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Effect of Age on Vascular ß₂-Adrenergic Receptor Desensitization Is Not Mediated by the Receptor Coupling to Gi Proteins

¹ Portland VA Medical Center, Research Service, Oregon.
² Oregon Health & Science University, School of Medicine, Portland.

Address correspondence to Scott L. Mader, MD, Portland VA Medical Center, Research Service–R&D 26, 3710 SW US Veterans Hospital Rd., Portland, OR 97201. E-mail: scott.mader{at}med.va.gov

	Abstract

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Beta-adrenergic receptor (ß-AR)-mediated vasorelaxation declines with age. In the vasculature, ß₂-AR undergoes protein kinase A-mediated desensitization that causes a switch in the G protein coupled to ß₂-AR; Gi links instead of Gs. We exposed Fischer 344 rat aortae of increasing age to a desensitizing dose of isoproterenol, and determined its effect on ß₂-AR-mediated vasorelaxation. Desensitization decreased ß₂-AR-mediated vasorelaxation in young aortae only. Subsequently, we used pertussis toxin to block Gi to determine whether changes in ß₂-AR/G protein coupling occurred. Gi inhibition did not reverse desensitization or the age-related change, but there appears to be a population of ß₂-AR linked to Gi, as pertussis toxin treatment improved ß₂-AR-mediated vasorelaxation in aortae from animals of all ages. These findings suggest aortic ß₂-AR in older animals may be maximally desensitized, which would explain impaired vasorelaxation. Our results also imply that protein kinase A-mediated ß₂-AR desensitization may not be responsible for the age-related decline.

One change associated with desensitization of the ß₂-AR is the G protein that participates in coupling. Recently, it has been found that PKA-mediated phosphorylation of the ß₂-AR causes switching of the G protein coupled to the receptor; the PKA-phosphorylated ß₂-AR preferentially couples to Gi rather than to Gs (7). This phenomenon has been documented in the heart (8) and in multiple cultured cell lines (9). When the ß₂-AR is linked to Gi, one cellular event that occurs is an inhibition of adenylyl cyclase activity that is manifested as a reduction of intracellular cAMP production (10).

Age-related changes in both PKA and Gi have been evaluated. Deisher and associates (11) determined that PKA activity declined with advancing age in aortic preparations. However, this change was entirely explained by the lack of cAMP generation, not an intrinsic change in PKA. Changes in the expression of Gi are equivocal. In rat aorta, Mader and colleagues (12) found a slight decrease in 32-P–linked-pertussis toxin (PTX) labeling (irreversibility binds and inhibits Gi) with age, but no change in Gi_1/2, or Gi₃ protein expression in aorta. In contrast, a 30% decrease in Gi_1/2 protein content between 6- and 24-month-old aortic preparations, without a concomitant change in respective messenger RNA (mRNA) transcripts of these genes, has been documented (13). In rat heart, a study by Kilts and colleagues (14) found that age-related Gi expression and activity were increased.

Current evidence suggests the age-related decline in ß-AR-mediated vasorelaxation is related to desensitization of the receptor. Therefore, we hypothesized that the ability of agonist to induce ß-AR desensitization would also decline in aorta isolated from old rats. In addition, we questioned whether desensitization would be associated with a switch in the coupling of the ß₂-AR from Gs to Gi. This switch could explain the decline in ß-AR-mediated vasorelaxation, regardless of changes in either PKA and/or Gi expression or activity. To evaluate this hypothesis, we used aortic rings isolated from Fischer 344 rats of increasing age and a physiologic tissue bath technique.

	METHODS

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Materials and Solutions
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. PTX was obtained from List Biological Laboratories (Campbell, CA), and activated per manufacturer-provided instructions. Composition of phosphate-buffered saline (PSS) is as follows (in mM): NaCl, 114; KCl, 4.7; KH₂PO₄, 1.15; Na₂HPO₄, 1.1; MgSO₄, 1.18; NaHCO₃, 15; CaCl₂, 1.25; and glucose, 5. Throughout the study, this solution was aerated with 95% O₂/5% CO₂.

Animals and Tissue Preparation
Male Fischer 344 rats (2-months, 6-months, 12-months, and 24-months old) obtained from the NIA colony at Harlan Sprague Dawley (Indianapolis, IN) were killed by pentobarbital sedation and exsanguination in accordance with the procedures approved by the Institutional Animal Care and Use Committee at the Portland VA Medical Center. Thoracic aortae were quickly removed, placed in ice-cold PSS, cleaned of adhering fat and connective tissue, and divided into ring segments 3- to 5-mm wide. Tissue from the aortic arch and abdominal aorta were excluded.

Vascular Reactivity Studies
Isometric tension development was determined in a muscle bath system containing PSS at 37°C with Grass Instruments (Quincy, MA) FT.03 force transducers as described by Chapman and colleagues (15). After equilibrium, vessels were contracted with increasing doses (1 nM–10 µM) of phenylephrine (PE) that yielded a tension plateau. Following, vessels were washed extensively with PSS until tension reached baseline. After 10 minutes at baseline, drugs were added based on specific protocols as listed.

Protocol 1: Isoproterenol-mediated and adenylyl cyclase-mediated vasorelaxation.-- A single calculated dose (approximately 300 nM) of PE was used that produced 80% maximum contraction. After a stable level of precontraction was achieved (approximately 10 minutes), vessels were relaxed with the nonselective ß-AR agonist isoproterenol (ISO), or the adenylyl cyclase-specific activator forskolin (FSK). Nine doses (1 nM–10 µM) of each drug were added (via cumulative addition) in 1-minute intervals.

Protocol 2: Determination of ß-AR subtype-mediating vasorelaxation.-- Thirty minutes prior to performing the procedures described in Protocol 1, the ß₁-AR antagonist metoprolol (1 µM) was added (16).

Protocol 3: Analysis of ß₂-AR desensitization.-- Aortae were incubated for 1 hour with either ISO (3.2 nM) or vehicle (H₂O), and during the final 30 minutes 1 µM antagonist metoprolol was added. This was followed by 10–15 washes with PSS over a 10-minute period. Thereafter, metoprolol (1 µM) was again added, and the vessels were again contracted with the same dose of PE that produced 80% maximum contraction. After a stable tone of contraction was achieved (approximately 10 minutes), a dose–response relationship was determined for ISO and for FSK.

Protocol 4 (a and b): Determination of the role of ß-AR/Gi coupling in ß₂-AR desensitization.-- Aortae were incubated in activated PTX (1 µg/mL; PTX functions as a Gi activity inhibitor by irreversibly ADP-ribosylating the protein) or vehicle for 2 hours prior to initiating the 80% maximal PE-mediated contraction. Following a procedure similar to Protocol 3 was used (Protocol 4a refers to no ISO pretreatment, and Protocol 4b refers to ISO pretreatment). PTX was maintained in the bath throughout the entire experimental period. The PTX dose and incubation time were chosen based on our previous experiments with this drug (12), as well as from the results presented in the literature. Miller and colleagues (17) and Sato and colleagues (18) both effectively used a low dose (100 ng/mL) and a 2-hour preincubation time in canine femoral arteries and rabbit coronary detector vessels, respectively. Petitcolin and colleagues (19) effectively used 1 µg/mL and 3 µg/mL doses, with a 2-hour preincubation time with rat aorta. Their results showed no difference between treatments of 1 µg/mL and 3 µg/mL.

Statistical Analysis
Results are expressed as mean values ± standard error. The experimental unit was the number of animals. Matched rings from each animal were used for each test. Relaxation data were determined by normalizing force (determined in Newtons [N]) on a percentage basis. The tone established just prior to addition of the first dose of relaxant agent was considered 100%. Treatment effect was determined by evaluating potency (the concentration of relaxing agent that produced 50% of maximal response; EC₅₀) and efficacy (the concentration of relaxing agent that produced maximal effect; MAX). Differences were determined by two-way analysis of variance (treatment by dose of relaxing agent) with Bonferroni's post hoc comparison. Both EC₅₀ and MAX were determined by computer nonlinear regression using a four-parameter logistic equation (Prism 4.0; GraphPad Software, Inc., San Diego, CA). Subsequently, age-related differences in EC₅₀ and MAX were determined again by two-way analysis of variance (Treatment vs Age) with Bonferroni's post hoc comparison. Statistical significance was considered at p <.05.

	RESULTS

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Age-Related Changes in Vasorelaxation Mediated by ß₁-AR, ß₂-AR, and Adenylyl Cyclase
Prior to initiating vasorelaxation, vessels were constricted with a single dose of PE as described in Methods (Protocol 1). There was no effect of age on the magnitude of this PE-mediated contraction. Aortae isolated from 2-, 6-, 12-, and 24-month-old animals generated 4.5 ± 0.8, 5.0 ± 1.1, 4.1 ± 1.0, and 5.2 ± 0.9 N/m², respectively. Similar to previous data by us and others (1), there was a significant reduction in the potency and efficacy of ISO to elicit half-maximal (EC₅₀) and maximal vasorelaxation, respectively, with advancing age, but there were no age-related changes in FSK effect (Figure 1). To determine the relative role of ß₁-AR- versus ß₂-AR-mediated vasorelaxation (Protocol 2), we used the selective ß₁-AR antagonist metoprolol (1 µM; 30-minute preincubation). Figure 1, C and D, shows that ß₁-AR blockade did not significantly effect the EC₅₀ for ISO, but did slightly reduce the maximum effect for ISO in aortae from all ages of rat. More importantly, the age-related decline in ISO effect was unchanged in the presence of ß₁-AR blockade. Table 1 presents complete EC₅₀, maximum relaxation, and relevant statistics.

View larger version (18K): [in this window] [in a new window]   Figure 1. The effect of age on isoproterenol (ISO)-mediated vasorelaxation. Aortae from 2-, 6-, 12-, and 24-month (M)-old Fischer 344 rats (n = 10 animals per age) were constricted with 300 nM phenylephrine, and then relaxed with increasing doses of ISO (A) or forskolin (FSK) (B). Data are expressed as percentage of PE-induced contraction. C, Dose required to elicit 50% response (EC50) is plotted for ISO alone (open bars) and for vessels pretreated for 30 minutes with 1 µM of the beta1-adrenergic receptor (ß1-AR) antagonist metoprolol (METO) (filled bars). D, Maximal effect of ISO alone (open bars) and for vessels pretreated for 30 minutes with 1 µM METO (filled bars). Each point represents the mean response to ISO ± standard error. *p <.05 for shown comparison via two-way analysis of variance with Bonferroni's post hoc comparison. See Tables for complete statistical results. The significance symbol shown (*) corresponds to the same symbol in Table 1. Within specific legends, the agents that directly follow "+" were pretreatments, whereas agents that directly follow "" stimulated vasorelaxation

View larger version (17K): [in this window] [in a new window]   Figure 2. The effect of age on beta-adrenergic receptor (ß2-AR) desensitization. Aortae from 2-, 6-, 12-, and 24-month (M)-old Fischer 344 rats (n = 10 animals per age; A–D, respectively) were incubated with vehicle (open symbols) or 3.2 nM isoproterenol (ISO) (closed symbols) for 1 hour, with 1µM of the ß1-AR antagonist metoprolol present during the last 30 minutes. Following washout, 300 nM phenylephrine (PE) was used for constriction. After consistent force was generated, increasing doses of ISO was used for relaxation. Data are expressed as percentage of PE-induced contraction. E, Dose required to elicit 50% response (EC50) for ISO plotted for vessels that were pretreated with vehicle (open bars) and for vessels pretreated with ISO (filled bars). F, Maximal response for ISO plotted for vessels that were pretreated with vehicle (open bars) and for vessels pretreated with ISO (filled bars). Each point represents the mean response to ISO ± standard error. p <.05 for shown comparison via two-way analysis of variance with Bonferroni's post hoc comparison. See Table 1 for complete statistical results. The significance symbol shown () corresponds to the same symbol in Table 1. Within the legends, agents that directly follow "+" were pretreatments, whereas agents that directly follow "" stimulated vasorelaxation

View larger version (9K): [in this window] [in a new window]   Figure 3. The effect of relaxing agent on beta2-adrenergic receptor (ß2-AR) desensitization. Aortae from 2-, 6-, 12-, and 24-month (M)-old Fischer 344 rats (n = 10 animals per age) were incubated with vehicle (open bars) or 3.2 nM isoproterenol (ISO) (filled bars) for 1 hour, with 1 µM of the ß1-AR antagonist metoprolol present during the last 30 minutes. Following washout, 300 nM phenylephrine (PE) was used for constriction. After consistent force was generated, increasing doses of forskolin (FSK) was used for relaxation. A, Dose required to elicit 50% response (EC50) for FSK plotted for vessels that were pretreated with vehicle (open bars) and for vessels pretreated with ISO (filled bars). B, Maximal response for FSK plotted for vessels that were pretreated with vehicle (open bars) and for vessels pretreated with ISO (filled bars). Each point represents the mean response to FSK ± standard error. See Table 2 for complete statistical results. Within the legends, agents that directly follow "+" were pretreatments, whereas agents that directly follow "" stimulated vasorelaxation

Effect of Gi Inhibition on Age-Related Changes in ß₂-AR Desensitization

To determine the possible role of ß₂-AR–Gi linkage in age-related desensitization (while still maintaining the power provided by physiologic whole tissue bath analysis), we used the Gi inhibitor PTX [1 µg/mL, 2-hour preincubation, as described by Petitcolin and colleagues (19)] as listed in Methods (Protocol 4). As expected, PTX did not affect the contractility of PE, as PE activates the _1D-AR in aorta that link to Gq [see also Petitcolin and colleagues (19)]. A single dose of 300 nM PE constricted control aortae 4.5 ± 1.3 N/m² (average for all ages); this compared to 4.9 ± 1.6 N/m² (average for all ages) for PTX-pretreated vessels.

PTX reduced EC₅₀ for ISO in nondesensitized preparations. PTX treatment enhanced ISO-mediated vasorelaxation (Figure 4A; see open vs horizontally hatched bars) 1.8 ± 0.8-, 1.5 ± 0.6-, 1.2 ± 0.7-, and 1.1 ± 0.8-fold for 2-, 6-, 12-, and 24-month-old animals, respectively. This change was apparent in all age groups, but reached statistical significance (p <.05) for preparations from 2- and 6-month-old animals only. Similar to the data from maximum relaxation following (Figure 4B), in desensitized preparations, PTX did not alter the ISO EC₅₀ (Figure 4A; see filled vs diagonally hatched bars).

View larger version (19K): [in this window] [in a new window]   Figure 4. The effect of Gi inhibition on beta-adrenergic receptor (ß2-AR) desensitization. Aortae from 2-, 6-, 12-, and 24-month (M)-old Fischer 344 rats (n = 10 animals per age) were pretreated with 1 µg/mL pertussis toxin (PTX) or vehicle for 2 hours. During the last 30 minutes, 1 µM ß1-AR antagonist metoprolol was included. Subsequently, 3.2 nM isoproterenol (ISO) (filled bars) or vehicle (open bars) was added for 1 hour. Following washout, 300 nM phenylephrine (PE) was used for constriction. After consistent force was generated, increasing doses of ISO were used for relaxation. A, Dose required to elicit 50% response (EC50) for ISO. B, Maximal response for ISO. Each point represents the mean response to ISO ± standard error. p <.05 for indicated comparison via two-way analysis of variance with Bonferroni's post hoc comparison. In A, for preparations from 12- and 24-month-old animals comparing ISO, versus PTX+ISO (open vs horizontally hatched bars), statistical significance was not reached, but the EC50 values tended to be reduced; the respective p values were.059 and.061. See Table 1 for complete statistical results. The significance symbol shown () corresponds to the same symbol in Table 1, and highlights only the effect of pertussis toxin. The statistical comparison of ISO-mediated desensitization (open bars vs filled bars) is shown in Figure 2. Within the legends, agents that directly follow "+" were pretreatments, whereas agents that directly follow "" stimulated vasorelaxation

In total these results suggest that, in the aorta, a population of ß₂-AR link to Gi. However, there is no age-related change in the proportion of this linkage. We suggest the lack of PTX-mediated enhancement in desensitized preparations represents a biological effect, rather than an experimental failure (i.e., the PTX treatment did not function), as a PTX-specific affect was detected experimentally. Table 1 presents complete EC₅₀, maximum relaxation, and relevant statistics.

	DISCUSSION

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The purpose of the present study was to evaluate physiological aspects of age-related changes in ß-AR desensitization in aortic ring sections from Fischer 344 rats. The primary findings of these studies were that, with ISO pretreatment—and thus ß₂-AR desensitization—ß₂-AR-mediated vasorelaxation was impaired in aortae from young animals only. ISO pretreatment did not affect ß-AR-mediated vasorelaxation in aortae from old animals. We used PTX to block coupling of the ß₂-AR to Gi and found that this did not reverse the age-related change. However, there appears to be a population of ß₂-AR coupled to Gi, as PTX treatment improved ß₂-AR-mediated vasorelaxation in aortae isolated from animals of each age group studied.

Multiple investigators have documented physiological ß-AR desensitization in a variety of vascular tissues, although none have evaluated age-related changes. Hayes and colleagues (20) and Vleeming and colleagues (21) infused rats with ISO and evaluated aortic ISO-mediated vasorelaxation with a tissue bath system. They found significant declines in ß-AR-mediated vasorelaxation in aorta isolated from ISO-infused rats. Fleisch and Titus (22) used a protocol similar to that of the present study and also found that in vitro incubation of aortic rings with ISO was associated with reduced vasorelaxation to a subsequent ß-AR activation. Both of these studies used tissue from rats that were younger than 6 months of age, and our results are in agreement with these studies; we also provide data using tissue from older animals. Martin and Broadley (23) did not detect agonist-induced ß-AR desensitization in in vitro preparations of pulmonary artery or aorta from rats younger than 6-months of age. This difference may be because their chosen time (6 hours vs 1 hour) of agonist incubation was significantly longer, their desensitizing dose (10 µM vs 320 nM) was significantly higher, or the fact that they did not include any ß-AR antagonists in their preparations.

Others have studied ß-AR desensitization with advancing age, although not with physiological endpoints. Scarpace and Abrass (5) evaluated heart membrane preparations from rats that were orally treated with either saline or the relatively ß₂-AR selective agonist metaproterenol. Their primary finding was that, in preparations from control animals, the quantity of ß-AR in membrane preparations remained the same across age groups. However, the percentage of ß-AR in the high-affinity state declined nearly 2-fold with advancing age. Metaproterenol treatment caused roughly a 40% reduction of membrane ß-AR in all age groups. Interestingly, metaproterenol treatment reduced the percentage of ß-AR in the high-affinity state only in preparations from young animals. These results were interpreted to mean that the ß-AR population in aorta from aged animals were already desensitized, and thus could not be further affected.

Conformation of this interpretation was provided by adenylyl cyclase activity results where, in control animals, activity was reduced with age. With metaproterenol treatment, a further reduction was apparent in both age groups; however, there was no age-to-age difference.

In aorta, similar age-related results were found by Gurdal and colleagues (6), using tissue from 1-, 6-, and 24-month-old Fischer 344 rats. Their strategy was to examine competition binding studies between ¹²⁵I-cyanopindolol and ISO, in the presence or absence of guanosine-5'-(ß-imido)triphosphate (GppNHp). GppNHp is a nonhydrolyzable GTP analogue that irreversibly activates Gs. This activation uncouples Gs from the ß-AR and produces low-affinity ß-ARs. Therefore, the proportion of ß-ARs in the high-affinity state can be determined by examining the age-related changes in the effect of GppNHp on ¹²⁵I-cyanopindolol binding. Results showed that, in aortic preparations from 1-month-old animals, 64% of the ß-ARs were in the high-affinity state. This result compared to 40% and 0% high-affinity ß-ARs for 6- and 24-month-old animals, respectively. The present findings agree with those of the previous studies, and add physiological validation. Taken together, our interpretation of the present results, in combination with those of other studies, is that in cardiovascular tissue from old animals, there is a change that appears as ß₂-AR desensitization. Therefore, with exposure to agonist, either in vivo or in vitro, the ß₂-AR cannot be further desensitized. This change is not ß₂-AR downregulation. Gurdal and colleagues (6), Gaballa and colleagues (24), and Mader (25) have all shown that, in aorta, ß-AR density remains constant with advancing age.

It has been suggested that the apparent age-related change in ß-AR sensitivity may be related to an age-related increase in catecholamines (26), and the decline in function is due to a normal ß-AR response to this milieu. Studies are equivocal regarding age-related changes in circulating catecholamine concentrations between young and old Fischer 344 rats; some reports suggest an increase (27), whereas others report no change (28,29). However, the literature does support an age-related increase in the concentration of circulating catecholamines in humans (30). Regardless, the local, rather than circulating, tissue concentration of catecholamines may be more relevant for receptor desensitization, and to our knowledge there are no reports about this. Nonetheless, vascular tissue isolated from rats or humans that is evaluated in vitro within catecholamine-free buffer shows an age-related decline in ß-AR-mediated vasorelaxation and cAMP production [see Figure 1, and Scarpace and colleagues (31)]. Therefore, it appears that the age-related change in ß-AR sensitivity may not specifically be caused by a change in chronic catecholamine exposure.

One initiator of ß₂-AR desensitization is agonist-stimulated activation and PKA-mediated phosphorylation of the ß₂-AR itself. In the heart (32), it has been well documented that the PKA phosphorylated ß₂-AR preferentially couples to Gi rather than to Gs (33), thus ß₂-AR-mediated cAMP accumulation is reduced, rather than enhanced (10). Our results are the first to suggest this process could occur in the vasculature, but may not be as pronounced. In the present study we acutely treated aortae in vitro with PTX, and we suggest there is a population of ß₂-AR linked to Gi as PTX treatment did cause a slight enhancement in vasorelaxation in all ages. However, PTX treatment did not reverse ISO-induced ß₂-AR desensitization (Figure 4). This finding is possibly in contrast to those in a study by Chin and colleagues (34) that chronically infused PTX in vivo and found no effect on the age-related change in ß-AR-mediated vasorelaxation in vitro. Our data suggest that the ß-AR impairment seen with advancing age is not likely PKA related. Presumably, if PKA-mediated desensitization and Gs/Gi switching caused the age-related decline in ß-AR-mediated vasorelaxation, then Gi inhibition would block the agonist-induced desensitization observed in 2- and 6-month-old animals.

With advancing age, ß-AR-mediated signaling (and thus cAMP production) is reduced (1). This decrease is related to a fundamental change at the receptor, or G protein, as adenylyl cyclase-mediated vasorelaxation (and cAMP production) remains intact in the aging mammalian vasculature (15). Studies to understand the biochemical/molecular basis of this apparent ß-AR/G protein uncoupling show little, if any age-related change in abundance of the ß-AR (35), Gs (12,13,36), adenylyl cyclase (37), or PKA (11). There appears to be a fundamental change in Gs that is not related to a change in expression, as Mader and colleagues (12) found a marked age-related decline in cholera toxin labeling of aortic Gs that was interpreted as a marker for age-related loss of Gs function. Changes in the expression of Gi are conflicting. Mader and colleagues (12) found a slight decrease in PTX labeling, but no change in Gi_1/2 or Gi₃ protein expression in aorta from aged Fisher 344 rats. This finding may be contrary to those in a study by Kilts and colleagues (14) where age-related Gi expression and activity were increased in a different tissue bed, the left ventricle, of the same rat strain. Overall, the age-related changes in ß-AR function suggest a desensitization mechanism, rather that a change in expression.

There are difficulties inherent in studies (present study included) that evaluate desensitization of G protein–coupled receptors (38). Most troublesome is the inability to directly determine the phosphorylation state of the ß-AR. To our knowledge, no accurate and reproducible technique is available to directly determine the phosphorylation state of the ß₂-AR in native rat tissue. Some phospho-ß-AR antibodies are available (39). However, these were produced against the epitopes of the human ß₂-AR, and there are sequence variations in the rat ß₂-AR. We have made multiple attempts under various conditions to use some of these antibodies in native tissue and have not yet been successful (data not shown).

Interpretation of the present results are hampered due to the overall complexity of the ß-AR signaling cascade. The present study evaluates only a focused number of proteins, and age-related changes in others may contribute to our results. For instance, it is now known that multiple kinases phosphorylate the ß₂-AR. Aside from PKA, G protein–coupled receptor kinases (GRKs) also desensitize the ß₂-AR in conjunction with ß-arrestin (40), which further associates the ß₂-AR with other regulatory elements such as Src, and ERK (41). Previous findings from this laboratory (42) and others (24) found age-related increases in multiple GRKs in aortic tissue. In addition, GRKs (43) and ß-AR (44) appear to be regulated by the scaffolding protein caveolin. Our previous findings suggest that the age-related decline in ß-AR-mediated vasorelaxation may be caused by changes in caveolin-1 that could lead to an age-related increase in ß-AR desensitization (45).

The decline in ß-AR-mediated vasorelaxation with preserved -AR-mediated vasoconstriction (15) may yield a chronic vasoconstricted state that could be associated with both hypertension and orthostatic hypotension (46). Also, depleted intracellular cAMP accumulation is associated with enhanced vascular smooth muscle proliferation that may promote atherosclerosis (47). Therefore, these biochemical changes are potentially of clinical significance in many aging patients (48). The results presented add detail to the possible mechanism(s) of the decline in ß-AR-mediated vasorelaxation with advancing age. First, we show that desensitization only occurs in vessels isolated from young animals. This finding suggests that ß₂-AR present in aorta from old animals may be maximally desensitized, which would explain impaired vasorelaxation. Second, our results further clarify our previous findings (42) that the age-related change is more than likely due to the action of GRK, rather than PKA. If the desensitization was caused by PKA-mediated ß-AR phosphorylation, PTX should have partially reversed the impaired vasorelaxation in the agonist-induced desensitized vessels. Further studies that directly evaluate the phosphorylation state of the ß-AR are necessary.

	Acknowledgments

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This work was supported by the Research Service, Department of Veterans Affairs and the Northwest Health Foundation (SLM).

We thank the laboratory of Sharon Anderson, MD (Oregon Health & Science University, Division of Nephrology and Hypertension), including Terry T. Oyama and Jessie Lindsley, for their technical expertise and editorial comments.

	Footnotes

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Decision Editor: James R. Smith, PhD

Received December 8, 2005

Accepted February 27, 2006

	References

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1. Schutzer WE, Mader SL. Age-related changes in adrenergic signaling: clinical and mechanistic implications. Ageing Res Rev. 2003;2:169-190.[Medline]
2. O'Donnell S, Wanstall J. Beta-1 and beta-2 adrenoceptor-mediated responses in preparations of pulmonary artery and aorta from young and aged rats. J Pharmacol Exp Ther. 1984;228:733-738.[Abstract/Free Full Text]
3. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature. 1994;372:231-236.[Medline]
4. Roth N, Campbell P, Caron M, Lefkowitz R, Lohse M. Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1991;88:6201-6204.[Abstract/Free Full Text]
5. Scarpace PJ, Abrass IB. Beta-adrenergic agonist-mediated desensitization in senescent rats. Mech Ageing Dev. 1986;35:255-264.[Medline]
6. Gurdal H, Friedman E, Johnson M. ß-adrenoceptor-Gs coupling decreases with age in rat aorta. Mol Pharmacol. 1995;47:772-778.[Abstract]
7. Daaka Y, Luttrell DK, Lefkowitz RJ. Switching of the coupling of the ß2-adrenergic receptor to different G proteins by protein kinase A. Nature. 1997;390:88-91.[Medline]
8. Xiao RP. Beta-adrenergic signaling in the heart: dual coupling of the beta2-adrenergic receptor to G(s) and G(i) proteins. Sci STKE. 2001;2001:RE15.[Medline]
9. Lefkowitz RJ, Pierce KL, Luttrell LM. Dancing with different partners: protein kinase A phosphorylation of seven membrane-spanning receptors regulates their G protein-coupling specificity. Mol Pharmacol. 2002;62:971-974.[Free Full Text]
10. Tepe NM, Liggett SB. Functional receptor coupling to Gi is a mechanism of agonist-promoted desensitization of the ß2-adrenergic receptor. J Recept Signal Transduct Res. 2000;20:75-85.[Medline]
11. Deisher TA, Mankani S, Hoffman BB. Role of cyclic AMP-dependent protein kinase in the diminished beta-adrenergic responsiveness of vascular smooth muscle with increasing age. J Pharmacol Exp Ther. 1989;249:812-819.[Abstract/Free Full Text]
12. Mader SL, Downing CL, Amos-Landgraf J, Swebjka P. Age-related changes in G proteins in rat aorta. J Gerontol Biol Sci. 1996;51A:B111-B116.[Abstract]
13. Johnson MD, Zhou Y, Friedman E, Roberts J. Expression of G-protein-alpha subunits in the aging cardiovascular system. J Gerontol Biol Sci. 1995;50A:B14-B19.[Abstract]
14. Kilts JD, Akazawa T, Richardson MD, Kwatra MM. Age increases cardiac Gi₂ expression, resulting in enhanced coupling to G protein-coupled receptors. J Biol Chem. 2002;277:31257-31262.[Abstract/Free Full Text]
15. Chapman J, Schutzer WE, Watts VJ, Mader SL. Impaired cholera toxin relaxation with age in rat aorta. J Gerontol Biol Sci. 1999;54A:B154-B159.[Abstract]
16. Fujii K, Onaka U, Goto K, Abe I, Fujishima M. Impaired isoproterenol-induced hyperpolarization in isolated mesenteric arteries of aged rats. Hypertension. 1999;34:222-228.[Abstract/Free Full Text]
17. Miller VM, Flavahan NA, Vanhoutte PM. Pertussis toxin reduces endothelium-dependent and independent responses to alpha-2- adrenergic stimulation in systemic canine arteries and veins. J Pharmacol Exp Ther. 1991;257:290-293.[Abstract/Free Full Text]
18. Sato K, Komaru T, Shioiri H, et al. Vasodilator signals from the ischemic myocardium are transduced to the coronary vascular wall by pertussis toxin-sensitive G proteins: a new experimental method for the analysis of the interaction between the myocardium and coronary vessels. J Am Coll Cardiol. 2002;39:1859-1865.[Abstract/Free Full Text]
19. Petitcolin MA, Vandeputte C, Spitzbarth-Regrigny E, Bueb JL, Capdeville-Atkinson C, Tschirhart E. Lack of involvement of pertussis toxin-sensitive G-proteins in norepinephrine-induced vasoconstriction of rat aorta smooth muscle. Biochem Pharmacol. 2001;61:485-491.[Medline]
20. Hayes JS, Wyss VL, Schenck KS, Cohen ML. Effects of prolonged isoproterenol infusion on cardiac and vascular responses to adrenoceptor agonists. J Pharmacol Exp Ther. 1986;237:757-763.[Abstract/Free Full Text]
21. Vleeming W, van Rooij HH, Wemer J, Porsius AJ. Modulation of adrenoceptor-mediated cardiovascular effects by short-term in vivo infusion of isoproterenol in rats. J Cardiovasc Pharmacol. 1990;16:584-593.[Medline]
22. Fleisch JH, Titus E. The prevention of isoproterenol desensitization and isoproterenol reversal. J Pharmacol Exp Ther. 1972;181:425-433.[Abstract/Free Full Text]
23. Martin SW, Broadley KJ. Relative resistance of functional beta2-adrenoceptor-mediated smooth muscle responses to in vitro desensitization. Can J Physiol Pharmacol. 1999;77:156-165.[Medline]
24. Gaballa MA, Eckhart AD, Koch WJ, Goldman S. Vascular ß-adrenergic receptor adenylyl cyclase system in maturation and aging. J Mol Cell Cardiol. 2000;32:1745-1755.[Medline]
25. Mader SL. Influence of animal age on the beta-adrenergic system in cultured rat aortic and mesenteric artery smooth muscle cells. J Gerontol Biol Sci. 1992;42A:B32-B36.
26. Lakatta EG. Catecholamines and cardiovascular function in aging. Endocrinol Metab Clin North Am. 1987;16:877-891.[Medline]
27. Cizza G, Pacak K, Kvetnansky R, et al. Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 344/N Fischer rats. J Clin Invest. 1995;95:1217-1224.[Medline]
28. McCarty R. Effects of 2-deoxyglucose on plasma catecholamines in adult and aged rats. Neurobiol Aging. 1984;5:285-289.[Medline]
29. McCarty R. Sympathetic-adrenal medullary and cardiovascular responses to acute cold stress in adult and aged rats. J Auton Nerv Syst. 1985;12:15-22.[Medline]
30. Petrie EC, Peskind ER, Dobie DJ, Veith RC, Raskind MA. Increased plasma norepinephrine response to yohimbine in elderly men. J Gerontol Med Sci. 2000;55A:M155-M159.[Abstract/Free Full Text]
31. Scarpace PJ, Tumer N, Mader SL. ß-adrenergic function in aging. Basic mechanisms and clinical implications. Drugs Aging. 1991;1:116-129.[Medline]
32. Xiao RP, Ji X, Lakatta EG. Functional coupling of the beta 2-adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol. 1995;47:322-329.[Abstract]
33. Zamah AM, Delahunty M, Luttrell LM, Lefkowitz RJ. Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system. J Biol Chem. 2002;277:31249-31256.[Abstract/Free Full Text]
34. Chin JH, Hiremath AN. Hoffman BB. cAMP signaling mechanisms with aging in rats. Mech Ageing Dev. 1996;86:11-26.[Medline]
35. Tsujimoto G, Lee CH, Hoffman BB. Age-related decrease in beta adrenergic receptor-mediated vascular smooth muscle relaxation. J Pharmacol Exp Ther. 1986;239:411-415.[Abstract/Free Full Text]
36. Schutzer WE, Watts VJ, Chapman J, et al. Viral-mediated gene delivery of constitutively activated Gs alters vasoreactivity. Clin Exp Pharmacol Physiol. 2000;27:9-13.[Medline]
37. Mader SL, Alley PA. Age-related changes in adenylyl cyclase activity in rat aorta membranes. Mech Ageing Dev. 1998;101:111-118.[Medline]
38. Clark RB, Rich TC. Probing the roles of protein kinases in G-protein-coupled receptor desensitization. Mol Pharmacol. 2003;64:1015-1017.[Free Full Text]
39. Tran TM, Friedman J, Qunaibi E, Baameur F, Moore RH, Clark RB. Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the ß₂-adrenergic receptor using phosphoserine-specific antibodies. Mol Pharmacol. 2004;65:196-206.[Abstract/Free Full Text]
40. Seibold A, Williams B, Huang ZF, et al. Localization of the sites mediating desensitization of the beta 2-adrenergic receptor by the GRK pathway. Mol Pharmacol. 2000;58:1162-1173.[Abstract/Free Full Text]
41. Lefkowitz RJ, Shenoy SK. Transduction of receptor signals by ß-arrestins. Science. 2005;308:512-517.[Abstract/Free Full Text]
42. Schutzer WE, Reed JF, Bliziotes M, Mader SL. Upregulation of G protein-linked receptor kinases with advancing age in rat aorta. Am J Physiol. 2001;280:R897-R903.
43. Carman CV, Lisanti MP, Benovic JL. Regulation of G-protein receptor kinases by caveolin. J Biol Chem. 1999;274:8858-8864.[Abstract/Free Full Text]
44. Schwencke C, Okumura S, Yamamoto M, Geng YJ, Ishikawa Y. Colocalization of ß-adrenergic receptors and caveolin within the plasma membrane. J Cell Biochem. 1999;75:64-72.[Medline]
45. Schutzer WE, Reed JF, Mader SL. Decline in caveolin-1 expression and scaffolding of G protein receptor kinase-2 with age in Fischer 344 aortic vascular smooth muscle. Am J Physiol Heart Circ Physiol. 2005;288:H2457-H2464.[Abstract/Free Full Text]
46. Streeten DH, Anderson GH, Jr. Mechanisms of orthostatic hypotension and tachycardia in patients with pheochromocytoma. Am J Hypertens. 1996;9:760-769.[Medline]
47. Smirnov VN, Voyno-Yasenetskaya TA, Antonov AS, et al. Vascular signal transduction and atherosclerosis. Ann N Y Acad Sci. 1990;598:167-181.[Medline]
48. Tumer N, Scarpace PJ, Lowenthal DT. Geriatric pharmacology: basic and clinical considerations. Ann Rev Pharmacol Toxicol. 1992;32:271-302.[Medline]

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