Len Best

Expression of volume-sensitive Cl(-) channels and ClC-3 in acinar cells isolated from the rat lacrimal gland and submandibular salivary gland.

1 The expression of ClC‐3 was examined in rat lacrimal gland and submandibular salivary gland cells using RT‐PCR and Western analysis. Whole‐cell patch clamp methods were used to investigate the expression of volume‐sensitive anion channels in acinar cells isolated from these tissues. 2 Expression of mRNA encoding ClC‐3, and ClC‐3 protein, was found in rat submandibular gland by RT‐PCR and Western analysis. Rat lacrimal gland cells, however, did not appear to express mRNA encoding for ClC‐3, nor the ClC‐3 protein. 3 Volume‐sensitive anion conductances were observed in both rat lacrimal gland and submandibular salivary gland acinar cells. The conductance was of a similar size in the two cell types, but was much slower to activate in the lacrimal cells. 4 The properties of the conductances in lacrimal and submandibular cells were similar, e.g. halide selectivity sequence (PI > PCl > Paspartate) and inhibition by 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid, 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate and tamoxifen. 5 The data suggest that the expression of ClC‐3 is not an absolute requirement for the activity of volume‐sensitive anion channels in rat lacrimal gland acinar cells.

A new role for aquaporin 7 in insulin secretion.

Bacgrouns/Aims: Several insulinotropic agents were recently reported to cause β-cell swelling. The possible participation of AQP7 to water transport was investigated in AQP7+/ + or AQP7-/- mice. Methods: Aquaporin expression, insulin secretion, cell swelling and electrical activity were investigated in pancreatic islets. Results: RT-PCR revealed the expression of AQP5 and AQP8 mRNA. Double immunofluorescent labeling indicated their presence in β-cells. Whilst basal insulin release from isolated pancreatic islets incubated at 2.8 mM D-glucose did not differ between AQP7+/ + or AQP7-/- mice, the secretion of insulin evoked by the omission of 50 mM NaCl, the substitution of 50 mM NaCl by 100 mM glycerol or a rise in D-glucose concentration to 8.3 mM and 16.7 mM was severely impaired in the islets from AQP7-/- mice. Yet, exposure of β-cells to either the hypotonic medium or a rise in D-glucose concentration caused a similar degree of swelling and comparable pattern of electrical activity in cells from AQP7+/ + and AQP7-/- mice. Both the cell swelling and change in membrane potential were only impaired in AQP7-/- cells when exposed to 50 mM glycerol. Conclusion: It is proposed, therefore, that AQP7 may, directly or indirectly, play a role at a distal site in the exocytotic pathway.

Stimulus-secretion coupling of hypotonicity-induced insulin release in BRIN-BD11 cells.

The stimulus-secretion coupling for hypotonicity-induced insulin release was investigated in BRIN-BD11 cells. A 50 mM decrease in extracellular NaCI caused a twofold increase in insulin release. The release of insulin evoked by hypotonicity progressively decreased in an exponential manner. The response to extracellular hypotonicity displayed a threshold value close to 20 mOsmol/L and amaximal response at about 70 mOsmol/L. Hypotonicity also caused a rapid increase in cell volume followed by a regulatory volume decrease (RVD), cell membrane depolarization with induction of spike activity, and a rise in cytosolic Ca2+ concentration. 5-Nitro-2-(3-phenylpropylamino) benzoate inhibited the secretory response to hypoosmolarity, failed to affect the early increase in cell volume but prevented the RVD, and suppressed the hypotonicity-induced plasma membrane depolarization. Insulin release provoked by hypotonicity was inhibited by verapamil, absence of Ca2+, thapsigargin, furosemide, tributyltin, and diazoxide. On the contrary, tolbutamide augmented modestly insulin release recorded in the hypoosmolar medium. Last, a rise in extracellular K+ concentration, while augmenting basal insulin output, failed to affect insulin release in the hypoosmolar medium. Thus, the insulin secretory response to hypotonicity apparently represents a Ca2+-dependent process triggered by the gating of volume-sensitive anion channels with subsequent depolarization and gating of voltage-sensitive Ca2+ channels.

Studies of the Mechanism of Activation of the Volume-Regulated Anion Channel in Rat Pancreatic β-Cells

There is evidence that depolarization of the pancreatic β cell by glucose involves cell swelling and activation of the volume-regulated anion channel (VRAC). However, it is unclear whether cell swelling per se or accompanying changes in intracellular osmolality and/or ionic strength are responsible for VRAC activation. VRAC activity was measured in rat β cells by conventional or perforated patch whole-cell recording. Cell volume was measured by video imaging. In conventional whole-cell recordings, VRAC activation was achieved by exposure of the cells to a hyposmotic bath solution, by application of positive pressure to the pipette, or by use of a hyperosmotic pipette solution. Increased concentrations of intracellular CsCl also caused channel activation, but with delayed kinetics. In perforated patch recordings, VRAC activation was induced by isosmotic addition of the permeable osmolytes urea, 3-Ο-methyl glucose, arginine, and NH4Cl. These effects were all accompanied by β-cell swelling. It is concluded that increased cell volume, whether accompanied by raised intracellular osmolality or ionic strength, is a major determinant of VRAC activation in the β cell. However, increased intracellular ionic strength markedly reduced the rate of VRAC activation. These findings are consistent with the hypothesis that the accumulation of glucose metabolites in the β cell, and the resultant increase in cell volume, provides a signal coupling glucose metabolism with VRAC activation.

Role of anion exchangers in Cl- and HCO3- secretion by the human airway epithelial cell line Calu-3.

Despite the importance of airway surface liquid pH in the lungs defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.

The glucokinase activator GKA50 causes an increase in cell volume and activation of volume-regulated anion channels in rat pancreatic β-cells

Glucokinase plays a key role in the metabolism of glucose by pancreatic β-cells. In this study the effects of the glucokinase activator GKA50 on cell volume and electrical activity in rat β-cells were examined. One micro molar GKA50 caused an increase in β-cell volume in the presence of 4mM glucose. GKA50 also caused a depolarisation of β-cell membrane potential and increased electrical activity. These changes were associated with the activation of inward whole-cell currents, and were attenuated by the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. In single channel experiments, the open probability of volume-regulated anion channels (VRAC) was increased from 0.03±0.01 to 0.19±0.04 (n=3) by the GKA50. The data suggest that a GKA50-evoked increase in glucose metabolism causes an increase in β-cell volume. This in turn activates VRAC leading to a depolarisation of the cell membrane potential.

Electrophysiological effects of osmotic cell shrinkage in rat pancreatic β-Cells

Electrical and secretory activity in the pancreatic β-cell can be elicited by hypotonic cell swelling, due largely to activation of a volume-regulated anion channel (VRAC) leading to depolarisation and electrical activity. However, β-cell responses to cell shrinkage are less well characterised. The present study has examined the effects of osmotic cell shrinkage on rat pancreatic β-cells. Electrical activity and whole-cell current were studied in isolated β-cells using the perforated patch and conventional whole-cell recording techniques. Insulin release was measured using intact islets by radioimmunoassay. Exposure to a 33% hypertonic bath solution resulted in an initial depolarisation and a period of electrical activity. In several cases, this depolarisation was transient and was followed by a hyperpolarisation. A similar pattern was observed with insulin release. In voltage-clamp experiments, osmotic shrinkage resulted in activation of a non-selective cation channel (NSCC) sensitive to inhibition by flufenamic acid and Gd3+. It is suggested that activation of this NSCC is responsible for the depolarisation evoked by hypertonic media. The secondary hyperpolarisation is likely to be the result of inhibition of VRAC activity. These opposing ionic effects could underlie the biphasic effect on insulin release following exposure to hypertonic media.

Effects of octane derivatives on activity of the volume-regulated anion channel in rat pancreatic β-cells

BACKGROUND Saturated free fatty acids (FFAs) have a dual action on pancreatic β-cells, consisting of an initial enhancement and subsequent suppression of glucose-induced electrical activity and insulin release. These stimulatory and inhibitory effects have been attributed, at least in part, to the activation and inhibition, respectively, of the volume-regulated anion channel (VRAC) by FFAs. Both effects were independent of their metabolism. We have now investigated the effects of related aliphatic compounds in order to further define the determinants of FFA interaction with VRAC. METHODS β-Cell VRAC and electrical activity were measured by conventional whole-cell and perforated patch recording, respectively. Cell volume was measured using a video-imaging technique. RESULTS In common with octanoic acid, addition of methyl octanoate or n-octanol resulted in a rapid, pronounced and reversible inhibition of VRAC activity. Addition of n-octane had no significant effect on VRAC activity. n-Octanol had a biphasic effect on β-cell membrane potential, namely a small transient depolarization followed by a marked hyperpolarization. n-Octanol was also found to prevent regulatory volume decrease in cells exposed to a hypotonic medium, consistent with VRAC inhibition. CONCLUSION It is suggested that methyl octanoate and n-octanol can mimic the effects of FFAs on the pancreatic β-cell via modulation of VRAC activity. The structural requirements for this effect appear to be a medium or long chain aliphatic compound containing at least one oxygen atom.