Hypoxic pulmonary vasoconstriction (HPV) is a unique physiological response of the lung that acts to optimize gas exchange by diverting blood flow from poorly ventilated regions. The endothelium has been thought to play a mostly modulatory role in this phenomenon through the synthesis of vasoactive agents such as nitric oxide (NO), prostacyclin, and endothelin. Data is provided showing that acute hypoxia induces increases in NO biosynthesis, promoting S-nitrosation of the metal binding protein metallothionein (MT), which resulted in intracellular release of zinc. Hypoxia released zinc induced contraction of pulmonary endothelial cells and contributed to vasoconstriction of small, non-muscularized intra-acinar arteries in isolated perfused mouse lungs (IPL). The relevance of this NO/MT/Zn pathway in HPV was illustrated by pharmacological inhibition of NO synthesis and analysis of the response in MT knockout (MT-/-) mice, both of which resulted in a blunted pressure response to hypoxia in IPL. Signaling pathways were delineated, indicating how changes in intracellular zinc can alter the actin cytoskeleton and promote cellular contraction. It was found that either hypoxia or exogenous zinc resulted in increases in the formation and alignment of actin stress fibers. These changes were mediated through the inhibition of myosin light chain phosphatase (MLCP), which promoted phosphorylation of myosin light chain (MLC) and tension generation. Activation of PKC appeared to play a role in this process, as indicated by activation and translocation of the enzyme in response to both hypoxia and/or increases in labile zinc, and by the blunted contractile response in isolated endothelial cells following pharmacological inhibition of PKC or utilization of a PCKε dominant negative construct. These data suggest that the NO released in response to hypoxia promotes increases in MLC phosphorylation through zinc-dependent pathways, which in turn are responsible for the force induction and cell stability necessary to elicit an active contractile response in pulmonary endothelium.

Ion channels are the molecular units that underlie electrical signaling in cells. Many physiological processes are dependent upon this signaling mechanism, as dysregulation often leads to severe pathophysiological consequences. The intermediate conductance calcium-activated potassium channel (KCa3.1) functions as heteromeric complexes with calmodulin (CaM), which is constitutively bound to the calmodulin-binding domain (CaMBD) of KCa3.1 located in the C-terminus, just distal to the sixth transmembrane domain (S6). This arrangement enables CaM to function as an intracellular Ca2+-sensor, coupling changes in the intracellular Ca2+ concentration to the regulation of channel activity. Understanding how channels gate or transition from the closed to the open conformation is a fundamental question in the field of ion channel biophysics. A chemomechanical gating model was proposed to explain how Ca2+-binding causes the channel to transition from a non-conducting to a conducting configuration. However, this model lacks a specific mechanism explaining how the conformational change in the CaMBD is coupled to the activation gate. Therefore, the goal of this dissertation was to investigate the role of S6 in the activation mechanism of KCa3.1. Specifically, I tested the hypothesis that the non-luminal residues in the C-terminal portion of S6 function as an interacting surface to couple CaM to the activation gate. Biochemical perturbation and site directed mutagenesis targeting predicted non-luminal residues in S6 act to shift the gating equilibrium toward the open state by increasing the apparent Ca2+ affinity and dramatically slowing the deactivation process. Kinetic modeling using a 6-state gating scheme showed these perturbations act to slow the transition between the open state back to the closed state. The modification in the steady-state and kinetic behavior of the channel in combination with the kinetic analysis indicate the shift in gating equilibrium is caused by slowing the closing transition, suggesting the non-luminal surface of S6 is allosterically coupled to the activation gate. Therefore, in addition to being a structural component of the pore; S6 is also a dynamic component of the activation mechanism. Continuing to identify regions of the channel participating in the activation mechanism is critical to understand how Ca2+ binding leads to channel opening.

Phosphatidylinositol (PI) and its phosphorylated derivatives, phosphatidylinositides (PIPs), are versatile cellular regulators participating in myriad events including signal transduction, cytoskeleton organization, protein targeting and many steps of membrane traffic. Different PIPs exhibit non-overlapping distributions on cellular membranes. This feature contributes to organelle identities and is tightly controlled by kinase/phosphatase-mediated PIP synthesis and turnover. Mechanisms regarding compartment-restriction and detailed functions of many PIPs and PI/PIP metabolizing enzymes remain largely unknown. My dissertation focuses on the cellular targeting mechanism of a PIP kinase and the pathogenesis of a disease caused by mutations in a PIP phosphatase. Phosphatidylinositol (4,5)-bisphosphate (PIP2), an apical-surface-enriched PIP in polarized epithelial cells, is primarily synthesized via phosphorylation of phosphatidylinositol 4-phosphate (PI4P) in the presence of type I PI 5-kinases (PI5KIs). Previous studies have suggested that the three isoforms of PI5KI (¦Á, ¦Â, and ¦Ã) exhibit distinct cellular functions. Data from our lab indicate that these three PI5KIs are differentially localized in polarized renal cells. While the majority of ¦Á and ¦Ã isoforms are present on lateral cell surface, the ¦Â isoform strikingly localizes to the apical plasma membrane. Using mutagenesis, immunofluorescence, and confocal microscopy, I have found that the apical surface distribution of PI5KI¦Â is nonsaturable and does not require catalytic activity or the presence of PIP2. These results provide useful information for future studies on PI5KI¦Â-regulated cellular activities. PIP2 turnover can be catalyzed by a variety of enzymes, one of which is OCRL1. OCRL1 is a PI 5-phosphatase that preferentially hydrolyzes PIP2, producing PI4P, and is associated with the trans-Golgi network, endosomes, and clathrin-coated-pits. Genetic defects of OCRL1 cause Lowe syndrome, a disease manifested by congenital cataracts, mental retardation, and renal tubular dysfunction. By examining cultured renal epithelial cells acutely depleted of OCRL1 via RNA interference, I have found that loss of OCRL1 does not interfere with endocytic trafficking of the multiligand receptor megalin, or uptake of megalin ligands. OCRL1 knockdown did appear to disrupt delivery of newly-synthesized lysosomal hydrolases and alter distribution of primary cilia length in renal epithelial cells. These findings suggest that multiple pathways may contribute to development of renal symptoms in Lowe patients.

Clathrin-mediated endocytosis is a selective pathway for the entry of transmembrane proteins into the cell through the generation of a short-lived vesicular intermediate. Cells and tissues depend on this process for obtaining nutrients, modulation of signaling and cell migration. The clathrin-coated structure intermediate is assembled on the plasma membrane from a cohort of 20-30 distinct proteins that aid in cargo selection, scaffolding, membrane bending and scission of the vesicle. Exactly how these complex assemblies are nucleated at the plasma membrane remains unclear although the lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) plays an important role by anchoring many of the endocytic components. The work in this thesis helps to clarify the nucleation phase by describing the molecular details of the interaction between a PtdIns(4,5)P2-generating lipid kinase PIPKIgamma and the heterotetrameric clathrin adaptor AP-2. By engaging a subdomain on the AP-2 beta2 subunit appendage, the kinase is strategically positioned at assembly sites to generate PtdIns(4,5)P2 and drive coat assembly forward. Clathrin binds to the same subdomain on the beta2 appendage but with a higher apparent affinity. I therefore invoke a model in which PtdIns(4,5)P2 production for nucleation is negatively regulated by PIPKIgamma displacement from AP-2 by clathrin at later stages of assembly. I also demonstrate that a cargo-sorting alternate adaptor that binds to the other subsite on the AP-2 beta2 appendage is not subject to displacement by clathrin during clathrin-coated vesicle budding, ensuring non-competitive cargo incorporation into the vesicle. Finally, the PtdIns(4,5)P2-binding EFC domain proteins FCHO1 and FCHO2 have been proposed to act as dedicated nucleators of clathrin-coated structures on the plasma membrane. I demonstrate in multiple cell lines that these proteins are not invariantly required for placement of clathrin-coated assemblies on the plasma membrane despite being early arriving components themselves. FCHO1/2 are involved in the regulation of the size and number of these assemblies in some cellular contexts. My data support the model of PtdIns(4,5)P2 regulated, not protein regulated, nucleation of clathrin-coated structures; however multiple parallel pathways may contribute to initiation of endocytic buds.

Most cells throughout their existence are constantly subjected to enormous amounts of endogenous and exogenous DNA damage. The cellular response to genotoxic stressors ultimately either leads to adaptive processes that mediate cellular repair and allow for continuous cellular function or leads to cell malfunction and death. In some theories of aging this cellular malfunction is due to accumulation of unrepaired DNA damage which over time leads to progressive deterioration of tissue/organ homoeostasis and function resulting in organismal aging. The overall goal of my studies is to understand the responses to DNA damage in corneal endothelial (CE) cells whose pump and barrier functions are essential for corneal transparency and which in vivo display age-related degeneration and accumulation of DNA damage. In three complementary and related studies I have focused on how the CE is affected by genotoxic stress. In the first study I have examined the clinical application of the DNA crosslinking agent mitomycin C during photorefractive keratectomy and documented its effects on the CE such as significant accumulation of DNA lesions and elevated levels of apoptosis. In the second study I have examined the long term consequences resulting from failure to repair endogenous DNA damage in vivo. Using a DNA repair-deficient mouse strain I have observed significant premature age-related dystrophic changes in the CE that only occur in very old mice. This suggests that the CE is sensitive and vulnerable to the effects of accumulating endogenous genotoxic stress and that DNA damage may drive CE aging. In the third study I have examined how CE cell-cell communication mediated by gap junctions is affected by acute genotoxic stress. Given that gap junction intercellular communication is essential for homeostasis and associated with cell proliferation, death and survival, alterations in the gap junction protein connexin-43 may be crucial for CE cell function and viability during genotoxic stress. The key findings of all my studies elucidate the role of genotoxic stress in CE aging and identify novel responses to stresses from DNA damage. Through a greater understanding of the responses to these stressors, efforts to preserve and improve CE viability and function can be achieved.

Objective: Obesity has become a major worldwide health issue over the past few years and often leads to insulin resistance (IR) and type 2 diabetes (T2D). Macrophage accumulation in adipose tissue (AT) during obesity contributes to inflammation and IR. In the decade since macrophages were shown to accumulate in AT, the majority of studies have focused on recruitment-dependent mechanisms for their accrual. However, recent evidence suggests that recruitment-independent mechanisms, including increased proliferation and decreased egress, may also regulate pro-inflammatory AT macrophage (ATM) numbers. Interestingly the regulation of longevity in ATM accrual in obesity had not been explored. The work in my dissertation shows that increased ATM survival during obesity is a recruitment-independent mechanism that contributes to ATM accumulation. Results: My studies demonstrated that cleaved caspase 3 activation is significantly reduced in the ATMs of diet-induced and genetically obese mice. This data suggests that activation of apoptotic pathways is significantly reduced in ATMs from diet-induced and genetically obese mice. Concurrently, pro-survival Bcl-2 family member protein levels and localization to the mitochondria was elevated in ATMs from obese mice. Conversely, the activities of pro-apoptotic proteins Bax and Bak were decreased in ATMs from obese compared to lean mice. Interestingly, this increased pro-survival signaling in obese ATMs was associated with elevated activation of the p65 subunit of the transcription factor, NF-κB. Furthermore, NF-κB was more nuclear localized in ATMs of obese mice, resulting in increased expression of NF-κB pro-survival target genes, XIAP and cIAP. Finally, an obesogenic milieu increased ATM viability only when NF-κB signaling pathways were functional. Conclusions: Our data demonstrate that obesity promotes survival of inflammatory ATMs, possibly through an NF-κB-regulated mechanism.

The heart must function properly to perform its essential role in supplying the body with the oxygen and nutrients required for survival. Over the course of a lifetime the heart will eventually be exposed to conditions of oxygen depletion or obstructed flow. At such times it is essential that the heart maintain its function and adapt to these conditions by altering its metabolism in response to the decrease in oxygen. Glucose, fatty acids, lactate and most other substrates require oxygen for full energy yield, making them unsuitable during an anoxic or ischemic period. Amino acids have a wide array of uses in the body, including as metabolic substrates. In particular, glutamine and glutamate can be easily converted to α-ketoglutarate and shuttled into the TCA cycle as metabolic substrates. For this reason we hypothesized that it may be possible to prolong cardiac function during and improve recovery after oxygen depletion by supplying the heart with excess glutamine and glutamate. Using action potential duration (APD) as a readout for the electrical function of the heart, we exposed rabbit hearts to anoxic and ischemic challenges and monitored their behavior. We show that elevated levels of glutamate and glutamine increased APD90 in anoxic hearts by 11% over controls. In ischemic hearts, however, the effects were even greater, as the enriched hearts had a 29% increase in APD90 and a 38% increase in APD50 compared to controls. We also demonstrate that blockage of amino acid transamination eliminates this effect and show that metabolism of these amino acids through the TCA cycle is the primary mechanism. These results are significant and conserved across both anoxia and ischemia, indicating that this could be a reliable and effective intervention for extending APD and possibly improving cardiac function.

Diabetes is characterized by excess levels of glucose and lipid in the blood, which have been proposed to directly and negatively impact islet β cells, exacerbating symptoms and promoting disease progression. Our understanding of glucotoxicity and lipotoxicity in human type 2 diabetes is limited, because most investigation has used either rodent islets or human islets in vitro. Based on these studies, numerous mechanisms have been proposed, but not tested, in human islets in vivo. To investigate the molecular mechanisms of excess glucose or lipid, we developed and employed three complementary models of nutrient excess, hyperglycemia (excess glucose), insulin resistance (excess lipid), or both, and examined direct effects on human islets in vivo. In response to hyperglycemia or insulin resistance, stimulated insulin secretion, the main indicator of β cell function, is severely reduced from human islet grafts. Surprisingly, in contrast to rodent islets, neither excess of lipid or glucose stimulated β cell proliferation or caused β cell apoptosis. In human islets in vivo, insulin resistance (1) blunted the unfolded protein response (UPR), (2) increased reactive oxygen species production and oxidative stress, (3) increased lipid droplets and amyloid deposits, and (4) reduced the key β cell transcription factors MAFB and NKX6.1. In contrast, hyperglycemia only blunted the UPR and reduced expression MAFB. Importantly, we found fundamental differences between human and mouse islets in response to identical metabolic conditions, such as stimulated insulin secretion, proliferative response, UPR induction, and transcription factor expression. To complement these studies, we performed a comprehensive, systematic, post-hoc analysis of variation of attributes and function across human islet preparations. Based on our data, we propose a model of glucotoxic and liptoxic consequences for human islets in vivo in which insulin resistance induces significantly more detrimental effects for human β cells in vivo than does hyperglycemia alone, but both conditions mechanistically converge to impair stimulated insulin secretion from human β cells in vivo. This work is the first to demonstrate and mechanistically investigate glucotoxic and lipotoxic consequences for human β cells in vivo, and the models developed will be useful to better understand and test interventions for gluco- and lipotoxicity.

Reduced pancreatic β cell mass is a hallmark of diabetes, which makes the ability to increase or restore β cell mass a major therapeutic goal. While testing the hypothesis that increased endothelial cell signaling would increase β cell mass using a model of inducible vascular endothelial growth factor-A (VEGF-A) overexpression in β cells (βVEGF-A mouse), we found that increased VEGF-A leads to reduced, not increased, β cell mass. Surprisingly, withdrawal of the VEGF-A stimulus is followed by robust β cell proliferation, leading to islet regeneration, normalization of β cell mass, and reestablishment of the intra-islet capillary network. Using islet and bone marrow transplantation approaches we found that β cell proliferation is dependent on the local microenvironment of endothelial cells, β cells, and bone marrow-derived macrophages recruited to the islets upon VEGF-A induction. Depleting macrophages greatly reduced β cell proliferation, indicating that these macrophages are required for the β cell proliferative response in regenerating islets. Based on these data, in addition to transcriptome analysis of FACS-sorted islet β cell, and islet-derived endothelial cell and macrophage populations during VEGF-A induction and normalization, we propose a new paradigm for β cell regeneration where β cell self-renewal is mediated by coordinated interactions between macrophages recruited to the site of β cell injury, intra-islet endothelial cells, and β cells. In this model, (1) increased growth factors produced by β cells, endothelial cells, macrophages, (2) increased production of growth factor receptors and integrins on β cells, and (3) increased integrin activation by the extracellular matrix cause simultaneous, and potentially synergistic, activation of the PI3K/Akt and MAPK signaling pathways, leading to β cell proliferation.

Loss-of-function mutations causing haploinsufficiency of the MC4R are the most common monogenic cause of severe early-onset obesity in humans. Research has primarily focused on how central MC4R contributes to energy homeostasis with the eventual goal of developing anti-obesity therapeutics. MC4R expression has been found in vago-vagal circuitry, as well as in enteroendocrine cell populations in the gastrointestinal tract. Multi-choice feeding studies in MC4R deficient mice revealed a surprising reduction in preference for palatable foods high in either fats or carbohydrates. This loss of macronutrient preference was also coupled with a homeostatic drive for hyperphagia. These complex defects in feeding behaviors were suggestive of defects in the gut-brain axis, a cascade of neural and hormonal signals which bi-directionally control feeding behaviors. Gastrointestinal expression of MC4R in L cells was shown to be functionally significant as MC4R agonism stimulated peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) secretion in vivo. These findings highlighted a complementary role for peripheral MC4R in the gut-brain axis that has implications for energy and glucose homeostasis. Furthermore, pharmacological action of melanocortin compounds at peripheral sites must now be considered when examining effects of the receptor on whole-animal physiology.