Browsing by Subject "ANP"

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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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    Localization of natriuretic peptide receptor A in the cochlea
    (2014-08) Prince, Sara Catherine
    Natriuretic peptides have been shown to alter fluid balance and ion levels in many tissues throughout the body including the renal and cardiovascular systems. When atrial natriuretic peptide (ANP) binds to its receptor (NPR-A), a dimerization event causes the activation of cGMP, initiating an internal second messenger cascade. Infusion of ANP causes improved hearing thresholds, theoretically stimulated by the secondary messengers initiated by the ANP pathway. The specific location of a natriuretic peptide receptor, NPR-A, in the inner ear including the exact mechanisms of the pathway stimulated by ANP binding, is still largely unknown.This study was designed to test the hypothesis that (1) NPR-A is in the cells of the cochlea critical for the regulation of endolymph and ion balance and (2) NPR-A is co-localized with the effector protein NKCC1, known for its role in the movement of potassium and other ions in the inner ear. Immunohistochemistry was performed on cryosections of cochlea collected from mice of normal hearing. Quantitative reverse-transcriptase polymerase chain reaction was also performed on kidney and cochlea homogenates, as well as laser capture microdissected cells to determine the mRNA levels of NPR-A in specific tissues.NPR-A was localized to six cell types in the cochlea including marginal and basal cells of the stria vascularis, root cells of the spiral ligament, interdental cells and limbal fibrocytes of the spiral limbus, and spiral ganglion neurons. NPR-A expression was found in gateways critical for K+ influx into a cellular network or efflux from a cell into the endolymph, a high K+ extracellular fluid unique to the cochlea. Furthermore, NPR-A was also found in type I neurons that are critical for neurotransmission, implicating a regulatory role for ANP directly in the sensory pathway.NPR-A was intermittently co-localized with NKCC1 in the marginal and interdental cells, which are both exit points for K+ from the recycling system into the endolymph. NKCC1 may be an effector in these cells responsible for K+ efflux into the endolymph, but is likely only one of several, as co-localization was sporadic. In other cells where NPR-A was expressed, NKCC1 was clearly not the effector activated in the ANP pathway as no co-localization was observed. These studies provide novel information about the location of NPR-A in the cochlea and its relationship to other proteins, indicating a regulatory role for natriuretic peptides in both neurotransmission and K+ recycling in the mouse inner ear. Globally, determining the location of NPR-A in the cochlea identifies cellular targets for future molecular study of ANP action and the mechanisms for its regulation of hearing thresholds.