The α1-ARs are members of the class I of the G-protein coupled receptors (GPCR) superfamily [1,2]. Three α1-ARs, α1A-AR, α1B-AR and α1D-AR have been cloned and characterized [1,3,4]. These receptors mediate responses to epinephrine and norepinephrine thus making a vital contribution to the control of blood flow and systemic arterial blood pressure. Abnormalities in the regulation of the α1-ARs may contribute to the development of hypertension and heart failure [5 - 8].
It is well known that the localization and trafficking properties of a receptor can modulate its physiological function . Results from heterologous expression systems have demonstrated that the α1B-AR is localized on the cell surface, as expected for a GPCR, while the α1A-AR is localized not only on the cell but also in intracellular compartments [10,11]. In contrast, we have shown that the α. In contrast, we have shown that the α1D-AR is localized intracellularly [11,12]. These results of nonconical cellular locatization are consistent with emerging data that show specific GPCR families can be localized not only to intracellular sites but also on the nuclear membrane .
In recent years, the concept of receptor dimerization has brought a new perspective to GPCR function [14 - 16]. Previous studies reported that the α1D-AR interacts with the α-AR interacts with the α1B-AR and the β2-AR  resulting in the cell surface expression of the α1D-AR. This has lead to the suggestion that these receptors are capable of heterodimerization [17 - 20]. These observations have been made in heterologous systems. However, the role of dimerization in the regulation of cells that natively express all three receptors such as vascular smooth muscle cells has not been well studied. This is due in part to the difficulty of tranfecting smooth muscle cells. To overcome this obstacle, we developed a recombinant adenovirus for the efficient expression of the human α1D-AR. We show that despite the presence of the other α1-AR family members, the α1D-AR is expressed mainly in intracellular compartments. We further show that while receptor dimerization may occur, it does not appear to alter the functional properties of the α1D-AR.
An adenoviral vector was constructed to drive the efficient expression of a GFP-labeled α1D-AR. Infection of aortic smooth muscle cells with virus expressing the α1D-AR/GFP resulted in approximately 80% receptor transfectional efficiency (not shown) demonstrating that adenovirus can be useful in cells that have been traditionally difficult to transfect with the α1-ARs. Following viral infection, the α1D-AR/GFP was detected in intracellular compartments of aortic smooth muscle cells (Figure 1A). A similar pattern of vascular smooth muscle intracellular expression was seen with immunocytochemistry studies using an α1D-AR selective antibody (Figure 1B). These localization results in smooth muscle cells are similar to our previous findings in HEK 293 cells transfected with an α1D-AR/GFP expression plasmid or immunohistochemistry studies in fibroblasts stably transfected with the α1D-AR [12,21]. Recently, it was proposed that the α1D-AR can dimerize with the α-AR can dimerize with the α1B-AR promoting its cell surface expression . Our results would argue that the presence of other ARs, particularly the α1B-AR, does not alter α1D-AR localization in vascular smooth muscle cells. To further substantiate that the presence of the α1B-AR does not affect α1D-AR localization, we infected fibroblasts that stably express the α1B-AR with the α-AR with the α-AR with the α-AR with the α1D-AR/GFP adenoviral construct (Figure 1C). Despite expression in a cell expressing the α1B-AR at high levels, α1D-AR was nonetheless expressed in intracellular compartments (Figure 1C). These data argue that the α1B-AR does not alter the cellular localization of the α1D-AR.
It is possible, however, that in these cultured smooth muscle cells the α1B-AR and/or α1A-AR are not expressed. To examine this possibility we used RT-PCR to assess message expression. The results of these experiments are shown in figure 2A. A very prominent PCR-product corresponding to the α1B-AR was detected in aortic smooth muscle cells. We were also able to detect an α1A- AR transcript. A very faint product corresponding to the α- AR transcript. A very faint product corresponding to the α1D-AR was also observed. Minute levels of tissue expression are typical for this receptor. A series of antibodies directed against each of the α1-ARs was used to determine the cellular localization of these receptors (Figure 2B). As has been shown by previous work the α). As has been shown by previous work the α1A- AR is expressed both intracellularly as well as on the cell surface while the α1B-AR is expressed predominately on the cell membrane. What is also apparent from comparing figures 1A–C and figure 2B is that the expression pattern of the α is that the expression pattern of the α1D- AR is markedly different from that of either the α- AR is markedly different from that of either the α1A- or α- or α1B-ARs. Therefore, in a mammalian cell where both the α1A- and α- and α1B-AR are natively expressed (as opposed to being transfected) the α1D-AR is nonetheless expressed intracellularly. Further, the data argue that while dimerization may occur in smooth muscle cells, it does not alter the localization of the α1D-AR.
Effects on intracellular calcium
In aortic smooth muscle cells phenylephrine produced a dose-dependent and statistically significant increase in intracellular calcium (see data for 25 uM presented in Figure 3). This increase was antagonized by 1 nM of the nonselective α1-AR blocker prazosin or 30 nM of the highly selective α1D-AR antagonist BMY 7378. To substantiate the selectivity of this dose of BMY 7378 we measured phenylephrine-induced increases in intracellular calcium levels in fibroblasts stably transfected with either the α1A-AR, α1B-AR or the α1D-AR. Despite pretreatment with 30 nM BMY 7378, phenylephrine maintained the ability to promote increases in intracellular calcium in the α1A-AR or α1B-AR expressing lines of fibroblasts (Figure 4). In contrast, BMY 7378 blocked the phenylephrine-induced increases in intracellular calcium in fibroblasts stably transfected with the α1D-AR (Figure 4). Therefore, BMY 7378 at 30 nM, the concentration used in the vascular smooth muscle cells (see above), can selectively block the α1D-AR. These data support the conclusion that the receptor that mediates increases in intracellular calcium in aortic smooth muscle cells is the α1D-AR.
Effects on reactive oxygen species
This type of specificity of coupling was also observed using a novel functional response to activation of the α1-AR-namely the generation of reaction oxygen species. In human aortic smooth muscle cells, phenylephrine produced a rapid, dose-dependent and statistically significant increase in the level reactive oxygen species (Figure 5). While we present the data with 10 uM, we could see statistically significant increases in ROS at 1 uM phenylephrine. This increase was blocked by 1 nM prazosin or 30 nM BMY 7378. Therefore, it is the α1D-AR that mediates increases in reactive oxygen species in these vascular smooth muscle cells.
Effects on smooth muscle contraction
Recent data from heterologous systems have suggested that the interaction between the α1D-AR and other G-protein coupled receptors alters the pharmacologic properties of the α1D-AR [17,19]. Our results from vascular smooth muscle cells that naturally express all three receptors indicate that functional responses from a receptor with α1D-AR characteristics can be detected.
To assess the relevance of the interaction between the α1D-AR and the other ARs in an intact blood vessel system, we studied contractile responses in the rat aorta. In previous work we have shown that the contractions of the rat aorta are mediated by the α1D-AR . In addition to potential dimerization among the α1-ARs, there is evidence of heterodimerization between the α1D-AR and β2-AR. Studies in heterologous systems also show that desensitization of the β2-AR with albuterol promotes the internalization and desensitization of the α1D-AR . We assessed responses in the rat aorta following a 12 hr exposure to albuterol. After this incubation period, the responses to albuterol were significantly decreased when compared to vehicle treated aorta (Figure 6). This indicates a desensitization of the β2-AR mediated response. The phenylephrine log dose response curves were the same in control and albuterol desensitized aorta. Therefore, desensitization of β2-AR does not lead to desensitization of the α1D-AR.
Previous work from our laboratory has shown that in heterologous systems the α1D-AR localizes intracellularly and does not undergo agonist-mediated internalization or desensitization [11,12,21]. Due in part to problems with transfection, it has been difficult to determine if this type of expression pattern occurs in cells that natively express the α1D-AR along with the other α1-AR family members. To facilitate its efficient expression, we developed an adenoviral vector expressing the human α1D-AR fused with the GFP. We then used the vector to infect human aortic smooth muscle cells. RT-PCR analysis showed that these cells express all three α1-ARs (Figure 2). The rank order of mRNA expression was α1B-AR> α1A-AR>>α1D-AR. This type of expression pattern is typical for the α1-ARs. Following infection of aortic smooth muscle cells we observed that the α1D-AR was localized to intracellular compartments (Figure 1A). An intracellular localization pattern was also observed when aortic smooth muscle cells were immunostained with an α1D-AR antibody (Figure 1B). Therefore, while dimerization between the α1D-AR and the other α1-ARs may occur in aortic smooth muscle cells, this does not alter the cellular localization of the α1D-AR.
If the α1D-AR forms heterodimers with the other α1-ARs, then it is possible that these complexes exhibit properties different from the α1D-AR alone [17,19]. We examined this possibility using the selective antagonist BMY 7378. In previous work we calculated that at 30 nM over 90 % of the α1D-ARs would be occupied by BMY 7378 while less than 10 % of either the α1A-AR or the α1B-AR would be occupied by this antagonist . Therefore, at this concentration BMY 7378 would be anticipated to be highly selective for the α1D-AR. This was substantiated in fibroblasts stably transfected with each of the α1-ARs. In these fibroblast cell lines phenylephrine treatment produced a significant increase in intracellular calcium. However, only in fibroblasts expressing the α1D-AR was BMY 7378 capable of antagonizing the calcium response to phenylephrine, indicating that BMY 7378 is selective for the α1D-AR (Figure 4). The phenylephrine-mediated increases in intracellular calcium in aortic smooth muscle cells were also blocked by this dose of BMY 7378 (Figure 3). In a similar fashion, we demonstrated that the generation of reactive oxygen species in aortic smooth muscle cells was antagonized by 30 nM BMY 7378 (Figure 5). There is no evidence that BMY 7378 at this concentration can block either the α1A- or the α1B-AR. Therefore the antagonism seen with BMY 7378 indicates that the observed increases in intracellular calcium and reactive oxygen species are mediated by a receptor of α1D-AR character. In aggregate, the data suggest that while heterodimerization may occur, it does not appear to alter the pharmacologic properties of the α1D-AR. We know that both increases in intracellular calcium and elevations in ROS are mediated by the α1D-AR. What we do not know is if the intracellularly expressed α1D-AR is signaling competent and responsible for these effects or whether it is a small population of cell surface expressed receptors. If signaling does indeed emanate from the intracellular α1D-AR, then there has to be a pathway that would allow agonist access to these receptors.
In addition to dimerization within the α1-AR family, there is also evidence that the α1D-AR can form dimers with the β2-AR. Studies in expression systems have shown that not only does the β2-AR promote the cell surface expression of the α1D-AR but that desensitization of the β2-AR also desensitized the α1D-AR response. We wished to determine if this type of activity could be obtained in a functional system that natively expresses these receptors without resorting to overexpression of cloned receptors in a model cell system. The contractile responses of phenylephrine in the rat aorta are due to interactions at the α1D-AR (see for example, Piascik et al. ). Therefore, we assessed potential β2-AR/α1D-AR interactions using this blood vessel. Overnight treatment of blood vessels with albuterol caused desensitization of the β2-AR as shown by diminished vasodilatory responses to this agent (Figure 6A). However, desensitization of the β2-AR did not cause a rightward shift of the phenylephrine dose response curve (see Figure 6B). Therefore, desensitization of the β2-AR does not alter contractile responses to the α1D-AR. These results show that cross desensitization between β2-AR and the α1D-AR does not occur in an intact, responding segment of vascular smooth muscle.
In summary, adenoviral vectors expressing the α1-ARs are a novel and efficient tool to investigate properties of these receptors in native cells. These vectors were used to show that in human smooth muscle cells expressing all three α1-ARs, the α1D-AR is localized in intracellular compartments. Therefore, despite recent reports of heterodimerization between the α1-ARs, in a human vascular smooth muscle cell line, the α1D-AR is still expressed intracellularly. Indeed none of the data we obtained in this work support the idea of α1D-AR heterodimerization. Using three independent measures (calcium levels, generation of reactive oxygen or vascular smooth muscle contraction) we also could not detect any evidence of altered pharmacologic properties of the α1D-AR.
Cell culture conditions
Human aortic smooth muscle cells were obtained from Cascade Biologics (Portland, OR) and grown in Medium 231 supplemented with smooth muscle growth supplement until they become confluent (Cascade Biologics, Portland, OR). Stably transfected Rat 1 fibroblast lines expressing each of the α1-AR subtypes were maintained in Dulbecco's modified Eagle's medium (Cellgro, Herdon, VA) supplemented with 10% fetal bovine serum and a 1% antibiotic/antimycotic cocktail (Invitrogen, Carlsbad, CA). All cells were grown in T75 flasks in a 37°C cell culture incubator with a humidified atmosphere (95% air and 5% CO2) and were fed every 2 to 3 days. After reaching confluence the cells were plated on plain untreated coverslips in 35 mm tissue culture dishes.
Construction of recombinant adenoviruses expressing the α1-ARs
A vector expressing the human α1D-AR coupled to the green fluorescent protein (α1D-AR/GFP) was provided by Dr. Gozoh Tsujimoto [23,24]. This vector was digested with EcoRI and XbaI enzymes and cloned into the pCI expression vector (Promega. Madison, WI). pCI was digested with BglII and ClaI. This fragment included the CMV I.E. promoter, the α1D-AR/GFP and the SV40 late poly (A). The BglII and ClaI fragment was cloned into the adenovirus recombination vector pAdLink. pAdLink and the wild type adenovirus vector, dl327, were linearized with NheI and ClaI respectively. Homologous recombination occurred by co-transfecting linearized pAdLink and the wild type adenovirus vector into HEK 293 cells. Positive plaques appeared 10 to 14 days after recombination and were then amplified. Plaques were purified by serial dilutions of a positive plaque (usually from 10-3 to 10-12) in 96 well plates using HEK 293 cells. After plaque purification, samples of viral DNA were analyzed for wild type virus contamination by PCR . Once a purified adenovirus was obtained, the plaque was amplified for large-scale production. Fifty 150 mm dishes of HEK 293 cells were used for amplification of adenovirus which was then purified using double cesium gradients. Adenovirus was tittered using the Adeno-X™ Rapid Titer Kit from BD Biosciences (Palo Alto, CA).
Infection of cells with recombinant adenovirus
Human aortic smooth muscle cells or Rat 1 fibroblasts were grown on glass coverslips. Two hours prior to infection, cells were placed in serum free medium and infected with adenovirus. Twenty four hours after infection the medium was changed and the virus free incubation was allowed to proceed for an additional 24 hours. Cells expressing the GFP labeled α1D-AR were fixed with 3.7% Formaldehyde in PBS for 10 mins. Cells were then mounted on slides with Vectashield (Vector Labs, Burlingame, CA). Cells were visualized using a Leica TCS SP 5 AOBS confocal microscope with a Plan-Apo 64X oil immersion objective lens (Leica, Wetzlar, Germany) using Leica TCS NT version 2.5 software. Images were transferred to a computer for reduction with Adobe Photoshop version 6.0 (Adobe Systems, Mountain View, CA).
Human aortic smooth muscle cells or grown on glass cover slips, were washed in PBS and fixed with 3.7% Formaldehyde in PBS for 10 min. Cells were then washed with .05% BSA in PBS and permeabilized with 0.1% Triton in PBS for 5 min. After permeabilization, the cells were washed and blocked with 10% lamb serum for 1 hour at room temperature. After washing polyclonal antibodies (Affinity Bioreagents, Golden, CO) against each of the α1-ARs, diluted 1:100 in 1% BSA in PBS, was added and incubated overnight at 4°C. Following this incubation, the cells were washed with .05% BSA in PBS and a Texas Red secondary antibody (Abcam, Cambridge MA), diluted 1:500 in PBS, was added and incubated in the dark at room temperature for 1 hr. Cells were washed with PBS and mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, CA). Cells were visualized with a confocal microscope as described above.
Total RNA from HASMCs was isolated and purified with the ChargeSwitch Total RNA kit from Invitrogen (Carlsbad, CA), and 0.5 μg samples were reverse transcribed at 45°C for one hour using Cloned Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Oligo(dT)20 primer (Invitrogen, Carlsbad, CA). After heating at 85°C for 5 min. to terminate the reaction, cDNA samples were stored at -20°C until used. Negative controls for the presence of genomic DNA were performed by replacing the reverse transcriptase enzyme with Taq DNA polymerase.
For PCR, primers were synthesized by Invitrogen based on those used by Esbenshade et al.  to detect and distinguish specific human AR subtypes. Sequences of the primers were as follows: α1A, 5'-ATGCTCCAGCCAAGAGTTCA-3' (sense, annealing to bases 1417–1437) and 5'-TCCAAGAAGAGCTGGCCTTC-3' (antisense, bases 1898–1918); α1B, 5'-CTGTGCGCCATCTCCATCGATCGCTAC-3' (sense, bases 406–432) and 5'-ATGAAGAAGGGTAGCCAGCACAAGATGAA-3' (antisense, bases 907–935); α1D, 5'-CTCTGCACCATCTCCGTGGACCGGTAC-3' (sense, bases 563–589) and 5'-AAAGAAGAAAGGGAACCAGCAGAGCACGAA-3' (antisense, bases 1073–1102). The receptor specific primers target sequences within the third intracellular loop (sense) and the carboxy terminus (antisense). Primers for β-actin were included as a positive control (RT-PCR Primer and Control Set, Invitrogen). The predicted sizes of the amplified human β-actin, α1A-, α1B-, and α1D-AR PCR products were 353, 502, 530, and 540 bp, respectively.
PCR was carried out with Platinum Taq DNA polymerase (Invitrogen) in a PCR Express (Hybaid Ltd., United Kingdom) thermal cycler. The amplification reactions, repeated for 35 cycles, consisted of denaturation at 94°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. PCR products were run on a 1.4% agarose gel.
Determination of intracellular calcium
Human aortic smooth muscle cells were loaded for 1 hour with 5:M Calcium Green ™ 1 AM (Molecular Probes, Eugene, OR). In certain experiments, Rat 1 fibroblasts were also loaded with the Calcium Green. Cells were then washed twice with serum containing medium and visualized with an inverted microscope with a Xe arc lamp with a Plan-Apo 60X oil immersion objective and an excitation filter of 480/15 nm and an emission filter of 535/20 nm. Images were taken using a CoolSnap HQ camera. Indicator dye-loaded cells underwent several drug treatments. Human aortic smooth muscle cells were pre-treated with vehicle, 1 nM prazosin or 30 nM BMY 7378 for 20 minutes, followed by 25 :M phenylephrine. Stably transfected fibroblasts were challenged with 10 :M phenylephrine. A higher phenylephrine concentration was required in smooth muscle to observe an equivalent increase in the calcium signal. Images from calcium measurements were processed using Metamorph software (Molecular Devices, Sunnyvale, CA). In the analysis, the nucleus was masked leaving the cytoplasm. The mean intensity of the cytoplasm was then taken for each cell. Images were prepared using Adobe Photoshop version 6.0 (Adobe Systems, Mountain View, CA). lmaging data were analyzed by one-way analysis of variance with Tukey's post-hoc test using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). In all figures, the data are expressed as the mean and standard error of the mean (S.E.). A value of P < 0.05 was considered statistically significant.
Reactive oxygen species
The generation of reactive oxygen species was measured using Mitotracker ROS (Invitrogen, Carlsbad, CA) diluted in DMSO. Human aortic smooth muscle cells, attached to glass coverslips, were incubated for 20 min at 37° with 5 nM Mitotracker ROS diluted in Serum Free Medium. Cells were washed twice with medium and fresh medium applied. After this time 10 μM phenylephrine was added and incubated with the cells for 20 min. Preliminary studies established this time as that optimal to record a significant increase in ROS levels. In certain experiments, cells were pretreated with 1 nM prazosin or 30 nM BMY 7378 prior to the addition of phenylephrine. After agonist incubation, the cells were fixed with 1.0% formaldehyde in PBS for 10 min and washed with PBS. Cells were mounted on slides with Vectashield (Vector Laboratories, Burlingame, CA). Cells were imaged by confocal microscopy as described above. Images were processed using Metamorph software (Molecular Devices, Sunnyvale, CA). Images were prepared using Adobe Photoshop version 6.0 (Adobe Systems, Mountain View, CA). lmaging data were analyzed by one-way analysis of variance with Tukey's post-hoc test using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). In all figures, the data are expressed as the mean and standard error of the mean (S.E.). A value of P < 0.05 was considered statistically significant.
Assessment of aortic contractile function
All animal protocols were reviewed and approved by the University of Kentucky Institutional Animal Care and Use Committee. Isolated blood vessels were prepared by techniques routinely used in our laboratory [21,22,27,28]. Aorta were removed from male Sprague-Dawley rats, cleaned of adventitia and extraneous tissue and then segmented into 1–2 mm rings. Following isolation, aortic rings were placed in a 37EC cell culture incubator and treated for 12 hr with 1 :M albuterol, a selective β2-AR agonist or a vehicle control. After this period, rings were placed in the tissue baths for the assessment of contractile activity. The water-jacketed muscle baths were filled with physiologic saline solution maintained at 37°C with constant oxygenation (95% O2, 5% CO2, pH 7.4) and under a passive force of 2.0 grams. Previous studies have shown that this passive force gives optimal responses. Aortic rings were contracted with 25 mM KCl and the ability of increasing amounts of albuterol to induce aortic relaxation was measured. Phenylephrine-dose response curves were also generated in a separate set of aortic rings treated with albuterol. Changes in the force generation were recorded using force displacement transducers (Astro-Med, Inc., Grass Instruments, West Warwick, RI) interfaced to a Dell computer. Data were retrieved using PolyVIEW version 2.5 and analyzed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA).
AR: Adrenergic receptor; GPCR: G-Protein-Coupled Receptor; HASMC: Human aortic smooth muscle cell; GFP: Green fluorescent protein.
MLG performed or assisted in many of the studies in the manuscript. Specifically, figures 1, 3, 4 and 6. Also, wrote the manuscript. JLS mantained cell culture, performed or assisted in studies reported in figures 1, 3, 4, 5. Edited the manuscript and performed statistical analysis. KAO performed the PCR analysis in figure 2. DFM supervised the PCR studies in figure 2. LAS assisted in calcium studies in figures 3 and 4. RWH supervised calcium imaging studies in figures 3 and 4. SDK supervised preparation and purification of adenoviral vectors. MTP supervised MLG and JLS and is senior author. Edited manuscript and oversaw all aspects of this work. All Authors read and approve of this work.