To define aspects of lipid composition and bilayer asymmetry critical to barrier function, we examined the permeabilities of liposomes that model individual leaflets of the apical membrane of a barrier epithelium, Madin-Darby canine kidney type 1 cells. Using published lipid compositions we prepared exofacial liposomes containing phosphatidylcholine, sphingomyelin, glycosphingolipids, and cholesterol; and cytoplasmic liposomes containing phosphatidylethanolamine, phosphatidylserine, and cholesterol. The osmotic permeability of cytoplasmic liposomes to water (P f ), solutes, and NH 3 was 18 -90-fold higher than for the exofacial liposomes (P f(ex) ‫؍‬ 2.4 ؎ 0.4 ؋ 10 ؊4 cm/s, P f(cy) ‫؍‬ 4.4 ؎ 0.3 ؋ 10 ؊3 cm/s; P glycerol(ex) ‫؍‬ 2.5 ؎ 0.3 ؋ 10 ؊8 cm/s, P glycerol(cy) ‫؍‬ 2.2 ؎ 0.02 ؋ 10 ؊6 cm/s; P NH3(ex) ‫؍‬ 0.13 ؎ 0.4 ؋ 10 ؊4 cm/s, P NH3(cy) ‫؍‬ 7.9 ؎ 1.0 ؋ 10 ؊3 cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes (P H؉(ex) ‫؍‬ 1.1 ؎ 0.1 ؋ 10 ؊2 cm/s, P H؉(cy) ‫؍‬ 2.7 ؎ 0.6 ؋ 10 ؊3 cm/s). By adding single leaflet permeabilities, we calculated a theoretical P f for a Madin-Darby canine kidney apical membrane of 4.6 ؋ 10 ؊4 cm/s, which compares favorably with experimentally determined values. In exofacial liposomes lacking glycosphingolipids or sphingomyelin, permeabilities were 2-7-fold higher, indicating that both species play a role in barrier function. Removal of cholesterol resulted in 40 -280-fold increases in permeability. We conclude: 1) that we have reconstituted the biophysical properties of a barrier membrane, 2) that the barrier resides in the exofacial leaflet, 3) that both sphingomyelin and glycosphingolipids play a role in reducing membrane permeability but that there is an absolute requirement for cholesterol to mediate this effect, 4) that these results further validate the hypothesis that each leaflet offers an independent resistance to permeation, and 5) that proton permeation was enhanced by sphingolipid/cholesterol interactions.

Lower urinary tract (LUT) symptoms become prevalent with aging and affect millions; however, therapy is often ineffective because the etiology is unknown. Existing assays of LUT function in animal models are often invasive; however, a noninvasive assay is required to study symptom progression and determine genetic correlates. Here, we present a spontaneous voiding assay that is simple, reproducible, quantitative, and noninvasive. Young female mice from eight inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ) were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within-individual and within-strain variations, but all parameters (spot number, total volume, percent area in primary void, corner voiding, and center voiding) exhibited significant variations between strains. Calculation of the intraclass correlation coefficient, an estimate of broad-sense heritability, for each time of day and for each voiding parameter revealed highly significant heritability [spot number: 61%, percent urine in primary void: 90%, and total volume: 94% (afternoon data)]. Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior-voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUT symptoms in several systems, including a demonstration of voiding abnormalities in older C57BL/6J mice (18-24 mo), in a model of protamine sulfate-induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUT symptoms using outbred mouse populations.

Aquaporins (AQPs) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP's transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore have characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To study the mechanism of Drosophila Malpighian tubule (MT)-facilitated water transport, we identified seven putative AQPs in the Drosophila genome and found that one of these, previously named DRIP, has the greatest sequence similarity to those vertebrate AQPs that exhibit the highest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult MTs, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles, and we found that significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy required for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mol. This low value is characteristic of AQP-mediated water transport, whereas the value in control vesicles was 16.4 kcal/mol. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system with which to investigate AQP function. fluid homeostasis; osmosis; channel; membrane IN ORDER FOR ORGANISMS to maintain fluid homeostasis between themselves and the external environment, they must be able to control water transport. Members of the aquaporin (AQP) family of water channels facilitate water transport and appear to play an essential role in regulating the flow of water between compartments and between the organism and its environment. For example, after hormonal signaling of dehydration, AQP2 levels in the apical membrane of kidney collecting duct cells rise, allowing more water to move into the cells and then out to the interstitium (via AQP3 and AQP4); a defect in AQP2 results in diabetes insipidus, which is characterized by a failure to concentrate urine (25). In contrast to the known function of AQP2, why seven different AQP family members need to be expressed in this organ is still not understood. In one hypothetical model, differences in substrate specificity explain the breadth of AQPs expressed in the kidney. It is also possible that AQP levels or localizations change during development and thus can be adjusted. It appears highly likely that many of the physiological roles of AQPs have yet to be discovered.To date, most AQPs have been found to share certain properties regarding permeability: the movement of water or othe...

The urothelium is proposed to be a sensory tissue that responds to mechanical stress by undergoing dynamic membrane trafficking and neurotransmitter release; however, the molecular basis of this function is poorly understood. Transient receptor potential (TRP) channels are ideal candidates to fulfill such a role as they can sense changes in temperature, osmolarity, and mechanical stimuli, and several are reported to be expressed in the bladder epithelium. However, their complete expression profile is unknown and their cellular localization is largely undefined. We analyzed expression of all 33 TRP family members in mouse bladder and urothelium by RT-PCR and found 22 specifically expressed in the urothelium. Of the latter, 10 were chosen for closer investigation based on their known mechanosensory or membrane trafficking functions in other cell types. Western blots confirmed urothelial expression of TRPC1, TRPC4, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, TRPML1, and polycystins 1 and 2 (PKD1 and PKD2) proteins. We further defined the cellular and subcellular localization of all 10 TRP channels. TRPV2 and TRPM4 were prominently localized to the umbrella cell apical membrane, while TRPC4 and TRPV4 were identified on their abluminal surfaces. TRPC1, TRPM7, and TRPML1 were localized to the cytoplasm, while PKD1 and PKD2 were expressed on the apical and basolateral membranes of umbrella cells as well as in the cytoplasm. The cellular location of TRPV1 in the bladder has been debated, but colocalization with neuronal marker calcitonin gene-related peptide indicated clearly that it is present on afferent neurons that extend into the urothelium, but may not be expressed by the urothelium itself. These findings are consistent with the hypothesis that the urothelium acts as a sentinel and by expressing multiple TRP channels it is likely it can detect and presumably respond to a diversity of external stimuli and suggest that it plays an important role in urothelial signal transduction.

BackgroundNormal urinary bladder function requires bidirectional molecular communication between urothelium, detrusor smooth muscle and sensory neurons and one of the key mediators involved in this intercellular signaling is ATP. Ectonucleotidases dephosphorylate nucleotides and thus regulate ligand exposure to P2X and P2Y purinergic receptors. Little is known about the role of these enzymes in mammalian bladder despite substantial literature linking bladder diseases to aberrant purinergic signaling. We therefore examined the expression and distribution of ectonucleotidases in the mouse bladder since mice offer the advantage of straightforward genetic modification for future studies.Principal FindingsRT-PCR demonstrated that eight members of the ectonucleoside triphosphate diphosphohydrolase (NTPD) family, as well as 5′-nucleotidase (NT5E) are expressed in mouse bladder. NTPD1, NTPD2, NTPD3, NTPD8 and NT5E all catalyze extracellular nucleotide dephosphorylation and in concert achieve stepwise conversion of extracellular ATP to adenosine. Immunofluorescent localization with confocal microscopy revealed NTPD1 in endothelium of blood vessels in the lamina propria and in detrusor smooth muscle cells, while NTPD2 was expressed in cells localized to a region of the lamina propria adjacent to detrusor and surrounding muscle bundles in the detrusor. NTPD3 was urothelial-specific, occurring on membranes of intermediate and basal epithelial cells but did not appear to be present in umbrella cells. Immunoblotting confirmed NTPD8 protein in bladder and immunofluorescence suggested a primary localization to the urothelium. NT5E was present exclusively in detrusor smooth muscle in a pattern complementary with that of NTPD1 suggesting a mechanism for providing adenosine to P1 receptors on the surface of myocytes.ConclusionsEctonucleotidases exhibit highly cell-specific expression patterns in bladder and therefore likely act in a coordinated manner to regulate ligand availability to purinergic receptors. This is the first study to determine the expression and location of ectonucleotidases within the mammalian urinary bladder.