Journal of Applied Physiology Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 84: 169-176, 1998;
8750-7587/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gao, X.-P.
Right arrow Articles by Rubinstein, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gao, X.-P.
Right arrow Articles by Rubinstein, I.

Ovalbumin increases macromolecular efflux from the in situ nasal mucosa of allergic hamsters

Xiao-Pei Gao, Syed R. Akhter, and Israel Rubinstein

Department of Medicine, University of Illinois at Chicago, and West Side Department of Veterans Affairs Medical Center, Chicago, Illinois 60612

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Gao, Xiao-Pei, Syed R. Akhter, and Israel Rubinstein. Ovalbumin increases macromolecular efflux from the in situ nasal mucosa of allergic hamsters. J. Appl. Physiol. 84(1): 169-176, 1998.---The purpose of this study was to determine whether bradykinin mediates ovalbumin-induced increase in macromolecular efflux from the nasal mucosa of ovalbumin-sensitized hamsters in vivo and, if so, whether the L-arginine/nitric oxide biosynthetic pathway transduces, in part, this response. We found that suffusion of ovalbumin onto the in situ nasal mucosa of ovalbumin-sensitized hamsters, but not of controls, elicited a significant time- and concentration-dependent increase in clearance of fluorescein isothiocyanate-labeled dextran (mol mass, 70 kDa; P < 0.05). HOE-140, but not des-Arg9,[Leu8]-bradykinin, and NG-L-arginine methyl ester (L-NAME), but not NG-D-arginine methyl ester, significantly attenuated ovalbumin-induced responses. L-Arginine, but not D-arginine, abolished the effects of L-NAME. L-NAME also significantly attenuated bradykinin-, but not adenosine- induced increase in macromolecular efflux from the in situ nasal mucosa. Overall, these data suggest that ovalbumin increases macromolecular efflux from the in situ nasal mucosa of ovalbumin-sensitized hamsters, in part, by producing bradykinin with subsequent activation of the L-arginine/nitric oxide biosynthetic pathway.

microcirculation; inflammation; bradykinin; nitric oxide; allergic rhinitis

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IT IS WELL ESTABLISHED that plasma exudation, a characteristic feature of inflammation, contributes appreciably to nasal mucosa congestion and nasal cavity obstruction in patients with allergic rhinitis (13, 19, 20). Current concepts suggest that bradykinin, a potent phlogistic mediator produced during the host inflammatory response to injury (3), mediates, in part, this process (2, 15, 20, 22, 26). However, the intracellular signaling pathway(s) transducing this response is uncertain.

Previous studies showed that stimulation of the L-arginine/nitric oxide (NO) biosynthetic pathway, which is expressed in the nasal mucosa (5, 10, 11), by bradykinin and other phlogistic mediators elicits plasma exudation from the microcirculation into the airway mucosa, thereby promoting tissue injury (1, 6, 16, 23). For instance, Mayhan (16) and Gao and Rubinstein (6) showed that bradykinin-induced increase in macromolecular efflux from the in situ hamster cheek pouch is mediated by NO or an NO-related compound(s). Nonetheless, the role of the L-arginine/NO biosynthetic pathway in transducing the edemagenic effects of bradykinin in allergic rhinitis is uncertain.

Hence, the purpose of this study was to begin to address this issue by determining whether bradykinin mediates ovalbumin-induced increase in macromolecular efflux from the in situ nasal mucosa of ovalbumin-sensitized hamsters and, if so, to probe whether the L-arginine/NO biosynthetic pathway transduces, in part, this response.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Immunization of Animals

The protocol to immunize hamsters with ovalbumin has been previously described in detail in the literature (4, 21). Briefly, male golden Syrian hamsters weighing 100-110 g were immunized by intraperitoneal injections of 10 µg ovalbumin in 0.2 ml saline containing 10 mg aluminum hydroxide [Al(OH)3] as adjuvant. Four weeks later, 1 µg of ovalbumin in Al(OH)3 was injected intraperitoneally, and experiments were conducted 10 days later. Saline or 10 mg Al(OH)3 were injected intraperitoneally in control animals. These animals were then used in studies outlined below.

Preparation of Animals

Hamsters weighing 138 ± 2 g (n = 82) were anesthetized with pentobarbital sodium (6 mg/100 g body wt ip). A tracheotomy was performed to facilitate spontaneous breathing. A femoral vein was cannulated to inject the intravascular tracer fluorescein isothiocyanate-labeled dextran (FITC-dextran; mol mass 70 kDa; 40 mg/100 g body wt dissolved in 1.0 ml saline and administered over 1 min) and supplemental anesthesia (2-4 mg · 100 g body wt-1 · h-1). A femoral artery was cannulated to record arterial blood pressure and to obtain blood samples. Body temperature was kept constant (37-38°C) during the experiments by using a heating pad.

A polyethylene tubing (PE-10, Clay Adams, Parsippany, NJ) was introduced ~3 mm into the left nostril along the nasal septum. The tongue was extruded from the mouth with forceps and secured to a pedestal by using a silk thread (4/0, Look, Norwell, MA). Another polyethylene tubing (PE-90) was introduced through the mouth into the left posterior concha under direct observation. The left nostril was covered with a laboratory film (PARAFILM "M," American National Can, Greenwich, CT) to prevent leakage of suffusate. The anterior nasal cannula was connected via a three-way valve to a reservoir containing warmed bicarbonate buffer (37-38°C) composed of 131.9 mM NaCl, 2.95 mM KCl, 1.48 mM CaCl2, 0.76 mM MgCl2, and 11.87 mM NaHCO3, which allowed continuous suffusion of the left nasal cavity. The cannula was also connected to an infusion pump (model 341B, Sage Instruments, Boston, MA) for continuous administration of ovalbumin and drugs into the suffusate. The posterior nasal cannula was connected through a peristaltic pump (Cole-Parmer Instruments, Chicago, IL) to a fraction collector (Cygnet, ISCO, Lincoln, NE). The flow rate of the pump was adjusted so that the entire volume of suffusate introduced into the nasal cavity through the anterior nasal cannula was collected into the fraction collector.

Determination of Clearance of Macromolecules

The suffusate was collected at 5-min intervals into glass test tubes throughout the experiment. The concentration of FITC-dextran was determined in all samples. Arterial blood samples were collected in heparinized capillary tubes (70-µl volume; Scientific Products, McGaw Park, IL) beginning 5 min before and 5, 60, 150, and 240 min after injection of FITC-dextran. The concentration of FITC-dextran was determined in all plasma samples.

To quantitate the concentration of FITC-dextran in plasma and suffusate, a standard curve for FITC-dextran concentrations vs. percent emission was performed on a spectrophotofluorometer (Photon Technology International, Princeton, NJ). The standard was FITC-dextran, which was prepared on a weight-per-volume basis. With the bicarbonate buffer used as background, a standard curve was generated for each experiment, and each curve was subjected to linear regression analysis. The percent emission for unknown samples (plasma and suffusate) was measured on the spectrophotofluorometer, and the concentration of FITC-dextran was calculated from the standard curve. In preliminary studies, minimal fluorescence signal (<2% above background) was detected when drugs were added to the buffer and when plasma and suffusate samples were examined before FITC-dextran was added. Clearance of FITC-dextran was determined by calculating the ratio of suffusate (ng/ml) to plasma (mg/ml) concentration of FITC-dextran and multiplying this ratio by the suffusate flow rate (2 ml/min).

Experimental Design

Effects of bradykinin on clearance of FITC-dextran. The purpose of these studies was to determine whether bradykinin increases clearance of FITC-dextran from the in situ hamster nasal mucosa and whether this response is mediated by bradykinin B2 receptors and transduced by the L-arginine/NO biosynthetic pathway. After suffusion of the bicarbonate buffer into the left nostril for 30 min (equilibration period), FITC-dextran was injected intravenously and the suffusate was collected for 40 min. Then, bradykinin (1.0 µM) was suffused into the left nostril for 5 min before and after suffusion of HOE-140 (1.0 µM), a selective bradykinin B2-receptor antagonist (2, 3, 7, 12, 27), or des-Arg9,[Leu8]-bradykinin (1.0 µM), a selective bradykinin B1-receptor antagonist (3, 7, 26), for 30 min. In another series of experiments, bradykinin (1.0 µM) was suffused for 5 min before and after suffusion of NG-L-arginine methyl ester (L-NAME; 10.0 µM), a NO synthase inhibitor (1, 6, 23, 24), for 30 min. Clearance of FITC-dextran was determined before and every 5 min for 60 min during each intervention. In preliminary studies, we determined that repeated suffusions of bradykinin (1.0 µM) were associated with reproducible results. In addition, suffusion of HOE-140 and des-Arg9,[Leu8]-bradykinin (each, 1.0 µM) alone had no significant effects on clearance of FITC-dextran. The concentrations of bradykinin, HOE-140, des-Arg9,[Leu8]-bradykinin, and L-NAME used in these studies were based on previous studies in our laboratory and reports in the literature (3, 6-8, 12, 16, 18, 23, 27, 31).

Effects of ovalbumin on clearance of FITC-dextran. The purpose of these studies was to determine whether ovalbumin increases clearance of FITC-dextran from the nasal mucosa of normal, Al(OH)3-treated, and ovalbumin-sensitized hamsters. After the equilibration period, FITC-dextran was injected intravenously and the suffusate was collected for 40 min. Then, two concentrations of ovalbumin (10 and 100 µg/ml) were suffused into the left nostril in an arbitrary order. Each concentration was suffused for 10 min. At least 50 min elapsed between subsequent suffusions of ovalbumin. Clearance of FITC-dextran was determined during each intervention as outlined in Effects of bradykinin on clearance of FITC-dextran. In preliminary studies, we determined that repeated suffusions of ovalbumin (10 and 100 µg/ml) in ovalbumin-sensitized hamsters were associated with reproducible results. The concentrations of ovalbumin used in these studies were based on previous reports in the literature (4, 21).

Effects of bradykinin-receptor antagonists on ovalbumin-induced responses. The purpose of these studies was to determine whether bradykinin mediates, in part, ovalbumin-induced increase in clearance of FITC-dextran from the in situ nasal mucosa of ovalbumin-sensitized hamsters. After the equilibration period, FITC-dextran was injected intravenously and clearance of FITC-dextran was determined for 40 min. Then, ovalbumin (10 and 100 µg/ml) was suffused for 10 min before and after suffusion of HOE-140 (1.0 µM) or des-Arg9,[Leu8]-bradykinin (1.0 µM) for 30 min. Clearance of FITC-dextran was determined during each intervention.

Effects of L-NAME on ovalbumin-induced responses. The purpose of these studies was to determine whether the L-arginine/NO biosynthetic pathway transduces ovalbumin-induced increase in clearance of FITC-dextran from the in situ nasal mucosa in ovalbumin-sensitized hamsters. After the equilibration period, FITC-dextran was injected intravenously and clearance of FITC-dextran was determined for 40 min. Then, ovalbumin (10 and 100 µg/ml) was suffused for 10 min before and after suffusion of L-NAME or NG-D-arginine methyl ester (D-NAME) (each, 10.0 µM) for 30 min. Clearance of FITC-dextran was determined during each intervention. In another series of experiments, ovalbumin (10 and 100 µg/ml) was suffused for 10 min before and after suffusion of L-arginine or D-arginine (each, 1.0 mM) together with L-NAME (10.0 µM) for 30 min. Clearance of FITC-dextran was determined during each intervention. The concentrations of D-NAME, L-arginine, and D-arginine used in these studies were based on previous studies in our laboratory (6, 23).

Effects of L-NAME on adenosine-induced responses. The purpose of these studies was to determine the specificity of L-NAME attenuation of ovalbumin-induced responses by determining its effects on adenosine-induced increase in clearance of FITC-dextran from the in situ nasal mucosa of normal hamsters. It is well established that adenosine, like bradykinin, increases macromolecular efflux from the in situ hamster microcirculation through a receptor-mediated mechanism(s) (6, 8, 18, 31). After the equilibration period, FITC-dextran was injected intravenously and clearance of FITC-dextran was determined for 40 min. Then, adenosine (1.0 µM) was suffused into the left nostril for 5 min as outlined in Effects of ovalbumin on clearance of FITC-dextran. Fifty minutes after suffusion of adenosine was stopped, L-NAME (10.0 µM) was suffused for 30 min followed by suffusion of adenosine (1.0 µM) for 5 min. Clearance of FITC-dextran was determined during each intervention. In preliminary studies, we determined that repeated suffusions of adenosine (1.0 µM) were associated with reproducible results. The concentration of adenosine used in these studies was based on previous studies in our laboratory and reports in the literature (6, 8, 18, 31).

Effects of phenylephrine on bradykinin- and adenosine-induced responses. The purpose of these studies was to determine whether bradykinin- and adenosine-induced increases in clearance of FITC-dextran from the in situ hamster nasal mucosa are mediated, in part, by agonist-induced vasodilation. To accomplish this goal, we determined the effects of phenylephrine, a potent endothelium-independent vasoconstrictor (25), suffused onto the nasal mucosa on bradykinin- and adenosine-induced responses. The experimental design was similar to that outlined in Effects of ovalbumin on clearance of FITC-dextran except that bradykinin (1.0 µM) or adenosine (1.0 µM) was now suffused with phenylephrine (0.2 or 0.5 µM) for 5 min. Clearance of FITC-dextran was determined during each intervention. In preliminary studies, we determined that suffusion of 0.2 and 0.5 µM phenylephrine onto the hamster cheek pouch for 5 min elicited an 18 and 24% decrease in arteriolar diameter from baseline, respectively. Suffusion of higher concentrations of phenylephrine was associated with a decrease of mean arterial pressure, indicative of systemic absorption. Suffusion of phenylephrine (0.2 and 0.5 µM) alone onto the nasal mucosa for 5 min was not associated with a significant increase in clearance of FITC-dextran.

Drugs

Ovalbumin (grade V), Al(OH)3 FITC-dextran, bradykinin, des-Arg9,[Leu8]-bradykinin, L-NAME, D-NAME, L-arginine, D-arginine, adenosine, and phenylephrine were obtained from Sigma Chemical (St. Louis, MO). HOE-140 was a gift from Hoechst-Roussel Pharmaceuticals (Somerville, NJ). All drugs were dissolved in saline and diluted to the desired concentrations on the day of the experiment.

Data and Statistical Analyses

When a test compound was suffused onto the nasal mucosa, we determined the maximal change in clearance of FITC-dextran and used it as the response to that compound. Data are expressed as means ± SE. Because clearance of FITC-dextran returned to baseline between successive applications of test compounds during each experiment, control data are expressed as a single mean value. Statistical analysis was performed by using two-way analysis of variance and the Newman-Keuls test for multiple comparisons. In this paper, n is the number of experiments, with each experiment representing a separate animal. P < 0.05 was considered significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

There was no significant change in mean arterial pressure during the experiments (105 ± 3 mmHg at the start and 107 ± 4 mmHg at the conclusion of the experiments; n = 82; P > 0.5).

Suffusion of saline (vehicle) in normal and ovalbumin-sensitized hamsters for the entire duration of the experiment was not associated with a significant change in clearance of FITC-dextran. Clearance of FITC-dextran was 10.3 ± 2.4 ml/min × 10-6 at the start and 11.8 ± 2.6 ml/min × 10-6 at the conclusion of the experiment in normal hamsters and 11.6 ± 0.5 ml/min × 10-6 at the start and 13.1 ± 3.1 ml/min × 10-6 at the conclusion of the experiment in ovalbumin-sensitized hamsters (each group, n = 4; P > 0.5).

Effects of Bradykinin on Clearance of FITC-Dextran

Bradykinin (1.0 µM) elicited a significant increase in clearance of FITC-dextran from the nasal mucosa (Fig. 1; P < 0.05). This response was observed within 4 min after the start of suffusion and was maximal within 10 min. Clearance of FITC-dextran returned to baseline 30 min after suffusion of bradykinin was stopped. Bradykinin-induced responses were significantly attenuated by HOE-140 (1.0 µM) and L-NAME (10.0 µM) but not by des-Arg9,[Leu8]-bradykinin (1.0 µM; Fig. 1; each group, n = 4; P < 0.05). Clearance of FITC-dextran decreased significantly from 39.0 ± 4.5 ml/min × 10-6 during suffusion of bradykinin (1.0 µM) alone to 23.9 ± 6.5 and 17.2 ± 6.0 ml/min × 10-6 during suffusion of HOE-140 (1.0 µM) and bradykinin (1.0 µM) and of L-NAME (10.0 µM) and bradykinin (1.0 µM), respectively (Fig. 1; each group, n = 4; P < 0.05). Suffusion of HOE-140 (1.0 µM) and L-NAME (10.0 µM) alone had no significant effects on clearance of FITC-dextran relative to suffusion of saline alone (9.1 ± 3.0 and 10.9 ± 2.9 vs. 10.3 ± 2.3 ml/min × 10-6, respectively; each group, n = 4; P > 0.5).


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 1.   Effects of des-Arg9,[Leu8]-bradykinin (B1-RA; 1.0 µM), a bradykinin B1-receptor antagonist; HOE-140 (1.0 µM), a bradykinin B2-receptor antagonist; and NG-L-arginine methyl ester (L-NAME; 10.0 µM), a nitric oxide synthase inhibitor, on bradykinin (1.0 µM)-induced increase in clearance of fluorescein isothiocyanate (FITC)-dextran from in situ hamster nasal mucosa. Values are means ± SE; each group, n = 4. * P < 0.05 compared with control (saline). dagger  P < 0.05 compared with bradykinin alone.

Effects of Ovalbumin on Clearance of FITC-Dextran

Figure 2 shows the time course of changes in clearance of FITC-dextran from the in situ nasal mucosa during suffusion of ovalbumin (100 µg/ml) in ovalbumin-sensitized hamsters and controls. Ovalbumin elicited a significant, time-dependent increase in clearance of FITC-dextran from the nasal mucosa in ovalbumin-sensitized hamsters but not in controls (Fig. 2; each group, n = 4; P < 0.05). This response was observed within 10 min after the start of suffusion and was maximal within 20 min. Clearance of FITC-dextran returned to baseline 30 min after suffusion of ovalbumin was stopped.


View larger version (9K):
[in this window]
[in a new window]
  		Fig. 2.   Time course of changes in clearance of FITC-dextran from in situ nasal mucosa during suffusion of ovalbumin (100 µg/ml) in control (A) and ovalbumin-sensitized (B) hamsters. Open bars, duration of suffusion. Values are means ± SE; each group, n = 4. * P < 0.05 compared with baseline.

Suffusion of ovalbumin elicited a concentration-dependent increase in clearance of FITC-dextran from the nasal mucosa of ovalbumin-sensitized hamsters (Figs. 3-6; P < 0.05). By contrast, ovalbumin (100 µg/ml) had no significant effects on clearance of FITC-dextran in control hamsters and in hamsters treated with Al(OH)3 relative to baseline (18.5 ± 4.9 and 17.5 ± 6.8 vs. 16.8 ± 4.5 ml/min × 10-6, respectively; each group, n = 4; P > 0.5).


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 3.   Effects of HOE-140 (1.0 µM; hatched bars) on ovalbumin (solid bars)-induced increase in clearance of FITC-dextran from in situ nasal mucosa of ovalbumin-sensitized hamsters. Values are means ± SE; each group, n = 4; * P < 0.05 compared with control (C; saline; open bars). dagger  P < 0.05 compared with ovalbumin alone.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of des-Arg9[Leu8]-bradykinin (1.0 µM; hatched bars) on ovalbumin (solid bars)-induced increase in clearance of FITC-dextran from in situ nasal mucosa of ovalbumin-sensitized hamsters. Values are means ± SE; each group, n = 4. * P < 0.05 compared with control (C; saline; open bars).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 5.   Effects of L-NAME (10.0 µM), NG-D-arginine methyl ester (D-NAME; 10.0 µM), and L-NAME (10.0 µM) with L-arginine (1.0 mM) on ovalbumin-induced increase in clearance of FITC-dextran from in situ nasal mucosa of ovalbumin-sensitized hamsters. Values are means ± SE; each group, n = 4. * P < 0.05 compared with control (saline). ¶ P < 0.05 compared with ovalbumin alone.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 6.   Effects of L-NAME (10.0 µM) and L-NAME (10.0 µM) with D-arginine (1.0 mM) on ovalbumin-induced increase in clearance of FITC-dextran from in situ nasal mucosa of ovalbumin-sensitized hamsters. Values are means ± SE; each group, n = 4. * P < 0.05 compared with control (saline). ¶ P < 0.05 compared with ovalbumin alone.

Effects of Bradykinin-Receptor Antagonists Ovalbumin-Induced Responses

Suffusion of HOE-140 (1.0 µM) alone had no significant effects on clearance of FITC-dextran in ovalbumin-sensitized hamsters relative to suffusion of saline alone (10.6 ± 2.7 vs. 11.8 ± 1.8 ml/min × 10-6, respectively; each group, n = 4; P > 0.5). However, it significantly attenuated ovalbumin-induced increase in clearance of FITC-dextran from the nasal mucosa of ovalbumin-sensitized hamsters (Fig. 3; each group, n = 4; P < 0.05). Clearance of FITC-dextran decreased significantly from 45.5 ± 10.3 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) alone to 24.7 ± 6.5 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) and HOE-140 (1.0 µM; Fig. 3; each group, n = 4; P < 0.05). By contrast, des-Arg9,[Leu8]-bradykinin (1.0 µM) had no significant effects on ovalbumin-induced responses (Fig. 4; each group, n = 4; P > 0.5). Clearance of FITC-dextran was 38.3 ± 6.7 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) alone and 37.9 ± 4.4 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) and des-Arg9,[Leu8]-bradykinin (1.0 µM; Fig. 4; each group, n = 4; P > 0.5).

Effects of L-NAME on Ovalbumin-Induced Responses

Suffusion of L-NAME (10.0 µM) alone had no significant effects on clearance of FITC-dextran in ovalbumin-sensitized hamsters relative to suffusion of saline alone (14.6 ± 2.9 vs. 15.4 ± 3.4 ml/min × 10-6, respectively; each group, n = 4; P > 0.5). However, it significantly attenuated ovalbumin-induced increase in clearance of FITC-dextran from the in situ nasal mucosa of ovalbumin-sensitized hamsters (Fig. 5; each group, n = 4; P < 0.05). Clearance of FITC-dextran decreased significantly from 50.2 ± 4.1 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) alone to 29.6 ± 7.0 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) and L-NAME (10.0 µM; Fig. 5; each group, n = 4; P < 0.05). D-NAME had no significant effects on ovalbumin-induced responses (Fig. 5; each group, n = 4; P > 0.5). Clearance of FITC-dextran was 56.0 ± 10.0 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) alone and 51.2 ± 4.8 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) and D-NAME (10.0 µM; Fig. 5; each group, n = 4; P > 0.5).

Suffusion of L-arginine (1.0 mM) abrogated L-NAME (10.0 µM) attenuation of ovalbumin-induced responses (Fig. 4; each group, n = 4; P < 0.05 in comparison to ovalbumin and L-NAME). Clearance of FITC-dextran increased from 29.6 ± 7.0 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) and L-NAME (10.0 µM) to 51.2 ± 4.8 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml), L-NAME (10.0 µM), and L-arginine (1.0 mM; Fig. 5; each group, n = 4; P < 0.05). Suffusion of D-arginine (1.0 mM) had no significant effects on L-NAME (10.0 µM)-induced responses (Fig. 6; each group, n = 4; P > 0.5). Clearance of FITC-dextran was 29.6 ± 7.0 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml) and L-NAME (10.0 µM) and 37.3 ± 7.6 ml/min × 10-6 during suffusion of ovalbumin (100 µg/ml), L-NAME (10.0 µM), and D-arginine (1.0 mM; Fig. 6; each group, n = 4; P > 0.5).

Effects of L-NAME on Adenosine-Induced Responses

Adenosine (1.0 µM) elicited a significant increase in clearance of FITC-dextran from the in situ nasal mucosa. This response was observed within 5 min after the start of suffusion and was maximal within 10 min. Clearance of FITC-dextran returned to baseline 40 min after suffusion of adenosine was stopped. L-NAME (10.0 µM) had no significant effects on adenosine (1.0 µM)-induced increase in clearance of FITC-dextran from the in situ nasal mucosa (each group, n = 6; P > 0.5). Clearance of FITC-dextran was 53.6 ± 16.0 ml/min × 10-6 during suffusion of adenosine (1.0 µM) alone and 57.2 ± 15.3 ml/min × 10-6 during suffusion of adenosine (1.0 µM) and L-NAME (10.0 µM; each group, n = 6; P > 0.5).

Effects of Phenylephrine on Bradykininand Adenosine-Induced Responses

Suffusion of phenylephrine (0.2 and 0.5 µM) had no significant effects on bradykinin (1.0 µM)- and adenosine (1.0 µM)-induced increases in clearance of FITC-dextran from the in situ nasal mucosa (each group, n = 4; P > 0.5). Clearance of FITC-dextran increased from 19.3 ± 7.3 ml/min × 10-6 during suffusion of saline (vehicle) to 64.5 ± 15.1 and 70.2 ± 20.7 ml/min × 10-6 during suffusion of bradykinin (1.0 µM) alone and phenylephrine (0.5 µM) with bradykinin (1.0 µM), respectively (each group, n = 4; P > 0.5). Similarly, clearance of FITC-dextran increased from 21.2 ± 4.2 ml/min × 10-6 during suffusion of saline (vehicle) to 33.3 ± 7.6 and 33.9 ± 9.1 ml/min × 10-6 during suffusion of adenosine (1.0 µM) alone and phenylephrine (0.5 µM) with adenosine (1.0 µM), respectively (each group, n = 4; P > 0.5).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

There are several new findings of this study. We found that ovalbumin elicits a significant time- and concentration-dependent increase in macromolecular efflux from the in situ nasal mucosa in ovalbumin-sensitized hamsters but not in controls. These effects are not related to nonspecific damage to the endothelium because clearance of FITC-dextran returns to baseline once suffusion of ovalbumin is stopped and because ovalbumin has no significant effects on clearance of FITC-dextran in normal and Al(OH)3-treated hamsters. Ovalbumin-induced responses are mediated, in part, by local production of bradykinin through stimulation of bradykinin B2 receptors, because HOE-140, a bradykinin B2-receptor antagonist, but not des-Arg9,[Leu8]-bradykinin, a bradykinin B1-receptor antagonist, significantly attenuates bradykinin- and ovalbumin-induced responses. The edemagenic effects of bradykinin and ovalbumin are transduced, in part, by the L-arginine/NO biosynthetic pathway because L-NAME significantly attenuates bradykinin- and ovalbumin-induced responses in a stereospecific fashion and because L-arginine, but not D-arginine, abrogates the effects of L-NAME.

Bradykinin- and ovalbumin-induced increases in clearance of FITC-dextran from the in situ hamster nasal mucosa are, most likely, unrelated to vasodilation because L-NAME has no significant effects on the increase in macromolecular efflux elicited by adenosine, an endothelium- and NO-independent vasodilator, and because phenylephrine, an endothelium-independent vasoconstrictor, has no significant effects on bradykinin- and adenosine-induced responses. Overall, these data suggest that ovalbumin increases macromolecular efflux from the in situ nasal mucosa of ovalbumin-sensitized hamsters, in part, by producing bradykinin and subsequent activation of the L-arginine/NO biosynthetic pathway.

Björk and Smedegård (4) and Raud et al. (21) showed that ovalbumin elicits a significant increase in macromolecular efflux from the in situ cheek pouch of ovalbumin-sensitized hamsters. The results of the present study extend these observations by showing that ovalbumin also increases macromolecular efflux from the in situ nasal mucosa of ovalbumin-sensitized hamsters and that this response is mediated, in part, by bradykinin. These effects are not related to regional variation in clearance of FITC-dextran, the intravascular tracer used in this study, because the entire left nasal cavity is suffused with ovalbumin at a constant flow rate, thereby minimizing the effects of regional tissue variability on the concentration of FITC-dextran in the suffusate. Moreover, we used 70-kDa FITC-dextran, not 150-kDa FITC-dextran, as the intravascular tracer because Gawlowski et al. (9) showed that in hamsters 150-kDa FITC-dextran activates circulating leukocytes, which are thought to play a role in the pathophysiology of allergic rhinitis (13), and promotes microvascular transport independent of subsequent exposure to phlogistic mediators. Overall, these data indicate that the ovalbumin-sensitized hamster is a suitable model to investigate the regulation of macromolecular efflux from the in situ nasal mucosa in allergic rhinitis.

Changes in vasomotor tone and/or venular driving pressure may have mediated, in part, the effects of bradykinin, ovalbumin, and L-NAME on macromolecular efflux from the in situ hamster nasal mucosa. However, this possibility seems unlikely for the following reasons. Suffusion of phenylephrine, a potent vasoconstrictor, onto the nasal mucosa has no significant effects on bradykinin- and adenosine-induced increases in clearance of FITC-dextran. In addition, L-NAME, which elicits vasoconstriction in the hamster cheek pouch (23), has no significant effects on adenosine-induced increase in macromolecular efflux from the nasal mucosa. These data are consistent with previous studies showing that agonist-induced increase in microvascular transport is not related to changes in microvascular diameter and/or venular driving pressure in other vascular beds and species (17, 18, 28-30). Moreover, isoproterenol, a potent vasodilator that increases venular pressure but has no significant effects on macromolecular efflux (18), has been shown to attenuate bradykinin-induced increase in clearance of FITC-dextran from the cheek pouch (28). On balance, these data suggest that the effects of bradykinin, ovalbumin, and L-NAME, at the concentrations used in the present study, on macromolecular efflux from the in situ hamster nasal mucosa could not be attributed to local changes in vasomotor tone and/or venular driving pressure.

Current concepts suggest that under physiological conditions mucosal NO or NO-related compound(s) tonically suppresses plasma exudation in laboratory animals, thereby conserving tissue integrity (1, 14, 24). We found, however, that suffusion of L-NAME, an NO synthase inhibitor, alone onto the in situ nasal mucosa of normal and ovalbumin-sensitized hamsters had no significant effects on clearance of FITC-dextran. These data suggest that under basal conditions the L-arginine/NO biosynthetic pathway does not contribute appreciably to the barrier function of nasal mucosa microcirculation in hamsters. Although the reasons behind this discrepancy are unknown, they might be related, in part, to differences in species and vascular beds used in these studies (1, 14).

Ricciardolo et al. (22) showed that ovalbumin-induced increase in macromolecular efflux from the nasal mucosa of ovalbumin-sensitized guinea pigs is mediated by bradykinin. However, the intracellular signaling pathway(s) transducing this response was not elucidated in this study. Other investigators showed that stimulation of the L-arginine/NO biosynthetic pathway by bradykinin and other phlogistic mediators elicits plasma exudation in the airway mucosa (1, 6, 16). The results of the present study support this contention by showing that the L-arginine/NO biosynthetic pathway transduces, in part, bradykinin- and ovalbumin-induced increases in macromolecular efflux from the in situ nasal mucosa of normal hamsters and ovalbumin-sensitized hamsters, respectively. Nonetheless, the cellular origin(s) of NO or NO-related compound(s) produced in the hamster nasal mucosa during suffusion of bradykinin and ovalbumin was not elucidated. Clearly, additional studies are warranted to address this issue.

In summary, we found that ovalbumin increases macromolecular efflux from the in situ nasal mucosa of ovalbumin-sensitized hamsters, in part, by producing bradykinin and subsequent activation of the L-arginine/NO biosynthetic pathway.

    ACKNOWLEDGEMENTS

This study was supported, in part, by grants from the National Institute of Dental Research (NIDR) (DE-10347), American Heart Association of Metropolitan Chicago, and Laerdal Foundation for Acute Medicine. I. Rubinstein is a recipient of NIDR Research Career Development Award DE-00386 and a University of Illinois Scholar Award.

    FOOTNOTES

Address for reprint requests: I. Rubinstein, Dept. of Medicine (M/C 787), Univ. of Illinois at Chicago, 840 South Wood St., Chicago, IL 60612-7323.

Received 11 November 1996; accepted in final form 4 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

  1. Bernareggi, M., J. A. Mitchell, P. J. Barnes, and M. G. Belvisi. Dual action of nitric oxide on airway plasma leakage. Am. J. Respir. Crit. Care Med. 155: 869-874, 1997[Abstract].
  2. Bertrand, C., P. Geppetti, J. Baker, G. Petersson, G. Piedimonte, and J. A. Nadel. Role of peptidases and NK1 receptors in vascular extravasation induced by bradykinin in rat nasal mucosa. J. Appl. Physiol. 74: 2456-2461, 1993[Abstract/Free Full Text].
  3. Bhoola, K. D., C. D. Figueroa, and K. Worthy. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol. Rev. 44: 1-80, 1992[Medline].
  4. Björk, J., and G. Smedegård. The microvasculature of the hamster cheek pouch as a model for studying acute immune-complex-induced inflammatory reactions. Int. Arch. Allergy Appl. Immunol. 74: 178-185, 1984[Medline].
  5. Furukawa, N., D. G. Harrison, D. Saleh, H. Shennib, F. P. Chagnon, and A. Giaid. Expression of nitric oxide synthase in the human nasal mucosa. Am. J. Respir. Crit. Care Med. 153: 847-850, 1996[Abstract].
  6. Gao, X.-p., and I. Rubinstein. The stable VIP analogue, Ro 24-9981, potentiates bradykinin-induced increase in clearance of macromolecules. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1648-H1655, 1995[Abstract/Free Full Text].
  7. Gao, X.-p., J. K. Vishwanatha, J. M. Conlon, C. O. Olopade, and I. Rubinstein. Mechanisms of smokeless tobacco-induced buccal mucosa inflammation. Role of bradykinin. J. Immunol. 157: 4624-4633, 1996[Abstract].
  8. Gawlowski, D. M., and W. N. Durán. Dose-related effects of adenosine and bradykinin on microvascular permeability to macromolecules in the hamster cheek pouch. Circ. Res. 58: 348-355, 1986[Abstract/Free Full Text].
  9. Gawlowski, D. M., N. R. Harding, and H. J. Granger. Leukocyte phagocytosis and alterations in microvascular integrity elicited by FITC-dextran 150 and epi-illumination in the microcirculation of the hamster cheek pouch. Microvasc. Res. 37: 1-15, 1989[Medline].
  10. Gerlach, H., R. Rossaint, D. Pappert, M. Knorr, and K. J. Falke. Autoinhalation of nitric oxide after endogenous synthesis in nasopharynx. Lancet 343: 518-519, 1994[Medline].
  11. Hanazawa, T., A. Konno, T. Kaneko, K. Tanaka, H. Ohshima, H. Esumi, and T. Chiba. Nitric oxide synthase-immunoreactive nerve fibers in the nasal mucosa of the rat. Brain Res. 657: 7-13, 1994[Medline].
  12. Hock, F. J., K. Wirth, U. Albus, W. Linz, H. J. Gerhards, G. Wiemer St. Henke, G. Breipohl, W. Konig, J. Knolle, and B. A. Scholkens. Hoe 140 a new potent and long acting bradykinin-antagonist: in vitro studies. Br. J. Pharmacol. 102: 769-773, 1991[Medline].
  13. Kopke, R. D., and R. L. Jackson. Rhinitis. In: Head and Neck Surgery-Otolaryngology (1st ed.), edited by B. J. Bailey. Philadelphia, PA: Lippincott, 1993, p. 269-289.
  14. Kubes, P., and D. N. Granger. Nitric oxide modulates microvascular permeability. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H611-H615, 1992[Abstract/Free Full Text].
  15. Lindell, E., M. E. Svensjö, L. Malm, and G. Petersson. Tachykinin-induced nasal fluid secretion and plasma exudation in the rat: effects of peptidase inhibition. Neuropeptides 28: 309-315, 1995[Medline].
  16. Mayhan, W. G. Role of nitric oxide in modulating permeability of hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin. Inflammation 16: 295-305, 1992[Medline].
  17. Miller, F. N., I. G. Joshua, and G. L. Anderson. Quantitation of vasodilator-induced macromolecular leakage by in vivo fluorescent microscopy. Microvasc. Res. 24: 56-67, 1982[Medline].
  18. Murray, M. A., D. D. Heistad, and W. G. Mayhan. Role of protein kinase C in bradykinin-induced increase in microvascular permeability. Circ. Res. 68: 1340-1348, 1990[Abstract/Free Full Text].
  19. Persson, C. G. A., I. Erjefält, U. Alkner, C. Baumgarten, L. Greiff, B. Gustafsson, A. Luts, U. Pipkorn, F. Sundler, C. Svensson, and P. Wollmer. Plasma exudation as a first line respiratory mucosal defence. Clin. Exp. Allergy 21: 17-24, 1991[Medline].
  20. Proud, D., C. R. Baumgarten, R. M. Naclerio, and P. E. Ward. Kinin metabolism in human nasal secretions during experimentally induced allergic rhinitis. J. Immunol. 138: 428-434, 1987[Abstract].
  21. Raud, J., S.-E. Dahlen, A. Sydbom, L. Lindbom, and P. Hedqvist. Enhancement of acute allergic inflammation by indomethacin is reversed by prostaglandin E2: apparent correlation with in vivo modulation of mediator release. Proc. Natl. Acad. Sci. USA 85: 2315-2319, 1988[Abstract/Free Full Text].
  22. Ricciardolo, F. L. M., J. A. Nadel, C. Bertrand, I. Yamawaki, B. Chan, and P. Geppetti. Tachykinins and kinins in antigen-evoked plasma extravasation in guinea-pig nasal mucosa. Eur. J. Pharmacol. 261: 127-132, 1994[Medline].
  23. Rubinstein, I., and W. G. Mayhan. L-Arginine dilates cheek pouch arterioles in hamsters with hereditary cardiomyopathy. J. Lab. Clin. Med. 125: 313-318, 1995[Medline].
  24. Schmidt, H. H. H. W., and U. Walter. NO at work. Cell 78: 919-925, 1994[Medline].
  25. Séjourné, F., H. Suzuki, H. Alkan-Önyüksel, X.-p. Gao, H. Ikezaki, and I. Rubinstein. Mechanisms of vasodilation elicited by VIP in sterically stabilized liposomes in vivo. Am. J. Physiol. 273 (Regulatory Integrative Comp. Physiol. 42): R287-R292, 1997[Abstract/Free Full Text].
  26. Shirasaki, H., K. Asakura, T. Kojima, S. Sohma, and A. Kataura. The roles of histamine, leukotriene C4 and bradykinin on nasal vascular permeability in experimental nasal allergy of guinea pigs. Rhinology 30: 41-48, 1992[Medline].
  27. Sung, C. P., A. J. Arleth, K. Shikano, and B. A. Berkowitz. Characterization and function of bradykinin receptors in vascular endothelial cells. J. Pharmacol. Exp. Ther. 247: 8-13, 1988[Abstract/Free Full Text].
  28. Svensjö, E., C. G. A. Persson, and G. Rutili. Inhibition of bradykinin induced macromolecular leakage from post-capillary venules by a beta 2-adrenoceptor stimulant, terbutaline. Acta Physiol. Scand. 101: 504-506, 1977[Medline].
  29. Tomeo, A. C., and W. N. Durán. Resistance and exchange microvessels are modulated by different PAF receptors. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H1648-H1652, 1991[Abstract/Free Full Text].
  30. Warren, J. B., A. J. Wilson, R. K. Loi, and M. L. Coughlan. Opposing roles of cyclic AMP in the vascular control of edema formation. FASEB J. 7: 1394-1400, 1993[Abstract].
  31. Yong, T., X.-P. Gao, S. Koizumi, J. M. Conlon, S. I. Rennard, W. G. Mayhan, and I. Rubinstein. Role of peptidases in bradykinin-induced increase in vascular permeability in vivo. Circ. Res. 70: 952-959, 1992[Abstract/Free Full Text].

The Journal of Applied Physiology 84(1):169-176
0161-7567/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
X.-P. Gao
Grain sorghum dust increases macromolecular efflux from the in situ nasal mucosa
J Appl Physiol, April 1, 1998; 84(4): 1431 - 1436.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gao, X.-P.
Right arrow Articles by Rubinstein, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gao, X.-P.
Right arrow Articles by Rubinstein, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online