Nucleotide-induced mucin release from primary hamster tracheal surface epithelial cells involves the P2u purinoceptor

Abstract

Mucin release by airway surface epithelial cells is regulated by extracellular adenosine triphosphate (ATP) via a P 2 purinoceptor-mediated mechanism. The objective of the present experiment was to examine the possible involvement of uridine triphosphate (UTP) in this purinergic signal transduction pathway.Using primary hamster tracheal surface epithelial cells, ATP and UTP were compared in their abilities: 1) to displace ATPγS 35 -binding to intact cells; 2) to accumulate inositol phosphates; and 3) to stimulate mucin release. Finally, the presence of a P 2u receptor message was examined.Our results showed that: 1) UTP was much less effective than ATP in displacing ATPγS 35 -binding (median inhibitory concentrations (IC50s) 240 vs 2.9 µM); 2) UTP was more potent than ATP in accumulating inositol phosphates (100 vs 43% increase at 2 mM); 3) UTP was equipotent with ATP in stimulating mucin release; 4) Northern blot analysis of messenger ribonucleic acids (mRNAs) with a mouse P 2u receptor complementary deoxyribonucleic acid (cDNA) probe revealed a single specific band (2.8 kb), partial sequencing of which showed a great homology with those of human or mouse P 2u receptors.We conclude that, although both ATP and UTP are equipotent in stimulating mucin release, their binding kinetics to the cell surface are quite different, suggesting the presence of a common binding domain which may be responsible for the mucin release by these nucleotides. We suggest that the P 2u purinoceptor is likely to be responsible for mucin release by these nucleotides, probably via activation of phospholipase C. Eur Respir J., 1996, 9, 542-548 P 2u receptors purely based on agonist potency orders in which UTP = ATP > ATPγS > 2-methylthio ATP > α,β-methylene ATP [6]. However, our recent binding study indicated that, whilst there appears to be a good correlation between the mucin-releasing potency and the ATPγ S 35 displacement potency among various nucleotides, the ATPγS 35 displacement potency of UTP was, surprisingly, lower than ATP [10].Despite a plethora of information regarding the P 2u receptor in various systems, great variations among these systems make it difficult to conclude whether or not these nucleotides indeed act through the same mechanism, especially in the regulation of airway mucin secretion. Therefore, in the present experiment, this problem was approached by directly comparing these two nucleotides using a primary airway epithelial cell culture system, which has been well characterized for secretion of mucin both biochemically and morphologically [11,12]. This is the first report which attempts to elucidate the biochemical mechanism of the nucleotide-induced mucin release at various levels, from receptor-binding to actual secretion.In the airway, mucus plays an important role in the host's defence, and its proper function is believed to be attributed mainly to the quality and quantity of mucus glycoproteins or mucins which are present in the mucus. These airway mucins are secreted by two types of cell; goblet...

Coupled Nucleotide and Mucin Hypersecretion from Goblet-Cell Metaplastic Human Airway Epithelium

“…As predicted on the basis of previous studies, apical treatment of control HBE cultures with UTP or ionomycin for 10 minutes resulted in increased secretion of mucins ( Figure 5A) (6,(22)(23)(24)(25). Mucin secretion in response to these agonists was markedly enhanced in RSV and IL-13-induced goblet-cell metaplastic cultures ( Figure 5A).…”

Airway goblet cell mucin: its structure and regulation of secretion

“…It is important to note that this PLC-PKC pathway, however, accounts for only 50% of ATP-induced mucin release [65], which suggests the presence of another, as yet unknown, mechanism. Both the binding kinetics of ATPγS 35 in cultured TSE cells [68], and the comparison of ATP and uridine triphosphate (UTP) in their mucin-releasing activity [69,70], indicate that mucin release by nucleotides is mediated by the P 2u receptor.…”

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

“…Expression of P2Y 2 receptor mRNA has been detected in human skeletal muscle, heart, brain, spleen, lymphocytes, macrophages, bone marrow, and lung, with lower expression levels detected in liver, stomach, and pancreas . Functional P2Y 2 receptors are expressed in epithelial, smooth muscle, and endothelial cells and in leukocytes, cardiomyocytes, osteoblasts, and cells derived from the peripheral and central nervous system, including Schwann cells, rat cortical neurons, oligodendrocytes, dorsal horn and cortical astrocytes, immortalized astrocytes, astrocytoma cells, and NG108-15 neuroblastoma ϫ glioma hybrid cells (Bowler et al, 1995;Ho et al, 1995;Kirischuk et al, 1995;Rice et al, 1995;Berti-Mattera et al, 1996;Kim et al, 1996;Kunapuli and Daniel, 1998;Weisman et al, 1999;Pillois et al, 2002;Seye et al, 2002;Gendron et al, 2003;Kumari et al, 2003).…”