An adenoviral vector was constructed to drive the efficient expression of a GFP-labeled α_1D-AR. Infection of aortic smooth muscle cells with virus expressing the α_1D-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 α_1D-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 α_1D-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 α_1D-AR/GFP expression plasmid or immunohistochemistry studies in fibroblasts stably transfected with the α_1D-AR 1221. Recently, it was proposed that the α_1D-AR can dimerize with the α-AR can dimerize with the α_1B-AR promoting its cell surface expression 19. Our results would argue that the presence of other ARs, particularly the α_1B-AR, does not alter α_1D-AR localization in vascular smooth muscle cells. To further substantiate that the presence of the α_1B-AR does not affect α_1D-AR localization, we infected fibroblasts that stably express the α_1B-AR with the α-AR with the α-AR with the α-AR with the α_1D-AR/GFP adenoviral construct (Figure 1C). Despite expression in a cell expressing the α_1B-AR at high levels, α_1D-AR was nonetheless expressed in intracellular compartments (Figure 1C). These data argue that the α_1B-AR does not alter the cellular localization of the α_1D-AR.

It is possible, however, that in these cultured smooth muscle cells the α_1B-AR and/or α_1A-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 α_1B-AR was detected in aortic smooth muscle cells. We were also able to detect an α_1A- AR transcript. A very faint product corresponding to the α- AR transcript. A very faint product corresponding to the α_1D-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 α_1A- AR is expressed both intracellularly as well as on the cell surface while the α_1B-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 α_1D- AR is markedly different from that of either the α- AR is markedly different from that of either the α_1A- or α- or α_1B-ARs. Therefore, in a mammalian cell where both the α_1A- and α- and α_1B-AR are natively expressed (as opposed to being transfected) the α_1D-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 α_1D-AR.

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 α_1D-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 α_1A-AR, α_1B-AR or the α_1D-AR. Despite pretreatment with 30 nM BMY 7378, phenylephrine maintained the ability to promote increases in intracellular calcium in the α_1A-AR or α_1B-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 α_1D-AR (Figure 4). Therefore, BMY 7378 at 30 nM, the concentration used in the vascular smooth muscle cells (see above), can selectively block the α_1D-AR. These data support the conclusion that the receptor that mediates increases in intracellular calcium in aortic smooth muscle cells is the α_1D-AR.

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 α_1D-AR that mediates increases in reactive oxygen species in these vascular smooth muscle cells.

Recent data from heterologous systems have suggested that the interaction between the α_1D-AR and other G-protein coupled receptors alters the pharmacologic properties of the α_1D-AR 1719. Our results from vascular smooth muscle cells that naturally express all three receptors indicate that functional responses from a receptor with α_1D-AR characteristics can be detected.

To assess the relevance of the interaction between the α_1D-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 α_1D-AR 22. In addition to potential dimerization among the α₁-ARs, there is evidence of heterodimerization between the α_1D-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 α_1D-AR 17. 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 α_1D-AR.

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.

A vector expressing the human α_1D-AR coupled to the green fluorescent protein (α_1D-AR/GFP) was provided by Dr. Gozoh Tsujimoto 2324. 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 α_1D-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 25. 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).

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 α_1D-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. 26 to detect and distinguish specific human AR subtypes. Sequences of the primers were as follows: α_1A, 5'-ATGCTCCAGCCAAGAGTTCA-3' (sense, annealing to bases 1417–1437) and 5'-TCCAAGAAGAGCTGGCCTTC-3' (antisense, bases 1898–1918); α_1B, 5'-CTGTGCGCCATCTCCATCGATCGCTAC-3' (sense, bases 406–432) and 5'-ATGAAGAAGGGTAGCCAGCACAAGATGAA-3' (antisense, bases 907–935); α_1D, 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, α_1A-, α_1B-, and α_1D-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.

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.

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.

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 21222728. 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).