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Incubation of airways from nonatopic patients with serum from patients with high IgE levels confers responsiveness to "specific" (allergen) and hyperresponsiveness to "nonspecific" (histamine) stimuli. We have tested the hypothesis that the level of IgE determines the degree of specific and nonspecific responsiveness. Bronchial rings from nonatopic patients were sensitized overnight with serum containing high levels of allergen-specific IgE, or with an allergen-specific chimeric IgE (JW8) in physiologic buffer. In vitro responsiveness to allergen and histamine was evaluated and compared with non-sensitized tissues from the same patients. Responses to specific allergen were demonstrated in all tissues sensitized with atopic serum or chimeric IgE, but not in nonsensitized tissues. Allergen responses were specific, since tissues sensitized using serum containing high Dermatohagoides farinae-specific IgE only, did not respond to either horse or dog allergens. The potency and magnitude of the maximal contraction to histamine was significantly (p < 0.05) increased in tissues sensitized using atopic serum with high total IgE concentrations compared with nonsensitized preparations, but was unchanged in tissues sensitized using chimeric IgE or serum with low total IgE levels. Therefore, specific IgE determines allergen responsiveness in passively sensitized human airways, but histamine hyperresponsiveness is independent of specific IgE and appears to be related to some other factor associated with serum containing high concentrations of total IgE.

The ability of G protein ␣ and ␤␥ subunits to activate the p110␥ isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110␥ form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTPactivated G s , G i , G q , or G o ␣ subunits were unable to activate PtdIns 3-kinase. Dimers containing G␤ 1-4 complexed with ␥ 2 -stimulated PtdIns 3-kinase activity about 26-fold with EC 50 values ranging from 4 to 7 nM. G␤ 5 ␥ 2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase C␤. A series of dimers with ␤ subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of ␤␥ with phosducin (␤1H311A␥2, ␤1R314A␥2, and ␤1W332A␥2) was tested, and only ␤1W332A␥2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the ␤ 1 subunit complexed with a panel of different G␥ subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The ␤ 1 ␥ 2 , ␤ 1 ␥ 10 , ␤ 1 ␥ 12 , and ␤ 1 ␥ 13 dimers all activated PtdIns 3-kinase about 26-fold with 4 -25 nM EC 50 values. The ␤ 1 ␥ 11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110␥ form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the ␥ subunit in determining the activation of PtdIns 3-kinase was examined using ␥ subunits with altered CAAX boxes directing the addition of farnesyl to the ␥ 2 subunit and geranylgeranyl to the ␥ 1 and ␥ 11 subunits. Replacement of the geranylgeranyl group of the ␥ 2 subunit with farnesyl inhibited the activity of ␤ 1 ␥ 2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the ␥ 1 and ␥ 11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all ␤ subunits, with the exception of ␤ 5 , interact equally well with PtdIns 3-kinase. In contrast, the composition of the ␥ subunit and its prenyl group markedly affects the ability of the ␤␥ dimer to stimulate PtdIns 3-kinase.The generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) 1 in the inner leaflet of the plasma membrane is critical to the regulation of cell function (1-3). The phosphorylated inositol head group provides a docking site for proteins containing pleckstrin homology domains (PH domains) and leads to activation of many enzymes including the phosphoinositol-dependent protein kinase and protein kinase B (Akt). Activation of protein kinase B regulates multiple cellular functions including differentiation, regulation of metabolic events, cell survival, and motility (1-3). In keeping with this central role, the level of PIP3 is tightly regulated, it can be elevated by multiple classes of receptors (1, 2), and there are specific phosphatidylinositol 5-phosphatases (SHIP and PTEN) that degrade the signal (4 -8).A large family of phosphatidylinositol 4,5-bisphosphate 3-kinases (PtdIns 3-kinases) is responsible for generating PIP3 by phosphorylating the D3 ...

1 Non-selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) block allergen-induced contraction of passively sensitized human airways in vitro by a dual mechanism involving a direct relaxant eect on smooth muscle and inhibition of histamine and cysteinyl leukotriene (LT) release from airways. We investigated the eects of non-selective PDE inhibitors and selective inhibitors of PDE3 and PDE4 in order to determine the involvement of PDE isoenzymes in the suppression of allergic bronchoconstriction. 2 Macroscopically normal airways from 76 patients were sensitized with IgE-rich sera (4250 u ml 71) containing speci®c antibodies against allergen (Dermatophagoides farinae). Contractile responses of bronchial rings were assessed using standard organ bath techniques. 3 Passive sensitization caused increased contractile responses to allergen, histamine and LTC 4 . Non-selective PDE inhibitors (theophylline, 3-isobutyl-1-methylxanthine [IBMX]), a PDE3-selective inhibitor (motapizone), PDE4-selective inhibitors (RP73401, rolipram, AWD 12-281) and a mixed PDE3/4 inhibitor (zardaverine) all signi®cantly relaxed inherent bronchial tone at resting tension and to a similar degree. Theophylline, IBMX, zardaverine and the combination of motapizone and RP73401 inhibited the contractile responses to allergen and LTC 4 . Pre-treatment with motapizone, RP73401, rolipram or the methylxanthine adenosine receptor antagonist, 8-phenyltheophylline, did not signi®cantly decrease responses to either allergen or LTC 4 . 4 We conclude that combined inhibition of PDE3 and PDE4, but not selective inhibition of either isoenzyme or antagonism of adenosine receptors, is eective in suppressing allergen-induced contractions of passively sensitized human airways. The relationship between allergen-and LTC 4 -induced responses suggests that PDE inhibitors with PDE3 and PDE4 selectivity are likely to act in part through inhibition of mediator release and not simply through direct relaxant actions on airway smooth muscle.