Beta(2)-agonists block tussive responses in guinea pigs via an atypical cAMP-dependent pathway.

beta(2)-Adrenoceptor agonists are the most effective bronchodilators currently available, and are used for symptom management in asthmatics. However, whether beta(2)-agonists are also antitussive is controversial. Identifying an antitussive role for beta(2)-agonists and dissecting the possible mechanism of action may help to explain the inconsistencies in the clinical literature and lead to the development of novel therapeutic agents. The aim of the present study was to determine whether or not beta(2)-agonists attenuate the tussive response in guinea pig and human models, and, if so, to identify the mechanism(s) involved. Depolarisation of vagal sensory nerves (human and guinea pig) was assessed as an indicator of sensory nerve activity. Cough was measured in a conscious guinea pig model. A beta(2)-agonist, terbutaline, dose-dependently inhibited the cough response to tussive agents in conscious guinea pigs. Terbutaline and another beta(2)-agonist, fenoterol, blocked sensory nerve activation in vitro. Using these mechanistic models, it was established that beta(2)-agonists suppress the tussive response via a nonclassical cyclic adenosine monosphosphate-dependent pathway that involves the activation of protein kinase G and, subsequently, the opening of large-conductance calcium-activated potassium channels. In conclusion, beta(2)-adrenoceptor agonists are antitussive, and this property occurs due to a direct inhibition of sensory nerve activation. These findings may help to explain the confusion that exists in the clinical literature, and could be exploited to identify novel therapies for the treatment of cough, which is a significant unmet medical need.

Can the anti-inflammatory potential of PDE4 inhibitors be realized: guarded optimism or wishful thinking?

PDE4 inhibitors have been in development as a novel anti-inflammatory therapy since the 1980s with asthma and chronic obstructive pulmonary disease (COPD) being primary indications. Despite initial optimism, none have yet reached the market. In most cases, the development of PDE4 inhibitors of various structural classes, including cilomilast, filaminast, lirimilast, piclamilast, tofimilast, AWD-12-281 (aka GSK 842470), CDP840, CI-1018, D-4418, IC485, L-826,141, SCH 351391 and V11294A has been discontinued due to lack of efficacy. A primary problem is the low therapeutic ratio of these compounds, which severely limits the dose that can be given. Indeed, for many of these compounds it is likely that the maximum tolerated dose is either sub-therapeutic or at the very bottom of the efficacy dose-response curve. Therefore, the challenge is to overcome this limitation. It is, therefore, encouraging that many 'new(er)' PDE4 inhibitors in development are reported to have an improved therapeutic window including tetomilast, oglemilast, apremilast, ONO 6126, IPL-512602 and IPL-455903 (aka HT-0712), although the basis for their superior tolerability has not been disclosed. In addition, other approaches are possible that may allow the anti-inflammatory activity of PDE inhibitors to be realized. Accordingly, this Commentary endorses the view of Spina (2008), published in the current issue of the British Journal of Pharmacology, that the therapeutic utility of PDE4 inhibitors to suppress inflammation still remains a viable concept.

A Holy Grail of asthma management: toward understanding how long-acting beta(2)-adrenoceptor agonists enhance the clinical efficacy of inhaled corticosteroids.

There is unequivocal evidence that the combination of an inhaled corticosteroid (ICS) -- i.e. glucocorticoid -- and an inhaled long-acting beta(2)-adrenoceptor agonist (LABA) is superior to each component administered as a monotherapy alone in the clinical management of asthma. Moreover, Calverley and colleagues (Lancet 2003, 361: 449-456; N Engl J Med 2007, 356: 775-789) reporting for the 'TRial of Inhaled STeroids ANd long-acting beta(2)-agonists (TRISTAN)' and 'TOwards a Revolution in COPD Health (TORCH)' international study groups also demonstrated the superior efficacy of LABA/ICS combination therapies over ICS alone in the clinical management of chronic obstructive pulmonary disease. This finding has been independently confirmed indicating that the therapeutic benefit of LABA/ICS combination therapies is not restricted to asthma and may be extended to other chronic inflammatory diseases of the airways. Despite the unquestionable benefit of LABA/ICS combination therapies, there is a vast gap in our understanding of how these two drugs given together deliver superior clinical efficacy. In this article, we review the history of LABA/ICS combination therapies and critically evaluate how these two classes of drugs might interact at the biochemical level to suppress pro-inflammatory responses. Understanding the molecular basis of this fundamental clinical observation is a Holy Grail of current respiratory diseases research as it could permit the rational exploitation of this effect with the development of new 'optimized' LABA/ICS combination therapies.

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.

Potentiation of NF-kappaB-dependent transcription and inflammatory mediator release by histamine in human airway epithelial cells.

BACKGROUND AND PURPOSE: In asthma, histamine contributes to bronchoconstriction, vasodilatation and oedema, and is associated with the late phase response. The current study investigates possible inflammatory effects of histamine acting on nuclear factor kappaB (NF-kappaB)-dependent transcription and cytokine release. EXPERIMENTAL APPROACH: Using BEAS-2B bronchial epithelial cells, NF-kappaB-dependent transcription and both release and mRNA expression of IL-6 and IL-8 were examined by reporter assay, ELISA and quantitative RT-PCR. Histamine receptors were detected using qualitative RT-PCR and function examined using selective agonists and antagonists. KEY RESULTS: Addition of histamine to TNFalpha-stimulated BEAS-2B cells maximally potentiated NF-kappaB-dependent transcription 1.8 fold, whereas IL-6 and IL-8 protein release were enhanced 7.3- and 2.7-fold respectively. These responses were, in part, NF-kappaB-dependent and were associated with 2.6- and 1.7-fold enhancements of IL-6 and IL-8 mRNA expression. The H(1) receptor antagonist, mepyramine, caused a rightward shift in the concentration-response curves of TNFalpha-induced NF-kappaB-dependent transcription (pA(2)=9.91) and release of IL-6 (pA(2)=8.78) and IL-8 (pA(2)=8.99). Antagonists of histamine H(2), H(3) and H(4) receptors were without effect. Similarly, H(3) and H(4) receptor agonists did not affect TNFalpha-induced NF-kappaB-dependent transcription, or IL-6 and IL-8 release at concentrations below 10 microM. The anti-inflammatory glucocorticoid, dexamethasone, inhibited the histamine enhanced NF-kappaB-dependent transcription and IL-6 and IL-8 release. CONCLUSIONS AND IMPLICATIONS: Potentiation of NF-kappaB-dependent transcription and inflammatory cytokine release by histamine predominantly involves receptors of the H(1) receptor subtype. These data support an anti-inflammatory role for H(1) receptor antagonists by preventing the transcription and release of pro-inflammatory cytokines.

Inhibitory effect of p38 mitogen-activated protein kinase inhibitors on cytokine release from human macrophages.

BACKGROUND AND PURPOSE: Macrophages release cytokines that may contribute to pulmonary inflammation in conditions such as chronic obstructive pulmonary disease. Thus, inhibition of macrophage cytokine production may have therapeutic benefit. p38 MAPK may regulate cytokine production, therefore, the effect of two p38 MAPK inhibitors, SB239063 and SD-282, on the release of TNF-alpha, GM-CSF and IL-8 from human macrophages was investigated. EXPERIMENTAL APPROACH: Cytokine release was measured by ELISA. Immunoblots and mRNA expression studies were performed to confirm p38 MAPK isoform expression and activity. Macrophages were isolated from lung tissue of current smokers, ex-smokers and emphysema patients and exposed to lipopolysaccharide. These cells then released cytokines in a concentration-dependent manner. KEY RESULTS: SB239063 only inhibited TNF-alpha release (EC50 0.3 +/- 0.1 microM). Disease status had no effect on the efficacy of SB239063. SD-282 inhibited both TNF-alpha and GM-CSF release from macrophages (EC50 6.1 +/- 1.4 nM and 1.8 +/- 0.6 microM respectively) but had no effect on IL-8 release. In contrast, both inhibitors suppressed cytokine production in monocytes. CONCLUSIONS AND IMPLICATIONS: The differential effects of p38 MAPK inhibitors between macrophages and monocytes could not be explained by differences in p38 MAPK isoform expression or activity. However, the stability of TNF-alpha mRNA was significantly increased in macrophages compared to monocytes. These data suggest a differential involvement for p38 MAPK in macrophage cytokine production compared with monocytes. These effects are not due to lack of p38 activation or p38alpha expression in macrophages but may reflect differential effects on the stability of cytokine mRNA.

Phosphodiesterase 4 and tolerance to beta 2-adrenoceptor agonists in asthma.

beta 2-Adrenoceptor agonists provide a mainstay in the treatment of asthma worldwide. However, despite their ability to provide symptomatic relief, chronic or repeated exposure to beta 2-adrenoceptor agonists does not resolve asthmatic inflammation, because of the rapid development of tolerance by pro-inflammatory and immune cells of the lung. The prevailing belief is that tolerance to the so-called non-bronchodilator actions of beta 2-adrenoceptor agonists is largely attributable to direct receptor desensitization mediated by G protein receptor-coupled kinases and/or cAMP-dependent protein kinase. Here, Mark Giembycz suggests another, largely ignored, explanation for beta 2-adrenoceptor desensitization that is based on the accelerated degradation of cAMP by phosphodiesterase.

Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation.

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.

The long-acting ß2 -adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner.

BACKGROUND AND PURPOSE: Inhaled glucocorticoid (ICS)/long-acting ß2 -adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. EXPERIMENTAL APPROACH: BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. KEY RESULTS: Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. CONCLUSIONS AND IMPLICATIONS: These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues.

Suppression of granulocyte/macrophage colony-stimulating factor release from human monocytes by cyclic AMP-elevating drugs: role of interleukin-10.

1. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory cytokine secreted by cells of the monocyte/macrophage lineage and has been implicated in the pathogenesis of bronchitis and asthma. 2. In the present study we have evaluated the effect of several cyclic AMP-elevating agents on lipopolysaccharide (LPS)-induced GM-CSF release from human monocytes and the extent to which the anti-inflammatory cytokine, interleukin (IL)-10, is involved. 3. LPS evoked a concentration-dependent generation of GM-CSF from human monocytes that was inhibited, at the mRNA and protein level, by 8-Br-cyclic AMP, cholera toxin, prostaglandin E2 (PGE2) and a number of structurally dissimilar phosphodiesterase (PDE) 4 inhibitors. 4. Pre-treatment of monocytes with a concentration of an anti-IL-10 monoclonal antibody that abolished the inhibitory action of a maximally effective concentration of exogenous human recombinant IL-10, significantly augmented LPS-induced GM-CSF generation. This effect was associated with a parallel upwards displacement of the concentration-response curves that described the inhibition of GM-CSF by PGE2, 8-Br-cyclic AMP and the PDE4 inhibitor, rolipram, without significantly changing the potency of any drug. Consequently, the maximum percentage inhibition of GM-CSF release was reduced. Further experiments established that the reduction in the maximum inhibition of GM-CSF release seen in anti-IL-10-treated cells was not due to functional antagonism as rolipram, PGE2 and 8-Br-cyclic AMP were equi-effective at all concentrations of LPS studied. 5. These data indicate that cyclic AMP-elevating drugs attenuate the elaboration of GM-CSF from LPS-stimulated human monocytes by a mechanism that is not mediated via IL-10. Suppression of GM-CSF from monocytes may explain, at least in part, the efficacy of PDE4 inhibitors in clinical trials of chronic obstructive pulmonary disease.

Leukotriene D4 receptors are not negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma.

1. The possibility that receptors for the peptide-containing leukotrienes may be negatively coupled to adenylyl cyclase in guinea-pig lung parenchyma was investigated by comparing the effect of leukotriene D4 (LTD4) on the intracellular cyclic nucleotide (cyclic AMP and cyclic GMP) content and on the activity of cyclic AMP-dependent protein kinase (PKA). In addition, the potential association between changes in the cyclic nucleotide content and the ability of LTD4 to increase lung parenchymal tone was also evaluated. 2. Non-cumulative challenge of parenchymal lung strips with LTD4 elicited concentration-dependent contractions (pD2 = 8.23) that were paralleled by concentration-related increases in the intracellular level of cyclic AMP and cyclic GMP, and in the activation state of PKA (Kact = 33 nM). Temporally, these biochemical effects of LTD4 were transient, peaking after approximately 5 min drug contact thereafter decaying, despite the continued generation of tone. Both the biochemical and mechanical effects of LTD4 were antagonized by the LTD4-receptor blocking drug, ICI 198,615 (1 microM for 60 min), indicating that they were receptor-mediated events. 3. Challenge of guinea-pig lung with LTD4 (200 nM; EC100 for tension generation) stimulated a 150 and 70 fold increase in the elaboration of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) respectively, relative to that generated spontaneously. 4. Pretreatment of lung strips for 60 min with an irreversible inhibitor of cyclo-oxygenase, flurbiprofen,at a concentration (8 microM) that abolished both basal and LTD4 (200 nM)-induced TXB2 and 6-keto-PGF1alpha release, relaxed rapidly the spontaneous tone of the tissues, reduced the cyclic AMP content by ~50%and lowered the PKA activity ratio from 29% to 17%. In addition, flurbiprofen abolished the ability of LTD4 (200 nM) to increase the cyclic AMP content and to activate PKA. Functionally, the magnitude of LTD4 (200 nM)-induced tone and the increase in cyclic GMP content were attenuated by approximately 20% and 50% respectively in flurbiprofen-treated tissues.5. In flurbiprofen-treated tissues, isoprenaline (10 microM for 10 min) increased the cyclic AMP content(from 4 to 27 pmol mg-1 protein) and activated PKA (from 15% to 26%). Preincubation (30 s or 5 min)of lung with LTD4 (200 nM) did not inhibit (or enhance) these isoprenaline-induced effects.6. Pretreatment of lung strips for 60 min with the thromboxane synthetase inhibitor, dazmegrel (10 microM),relaxed the spontaneous tone of the tissues, abolished the LTD4 (200 nM)-stimulated release of TXB2 and significantly enhanced (~two fold) the elaboration of 6-keto-PGF1alpha. In addition, dazmegrel attenuated (by ~50%) LTD4 (200 nM)-induced cyclic GMP accumulation but approximately doubled both the cyclic AMP content and PKA activity ratio. LTD4-induced contractions, in contrast, were not affected by dazmegrel.7. EP 092 (1 microM for 60 min), a selective TP-receptor blocking drug, had no effect on spontaneous tone,eicosanoid formation or on the cyclic GMP content of guinea-pig lung parenchymal strips. Likewise,EP 092 exerted no significant mechancial effect in lung challenged with LTD4 (200 nM) although it did potentiate, to a small extent, the ability of LTD4 (200 nM) to increase the cyclic AMP content.8. It is concluded that LTD4 can increase the intracellular level of cyclic AMP in guinea-pig parenchyma and activate PKA by a leukotriene-receptor-mediated mechanism sensitive to ICI 198,615. However,these biochemical actions of LTD4 are induced indirectly by an arachidonic acid-derived cyclo-oxygenase product(s) other than TXA2. Thus, contrary to reports of other investigators, no evidence was found to corroborate the finding that stimulation of leukotriene receptors on guinea-pig lung parenchyma results in a rapid lowering of the cyclic AMP content even in cyclo-oxygenase-blocked tissues. These data,therefore, do not support the hypothesis that leukotriene-induced tension generation is dependent upon a prior reduction in the cyclic AMP content.

Characterization of the prostanoid receptor(s) on human blood monocytes at which prostaglandin E2 inhibits lipopolysaccharide-induced tumour necrosis factor-alpha generation.

1. The prostanoid receptor(s) that mediates inhibition of bacterial lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) generation from human peripheral blood monocytes was classified by use of naturally occurring and synthetic prostanoid agonists and antagonists. 2. In human monocytes that were adherent to plastic, neither prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F(2 alpha) (PGF(2 alpha)) nor the stable prostacyclin and thromboxane mimetics, cicaprost and U-46619, respectively, promoted the elaboration of TNF alpha-like immunoreactivity, as assessed with a specific ELISA, indicating the absence of excitatory prostanoid receptors on these cells. 3. Exposure of human monocytes to LPS (3 ng ml-1, approximately EC84) resulted in a time-dependent elaboration to TNF alpha which was suppressed in cells pretreated with prostaglandin E1 (PGe1), PGE2 and cicaprost. This effect was concentration-dependent with mean pIC50 values of 7.14, 7.34 and 8.00 for PGE1, PGE2 and cicaprost, respectively. PGD2, PGF(2 alpha) and U-46619 failed to inhibit the generation of TNF alpha at concentrations up to 10 microM. 4. With respect to PGE2, the EP-receptor agonists, 16,16-dimethyl PGE2 (non-selective), misoprostol (EP2/EP3-selective), 11-deoxy PGE1 (EP2-selective) and butaprost (EP2-selective) were essentially full agonists as inhibitors of LPS-induced TNF alpha generation with mean pIC50 values of 6.21, 6.02, 5.67 and 5.59, respectively. In contrast to the results obtained with butaprost and 11-deoxy PGE1, another EP2-selective agonist, AH 13205, inhibited TNF alpha generation by only 21% at the highest concentration (10 microM) examined. EP-receptor agonists which have selectively for the EP1- (17-phenyl-omega-trinor PGE2) and EP3-receptor (MB 28,767, sulprostone) were inactive or only weakly active as inhibitors of TNF alpha generation. 5. Pretreatment of human monocytes with the TP/EP4-receptor antagonist, AH 23848B, at 10, 30 and 100 microM suppressed LPS-induced TNF alpha generation by 10%, 28% and 77%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves. 6. Given that AH 13205 was a poor inhibitor of TNF alpha generation, studies were performed to determine if it was a partial agonist and whether it could antagonize the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 and 30 microM AH 13205 inhibited the generation of TNF alpha by 31% and 53%, respectively, but failed to shift significantly the location of the PGE2 concentration-response curves at either concentration examined. 7. Since PGD2 and 17-phenyl-omega-trinor PGE2 (EP1-agonist) did not suppress TNF alpha generation, the EP1/EP2/DP-receptor antagonist, AH 6809, was employed to assess if EP2-receptors mediated the inhibitory effect of PGE2. Pretreatment of human monocytes with 10 microM AH 6809 did not affect LPS-induced TNF alpha generation but produced a parallel 3.5 fold rightwards shift of the PGE2 concentration-response curve. 8. Collectively, these data suggest that human peripheral blood monocytes express at least two distinct populations of inhibitory prostanoid receptors that mediate inhibition of LPS-induced TNF alpha generation. One of these probably represents i.p. receptors based upon the selectivity of cicaprost for this subtype. The other population has the pharmacology of EP-receptors, but the rank of potency for a range of synthetic EP-receptor agonists was inconsistent with an interaction with any of the currently defined subtypes. Given the pharmacological behaviour of butaprost, AH 6809 and AH 23848B in these cells, we propose that multiple (EP2- and/or EP-4- and/or i.p.) or novel EP-receptors mediate the inhibitory effect of PGE2 on TNF alpha generation.

Prostaglandin E2 suppression of acetylcholine release from parasympathetic nerves innervating guinea-pig trachea by interacting with prostanoid receptors of the EP3-subtype.

1. We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2. In epithelium-denuded tracheal strips pretreated with indomethacin (10 microM), PGE2 (0.1 nM-1 microM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2alpha and the stable thromboxane mimetic, U46619 (each at 1 microM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 microM) had no significant effect on [3H]-ACh release. 3. The selective TP-receptor antagonist, ICI 192,605 (0.1 microM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2alpha. 4. A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective) > PGE2 > M&B 28,767 (EP3 selective) > 17-phenyl-omega-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 microM), did not affect EFS-induced [3H]-ACh release. 5. AH6809 (10 microM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-omega-trinor PGE2 on [3H]-ACh release. 6. These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.

Characterization of phosphodiesterase 4 in guinea-pig macrophages: multiple activities, association states and sensitivity to selective inhibitors.

1. The cyclic AMP phosphodiesterases (PDE) in guinea-pig peritoneal macrophages were isolated, partially characterized and their role in regulating the cyclic AMP content in intact cells evaluated. 2. Differential centrifugation of macrophage lysates revealed that approximately 90% of the PDE activity was membrane-bound and exclusively hydrolyzed cyclic AMP. This activity was not removed by KCl (200 mM) but was readily solubilized by the non-ionic detergent, Triton X-100 (1% v/v). Greater than 80% of the hydrolytic activity was suppressed by the PDE4 inhibitors, R-rolipram and nitraquazone with IC50s of 240 and 540 nM, respectively. 3. Anion-exchange chromatography of the total protein extracted from macrophages resolved two major peaks of cyclic AMP PDE activity that were insensitive to cyclic GMP (10 microM), calmodulin (50 units plus 2 mM CaCl2) and a PDE3 inhibitor, SK&F 95654 (10 microM), but were markedly suppressed by RS-rolipram (10 microM). The two peaks of PDE activity were arbitrarily designated CPPDE4alpha and CPPDE4beta with respect to the order from which they were eluted from the column where the prefix, CP, refers to the species, Cavia porcellus. 4. The hydrolysis of cyclic AMP catalyzed by CPPDE4alpha and CPPDE4beta conformed to Michaelis-Menten kinetic behaviour with similar K(m)s (13.4 and 6.4 microM, respectively). 5. Thermal denaturation of membrane-bound PDE4 at 50 degrees C followed bi-exponential kinetics with t1/2 values of 1.5 and 54.7 min for the first and second components, respectively. In contrast, CPPDE4alpha and CPPDE4beta each decayed mono-exponentially with significantly different thermostabilities (t1/2 = 2.77 and 1.15 min, respectively). 6. Gel filtration of CPPDE4beta separated two peaks of rolipram-sensitive PDE activity. The main peak eluted at a volume indicative of a approximately 180 kDa protein but was preceded by a much larger form of the enzyme that had an estimated weight of 750 kDa. Size exclusion chromatography of CPPDE4alpha resolved a broad peak of activity with molecular weights spanning 50 to 200 kDa. 7. Of ten PDE inhibitors examined, none distinguished CPPDE4alpha from CPPDE4beta with respect to their IC50 values or their rank order of potency. RS-rolipram acted as a purely competitive inhibitor of cyclic AMP hydrolysis with K(i)s of 2 microM and 1.5 microM for CPPDE4alpha and CPPDE4beta, respectively. In contrast to the membrane-associated enzyme(s), R-rolipram and nitraquazone were 4 to 19 fold less potent as inhibitors of CPPDE4alpha and CPPDE4beta. 8. In intact macrophages, Ro 20-1724 and RS-rolipram potentiated isoprenaline-induced cyclic AMP accumulation under conditions where a PDE3 inhibitor, SK&F 94120, was essentially inactive. 9. These data demonstrate that the predominant cyclic AMP hydrolyzing activity in guinea-pig macrophages is a PDE4. Moreover, thermostability studies and size exclusion chromatography indicates the possible expression of two intrinsic, membrane-associated isoenzymes which can regulate the cyclic AMP content in intact cells. The finding that soluble and particulate forms of the same enzyme exhibit different sensitivities to rolipram and nitraquazone implies that PDE4 can change conformation. Finally, the identification of multiple molecular weight species of CPPDE4 suggests that this enzyme(s) might form multimeric complexes of variable association states.

Inhibition of eosinophil cyclic nucleotide PDE activity and opsonised zymosan-stimulated respiratory burst by 'type IV'-selective PDE inhibitors.

1. The cyclic nucleotide phosphodiesterase (PDE) of guinea-pig eosinophils was partially characterized and the effects of selective inhibitors of PDE isoenzymes upon opsonized zymosan (OZ)-stimulated respiratory burst were studied. 2. PDE activity in eosinophil lysates appeared to be membrane-associated, displayed substrate specificity for adenosine 3':5' cyclic monophosphate (cyclic AMP) versus guanosine 3':5' cyclic monophosphate (cyclic GMP) and was insensitive to cyclic GMP or Ca2+ and calmodulin. 3. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine caused a concentration-dependent inhibition of both OZ-stimulated hydrogen peroxide (H2O2) generation and cyclic AMP hydrolysis. The type IV-selective PDE inhibitors, rolipram and denbufylline, also inhibited H2O2 generation and cyclic AMP hydrolysis in a concentration-dependent manner whilst SK&F 94120 and Org 9935 (type III-selective) and zaprinast (type Ia or V-selective) were ineffective. 4. Dibutyryl cyclic AMP, a cell-permeable, non-hydrolysable analogue of cyclic AMP, caused a concentration-dependent inhibition of H2O2 generation stimulated by OZ. Dibutyryl cyclic GMP was ineffective. 5. It is concluded that eosinophil respiratory burst activity induced by OZ can be regulated by intracellular cyclic AMP and that the levels of cyclic AMP are controlled exclusively by a rolipram- and denbufylline-sensitive PDE isoenzyme that resembles a type IV species.

Extracellular signal-regulated kinase 1/2 control Ca(2+)-independent force development in histamine-stimulated bovine tracheal smooth muscle.

The role of extracellular signal-regulated kinase (ERK)-1 and ERK-2 in controlling histamine-induced tone in bovine trachealis was investigated. PD 098059, an inhibitor of mitogen-activated protein kinase kinase (MKK)-1, had no effect on the histamine concentration-response relationship that described contraction. However, in the presence of EGTA, PD 098059 produced a parallel 5 fold rightwards shift of the histamine concentration-response curve without reducing the maximum response. The beta(2)-adrenoceptor agonist, procaterol, also displaced the histamine-concentration response curve to the right but the effect was much greater than that evoked by PD 098059, non-competitive and seen in the absence and presence of EGTA. A low basal level of pERK-1 and pERK-2 was always detected in untreated trachealis, which was significantly higher in EGTA-treated tissues and inhibited by PD 098059 and procaterol. Histamine markedly enhanced the phosphorylation of ERK-1 and ERK-2 by a mechanism that was also enhanced by EGTA and significantly attenuated by procaterol and PD 098059. Neither cholera toxin nor SP:-8-Br-cAMPS mimicked the ability of procaterol to dephosphorylate ERK. Similarly, neither pertussis toxin (PTX) nor RP:-8-Br-cAMPS, an inhibitor of cyclic AMP-dependent protein kinase (PKA), affected basal pERK levels or antagonized the inhibitory effect of procaterol. These data implicate the MKK-1/ERK signalling cascade in Ca(2+)-independent, histamine-induced contraction of bovine trachealis. In addition, the ability of procaterol to dephosphorylate ERK in an RP:-8-Br-cAMPS- and PTX-insensitive manner suggests that this may contribute to the anti-spasmogenic activity of beta(2)-adrenoceptor agonists by activating a novel PKA-independent pathway.

Possible role of cyclic AMP phosphodiesterases in the actions of ibudilast on eosinophil thromboxane generation and airways smooth muscle tone.

1. The possible role of cyclic AMP phosphodiesterase (PDE) in the inhibitory actions of ibudilast on tracheal smooth muscle contractility and eosinophil thromboxane generation was investigated. 2. Ibudilast was a non-selective inhibitor of partially purified cyclic nucleotide PDE isoenzymes from pig aorta and bovine tracheal smooth muscle, exhibiting only moderate potency against bovine tracheal PDE IV (IC50 = 12 +/- 4 microM, n = 3). Similar or slightly lower potencies were displayed against PDEs I, II, III and V. In contrast, rolipram exhibited selectivity for PDE IV (3 +/- 0.5 microM, n = 3). 3. Ibudilast (IC50 = 0.87 +/- 0.37 microM, n = 3), like rolipram (IC50 = 0.20 +/- 0.04 microM, n = 3), was a more potent inhibitor of membrane-bound PDE IV from guinea-pig eosinophils than of partially purified PDE IV from bovine tracheal smooth muscle. The potency of ibudilast increased when the eosinophil enzyme was solubilised with deoxycholate and NaCl (IC50 = 0.11 +/- 0.05 microM, n = 3) or exposed to vanadate/glutathione complex (V/GSH) (IC50 = 0.11 +/- 0.02 microM, n = 3). The potency of rolipram was also increased by solubilization (IC50 = 0.012 +/- 0.003, n = 3) or V/GSH (IC50 = 0.012 +/- 0.003, n = 3). 4. In intact eosinophils, ibudilast (0.032 microM-20 microM) potentiated isoprenaline-induced cyclic AMP accumulation in a concentration-dependent manner, being approximately 20 fold less potent than rolipram. Little or no effect on basal cyclic AMP levels was observed with either compound. The cyclicAMP-dependent protein kinase activity ratio was significantly increased following incubation of eosinophils with either ibudilast (20 MicroM) or rolipram (20 MicroM) in the absence or presence of isoprenaline.5. Leukotriene B4 (300 nM)-induced thromboxane generation from guinea-pig eosinophils was inhibited by ibudilast (IC50 = 11.3 +/- 3.7 MicroM, n = 5) and rolipram (IC50 = 0.280 +/- 0.067 MicroM, n = 5) in a concentration-dependent manner.6. Ibudilast (10 nM-1 MicroM), whilst generally less potent than rolipram (1 nM- 1 MicroM), produced concentration-dependent relaxation of spasmogen (methacholine, histamine, LTD4)-induced tone in the guinea pig isolated tracheal strip. Ibudilast was less potent in reversing the methacholine (IC50 = 1.95 +/- 0.40 JM,n =6)-induced contraction than those of histamine (IC50 = 0.18 +/- 0.70 MicroM, n =6) or leukotriene D4(LTD4, IC50 = 0.12 +/- 0.05 MicroM, n = 6). Rolipram also exhibited a similar pattern of activity, although the difference in potency against methacholine (IC50 = 0.1 +/- 0.01 MicroM, n = 6) compared with the other two spasmogens, histamine (IC50 = 0.034 +/- 0.017 MicroM, n = 7) and LTD4 (IC50 = 0.026 +/- 0.008 MicroM, n = 7), was not as great.7. These results demonstrate that ibudilast, like rolipram, has several biological actions on the eosinophil and airways smooth muscle which may be attributed to inhibition of cyclic AMP PDE. These actions may account, at least in part, for the recently reported anti-asthma effects of ibudilast.

Cyclic AMP-elevating agents prolong or inhibit eosinophil survival depending on prior exposure to GM-CSF.

1. Purified human eosinophils survived for up to 7 days when cultured in vitro in the presence of 1 ng ml-1 granulocyte-macrophage colony stimulating factor (GM-CSF) with a viability of 73%. In the absence of GM-CSF, eosinophil viability decreased after one day in culture, and only 4% of cells were viable by day 4. 2. Culture of eosinophils with cholera toxin produced a concentration-dependent decrease in GM-CSF-induced survival at 7 days (IC50 = 7 ng ml-1) which was associated with a 6 fold increase in the intracellular cyclic AMP concentration. This inhibition of cell survival could be prevented by the addition of the protein kinase A inhibitor, H89 (10(-6)M). 3. When eosinophils were cultured with dibutyryl cyclic AMP, there was a concentration-dependent inhibition of GM-CSF-induced survival at 7 days with an IC50 of 200 microM. The related cyclic nucleotide analogue, dibutyryl cyclic GMP did not inhibit GM-CSF-induced eosinophil survival over the same concentration range. 4. Culture of eosinophils with forskolin, or with the phosphodiesterase inhibitors, rolipram and SK&F94120, had no effect on GM-CSF-induced eosinophil survival at any concentration examined. 5. After 7 days' culture in the absence of GM-CSF, fractionation of eosinophil DNA on agarose gels demonstrated a 'ladder' pattern characteristic of apoptosis. GM-CSF prevented DNA fragmentation and this protection could be overcome by both cholera toxin and dibutyryl cyclic AMP. 6. GM-CSF did not affect intracellular cyclic AMP concentrations in unstimulated eosinophils or in cells stimulated by cholera toxin. Thus, GM-CSF does not apparently increase eosinophil survival by affecting cyclic AMP levels. 7. In the absence of GM-CSF both cholera toxin and dibutyryl cyclic AMP decreased the rate of eosinophil death, when compared to cells cultured with medium alone. The t1/2 values for cell death were 1.63 +/- 0.3, 2.46 +/- 0.3 and 4.62 +/- 1.0 days for cells cultured in the presence of medium, cholera toxin and dibutyryl cyclic AMP respectively. 8. In conclusion, cyclic AMP exerts opposing effects on eosinophil survival depending on prior exposure of the cells to GM-CSF.

Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2.

1. The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-gamma evaluated. 2. In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (approximately 65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3. Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4. Functionally, rolipram inhibited phytohaemagglutinin- (PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA- and anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5. When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL-2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6. Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL-2 biosynthesis and proliferation in human CD4+ and CD8+ T-lymphocytes. However, the ability of rolipram to suppress markedly mitogen-induced IL-2 generation without affecting T-cell proliferation suggests that growth and division of T-lymphocytes may be governed by mediators in addition to IL-2. Finally, T-cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.

Effects of agents which elevate cyclic AMP on guinea-pig eosinophil homotypic aggregation.

1. Eosinophil recruitment and activation in inflamed tissue is thought to play an important role in the pathophysiology of allergic diseases. Experimental evidence suggests that elevating cyclic AMP is an effective means of reducing eosinophil recruitment in vivo and may therefore have therapeutic benefit. In the present study, we have assessed the capacity of cyclic AMP-elevating agents to modulate guinea-pig eosinophil homotypic aggregation, a CD18-dependent process, which may be an important component of eosinophil function in vivo. 2. Prostaglandin E1 (PGE1, 10(-16) to 10(-6) M) inhibited platelet activating-factor (PAF)- and C5a-induced eosinophil aggregation in a concentration-dependent manner. However, PAF-induced responses were more potently and more effectively inhibited by PGE1. The inhibitory effects of PGE1 on PAF-induced aggregation were reversed by pretreatment of eosinophils with the protein kinase A inhibitors H89 and KT5720. 3. The beta 2-adrenoceptor agonists, salbutamol and salmeterol, concentration-dependently inhibited eosinophil aggregation induced by C5a and PAF and, again. PAF-induced responses were more effectively reduced. The inhibitory effect of salmeterol was mediated by beta-adrenoceptors, as assessed by the reversal after pretreatment with propranolol. 4. Rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, also attenuated PAF- and C5a-induced aggregation and at a low concentration which did not affect aggregation per se, had a synergistic effect with PGE1 and salbutamol to suppress this response. 5. Activation of eosinophils with PAF or C5a induced a small but significant increase in the level of CD18 expression on the eosinophil surface. PGE1 (10(-7) M) decreased PAF- and C5a-induced upregulation of CD18 by 93% and 62%, respectively. 6. These results demonstrate that cyclic AMP-elevating agents effectively inhibit eosinophil aggregation, a CD18-dependent functional response. Because CD18 has been shown to be important for eosinophil recruitment to inflamed tissue in vivo, our findings may be of relevance to the efficacy of cyclic AMP-elevating agents at inhibiting eosinophil trafficking.