Rationale: Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline. Objectives: To determine the role of phosphoinositide-3-kinase-delta (PI3K-d) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline. Methods: Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-a-induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n 5 17) and compared with that of nonsmoking (n 5 8) and smoking (n 5 7) control subjects. The effect of theophylline and a selective PI3K-d inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke-exposed mice. Measurements and Main Results: Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 6 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 6 10.5 nM; P 5 0.018) and smoking control subjects (47.5 6 19.6 nM; P 5 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-d, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-d failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette-smoke-exposed mice in vivo. Conclusions: Inhibition of oxidative stress dependent PI3K-d activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid insensitivity in COPD.

1 The pl protein, which we previously cloned from retina, assembles as a homooligomer that transduces the binding of y-aminobutyric acid (GABA) into robust chloride currents. However, its insensitivity to bicuculline, pentobarbitone and benzodiazepines, all potent agents at typical GABAA receptors, suggested that it may react atypically to other GABA agonists and antagonists.2 cDNAs for the pl and the a5p1 receptors for GABA were expressed as homo-and heterooligomers, respectively, in Xenopus oocytes. The selectivities of the respective receptors for various agonists were investigated using concentration-response experiments in voltage clamped cells. 3 The most potent agonists at the p1 receptor were trans-4-aminocrotonic acid (TACA) > GABA > muscimol; at the a5p, receptor the rank order was muscimol > GABA > 4,5,6,7-tetrahydroisoxazole[4,5-c]pyridine-3-ol (THIP). The most specific agonists were cis-(2-(aminomethyl)-cyclopropylcarboxylic acid (CAMP) and THIP for the pl and the a53, receptors, respectively. 4 Comparing GABA, TACA and cis-aminocrotonic acid (CACA) at pl receptors expressed in COS cells gave results almost indistinguishable from those found at oocytes; the pharmacology of p1 seems independent of the expression system. 5 Agonists THIP, piperidine-4-sulphonic acid (P4S), and isoguvacine, whose C-C-C-N chains are constrained by rings into a folded conformation and were potent at the a501 receptor, were among the weakest at the pl receptor. However CACA and CAMP, which align better with the extended than the folded conformation, were weakest at the a5pI receptor but moderately potent at the pl receptor. These findings suggest that the pl receptor recognizes agonists in the extended conformation, in contrast to GABAA receptors, which are believed to recognize agonists in the partially folded conformation.6 In contrast to the o5p1 receptor, gradations in maximum responses were apparent in the pl receptor, suggesting various degrees of partial agonism. In particular, imidazole-4-acetic acid (I4AA), whose maximum response was only 3% of GABA's maximum, had an apparent Kd for activating the pl receptor of 16f1M; but it had an apparent Kd for competitively blocking the receptor of 0.64 1AM. This difference suggests that steric constraints in the activated (open channel) receptor are tighter than in the resting receptor.7 Hill coefficients approached 2 at the pl receptor, but were closer to unity at the (0I1 receptor. Thus, the pl receptor displayed higher cooperativity. 8 Unlike typical GABAA receptors, the pl receptor was insensitive to the competitive antagonists bicuculline, SR95531, securinine, and (+)-tubocurarine.

Cycling of polyamines (spermine and spermidine) in the brain was examined by measuring polyamine transport in synaptic vesicles, synaptosomes and glial cells, and the release of spermine from hippocampal slices. It was found that membrane potential-dependent polyamine transport systems exist in synaptosomes and glial cells, and a proton gradientdependent polyamine transport system exists in synaptic vesicles. The glial cell transporter had high affinities for both spermine and spermidine, whereas the transporters in synaptosomes and synaptic vesicles had a much higher affinity for spermine than for spermidine. Polyamine transport by synaptosomes was inhibited by putrescine, agmatine, histidine, and histamine. Transport by glial cells was also inhibited by these four compounds and additionally by norepinephrine. On the other hand, polyamine transport by synaptic vesicles was inhibited only by putrescine and histamine. These results suggest that the polyamine transporters present in glial cells, neurons, and synaptic vesicles each have different properties and are, presumably, different molecular entities. Spermine was found to be accumulated in synaptic vesicles and was released from rat hippocampal slices by depolarization using a high concentration of KCl. Polyamines, in particular spermine, may function as neuromodulators in the brain.

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