Gerard Apodaca

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1−/− mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1−/− mice, as was hypoosmolality-evoked ATP release from cultured trpv1−/− urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton.

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.

Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder

Nitric oxide (NO) has been implicated in the regulation of the lower urinary tract. However, the source(s) of NO production in the urinary bladder (UB) has not been determined. Accordingly, we used a porphyrinic microsensor placed on the surface of UB strips in vitro to directly measure endogenous NO production. The afferent neurotoxin, capsaicin, and the mixed alpha/beta-adrenergic agonist, norepinephrine (NE), both evoked transient (1-3 s) NO release (range 50 nM to 1.4 microM). Adrenergic-mediated release was not decreased following denervation of the UB but was abolished following selective removal of the mucosa. On the other hand, release evoked by capsaicin (range 50-900 nM) was significantly decreased after UB denervation. These data indicate that NE releases NO from UB epithelium, and capsaicin releases NO from epithelium as well as nervous tissue in the UB. In light of reports that NO may regulate epithelial integrity and function in other tissues, agonist regulation of a constitutive nitric oxide synthase activity in the UB may provide a novel mechanism for modulation of bladder and urothelial function.Nitric oxide (NO) has been implicated in the regulation of the lower urinary tract. However, the source(s) of NO production in the urinary bladder (UB) has not been determined. Accordingly, we used a porphyrinic microsensor placed on the surface of UB strips in vitro to directly measure endogenous NO production. The afferent neurotoxin, capsaicin, and the mixed α/β-adrenergic agonist, norepinephrine (NE), both evoked transient (1-3 s) NO release (range 50 nM to 1.4 μM). Adrenergic-mediated release was not decreased following denervation of the UB but was abolished following selective removal of the mucosa. On the other hand, release evoked by capsaicin (range 50-900 nM) was significantly decreased after UB denervation. These data indicate that NE releases NO from UB epithelium, and capsaicin releases NO from epithelium as well as nervous tissue in the UB. In light of reports that NO may regulate epithelial integrity and function in other tissues, agonist regulation of a constitutive nitric oxide synthase activity in the UB may provide a novel mechanism for modulation of bladder and urothelial function.

The uroepithelium: not just a passive barrier.

The uroepithelium lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, where it forms a tight barrier that allows for retention of urine, while preventing the unregulated movement of ions, solutes, and toxic metabolites across the epithelial barrier. In the case of the bladder, the permeability barrier must be maintained even as the organ undergoes cyclical changes in pressure as it fills and empties. Beyond furthering our understanding of barrier function, new analysis of the uroepithelium is providing information about how detergent‐insoluble membrane/protein domains called plaques are formed at the apical plasma membrane of the surface umbrella cells, how mechanical stimuli such as pressure alter exocytic and endocytic traffic in epithelial cells such as umbrella cells, and how changes in pressure are communicated to the underlying nervous system.

Immunoglobulin transport across polarized epithelial cells

IgA, IgG and IgM are transported across epithelial cells in a receptor-mediated process known as transcytosis. In addition to neutralizing pathogens in the lumen of the gastrointestinal, respiratory and urogenital tracts, these antibody–receptor complexes are now known to mediate intracellular neutralization of pathogens and might also be important in immune activation and tolerance. Recent studies on the intracellular transport pathways of antibody–receptor complexes and antibody-stimulated receptor-mediated transcytosis are providing new insight into the nature and regulation of endocytic pathways.

Cell biology and physiology of the uroepithelium

The uroepithelium sits at the interface between the urinary space and underlying tissues, where it forms a high-resistance barrier to ion, solute, and water flux, as well as pathogens. However, the uroepithelium is not simply a passive barrier; it can modulate the composition of the urine, and it functions as an integral part of a sensory web in which it receives, amplifies, and transmits information about its external milieu to the underlying nervous and muscular systems. This review examines our understanding of uroepithelial regeneration and how specializations of the outermost umbrella cell layer, including tight junctions, surface uroplakins, and dynamic apical membrane exocytosis/endocytosis, contribute to barrier function and how they are co-opted by uropathogenic bacteria to infect the uroepithelium. Furthermore, we discuss the presence and possible functions of aquaporins, urea transporters, and multiple ion channels in the uroepithelium. Finally, we describe potential mechanisms by which the uroepithelium can transmit information about the urinary space to the other tissues in the bladder proper.

β-Adrenoceptor Agonists Stimulate Endothelial Nitric Oxide Synthase in Rat Urinary Bladder Urothelial Cells

We have investigated the intracellular signaling mechanisms underlying the release of nitric oxide (NO) evoked by β-adrenoceptor (AR) agonists in urinary bladder strips and cultured bladder urothelial cells from adult rats. Reverse transcription-PCR revealed that inducible NO synthase and endothelial NOS but not neuronal NOS genes were expressed in urothelial cells. NO release from both urothelial cells and bladder strips was decreased (37–42%) in the absence of extracellular Ca2+ (100 μmEGTA) and was ablated after incubation with BAPTA-AM (5 μm) or caffeine (10 mm), indicating that the NO production is mediated in part by intracellular calcium stores. NO release was reduced (18–24%) by nifedipine (10 μm) and potentiated (29–32%) by incubation with the Ca2+ channel opener BAYK8644 (1–10 μm). In addition, β-AR-evoked NO release (isoproterenol; dobutamine; terbutaline; 10−9 to 10−5m) was blocked by the NOS inhibitors NG-nitro-l-arginine methyl ester (30 μm) orNG-monomethyl-l-arginine (50 μm), by β-adrenoceptor antagonists (propranol, β1/β2; atenolol, β1; ICI 118551; β2; 100 μm), or by the calmodulin antagonist trifluoperazine (50 μm). Incubating cells with the nonhydrolyzable GTP analog GTPγS (1 μm) or the membrane-permeant cAMP analog dibutyryl-cAMP (10–100 μm) directly evoked NO release. Forskolin (10 μm) or the phosphodiesterase IBMX (50 μm) enhanced (39–42%) agonist-evoked NO release. These results indicate that β-adrenoceptor stimulation activates the adenylate cyclase pathway in bladder epithelial cells and initiates an increase in intracellular Ca2+ that triggers NO production and release. These findings are considered in light of recent reports that urothelial cells may exhibit a number of “neuron-like” properties, including the expression of receptors/ion channels similar to those found in sensory neurons.

ATP and purinergic receptor–dependent membrane traffic in bladder umbrella cells

The umbrella cells that line the bladder are mechanosensitive, and bladder filling increases the apical surface area of these cells; however, the upstream signals that regulate this process are unknown. Increased pressure stimulated ATP release from the isolated uroepithelium of rabbit bladders, which was blocked by inhibitors of vesicular transport, connexin hemichannels, ABC protein family members, and nucleoside transporters. Pressure-induced increases in membrane capacitance (a measure of apical plasma membrane surface area where 1 microF approximately equals 1 cm2) were inhibited by the serosal, but not mucosal, addition of apyrase or the purinergic receptor antagonist PPADS. Upon addition of purinergic receptor agonists, increased capacitance was observed even in the absence of pressure. Moreover, knockout mice lacking expression of P2X2 and/or P2X3 receptors failed to show increases in apical surface area when exposed to hydrostatic pressure. Treatments that prevented release of Ca2+ from intracellular stores or activation of PKA blocked ATPgammaS-stimulated changes in capacitance. These results indicate that increased hydrostatic pressure stimulates release of ATP from the uroepithelium and that upon binding to P2X and possibly P2Y receptors on the umbrella cell, downstream Ca2+ and PKA second messenger cascades may act to stimulate membrane insertion at the apical pole of these cells.

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa‐induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon‐tagged non‐cytotoxic mutants of a cytotoxic and lung‐virulent strain of P. aeruginosa (PA103). The transposon‐insertion site was determined by using an inverse polymerase chain reaction followed by DNA‐sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pili, based on their resistance to phage PO4 and/or loss of twitching motility (twt−). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein‐secretion apparatus appears to be involved in this process.

Role of membrane traffic in the generation of epithelial cell asymmetry

Epithelial cells have an apical–basolateral axis of polarity, which is required for epithelial functions including barrier formation, vectorial ion transport and sensory perception. Here we review what is known about the sorting signals, machineries and pathways that maintain this asymmetry, and how polarity proteins interface with membrane-trafficking pathways to generate membrane domains de novo. It is becoming apparent that membrane traffic does not simply reinforce polarity, but is critical for the generation of cortical epithelial cell asymmetry.