Giuliano Meyer

Mechanisms Sensing and Modulating Signals Arising From Cell Swelling

Cell volume alterations are involved in numerous cellular events like epithelial transport, metabolic processes, hormone secretion, cell migration, proliferation and apoptosis. Above all it is a need for every cell to counteract osmotic cell swelling in order to avoid cell damage. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. De-spite the large amount of experimental data that has accumulated, the intracellular mechanisms underlying the sensing of cell volume perturbations and the activation of volume compensatory processes, commonly summarized as regulatory volume decrease (RVD), are still only partly revealed. Moving into this field opens a complex scenario of molecular rearrangements and interactions involving intracellular messengers such as calcium, phosphoinositides and inositolphosphates as well as phosphoryla-tion/dephosphorylation processes and cytoskeletal reorganization with marked cell type- and tissue specific variations. Even in one and the same cell type significant differences regarding the activated pathways during RVD may be evident. This makes it virtually im-possible to unambigously define common sensing- and sinaling pathways used by differ-ent cells to readjust their celll volume, even if all these pathways converge to the activa-tion of comparatively few sets of effectors serving for osmolyte extrusion, including ion channels and transporters. This review is aimed at providing an insight into the manifold cellular mechanisms and alterations occuring during cell swelling and RVD.

Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred syndrome and nonsyndromic EVA

Pendred syndrome is an autosomal recessive disorder characterized by sensorineural hearing loss, with malformations of the inner ear, ranging from enlarged vestibular aqueduct (EVA) to Mondini malformation, and deficient iodide organification in the thyroid gland. Nonsyndromic EVA (ns-EVA) is a separate type of sensorineural hearing loss showing normal thyroid function. Both Pendred syndrome and ns-EVA seem to be linked to the malfunction of pendrin (SLC26A4), a membrane transporter able to exchange anions between the cytosol and extracellular fluid. In the past, the pathogenicity of SLC26A4 missense mutations were assumed if the mutations fulfilled two criteria: low incidence of the mutation in the control population and substitution of evolutionary conserved amino acids. Here we show that these criteria are insufficient to make meaningful predictions about the effect of these SLC26A4 variants on the pendrin-induced ion transport. Furthermore, we functionally characterized 10 missense mutations within the SLC26A4 ORF, and consistently found that on the protein level, an addition or omission of a proline or a charged amino acid in the SLC26A4 sequence is detrimental to its function. These types of changes may be adequate for predicting SLC26A4 functionality in the absence of direct functional tests.

Functional characterization of wild-type and mutated pendrin (SLC26A4), the anion transporter involved in Pendred syndrome

Pendred syndrome (PS) is the most frequent form of genetically related syndromic hearing loss, and is associated with mutations of pendrin, encoded by the SLC26A4 gene. This protein localizes to the cellular membrane and permits the exchange of anions between the cytosol and extracellular space. In the inner ear, pendrin conditions the endolymph, allowing for the proper function of sensory cells. Understanding the relationship between the genotype and phenotype of pendrin mutations would aid clinicians to better serve PS patients-however, little is known. Here, we summarize the available data concerning SLC26A4 mutations and how they relate to transporter function. The main findings suggest that all the truncation mutations tested annihilate pendrin function, and that the addition or omission of proline, or the addition or omission of charged amino acids in the sequence of SLC26A4 result in a substantial to dramatic reduction in pendrin function.

Expression and subcellular localization of the AQP8 and AQP1 water channels in the mouse gall-bladder epithelium

Background information. Transepithelial transport of water is one of the most distinctive functions by which the gall‐bladder rearranges its bile content. Water is reabsorbed from the gall‐bladder lumen during fasting, whereas it is secreted into the lumen following meal ingestion. Nevertheless, the molecular mechanism by which water is transported across the gall‐bladder epithelium remains mostly unclear.

Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension

We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

Stimulation by HCO3- of Na+ transport in rabbit gallbladder.

SummaryBicarbonate presence in the bathing media doubles Na+ and fluid transepithelial transport and in parallel significantly increases Na+ and Cl− intracellular concentrations and contents, decreases K+ cell concentration without changing its amount, and causes a large cell swelling. Na+ and Cl− lumen-to-cell influxes are significantly enhanced, Na+ more so than Cl−. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO3−-ATPase in the brush border. The stimulation goes together with a large increase in a Na+-dependent H+ secretion into the lumen. All of these data suggests that HCO3− both activates Na+−Cl− cotransport and H+−Na+ countertransport at the luminal barrier.Thiocyanate inhibits Na+ and fluid transepithelial transport without affecting H+ secretion and HCO3−-dependent Na+ influx. It reduces Na+ and Cl− concentrations and contents, increases the same parameters for K+, causes a cell shrinking, and abolishes the lumen-to-cell Cl− influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na+-dependent and HCO3−-activated. Thus, SCN− is likely to compete for the Cl− site on the cotransport carrier and to be slowly transferred by the cotransport system itself.

The nature of the neutral Na+-Cl(-)-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl-/HCO3- double exchange and Na+-Cl- symport

SummaryCl− influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to36Cl− and3H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN− which immediately and completely inhibits Cl− entry into the cell. Cellular influx was equal to 16.7μeq cm−2 hr−1 and decreased to 8.5μeq cm−2 hr−1 upon removal of HCO3− from the bathing media and by bubbling 100% O2 for 45 min. When HCO3− was present, cellular influx was again about halved by the action of 10−4m acetazolamide, 10−5 to 10−4m furosemide, 10−5 to 10−4m 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS), 10−3m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO3− was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO3− was present in the salines, Na+ removal from the mucosal side caused a slow decline of cellular Cl− influx; conversely, it immediately abolished cellular Cl− influx in the absence of HCO3−. In conclusion, about 50% of cellular influx is sensitive to HCO3−, inhibitable by SCN−, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na+. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN−, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na+. Thus, about 50% of apical membrane NaCl influx appears to result from a Na+/H+ and Cl−/HCO3− exchange, whereas the residual influx seems to be due to Na+−Cl− contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.

Different sodium chloride cotransport systems in the apical membrane of rabbit gallbladder epithelial cells

SummaryThe kinetics of Cl− influx from the lumen to the cell and the paracellular pathway was examined in isolated rabbit gallbladder by measuring36Cl uptake (45 s) and by correcting it for the extracellular space with3H-sucrose. The paracellular fraction of the influx was studied by incubating the tissue in Na+-free saline or in solutions containing 25mm SCN−; the kinetics turned out to be hyperbolic. The cellular fraction of the influx comprised three components. The first was immediately Na+-dependent and insensitive both to exogenous and endogenous cell bicarbonate; its sigmoidal kinetics revealed the presence of a carrier with three Cl− binding sites cooperating positively with one another, with strong interaction factors. The second cellular component was immediately Na+-dependent and sensitive to endogenous cell bicarbonate; the kinetics was hyperbolic with a maximum at 20mm Cl− concentration and a substrate inhibition from 20 to 130mm; it was completely inhibited by 10−4m acetazolamide. The third cellular component was slowly Na+-dependent and slowly sensitive to exogenous bicarbonate; its kinetics was hyperbolic, without substrate inhibition in the tested Cl− concentration range. On this basis, the presence of three Na+−Cl− cotransports is suggested: i) on a single carrier without any exchange with H+ and HCO3−, ii) on a single carrier with an exchange with H+ and HCO3−, and iii) on two separate carriers in exchange with H+ and HCO3−.

Electrical parameters in gallbladders of different species. Their contribution to the origin of the transmural potential difference.

SummaryAmphotericin B enhances Na+ conductance of the mucosal membrane of gallbladder epithelial cells and in such a way it modifies the brush border electromotive force. On this basis a method to measure cell and shunt resistances by comparing changes of the mucosal membrane potential (Vm) and of the transmural p.d. (Vms) is developed. This method is applied in gallbladders of different vertebrate species (i.e. rabbit, guinea pig, goose, tortoise, toad, trout). The two tested mammals, rabbit and guinea pig, exhibited a lower shunting percentage (89–93%) than the nonmammals (96–97%), but this fact did not bring about a homogeneous positiveVms. This means that shunting percent contributes, but it is not the only source of differences inVms, in accordance with that reported by Gelarden and Rose (J. Membrane Biol.19:37, 1974). Moreover, mammals exhibited a lower luminal resistance and a lower ratio between luminal and basolateral resistance than nonmammals. Possible causes of these differences are discussed.

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.