Chiara Sironi

Fast Fluorometric Method for Measuring Pendrin (SLC26A4) Cl-/I- Transport Activity

Malfunction of the SLC26A4 protein leads to Pendred syndrome, characterized by sensorineural hearing loss, often associated with mild thyroid dysfunction and goiter. It is generally assumed that SLC26A4 acts as a chloride/anion exchanger, which in the thyroid gland transports iodide, and in the inner ear contributes to the conditioning of the endolymphatic fluid. Here we describe a fast fluorometric method able to be used to functionally scrutinize SLC26A4 and its mutants described in Pendred syndrome. The validation of the method was done by functionally characterizing the chloride/iodide transport of SLC26A4, and a mutant, i.e. SLC26A4S28R, which we previously described in a patient with sensorineural hearing loss, hypothyroidism and goiter. Using the fluorometric method we describe here we can continuously monitor and quantify the iodide or chloride amounts transported by the cells, and we found that the transport capability of the SLC26A4S28R mutant protein is markedly reduced if compared to wild-type SLC26A4.

Functional Characterization of Wild-Type and a Mutated Form of SLC26A4 Identified in a Patient with Pendred Syndrome

Background: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. Methods: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4S28R) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. Results: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. Conclusions: The functional characteristics of SLC26A4S28R we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived.

A creatine transporter is operative at the brush border level of the rat jejunal enterocyte

Although ergogenic effects and health benefits have been reported for creatine used as nutritional supplement, to date little is known about the mechanism of creatine absorption in the small intestine. Thus the current study was undertaken to elucidate the mechanism of creatine intake in rat jejunum with the use of well-purified brush border membrane vesicles, isolated from jejunal enterocyte. Creatine uptake was found markedly stimulated by inwardly directed Na+ and Cl− gradients, potential-sensitive, strongly reduced by the substitution of Na+ and Cl− with various cations and anions and positively affected by intravesicular K+. Moreover, creatine uptake is: 1) significantly inhibited by creatine stuctural analogs, 2) abolished by low concentrations of 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), 3) saturable as a function of creatine concentration with an apparent Michaelis-Menten constant of 24.08 ± 0.80 μM and a maximal velocity of 391.30 ± 6.19 pmoles mg protein−1 30 s−1. The transport is electrogenic since at least two Na+ and one Cl− are required to transport one creatine molecule. Western blot analysis showed the same amount of creatine transport protein in the jejunal apical membrane when compared to ileum. Thus, these data demonstrate the existence of a Na+- and Cl−-dependent, membrane potential-sensitive, electrogenic carrier-mediated mechanism for creatine absorption in rat jejunal apical membrane vesicles, which is biochemically and pharmacologically similar to those observed in other tissues. However, in other cell types the stimulatory effect of intravesicular K+ was never detected.

Jejunal creatine absorption: what is the role of the basolateral membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

Volume-regulated Cl− channels in human pleural mesothelioma cells

Anion channels in human mesothelial and mesothelioma cell lines were characterized by patch‐clamp and biomolecular approaches. We found an outwardly rectifying anionic current which was inactivated at positive voltages and inhibited by extracellular adenosine 5′‐triphosphate (ATP). Mesothelial and mesothelioma cells behaved differently concerning current inactivation properties. Inactivation is more pronounced and has a steeper onset in mesothelial cells. Different reversal potentials, in asymmetrical Cl− solutions, that could be attributed to a different selectivity of the channel, have been observed in the two cell lines. Mesothelioma cell single‐channel analysis indicates that the number of the same active anion channel (3–4 pS) increased under hypoosmotic conditions. Immunocytochemistry experiments showed the presence of ICln protein in the cytosol and in the plasma membrane. Western blot analysis revealed an increase of ICln in the membrane under hypotonic conditions, an event possibly related to the activation of Cl− channels.

Membrane Thickness Changes Ion-Selectivity of Channel-Proteins

The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become.

Lubricating effect of sialomucin and hyaluronan on pleural mesothelium

Coefficient of kinetic friction (μ) between rabbit visceral and parietal pleura, sliding in vitro at physiological velocities and load, increases markedly after blotting mesothelial surface with filter paper; this increase is only partially reduced by wetting blotted mesothelium with Ringer solution. Given that mesothelial surface is covered by a thick coat with sialomucin and hyaluronan, we tested whether addition of sialomucin or hyaluronan solution after blotting lowers μ more than Ringer alone. Actually, these macromolecules lowered μ more than Ringer, so that μ was no longer significantly higher than its preblotting value. Moreover, Ringer addition, after washout of macromolecule solution, increased μ, in line with their dilution. These findings indicate that mesothelial blotting removes part of these molecules from the coat covering mesothelial surface, and their relevance for pleural lubrication. Transmission electron micrographs of pleural specimens after mesothelial blotting showed that microvilli were partially or largely removed from mesothelium, consistent with a substantial loss of macromolecules normally entrapped among them.

Evidence for Na+–glucose cotransporter in type I alveolar epithelium

Functional evidence of Na+–glucose cotransport in rat lung has been provided by Basset et al. (J. Physiol. 384:325–345, 1987). By autoradiography [3H]phloridzin binding has been found confined to alveolar epithelial type II cells in mouse and rabbit lungs (Boyd, J. Physiol. 422: 44P, 1990). In this research we checked by immunofluorescence whether Na+–glucose cotransporter (SGLT1) is also expressed in alveolar type I cells. Lungs of anesthetized rats and lambs were fixed by paraformaldehyde, perfused in pulmonary artery, or instilled into a bronchus, respectively. Tissue blocks embedded in paraffin or frozen were sectioned. Two specific anti-SGLT1 antibodies for rat recognizing aminoacid sequence 402–420, and 546–596 were used in both species. Bound primary antibody was detected by secondary antibody conjugated to fluorescein isothiocianate or Texas red, respectively. In some sections cellular nuclei were also stained. In rats alveolar type I cells were identified by fluorescent Erythrina cristagalli lectin. Sections were examined by confocal laser-scanning microscope. Both in rats and lambs alveolar epithelium was stained by either antibody; no labeling occurred in negative controls. Hence, SGLT1 appears to be also expressed in alveolar type I cells. This is functionally relevant because type I cells provide 95–97% of alveolar surface, and SGLT1, besides contributing to removal of lung liquid under some circumstances, keeps low glucose concentration in lining liquid, which is useful to prevent lung infection.

Expression of Na+–glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura

Indirect evidence for a solute-coupled liquid absorption from rabbit pleural space indicated that it should be caused by a Na(+)/H(+)-Cl(-)/HCO(3)(-) double exchanger and a Na(+)-glucose cotransporter [Agostoni, E., Zocchi, L., 1998. Mechanical coupling and liquid exchanges in the pleural space. In: Antony, V.B. (Ed.), Clinics in Chest Medicine: Diseases of the Pleura, vol. 19. Saunders, Philadelphia, pp. 241-260]. In this research we tried to obtain molecular evidence for Na(+)-glucose cotransporter (SGLT1) in visceral and parietal mesothelium of rabbit pleura. To this end we performed immunoblot assays on total protein extracts of scraped visceral or parietal mesothelium of rabbits. These showed two bands: one at 72kDa (m.w. of SGLT1), and one at 55kDa (which should also provide Na(+)-glucose cotransport). Both bands disappeared in assays in which SGLT1 antibody was preadsorbed with specific antigen. Molecular evidence for Na(+)/K(+) ATPase (alpha1 subunit) was also provided. Immunoblot assays for SGLT1 on cultured mesothelial cells of rabbit pleura showed a band at 72kDa, and in some cases also at 55kDa, irrespectively of treatment with a differentiating agent. Solute-coupled liquid absorption hinders liquid filtration through parietal mesothelium caused by Starling forces, and favours liquid absorption through visceral mesothelium caused by these forces.

Diphenylamine-2-carboxylic acid (DPC), usually an inhibitor of Cl– and non-selective cation channels, inhibits Cl–/HCO3– exchange and opens Cl– and cation conductances in rabbit gallbladder epithelium

Abstract. In the apical plasma membrane of rabbit gallbladder epithelium various drugs (hydrochlorothiazide, phlorizin, phenylglyoxal) inhibit Cl–/HCO3– exchange and probably enhance the almost negligible intrinsic anion conductance of the exchanger. By radiochemical measurements of apical Cl– influx, the anion exchange is shown here to be directly and immediately inhibited by diphenylamine-2-carboxylic acid (DPC) too. Using conventional microelectrode techniques in intact tissue, DPC, with same dose/response curve, is shown to activate an apical anion conductance (GCl) that has similar properties and amplitude to the GCl activated by the other exchange inhibitors so far tested; the actions are not additive. Patch-clamp methods (cell-attached and excised inside-out patch configurations) reveal that GCl is due to anion channels that are non-rectifying, cytoplasm independent, sensitive to stilbene and dipyridamole and have conductance of a few picosiemens. All this strengthens the correlation between inhibition of anion exchange and the activation of GCl and channels with features similar to those of the almost negligible intrinsic anion conductance of the exchanger. Among the drugs tested, the effects of DPC and hydrochlorothiazide are even more similar, such that even their dose/response curves overlap. Moreover, both drugs also directly activate some verapamil-sensitive Ca2+ channels and consequently apamin-sensitive, Ca2+-activated K+ channels. Thus DPC, usually an inhibitor of Cl– and non-selective cation channels, is shown here to be capable of activating Cl– and cation conductances.