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CARDIOVASCULAR
1-Adrenergic Receptor Subtypes along the Rat Tail Artery
Departments of Pharmacology (S.Y.K., A.M., V.L., A.R.T.S., A.S.P.) and Physiology (I.B.d.C., J.B.), Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil
Received for publication
April 6, 2005
Accepted
April 29, 2005.
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Abstract |
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1A-adrenoceptors (ARs). However, rings from proximal segments of the tail artery (within the initial 4 cm, PRTA) were at least 3-fold more sensitive to methoxamine and phenylephrine (n = 6–12; p < 0.05) than rings from distal parts (between the sixth and 10th cm, DRTA). Interestingly, the imidazolines N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively 1A-ARs, were equipotent in PRTA and DRTA (n = 4–12), whereas buspirone, which activates selectively 1D-AR, was 
70-fold more potent in PRTA than in DRTA (n = 8; p < 0.05). The selective 1D-AR antagonist 8-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) was 70-fold more potent against the contractions induced by phenylephrine in PRTA (pKB of 8.45; n = 6) than in DRTA (pKB of 6.58; n = 6), although the antagonism was complex in PRTA. 5-Methylurapidil, a selective 1A-antagonist, was equipotent in PRTA and DRTA (pKB of 8.4), but the Schild slope in DRTA was 0.73 ± 0.05 (n = 5). The noncompetitive 1B-antagonist conotoxin 
-TIA reduced the maximal contraction induced by phenylephrine in DRTA, but not in PRTA. These results indicate a predominant role for 1A-ARs in the contractions of both PRTA and DRTA but with significant coparticipations of 1D-ARs in PRTA and 1B-ARs in DRTA. Semiquantitative reverse transcription-polymerase chain reaction revealed that mRNA encoding 1A- and 1B-ARs are similarly distributed in PRTA and DRTA, whereas mRNA for 1D-ARs is twice more abundant in PRTA. Therefore, 1-ARs subtypes are differentially distributed along the tail artery. It is important to consider the segment from which the tissue preparation is taken to avoid misinterpretations on receptor mechanisms and drug selectivities.
1-adrenoceptor (AR) subtypes (1A-, 1B-, and 1D-ARs) are involved in the contractions of vascular smooth muscle in response to AR agonists. However, depending on the vessel, the participation of one particular subtype may predominate over the other two 1-ARs. The use of subtype selective 1-AR agonists and antagonists has allowed the identification of a predominant role for 1A-ARs in the contractions of the rat tail artery in response to norepinephrine (Gisbert et al., 2003
), phenylephrine (Piascik et al., 1995, 1997), methoxamine (Villalobos-Molina and Ibarra, 1996), NS-49 (Murata et al., 1999), and A-61603 (Lachnit et al., 1997; Chang et al., 2000; Jahnichen et al., 2004), suggesting that this artery is an interesting model for the study of the mechanisms involved in the vasoconstriction mediated by 1A-ARs and for the characterization of the properties of selective ligands.
Interestingly, studies evaluating the expression of mRNA encoding 1-ARs reveal that the 1A-AR is probably not the unique subtype expressed in the rat tail artery, since mRNA encoding 1B- and 1D-AR has been detected in this vessel (Piascik et al., 1995; Taki et al., 2004). In fact, radioligand binding studies have shown the presence of 1B-ARs in addition to 1A-ARs (Taki et al., 2004; Tanaka et al., 2004), and functional studies have identified a minor but definite component in the contractions in response to norepinephrine as being mediated by the activation of 1B-ARs (Jahnichen et al., 2004).
While studying the contractions of rings of the rat tail artery in response to phenylephrine in an attempt to establish in our laboratory a model of vasoconstriction mediated by 1A-ARs, we noticed considerable variation in the sensitivity of preparations taken from different segments along this artery. The rings cut from segments located within the initial 4 cm of the tail (proximal segments) where much more sensitive to phenylephrine than rings taken from segments more distally located (within the sixth and the 10th cm of the tail). This led us to investigate the identity of the 1-ARs mediating the contractions of arterial rings isolated from proximal (PRTA, rings of the tail artery isolated from proximal segments) and distal (DRTA, rings of the tail artery isolated from distal segments) regions of the tail of the rat. Experiments using selective agonists and antagonists reveal that the subtypes of 1-ARs are differentially distributed along the rat tail artery. In addition, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using primers for the specific amplification of mRNAs encoding each of the 1-ARs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed variation in the amount of transcripts for 1-ARs between these two regions. Although some studies have described heterogeneous distribution of 1- and 2-ARs along the rat tail artery (Rajanayagam and Medgett, 1987), this is, to our knowledge, the first report on differential distribution of functional 1-AR subtypes along an artery.
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Materials and Methods |
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2-ARs, and 
1-and 2-ARs, respectively. After an equilibration period of 60 min with adjustments of basal tension and changes of physiological solution at each 20 min, the arteries were repeatedly challenged with 10 µM phenylephrine at 20-min intervals until contractions of similar magnitude were obtained (usually three times). Then, concentration response-curves to 1-AR agonists were obtained in absence or presence of increasing concentrations of the 1-antagonists prazosin, BMY-7378 and 5-methylurapidil previously incubated for at least 45 min. Not more than four consecutive concentration-response curves to agonists were obtained from each preparation, since preliminary experiments showed there are significant changes in the sensitivity of the tail artery to AR agonists.
The pA2 values for competitive antagonists were calculated by Schild regression analysis (Arunlakshana and Schild, 1959). The ratios between the half-maximal concentrations of agonists (concentration ratios, r) were calculated only when the maximal amplitude of the concentration-response curve in the presence of the competitive antagonists was similar to that obtained in its absence. Data were plotted as log antagonist concentrations (molar) versus log (r - 1). For calculation purposes, the slope parameter was constrained to 1.0 when statistically not different from unity. When the slope parameter differed from the theoretical unity or when only one antagonist concentration was tested, the antagonist affinity was estimated as pKB calculated through the formula pKB = -log [lowest effective concentration of the antagonist (molar)] + log (r - 1).
Curve fitting and pD2 calculation was performed with the software package GraphPad Prism version 3.00 (GraphPad Software Inc., San Diego, CA). All values are shown as means ± S.E.M. of n experiments. Differences between mean values were tested for statistical significance (p 
0.05) using Student's paired or unpaired t-tests or analysis of variance followed by Newman-Keuls for multiple comparisons.
Drugs were obtained from the following sources: A-61603 (Tocris Cookson Inc., Bristol, UK); cocaine (Cocainum hydrochloricum puriss.) (Boehringer Ingelheim GmbH, Ingelheim, Germany); buspirone hydrochloride, corticosterone, and noradrenaline [(±)-arterenol HCl] (all from Sigma-Aldrich, St. Louis, MO); and BMY-7378, oxymetazoline HCl, prazosin HCl, (±)-propranolol HCl, and yohimbine HCl (all from Sigma/RBI, Natick, MA). Conotoxin -TIA was a generous gift from Dr. Richard J. Lewis (Xenome Ltd., Indooroopilly, Queensland, Australia).
Total RNA Extraction. The ventral artery of the rat tail was rapidly dissected, cleaned of connective and fat tissue in ice-cold saline (0.9% NaCl), and the endothelium was removed by gentle rubbing with stainless steel wire in the lumen.
A proximal segment of the tail artery was taken from the initial 4 cm of the rat tail, whereas a distal segment was taken from a region located between the sixth and 10th cm of the tail. These segments corresponded to those isolated for functional studies.
Proximal and distal segments of the tail artery were homogenized in TRIzol (Invitrogen, São Paulo, Brazil) with a Polytron, and total RNA was extracted according to the recommended protocol from a pool of samples composed by tissues from four animals. Total RNA concentration was measured by absorbance at 260 nm (BioPhotometer; Eppendorf - 5 Prime, Inc., Boulder, CO).
Semiquantitative RT-PCR. The sequence of the specific primers were described by Queiroz et al. (2002) as follows: 1a sense, GTA GCC AAG AGA GAA AGC CG (628–647) and 1a antisense, CAA CCC ACC ACG ATG CCC AG (820–839); 1b sense, GCT CCT TCT ACA TCC CGC TCG (629–649), and 1b antisense, AGG GGA GCC AAC ATA AGA TGA (908–928); 1d sense, CGT GTG CTC CTT CTA CCT ACC (759–779), and 1d antisense, GCA CAG GAC GAA GAG ACC CAC (1042–1062); GAPDH sense, CGG GAA GCT TGT GAT CAA TGG (258–277) and GAPDH antisense, GGC AGT GAT GCC ATG GAC TG (614–595). The size expected for the amplified products are 212, 300, 304, and 357 base pairs for 1a, 1b, 1d, and GAPDH genes, respectively. The GAPDH gene was used as an internal control for normalization of the data. To remove contaminating genomic DNA, 1 µg of total RNA was treated with DNase I (Invitrogen), and reverse transcription was then performed with 8 µl of the DNase-treated RNA solution using a SUPERScript III RT kit (Invitrogen) according to manufacturer's instructions.
PCR was performed on 2 µl (each of the 1-ARs subtypes) or 0.5 µl (for GAPDH) of cDNA in a PCR mastermix containing 1.5 mM MgCl2, 0.2 mM deoxyribonucleotides (dNTPs), 0.4 µM (for 1-ARs subtypes) or 1.6 µM (for GAPDH) specific primers, 1.25 units TaqDNA polymerase (Invitrogen, São Paulo, Brazil) and H2O in a total volume of 25 µl. PCR conditions for 1-ARs subtypes were adapted from Queiroz et al. (2002) as follows: one cycle of denaturation 96°C, 30 s, followed by 30 (1a, 1b) or 29 (1d) cycles of denaturation 96°C, 10 s; annealing 58°C (1a) or 55°C (1b, 1d), 10 s; extension 74°C, 40 s (1b, 1d) or 15 s (1a). A final extension of 74°C, 2 min was performed for all samples. GAPDH was amplified under one cycle of denaturation 94°C, 3 min; followed by 33 cycles of denaturation 94°C, 45 s; annealing 60°C, 45 s; extension 70°C, 1 min.
PCR products (15 µl) were separated in 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light and digitalized with Image Master VDS (Amersham Biosciences Inc., Piscataway, NJ). Band intensities were measured by computerized densitometry using the Image Gauge version 3.12 software (FujiFilm, Tokyo, Japan) and are expressed as arbitrary units.
Semiquantitative RT-PCR was validated by choosing the number of PCR cycles and amount of RNA within the linear range of the amplification curve for each gene. Experiments were performed on three independent pools, and the mean ± S.E.M. values were compared.
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Results |
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1A-AR agonists the imidazolines A-61603 and oxymetazoline (Minneman et al., 1994; Knepper et al., 1995) (Fig. 1B), and to the selective 1D-AR agonist buspirone (Eltze et al., 1999, Yamamoto and Koike, 2001) (Fig. 1C). The respective pD2, maximal contractions (in millinewtons) and relative intrinsic activities (calculated as a fraction of the absolute maximal response to phenylephrine in each segment) are shown in Table 1.
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The phenethylamine agonists phenylephrine and methoxamine were approximately 3 to 5 times more potent in PRTA than in DRTA (Fig. 1A; Table 1), whereas the imidazolines A-61603 and oxymetazoline were roughly equipotent in rings from both regions (Fig. 1B; Table 1). Buspirone was almost 70 times more potent in PRTA than in DRTA where it was only a very weak agonist as indicated by its low intrinsic activity (Fig. 1C; Table 1).
The rank order of potency was the same in PRTA and DRTA (A-61603 > oxymetazoline > phenylephrine > methoxamine = buspirone). However, some differences were observed in the efficacies of oxymetazoline and methoxamine, which behaved as partial agonists in PRTA but as full agonists in DRTA (Table 1).
It was not possible to include experiments using norepinephrine as an 1-AR agonist in both PRTA and DRTA due to strong participation of 2-ARs in the contractions. This participation was indicated by the complex antagonism shown by prazosin, which resulted in Schild plots with slopes much lower than 1.0 (data not shown).
Effects of AR Antagonists on Contractions Induced by Phenylephrine in Different Segments of the Rat Tail Artery. The effects of prazosin, 5-methylurapidil, and BMY-7378 where tested against the contractions induced by phenylephrine in PRTA and DRTA. The contractions of both PRTA and DRTA in response to phenylephrine were competitively antagonized by prazosin (Fig. 2, A and B) with similar affinities (Table 2). Similarly, 5-methylurapidil, a selective antagonist of 1A-ARs, was equipotent in PRTA and DRTA (Fig. 2, C and D), although the antagonism in DRTA was complex, since the slope in the Schild plot was lower than the theoretical unity (Fig. 3; Table 2). The 1D-AR selective antagonist BMY-7378 antagonized the contractions of the PRTA and DRTA in response to phenylephrine (Fig. 2, E and F). However, BMY-7378 was much more potent in PRTA than in DRTA, as characterized by the effectiveness of the low concentrations of BMY-7378 in PRTA (10–300 nM; Fig. 2E), which induced rightward shifts in the concentrations-response curves to phenylephrine that where concentration-dependent but not linearly related; i.e., in this concentration range, a 3-fold increase in BMY-7378 did not result in a 3-fold reduction in the potency of phenylephrine, as predicted by the dynamics of the competitive antagonism at a single receptor. The resulting slope in the Schild plot was lower than 1.0 (Fig. 3; Table 2). On the other hand, the contractions of DRTA in response to phenylephrine were not affected by low concentrations of BMY-7378 (10–300 nM; Fig. 2F), and the resulting straight line in the Schild plot had slope not different from 1.0 (Figs. 2F and 3; Table 2).
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Effects of BMY-7378 on Contractions Induced by Other AR Agonists in PRTA. We tested the effects of BMY-7378 on contractions induced by other AR agonists in PRTA to investigate whether the shallow slope in the Schild plot observed for this antagonist against phenylephrine is related to the presence of multiple functional 1-AR subtypes in proximal segments of the rat tail artery. The contractions induced by methoxamine, another phenethylamine agonist, were also sensitive to low concentrations of BMY-7378 (10–300 nM; Fig. 4A) and similarly to what was observed when the contractions where induced by phenylephrine, the resulting slope in the Schild plot was much lower than the unity (Fig. 4E; Table 3).
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In contrast, when the contractions were induced by the imidazolines A-61603 and oxymetazoline, the antagonism presented by BMY-7378 was competitive (Fig. 4, B, C, and E) and yielded low pA2 values (6.4; Table 3). Interestingly, the contractions induced by buspirone where also competitively antagonized by BMY-7378 (Fig. 4D) but with affinity approximately 80-fold higher (pA2 8.3; Table 3).
The Nature of the Receptor Activated by Buspirone. Buspirone is classically known as a partial agonist at 5-hydroxytryptamine 1A receptors. However, it is also known that most of the vasoconstriction induced by buspirone results from the activation of 1-ARs (Gurdal et al., 1992; Terron et al., 1996; Osei-Owusu and Scrogin, 2004) and that this drug activates selectively 1D-ARs (Eltze et al., 1999; Yamamoto and Koike, 2001). In spite of this, it is important to determine whether the contractions of the rat tail artery to buspirone recorded in the present study are due to activation of 1-ARs. To determine this, the potency of prazosin was determined against the contractions induced by buspirone in both PRTA and DRTA. Prazosin (1–10 nM) antagonized with high affinity the contractions of the PRTA in response to buspirone (pA2 = 9.39 ± 0.05, Schild slope = 1.0 ± 0.1; n = 4; data not shown), suggesting that 1-ARs are involved. Buspirone was a much weaker agonist in the DRTA, precluding construction of Schild plots. However the pKB estimated with single concentrations of prazosin (1 nM) was 9.2 ± 0.1 (n = 4), and of 5-methylurapidil (30 nM) was 8.0 ± 0.1 (n = 4), whereas BMY-7378 (up to 30 nM) was not effective (data not shown). These results indicate that the small contractions induced by buspirone in DRTA also result from the activation of 1-ARs.
Effects of Conotoxin -TIA on Contractions Induced by Phenylephrine in PRTA and DRTA. It has been recently shown that conotoxin -TIA interacts differently with 1-AR subtypes. It is a competitive antagonist of 1A- and 1D-AR but a noncompetitive antagonist of 1B-ARs; in addition, the toxin shows slight selectivity for 1B-ARs (10–25-fold) over the other two subtypes (Chen et al., 2004; Lima et al., 2005). Therefore, the effects of different concentrations of conotoxin -TIA on contractions induced by phenylephrine in PRTA and DRTA were investigated. In PRTA, conotoxin -TIA (0.3–3 µM) induced concentration-dependent rightward shifts in the concentration-response curves to phenylephrine without affecting the maximal contraction induced by this agonist (Fig. 5A). The resulting Schild plot had slope = 0.92 ± 0.04 and pA2 = 7.34 ± 0.08 (n = 4). In DRTA, however, although 0.3 and 1 µM conotoxin -TIA induced concentration-dependent rightward shifts in the concentration-response curves to phenylephrine, 3 µM conotoxin -TIA reduced the maximal contraction induced by phenylephrine by approximately 15% (p < 0.05; Fig. 5B).
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1B-ARs or to some nonspecific event, we examined the effect of 3 µM conotoxin -TIA on contractions induced by the selective 1A-AR agonist A-61603. Although 3 µM conotoxin -TIA induced a large reduction in the potency of A-61603 in DRTA (pKB = 7.50 ± 0.07; n = 4), the toxin was not able to affect the maximal contraction induced by this agonist (Fig. 5C).
Expression of mRNA for 1-AR Subtypes in Different Segments of the Rat Tail Artery. The mRNA encoding each of the 1-AR subtypes was amplified by RT-PCR from total RNA extracted from proximal and distal segments of the rat tail artery using specific primers for each subtype. The amplified products were separated by agarose gel electrophoresis, quantified by densitometry, and compared with the mRNA encoding GAPDH. This semiquantitative procedure allows convenient comparisons of the expression levels of mRNA for the same receptor subtype in different segments. However, this procedure does not allow comparisons of the expression levels among different receptor subtypes. Figure 6A shows representative gels with bands corresponding to the products amplified using primers specific for GAPDH, 1A-AR, 1B-AR, and 1D-AR mRNAs, whereas Fig. 6B shows the expression of each 1-AR subtype mRNA in relation to the respective GAPDH mRNA. The transcripts for 1A- and 1B-ARs are similarly distributed in proximal and distal segments of the rat tail artery (Fig. 6B). However, the relative expression of 1D-AR mRNA in proximal segments is 2.4-fold higher than in distal segments (Fig. 6B; p < 0.05).
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Discussion |
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1-AR subtypes involved in the contractions of rings from proximal and distal segments of the rat tail artery was investigated in the present study. The hypothesis that there might be a differential distribution of functional 1-AR subtypes along the rat tail artery was raised from the observation that rings taken from proximal segments of this vessel were much more sensitive to some, but not all AR agonists, than rings taken from more distal segments. This was observed during preliminary experiments carried out in an attempt to standardize in our laboratory a model of vasoconstriction mediated by the activation of 1A-ARs, which would be suitable for the study of the processes resultant from the activation of this subtype and of the properties of selective ligands.
The phenethylamine agonists phenylephrine and methoxamine were 3- to 5-fold more potent in PRTA than in DRTA. Importantly, the selective 1A-AR imidazoline agonists A-61603 and oxymetazoline were equipotent in PRTA and DRTA, suggesting that the higher sensitivity to AR agonists is not a general phenomena but related to which receptor subtype is activated. Strikingly, buspirone, a partial agonist at 5-hydroxytryptamine 1A receptors and selective agonist at 1D-AR (Eltze et al., 1999; Yamamoto and Koike, 2001), was almost 70-fold more potent in PRTA than in DRTA where its maximal contraction represented only 30% of that induced by the full agonist phenylephrine. Based on the similar potency presented by the selective 1A-AR agonists A-61603 and oxymetazoline and the higher potency and/or efficacy shown by phenylephrine, methoxamine, and buspirone, it is tempting to speculate that PRTA is endowed with functional 1A- and 1D-ARs, whereas this latter subtype is absent in DRTA. However, it is difficult to conclude about distributions of receptor subtypes relying only on evidence obtained with agonists, mainly because of the hyperbolic nature of the occupancy-response relationships often observed for 1-ARs and their agonists. Therefore, this heterogeneous distribution was further investigated using the selective 1D-AR competitive antagonist BMY-7378.
The selective antagonist BMY-7378 discriminated two distinct components in the contractions of PRTA in response to phenylephrine and methoxamine: the first component was similarly blocked by "low" concentrations of BMY-7378 (10–300 nM), whereas the second was competitively antagonized by higher concentrations (1–10 µM); such behavior precluded the determination of a single consistent affinity for this antagonist in PRTA since the slope in the Schild plots was much lower than the theoretical unity. However, the pKB estimated from the lowest effective concentration of BMY-7378 (10 nM) through the formula pKB = -log [BMY-7378 (molar)] + log (r - 1) against either phenylephrine (8.4 ± 0.1; n = 6) or methoxamine (8.4 ± 0.1; n = 6) indicates that part of the contractions of PRTA in response to these two phenethylamines is due to activation of 1D-ARs. On the other hand, if only the effects of 0.3 to 10 µM are used to construct the Schild plots, the resulting slopes are not different from unity (1.0 ± 0.1 versus phenylpephrine and 1.0 ± 0.1 versus methoxamine), but the derived pA2 value (6.4 ± 0.1 versus phenylpephrine and 6.5 ± 0.1 versus methoxamine) is much lower than that expected for the involvement of 1D-ARs. These pA2 values indicate participation of either 1A- or 1B-ARs. However, the high pA2 values for 5-methylurapidil estimated against either phenylephrine (
8.3) or methoxamine (8.3; data not shown) suggest that the subtype sensitive only to the higher concentrations of BMY-7378 is the 1A-AR. Therefore, these results corroborate those found with agonists suggesting that both 1A- and 1D-ARs are functional in PRTA and that phenylephrine and methoxamine contract these arterial rings through the activation of these two subtypes.
The competitive behavior and the high pA2 values found for BMY-7378 against the contractions induced by buspirone in PRTA further support the presence of functional 1D-ARs. Conversely, the results obtained with oxymetazoline and A-61603 indicate that 1A-ARs are functional in PRTA since these imidazolines are selective 1A-AR agonists (Minneman et al., 1994; Knepper et al., 1995), and the contractions induced by these drugs were competitively antagonized with low affinity by BMY-7378 (pKB 6.5).
The interpretation of the behavior shown by BMY-7378 against the contractions induced by phenylephrine in DRTA is much more straightforward. These data suggest that 1D-ARs are not involved in the contractions of rings from distal segments since there was no component sensitive to low concentrations of BMY-7378, and the Schild plot had a slope not different from unity, resulting in the estimation of low affinity for this antagonist. The absence of functional 1D-ARs in DRTA is further supported by the very weak agonist activity shown by buspirone in these arterial rings. Therefore, functional 1D-ARs are differentially distributed along the rat tail artery mediating part of the contractions induced by phenylephrine and methoxamine in PRTA but not in DRTA.
The antagonism presented by the selective 1A-AR competitive antagonist 5-methylurapidil also differed in PRTA and DRTA. Although the contractions of both PRTA and DRTA were antagonized by the same concentrations of 5-methylurapidil, the slope of the straight line in the Schild plot derived in DRTA was lower than the theoretical unity. Interestingly, the largest selectivity for 1A-ARs presented by 5-methylurapidil is over the 1B-AR subtype (30–100-fold), whereas that over 1D-ARs is only marginal (3–10-fold). Therefore, the complex antagonism presented by 5-methylurapidil in DRTA could be related to the presence of functional 1B-ARs in these rings. We took advantage of the differential antagonism presented by -conotoxin TIA at 1-ARs to further check for the presence of functional 1B-ARs in DRTA. -Conotoxin TIA is a competitive antagonist of 1A- and 1D-AR but a noncompetitive antagonist of 1B-ARs, against which the toxin shows slight selectivity (10–25-fold) over the other two subtypes (Chen et al., 2004; Lima et al., 2005). In fact, high concentrations of -conotoxin TIA reduced the maximal contraction induced by phenylephrine in DRTA but not in PRTA where it was a competitive antagonist. In addition, -conotoxin TIA was not able to reduce the maximal contraction induced by the selective 1A-AR agonist A-61603, suggesting that the inhibition of the maximal contraction induced by phenylephrine is related to the involvement of 1B-ARs. These data suggest that 1B-ARs are also differentially distributed in the rat tail artery, taking part in the contractions of rings from distal segments but not from proximal ones. The presence of functional 1B-ARs contracting the rat tail artery has already been shown. Recently, Jahnichen et al. (2004) described that both 1A- and 1B-ARs are involved in the contractions of rings from the rat tail artery and that a receptor protection protocol using the selective 1A-AR B8805-033 and the alkylating agent chloroethylclonidine is necessary to obtain contractions mediated by 1A-ARs, otherwise mixed receptor populations are uncovered.
The pattern of expression of the mRNA encoding each of the 1-ARs was investigated by semiquantitative RT-PCR using specific primers. Interestingly, mRNA encoding 1D-ARs is approximately twice as abundant in proximal than in distal segments of the tail artery, whereas mRNA encoding 1A- and 1B-ARs are similarly distributed. Therefore, these results show that both functional 1D-ARs and their mRNAs are differentially distributed along the rat tail artery. It is difficult to correlate mRNA abundance with functional response, but the similar distribution of mRNA encoding 1B-ARs suggests that these receptors, if present in proximal segments, are not efficiently coupled to contraction. Alternatively, semiquantitative RT-PCR might not be sensitive enough to detect subtle differences in the expression of 1B-ARs transcripts.
We are not aware of other studies describing heterogeneous distribution of 1-AR subtypes along different segments of the same vessel. However, differences in the potencies of prazosin and idazoxan in antagonizing contractions induced by epinephrine in proximal and distal segments of the rat tail artery led Rajanayagam and Medgett (1987) to conclude that the participation of 2-ARs is greater in distal parts.
The idea that the rat tail artery contracts in response to some AR agonists via activation of multiple 1-AR subtypes is not new. For example, Lachnit et al. (1997) concluded that one of these subtypes displays the pharmacology of the 1A-AR, whereas the other remained to be defined. This seemed to be resolved by the previously mentioned study from Jahnichen et al. (2004) describing that 1A- and 1B-ARs are functional in this vessel. Hence, one important contribution of the present study is the observation that depending on the segment of the rat tail artery from which the arterial preparation is isolated, either 1B- or 1D-AR can be found to coparticipate with 1A-ARs in the contractions induced by AR agonists. It will be interesting to investigate whether such a pattern of differential distribution of receptor subtypes also occurs in other vessels.
It is important to acknowledge the existence of multiple functional 1-AR subtypes in the rat tail artery mainly because this tissue has been extensively used for the study of the mechanisms involved in the vasoconstriction mediated by 1A-ARs and for the characterization of the properties of selective ligands. Therefore, even the minor participation of 1-ARs other than the 1A-AR must be taken into account to avoid misinterpretations.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: AR, adrenoceptor; NS-49, (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride; A-61603, N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide; PRTA, rings from proximal segments of the tail artery; DRTA, rings from distal segments of the tail artery; RT-PCR, reverse transcription-polymerase chain reaction; BMY-7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; B8805-033, (±)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodio xin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione.
Address correspondence to: Dr. André Sampaio Pupo, Department of Pharmacology, Instituto de Biociências, UNESP, Botucatu, São Paulo, Brazil, 18618-000. E-mail: aspupo{at}ibb.unesp.br
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