Insulin secretion is the natural response to hyperglycemia, and it is crucial to maintain glucose homeostasis in healthy individuals. Impairment in this regulation eventually results in Type-2 diabetes. From the perspective of finding new drug targets, it is fundamental to investigate stimuli other than glucose that regulate insulin secretion. I focused my study on dopamine, based on previous literature that showed its role as an inhibitor of insulin secretion. I tested the hypothesis that the islets synthesize dopamine from the circulating precursor L dopa. I observed a dose-dependent inhibition of glucose-stimulated insulin secretion (GSIS) by combining L-dopa and glucose. The inhibition was attributed to increased intracellular dopamine content. I measured a dose dependent decrease in the frequency of the [Ca2+]i oscillations when the islets were treated with dopamine. Also, I investigated which dopamine receptor (DR) is active in the islets. The data show that while DRD2 and DRD3 are both present in the islets, only the antagonism of DRD3 abolished the effects of dopamine, enhanced GSIS in untreated islets, and increased the frequency of [Ca2+]i oscillations. I verified that dopamine transporter also is expressed in the islets, and its pharmacological inhibition impairs GSIS. These results depict a dopaminergic negative feedback acting on insulin secretion. Blocking this dopaminergic feedback increases GSIS. Therefore DRD3 is a potential target for new drugs for the treatment of type 2 diabetes.

The endocytic and postendocytic trafficking pathways in polarized epithelial cells was examined. First, sorting of fluid and membrane internalized from the apical plasma membrane was analayzed. Internalized fluid and membrane were intially delivered to apical early endosomes (AEE). Fluid remained in the AEE while membrane was rapidly sorted and delivered to the Rab11+ apical recycling endosome (ARE). The delivery of membrane markers to the ARE was microtubule-dependent. Transferrin, a marker of basolateral recycling pathway, had access to the AEE but not the ARE. Next, the role of Rac1 and RhoA in endocytosis and postendocytic was determined. Both Rac1 and RhoA were involved in regulation of endocytosis from both plasma membrane domains. Furthermore, Rac1 was implicated in regulation of apically targeted traffic from both the endocytic and secretory pathways. Expression of dominant active Rac1 (Rac1V12) caused formation of a central aggregate of membranes composed in part of the ARE. Markers targeted for the apical plasma membrane were trapped within this aggregate. RhoA was involved in the regulation of traffic from basolateral early endosomes (BEE). Expression of dominant active RhoA (RhoAV14) trapped ligands internalized from the basolateral plasma membrane in BEE that were also associated with filamentous actin (F-actin). A subset of BEE from control cells were also f-actin positive. Colocalization of BEE with proteins involved in actin polymerization based propulsion (APBP) and myosin motor-based propulsion was determined. Proteins involved in APBP were not associated with BEE bu MIc, a type I myosin, did colocalize with a subset of BEE. This suggests that BEE are transported along associated f-actin to the level of the stress fibers carried by MIc. Finally, the role of SNAP-23 in basolateral recycling of transferrin was determined. SNAP-23 was localized to the endosomes throughout the cell and to the basolateral plasma membrane. Treatment of permeabilized MDCK cells with botulinum neurotoxin E or addition of exogenous SNAP-23 or anti-SNAP-23 antibodies all inhibited transferrin recycling. This suggests that SNAP-23 is important for basolateral recycling in polarized epithelial cells.

Mechanical forces affect many cellular functions. How cells respond to mechanical stimuli and how the stimuli are transduced into cellular signals are important questions in cell biology. To this end, the effects of hydrostatic pressure (filling) on bladder uroepithelium were examined by exposing isolated pieces of uroepithelium to hydrostatic pressure in modified Ussing chambers, thus mimicking the filling process of rabbit urinary bladders. Filling had profound effects on both ion and membrane transport in the uroepithelium. Membrane distention activated mechanosensitive ion channels, stimulating Na+ absorption across the umbrella cells via epithelial Na+ channels, K+ secretion via nonselective cation channels, and Cl- secretion via Cl- channels. In addition to elevated ion transport, pressure also stimulated discoidal vesicle exocytosis in umbrella cells, resulting in ~55% increase in apical surface area of the umbrella cells. Exocytosis in these cells also increased the amount of uroplakin III at the apical surface by 67%, and caused release of secretory proteins into the bladder lumen. One pressure-sensing mechanism that could regulate umbrella cell exocytosis is ATP and purinergic receptor signaling. Significant amounts of ATP were released from the uroepithelium during filling. However, when the serosal surface of the uroepithelium was treated with the ATPase apyrase or hexokinase, or incubated with the purinergic receptor antagonist PPADS, pressure-activated exocytosis was blocked. More importantly, filling-induced exocytosis was blocked when P2X2 or P2X3 receptors were absent from mice bladders. The exocytic events were mediated by Ca2+, cAMP, and PKA-dependent mechanisms. In addition to exocytosis, hydrostatic pressure induced endocytosis of 100% of a biotinylated membrane pool within 5 minutes of stimulation. The endocytosed membrane was delivered to lysosome and degraded by a leupeptin-sensitive pathway. The endocytic event could be activated by the purinergic receptor agonist ATP?S, indicating that filling-induced endocytosis may depend on purinergic receptor signaling as well. These results have shed light on how hydrostatic pressure regulates ion and membrane transport in uroepithelial cells and may provide insights to how other mechanosensitive cell types respond to external forces.

The elucidation of mechanisms for epithelial maintenance and renewal after injury are central to understanding aspects of normal airway diversity and the pathobiology of lung diseases including asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and cancer. Due to the low steady state turnover of the airway epithelium, it has been proposed that epithelial remodeling following chronic lung injury or disease may be the result of aberrant epithelial stem cell activation. Previous results indicated that intrapulmonary conducting airways contain rare populations of stem cells that localized to neuroepithelial body (NEB) microenvironments, and that these cells are activated following injury involving depletion of airway Clara cells. These airway cells were uniquely pollutant resistant, exhibited robust mitotic and differentiation potential, and exhibited the molecular property of Clara cell secretory protein (CCSP) expression. Despite this recent progress, many aspects of airway stem cell maintenance, initiation, and regulation remain elusive. Studies presented in this dissertation were undertaken (1) to investigate the existence of alternate, regionally distinct airway stem cell populations, (2) to elucidate mechanisms of airway stem cell pollutant resistance, and (3) to identify signaling pathways associated with stem cell-associated repair. Results of these studies demonstrate the existence of unique, NEB microenvironment-independent CCSP expressing stem cells restricted to airway bronchoalveolar duct junction (BADJ) microenvironments. Results also identify likely mechanisms of CCSP expressing stem cell pollutant resistance that include reduced levels of Cytochrome P450 expression and robust drug / pollutant efflux systems. Finally, results of these studies indicate that activation of the b-catenin signaling pathway and definitive downstream target genes occurs within NEB and BADJ microenvironments during airway regeneration. Together, these findings demonstrate that regionally distinct, pollutant resistant airway stem cell populations are responsible for the maintenance of appropriate epithelial diversity and facilitate renewal processes after injury. Furthermore, these studies support the notion that b-catenin signaling and downstream target gene activation are important mediators of stem cell-associated epithelial renewal.

Na+ transport through epithelial cells mediated by the epithelial Na+ channel (ENaC) is important for maintaining body fluid Na+ homeostasis, alveolar fluid clearance and normal airway mucocilliary function. A large body of evidence shows significant correlation between serine protease activity, channel fragmentation and transepithelial movement of Na+. The extracellular protease dependent regulation may play an important role in epithelial cells where the channel activity is intended more for the control of the extracellular environment, such as in airway cells, than in the control of the internal fluid status of the organism. Presented here is evidence supporting the hypothesis that Na+ channels are inserted into the apical membrane as inactive precursors whereupon they are acted upon by membrane resident serine proteases resulting in active channels. The effects of the serine protease inhibitor, aprotinin, on ENaC single channel properties were studied using transepithelial fluctuation analysis in the A6 amphibian kidney epithelium. Aprotinin causes a potent specific time-dependent inhibition of Na+ transport. Analysis of blocker induced fluctuations in Na+ current (INa) showed linear rate-concentration plots with the same blocker on- and off-rates in control and aprotinin inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. It was shown that protease regulation of INa is mediated by increasing the number of active channels in the apical membrane. To test the hypothesis that residues on ENaC mediate protease dependent channel activation ENaC was subjected to site-directed mutagenesis and heterologously expressed in Fisher rat thyroid (FRT) model epithelial cells. Activation by exogenous proteases depended on the presence of substrate specific residues in ENaC which dictated the rates of activation and the steady-state current levels.

The Duchenne and Limb Girdle Muscular Dystrophies (DMD, LGMD) are a heterogeneous group of genetic disorders. Primary mutations in the dystrophin gene result in the absence of the protein in DMD, and mutations in any one of four sarcoglycan (á, â, ä, ã) genes results in a loss of the entire sarcoglycan complex in LGMD. Mutations of the á-sarcoglycan gene are clinically the most frequently observed, and of these cases, one-third have a missense substitution of a cysteine for an arginine at residue 77 (R77C) of the á-sarcoglycan protein. The function of á-sarcoglycan and the implications of the R77C mutation on protein traffic are currently unknown. Here a model system has been developed to study dystrophin protein complex (DPC) assembly in living cells. We report that a minidystrophin gene construct, currently the most promising avenue for adeno-associated virus mediated gene therapy, properly assembles and integrates into the DPC in vivo, utilizing similar mechanisms as wild type dystrophin. We also demonstrate by a variety of assays that in the absence of sarcoglycan complex assembly, á-sarcoglycan is recycled from the plasma membrane. Furthermore, I provide evidence that R77C, the most commonly occurring LGMD mutation, causes a fundamental defect in protein biosynthesis, trapping the mutant protein in the endoplasmic reticulum in vitro and in vivo. Additionally, I show through re-introduction of selected sarcoglycans that the sarcoglycans are able to associate intracellularly to form specific sub-complexes. Central to sarcoglycan complex assembly is the formation of a â-ä-core complex which promotes the deposition of both the core complex and á-sarcoglycan at the plasma membrane, as seen clinically in the microscopic pathology of some cases of LGMD-2C (ã-sarcoglycan deficiency). Taken together these data show the DPC follows a systematic and sequential assembly process, where proper integration, delivery and deposition of each protein into the complex is dependent on several protein-protein associations that in turn allow appropriate trafficking and assembly at the plasma membrane. The multi-factorial reconstruction of the DPC must therefore be carefully evaluated when treating the muscular dystrophies in humans.

Clathrin-mediated endocytosis is the major process by which cells internalize nutrients, extracellular macromolecules, and membrane constituents to regulate such diverse processes as cell polarity, development, and motility. Over twenty proteins comprise a large protein interaction web that is pertinent to this process. This work investigates proteins that act as clathrin-associated sorting proteins (CLASPs), and their interactions with other endocytic components. Epsin 1 is shown to be a CLASP that engages components of the endocytic clathrin coat and selects for polyubiquitinated cargo. The interaction with polyubiquitin is enabled through epsin 1s UIM domains. I show that polyubiquitin is an efficient endocytic signal, which is relevant for physiological mammalian substrates such as the epithelial sodium channel (ENaC). Stonin 2 behaves as an unconventional CLASP, as it doesnt directly engage clathrin or the plasma membrane. My work uses this protein to biochemically characterize the WXXF motif and identify a privileged binding site located on the sandwich subdomain of the AP-2 ? appendage. This work supports a model in which arrays of binding motifs and multiple engagement sites on the ? appendage allow for an increase in binding affinity, which affects the temporal ordering of endocytic accessory protein interactions during clathrin-mediated endocytosis. These studies have defined important protein interactions that have improved our understanding of the molecular mechanism of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is a critical process through which a wide variety of extracellular material is internalized. The primary component, clathrin, forms a cargo-selective lattice at the plasma membrane, as well as on endosomes and the TGN, though the cargo-selective components are incompletely defined. An ideal tool for understanding the spatio-temporal dynamics of both the clathrin coat and the cargo selected is total internal reflection fluorescence microscopy (TIR-FM), which permits selective imaging of events closely apposed to the ventral plasma membrane. Previously, observation of the clathrin coat has shown both static and dynamic populations, with some dynamic structures undergoing microtubule-dependent motion; the 70-110 nm decay constant of the TIR-FM field has led to the assumption that these are all representative of coated pits. Here, I demonstrate that the dynamic population of clathrin is primarily endosomal, as it lacks colocalization with the plasma membrane-specific endocytic adaptor AP-2, but colocalizes with large, internalized low density lipoprotein (LDL) and transferrin positive structures. Other clathrin-associated sorting proteins (CLASPs) remain in relatively static structures as well. One such CLASP, autosomal recessive hypercholesterolemia (ARH) protein, is the defective protein in ARH, which is typified by the failure of hepatic LDL receptor internalization, despite no LDL receptor mutations. ARH interacts with AP-2 via the novel, helical FXX[FL]XXXR motif present in its C-terminus. Here, I demonstrate the importance of this motif for targeting ARH to coated pits in cells and LDL uptake. As knockdown of ARH is insufficient to block LDL receptor endocytosis in fibroblasts, I show that the CLASP Disabled-2 (Dab2) works with ARH to sort the LDL receptor. Ablation of these two components using RNAi halts LDL receptor endocytosis, and either exogenous ARH or Dab2 rescue this phenotype. The endocytic defect in the liver of ARH patients is due to the lack of Dab2 expression in hepatocytes, making this cell type sensitive to ARH levels for LDL uptake. This work formally validates the CLASP hypothesis, and demonstrates that these CLASPs are general components of the clathrin-coated pit that are regulated in a tissue-specific fashion.

In response to changes in intralumenal pressure, the urinary bladder modulates its mucosal surface to accommodate a wide range of urine volumes upon filling and voiding. During bladder stretch, umbrella cells that line the mucosal surface of the bladder undergo changes in surface area mediated by the exocytosis and endocytosis of subapical discoidal vesicles. While a number of signaling factors are required for this process, how these signals interact with each other and whether they are integrated in any manner is not known. The identification of the epidermal growth factor receptor (EGFR) as an apical receptor for stretch-induced HB-EGF signaling provides a previously unrecognized function for this versatile receptor in bladder physiology. It appears that the EGFR signaling is able to regulate protein synthesis via a MAPK signaling pathway, which is required for the late response of the tissue to prolonged stretch. The transactivation of EGFR via a metalloproteinase-dependent pathway opens the possibility that several previously recognized stretch-induced signals function upstream to stimulate EGFR activity in an integrated signaling pathway. The importance of tight regulation of EGFR activity in the bladder is highlighted by its role in bladder carcinoma pathophysiology, and future studies of this pathway may provide insights that lead to diagnostic and therapeutic advances.

The conducting airway epithelium is lined with a heterogeneous population of secretory and ciliated cells. Inflammation and inhaled toxicants can damage the epithelial lining, which is rapidly repaired through a tissue stem cell-mediated process. Incomplete and disrupted repair contribute to the development of chronic lung disease. The signaling pathways that orchestrate airway epithelial repair are largely unknown. In the first part of the dissertation, the necessity for Wnt/â-catenin signaling in airway epithelial homeostasis and repair was determined. Wnt/â-catenin signaling regulates epithelial homeostasis in the intestine and skin, but the role of this pathway in the airways is unknown. This hypothesis was tested through the generation of mice airway-specific loss of â-catenin. A â-catenin null airway epithelium repaired normally in an in vivo injury model system. No defects in epithelial homeostasis or differentiation were seen in the absence of â-catenin. We concluded that â-catenin is not a principal regulator of airway homeostasis in the adult conducting airway epithelium. The second project determined the role of TGFâ/BMP signaling in airway branching morphogenesis, tumor suppression, and epithelial repair. Genetic deletion of Smad4 in the epithelium was used to block signaling through both the TGFâ and BMP pathways. Loss of Smad4-dependent signaling during embryogenesis markedly increased airway branching. These mice later developed adenomas, reflecting the role of Smad4 in lung tumor suppression. Surprisingly, epithelial repair was not influenced by loss of Smad-dependent signaling. These date indicated a role for TGFâ/BMP signaling in branching morphogenesis and tumor suppression, but not epithelial repair. The third project of this dissertation further characterized the molecular phenotype of the airway secretory cell population. Two ablation models were used to deplete secretory cells in vivo. Microarray analysis was performed following secretory cell ablation to identify genes that might be expressed within the secretory population. Four novel secretory cell markers were identified by this approach. In conclusion, this dissertation determined roles for Wnt/â-catenin and TGFâ/BMP signaling in the airway epithelium and further characterized the molecular repertoire of the airway secretory cell population.