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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 746-751, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0030OC

Systemic Ovalbumin Sensitization Downregulates Norepinephrine Uptake by Rabbit Aortic Smooth Muscle Cells

Gabor Horvath, Aliza Torbati, Gregory E. Conner, Matthias Salathe and Adam Wanner

Division of Pulmonary and Critical Care Medicine, and Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida; and Department of Respiratory Medicine, Semmelweis University, Budapest, Hungary

Address correspondence to: Adam Wanner, M.D., Division of Pulmonary and Critical Care Medicine, University of Miami School of Medicine, P.O. Box 016960 (R-47), Miami, FL 33101. E-mail: awanner{at}miami.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Norepinephrine (NE) concentration at {alpha}-adrenergic receptors is partially regulated by steroid-sensitive, extraneuronal catecholamine uptake (uptake-2). Because {alpha}1-adrenergic agonist– and glucocorticosteroid (GS)-induced bronchial vasoconstriction is enhanced in individuals with asthma, atopy could be associated with decreased uptake-2 by vascular smooth muscle cells (SMCs). We therefore evaluated whether NE uptake and its specific transporter messenger RNA (mRNA) were reduced in aortic SMCs of rabbits systemically sensitized with ovalbumin (OVA). NE uptake was measured using a semiquantitative fluorescence microscopic method. Corticosterone and O-methyl-isoprenaline, but not desipramine, co-incubation (1 µM each) for 20 min decreased NE uptake into SMCs, an inhibitor profile indicative of extraneuronal monoamine transporter (EMT). In OVA-sensitized rabbits, NE uptake was 25.9 ± 4.5% (mean ± SEM) lower than in control animals (P < 0.05). Sensitized serum had no effect on NE uptake into naive SMCs. EMT mRNA expression was measured in aortic smooth muscle, using multiplex reverse transcriptase-polymerase chain reaction. In OVA-sensitized rabbits, expression was 61.1 ± 16.4% lower than in control animals (P < 0.05). These data demonstrate that NE uptake by aortic SMCs is impaired in atopic rabbits, and associated with a decreased transporter mRNA expression. The same mechanism may operate in bronchial arteries in individuals with asthma.

Abbreviations: complementary DNA, cDNA • catecholamine-O-methyltransferase, COMT • extraneuronal monoamine transporter, EMT • mean fluorescence intensity value, FN • glyceraldehydes-3-phosphate dehydrogenase, GAPDH • glucocorticosteroid, GS • inhibition constant, Ki • monoamine oxidase, MAO • messenger RNA, mRNA • norepinephrine, NE • neuronal norepinephrine transporter, NET • organic cation transporter, OCT • ovalbumin, OVA • phosphate-buffered saline, PBS • reverse transcriptase–polymerase chain reaction, RT-PCR • smooth muscle cell, SMC • sucrose-potassium phosphate-glyoxylic acid, SPG


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The main nervous control of the bronchial circulation is provided by the sympathetic nervous system (1). Sympathetic nerve endings release norepinephrine (NE), thereby causing vasoconstriction through activation of {alpha}-adrenoceptors on vascular smooth muscle. This signal is terminated by the uptake of released NE into neurons (reuptake or uptake-1) and non-neuronal cells (extraneuronal uptake or uptake-2) (2). In addition to endogenous NE, uptake-2 handles many other catecholamines, thereby terminating their cell surface receptor action (3). We have shown that isolated human bronchial arterial smooth muscle cells (SMCs) display glucocorticosteroid (GS)-inhibitable uptake of NE (4) and express messenger RNA (mRNA) of the recently identified, GS-sensitive, extraneuronal monoamine transporter (EMT or organic cation transporter [OCT] 3) (5, 6). We have also shown that inhaled GSs cause a rapid bronchial vasoconstriction in vivo (7), presumably by inhibiting the extraneuronal NE uptake and thereby increasing NE concentration at {alpha}-adrenoceptors of bronchial vessels.

Inhaled {alpha}1-adrenergic agonist- and GS-induced bronchial vasoconstriction is potentiated in individuals with asthma compared with healthy subjects (7, 8). Because extraneuronal uptake, which is uniquely sensitive to GSs, functionally inactivates a wide variety of adrenergic agonists (9), the vascular hyperresponsiveness in asthma raises the possibility that extraneuronal uptake is downregulated. As bronchial arteries from subjects with asthma were not available to test this hypothesis, we evaluated whether systemic ovalbumin (OVA) sensitization of rabbits alters NE uptake and EMT mRNA expression in aortic SMCs. This approach was supported by our previous observations that rabbit aortic SMCs exhibit GS-sensitive NE uptake and express EMT mRNA (4), and that systemic OVA sensitization induces hyperresponsiveness to {alpha}1-adrenergic agonists in isolated rabbit bronchial arteries (10).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
All materials were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Rabbit Sensitization and Aortic Smooth Muscle and SMC Isolation
New Zealand female albino rabbits were used in these experiments according to protocols approved by the University of Miami Institutional Animal Care and Use Committee. Rabbits (weight 3–4 kg) were systemically sensitized with OVA as described previously (10). Briefly, the animals received an injection of 1 ml of an OVA-containing emulsion (2.5 mg OVA in 1 part phosphate-buffered saline [PBS] and 1 part of Freund's complete adjuvant) given in 50–100 µl portions subcutaneously on the rabbit's back on either side of the vertebral column. After 4 wk, OVA injection was repeated using Freund's incomplete adjuvant. The control animals were injected with 1 ml of PBS subcutaneously instead of OVA.

Eight weeks after the first injection, rabbits were killed by an overdose of pentobarbital sodium. The thoracic aorta was excised, adhering fat and connective tissue removed, and the vessel was opened longitudinally. Endothelial cells were removed by carefully scraping the inside surface of the vessel, and 1-mm-wide strips of the smooth muscle layer were separated from the adventitia. Fifteen to twenty strips (~ 1 x 10-mm pieces) were cut transversely from the muscle preparation and were used immediately for SMC isolation or RNA extraction.

SMC isolation was performed based on the method of Clapp and Gurney (11) with some modifications as described previously (4). Muscle strips were transferred to a constantly oxygenated incubation solution (137 mM NaCl, 10 mM NaHCO3, 0.2 mM NaH2PO4, 5.4 mM KCl, 0.5 mM KH2PO4, 6 mM glucose, 0.15 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 0.02% bovine serum albumin, pH 7.0) containing papain (4 mg/ml) and dithiothreitol (2 mM), and were incubated at 37°C for 60 min with shaking. At the end of the dissociation period, individual SMCs were obtained by gentle tituration followed by filtration through a wire sieve (500 µm pore size). Finally, cells were collected by centrifugation at 1,000 x g for 3 min and resuspended in fresh incubation solution.

NE Uptake Experiments
For NE uptake measurement, cells were exposed to incubation solution containing NE with or without the uptake-2 inhibitors, corticosterone (1 µM) or O-methyl-isoprenaline (1 µM) and the uptake-1 inhibitor desipramine (1 µM) at 37°C. To inhibit the intracellular NE-metabolizing enzymes (catecholamine-O-methyltransferase [COMT] and monoamine oxidase [MAO]), 500 µM pargyline (MAO inhibitor) (12) and 1 µM Ro-41–0960 (COMT inhibitor) (13) were added into the incubation solution 30 min before the NE uptake experiments. Corticosterone was dissolved in ethanol and freshly diluted into the incubation solution just before use. The final concentration of ethanol <= 0.01%. This ethanol concentration had no significant effects on uptake measurements as confirmed in control experiments using vehicle only. At the end of the incubation period, SMCs were centrifuged at 1,000 x g for 3 min at 4°C followed by resuspension in 0.3 ml ice-cold, high KCl (20 mM) containing incubation solution. Then, the SMC suspension was deposited onto a poly-L-lysine-coated slide and the cells were allowed to settle for 5 min at 4°C before estimating the intracellular NE concentration.

Intracellular NE was visualized using a sucrose-potassium phosphate-glyoxylic acid (SPG) method described for tissue slices (14, 15) and adapted by us for use in isolated cells (4). Briefly, slides with SMCs were dipped into SPG solution (0.2 M sucrose, 236 mM KH2PO4, 1% glyoxylic acid monohydrate, pH 7.4) at room temperature for 3 s. After 5 min of air-drying, the specimen was covered with a drop of light mineral oil. Then, the slide was sealed with a coverslip and put into an oven at 95°C for 2.5 min. To quantitate fluorescence, a Nikon Eclipse E600FN microscope (Nikon, Melville, NY) with a Lambda DG-4 excitation system (Sutter Instruments, Novato, CA), a cooled CCD camera (Quantix; Photometrics, Tucson, AZ), and "Isee" software from Inovision Inc. (Durham, NC) were used. Cells were imaged at x600 (5–25 cells per field) with differential-interference-contrast microscopy and individual cells were identified as regions of interest. For quantification of the SPG fluorescence (or intracellular NE concentration) in these cells (or regions of interest), a 10-nm-wide filter centered on 405 nm was used for excitation and the emission measured at > 455 nm using a long pass filter (emission maximum: 480 nm) integrating the signal for 1 s. The cooled CCD camera was always set to a predefined gain, which was held constant throughout the experiments. SMCs were measured by selecting six well-separated regions on each slide (8–10 cells per region). Each single cell's mean fluorescence intensity value (FN), expressed in arbitrary units, was corrected for background fluorescence by subtracting the mean FN of SMCs from the same tissue not exposed to NE. Average NE uptake of each experimental group was calculated using the mean normalized FN of all cells.

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA from rabbit aortic smooth muscle strips (for OVA sensitization experiments) as well as lung, kidney, and liver samples (for EMT tissue distribution studies) was extracted using the RNeasy Midi Kit (Qiagen, Valencia, CA), treated with DNase (DNase I Amplification Grade; Life Technologies, Rockville, MD), precipitated with ethanol, and quantified spectrophotometrically at 260 nm. RNA quality was evaluated using an RNA 6000 LabChip Kit (Agilent Technologies, Palo Alto, CA) and an Agilent 2100 Bioanalyzer (Agilent Technologies) provided by the University of Miami DNA Microarray Facility. Multiplex reverse transcriptase–polymerase chain reaction (RT-PCR) was used to amplify a 265-base pair (bp) EMT complementary DNA (cDNA) fragment simultaneously with a 372-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment serving as an internal standard of each sample (16). RNA (0.5 µg per sample) was used for first strand cDNA synthesis with Superscript II RT (Life Technologies) using oligo-dT16 primers. Duplex PCR amplification (18–28 cycles of 30 s at 94°C, 45 s at 60°C, and 30 s at 72°C) was performed using Taq DNA polymerase (Life Technologies) and the following primers: 5'-CTG GGT GGT CCC TGA GTC TCC-3' (EMT, forward), 5'-TCC CAG GCG CAT GAC AAG TCC-3' (EMT, reverse), 5'-TAA TAC GAC TCA CTA TAG GAC TTC AAC AGT GCC ACC CAC-3' (GAPDH, forward), and 5'-ATT TAG GTG ACA CTA TAG ATT CAT GAC AAG GTA GGG CTC C-3' (GAPDH, reverse).

RT-PCR products were electrophoresed on 2% SeaKem agarose (BMA, Rockland, ME) gels and stained with a 1:10,000 dilution of SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR) for 40 min (17). The PCR product's signal intensities were quantified using an AlphaImager 3300 Gel Documentation and Image Analysis System (Alpha Innotech, San Leandro, CA). The linear range for the PCR was established by plotting cycle number against the log of the signal intensity. EMT signal intensities were normalized against the corresponding GAPDH signal at this range. Control reactions were performed in the absence of RT to verify that the amplified products were from mRNA and not from genomic DNA contamination. In the absence of RT, no PCR products were observed. Specific amplification of EMT and GAPDH mRNAs was confirmed by sequencing (University of Miami DNA Core Laboratory) of PCR fragments. Sequences were compared with the published rabbit EMT (GenBank accession # AF294824) and GAPDH (GenBank accession # L23961) cDNA sequences by PileUp (Wisconsin Package; GCG, Madison, WI).

Statistics
Results were expressed as mean ± SEM with n representing number of animals or experiments. Statistical significance was determined with an unpaired Student's t test. A P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pharmacologic Inhibition of NE Uptake by Rabbit Aortic SMCs
NE uptake into non-neuronal cells can be mediated through neuronal (NET) or non-neuronal (OCT1, OCT2, or EMT) membrane-associated transporters (9, 18). As these transport processes can be distinguished pharmacologically (2, 19, 20), freshly dissociated rabbit aortic SMCs were exposed to 50 µM NE for 20 min in the presence or absence of inhibitors of NE transporters. Following enzymatic dispersion, SMC viability was > 95% as determined by trypan blue exclusion. This was confirmed by observing SMC contraction responses induced by 50 µM NE or 20 mM KCl. After 20 min of co-incubation with 50 µM NE, 1 µM desipramine decreased NE uptake by only 4.2 ± 5.7%, a value not significantly different from controls (mean ± SEM for n = 3 experiments; P > 0.05 versus NE-exposed control animals). Desipramine inhibits only NET (Ki = 4 nM) (21). After 20 min of co-incubation with 50 µM NE, 1 µM corticosterone and 1 µM O-methyl-isoprenaline (Boehringer Ingelheim, Germany) inhibited NE uptake by 63.2 ± 6.9% and 57.8 ± 2.9%, respectively (mean ± SEM for n = 3 experiments; P < 0.05 versus NE-exposed control animals for both) (Figure 1). Corticosterone inhibits OCT2 (Ki = 200 nM) and EMT (Ki = 1 µM), but less potently OCT1 (Ki = 72 µM) (5, 19, 22). O-methyl-isoprenaline inhibits EMT (Ki = 1.9 µM), but less potently OCT2 (Ki = 580 µM) (5, 22). Together with the Km value of 245 µM for NE uptake reported in our prior study (4), these pharmacologic data confirmed our hypothesis that NE uptake is mainly mediated by EMT in rabbit aortic SMCs.



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  		Figure 1. NE uptake and its sensitivity to NE transporter inhibitors in aortic SMCs of nonsensitized rabbits. Cells were exposed to 50 µM NE for 20 min without (NE) or with 1 µM desipramine (D+NE), 1 µM corticosterone (C+NE), or 1 µM O-methyl-isoprenaline (O+NE). NE uptake was measured at the single cell level, using fluorescence microscopy. None of the inhibitors in the absence of NE revealed significant intracellular fluorescence (D, C, O). Shown are means ± SEM (n = 3 experiments). *P < 0.05 versus NE.

 
NE Uptake in OVA-Sensitized Rabbits
To examine whether OVA sensitization alters NE uptake, freshly isolated aortic SMCs of control and OVA-sensitized rabbits were exposed to 50 µM NE-containing incubation solution for 20 min. The NE concentration used in these experiments was in the estimated concentration range found at the sympathetic neuromuscular junction (23). NE uptake was 19.8 ± 0.7 arbitrary units in control and 14.7 ± 0.9 arbitrary units in OVA-sensitized rabbits (mean ± SEM for n of rabbits = 6 in both groups; P < 0.05) (Figure 2A, solid bars). Thus, OVA sensitization decreased NE uptake into aortic SMCs by 25.9 ± 4.5%.



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  		Figure 2. NE uptake in OVA sensitization. (A) NE uptake and its inhibition by corticosterone in aortic SMCs of control and OVA-sensitized rabbits. Cells were exposed to 50 µM NE for 20 min without (NE) or with 1 µM corticosterone (C+NE). (B) NE uptake and its inhibition by corticosterone in naive aortic SMCs exposed to OVA-sensitized rabbit serum. Aortic SMCs of nonsensitized rabbits were exposed to 50 µM NE for 20 min in whole serum of control or OVA-sensitized rabbits (OVA serum) as an incubation medium and without (NE) or with 1 µM corticosterone (C+NE). Shown are means ± SEM (n = 6 rabbits in both groups). *P < 0.05 versus corresponding NE, ** P < 0.05 versus NE in controls.

 
To see whether OVA sensitization potentiates the inhibitory effects of GS on NE uptake, aortic SMCs of control and OVA-sensitized rabbit were exposed to 50 µM NE for 20 min in the presence of 1 µM corticosterone. This concentration of corticosterone has been shown to inhibit ~ 65% of NE uptake into dissociated aortic SMCs (4). After 20 min of co-incubation with 50 µM NE, 1 µM corticosterone inhibited NE uptake into SMCs in control and sensitized animals by 67.6 ± 8.2% and 68.9 ± 6.6%, respectively (mean ± SEM for n of rabbits = 6 in both groups; P < 0.05 versus corresponding NE-exposed controls), suggesting that GS inhibition of NE uptake was not potentiated in OVA-sensitized animals. When expressed in absolute numbers, corticosterone inhibition had a tendency to result in lower NE uptake in sensitized animals in comparison with control animals (4.56 ± 0.57 versus 6.43 ± 0.93 in arbitrary units, respectively); however, this difference did not reach statistical significance (Figure 2A, open bars).

Because systemic OVA sensitization decreased NE uptake, indicative of reduced transporter function, expression, or both, we evaluated NE uptake by naive aortic SMCs in the presence of serum obtained from sensitized animals to see whether sensitization-associated, circulating factors were responsible for reduced NE transporter function. Using whole serum of control or OVA-sensitized rabbits as an incubation medium, freshly dissociated aortic SMCs of nonsensitized rabbits were exposed to 50 µM NE for 20 min in the presence or absence of 1 µM corticosterone. NE uptake was 19.7 ± 1.2 arbitrary units in nonsensitized and 20.7 ± 0.6 arbitrary units in OVA-sensitized rabbit serum (mean ± SEM for n of rabbits = 6 in both groups; P > 0.05) (Figure 2B, solid bars). After 20 min of co-incubation with 50 µM NE, 1 µM corticosterone inhibited NE uptake into SMCs in nonsensitized and OVA-sensitized rabbit serum by 60.9 ± 6.5% and 56.1 ± 3.2%, respectively (mean ± SEM for n of rabbits = 6 in both groups; P < 0.05 versus corresponding NE-exposed controls) (Figure 2B, open bars). These data indicated that the effect of systemic sensitization was not transferable by serum, suggesting that sensitization-associated circulating factors did not interfere functionally with EMT.

EMT mRNA Expression in Sensitized and Control Rabbits
Because NE uptake by aortic SMC of OVA-sensitized animals was reduced, it was possible that expression of EMT was reduced. To examine whether EMT mRNA was downregulated in sensitized animals, multiplex RT-PCR reactions were initiated as described in MATERIALS AND METHODS. Following RT and duplex PCR amplification with EMT- and GAPDH-specific primers, gel electrophoresis of products showed bands of expected size. Gel-purification and sequence analysis confirmed that the two amplicons were fragments of the published rabbit EMT and GAPDH cDNAs, respectively (4, 24). The isolated fragments contained only exon sequences of the corresponding human EMT (25) and GAPDH genes (26) confirming the amplification of mRNA rather than genomic DNA.

To show that EMT mRNA can be measured semiquantitatively using this RT-PCR technique, EMT mRNA expression was evaluated in samples from nonsensitized rabbit lung, aorta, kidney, and liver and those levels compared with recently published results obtained in rats and humans (27, 28). EMT and GAPDH ratios of different samples were compared in the linear range of PCR (up to 22 cycles for GAPDH and up to 26 cycles for EMT) (Figures 3A–3D). EMT mRNA expression was found to be highest in the aorta, more than 3-fold lower expression was found in the kidney, and almost 5-fold lower expression in the lung (Figure 3E). No EMT mRNA could be detected in liver cells, suggesting an over 10-fold lower expression level (Figure 3D). This distribution pattern was in keeping with the results of recent studies using rat and human tissues (27, 28).



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  		Figure 3. EMT mRNA expression in nonsensitized rabbits. (A–D) Cycle number against the log of EMT (open symbols) and GAPDH (solid symbols) products' signal intensities (F) following RT and duplex PCR amplification. In the liver (D), EMT-specific product was not detectable up to 26 PCR cycles. Shown are means ± SEM (error bars are inside the symbols) for triplicate measurements. (E) EMT mRNA expression levels in rabbit tissues calculated as the EMT/GAPDH ratios in the linear range of PCR amplification. Shown are means ± SEM for triplicate measurements.

 
To examine whether the reduced NE uptake shown above was associated with an altered transporter expression in OVA-sensitized animals, EMT mRNA levels of aortic smooth muscle strips were measured in OVA-sensitized and control rabbits. In OVA-sensitized animals, EMT mRNA expression in aortic smooth muscle was 61.1 ± 16.4% lower than in control animals (mean ± SEM for n = 6 animals in both groups; P < 0.05) (Figure 4).



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  		Figure 4. EMT mRNA expression in aortic smooth muscle of OVA-sensitized and control rabbits. (A) Co-amplification of a GAPDH (372-bp) and an EMT (265-bp) mRNA fragment using RT and duplex PCR in six sensitized and six control animals. Agarose gel was stained with SYBR Gold nucleic acid gel stain. (B) EMT mRNA expression levels calculated as the EMT/GAPDH ratios in OVA-sensitized and control rabbits. Shown are means ± SEM. *P < 0.05 versus controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that systemic OVA sensitization decreased NE uptake into rabbit aortic SMCs. Corticosterone inhibited NE uptake in both control and sensitized animals. In the presence of corticosterone, NE uptake appeared to be slightly lower in sensitized animals compared with control animals, although this difference did not reach statistical significance. Sensitization of rabbits was also found to significantly decrease EMT mRNA expression in aortic smooth muscle. These data suggest that NE uptake into vascular smooth muscle is downregulated in atopy, and thus provide a possible mechanism for the observed enhanced bronchial vasoconstrictor response to inhaled {alpha}-adrenergic agonists and GSs in individuals with asthma (7, 8).

Rabbit sensitization was performed by systemic administration of OVA as described previously (10). In a previous study, we showed that OVA sensitization of rabbits increased contraction responses of isolated bronchial arteries to {alpha}-adrenergic agonists (10), similar to the potentiated bronchial vasoconstrictor response to inhaled {alpha}-adrenergic agonists seen in individuals with asthma (29). This effect of OVA sensitization has also been found by others in dog pulmonary arteries (30), suggesting a generalized, vascular hyperresponsiveness to {alpha}-adrenergic agonists in atopy as seen in individuals with asthma (8). To study the mechanism of how OVA sensitization leads to {alpha}-adrenergic vascular hyperresponsiveness, we investigated changes of NE uptake in atopy. NE uptake into vascular SMCs was measured using a single-cell fluorescence assay developed in our laboratory (4). We found that systemic OVA sensitization decreased NE uptake into aortic SMCs by ~ 25%. The effect of sensitization was not transferable by serum, suggesting that sensitization-associated circulating factors did not interfere functionally with EMT. These data suggested that downregulated EMT expression rather than functional inhibition was responsible for decreased NE uptake after sensitization, a hypothesis formulated after obtaining our in vivo results in individuals with asthma (7, 8).

The effects of sensitization on isoprenaline transport, which uses the same uptake mechanism as NE (9), have been investigated before in rat tracheal smooth muscle (31). The results of that study showed that sensitization increased uptake at low isoprenaline concentrations, but had no effect at high isoprenaline concentrations. Thus, these results are in direct contradiction with the ones obtained by us. However, the differences may be explained by the different origin of the smooth muscle (bronchial versus vascular). Furthermore, as formation of the metabolite O-methylisoprenaline was used in that study to calculate isoprenaline uptake, altered metabolizing activity induced by sensitization might have had an influence on the results. In our assays, NE was directly measured and the metabolizing enzymes were inhibited in order not to interfere with the measurements.

Expression of EMT mRNA was about one third in vascular smooth muscle obtained from sensitized animals compared with control animals, further supporting our hypothesis of EMT downregulation. Protein expression of EMT could not be tested directly, as specific antibodies against rabbit EMT are not available at present. Although other transporters (OCT1 and OCT2) have been shown to transport NE into non-neuronal cells in addition to EMT (32–34), these are mainly expressed in the kidney and the liver. In rat blood vessels, OCT1 mRNA is expressed at > 15-fold lower levels compared with the kidney, whereas OCT2 mRNA expression is nearly undetectable (27). In contrast to its low kidney level, EMT mRNA expression has been reported to be highest in blood vessels (27, 28). Our results on EMT mRNA expression levels in lung, aorta, kidney, and liver in rabbits support this notion. Because NE uptake into rabbit aorta has been shown to be mediated by EMT (5, 19), high levels of EMT mRNA expression demonstrated here further confirm its dominant role in NE uptake in this tissue. The downregulated EMT mRNA expression in our OVA-sensitized animals suggests that impaired NE uptake in atopy is might be due to decreased transporter expression, although other mechanisms cannot be ruled out (35).

The rabbit EMT gene sequence and thus the transcriptional regulatory mechanisms have not been identified yet. However, the human EMT gene is known to contain a prototype of a TATA (CCAAT)-less promoter (25). In genes lacking these promoter elements, proximally positioned Sp1 sites serve as the critical determinants of promoter activity (36, 37). The large number of potential binding sites for Sp1 likely provide for substantial transcription levels of the EMT. As Sp1 binding/activity can be regulated through several pathways in SMCs (for review, see Ref. 38), there could be multiple mechanisms to suppress transcription of EMT. In recent studies, inflammation-associated substances (NO, cyclic nucleotides, lipopolysaccharide, and TNF-{alpha}) have been shown to inhibit Sp1 activity on TATA-less gene promoters (39). This mechanism could explain EMT mRNA downregulation in allergic inflammation.

Neuronal and extraneuronal uptake mechanisms are responsible for the functional inactivation of NE and other catecholamines, as the metabolizing enzymes are located intracellularly. Pharmacologic inhibition of NE uptake has been shown to facilitate sympathetic neuromuscular transmission (23, 40) and to potentiate the effect of exogenous NE on several systemic vascular beds (41–45). However, little is known about a possible NE uptake dysfunction associated with pathologic conditions (46). In recent studies, impaired neuronal NE uptake has been proposed to be responsible for the increased sympathetic tone seen in essential hypertension (47), congestive heart failure (48), and the postural tachycardia syndrome (49). Those studies also demonstrated that a < 10% decrease in uptake efficiency in cardiac sympathetic nerves resulted in a near doubling of the amount of NE that escapes reuptake (48). In general, extraneuronal uptake is quantitatively less than neuronal reuptake; however, the closer proximity of neuronal reuptake to sites of NE release implies that the NE removed by extraneuronal uptake has a longer duration and wider range of action than the NE removed by reuptake (50). Thus, the 25% decrease in NE uptake, together with a reduced NE transporter mRNA expression in vascular smooth muscle shown here, is expected to have a significant physiologic effect in atopy. We submit that the decrease in NE uptake in vascular smooth muscle could be responsible for, or contribute to, the increased bronchial vasoconstrictor responsiveness to inhaled GSs and {alpha}-adrenergic agonists seen in individuals with asthma.


   Acknowledgments
 
This work was supported in part by grants from the National Heart, Lung, and Blood Institute (HL-58086 to A.W.; HL-60644 & 67206 to M.S.; and HL-66125 to G.C), and an award from the American Heart Association, Florida/Puerto Rico Affiliate (to G.H.).

Received in original form March 5, 2002

Received in final form June 21, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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