Smooth muscle cells from human bronchi were purchased from Invitrogen-Cambrex (Walkersville, MD). Cell culture supplies, forskolin, PMA, isobutylmethylxanthine (IBMX) and isoproterenol were purchased from Sigma Chemical Co (St. Louis, MO); LTD₄ and cAMP EIA kit from Cayman Chemical Co. (Ann Arbor, MI); montelukast was a gift from Merck & Co. (West Point, PA). GF109203X and H89 were from Calbiochem (La Jolla, CA). DC™Protein assay from Bio-Rad Laboratories (Richmond, CA). Bronchial rings for functional studies were obtained from 6 non-asthmatic patients undergoing thoracotomy for lung cancer.

Monolayers of HASMC from human bronchi were grown in Minimum Essential Medium supplemented with 10% FBS, 100-U/ml penicillin, and 100-μg/ml streptomycin, as previously described in detail 25. Cells were used between 3^rd and 8^th passage at a 1:3 ratio in 75-cm² culture flasks. At least two different cell line have been used.

Accumulation of cAMP was measured in cells grown to confluence in 12-well plates and serum-starved for 24 h. Cells were incubated at 37°C for 10 min in 1-ml PBS containing 3 × l0^-4M ascorbic acid and 10^-3M isobutylmethylxanthine. Reactions were stopped by placing the plates on ice, cells were then washed once with cold PBS and 150 μl of l0^-1M HC1 were added to each well. After 20-min incubation, cells were scraped and centrifuged 12000 × g for 10 min. Supernatant solutions were first assayed for protein concentration and then for cAMP content using a cAMP EIA-kit following manufacturer's instructions. cAMP concentrations of unknown samples were determined by computer-assisted interpolation from a standard curve.

Concentration-response curves of cAMP accumulation in response to isoproterenol (10^-9M to 10^-4M) were obtained in HASMC at control (vehicle treated) or after exposure to LTD₄ (10^-6M for 30 min), with or without 30-min pre-incubation with 10^-6M GF109203X. The increase of cAMP above baseline in response to 10^-5M isoproterenol was studied in HASMC at control and after 30-min exposure to 10^-6M LTD₄ or 5 × l0^-7M PMA, with or without 10^-6M montelukast, GF109203X, or H89. The effect of 10^-4M forskolin was studied by 10-min incubation after 30-min exposure to either vehicle or LTD₄.

24 bronchial rings from surgical specimens were passively sensitized against dust mites by an overnight incubation (18 h) at room temperature with serum pooled from three atopic subjects diluted 1:9 in aerated (95% O₂, 5 % CO₂) PSS of the following composition (mM): NaCl 110.5, KC1 3.4, CaCl₂ 2.4, MgSO₄ 0.8, KH₂PO₄ 1.2, NaHCO₃ 25.7, and dextrose 5.6, as previously described in details 26. The serum specific concentrations of specific IgE for Dermatophagoides Pteronyssinus and D. Farinae were larger than 13.2 Phadebast RAST units/ml (Pharmacia, Uppsala, Sweden) and the total serum concentration was 180 ± 33 international units/ml. Nineteen sensitized rings were incubated with montelukast (10^-7M, n = 5 and 10^-6M, n = 5), or GF109203X (10^-7M, n = 2 and 10^-6M, n = 1), or PSS (n = 6) for 30 min and then challenged by a 60-min incubation with 200 AU/ml of Dermatophagoides mix at 37°C. Challenged rings incubated with PSS alone served as control (n = 6). Rings were then suspended in water-jacketed 25-ml tissue baths containing aerated PSS at 37°C using two stirrups connected to a fixed hook at the bottom of the tissue bath and to a force transducer via a silk string, respectively. Rings were gradually stretched until a steady reference length of 1 gr was achieved. PSS was changed every 20 min. All rings were contracted with 10^-6M carbachol and, after a steady contraction was achieved, relaxed with salbutamol added cumulatively from 10^-9M to 10^-4M with half-Log increments. Each concentration-response curve was fitted by sigmoid least-square interpolation between extreme values constrained at 100% (maximal carbachol-induced force) and 0 (minimal force at 10^-4M salbutamol).

All curves shown were analyzed by Prism-4 software using the four parameters logistic equation and parameters compared using the extra sum of square principle 27. Parameter errors are expressed as percentage coefficient of variation (%CV) and calculated by simultaneous analysis of at least two different and independent experiments performed in duplicate or triplicate (for HASMC). One-way independent or two-way repeated-measure analysis of variance (ANOVA) were used whenever appropriate with Dunnett or Bonferroni post-hoc tests for multiple comparisons. P values < 0.05 were considered statistically significant. Data are expressed as means ± S.E.M.

Increasing concentrations of isoproterenol caused a concentration-dependent accumulation of cAMP in all experiments.

After challenge with LTD₄ (Fig. 1A) the maximum cAMP accumulation was significantly (P < 0.05) reduced (33%) from 4109 pmol/mg prot (CV 10%) to 2760 pmoles/mg prot (CV 13%), whereas EC₅₀ was substantially unaffected (from 0.68 μM, CV 59% to 0.69 μM, CV 82%). In montelukast-treated and LTD₄-challenged HASMC (Fig. 1B), isoproterenol-induced cAMP accumulation was not significantly different from unchallenged HASMC and significantly greater than in untreated LTD₄-challenged HASMC (P < 0.01).

After challenge with PMA (Fig. 1C), the maximum isoproterenol-induced cAMP accumulation was significantly (P < 0.01) reduced to 52% ± 12 SEM of the maximal stimulation, suggesting that PKC plays a pivotal role in the regulation of β₂-AR in HASMC. In GF109203X-treated and PMA-challenged HASMC, isoproterenol-induced cAMP accumulation was not significantly different from unchallenged HASMC and significantly greater than in untreated PMA-challenged HASMC (P < 0.01).

More importantly, in GF109203X-treated and LTD₄-challenged HASMC (Fig. 2) the maximal isoproterenol-induced cAMP accumulation was 3417 pmoles/mg prot (CV 5%), significantly (P < 0.01) greater than 2464 pmoles/mg prot (CV 7%) in untreated LTD₄-challenged HASMC and insignificantly different from 3632 pmol/mg prot (CV 5%) in unchallenged HASMC, confirming a critical role for PKC in the LTD₄-induced β₂-AR desensitization.

Pre-treatment with H89 did not alter the effect of LTD₄ challenge on isoproterenol-induced maximal cAMP accumulation (Fig. 3A), suggesting that LTD₄-induced β₂-AR desensitization does not involve PKA activation. Moreover, LTD₄ challenge did not affect the forskolin-induced maximal cAMP accumulation (Fig. 3B), suggesting that the adenylyl cyclase was not directly affected by LTD₄.

The mean weight of the 24 bronchial rings was 91 ± 5 mg. The mean resting force and the mean normalized-response to carbachol were 0.83 ± 0.05 g and 14 ± 2 gr/gr of tissue, without significant differences between sensitized, challenged, and treated rings Table 1.

Salbutamol relaxed bronchial rings significantly (P < 0.01) in a concentration-dependent manner (Fig. 4). The salbutamol concentration-response curve of challenged rings was significantly (P < 0.01) shifted to the right of the dose response curve of sensitized unchallenged rings, with significant differences (P < 0.01) at salbutamol concentrations from 10^-6M to 10^-5M. Pre-treatment with either 10^-6M or 10^-7M montelukast displaced significantly (P < 0.01) to the left of the concentration-response curves of challenged rings, with significant differences (P < 0.05) at salbutamol concentrations from 10^-6M to 10^-5M.

The mean values for IC₅₀ of challenged rings was -5.49 ± 0.12 Log M significantly (P < 0.05) higher than -6.07 ± 0.15 Log M of sensitized untreated rings (Fig. 5). The IC₅₀ values of challenged rings treated with 10^-6M and 10^-7M montelukast were -6.05 ± 0.03 and 5.96 ± 0.19, respectively, which were not significantly different from those of sensitized untreated rings. The IC₅₀ values of challenged rings treated with montelukast were lower than those of challenged rings (P < 0.05 for 10^-6M and P = 0.07 for 10^-7M).

In challenged rings treated with either 10^-7M or 10^-6M GF109203X, the concentration-response curves to salbutamol were significantly (P < 0.01) shifted to the left of the concentration-response curve of challenged rings (Fig. 6).

We first constructed concentration response curves of isoproterenol-induced cAMP accumulation in HASMC utilizing a non-cumulative protocol. A maximum effect was clearly observed at isoproterenol concentration of 10^-5M, and this was therefore used for subsequent single-concentration experiments. Isoproterenol was used in cAMP accumulation experiments because, as a full β-AR agonist, is more suited for the desensitization studies. The β₂-AR selective partial agonist salbutamol was used for bronchial rings studies because it is the reference drug generally used for clinical studies. However, in two separate experiments we found that the effect of salbutamol on cAMP accumulation was much weaker than that of isoproterenol, while the relative reduction caused by LTD₄ challenge was similar to that observed using isoproterenol, being even slightly more pronounced (Fig. 7). Therefore, we are confident that the results of our HASMC and bronchial rings studies are comparable.

Furthermore, the fact that after LTD₄ challenge in HASMC only the maximal cAMP accumulation was reduced, whereas only the IC₅₀ of salbutamol-induced relaxation was reduced might be explained by the fact that the relaxing effect of a β2 agonist is a far more downstream response than a second messenger (i.e. cAMP) production, and certainly involve the activation of other components downstream of the receptor, while the β2-AR may perform functions other than adenylyl cyclase activation 28, yet equally involved in bronchial relaxation.

As in our previous studies 2326293031, human bronchial rings were passively sensitized by using a pool of sera containing high levels of specific IgEs but low levels of total IgEs. With this method of passive sensitization and allergen challenge, followed by repeated washouts, the force generation capacity of airway smooth muscle was not altered 23, which makes us confident that the reference force of 1 g and the level of pre-contraction induced by carbachol 10^-6M were similar in all experimental conditions. Furthermore, the relaxant responses to either theophylline 26 or forskolin 30 remained unaltered in previous studies using the same methodology. Therefore, the use of sensitized unchallenged rings as a control seems justified and any difference in response to salbutamol can be attributed to changes in the β₂-AR pathway.

For relaxation studies, bronchial rings were pre-contracted with the non-selective muscarinic agonist carbachol, thus activating both M₃ and M₂ receptors on smooth muscle cell membrane. M₂ receptors are coupled to G_i-protein, which inhibits adenylyl cyclase. Thus, had sensitization or allergen challenge changed G_i-protein expression or activity, the response to a β₂-agonist would have been affected. In this model, however, both expression and activity of G_i-protein were similar in sensitized and challenged rings 29.

In bronchial tissue studies, the effects of allergen challenge were presumably due to mediator release from resident inflammatory cells 23. Thus, it cannot be excluded that the protective effects of GFX and montelukast against β₂-AR dysfunction were in part due to inhibition of mediator release. However, the observation that GFX and montelukast also protected against β₂-AR dysfunction in HASMC does suggest that airway smooth muscle PKC was directly involved

The response to β₂-AR has been found to be reduced in airways from subjects with fatal asthma 32. A reduced β₂-AR responsiveness in asthma may be the result of activation of the β₂-AR by specific agonists (homologous desensitization) or activation of other receptors by the inflammatory mediators, which are present in the asthmatic airways (heterologous desensitization) 33. β₂-AR desensitization induced by agents that increase cAMP levels, such as bradykinin 34 and some cytokines 35 acting through the elevation of prostaglandin E₂ 36, is probably regulated by PKA 633. On the contrary, muscarinic agonists 37, phorbol esters, and other inflammatory mediators may attenuate responses to β-agonists through the activation of PKC 38, as also recently suggested in bovine tracheal smooth muscle preparations 3940. However, it appears that these mechanisms of desensitization are cell-type specific 41 and may depend on kinase expression levels 42.

Among the inflammatory mediators involved in asthma, cysteinyl-LTs seem to play a key role in the bronchoconstrictor response to allergen 151617 through activation of CysLT₁R. Though preferentially coupled to G_q/11-protein, constitutively expressed CysLT₁ also activates pertussis toxin (PTX)-sensitive and -insensitive G-proteins 4344. In HASMC, we have previously found that CysLT₁ stimulation activates PKC 25 and mitogen-activated protein kinases ERK1/2 through mechanisms that involve a PTX-sensitive G-protein 19. Thus, it is possible that cysteinyl-LTs may contribute to β₂-AR desensitization not only by a PKC-dependent mechanism, but also by modulating the adenylyl cyclase-PKA pathway.

The results of the present study show that the cAMP accumulation in response to isoproterenol is reduced in HASMC treated with exogenous LTD₄ or the PKC activator PMA and the relaxant response to salbutamol is reduced in human bronchi challenged with the sensitizing allergen. The effects of LTD₄ in HASMC and allergen challenge in bronchial rings were prevented by the CysLT₁R antagonist montelukast and the PKC specific inhibitor GF109203X. Altogether, these findings strongly suggest that in the models used in the present study β₂-AR desensitization was the result of PKC activation by LTD₄.

In HASMC, exogenous LTD₄ did not alter the cAMP accumulation induced by forskolin, thus excluding that the reduced response of β₂-AR to isoproterenol was due to adenylyl cyclase dysfunction. The PKA inhibitor H89 also failed to prevent the LTD₄-induced β₂-AR desensitization in HASMC, thus ruling out the possibility of the involvement of this protein kinase. Indeed, H89 tended to enhance the response to isoproterenol both in LTD₄-challenged and -unchallenged HASMC, suggesting the presence of the well known G_S/G_i switch phenomenon of β₂-AR coupling due to PKA phosphorylation 45, which was not enhanced by LTD₄. This finding suggests that the β₂-AR function is independently modulated by PKA and PKC mechanisms and it is consistent with the observations by Penn et al. 6 who showed that inhibition of PKC did not alter β₂-AR desensitization induced by PKA activation.

In human bronchi, allergen challenge may cause β₂-AR desensitization through different mechanisms involving inflammatory mediators other than LTs, thus possibly involving PKA. However, in previous studies we found that the reduction of relaxant response to salbutamol in allergen-challenged rings was not prevented by inhibition of prostaglandins 23, IL-lβ, or TNFα 30, which are known to cause β₂-AR dysfunction/desensitization through the activation of PKA 6333536.