### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

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Receptors for Purines and Pyrimidines

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.

https://pharmrev.aspetjournals.org/content/pharmrev/58/3/281.full.pdf

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

This review is aimed at providing readers with a comprehensive reference article about the distribution and function of P2 receptors in all the organs, tissues, and cells in the body. Each section provides an account of the early history of purinergic signaling in the organ/cell up to 1994, then summarizes subsequent evidence for the presence of P2X and P2Y receptor subtype mRNA and proteins as well as functional data, all fully referenced. A section is included describing the plasticity of expression of P2 receptors during development and aging as well as in various pathophysiological conditions. Finally, there is some discussion of possible future developments in the purinergic signaling field.

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Cellular distribution and functions of P2 receptor subtypes in different systems.

Buccal bioadhesive drug delivery--a promising option for orally less efficient drugs.

IL-33, a new member of the IL-1 cytokine family, promotes Th2 inflammation, but evidence on the implications of this cytokine in asthma is lacking IL-33 would be mainly expressed by structural cells, but whether proinflammatory cytokines modulate its expression in airway smooth muscle cells (ASMC) is unknown Endobronchial biopsies were obtained from adults with mild (n = 8), moderate (n = 8), severe (n = 9), asthma and from control subjects (n = 5) Immunocytochemistry, laser-capture microdissection, reverse transcriptase, and real-time quantitative PCR were used for determining IL-33 expression in the lung tissues ASMC isolated from resected lung specimens were cultured with proinflammatory cytokines and with dexamethasone IL-33 expression by ASMC was determined by PCR, ELISA, and Western blotting Higher levels of IL-33 transcripts are detected in biopsies from asthmatic compared with control subjects, and especially in subjects with severe asthma ASMC show IL-33 expression at both protein and mRNA levels IL-33 and TNF-alpha transcript levels correlate in the lung tissues, and TNF-alpha up-regulates IL-33 expression by cultured ASMC in a time- and dose-dependent manner IFN-gamma also increases IL-33 expression and shows synergistic effect with TNF-alpha Dexamethasone fails to abolish TNF-alpha-induced IL-33 up-regulation IL-33 expression increases in bronchial biopsies from subjects with asthma compared with controls, as well as subjects with asthma severity ASMC are a source of the IL-33 cytokine Our data propose IL-33 as a novel inflammatory marker of severe and refractory asthma

Increased Expression of IL-33 in Severe Asthma: Evidence of Expression by Airway Smooth Muscle Cells

1. Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo- and 16HBE14o- epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU-3 serous cells, the 6CFSMEo- submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC-1. These experiments revealed that P2Y2 and P2Y6 mRNA are co-expressed in the IHAEo-, 16HBE14o- and A549 epithelial cell lines. The CFPAC-1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors. 2. We have then performed RT-PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo-, 16HBE14o-, 6CFSMEo-, HASMSC1 and CFPAC-1. P2Y1 mRNA was only detected in the CALU-3 cell line. 3. Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo- and the 16HBE14o- airway epithelial cell lines which co-express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo- submucosal cells expressed a UTP-specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

https://bpspubs.onlinelibrary.wiley.com/doi/pdf/10.1038/sj.bjp.0702560

Expression of P2Y receptors in cell lines derived from the human lung.

Interleukin 8 and monocyte chemoattractant protein 1 production by cultured human airway smooth muscle cells

Human bronchial surface epithelial cells were maintained in secondary culture on a collagen gel substrate in a defined, serum-free medium. These conditions have previously been reported to promote mucous cell differentiation. After 3 wk in culture, approximately 40% of the cells were stained by an antibody directed against human respiratory mucin. Analysis of media from cells cultured in the presence of the radioactive precursors [3H]glucosamine and [35S]sulfate revealed that the cells secreted high molecular weight glycoproteins with properties of typical respiratory mucins. In addition, hyaluronic acid and proteoglycans containing chondroitin sulfate and/or heparan sulfate glycosaminoglycans were identified in cell conditioned media. Finally, Western blot analyses showed that the cells secreted lysozyme and mucous proteinase inhibitor, proteins that are generally considered to be markers for submucosal gland serous cells. These results show that human bronchial cells from the surface epithelium in secon...

Mucous and serous secretions of human bronchial epithelial cells in secondary culture.

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (M w ) for three different preparations ranged from 3.3×106 to 4.7×106 g/mol (corresponding to an average structure of 1 – 2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5×10–8 to 6.4×10–8 cm2/s were calculated using the Svedberg equation. A polydispersity index (M z /M w ) of ∼1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05×10–8 cm2/s (inter-quartile range = 1.11×10–7 to 6.08×10–8 cm2/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98×10–9 cm2/s (inter-quartile range = 1.47×10–8 to 3.25×10–9 cm2/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D 20,w ) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D 37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell.

Hydrodynamic properties of mucins secreted by primary cultures of guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution.

We have prepared a monoclonal antibody (MAb), 3D3, raised against purified human respiratory mucins. This antibody recognized mucins and proteolytically derived glycopeptides. The epitope recognized by the antibody was destroyed by alpha-L-fucosidase, indicating that it was present on the carbohydrate moieties. Structural specificity was determined by adsorption on a variety of synthetic, insolubilized oligosaccharides. Several lines of evidence indicate that the 3D3 MAb reacted strongly with the Lewis (Leb) antigen, but also recognized Le(a) and Le(y) determinants. This antibody might be useful to study mucin secretion.

Graham A. Place

Papers

Expression of P2Y receptors in cell lines derived from the human lung.

Interleukin 8 and monocyte chemoattractant protein 1 production by cultured human airway smooth muscle cells

Mucous and serous secretions of human bronchial epithelial cells in secondary culture.

Hydrodynamic properties of mucins secreted by primary cultures of guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution.

A new monoclonal antibody (3D3) generated with human respiratory mucins and directed against Lewis determinants.