The properties of the secretagogue-evoked chloride current in mouse pancreatic acinar cells.

In this paper we describe the properties of the secretagogue-evoked chloride current from mouse pancreatic acinar cells. Single cells were patch-clamped in the whole-cell configuration with solutions that excluded cation currents and then stimulated with 1 mM carbachol (CCh). This resulted in a current that rose to a peak and then decayed to a plateau level. The current/voltage relationship of the peak current was linear whereas that of the plateau phase was rectified and showed time and voltage dependence. To determine if the CCh evoked current was strictly Ca2+ dependent, we compared the properties of the plateau current with those of currents evoked by directly raising cytosolic [Ca2+] The properties of the two currents were the same, with both currents showing outward rectification, development at depolarised potentials, similar time constants of activation, permeability sequences and sensitivity to the Cl- channel inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS). We conclude that the agonist-evoked rise in Ca2+ is alone a sufficient signal to activate the CL- current.

Mechanisms underlying InsP3-evoked global Ca2+ signals in mouse pancreatic acinar cells.

In secretory epithelial cells, complex patterns of Ca2+ signals regulate physiological processes. How these patterns are generated is still not fully understood. In particular, the basis of global Ca2+ waves is not clear. We have studied regional differences in InsP3-evoked Ca2+ release in single mouse pancreatic acinar cells, using high-speed (approximately 90 frames s-1), high-sensitivity Ca2+ imaging combined with rapid (10 ms) spot photolysis (2 micrometer diameter) of caged InsP3. Within a single region we measured Ca2+ response latency and rate of rise to construct an InsP3 dose-response relationship. Spot InsP3 liberation in the secretory pole region consistently elicited a dose-dependent, rapid release of Ca2+. Spot InsP3 liberation in the basal pole region of approximately 50% of cells elicited a similar dose-response relationship but with a lower apparent InsP3 affinity than in the secretory pole. In the other cells, basal pole InsP3 liberation did not elicit active Ca2+ release, even at the highest stimulus intensities we employed, although these same cells did respond when the stimulus spot was moved to different regions. We conclude that in the basal pole active sites of rapid Ca2+ release have a lower functional affinity for InsP3 than those in the secretory pole and are spread out in discrete sites across the basal pole. These properties explain the propagation of Ca2+ waves across the basal pole that are only observed at higher stimulus levels.

Intracellular Ca2+ and Cl- channel activation in secretory cells.

Molecular and functional evidence indicates that a variety of Ca(2+)-dependent chloride (Cl(Ca)) channels are involved in fluid secretion from secretory epithelial cells in different tissues and species. Most Cl(Ca) channels so far characterized have an I- permeability greater than Cl-, and most are sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Whole-cell Cl(Ca) currents show outward rectification. Single-channel current voltage relationships are linear with conductances ranging from 2 to 30 pS. Some Cl(Ca) channels are blocked by Ca(2+)-calmodulin-dependent protein kinase (CAMKII) inhibitors. Others, such as the Cl(Ca) channels of parotid and submandibular acinar cells, appear to be directly regulated by Ca2+. In native cells, the Cl(Ca) channels are located on the apical plasma membrane and activated by localized mechanisms of Ca2+ release. This positioning allows the Cl(Ca) channel to respond specifically to localized Ca2+ signals that do not invade other regions of the cell. The Cl(Ca) follows the rising phase of the Ca2+ signal, but in the falling phase hysteresis occurs where the Cl(Ca) current decays more rapidly than the underlying Ca2+. The future elucidation of the identity and mechanisms of regulation of Cl(Ca) channels will be critical to our understanding of stimulus-secretion coupling.

Microtubules regulate local Ca2+ spiking in secretory epithelial cells.

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.

Two mechanisms of genistein inhibition of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in murine cell line.

1. The 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. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel 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. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The 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.

A bimodal pattern of InsP(3)-evoked elementary Ca(2+) signals in pancreatic acinar cells.

InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have quantified these responses and compared the Ca(2+) signals to the elementary events shown in Xenopus oocytes. The results demonstrate, at the same concentrations of InsP(3), Ca(2+) signals consisting of one population of small transient Ca(2+) release events and a second distinct population of larger Ca(2+) spikes. The signal mass amplitudes of both types of events are within the range of amplitudes for the elementary events in Xenopus oocytes. However, the bimodal Ca(2+) distribution of Ca(2+) responses we observe is not consistent with the continuum of event sizes seen in Xenopus. We conclude that the two types of InsP(3)-dependent events in acinar cells are both elementary Ca(2+) signals, which are independent of one another. Our data indicate a complexity to the organization of the Ca(2+) release apparatus in acinar cells, which might result from the presence of multiple InsP(3) receptor isoforms, and is likely to be important in the physiology of these cells.

The role of Ca2+ feedback in shaping InsP3-evoked Ca2+ signals in mouse pancreatic acinar cells.

1. Cytosolic 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. 2. Mouse 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 microM EGTA 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. 3. At higher buffer concentrations (200-500 microM), increasing concentrations of EGTA increased spike frequency and reduced spike amplitude. In contrast, BAPTA decreased spike frequency and maintained large spike amplitudes. 4. We conclude that, during InsP3-evoked spiking, long-range Ca2+ feedback ( approximately 2-4 microm) shapes the rising phase of the Ca2+ signal by acting to co-ordinate discrete Ca2+ release events and short-range ( approximately 40 nm) Ca2+ feedback acts to inhibit further Ca2+ release.

Regulation of murine cystic fibrosis transmembrane conductance regulator Cl- channels expressed in Chinese hamster ovary cells.

1. We 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. 2. The 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. 3. Fluoride, 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. 4. The 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. 5. As murine CFTR had a low Po and 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 Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.

Endobronchial cuff pressures.

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.

Successful difficult intubation. Use of the gum elastic bougie.

The reliability of two signs of tracheal placement of a gum elastic bougie was studied. These signs were clicks (produced as the tip of the bougie runs over the tracheal cartilages) and hold up of the bougie as it is advanced (when the tip reaches the small bronchi). Ninety-eight simulated and two genuine Grade 3 difficult intubations were attempted with the aid of a gum elastic bougie. Seventy-eight tracheal and 22 oesophageal placements of the bougie resulted. No clicks or hold up occurred with the bougie in the oesophagus. Clicks were recorded in 89.7% of tracheal placements of the bougie. Hold up at between 24-40 cm occurred in all tracheal placements. We conclude that these signs are reliable and that they should be taught as part of any difficult intubation drill in which the gum elastic bougie is used.