Introduction

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Formoterol (FOR) and salmeterol (SLM) are highly selective ₂ adrenoceptor (AR) agonists characterized by a long duration of action. Whereas the long-acting bronchodilation by FOR was found by chance in clinical studies (Löfdahl and Svedmyr, 1989; Anderson, 1993), SLM was the result of a specific research program to design long-acting drugs through molecular modification of the ₂AR agonist salbutamol (for review, see Jack, 1991; Johnson et al., 1993; Johnson, 1995).

FOR, which is characterized by high potency for relaxation of airway smooth muscle (Ida, 1976; Lemoine and Overlack, 1992), high-affinity binding to the high- and low-affinity states of pulmonary ₂AR (Lemoine, 1992; Lemoine et al., 1992) and of ₂AR expressed in COS-7 cells (Green et al., 1996), and high efficacy for stimulation of ₂AR-coupled adenylyl cyclase (AC), acts as a full agonist for ₂AR (Lemoine and Overlack, 1992). Based on radioligand-binding studies with (±)-[³H]FOR and on the resistance of (±)-[³H]FOR to displacement by high concentrations of AR agonists or antagonists, it was postulated that the stability of the FOR-AR complexes may contribute to its long-lasting therapeutical action (Lemoine, 1992).

The SLM-induced relaxation of airway smooth muscle is atypical in that it is slow in onset of action, prolonged in duration, and almost resistant to washout (Ball et al., 1991; Lindén et al., 1991). The tenacity of the action of SLM was first observed in vitro in isolated airway smooth muscle, in which SLM relaxation reappears after blockade by AR antagonist and successive washout of antagonist without the readdition of SLM, a phenomenon termed "reassertion" (Ball et al., 1991). This unique property of a AR agonist led to the hypothesis that SLM binds to two sites of the ₂AR: the classic AR site (active site) interacting with the saligenin moiety of SLM and a new site, called the "exo-site," to which the hydrophobic tail of the molecule is supposed to bind quasi-irreversibly. As a consequence of this hypothesis, a high-affinity binding of the hydrophobic tail to the exo-site is thought 1) to keep SLM in the vicinity of the receptor, thus improving the likelihood that the saligenin head interacts with the active site; and 2) to restore its action by flipping in and out of the active site after withdrawal of antagonists (Johnson et al., 1993).

The high lipophilicity of the compound (Rhodes et al., 1992) led to another plausible hypothesis, the "diffusion microkinetic model" of Anderson et al. (1994). The essential feature of the microkinetic model is that after the inhalation of SLM, a bulk concentration of the drug enters the plasmalemma lipid bilayer of airway smooth muscle cells and acts as an agonist depot even after withdrawal of the drug. In this model, drug access to the active site of the ₂AR occurs via lateral diffusion between the  helices into the ₂AR rather than via a direct approach from the extracellular aqueous biophase, thus accounting for the slow onset and long duration of action.

More recently, evidence in favor of the exo-site binding of SLM was published based on site-directed mutagenesis of ₂AR by replacing ₂AR amino acids 149 through 173 of the transmembrane spanning domain IV with the corresponding ₁AR sequences (Green et al., 1996). In the resultant constructs expressed in COS-7 cells and assayed by radioligand binding, SLM binding under washout conditions was reduced by 67%. In contrast were findings with structural analogs of SLM characterized by the same aliphatic side chain known to be responsible for exo-site binding of SLM, which, however, did not compete with SLM for the exo-site (Bergendal et al., 1996). All of the studies supporting exo-site binding and prolonged action cited above were performed in isolated tissues and superflow organ baths or with cell culture techniques that use large volumes of buffer and high amounts of SLM. We decided to use isolated receptor membranes that were pretreated in small volumes with low amounts of SLM and used the AR-coupled AC activity to assess SLM binding to the receptor. Some of these results have been published in preliminary form (Teschemacher and Lemoine, 1998).

	Materials and Methods

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Preparation of Rat Lung Membranes

Rat lung membranes were prepared as described by Lemoine et al., (1992). Briefly, lungs were placed in 1 mM ice-cold KHCO₃, minced, homogenized with a Polytron (1 × 2-s setting 7, 2 × 20-s setting 3.5), and filtered through Japanese silk. The crude homogenate was centrifuged at 120g for 10 min, the pellet was discarded, and the supernatant was collected at 40,000g for 30 min. Lung membranes were further purified by a sucrose density gradient (20:40% w/w) followed by a 2-h centrifugation at 100,000g to collect membrane particles. The AR density in purified membranes was approximately 1000 fmol/mg, as estimated in saturation binding experiments with [¹²⁵I]iodopindolol (IPIN).

Isolation of Smooth Muscle Cells from Calf Trachea

Cells were isolated by enzymatic disaggregation as detailed in Lemoine et al., (1989). Briefly summarized, tracheal strips were freed from cartilage, mucosa, and connective tissue at room temperature in Krebs' solution containing 89 mM NaCl, 30 mM NaHCO₃, 5 mM KCl, 1 mM Na₂HPO₄, and 0.5 mM MgSO₄, oxygenated with carbogen (95% O₂/5% CO₂). The ring muscle layer was then cut into narrow strips and incubated in an isotonic (130 mM) potassium-methanesulfonate solution, buffered with 10 mM HEPES at pH 7.4, and supplemented with 5 mM pyruvate, 5 mM creatine, 0.7 g/liter collagenase D (Boehringer Mannheim, Mannheim, Germany), and 0.1 g/liter Pronase E (Serva, Heidelberg, Germany). During enzymatic disaggregation, muscle strips were gently moved by magnetic stirring. Cells were harvested every 30 min and washed once in isotonic buffer, yielding fusiformed smooth muscle cells. Membranes were obtained after gentle homogenization of cells with a glass-glass homogenizer and centrifugation with 40,000g for 30 min at 4°C. The pellet was resuspended in 3 mM EGTA (pH 7.4) with 0.3 mM ascorbic acid and stored at 80°C until use. The protein content was determined according to Bradford (1976) using BSA as a standard.

Radioligand Binding

Experiments were carried out in rat lung membranes at 37°C in 50 mM Tris·HCl (pH 7.4), 2 mM MgCl₂, 1 mM EGTA, and 0.1 mM ascorbic acid and in the presence of a nonhydrolyzable GTP analog {10 µM guanosine-5'-(,-imido)triphosphate [Gpp(NH)p]}) to convert the receptors in the low-affinity state for agonist binding. In addition, 1 µM CGP 20,712 A was added, a concentration known to selectively block ₁AR (Lemoine and Kaumann, 1991; Lemoine and Overlack, 1992). To measure slow dissociation of agonists, lung membranes were pretreated with a nonradioactive agonist and used, after dilution, for association binding with a radioligand ([¹²⁵I]IPIN). Untreated membranes were used as a control. A delayed association of the radioligand (L*) with the receptors (R) was expected for agonists (A) with slow dissociation (Fig. 1). Membranes were preincubated for 30 min with agonist in a small volume. After 30 min, 3-µl samples of pretreated membranes were transferred into a volume of 150 µl (dilution of 1:50) containing radioligand (0.2-0.4 nM [¹²⁵I]IPIN), 10 µM Gpp(NH)p, and 1 µM CGP 20,712 A. Thus, dissociation of the agonist was induced by a 50-fold dilution and the simultaneous addition of the radioligand. Binding observed in the presence of 0.2 mM ()-isoproterenol was regarded to be nonspecific (B_ns 10%). Bound radioligand was separated from free radioligand by rapid vacuum filtration through Whatman GF/A glass-fiber filters. Bound radioligand was counted in a gamma counter with an efficiency of 70%. Association kinetics of [¹²⁵I]IPIN (L*) were analyzed (see also Lemoine, 1992) by nonlinear regression as a pseudo-first order kinetic according to

(1)

where B_s, B_ns, and B_eq represent specific, nonspecific, and equilibrium binding, respectively, and k_app is related to the apparent time constant of association by T_[1/2] = ln 2/k_app. Dissociation of [¹²⁵I]IPIN induced by a maximum effective concentration of isoprenaline (ISO; 0.2 mM) was analyzed according to

(2)

where k_off represents the time constant of the dissociation. The time constant of association k_on was calculated according to

(3)

The ratio of time constants k_off/k_on should match the equilibrium dissociation constant K_D.

To determine equilibrium dissociation constants K_D of agonists for binding (B) to ₂AR, competition binding experiments were performed with [¹²⁵I]IPIN (L*) in the presence of increasing concentrations of agonists (A) and analyzed by nonlinear regression (Ehle et al., 1985; Lemoine et al., 1985) according to

(4)

where K_L* and K_C are the equilibrium dissociation constants of the radioligand L* and of the ₁AR antagonist CGP 20,712 A, respectively.

AC Assay

Assays were carried out in 60 µl of a reagent (MIX) containing 0.04 mM [-³²P]ATP (250-350 cpm·pmol¹), 100 mM Tris·HCl (pH 7.4), 2 mM MgCl₂, 1 mM EGTA, 0.1 mM ascorbic acid, 0.01 mM GTP, 1 mM [³H]cyclic AMP (cAMP; 10.000 dpm/tube), 20 mM creatine phosphate as substrate of the ATP-regenerating enzyme system, and the enzymes (IU·ml¹) 15 mM creatine phosphokinase and 9.8 mM myokinase. The reaction was performed at 32.5°C and terminated by a quick freeze stop in liquid nitrogen and the addition of 50 µl of a stop solution containing 10 mM cAMP, 40 mM ATP, and 1% SDS. Cyclic [³²P]AMP was purified by double-column chromatography as described by Salomon et al. (1974) and measured by liquid scintillation counting.

AC Kinetics

For the analysis of receptor interactions with long-acting SLM in comparison to other ₂ SYM, kinetic experiments were designed based on the close coupling of receptors to AC; the association of an agonist with a receptor is directly translated to an increase in cyclase activity and after the dissociation of the agonist AC activity immediately ceases. Thus, the dissociation of SLM was analyzed under several conditions and compared with that of other agonists.

Dissociation by Addition of a Blocker. The assay was started by addition of 20 µl of native receptor membranes to 40 µl of AC buffer supplemented with the indicated agonists (see Fig. 3). The AR antagonist ()-bupranolol (BU; 1 µM) was added to the test tubes 5 min after the start of the reaction to induce the dissociation of agonists.

Dissociation by Dilution. Dissociation of drug/receptor complexes was induced by a 100-fold dilution of membranes preincubated with various agonists (ISO, FOR, SLM) in a small volume (20 µl, Fig. 4A step 1) (see Figs. 4A, 5, and 6). Drug concentration was 10 K (condition HIGH and Dissociation) and 0.1 K (condition LOW). After a 15-min preincubation at 32.5°C, a 100-fold volume (2 ml) of MIX reagent was added containing a drug concentration of 10, 0.1, or 0 K for the conditions HIGH, LOW, and Dissociation (Fig. 4A step 2), respectively. Aliquots of 50 µl were taken at the times indicated in the Figs. 5 and 6.

Dissociation by Dilution after SLM Loading. SLM-loaded membranes were obtained by pretreatment with 20 nM SLM in a large volume of 40 ml for 1 h at 10°C (Fig. 4B step 1) (see Figs. 7 and 8). Membranes were collected by centrifugation at 40,000g and 4°C for 30 min. After resuspension of pellets, membranes were preincubated with 20 nM SLM in the absence (condition HIGH) or presence of 100 K_B blocker [80 nM BU; condition LOW] or in the absence of all drugs (condition Dissociation) in a volume of 20 µl (step 2). After 15 min at 32.5°C, AC reaction was started by the addition of 2 ml of MIX reagent (Fig. 4B step 3) containing the same concentrations of SLM and BU as used for preincubation. Aliquots were taken and stopped at the times indicated.

Comparison of Differently Pretreated Membranes. Membranes were loaded with SLM as described in Fig. 4B and compared with untreated controls (sham membranes) (see Figs. 8 and 9). As a third condition to test a putative wash-out effect, SLM-loaded membranes were washed by incubation in 40 ml of fresh buffer (3 mM Tris, 3 mM EGTA, pH 7.4) before being collected by centrifugation. Membrane pellets in all three conditions had a volume of 100 µl and were resuspended in 10 ml. AC assays were started without preincubation by the addition of 20 µl of membrane suspensions to 40 µl of AC buffer in the presence or absence of drugs. The reaction was stopped after 20 min by freeze stop.

Estimation of Free SLM Concentration

Bioassay for SLM Concentration. The free concentration of SLM in SLM-loaded membranes can be estimated 1) directly by comparison of preloaded membranes to native membranes stimulated with SLM in a concentration-dependent manner and 2) indirectly from the inhibition of AC activity in preloaded membranes by an antagonist. For this purpose, the concentration-effect curves for agonists and antagonists must be fitted by nonlinear regression according to the following set of equations. Concentration-effect curves for AC stimulation by SLM (see Fig. 9, top) were analyzed according to:

(5)

where [cAMP]_max and [cAMP]_bas represent maximal and basal rates of cAMP production, respectively, and [cAMP]_SLM is the rate depending on SLM concentration [SLM]. The EC₅₀ value indicates the SLM concentration at which cAMP production is half-maximal. Inhibition curves with the  blocker BU (see Fig. 9, bottom) were analyzed according to:

(6)

where IC₅₀ stands for the BU concentration [BU] at which inhibition of cAMP production is half-maximal in a SLM-prestimulated system. Using the EC₅₀ value for stimulation by SLM (EC_50,SLM) and the IC₅₀ value for inhibition of SLM-prestimulated membranes by BU, the dissociation constant of BU (K_B) is

(7)

The unknown effective concentration of SLM in SLM-loaded membranes can be calculated by rearranging eq. 7:

(8)

Estimation of the SLM Concentration with Partition Coefficients. Rhodes et al. (1992) determined membrane partition coefficients K_p[mem] according to

(9)

where m(lipid) and m(water) represent the mass of liposomes and water, respectively, and m(drug) is the mass of drug in the prevailing condition. We adapted eq. 9 to our system and estimated the fraction of SLM trapped in the membranes after the loading process according to

(10)

where m(membrane lipids) and m(buffer) represent the mass of membrane lipids and buffer, respectively, and m(SLM) is the mass of SLM in the prevailing condition.

Drugs and Materials

[-³²P]ATP (22.2 TBq/mmol) and [¹²⁵I]IPIN (81.4 TBq/mmol) were purchased from Du Pont de Nemours (Dreieich, Germany). Cyclic [³H]AMP (1.33 TBq/mmol) was purchased from the Radiochemical Center (Amersham, UK). ATP, cAMP, GTP, Gpp(NH)p, creatine phosphate, myokinase, ()-ISO HCl, and forskolin were obtained from Sigma Chemical Co. (St. Louis, MO). Creatine phosphokinase was obtained from Calbiochem (La Jolla, CA). The antagonists BU and CGP 20,712 A (1-[2(3-carbamoyl-4-hydroxy phenoxy)-ethylamino]3-[4-(1-methyl-4-trifluoromethyl-2 imidazolyl)phenoxy]-2-propanol methanesulfonate) were purchased from Schwarz-Sanol (Monheim, Germany) and Ciba-Geigy (Basel, Switzerland). ()-FOR and (±)-SLM were generous gifts from Ciba-Geigy (Basel, Switzerland) and Glaxo (Hamburg, Germany), respectively.

	Results

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Radioligand Binding. The dissociation of SLM, which is not a promising candidate to be developed as a radioligand because of its high lipophilicity, can be studied only indirectly (i.e., by a delayed association of a radioligand that itself is characterized by fast association kinetics) as shown in Fig. 1. We chose [¹²⁵I]IPIN for this purpose and studied its receptor binding kinetics in rat lung membranes that possess a high density of AR. The receptor density was measured with saturation binding, resulting in a B_max value of 1093 ± 24 fmol/mg protein and a dissociation constant (log, M) of 9.67 ± 0.09 for [¹²⁵I]IPIN (experiment not shown). To study receptor -binding kinetics, the subdominant ₁ subtype of lung AR (Lemoine, 1992) was antagonized with 1 µM CGP 20,712 A, a concentration known to prevent binding to ₁AR without influencing binding to ₂AR (Lemoine and Kaumann, 1991). A nonhydrolyzable GTP analog [10 µM Gpp(NH)p] was used to convert the receptors from the high-affinity to the low-affinity binding state for agonists. Thus, it was possible to study the association and dissociation kinetics of [¹²⁵I]IPIN to a homogeneous class of low-affinity ₂AR. The kinetics followed a simple exponential relation and were fitted by nonlinear regression to eqs. 1 and 2, respectively. The parameter estimates listed in Table 1 illustrate fast association and slow dissociation of [¹²⁵I]IPIN with half-times (T_1/2) of approximately 1.0 and 5.2 min, respectively. The ratio of kinetic constants (Log k_off/k_on = 10.25 ± 0.24) matched the dissociation constant pK_D of 9.67 ± 0.09 determined by saturation binding, confirming the high affinity of [¹²⁵I]IPIN for ₂AR. For the agonists of interest (ISO, FOR, SLM), competition binding experiments (not shown) were performed in the presence of 10 µM Gpp(NH)p. The dissociation constants (pK_D) estimated by nonlinear regression (eq. 4) were 6.62 ± 0.04 for ISO, 8.06 ± 0.04 for FOR, and 8.44 ± 0.04 for SLM.

View this table: [in this window] [in a new window]   TABLE 1 Comparison of kinetic constants for the association of [125I]PIN in rat lung membranes with and without preincubation of AR ligands

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Dissociation of preassociated receptor-agonist complexes (R-A) induced by dilution and simultaneous addition of radioligand (L*).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Association of [125I]IPIN after preincubation of receptor membranes with ISO and SLM. Membranes of rat lung (2.78 ± 0.04 µg/tube) were preincubated with 5 µM ISO (top) and 100 nM SLM (bottom). Dissociation of drugs was induced by 50-fold dilution of preincubated membranes with Tris buffer containing 0.39 nM [125I]IPIN. Experiments were carried out in the presence of 1 µM CGP 20,712 A to prevent binding to 1AR and 10 µM Gpp(NH)p to prevent binding to the high-affinity state of 2AR. Symbols and bars represent mean ± S.E.M. of quadruplicate determinations. Data were analyzed by nonlinear regression according to eq. 1. The apparent association constant (kapp) of [125I]IPIN was 0.835 ± 0.054 min1 after preincubation with ISO and 0.486 ± 0.031 min1 after preincubation with SLM. The association of [125I]IPIN after preincubation with SLM was only slightly delayed compared with ISO. See also Table 1.

AC Assay

In regard to the exo-site hypothesis (Johnson et al., 1993), the fast dissociation of SLM induced by a radioligand is not sufficient to disprove the hypothesis; that is why AC experiments were designed that also allow the dissociation of SLM to be determined in the absence of antagonists. Thus, dissociation of SLM was induced by a 100-fold dilution of membranes preincubated in a small (20 µl) or a large (40 ml) volume of buffer containing 20 nM SLM and compared with dissociation kinetics induced by a AR antagonist. For AC experiments, membranes of disaggregated tracheal cells were used that can be stimulated maximally up to 10-fold over basal by AR agonists (Lemoine et al., 1989).

Blocker-Induced Dissociation of SLM. Stimulation of AC activity by maximum effective concentrations of ISO and SLM induced an immediate increase of cAMP production that was linear with time up to 20 min (Fig. 3). Maximal AC stimulation by ISO was 4.7 times higher than basal activity, and that by SLM was 2.5 times higher. Thus, SLM was characterized as a partial agonist. Dissociation of SLM was induced by the addition of a surplus (10 µM) of the AR blocker BU added 5 min after the start of the reaction with SLM. With the kinetic constants measured with the [³H] derivative of BU (k_on = 1.21 min¹·nM¹ of the radioligand, k_off = 0.26 min¹; Lemoine et al., 1985), it could be calculated that the association of BU at a concentration of 10 µM would occur with a half-time of less than 30 ms (i.e., the association of the competing ligand with the receptors was not rate limiting). The experiment depicted in Fig. 3 (triangles) shows that the dissociation of SLM was fast and complete, resulting in a line parallel to basal activity. Thus, it had to be concluded that the dissociation of SLM from the ₂AR was complete within less than 1 min. Similar experiments (not shown) performed with 100 nM ()-FOR and 5000 nM ()-ISO showed an immediate association by the addition of the respective agonist and an immediate dissociation by the addition of BU.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Immediate interruption of SLM-induced AC stimulation by blocker addition. Kinetics of 2AR-induced AC stimulation were measured in membranes (4.4 ± 0.1 µg/tube) derived from disaggregated cells of calf trachea. Basal () and maximum activity () were determined in the absence of ISO and in the presence of 10 µM ISO. The reaction induced by 1 µM SLM () was antagonized by the addition of 10 µM BU () at 5 min (small arrow). The broken line is parallel to the basal line and represents the immediate interruption of AC activity by the blocker. The small difference between the triangles and the broken line indicates that the dissociation of SLM is complete within less than 1 min. Symbols and bars represent mean ± S.E.M. of duplicate determinations. All conditions showed a linear increase of cAMP formation.

Dissociation of SLM Induced by Dilution. Membranes of tracheal myocytes were preincubated with 20 nM SLM in a very small volume (20 µl; Fig. 4A); dissociation was induced by a 100-fold dilution with MIX reagent containing [-³²P]ATP to start the AC reaction simultaneously (Fig. 5). A concentration of 20 nM (10 K) was chosen to cause a submaximal (about 90%) stimulation of AC activity. The basal rate of cAMP formation of 98.4 ± 3.7 pmol·mg¹·min¹ was stimulated to 368.8 ± 5.2 pmol·mg¹·min¹ with 200 µM ISO. With 10 K SLM, the cAMP formation rate was 314.4 ± 10.6, and that with 0.1 K SLM was 131.0 ± 4.0 pmol·mg¹·min¹. As with dissociation by adding a blocker (Fig. 3), the dissociation by dilution (Fig. 5) caused an immediate cessation of AC stimulation by SLM, in contrast to the predictions of the exo-site hypothesis. The kinetic after dilution matched the kinetic stimulated with 0.2 nM SLM, corresponding to the 0.1 K condition. Both kinetics could be fitted with straight lines. It can be concluded from the small difference between the two lines that the dissociation of SLM is complete within 1 min and that there is no hint at a prolonged action of SLM on the level of receptor-coupled AC.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Incubation scheme for preincubation (A) and preloading (B) of membranes with HIGH (10 K) or LOW (0.1 K) concentration of drugs. A, membranes were preincubated in a small volume (20 µl) with 10 K of drugs before the AC reaction was started by adding the [-32P]ATP-containing reaction mixture. B, membranes were preloaded for 60 min in a large volume (40 ml) with 20 nM ( 10 K) of SLM before the AC reaction was started by adding the [-32P]ATP-containing reaction mixture.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Dilution induced a complete reversal of AC stimulation by SLM. Cell membranes of tracheal smooth muscle cells (4.2 ± 0.1 µg/tube) were preincubated according to Fig. 4A in a small volume (20 µl) with a high SLM concentration (20 nM = 10 K). The AC reaction was started by adding the [-32P]ATP-containing reaction mixture, resulting in a 100-fold dilution of SLM to induce dissociation (). As control conditions [-32P]ATP-containing reaction mixtures supplemented with 0.2 (0.1 K, ) and 20 nM () SLM were added to membranes pretreated with either 0.2 or 20 nM SLM. Symbols and bars represent mean ± S.E.M. of duplicate measurements ( and ); triangles are single determinations. Dotted lines indicate basal and maximal rates of cAMP formation in the absence or presence of 200 µM ISO. All conditions followed a linear relationship between cAMP production and reaction time. The slight difference between the time course after dissociation () and that in the presence of 0.2 nM SLM () indicates that the dissociation of SLM is complete within less than 1 min.

Dissociation of ()-FOR and ()-ISO Induced by Dilution. As with SLM, the dissociation of the long-acting ₂SYM ()-FOR and of the short-acting catecholamine ISO was investigated by dilution of pretreated membranes of tracheal myocytes. Membranes were pretreated with concentrations of 500 nM ISO (Fig. 6, bottom) and 50 nM ()-FOR (Fig. 6, top), which were submaximally effective with V_max values approximately 3.5 times basal rates. As in the previous experiments, a 100-fold dilution caused an immediate cessation of AC activity by AR agonists; reaction kinetics after dilution were linear and matched linear kinetics stimulated with 0.1 K concentrations.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Dilution induced a complete reversal of AC stimulation by ISO and FOR. Cell membranes of tracheal smooth muscle cells (4.6 ± 0.1 µg/tube) were preincubated according to Fig. 4A in a small volume (20 µl). Experiments were performed as described in the legend to Fig. 5. A high drug concentration (10 K; i.e., 50 nM FOR and 500 nM ISO) was used to pretreat receptor membranes before dissociation () was induced by a 100-fold dilution of agonist-pretreated membranes. Acute stimulation was done at high (10 K, ) and low (0.1 K, ) concentrations of agonists as indicated. Symbols and bars represent mean ± S.E.M. of duplicate measurements ( and ); triangles are single determinations. Dotted lines indicate basal and maximal rate of cAMP formation in the absence or presence of 200 µM ISO. All conditions followed a linear relationship between cAMP production and reaction time.

Absence of Dissociation after Loading of Membranes with SLM. To examine whether the total amount of SLM offered to the membranes during pretreatment has an influence on the dissociation of SLM, membranes were incubated in 40 ml of Tris buffer with 20 nM SLM for 60 min at 10°C, a process termed "loading". After collection of the membranes by centrifugation, the AC assay was performed as depicted in Fig. 4B. In addition to the submaximal stimulation (474.9 ± 3.8 pmol cAMP·min¹·mg protein¹) at a concentration equivalent to 10 K (20 nM SLM) and to the 100-fold dilution, a third condition was chosen, which showed that 80 nM BU nearly completely reversed prestimulation with SLM (Fig. 7), reducing activity to basal rates (137.2 ± 2.4 pmol cAMP·min¹·mg protein¹). In contrast to the blocker-induced reversal of AC stimulation in preloaded membranes and to the reversal by a 100-fold dilution after pretreatment of membrane in a small volume (Fig. 5), a 100-fold dilution of preloaded membranes was quite ineffective (Fig. 7). The cAMP production after dilution was almost as high as that in the membranes stimulated with 20 nM SLM. From this experiment, it becomes apparent that the exo-site binding thought to be accompanied by slow dissociation kinetics can be mimicked by incubating the membranes in a large volume of buffer containing SLM, presumably by trapping of SLM in the phospholipid bilayer of the receptor membranes.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Absence of dissociation by dilution after preloading of membranes with (±)-SLM. Membranes of smooth muscle cells were preincubated ("preloaded") with SLM (20 nM) for 1 h at 10°C in a final volume of 40 ml (Fig. 4B). After preloading, membranes were collected by centrifugation at 40,000g for 30 min and then resuspended in reaction buffer and assayed under three different conditions. The first condition was designed to measure dissociation by 100-fold dilution (), and the second () and third () conditions were assayed in the presence of 20 nM SLM with or without AR antagonist (80 nM BU). The second and third conditions resulted in an occupation of the available 2AR by SLM of approximately 90% and 10%, respectively. Broken lines represent basal and maximal activation in the absence or presence of 200 µM ISO. The protein content per test tube was 3.9 ± 0.1 µg. Symbols and bars represent mean ± S.E.M. of duplicates ( and ); triangles are single determinations. All conditions followed a linear relationship between cAMP production and reaction time.

Ineffectiveness of Washing of Preloaded Membranes. Sham membranes (Fig. 8) were compared with membranes submitted to the loading procedure with SLM ("preloaded") and with preloaded membranes that were washed by centrifugation ("loaded & washed"). AC activity was stimulated via AR using ISO or SLM, or directly with forskolin and was compared with basal conditions in the absence and presence of the AR antagonist BU. The experiment clearly indicates 1) that the basal rate in sham membranes matched the rate in the presence of the AR antagonist in preloaded membranes; 2) that the elevated rate of cAMP formation in SLM-preloaded membranes persisted without the addition of SLM during incubation, thereby confirming the results of Fig. 7; and 3) that the washing procedure did not reduce the rate of cAMP formation in preloaded membranes, hinting at the effectiveness of trapping of SLM by the membranes.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   SLM-preloaded membranes showing the ineffectiveness of washing. Membranes were preloaded with SLM as described in the legend to Fig. 7. After centrifugation, pellets were resuspended at a ratio of 1:100 (v/v) with buffer. A portion of the SLM-preloaded membranes was reincubated for 10 min in SLM-free buffer and recollected by centrifugation. Sham membranes were treated in the same manner but remained without SLM. AC assays were started by the addition of membrane suspensions (4.5 µg/test tube) and continued for 20 min at 32.5°C. Columns and bars represent mean ± S.E.M. of quadruplicate determinations. Basal AC activities determined in the presence of BU were 94 ± 2 (sham), 103 ± 2 (preloaded), and 107 ± 1 (preloaded and washed) pmol·mg1·min1. The AC activity induced by a maximal effective concentration of ISO was 326 ± 7 (sham), 317 ± 6 (preloaded), and 320 ± 14 (preloaded and washed) pmol·mg1·min1. Forskolin-induced AC activity was 457 ± 12 (sham), 474 ± 33 (preloaded), and 547 ± 2 (preloaded and washed) pmol·mg1·min1. No difference between control and BU-blocked AC activity could be found in sham membranes, whereas in preloaded membranes, basal AC activity was 21.5 ± 0.7% (preloaded) and 20.3 ± 0.9% (preloaded and washed), higher than in the presence of antagonist.

Estimation of Unknown SLM Concentration in Preloaded Membranes by Bioassay. A bioassay was designed to determine the unknown SLM concentration of preloaded membranes with the help of the SLM-induced AC stimulation. As detailed in Materials and Methods, the unknown SLM concentration was determined (Fig. 9) as a shift of inhibition curves by an antagonist (BU), comparing the inhibition in preloaded membranes (pIC₅₀ = 8.46 ± 0.08) with sham membranes in the presence of 20 nM SLM (pIC₅₀ = 8.08 ± 0.07). Taking into account the estimated dissociation constant of BU (pK_B = 9.16, eq. 7), the pEC₅₀ value for SLM of 8.74 (Fig. 9, top), and the volume of buffer used for resuspension of pelleted membranes (100 µl of membranes were resuspended in 30 ml of buffer), the unknown SLM concentration in preloaded membranes was approximately 7.3 nM (eq. 8). This is the concentration that is effective under AC assay conditions, and it corresponds to a SLM concentration of 2.4 µM (i.e., 240 pmol in 100 µl) in the membrane pellet and to a 120-fold accumulation during the loading process (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Bioassay for estimating of the effective SLM concentration in SLM-preloaded membranes. AC assays were performed with the same membranes (sham and SLM-preloaded) as for Fig. 8. The dose-response curve with SLM (top) was determined in sham membranes. Inhibition curves by BU were measured in sham membranes after the addition of 20 nM SLM (bottom, ) and in the SLM-preloaded membranes without adding further SLM (bottom, ). Symbols and bars represent mean ± S.E.M. of triplicate determinations. Curves were fitted by nonlinear regression according to eq. 5 (stimulation) and eq. 6 (inhibition). Top, in sham membranes, estimated values of pEC50 and intrinsic activity were 8.74 ± 0.06% (log, M) and 70.7 ± 2.9%. Basal and maximal rates (pmol·mg1·min1) of cAMP formation were 124.9 ± 3.7 and 310.2 ± 6.7. Bottom, sham membranes were assayed in the presence of 20 nM SLM. The pIC50 values for the inhibition of SLM by BU were 8.08 ± 0.07 in sham membranes and 8.46 ± 0.08 in SLM-preloaded membranes. Basal AC activities were 133.6 ± 5.6 () and 140.6 ± 5.0 () pmol·mg1·min1, and activities in the absence of BU were 300.2 ± 3.5 (sham, 89.9 % of maximum) and 243.4 ± 3.3 (preloaded, 55.5 % of maximum) pmol·mg1·min1.

Estimation of Unknown SLM Concentration in Preloaded Membranes Using Partition Coefficients. Partition coefficients had been reported to be 22,500 for the membrane partition coefficient K_p[mem] and 7600 for the octanol-water coefficient K_p[o/w] (Rhodes et al., 1992). Assuming as a first approximation that the mass of membrane lipids equals that of membrane proteins, the mass of membrane lipids that contributed to the trapping of SLM during the loading process (Fig. 4B) was about 2.5 mg. Using this assumption and with a value for K_p[mem] = 22,500, eq. 10 shows that about 58% of the free SLM concentration was trapped in membrane lipids. Given a concentration of 20 nM SLM in a volume of 40 ml (corresponding to a total of 800 pmol of SLM) offered to the membranes during loading, the mass of trapped SLM was approximately 470 pmol. On the basis of octanol-water coefficient, eq. 8 yields an estimate of 32% (260 pmol of SLM) accumulated in the membrane lipids. This latter value for the amount of trapped SLM matches the amount of SLM estimated by bioassay (30% and 240 pmol).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our results using AR-coupled AC activity as a measure of the occupancy of ₂AR by long-acting ₂SYM clearly indicate that in isolated receptor membranes 1) the onset of receptor occupation and the on kinetics of AC begin as soon as the agonist is added and 2) the off kinetics of AC, which represent dissociation of receptor-agonist complexes, immediately follow dilution or the addition of antagonist. These findings were made with a hydrophilic catecholamine (ISO, log K_ow = 0.64), a weak lipophilic agonist (FOR, log K_ow = 1.40), and a strong lipophilic partial agonist (SLM, log K_ow = 4.15; K_ow values are octanol/water partition coefficients calculated according to Meylan and Howard, 1995). In the case of SLM, these findings contradict the exo-site hypothesis, which essentially predicts a delay of dissociation from the exo-site combined with sustained activation of ₂AR and receptor-mediated signal transduction.

To elucidate what may have led to the postulation of the exo-site hypothesis, we tried to imitate the experimental conditions that underlaid its postulations. These experiments were done in organ baths with superfused tissues (Ball et al., 1991; Nials et al., 1993) or in cell culture systems (McCrea and Hill, 1993; Green et al., 1996) characterized by the use of high amounts of SLM. For example, in experiments with isolated tracheal strips, 100 nM SLM were superfused for 20 min at a flow rate of 2 ml/min, resulting in a total application of 4000 pmol of SLM to the tissue (Ball et al., 1991); or in cell culture experiments, 1 µM SLM was incubated for 10 min in 35-mm dishes, resulting in a total application of about 2000 pmol of SLM to the cell monolayers (Green et al., 1996). In the experiments we designed to mimic exo-site binding of SLM with receptor membranes, 40 ml with only 20 nM SLM (i.e., 800 pmol of SLM) was preloaded for 60 min at 10°C; nevertheless, the dissociation by dilution failed (Fig. 7). This is in stark contrast to the experiments with membranes preloaded in a small volume of 20 µl (i.e., 0.4 pmol of SLM, Fig. 5) and indicates that it was the manipulation of experimental conditions and not a characteristic behavior of the receptor that was responsible for the persistence of SLM effects.

The same line of evidence was found with radioligand-binding techniques applied to rat lung membranes. Whereas the pretreatment with high concentrations of SLM (10 µM, corresponding to about 1000 K_D of the drug) completely prevents the access of [¹²⁵I]IPIN to ₂AR (Jack, 1991; Johnson, 1991; Nials et al., 1993), smaller concentrations (100 nM, corresponding to about 10 K_D), which are supposed to be achieved during therapy of bronchial asthma, have no effect on the access of [¹²⁵I]IPIN to ₂AR (this report). Based on these results, it was concluded that [¹²⁵I]IPIN, by virtue of its 2-iodo substituent, acquires a molecular size sufficient to deny its access to the binding sites of ₂AR when SLM is present (Jack, 1991). In contrast, other groups suppose that [¹²⁵I]iodocyanopindolol, which is an even bigger molecule than [¹²⁵I]IPIN, freely interacts with the receptor binding site even though SLM is quasi-irreversibly bound to the exo-site (Clark et al., 1996; Green et al., 1996), hinting at some inconsistencies in exo-site theory.

Recently, the exo-site hypothesis was supported by experiments using site-directed mutagenesis to identify amino acids of the ₂ AR involved in the exo-site binding of SLM (Green et al., 1996). The replacement of these amino acids by the analogous ₁AR sequences, which are not suspected of contributing to exo-site binding, resulted in mutants that were expressed in COS-7 cells and assayed by radioligand binding with [¹²⁵I]iodocyanopindolol. However, the reduction of radioligand binding by SLM in cells with wild-type ₂AR was only 38.2% [in contrast to a 90% inhibition of binding in rat lung membranes reported by Jack (1991) and Nials et al. (1993)], and only part of the 38.2% fraction disappeared in experiments with mutant receptors. Taking into account that these data were derived from saturation binding in different cell cultures with strongly varying receptor densities and normalized to the protein content of the cell preparations, they should not be taken as unequivocal evidence.

Another line of evidence casting doubt on the exo-site hypothesis is based on the failure of SLM analogs to block the exo-site binding of SLM (Bergendal et al., 1996). SLM analogs were designed and synthesized to interact with SLM at the putative high-affinity binding site of the aliphatic side chain. A reasonable assumption of this experimental approach was that a high specificity of a recognition site (exo-site), characterized by high affinity, should constitute the structural basis for competition by related structural analogs. A second line of theoretical considerations focuses on the point that a drug that binds with high affinity to a site near a receptor with a slow onset of association (Jack, 1991; Johnson, 1991) will not interact with the receptor binding sites in a simple competitive manner following the law of mass action but rather will be characterized by steep concentration-effect curves (Hill slope 1) and other nonequilibrium effects as observed in isolated tissues of guinea pig trachea (Dougall et al., 1991) and human bronchus (Anderson et al., 1994; Naline et al., 1994). Despite these conclusions drawn from the exo-site hypothesis, concentration-effect curves for AC stimulation (Fig. 9; Clark et al., 1996) and binding inhibition curves (this report; Clark et al., 1996) have Hill slopes close to 1.0.

In conclusion, we have found that in receptor membranes incubated (but not flooded) with appropriate concentrations of SLM, neither a slow onset nor a long duration of action can be found. Clinical effects of SLM such as slow onset and long duration of action over 12 h, however, distinguish it from traditional inhaled AR agonists and qualify the drug for the maintenance treatment of reversible airway obstruction (Ullman and Svedmyr, 1988; Pearlman et al., 1992; Lötvall et al., 1994). Thus, we conclude by exclusion that partitioning of the drug in lipophilic compartments after inhalation along with its high affinity for lung ₂AR underlies its long duration of action as outlined in the "diffusion microkinetic model" (Anderson et al., 1994; reviewed by Lindén et al., 1996; and Waldeck, 1996).