The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization.

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

Differential role of Na+/H+ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line.

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.

Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187.

The regulated Cl(-) secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca(2+), cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux (J(w) = -0.16 +/- 0.02 microl.min(-1).cm(-2)) in parallel with moderate short-circuit current values (I(sc) = 1.55 +/- 0.23 microA/cm(2)). The secretory J(w) reversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I(sc), but, unexpectedly, J(w) was not affected. Bumetanide, an inhibitor of basolateral Na(+)-K(+)-2Cl(-) cotransporter, completely blocked secretory J(w) with no change in I(sc). Conversely, serosal forskolin increased I(sc), but J(w) switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J(w) and I(sc). No difference between the absorptive and secretory unidirectional Cl(-) fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl(-) flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J(w) values cannot be shown by I(sc) and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.

The water channel aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cyclic AMP.

We previously found that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected an N-glycosylated band of approximately 34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt(2)cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt(2)cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt(2)cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.

Vasopressin regulates water flow in a rat cortical collecting duct cell line not containing known aquaporins.

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD(1)). Transepithelial net water fluxes (J(w)) were recorded every minute in RCCD(1) monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10(-5) m), apical glibenclamide (10(-4) m) and apical BaCl(2) (5 x 10(-3) m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD(1) cells. AVP stimulates cAMP production and sodium reabsorption in RCCD(1) cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I(sc)) was paralleled by a simultaneous modification of the observed J(w): both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J(w) induced by serosal hypertonicity. We can conclude that: (i) transepithelial J(w) occurs in RCCD(1) cells in the absence of known renal aquaporins; (ii) the "water secretory component" observed could be linked to Cl- and K = secretion; (iii) the natriferic response to AVP, preserved in RCCD(1) cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost.

Effects of medium hypertonicity on water permeability in the mammalian rectum: ultrastructural and molecular correlates.

Minute-by-minute net water fluxes (Jw) were measured across the isolated rectal epithelium in rats and rabbits. Five minutes after a serosal (but not mucosal) hypertonic challenge (plus 200 mosmol/l) a significant increase in the basal Jw was recorded in both species [deltaJw, microl min(-1) cm(-2): 0.40+/-0.06 (rats); 0.45+/-0.10 (rabbits)]. At the same time, most epithelial cells shrank markedly while the intercellular spaces were wide open (electron microscopy studies). In freeze-fracture studies multi-strand tight-junction structures (only slightly modified by serosal hypertonicity in rabbits) were observed in control conditions. No structural changes were observed after mucosal hypertonicity (both in rats and rabbits). Immunohistochemical studies showed the expression of aquaporin 3 (AQP3) at the basolateral membrane of epithelial cells in the rat. A first conclusion is that the epithelium of the mammalian rectum is a highly polarized, aquaporin-3-containing, water permeability structure. The Jw increase induced by serosal hypertonicity was sensitive to mercurial agents in both species and no changes in unidirectional [14C]mannitol fluxes (Ps) or transepithelial resistance (RT) were observed during this Jw increase. These observations suggest a transcellular route for the osmotically induced increase in water fluxes. In the rabbit rectum the initial Jw response, associated with serosal hypertonicity, was a transient one. It was followed by a second, slow and HgCl2-sensitive Jw increase (a transient peak in paracellular mannitol permeability was also observed). A second conclusion is that serosal hypertonicity induces an increase in transcellular water permeability in both rat and rabbit rectum.

Functional characterization and localization of AQP3 in the human colon.

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

Functional characterization and localization of AQP3 in the human colon

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98 percent identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells

A new data-acquisition system for the measurement of the net water flux across epithelia.

We describe a computer-based data-acquisition system designed for the measurement of net water flux across epithelia. It is based on the detection of a liquid meniscus position in a capillary tube using an electro-optical device. Then the data are digitized, transferred to the computer main memory and saved in a file. The advantages of the system are: (a) to measure net water transfer with an accuracy of 50 nl; (b) to switch from absorptive to secretory conditions in real time; (c) data are directly digitized to be analytically, graphically and statistically treated; (d) the specially developed software allows change of time intervals for data acquirement and the scale in graphic events; (e) the software also allows recall to the monitor screen of graphics from previous tests or to print them on paper; (f) a simple mechanical structure, saving maintenance and time; and (g) high reliability.

Water permeability properties of the human small intestine in vitro: effects of Escherichia coli heat-stable enterotoxin.

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (delta Osm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 +/- 0.049 cm/s vs. 0.156 +/- 0.022 cm/s and 0.0012 +/- 0.0001 cm/s vs. 0.0019 +/- 0.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 micrograms/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48% decrease in the absorptive Jwt values.