Primary pulmonary hypertension is characterized by increased pulmonary vascular resistance and smooth muscle proliferation. Stable analogs are increasingly being used to treat this disease, although no data exists comparing their effects on proliferation. We therefore investigated the antiproliferative activity of several prostacyclin (PGI(2)) analogs on human pulmonary arterial smooth muscle cells, including UT-15 and iloprost, analogs that have recently completed successful clinical trials. Serum-induced proliferation, as assessed by [(3)H]thymidine incorporation (30 h) or cell number (48 h), was significantly inhibited with a 10-fold difference in potency, ranking in effectiveness UT-15 > iloprost > cicaprost > beraprost. Effects were reversed by the adenylyl cyclase inhibitor, 2,5'dideoxyadenosine (DDA) but not SQ22536. Intracellular cyclic AMP (cAMP) was elevated by all analogs and inhibited by DDA, although SQ22536 was a highly variable inhibitor, suggesting that different pathways might mediate cAMP generation. UT-15 produced a significantly larger and more sustained increase in cAMP compared with other analogs, with iloprost being the weakest elevator. Thus, PGI(2) analogs potently inhibit proliferation of human pulmonary artery, probably via a cAMP-dependent pathway, although cAMP elevation in itself is not a good predictor of antiproliferative potency.

In this study, a potential mechanism of ␤ 2 -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 E 2 increased PDE3 and PDE4 activity in an actinomycin Dand 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 vehicleand 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 cAMPelevating agents compromises ␤ 2 -adrenoreceptor-mediated cAMP accumulation by increasing the expression of HSPDE3B and HSPDE4D gene products. Cyclic nucleotide phosphodiesterases (PDEs)1 are a heterogeneous group of immunologically distinct enzymes whose sole function is to metabolize the second messenger purine nucleotides, cAMP and cGMP, to their biologically inactive nucleotide 5Ј-monophosphates. Currently, PDEs are categorized in to seven broad families (see Ref. 1 for nomenclature) that are distinguished by a number of criteria including substrate specificity, kinetic properties, sensitivity to allosteric modulators and synthetic inhibitors, and primary amino acid sequence (2, 3). In many cases, these families comprise multiple subtypes, which suggests that the degradation of cAMP and cGMP is a highly complex and tightly regulated process. Over the last decade, the possible applications of PDE inhibitors to the treatment of an array of inflammatory diseases including asthma and atopic dermatitis has been realized. In particular, the cAMP-specific PDE, or PDE4, is viewed by the pharmaceutical industry as a viable molecular locus amenable to therapeutic intervention with selective inhibitors. The selection of this enzyme family as a possible drug target comes from the knowledge that PDE4 is the predominant, if not exclusive, regulator of cAMP homeostasis in essentially all pro-inflammatory and immune cells. Moreover, PDE4 inhibitors suppress many functional indices of cell activation that are considered pro-inflammatory (4 -7).Currently, four genes (for PDE4A, PDE4B, PDE4C, and PDE4D) have been identified in rat (8 -11), in mouse (12), ...

Figure 2Effect of chronic treatment of rats with albuterol (a-d) or PGE 2 (e and f) on lung function in anesthetized rats. Animals were given albuterol, PGE 2 (open bars; both 40 µg/kg/h) or vehicle (filled bars) for 7 days and then instrumented for the measurement of lung function. ACh (500 µg/kg intravenously) was administered and the maximum increase in overflow pressure was measured. When baseline lung function was reestablished, albuterol (Alb; 100 µg/kg; a and f), PGE 2 (PG; 300 µg/kg; b and e), forskolin (F; 300 µg/kg; c), or IBMX (300 µg/kg; d) was given intravenously, and 5 minutes later, ACh was administered again and any change in overflow pressure was noted. Each bar represents the mean ± SEM of four independent determinations. A P < 0.05, significant protection of ACh-induced bronchoconstriction.

1 The aim of the present study was to examine the eects of chronic infusion of the long-acting agonist salmeterol on pulmonary b 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 AChinduced bronchoconstriction when compared to rats treated identically with vehicle. 3 b 1 -and b 2 -adrenoceptor density was signi®cantly 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 sa 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 o *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 eect is a reduction in membrane-associated G sa . The short-acting nature of salmeterol, when administered systemically, and the reduction in b-adrenoceptor number may be due to metabolism to a biologically-active, short-acting and non-selective b-adrenoceptor agonist. British Journal of Pharmacology (2001) 132, 1261 ± 1270 Keywords: b 2 -adrenoceptor desensitization in vivo; salmeterol; down-regulation of G sa Abbreviations: FEF 25 ± 75 , forced expiratory¯ow at 25% and 75% of vital capacity; FEV 1 , forced expiratory volume in 1 s;