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J Appl Physiol 87: 1877-1886, 1999;
8750-7587/99 $5.00
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Vol. 87, Issue 5, 1877-1886, November 1999

Sensory nerve-mediated immediate nasal responses to inspired acrolein

John B. Morris, John Stanek, and Gerald Gianutsos

Toxicology Program, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269-2092


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the role of sensory C-fiber stimulation and tachykinin release in the immediate nasal responses to the sensory irritant acrolein, the upper respiratory tract of the urethan-anesthetized male Fischer 344 rat was isolated via insertion of an endotracheal tube, and acrolein-laden air [2, 5, 10, or 20 parts/million (ppm)] was drawn continuously through that site at a flow rate of 100 ml/min for 50 min. Uptake of the inert vapor acetone was measured throughout the exposure to assess nasal vascular function. Plasma protein extravasation into nasal tissue and nasal lavage fluid was also assessed via injection of Evans blue dye. At 20 ppm, acrolein induced 1) a twofold increase in acetone uptake, indicative of vasodilation, followed by a progressive decline toward basal levels and 2) increased plasma protein extravasation, as indicated by dye leakage into nasal tissue and nasal lavage. These responses were inhibited by capsaicin pretreatment and the neurokinin type 1 antagonist N-acetyltrifluoromethyl tryptophan benzyl ester and were potentiated by the peptidase inhibitors phosphoramidon and captopril, suggesting that these responses were mediated by tachykinin. At lower exposure concentrations, acrolein was without effect on dye leakage but produced vasodilation, as indicated by increased acetone uptake. The responses at the lower concentrations were inhibited by capsaicin pretreatment, implicating nasal sensory C-fiber involvement, but were not influenced by N-acetyltrifluoromethyl tryptophan benzyl ester, phosphoramidon, or captopril, suggesting the involvement of a mediator other than the tachykinins substance P and neurokinin A.

substance P; sensory irritation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACROLEIN IS A HIGHLY reactive aldehyde that is produced during combustion. Tobacco smoke represents a common source of acrolein vapor in indoor air. Acrolein concentrations in mainstream smoke may reach >= 50 parts/million (ppm) (10). In smoky rooms, concentrations of 0.02 ppm have been observed (26). Acrolein is also produced in structural fires. In 10% of the fires examined in a recent study in Boston, MA, acrolein concentrations were >3 ppm (49). Acrolein is a potent nasal toxicant. Short-term inhalation exposure of rats to >= 0.67 ppm acrolein results in respiratory and olfactory mucosal damage (15, 20, 25). Acrolein is ciliastatic at >= 15 ppm (17).

Previous work in our laboratory has focused on nasal dosimetry of acrolein and its effects on the uptake of other vapors (34, 35). At exposure concentrations of 9 ppm (20 µg/l), acrolein alters the nasal uptake of acetone and acetaldehyde vapors. Two effects were observed on acetone uptake during a 40-min exposure: an immediate twofold increase in uptake efficiency, followed by a progressive decline in uptake efficiency throughout the exposure (34). Acetone is a nonreactive, nonmetabolized vapor, the uptake of which is dependent on nasal blood flow (37, 38). The mechanisms of the acrolein-induced changes in acetone uptake are not known, but on the basis of acetone uptake mechanisms, it was hypothesized that this response was due to immediate nasal vasodilation and a progressive increase in the thickness of the air-blood barrier due perhaps to excess mucus secretion and/or tissue swelling (34).

Acrolein is a potent sensory irritant. The concentration of acrolein that elicits a 50% decrease in respiratory rate (RD50) is 6 ppm in the Fischer 344 (F-344) rat and 4.6-9.2 ppm in the Wistar rat (3, 8, 14). Sensory irritation is a response that occurs immediately after the onset of exposure and is detected in animal models as a decrease in respiratory frequency due to a pause during expiration (1). This response is thought to be is evoked via stimulation of nasal trigeminal sensory C fibers (1, 40, 47, 51). Much basic research has shown that stimulation of sensory C fibers results in the antidromal release of neuropeptides, including the tachykinins substance P and neurokinin A (NKA), in the airway mucosa (5, 43). This suggests that sensory irritants, in addition to depressing respiration, should cause the local release of neuropeptides in the nasal mucosa. Although the role of sensory C-fiber stimulation in the nasal response to a few irritants has been examined (28), the relationships between respiratory depression and local neuropeptide release are poorly studied.

Sensory C-fiber stimulation may be involved in the lower respiratory tract response to acrolein. Acute exposure to acrolein (>= 20 ppm) depletes tracheal nerve substance P content (48), and C-fiber stimulation, and release of neuropeptides by acrolein (1.6 ppm) has been suggested to be a protective mechanism against the lower respiratory injury produced by this irritant in the guinea pig (50). To our knowledge, direct evidence of tachykinin involvement in the immediate nasal response to acrolein is lacking. Thus the present research was aimed at testing the hypothesis that acrolein exposure produces immediate nasal responses via stimulation of sensory fibers and release of physiologically significant quantities of tachykinins. The focus of this study was on tachykinins, in particular substance P, because its known effects, vasodilation, neurogenic edema, and mucus/fluid hypersecretion (5, 43), are precisely the mechanism previously hypothesized to play a role in the effect of acrolein on acetone uptake (34).

The response to substance P is mediated primarily through the neurokinin type 1 (NK1) receptor, although it may also exert effects through the NK2 and NK3 receptors (43). After release, substance P is rapidly destroyed by two tissue peptidases: neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) (5, 43). It is thought that the physiological effects of substance P are strongly modulated by the levels of the peptidases that are present (43). Studies using capsaicin-, substance P-, and NK1-selective agonists and antagonists have provided strong evidence that substance P may play a role in sensory C-fiber-induced nasal vasodilation in the rat (44, 45). Although not as well characterized, it is also thought that sensory C-fiber-induced neurogenic edema and fluid secretion in the rat nose are also mediated via substance P and the NK1 receptor (27, 41). NKA is another tachykinin that is released from sensory nerves. It can bind to the NK1 receptor, but with lower affinity than substance P. NKA is degraded by NEP, but not by ACE (5, 43).

To investigate the role of sensory nerves and tachykinins in the immediate nasal response to acrolein, several studies were performed. First, a concentration-response study was performed to more precisely define the immediate response to acrolein. The potential modulations of the response by the NK1 receptor antagonist N-acetyltrifluoromethyl tryptophan benzyl ester (AFTB) (30), the NEP inhibitor phosphoramidon, and the ACE inhibitor captopril (41, 43) were also studied. AFTB is an NK1-selective agonist that is effective against substance P-induced plasma extravasation (13, 30). It was anticipated that the antagonist would diminish and both peptidase inhibitors would augment the responses to acrolein if they were mediated by substance P. A potentiation by NEP, but not by ACE, inhibition would implicate a role for NKA. The use of an NK1 receptor antagonist does not examine the potential role of NK2 or NK3 receptor-mediated responses to tachykinins. It was reasoned that the peptidase inhibitors would result in elevated tachykinin levels, thereby potentiating their effects regardless of the precise receptors (NK1, NK2, or NK3) involved. Finally, adult capsaicin pretreatment was used to defunctionalize sensory nerves (23, 28) with the aim of confirming and extending results. Inhibition of the responses suggested to be mediated via tachykinins (by the NK1 antagonist and/or peptidase inhibitors) would confirm that the responses were due to a sensory fiber-dependent mediator. Conversely, examination of the effects of capsaicin pretreatment on any effects that were not influenced by the NK1 antagonist and/or the peptidase inhibitors would provide information on whether such effects were due to other sensory nerve-dependent mediators.

Several responses were examined. The uptake of acetone vapor was used as a continuous measure of nasal vascular responses. Use of inspired vapor uptake to assess respiratory tract tissue volumes and/or blood flow is well established (12, 16, 42). This laboratory has studied nasal acetone uptake extensively and has developed a physiologically based pharmacokinetic model that relates nasal acetone uptake to nasal perfusion rates and tissue depth (38). Nasal airflow resistance was also continuously monitored throughout the exposure. Evans blue dye leakage into nasal tissue and the nasal air space (as sampled by nasal lavage) was used as a measure of plasma protein extravasation (neurogenic edema) and mucus secretion. Finally, the present study also included measurement of nasal NEP activity, inasmuch as indirect evidence suggests that acrolein may inhibit this enzyme (7).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals, exposure, and tissue collection. Specific pathogen-free male F-344 rats (VAF/Plus Crl:CDBR; Charles River Laboratories) were acclimitized for >= 1 wk before use. At the time of use, the animals weighed 170-230 g and were 45-70 days of age. All animals were anesthetized with urethan (1.3 g/kg). After the onset of anesthesia, the trachea was isolated and an endotracheal tube was inserted in an anterior direction until its tip was at the larynx; it was tied in place to isolate the upper respiratory tract (URT). Animals were then placed in a nose-only inhalation chamber and exposed to acrolein for 50 min by drawing chamber air continuously through the isolated URT; a schematic of the exposure system has been published previously (36). At the end of exposure the animal was removed from the chamber, the endotracheal tube was removed and replaced with a clean tube, and 3 ml of saline were gently pushed through the URT and collected at the external nares to obtain secretions from the nasal airway lumen. Recovery of fluid exceeded 2.5 ml and was not influenced by any exposure and/or pretreatment protocol. After lavage, animals were killed by exsanguination by cutting the abdominal vena cava.

When administered, Evans blue dye (30 mg/ml iv in saline) was given just before uptake measurement. After exposure and exsanguination, the thorax was opened and the vascular bed was perfused with 30 ml of saline via injection into the left ventricle. The animal was then decapitated, the skull was split open sagittally, and nasal tissues were collected and homogenized in Krebs-Ringer buffer (total volume 9 ml). The homogenate was spun at 600 g for 5 min to remove debris.

Drug/chemical pretreatments. For capsaicin pretreatment, animals received 50 mg/kg of the toxin dissolved in 1:1:8 ethanol-Tween 80-saline (injection volume 5 ml/kg) by dorsal subcutaneous injection (2). Before injection, animals were anesthetized with pentobarbital sodium (60 mg/kg ip) and received theophylline (10 mg/kg sc) and terbutaline (0.1 mg/kg) (2). Control animals were anesthetized, given theophylline and terbutaline, and then injected with the capsaicin vehicle. For the NK1 antagonist experiments, animals were pretreated with 6 mg of AFTB (10 mg/ml in propylene glycol sc) 30 min before exposure (30). Because AFTB is an ester and rat nasal tissues contain high carboxylesterase activities, animals were pretreated with the carboxylesterase inhibitor bis(p-nitrophenyl)phosphate (20 mg/ml in water, 5 ml/kg sc) 90 min before they received AFTB. This carboxylesterase inhibitor regimen has been used previously in our laboratory and is without effect on URT acetone uptake (32). Control animals received bis(p-nitrophenyl)phosphate and propylene glycol by the same regimen. The NEP inhibitor phosphoramidon (2.5 mg/kg, 2.5 mg/ml in saline) or the ACE inhibitor captopril (0.625 mg/kg, 0.625 mg/ml in saline) was administered intravenously 15 min before measurement of uptake. Control animals received saline intravenously. Evans blue dye (30 mg/ml in saline) was administered intravenously at 30 mg/kg 5 min before the start of exposure. All drugs were obtained from Sigma Chemical (St. Louis, MO).

Exposure conditions. Exposure atmospheres were generated in a stainless steel Jaeger-NYU directed-flow nose-only inhalation chamber (C. H. Technologies, Westwood, NJ), as described previously (34, 35). Chamber airflow rates were maintained at 5 l/min with heated, humidified air to prevent nasal dehydration. Animals were exposed to 0, 2, 5, 10, or 20 ppm acrolein (0, 5, 10, 22, or 44 µg/l). These concentrations match those used in our earlier studies (34). Chamber air also contained 35 ppm acetone (80 µg/l); acetone uptake was used as a measure of nasal vascular function (see below). To generate the vapors, aqueous solutions of acetone and/or acrolein were fed into a heated flask (80°C) that was continuously flushed with 0.6 l/min of air. Vapor-laden and diluting air were mixed before entering the chamber. All air lines and the chamber walls were heated to prevent condensation.

For exposure, the animal was placed in the nose-only chamber, and the endotracheal tube was connected to an air-sampling system (containing a 6-ml trap) that was connected to a vacuum source (see below). Chamber air was continuously drawn through the URT and through the trap at a flow rate of 100 ml/min for 50 min (Fig. 1). Flow rate was controlled with a rotameter that was previously calibrated in the sample line. The endotracheal tube was connected via a T connector to a differential pressure transducer (model DP45, Validyne, Northridge, CA). The transducer was connected to an amplifier/carrier demodulator (model CD23, Validyne), and differential pressure was recorded at 5-min intervals during the exposure.


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  		Fig. 1.   Acetone uptake efficiency vs. exposure time. Values are means ± SD of 14 control animals [0 parts/million (ppm) acrolein], 11 animals treated with 10 ppm acrolein, and 15 animals treated with 20 ppm acrolein.

Acetone uptake. Acetone uptake was measured as described previously (34). For analysis, air was continuously bled off the trap and through an eight-port, two-loop, gas-sampling valve connected to a gas chromatograph. Air samples were injected into the gas chromatograph at 3-min intervals. This provided a measure of acetone concentration in air exiting the URT every 3 min during the exposure. Chamber acetone (inspired) concentrations were measured by gas chromatography immediately before and immediately after exposure of each animal. Chamber air acetone concentrations remained steady, the ratio of the "before" to "after" concentrations was 0.994 ± 0.034 (SD). Acetone uptake was calculated from the inspired air and URT exiting air concentrations by the formula
uptake = (inspired concentration − exiting concentration)

/inspired concentration
and expressed as a percentage.

Analytic methods. Airborne vapor concentrations were measured with a gas chromatograph (model 3400, Varion) with a flame ionization detector. A 15-m DB-Wax megabore column was used with a carrier gas flow rate of 30 ml/min and a column temperature of 33°C. The acetone and acrolein peaks eluted at 0.36 and 0.41 min, respectively. Vapor concentration was calculated on the basis of peak height. Standard curves were prepared by placing acetone and acrolein in Teflon gas-sampling bags, allowing >= 1 h for evaporation, and drawing air samples from the bags by sampling system used for the uptake measurements. Acrolein had no effect on the quantitation of acetone vapor. Acetone had no effect on the detection and quantitation of >= 4 ppm acrolein; however, at <4 ppm acrolein, the tail on the acetone peak made quantitation of the acrolein peak height difficult.

Therefore, the exposure concentration in the 2 ppm exposure groups was estimated on the basis of the mass ratio of acrolein to acetone in the aqueous generation fluid and the observed acetone concentration in the chamber. The average chamber acetone concentration was 36 ppm. The actual chamber acrolein concentrations for the groups exposed to 20, 10, and 5 ppm were 20 ± 1, 10 ± 1, and 4.5 ± 0.2 (SE) ppm, respectively. For the group exposed to 2 ppm the acrolein concentration was estimated to be 1.9 ppm.

To measure tissue Evans blue dye concentrations, homogenates were diluted 1:10 in formamide, incubated overnight at 60°C, and then read in a fluorometer at an excitation wavelength of 620 nm and emission wavelength of 645 nm (41). Nasal homogenate NEP activity was measured as described by Yandle et al. (52), with succinyl-Ala-Ala-Phe-7-amido-4-methyl-coumarin as a substrate. Substrate hydrolysis rates were linear with time and protein concentration.

Statistical analysis. All statistical calculations were performed using Statistica software (StatSoft, Tulsa, OK). Groups of data were compared by ANOVA and then by Newman-Keuls test. Repeated-measures ANOVA was used to analyze the flow resistance data. Data were logarithmically transformed when variances were unequal. Linear trends were analyzed by linear regression analysis, and correlations between various biological measures were assessed by Spearman's rank correlation coefficient. P < 0.05 was required for significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Concentration-response relationships. Acetone uptake vs. time of exposure for animals exposed to acetone alone or acetone with 10 or 20 ppm acrolein is shown in Fig. 1. In animals exposed to acetone alone, uptake remained steady during the exposure and averaged ~22%. In animals exposed to 10 ppm acrolein, acetone uptake also remained steady throughout the exposure and was increased to ~40%. The response was fully developed by 6 min after the onset of exposure. In animals exposed to 20 ppm acrolein, uptake was increased over control levels but did not remain steady during the exposure; rather, a progressive decline in uptake was observed during the entire 50-min exposure.

To quantitate the rate of decline in uptake, the slope of the uptake vs. time relationship was obtained for every animal by linear regression analysis. These slopes were then compared by ANOVA. In animals exposed to acetone alone or acetone with 2, 5, or 10 ppm acrolein, uptake tended to increase slightly during the exposure (positive change in uptake; Fig. 2), but for no group was the average change per minute different from zero, indicating that statistical steady-state uptake was maintained. In animals exposed to 20 ppm acrolein, uptake decreased by ~0.3%/min, an average value significantly different from that of any other exposure group and different from zero, indicating that steady state was not maintained in this group.


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  		Fig. 2.   Effect of acrolein on acetone steady-state uptake behavior. A change in uptake vs. time of 0%/min is reflective of steady state. Values are means ± SD of 14 control animals (0 ppm), 5 animals treated with 2 ppm acrolein, 7 treated with 5 ppm, 11 treated with 10 ppm, and 15 treated with 20 ppm. Data were compared by ANOVA and then by Newman-Keuls test; groups with different letters (a and b) are significantly different from each other (P < 0.05).

The total mass of acetone removed from the air in the URT during 6-50 min of exposure was calculated from the flow rate, inspired concentration (normalized to 35 ppm for each animal), and percent uptake at each sampling period. These data were then compared by ANOVA. Acrolein induced a concentration-dependent increase in uptake (P < 0.05; Table 1), with total uptake being elevated over control levels at all exposure concentrations. At 20 ppm, average uptake was lower than at 10 ppm; however, direct comparisons between these groups should be made with caution, because in animals exposed to 20 ppm acrolein, acetone uptake did not maintain a steady state.

                              
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  		Table 1.   Concentration-response relationships for lavage and tissue responses to acrolein

The nasal flow resistance in each group is shown in Fig. 3. Flow resistance increased with time during the exposure to ~6 cmH2O · l-1 · min. Because the increase in flow resistance vs. time was not always linear, data were compared by repeated-measures ANOVA. A significant effect of acrolein and of time were detected as well as an interaction between acrolein and time. Newman-Keuls test revealed that the flow resistance at 20 ppm acrolein was higher than in the other groups (P < 0.05).


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  		Fig. 3.   Effect of acrolein on upper respiratory tract airflow resistance. Airflow resistance is plotted vs. exposure time. SD bars are excluded for clarity. SDs ranged from 1 to 4 cmH2O · l-1 · min. Data were compared by repeated-measures ANOVA and then by Newman-Keuls test. A significant (P < 0.05) effect of acrolein concentration and exposure time was detected. A statistical interaction between concentration and time was also detected. Newman-Keuls test revealed that airflow resistance was higher in group treated with 20 ppm acrolein than in any other group.

The dose-response studies were performed in two phases. Animals in the first phase were not injected with Evans blue dye, and tissue parameters were not measured. In the second phase (4 animals per dose level: control and 5, 10, and 20 ppm), Evans blue dye was injected and tissue parameters were measured. (Evans Blue dye injection had no effect on acetone uptake parameters.) Lavage Evans blue dye content was elevated roughly threefold over control levels in animals exposed to 20 ppm acrolein (P < 0.05; Table 1). A correlation was observed between the degree of non-steady-state behavior and the lavage Evans blue dye content of each animal (Spearman rank correlation coefficient = 0.64, n = 16, P < 0.01). No difference was detected among exposure groups in tissue Evans blue dye content (P = 0.15; Table 1). NEP activities averaged 55 ± 22 in control nasal tissue homogenates and were not statistically altered in any exposure group (P > 0.5).

NK1 antagonist studies. The steady-state acetone uptake behavior in animals exposed to 0, 10, or 20 ppm acrolein and pretreated with vehicle or AFTB is shown in Fig. 4. Data were analyzed by ANOVA and then by Newman-Keuls test. Acetone uptake demonstrated steady-state behavior in animals exposed to acetone alone or acetone + 10 ppm acrolein, and AFTB was without effect on this behavior (P > 0.05). Vehicle-treated animals exposed to 20 ppm acrolein demonstrated a continuous decline in uptake at a rate of 0.3%/min, similar to that observed in the dose-response study (Fig. 2). This response was abolished by AFTB. Total URT uptake of acetone during 6-50 min averaged 86 ± 5, 137 ± 4, and 120 ± 9 (SD) µg in the control, 10 ppm, and 20 ppm vehicle groups and was not influenced by AFTB in any exposure group. Thus AFTB abolished the non-steady-state response to acrolein without influencing the enhanced uptake response.


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  		Fig. 4.   Effect of the neurokinin type 1 (NK1) receptor antagonist N-acetyltrifluoromethyl tryptophan benzyl ester (AFTB) on acrolein-induced changes in acetone uptake steady-state behavior. A change in uptake vs. time of 0%/min is reflective of steady state. Values are means ± SD of 3-5 animals in each group. Data were compared by ANOVA and then by Newman-Keuls test; groups with different letters (a and b) are significantly different from each other (P < 0.05).

The effects of acrolein and AFTB on nasal flow resistance are shown in Fig. 5. Repeated-measures ANOVA revealed a significant effect of acrolein and exposure time. The effect of AFTB did not achieve statistical significance (P = 0.052). No statistical interactions were observed. As in the dose-response studies, Newman-Keuls tests indicated that flow resistance was significantly increased over control levels in only the 20 ppm acrolein groups. The maximal value was ~6-10 cmH2O · l-1 · min. Tissue parameters were not measured in this study, because the study was done before the completion of the dose-response and capsaicin studies and the need for tissue data was not appreciated.


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  		Fig. 5.   Effect of NK1 receptor antagonist AFTB on acrolein-induced changes in upper respiratory tract airflow resistance. Airflow resistance is plotted vs. exposure time. SD bars are excluded for clarity. SDs ranged from 1 to 4 cmH2O · l-1 · min. Data were compared by repeated-measures ANOVA and then by Newman-Keuls test, which revealed a significant (P < 0.05) effect of acrolein concentration and exposure time but not AFTB (P = 0.052). No statistical interactions were detected. Newman-Keuls test revealed that airflow resistance was higher in group treated with 20 ppm acrolein than in any other group.

Effect of peptidase inhibition. These studies focused on the potential for the peptidase inhibitors to potentiate the non-steady-state uptake response to acrolein and, therefore, utilized an acrolein exposure concentration of 10 ppm. This was the highest concentration that did not induce non-steady-state behavior in the concentration-response studies (Fig. 2). The effects of the peptidase inhibitors on the response to lower concentrations of acrolein were not examined in detail, because the NK1 antagonist studies did not suggest a role for substance P in the response at these lower concentrations. Moreover, in pilot studies, total URT uptake of acetone averaged 88 ± 9 and 106 ± 13 µg in captopril- or phosphoramidon-pretreated animals exposed to 2 ppm acrolein, values similar to those observed in nonpretreated animals (Table 1), suggesting that peptidase inhibition does not potentiate the acetone uptake response at lower acrolein exposure concentrations.

The effect of acrolein and peptidase inhibition on steady-state acetone uptake behavior is shown in Fig. 6. Steady-state uptake behavior (no change in uptake vs. time) was observed in all control groups. Animals exposed to 10 ppm acrolein and pretreated with vehicle demonstrated steady-state behavior as well; however, at this exposure concentration, phosphoramidon and captopril induced non-steady-state behavior (P < 0.05, Newman-Keuls test). Average acetone uptake was increased from 93 ± 4 to 145 ± 8 µg by 10 ppm acrolein (P < 0.05), an effect not significantly altered by phosphoramidon or captopril. Thus captopril and phosphoramidon potentiated the non-steady-state response to acrolein but did not influence the enhanced uptake response.


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  		Fig. 6.   Effect of peptidase inhibitors phosphoramidon and captopril on acrolein-induced changes in acetone uptake steady-state behavior. A change in uptake vs. time of 0%/min is reflective of steady state. Values are means ± SD of 3-5 animals in each group. Data were compared by ANOVA and then by Newman-Keuls test; groups with different letters (a and b) are significantly different from each other (P < 0.05).

The effects of peptidase inhibition on nasal flow resistance are shown in Fig. 7. Repeated-measures ANOVA revealed a significant effect of acrolein vs. control, no effect of captopril or phosphoramidon (P = 0.8), and no effect of time. No significant statistical interactions were observed. Flow resistance was increased to ~2 cmH2O · l-1 · min in the 10 ppm groups. This is similar to the response to 10 ppm acrolein observed in the dose-response studies (Fig. 3) and somewhat less than that observed in the capsaicin studies (see Fig. 10). Tissue parameters were not measured in this study, because the study was done before the completion of the dose-response and capsaicin studies and the need for tissue data was not appreciated.


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  		Fig. 7.   Effect of peptidase inhibitors phosphoramidon and captopril on acrolein-induced changes in upper respiratory tract airflow resistance. Airflow resistance is plotted vs. exposure time. SD bars are excluded for clarity. SDs ranged from 1 to 2 cmH2O · l-1 · min. Data were compared by repeated-measures ANOVA and then by Newman-Keuls test, which revealed a significant (P < 0.05) effect of acrolein concentration and exposure time but not of inhibitors (P = 0.82). No statistical interactions were detected. Newman-Keuls test revealed that airflow resistance was higher in group treated with 10 ppm acrolein than in control group.

Capsaicin-pretreatment studies. The effect of capsaicin pretreatment on steady-state acetone uptake behavior is shown in Fig. 8. As observed previously (Figs. 2 and 4), uptake decreased with time in the 20 ppm exposure group. This effect was abolished by pretreatment with capsaicin. Capsaicin was without effect on steady-state behavior in the control and 10 ppm exposure groups.


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  		Fig. 8.   Effect of capsaicin pretreatment on acrolein-induced changes in acetone uptake steady-state behavior. A change in uptake vs. time of 0%/min is reflective of steady state. Values are means ± SD of 6-7 animals in each group. Data were compared by ANOVA and then by Newman-Keuls test; groups with different letters (a and b) are significantly different from each other (P < 0.05).

Capsaicin pretreatment attenuated the acrolein-induced increase in total acetone uptake (Fig. 9). Specifically, in the 10 ppm exposure group, uptake averaged 111 µg in capsaicin-pretreated animals compared with 146 µg in the vehicle controls (P < 0.05, Newman-Keuls test). Total acetone uptake was similar in the 20 ppm control and capsaicin-pretreated groups, but the control groups exhibited non-steady-state uptake (Fig. 8), making precise quantitative comparisons impossible. Interestingly, total acetone uptake in the capsaicin-pretreated groups exposed to 10 or 20 ppm acrolein was significantly higher than control levels, indicating that the effects of acrolein were not abolished by capsaicin.


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  		Fig. 9.   Effect of capsaicin pretreatment on acrolein-induced changes in total acetone uptake. Values are means ± SD of 6-7 animals in each group. Data were compared by ANOVA and then by Newman-Keuls test; groups with different letters (a-c) are significantly different from each other (P <0.05).

The effects of capsaicin pretreatment on nasal flow resistance are shown in Fig. 10. Repeated-measures ANOVA revealed a significant effect of exposure concentration and time but no effect of capsaicin (P = 0.94). An interaction between acrolein concentration and time was detected, but no other significant interactions were observed. Peak flow resistance values were ~4-6 and 8-11 cmH2O · l-1 · min in the 10 and 20 ppm groups, respectively.


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  		Fig. 10.   Effect of capsaicin pretreatment on acrolein-induced changes in upper respiratory tract airflow resistance. Airflow resistance is plotted vs. exposure time. SD bars are excluded for clarity. SDs ranged from 1 to 6 cmH2O · l-1 · min. Data were compared by repeated-measures ANOVA and then by Newman-Keuls test, which revealed a significant (P < 0.05) effect of acrolein concentration and exposure time but not capsaicin (P = 0.94). A statistically significant interaction between concentration and time was observed, but no other interactions were detected.

Lavage and tissue parameters are shown in Table 2. Data were analyzed by ANOVA and then by Newman-Keuls test. Exposure to 20 ppm, but not 10 ppm, acrolein resulted in a significant increase in nasal lavage Evans blue dye content. The response to 20 ppm was attenuated but not abolished by pretreatment with capsaicin. Tissue Evans blue dye content was also significantly increased by exposure to 20 ppm acrolein. The larger sample size (6-7 rats/group) in this study than in the dose-response study (4 rats/group) is probably responsible for an enhanced statistical sensitivity of the capsaicin study. Although tissue Evans blue dye content was lower in capsaicin- than in vehicle-pretreated animals exposed to 20 ppm acrolein, the difference between the groups did not attain statistical significance (P = 0.08). Lavage urea was not increased by acrolein in the vehicle-pretreated groups but was significantly elevated over the respective control (no acrolein) level in the capsaicin-pretreated animals.

                              
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  		Table 2.   Effect of capsaicin pretreatment on tissue responses to acrolein


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Acetone is a nonreactive nonmetabolized vapor, the uptake of which in the rat URT has been extensively studied in this laboratory (33, 37). Continued uptake throughout many minutes of exposure is dependent on diffusion of acetone through the nasal air-blood barrier and removal via the bloodstream. With the assumption of a rat nasal tissue volume of 0.2 ml and a tissue-air partition coefficient of 260 [equal to the blood-air partition coefficient (10)], the maximal amount of acetone that can accumulate in nasal tissue at an exposure concentration of 35 ppm (80 µg/l) is 4 µg. That >150 µg of acetone were scrubbed from the airstream in the URT during the exposure highlights the importance of nasal perfusion in controlling overall uptake in this experimental paradigm.

Exposure to acrolein vapor produced consistent effects on acetone uptake: a prolonged increase in uptake efficiency and non-steady-state uptake behavior (at the highest exposure concentration). This irritant also produced a dose-dependent steady increase in nasal flow resistance and an increase in plasma protein extravasation (as measured by Evans blue dye leakage). These results are summarized in Table 3. The effects were reproducible, being observed in the dose-response studies and in the vehicle controls of the AFTB, peptidase inhibitor, and/or capsaicin-pretreatment studies. The acrolein-induced enhancement in nasal acetone uptake and induction of non-steady-state uptake behavior were observed in our previous study on acrolein uptake (34), but concentration-response and mechanistic information were not provided.

                              
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  		Table 3.   Summary of results

At >= 2 ppm, acrolein produced a prolonged increase in acetone uptake efficiency, indicating that it produced nasal vasodilation and increased blood flow. The physiologically based pharmacokinetic model for acetone uptake predicts that, in normal animals, nasal uptake can be explained by a nasal perfusion rate of 0.18 ml/min (38). This model predicts that nasal perfusion rates of 0.5 ml/min are necessary to account for acetone uptake efficiencies of 40%, such as those observed in the present study suggesting that acrolein produced as much as a threefold increase in nasal blood flow. This response was produced in the absence of a change in nasal flow resistance at exposure concentrations of 2 or 5 ppm acrolein, suggesting that the vasodilation was independent of changes in nasal blood congestion and airflow obstruction.

Many biological mediators have been shown to produce this type of response (e.g., substance P, vasoactive intestinal peptide, and prostaglandin E2) (29). The large increases in blood flow in the absence of nasal airflow obstruction suggest that the response may be due to dilation of the arteriovenous anastomoses rather than the cavernous sinuses (4, 18, 28). The mechanisms through which this response is mediated are not known with certainty. That the response is significantly diminished by capsaicin pretreatment provides strong evidence that it is mediated, at least in part, via sensory nerve stimulation. The failure of the capsaicin pretreatment to abolish the vasodilatory response may indicate that not all sensory nerves were defunctionalized by the capsaicin pretreatment and/or that nonneuronal mechanisms also play a role in this response. Even though tachykinins are released from sensory nerves on stimulation and are known to induce increased blood flow in the rat nose (5, 43, 44), they do not appear to be involved in the vasodilatory response to acrolein, as evidenced by the fact that total acetone uptake was not influenced by the NK1 antagonist AFTB or the peptidase inhibitors. NEP inhibition would be expected to enhance any tachykinin-dependent response regardless of the precise receptors (NK1, NK2, or NK3) involved. The lack of effect of the peptidase inhibitors also suggests that other peptides, such as bradykinin, do not play a role (9). Nasal intraepithelial sensory nerves in the rat express calcitonin gene-related peptide (21) and neuronal nitric oxide synthase (22, 24). Initial studies in our laboratory suggest that these mediators may play a role in the vasodilatory response to acrolein (39). Further studies are needed to elucidate the precise mechanisms of acrolein-induced vasodilatory response.

Exposure to the highest concentration of acrolein (20 ppm) produced non-steady-state uptake behavior and plasma protein extravasation, as indicated by increases in lavage and/or tissue Evans blue dye content. Results of the present study suggest that these responses are mediated by substance P. Specifically, the non-steady-state behavior was abolished by capsaicin pretreatment, implicating sensory nerve involvement. This behavior was inhibited by pretreatment with the NK1 antagonist AFTB and potentiated by the NEP or the ACE peptidase inhibitors, suggesting that it is mediated via a sensory nerve peptide that interacts with the NK1 receptor and is degraded by both peptidases. Because NKA is not a substrate for ACE (43), substance P is the most likely candidate (5, 43). The dye-leakage response, particularly in the nasal lavage fluid, was also diminished by capsaicin pretreatment (Table 2), implicating a role for sensory nerves. Petersson et al. (41) showed that sensory C-fiber stimulation causes substance P-mediated neurogenic edema in the F-344 rat nose, and, as indicated by modulation of the non-steady-state uptake behavior by the NK1 antagonist and peptidase inhibitors, physiologically significant levels of substance P appear to be released during exposure to 20 ppm acrolein.

Ben-Jebria et al. (7) provide indirect evidence that, in vitro, acrolein at concentrations as low as 0.1 ppm inhibits tracheal NEP. Thus, in addition to stimulating tachykinin release, acrolein might also inhibit its degradation via NEP, leading to complex concentration-response behavior. The present study, with use of direct measurement of NEP activity, failed to detect irreversible inhibition of nasal NEP at exposure concentrations as high as 20 ppm, suggesting that NEP inhibition by acrolein does not play a role in the in vivo responses that were observed in this animal model. However, the possibility of selective inhibition of NEP at discrete compartments (epithelial vs. submucosal) cannot be eliminated.

The precise physiological mechanisms for the non-steady-state uptake response are not known. It is possible that the tachykinin-mediated non-steady-state response is the result of depletion of mediator and/or receptor desensitization during the exposure. We previously hypothesized on strong theoretical grounds that non-steady-state uptake behavior is reflective of an acrolein-induced steady thickening of the nasal air-blood barrier leading to increased tissue phase diffusional resistances (34). The present results support this hypothesis. The increase in tissue and nasal lavage Evans blue dye content caused by acrolein suggests that enhanced plasma protein extravasation and tissue swelling occurred with leakage and/or secretion into the air spaces due, perhaps, to increased epithelial permeability. At 20 ppm, acrolein is probably ciliastatic (17); this may play a role in the increased Evans blue dye content in the lavage sample. Were the increased lavage Evans blue dye the result of hypersecretion, it might represent a protective response to acrolein. The acrolein-induced increase in nasal airflow resistance does not appear to be involved in the non-steady-state uptake response, because 1) airflow resistance was increased at an exposure concentration of 10 ppm, a concentration that does not induce non-steady-state uptake behavior; and 2) the non-steady-state uptake behavior was significantly modulated by capsaicin, the peptidase inhibitors, and the NK1 antagonist, and the airflow resistance response was not.

Acrolein also induced a steady increase in nasal airflow resistance throughout the 50-min exposure. A ~6- to 10-fold increase in airflow resistance was observed in animals exposed to 20 ppm acrolein (Figs. 3, 7, and 10). A smaller increase in airflow resistance (2- to 6-fold) was observed in animals exposed to 10 ppm in the peptidase inhibitor and capsaicin studies. A similar increase was seen in the dose-response and AFTB studies, but it did not achieve statistical significance. The mechanisms of this response are elusive. The response was not significantly altered by capsaicin pretreatment, by the peptidase inhibitors, or by the NK1 antagonist. The flow rate response did not correlate with the non-steady-state response or the vasodilatory response. Further studies are needed to better define this response.

It is important to note that the responses observed in the present study were only observed at relatively high exposure concentrations and, therefore, may be important only in circumstances where acrolein exposures are quite high, such as in fire-fighting operations. Respiratory depression is another immediate nasal response thought to be mediated by sensory fiber stimulation (1, 40, 47, 51). The RD50 for acrolein in the F-344 rat is 6 ppm (3). Acrolein concentrations approaching or in excess of the RD50 were necessary to produce the local responses described in the present study. Perhaps a similar degree of nerve stimulation is needed to cause local responses and respiratory depression. Whether similar relationships are seen for other sensory irritants is not known.


    ACKNOWLEDGEMENTS

The expert technical assistance of Barbro Simmons and Michael Lewis is gratefully acknowledged.


    FOOTNOTES

This publication was made possible by National Institute of Environmental Health Sciences Grants ES-03676 and ES-08765. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Environmental Health Sciences.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. B. Morris, School of Pharmacy, Box U-92, 372 Fairfield Rd., Univ. of Connecticut, Storrs, CT 06269-2092 (E-mail: morris{at}uconnvm.uconn.edu).

Received 13 August 1998; accepted in final form 22 July 1999.


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