UVM Theses and Dissertations
Format:
Print
Author:
Held, Kara F.
Dept./Program:
Cell and Molecular Biology Program
Year:
2010
Degree:
Ph. D.
Abstract:
The intracellular second messenger, cyclic guanosine-3',5'-monophosphate (cGMP), is a critical modulator of vascular smooth muscle (VSM) in the regulation of arterial vasodilation, essential for the maintenance of blood flow and pressure. cGMP is synthesized through activation of soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC) by nitric oxide (NO) and natriuretic peptides (NPs), respectively (Friebe et al., 2003; Kuhn, 2003), and degraded by phosphodiesterases, or PDEs (Conti et al., 2007). This thesis addresses the poorly understood dynamics of intracellular cGMP in isolated cells and the physiological consequences in arteries from the resistance vasculature. NO application has been shown to stimulate transient changes in cytosolic cGMP as well as vasomotor reactivity and, but whether these transient responses are due to the chemical properties of NO or to the regulation of VSM-specific intracellular signaling pathways is not known (Ignarro et al., 1987; Palmer et al., 1987; Nausch et a1., 2008). In addition, recent results from our laboratory suggested that atrial NP (ANP) induces sustained and exclusively membrane associated increase of cGMP (Nausch et al., 2008). It is thus hypothesized that NO and ANP give rise to distinct spatial and temporal patterns of the cGMP regulation in VSM cells.
We have employed several methods for determining cGMP regulation in VSM cells and tissue. The alteration of the kinetic profile of NO into pulsed or sustained (clamped) doses (Griffiths et al., 2003; Roy et al., 2006) were applied to intact, pre-constricted resistance arteries in a pressure myograph and isolated VSM cells transfected with our cGMP-biosensor, FlincG (Nausch et al., 2008), using epi-fluorescence and confocal microscopy. Pharmacological inhibitors as well as comprehensive mathematical modeling of the essential enzymatic components of cGMP turnover in VSM cells were also used to assess cGMP regulation and localization in response to both NO and ANP.
Here we show ANP stimulated cGMP to be spatially distinct from the NO-induced population and each pool can be temporally separated within individual cells. This membrane-localization of the ANP-induced cGMP was spatially regulated by PDE5 and phospho-PDE5. The alteration of the NO kinetic profile into pulsed and clamped NO led to the conclusion that NO directly dictates both vasodilation and cGMP synthesis, and that the kinetic profiles of each are linked, with cGMP acting as the direct intracellular mediator in the propagation of the NO-vasodilation signaling pathway. Finally, the regulation of this response was shown to be mediated through sGC synthesis and PDE5, phosphorylated PDE5, and PDEI hydrolysis of cGMP. This occurred in the absence of a desensitization mechanism of any of the enzymes involved and with a very high potency for NO in both isolated VSM cells and intact arteries.
Together, these results demonstrate two separate mechanisms by which NO and ANP regulate intracellular cGMP and consequently vasodilation, which may provide distinct regulation in the tight control of blood pressure.
We have employed several methods for determining cGMP regulation in VSM cells and tissue. The alteration of the kinetic profile of NO into pulsed or sustained (clamped) doses (Griffiths et al., 2003; Roy et al., 2006) were applied to intact, pre-constricted resistance arteries in a pressure myograph and isolated VSM cells transfected with our cGMP-biosensor, FlincG (Nausch et al., 2008), using epi-fluorescence and confocal microscopy. Pharmacological inhibitors as well as comprehensive mathematical modeling of the essential enzymatic components of cGMP turnover in VSM cells were also used to assess cGMP regulation and localization in response to both NO and ANP.
Here we show ANP stimulated cGMP to be spatially distinct from the NO-induced population and each pool can be temporally separated within individual cells. This membrane-localization of the ANP-induced cGMP was spatially regulated by PDE5 and phospho-PDE5. The alteration of the NO kinetic profile into pulsed and clamped NO led to the conclusion that NO directly dictates both vasodilation and cGMP synthesis, and that the kinetic profiles of each are linked, with cGMP acting as the direct intracellular mediator in the propagation of the NO-vasodilation signaling pathway. Finally, the regulation of this response was shown to be mediated through sGC synthesis and PDE5, phosphorylated PDE5, and PDEI hydrolysis of cGMP. This occurred in the absence of a desensitization mechanism of any of the enzymes involved and with a very high potency for NO in both isolated VSM cells and intact arteries.
Together, these results demonstrate two separate mechanisms by which NO and ANP regulate intracellular cGMP and consequently vasodilation, which may provide distinct regulation in the tight control of blood pressure.