The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P 2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ), in contrast, are not. We report here activation of the epithelial Na ؉ channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na ؉ reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns-(3,4)P 2 /PtdIns(3,4,5)P 3 , as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P 2 and PtdIns-(3,4,5)P 3 activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3 . This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.

BACKGROUND Anecdotal cases of chromosomal abnormalities in Philadelphia chromosome (Ph)‐negative metaphases have been reported in patients with chronic myelogenous leukemia (CML) in the chronic phase during treatment with interferon and, more recently, with imatinib. This phenomenon is different from true clonal evolution in that the additional cytogenetic abnormality occurs in Ph‐negative cells. METHODS The authors analyzed their experience with 342 patients with CML in chronic phase treated with imatinib to investigate the frequency and significance of this event. RESULTS After a median follow‐up of 30 months (range, 16–35 months), 21 patients (6%; 95% confidence interval, 0.04, 0.09) developed 25 chromosomal abnormalities in Ph‐negative cells. Thirteen (54%) of these abnormalities were seen in 2 or more metaphases. The median time from the start of treatment with imatinib to the appearance of the abnormalities was 6 months (range, 3–22 months). The most common cytogenetic abnormality detected was trisomy 8 (33%). Twenty of 21 patients (95%) achieved a major (Ph < 35%) cytogenetic response (complete cytogenetic response in 13–62%). After a median follow‐up of 22 months (range, 4–33 months), all 21 patients were alive, 20 of them in chronic phase and in complete hematologic response. None of the patients showed features of myelodysplasia. CONCLUSIONS Cytogenetic abnormalities occur in Ph‐negative cells in a fraction of patients with CML in chronic phase treated with imatinib. With a short follow‐up, no clear clinical consequences can be identified. Cancer 2003. © 2003 American Cancer Society.

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na ϩ channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane ETB but not ETA receptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.salt-sensitive hypertension; systemic blood pressure ENDOTHELIN-1 (ET-1) is a powerful vasoconstricting peptide hormone that is an important regulator of systemic blood pressure (53). Independent of its vascular effects, ET-1 also affects renal Na ϩ and water handling favoring natriuresis and diuresis. While circulating ET-1 arises from endothelial cells, local ET-1 systems also exist. For instance in the kidney, the collecting duct produces significant amounts of 25,38,51). ET-1 targets cells through two distinct receptor subtypes, ET A and ET B (32, 41). Renal collecting duct cells have both types of receptors and are able to bind 49,50). Thus, collecting duct-derived ET-1, acting in a paracrine/ autocrine manner, is an important regulator of renal Na ϩ handling (2,20,26,42).Regulated Na ϩ reabsorption in the renal collecting duct, in part, controls blood pressure. Here, activity of the aldosteronesensitive epithelial Na ϩ channel (ENaC) is limiting for Na ϩ transport (reviewed in Refs. 19,30,31). Dysfunction and inappropriate regulation of ENaC consequently result in improper renal Na ϩ handling and thus, blood pressure disorders. For instance, gain of ENaC function in rodents and humans is causative for hypertension associated with the hallmarks of low plasma renin activity and aldosterone levels (1,22,23,45,46). Amiloride, an ENaC blocker, ameliorates this hypertension.Spotting lethal (sl) rats have a naturally occurring null mutation of ET B (17). These rats, when rescued from lethal intestinal aganglionosis by directed ET B transgene expression in the enteric nervous system, are particularly sensitive to DOCA and salt-induced hypertension (18,33,34). Similarly, mice with collecting duct-specific knockout of the ET B receptor have elevated blood pressure that further increases with high salt feeding (20). Collecting duct-specific ET-1 knockout, moreover, leads to hypertension exacerbated by high salt (2, 42). Plasma renin activity and aldoste...

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) are physiologically important second messengers. These molecules bind effector proteins to modulate activity. Several types of ion channels, including the epithelial Na+ channel (ENaC), are phosphoinositide effectors capable of directly interacting with these signaling molecules. Little, however, is known of the regions within ENaC and other ion channels important to phosphoinositide binding and modulation. Moreover, the molecular mechanism of this regulation, in many instances, remains obscure. Here, we investigate modulation of ENaC by PI(3,4,5)P3 and PI(4,5)P2 to begin identifying the molecular determinants of this regulation. We identify intracellular regions near the inner membrane interface just following the second transmembrane domains in β- and γ- but not α-ENaC as necessary for PI(3,4,5)P2 but not PI(4,5)P2 modulation. Charge neutralization of conserved basic amino acids within these regions demonstrated that these polar residues are critical to phosphoinositide regulation. Single channel analysis, moreover, reveals that the regions just following the second transmembrane domains in β- and γ-ENaC are critical to PI(3,4,5)P3 augmentation of ENaC open probability, thus, defining mechanism. Unexpectedly, intracellular domains within the extreme N terminus of β- and γ-ENaC were identified as being critical to down-regulation of ENaC activity and Po in response to depletion of membrane PI(4,5)P2. These regions of the channel played no identifiable role in a PI(3,4,5)P3 response. Again, conserved positive-charged residues within these domains were particularly important, being necessary for exogenous PI(4,5)P2 to increase open probability. We conclude that β and γ subunits bestow phosphoinositide sensitivity to ENaC with distinct regions of the channel being critical to regulation by PI(3,4,5)P3 and PI(4,5)P2. This argues that these phosphoinositides occupy distinct ligand-binding sites within ENaC to modulate open probability.

Mycoplasma pneumoniae causes acute and chronic lung infections in humans, leading to a variety of pulmonary and extrapulmonary sequelae. Of the airway complications of M. pneumoniae infection, M. pneumoniae-associated exacerbation of asthma and pediatric wheezing are emerging as significant sources of human morbidity. However, M. pneumoniae products capable of promoting allergic inflammation are unknown. Recently, we reported that M. pneumoniae produces an ADP-ribosylating and vacuolating toxin termed the community-acquired respiratory distress syndrome (CARDS) toxin. Here we report that naive mice exposed to a single dose of recombinant CARDS (rCARDS) toxin respond with a robust inflammatory response consistent with allergic disease. rCARDS toxin induced 30-fold increased expression of the Th-2 cytokines IL-4 and IL-13 and 70-to 80-fold increased expression of the Th-2 chemokines CCL17 and CCL22, corresponding to a mixed cellular inflammatory response comprised of a robust eosinophilia, accumulation of T cells and B cells, and mucus metaplasia. The inflammatory responses correlate temporally with toxin-dependent increases in airway hyperreactivity characterized by increases in airway restriction and decreases in lung compliance. Furthermore, CARDS toxin-mediated changes in lung function and histopathology are dependent on CD4 1 T cells. Altogether, the data suggest that rCARDS toxin is capable of inducing allergic-type inflammation in naive animals and may represent a causal factor in M. pneumoniae-associated asthma.Keywords: Mycoplasma pneumoniae; asthma; rCARDS toxin; eosinophilia; T cell Mycoplasma pneumoniae is a common human bacterial pathogen that causes acute and chronic infections of the respiratory tract and extrapulmonary pathology (1, 2). With the exception of mycoplasma adherence to the host epithelium, molecular mechanisms of virulence associated with the pathogenesis of M. pneumoniae infection are not well understood (1, 3). M. pneumoniae is predominantly an extracellular pathogen that binds to respiratory epithelial cells using a polarized tip organelle (1, 3-5). Interaction of M. pneumoniae with the respiratory epithelium results in significant cytopathology in cell culture and in vivo (4, 5). Previously, the cytopathology was attributed in part to the cytotoxic effects of hydrogen peroxides produced by M. pneumoniae (3). However, recently, we identified an ADP-ribosylating and vacuolating toxin produced by M. pneumoniae that is capable of inducing cytopathology in vitro and in vivo and that reproduces the infectious process (6-9).The community-acquired respiratory distress syndrome (CARDS) toxin encoded by the MPN372 gene was functionally identified as a human surfactant protein A binding protein (7). Upon further investigation, we discovered that CARDS toxin possesses structurally and functionally important regions of identity to the pertussis toxin S1 protein. Furthermore, highly purified rCARDS toxin causes extensive dose-dependent cytopathology in mammalian cell and organ culture, suggestin...