Search

1The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl−channels in excised inside‐out membrane patches from cells expressing wild‐type human CFTR.2Addition of genistein (100 μM) to the intracellular solution caused a small decrease in single‐channel current amplitude (i),but a large reduction in open probability (Po).3Single‐channel analysis of channel block suggested that genistein (100 μM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels.4Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR.5Genistein inhibition of CFTR Cl−currents was weakly voltage dependent and unaffected by changes in the extracellular Cl−concentration.6Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide‐binding domains (NBDs) of CFTR to greatly stimulate channel activity.7ATP (5 mM) prevented the genistein‐induced decrease in Po, but was without effect on the genistein‐induced decrease in i.8The genistein‐induced decrease in iwas voltage dependent, whereas the genistein‐induced decrease in Powas voltage independent.9The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl−permeation.

1Cytosolic Ca2+has been proposed to act as both a positive and a negative feedback signal on the inositol trisphosphate (InsP3) receptor. However, it is unclear how this might affect the Ca2+response in vivo.2Mouse pancreatic acinar cells were whole‐cell patch clamped to record the Ca2+‐dependent chloride (Cl(Ca)) current spikes and imaged to record the cytosolic Ca2+spikes elicited by the injection of Ins(2,4,5)P3. Increasing concentrations of Ca2+buffer (up to 200 μmEGTA or BAPTA) were associated with the appearance of steps in the current activation phase and a prevalence of smaller‐amplitude Cl(Ca)spikes. Imaging experiments showed that with increased buffer the secretory pole cytosolic Ca2+signal became fragmented and spatially discrete Ca2+release events were observed.3At higher buffer concentrations (200‐500 μm), increasing concentrations of EGTA increased spike frequency and reduced spike amplitude. In contrast, BAPTA decreased spike frequency and maintained large spike amplitudes.4We conclude that, during InsP3‐evoked spiking, long‐range Ca2+feedback (∼2–4 μm) shapes the rising phase of the Ca2+signal by acting to co‐ordinate discrete Ca2+release events and short‐range (∼40 nm) Ca2+feedback acts to inhibit further Ca2+release.

1We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside‐out configuration of the patch‐clamp technique.2The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP‐stimulated iodide efflux. However, PKC did not augment the single‐channel activity of either human or murine CFTR Cl−channels that had previously been activated by protein kinase A.3Fluoride, a non‐specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl−channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP‐stimulated iodide efflux.4The alkaline phosphatase inhibitor, (−)‐bromotetramisole augmented cAMP‐stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)‐bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single‐channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR.5As murine CFTR had a low Poand was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single‐channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Pogreater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.

In 20 adult patients (18 male) who presented for thoracotomy, the trachea was intubated using Mallinckrodt disposable double-lumen tubes (18 large and two medium). The endobronchial cuff was inflated by a trained operating department assistant using an air-filled syringe. The volume of air and the initial endobronchial cuff pressure were measured. The minimum cuff pressure required to prevent respiratory gas leakage from the isolated lung was measured also and maintained using the Cardiff Cuff Controller. Mean initial cuff pressure was 69.3 (SEM 6.0) mm Hg, whereas the mean minimum cuff pressure was 29.5 (4.0) mm Hg (P < 0.0001). The results suggest that the method described of inflating the endobronchial cuff may lead to overinflation and subsequent excessive pressure on the endobronchial wall.