A novel, orally active LPA(1) receptor antagonist inhibits lung fibrosis in the mouse bleomycin model.

BACKGROUND AND PURPOSE: The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA(1), in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA(1)-antagonist (4'-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966). EXPERIMENTAL APPROACH: The potency and selectivity of AM966 for LPA(1) receptors was determined in vitro by calcium flux and cell chemotaxis assays using recombinant and native cell cultures. The in vivo efficacy of AM966 to reduce tissue injury, vascular leakage, inflammation and fibrosis was assessed at several time points in the mouse bleomycin model. KEY RESULTS: AM966 was a potent antagonist of LPA(1) receptors, with selectivity for this receptor over the other LPA receptors. In vitro, AM966 inhibited LPA-stimulated intracellular calcium release (IC(50)= 17 nM) from Chinese hamster ovary cells stably expressing human LPA(1) receptors and inhibited LPA-induced chemotaxis (IC(50)= 181 nM) of human IMR-90 lung fibroblasts expressing LPA(1) receptors. AM966 demonstrated a good pharmacokinetic profile following oral dosing in mice. In the mouse, AM966 reduced lung injury, vascular leakage, inflammation and fibrosis at multiple time points following intratracheal bleomycin instillation. AM966 also decreased lactate dehydrogenase activity and tissue inhibitor of metalloproteinase-1, transforming growth factor beta1, hyaluronan and matrix metalloproteinase-7, in bronchoalveolar lavage fluid. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that AM966 is a potent, selective, orally bioavailable LPA(1) receptor antagonist that may be beneficial in treating lung injury and fibrosis, as well as other diseases that are characterized by pathological inflammation, oedema and fibrosis.

Compartmentation of G-protein-coupled receptors and their signalling components in lipid rafts and caveolae.

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

Homologous and heterologous uncoupling of muscarinic M(3) and alpha(1B) adrenoceptors to Galpha(q/11) in SH-SY5Y human neuroblastoma cells.

1. The present study employed a [(35)S]-GTPgammaS binding protocol in conjunction with immunoprecipitation (IP) of the Galpha subunits to investigate the desensitization of G(q/11)-coupled receptors at the level of the G-protein activation. Membranes from SH-SY5Y cells expressing the recombinant human alpha(1B)-adrenoceptor (alpha(1B)-AR) (and endogenously expressing the M(3) muscarinic acetylcholine receptor (M(3)-AChR)) exhibited G(q/11) activation in a concentration-dependent manner in response to noradrenaline or methacholine. 2. Pre-treatment of intact cells with agonist prior to membrane preparation and use in the [(35)S]-GTPgammaS IP assay demonstrated that both receptors were homologously desensitized by pre-treatment with agonist since the G(q/11) activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of alpha(1B)-AR was effective at heterologously desensitizing the M(3)-AChR. The PKC inhibitor, Ro-31-8220 (10 microM) was ineffective at preventing the agonist-mediated receptor desensitization. 3. [(32)P]P(i)-labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 microM) was effective at producing a Ro-31-8220 (10 microM)-sensitive, detectable increase in alpha(1B)-AR but not M(3)-AChR phosphorylation. Noradrenaline (30 microM) stimulated alpha(1B)-AR phosphorylation, which could be partially inhibited by Ro-31-8220 (10 microM). The phosphorylation of M(3)-AChR was increased by methacholine (100 microM) incubation and this effect appeared to be insensitive to Ro-31-8220 (10 microM). 4. These findings demonstrate that [(35)S]-GTPgammaS-Galpha-subunit IP can be used to estimate receptor desensitization as a decline in receptor-G-protein coupling. Both the alpha(1B)-AR and M(3)-AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M(3)-AChR produced by alpha(1B)-AR stimulation is not associated with a detectable increase in M(3)-AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.

An investigation of noradrenaline uptake and release by the CATH.a cell line.

The cell bodies of ascending noradrenergic neurons in the brain are located predominantly in the locus coeruleus. An in vitro model of locus coeruleus neurons could prove to be a useful tool in the investigation of noradrenergic neural networks and their associated pathophysiologies. The CATH.a cell line demonstrates some of the properties expected of locus coeruleus neurons, and the present study investigated the neurotransmitter uptake and release properties of the CATH.a cells. It was surprising that the CATH.a cells failed to accumulate [3H]noradrenaline ([3H]NA), suggesting the lack of a functional NA transporter. RT-PCR supported this finding by demonstrating the absence of NA transporter mRNA. Treatment of CATH.a cells with various differentiating agents failed to increase the [3H]NA uptake. Endogenous NA release was studied using HPLC detection, which revealed a lack of depolarisation-induced increases in endogenous NA release. A human NA transporter-transfected CATH.a cell line was generated (termed RUNT), and a study of the [3H]NA uptake revealed that the RUNT cells displayed significant uptake that could be blocked by cocaine (10 microM). Furthermore, the uptake capacity could be dramatically increased by differentiation of the cells with dibutyryl cyclic AMP (1 mM) for 24 h. Using dibutyryl cyclic AMP-differentiated RUNT cells, high K+ concentrations (50 mM) significantly increased [3H]NA release above basal levels.

Inhibition of receptor-mediated calcium responses by corticotrophin-releasing hormone in the CATH.a cell line.

A region of the brain believed to be important in the CNS response to stress is the locus coeruleus, the predominant site of noradrenergic cell bodies. Corticotrophin releasing hormone (CRH) is the primary hypothalamic releasing hormone responsible for the activation of the pituitary-adrenal axis in response to stress and, in this study, we employed a locus coeruleus-like cell line, CATH.a, to investigate the modulation of receptor signalling pathways by CRH. Pituitary adenylyl cyclase-activating polypeptide (PACAP) (10 nM), vasoactive intestinal peptide (VIP) (1 microM) and carbachol (1 mM) produced transient increases in intracellular [Ca2+]. The inhibition of the carbachol (1 mM) response by CRH was concentration-dependent (EC50 = 154 +/- 1.8 nM). Calcium responses to sub-maximally effective concentrations of PACAP (5 nM), VIP (400 nM) and carbachol (1 mM) were abolished by prior exposure to CRH (1 microM). At the concentrations employed, CRH and VIP both substantially increased intracellular [3H]-cyclic AMP accumulation. The adenylyl cyclase activator forskolin (10 microM) was also effective at eliminating the agonist-induced calcium responses. Incubation with the cell permeant cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) (1 mM), an activator of protein kinase A (PKA), for 12 min prior to agonist exposure similarly abolished the intracellular calcium response to carbachol. Carbachol increased [3H]-inositol phosphate ([3H]-IP) accumulation to a maximum of 2.4 +/- 0.11-fold basal (EC50 = 6.75 +/- 0.26 microM). PACAP produced a much greater accumulation (19.9 +/- 2.1 fold basal; EC50 = 24 nM). In the presence of forskolin (10 microM), neither carbachol- nor PACAP-induced [3H]-IP accumulation was significantly different from in its absence. These results demonstrate that CRH inhibits receptor-mediated intracellular calcium responses in a locus coeruleus-like cell line possibly via activation of PKA. This modulation could be important in controlling neuronal function in vivo in stressful situations in which the levels of CRH are increased in the locus coeruleus.

Corticosteroid modulation of signal transduction in the CATH.a cell line.

Noradrenergic neuronal networks originating in the locus coeruleus have been implicated in the stress response. In order to study this system in vitro, we have employed a locus coeruleus-like cell line, CATH.a, and have determined the effect of dexamethasone on receptor-mediated second messenger responses. The CATH.a cell line produced increases in intracellular cyclic AMP conversion in response to corticotrophin-releasing factor (EC50 = 6.93 +/- 1.26 nM, maximum conversion = 4.11 +/- 0.20%) and vasoactive intestinal polypeptide (EC50 = 240 +/- 40 nM, maximum conversion = 8.92 +/- 1.24%). Forskolin (10 microM) increased conversion from 0.48 +/- 0.05 to 6.39 +/- 0.38%. The alpha 2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) inhibited the forskolin response with an IC50 of 6.76 +/- 0.11 nM. Carbachol increased total 3H-labelled inositol phosphate accumulation to a maximum of 3.01 +/- 0.79 fold basal (EC50 = 7.94 +/- 0.14 microM). Bradykinin produced a maximum 1.81 +/- 0.05 fold basal stimulation of phosphoinositide hydrolysis (EC50 = 9.12 +/- 0.16 nM). Both carbachol and bradykinin increased intracellular Ca2+ concentration probably via a combination of mobilisation of intracellular stores and gating of extracellular Ca2+. Incubation for 24 h with the glucocorticoid receptor agonist, dexamethasone (1 microM), significantly potentiated the receptor-mediated phosphoinositide responses to all the agents tested; however, of the receptor-mediated increases in cyclic AMP conversion, only the vasoactive intestinal polypeptide response was potentiated. These results show that the CATH.a cell line displays some of the properties expected of locus coeruleus neurons and that glucocorticoid receptor stimulation selectively modulates receptor-mediated increases in second messenger formation.