Chronic systemic administration of salmeterol to rats promotes pulmonary beta(2)-adrenoceptor desensitization and down-regulation of G(s alpha).

1. The aim of the present study was to examine the effects of chronic infusion of the long-acting agonist salmeterol on pulmonary beta(2)-adrenoceptor function in Sprague-Dawley rats in vivo and to elucidate the molecular basis of any altered state. 2. Systemic administration of rats with salmeterol for 7 days compromised the ability of salmeterol and prostaglandin E(2) (PGE(2)), given acutely by the intravenous route, to protect against ACh-induced bronchoconstriction when compared to rats treated identically with vehicle. 3. beta(1)- and beta(2)-adrenoceptor density was significantly reduced in lung membranes harvested from salmeterol-treated animals, which was associated with impaired salmeterol- and PGE(2)-induced cyclic AMP accumulation ex vivo. 4. Three variants of G(s alpha) that migrated as 42, 44 and 52 kDa peptides on SDS polyacrylamide gels were detected in lung membranes prepared from both groups of rats but the intensity of each isoform was markedly reduced in rats that received salmeterol. 5. The activity of cytosolic, but not membrane-associated, G-protein receptor-coupled kinase was elevated in the lung of salmeterol-treated rats when compared to vehicle-treated animals. 6. The ability of salmeterol, administered systemically, to protect the airways of untreated rats against ACh-induced bronchoconstriction was short-acting (t(off) approximately 45 min), which contrasts with its long-acting nature when given to asthmatic subjects by inhalation. 7. These results indicate that chronic treatment of rats with salmeterol results in heterologous desensitization of pulmonary G(s)-coupled receptors. In light of previous data obtained in rats treated chronically with salbutamol, we propose that a primary mechanism responsible for this effect is a reduction in membrane-associated G(s alpha). The short-acting nature of salmeterol, when administered systemically, and the reduction in beta-adrenoceptor number may be due to metabolism to a biologically-active, short-acting and non-selective beta-adrenoceptor agonist.

Albuterol-induced downregulation of Gsalpha accounts for pulmonary beta(2)-adrenoceptor desensitization in vivo.

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

Induction of phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T-lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists. Potential role in beta2-adrenoreceptor desensitization.

In this study, a potential mechanism of beta2-adrenoreceptor desensitization has been explored that is based upon the enhanced degradation of cAMP by phosphodiesterase (PDE). Pretreatment of Jurkat T-cells with 8-bromo cAMP (8-Br-cAMP) or prostaglandin E2 increased PDE3 and PDE4 activity in an actinomycin D- and cycloheximide-sensitive manner. This effect was associated with increased expression of HSPDE3B, HSPDE4A4, HSPDE4D1, HSPDE4D2, and HSPDE4D3 mRNA transcripts. Western analysis reproducibly labeled a band of immunoreactivity in vehicle-treated cells that corresponded to HSPDE4A4 (125 kDa). Although the intensity of this band was unchanged in cells treated with 8-Br-cAMP, additional 68-72-kDa proteins (HSPDE4D2, HSPDE4D1) were labeled that were not detected after vehicle. Similar results were obtained with T-lymphocytes exposed to 8-Br-cAMP and fenoterol. However, in those experiments HSPDE4A4 and HSPDE4D1 appeared to be equally expressed in vehicle- and treated cells, whereas HSPDE4D2 (72 kDa) was detected only after 8-Br-cAMP. The up-regulation of PDE activity in Jurkat T-cells abolished the ability of isoproterenol to elevate cAMP, which was partially reversed by the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine, and by the PDE3 and PDE4 inhibitors, Org 9935 and rolipram, respectively. Collectively, these data suggest that chronic treatment of T-cells with cAMP-elevating agents compromises beta2-adrenoreceptor-mediated cAMP accumulation by increasing the expression of HSPDE3B and HSPDE4D gene products.