Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

The University of Tennessee Health Science Center

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery.

Hyporeactivity to vasoconstrictors is one of the clinical manifestations of sepsis in man and experimental animals. The objective of the investigation was to examine whether atorvastatin can prevent hyporeactivity to norepinephrine (NE) in mouse aorta in sepsis, and if so, what are the mechanisms involved. Sepsis in mice was induced by cecal ligation and puncture. The aorta was harvested for tension experiment, nitric oxide (NO) and cyclic guanosine monophosphate measurements, and inducible NO synthase (iNOS) and α(1D)-adrenoceptor mRNA expression studies. In comparison with sham-operated controls, sepsis significantly decreased the contractile response to NE in the mouse aorta. Pretreatment with atorvastatin of septic animals completely restored NE-induced contractions to levels similar to those of sham-operated controls and significantly increased survival time and mean arterial pressure. Atorvastatin also attenuated iNOS-induced overproduction of NO, as well as iNOS mRNA expression. Accordingly, hyporeactivity to NE was not evident in tissues pretreated with selective iNOS inhibitor 1400W in sepsis. Although basal cyclic guanosine monophosphate accumulation in the aorta was reduced in sepsis, pretreatment of the tissues with soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) partially restored the reactivity to NE. Interestingly, hyporeactivity to NE in sepsis was associated with a decreased α(1D)-adrenoceptor mRNA expression in the mouse aorta. Atorvastatin pretreatment, however, prevented the decrease in α(1D)-adrenoceptor mRNA expression in septic animals. In conclusion, atorvastatin seems to prevent hyporeactivity to vasoconstrictor NE in the aorta from septic mice through attenuation of overproduction of NO as well as improved α(1D)-adrenoceptor mRNA expression. The findings of the present study may explain the beneficial effects of atorvastatin on improved hemodynamic functions in sepsis.

Atorvastatin prevents vascular hyporeactivity to norepinephrine in sepsis: role of nitric oxide and α₁-adrenoceptor mRNA expression.

Both hyperoxia and mechanical ventilation can independently cause lung injury. In combination, these insults produce accelerated and severe lung injury. We recently reported that pre-exposure to hyperoxia for 12 hours, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone. We also reported that such injury and apoptosis are inhibited by antioxidant treatment. In this study, we hypothesized that apoptosis signal-regulating kinase-1 (ASK-1), a redox-sensitive, mitogen-activated protein kinase kinase kinase, plays a role in lung injury and apoptosis in this model. To determine the role of ASK-1 in lung injury, the release of inflammatory mediators and apoptosis, attributable to 12 hours of hyperoxia, were followed by large tidal volume mechanical ventilation with hyperoxia. Wild-type and ASK-1 knockout mice were subjected to hyperoxia (Fi(O(2)) = 0.9) for 12 hours before 4 hours of large tidal mechanical ventilation (tidal volume = 25 μl/g) with hyperoxia, and were compared with nonventilated control mice. Lung injury, apoptosis, and cytokine release were measured. The deletion of ASK-1 significantly inhibited lung injury and apoptosis, but did not affect the release of inflammatory mediators, compared with the wild-type mice. ASK-1 is an important regulator of lung injury and apoptosis in this model. Further study is needed to determine the mechanism of lung injury and apoptosis by ASK-1 and its downstream mediators in the lung.

Deletion of apoptosis signal-regulating kinase-1 prevents ventilator-induced lung injury in mice.

Endotoxemia, a major feature of sepsis, is a common cause of acute lung injury and initiates rapid accumulation of leukocytes in the lung vasculature. Endothelial mechanisms that underlie this accumulation remain unclear, as current experimental models of endotoxemia are less suitable for targeted activation of the endothelium. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. With a microcatheter inserted through a left atrial cannula, we cleared blood cells from a small lung region and then infused lipopolysaccharide (LPS) into microvessels. After a Ringer's wash to remove residual LPS, we infused fluorescently-labeled autologous leukocytes and imaged their transit through the treated microvessels. Image analysis revealed that leukocytes infused 90 min after LPS treatment were retained more in treated venules and capillaries than untreated vessels. Further, pretreatment with either the intercellular adhesion molecule-1 (ICAM-1) mAb or polymyxin-B blunted LPS-induced leukocyte retention in both microvessel segments. In addition, retention of leukocytes treated ex vivo with LPS in LPS-treated microvessels was higher compared to retention of untreated leukocytes. In situ immunofluorescence experiments revealed that LPS significantly increased microvessel ICAM-1 expression at 90 min post treatment. Polymyxin pretreatment inhibited this increase. Taken together, the data suggest that LPS increased leukocyte retention in both venules and capillaries and this response was mediated by the increased expression of endothelial ICAM-1. Thus, endothelial mechanisms may themselves play a major role in LPS-induced leukocyte retention in lung microvessels. Blunting the endothelial responses may mitigate endotoxin-induced morbidity.

Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.

Nitric oxide synthesized from inducible nitric oxide synthase (iNOS) plays role in acetaminophen (APAP)-induced liver damage. The present study was undertaken to evaluate the effect of iNOS inhibitor S-methylisothiourea (SMT) in APAP-induced hepatotoxicity in rats (1 g/kg, i.p.). SMT was (10, 30, and 100 mg/kg; i.p.) given 30 min before and 3 h after APAP administration. At 6 and 24 h, blood was collected to measure alanine transaminase (ALT), aspartate transaminase (AST), and nitrate plus nitrite (NOx) levels in serum. At 48 h, animals were sacrificed, and blood and liver tissues were collected for biochemical estimation. SMT reduced significantly the serum ALT, AST, and NOx levels at 24 and 48 h and liver NOx levels at 48 h as compared with APAP-treated control. The amount of peroxynitrite measured by rhodamine assay was significantly reduced by SMT, as compared with APAP-treated control group. SMT treatment (30 mg/kg) has significantly reduced the lipid peroxidation and protein carbonyl levels, increased SOD and catalase, and reduced glutathione and total thiol levels significantly as compared with APAP-treated control. SMT 30 mg/kg dose has protected animals from APAP-induced hypotension and reduced iNOS gene expression. Hepatocytes were isolated from animals, and effect of SMT on apoptosis, MTP, and ROS generation was studied, and their increased value in APAP intoxicated group was found to be significantly decreased by SMT (30 mg/kg) at 24 and 48 h. In conclusion, nitric oxide produced from iNOS plays important role in toxicity at late hours (24 to 48 h), and SMT inhibits iNOS and reduces oxidative and nitrosative stress.

Effect of S-methylisothiourea in acetaminophen-induced hepatotoxicity in rat.

The pulmonary microvasculature plays a critical role in endotoxin-induced acute lung injury. However, the relevant signaling remain unclear. Specifically the role of endothelial Ca(2+) in the induction of endotoxin-mediated responses in lung microvessels remains undefined. Toward elucidating this, we used the isolated blood-perfused rat lung preparation. We loaded microvessels with the Ca(2+) indicator, Fura 2 AM and then determined Ca(2+) responses to infusions of lipopolysaccharide (LPS) into the microvessels. LPS induced a more than two-fold increase in the amplitude of cytosolic Ca(2+) oscillations. Inhibiting inositol 1,4,5 trisphosphate receptors on endoplasmic reticulum (ER) Ca(2+) stores with Xestospongin C (XeC), blocked the LPS-induced increase in the Ca(2+) oscillation amplitude. However, XeC did not affect entry of external Ca(2+) via plasma membrane Ca(2+) channels in lung microvascular endothelial cells. This suggested that LPS augmented the oscillations via release of Ca(2+) from ER stores. In addition, XeC also blocked LPS-mediated activation and nuclear translocation of nuclear factor-kappa B in lung microvessels. Further, inhibiting ER Ca(2+) release blunted increases in intercellular adhesion molecule-1 expression and retention of naïve leukocytes in LPS-treated microvessels. Taken together, the data suggest that LPS-mediated Ca(2+) release from ER stores underlies nuclear factor-kappa B activation and downstream inflammatory signaling in lung microvessels. Thus, we show for the first time a role for inositol 1,4,5 trisphosphate-mediated ER Ca(2+) release in the induction of LPS responses in pulmonary microvascular endothelium. Mechanisms that blunt this signaling may mitigate endotoxin-induced morbidity.

Kathirvel Kandasamy

Department of Physiology

The University of Tennessee Health Science Center

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Kathirvel Kandasamy

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

The University of Tennessee Health Science Center

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Department of Physiology