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In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin‐luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing‐type chamber. We developed a LL pulse protocol to determine the rate of ATP release (RATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure RATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 μl of A6 cells released ATP at a rate of 66 ± 8 fmol min−1. A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H2O)−1 elevated RATP rapidly to a peak value of 1.89 ± 0.11 pmol min−1 (RATPpeak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min−1 (RATPplat). Both RATPpeak and RATPplat values increased with the degree of dilution. The magnitude of RATPplat remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 ± 0.08 pmol min−1 was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H2O)−1. This RATP value, induced in the absence of cell swelling, is comparable to RATPplat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose‐response curves for peak [Ca2+]i increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with RATPpeak. The link between the ATP release and the fast [Ca2+]i transient was also demonstrated by the depression of both phenomena by Cl− removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin‐sensitive rise of [Ca2+]i during the hyposmotic shock.

Background:The ubiquitous sarco/endoplasmic reticulum Ca 2ϩ ATPase SERCA2b has a C-terminal extension that increases the Ca 2ϩ affinity. It consists of a transmembrane helix (TM11) and a luminal extension. Results: Both parts control Ca 2ϩ affinity independently. Conclusion: TM11 is an independent and highly conserved functional region of SERCA2b. Significance: This study shows that TM11 acts as a genuine regulator of the Ca 2ϩ pump.

germont. Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells. Am J Physiol Cell Physiol 290: C1287-C1296, 2006. First published December 7, 2005 doi:10.1152/ajpcell.00545.2005.-Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K ϩ , Cl Ϫ , and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl Ϫ current (ICl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl Ϫ or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl Ϫ , taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel WHEN EXPOSED TO AN ACUTE hypotonic stimulus (HTS), mammalian cells typically release K ϩ , Cl Ϫ , and organic osmolytes, such as taurine, D-myo-inositol, and sorbitol, in an attempt to counteract the volume increase and to avoid swelling-induced cell lysis (27,33,51). The cellular loss of osmolytes drives the osmotic efflux of water, thereby restoring (partially) the initial cell volume, a phenomenon also known as regulatory volume decrease (RVD). In some cell types (e.g., astrocytes, airway and intestinal epithelial cells, and pancreatic acinar cells), hypotonic swelling also provokes the release of ATP, which has been proposed to assist RVD in an autocrine/paracrine way (7,8,21,39).The ion channel that is responsible for the swelling-induced release of Cl Ϫ is known as the volume-regulated anion channel (VRAC) (also known as volume-sensitive organic osmolyteanion channel or volume-sensitive outwardly rectifying channel). VRAC is a ubiquitously expressed plasma membrane anion-selective channel that has been extensively characterized at the biophysical and pharmacolo...

The intracellular fluorescence of 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ), a Cl − -sensitive fluorescent dye, is quenched by intracellular organic anions and proteins of unknown identity. The concentration of these intracellular quenchers (ICQs), however, is dependent on cell volume. In the absence of Cl − , changes in the observed SPQ fluorescence may therefore reflect changes in cell volume. This concept has been applied to determine relative changes in cell volume of cultured corneal endothelium in response to anisosmotic shocks, using as the Cl − substituent. SPQ fluorescence increased with decreasing osmolarity and vice versa. A 20 mosM hypertonic shock was needed to detect a change in SPQ fluorescence with a signal-to-noise ratio of >25. Assuming dynamic quenching by ICQs, we applied an extension of the Stern-Volmer equation to develop a simple relationship between the measured SPQ fluorescence and relative changes in cell volume. For large hyposmotic shocks, regulatory volume decrease (RVD) was observed. The rate of RVD could be enhanced by exposure to 0.5 μM gramicidin in low-Na+ Ringer solution (i.e., efflux), indicating that K+ conductance is rate limiting for RVD. These results demonstrate the principle of using fluorescence quenching to measure changes in cell volume in real time. Because SPQ is sensitive to Cl − , its usefulness as a quenching probe is limited. However, a structure-activity study can be expected to yield useful Cl − -insensitive analogs.Keywords corneal endothelium; 6-methoxy-N-(3-sulfopropyl)quinolinium; volume regulation Measurement of cell volume, with high temporal resolution and sensitivity, is critical for estimating osmotic water permeability (P f ) (12,14), investigating mechanisms of cell volume regulation (1, 2,9,10,18,23), and ascertaining transient changes in cell volume on stimulation of ion transport mechanisms under isosmotic conditions (11,25). Many techniques to measure cell volume have been developed based on light scattering (11, 12,