Hypoxia upregulates lung microvascular neurokinin-1 receptor expression

Eric D. Zee, Stacey Schomberg, Todd C. Carpenter

Abstract

Subacute exposure to moderate hypoxia can promote pulmonary edema formation. The tachykinins, a family of proinflammatory neuropeptides, have been implicated in the pathogenesis of pulmonary edema in some settings, including the pulmonary vascular leak associated with exposure to hypoxia. The effects of hypoxia on tachykinin receptor and peptide expression in the lung, however, remain poorly understood. We hypothesized that subacute exposure to moderate hypoxia increases lung neurokinin-1 (NK-1) receptor expression as well as lung substance P levels. We tested this hypothesis by exposing weanling Sprague-Dawley rats to hypobaric hypoxia (barometric pressure 0.5 atm) for 0, 24, 48, or 72 h. Hypoxia led to time-dependent increases in lung NK-1 receptor mRNA expression and lung NK-1 receptor protein levels at 48 and 72 h of exposure (P < 0.05). Immunohistochemistry and in situ NK-1 receptor labeling with substance P-conjugated fluorescent nanocrystals demonstrated that hypoxia increased NK-1 expression primarily in the pulmonary microvasculature and in alveolar macrophages. Hypoxia also led to increases in lung substance P levels by 48 and 72 h (P < 0.05) but led to a decrease in preprotachykinin mRNA levels (P < 0.05). We conclude that subacute exposure to moderate hypoxia upregulates lung NK-1 receptor expression and lung substance P peptide levels primarily in the lung microvasculature. We speculate that this effect may contribute to the formation of pulmonary edema in the setting of regional or environmental hypoxia.

  • tachykinin
  • pulmonary edema
  • acute respiratory distress syndrome

acute lung injury and acute respiratory distress syndrome (ARDS) are clinical syndromes associated with significant morbidity and mortality in humans. The hallmarks of these syndromes are increased pulmonary capillary permeability, pulmonary edema formation, and profound hypoxemia (22). The precise mechanisms that lead to this pulmonary vascular leak remain uncertain (3, 21, 27), although many inflammatory and vasoactive substances have been implicated.

Among the mediators implicated in the development of pulmonary edema are the tachykinins. These molecules, a part of the nonadrenergic, noncholinergic nervous system, are a family of peptides that are expressed in the lung in and around the pulmonary vasculature as well as in sensory nerves located primarily near the airways. Substance P, the tachykinin peptide most highly expressed in the lung, exerts its effects primarily through the neurokinin-1 (NK-1) receptor. This ligand-receptor pair mediates neurogenic inflammation in the airways via cytokines including IL-1, IL-6, and TNF-α (14, 20). In addition, substance P levels are markedly elevated in pulmonary edema fluid from ARDS patients (6), and the NK-1 receptor has been implicated in the pathophysiology of lung injury associated with pancreatitis (13), smoke inhalation (30), and viral infections (23) including respiratory syncytial virus (11).

Previous work has also suggested that the tachykinin system may be altered in response to hypoxia. In particular, we previously demonstrated (5) that increases in pulmonary vascular albumin extravasation associated with short-term exposures to hypoxia are reversible with NK-1 receptor antagonism, suggesting a role for tachykinins in vascular permeability changes in the lung associated with brief exposures to hypoxia. Whether subacute exposures to hypoxia alter lung NK-1 receptor expression or lung substance P content, and in which cells those changes occur, however, remains uncertain.

We hypothesized, then, that subacute exposure to moderate hypoxia leads to upregulation of lung NK-1 receptor expression as well as increases in lung substance P content. We studied these questions in weanling rats exposed to hypobaric hypoxia. Lung NK-1 receptor mRNA and protein expression were evaluated by RT-PCR and Western blot, respectively. Changes in expression and location of lung NK-1 receptors were evaluated with standard immunohistochemistry techniques as well as a novel in situ receptor binding technique using fluorescent semiconductor nanocrystals conjugated to substance P. Lung substance P peptide content was measured by ELISA, and lung preprotachykinin (PPT) mRNA expression was measured by RT-PCR.

MATERIALS AND METHODS

Care of animals.

Experimental animals were pathogen-free weanling male Sprague-Dawley rats purchased from a commercial vendor (Harlan Sprague Dawley, Indianapolis, IN). The animals arrived in Denver at age 20–22 days, weighed 50–60 g each, and acclimated to Denver altitude (1,600 m) for 7 days before hypoxic exposure. All rats were allowed free access to food and water and were subjected to a day-night cycle. The University of Colorado Health Sciences Center Institutional Animal Care and Use Committee approved all animal protocols.

Exposure to hypoxia.

Experimental animals were exposed to hypobaric hypoxia (barometric pressure of 0.5 standard atmospheres, equivalent to an altitude of 17,000 feet above sea level) for 24, 48, or 72 h. Control animals were maintained at Denver altitude. At the end of the experimental hypoxic exposure, animals were rapidly anesthetized with halothane, the lungs were flushed clear of blood with PBS infused via the pulmonary artery, and the heart-lung block was removed. The right lung was snap frozen in liquid nitrogen for later assays of protein and mRNA levels. The left lung was inflated with optimum cutting temperature (OCT) compound (Tissue-Tek, Sakura, Torrance, CA) via the trachea and stored frozen until being used for histological studies. Lungs from normoxic animals and 72-h hypoxic animals were also fixed by tracheal perfusion with zinc formalin at 25 cmH2O pressure for 24 h and then paraffin embedded and sectioned for immunohistochemical studies.

Western blotting for NK-1 receptor.

To determine whether hypoxia alters lung NK-1 receptor protein expression, lung homogenates were studied by standard Western blot techniques. To reduce the multiple nonspecific bands obtained on the blots with whole lung homogenates for these studies (data not shown), crude membrane fractions were prepared by differential centrifugation and used as samples for the Western blotting studies. Membrane fractions were isolated as previously described (4). Samples were separated on a precast 4–12% Bis-Tris acrylamide gel (NuPAGE, Invitrogen Life Technologies, Carlsbad, CA) by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ) with a wet transfer apparatus. The membranes were probed with a rabbit polyclonal antibody to the NK-1 receptor (anti-substance P receptor, Sigma, St. Louis, MO). The primary antibody was detected with an anti-rabbit peroxidase-conjugated secondary antibody. The blots were visualized with a chemiluminescent reagent (Western Lightning, Perkin Elmer Life Sciences, Boston, MA) and exposure to film. The membranes were reprobed with a monoclonal antibody to β-actin (Sigma) to confirm equal protein loading. The resulting images were scanned into a computer and analyzed by optical densitometry. Results are shown in arbitrary units, expressed as a ratio of NK-1 receptor to β-actin in each lane.

RT-PCR studies.

The expression of NK-1 receptor and PPT mRNA was assessed by relative RT-PCR. Total lung mRNA was isolated with Tri reagent (Sigma), following the manufacturer's instructions. Reverse transcription to cDNA was performed by using oligo(dT) primers and a Superscript II reverse transcriptase kit (Invitrogen Life Technologies). The resulting cDNA was then treated with RNase H to remove untranscribed RNA. The cDNA was diluted 1:10 in nuclease-free water, and PCR amplification was performed on 2 μl of diluted cDNA from each sample. The primers used to amplify NK-1 receptor mRNA were 5′-TGAGCAAGTCTCTGCCAAACG-3′ and 5′-GGTATCGGGTGGATTTCATTTCC-3′. In the case of PPT, four alternatively spliced products for the PPT-A gene have been described. Substance P is encoded by all four transcripts, but the β- and γ-transcripts also encode neurokinin A. The primers used to amplify PPT mRNA (5′-CCTTTGAGCATCTTCAGAG-3′ and 5′-GTAGTTCTGCATTGCGCTTCT-3′) were previously described (16) to amplify all four alternative transcripts of the PPT-A gene. We identified a band representing the β-isoform (235 bp) and a single band representing both the α- and γ-isoforms (181 and 190 bp, respectively) in all samples tested. We did not find the smaller band representing the δ-isoform in any samples.

As an internal control for each PCR reaction, RT-PCR amplification of β-actin mRNA was performed as previously described (5). The primers used spanned intron 3 of the rat β-actin gene, providing a control for genomic DNA contamination of the cDNA templates. No genomic DNA contamination was noted in any of the samples used in these studies (data not shown). For all three targets, preliminary control reactions with increasing cycle numbers confirmed that the reaction for the given product was in the exponential phase under the cycling conditions used (data not shown). PCR products were loaded onto a 1.5% agarose-Tris-acetate-EDTA gel, separated by electrophoresis, and visualized with ethidium bromide. Gels were photographed under UV illumination, and the images were scanned into the computer for optical densitometry analysis. Results were expressed as the ratio of NK-1 receptor or PPT PCR product to β-actin for each sample.

Immunohistochemistry.

To determine the anatomic location of NK-1 receptor expression changes within the lung, zinc formalin-fixed, paraffin-embedded whole lung sections were stained for the NK-1 receptor with standard immunohistochemical techniques. Briefly, lung sections were rehydrated with graded ethanol washes, blocked with 5% BSA, and incubated with a goat polyclonal antibody to the amino terminus of the NK-1 receptor (N-19, Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. To confirm the specificity of the antibody staining, additional sections were stained with the primary antibody preincubated with blocking peptide (N-19P, Santa Cruz Biotechnology) before application to the lung sections. The secondary antibody was a biotinylated chicken anti-goat IgG (chicken anti-goat IgG-B, Santa Cruz Biotechnology). Slides were developed with a commercially available kit and substrate (VECTASTAIN kit and Vector Red substrate, Vector Laboratories, Burlingame, CA) and were counterstained with hematoxylin, rehydrated, and permanently mounted.

To determine the anatomic location of changes in substance P peptide levels within the lung, additional sections from normoxic and hypoxic lungs were incubated with a polyclonal rabbit antibody specific for substance P (Chemicon, Temecula, CA) followed by detection and visualization with a commercial peroxidase immunostaining kit (Vector Laboratories).

Substance P-labeled fluorescent nanocrystal in situ tissue binding.

To confirm the findings of the immunohistochemical studies and eliminate possible confounding effects of nonspecific cross-reactive epitopes in the lung, we devised a technique to detect NK-1 receptors in tissue sections using in situ binding of substance P peptide conjugated to fluorescent semiconductor nanocrystals (EviTags, Evident Technologies, Troy, NY). Substance P (AnaSpec, San Jose, CA) was conjugated on its amino terminus to carboxy-terminated EviTags with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysulfosuccinamide (Sigma) linking according to the suggested protocol of the manufacturer, using a substance P-to-EviTag molar ratio of 185:1 for the binding reaction. We chose to conjugate substance P on its amino terminus to avoid possible steric hindrance of the binding region, which is located on the carboxy terminus of the peptide (28). The substance P-EviTag conjugates were then incubated with unfixed frozen tissue sections with a modification of a previously published technique (24). The binding procedure was accomplished by using three buffers: buffer 1 (170 mM Tris·HCl, pH 7.4 with 0.02% BSA), buffer 2 (buffer 1 with 3 mM MnCl2) and buffer 3 (buffer 2 with 40 μg/ml bacitracin, 4 μg/ml leupeptin, and 5 mM phosphoramidon). The freshly cut sections were allowed to air dry at 4°C, rinsed in cold PBS for 5 min, and then rinsed in buffer 1 for 5 min to block the sections and to wash out endogenous substance P. The sections were subsequently incubated with substance P-labeled EviTags at a 1:100 dilution in buffer 3 for 60 min and then washed in buffer 2 for 15 min. The slides were then rinsed briefly in H2O and allowed to air dry. To identify specific NK-1 receptor binding, serial sections were preincubated with a specific NK-1 receptor antagonist (RP67580, Tocris, Ellisville, MO) for 30 min and then incubated with 7 μM RP67580 and substance P-labeled EviTags in buffer 3 as above. As an additional control for nonspecific binding, sections were also incubated with substance P-EviTags in the presence of excess unlabeled substance P. After the washes, sections were fixed with 2% paraformaldehyde for 5 min. To better visualize the underlying tissue structure, some sections were also poststained with a nuclear dye (100 μM Hoechst 33342, Molecular Probes) for 2 min and then rinsed again in PBS before visualization. The sections were then visualized under UV illumination, and images were captured with a digital camera.

Quantification of substance P.

To determine whether subacute hypoxia also changes the lung expression of the primary ligand for the NK-1 receptor, substance P peptide was extracted from whole lung homogenates and quantified with a commercial substance P enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, MI). Frozen lung was homogenized in 2 M acetic acid (100 mg tissue/ml). The homogenate was boiled for 30 min and then cooled at room temperature for 3 min. After cooling, the homogenate was centrifuged for 15 min at 13,000 rpm. The supernatant was collected and placed in a separate tube. The pellet was resuspended in 2 M acetic acid and boiled again for 30 min. After cooling, the homogenate was centrifuged again at 13,000 rpm for 15 min. The resulting supernatant was removed and pooled with the previous supernatant. The pooled supernatants were dried under vacuum centrifugation and were resuspended with EIA buffer from the kit to the original volume. Substance P was then quantified per the manufacturer's instructions. Results are expressed in picograms of substance P per gram of lung tissue.

Measurement and localization of vascular leak.

To confirm our previous findings that hypoxia increases vascular leak in the distal lung, leak was measured as extravasation of Evans blue-labeled albumin into the distal lung as previously described (5). To determine whether NK-1 receptor activation still contributes to the ongoing vascular leak seen after 72 h of hypoxia, animals were exposed to hypoxia for 72 h and then injected subcutaneously with either an NK-1 receptor antagonist (RP67580, 15 mg/kg) or vehicle control. The animals were then returned to hypoxia, and albumin extravasation was measured 2 h later as above.

To determine whether sites of increased NK-1 receptor expression in the hypoxic lung are also sites of vascular leak, we first determined the sites of vascular leak with a modification of a previously published technique for labeling sites of vascular leak by fluorescent microsphere extravasation (2). Animals exposed to either normoxia (n = 3) or hypoxia for 72 h (n = 3) were injected via the tail vein under ketamine anesthesia with 0.5 cm3/kg of 100-nm-diameter red fluorescent microspheres (Duke Scientific, Palo Alto, CA). After the microspheres were allowed to circulate for 2 min, the animals were heparinized and the pulmonary circulation was flushed clear of blood via the pulmonary artery with PBS. The pulmonary circulation was then fixed by perfusion with 1% paraformaldehyde in PBS, the lungs were inflated via the trachea with OCT compound, and frozen sections were cut for microscopic visualization of the microspheres. NK-1 receptor expression in those same sections was then assessed by incubation with goat anti-NK1 antibody and detection with an anti-goat secondary antibody conjugated to green fluorescent nanocrystals (EviTags). Sections were postfixed in buffered formalin for 2 min and visualized under UV illumination.

Statistical analysis.

All results are expressed as means ± SD unless otherwise noted. Comparisons between multiple groups were analyzed with one-way ANOVA and Tukey's multiple-comparison posttesting. Statistical analyses were done with Prism 4 statistical analysis software (GraphPad Software, San Diego, CA) on an Apple Macintosh computer. Results were considered significant when P < 0.05.

RESULTS

Hypoxia increases lung NK-1 receptor mRNA expression.

To determine whether exposure to hypoxia led to increases in NK-1 receptor gene transcription, lung NK-1 receptor mRNA expression was studied by relative RT-PCR. As shown in Fig. 1, hypoxia led to a progressive increase over time in lung NK-1 receptor mRNA levels. When compared with normoxic control animals (n = 4), animals exposed to 48 (n = 3) or 72 (n = 5) h of hypoxia expressed significantly more lung NK-1 receptor mRNA (P < 0.01 and P < 0.001, respectively).

Fig. 1.

Hypoxia increases lung neurokinin-1 (NK-1) receptor (NK-1R) mRNA expression as assessed by relative RT-PCR. A: representative NK-1R PCR products on agarose gel. B: densitometry of NK-1R mRNA bands normalized to β-actin, expressed as arbitrary units. C, normoxic control. *P < 0.01, **P < 0.001 vs. normoxic controls.

Hypoxia increases lung NK-1 receptor protein expression.

To determine whether exposure to hypoxia alters lung NK-1 receptor protein expression, NK-1 receptor protein content was measured on crude membrane fractions of lung homogenates by Western blotting. As shown in Fig. 2, animals exposed to hypoxia for 48 (n = 4) or 72 (n = 5) h expressed significantly more lung NK-1 receptor protein than normoxic control animals (n = 4). Differences were statistically significant between all groups studied: between control and 48-h hypoxic (P < 0.01), control and 72-h hypoxic (P < 0.001), and 48-h and 72-h hypoxic animals (P < 0.01).

Fig. 2.

Hypoxia increases lung NK-1R protein expression. A: representative Western blot of NK-1R. B: densitometry of NK-1R bands normalized to β-actin, expressed as arbitrary units. *P < 0.01, **P < 0.001 vs. normoxic controls.

Hypoxia increases lung NK-1 receptor immunoreactivity and receptor binding.

To determine the anatomic location of changes in NK-1 receptor upregulation in the lung, NK-1 receptor expression was also studied by immunohistochemistry in normoxic control and 72-h hypoxic animals. As shown in Fig. 3, in normoxic lung only weak NK-1 receptor immunoreactivity was observed in alveolar septa, in alveolar macrophages, and in the endothelium of occasional small pulmonary vessels. Weak staining of large airway epithelium was also noted. After 72-h exposure to hypoxia, NK-1 receptor immunoreactivity was markedly increased in alveolar septa and alveolar macrophages, although the increase was patchy in distribution. Those areas most affected also often demonstrated thickened, edematous-appearing alveolar septa. Increased staining was also noted in some small pulmonary vessels in those same areas, although no change was noted in NK-1 staining in larger vessels or airways. Sections coincubated with NK-1 receptor antibody blocking peptide showed a near-complete lack of staining, confirming the specificity of the staining for the NK-1 receptor primary antibody.

Fig. 3.

Increased NK-1R immunoreactivity (red) in lungs of animals exposed to hypoxia for 72 h. A: normoxia. Note weak endothelial NK-1 staining in small pulmonary artery. B: normoxia. Note weak NK-1 staining in alveolar septae and alveolar macrophage (arrowhead). C: hypoxia 72 h. Note increased NK-1 staining in alveolar septa and small pulmonary vessels (arrow). D: hypoxia 72 h. Note increased NK-1 staining in alveolar septa and alveolar macrophage (arrowhead) and thickened alveolar septa. E: hypoxia 72 h, NK-1 staining after incubation with antibody blocking peptide. Scale bars = 50 μm.

As an alternative approach to identifying NK-1 receptor location in the lung, we developed a novel technique to identify NK-1 receptors in tissue sections in situ by using substance P conjugated to fluorescent semiconductor nanocrystals. As shown in Fig. 4 and consistent with our immunostaining results, normoxic lungs showed only very sparse fluorescent substance P binding along alveolar septa (Fig. 4, A and D) and in the endothelium of occasional small pulmonary vessels. Weak receptor binding was also noted in large airway epithelium. As with the immunostaining studies, however, exposure to 72 h of hypoxia led to patchy but marked increases in substance P binding, most notably in alveolar septa (Fig. 4, B, E, and H). In addition, some perivascular cells and small pulmonary vessels in those areas of increased alveolar signal also demonstrated intense substance P binding (Fig. 4G). No consistent change in large airway or large vessel labeling was noted with hypoxia. Sections preincubated with NK-1 receptor antagonist (RP67580) showed marked attenuation of the substance P signal (Fig. 4C), confirming the specificity of binding for the NK-1 receptor. Additional sections coincubated with excess unlabeled substance P also showed marked attenuation of signal (Fig. 4I). Sections poststained with Hoechst 33342 nuclear dye allowed more clear anatomic localization of the bound nanocrystals in the tissue section, although the nuclear dye staining and additional wash appeared to reduce the signal intensity from the nanocrystals (Fig. 4, D–G).

Fig. 4.

Lung NK-1R expression localized by in situ tissue binding of substance P labeled with fluorescent nanocrystals. A: normoxic control. B: 72-h hypoxia. C: 72-h hypoxia, section incubated with NK-1 antagonist RP67580. Sections in D–G were poststained with Hoechst 33342 nuclear dye. D: normoxia, alveoli. E: 72-h hypoxia, alveoli. F: normoxia, small pulmonary vessel. G: 72-h hypoxia, small pulmonary vessel. H: 72-h hypoxia. I: 72-h hypoxia, section incubated with excess unlabeled substance P. Scale bars = 50 μm.

Hypoxia increases lung substance P peptide levels.

Having determined that hypoxia increases lung NK-1 receptor expression, we sought to determine whether hypoxia might also alter lung levels of its peptide ligand, substance P. To answer this question, whole lung substance P content was determined by EIA. As shown in Fig. 5A, compared with normoxic control animals (661 ± 96.4 pg/mg lung tissue; n = 4), animals exposed to hypoxia for 48 h (1,185 ± 215.6 pg/mg lung tissue; n = 4) or for 72 h (1,511 ± 346.3 pg/mg lung tissue; n = 4) displayed a significantly greater lung substance P content (P < 0.05 and P < 0.01, respectively). Lung substance P content in animals exposed to hypoxia for 24 h (n = 3) was not significantly different from the controls.

Fig. 5.

Hypoxia increases lung substance P peptide content. A: lung substance P peptide content as assessed by ELISA. *P < 0.05, **P < 0.01 vs. normoxic controls. B: immunohistochemistry for substance P peptide. neg ctrl IgG, negative control IgG. Arrowhead, apparent alveolar macrophages; arrows, alveolar septa.

To determine in which anatomic compartment within the lung substance P levels were increased by hypoxia, immunohistochemical studies of substance P expression were conducted on sections of normoxic and hypoxic lung. As shown in Fig. 5B, very little substance P immunoreactivity was visible in the normoxic lung, most of it located in airway epithelial cells. In contrast, in lungs from animals exposed to 72 h of hypoxia, substance P immunoreactivity was markedly increased. This increase was most notable in cells appearing to be alveolar macrophages and in the alveolar septae as well as, to a lesser degree, in the airway epithelium.

Hypoxia decreases β-PPT mRNA expression.

To determine whether the increase in substance P peptide content in the hypoxic lung was a result of increased transcription of the PPT gene, lung PPT mRNA expression was studied via relative RT-PCR. As shown in Fig. 6, hypoxia did not alter the total expression of the α- and γ-PPT mRNA species. The expression of the β-PPT mRNA transcript, however, declined rapidly with exposure to hypoxia and after 24 h was significantly lower than in normal controls [ANOVA P = 0.04, multiple-comparison posttest P < 0.05; n = 4 (control) or 3 (24-h hypoxia)]. With additional time in hypoxia, the level of expression of β-PPT mRNA returned toward normal and was not statistically different from control levels by 48 h of hypoxia [n = 3 (48-h hypoxia) or 5 (72-h hypoxia)].

Fig. 6.

Hypoxia downregulates lung β-preprotachykinin (PPT) mRNA expression as assessed by relative RT-PCR. Densitometric analysis of PPT mRNA normalized to β-actin, expressed as arbitrary units. H24, H48, H72, 24-h, 48-h, and 72-hypoxia, respectively. Filled bars, β-PPT mRNA; gray bars, α/γ-PPT mRNA. *P < 0.05 vs. normoxic controls.

NK-1 receptor activation contributes to hypoxia-induced vascular leak.

We showed previously (5) that hypoxia increases vascular leak in the lung and that this leak can be blocked by administration of an NK-1 receptor antagonist before hypoxic exposure. To confirm that finding and to determine whether NK-1 receptor activation contributes to the ongoing leak after 72 h of hypoxia, albumin extravasation into the distal lung was measured in animals exposed to normoxia, hypoxia alone, or hypoxia plus an NK-1 receptor antagonist (RP67580) administered 2 h before the end of hypoxic exposure. Consistent with our previous report, exposure to 72 h of hypoxia led to a marked increase in albumin extravasation into the distal lung (normoxia 96 ± 20 vs. hypoxia alone 280 ± 70 ng Evans blue/mg dry lung; n = 5 per group, P < 0.01). Administration of RP67580 2 h before the end of the hypoxic exposure led to a partial reversal of the leak (191 ± 76 ng Evans blue/mg dry lung; n = 5, P < 0.05 vs. hypoxia alone), although these animals still leaked significantly more albumin than normoxic controls (P < 0.05).

To determine the anatomic site of the vascular leak in the hypoxic lung, normoxic (n = 3) and 72-h hypoxic (n = 3) animals were injected with 100-nm-diameter fluorescent red microspheres and frozen sections of lung were examined to identify sites of microsphere extravasation. In the normoxic animals, only sporadic isolated microspheres were identified in alveolar septa (Fig. 7, left). In hypoxic animals, in contrast, frequent patches of microspheres were seen in the alveolar septa, suggesting that those were sites of microvascular endothelial disruptions (Fig. 7, right). Microspheres were not visible in the lumens of either pulmonary vessels or airway submucosal vessels in either normoxic or hypoxic animals. When sections from these animals were also immunostained for the NK-1 receptor, NK-1 receptor immunoreactivity was minimal in the normoxic animals but patchy increases in immunoreactivity were observed in the hypoxic lung (Fig. 7). The NK-1 receptor immunoreactivity in the hypoxic lung generally correlated with areas of microsphere extravasation, although some patches of microspheres appeared in the absence of NK-1 staining and rare areas of NK-1 staining without extravasated microspheres were identified.

Fig. 7.

Hypoxia increases vascular leak in the distal lung as marked by fluorescent microsphere extravasation (yellow), and hypoxia increases NK-1R immunoreactivity (green) in the same areas. Scale bars = 100 μm.

DISCUSSION

The major finding of this study is that the expression of the NK-1 receptor in the lung is upregulated with exposure to moderate hypoxia. This upregulation was seen in both mRNA expression and protein content by 48 and 72 h of hypoxia exposure. Both immunohistochemistry and in situ receptor binding studies localized the increase in NK-1 receptor expression to the alveolar septa in a location most consistent with the pulmonary microvasculature. In addition, exposure to hypoxia also increased lung levels of substance P peptide, the primary ligand for the NK-1 receptor. These results suggest that hypoxia can upregulate the tachykinin system in the lung, which could then play a role in hypoxia-induced changes in pulmonary fluid balance.

Hypoxia has been shown to promote pulmonary vascular protein leakage in a variety of settings (3, 21, 27), and some evidence suggests that the effects of hypoxia in the lung are mediated at least in part by tachykinins (5). The effects of acute exposure to moderate hypoxia on NK-1 receptor expression in the lung, however, have not previously been described. We found a nearly threefold increase in lung NK-1 receptor protein expression in 72 h of hypoxia, accompanied by substantial increases in lung NK-1 receptor mRNA expression as well, suggesting that even moderate hypoxia rapidly and robustly increases lung NK-1 receptor expression. These results are consistent with the single previous report of the effect of hypoxia on lung NK-1 receptor expression, which showed smaller increases (20–30%) in lung NK-1 expression in adult rats exposed to chronic intermittent hypoxia for 1–4 wk (12). Although the precise reason for the more pronounced and more rapid increases in NK-1 receptor expression observed in our study remains unclear, potential explanations include the different techniques and time courses of hypoxic exposure as well as age-related differences in control of NK-1 receptor expression. Some support for the latter idea exists in the form of a report that NK-1 receptor expression in the brain decreases with age, suggesting that developmental influences may be important factors in determining NK-1 receptor expression levels (25).

Having identified an increase in NK-1 receptor expression in the whole lung, we next attempted to identify the specific anatomic location of the receptor in the normoxic lung. Previous studies of NK-1 receptor localization have relied on either immunohistochemistry or traditional receptor autoradiography. Immunohistochemical analysis can be complicated by wide variations in antibody sensitivity and specificity, even for antibodies ostensibly raised against the same peptide (7). Receptor autoradiography provides an alternate means to assess receptor location in tissue sections that is not dependent on antibody-based detection, but this technique can be limited in terms of the degree of resolution possible. We modified previously published techniques of receptor autoradiography by using ligand labeled with fluorescent semiconductive nanocrystals. This approach not only provided an alternate means of confirming the results of our immunostaining studies but also had the advantage of a relatively high resolution, allowing clear localization of labeled ligand to structures in the distal lung.

Using routine immunohistochemical techniques as well as in situ ligand binding studies, then, we found evidence in normoxic animals of only weak NK-1 receptor expression in alveolar macrophages, in alveolar septae, and in the endothelium of occasional small (<50-μm diameter) pulmonary vessels. Although previous studies of NK-1 receptor localization in the normoxic lung have generated conflicting results, our findings are consistent with previous work suggesting that very few detectable NK-1 receptors are present in the normal distal lung in rats, guinea pigs, or humans (8, 17, 29).

Using the same two techniques, we next studied the effect of hypoxia on NK-1 receptor localization. In the lungs of animals exposed to 72 h of hypoxia, we found patchy but dramatic increases in NK-1 receptor staining and ligand binding that were almost entirely localized to the alveolar septa and to alveolar macrophages. In addition, some small pulmonary vessels demonstrated increased NK-1 labeling both in the vessel wall and in cells accumulating around the vessel. Although no previous studies have examined the anatomic location of increased NK-1 receptor expression in hypoxia, the increase in alveolar wall NK-1 receptor expression is strikingly similar to the pattern of NK-1 receptor upregulation reported in human sarcoidosis patients (19). These results not only suggest that the pulmonary microvasculature is the primary location in which hypoxia exerts its effects on NK-1 receptor expression in the lung but also add to the evidence that similarities exist between hypoxia and inflammatory lung disease in terms of their effects on the distal lung (15).

Having identified increased levels of NK-1 receptor in the hypoxic lung, we next assayed lung substance P peptide content. We found a marked increase in lung substance P levels in hypoxic lung as measured by ELISA, and immunohistochemistry showed the increase in substance P to be localized to apparent alveolar macrophages and the alveolar septa. The finding of increased substance P peptide levels in the hypoxic lung demonstrated the presence of increased levels of the principal ligand for the NK-1 receptor in the lung at the same time that NK-1 receptor expression itself is increasing. Although we did not definitively identify the source of substance P in the hypoxic lung, several possibilities exist. Substance P is produced by peptidergic nerve fibers in the airways, although the extent to which those fibers are distributed in the distal lung in different species remains uncertain (20). Of interest, however, and in agreement with our immunostaining results, substance P peptide production or mRNA expression has previously been demonstrated in alveolar macrophages (10), neutrophils (9), as well as endothelial cells (1). Within the environment of the lung microcirculation, then, multiple sources could release substance P in proximity to the NK-1 receptor. Interestingly, in this study we found that lung substance P levels increased with exposure to hypoxia, whereas PPT mRNA appeared to decrease. Although this result was surprising, it is consistent with previous data showing that the enzyme neutral endopeptidase (NEP) regulates substance P peptide levels (18) and that NEP expression is rapidly reduced in the hypoxic lung (5). In the setting of hypoxia, then, increasing lung substance P levels seem most likely to result from decreased enzymatic degradation of the peptide rather than increased gene transcription. These experiments, however, do not rule out either increased PPT transcription in a specific subset of lung cells or alterations in the processing of PPT to mature substance P.

Previous work has shown that moderate hypoxia alone can lead to significant increases in vascular leak in the lung and that NK-1 receptor antagonism given before exposure to hypoxia markedly ameliorates that leak (5). In this study, not only did we confirm that hypoxia alone increases albumin extravasation into the distal lung but we were also able to show that an NK-1 receptor antagonist given after exposure to 72 h of hypoxia was able to partially reverse the leak seen in those animals. These results suggest that ongoing activation of NK-1 receptors contributes to vascular leak even after 72 h, although the partial response strongly suggests that additional factors contribute as well. Our studies using fluorescent microspheres to label sites of vascular leak in the hypoxic lung provide further evidence in support of this idea. The sites of microsphere extravasation in the hypoxic lung suggest that the pulmonary microcirculation is the major site of leak under those conditions. Increases in NK-1 receptor expression appear to predominantly—though not always—occur in those same areas. Coupled with the quantitative reduction in albumin extravasation with NK-1 antagonism, these findings strongly implicate the NK-1 receptor in hypoxia-induced vascular leak in the lung.

Finally, the likely mechanism by which the NK-1 receptor might contribute to edema formation in the hypoxic lung deserves mention. Although NK-1 receptor antagonism ameliorates many experimental lung injuries, including the vascular leak associated with subacute exposure to hypoxia, the physiological basis of this effect is uncertain. Pulmonary edema formation is widely conceived of as occurring because of increased vascular pressures, increased vascular permeability, or both. Substance P has been shown to alter both of these parameters. For example, in isolated, perfused guinea pig lungs, substance P has been described to cause increased pulmonary capillary pressure primarily as a result of postcapillary vasoconstriction without significant effect on endothelial permeability per se (26). In isolated, perfused rat lungs, however, substance P acutely raises capillary permeability as well as vascular pressures (12). Although our data do not directly address this question, the primarily microvascular location of the hypoxia-associated increase in NK-1 receptor expression suggests that NK-1 receptor activation in the hypoxic lung could have a greater effect on permeability than on vascular pressure.

In summary, this study shows that subacute exposure of young animals to moderate hypoxia strongly upregulates the tachykinin system in the lung. NK-1 receptor expression increases in the hypoxic lung in a tissue distribution most consistent with increased expression in microvascular endothelium and in alveolar macrophages, and hypoxia also increases lung substance P peptide levels. In addition, NK-1 receptors colocalize with sites of vascular leak in the hypoxic lung, and NK-1 antagonism reduces lung albumin extravasation under those conditions. These findings suggest that tachykinins contribute to hypoxia-induced alterations in lung fluid balance and thus may be important in the pathophysiology of lung injury.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-04484 and HL-07743 (T. C. Carpenter).

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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