UVM Theses and Dissertations
Format:
Online
Author:
Nausch, Lydia Waltraud Maria
Dept./Program:
Pharmacology
Year:
2008
Degree:
PhD
Abstract:
Cyclic 3',5'-guanosine monophosphate (cGMP) is a key signaling molecule involved in a myriad of physiological processes, including vascular smooth muscle (VSM) tone, water- and electrolyte homeostasis, platelet aggregation, airway smooth muscle tone, smooth muscle proliferation and bone formation. Increased occurrence of vascular disorders including erectile dysfunction, hypertension, stroke and coronary artery disease, have made it increasingly important to study the dynamic interplay between cGMP synthesis and hydrolysis in VSM cells.
This dissertation examines the spatial distribution of intracellular cGMP, [cGMP][subscript i], in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP][subscript i], we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP][subscript i] in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for realtime, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG.
We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP][subscript i] formation.
This dissertation examines the spatial distribution of intracellular cGMP, [cGMP][subscript i], in response to NO and atrial natriuretic peptide (ANP) in VSM cells. To investigate the spatial patterning of [cGMP][subscript i], we have developed a new generation of non-FRET (fluorescence resonance energy transfer) cGMP biosensors that are suitable to monitor [cGMP][subscript i] in response to physiological (low-nanomolar) NO and ANP concentrations and that qualify for realtime, confocal imaging techniques. We have termed these indicators FlincGs, for green fluorescent indicators of cGMP. For the development of FlincGs, we made use of the specific cGMP binding characteristics of PKG.
We utilized site-specific mutagenesis, kinetic cGMP binding, dissociation and kinase assays, as well as crystallography, in order to investigate PKG activation and cGMP binding dynamics in greater detail. Based on these studies, our novel, non-FRET cGMP biosensors were designed by attaching cGMP binding fragments of PKG to the N-terminus of circular permutated green fluorescent protein. We applied FlincGs in cultured VSM cells as well as in intact tissue to determine whether two spatially distinct populations of guanlylyl cyclase (cytosolic versus membrane bound) underlie the generation of spatiotemporally-specific patterns of [cGMP][subscript i] formation.