Background: Asthma is associated with airway hyperresponsiveness and enhanced T-cell number/ activity on one hand and increased levels of exhaled nitric oxide (NO) with expression of inducible NO synthase (iNOS) on the other hand. These findings are in paradox, as NO also relaxes airway smooth muscle and has immunosuppressive properties. The exact role of the endothelial NOS (eNOS) isoform in asthma is still unknown. We hypothezised that a delicate regulation in the production of NO and its bioactive forms by eNOS might be the key to the pathogenesis of asthma.

Airway hyperresponsiveness and epithelial damage are key features in asthma, a disease reaching epidemic proportions in the developed world. To investigate and modulate airway hyperresponsiveness, we used two novel peptides, CALP1 and CALP2, which interact with calcium-binding EF hand motifs and regulate calcium channels. We found that CALP1 induced, whereas CALP2 blocked, airway hyperresponsiveness. Both effects were epithelium-dependent. In epithelial cells, CALP1 blocked agonist-induced increase in [Ca 2+ ] i , whereas CALP2 increased agonist-induced elevation in [Ca 2+ ] i . CALP2 causes the release of nitric oxide (NO), a mediator that relaxes airway smooth muscle. We conclude that epithelial calcium channels play a key role in regulating airway hyperresponsiveness by controlling [Ca 2+ ] i and consequently the production of NO. These results bring new insights into the mechanism of airway hyperresponsiveness and suggest new therapeutic targets for asthma.Key words: calcium-like peptide • nitric oxide • epithelium • tracheal reactivity sthma is a chronic airway disease characterized by infiltration of inflammatory cells, epithelial cell damage, and airway hyperresponsiveness to a variety of specific and nonspecific stimuli. The degree of epithelial damage is associated with the degree of airway hyperresponsiveness (1). The airway epithelium is a physical barrier that protects sensory nerves and smooth muscle from stimulation by inhaled irritants (2). Epithelial cells also release mediators that can inhibit bronchoconstriction by relaxing the underlying smooth muscle. These "epithelium-derived relaxing factors," such as prostaglandin E 2 and nitric oxide (NO), protect the airways from excessive bronchoconstriction (reviewed in 3).Inhaled NO causes bronchodilation in asthmatic patients (4), and inhibition of NO production by NO synthase inhibitors increases airway responsiveness in patients with mild asthma (5, 6). Viral A respiratory infections induce airway hyperresponsiveness in healthy individuals and exacerbate it in asthmatic subjects. These infections are estimated to underlie about 85% and 45% of asthma exacerbations in children and adults, respectively (7-9). NO plays a protective role after an experimental viral infection in asthmatics (10). These results are consistent with our earlier findings that, in guinea pigs, NO synthase inhibitors induce airway hyperresponsiveness in vivo and in vitro (11) and that the NO production in tracheal tubes from virus-inoculated guinea pigs is reduced by 75% (12).Calmodulin (CaM), the ubiquitous calcium-sensing regulatory protein, binds and regulates different target proteins, including protein kinases, ion channels, and NO synthases (13). The constitutively expressed NO synthase (cNOS), present in the airway epithelium (14, 15), is activated by CaM in a calcium-dependent manner (16). NO produced by cNOS is an important regulator of tone and responsiveness of airway smooth muscle (reviewed in (14)). Agonist stimulation increases the intracellular calcium concentra...

Release of allergic mediators from mast cells is enhanced by very late Ag (VLA)-5-mediated interaction of these cells with fibronectin. In this report, we show that VLA-5-mediated adhesion of bone marrow-derived mast cells to fibronectin can be induced by two different pathways: first, FcεRI clustering, which depends on calmodulin activation and extracellular Ca2+, and, second, by Mn2+ stimulation, which is independent of calmodulin activation and antagonized by Ca2+. Previous studies have shown the presence of several cation-binding domains in VLA-5 that are homologous to the calcium-binding EF-hands of calmodulin. To show a role for EF-hands of different proteins in VLA-5-mediated adhesion, we used calcium-like peptides (CALP), CALP1 and CALP2, designed to bind to EF-hands based on inverted hydropathy. CALP1 and, more potently, CALP2 inhibited FcεRI-induced adhesion to fibronectin via different mechanisms. The target for the effects of CALP1 and 2 on FcεRI-induced adhesion and degranulation was intracellular and likely involved calmodulin. Interestingly only CALP2 was able to inhibit Mn2+-induced calmodulin-independent adhesion by interfering with an extracellular target, which is probably VLA-5. We conclude that CALP1 and 2 can inhibit VLA-5-mediated adhesion of mast cells to fibronectin through binding to EF-hands of multiple proteins, and that these peptides can be used as lead compounds for the development of future therapy against allergy.