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Biomedical subjects

G Apodaca

Publications and source records attributed to G Apodaca.

At least 19 recordsLinked to original sources
PubMed2002

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.

Acetic Acid↗
PubMed2001

A novel clathrin homolog that co-distributes with cytoskeletal components functions in the trans-Golgi network.

A clathrin homolog encoded on human chromosome 22 (CHC22) displays distinct biochemistry, distribution and function compared with conventional clathrin heavy chain (CHC17), encoded on chromosome 17. CHC22 protein is upregulated during myoblast differentiation into myotubes and is expressed at high levels in muscle and at low levels in non-muscle cells, relative to CHC17. The trimeric CHC22 protein does not interact with clathrin heavy chain subunits nor bind significantly to clathrin light chains. CHC22 associates with the AP1 and AP3 adaptor complexes but not with AP2. In non-muscle cells, CHC22 localizes to perinuclear vesicular structures, the majority of which are not clathrin coated. Treatments that disrupt the actin-myosin cytoskeleton or affect sorting in the trans-Golgi network (TGN) cause CHC22 redistribution. Overexpression of a subdomain of CHC22 induces altered distribution of TGN markers. Together these results implicate CHC22 in TGN membrane traffic involving the cytoskeleton.

Cells, Cultured↗
PubMed2001

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.

Actins↗
PubMed2001

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells.

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.

Actins↗
PubMed2000

Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1.

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.

Actins↗
PubMed2000

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state.

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.

Animals↗
PubMed2000

Sorting of membrane and fluid at the apical pole of polarized Madin-Darby canine kidney cells.

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (</=2.5 min at 37 degrees C) and are then rapidly segregated from one another. The dextran remains in the large supranuclear EEA1-positive early endosomes while recycling polymeric immunoglobulin receptor-bound immunoglobulin A is delivered to a Rab11-positive subapical recycling compartment. This latter step requires an intact microtubule cytoskeleton. Receptor-bound transferrin, a marker of the basolateral recycling pathway, has limited access to the fluid-rich apical early endosome but is excluded from the subapical elements of the Rab11-positive recycling compartment. We propose that the term ARE be used to describe the subapical Rab11-positive compartment and that the ARE is distinct from both the transferrin-rich common recycling endosome and the fluid-rich apical early endosome.

Animals↗
PubMed2000

Urothelial pathophysiological changes in feline interstitial cystitis: a human model.

Unique barrier properties of the urothelial surface membrane permit urine storage. Interstitial cystitis causes disabling dysuria, and frequency. Similarly, feline interstitial cystitis (FIC) occurs in cats. These studies define the permeability and structural properties of normal and FIC urothelium. To determine the effects of bladder filling, groups were studied before and after hydrodistention. Normal urothelium with or without hydrodistention exhibited high transepithelial resistances (TER) and low water and urea permeabilities, resembling other species. Fluorescence confocal microscopy revealed localization of the marker AE-31 to the apical surface of all umbrella cells in normal urothelium, with the tight junction protein ZO-1 localized to tight junctions. Scanning and transmission electron microscopy revealed uniform distribution of luminal cells with characteristic apical membrane and tight junction morphology. Urothelium in FIC animals displayed reduced TER and increased water and urea permeability following hydrodistention. Structural studies in FIC revealed denuded urothelium, with appearance of AE-31 in underlying epithelial cells. The results demonstrate severe epithelial damage and dysfunction in FIC and suggest novel approaches toward examining the etiology and therapy of IC.

Animals↗
PubMed1999

ADP-ribosylation factor 6 and endocytosis at the apical surface of Madin-Darby canine kidney cells.

We report that the small GTPase, ADP-ribosylation factor 6 (ARF6), is present only on the apical surface of polarized MDCK epithelial cells. Overexpression of a mutant of ARF6, ARF6-Q67L, which is predicted to be in the GTP-bound form, stimulates endocytosis exclusively at this surface. Surprisingly, overexpression of the mutant ARF6-T27N, which is predicted to be in the GDP-bound form, also stimulated apical endocytosis, though to a lesser extent. ARF6-stimulated endocytosis is inhibited by a dominant-negative form of dynamin, or a dominant-negative hub fragment of clathrin heavy chain, indicating that it is mediated by clathrin. Correspondingly, overexpression of either mutant of ARF6 leads to an increase in the number of clathrin-coated pits at the apical plasma membrane. When ARF6-Q67L is overexpressed in the presence of the dominant-negative dynamin, the ARF6-Q67L colocalizes with clathrin and with IgA bound to its receptor. We conclude that ARF6 is an important modulator of clathrin-mediated endocytosis at the apical surface of epithelial cells.

ADP-Ribosylation Factor 6↗
PubMed1999

Primary uroepithelial cultures. A model system to analyze umbrella cell barrier function.

Despite almost 25 years of effort, the development of a highly differentiated and functionally equivalent cell culture model of uroepithelial cells has eluded investigators. We have developed a primary cell culture model of rabbit uroepithelium that consists of an underlying cell layer that interacts with a collagen substratum, an intermediate cell layer, and an upper cell layer of large (25-100 micrometer) superficial cells. When examined at the ultrastructural level, the superficial cells formed junctional complexes and had an asymmetric unit membrane, a hallmark of terminal differentiation in bladder umbrella cells. These cultured "umbrella" cells expressed uroplakins and a 27-kDa uroepithelial specific antigen that assembled into detergent-resistant asymmetric unit membrane particles. The cultures had low diffusive permeabilities for water (2.8 x 10(-4) cm/s) and urea (3.0 x 10(-7) cm/s) and high transepithelial resistance (>8000 Omega cm2) was achieved when 1 mM CaCl2 was included in the culture medium. The cell cultures expressed an amiloride-sensitive sodium transport pathway and increases in apical membrane capacitance were observed when the cultures were osmotically stretched. The described primary rabbit cell culture model mimics many of the characteristics of uroepithelium found in vivo and should serve as a useful tool to explore normal uroepithelial function as well as dysfunction as a result of disease.

Animals↗
PubMed1999

Modulation of endocytic traffic in polarized Madin-Darby canine kidney cells by the small GTPase RhoA.

Efficient postendocytic membrane traffic in polarized epithelial cells is thought to be regulated in part by the actin cytoskeleton. RhoA modulates assemblies of actin in the cell, and it has been shown to regulate pinocytosis and phagocytosis; however, its effects on postendocytic traffic are largely unexplored. To this end, we expressed wild-type RhoA (RhoAWT), dominant active RhoA (RhoAV14), and dominant inactive RhoA (RhoAN19) in Madin-Darby canine kidney (MDCK) cells expressing the polymeric immunoglobulin receptor. RhoAV14 expression stimulated the rate of apical and basolateral endocytosis, whereas RhoAN19 expression decreased the rate from both membrane domains. Polarized basolateral recycling of transferrin was disrupted in RhoAV14-expressing cells as a result of increased ligand release at the apical pole of the cell. Degradation of basolaterally internalized epidermal growth factor was slowed in RhoAV14-expressing cells. Although apical recycling of immunoglobulin A (IgA) was largely unaffected in cells expressing RhoAV14, transcytosis of basolaterally internalized IgA was severely impaired. Morphological and biochemical analyses demonstrated that a large proportion of IgA internalized from the basolateral pole of RhoAV14-expressing cells remained within basolateral early endosomes and was slow to exit these compartments. RhoAN19 and RhoAWT expression had little effect on these postendocytic pathways. These results indicate that in polarized MDCK cells activated RhoA may modulate endocytosis from both membrane domains and postendocytic traffic at the basolateral pole of the cell.

3,3'-Diaminobenzidine↗
PubMed1998

SNAP-23 requirement for transferrin recycling in Streptolysin-O-permeabilized Madin-Darby canine kidney cells.

Fusion of recycling and transcytotic vesicles with the apical and basolateral plasma membrane domains of Madin-Darby canine kidney (MDCK) cells requires the N-ethylmaleimide-sensitive factor and is sensitive to botulinum neurotoxin serotype E (BoNT/E). BoNT/E is thought to selectively proteolyze the 25,000-dalton synaptosomal associated protein (SNAP-25), a protein found in neurons or cells of neuroendocrine origin. However, SNAP-25 is not found in MDCK cells. One possible target for BoNT/E in MDCK cells is SNAP-23, a newly described SNAP-25 homolog that is found in several organs including kidney. Currently, the function of SNAP-23 is unknown. We have reconstituted transferrin recycling in permeabilized MDCK cells to assess the role of SNAP-23 in the endocytic traffic of this protein. We find that: (i) SNAP-23 is expressed in MDCK cells and is found both at the basolateral plasma membrane and associated with apical and basolateral vesicles, (ii) canine SNAP-23 is cleaved by BoNT/E, (iii) transferrin recycling is N-ethylmaleimide-sensitive factor-dependent and BoNT/E-sensitive, and (iv) addition of either exogenous SNAP-23 or anti-SNAP-23 antibodies inhibits ligand recycling. Our observations suggest that SNAP-23 may be required for fusion of recycling vesicles with the basolateral membrane of polarized MDCK cells.

Animals↗
PubMed1998

Selective perturbation of apical membrane traffic by expression of influenza M2, an acid-activated ion channel, in polarized madin-darby canine kidney cells.

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin-Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

Animals↗
PubMed1998

Disruption of guinea pig urinary bladder permeability barrier in noninfectious cystitis.

Although most cell membranes permit rapid flux of water, small nonelectrolytes, and ammonia, the apical membranes of bladder epithelial umbrella cells, which form the bladder permeability barrier, exhibit strikingly low permeabilities to these substances. In cystitis, disruption of the bladder permeability barrier may irritate the bladder wall layers underlying the epithelium, causing or exacerbating inflammation, and increasing urinary frequency, urgency, and bladder pain. To determine the effects of inflammation on the integrity of the permeability barrier, guinea pigs were sensitized with ovalbumin, and the bladders were exposed subsequently to antigen by instillation on the urinary side. Inflammation of the bladder wall markedly reduced transepithelial resistance of dissected epithelium mounted in Ussing chambers and increased water and urea permeabilities modestly at 2 h and more strikingly at 24 h after induction of the inflammation. Transmission and scanning electron microscopy of bladders at 30 min and 24 h after antigen exposure revealed disruption of tight junctions, denuding of patches of epithelium, and occasional loss of apical membrane architecture. These permeability and structural effects did not occur in nonsensitized animals in which the bladders were exposed to antigen and in sensitized animals exposed to saline vehicle rather than antigen. These results demonstrate that inflammation of the underlying muscle and lamina propria can disrupt the bladder permeability barrier by damaging tight junctions and apical membranes and causing sloughing of epithelial cells. Leakage of urinary constituents through the damaged epithelium may then exacerbate the inflammation in the underlying muscle layers.

Animals↗
PubMed1998

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.

Adrenergic Agonists↗
PubMed1997

Both microtubules and actin filaments are required for efficient postendocytotic traffic of the polymeric immunoglobulin receptor in polarized Madin-Darby canine kidney cells.

It has been postulated that membrane traffic in polarized epithelial cells requires both actin filaments and microtubules. We have tested this hypothesis by analyzing the effect of cytochalasin D (cytoD; an actin-disrupting agent), by itself or in combination with nocodazole (a microtubule depolymerizing agent), on postendocytic traffic in Madin-Darby canine kidney cells. CytoD treatment inhibited basolateral to apical transcytosis of IgA in polymeric immunoglobulin receptor-expressing cells by approximately 45%, but had little effect on basolateral recycling of transferrin. Apical recycling of IgA was also inhibited by approximately 20%. Like nocodazole, cytoD acted at an early step in transcytosis, and inhibited translocation of IgA between the basolateral early endosomes and the apical recycling endosome. There was little inhibition of the subsequent release of IgA from the apical recycling endosome of cytoD- or nocodazole-treated cells. Order-of-addition experiments suggest that the cytoD-sensitive step preceded the nocodazole-sensitive step. Treatment with both cytoD and nocodazole inhibited transcytosis 95%. These results suggest that in addition to microtubules, efficient postendocytic traffic in polarized epithelial cells also requires actin filaments.

Actin Cytoskeleton↗
PubMed1997

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 pill, 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.

Amino Acid Sequence↗