1 A de®ciency of constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO), due to reduced availability of L-arginine, importantly contributes to allergen-induced airway hyperresponsiveness (AHR) after the early asthmatic reaction (EAR). Since cNOS and arginase use L-arginine as a common substrate, we hypothesized that increased arginase activity is involved in the allergeninduced NO de®ciency and AHR. 2 Using a guinea-pig model of allergic asthma, we addressed this hypothesis by examining the e ects of the speci®c arginase inhibitor N o -hydroxy-nor-L-arginine (nor-NOHA) on the responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals and from animals 6 h after ovalbumin challenge. Arginase activity in these preparations was investigated by measuring the conversion of L-[ 14 C]arginine to [ 14 C]urea. 3 Airways from allergen-challenged animals showed a 2 fold (P50.001) increase in responsiveness to intraluminal (IL) administration of methacholine compared to controls. A similar hyperresponsiveness (1.8 fold, P50.01) was observed in control airways incubated with the NOS inhibitor N onitro-L-arginine methyl ester (L-NAME, 0.1 mM, IL), while L-NAME had no further e ect on the airways from challenged animals. 4 Remarkably, 5 mM nor-NOHA (IL) normalized the hyperresponsiveness of challenged airways to basal control (P50.001), and this e ect was fully reversed again by 0.1 mM L-NAME (P50.05). Moreover, arginase activity in homogenates of the hyperresponsive airways was 3.5 fold (P50.001) enhanced compared to controls. 5 The results indicate that enhanced arginase activity contributes to allergen-induced de®ciency of cNOS-derived NO and AHR after the EAR, presumably by competition with cNOS for the common substrate, L-arginine. This is the ®rst demonstration that arginase is involved in the pathophysiology of asthma. British Journal of Pharmacology (2002) 136, 391 ± 398 Keywords: Arginase; constitutive nitric oxide synthase; nitric oxide; methacholine; N o -hydroxy-nor-L-arginine; allergic asthma; early asthmatic reaction; airway hyperresponsiveness; tracheal perfusion; guinea-pig Abbreviations: AHR, airway hyperreactivity; cNOS, constitutive nitric oxide synthase; EAR, early asthmatic reaction; EL, extraluminal; E max , maximal e ect; eNOS, endothelial nitric oxide synthase; IL, intraluminal; iNANC, inhibitory nonadrenergic noncholinergic; iNOS, inducible nitric oxide synthase; KH, Krebs-Henseleit; LAR, late asthmatic reaction; L-NAME, N o -nitro-L-arginine methyl ester; L-NMMA, N G -monomethyl-L-arginine; nNOS, neuronal nitric oxide synthase; nor-NOHA, N o -hydroxy-nor-L-arginine; DP, di erential (hydrostatic) pressure; P inlet , (hydrostatic) pressure at the inlet; P outlet , (hydrostatic) pressure at the outlet; pEC 50 , 7log 10 of the concentration causing 50% of the e ect

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [(3)H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M(3)-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 microM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1-10 microM), but only in cells having functional muscarinic M(3)-receptors. This synergism could be suppressed significantly by the selective M(3)-receptor antagonists DAU 5884 (0.1 microM) and 4-DAMP (10 nM), but not at all by the M(2)-subtype selective antagonist gallamine (10 microM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M(3)-receptors in bovine tracheal smooth muscle cells.

In the developing world major public health issues such as malnutrition and compromised physical development are intimately linked to altered gut morphology and function with underlying chronic inflammatory responses. In these societies the downward spiral of malnutrition and infections does not seem to be remedied by well-informed nutritional interventions that supplement the identified nutrient deficiencies, suggesting that additional strategies are needed. The aim of this scientific opinion paper is to consider how a child from the developing world might benefit, separately and additively, from interventions targeted to impact hygiene, nutritional status, disease resistance and gut function, if successful interventions could be found. A failure to tackle environmental enteropathy (EE) may be a critical limiting factor that can explain the relative lack of success of interventions focussed on micronutrient supplementation so far. Therefore this paper starts with a summary of the aetiology and consequences of EE on child health and the current recommendations aimed at tackling this problem. Then a number of hypotheses will be considered in terms of research strategy to positively affect nutritional status, intestinal health and growth of children with EE, with the aim of inspiring future innovative strategies, for both the food industry and the public health sector, which could benefit millions of children.

1 The present study aims to investigate whether phenotypic changes, reported to occur in cultured isolated airway smooth muscle (ASM) cells, are of relevance to intact ASM. Moreover, we aimed to gain insight into the signalling pathways involved. 2 Culturing of bovine tracheal smooth muscle (BTSM) strips for up to 8 days in the presence of 10% foetal bovine serum caused a time-dependent (t 1/2 =2.8 days) decrease in maximal contraction (E max ) to methacholine compared to serum-deprived controls (E max =74+4% at day 8). A reduced E max was also found using insulin-like growth factor-1 (30 ng ml 71 ) and platelet-derived growth factor (30 ng ml 71 ), but not using epidermal growth factor (10 ng ml 71 ) (E max =83+3, 67+8, 100+4%, respectively). Similar serum and growth factor-induced changes in E max were found for KCl-induced contraction (65+9, 80+7, 64+11% and 107+2%, respectively). 3 Strong correlations were found between the growth factor-induced reductions in E max and their proliferative responses, assessed by [ 3 H]-thymidine-incorporation, in BTSM cells. (r=0.97, P=0.002 for methacholine and r=0.93, P=0.007 for KCl). 4 The PDGF-induced reduction in E max was inhibited completely by combined treatment with either PD 98059 (30 mM) or LY 294002 (10 mM). 5 These results indicate that serum and growth factors may cause a functional shift towards a less contractile phenotype in intact BTSM, which is associated with their proliferative response and dependent on signalling pathways involving the mitogen-activated protein kinase pathway and the phosphatidylinositol-3-kinase pathway.

Increased smooth muscle mass due to hyperplasia and hypertrophy of airway smooth muscle (ASM) cells is a common feature in asthma. Angiotensin II (Ang II), a potent vasoconstrictor and mitogen for a wide variety of cells, has recently been implicated in bronchoconstriction in asthmatics. However, a possible mitogenic role as well as underlying molecular mechanisms of this octapeptide in human ASM cells are not yet known. We studied the effects of Ang II on ASM cell proliferation and growth and on the expression of three transcription factors, egr-1, c-fos, and c-jun, as well as a cytokine, transforming growth factor-beta1 (TGF-beta1). Human ASM cells were isolated by enzymatic digestion of bronchial smooth muscle obtained from lung resection tissue. Confluent cells were growth-arrested and subsequently incubated with Ang II (100 nM) for different time periods and processed for the measurement of cell growth and gene expression. Ang II significantly induced DNA and protein synthesis in human ASM cells at 8 h, resulting in a net increase in the accumulation of protein over DNA (i.e., cellular hypertrophy) at 16 h of incubation. Cell counts and MTT-reduction assay, however, showed no increase in cell number as a result of Ang II stimulation. Ang II stimulated the expression of egr-1 and c-fos as early as 15 min, reaching maximum levels at 45 min, whereas the expression of c-jun peaked at 2 h of Ang II exposure. Furthermore, steady-state mRNA levels of TGF-beta1 were upregulated by Ang II after 4 h and reached peak levels at 16 h of incubation. Secretion of biologically active TGF-beta1 from human ASM cells was significantly (P <= 0.02) enhanced by Ang II incubation after 8 h, which remained elevated until 24 h. Our results suggest that the Ang II-induced transient early expression of transcription factors may regulate autocrine genes like TGF-beta1, of which the subsequent late upregulation could contribute to cellular hypertrophy during, for example, airway remodeling in asthma.