Browsing by Subject "ATP"

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    Activation and Inhibition Mechanisms of Membrane Guanylyl Cyclases
    (2013-04) Robinson, Jerid
    Guanylyl cyclase (GC)-A and GC-B are homologous enzymes that catalyze the formation of cyclic guanosine monophosphate and pyrophosphate from GTP. GC-A is activated by atrial natriuretic peptide and B-type natriuretic peptide and regulates the cardiovascular system. GC-B is activated by C-type natriuretic peptide and regulates the skeletal and female reproductive systems. Activation of GC-A and GC-B was hypothesized to occur through two steps, binding of natriuretic peptide and subsequent binding of ATP to the kinase homology domain. However, our group reported that ATP binding does not increase maximal velocity but reduces the Michaelis constant. My work revealed an allosteric ATP binding site in the catalytic domain. Mutation and structure/function studies indicated that the allosteric and catalytic sites are different and that GC-A and GC-B are asymmetric homodimers, not symmetric homodimers as had been previously suggested. Interestingly, a constitutively active mutant of GC-B mimicked an ATP bound state. ATP inhibits soluble guanylyl cyclases (sGC), and I demonstrated that physiological concentrations of ATP inhibit GC-A and GC-B. I went on to determine that the mechanism of inhibition was through binding the pyrophosphate-product site. Together, these data revealed how low and high concentrations of ATP activate and inhibit GC-A and GC-B, respectively.
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    Mechanisms and functional consequences of glial signaling in the retina.
    (2009-07) Kurth-Nelson, Zebulun Lloyd
    Twenty years ago, glia were viewed as passive support cells for neurons. Since then, experiments have shown that glial cells have their own form of excitability with precise intracellular spatiotemporal dynamics, intercellular communication among themselves, a bidirectional dialog with neurons and synapses, and a key role in mediating blood flow changes in response to neuronal activity. Most of these experiments have been conducted in brain regions such as hippocampus, cortex, hypothalamus, and cerebellum. However, as work from our laboratory has shown, the mammalian retina is also an excellent preparation to study the active functions of glial cells. Here, we describe two forms of active glial signaling in the retina. First, we tested the hypothesis that glial cells modulate synaptic activity in the retina. We measured synaptic strength by evoking excitatory postsynaptic currents (EPSCs) in ganglion cells with either light or an electrical stimulus. We then excited glial cells through several methods, including agonist ejection, photolysis of caged Ca2+, and depolarization. The amplitude of the synaptic currents was altered by some, but not all, of these glial stimuli, leaving us unable to draw a definitive conclusion as to whether glial excitation alone is sufficient to modulate synaptic transmission in the retina. Second, we characterized spontaneous intercellular glial Ca2+ waves in the retina. Glial cell excitability takes the form of transient intracellular Ca2+ elevations. One of the first recognized active properties of glia was their ability to propagate these Ca2+ elevations from cell to cell in a wave-like pattern. In most previous experiments, glial Ca2+ waves were initiated by an experimenter-driven stimulus, raising doubts about whether these waves occurred naturally in the organism. We demonstrate here that these waves occur spontaneously both in intact tissue and in vivo, and that the rate of spontaneous wave generation increases as animals age. These spontaneous waves propagate by glial release of ATP and activation of ATP receptors on neighboring cells. Finally, spontaneous waves cause changes in blood vessel diameter. This is the first demonstration of a functional effect of spontaneous intercellular glial signaling. These results suggest a functional role for glial cell signaling in the retina and raise the possibility that glial signaling may actively participate in the aging of the nervous system.
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    Nanomaterial solutions for the protection of insulin producing beta cells
    (2013-11) Atchison, Nicole Ann.
    Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) toβ cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat β cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.