Albuterol-induced downregulation of Gsα accounts for pulmonary β₂-adrenoceptor desensitization in vivo

The aim of the present study was to develop a chronic in vivo model of pulmonary β₂-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E₂, but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. β₂-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsα expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin–induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein–coupled receptor kinase-2, but the extent to which these events contributed to β₂-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsα representing a primary molecular etiology.

β₂-Adrenoceptor agonists are the most effective bronchodilators currently available and exhibit efficacy irrespective of the mediator(s) evoking bronchospasm. However, there has been concern that regular use of this group of drugs may render susceptible individuals tolerant to their beneficial effects in asthma. Lipworth and colleagues (1, 2) have reported that 4 weeks of treatment of asthmatic subjects with inhaled formoterol produced tachyphylaxis to the bronchodilator effects of these drugs. In addition, the results of several studies with terbutaline and albuterol have demonstrated a loss of protection against various bronchoconstrictor challenges (3, 4). This effect renders the airways twice as sensitive to allergen and exercise and increases the late-phase asthmatic response and associated inflammation (5) and may be relevant to the reduced asthma control seen with the regular use of high doses of inhaled β₂-adrenoceptor agonists (6, 7). The cause(s) of this effect is unclear. It has been reported that airways resected from asthmatic patients fail to relax normally to isoproterenol, supporting a possible defect in β-adrenoceptor function (8–10), although it is unknown whether this is the result of treatment or a consequence of the disease process itself. Nevertheless, downregulation of β-adrenoceptor number in lung and airways smooth muscle has been reported in animals given isoproterenol and norepinephrine chronically by infusion (11–13) and is associated with a reduced functional responsiveness toward β-adrenoceptor agonists ex vivo (12, 13). Thus, it is possible that the loss of protection against various bronchoconstrictor challenges in humans is due, at least in part, to pulmonary β₂-adrenoceptor desensitization.

Two major molecular mechanisms have been elucidated in isolated cells that result in β₂-adrenoceptor desensitization. One of these promotes short-term homologous refractoriness and involves the uncoupling of the agonist-occupied form of the receptor from the stimulatory guanine nucleotide binding protein, Gs, by mechanisms that require phosphorylation of serine and threonine residues at the COOH-terminus of the receptor (14, 15). This reaction can be catalyzed by at least three members of the G protein–coupled receptor (GPCR) kinase (GRK) superfamily (GRK-2, GRK3, GRK5) (14, 15). The subsequent binding of β-arrestin, a soluble protein that prevents further coupling to Gs (16), then halts signaling through the receptor. The β₂-adrenoceptor is similarly desensitized by cyclic adenosine monophosphate–dependent (cAMP-dependent) protein kinase (PKA) following phosphorylation of serine and threonine residues present within the third intracellular loop of the protein in response to an increase in intracellular cAMP (17). The other established process, which promotes prolonged periods of desensitization, involves the physical internalization and subsequent degradation of receptors due to an inhibition of transcription and/or increased post-transcriptional processing of β₂-adrenoceptor mRNA (16, 17). Although the aforementioned processes are believed to account for desensitization of many GPCRs, other mechanisms have also been described, including induction of cAMP phosphodiesterases (PDE) (18) and downregulation of Gsα (19), although the functional significance of these effects has not been rigorously explored.

Although desensitization of GPCRs has been studied extensively, most of the information to date has been gathered from cultured cells, and the extent to which this applies to the in vivo situation is little investigated. Thus, the aim of the present study was to develop an in vivo model of pulmonary β₂-adrenoceptor desensitization and investigate the nature and molecular basis of this phenomenon.

Effect of chronic treatment of rats with albuterol on the ability of albuterol, PGE₂, forskolin, and IBMX to protect against ACh-induced bronchoconstriction. Intravenous administration of albuterol (100 μg/kg) or PGE₂ (300 μg/kg) to saline-treated rats significantly reduced (by 52% and 47%, respectively) the magnitude of ACh-induced bronchoconstriction, whereas in the albuterol-treated group of animals, no significant protection was observed with either agonist (Figure 2, a and b), indicating that the desensitization was heterologous. In contrast, forskolin (300 μg/kg intravenously) and IBMX (300 μg/kg intravenously) inhibited, equally, ACh-induced bronchoconstriction in both groups of rats (Figure 2, c and d), suggesting that the intracellular site(s) of desensitization was upstream of adenylyl cyclase. In none of the experiments did albuterol, PGE₂, forskolin, or IBMX affect resting bronchial tone.

Figure 2

Effect of chronic treatment of rats with albuterol (a–d) or PGE₂ (e and f) on lung function in anesthetized rats. Animals were given albuterol, PGE₂ (open bars; both 40 μg/kg/h) or vehicle (filled bars) for 7 days and then instrumented for the measurement of lung function. ACh (500 μg/kg intravenously) was administered and the maximum increase in overflow pressure was measured. When baseline lung function was reestablished, albuterol (Alb; 100 μg/kg; a and f), PGE₂ (PG; 300 μg/kg; b and e), forskolin (F; 300 μg/kg; c), or IBMX (300 μg/kg; d) was given intravenously, and 5 minutes later, ACh was administered again and any change in overflow pressure was noted. Each bar represents the mean ± SEM of four independent determinations. ^AP < 0.05, significant protection of ACh-induced bronchoconstriction.

Effect of chronic treatment of rats with PGE₂ on the ability of PGE₂ and albuterol to protect against ACh-induced bronchoconstriction. Figure 2 shows the results of experiments designed to determine whether chronic treatment of rats with PGE₂, an agonist at Gs-coupled receptors of the EP₂- and EP₄-subtype, also promoted heterologous desensitization. Intravenous administration of PGE₂ (300 μg/kg) to saline-treated animals significantly reduced (by 48%) the magnitude of ACh-induced bronchoconstriction, whereas in PGE₂-treated rats, no significant protection was observed (Figure 2e). Identical results were obtained when the bronchoprotective effect of albuterol (100 μg/kg intravenously) was examined in saline- and PGE₂-treated animals (Figure 2f).

Effect of chronic treatment of rats with albuterol on β-adrenoceptor density in lung. Chronic treatment of rats with albuterol (40 μg/kg/h) produced a significant reduction in β-adrenoceptor density when compared with naive animals, without affecting the affinity of the nonselective ligand, [¹²⁵I]ICYP (Table 1). This effect was selective for the β₂-adrenoceptor subtype and resulted in a 30% reduction in B_max, which lowered the β₂/β₁ subtype ratio from 2:1 in saline-treated rats to 1.45:1 in animals given albuterol (Table 1).

Table 1

Effect of chronic treatment of rats with albuterol on the density of β-adrenoceptors on rat lung membranes and on the affinity of [¹²⁵I]ICYP

Effect of chronic treatment of rats with albuterol on the ability of albuterol and PGE₂ to increase the cAMP content and activate PKA ex vivo. To determine whether downregulation of β₂-adrenoceptor number was accompanied by compromised signal transduction, the ability of albuterol to increase cAMP and activate PKA in lung parenchyma was assessed ex vivo. Lung taken from saline-treated animals responded to albuterol with a robust increase in the cAMP content (EC₅₀ = 4.1 ± 1.7 μM). This effect was concentration-dependent and resulted, maximally, in a sixfold increase in cAMP over the baseline level (Figure 3a). In contrast, albuterol failed to elevate the cAMP content in lung excised from rats given albuterol chronically for 7 days at any concentration examined (Figure 3a). No significant difference in basal cAMP content in lung excised from saline- and albuterol-treated rats was found. Consistent with the cAMP results already described here, the resting PKA activity ratio in lung taken from saline- and albuterol-treated rats was not significantly different (Figure 4a). However, the ability of albuterol to activate PKA in lung excised from rats desensitized to the bronchoprotective effect of albuterol was markedly impaired when compared with lung taken from saline-treated animals (Figure 4a). Albuterol-induced cAMP accumulation was also blunted in tracheae taken from albuterol-treated rats (data not shown), indicating that β₂-adrenoceptors were desensitized in the relevant tissue. No difference in the basal cAMP content was detected.

Figure 3

Effect of chronic treatment of rats with albuterol on albuterol-induced cAMP accumulation ex vivo. Rats were given saline (filled circles) or albuterol (open circles; 40 μg/kg/h) for 7 days, and the ability of albuterol to increase the cAMP content in lung was determined in the absence (a) or presence (b) of IBMX (100 μM). Each data point represents the mean ± SEM of eight determinations. Basal cAMP levels were 81 ± 75 and 57 ± 36 pmol/mg protein in saline- and albuterol-treated rats, respectively, in the absence of IBMX, and 803 ± 57 and 891 ± 78 pmol/mg protein in saline- and albuterol-treated rats, respectively, in the presence of IBMX. ^AP < 0.05, significant increase in cAMP formation induced by isoproterenol in IBMX-treated tissue.

Figure 4

Effect of chronic treatment of rats with albuterol on β₂-adrenoceptor signaling in lung ex vivo. Rats were given saline (filled bars) or albuterol (open bars; 40 μg/kg/h) for 7 days, and albuterol-induced PKA activation (a) and PGE₂-induced cAMP accumulation (b) in lung were determined. Each bar represents the mean ± SEM of eight determinations. ^AP < 0.05, cAMP accumulation and PKA activation significantly attenuated in lung from albuterol-treated rats.

Consistent with the in vivo results, the desensitization of β-adrenoceptor–mediated cAMP accumulation produced in rats treated chronically with albuterol was heterologous in that the ability of PGE₂ to elevate the cAMP content in lung also was profoundly impaired (Figure 4b).

Effect of chronic treatment of rats with albuterol on the ability of CTX to increase the cAMP content in lung ex vivo. A pharmacologic approach was adopted to determine whether pulmonary β₂-adrenoceptor desensitization and compromised cAMP formation and PKA activation in lung were associated with any change in the ability of Gs to couple to adenylyl cyclase. This was achieved by making use of CTX, which directly activates Gs by catalyzing the NAD⁺-dependent ADP-ribosylation of the α-subunit. As shown in Figure 5, CTX elicited concentration-dependent cAMP accumulation in lung taken from saline-treated animals with an EC₅₀ of 1.83 ± 0.17 μg/mL. CTX also increased cAMP in lung from desensitized rats, but the concentration-response curve was significantly (P < 0.05; ANCOVA) displaced 5.9-fold to the right (EC₅₀ = 10.8 ± 4.4 μg/mL) in an apparently parallel fashion without a reduction in the maximum response.

Figure 5

Effect of chronic treatment of rats with albuterol on the ability of CTX to promote cAMP formation in lung ex vivo. Rats were given saline (open circles) or albuterol (filled circles; 40 μg/kg/h) for 7 days, and CTX-induced cAMP accumulation in lung was determined. Each point represents the mean ± SEM of six determinations. Basal cAMP levels were 108 ± 28 and 84 ± 21 pmol/mg protein in saline- and albuterol-treated rats, respectively.

Effect of chronic treatment of rats with albuterol on the expression of Gsα in lung and trachea. Western blotting was performed to determine whether the compromised ability of CTX to elevate the cAMP content in lung taken from albuterol-treated rats was associated with a reduction in the expression of Gsα. A primary antibody, raised against the α-subunit of Gs, identified three bands in membranes prepared from the lung of saline-treated rats that migrated as 42-, 44-, and 52-kDa peptides on SDS polyacrylamide gels. Treatment of rats for 7 days with albuterol significantly reduced the intensity of each of these bands by 40–60% relative to the “housekeeping” protein, actin (Figure 6a). Identical results were obtained with tracheae (data not shown). A primary antibody raised against the common Gβ-subunit, recognized a single 35-kDa band in lung and tracheal membranes prepared from saline-treated rats. However, relative to actin, Gβ was not altered in tissue taken from albuterol-treated animals (data not shown). Neither α nor β subunits of Gs were detected in the cytosolic fraction of tissue taken from either group of animals at 7 days.

Figure 6

Effect of chronic treatment of rats with albuterol on Gsα expression in lung membranes. Rats were given saline (filled bars) or albuterol (gray bars; 40 μg/kg/h) for 7 days, and the expression of membrane-associated Gsα was determined. (a) Representative Western blot and bar charts of the mean (± SEM) results of nine determinations. (b) Representative digitized phosphorimage of the amount of Gsα assessed by CTX-catalyzed NAD⁺-dependent ADP ribosylation. Equal loading was confirmed by staining of the gels with Coomassie blue. Sal, animals treated with saline; Alb, animals treated with albuterol. ^AP < 0.05, significant reduction in Gsα expression.

Pretreatment of lung membranes with CTX resulted in the [α³²P]NAD⁺-dependent ADP ribosylation of a single broad 44-kDa band of Gsα (probably reflecting poor resolution of radiolabeled CTX substrates) that corresponded to the predominant species detected by Western blotting. However, a significant (P < 0.05) loss of CTX substrate (51.3 ± 12.6%; n = 6) was found in membranes taken from albuterol-treated rats relative to their saline-treated counterparts (Figure 6b).

Effect of chronic treatment of rats with albuterol on cAMP PDE activity in lung. In many cells, agonist-induced desensitization of receptors that couple through Gs is attributable, in part, to upregulation of cAMP PDE (18, 22). To determine the extent to which this effect accounted for the desensitization of β₂-adrenoceptor–mediated cAMP accumulation reported in the current study, the ability of albuterol to elevate cAMP in lung was assessed ex vivo in the absence and presence of the PDE inhibitor, IBMX. As shown in Figure 3, albuterol-induced cAMP accumulation in lung excised from saline-treated rats was markedly potentiated (approximately sixfold) by IBMX (100 μM) with no change in potency (EC₅₀ = 1.1 ± 0.3 μM). Significantly, in lung excised from albuterol-treated rats, IBMX conferred sensitivity to albuterol ex vivo. Thus, the cAMP content increased in a concentration-dependent manner with an EC₅₀ of 1.3 ± 0.4 μM and a maximum response equivalent to that achieved by albuterol in naive tissue in the absence of IBMX (compare Figure 3, a with b).

cAMP PDE activity was significantly elevated (1.5-fold) in lung tissue harvested from albuterol-treated rats when compared with animals that received saline [(B + C + D)/(A + D) in Figure 7]. Rolipram (30 μM) markedly attenuated cAMP hydrolysis in lung excised from both groups of animals by approximately 50%, and quantification of this effect (B/A in Figure 7a) demonstrated a 1.43 increase in PDE4 activity. However, a residual activity remained in albuterol-treated lung in the presence of rolipram (C + D in Figure 7a) that was consistently greater than that seen in control tissue (D in Figure 7a), indicating that a rolipram-insensitive isoenzyme was also upregulated [(C + D)/D in Figure 7a]. This activity was attributable solely to PDE3. Indeed, the PDE3 inhibitor, Org 9935 (30 μM) suppressed cAMP hydrolysis in lung excised from saline- and albuterol-treated rats by 56% and 57%, respectively (A and B in Figure 7b), which equated to a 1.5-fold increase in activity (B/A in Figure 7b). Furthermore, when rolipram and Org 9935 were used in combination, the remaining PDE activity (D in Figure 7c) was the same in lung taken from both groups of animals.

Figure 7

Effect of chronic treatment of rats with albuterol on cAMP PDE activity in lung. The ability of rolipram (30 μM; a), Org 9935 (30 μM; b), and a combination of rolipram and Org 9935 (c) to suppress cAMP hydrolysis was assessed in lung taken from rats treated chronically with saline and albuterol. Open and filled bars show PDE activity in the absence and presence of PDE inhibitor, respectively. In each panel, B/A corresponds to the fold-increase in activity attributable to the PDE isoenzyme(s) defined with rolipram, Org 9935, or rolipram/Org 9935. Total induction is given by (B + C + D)/(A + D). Data represent the mean ± SEM of eight determinations. ^EP < 0.05, significant inhibition of PDE activity.

Effect of chronic treatment of rats with albuterol on GRK activity and GRK-2 expression in lung. Chronic infusion of rats with albuterol produced a significant (56%) increase in cytosolic GRK activity in lung parenchyma when compared with saline-treated animals (Figure 8a). This effect was associated with a 1.95-fold increase in the expression of GRK-2 as assessed by Western blotting using an antibody directed against the COOH-terminus of the protein (Figure 8, b and c). In contrast, no change in GRK activity or GRK-2 expression was detected in the particulate fraction of albuterol-treated rats at 7 days (data not shown).

Figure 8

Effect of chronic treatment of rats with albuterol on GRK activity and GRK2 expression in lung. Rats were given saline or albuterol (40 μg/kg/h) for 7 days, and GRK activity and the expression of GRK2 protein in lung were determined by measuring the phosphorylation of rhodopsin (a) and by Western analysis (b and c), respectively. Each bar represents the mean ± SEM of 12 determinations. ^AP < 0.05, significant increase in GRK2 activity and expression.

Intravenous administration of ACh to saline- and albuterol-treated rats provoked bronchoconstriction that was submaximal, highly reproducible with repeated challenges, and of equivalent magnitude. Given that chronic treatment of rats with albuterol resulted in profound pulmonary β₂-adrenoceptor desensitization, these data suggest that neither sympathetic tone nor endogenous catecholamines regulate baseline airway caliber, which is consistent with the sparse innervation of airway smooth muscle with catecholamine-containing varicosities in this species (28, 29).

An in vivo model of pulmonary β₂-adrenoceptor desensitization was established in rats by administering albuterol continuously to mimic the effect of regular therapy of asthmatic subjects with β₂-adrenoceptor agonists. In these animals, the ability of albuterol, given acutely, to protect against ACh-induced bronchoconstriction was abolished, indicating that functional desensitization had occurred. Although desensitization of β₂-adrenoceptor–mediated relaxation has been demonstrated ex vivo in tracheae and lung taken from rats treated chronically with isoproterenol and norepinephrine (12, 30), this is one of the first reports to our knowledge to demonstrate this phenomenon in vivo.

Albuterol-induced desensitization was studied in more detail by examining the effect of PGE₂, which relaxes rat airways by stimulating adenylyl cyclase through EP₄-like prostanoid receptors (31). The demonstration that the antispasmogenic activity of PGE₂ was also impaired indicates that the desensitization was heterologous. Furthermore, rats rendered tolerant to PGE₂ were also insensitive to albuterol, implying that a common mechanism(s) may account for β₂-adrenoceptor- and EP₄-receptor–mediated cross-desensitization. Although few reports document heterologous desensitization of GPCRs in vivo, the data reported here are consistent with results of Zeiders et al. (32), who demonstrated that chronic treatment of rats with isoproterenol attenuated isoproterenol- and glucagon-stimulated adenylyl cyclase activity in cardiac membranes ex vivo.

A clue to the mechanism of pulmonary β₂-adrenoceptor desensitization in vivo was that the antispasmogenic activity of forskolin was preserved in albuterol-treated rats, implying that a defect in signal transduction had occurred upstream of adenylyl cyclase. Several possibilities were considered as follows.

Downregulation of β₂-adrenoceptor number. Treatment of rats for 7 days with albuterol produced a significant (30%) reduction in pulmonary β₂-adrenoceptor number when compared with animals that were given saline. A similar loss in pulmonary β-adrenoceptor density has also been reported in rats after in vivo treatment with isoproterenol (11, 21, 33, 34) and terbutaline (35), and in guinea pigs given norepinephrine (12). Collectively, these findings support the general concept that prolonged in vivo exposure of animals to β₂-adrenoceptor agonists promotes desensitization by stimulating the internalization and degradation of the cognate receptor (17). Thus, such a process could account for the in vivo desensitization seen in the present study. However, this mechanism probably plays a relatively minor role for at least two reasons (15, 36). First, the reduction in β₂-adrenoceptor density was quite small and of questionable functional significance given that a receptor reserve is thought to exist on airways smooth muscle for many β₂-adrenoceptor agonists (5). Second, in vivo treatment of rats with albuterol produced heterologous desensitization.

Activation of GRKs. Another mechanism of desensitization involves phosphorylation of the β₂-adrenoceptor by GRKs, in particular GRK2. Unlike results obtained in other systems (14, 15), GRK activity was unchanged in lung membranes prepared from albuterol-treated rats, which is, perhaps, not unexpected, as translocation of GRK2 to the plasma membrane is rapid and transient, occurs concurrently with receptor phosphorylation, and precedes desensitization and receptor sequestration. Moreover, GRKs show considerable substrate specificity and effect homologous desensitization by phosphorylating only the agonist-occupied form of the receptor (15). Clearly, therefore, this mechanism cannot readily explain why PGE₂-induced responses were also desensitized by albuterol.

It is noteworthy that cytosolic GRK activity was significantly increased in the lung of albuterol-treated rats when compared with those animals that received saline. This effect was associated with increased protein expression suggesting that GRK2 mRNA stability and/or gene transcription had been increased. This interpretation is supported by the finding that long term infusion of mice with isoproterenol increased mRNA, protein and activity of GRK2 in the heart by a mechanism that was attributed to a cAMP-independent increase in GRK2 gene transcription (37).

Downregulation of membrane-bound Gsα. One mechanism that could account for the ability of albuterol to promote heterologous desensitization is a reduction in the abundance of Gsα (19). Three molecular weight species of Gsα were detected in lung and tracheal membranes prepared from both groups of rats. Based on electrophoretic mobility on SDS polyacrylamide gels, two of these proteins represent so-called “long” and “short” Gsα variants that arise through alternative mRNA splicing and are ubiquitously expressed (38, 39). The other isoform is probably one of several other splice variants that have a more tissue-specific distribution (40). Significantly, the expression of Gsα (assessed by Western blotting and CTX-catalyzed ADP ribosylation) was reduced (∼50%) in the lung of albuterol-treated rats. Moreover, the ability of CTX to increase the cAMP content in lung harvested from albuterol-treated rats was also compromised when compared with animals that received saline. Collectively, these results provide a compelling explanation for heterologous desensitization in this model. Indeed, downregulation of Gsα would blunt the ability of albuterol, and other agonists that utilize the same pool of Gsα, to activate adenylyl cyclase and protect the airways against ACh-induced bronchoconstriction. A similar explanation has been advanced to account for the impaired activation of adenylyl cyclase by isoproterenol and glucagon ex vivo in cardiac membranes purified from rats treated chronically with isoproterenol (32).

Albuterol could downregulate Gsα by at least three mechanisms: decreased Gsα gene transcription, destabilization of preexisting Gsα mRNA transcripts, or redistribution/degradation of membrane-associated Gsα. McKenzie and Milligan (41) have reported that downregulation of Gsα after treatment of NG108-15 cells with PGE₁ is not associated with a reduction in Gsα mRNA or prevented by cycloheximide. Thus, the decrease of Gsα may not reflect a reduction in transcription or in mRNA stability. Concordant with this interpretation is that isoproterenol promotes a rapid translocation of Gsα from the membrane to a cytosolic pool in a number of different cells (42, 43) that is followed, after chronic exposure, by degradation of the protein (19). In this study, Gsα was not detected in the cytosol of lung or tracheae taken from albuterol-treated rats, suggesting that degradation of Gsα may have occurred. It is unclear exactly how albuterol reduces the abundance of Gsα at the membrane but it might involve rapid depalmitoylation at Cys³, which prevents the association of Gsα with the membrane (44, 45). Indeed, isoproterenol-induced depalmitoylation of Gsα correlates with a redistribution of the G-protein from membrane to cytosol (44), which is consistent with the finding that that membrane-bound, but not cytosolic, Gsα contains palmitic acid (46). The molecular basis of this depalmitoylation is unknown, but it is independent of cAMP (45).

It is unclear whether a 50% reduction in Gsα reduced signaling through pulmonary β₂-adrenoceptors in this study. However, agonist-induced activation of adenylyl cyclase is attenuated in GH₃ rat pituitary tumor cells transfected with a plasmid-encoding antisense Gsα mRNA under conditions in which Gsα protein was reduced by approximately 50% (47). It is likely, therefore, that the loss of membrane-bound Gsα in lung and tracheae accounts for compromised β₂-adrenoceptor signaling in rats treated chronically with albuterol. Furthermore, the magnitude of Gsα downregulation in GH₃ cells is agonist-dependent, indicating differences in the efficiency at which GPCRs utilize the available pools of Gsα for the stimulation of adenylyl cyclase (47). This finding could have important clinical implications for the design of new β₂-adrenoceptor agonists that produce less desensitization, as depalmitoylation of Gsα is positively related to the intrinsic activity of the agonist (45). Thus, given that a large receptor reserve exists in airway smooth muscle for most β₂-adrenoceptor agonists (5), a drug with low intrinsic activity would produce less downregulation of Gsα and so limit β₂-adrenoceptor desensitization without significantly affecting bronchodilator activity.

Another mechanism that can produce heterologous desensitization is upregulation of cAMP PDE (18). In leukocytes, β₂-adrenoceptor agonists increase PDE activity through the induction of specific PDE isogenes, thereby compromising signaling through all GPCRs that couple positively to adenylyl cyclase (18, 22). Although the in vivo results obtained with forskolin in albuterol-treated rats imply that the site of desensitization was upstream of adenylyl cyclase, the activity of PDE3 and PDE4 was elevated in lung taken from these animals. It is possible, therefore, that induction of PDE occurred over a time frame when albuterol-induced adenylyl cyclase activation was not impaired. The additional finding that IBMX partially restored albuterol-induced cAMP accumulation in “desensitized” lung implies that upregulation of PDE conferred this effect. However, whether the enhanced rate of cAMP metabolism contributed to the loss of antispasmogenic activity of albuterol and PGE₂ is unclear given that the protection afforded by forskolin against ACh-induced bronchoconstriction was preserved in both groups of animals. Therefore, we conclude that the increase in PDE in the lung is either functionally irrelevant at 7 days or occurred in nonairway smooth muscle cells. Alternatively, as forskolin can evoke large increases in cAMP, the upregulation of PDE may be insufficient to alter PKA activity.

In conclusion, the results of this study demonstrate that chronic treatment of rats with albuterol produced pulmonary β₂-adrenoceptor desensitization. Although a number of processes could contribute to this effect, the heterologous nature of the desensitization and the finding that the antispasmogenic activity of forskolin was preserved suggest that the primary molecular etiology is a reduction in the abundance of membrane-associated Gsα. It is important to appreciate that these studies were performed in healthy rats and that the applicability of the model to patients with asthma, in whom the airways are inflamed and hyper-reactive, is unknown. However, it has been reported that segmental allergen challenge of asthmatic subjects does not impair β₂-adrenoceptor function in epithelial cells obtained by bronchoscopic brushings, suggesting that the disease itself does not predispose to desensitization (48). Thus, downregulation of Gsα may have direct relevance to the treatment of asthma in circumstances in which susceptible individuals become tolerant to the beneficial effects of regular, high-dose β₂-adrenoceptor agonists.

The authors gratefully acknowledge M. Johnson for constructive criticism of the manuscript and GlaxoWellcome and Monsanto/Searle for financial support.

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