Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Inhaled -agonists are the most widely used agents in asthma therapy and are universally recognized as the treatment of choice for acute asthma attacks. When used in asthma treatment, -agonists mediate their effect primarily by activating ₂-adrenergic receptors (₂ARs) on airway smooth muscle. ₂AR activation promotes G-protein (G_s) activation of adenylyl cyclase, an increase in intracellular cyclic adenosine monophosphate (cAMP), and subsequent activation of protein kinase A (PKA). PKA activation results in a host of intracellular effects that contribute to the relaxation of airway smooth muscle. Despite the widespread use of -agonists and a deep understanding of their mechanism of action in airway smooth muscle relaxation, surprisingly little is known about the manner in which the ₂AR signaling pathway is regulated in human airway smooth muscle (HASM). Alterations in ₂AR responsiveness can be effected by mediators of inflammation, glucocorticoids, and -agonists themselves, in a time-dependent and cell-specific manner (1). Indeed, the issue of tachaphalaxis and the phenomenon of agonist-specific desensitization have been important considerations in -agonist drug design (2, 3), and may also underlie important clinical observations, such as the loss of prophylactic bronchoprotection and deterioration of asthma control (4).

Although numerous studies have identified impairment of ₂AR-mediated functions in human and nonhuman airway smooth muscle, only a handful of studies have examined the regulation of the HASM ₂AR-adenylyl cyclase (AC) signaling pathway per se. Mechanisms of acute (short-term) regulation of ₂AR signaling identified in other systems have shown important roles for intracellular kinases (G protein-coupled receptor kinases [GRKs], PKA, and/or protein kinase C [PKC]) in modulating the responsiveness of this system. ₂AR phosphorylation by PKA (induced by agents that increase intracellular cAMP levels, and associated with heterologous desensitization) and by GRK2 (specific for the agonist-occupied receptor and associated with homologous desensitization) promote rapid uncoupling of the ₂AR from the stimulatory G protein G_s. However, the extent to which either of these desensitization mechanisms is used appears to be highly cell-specific (8), and may be related to kinase expression levels (9). In this report we characterize the salient features of the acute regulation of the ₂AR-AC pathway in HASM cultures, and provide insight into the specific roles of GRKs and PKA in these processes. In addition, we present a technique enabling highly efficient transfection of HASM cultures, which should facilitate the further analysis of molecular mechanisms of G protein-coupled receptor-pathway regulation in HASM.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Development of HASM Cultures

HASM cultures were established as described by Panettieri and colleagues (10) from human tracheae obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. Briefly, a segment of trachea superior to the carina was removed under sterile conditions, and the trachealis muscle was isolated. Approximately 0.5 g (wet tissue) of trachealis muscle was isolated from the distal trachea, minced, centrifuged, and resuspended in 10 ml of buffer containing 0.2 mM CaCl₂, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor, and 10 U/ml elastase. Enzymatic dissociation of the tissue was done for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105-µm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F12 medium supplemented with 10% fetal bovine serum (FBS). Aliquots of the cell suspension were plated at a density of 10⁴ cells/cm² in Ham's F12 medium supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 µg/ml amphotericin B. The medium was replaced every 72 h. Confluent cells in flasks were passaged with 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) at a plating density of 10⁴ cells/cm². Cells were maintained in serum-supplemented Ham's F12 medium for 12 d after passage. The medium was changed to serum-free medium (IT medium) containing 5 µg/ml transferrin and 5 µg/ml insulin 24 h before experimentation.

HASM cultures in subculture during the second to sixth cell passages were used in the described experiments. During these cell passages, HASM cells retain native contractile protein expression, as shown by immunocytochemical staining for smooth muscle actin and myosin (11), as well as retaining functional cell-excitation coupling systems as determined by fura-2 measurements of agonist-induced changes in cytosolic calcium (11, 12). Importantly, the stability of the ₂AR-G_s-AC signaling pathway with repeated cell passaging has been previously demonstrated (13).

Pretreatment of Cells

Cells grown to confluence on tissue-culture plates (12-well, 24-well, or 150-mm plates) were pretreated with IT medium ± vehicle (control) or the potential regulatory agent (isoproterenol [ISO], forskolin [FSK], or prostaglandin E₂ [PGE₂]) for 30 min. In selected experiments, staurosporine (a potent inhibitor of both PKA and PKC), bisindolylmaleimide IX (Bis IX, a potent, specific inhibitor of PKC), or concanavalin A (Con A, a lectin that blocks ₂AR sequestration) were added to cultures 1 h (20 min for Con A) before the 30-min pretreatment period. For studies examining ₂AR resensitization, cells were washed thrice in IT medium at 37°C and returned to the incubator for 1 h.

Following pretreatment or after recovery, cells in 12- or 24-well plates were placed on ice and washed thoroughly with cold phosphate-buffered saline (PBS), then stimulated as described subsequently. For studies assessing ₂AR distribution in intact cells (receptor sequestration studies), cells in 150-mm dishes were harvested by incubation with 0.25% trypsin/0.53 mM EDTA (± 1 µM ISO) during the final 2 min of the pretreatment period. Cells were then washed extensively in ice-cold PBS and resuspended in PBS for subsequent binding assay (see the following discussion).

Homogenate, Membrane Preparations

Cells were scraped from tissue-culture plates into ice-cold homogenization buffer consisting of 25 mM Tris, pH 7.5; 5 mM EDTA; 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid (EGTA); 0.02 mg/ml leupeptin; 0.2 mg/ml benzamidine; and 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and were homogenized by Polytron disruption. Membranes were prepared by centrifugation of homogenates at 19,000 × g for 20 min at 4°C. Pellets were resuspended in homogenization buffer using a glass-glass dounce homogenizer, and were then washed twice by repeating the prior step of resuspension and centrifugation. The final pellet was resuspended in homogenization buffer and assayed immediately.

Whole-cell lysates used in immunoblot analysis were prepared by lysing cells in the homogenization buffer described previously containing 1% Triton X-100.

Protein concentrations from 12-well plates, cell suspensions, membrane preparations, and homogenates/lysates were determined with the Bradford assay (14).

₂AR-binding Assays

For preliminary determination of ₂AR density in HASM cultures, approximately 100 µg of membrane protein were incubated in a total volume of 500 µl of 25 mM Tris, pH 7.5, plus 2 mM MgCl₂, containing ~ 5 to 200 pM [¹²⁵I]iodopindolol (PIN) (2,200 Ci/mmol; NEN, Boston, MA) at 37°C for 1 h. Data were transformed according to Scatchard (15) for estimation of receptor density and affinity for antagonist. In subsequent experiments, ₂AR density in control and pretreated HASM cells was assessed by single-point (200 pM [¹²⁵I]PIN) analysis as previously described (16). Nonspecific binding was determined by displacement of [¹²⁵I]PIN binding with 1 µM alprenolol.

In binding studies examining ₂AR distribution in intact cells, 100 µg of harvested cells were incubated at 14°C for 3.5 h with 200 pM [¹²⁵I]PIN in the presence or absence of 1 µM alprenolol (for determination of whole-cell ₂AR density) or 100 nM CGP 12177 (cell surface ₂ARs), as previously described (17).

All binding reactions were terminated by addition of five aliquots each 4 ml ice-cold 25 mM Tris-HCl, pH 7.5, plus 2 mM MgCl₂, and filtration through Whatman GF/C filters, using a Brandel Cell Harvester (Gaithersburg, MD).

Measurement of cAMP Production

Experiments examining cAMP accumulation were done with cells grown to confluence in 12- or 24-well plates in a modification of the method described by Kong and coworkers (18). Individual wells were stimulated at 37°C for 10 min with 500 µl PBS containing 300 µM ascorbic acid and 1 mM isobutylmethylxanthine (IBMX) (excepting time-course studies) in the absence (basal) or presence of either 10⁹ to 10⁴ M ()ISO or 100 µM FSK. Preliminary studies were also done, using 1 mM RO-20-1724 (a Type IV phosphodiesterase inhibitor); experimental effects of pretreatment with agonist and kinase inhibitor were not influenced by the mode of phosphodiesterase inhibition (data not shown). Reactions were stopped by placing the plates on ice, aspirating the media, and adding 500 µl of ice-cold ethanol. After a 1-h incubation at room temperature, the ethanol from each well was transferred to an Eppendorf tube. Samples were then lyophilized for 1 h, and the pellet was resuspended in 500 µl H₂O and stored at 4°C. We have determined that this method of cAMP isolation slightly improves the reproducibility of results over that with methods using acid lysis and bicarbonate neutralization. Samples were assayed for cAMP content on the following day with a radioimmunoassay using a [¹²⁵I]cAMP tracer and cAMP antibody (provided by Dr. M. Ascoli, University of Iowa) as previously described (19). cAMP concentrations were determined by interpolation from a standard curve.

Transfection of HASM Cultures

HASM cells were transfected through a modification of the procedure described by Forsayeth and Garcia (20), in which a replication-deficient adenovirus (human adenovirus 5 dl343) is utilized to enhance 2-diethylaminoethanol (DEAE)-dextran-mediated transfection of cells that are typically resistant to conventional methods of transfection. For the present study we used the Ad5-guanine phosphotransferase (GPT) version of this virus (provided by P. Garcia) in which the entire E1A and E1B coding regions are replaced by a GPT coding sequence. Briefly, 4 × 10⁶ cells were harvested and resuspended in 5 ml of Dulbecco's minimal essential medium (DMEM) containing 200 µg of DEAE-dextran, 3 × 10⁸ plaque-forming units of Ad5-GPT, and 2 µg of each DNA to be transfected. The mixture was plated onto 10-cm tissue-culture plates and incubated for 2 h at 37°C. The medium was then removed, the cells were washed for 1 min with 2 ml of 10% dimethylsulfoxide (DMSO) in Ca²⁺- and Mg²⁺-free PBS, and the medium was replaced with serum-supplemented Ham's F12 medium. From 20 to 24 h later the transfected cells were harvested and replated at high density (2 × 10⁴ cells/ cm²) in 12-well plates or 150-mm plates, and were grown in serum-supplemented medium. Transfection efficiency was determined from 2 to 6 d after transfection by assessment of -galactosidase activity (21) in cells transfected with pCMVneolacz, or by fluorescence-activated cell sorting (FACS) analysis of expression of cotransfected pcDNA3- GFPS65T.

Preparation of Plasmids for Transfection

pcDNA3₂AR-flag was constructed by excising the open reading frame of pBC-₂ARflag (provided by Dr. B. Kobilka, Stanford University) with SalI/HindIII, and the ~ 2-kb fragment was directionally cloned into pcDNA3.

pCMVneolacz was constructed by excising a 3.7-kb fragment from pAP-lacz (provided by A. Pronin, Thomas Jefferson University) with HindIII/XbaI, blunting the fragment with Klenow, and ligating the blunted fragment into pCMVneo. Correct orientation was confirmed by restriction analysis.

pcDNA3ARK was constructed by excising an ~ 3-kb HindIII fragment from the bovine ARK clone ARK3A (22) and ligating into the HindIII site of pcDNA3.

pcDNA3-GFPS65T, encoding a variant of Aequorea victoria green fluorescent protein (Clontech, Palo Alto, CA), was provided by L. Kallal (Thomas Jefferson University).

Immunoblotting

From 20 to 30 mg of homogenate were electrophoresed on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a nitrocellulose membrane as previously described (23). The blot was washed in 20 mM Tris, pH 7.5, plus 150 mM NaCl (Tris-buffered saline; TBS), and was blocked for 1 h in TBS plus 0.05% Tween 20 (TTBS) with 5% (wt/vol) dried nonfat milk. The blot was then incubated for several hours with the primary monoclonal antibody 3A10 (which specifically recognizes GRK2) (23), and was subsequently washed thrice for 10 min each time with TTBS. The blot was subsequently incubated for 1 h with affinity-purified goat antimouse IgG conjugated to horseradish peroxidase, in 5% milk-TTBS. The blot was then washed thrice for 10 min each in TTBS, and was visualized through the enhanced chemiluminescence (ECL) technique (Amersham, Arlington Heights, IL).

Data Analysis

Data points from individual assays represent the mean values of duplicate or triplicate measurements. Dose-response curves for ISO-mediated cAMP production were analyzed by Sigmaplot (Jandel Corp., Chicago, IL) to obtain V_max and EC₅₀ values. Except where indicated, cAMP generation was calculated as the increase in cAMP over basal levels and was expressed as a percentage of the values resulting from 100 µM FSK stimulation, to provide an index of ₂AR coupling capacity (24). Statistically significant differences among groups were assessed with the t test for paired samples, with values of P < 0.05 sufficient to reject the null hypothesis. All experiments were designed such that matched control conditions existed within each experiment to enable statistical comparison of paired samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of ₂AR Expression and Signaling in HASM Cultures

Preliminary studies were done to establish the levels of ₂AR receptor density and responsiveness to -agonist of HASM cultures. Saturation binding analyses of [¹²⁵I]PIN binding to HASM membranes from four separate cultures demonstrated high-affinity binding (Kd values ranging from 13 to 22 pM), with ₂AR density ranging from 13 to 23 fmol/mg proteinvalues similar to those recently reported by Green and associates (25). ₂AR density (fmol/ mg protein) in both intact whole-cell preparations and whole-cell homogenate preparations was approximately one-third that measured in membrane preparations from the same culture (data not shown). Figure 1 shows the time-dependent cAMP response of HASM to stimulation with 1 µM ISO, 1 µM PGE₂, and 100 µM FSK, which directly activates AC (independently of ₂AR activation). ISO elicits rapid cAMP production in a linear manner for up to 7.5 to 10 min, after which the rate of cAMP accumulation diminishes significantly, suggesting receptor desensitization. A similar profile is observed for stimulation with PGE₂, although absolute cAMP production is ~ 2-fold that observed with ISO. In contrast, FSK-stimulated cAMP accumulation is more linear throughout 20 min of stimulation. Absolute values of ISO- and PGE₂-stimulated cAMP production among cultures were fairly consistent, whereas values of FSK-stimulated cAMP displayed slightly greater variability. For the 15 different cultures utilized in these studies, both ₂AR density and cAMP responses were highly reproducible with repeated cell passaging (Passages two through six were examined), as previously noted (13, 26).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Time course of cAMP production in HASM cultures. Individual wells of HASM cells grown to confluence on 12-well plates were stimulated with either vehicle (basal), 1 µM ISO, 1 µM PGE2, or 100 µM FSK. cAMP was isolated and measured by radioimmunoassay as described in MATERIALS AND METHODS. Data represent mean ± SEM of four experiments.

Homologous and Heterologous Desensitization of HASM ₂AR

We next examined acute regulation of ₂AR responsiveness by pretreating HASM cells for 30 min with 1 µM ISO, 0.1 µM PGE₂, or 10 µM FSK, and observing the cAMP response to subsequent ISO challenge (Figure 2). ISO-pretreated cells displayed a large (~ 60%) reduction in maximal ISO-stimulated cAMP production, with a ~ 3-fold increase in the EC₅₀ value for ISO, an agonist-specific desensitization similar to that observed in the human airway epithelial cell line BEAS-2B (16). FSK- and PGE₂-pretreated cells each displayed a ~ 2-fold increase in EC₅₀ values and a significant reduction (21% and 28%, respectively) in maximal cAMP production, suggesting that acute PKA-mediated heterologous desensitization also occurs in HASM cultures. FSK-stimulated cAMP production was relatively unaffected by pretreatment with ISO, FSK, or PGE₂, suggesting that desensitization is effected primarily at the ₂AR locus under these conditions (see Figure 2 legend). Although no other studies of acute ₂AR regulation in HASM cells exist for comparison, these results are in general agreement with those of Hall and associates (13), who demonstrated both homologous and heterologous ₂AR desensitization following more chronic (1- to 24-h) pretreatment conditions. Thus, our data suggest that both forms of desensitization may be invoked in HASM, in contrast to our previous observations in both acutely dissociated (27) and immortalized (16) human airway epithelium, in which mechanisms of heterologous desensitization were shown to have minimal effects.

View larger version (23K): [in this window] [in a new window]   Figure 2.   Short-term 2AR desensitization in HASM cells. Confluent cells in 24-well plates were pretreated with vehicle (CON), 10 µM forskolin (FSK-D), 0.1 µM PGE2 (PGE-D), or 1 µM isoproterenol (ISO-D) for 30 min, washed four times with ice-cold PBS, and subsequently challenged with 109 to 104 M ISO, as described in MATERIALS AND METHODS. Data points represent increase over basal stimulation normalized to 104 M FSK-stimulated values (mean ± SEM of 7 to 19 experiments). Vmax values are compared with those for CON group: FSK-D: 79 ± 4% (mean ± SEM), n = 7 paired observations; PGE-D: 72 ± 3%, n = 7 paired observations; ISO-D: 41 ± 4%, n = 12 paired observations. EC50 values (nM): FSK-D: 90 ± 12 versus CON: 45 ± 6; PGE-D: 81 ± 11 versus CON: 46 ± 7; ISO-D: 157 ± 45 versus CON: 52 ± 6). Statistically significant (P < 0.05) differences were determined by paired t test for both Vmax and EC50 values for FSK-D, PGE-D, and ISO-D versus CON group. FSK-stimulated cAMP production did not differ among groups (FSK-D: 98 ± 5% of CON value; PGE-D: 102 ± 10%; ISO-D: 99 ± 5%).

Role of Sequestration in HASM ₂AR Regulation

The homologous desensitization shown in Figure 2 was associated with significant sequestration of cell-surface ₂ARs. Figure 3A depicts the time-dependent loss of cell surface ₂ARs associated with acute exposure of HASM cultures to 1 µM ISO. ₂AR density was measured in intact cells from [¹²⁵I]iodopindolol binding, using either a hydrophobic or hydrophilic competitive antagonist to determine total and cell surface ₂AR density, respectively. Cell surface ₂AR density was calculated to be 82% of the total receptor number. A rapid loss (~ 35%) of cell surface ₂ARs was observed within 10 min of agonist exposure, with a loss of ~ 45% observed at 30 min. Only a minor (< 10%) loss of total cell ₂ARs was measured within the 30-min treatment period, showing that cell surface receptor loss was due to sequestration and not downregulation (the latter of which is typically associated with chronic exposure to -agonist). Several studies of numerous cell types (28, 29) have shown that sequestration may be blocked without affecting the initial rapid uncoupling of the receptor from AC stimulation (and thus not contributing to desensitization per se). However, these studies typically examined cell types in which ₂ARs were expressed at high levels, in which case a significant receptor reserve might confer tolerance of cell surface receptor loss. Because HASM cells express relatively low levels of ₂ARs, and ₂AR signaling might therefore be affected by ₂AR sequestration, we investigated whether blockage of ₂AR sequestration by pretreatment with Con A influenced the pattern of homologous desensitization. As shown in Figure 3B, pretreatment with Con A had no significant effect on the dose-dependent response to ISO in either vehicle- (CON-) or ISO-pretreated cells. However, blockage of ₂AR sequestration with Con A produced a profound effect on the ability of HASM cells to recover from homologous desensitization. Following 30 min of pretreatment with ISO, cells that were washed and allowed to incubate an additional 60 min at 37°C (Figure 3B; RECOV) showed a significantly increased V_max value (~ 75% of CON) compared with the ISO-pretreated group. However, those cells pretreated with Con A prior to the pretreatment/recovery phases (RECOV + Con A) exhibited little if any resensitization. This finding suggests an apparent requirement of receptor recycling for rapid recovery from acute homologous desensitization, and is consistent with the finding in recent studies that implicate sequestration as important in ₂AR dephosphorylation and resensitization (30, 31).

View larger version (20K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   Figure 3.   (A) Alterations in HASM 2AR cellular distribution upon exposure to 1 µM ISO. HASM cells grown to confluence on 15-cm tissue-culture plates were treated with 1 µM ISO for 0 to 30 min. Cells were harvested with 2 ml 0.25 mM trypsin/1 mM EDTA ± 1 µM ISO, quenched with 10 ml ice-cold PBS containing 0.1 mg/ml soybean trypsin inhibitor, washed four times, and resuspended in ice-cold PBS. Binding studies were done with 200 pM [125I]PIN in the presence and absence of 1 µM alprenolol (for determination of whole-cell 2AR density), or with 100 nM CGP 12177 (cell surface 2AR) at 14°C for 3.5 h, as previously described (17). Data represent mean ± SD of three experiments (5-min data point is mean of two experiments). (B) Effect of Con A on homologous desensitization and resensitization of HASM 2AR. HASM cultures were pretreated with (+ Con A) or without 0.25 mg/ml Con A for 20 min, prior to 30-min pretreatment with ± 1 µM ISO, and 2AR responsiveness was subsequently assessed as described in MATERIALS AND METHODS. Selected cells were washed thrice with 37°C IT medium after ISO pretreatment, and were returned to the incubator for 1 h prior to ISO challenge (RECOV). Con A pretreatment had no significant effect on ISOD Vmax values (ISO-D: 40 ± 5% versus ISO-D + Con A: 45 ± 5%), but did have a significant effect on RECOV (RECOV: 75 ± 11% versus RECOV + Con A: 48 ± 6%; P < 0.05). Values presented are means of three paired observations; error bars are removed for graph clarity.

Role of Protein Kinases in Acute ₂AR Desensitization

To explore the potential role of intracellular kinases in acute desensitization of the HASM ₂AR, cells were pretreated with 1 µM staurosporine, a potent, membrane-permeable inhibitor of both PKA and PKC, for 1 h prior to 30 min pretreatment with FSK, PGE₂, or ISO. As depicted by the ISO dose-response curve in Figures 4A and 4B, staurosporine pretreatment largely reversed the heterologous desensitization observed in cells pretreated with FSK (V_max increased to 91 ± 3% from 81 ± 4% of CON value, n = 4 paired observations) and PGE₂ (92 ± 6% from 73 ± 3% of CON values; n = 5 paired observations). Staurosporine also had a small but statistically significant effect on homologous desensitization (Figure 4C), increasing the V_max of the ISO dose-response curve to 50 ± 6% from 41 ± 4% of CON values (n = 6 paired observations). Significant shifts in EC₅₀ values were also observed in the staurosporine-pretreatment groups for each of the pretreatment conditions (FSK-D, PGE-D, or ISO-D; see Figure 4 legend).

View larger version (23K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   Figure 4.   Effects of staurosporine on acute 2AR desensitization in HASM. HASM cells were grown to confluence in 24-well plates and pretreated with vehicle or 1 µM staurosporine (+ ST) for 60 min, followed by an additional 30 min of pretreatment with vehicle (CON), 10 µM FSK (FSK-D), 0.1 µM PGE2 (PGE-D), or 1 µM ISO (ISO-D). Cells were then washed thoroughly in ice-cold PBS and subsequently challenged with vehicle, 109 to 104 M ISO, or 100 µM FSK, as described in MATERIALS AND METHODS. Staurosporine pretreatment significantly (P < 0.05, paired t test) increased Vmax values in HASM pretreated with FSK ([A] FSK-D: 81 ± 4% [mean ± SEM] versus FSK-D + ST: 91 ± 3%; n = 4), PGE2 ([B] PGE-D: 73 ± 3% versus PGE-D + ST: 92 ± 7%; n = 5), or ISO ([C] ISO-D: 41± 4% versus ISO-D + ST: 50 ± 6%; n = 6). Staurosporine pretreatment also significantly (P < 0.05, paired t test) decreased EC50 values for ISO in each desensitization group (FSK-D: 80 ± 10 nM versus FSK-D + ST: 49 ± 6 nM; PGE-D: 75 ± 8 nM versus PGE-D ± ST: 40 ± 3 nM; ISO-D: 142 ± 52 nM versus ISO-D ± ST: 81 ± 18 nM). FSK-stimulated cAMP values did not vary among groups (data not shown).

Because staurosporine is a potent inhibitor of both PKA and PKC, we further examined the potential role of PKC in FSK-, PGE₂-, and ISO-treated cells by pretreating HASM cultures with Bis IX. Pretreatment with 1 µM Bis IX had essentially no effect on either V_max or EC₅₀ values in control or in FSK-, PGE₂, and ISO-treated cells, with dose-response curves for the four conditions being virtually superimposable (data not shown). These data suggest that the observed experimental effects of staurosporine are mediated through inhibition of PKA, and that PKC has no effect on either the heterologous ₂AR desensitization mediated by activations of AC or on homologous desensitization in HASM.

Effect of GRK2 Overexpression on Agonist-specific ₂AR Desensitization

In a comparison of the time course of ISO-stimulated cAMP production in multiple types of lung cells, McGraw and Liggett (9) recently demonstrated that a relatively low level of GRK2 expression in HASM is associated with an attenuated rate of short-term agonist-promoted ₂AR desensitization. This finding suggests that GRK2 levels may be limiting in agonist-specific ₂AR desensitization in HASM. We therefore hypothesized that increased expression of GRK2 in HASM cells would enhance agonist-specific ₂AR desensitization.

To pursue this question it was first necessary to establish an effective technique for transient transfection of HASM cultures. Traditionally, HASM cultures have been difficult to transfect, because conventional methods (e.g., lipid-assisted or DEAE-dextran) produce extremely low levels (1%) of transfection. We therefore used a modified version of a technique recently described by Forsayeth and Garcia (20), in which DEAE-dextran is used to target unmodified plasmid DNA to the cellular endocytotic pathway while a replication-deficient adenovirus facilitates lysis of the endocytotic vesicles. Application of this method proved successful, resulting in high transfection efficiency (typically ~ 50%) as assessed either from the activity of expressed -galactosidase (data not shown) or from cellular fluorescence of transfected green fluorescent protein as determined by FACS (Figure 5A).

View larger version (20K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 5.   Analysis of adenovirus-facilitated transient transfection of HASM. (A) FACS analysis of transfection efficiency. Populations of 4 × 106 HASM cells were transfected in 10-cm plates at Day 0, using 40 µg/ml DEAE-dextran, 7.5 × 108 pfu of Ad5-GPT, and 2 µg of pcDNA3GFPS65T (encoding Aequorea victoria green fluorescent protein) as described in MATERIALS AND METHODS. Cells were subsequently harvested on Day 3, and transfection efficiency was assessed with FACS. FACS analysis demonstrated a significant population (> 50% of total) of cells expressing GFP (right panel ) by comparison with untransfected cells (left panel) (B). ARK (GRK2) expression in pcDNA3vector and pcDNA3ARK-transfected HASM cells. Samples represent 20 µg of protein from cell lysates prepared from cells 3 and 6 d after transfection. Immunoblotting was done as described in MATERIALS AND METHODS, using the monoclonal antibody 3A10, which is specific for GRK2. Figure represents 2-s exposure of ECL blot; endogenous GRK2 expression in pcDNA3vector-transfected cells was visible with exposures of longer duration (not shown).

Transfection of HASM with pcDNA3ARK, encoding bovine GRK2, resulted in very high expression 3 d after transfection, with somewhat attenuated expression still observed 6 d after transfection (Figure 5B). The effect of GRK2 overexpression on agonist-specific ₂AR desensitization was subsequently investigated in multiple experiments, using transfected cells examined 3 d after transfection (Figure 6). GRK2 overexpression resulted in a small but statistically significant increase in the loss of ISO-stimulated cAMP following ISO pretreatment. Overexpression of GRK2 in various cell types has been used to demonstrate a role for GRK2 in agonist-specific desensitization of numerous receptors, including ₂AR (32) and ₁AR (33). The present data support the notion advanced by McGraw and Liggett (9) that heterogeneity of GRK2 expression among cell types as well as dynamic regulation of endogenous GRK2 levels may influence desensitization patterns.

View larger version (24K): [in this window] [in a new window]   Figure 6.   Effects of GRK2 overexpression on agonist-specific desensitization. HASM were transfected with either pcDNA3vector (control) or pcDNA3ARK, and experiments characterizing agonist-specific desensitization were performed 3 d after transfection. Cells overexpressing GRK2 exhibited a more profound loss of 2AR responsiveness than did control cells (pcDNA3vector: 48 ± 4% loss, versus pcDNA3ARK: 62 ± 3% loss; P < 0.05, n = 8 paired observations) as a result of ISO pretreatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The purpose of this study was to characterize acute regulation of the ₂AR-AC system in HASM cultures, and to elucidate mechanisms by which desensitization of ₂ARs occurs. Thirty-minute pretreatment of HASM with either: (1) -agonist (inducing homologous desensitization); or (2) agents that increased intracellular cAMP independently of ₂AR activation (inducing heterologous desensitization) resulted in a loss of ₂AR responsiveness to subsequent ISO challenge. Minimal alterations in FSK-stimulated cAMP production were observed with both modes of desensitization, suggesting that the ₂AR is the principal locus of regulation in HASM. Homologous desensitization was associated with but not dependent on a ~ 45% loss of cell surface ₂ARs. However, rapid recovery of ₂AR responsiveness from homologous desensitization was highly dependent on ₂AR sequestration. Heterologous desensitization could be largely reversed by pretreatment of cells with staurosporine, whereas a small but significant attenuation of homologous desensitization was observed with staurosporine pretreatment. Bis IX, a potent PKC inhibitor, had no effect on ₂AR desensitization, suggesting that the effects of staurosporine were mediated through inhibition of PKA. Overexpression of GRK2 in HASM cells enhanced ISO-mediated desensitization, implicating GRK2 as a mediator of agonist-specific desensitization whose low level of endogenous expression may limit the magnitude of desensitization in HASM cells.

Desensitization is a phenomenon in which cellular responsiveness is ultimately diminished when cells are subjected to a continuous stimulus. Desensitization of the ₂AR-AC system characterized by diminished cAMP production has been studied in numerous expression systems and cell lines. The mechanisms involved in regulating the responsiveness of ₂AR (and other G protein-coupled receptors) are cell-specific and complex; they include rapid alterations such as receptor phosphorylation (by PKA, GRKs, or PKC), uncoupling from G protein, and sequestration, which occur within minutes of agonist activation, as well as slower changes that may involve increased receptor degradation and decreased receptor synthesis. Although changes in receptor responsiveness may serve as the primary means of desensitization, there is also evidence that G proteins and effector enzymes such as AC may be directly regulated (for a comprehensive review of mechanisms of G protein-coupled receptor regulation, see Penn and Benovic [1]).

Despite the physiologic importance of ₂AR stimulation in HASM function, the regulation of ₂AR-mediated signal transduction in HASM cells remains poorly understood. Alterations in ₂AR-AC system function may occur in vivo during airway inflammation (through PKA- or PKC-mediated mechanisms in association with heterologous desensitization) or with the repeated use of -agonist inhalers (agonist-specific or homologous desensitization) in asthmatic individuals. Indeed, for years, speculation about HASM ₂AR desensitization has been a by-product of clinical studies and pulmonary function analyses of the effects of chronic -agonist treatment on various measures of airway responsiveness. Reports that systemic administration of -agonists promotes ₂AR downregulation in peripheral blood cells, and more recent studies indicating that inhaled -agonists may also desensitize and/or downregulate ₂ARs in peripheral blood (34), whole lung (35), and airway epithelium (27, 36) have further advanced this hypothesis. However, only a handful of studies have examined regulation of ₂AR-G_s-AC cellular signaling in HASM. In an interesting study that predated the identification of many of the mechanisms involved in G protein-coupled receptor regulation, Davis and Conolly (37) noted reductions in both ISO-mediated relaxation responses and cAMP generation following 1 h pretreatment with ISO in bronchial strips obtained from carcinoma patients. In an analysis of effects of chronic -agonist pretreatment in HASM cultures, Hall and colleagues (13) observed a loss of AC responsiveness (cAMP generation) to subsequent ISO stimulation. Because pretreatment with FSK resulted in similar but less impressive effects, the authors concluded that mechanisms associated with both homologous and heterologous desensitization were probably involved. Green and coworkers (25) have in part confirmed the findings of Hall and colleagues in their characterization of chronic, agonist-specific desensitization and ₂AR downregulation in HASM cultures expressing different ₂AR polymorphisms. More recent studies by Nogami and coworkers showed that chronic exposure of HASM cultures to inflammatory mediators can both desensitize (38) and sensitize (39) -agonist-mediated signaling, whereas phorbol ester activation of PKC sensitizes FSK stimulation of AC (39). However, the mechanisms of both acute and chronic -agonist-induced ₂AR desensitization in HASM have so far remained largely uncharacterized, with the respective contributions of intracellular kinases to these processes unknown.

The present study identifies both heterologous and homologous ₂AR desensitization following short-term treatment of HASM cultures. HASM cultures pretreated with agents that increased intracellular cAMP either by direct activation of AC (FSK) or via a G_s-coupled receptor (PGE₂) exhibited a significant loss of ₂AR responsiveness (20% to 30% loss of V_max and a 2-fold increase in EC₅₀ values for ISO stimulation). These findings contrast with those previously made in our studies of BEAS-2B cells, in which pretreatment with either FSK or PGE₂ elicited no change in V_max and very small shifts in EC₅₀ values (16). Collectively, these findings suggest that airway cells differ in their susceptibility to ₂AR desensitization potentially elicited by local agents (e.g., inflammatory mediators) that activate AC.

A somewhat classical homologous ₂AR desensitization was seen in HASM cultures, with a large reduction in ₂AR responsiveness (~ 60% decrease in V_max, ~ 3-fold increase in EC₅₀) being observed after 30 min pretreatment with ISO. Minimal alterations in FSK-stimulated cAMP (reflecting inherent AC activity) were observed, suggesting that desensitization of the pathway occurs primarily at the level of ₂AR-G_s coupling. Although alterations in G_s expression or function may contribute to the observed desensitization, they seem unlikely for the following reasons: First, we have not observed any discernible change in G_s expression as assessed by Western blotting of either whole-cell homogenates or membrane preparations derived from HASM cells treated either acutely or chronically with -agonist (data not shown). Second, Hall and colleagues (13) observed that pretreatment of HASM cultures with ISO did not significantly alter PGE₂-mediated cAMP production, suggesting that G_s function remains unaltered in ISO-treated HASM cells. Thus, the principal locus of regulation appears to be the ₂AR.

Acute homologous desensitization of HASM ₂ARs was shown to be associated with a ~ 45% loss of cell surface ₂ARs. Although the relatively low level of ₂AR expression in HASM raises the intriguing possibility that sequestration may play a causal role in homologous desensitization in these cells, we were unable to detect any significant change in desensitization in cells pretreated with Con A. However, we were able to determine a significant effect of Con A pretreatment on the resensitization of ₂AR signaling in HASM cultures. Cells allowed to recover for 1 h from the 30-min ISO treatment demonstrated a clear enhancement of responsiveness (to 75% from 40% of CON V_max values), whereas recovering cells previously treated with Con A exhibited little if any recovery of responsiveness. Thus, despite extremely low levels of ₂AR expression, HASM cells appear similar to many cell lines/ expression systems expressing high levels of ₂AR in demonstrating no requirement for ₂AR sequestration for acute homologous desensitization, but an apparent requirement of receptor recycling in order to facilitate rapid recovery of ₂AR responsiveness.

The most extensively studied mechanisms of rapid ₂AR desensitization involve receptor phosphorylation by GRKs and PKA, which serve to uncouple the receptor from G_s. However, each kinase has distinct mechanisms of activation. PKA activation can be effected by any agent that increases intracellular cAMP and may thus promote ₂AR phosphorylation and desensitization independent of ₂AR occupancy. ARK, or GRK2, the best-characterized member of a recently established family of related G protein-coupled receptor kinases (1, 40), specifically phosphorylates the agonist-occupied form of ₂AR. Agonist activation of the ₂AR invokes G protein subunit-mediated translocation of GRK2 from the cytosol to the plasma membrane (41). In addition to promoting ₂AR uncoupling, ARK phosphorylation of ₂AR initiates the subsequent binding of arrestin proteins to ₂AR (42, 43), further uncoupling the receptor and facilitating ₂AR sequestration via clathrin-mediated endocytosis (17, 44).

Although many cells appear capable of invoking both PKA- and GRK2-mediated mechanisms of desensitization, early studies suggested that although PKA appeared to be the fundamental regulator of heterologous desensitization, its role in -agonist-mediated desensitization was minimal if not inconsequential. This notion stemmed from the observations that the extent of -agonist-mediated ₂AR phosphorylation (45) and desensitization (45) were similar among wild-type, cyc- (which lack G_s), and kin- (which lack PKA) s49 lymphoma cells. A subsequent study by Roth and associates (50) of the kinetics of ₂AR phosphorylation and desensitization in A431 cells concluded that GRK2-mediated phosphorylation was the most rapid and quantitatively important process contributing to rapid desensitization. However, these conclusions were not entirely supported by other studies using other cell types or expression systems with mutant ₂ARs that lack consensus PKA or GRK2 phosphorylation sites (24, 51, 52). Using antisense oligonucleotides targeting PKA and GRK2 in multiple cell types, Shih and Malbon (8) recently reported that agonist-induced ₂AR desensitization in Chinese hamster ovary cells and DDT₁MF-2 smooth muscle cells is largely mediated by ARK, desensitization in rat osteosarcoma cells appears to depend predominantly on PKA activity, and PKA and ARK appear equally effective in A431 cells. Utilizing an alternative approach to assess ₂AR-G_s coupling, Post and colleagues (53) recently showed that high-affinity [³H]FSK binding (an index of receptor-activated G_s-AC association) is significantly greater in kin-s49 lymphoma cells than in wild-type cells following -agonist pretreatment, suggesting that PKA plays an important role in the agonist-promoted uncoupling of ₂AR.

Our data suggest that PKA is probably the principal regulator of FSK- and PGE₂-mediated ₂AR desensitization in HASM as evidenced by the ability of staurosporine (a potent inhibitor of PKA and PKC), but not Bis IX (a potent PKC-specific inhibitor), to attenuate the observed desensitization. Staurosporine (but not Bis IX) also significantly, albeit to a much lesser degree, attenuated ISO-induced ₂AR desensitization. We can therefore attribute to PKA a small but decided role in modulating homologous ₂AR desensitization in HASM cells. One potential explanation for an observable role of PKA in the homologous desensitization of HASM ₂AR may be a relatively lesser capacity (compared with that in other cell types) of GRK2 to invoke agonist-mediated desensitization. McGraw and Liggett (9) have recently suggested that relatively low levels of GRK2 expression in HASM contribute to a relatively slow rate of HASM ₂AR desensitization. In the present study, overexpression of GRK2 in HASM cultures resulted in a small but significant enhancement of ISO- induced desensitization, consistent with the hypothesis that GRK2 may be limiting in HASM cells and thus may contribute to a small degree of resistance to homologous desensitization of ₂AR. Our results, consistent with findings in previous studies (9, 54, 55), suggest that regulation of GRK2 expression in HASM (perhaps via chronic use of -agonist inhalers) may modulate the degree of ₂AR desensitization. On the other hand, the possibility also exists that other protein kinases (such as PKA or other GRKs) confer plasticity on this system and adequately compensate for a low level of GRK2 activity. To date, we have been able to identify proteins in HASM lysates immunoreactive to two separate antibodies, one specifically directed against GRK5/ GRK6, and another specifically directed against GRK6 (unpublished observations). Thus, GRK5 and/or GRK6 also may play important roles in homologous desensitization of the HASM ₂AR. Future studies of the specificity of GRKs in HASM should help delineate further the respective roles of protein kinases in the regulation of G-protein-coupled receptors in HASM.