Regional transcapillary albumin exchange in rodent endotoxaemia: effects of fluid resuscitation and inhibition of nitric oxide synthase.

Sepsis is characterized by increased microvascular permeability and regional variations in capillary perfusion, which may be modulated by nitric oxide (NO) and reversed by fluid resuscitation (FR). The effects of saline FR and NO synthase blockade [by N(G)-nitro-L-arginine methyl ester (L-NAME)] on microvascular albumin transport and perfused capillary density were assessed in anaesthetized Wistar rats with acute normodynamic endotoxaemia. Separate dual-isotope techniques were employed to measure the permeability index (PI(A)) and the permeabilityxsurface area product index (PI(B)), which provide different and complementary information regarding blood-tissue albumin exchange. PI(A) represents the tissue/blood distribution volume ratio of albumin. PI(B) is a composite measure of endothelial permeability and the vascular surface area available for albumin exchange, and therefore takes into account the effect of altered blood volume. Capillary density was quantified by fluorescence microscopy following circulation of Evans Blue-labelled albumin. Compared with controls, PI(A) was reduced significantly in lipopolysaccharide (LPS)-treated animals in skeletal muscle and skin, probably due to blood volume redistribution rather than to changes in permeability. PI(B) was increased significantly in LPS-treated animals in the kidney, mesentery, skeletal muscle, skin and lung, and in the small bowel following FR. FR also improved the LPS-induced metabolic base deficit, but did not alter capillary density. L-NAME significantly attenuated the LPS-induced rise in PI(B) in the lung. In conclusion, acute endotoxaemia induces tissue-dependent variations in microvascular albumin exchange. FR improves acid-base disturbance in endotoxaemia, through mechanisms other than microvascular recruitment. NO appears to increase microvascular permeability in endotoxaemia, an effect that may be attenuated by L-NAME, particularly in the lung.

Effects of nitric oxide-modulating amino acids on coronary vessels: relevance to sepsis.

Excessive nitric oxide (NO) production in septic shock is thought to contribute to the associated profound hypotension. Here we show that despite induction of NO synthase (NOS) in the hearts of endotoxin-treated rats, coronary vascular responses to the contractile peptide endothelin-1, were not modified. This was not due to any change in the expression of endothelin receptors. However, when the substrate for NOS, L-arginine, was added to the perfusate, increases in coronary perfusion pressure stimulated by endothelin were reduced in hearts from endotoxin-treated animals compared to those from controls. In addition, L-glutamine, which blocks the generation of L-arginine from intracellular stores, enhanced the increase in perfusion pressure stimulated by endothelin-1. These data suggest that L-arginine becomes rate limiting for the production of NO in the coronary vessels during septic shock. Moreover, it suggests that vascular reactivity may be modulated positively or negatively by supplementation with the relevant amino acids.

Haem oxygenase shows pro-oxidant activity in microsomal and cellular systems: implications for the release of low-molecular-mass iron.

Haem oxygenase-1 (HO-1) is a highly inducible stress protein that removes haem from cells with the release of biliverdin, carbon monoxide and low-molecular-mass iron (LMrFe). Several antioxidant functions have been ascribed to HO; its induction is considered to be a protective event. However, LMrFe produced during haem catabolism might elicit a pro-oxidant response, with deleterious consequences. We therefore investigated the delicate balance between pro-oxidant and antioxidant events with the use of a microsomal lipid peroxidation (LPO) system. By using microsomal-bound HO in an NADPH-dependent LPO system, we assessed the pro-oxidant nature of the released LMrFe and the antioxidant effect of the released bilirubin. Hb, a biologically relevant substrate for HO, was included with the microsomes to supplement the source of haem iron and to promote LPO. We found significant increases in microsomal LPO, by using the thiobarbituric acid (TBA) test, after incubation with Hb. This Hb-stimulated peroxidation was inhibited by HO inhibitors and by iron chelators, suggesting a HO-driven, iron-dependent mechanism. GLC-MS was employed to measure the specific LPO product 4-hydroxy-2-nonenal and to confirm our TBA test results. A HO inhibitor attenuated an increase in intracellular LMrFe that occurred after treatment of rat pulmonary artery smooth-muscle cells with Hb. Additionally, exogenously added bilirubin at an equimolar concentration to the LMrFe present in both microsomal and liposomal systems was unable to prevent the pro-oxidant effect of the iron. Under certain circumstances HO can act as a pro-oxidant and seems to have a role in stimulating microsomal LPO.

Surfactant replacement therapy in late-stage adult respiratory distress syndrome.

In four adult patients with late-stage acute respiratory distress syndrome (ARDS), a single dose of the artificial surfactant ALEC was given by intrabronchial instillation. There was no sustained clinical improvement, but bronchoalveolar lavage measurements indicated that phosphatidylcholine (PC) at 24 h after treatment had increased up to 4.4 fold and phosphatidylglycerol up to 34.7 fold. However, PC relative to total phospholipid remained below normal, and protein contamination relative to PC remained above normal. Thus, therapeutic formulations and regimens to achieve greater and more sustained supplementation of PC may be required in patients with late-stage ARDS.

Action of carbon dioxide on hypoxic pulmonary vasoconstriction in the rat lung: evidence against specific endothelium-derived relaxing factor-mediated vasodilation.

OBJECTIVES: The effect of hypercapnia on pulmonary vascular tone is controversial with evidence for both a vasoconstrictor and vasodilator action. The objective of this study was to investigate the possibility that this dual response to CO2 could be explained by a direct constrictor action on smooth muscle and an indirect dilator action via the release of endothelium-derived relaxing factor. The effect of ventilation with hypercapnia (FICO2 0.15) on pulmonary pressor response to hypoxia (FIO2 0.3) was investigated. DESIGN: Prospective, randomized study. SETTING: The National Heart and Lung Institute, UK. SUBJECTS: The isolated, blood-perfused rat lung. INTERVENTIONS: Angiotensin-II and a blocker of endothelium-derived relaxing factor synthesis, NG-monomethyl-L-arginine (L-NMMA). MEASUREMENTS AND MAIN RESULTS: The vasomotor effect of hypercapnia depended on pulmonary arterial pressure. Under resting tone, CO2 acted as a mild constrictor (change in mean pulmonary arterial pressure from 14 +/- 2 to 15 +/- 2 mm Hg, n = 4; p < .05. At increased tone, induced either by hypoxia or Angiotensin-II, CO2 was a vasodilator. Thus, hypoxia increased mean pulmonary arterial pressure from 17 +/- 2 to 32 +/- 2 mm Hg (n = 8; p < .01), but simultaneous ventilation with hypoxia and hypercapnia reduced this by 16 +/- 1% (p < .01). Angiotensin-II (1 microgram) increased pulmonary arterial pressure from 14 +/- 2 to 39 +/- 5 mm Hg (n = 8; p < .01), but with hypercapnia, this angiotensin-induced pulmonary vasoconstriction was reduced by 18 +/- 6% (p < .001). The reduction in hypoxic pulmonary vasoconstriction induced by hypercapnia was not significantly different from that seen with Angiotensin-II hypercapnia. Blocking endothelium-derived relaxing factor synthesis using 30 microM NG-monomethyl-L-arginine did not significantly change either basal pulmonary arterial pressure or the response to hypercapnia, but increased hypoxic pulmonary vasoconstrictor by 24 +/- 4% (n = 4; p < .01). There was no significant difference between the change in hypoxic pulmonary vasoconstriction induced by hypercapnia after saline control (21 +/- 8% decrease) and the change in hypoxic pulmonary vasoconstriction caused by CO2 after 30 microM L-NMMA (25 +/- 10% decrease, p < .05, n = 8). CONCLUSION: Endothelium-derived relaxing factor seems unlikely to specifically modulate CO2-induced vasodilation in the rat pulmonary circulation.

Ligustrazine is a vasodilator of human pulmonary and bronchial arteries.

We have investigated the dilator effect of ligustrazine, the semisynthetic principle of a traditional Chinese herbal remedy, on human pulmonary and bronchial arteries in vitro. Ligustrazine caused a concentration-dependent relaxation of human small pulmonary arteries, which was independent of endothelium. Although ligustrazine was equally potent in inducing dilatation of pulmonary and bronchial arteries, it was about 10 times more potent in relaxing small pulmonary arteries (300-500 microns i.d.) compared with lobar pulmonary arteries (7-8 mm i.d.). By contrast, the relaxant responses of small and lobar pulmonary arteries to sodium nitroprusside was not significantly different. Ligustrazine was equally potent in relaxing prostaglandin F2 alpha- or 5-hydroxytryptamine-precontracted pulmonary arteries, suggesting that it is not a prostaglandin F2 alpha or 5-hydroxytryptamine antagonist. Preincubating the vessels with propranolol (1 microM) or indomethacin (10 microM) had no significant effect on the ligustrazine-induced vasodilatation. However, ligustrazine caused concentration-dependent inhibition of calcium-evoked contraction when applied to rat aorta in calcium-free K(+)-depolarizing medium. We conclude that ligustrazine is a dilator of human pulmonary and bronchial arteries, which is endothelium-independent and that ligustrazine preferentially relaxes pulmonary resistance vessels rather than large conduit pulmonary arteries.

Inflammatory mediators involved in antigen-induced airway microvascular leakage in guinea pigs.

Antigen challenge of ovalbumin (OA)-sensitized guinea pigs results in significant (p less than 0.05) increases in vascular permeability to Evans blue (EB) dye in the airways, esophagus, and bladder. Mean values +/- SEM in ng EB/mg wet weight tissue for unsensitized versus sensitized animals were: trachea, 23.6 +/- 6.6 versus 92.5 +/- 11.1; main bronchi, 31.1 +/- 12.2 versus 153.1 +/- 14.9; "central" intrapulmonary airways (ipa), 34.6 +/- 11.2 versus 101.3 +/- 6.2; and "peripheral" ipa, 26.2 +/- 6.8 versus 93.5 +/- 13.6. We investigated the involvement of several mediators of inflammation in this process. FPL 55712, a sulfidopeptide leukotriene receptor antagonist, caused significant inhibition of leakage in trachea (to 55.1 +/- 9.8) and main bronchi (91.7 +/- 15.8). Blockade of the cyclooxygenase and lipoxygenase pathways with BW 755C, but not of the cyclooxygenase pathway alone with indomethacin, also significantly reduced EB dye extravasation in trachea (55.1 +/- 18.0), main bronchi (71.7 +/- 23.0), and "central" ipa (62.7 +/- 16.4). The histamine antagonists, chlorpheniramine and cimetidine, only inhibited microvascular leakage in main bronchi (94.4 +/- 20.0). PAF-receptor blockade with the ginkgolide mixture BN 52063 had no effect. Nedocromil sodium, a mast cell stabilizer and an inhibitor of inflammatory cell activation, caused significant inhibition throughout the airways: trachea, 50.4 +/- 10.6; main bronchi, 72.0 +/- 15.3; "central" ipa 61.0 +/- 8.6; "peripheral" ipa 41.9 +/- 12.2. Thus, histamine and lipoxygenase products (in particular, leukotrienes), but not PAF, may mediate the antigen-induced increase in vascular permeability to different degrees in differing regions of the respiratory tract in guinea pigs.

Effect of platelet-activating factor on airway vascular permeability: possible mechanisms.

We studied the effects of the potent inflammatory mediator, platelet-activating factor (PAF), on vascular permeability in airways (and other tissues) of guinea pigs by measuring extravasation of circulating Evans blue dye. PAF caused a dose-dependent increase in vascular permeability. At 1 ng/kg iv, PAF caused an increase in Evans blue extravasation of 220% (P less than 0.05) in the trachea, with the greatest effect at a dose of 100 ng/kg (858%; P less than 0.01). Histamine (150 micrograms/kg iv) caused a 320% increase over base line in the trachea and 200% in main bronchi; this effect was equivalent to that induced by 10 ng/kg PAF in the trachea and 1 ng/kg in main bronchi. The duration of effect of PAF was greatest in main bronchi (less than 10 min). Platelet depletion with a cytotoxic antibody, or the cyclooxygenase inhibitor, indomethacin, or the cyclooxygenase-lipoxygenase inhibitor, BW 7556, did not affect the vascular permeability response to PAF. The PAF-receptor antagonist, BN 52063, inhibited Evans blue extravasation in the airways in a dose-dependent manner, with complete inhibition at 5 mg/kg. Thus PAF-induced airway vascular leakage is mediated by specific receptors but not by products of arachidonic acid metabolism or by platelets. Increased airway microvascular leakage induced by PAF may lead to plasma extravasation and airway edema, factors that may contribute to the airway narrowing and hyperresponsiveness induced by PAF.

Histamine release and circulating cells after antigen inhalation in allergic dogs.

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.