Early Changes in Alveolar Fluid Clearance by Nitric Oxide after Endotoxin Instillation in Rats
Department of Thoracic Surgery, Institute of Development, Aging and Cancer, Tohoku University; Department of Pathology, Tohoku University School of Medicine, Sendai; and Laboratory of Applied Biomedicinal Chemistry, Institute for Life Support Technology, Yamagata Public Corporation for the Development of Industry, Yamagata, Japanh%tj, 百拇医药
ABSTRACTh%tj, 百拇医药
TOPh%tj, 百拇医药
ABSTRACTh%tj, 百拇医药
INTRODUCTIONh%tj, 百拇医药
METHODSh%tj, 百拇医药
RESULTSh%tj, 百拇医药
DISCUSSIONh%tj, 百拇医药
REFERENCESh%tj, 百拇医药
Alveolar fluid clearance may be inhibited and/or stimulated under pathologic conditions. We examined the early change of alveolar fluid clearance after endotoxin instillation in adult rats. We employed electron paramagnetic resonance nitric oxide (NO) trapping technique with iron complex with N,N-diethyldithiocarbamate as an NO trapping agent. We found that lung NO signals reached the highest magnitude by 6 hours after endotoxin instillation. NO production was accompanied by increases in lung cyclic guanosine monophosphate levels. Alveolar fluid clearance decreased significantly 6 hours after the administration of the endotoxin and increased further at 24 hours. These changes were shown to be related to the function of amiloride-sensitive sodium ion channels. Treatment with gadolinium chloride and aminoguanidine significantly decreased lung NO and cyclic guanosine monophosphate levels and completely ameliorated the decrease in alveolar fluid clearance. In addition, the increase in alveolar fluid clearance at 24 hours returned to normal levels after treatment with gadolinium chloride and aminoguanidine. We found immunoreactive inducible nitric oxide synthase to be abundantly expressed in the cytoplasm of alveolar macrophages. Our results suggest that alveolar endotoxin inhibits alveolar fluid clearance at 6 hours by NO. NO is produced via inducible NO synthase in endotoxin-stimulated alveolar macrophages and was also shown to increase alveolar fluid clearance at 24 hours.
Key Words: inducible nitric oxide synthase • nitric oxide • electron paramagnetic resonance • alveolar fluid clearance • epithelial Na+ channelqc'r, 百拇医药
INTRODUCTIONqc'r, 百拇医药
TOPqc'r, 百拇医药
ABSTRACTqc'r, 百拇医药
INTRODUCTIONqc'r, 百拇医药
METHODSqc'r, 百拇医药
RESULTSqc'r, 百拇医药
DISCUSSIONqc'r, 百拇医药
REFERENCESqc'r, 百拇医药
Alveolar epithelium is the most likely site at which absorption of excess fluid from the alveolar space might take place (1–3). Alveolar epithelial cells express amiloride-sensitive sodium ion (Na+) channels (ENaCs) on the apical surface and Na+/potassium ion-ATPase on the basolateral side, which in turn results in the unidirectional movement of Na+ and fluids out of the alveolar space. It has been shown that alveolar epithelial active Na+ transport functions in many species, including humans (4) and plays a very important role in keeping the alveolar space relatively fluid-free for adequate gas exchange (5).
Alveolar fluid clearance may be inhibited and/or stimulated under pathologic conditions. It has been previously reported that alveolar endotoxin increased alveolar fluid clearance by stimulating alveolar ENaC function at 24 or 40 hours (6). However, alveolar endotoxin may also stimulate alveolar macrophages and produce nitric oxide (NO), which impairs lung epithelial Na+ transport in a very short period of time (minutes to hours) (7–9), via inducible nitric oxide synthase (iNOS) (10). Lipopolysaccharide-stimulated alveolar macrophages have been shown to inhibit the function of alveolar ENaCs by NO as early as 4 hours (11). However, the early changes in alveolar fluid clearance after endotoxin instillation has not been specifically addressed.2n6, http://www.100md.com
In the present study, we determined the early time course of lung NO levels and alveolar fluid clearance after endotoxin instillation in rats. We then examined whether the inhibition of lung NO production would influence alveolar fluid clearance. Our results appear to suggest that alveolar endotoxin inhibits alveolar fluid clearance at 6 hours by NO, which is produced via iNOS in endotoxin-stimulated alveolar macrophages. In addition, the increase in alveolar fluid clearance 24 hours after endotoxin instillation was also mediated by alveolar macrophage-derived NO.
METHODSoh#z, http://www.100md.com
TOPoh#z, http://www.100md.com
ABSTRACToh#z, http://www.100md.com
INTRODUCTIONoh#z, http://www.100md.com
METHODSoh#z, http://www.100md.com
RESULTSoh#z, http://www.100md.com
DISCUSSIONoh#z, http://www.100md.com
REFERENCESoh#z, http://www.100md.com
Animalsoh#z, http://www.100md.com
Specific, pathogen-free male Sprague–Dawley rats (250–350 g) were used in this study (n = 157). All animals received humane care, in compliance with the guidelines of the University Committee on Animal resources, Tohoku University, Sendai, Japan. All animal experiments were conducted in accordance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication 86-23 1985).oh#z, http://www.100md.com
Endotoxin Instillationoh#z, http://www.100md.com
Rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally). Endotoxin (100 µg/kg of O55:B5 Escherichia coli; Difco Laboratories, Detroit, MI) was suspended in 0.2 ml of sterilized saline and introduced directly into the trachea via a 26-gauge needle. To examine whether endotoxin altered alveolar fluid clearance via alveolar macrophages, rats were pretreated with an intraperitoneal injection of 60 mg/kg of gadolinium chloride (GdCl3) (Wako, Osaka, Japan) in 0.5 ml of sterilized saline 72 hours before endotoxin instillation. GdCl3 was reported to induce apoptosis of macrophages (12) and inhibit the inflammatory response of alveolar macrophages (13). To examine whether endotoxin altered alveolar fluid clearance via iNOS, rats were treated with a subcutaneous injection of 75 mg/kg of aminoguanidine (Sigma Chemical, St. Louis, MO), an iNOS inhibitor (14), in 0.5 ml of sterilized saline 10 minutes before endotoxin instillation.
Measurements1}aev{}, http://www.100md.com
Lung NO production was determined by recording a specific signal from the NO adduct of iron complex with N,N-diethyldithiocarbamate via electron paramagnetic resonance (15). Electron paramagnetic resonance spectra were recorded as three lines typical of NO–iron–N, N-diethyldithiocarbamate complex (16) using an X-band spectrometer (TE-200 type; JEOL, Tokyo, Japan) at room temperature. Cyclic guanosine monophosphate (cGMP) was measured using a cGMP enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Additional details on the method for lung tissue preparation and assay conditions are provided in the online supplement.1}aev{}, http://www.100md.com
Alveolar fluid clearance was measured in isolated rat lungs and anesthetized rats by determining the increase in albumin concentration in the instillate solution (sodium chloride 140 mM, potassium chloride 5 mM, calcium chloride 1 mM, magnesium chloride 1 mM, D-glucose 5 mM, HEPES 6 mM, bovine serum albumin 4 g/dl, pH 7.1) as described elsewhere (17). In addition, the effects of 0.1 mM amiloride (Sigma) and 1 mM terbutaline (Sigma) in the instillate solution were also examined in isolated rat lungs. Anesthetized rats were used to examine the impact of reduced alveolar fluid clearance on arterial blood gases. Additional details on the method for lung tissue preparation and calculation of alveolar fluid clearance are provided in the online supplement.
Lung extravascular water content was assessed by calculating the wet/dry weight ratio corrected for lung hemoglobin concentration (18).|5, 百拇医药
For histologic examinations, lungs were inflation-fixed with 4% paraformaldehyde (PFA) at 10 cm H2O of airway pressure. Immunohistochemistry was also performed using a polyclonal anti-mouse macrophage iNOS antibody (N32030; Transduction Laboratories, Lexington, KY). Electron microscopic examination was also performed after the standard tissue fixation and staining steps.|5, 百拇医药
Statistics|5, 百拇医药
All data are presented as mean ± SEM. Statistical analyses were performed by using analysis of variance and the Fisher's exact test. Statistical significance was defined as p values less than 0.05.|5, 百拇医药
RESULTS|5, 百拇医药
TOP|5, 百拇医药
ABSTRACT|5, 百拇医药
INTRODUCTION|5, 百拇医药
METHODS|5, 百拇医药
RESULTS6., 百拇医药
DISCUSSION6., 百拇医药
REFERENCES6., 百拇医药
A specific NO signal was detected with the highest magnitude 6 hours after endotoxin instillation and then decreased toward baseline levels . The vehicle control experiment (n = 3) showed no significant difference in lung NO levels 6 hours from levels at time 0. There was a significant increase in lung cGMP levels as early as 3 hours, which increased to a maximal level at 6 hours, after which lung cGMP levels decreased toward baseline levels . Tissues obtained from vehicle-treated animals (n = 4) showed no significant difference in lung cGMP levels at 6 hours from baseline values. The increase in lung NO and cGMP levels at 6 hours after endotoxin instillation were significantly reduced by treatment with GdCl3 and aminoguanidine .6., 百拇医药
fig.ommitted6., 百拇医药
Figure 1. Changes in lung NO levels after endotoxin instillation. (A) Specific signals of NO–iron–N,N-diethyldithiocarbamate complex were recorded 6 hours after endotoxin instillation with the highest magnitude. All spectra were measured in the same instrumental settings as shown in the online supplement. (B) Quantification of lung NO levels showed a rapid increase between 3 and 6 hours and a rapid decrease 12 hours after endotoxin instillation. Data are presented as mean ± SEM, n = 5 for each time point. *p Values less than 0.05 versus values at time 0 (without endotoxin) by analysis of variance.
fig.ommitted%)slt, http://www.100md.com
Figure 2. Changes in lung cGMP levels after endotoxin instillation. Lung cGMP concentrations increased to maximum levels at 6 hours after endotoxin instillation. A significant increase was observed as early as 3 hours. Data are presented as mean ± SEM, n = 5 for each time point. *p Values less than 0.05 versus values at time 0 (without endotoxin) by analysis of variance.%)slt, http://www.100md.com
fig.ommitted%)slt, http://www.100md.com
TABLE 1. Effects of gadolinium chloride and aminoguanidine on lung nitric oxide and cyclic guanosine monophosphate levels%)slt, http://www.100md.com
There was no significant change in alveolar fluid clearance determined in isolated rat lungs in the first 3 hours; however, a significant decrease was observed at 6 hours . At 12 hours after endotoxin instillation, alveolar fluid clearance had returned to baseline levels and further increased at 24 hours . Alveolar fluid clearance in vehicle-treated control specimens (n = 5) did not change significantly from baseline values at 6 hours. There was no significant change in alveolar fluid clearance determined in the presence of 0.1 mM amiloride (amiloride-insensitive component) over 24 hours, and there was no significant difference in alveolar fluid clearance determined in the presence or absence of 0.1 mM amiloride at 6 hours . Terbutaline significantly increased alveolar fluid clearance in the control specimens and also at 6 hours after endotoxin instillation . The terbutaline-stimulated alveolar fluid clearance was significantly inhibited by 0.1 mM amiloride . When alveolar fluid clearance was plotted versus amiloride concentrations, there were no significant differences in alveolar fluid clearance determined in the presence of amiloride at 0.01, 0.1, and 1 mM between control experiment and 6 hours after endotoxin instillation , indicating that amiloride had similar effects on alveolar fluid clearance in both experimental conditions. The amiloride half-maximal inhibitory concentration in the control experiment was estimated to be less than 0.01 mM ). In contrast, 0.01 mM amiloride showed no significant effect on alveolar fluid clearance at 6 hours after endotoxin instillation .
fig.ommitted!%(, 百拇医药
Figure 3. Changes in alveolar fluid clearance after endotoxin instillation. Alveolar fluid clearance determined in isolated rat lungs decreased significantly at 6 hours, and returned to control levels, and further increased at 24 hours. In contrast, there were no significant changes in alveolar fluid clearance determined in the presence of amiloride over the 24 hours. There was no inhibitory effect of amiloride on alveolar fluid clearance at 6 hours. Terbutaline significantly increased alveolar fluid clearance in control specimens and also at 6 hours after endotoxin instillation. The terbutaline-stimulated alveolar fluid clearance was inhibited by amiloride. Data are presented as mean ± SEM, n = 5 for each experiment at each time point, excepting n = 3 for terbutaline + amiloride experiments. *p Values less than 0.05 versus values without endotoxin by analysis of variance. p Values less than 0.05 versus values of no inhibitor by analysis of variance. p Values less than 0.05 versus values of terbutaline by analysis of variance.!%(, 百拇医药
fig.ommitted!%(, 百拇医药
Figure 4. Effects of amiloride on alveolar fluid clearance. There were no significant differences in alveolar fluid clearance determined in the presence of amiloride at 0.01, 0.1, and 1 mM between control experiment and 6 hours after endotoxin instillation. The amiloride half-maximal inhibitory concentration was found to be less than 0.01 mM M in control experiment; however, 0.01 mM amiloride did not show any significant inhibition of alveolar fluid clearance in endotoxin experiments. Data are presented as mean ± SEM, n = 3 for each experiment at each time point, excepting n = 5 for the values of no amiloride and 10-4 M amiloride.
Neither GdCl3 nor aminoguanidine treatment altered alveolar fluid clearance determined in isolated rat lungs in vehicle-treated control specimens; however, both agents ameliorated the decrease in alveolar fluid clearance at 6 hours . In addition, the increase at 24 hours had returned to baseline levels by pretreatment with GdCl3 and aminoguanidine .c?x, 百拇医药
fig.ommittedc?x, 百拇医药
Figure 5. Effects of treatment with GdCl3 and aminoguanidine on alveolar fluid clearance. Both GdCl3 and aminoguanidine ameliorated the decrease in alveolar fluid clearance at 6 hours after endotoxin instillation. At 24 hours, GdCl3 and aminoguanidine changed alveolar fluid clearance to values seen at time 0. Data are presented as mean ± SEM, n = 5 for each time point. *p Values less than 0.05 versus values of no treatment by analysis of variance.c?x, 百拇医药
There was no significant difference in alveolar fluid lactate dehydrogenase levels or lung wet/dry weight ratio between the vehicle control and endotoxin experiments .
fig.ommitted@?5u, 百拇医药
TABLE 2. Alveolar fluid lactate dehydrogenase levels and lung wet/dry weight ratio@?5u, 百拇医药
In anesthetized rats, instillation of the solution into the left lung caused a decrease in PaO2 in both control and endotoxin experiments. However, the difference in PaO2 before introduction compared to that 1 hour after introduction of the instillate solution was significantly larger in endotoxin experiments . Treatment with GdCl3 and aminoguanidine ameliorated the arterial blood gas tension and alveolar fluid clearance .@?5u, 百拇医药
fig.ommitted@?5u, 百拇医药
TABLE 3. Alveolar fluid clearance and the difference in paO2 between before introduction and 1 hour after introduction of the instillate solution@?5u, 百拇医药
The lungs at 6 hours after endotoxin instillation were of normal appearance macroscopically. Light microscopic examinations (n = 2) showed no considerable fluid accumulation or neutrophil infiltration in the alveolar space and/or interstitium. Electron microscopic examinations (n = 2) also showed no ultrastructural evidence for damages in alveolar epithelial cells and capillary endothelial cells. Positive immunoreactivity for iNOS was predominantly detected in the cytoplasm of alveolar macrophages but was not detectable in alveolar epithelial cells or mesenchymal cells, at 6 hours after endotoxin instillation (n = 2) . In the control specimens, immunoreactivity for iNOS was not detected in the alveolar area or airway epithelia (n = 2).
fig.ommitted-c, http://www.100md.com
Figure 6. Immunohistochemistry for iNOS. No immunoreactivity for iNOS was observed in control experiments (A). At 6 hours after endotoxin instillation, positive immunoreactivity for iNOS was detected in the cytoplasm of alveolar macrophages, whereas no detectable immunoreactivity was observed in alveolar epithelial cells or mesenchymal cells (B). Results were representative of two different lung specimens. Original magnification: x400.-c, http://www.100md.com
DISCUSSION-c, http://www.100md.com
TOP-c, http://www.100md.com
ABSTRACT-c, http://www.100md.com
INTRODUCTION-c, http://www.100md.com
METHODS-c, http://www.100md.com
RESULTS-c, http://www.100md.com
DISCUSSION-c, http://www.100md.com
REFERENCES-c, http://www.100md.com
Tissue NO levels may be examined by detecting nitrogen dioxide and nitrate ions, stable end products of NO, using commercially available detection kits. However, the results may only be estimates of the amount of NO that had been produced before tissue preparation, and thus may not represent the exact NO levels at a specific time point. We therefore decided to use electron paramagnetic resonance to determine the time course of NO production after endotoxin instillation. It was previously reported that activated peritoneal macrophages exhibit a characteristic electron paramagnetic resonance signal at cryogenic temperatures (19), and the signal was identified as that of the dinitrosyl dithiolato iron complex, which was formed by the reaction of NO with intracellular iron–sulfur proteins (20). In this study, we decided to employ a direct detection of NO that is trapped with the iron–N,N-diethyldithiocarbamate complex, and the resultant NO complex exhibits a three-line electron paramagnetic resonance signal at room temperature as shown in Figure 1A. A similar technique with the iron–N,N-diethyldithiocarbamate complex as an NO trapping agent has been used at cryogenic temperatures in lung tissues (21). We found that lung NO levels reached the highest levels at 6 hours and rapidly returned toward baseline values over the next 18 hours. This time course was very similar to that of cGMP, the most potent intracellular second messenger of NO (22). These results, therefore, suggest that endotoxin instillation influences local lung cellular functions via NO and cGMP, although a significant increase in lung cGMP levels at 3 hours may be related to a direct action of endotoxin on guanylate cyclase (23).
It is yet to be found what cell type is responsible for the production of NO in the lung remains unknown. We found that the increase in lung NO and cGMP levels at 6 hours after endotoxin instillation were significantly inhibited by treatment with GdCl3. GdCl3 was reported to induce apoptosis of macrophages (12) and inhibit the inflammatory response of alveolar macrophages (13). We examined the effect of GdCl3 on the cell count of alveolar macrophages in bronchoalveolar lavage fluid and found a significant decrease (approximately 25% of that of control specimens) at 72 hours (data not shown). This finding therefore appears to suggest that alveolar macrophages may play an important role in lung NO production. In addition, treatment with aminoguanidine displayed a similar inhibitory effect on lung NO and cGMP levels. Moreover, histochemical examination revealed abundant expression of iNOS in the cytoplasm of alveolar macrophages 6 hours after endotoxin instillation, whereas no detectable immunoreactivity was observed in other cell types such as alveolar epithelial cells or mesenchymal cells. It is very likely, therefore, that alveolar macrophages expressing iNOS are the most important source of endotoxin-induced lung NO production.
Alveolar fluid clearance was significantly decreased at 6 hours after endotoxin instillation, at a time when lung NO and cGMP levels were highest. Because amiloride did not show any inhibitory effect, the decrease in alveolar fluid clearance may be related to an impairment in alveolar ENaC function and/or a change in the amiloride half-maximal inhibitory concentration. We examined the effect of amiloride on alveolar fluid clearance at higher and lower concentrations. We found a similar dose-dependent inhibition of alveolar fluid clearance in endotoxin experiments. This suggests that endotoxin did not alter half-maximal inhibitory concentration for amiloride. The endotoxin-induced decrease in alveolar fluid clearance was completely ameliorated by treatment with GdCl3 and aminoguanidine. This finding suggests that NO produced via probably iNOS in alveolar macrophages may be responsible for impairing alveolar ENaC function. There are several lines of evidence indicating that ENaC function is inhibited by NO in a short period of time (8, 9, 11). NO increases cGMP levels in target cells, which in turn alters the probability that ion channels may be found in the open state (24). In addition, the decrease in alveolar fluid clearance was reversed by intra-alveolar administration of terbutaline, and the terbutaline-stimulated alveolar fluid clearance was predominantly related to the amiloride-sensitive component. Terbutaline has been shown to increase intracellular adenosine 3',5'-cyclic monophosphate, stimulate alveolar ENaC (25, 26), and increase alveolar fluid clearance in rats (2, 27). The recovery of alveolar fluid clearance with terbutaline suggests that the decrease in the alveolar ENaC function is related to a change in individual channel activity rather than a downregulation in the expression of channel proteins.
It is important to consider that aminoguanidine at high concentrations may inhibit not only iNOS but also other types of NOS. Although aminoguanidine has been used as a specific iNOS inhibitor (14), the specificity on iNOS is lower than that of 1400W, a newly synthesized potent and selective iNOS inhibitor (28). In addition, it has been shown that iNOS-deficient mice did not display amiloride-sensitive alveolar fluid clearance, and upregulation of iNOS by hyperoxia in iNOS (+/+) mice had similar levels of alveolar fluid clearance in iNOS (-/-) mice (29). iNOS (-/-) mice are also able to produce nitrothyosine in the lungs when infected with mycoplasmas, suggesting that other types of NOS may play a role in the generation of reactive oxygen–nitrogen intermediates (30). In addition, instillation of DETANONOate, an NO donor, reduced the amiloride-sensitive component of alveolar fluid clearance; however, amiloride-insensitive fraction was increased, and the total alveolar fluid clearance was preserved at the control levels (31). Therefore, it may be possible that multiple mechanisms in addition to NO production via iNOS are involved in the modulation of alveolar fluid clearance by endotoxin.
After an early decrease, alveolar fluid clearance had recovered by 12 hours, and increased further over baseline levels by 24 hours. This late increase was related to the amiloride-sensitive component of alveolar fluid clearance. However, Garat and coworkers reported that the increase in sodium influx was not inhibited by propranolol, suggesting that endogenous catecholamines were not responsible for stimulating alveolar ENaCs (6). In this study, we found that treatment with GdCl3 and aminoguanidine returned alveolar fluid clearance at 24 hours to baseline levels. This observation may suggest that NO also plays a role in the mechanism leading to an increment in sodium influx in the alveolar epithelium. However, it is unlikely that NO and cGMP display such dual actions on channel function, such as early inhibition followed later by stimulation. In addition, lung NO and cGMP levels 24 hours after endotoxin instillation were low.'jp, 百拇医药
NO is well known to influence pulmonary circulation, reducing pulmonary vascular resistance (32) and coefficient filtration (33). However, these protective effects of NO in opposing lung fluid filtration in vivo do not explain the changes in alveolar fluid clearance examined in isolated rat lungs. A previous study has shown that cGMP may be a competitive signaling molecule with respect to the transport of adenosine 3',5'-cyclic monophosphate out of the cell via a membrane-bound transporter (34). An increase in cGMP may limit adenosine 3',5'-cyclic monophosphate transport out of the cell, thereby trapping this molecule within the cell, which in turn may stimulate channel function and alveolar fluid clearance. However, it is difficult to measure exact adenosine 3',5'-cyclic monophosphate concentrations in alveolar epithelial cells obtained from lung tissue homogenates of endotoxin-challenged lungs. Moreover, the direct measurement of in vivo intracellular concentrations of adenosine 3',5'-cyclic monophosphate also presents the investigator with an incredibly challenging task.
In this study, we administrated endotoxin at a dose of 100 µg/kg. This dose was high enough to stimulate alveolar macrophages, but more importantly, low enough to avoid structural abnormalities in lung tissues. The fluid-filled lung model, using albumin concentration as an indicator of alveolar fluid clearance, does not measure unidirectional fluid transport separately, and the results may be influenced in the case that endotoxin instillation caused pulmonary edema. When we administrated the same E. coli endotoxin at 200 µg/kg, some rats did not survive beyond 12 hours, and when the lungs were examined, they were found to be reddish in color and edematous macroscopically. All rats receiving 100 µg/kg of endotoxin survived more than 24 hours, and when the lungs were examined in these animals, the lungs were of normal appearance. We were not able to demonstrate any considerable fluid accumulation in the interstitium and/or alveolar space of the lungs by light microscopy. Electron microscopic examination also showed no abnormalities in lung cells including alveolar epithelial cells. There was no significant difference in lung wet/dry weight ratio or alveolar epithelial lactate dehydrogenase levels at the completion of 2 hours of the incubation period between control and endotoxin experiments. In addition, we examined possible leak of blood-borne particles into the alveolar apace by injecting Evans blue dye before lung isolation and found that endotoxin did not increase the concentration of Evans blue dye in the recovered alveolar fluids (data not shown). It is very unlikely, therefore, that endotoxin altered alveolar epithelial and endothelial barrier function.
In summary, our results appear to suggest that endotoxin administered into rat lungs inhibits alveolar fluid clearance followed by an increase at a later phase. Both the early decrease and late increase in alveolar fluid clearance may be mediated by NO, which is produced probably via iNOS in endotoxin-stimulated alveolar macrophages. We speculate that the early decrease in alveolar fluid clearance in endotoxin-induced pulmonary inflammation may play a role in keeping and/or recovering intact alveolar barrier functions. A decrease in alveolar fluid clearance may allow for the fast diffusion of growth factors into alveolar fluids, which in turn may modulate alveolar epithelial proliferation and differentiation, and thereby accelerate the recovery of an injured alveolar epithelium.37c1u|, 百拇医药
REFERENCES37c1u|, 百拇医药
TOP37c1u|, 百拇医药
ABSTRACT37c1u|, 百拇医药
INTRODUCTION37c1u|, 百拇医药
METHODS37c1u|, 百拇医药
RESULTS
DISCUSSION2, 百拇医药
REFERENCES2, 百拇医药
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Wu CC, Chen SJ, Yen MH. Nitric oxide-independent activation of soluble guanylyl cyclase contributes to endotoxin shock in rats. Am J Physiol 1998;275:H1148–H1157.
Light DB, Corbin JD, Stanton BA. Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase. Nature 1990;344:336–339.3p, http://www.100md.com
Goodman BE, Brown SES, Crandall ED. Regulation of transport across pulmonary alveolar epithelial cell monolayers. J Appl Physiol 1984;57:703–710.3p, http://www.100md.com
Yue G, Shoemaker RL, Matalon S. Regulation of low-amiloride-affinity sodium channels in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 1994;267:L94–L100.3p, http://www.100md.com
Sakuma T, Folkesson HG, Suzuki S, Okaniwa G, Fujimura S, Matthay MA. Beta-adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am J Respir Crit Care Med 1997;155:506–512.3p, http://www.100md.com
László F, Whittle BJR. Actions of isoform-selective and non-selective nitric oxide synthase inhibitors on endotoxin-induced vascular leakage in rat colon. Eur J Pharmacol 1997;334:99–102.3p, http://www.100md.com
Hardiman KM, Lindsey JR, Matalon S. Lack of amiloride-sensitive transport across alveolar and respiratory epithelium of iNOS(-/-) mice in vivo. Am J Physiol Lung Cell Mol Physiol 2001;281:L722–L731.
Hickman-Davis JM, Lindsey JR, Matalon S. Cyclophosphamide decreases nitrotyrosine formation and inhibits nitric oxide production by alveolar macrophages in Mycoplasmosis. Infect Immun 2001;69:6401–6410.pw, 百拇医药
Nielsen VG, Baird MS, Chen L, Matalon S. Detanonoate, a nitric oxide donor, decreases amiloride-sensitive alveolar fluid clearance in rabbits. Am J Respir Crit Care Med 2000;161:1154–1160.pw, 百拇医药
Berger J, Gibson RL, Redding GJ, Standaert TA, Clarke WR, Truog WE. Effects of inhaled nitric oxide during group B streptococcal sepsis in piglets. Am Rev Respir Dis 1993;147:1080–1086.pw, 百拇医药
Bjertnaes LJ, Koizumi T, Newman JH. Inhaled nitric oxide reduces lung filtration after endotoxin in awake sheep. Am J Respir Crit Care Med 1998;158:1416–1423.pw, 百拇医药
Hamet P, Pang SC, Tremblay J. Atrial natriuretic factor-induced egression of cyclic guanosine 3':5'-monophosphate in cultured vascular smooth muscle and endothelial cells. J Biol Chem 1989;264:12364–12369.(Hiroyoshi Tsubochi, Satoshi Suzuki, Hiroshi Kubo, Takaharu Ueno, Tetsuhiko Yoshimura, Takashi Suzuki)
ABSTRACTh%tj, 百拇医药
TOPh%tj, 百拇医药
ABSTRACTh%tj, 百拇医药
INTRODUCTIONh%tj, 百拇医药
METHODSh%tj, 百拇医药
RESULTSh%tj, 百拇医药
DISCUSSIONh%tj, 百拇医药
REFERENCESh%tj, 百拇医药
Alveolar fluid clearance may be inhibited and/or stimulated under pathologic conditions. We examined the early change of alveolar fluid clearance after endotoxin instillation in adult rats. We employed electron paramagnetic resonance nitric oxide (NO) trapping technique with iron complex with N,N-diethyldithiocarbamate as an NO trapping agent. We found that lung NO signals reached the highest magnitude by 6 hours after endotoxin instillation. NO production was accompanied by increases in lung cyclic guanosine monophosphate levels. Alveolar fluid clearance decreased significantly 6 hours after the administration of the endotoxin and increased further at 24 hours. These changes were shown to be related to the function of amiloride-sensitive sodium ion channels. Treatment with gadolinium chloride and aminoguanidine significantly decreased lung NO and cyclic guanosine monophosphate levels and completely ameliorated the decrease in alveolar fluid clearance. In addition, the increase in alveolar fluid clearance at 24 hours returned to normal levels after treatment with gadolinium chloride and aminoguanidine. We found immunoreactive inducible nitric oxide synthase to be abundantly expressed in the cytoplasm of alveolar macrophages. Our results suggest that alveolar endotoxin inhibits alveolar fluid clearance at 6 hours by NO. NO is produced via inducible NO synthase in endotoxin-stimulated alveolar macrophages and was also shown to increase alveolar fluid clearance at 24 hours.
Key Words: inducible nitric oxide synthase • nitric oxide • electron paramagnetic resonance • alveolar fluid clearance • epithelial Na+ channelqc'r, 百拇医药
INTRODUCTIONqc'r, 百拇医药
TOPqc'r, 百拇医药
ABSTRACTqc'r, 百拇医药
INTRODUCTIONqc'r, 百拇医药
METHODSqc'r, 百拇医药
RESULTSqc'r, 百拇医药
DISCUSSIONqc'r, 百拇医药
REFERENCESqc'r, 百拇医药
Alveolar epithelium is the most likely site at which absorption of excess fluid from the alveolar space might take place (1–3). Alveolar epithelial cells express amiloride-sensitive sodium ion (Na+) channels (ENaCs) on the apical surface and Na+/potassium ion-ATPase on the basolateral side, which in turn results in the unidirectional movement of Na+ and fluids out of the alveolar space. It has been shown that alveolar epithelial active Na+ transport functions in many species, including humans (4) and plays a very important role in keeping the alveolar space relatively fluid-free for adequate gas exchange (5).
Alveolar fluid clearance may be inhibited and/or stimulated under pathologic conditions. It has been previously reported that alveolar endotoxin increased alveolar fluid clearance by stimulating alveolar ENaC function at 24 or 40 hours (6). However, alveolar endotoxin may also stimulate alveolar macrophages and produce nitric oxide (NO), which impairs lung epithelial Na+ transport in a very short period of time (minutes to hours) (7–9), via inducible nitric oxide synthase (iNOS) (10). Lipopolysaccharide-stimulated alveolar macrophages have been shown to inhibit the function of alveolar ENaCs by NO as early as 4 hours (11). However, the early changes in alveolar fluid clearance after endotoxin instillation has not been specifically addressed.2n6, http://www.100md.com
In the present study, we determined the early time course of lung NO levels and alveolar fluid clearance after endotoxin instillation in rats. We then examined whether the inhibition of lung NO production would influence alveolar fluid clearance. Our results appear to suggest that alveolar endotoxin inhibits alveolar fluid clearance at 6 hours by NO, which is produced via iNOS in endotoxin-stimulated alveolar macrophages. In addition, the increase in alveolar fluid clearance 24 hours after endotoxin instillation was also mediated by alveolar macrophage-derived NO.
METHODSoh#z, http://www.100md.com
TOPoh#z, http://www.100md.com
ABSTRACToh#z, http://www.100md.com
INTRODUCTIONoh#z, http://www.100md.com
METHODSoh#z, http://www.100md.com
RESULTSoh#z, http://www.100md.com
DISCUSSIONoh#z, http://www.100md.com
REFERENCESoh#z, http://www.100md.com
Animalsoh#z, http://www.100md.com
Specific, pathogen-free male Sprague–Dawley rats (250–350 g) were used in this study (n = 157). All animals received humane care, in compliance with the guidelines of the University Committee on Animal resources, Tohoku University, Sendai, Japan. All animal experiments were conducted in accordance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication 86-23 1985).oh#z, http://www.100md.com
Endotoxin Instillationoh#z, http://www.100md.com
Rats were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally). Endotoxin (100 µg/kg of O55:B5 Escherichia coli; Difco Laboratories, Detroit, MI) was suspended in 0.2 ml of sterilized saline and introduced directly into the trachea via a 26-gauge needle. To examine whether endotoxin altered alveolar fluid clearance via alveolar macrophages, rats were pretreated with an intraperitoneal injection of 60 mg/kg of gadolinium chloride (GdCl3) (Wako, Osaka, Japan) in 0.5 ml of sterilized saline 72 hours before endotoxin instillation. GdCl3 was reported to induce apoptosis of macrophages (12) and inhibit the inflammatory response of alveolar macrophages (13). To examine whether endotoxin altered alveolar fluid clearance via iNOS, rats were treated with a subcutaneous injection of 75 mg/kg of aminoguanidine (Sigma Chemical, St. Louis, MO), an iNOS inhibitor (14), in 0.5 ml of sterilized saline 10 minutes before endotoxin instillation.
Measurements1}aev{}, http://www.100md.com
Lung NO production was determined by recording a specific signal from the NO adduct of iron complex with N,N-diethyldithiocarbamate via electron paramagnetic resonance (15). Electron paramagnetic resonance spectra were recorded as three lines typical of NO–iron–N, N-diethyldithiocarbamate complex (16) using an X-band spectrometer (TE-200 type; JEOL, Tokyo, Japan) at room temperature. Cyclic guanosine monophosphate (cGMP) was measured using a cGMP enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Additional details on the method for lung tissue preparation and assay conditions are provided in the online supplement.1}aev{}, http://www.100md.com
Alveolar fluid clearance was measured in isolated rat lungs and anesthetized rats by determining the increase in albumin concentration in the instillate solution (sodium chloride 140 mM, potassium chloride 5 mM, calcium chloride 1 mM, magnesium chloride 1 mM, D-glucose 5 mM, HEPES 6 mM, bovine serum albumin 4 g/dl, pH 7.1) as described elsewhere (17). In addition, the effects of 0.1 mM amiloride (Sigma) and 1 mM terbutaline (Sigma) in the instillate solution were also examined in isolated rat lungs. Anesthetized rats were used to examine the impact of reduced alveolar fluid clearance on arterial blood gases. Additional details on the method for lung tissue preparation and calculation of alveolar fluid clearance are provided in the online supplement.
Lung extravascular water content was assessed by calculating the wet/dry weight ratio corrected for lung hemoglobin concentration (18).|5, 百拇医药
For histologic examinations, lungs were inflation-fixed with 4% paraformaldehyde (PFA) at 10 cm H2O of airway pressure. Immunohistochemistry was also performed using a polyclonal anti-mouse macrophage iNOS antibody (N32030; Transduction Laboratories, Lexington, KY). Electron microscopic examination was also performed after the standard tissue fixation and staining steps.|5, 百拇医药
Statistics|5, 百拇医药
All data are presented as mean ± SEM. Statistical analyses were performed by using analysis of variance and the Fisher's exact test. Statistical significance was defined as p values less than 0.05.|5, 百拇医药
RESULTS|5, 百拇医药
TOP|5, 百拇医药
ABSTRACT|5, 百拇医药
INTRODUCTION|5, 百拇医药
METHODS|5, 百拇医药
RESULTS6., 百拇医药
DISCUSSION6., 百拇医药
REFERENCES6., 百拇医药
A specific NO signal was detected with the highest magnitude 6 hours after endotoxin instillation and then decreased toward baseline levels . The vehicle control experiment (n = 3) showed no significant difference in lung NO levels 6 hours from levels at time 0. There was a significant increase in lung cGMP levels as early as 3 hours, which increased to a maximal level at 6 hours, after which lung cGMP levels decreased toward baseline levels . Tissues obtained from vehicle-treated animals (n = 4) showed no significant difference in lung cGMP levels at 6 hours from baseline values. The increase in lung NO and cGMP levels at 6 hours after endotoxin instillation were significantly reduced by treatment with GdCl3 and aminoguanidine .6., 百拇医药
fig.ommitted6., 百拇医药
Figure 1. Changes in lung NO levels after endotoxin instillation. (A) Specific signals of NO–iron–N,N-diethyldithiocarbamate complex were recorded 6 hours after endotoxin instillation with the highest magnitude. All spectra were measured in the same instrumental settings as shown in the online supplement. (B) Quantification of lung NO levels showed a rapid increase between 3 and 6 hours and a rapid decrease 12 hours after endotoxin instillation. Data are presented as mean ± SEM, n = 5 for each time point. *p Values less than 0.05 versus values at time 0 (without endotoxin) by analysis of variance.
fig.ommitted%)slt, http://www.100md.com
Figure 2. Changes in lung cGMP levels after endotoxin instillation. Lung cGMP concentrations increased to maximum levels at 6 hours after endotoxin instillation. A significant increase was observed as early as 3 hours. Data are presented as mean ± SEM, n = 5 for each time point. *p Values less than 0.05 versus values at time 0 (without endotoxin) by analysis of variance.%)slt, http://www.100md.com
fig.ommitted%)slt, http://www.100md.com
TABLE 1. Effects of gadolinium chloride and aminoguanidine on lung nitric oxide and cyclic guanosine monophosphate levels%)slt, http://www.100md.com
There was no significant change in alveolar fluid clearance determined in isolated rat lungs in the first 3 hours; however, a significant decrease was observed at 6 hours . At 12 hours after endotoxin instillation, alveolar fluid clearance had returned to baseline levels and further increased at 24 hours . Alveolar fluid clearance in vehicle-treated control specimens (n = 5) did not change significantly from baseline values at 6 hours. There was no significant change in alveolar fluid clearance determined in the presence of 0.1 mM amiloride (amiloride-insensitive component) over 24 hours, and there was no significant difference in alveolar fluid clearance determined in the presence or absence of 0.1 mM amiloride at 6 hours . Terbutaline significantly increased alveolar fluid clearance in the control specimens and also at 6 hours after endotoxin instillation . The terbutaline-stimulated alveolar fluid clearance was significantly inhibited by 0.1 mM amiloride . When alveolar fluid clearance was plotted versus amiloride concentrations, there were no significant differences in alveolar fluid clearance determined in the presence of amiloride at 0.01, 0.1, and 1 mM between control experiment and 6 hours after endotoxin instillation , indicating that amiloride had similar effects on alveolar fluid clearance in both experimental conditions. The amiloride half-maximal inhibitory concentration in the control experiment was estimated to be less than 0.01 mM ). In contrast, 0.01 mM amiloride showed no significant effect on alveolar fluid clearance at 6 hours after endotoxin instillation .
fig.ommitted!%(, 百拇医药
Figure 3. Changes in alveolar fluid clearance after endotoxin instillation. Alveolar fluid clearance determined in isolated rat lungs decreased significantly at 6 hours, and returned to control levels, and further increased at 24 hours. In contrast, there were no significant changes in alveolar fluid clearance determined in the presence of amiloride over the 24 hours. There was no inhibitory effect of amiloride on alveolar fluid clearance at 6 hours. Terbutaline significantly increased alveolar fluid clearance in control specimens and also at 6 hours after endotoxin instillation. The terbutaline-stimulated alveolar fluid clearance was inhibited by amiloride. Data are presented as mean ± SEM, n = 5 for each experiment at each time point, excepting n = 3 for terbutaline + amiloride experiments. *p Values less than 0.05 versus values without endotoxin by analysis of variance. p Values less than 0.05 versus values of no inhibitor by analysis of variance. p Values less than 0.05 versus values of terbutaline by analysis of variance.!%(, 百拇医药
fig.ommitted!%(, 百拇医药
Figure 4. Effects of amiloride on alveolar fluid clearance. There were no significant differences in alveolar fluid clearance determined in the presence of amiloride at 0.01, 0.1, and 1 mM between control experiment and 6 hours after endotoxin instillation. The amiloride half-maximal inhibitory concentration was found to be less than 0.01 mM M in control experiment; however, 0.01 mM amiloride did not show any significant inhibition of alveolar fluid clearance in endotoxin experiments. Data are presented as mean ± SEM, n = 3 for each experiment at each time point, excepting n = 5 for the values of no amiloride and 10-4 M amiloride.
Neither GdCl3 nor aminoguanidine treatment altered alveolar fluid clearance determined in isolated rat lungs in vehicle-treated control specimens; however, both agents ameliorated the decrease in alveolar fluid clearance at 6 hours . In addition, the increase at 24 hours had returned to baseline levels by pretreatment with GdCl3 and aminoguanidine .c?x, 百拇医药
fig.ommittedc?x, 百拇医药
Figure 5. Effects of treatment with GdCl3 and aminoguanidine on alveolar fluid clearance. Both GdCl3 and aminoguanidine ameliorated the decrease in alveolar fluid clearance at 6 hours after endotoxin instillation. At 24 hours, GdCl3 and aminoguanidine changed alveolar fluid clearance to values seen at time 0. Data are presented as mean ± SEM, n = 5 for each time point. *p Values less than 0.05 versus values of no treatment by analysis of variance.c?x, 百拇医药
There was no significant difference in alveolar fluid lactate dehydrogenase levels or lung wet/dry weight ratio between the vehicle control and endotoxin experiments .
fig.ommitted@?5u, 百拇医药
TABLE 2. Alveolar fluid lactate dehydrogenase levels and lung wet/dry weight ratio@?5u, 百拇医药
In anesthetized rats, instillation of the solution into the left lung caused a decrease in PaO2 in both control and endotoxin experiments. However, the difference in PaO2 before introduction compared to that 1 hour after introduction of the instillate solution was significantly larger in endotoxin experiments . Treatment with GdCl3 and aminoguanidine ameliorated the arterial blood gas tension and alveolar fluid clearance .@?5u, 百拇医药
fig.ommitted@?5u, 百拇医药
TABLE 3. Alveolar fluid clearance and the difference in paO2 between before introduction and 1 hour after introduction of the instillate solution@?5u, 百拇医药
The lungs at 6 hours after endotoxin instillation were of normal appearance macroscopically. Light microscopic examinations (n = 2) showed no considerable fluid accumulation or neutrophil infiltration in the alveolar space and/or interstitium. Electron microscopic examinations (n = 2) also showed no ultrastructural evidence for damages in alveolar epithelial cells and capillary endothelial cells. Positive immunoreactivity for iNOS was predominantly detected in the cytoplasm of alveolar macrophages but was not detectable in alveolar epithelial cells or mesenchymal cells, at 6 hours after endotoxin instillation (n = 2) . In the control specimens, immunoreactivity for iNOS was not detected in the alveolar area or airway epithelia (n = 2).
fig.ommitted-c, http://www.100md.com
Figure 6. Immunohistochemistry for iNOS. No immunoreactivity for iNOS was observed in control experiments (A). At 6 hours after endotoxin instillation, positive immunoreactivity for iNOS was detected in the cytoplasm of alveolar macrophages, whereas no detectable immunoreactivity was observed in alveolar epithelial cells or mesenchymal cells (B). Results were representative of two different lung specimens. Original magnification: x400.-c, http://www.100md.com
DISCUSSION-c, http://www.100md.com
TOP-c, http://www.100md.com
ABSTRACT-c, http://www.100md.com
INTRODUCTION-c, http://www.100md.com
METHODS-c, http://www.100md.com
RESULTS-c, http://www.100md.com
DISCUSSION-c, http://www.100md.com
REFERENCES-c, http://www.100md.com
Tissue NO levels may be examined by detecting nitrogen dioxide and nitrate ions, stable end products of NO, using commercially available detection kits. However, the results may only be estimates of the amount of NO that had been produced before tissue preparation, and thus may not represent the exact NO levels at a specific time point. We therefore decided to use electron paramagnetic resonance to determine the time course of NO production after endotoxin instillation. It was previously reported that activated peritoneal macrophages exhibit a characteristic electron paramagnetic resonance signal at cryogenic temperatures (19), and the signal was identified as that of the dinitrosyl dithiolato iron complex, which was formed by the reaction of NO with intracellular iron–sulfur proteins (20). In this study, we decided to employ a direct detection of NO that is trapped with the iron–N,N-diethyldithiocarbamate complex, and the resultant NO complex exhibits a three-line electron paramagnetic resonance signal at room temperature as shown in Figure 1A. A similar technique with the iron–N,N-diethyldithiocarbamate complex as an NO trapping agent has been used at cryogenic temperatures in lung tissues (21). We found that lung NO levels reached the highest levels at 6 hours and rapidly returned toward baseline values over the next 18 hours. This time course was very similar to that of cGMP, the most potent intracellular second messenger of NO (22). These results, therefore, suggest that endotoxin instillation influences local lung cellular functions via NO and cGMP, although a significant increase in lung cGMP levels at 3 hours may be related to a direct action of endotoxin on guanylate cyclase (23).
It is yet to be found what cell type is responsible for the production of NO in the lung remains unknown. We found that the increase in lung NO and cGMP levels at 6 hours after endotoxin instillation were significantly inhibited by treatment with GdCl3. GdCl3 was reported to induce apoptosis of macrophages (12) and inhibit the inflammatory response of alveolar macrophages (13). We examined the effect of GdCl3 on the cell count of alveolar macrophages in bronchoalveolar lavage fluid and found a significant decrease (approximately 25% of that of control specimens) at 72 hours (data not shown). This finding therefore appears to suggest that alveolar macrophages may play an important role in lung NO production. In addition, treatment with aminoguanidine displayed a similar inhibitory effect on lung NO and cGMP levels. Moreover, histochemical examination revealed abundant expression of iNOS in the cytoplasm of alveolar macrophages 6 hours after endotoxin instillation, whereas no detectable immunoreactivity was observed in other cell types such as alveolar epithelial cells or mesenchymal cells. It is very likely, therefore, that alveolar macrophages expressing iNOS are the most important source of endotoxin-induced lung NO production.
Alveolar fluid clearance was significantly decreased at 6 hours after endotoxin instillation, at a time when lung NO and cGMP levels were highest. Because amiloride did not show any inhibitory effect, the decrease in alveolar fluid clearance may be related to an impairment in alveolar ENaC function and/or a change in the amiloride half-maximal inhibitory concentration. We examined the effect of amiloride on alveolar fluid clearance at higher and lower concentrations. We found a similar dose-dependent inhibition of alveolar fluid clearance in endotoxin experiments. This suggests that endotoxin did not alter half-maximal inhibitory concentration for amiloride. The endotoxin-induced decrease in alveolar fluid clearance was completely ameliorated by treatment with GdCl3 and aminoguanidine. This finding suggests that NO produced via probably iNOS in alveolar macrophages may be responsible for impairing alveolar ENaC function. There are several lines of evidence indicating that ENaC function is inhibited by NO in a short period of time (8, 9, 11). NO increases cGMP levels in target cells, which in turn alters the probability that ion channels may be found in the open state (24). In addition, the decrease in alveolar fluid clearance was reversed by intra-alveolar administration of terbutaline, and the terbutaline-stimulated alveolar fluid clearance was predominantly related to the amiloride-sensitive component. Terbutaline has been shown to increase intracellular adenosine 3',5'-cyclic monophosphate, stimulate alveolar ENaC (25, 26), and increase alveolar fluid clearance in rats (2, 27). The recovery of alveolar fluid clearance with terbutaline suggests that the decrease in the alveolar ENaC function is related to a change in individual channel activity rather than a downregulation in the expression of channel proteins.
It is important to consider that aminoguanidine at high concentrations may inhibit not only iNOS but also other types of NOS. Although aminoguanidine has been used as a specific iNOS inhibitor (14), the specificity on iNOS is lower than that of 1400W, a newly synthesized potent and selective iNOS inhibitor (28). In addition, it has been shown that iNOS-deficient mice did not display amiloride-sensitive alveolar fluid clearance, and upregulation of iNOS by hyperoxia in iNOS (+/+) mice had similar levels of alveolar fluid clearance in iNOS (-/-) mice (29). iNOS (-/-) mice are also able to produce nitrothyosine in the lungs when infected with mycoplasmas, suggesting that other types of NOS may play a role in the generation of reactive oxygen–nitrogen intermediates (30). In addition, instillation of DETANONOate, an NO donor, reduced the amiloride-sensitive component of alveolar fluid clearance; however, amiloride-insensitive fraction was increased, and the total alveolar fluid clearance was preserved at the control levels (31). Therefore, it may be possible that multiple mechanisms in addition to NO production via iNOS are involved in the modulation of alveolar fluid clearance by endotoxin.
After an early decrease, alveolar fluid clearance had recovered by 12 hours, and increased further over baseline levels by 24 hours. This late increase was related to the amiloride-sensitive component of alveolar fluid clearance. However, Garat and coworkers reported that the increase in sodium influx was not inhibited by propranolol, suggesting that endogenous catecholamines were not responsible for stimulating alveolar ENaCs (6). In this study, we found that treatment with GdCl3 and aminoguanidine returned alveolar fluid clearance at 24 hours to baseline levels. This observation may suggest that NO also plays a role in the mechanism leading to an increment in sodium influx in the alveolar epithelium. However, it is unlikely that NO and cGMP display such dual actions on channel function, such as early inhibition followed later by stimulation. In addition, lung NO and cGMP levels 24 hours after endotoxin instillation were low.'jp, 百拇医药
NO is well known to influence pulmonary circulation, reducing pulmonary vascular resistance (32) and coefficient filtration (33). However, these protective effects of NO in opposing lung fluid filtration in vivo do not explain the changes in alveolar fluid clearance examined in isolated rat lungs. A previous study has shown that cGMP may be a competitive signaling molecule with respect to the transport of adenosine 3',5'-cyclic monophosphate out of the cell via a membrane-bound transporter (34). An increase in cGMP may limit adenosine 3',5'-cyclic monophosphate transport out of the cell, thereby trapping this molecule within the cell, which in turn may stimulate channel function and alveolar fluid clearance. However, it is difficult to measure exact adenosine 3',5'-cyclic monophosphate concentrations in alveolar epithelial cells obtained from lung tissue homogenates of endotoxin-challenged lungs. Moreover, the direct measurement of in vivo intracellular concentrations of adenosine 3',5'-cyclic monophosphate also presents the investigator with an incredibly challenging task.
In this study, we administrated endotoxin at a dose of 100 µg/kg. This dose was high enough to stimulate alveolar macrophages, but more importantly, low enough to avoid structural abnormalities in lung tissues. The fluid-filled lung model, using albumin concentration as an indicator of alveolar fluid clearance, does not measure unidirectional fluid transport separately, and the results may be influenced in the case that endotoxin instillation caused pulmonary edema. When we administrated the same E. coli endotoxin at 200 µg/kg, some rats did not survive beyond 12 hours, and when the lungs were examined, they were found to be reddish in color and edematous macroscopically. All rats receiving 100 µg/kg of endotoxin survived more than 24 hours, and when the lungs were examined in these animals, the lungs were of normal appearance. We were not able to demonstrate any considerable fluid accumulation in the interstitium and/or alveolar space of the lungs by light microscopy. Electron microscopic examination also showed no abnormalities in lung cells including alveolar epithelial cells. There was no significant difference in lung wet/dry weight ratio or alveolar epithelial lactate dehydrogenase levels at the completion of 2 hours of the incubation period between control and endotoxin experiments. In addition, we examined possible leak of blood-borne particles into the alveolar apace by injecting Evans blue dye before lung isolation and found that endotoxin did not increase the concentration of Evans blue dye in the recovered alveolar fluids (data not shown). It is very unlikely, therefore, that endotoxin altered alveolar epithelial and endothelial barrier function.
In summary, our results appear to suggest that endotoxin administered into rat lungs inhibits alveolar fluid clearance followed by an increase at a later phase. Both the early decrease and late increase in alveolar fluid clearance may be mediated by NO, which is produced probably via iNOS in endotoxin-stimulated alveolar macrophages. We speculate that the early decrease in alveolar fluid clearance in endotoxin-induced pulmonary inflammation may play a role in keeping and/or recovering intact alveolar barrier functions. A decrease in alveolar fluid clearance may allow for the fast diffusion of growth factors into alveolar fluids, which in turn may modulate alveolar epithelial proliferation and differentiation, and thereby accelerate the recovery of an injured alveolar epithelium.37c1u|, 百拇医药
REFERENCES37c1u|, 百拇医药
TOP37c1u|, 百拇医药
ABSTRACT37c1u|, 百拇医药
INTRODUCTION37c1u|, 百拇医药
METHODS37c1u|, 百拇医药
RESULTS
DISCUSSION2, 百拇医药
REFERENCES2, 百拇医药
Mason RJ, Williams MC, Widdicombe JH, Sanders MJ, Misfeldt DS, Berry LC. Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc Natl Acad Sci USA 1982;79:6033–6037.2, 百拇医药
Effros RM, Mason GR, Sietsema K, Silverman P, Hukkanen J. Fluid reabsorption and glucose consumption in edematous rat lungs. Circ Res 1987;60:708–719.2, 百拇医药
Matthay MA, Folkesson HG, Verkman AS. Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am J Physiol Lung Cell Mol Physiol 1996;270:L487–L503.2, 百拇医药
Sakuma T, Okaniwa G, Nakada T, Nishimura T, Fujimura S, Matthay MA. Alveolar fluid clearance in the resected human lung. Am J Respir Crit Care Med 1994;150:305–310.2, 百拇医药
Matthay MA, Wiener-Kronish JP. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am Rev Respir Dis 1990;142:1250–1257.2, 百拇医药
Garat C, Rezaiguia S, Meignan M, D'Ortho MP, Harf A, Matthay MA, Jayr C. Alveolar endotoxin increases alveolar liquid clearance in rats. J Appl Physiol 1995;79:2021–2028.
Compeau CG, Rotstein OD, Tohda H, Marunaka Y, Rafii B, Slutsky AS, O'Brodovich H. Endotoxin-stimulated alveolar macrophages impair lung epithelial Na+ transport by an L-Arg-dependent mechanism. Am J Physiol Cell Physiol 1994;266:C1330–C1341.l, http://www.100md.com
Guo Y, DuVall MD, Crow JP, Matalon S. Nitric oxide inhibits Na+ absorption across cultured alveolar type II monolayers. Am J Physiol Lung Cell Mol Physiol 1998;274:L369–L377.l, http://www.100md.com
Jain L, Chen XJ, Brown LA, Eaton DC. Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels. Am J Physiol Lung Cell Mol Physiol 1998;274:L475–L484.l, http://www.100md.com
Warner RL, Paine R III, Christensen PJ, Marletta MA, Richards MK, Wilcoxen SE, Ward PA. Lung sources and cytokine requirements for in vivo expression of inducible nitric oxide synthase. Am J Respir Cell Mol Biol 1995;2:649–661.l, http://www.100md.com
Ding JW, Dickie J, O'Brodovich H, Shintani Y, Rafii B, Hackam D, Marunaka Y, Rotstein OD. Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide. Am J Physiol Lung Cell Mol Physiol 1998;274:L378–L387.
Mizgerd JP, Molina RM, Stearns RC, Brain JD, Warner AE. Gadolinium induces macrophage apoptosis. J Leukoc Biol 1996;59:189–195.&&, http://www.100md.com
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