role of thromboxane A2.

Macrophages, neutrophils, and platelets may play a role in acute edematous lung injury, such as that seen in the adult respiratory distress syndrome (ARDS), but their potential actions and interactions are unclear. Because stimulated human macrophages and neutrophils can release acetyl glyceryl ether phosphorylcholine (AGEPC), a potent platelet activator, we hypothesized that in ARDS, leukocyte release of AGEPC might stimulate platelets to release thromboxane A2 (TXA2), which then produces pulmonary hypertension and lung edema. In support of this premise, we found that pulmonary hypertension and edema occurred in isolated rabbit lungs perfused with human platelets and AGEPC, but not with platelets or AGEPC alone. Infusion of a vasodilator (nitroglycerin) to maintain base-line pulmonary artery pressures in lungs perfused with platelets and AGEPC prevented the development of lung edema suggesting that platelet and AGEPC-induced edema was hydrostatic in nature. Additional experiments suggested that the increased pressure was a result of TXA2 release from platelets stimulated by AGEPC. Specifically, preincubation of platelets with imidazole, a thromboxane synthetase blocker, prior to infusion with AGEPC significantly diminished pulmonary hypertension and prevented lung edema. Furthermore, pretreating lung preparations with 13-azaprostanoic acid, a TXA2 antagonist, before infusion of AGEPC and untreated platelets also reduced the pulmonary hypertension and blocked the lung edema. The role of TXA2 was further suggested when perfusates from lungs infused with platelets and AGEPC developed high levels of TXA2, whereas perfusates from controls did not. These results suggest that platelet aggregation induced by AGEPC may contribute to ARDS by releasing TXA2, which raises microvascular pressure and increases edema formation, especially when an underlying permeability defect is present.     

Acetylcholine-induced contractions in isolated rabbit pulmonary arteries: role of thromboxane A2

Acetylcholine has been reported to produce vasodilation or vasoconstriction in the pulmonary circulation of different species. In rabbit lungs, acetylcholine is a potent vasoconstrictor. The present study was undertaken to examine the contractile effects of acetylcholine in arteries isolated from various regions of the rabbit pulmonary vascular bed and in thoracic aorta. Arteries isolated from within the lung were more responsive than extrapulmonary arteries (main pulmonary artery and aorta) to the contractile effects of acetylcholine. In vessels precontracted with norepinephrine, acetylcholine caused biphasic (relaxation-contraction) concentration-response curves. Atropine inhibited acetylcholine-induced contractions in all vessels, whereas pretreatment with cyclooxygenase or thromboxane synthetase inhibitors abolished contractile responses to acetylcholine only in intrapulmonary arteries. In accordance with these findings, acetylcholine caused a 3-fold increase in thromboxane A2 release from intrapulmonary arteries but not from extrapulmonary arteries. Inhibition of thromboxane synthetase abolished this effect of acetylcholine. Endothelium removal decreased contractile responses in intrapulmonary arteries but it did not decrease contractions in extrapulmonary arteries, suggesting that endothelium may contribute to acetylcholine-induced, thromboxane-mediated contractions in intrapulmonary arteries. Indomethacin did not inhibit contractile responses in endothelium-denuded main pulmonary artery or aorta but it abolished the weak contractile responses in intrapulmonary arteries without endothelium, indicating that arterial smooth muscle also was a source of contractile prostanoid biosynthesis enhanced by acetylcholine. These results demonstrate that acetylcholine contracts rabbit intrapulmonary arteries through generation of thromboxane A2 but that a different mechanism is responsible for mediating weaker acetylcholine-induced contractions in extrapulmonary artery and aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume-dependent compliance and ventilation-perfusion mismatch in surfactant-depleted isolated rabbit lungs

OBJECTIVE: Volume-dependent alterations of lung compliance are usually studied over a very large volume range. However, the course of compliance within the comparably small tidal volume (intratidal compliance-volume curve) may also provide relevant information about the impact of mechanical ventilation on pulmonary gas exchange. Consequently, we determined the association of the distribution of ventilation and perfusion with the intratidal compliance-volume curve after modification of positive end-expiratory pressure (PEEP). DESIGN: Repeated measurements in randomized order. SETTING: An animal laboratory. SUBJECTS: Isolated perfused rabbit lungs (n = 14). INTERVENTIONS: Surfactant was removed by bronchoalveolar lavage. The lungs were ventilated thereafter with a constant tidal volume (10 mL/kg body weight). Five levels of PEEP (0-4 cm H2O) were applied in random order for 20 mins each. MEASUREMENTS AND MAIN RESULTS: The intratidal compliance-volume curve was determined with the slice method for each PEEP level. Concurrently, pulmonary gas exchange was assessed by the multiple inert gas elimination technique. At a PEEP of 0-1 cm H2O, the intratidal compliance-volume curve was formed a bow with downward concavity. At a PEEP of 2 cm H2O, concavity was minimal or compliance was almost constant, whereas higher PEEP levels (3-4 cm H2O) resulted in a decrease of compliance within tidal inflation. Pulmonary gas exchange did not differ between PEEP levels of of 0, 1, and 2 cm H2O. Pulmonary shunt was lowest and perfusion of alveoli with a normal ventilation-perfusion was highest at a PEEP of 3-4 cm H2O. Deadspace ventilation did not change significantly but tended to increase with PEEP. CONCLUSIONS: An increase of compliance at the very beginning of tidal inflation was associated with impaired pulmonary gas exchange, indicating insufficient alveolar recruitment by the PEEP level. Consequently, the lowest PEEP level preventing alveolar atelectasis could be detected by analyzing the course of compliance within tidal volume without the need for total lung inflation.     

Effects of haemoconcentration and haemodilution on acute hypoxia-induced pulmonary hypertension and changes in vascular compliance of isolated rabbit lungs

Objective: Erythrocytes influence the magnitude of hypoxia-induced pulmonary artery pressure increase. It is, however, unknown to what extent haemoconcentration and haemodilution affect this response and whether intrapulmonary blood volume (and thus vessel dimensions) alters the magnitude of pressure increase. Furthermore, it is unclear whether the haemodilution/haemoconcentration-dependent pressure increase is flow-related, via flow-dependent changes in vasomotor tone or rheologic effects, or can also be observed under no-flow conditions. Design: Experimental study in isolated rabbit lungs (n = 12) perfused with autologous blood at constant flow (100 ml/min) and ventilated with 5 % carbon dioxide in air. Setting: Laboratory for experimental studies. Interventions: Haemoconcentration (centrifugation) and haemodilution (Krebs-Henseleit/albumin) were carried out, resulting in haematocrits between 50 % and 0 %. During hypoxic ventilation, inspiratory oxygen fraction was reduced from 0.20 to 0.03. Measurements and results: Under constant flow conditions, haemodilution (from a Hct of 34–36 % to 0–1 %) decreased hypoxic pulmonary artery pressure response to one-third (from 10.8 ± 2.3 cmH₂O to 3.1 ± 1.0 cmH₂O, P < 0.05), while haemoconcentration did not affect the magnitude of hypoxic response (10.5 ± 2.0 cmH₂O). For all haematocrit values an increase in pulmonary blood volume (by 5 ml) decreased the magnitude of pressure response. Hypoxia-induced changes in static vascular filling pressure (double occlusion pressure) and vascular compliance were used to assess the strength of hypoxic vasoconstriction under static conditions. Neither haemoconcentration nor haemodilution altered hypoxia-induced changes in either variable. Conclusions: The magnitude of the acute hypoxic pressure response is not altered by haemoconcentration, but significantly reduced by haemodilution. In contrast, neither haemoconcentration nor haemodilution influenced hypoxia-induced changes in static vascular filling pressure and compliance. This suggests that the degree of hypoxic pulmonary vasoconstriction is not affected under static conditions and that the red blood cell-dependence of the magnitude of hypoxic pressure response is based on flow-related mechanisms. Received: 19 October 1999 Final revision received: 4 April 2000 Accepted: 28 April 2000     

Effects of capsaicin on the endothelial permeability in isolated and perfused rabbit lungs

Summary— Changes in pulmonary endothelial permeability and in microvascular hemodynamics in response to capsaicin (10^?4M) were investigated in isolated, perfused rabbit lungs. Blood-free perfusate was recirculated through ventilated lungs in an isogravimetric state, under zone III conditions with a constant flow. Using the occlusions method, the total pressure gradient between the arterial and the venous levels (δPt) was partitioned into four components: arterial (δPa), pre- (δPa') and post-(δPv') capillary, and venous (δPv). The capillary filtration coefficient (Kf,c) was evaluated by measuring the amount of fluid filtering through the endothelium when arterial and venous pressures were suddenly increased. Capsaicin caused no changes in the vascular pressures at any level of the pulmonary circulation but induced a significant 3-fold increase in the Kf,c (P < 0.05). This reaction was accompanied by pulmonary oedema. The mechanisms involved in the permeability changes were investigated by testing the capacity of different drugs to block the response to capsaicin. Clonidine (10^?7M to 10^?5M), morphine (10^?6M), aspirin (10^?3M), ketanserin (10^?8M) and (±) CP 96,345 (10^?6M), an antagonist of neurokinin NK₁ receptor, completely prevented the effects of capsaicin on the Kf,c. In contrast, terfenadine (10^?7) together with cimetidine (10^?5M) had no protective effect against capsaicin. It was concluded that capsaicin-induced pulmonary oedema was due to an increase in the capillary filtration coefficient and not to hemodynamic changes. This alteration in the endothelium permeability is mediated by the release of endogenous peptides from C-fibers upon the action of capsaicin and subsequent activation of NK₁ receptors, probably by substance P. Moreover, 5-hydroxytryptamine receptors and arachidonic acid derivates are also involved in this reaction.     

Effects of calcium on vasopressin-mediated cyclic adenosine monophosphate formation in cultured rat inner medullary collecting tubule cells. Evidence for the role of intracellular calcium

We explored the effects of alterations in extracellular and intracellular calcium concentration on arginine vasopressin (AVP)-stimulated cAMP formation in cultured rat inner medullary collecting tubule cells. cAMP formation remains constant at extracellular calcium concentrations between 0.5 and 4.0 mM, which did not change intracellular calcium. Maneuvers that alter intracellular calcium concentration are associated with marked changes in cAMP generation. EGTA decreases intracellular calcium and enhances AVP-stimulated cAMP formation, while increasing cellular calcium with 2 microM A23187 decreases AVP-stimulated cAMP formation in the presence, but not in the absence, of extracellular calcium. The changes in cAMP formation observed when intracellular calcium is altered are associated with reciprocal changes in prostaglandin E2 (PGE2) synthesis. Despite greater than 95% inhibition of PGE2 synthesis with 5 microM meclofenamic acid, the changes in cAMP formation accompanying alterations in intracellular calcium concentration are still evident. These studies suggest that intracellular calcium critically influences AVP-stimulated cAMP formation. It does so by a mechanism independent of PG that is probably mediated by a direct effect of the cation on the adenylate cyclase complex.     

Pharmacokinetics and metabolism of infused versus inhaled iloprost in isolated rabbit lungs

Iloprost is a potent prostacyclin analog, which has been shown to exert beneficial effects in several vascular disorders. Inhalation of aerosolized iloprost was found to cause selective pulmonary vasodilatation, and this approach is under current investigation for treatment of chronic pulmonary hypertension. The present study investigated pharmacokinetics and metabolism of aerosolized iloprost in isolated buffer-perfused rabbit lungs, compared with intravascular administration of the prostanoid. After buffer admixture of iloprost, a steady decline of perfusate concentrations of the intact prostanoid was noted (half-life approximately 3.5 h), mostly attributable to progressive metabolism to dinor- and tetranoriloprost. Inhaled iloprost rapidly entered the intravascular compartment, with peak buffer concentrations being noted after 30 min (bioavailability approximately 63%). Compared with infused iloprost, significantly more rapid metabolism to dinor- and tetranoriloprost was noted for iloprost administered via the inhalative route of application. However, the percentage of the nebulized agent that enters the intravascular space as intact iloprost displays the same clearance rate as directly perfusate-admixed prostanoid. We conclude that a high percentage of inhaled iloprost rapidly enters the intravascular compartment in intact rabbit lungs. The lung is capable of metabolizing iloprost via beta-oxidation, and more rapid appearance of dinor- and tetranoriloprost is noted for the inhalative as compared with the intravascular route of iloprost administration.     

Hypoxic contractile response in isolated rat thoracic aorta: role of endothelium, extracellular calcium and endothelin

Summary— The effects of hypoxia on isolated arteries remain controversial, depending on the species, vascular beds and protocols. The aims of the study were to characterize the response of rat thoracic aorta to hypoxia and to examine the roles of endothelium, extracellular calcium and endothelin in this response. Hypoxia was induced by bubbling Krebs solution with 95% N₂ and 5% CO₂ instead of 95% O₂ and 5% CO₂. Experiments were performed during 1 h in norepinephrine (0.01 μM) precontracted rings. Hypoxia produced a biphasic response consisting of an initial transient partial relaxation (67% at 14 min) followed by a slow but sustained contraction (27% from 40 to 60 min). After endothelium removal, relaxation appeared faster with increased magnitude (82% at 12 min) and was followed by a weak transient contraction (16% at 25 min). In endothelium-intact rings, Ca²⁺ free medium (EGTA, 0.1 mM) and Ca²⁺ channel blockers, verapamil (0.05, 0.5 and 5 μM) or nicardipine (0.1, 1 and 10 μM), had no effect on relaxation but inhibited the contraction, the effects of both calcium antagonists being concentration-dependent. Similarly, the ET_A/ET_B receptor antagonist, bosentan (0.1, 10 and 1,000 nM), induced a concentration-dependent decrease in the contraction. We conclude that 1) the response of rat thoracic aorta during 1 h of hypoxia is biphasic (relaxation followed by contraction); 2) the endothelium is involved in the contraction whereas its role in the relaxation remains to be elucidated; 3) extracellular calcium is involved in the contraction; and 4) endothelin may play a role in the contraction.     

改良腹腔高压（兔）液体动物模型心脏和肺脏病理改变

目的通过制作改良腹腔高压（兔）液体动物模型,探索腹腔间隔室综合征（ACS）继发心脏和肺脏病理变化的特点。 方法将25只新西兰兔随机分为3组,对照组5只,实验1组10只,实验2组10只,分别制作改良腹腔高压液体动物模型。对照组加压水囊不注液,维持压力为0 mmHg（1 mmHg=0.133kPa）,观察48 h;实验组记录腹腔压力和腹腔增容量测定值,绘制腹腔压力-腹腔增容量曲线,腹腔压力调节在25 mmHg,实验1组观察24 h,实验2组观察48 h。在到达观察时限后处死实验兔,完整取出心脏和肺脏,用10%甲醛溶液固定24 h后常规石蜡包埋、切片、HE染色,在生物光学显微镜下观察病理改变。对实验组兔腹腔压力与腹腔增容量的关系进行简单线性回归分析。 结果对照组5只实验兔均存活,实验1组10只兔死亡1只,实验2组10只兔死亡2只。实验组兔腹腔压力与腹腔增容量存在正相关关系（r²=0.8023,P=0.0064）,腹腔压力-腹腔增容量曲线函数方程：Y=0.1642X-132.0000。各实验组兔心脏病理变化：对照组：心肌纤维呈短圆柱状,有分支,相互连接成网;可见闰盘,呈着色较深的横行粗线。实验1组：心肌结构尚正常,心肌纤维变细,心肌间小血管扩张充血,心肌肌束萎缩,萎缩的肌细胞核聚集。实验2组：心肌间小动脉扩张充血,管腔内可见均匀一致的粉染物,心肌间静脉扩张出血,血管壁玻璃样变。各实验组兔肺脏病理变化：对照组：正常肺脏内可见终末细支气管和大量肺泡,间质内小血管扩张。实验1组：肺泡上皮增生,肺泡间隔大小不一,小灶肺泡腔融合;肺泡组织间出血,可见暗褐色物质沉积。血管内血栓,可见机化再通。实验2组：肺泡明显扩张,部分肺泡腔融合,肺泡间隔不一;终末细支气管腔内可见大量红细胞及纤维素样渗出物,其中有暗褐色物质沉积。 结论ACS可造成严重的心脏和肺脏损伤,随着时间延长损伤加重,这可能是ACS直接致死因素。     

The influence of mecamylamine on contractions induced by different agonists and on the role of calcium ions in the isolated rabbit aorta.

The exposure of helically cut strips of rabbit aorta to mecamylamine (1 mM) for 5 minutes blocked the histamine (3.25 muM)-induced contractions completely and reduced those induced by potassium (25mM, 68%) without affecting contractions induced by norepinephrine (3.0 muM, NE) or acetylcholine (10 muM). Mecamylamine, by itself, did not exhibit agonist activity, but it increased the contractile tension of the muscle which was contracted by NE. The responses to NE were either enhanced or not affected by small doses of mecamylamine (1 mM), but were partially blocked by large doses of mecamylamine (10 mM). The antagonism exhibited by mecamylamine in low doses (1 mM) against the above agonists was reversible. The shape of the response to NE on the muscle, which was treated with a high dose of mecamylamine (20 mM) and washed, was significantly different from that of the control. Our results are consistent with the following conclusions: 1) Mecamylamine in low doses (1 mM) blocks the histamine receptor and therefore the histamine-induced contractions. Two molecules of mecamylamine are competitive with 1 molecule of histamine. 2) Mecamylamine blocks the potassium-induced contractions, but does not block completely the calcium influx in the potassium-depolarized muscles. Therefore, it may interfere partially with the utilization of extracellular or loosely bound calcium during potassium-induced contractions. 3) Mecamylamine in high doses (10 mM) partially blocks norepinephrine-induced contractions by affecting firmly bound calcium stores. 4) Procaine antagonizes the effects of NE, histamine and acetylcholine by affecting, at least partially, the firmly bound calcium stores.     

Evidence for a role in thrombus stabilization for thromboxane A2 in human platelet deposition on collagen.

The role of thromboxane A2 (TxA2) in platelet deposition onto collagen was studied in flowing whole heparinized human blood in vitro by using a cyclooxygenase inhibitor, aspirin, and a TxA2 receptor antagonist, GR32191B. Previous studies have demonstrated a role for TxA2 in platelet aggregation in citrated plasma, and for platelet deposition in flowing citrated human and rabbit blood, but not in flowing heparinized rabbit blood. In contrast with the literature regarding rabbit blood, aspirin was demonstrated to be effective in reducing platelet accumulation in heparinized human blood, as was GR32191B. The temporal pattern of the platelet deposition, which reached an asymptote with TxA2 inhibition at 2.0 minutes but did not do so without inhibition, suggested that TxA2 plays a role in thrombus stabilization. The reduction in platelet deposition (which included some aggregation) seen with aspirin and GR32191B in the first 1.5 minutes of perfusion indicated that some inhibition of platelet recruitment occurred. Scanning electron microscopy revealed that less fibrin was present in thrombi derived from GR32191B-treated heparinized blood than in thrombi derived from control heparinized blood. No fibrin formation was observed in citrated blood with or without TxA2 inhibition. It is proposed that, in addition to its role as a mediator of platelet recruitment, TxA2 is involved in the stabilization of platelet-platelet interactions in the thrombus, perhaps by enhancing local fibrin formation or binding.     

Evidence for role of cytosolic free calcium in hypoxia-induced proximal tubule injury.

The role of cytosolic free Ca2+ ([Ca2+]i) in hypoxic injury was investigated in rat proximal tubules. [Ca2+]i was measured using fura-2 and cell injury was estimated with propidium iodide (PI) in individual tubules using video imaging fluorescence microscopy. [Ca2+]i increased from approximately 170 to approximately 390 nM during 5 min of hypoxia. This increase preceded detectable cell injury as assessed by PI and was reversible with reoxygenation. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 100 microM) reduced [Ca2+]i under basal conditions (approximately 80 nM) and during hypoxia (approximately 120 nM) and significantly attenuated hypoxic injury. When [Ca2+]i and hypoxic cell injury were studied concurrently in the same individual tubules, the 10 min [Ca2+]i rise correlated significantly with subsequent cell damage observed at 20 min. 2 mM glycine did not block the rise in [Ca2+]i, yet protected the tubules from hypoxic injury. These results indicate that in rat proximal tubules, hypoxia induces an increase of [Ca2+]i which occurs before cell damage. The protective effect of BAPTA supports a role for [Ca2+]i in the initiation of hypoxic proximal tubule injury. The glycine results, however, implicate calcium-independent mechanisms of injury and/or blockade of calcium-mediated processes of injury such as activation of phospholipases or proteases.     

Thromboxane-mediated hypertension and vascular leakage evoked by low doses of Escherichia coli hemolysin in rabbit lungs.

Escherichia coli hemolysin has been implicated as a pathogenicity factor in extraintestinal E. coli infections including sepsis. In the present study the effects of intravascular administration of hemolysin were investigated in isolated blood-free perfused rabbit lungs. Low concentrations of the toxin in the perfusate (0.05-5 hemolytic units/ml, corresponding to approximately 5-500 ng/ml), caused a dose- and time-dependent release of potassium, thromboxane A2, and prostaglandin I2, but not of lactate dehydrogenase, into the recirculating medium, as well as a dose-dependent liberation of the prostanoids into the bronchoalveolar space. These events were paralleled by a dose-dependent pulmonary hypertension, and studies with different inhibitors collectively indicated that the vasoconstrictor response was mediated predominantly by pulmonary thromboxane generation. In addition, E. coli hemolysin elicited a protracted, dose-dependent increase in the lung capillary filtration coefficient, which was independent of the prostanoid-mediated pressor response and resulted in severe pulmonary edema formation. We conclude that E. coli hemolysin can elicit thromboxane-mediated pulmonary hypertension combined with severe vascular leakage in isolated lungs in the absence of circulating inflammatory cells and humoral mediator systems, mimicking the key events in the development of acute respiratory failure in states of septicemia.     

Mechanism of cyclosporin potentiation of vasoconstriction of the isolated rat mesenteric arterial bed: role of extracellular calcium

Hypertension is a serious side-effect of the clinical use of Cyclosporin A (CsA). One notion is that alterations of vascular reactivity contribute to this hypertension. In this study we used the isolated rat mesenteric vascular bed to test the specific hypothesis that CsA modifies vascular calcium regulation to potentiate vascular contractility. Mesenteric vessels from CsA-treated rats (10 mg/kg/day i.m. for 7 days) exhibited significantly greater vasoconstrictor responses to exogenous norepinephrine (NE) and potassium-induced depolarization than those of vehicle-treated animals. Similarly, in vitro CsA (8.3 X 10(-8) to 8.3 X 10(-6) M) augmented in a concentration-dependent manner both the sensitivity and the maximum response to NE and potassium. This effect of CsA on vasoconstriction was critically dependent on the presence of external calcium [( Ca++]o). The degree of vasoconstriction potentiation correlated significantly with the [Ca++]o which was used during exposure of the mesenteric bed to CsA. Reapplication of external calcium in the presence of CsA to "calcium-depleted" preparations increased significantly the amplitude of subsequent NE responses in a calcium-free medium. Thus, CsA-potentiated NE responses, once established, were not reversed by removing external calcium; however, attenuation occurred with the intracellular calcium antagonist dantrolene. Verapamil and nifedipine blocked potassium-elicited responses, but failed to prevent the CsA effect on NE-induced vasoconstriction. We conclude that CsA potentiation of vasoconstriction depends on extracellular calcium, and results from an enhanced transmembrane calcium transport. We speculate that CsA also increases the filling of intracellular stores of releasable calcium. These effects lead to greater calcium influx and greater intracellular calcium release upon stimulation.     

Indomethacin secretion in the isolated perfused proximal straight rabbit tubule. Evidence for two parallel transport mechanisms.

We studied indomethacin as a probe of anion transport across the isolated perfused proximal straight tubule of the rabbit and discovered that a substantial component of transport may occur by anion exchange at the basolateral membrane. Various perturbations involving direct or indirect dissipation of the cellular sodium gradient (ouabain, sodium- or potassium-free solutions, cooling to 18 degrees C) resulted in only a 50% inhibition of indomethacin transport, which raised the question of a co-existent alternative pathway for secretion. Similarly, the anion exchange inhibitor, 4,4'-diisothiocyanostilbene (DIDS), diminished indomethacin secretion by only 50%. Cooling followed by DIDS or the reverse sequence resulted in additive inhibition such that the combination abolished active secretion of indomethacin. We conclude that active secretion of indomethacin by the proximal straight tubule appears to be in part sodium gradient dependent; the remainder may be driven by an anion exchanger on the basolateral membrane.     

Evidence that peptidoleukotriene is a prerequisite for antigen-dependent thromboxane synthesis in IgG1-passively sensitized guinea pig lungs

Lungs from guinea pigs passively sensitized with an affinity-purified IgG1 antibody produce both leukotriene (LT)D4 and thromboxane (Tx)B2 upon ex vivo antigen challenge. This study was undertaken to determine the possibility of endogenously generated peptido-LTs being a prerequisite for Tx synthesis. In immunoglobulin G1-sensitized lungs, exogenous LTD4 induced TxB2 production with a median effective dose of 4.1 nM, whereas the response to LTE4, LTB4 or platelet-activating factor was relatively weak. Although LTC4 was as potent as LTD4 in stimulating TxB2 generation, LTC4's dose-response curve was shifted significantly to the right by AT-125, an irreversible gamma-glutamyl transpeptidase inhibitor, suggesting that at least a part of LTC4 sensitized lungs with antigen (0.01-30 micrograms/ml ovalbumin) for 20 min precipitated a significant amount of LTD4 production. The levels of LTD4 range from 8 to 26 nM (without taking LTD4 recovery into consideration). This level is 2- to 7-fold greater than the median effective dose value observed with exogenous LTD4. Moreover, pretreatment of sensitized lungs with ICI-198,615 a specific LTD4 antagonist, blocked equally both antigen (IC50 = 0.01 microM)- and LTD4 (IC50 = 0.017 microM)-induced TxB2 production. When sensitized lung fragments were treated with 5 mM AT-125, ICI-198,615 was effective in preventing not only antigen-but also LTC4-dependent production of TxB2 (IC50 = 0.018 and 0.021 microM, respectively). In contrast, neither WEB-2086, a platelet-activating factor antagonist, nor pyrilamine, a histamine antagonist, inhibited antigen and LTD4 responses (IC50 greater than 30 microM). Unlike its effect on antigen response, ICI-198,615 was unable to block Ca2+ ionophore-induced TxB2 production.2     

Differential influence of calcium channel-blockers on prostanoid generation and thromboxane-mediated vasoconstriction in rabbit lungs

In blood-free perfused and ventilated rabbit lungs, the influence of calcium channel-blockers on pulmonary prostanoid generation and thromboxane-mediated vasoconstriction was investigated. The specific pathways of arachidonic acid (AA) metabolism were stimulated by repetitive pulmonary artery injection of the calcium-ionophore A 23187, which induces the liberation of endogenous AA, or by repetitive application of exogenous AA. In control lungs, both stimuli provoked reproducible phasic vascular pressor responses, accompanied by the release of thromboxane A2 and prostaglandin I2 into the perfusion fluid. Repeated stimulus application in the presence of increasing concentrations of different calcium antagonists showed a dose-dependent inhibition of the pressor responses by all agents. The rank order of potency was nimodipin greater than LU-40883 (verapamil derivative) greater than diltiazem and D-888 (verapamil derivative) greater than verapamil for the ionophore-induced pressure increase, whereas diltiazem was nearly ineffective in the presence of exogenously applied AA. The prostanoid release after application of A 23187 was influenced differentially by the calcium channel-blockers. It was not affected by nimodipin and verapamil, it was slightly depressed by the verapamil derivatives D-888 and LU-40883 and it was dose-dependently and in higher concentrations nearly inhibited completely by diltiazem. We conclude that the thromboxane-mediated pulmonary vasoconstriction is inhibited by different-type calcium channel-blockers with a marked rank order of potency. The prostanoid generation induced by stimulation of the lung vascular AA metabolism is influenced differentially by these agents.