Introduction

Top Summary Introduction Procedures Results Discussion References

Three ₁-AR subtypes (_1a, _1b, and _1d) have been cloned, and mRNA for each has been detected in the rat aorta (1, 2). The specific role of each ₁-AR subtype in regulating vascular smooth muscle function has not been completely established. When overexpressed in cultured cells, each of the subtypes has been shown to be capable of eliciting characteristic ₁-adrenergic responses, including activation of PLC and increased intracellular calcium (3, 4). Several studies have shown that ₁-ARs can couple to G_q and activate phospholipase C, resulting in production of inositol trisphosphate and release of intracellular calcium (5). However, it has been shown that ₁-AR-mediated contractile responses in vascular smooth muscle are partially inhibited by PTX treatment, suggesting the involvement of both PTX-sensitive and -insensitive G proteins in the contractile response (6-8). Thus, in vascular smooth muscle, it is not clear what combination of endogenous ₁-AR subtypes and G proteins is responsible for activating PLC and eliciting the contractile response.

In previous studies, we showed that although aortas from 1-month-old Fischer 344 rats express _1a-, _1b-, and _1d-ARs, the contractile response to NE is mediated by stimulation of the _1b- and _1d-ARs (9, 10). NE produced a more potent contractile response in these aortas compared with aortas of older rats, in which the expression and functional role of the _1a-AR increase (9, 11). The determination of the coupling of ₁-AR subtypes to specific G proteins will facilitate understanding of the functional roles of ₁-AR subtypes in the aorta; therefore, the aim of the current study was to define ₁-AR/G protein coupling in aortic membranes. This was achieved by examining the sensitivity of ₁-AR-mediated responses to PTX treatment; assessing ₁-ARs that coimmunoprecipitated with G proteins using specific antisera directed against G subunits, G proteins that coimmunoprecipitated with ₁-AR subtypes using specific antisera directed against ₁-AR subtypes; and examining ₁-AR-stimulated palmitoylation of  subunits or receptor-stimulated changes in coprecipitation of ₁-AR binding sites with G subunits in aortic membranes.

	Experimental Procedures

Top Summary Introduction Procedures Results Discussion References

Animals. One-month-old male Fischer 344 rats were obtained from National Center for Toxicological Research (Jefferson, AR), where they are bred and maintained under the auspices of the National Institute on Aging. On receipt at our institution, animals were maintained for 1-2 weeks under barrier conditions comparable to those under which they were raised.

Contraction. Rats were killed through decapitation, and the aorta was removed and placed in ice-cold PSS composed of 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1.0 mM NaH₂PO₄, 25 mM NaCO₃, 1.8 mM CaCl₂, 11 mM glucose, and 0.024 mM EDTA. Vessels were cleansed of fat and connective tissue and cut into 3-mm-wide rings. Aortic ring segments were mounted at 37° in 15-ml organ baths using stainless steel hooks connected by fine gold chain at the bottom to a stationary glass rod attached to the bath and at the top to a Grass Instruments (Quincy, MA) model FT0.03 force-displacement transducer and bubbled continuously with 95% O₂/5% CO₂. Responses were recorded on a Grass model 7 polygraph. Rings were equilibrated for 1 hr at a previously optimal resting tension of 1.5 g. Concentration-response curves were determined through cumulative increases in the concentration of agonist. Rings then were washed extensively by several changes of PSS over 1-2 hr until tension stabilized at the precontraction level. For experiments with PTX, rings were incubated with the toxin for 4 hr in PSS. Then, the rings were washed for 1 hr with several changes of PSS, and cumulative concentration-response curves to PHE were obtained.

IP accumulation. The method used for measuring [³H]inositol metabolism was described previously (12, 13). Aortic rings were prepared as described above and then preincubated in oxygenated buffer composed of 122 mM NaCl, 4.9 mM KCl, 1.2 mM MgCl₂, 1.2 mM KH₂PO₄, 3.6 mM NaCO₃, 1.3 mM CaCl₂, 11 mM glucose, and 30 mM HEPES, pH 7.4, at 37° for 1 hr. Subsequently, artery segments were incubated for 1.5 hr in buffer containing 20 µCi/ml of myo-[³H]inositol (17 Ci/mmol; New England Nuclear Research Products, Boston, MA) under the same conditions. Labeled artery segments were washed four times and placed inot individual tubes containing buffer with 10 mM LiCl (total assay volume, 300 µl). Treatment with PTX was as described above. Incubation with agonist was for 60 min with oxygenation at 15-min intervals and was stopped by the addition of 300 µl of ice-cold 15% trichloroacetic acid and then left on ice for 15 min. The tubes were centrifuged (1500 × g for 10 min), and aliquots (350 µl) of supernatant were added to 125 µl of 10 mM EDTA in 1.5-ml microcentrifuge tubes, followed by 500 µl of 1:1 Freon/tri-n-octylamine. The samples were vortexed and left to stand for 10 min before centrifugation (12,000 × g for 10 min), and 350 µl of upper aqueous phase was taken for analysis of IPs. Samples were loaded onto Dowex-1(X8) ion exchange columns (formate form, 100-200 mesh, 1 ml). The columns were washed initially with 16 ml of myo-[³H]inositol (5 mM). Then, IPs were eluted with 4 ml of 0.1 M formic acid/1 M ammonium formate. Radioactivity was measured by liquid scintillation counting.

Preparation of aortic membrane. Rats were killed by decapitation, and the aorta was removed; placed in 20 mM NaH₂PO₄-Na₂HPO₄ buffer (pH 7.6) containing 154 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 20 µg/ml aprotinin, and 25 µg/ml pepstatin; cleansed of fat and connective tissue; homogenized using a glass-to-glass homogenizer; and centrifuged at 500 × g for 10 min at 4°. The supernatant was centrifuged at 100,000 × g for 60 min at 4°. The resulting pellet was resuspended, rehomogenized, and then recentrifuged under the same conditions. The final pellet was resuspended in PBS. Protein content was measured according to the method of Bradford (14).

Solubilization of aortic membrane. Aortic membranes were solubilized by modification of a previously described procedure (15). Briefly, aortic membranes were prepared as described above. They were then solubilized by gentle end-over-end shaking for 60 min at 4° in PBS containing 1.5% digitonin, 0.5 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 20 µg/ml aprotinin, and 25 µg/ml pepstatin. The sample was centrifuged at 100,000 × g for 60 min at 4°, and the supernatant was used for the soluble fraction of the membrane. The pellet was also collected for determination of the insoluble ₁-ARs and G protein  subunits remaining in the membranes. The solubilized ₁-ARs were detected by measuring [¹²⁵I]HEAT binding. The samples were incubated with a 300-400 pM concentration of [¹²⁵I]HEAT for 60 min at 37°. Reactions were terminated by rapid filtration using a Brandel (Montreal, Quebec, Canada) cell harvester and Whatman (Clifton, NJ) DE81 filters to trap the solubilized proteins. Filters were washed four times with 4 ml of ice-cold PBS. The filter-bound radioactivity was determined in a Beckman Instruments (Palo Alto, CA) -counter. Nonspecific binding was defined as binding in the presence of 0.1 µM prazosin or 1 mM NE with identical results. Assays were conducted in duplicate. After solubilization of aortic membranes, 20-30% of the initial ₁-AR binding was detected in soluble fraction.

Immunoprecipitation of G protein  subunits and ₁-AR subtypes. Solubilized G protein  subunits were immunoprecipitated as described previously (15, 16). Soluble membrane protein (10-15 fmol of ₁-AR) was incubated with an appropriate dilution of G-specific antiserum overnight in a rotatory shaker at 4°C. Nonimmune serum at the same dilution was used as a control. Appropriate dilution was determined when no further immunoprecipitation was observed at a higher concentration of the antiserum (maximum concentration, 1:50). Then, 100 µl of a 1:1 suspension of protein A/Sepharose beads (CL-4B: Sigma Chemical, St. Louis, MO), prewashed three times and diluted in PBS, was added to the samples and incubated overnight in a rotary shaker at 4°C. The samples were centrifuged at 10,000 × g for 3 min; the supernatant was collected to measure remaining 1-ARs; and the pellet was resuspended in PBS and recentrifuged as described above. The immunoprecipitate was resuspended in PBS, and ₁-ARs were detected by measuring [¹²⁵I]HEAT binding as described above. In some samples, the immunoprecipitate was separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membrane, and immunoblotted with anti-G antisera to confirm the identity of the precipitated G protein  subunits. In several experiments, the immunoprecipitate was incubated with 0.1 mM guanosine-5-(,-imido)triphosphate for 60 min at 25° and then centrifuged at 10,000 × g for 3 min. The pellet was resuspended in PBS, and ₁-ARs in the immunoprecipitate were determined using the radioligand binding assay described above.

Immunoblots. Aortic membranes, solubilized membranes, or membrane immunoprecipitates were subjected to 10% SDS-polyacrylamide gel electrophoresis (18) and then transferred electrophoretically to nitrocellulose. Immunoblotting was performed using antisera for  subunits of G proteins [RM/1 (G_s), AS/7 (G_i), GC/2 (G_o), QL (G_q/11); dilution 1:2000; New Nuclear England Research Products] and ECL as described previously (15, 19). Briefly, nitrocellulose membranes were incubated overnight at 4° in PBS containing 3% bovine serum albumin and 8% nonfat dry milk. Blots were washed several times with 0.1% TBS and then incubated with antisera at room temperature for 1-2 hr with shaking. Blots were then washed four times (10 min each) with 0.1% TBS and then incubated with 1:10,000 dilutions of horseradish peroxidase-labeled donkey anti-rabbit IgG or rabbit anti-goat (_1a-AR) in 0.1% TBS for 1 hr at room temperature. Blots were washed once with 0.3% TBS for 30 min followed by four 5-min washes with 0.1% TBS and then incubated with ECL Western blotting reagent (SuperSignal substrate, Western Blotting; Pierce, Rockford, IL) for 4 min and exposed to X-ray film for 15-45 sec.

Agonist-induced palmitoylation of G proteins. Aortas were homogenized and centrifuged (500 × g for 10 min at 4°) as described above except with HEPES buffer containing 25 mM HEPES, pH 7.4, and 2 mM EGTA. The supernatant was centrifuged at 100,000 × g for 60 min at 4°. The resulting pellet was resuspended, rehomogenized, and then recentrifuged under the same conditions. The final pellet was resuspended in oxygenated Krebs-HEPES buffer containing 25 mM HEPES, pH 7.4, 154 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgCl₂, 0.2% 2-mercaptoethanol, 25 µg/ml leupeptin, 25 µg/ml pepstatin A, 0.01unit/ml soybean trypsin inhibitor, and 0.05 mM phenylmethylsulfonyl fluoride and used as the crude membrane preparation. The assay mixture (250 µl) containing 200 µg of membrane protein, 800 µCi/ml [9,10-³H]palmitic acid (specific activity, 50 Ci/mmol; New Nuclear England Research Products) was incubated at 37° for 10 min followed by an additional 5-min incubation with either buffer or agonist. To test the specificity of this receptor mediated response, membranes were incubated with a selective ₁-AR antagonist for 5 min before the addition of the agonist. The reaction was terminated by dilution with 750 µl of ice-cold Krebs-HEPES containing 1 mM EGTA, mixed, placed on ice, and immediately centrifuged at 16,000 × g for 30 min in microcentrifuge. The pellets were solubilized in 1.5 ml of Krebs-HEPES buffer containing 1.5% digitonin, and soluble membrane proteins were immunoprecipitated using antisera directed against the G proteins as described above. The radioactivity in the immunoprecipitate was measured by liquid scintillation counting. The radioactivity precipitated by the normal rabbit serum was considered background and subtracted from all values.

Data analysis. Differences were determined by ANOVA and post hoc analysis for multiple comparisons. A value of p < 0.05 was considered significant.

Materials. For these studies, pargyline HCl, soybean trypsin inhibitor, and the buffer reagents were purchased from Sigma. The chemicals used for IP isolation and determination were purchased from Fisher Scientific (Pittsburgh, PA). PHE was purchased from Research Biochemicals (Natick, MA). Normal rabbit serum and Pansorbin were purchased from Calbiochem. Prazosin HCl was generously supplied by Pfizer (New York, NY). [9,10-³H]Palmitic acid (50 Ci/mmol), [¹²⁵I]HEAT (2200 Ci/mmol), and the antisera to G_s (RM/1), G_i(1,2) (AS/7), G_o (GC/2), and G_q/11 (QL) were purchased from New Nuclear England Research Products. Antisera to _1b- and _1d-ARs were produced by one of the authors (R.D.B.). Antiserum to _1a was purchased from Santa Cruz Biochemicals (Santa Cruz, CA). Horseradish peroxidase-labeled donkey anti-rabbit IgG or rabbit anti-goat (SuperSignal substrate, Western Blotting; Pierce).

	Results

Top Summary Introduction Procedures Results Discussion References

Effect of PTX on contraction and IP accumulation. PTX treatment maximally inhibited PHE-induced contractile response at a concentration of 2.5 µg/ml (Fig. 1). Higher PTX concentrations did not cause further inhibition in the responses to PHE, and KCl-induced contraction was not altered by PTX treatment (data not shown). PHE-induced IP accumulation was also inhibited (52 ± 8%) by 2.5 µg/ml PTX treatment (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   PHE-induced contraction of aortic ring segments in 1-month-old Fischer 344 rats before () and after () treatment with PTX (2.5 µg/ml, 4 hr at 37°). Data represent mean ± standard error of determinations obtained from five or six animals. A significant reduction in PHE concentration-response curve was obtained after PTX treatment as determined by ANOVA followed by Newman-Keuls test for multiple comparisons (*, p < 0.05).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   PHE-stimulated IP accumulation in aortic ring segments from 1-month-old aorta before and after treatment with PTX (2.5 µg/ml, 4 hr at 37°). Aortic rings were labeled with myo-[3H]inositol and subjected to incubation with agonist ,and IPs were separated by ion exchange chromatography. Data represent mean ± standard error of determinations obtained from five or six animals. A significant reduction in PHE-activated IP accumulation was obtained after PTX treatment as determined by ANOVA followed by Newman-Keuls test for multiple comparisons (*, p < 0.05).

Coupling of G proteins and ₁-ARs. The possibility that ₁-ARs directly couple to G proteins was tested by coimmunoprecipitation of ₁-ARs with anti-G protein antibodies. We have previously shown by immunoblot analysis the presence of single bands for G_o (39 kDa), G_i (41 kDa), and G_q (42 kDa) and two bands for G_s (45 and 52 kDa) in aortic membranes (15, 19). The results summarized in Fig. 3 demonstrate that antisera against G_q and G_o but not G_s or G_i, at dilutions of 1:200, precipitated specific ₁-AR binding sites labeled by the selective ₁-AR ligand [¹²⁵I]HEAT. To confirm the specificity of the immunoprecipitation, the precipitates obtained using G_q and G_o antisera were subjected to immunoblot analyses. G_q and G_o antisera selectively precipitated the respective proteins (data not shown). The coupling of G proteins to ₁-AR subtypes _1a, _1b, and _1d was investigated by monitoring the G proteins that were coimmunoprecipitated with specific antisera directed against the ₁-AR subtypes. Immunoprecipitates of the ₁-AR subtypes derived from 400 µg of solubilized aortic membranes were separated on SDS-polyacrylamide gels and blotted with antibodies against the G proteins. Fig. 4, A and B, illustrates that _1a-, _1b-, and _1d-AR antibodies coimmunoprecipitated G_q protein, whereas the _1b-AR antibody also immunoprecipitated G_o protein. Densitometric analysis of the results indicate that ~4.5-6% of membrane G_q was found to be associated with _1a-, _1b-, and _1d-ARs, whereas only 2.8% of G_o was linked to _1b-AR. The specificity of the anti-receptor antisera and the effectiveness of the immunoprecipitation are presented in Fig. 5. The figure demonstrates that the antiserum for each of the receptor subtypes completely and selectively precipitated the respective protein. Furthermore, incubation of aortic membranes with 1 µM PHE was found to increase [¹²⁵I]HEAT binding in immunoprecipitates of G_q and G_o proteins by 370% and 350%, respectively (Fig. 6). These receptor stimulation-induced increases in receptor/G coupling were inhibited 70-80% by an equimolar concentration of the ₁-AR antagonist prazosin (Fig. 6). Thus, the data indicate that in aortic membranes, the _1a- and _1d-ARs are coupled to G_q protein and the _1b-AR subtype is linked to both G_q and G_o proteins.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Immunoprecipitation of 1-ARs by antisera to Gs, Go, Gq, and Gi and by nonimmune serum (NIS) in solubilized aortic membranes from 1-month-old rat. Soluble membrane proteins were incubated with antisera directed against the G proteins, and immuncomplexes were precipitated with protein A/Sepharose beads. The 1-ARs were detected by measuring [125I]HEAT binding in the precipitate. Each value represents the mean ± standard error of six or seven individual experiments. *, p < 0.05, significant difference by ANOVA followed by Newman-Keuls test for multiple comparisons.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Coimmunoprecipitation of 1-AR subtypes with G proteins. Rat aortic membranes were solubilized and subjected to immunoprecipitation with anti-peptide antisera raised against 1a-, 1b-, or 1d-AR (1:250 dilution). The immunocomplexes derived from 400 µg of solubilized aortic membranes were then solubilized and separated on 10% SDS-polyacrylamide gels, transferred onto nitrocellulose membranes, immunoblotted with specific G antisera (1:2000 dilution), and detected by ECL. Gq was detected in 1a-, 1b-, and 1d-AR immunoprecipitates (A and B), whereas Go was observed only in the 1b-AR immunoprecipitate (B). Lane C, immunoblots of 25 µg of solubilized aortic membranes. The immunoblots shown are representatives of four individual experiments that yielded similar results.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Specificity of the antisera to the 1a-, 1b-, and 1d-ARs. The specificity of each of the 1-AR antisera was tested in rat aortic membranes. Rat aortic membranes were solubilized, and 400 µg of protein was subjected to immunoprecipitation with antisera raised against 1a-, 1b-, or 1d-ARs (1:250 dilution). Aliquots of solubilized immunocomplexes representing 50 µg of the original solubilized membrane preparation were separated on 10% SDS-polyacrylamide gels; transferred onto nitrocellulose membranes; immunoblotted with specific 1a-, 1b-, or 1d-AR antisera (0.25 µg/ml for 1a, 1:1000 dilution for 1b or 1d); and detected by ECL. Data indicate there was no cross-reactivity among the three antisera tested. Furthermore, the data indicate that immunoprecipitation with each of the antiserum resulted in >90% recovery of the specific 1-AR subtype compared with the signal obtained from 50 µg of solubilized aortic membranes (lane C). Immunoblots are representative of three individual experiments that yielded comparable results.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Coupling of 1-ARs to Go and Gq in PHE-stimulated aortic membranes from 1-month-old rats. Aortic membranes were incubated with buffer or 1 µM PHE for 5 min. Tissues were solubilized, and membrane proteins were incubated with antisera directed against Go or Gq proteins. Immuncomplexes were precipitated with protein A/Sepharose beads. In some experiments, aortic membranes were incubated with 1 µM concentrations of the selective 1-AR antagonist prazosin for 5 min before the addition of buffer or PHE. The 1-ARs in the immunoprecipitates were detected by measuring specific [125I]HEAT binding. Value represent the mean ± standard error of five individual experiments. PHE stimulation caused 370% and 350% increases in coupling of Gq and Go proteins to 1-ARs. Prazosin inhibited the coupling of Go and Gq to 1-ARs by 70% and 80%, respectively. Statistical significance was determined by ANOVA followed by Newman-Keuls test for multiple comparisons. *, p < 0.05 compared with controls. +, p < 0.05 compared with PHE-induced response.

Receptor-activated palmitoylation of G proteins. To further examine the coupling of ₁-AR to G proteins, PHE-stimulated palmitoylation of G proteins was assessed. Incubation of aortic membranes with PHE resulted in significant increases in [³H]palmitoylation of G_q and G_o proteins (Fig. 7) without affecting G_s and G_i proteins. In addition, pretreatment of membranes with 1 µM of the ₁-AR antagonist prazosin blocked the PHE-stimulated incorporation of palmitic acid into G_q and G_o proteins by 76% and 73%, respectively (Fig. 7). These results therefore support the above data, which indicate that in aorta of 1-month-old Fischer 344 rats, ₁-ARs are coupled to both G_q and G_o proteins.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Palmitoylation of Gq and Go proteins in PHE-stimulated aortic membranes. Aortic membranes were incubated with 800 µCi/ml [9,10-3H]palmitic acid, followed by stimulation with buffer or 1 µM PHE. In some experiments, aortic membranes were incubated with 1 µM concentration of the selective 1-AR antagonist prazosin for 5 min before the addition of buffer or PHE. The membranes were solubilized and subjected to immunoprecipitation with antisera directed against a G protein  subunit (1:200 dilution). The radioactivity in the immunoprecipitates was measured. Data represent mean ± standard error of determinations obtained from four animals. PHE stimulation caused significant increases in the palmitoylation of Gq and Go proteins as determined by ANOVA followed by Newman-Keuls test for multiple comparisons. *, p < 0.05 compared with controls. +, p < 0.05 compared with PHE-induced response.

	Discussion

Top Summary Introduction Procedures Results Discussion References

Activation of PLC and increased intracellular Ca²⁺ are important elements of ₁-AR-mediated signaling, which result in the contractile response of vascular smooth muscle (20, 21). Despite considerable progress in elucidating the structure and signaling mechanisms of ₁-ARs, it is not clear which G protein or proteins are responsible for ₁-AR-mediated effects in blood vessels. Many G protein-coupled receptors were shown to activate PLC and increase the intracellular concentration of inositol-(1,4,5)-trisphosphate that eventually lead to vasoconstriction. Two ₁-AR-mediated pathways are known to activate PLC and to produce vascular contraction based on their sensitivity to PTX (5, 22). The _1a, _1b, and _1d subtypes of the ₁-AR have been shown to activate PLC in transfected COS-7 cells via coupling to the PTX-insensitive G proteins G_q/G₁₁ (5). On the other hand, several studies have shown that PTX pretreatment only partially inhibits ₁-AR-mediated contraction in blood vessels (6, 7), implicating the existence of ₁-ARs that are linked to a PTX-sensitive G protein in vessels. G_o protein was proposed to be that PTX-sensitive G protein based on the observation that _1b-ARs, expressed in Xenopus laevis oocytes, use G_o protein in activating PLC-mediated Cl current (22, 23).

The current study confirms the observation that PTX partially inhibits both ₁-AR-elicited contraction and IP accumulation in the aorta and suggests that both PTX-sensitive and -insensitive G proteins are involved in ₁-AR-mediated signal transduction in vascular smooth muscle. To identify these G proteins, we coimmunoprecipitated ₁-ARs with their associated G proteins in the rat aorta. Specific antiserum directed against G_q protein coimmunoprecipitated ₁-ARs that are specifically labeled by [¹²⁵I]HEAT, suggesting a linkage between ₁-AR and G_q protein. Antiserum specific for G_o also coimmunoprecipitated [¹²⁵I]HEAT binding sites, indicating that endogenous ₁-ARs in aorta membranes also couple to G_o. These data are therefore in agreement with previous studies that indicated functional coupling between ₁-ARs and G_q in transfected cells (5) and between _1b-AR and G_o in X. laevis oocytes that express these proteins (22). In the current study, functional relevance of these linkages is also supported by the ability of the ₁-AR agonist PHE to increase the coupling of labeled receptors with both G_q and G_o proteins and to stimulate palmitoylation of G_q and G_o proteins in aortic membranes in a prazosin-sensitive manner. Palmitoylation of G protein  subunits is a reversible post-translational modification that is regulated by receptor stimulation (24-26). No linkage was detected between ₁-AR and G_s or G_i proteins using either coimmunoprecipitation or the palmitoylation approach. Thus, the data suggest that G_o, not G_i, is responsible for the PTX-sensitive responses to ₁-AR stimulation in aorta of the 1-month-old Fischer 344 rat.

It has been shown that rat aorta express three ₁-AR subtypes: _1a-, _1b-, and _1d-ARs (1, 2). The expression level of these subtypes change with age, coincident with changes in the magnitude of aortic contraction (9, 10). This raises questions about possible differences in the functional roles of the ₁-AR subtypes. Although several studies have shown that the three subtypes are capable of stimulating the same signal transduction pathways when they are expressed in cultured cells (3, 4), data from the current study indicate that antisera directed against _1a- and _1d-ARs coimmunoprecipitated G_q protein, whereas anti-_1b-AR antiserum coimmunoprecipitated both G_q and G_o proteins. Furthermore, stimulation of ₁-AR with PHE elicited an increase in the coupling of ₁-ARs to both G_q and G_o proteins. Because G proteins determine the specificity and functional diversity of downstream intracellular effectors, identification of the interaction of a particular ₁-AR subtype with its G proteins represents an important step in unraveling the signal transduction cascades by which specific ₁-AR subtypes exert their effects.

In summary, the current results suggest that ₁-ARs are coupled to the PTX-insensitive G_q and the PTX-sensitive G_o in aorta derived from 1-month-old Fischer 344 rat. These G proteins therefore seem to couple to PLC and to mediate ₁-AR-stimulated contraction. It has been shown that the three subtypes of the ₁-AR activate PLC and cause increases in inositol trisphosphate level through G_q/G₁₁ proteins (5). This and a previous study (22) demonstrate that _1b-ARs stimulate PLC by coupling, in addition, to the PTX-sensitive G_o protein. The ultimate definition of the coupling of ₁-AR subtypes with specific G proteins will help to understand the functional roles of the different ₁-AR subtypes. This study for the first time demonstrates the specific coupling between ₁-AR subtypes and G proteins in vascular smooth muscle membranes and implicates these G proteins in coupling of ₁-AR subtypes to the contractile response elicited by ₁-AR stimulation in 1-month-old Fischer 344 rat aorta.