Introduction

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The sympathetic nervous system controls peripheral vascular resistance by release of norepinephrine (NE) and epinephrine, which stimulate ₁-adrenergic receptors (ARs) on vascular smooth muscle cells. Molecular cloning and radioligand binding studies using selective antagonists have identified three distinct ₁-AR subtypes (_1A-, _1B-, and _1D-AR). A number of ₁-AR subtype-selective antagonists are available that can distinguish between these subtypes. WB4101, 5-methylurapidil, and niguldipine have been reported to bind with 10- to 100-fold higher affinity to the _1A-AR than to the _1B- and _1D-ARs (Zhong and Minneman, 1999), and are therefore useful tools to distinguish _1A-AR-mediated responses. BMY 7378 has been shown to bind to the _1D-AR with 100-fold higher affinity compared with the _1A-AR and _1B-AR subtypes (Piascik et al., 1995), allowing selective inhibition of _1D-AR- mediated responses. Of the ₁-AR subtypes, the _1B-AR has the least number of selective antagonists. The alkylating agent chloroethylclonidine has been reported to selectively block the _1B-AR but is a difficult tool to use due to the noncompetitive nature of the ligand and dependence on experimental conditions (Xiao and Jeffries, 1998). A prazosin derivative, cyclazosin, has been reported to have 10- to 15-fold higher affinity for the _1B-AR compared with the _1A-AR subtype in radioligand binding studies (Giardina et al., 1995).

Upon agonist stimulation, ₁-ARs increase vascular tone through activation of the G_q/11 signal transduction pathway. G_q/11 activates phospholipase C, which cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. The combination of inositol 1,4,5-trisphosphate-induced Ca²⁺ release from intracellular stores and diacylglycerol activation of protein kinase C results in activation of the contractile machinery within the smooth muscle cell (Zhong and Minneman, 1999). Recent studies have found that ₁-ARs can also activate the mitogenic signaling intermediate, extracellular signal-regulated kinase (ERK) (Thorburn and Thorburn, 1994; Williams et al., 1998). ERK is a member of the mitogen-activated protein kinase (MAPK) family, which comprises a number of serine-threonine kinases that are activated by phosphorylation of conserved tyrosine-X-threonine motifs (Robinson and Cobb, 1997). Upon phosphorylation, ERK activates a variety of target substrates including cytoskeletal proteins (myelin basic protein, microtubule-associated protein), contractile proteins (caldesmon), kinases (MAPK-activated proteins), and transcription factors c-fos, elk-1, and c-myc (Post et al., 1996; Dessy et al., 1998). In primary smooth muscle cell cultures, activation of transcription factors via ₁-AR stimulation of ERK has been shown to affect pathways associated with cell growth and hypertrophy, including increases in DNA and mRNA synthesis (Chen et al., 1995; Hu et al., 1999). Thus, these reports suggest an important role for ₁-ARs in regulating both contraction and mitogenic responses in vascular tissues.

This study was designed to compare ₁-AR-induced contraction and ERK activation in an intact blood vessel. In these studies we used the bovine inferior alveolar artery (BIAA), which is located inside the mandible and supplies the teeth and oral tissues with blood. During aging, the human inferior alveolar artery displays morphological characteristics of excessive growth factor pathway activation, including formation of atherosclerotic lesions and luminal narrowing (Bradley, 1972; Zoud and Doran, 1993). Using contraction experiments and immunoblotting, we determined the ₁-AR subtypes mediating contraction and ERK activation in the BIAA. We found that only one ₁-AR subtype mediates contraction, whereas more then one subtype activates ERK. We also found that concentrations of NE that caused contraction also activated ERK. Finally, transmural electrical stimulation (TES) of artery segments caused ERK to be activated through stimulation of ₁-ARs. These data suggest that, in vivo, the sympathetic nervous system via stimulation of ₁-ARs can simultaneously activate ERK and cause vasoconstriction. Thus, stimulation of ERK may play a role in sympathetic nervous system regulation of vascular function.

	Materials and Methods

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Chemicals and Reagents. Agents used were obtained from the following sources: desipramine, hydrocortisone, NE, propranolol, aprotonin, phenylmethylsulfonyl fluoride, and okadaic acid (Sigma-Aldrich, St. Louis, MO); 5-methylurapidil, BMY 7378, cyclazosin, rauwolscine, niguldipine, phenylephrine, and WB4101 (RBI/Sigma, Natick, MA); BE 2254 (Tocris Cookson Inc., Ballwin, MO); rabbit anti-phospho-ERK1/2 antibody and rabbit anti-total ERK1/2 antibody (Cell Signaling Technology Inc., Beverly, MA); anti-rabbit IgG-horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Phenylephrine and NE were dissolved in 0.9% saline containing 0.2% ascorbic acid. Phenylmethylsulfonyl fluoride was dissolved in 95% ethanol. All other drugs were dissolved in water.

Contraction Experiments. Bovine lower jaws were obtained from a local slaughterhouse and immediately put into ice-cold phosphate-buffered saline (pH 7.6), containing 137 mM NaCl, 9.6 mM NaH₂PO₄, 2.7 mM KCl, 1.7 mM KH₂PO₄, for transportation to the laboratory (5 min). The BIAA was removed from the jaw and stripped of surrounding fat and connective tissue. To eliminate the release of nitric oxide or other factors from the endothelium, endothelium was removed by gentle rubbing with a stainless steel wire (Bockman et al., 1996). The absence of endothelium was confirmed by the loss of relaxation in response to 1 µM acetylcholine in arteries precontracted with 60 mM potassium chloride. Arteries were used immediately or were stored overnight in Krebs' solution (pH 7.4), containing 120 mM NaCl, 5.5 mM KCl, 2.5 mM CaCl₂, 1.4 mM NaH₂PO₄, 1.2 mM MgCl₂, 20 mM NaHCO₃, 11.1 mM dextrose, and 0.027 mM CaNa₂-EDTA, at 4°C for use the following day. No differences were observed in contractile responses between arteries used immediately and those stored overnight. The artery was cut into 3-mm-long ring segments, and the rings were attached to Grass FT 0.03 isometric force transducers (Grass Instruments, Quincy, MA) using stainless steel pins inserted through the lumen of the artery. The transducers were connected to a Grass polygraph for tension recordings. Rings were placed in Krebs' solution in glass muscle chambers, gassed with 95% O₂/5% CO₂, and maintained at 37°C. After 1 h equilibration at 2 g resting tension, tissues were contracted with 60 mM potassium chloride and then washed with Krebs' solution for 30 min. The potassium chloride contraction and washing procedure was then repeated twice more. After a final 30-min wash period, cumulative concentration-response curves for contraction were generated in each ring.

Measurement of Antagonist Affinity Values. After initial NE concentration-response curves were obtained, the rings were washed with Krebs' solution and allowed to relax to the resting tension. Each ring was equilibrated for 1 h with a single concentration of antagonist, and NE concentration-response curves were then repeated. The concentrations of ₁-AR antagonists used were from 5 to 250 times the average literature K_B value for an antagonist at its favored (highest affinity) ₁-AR (Table 1). In each experiment, NE concentration-response curves were also generated in a control ring that was not treated with antagonist, to determine whether there were changes in agonist potency or maximal contraction over time. Concentration-response curves were plotted and EC₅₀ values were obtained using nonlinear regression curve fitting (GraphPad Prism 3.0; GraphPad Software, San Diego, CA). Schild plots were constructed by the method of Arunlakshana and Schild (1959). Linear regression of the Schild plot gave pA₂ values from the x-intercept of the regression line. Slopes of the Schild plots were tested for difference from unity using a one-sample t test.

View this table: [in this window] [in a new window]   TABLE 1 Affinities of -AR antagonists for inhibiting norepinephrine-mediated contraction of the BIAA Each value is the mean ± S.E.M. of four to six experiments, with each experiment using an artery from a different animal.

ERK1/2 Activation. Inferior alveolar arteries were cut into 10-mm-long artery segments and placed in glass test tubes containing 10 ml of Krebs' solution maintained at 37°C and gassed with 95% O₂/5% CO₂. As in contraction studies, artery segments were equilibrated for 1 h, treated with 60 mM potassium chloride, and washed for 30 min. This process was then repeated once more. ERK1/2 was stimulated by the addition of adrenergic agonists or by TES. For TES experiments, artery segments were placed between two platinum electrodes and stimulated by applying square-wave pulses (10 Hz, 0.3 ms, 60 V) using a Grass S44 stimulator. In preliminary experiments, we have found that these stimulation parameters also cause ₁-AR-mediated contraction of the BIAA (data not shown). For each experiment, one artery segment was not treated with agonist or other drugs and was used for determination of basal ERK1/2 activity. For experiments involving antagonists, artery segments were incubated with antagonist for 1 h before TES or addition of agonist drugs. The concentrations of ₁-AR-selective antagonists used were approximately 13 (10 nM antagonist) or 130 nM (100 nM antagonist) times the average literature K_B value for an antagonist at its favored (highest affinity) ₁-AR subtype (Table 1; see Fig. 3 legend). Assays were terminated by placing artery segments in 0°C lysis buffer containing 137 mM NaCl, 20 mM Tris HCl, 1 mM CaCl₂, 1 mM MgCl₂, 1% Nonidet P-40, 1 mM EDTA, 1 µM aprotonin, 100 µM phenylmethylsulfonyl fluoride, and 10 nM okadaic acid. Tissues were minced with scissors and homogenized 3 times for 10-s intervals at 13,500 rpm with a Tissumizer using an SDT-080EN probe (Tekmar, Cincinnati, OH). Samples were centrifuged at 12,000g for 15 min to remove particulate, and the supernatants containing soluble cytosolic protein, including ERK, were aliquoted and stored at -20°C. Protein concentrations of the supernatant samples were determined using the Coomassie Plus Protein Assay (Pierce, Rockford, IL).

Immunoblotting. Samples were thawed and boiled for 5 min in loading buffer (pH 6.8) containing 60 mM Tris-HCl, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, and 0.1% bromphenol blue. Protein samples (20 µg) were electrophoresed for 45 min on a 4 to 15% Tris-HCl/polyacrylamide gel (Bio-Rad, Hercules, CA) at a constant voltage of 200 V for 50 min on a Mini-PROTEAN electrophoresis apparatus (Bio-Rad). A colorimetric ladder (Bio-Rad) was included in each gel to identify protein size. Following electrophoresis, the contents of the gel were transferred to a nitrocellulose membrane (Osmonics, Westborough, MA) using a semidry transfer method (Bio-Rad). Nitrocellulose membranes were then washed three times for 10-min intervals in Tween 20 buffer containing 150 mM NaCl, 23 mM Tris-HCl, 2 mM Tris-Base, and 0.1% Tween 20. These membranes were blocked and probed with either anti-phospho (1:5000 diluted in 5% BSA/Tween 20 buffer) or anti-total (1:1000 diluted in 5% BSA/Tween 20 buffer) ERK1/2 antibodies overnight at 4°C. Nitrocellulose membranes were washed five times for 10-min intervals with Tween 20 buffer and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:1000 diluted in 1% BSA-1% blotting grade nonfat dry milk/Tween 20 buffer). Nitrocellulose membranes were then washed five times for 10- min intervals with Tween 20 buffer, treated with West Pico enhancer and substrate solutions (Pierce, Rockford, IL) for 3 min to detect protein bands by chemiluminescence, and exposed to enhanced chemiluminescent Hyperfilm (Amersham Biosciences Inc., Piscataway, NJ). The film was developed and used for quantitation of the Western blots.

Western Blot Analysis. A representative Western blot displaying the results from a single experiment and its analysis is shown in Fig. 1. Protein bands (Fig. 1B) were subjected to densitometry analysis (Molecular Analyst for Windows; Bio-Rad) and quantified as optical densitometry units. Each sample value was normalized by dividing the phospho-ERK1/2 densitometry volume by the total ERK1/2 densitometry volume. For each drug treatment, ERK1/2 stimulation over basal was calculated by dividing the treated normalized optical densitometry volume by the basal normalized optical densitometry volume. Each value was then expressed as percentage stimulation, with 100 µM agonist made equal to 100% stimulation of ERK1/2. Figure 1A shows a bar graph of the normalized percentage stimulation values calculated from the Western blot shown in Fig. 1B. Means ± S.E.M. of the data were calculated and were statistically compared using the one-sample t test. A p value less then 0.05 was used to indicate a significant difference between groups.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Representative analysis of a single Western blot experiment of ERK1/2 activation in the BIAA. Panel A, bar graph of quantitation of the Western blot shown in panel B. Percentage of maximal ERK1/2 stimulation values inside each bar were calculated as described under Materials and Methods. Panel B, representative Western blot of ERK1/2 activation by NE in the absence and presence of the 1-AR antagonist BE 2254. Ab: Total-ERK1/2 indicates bands identified by a primary antibody that recognizes both the phosphorylated and unphosphorylated forms of ERK1/2. Ab: p-ERK1/2 indicates bands identified by a primary antibody that recognizes the phosphorylated form of ERK1/2. p44 and p42 arrows indicate 44- and 42-kDa bands corresponding to ERK1 and ERK2, respectively. Band intensities were determined by densitometry and are expressed as optical density (OD) units, which are shown under each set of bands.

	Results

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Time Course and Concentration-Response Relationship for ERK1/2 Activation. To determine the conditions that would provide maximal ERK1/2 stimulation, time course and concentration-response relationships of adrenergic agonist activation of ERK1/2 were investigated. Maximal ERK1/2 activation was reached at 10 min with 100 µM NE or phenylephrine (data not shown). Therefore, in the following experiments, artery segments were stimulated with 100 µM agonist for 10 min to maximize the amount of ERK1/2 activation.

Identification of the AR Types Causing Phenylephrine-Mediated ERK1/2 Activation. To isolate the ₁-AR component of ERK1/2 activation, we used the ₁-AR-selective agonist phenylephrine to stimulate ERK1/2. To confirm that ₁-ARs were mediating phenylephrine activation of ERK1/2, we used selective antagonists of ₁-ARs (BE 2254), ₂-ARs (rauwolscine), and -ARs (propranolol). BIAA segments were incubated with receptor type-selective concentrations of each antagonist for 1 h and then stimulated with 100 µM phenylephrine for 10 min. As shown in Fig. 2, the ₁-AR antagonist BE 2254 significantly inhibited ERK1/2 activation, with 100 nM BE 2254 reducing ERK1/2 activity to basal levels. Rauwolscine and propranolol were unable to significantly inhibit ERK1/2 activation. These data demonstrate that phenylephrine increases ERK1/2 activity in the BIAA through stimulation of ₁-ARs.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of adrenergic receptor antagonists on phenylephrine (PHE)-stimulated ERK1/2 activation in the BIAA. Artery segments were incubated with antagonists for 1 h before stimulation with PHE. Bar graph of mean data shows the effect of BE 2254 (BE; 1-AR-selective), rauwolscine (Rau; 2-AR-selective), or propranolol (Pro; -AR-selective) on phenylephrine activation of ERK1/2. The dashed line represents the basal level of ERK1/2 in the absence of drugs. Bars are the percentage of 100 µM phenylephrine stimulation and are the mean ± S.E.M. of four artery segments, each taken from a different animal. , significantly different from 100 µM phenylephrine, p < 0.05.

Characterization of the ₁-AR Subtype Causing ERK1/2 Activation. To identify the ₁-AR subtypes that mediate ERK1/2 activation, artery segments were incubated with receptor subtype-selective concentrations of the antagonists niguldipine, 5-methylurapidil, WB4101 (_1A-AR-selective), cyclazosin (_1B-AR-selective), and BMY 7378 (_1D-AR-selective). Except for BMY 7378, concentrations of these antagonists up to 100 nM were used to ensure selectivity for receptor subtypes. When used individually, none of the antagonists inhibited phenylephrine stimulation of ERK1/2, suggesting that more than one ₁-AR subtype may be mediating this response (data not shown). Therefore, we incubated BIAA segments with different combinations of two of the ₁-AR subtype-selective antagonists niguldipine, cyclazosin, and BMY 7378. Incubating BIAA segments with two of these antagonists (100 nM each) did not cause a significant decrease in phenylephrine-induced ERK1/2 activation (Fig. 3). However, addition of all three antagonists simultaneously, at 10 nM concentrations of each, caused a moderate decrease in phenylephrine-mediated ERK1/2 activation, whereas 100 nM concentrations caused a marked inhibition (Fig. 3). These data suggest that all three ₁-AR subtypes can couple to ERK1/2 activation in the BIAA.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of combinations of 1-AR subtype-selective antagonists on phenylephrine (PHE)-stimulated ERK1/2 activation in the BIAA. Artery segments were incubated with combinations of antagonists for 1 h before stimulation with PHE. The concentrations of antagonists used were approximately 13 (10 nM antagonist) or 130 (100 nM antagonist) times the KB value for an antagonist at its favored (highest affinity) 1-AR subtype. Literature values for antagonists other than niguldipine are listed in Table 1. The average literature value for niguldipine (0.7 nM) was from Minneman et al. (1994) and Buscher et al. (1996). The bar graph is of mean data showing the inhibition of phenylephrine activation of ERK1/2 by combinations of niguldipine (Nig), cyclazosin (Cyc), and BMY 7378 (BMY). Antag indicates combinations of either 10 nM or 100 nM of all three 1-AR antagonists listed above. The dashed line represents the basal level of ERK1/2 in the absence of drugs. Bars are the percentage of 100 µM phenylephrine stimulation and are the mean ± S.E.M. of four artery segments, each taken from a different animal. , significantly different from 100 µM phenylephrine, p < 0.05.

Identification of the AR Types Causing NE-Mediated ERK1/2 Activation. To identify the adrenergic receptor types mediating activation of ERK1/2 by the endogenous agonist NE, artery segments were incubated for 1 h with the adrenergic receptor type-selective antagonists BE 2254, rauwolscine, and propranolol. Segments were then stimulated with 100 µM NE for 10 min. As shown in Fig. 4, a 100 nM concentration of the ₁-AR antagonist BE 2254 significantly inhibited NE-mediated ERK1/2 activation, reducing activation levels by 94 ± 1.3%. Rauwolscine was unable to significantly inhibit ERK1/2 activation by NE, whereas 1 µM propranolol caused a small, nonsignificant decrease in NE-induced ERK1/2 activation. These data demonstrate that NE increases ERK1/2 activity in the BIAA through stimulation of ₁-ARs, and that ₂-ARs or -ARs do not appear to be involved in mediating this response.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effects of adrenergic receptor antagonists on NE-stimulated ERK1/2 activation in the BIAA. Artery segments were incubated with antagonists for 1 h before stimulation with NE. Bar graph of mean data showing the effect of BE 2254 (BE; 1-AR-selective), rauwolscine (Rau; 2-AR-selective), or propranolol (Pro; -AR-selective) on NE activation of ERK1/2. The dashed line represents the basal level of ERK1/2 in the absence of drugs. Bars are the percentage of 100 µM NE stimulation and are the mean ± S.E.M. of four artery segments, each taken from a different animal. , significantly different from 100 µM norepinephrine, p < 0.05.

Characterization of the ₁-AR Subtype Causing Contraction. Mean concentration-response curves for adrenergic agonist-induced contraction were generated in BIAA rings and are shown in Fig. 5. NE contracted BIAA rings with low potency (EC₅₀ = 11 ± 2.0 µM). To determine whether extraneuronal uptake, neuronal uptake, or stimulation of -ARs contributed to the low potency of NE, concentration-response curves were generated in the presence of hydrocortisone, desipramine, and propranolol. These drugs had no significant effect on the potency of NE, suggesting that there are little or no effects of extraneuronal uptake, neuronal uptake, or -AR stimulation on NE-mediated contraction. Concentration-response curves were also generated for the ₁-AR subtype-selective agonist phenylephrine and the ₂-AR subtype-selective agonist UK 14304. Phenylephrine contracted the BIAA with slightly higher potency (EC₅₀ = 5.12 ± 1.8 µM), but lower intrinsic activity (0.87 ± 0.09) compared with NE, whereas UK 14304 did not cause contraction. These data suggest that stimulation of ₁-ARs causes contraction of the BIAA and that ₂-AR stimulation does not.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Contraction of BIAA ring segments by adrenergic agonists. Concentration-response curves were generated for the 1-AR agonist phenylephrine (PHE), the 2-AR agonist UK14304, and the nonselective AR agonist NE. NE concentration-response curves were generated in the absence and presence of 0.1 µM desipramine (DMI), 1 µM hydrocortisone (HC), and 1 µM propranolol (PRO). Contraction is expressed as the percentage of maximal contraction produced by NE in each ring segment. Data are the mean ± S.E.M. of four arteries, each taken from different animals.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   BE 2254 antagonism of NE-induced contractions of BIAA ring segments. A, NE concentration-response curves in the absence and presence of the 1-AR antagonist BE 2254. Contraction is expressed as the percentage of maximal contraction produced by NE in each ring segment. Data are the mean of responses from four arteries, each taken from different animals. B, Schild plot of the data shown in A. The mean ± S.E.M. pA2 and slope value of the Schild regression are listed.

Comparison of the Potency of NE Activation of ERK1/2 and Contraction. NE mean concentration-response curves for contraction and ERK1/2 activation were generated and are shown in Fig. 7. The EC₅₀ for NE in stimulating ERK1/2 activation was 21 ± 23 µM. Increases in ERK1/2 activation over basal were first observed at 1 µM NE with maximal ERK1/2 stimulation at 100 µM NE. NE also stimulated contraction with low potency (EC₅₀ = 11 ± 2 µM), with responses first observed at 1 µM NE. Thus, the potency of NE in causing contraction and ERK1/2 activation was nearly the same.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Comparison of NE concentration-response curves for stimulation of contraction and ERK1/2 activation in BIAA. Each point is expressed as the percentage of maximal contraction or maximal ERK1/2 activation produced by NE in each ring. All data are the mean ± S.E.M. of three to four arteries, each taken from different animals.

Effect of TES on ERK1/2. As shown in Fig. 8, TES of BIAA segments resulted in increases in ERK1/2 activation. TES for 2, 5, and 10 min caused progressive stimulation of ERK1/2 by 2.1-, 3.0-, and 4.3-fold over basal, respectively. We also compared ERK1/2 activation produced by TES to ERK1/2 activation caused by 100 µM phenylephrine for 10 min. TES for 5 min produced similar levels of ERK1/2 activation (93 ± 24.3%) relative to phenylephrine, whereas after 10 min of TES, ERK1/2 activation was slightly greater (133 ± 41.2%) compared with phenylephrine. To determine whether ₁-ARs were mediating ERK1/2 activation produced by TES, artery segments were incubated with 100 nM concentrations of the ₁-AR-selective antagonist BE 2254 1 h before TES. BE 2254 inhibited 10-min TES ERK1/2 activation to basal levels, suggesting that ₁-ARs are mediating this response. Taken together, these data suggest that in the BIAA, sympathetic nerves release norepinephrine to stimulate ₁-ARs, resulting in activation of ERK1/2.

View larger version (36K): [in this window] [in a new window]   Fig. 8.   Effect of TES on ERK1/2 activation in the BIAA. Artery segments were stimulated for 2, 5, and 10 min. A, bar graph of mean data showing activation of ERK1/2 by 100 µM phenylephrine (PHE) for 10 min and 2, 5, and 10 min of TES. The effect of 1 h of treatment with the 1-AR-selective antagonist BE 2254 on 10 min of TES ERK1/2 activation is also shown. The dashed line represents the basal level of ERK1/2 in the absence of drugs. Bars are the percentage of 100 µM phenylephrine stimulation and are the mean ± S.E.M. of three to six artery segments, each taken from a different animal. B, representative Western blot of ERK1/2 activation by 100 µM phenylephrine and 2, 5, and 10 min of TES. Ab: Total ERK1/2 indicates bands identified by a primary antibody that recognizes both the phosphorylated and unphosphorylated forms of ERK1/2. Ab: p-ERK1/2 indicates bands identified by a primary antibody that recognizes the phosphorylated form of ERK1/2. p44 and p42 arrows indicate 44- and 42-kDa bands corresponding to ERK1 and ERK2, respectively. , significantly different from 100 µM phenylephrine, p < 0.05.

	Discussion

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In this study we compared adrenergic agonist stimulation of ERK activation and contraction in the BIAA. We identified the adrenergic receptor types that are stimulated by NE to cause contraction and ERK activation. Both the ₂-AR antagonist rauwolscine and the -AR antagonist propranolol had no effect on NE or phenylephrine activation of ERK- or NE-induced contraction, whereas the ₁-AR antagonist BE 2254 significantly antagonized both responses, suggesting that ₁-ARs mediate both contraction and ERK activation in the BIAA. We also determined the ₁-AR subtypes mediating these responses in the BIAA, by using ₁-AR subtype-selective antagonists that can distinguish between the _1A-, _1B-, and _1D-AR subtypes. We used a variety of ₁-AR subtype-selective antagonists to inhibit ₁-AR-mediated ERK activation and contraction. Using concentrations of antagonists reported to be selective for each subtype, we found that all three ₁-ARs appear to participate in the stimulation of ERK activation. However, affinity values generated for ₁-AR subtype-selective antagonists in blocking NE-induced contraction showed that only the _1A-AR mediates this response. We also compared the relative potencies of NE in activating ERK and causing contraction, and found that NE activates each response with almost equal potency. In addition, TES of the BIAA caused activation of ERK through stimulation of ₁-ARs. These data suggest that in some vascular beds in vivo, the sympathetic nervous system activates ERK while also regulating vascular tone.

To understand the functional roles of ₁-ARs in the BIAA, we first identified the ₁-AR subtype mediating contraction. To do this, affinity values of ₁-AR subtype-selective antagonists in blocking contraction were determined and compared with their reported affinities at ₁-AR subtypes reported in previous studies. The _1A-AR-selective antagonists 5-methylurapidil and WB4101 inhibited NE-induced contraction with high affinity. WB4101 had a slightly higher affinity then 5-methylurapidil, which is consistent with previous reports of the relative affinities of these compounds at the _1A-AR (Hanft and Gross, 1989). The _1B-AR-selective antagonist cyclazosin antagonized NE contractions with relatively low affinity (5 nM), similar to its affinity (11 nM) in inhibiting _1A-AR-mediated contractions of rat small mesenteric artery (Stam et al., 1998). This result, combined with the high affinity of _1A-AR-selective antagonists, suggests that the _1B-AR does not mediate contraction of the BIAA. The _1D-AR-selective antagonist BMY 7378 did not inhibit NE-induced contraction, indicating that the _1D-AR does not contract the BIAA. This result is not surprising, as the _1D-AR mediates contraction in only a few vascular tissues, including the rat (Piascik et al., 1995) and mouse (Yamamoto and Koike, 2001) aorta. The high affinity of WB4101 and 5-methylurapidil, and the low affinities of cyclazosin and BMY 7378 suggest that only _1A-ARs mediate NE-induced contraction of the BIAA. This is consistent with other reports that _1A-ARs are the primary receptor subtype mediating contraction of many blood vessels (Docherty, 1998).

We also identified the ₁-AR subtype mediating ERK activation in the BIAA, to determine whether the _1A-AR receptor subtype is mediating both contraction and ERK activation or whether other ₁-AR subtypes are also involved in activation of ERK. We found that concentrations of individual ₁-AR subtype-selective antagonists reported to be selective for each subtype were unable to inhibit ERK activation. Combinations of two ₁-AR subtype-selective antagonists also failed to inhibit ERK activation. However, when using _1A-, _1B-, and _1D-AR-selective antagonists together, ERK activation was inhibited significantly. These data suggest that more than one ₁-AR subtype is coupled to ERK activation in the BIAA. Previous studies have reported that all three ₁-AR subtypes can activate ERK when they are transfected separately into rat PC12 cells (Zhong and Minneman, 1999). However, few reports have attempted to determine the ₁-AR subtype that activates ERK in native tissues that endogenously express ₁-ARs. In contrast to our findings, Xin et al. (1997) found that only the _1D-AR stimulated ERK in rat aorta vascular smooth muscle cells. However, that study used a primary cell culture of aortic smooth muscle cells, which did not contain the various cell types found in an intact blood vessel (i.e., smooth muscle cells, fibroblasts, macrophages, monocytes, nerves). ₁-ARs are present in both the medial (primarily smooth muscle cells) and adventitial (primarily fibroblasts and other cell types) layers of arteries (Faber et al., 2001). Thus, _1B- and _1D-ARs may mediate ERK activation in the fibroblast-containing adventitial layer, whereas the _1A-AR may mediate ERK activation in the smooth muscle cell-containing medial layer of the BIAA. These data may explain why mRNA (Scofield et al., 1995; Faber et al., 2001) and protein (Piascik et al., 1997; Hrometz et al., 1999; Faber et al., 2001) for each ₁-AR subtype can be detected in most blood vessels, but only one ₁-AR subtype is typically responsible for mediating contraction. It may be possible that ₁-ARs that are expressed but not linked to contraction can activate ERK or other signaling pathways in the supporting adventitial layer of blood vessels.

We also compared the potencies of NE in causing contraction and ERK activation. We found that NE had nearly the same potency for causing contraction of the BIAA as it does for activating ERK, suggesting that in vivo, the degree of ERK activation is directly proportional to the level of vasoconstriction. Furthermore, we found that TES resulted in activation of ERK through the stimulation of ₁-ARs. These data suggest that during moment-to-moment control of vas-cular tone, sympathetic nerves release NE to cause contraction and to activate ERK. However, the physiological role of ₁-AR-mediated ERK activation in vascular tissues is unknown. Numerous in vivo and in vitro studies have suggested that the sympathetic nervous system regulates mitogenic responses in vascular tissues. For example, increased sympathetic innervation (Head, 1991) or exogenous elevation of plasma catecholamines (Stewart et al., 1992) has been associated with vascular smooth muscle hypertrophy in vivo. In addition, sympathetic denervation of blood vessels through mechanical (Bevan, 1975), chemical (Fronek et al., 1978), or immunological (Lee et al., 1987) methods results in decreases in DNA synthesis and cell number. The addition of catecholamines to vascular smooth muscle cell cultures increases DNA (Nakaki et al., 1990) and protein synthesis (Chen et al., 1995; Hu et al., 1999), as well as cell growth (Blase and Boissel, 1993). Previous reports have also suggested that ERK is involved in regulating agonist-induced vasoconstriction. For example, inhibition of MAPK activation has been shown to attenuate not only 5-hydroxytryptamine_2A (Florian and Watts, 1998; Banes et al., 1999) but also angiotensin (Epstein et al., 1997)- and ₁-AR (DiSalvo et al., 1993) mediated vasoconstriction. Possible mechanisms by which ERK activation has been proposed to affect contractile responses include phosphorylation of caldesmon (Adam et al., 1989; Hedges et al., 2000) or myosin (Jin et al., 1996). Although evidence exists for ERK-regulating mitogenic responses and contractile function in blood vessels, the role of ₁-AR-stimulated ERK activation may be dependent on cell type. For example, ₁-AR activation of ERK in smooth muscle cells within the medial layer may regulate contraction, whereas ₁-AR activation of ERK in fibroblasts within the adventitial layer may be involved in vascular remodeling.

In conclusion, we found that only ₁-ARs mediate contraction and ERK activation in the BIAA. Activation of the _1A-AR subtype causes contraction, whereas _1A-, _1B-, and _1D-ARs can all activate ERK. The endogenous adrenergic agonist NE was equipotent in stimulating both contraction and ERK activation. Our data also suggest that ERK is activated following sympathetic stimulation of ₁-ARs in intact blood vessels. The activation of ERK in the BIAA may play a role in modifying ₁-AR-induced contraction, or ERK may be responsible for regulating growth or other mitogenic pathways in vascular smooth muscle cells or in other cell types found in intact blood vessels. The human inferior alveolar artery is the primary source of blood supply to the mandible and mandibular teeth. During aging, this artery slowly degenerates until it no longer contributes the major source of blood to the mandible, teeth, and oral tissues (Zoud and Doran, 1993). The human inferior alveolar artery also develops luminal narrowing much earlier in life than other cranial arteries (Bradley, 1975). One explanation for these changes is that vascular remodeling or arteriosclerotic narrowing of the inferior alveolar artery develops due to excessive growth factor pathway activation. Thus, sympathetic nervous system activation of ERK via ₁-ARs may be important for regulation of normal blood vessel growth and/or contraction as well as abnormal growth and remodeling associated with aging or other conditions.