ARTICLE IN PRESS
Pulmonary Pharmacology & Therapeutics 20 (2007) 149–156 www.elsevier.com/locate/ypupt
a2-adrenoceptor agonists as nasal decongestants M.R. Corboz, J.C. Mutter, M.A. Rivelli, G.G. Mingo, R.L. McLeod, L. Varty, Y. Jia, M. Cartwright, J.A. Hey Pulmonary and Peripheral Neurobiology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA Received 16 March 2006; accepted 17 March 2006
Abstract Nasal congestion, one of the major disease features of rhinitis, is induced by the filling of venous sinusoids causing mucosal engorgement with resultant obstruction of nasal airflow. The only available drugs that directly target the underlying vascular features driving nasal obstruction are the sympathomimetic a-adrenoceptor agonists due to their vasoconstrictor action. However, standard decongestants are nonselective a-adrenoceptor agonists, which have the potential for side-effects liabilities such as hypertension, stroke, insomnia and nervousness. In the present study, the effects of nonsubtype selective a2-adrenoceptor agonists BHT-920 and PGE-6201204 were evaluated in several isolated nasal mucosa contractile bioassays including dog, pig and monkey, and in a real-time tissue contractility assay using isolated pig nasal explants for BHT-920. The decongestant activity of PGE-6201204 was evaluated in vivo in a cat model of experimental congestion. Our results showed that a2-adrenoceptor agonists (1) contract nasal mucosa of different species, (2) exert a preferential vasoconstrictor effect on the capacitance vessels (veins and sinusoids), and (3) elicit decongestion. In conclusion, a selective a2-adrenoceptor agonist causing constriction preferentially in the large venous sinusoids and veins of nasal mucosa and producing nasal decongestion is expected to show efficacy in the treatment of nasal congestion without the characteristic arterioconstrictor action of the standard nonselective sympathomimetic decongestants. r 2006 Elsevier Ltd. All rights reserved. Keywords: Nasal mucosa; Decongestion; a2-Adrenoceptor agonist; BHT-920
1. Introduction Nasal congestion, one of the symptoms of rhinitis, results from vascular reactions such as vasodilatation, increased blood flow and increased vascular permeability with resultant engorgement of venous sinusoids [1]. These events lead to the swelling of the anterior and inferior turbinates, and finally to the obstruction of nasal airflow. The nose is one of the most vascularized organs of the body with a total blood flow per cubic centimeter of tissue exceeding that in muscle, brain and liver [2]. Nasal blood flows typically from arteries (resistance vessels) to capillaries and into veins (capacitance vessels), at a rate of 42 mL/100 g of tissue per minute in the superficial nasal mucosa [3], but numerous arteriovenous anastomoses are also found in the nasal mucosa with cavernous sinusoids Corresponding author. Tel.: +1 908 740 7238; fax: +1 908 740 7175.
E-mail address:
[email protected] (M.R. Corboz). 1094-5539/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2006.03.012
between the capillaries and venules [4,5]. A very dense sympathetic innervation is found around arteries, venules and venous sinusoids [6]. The cavernous sinuses are normally contracted, but under conditions of reduced sympathetic tone, this erectile tissue becomes engorged and causes nasal obstruction [4]. The only drugs specifically used to relieve vascular nasal obstruction are the a-adrenoceptor agonist sympathomimetic agents because of their vasoconstrictor action, which opposes mucosal engorgement in the highly vascular nasal mucosa [1]. Standard selective a1- and/or nonselective aadrenoceptor agonists such as phenylephrine, pseudoephedrine and phenylpropanolamine produce decongestion by constricting both capacitance and resistance vessels in the nasal mucosa, by selective activation of a1-adrenergic receptors only (phenylephrine) or by nonselective activation of a1- and a2-adrenergic receptors (pseudoephedrine, phenylpropanolamine). Administration of phenylephrine and the standard topical decongestant oxymetazoline
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(nonselective a-adrenoceptor) to the nasal mucosa constricts vessels and decreases mucosal engorgement leading to the relief of congestion in animals [6,7] and humans [8]. Previous studies reported that a2-adrenoceptors are distributed in nasal mucosa of dog [9], pig [10] and human [8,11] and a2-mechanism predominates in the regulation of venous sinusoids in pig [6] and nasal venous vessels in dog [12]. Indeed, we previously showed that a2-adrenoceptors are present and mediate vasoconstriction in pig [10] and human [11] nasal mucosa with a preferential constrictor effect on the venular side in human [11]. Because the pathophysiology of nasal congestion predominantly affects the large venous sinusoids and collecting veins, vascular a2adrenoceptors represent an attractive agonist target for nasal decongestion based on their localization within the nasal mucosa. Moreover, an a2-adrenoceptor message was also demonstrated in arteriovenous anastamoses and venous sinusoids of human nasal turbinate by in situ hybridization [13] and higher levels of a2-adrenoceptor protein than a1-adrenoceptor protein was showed in human nasal mucosa of nonallergic and allergic patients by using binding technique [14]. We also reported that a2adrenoceptor proteins were expressed in human nasal mucosa [11]. In addition, a recent human study in an allergen exposure unit demonstrated that a nonselective a2adrenoceptor agonist produced clinically and statistically significant relief of nasal congestion in human subjects with moderate to severe seasonal allergic rhinitis to ragweed allergen [15]. The decongestant effects observed in this study were equivalent to the positive comparator pseudoephedrine [15]. The aims of the present study were: (1) to characterize the a2-adrenoceptors mediating vasoconstriction in nasal mucosa of different species by using the standard a2adrenoceptor agonists BHT-920 and PGE-6201204, (2) to define the relative contribution of arteries and veins in pig nasal vessels challenged with BHT-920, (3) to determine the decongestant activity of PGE-6201204 in the cat congestion model.
bath solution was maintained at 37 1C with a pH of 7.4 and continuously gassed with 95% O2 and 5% CO2. The transducers were connected to a physiograph (model WR3310, Western Graphtech, Inc., Irvine, CA, USA) for continuous recording of isometric tension. Initial tension of 1.0 g was applied to dog and monkey mucosal strips, and 2.0 g to pig mucosal strips and tissues were then equilibrated for 1 h. 2.1.2. Experimental protocol Nasal mucosa strips were first tested for responsiveness with the nonselective a-adrenoceptor agonist norepinephrine (10 mM for dog, monkey and 100 mM for Yucatan pig) that provided a maximal contraction. The tissues were then washed 3 times during a period of 1 h. Concentration– response curves to the a2-adrenoceptor agonists (log increments) BHT-920 and PGE-6201204 were then performed in the absence or the presence of the a1- or a2adrenoceptor antagonists (prazosin and yohimbine respectively) added to the bath 1 h before the cumulative additions of BHT-920 and PGE-6201204. Data were presented as a percent of norepinephrine-induced contraction.
2. Methods
2.1.3. Data analysis and statistics Agonist activity was estimated as gram tension increase over baseline and normalized as a percent of maximum norepinephrine response. Agonist potency was expressed as a pD2 (pD2 ¼ log10 EC50). EC50 is the agonist concentration causing 50% of the maximal response to the a2adrenoceptor agonists and was estimated using linear regression analysis of the concentration response curves. Antagonist activity was estimated by the agonist dose ratio (DR). DR is equal to A0 /A, where A0 and A are the agonist EC50 values in the presence and absence of the antagonist. Antagonist potency was expressed as an apparent pKb ( ¼ log10 of Kb) [15]. Kb ¼ [B]/(A0 /A–1), where [B] is the molar concentration of the antagonist tested [16]. Apparent pKb was calculated using individual DR values X2. All results are expressed as means7standard error of the mean (S.E.M.). Numbers of observations (n) indicate the number of tissues, at least from 4 animals in each group.
2.1. Nasal mucosa contractility assay
2.2. Pig nasal mucosa histology
2.1.1. Tissue preparation Nasal mucosa of dog (10–15 kg), Yucatan mini-pig (75–80 kg), Cynomolgus monkey (5–10 kg) and Rhesus monkey (7–11 kg) was dissected from the turbinates after euthanasia by LAMS, Schering Plough Research Institute. Mucosa was cut into pieces of approximately 0.8–1.5 cm in length and 0.2–0.5 cm in width. The tissues were gently wiped with a cotton-tip applicator to remove excess mucus. Each mucosal strip was then fixed at the lower end of an organ bath (Q-bath, Radnoti Glass Technology, Inc., Monrovia, CA, USA) containing 25 mL Krebs–Ringer solution and attached to an isometric transducer (Grass FT-03, Astro-Med., Inc., West Warick, RI, USA). The
Histology studies were performed to identify the architecture of the pig nasal mucosa and to evaluate the reactivity of nasal arteries and veins to the a2-adrenoceptor agonist BHT-920. The preparation of tissues has been previously described (see above). However, the tissues in organ baths were challenged only with a single dose (10 mM) of the a2-adrenoceptor agonist BHT-920. Tissues were then removed from the baths at the contraction peak and fixed into neutral formalin (10% phosphate-buffered formalin). Formalin-fixed tissues were processed through a graded series of alcohols and finally in xylene, and then embedded in paraffin. Paraffin-embedded tissues were sliced to 5 mm thick sections, mounted on glass slides,
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and stained with either hematoxylin and eosin or Verhoeff’s Van Gieson stain. 2.3. Pig nasal mucosa real-time contractility assay 2.3.1. Tissue preparation Pig snouts from male and female domestic pigs (110–230 kg) were provided by local abattoir, Animal Parts (Scotch Plains, NJ, USA). Fresh snout tissue was shipped on wet ice in Leibovitz’s L-15 solution (Cellgro, VA, USA) and was received at the Schering Plough Research Institute 2–4 h post-removal. Turbinate strips were isolated from the pig snouts and turbinate mucosa was then dissected free of cartilage and cut into 5 mm2 sections. Mucosal sections were placed in a 3 cm3 syringe containing 6% agarose at 40 1C then cooled to 4 1C for 60 min. Each agarose cylinder was removed from the syringe and sliced to 100 mm thickness with the Krumdieck Tissue Slicer (Alabama Research and Development, Munford, AL, USA) in Krebs buffer at 4 1C. Each slice, freed of agarose, was then placed in a 6-well tissue culture dish with Clonetics SmGM-2 culture media (Biowittaker, Walkersville, MD, USA) in the presence of 1% penicillin/streptomycin (Biowittaker, Walkersville, MD, USA). The tissue culture dish was then placed in a Precision shaker incubator maintained at 37 1C and aerated with 95% O2–5% CO2 overnight. 2.3.2. Experimental protocol The following day, nasal slices were equilibrated for 30 min at 37 1C in 100 mL of Krebs buffer in the presence and absence of a-adrenoceptor antagonists. Tissues were then challenged with the nonselective a-adrenoceptor agonist epinephrine (0.01–100 mM) in the absence and the presence of the a1-adrenoceptor antagonist prazosin (0.1 mM) and the a2-adrenoceptor antagonist yohimbine (1 mM). Because epinephrine can interact with b-adrenoceptors, the b-adrenoceptor antagonist propanolol (1 mM) was present at all times in tissue dishes to block badrenoceptors. Images of nasal mucosa slices were recorded using the Zeiss Axiovert 100 microscope (Carl Zeiss Microimaging, Thornwood, NY, USA) before and after each epinephrine challenge. 2.3.3. Data analysis and statistics Cross-sectional area of nasal arteries and veins were analyzed using Image J Software and vessel contraction was expressed as a percent decrease of cross-sectional area from baseline. All results are expressed as means7 S.E.M. Number of observations (n) indicates the number of nasal arteries and veins, at least from 5 animals in each group. 2.4. Cat congestion model 2.4.1. Animal preparation Harlan short-haired male cats (1.5–3.0 kg) were anesthetized with methohexital sodium (5 mg/kg, intravenously
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(i.v.)). Supplemental doses of methohexital sodium (0.5–1 mg/kg, i.v.) were given if necessary to maintain a steady depth of anesthesia. The right femoral artery and vein were cannulated for measurement of mean blood pressure and administration of i.v. drugs, respectively. 2.4.2. Acoustic rhinometry and blood pressure measurement Noninvasive measurements of nasal volume were performed using acoustic rhinometry method as previously described [17,18]. Briefly, measurements of nasal crosssectional area and estimates of nasal volume from a distance of 0–3 cm into the nasal cavity were performed using an acoustic rhinometer (NADAR, Aahus, Denmark). Reflected acoustic waves from the left and right nasal cavities were amplified, recorded and data obtained were converted to area–distance curves. The generated area–distance curves provided estimates of cross-sectional areas, nasal volume, Amin (minimum cross-sectional area) and Dmin (distance to the Amin) values. Systemic blood pressure was measured from the hind leg using an ultrasonic Doppler flow detector (model 811-B, Park Medical Electronics, Inc., Plymouth, MN, USA). 2.4.3. Pharmacology studies Compound 48/80 (1%, 50 mL) was given topically into the left naris to produce nasal congestion while the right naris received saline. The nasal effects of the selective a2adrenoceptor agonist PGE-6201204 (0.1%, 50 mL) were evaluated on the decrease in nasal cavity volume produced by nasally instilled compound 48/80. PGE-6201204 was administered into both the left and right nasal cavity 10 min before compound 48/80 challenges. Acoustic measurements of both the left and the right nasal cavity were evaluated at 0, 30 and 60 min after compound 48/80 challenges. Each evaluation consisted of the average of five 200 ms measurements. Systolic blood pressure measurements were determined at 0, 30 and 60 min after challenge with compound 48/80. 2.4.4. Data analysis and statistics The nasal cavity volume data are expressed as the ratio of the volume of the left, treated nares versus the right, untreated nares. The values represent the mean7S.E.M. of 5–8 animals per group. Statistical significance was evaluated by a one-way ANOVA in conjunction with a Dunnett’s two tailed t test. Probability ðPÞo0:05 was accepted as the level of statistical significance. 2.5. Solution and drugs 2.5.1. Nasal contractility assays, pig nasal real-time contractility assay and pig nasal mucosa histology Krebs solution was composed of 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24.9 mM NaHCO3, 11.1 mM glucose, 2.55 mM CaCl2. PGE-6201204 was synthesized by the Chemical Research, Schering-Plough
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Research Institute, (Kenilworth, NJ, USA). Norepinephrine, BHT-920, yohimbine and prazosin were obtained from Sigma/RBI (Natick, MA, USA). Formalin, paraffin, xylene, hematoxylin and eosin, Verhoeff’s Van Gieson stain were obtained from Fisher (Pittsburgh, PA, USA), Norepinephrine, BHT-920, yohimbine and prazosin were prepared as concentrated stocks in deionized water before dilution to final concentration in the nasal mucosa bioassay buffer. PGE-6201204 ((4,5-dihydro-1H-imidazol2-yl)-(4-methyl-1H-benzoimidazol-5-yl)-amine) was prepared as 10 mM stocks in DMSO and then diluted further in deionized water, before addition to the baths in the nasal mucosa assays. The final concentration of DMSO did not exceed 0.1% in the bath.
3. Results 3.1. Nasal mucosa contractility assay The a2-adrenoceptor agonists BHT-920 (pD2 range of 6.2–7.0) and PGE-6201204 (pD2 range of 6.1–7.4) produced concentration-dependent contractions in nasal mucosa strips of different species (Fig. 1A–C). The a2adrenoceptor antagonist yohimbine (0.1 mM), but not the a1-adrenoceptor antagonist prazosin (0.03 mM in pig preparation and 0.3 mM in dog and monkey preparations) inhibited BHT-920- and PGE-6201204-induced contraction (Fig. 1A–C). Yohimbine caused parallel shifts in the concentration response curve to BHT-920 and PGE6201204 (Fig. 1C) and was a potent inhibitor with a pKb range of 7.5–9.0 for BHT-920 and with a pKb range of 6.9–7.9 for PGE-6201204. We previously demonstrated that BHT-920 (10 mM) produced contractions in human nasal mucosa that were blocked by yohimbine (0.1–1 mM) while prazosin (0.1 mM) had no effect [11].
2.5.2. Cat congestion model Compound 48/80, BHT-920 dihydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Drug doses refer to their respective free bases. All drugs were dissolved in physiological saline (0.9%).
Dog Nasal Mucosa BHT-920 (n = 24) BHT-920 +100 nM Yohimine (n =10) BHT-920 +300 nM Prazosin (n = 6)
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6.7 ± 0.1
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pD2 PGE 6201204
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-
pKb Yohimbine
7.9 ± 0.2
7.8 ± 0.1*
7.9 ± 0.2
6.9 ± 0.3
-
* Values reported from Corboz et al. (Auton & Autac Pharmacol 2003;23:201-74) (C) Fig. 1. (A) Contractile responses to the a2-adrenoceptor agonist BHT-920 in dog nasal mucosa in the absence (closed circle, n ¼ 24 from 10 dogs), and the presence of the a1-adrenoceptor antagonists prazosin (closed diamond, n ¼ 6 from 4 dogs) at the concentration of 300 nM, and the a2-adrenoceptor antagonists yohimbine (closed square, n ¼ 10 from 4 dogs) at the concentration of 100 nM. Data are normalized to the maximal contraction response induced by 10 mM norepinephrine (NE) and values are means7S.E.M. (B) Contractile responses to the a2-adrenoceptor agonist BHT-920 in Yucatan pig nasal mucosa in the absence (closed circle, n ¼ 7 from 6 pigs), and the presence of the a1-adrenoceptor antagonists prazosin (closed diamond, n ¼ 7 from 6 pigs) at the concentration of 30 nM, and the a2-adrenoceptor antagonists yohimbine (closed square, n ¼ 7 from 6 pigs) at the concentration of 100 nM. Data are normalized to the maximal contraction response induced by 100 mM norepinephrine (NE) and values are means7S.E.M. (C) Agonist potency (pD2) for the a2-adrenoceptor agonists BHT-920 and PGE-6201204 and affinity (pKb) estimates for the a2-adrenoceptor antagonist yohimbine against contractions induced by BHT-920 and PGE-6201204.
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3.2. Pig nasal mucosa histology Various tissue components were present in the specimens of pig nasal mucosa and included mucosal and glandular epithelium, arteries, venous sinusoids and veins (Fig. 2A). With no therapeutic intervention, the nasal mucosa has a large number of dilated, thin-walled venous sinusoids that contain a great volume of blood (Fig. 2A). Arteries have a thick layer of smooth muscle. We found that the a2adrenoceptor-agonist BHT-920 preferentially contracts veins (Fig. 2C). The a1-adrenoceptor-agonist phenylephrine preferentially acts on arteries, but also has some constrictor effect on veins of pig nasal mucosa [19]. Arteries and veins are opened when no drugs are applied (Fig. 2B). Verhoeff’s Van Gieson stain was used to confirm the microscopic identification of arteries versus veins and sinusoids. 3.3. Pig nasal mucosa real-time tissue contractility assay The nonselective a-adrenoceptor epinephrine (0.01– 100 mM, n ¼ 15) caused concentration-dependent contractions in veins and arteries of the nasal slices (Fig. 3A). When tissues were pretreated with the a2-adrenoceptorantagonist yohimbine (1 mM, n ¼ 5), epinephrine preferentially induced contractions on the arteriolar side while venous contractions were greatly inhibited (Fig. 3C). In contrast, the a1-adrenoceptor-antagonist prazosin (0.1 mM,
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n ¼ 4) predominantly inhibited the contraction of the nasal arteries while the venous contractions were largely observed (Fig. 3B). A combination of yohimbine (1 mM) and prazosin (0.1 mM) (n ¼ 4) blocked the contractions due to epinephrine on both vascular sides (data not shown). The muscarinic cholinergic antagonist atropine (1 mM, n ¼ 3) did not block the constriction of the pig nasal arteries and veins (data not shown) indicating that the a-adrenoceptor antagonists produced a specific action. All functional realtime studies were performed in 4–9 pigs with the n describing the number of tissue slices used per experiment. 3.4. Cat congestion model Nasal congestion induced by compound 48/80 was dosedependently reversed after topical administration of 0.1% PGE-6201204 (Fig. 4). Increase in blood pressure was observed only at 30 min post-compound 48/80 challenge (data not shown). 4. Discussion Post-junctional a-adrenoceptor contractile responses of nasal blood vessels elicited by the a2-adrenoceptor agonists BHT-920 and PGE-6201204 are mediated by the a2adrenoceptors in nasal mucosa of different species including dog, Yucatan mini-pig, Rhesus monkey and Cynomolgus monkey. These results confirm our previous studies
Fig. 2. Light microscopy of the pig nasal mucosa. (A) Section of nasal mucosa clearly shows the epithelium (E), the submucosal glands (G), arteries (A), veins (V) and partially collapsed to distended thin-walled venous sinusoids (S). H&E stain; 10 magnification. (B) Section of lamina propria from vehicle control group demonstrating distended and collapsed thin-walled venous sinusoids (S) and arteries (A). Verhoeff’s Van Gieson stain; 10 magnification. The lumen of these arteries and sinusoids are filled primarily with erythrocytes. (C) Section of lamina propria following treatment with the a2-adrenoceptor agonist BHT-920 (10 mM) illustrating collapse (contraction) of thin-walled venous sinusoids (S) and minimal to no effect on small arteries (A). Hematoxylin an eosin stain; 10 magnification.
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100
% Decrease from Baseline
% Decrease from Baseline
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75 50 25 0
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75 50 25 0
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-7
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Yohimbine + Epinephrine (α2-blockade) Vein Artery
75 50 25 0
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-7 -6 -5 Epinephrine (μM)
-4
Fig. 3. Real-time contractility studies. Effect of the nonselective a-adrenoceptor agonist epinephrine in pig nasal mucosa in the absence (A) and the presence of the a1-adrenoceptor antagonist prazosin (0.1 mM) (B) and the a2-adrenoceptor-antagonist yohimbine (1 mM) (C). Epinephrine alone contracts both arteries and veins through a1- and a2-adrenoceptors (A), but in presence of prazosin (a1-blockade), epinephrine preferentially constrict nasal veins (B), indicating the preferential effect of a2-adrenoceptors on capacitance vessels. Data are expressed as % decrease of cross-section area from baseline and values are means7S.E.M.
+30 min
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1.00 0.75 0.50 0.25
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62 PG
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c 01 ont ro 20 ls 4 (0 .1 % )
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ro
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(TREATED/UNTREATED)
NASAL CAVITY VOLUME RATIO
Baseline
TIME (MIN) Fig. 4. Nasal decongestant effect of topical PGE-6201204 in the cat congestion model. This figure illustrates the effect of PGE-6201204 (0.1%) on nasal cavity volume ratio before, 30 and 60 min after nasal exposure to compound 48/80 (1%). Each bar represents the mean7S.E.M. of 4 animals (*Po0:05 compared with time 0; **Po0:05 compared with compound 48/80).
in pig [10] and human [11] nasal mucosa in which standard a2-adrenoceptor agonists induced contractility. Histology analysis showed that BHT-920 preferentially contracts the venular side of pig nasal mucosa indicating the predominant role of a2-adrenoceptors in nasal veins. By using realtime image analysis, we also demonstrated that a-adrenoceptor agonist-induced contractions were predominantly mediated by the a2-adrenoceptors in pig nasal veins. Finally, PGE-6201204, a peripherally selective and orally active a2-adrenoceptor agonist [20], produced decongestant activity in the cat congestion model by blocking the decrease in nasal cavity volume produced by compound 48/80. The two a2-adrenoceptor agonists BHT-920 and PGE6201204 produced concentration-dependent contractions in nasal strips of all species tested, including dog, Yucatan mini-pig, Rhesus monkey and Cynomolgus monkey with a potency (pD2) range of 6.2–7.0 for BHT-920 and 6.1–7.4 for PGE-6201204. These contractions were specifically inhibited by the a2-adrenoceptor-antagonist yohimbine with potency range (pKb) of 7.5–9.0 for BHT-920 and 6.9–7.9 for PGE-6201204; the a1-adrenoceptor antagonist prazosin had no effect on these contractions. The present potency estimates for BHT-920 and PGE-6201204 are in
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agreement with functional potency obtained in different vascular beds and species, such as pig nasal mucosa [10], rat aortic segments [21], human saphenous vein [22,23], human internal mammary artery [24]. Similar to the histology study on human nasal mucosa [11], a2-adrenoceptors play a major role in the contraction of pig nasal veins as demonstrated by the preferential constrictor effect of BHT-920 on veins. This histological finding was confirmed by results obtained with the realtime image analysis that showed a predominant action of a2-adrenoceptors on pig nasal veins. Also the architecture of pig nasal mucosa defined by histology in the present study resembles to the architecture of human nasal mucosa previously reported [4,25]. Indeed, cross-section of human nasal mucosa showed superficially the epithelium, then the basement membrane, lamina propria, glandular area, arteries and veins with the cavernous sinusoids extending in the deeper portion of the nasal mucosa [4]. Seromucous glands are present more superficially in the human normal inferior turbinate while a rich network of thin-walled venous sinusoids is distributed more deeply [4]. In the present study, a similar distribution is observed in the pig nasal mucosa, including from the superficial to the deep portions of the mucosa, the epithelium, glandular area, arteries, venous sinusoids and veins. Congestion of venous sinusoid plays a major role in pathophysiological conditions of the inferior turbinate by causing enlargement of the inferior turbinate. Human nasal valve area has very high concentration of venous sinusoids related to arteries [11], and the venous area is 3–4 times greater than the arterial area. It is interesting to note that in the study of Berger [25], no significant differences in terms of thickness of mucosal layers, basement membrane, lamina propria, were observed between inferior turbinates removed from cadavers and inferior turbinates obtained from patients that underwent nasal surgery to correct a deviated septum. However, the only difference was the area fraction of venous sinusoids that was greater in all mucosa layers of cadavers. The authors explained this difference by the vasoconstrictor effect of adrenaline, a substance injected with the local anesthetic in patients undergoing turbinate reduction, causing shrinkage of the vascular bed [25]. This observation provides further support to the hypothesis that a vasoconstrictor drug, such as an a2adrenoceptor agonist, by targeting the venous sinusoids will reduce nasal congestion. We previously demonstrated that the standard a2adrenoceptor agonist BHT-920 produced decongestion in the cat congestion model [18]. The congestant effect induced by compound 48/80 in this study was blocked by topical administration of the selective a2-adrenoceptor agonist PGE-6201204 (0.1%) to a degree similar to those previously reported for BHT-920 (0.3–1%) and the mixed a1- and a2-adrenoceptor agonist decongestant oxymetazoline [18]. However, oxymetazoline at all doses (0.01–0.3%) produced hypertension 30 min after inducing nasal congestion with compound 48/80 while BHT-920 had no effect on
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blood pressure at any time [18]. Hypertension was observed with oxymetazoline at doses (0.01% and 0.03%) that did not produce decongestion [18]. In the present study, PGE6201204 caused increase in blood pressure only at 30 min post-compound 48/80 challenges. In conclusion, a2-adrenoceptors are present and functional in nasal mucosa of different species. Histological analysis of pig nasal mucosa showed a predominance of venous sinusoids deep in the mucosal structure similar to the human nasal mucosa. We also demonstrated by using the real-time contractility assay that a2-adrenoceptor mechanisms predominate in the regulation of pig venous sinusoids, a component of the nasal vasculature that becomes engorged during nasal congestion. The fact that the topical a2-adrenoceptor agonists displayed decongestant activity in cat congestion model, support our hypothesis that, by targeting specifically the venous component of nasal circulation, an a2-adrenoceptor agonist will reduce engorgement of large venous sinusoids and collecting veins. Acknowledgment The authors would like to thank Kevin Jordan for his histology support. References [1] O’Donnell SR. Sympathomimetic vasoconstrictors as nasal decongestants. Med J Aust 1995;162:264–7. [2] Drettner B, Aust R. Plethysmographic studies of the blood flow in the mucosa of the human maxillary sinus. Acta Otolaryngol 1974;74:259–63. [3] Druce HM, Bonner RF, Patow C, Choo P, Kaliner MA. Response of nasal blood flow to neurohormones as measured by laser-Doppler velocimetry. J Appl Physiol 1984;57:1276–83. [4] White MV, Kaliner MA. Mediators of allergic rhinitis. J Allergy Clin Immunol 1992;90:699–704. [5] Widdicombe J. Microvascular anatomy of the nose. Allergy 1997;52: 7–11. [6] Lacroix JS. Adrenergic and non-adrenergic mechanisms in the sympathetic vascular control of the nasal mucosa. Acta Physiol Scand 1989;136:1–63. [7] Kristiansen AB, Heyeraas KJ, Kirkebo A. Increased pressure in venous sinusoids during decongestion of rat nasal mucosa induced by adrenergic agonists. Acta Physiol Scand 1993;147:151–61. [8] Andersson K-E, Bende M. Adrenoceptors in the control of human nasal mucosal blood flow. Ann Otol Rhino Laryngol 1984;93:179–82. [9] Berridge TL, Roach AG. Characterization of the a-adrenoceptors in the vasculature of the canine nasal mucosa. Br J Pharmacol 1986;88: 345–54. [10] Corboz MR, Varty LM, Rizzo CA, Mutter J, Rivelli MA, Wan Y, et al. Pharmacological characterization of a2-adrenoceptor-mediated responses in pig nasal mucosa. Aut Autac Pharmacol 2004;23:208–19. [11] Corboz MR, Rivelli MA, Varty LM, Mutter J, Cartwright M, Rizzo CA, et al. Pharmacological characterization of postjunctional aadrenoceptor in human nasal mucosa. Am J Rhinol 2005;19:495–502. [12] Wang M, Lung MA. Adrenergic mechanisms in canine nasal venous systems. Br J Pharmacol 2003;138:145–55. [13] Stafford Smith M, Baraniuk J, Wilson K, Schwinn DA. a2Adrenergic receptor subtypes in human nasal turbinate: expression of mRNA encoding specific a2-adrenergic receptor subtypes in nasal epithelium, gland and vascular cells. Anesthesiology 1997;87:A1078.
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