Increased arginase activity contributes to airway nitric oxide (NO) deficiency in cystic fibrosis (CF). Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation.

Asymmetric dimethylarginine is increased in cystic fibrosis airways and may contribute to airway obstruction in patients with cystic fibrosis by reducing nitric oxide formation.

The ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M 2 muscarinic receptor knockout mice. Forskolin (5.0 M) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M 2 muscarinic receptor knockout mice. This reduction in contractile activity was characterized by decreases in potency or maximal response, but not always both. Similar results were obtained with isoproterenol (1.0 M). The relaxant effects of forskolin in ileum, trachea, and urinary bladder from M 2 receptor knockout mice were approximately 3-to 9-fold greater than those observed in the same tissues from wild-type mice. Similar results were obtained with isoproterenol in ileum and urinary bladder, although the differences between wild-type and M 2 receptor knockout tissues were less than those observed with forskolin. In contrast, there was no significant difference between the relaxant effect of isoproterenol in trachea from wild-type and M 2 receptor knockout mice. In contrast to the results observed with oxotremorine-M as the contractile agent, forskolin and isoproterenol did not exhibit greater relaxant activity against KCl-induced contractions in M 2 receptor knockout mice compared with wild-type mice. These results suggest that a component of the contractile response to muscarinic agonists in smooth muscle involves an M 2 muscarinic receptor-mediated inhibition of the relaxant effects of agents that increase cAMP levels.

1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.