UVM Theses and Dissertations
Format:
Print
Author:
Cawley, Sharon M.
Dept./Program:
Cell and Molecular Biology Program
Year:
2007
Degree:
Ph. D.
Abstract:
Cyclic-3',5'-guanosine monophosphate (cGMP) mediates the intracellular signaling cascade responsible for nitric oxide (NO) initiated relaxation of vascular smooth muscle (VSM). However, the temporal dynamics of this molecule, including the regulation of cGMP synthesis and hydrolysis, are largely unknown. Our laboratory has previously developed genetically encoded, fluorescence resonance energy transfer (FRET)-based biosensors known as Cygnets, for the detection of intracellular cGMP in living cells. The work in this thesis represents the utilization of Cygnet-2.1 in primary vascular smooth muscle cells in order to characterize the dynamics of NO-mediated cGMP responses. Physiological concentrations of NO induced rapid and transient cGMP responses ("peaks"). As demonstrated by their respective inhibition using 1H -[1,2,4]oxadiazolo[4,3-alquinoxalin-1 - one (ODQ) and Sildenafil, cGMP transients are governed by the orchestrated activationlinactivation of the NO-sensitive guanylyl cyclase (GC) and phosphodiesterase type V (PDES).
These responses occurred in the presence of elevated cGMP (515% emission ratios), and thus activated PKG and phosphorylated PDE5, suggesting a prominent role for GC in the maintenance and termination of cGMP peaks. Furthermore, cGMP transients could be elicited repeatedly without apparent desensitization of GC or by suppression of cGMP via long-term PDES activity. This work demonstrates the continuous sensitivity of the NOIcGMP signaling system, inherent to the phasic nature of smooth muscle physiology.
PDES phosphorylation by PKG as well as dephosphorylation by the SerJThr phosphatase PPl has been suggested to regulate the relaxation/contraction cycle of smooth muscle. By utilizing the FRET-based cGMP indicator Cygnet 2.1 in primary isolated VSM cells, we examined alterations in cGMP duration and amplitude following inhibition of PKG with DT-2 or SerIThr phosphatases using okadaic acid. Our results show that PKG as well as phosphatases PPIlPP2A impact the cGMP response in VSM. However, the phosphorylation status of PDES was unaltered by PKG or phosphatase inhibition, therefore the presence of phospho-PDE5 does not control the resensitization of the NO response in VSM cells.
In vasoconstrictive diseases such as atherosclerosis, the loss of NOIcGMP-mediated smooth muscle relaxation has been attributed to downregulation of NO-sensitive GC activity. It has been proposed that GC is redox-regulated, specifically by the oxidation status of the heme group, but it is unclear whether increased oxidants contribute to the loss of vasodilation by directly oxidizing GC in vivo. In this study, we have characterized the cGMP response to HMR 1766, a novel activator of GC, in primary vascular smooth muscle cells. This compound has been previously suggested to specifically activate the oxidized, ferric form of GC. We have evaluated the cGMP response to NO and HMR under both oxidative and antioxidant conditions. The NOsensitive GC can be oxidized by ODQ, and by reactive oxygen species (ROS) in vivo.
By treating VSM cells with the antioxidant combination Ebselen and Vitamin E, we have shown an enhanced response to NO and an attenuated response to HMR 1766. In contrast, the cGMP response to HMR 1766 was enhanced by ODQ, as well as by pro-oxidant signaling via Angiotensin 11. Accordingly, under oxidative conditions, NO responsiveness was attenuated, confirming that the relative abundance of the ferrous and ferric forms of GC was modulated by the redox status of the cell. This work validates that the redox isoform of GC is specifically activated by HMR 1766. This also provides evidence that GC is a redox-regulated enzyme and that its oxidation may contribute to the loss of smooth muscle relaxation associated with inflammatory vascular diseases. The specific activation of the oxidized GC demonstrates the potential of this compound as a treatment for atherosclerotic and other vasoconstrictive diseases.
These responses occurred in the presence of elevated cGMP (515% emission ratios), and thus activated PKG and phosphorylated PDE5, suggesting a prominent role for GC in the maintenance and termination of cGMP peaks. Furthermore, cGMP transients could be elicited repeatedly without apparent desensitization of GC or by suppression of cGMP via long-term PDES activity. This work demonstrates the continuous sensitivity of the NOIcGMP signaling system, inherent to the phasic nature of smooth muscle physiology.
PDES phosphorylation by PKG as well as dephosphorylation by the SerJThr phosphatase PPl has been suggested to regulate the relaxation/contraction cycle of smooth muscle. By utilizing the FRET-based cGMP indicator Cygnet 2.1 in primary isolated VSM cells, we examined alterations in cGMP duration and amplitude following inhibition of PKG with DT-2 or SerIThr phosphatases using okadaic acid. Our results show that PKG as well as phosphatases PPIlPP2A impact the cGMP response in VSM. However, the phosphorylation status of PDES was unaltered by PKG or phosphatase inhibition, therefore the presence of phospho-PDE5 does not control the resensitization of the NO response in VSM cells.
In vasoconstrictive diseases such as atherosclerosis, the loss of NOIcGMP-mediated smooth muscle relaxation has been attributed to downregulation of NO-sensitive GC activity. It has been proposed that GC is redox-regulated, specifically by the oxidation status of the heme group, but it is unclear whether increased oxidants contribute to the loss of vasodilation by directly oxidizing GC in vivo. In this study, we have characterized the cGMP response to HMR 1766, a novel activator of GC, in primary vascular smooth muscle cells. This compound has been previously suggested to specifically activate the oxidized, ferric form of GC. We have evaluated the cGMP response to NO and HMR under both oxidative and antioxidant conditions. The NOsensitive GC can be oxidized by ODQ, and by reactive oxygen species (ROS) in vivo.
By treating VSM cells with the antioxidant combination Ebselen and Vitamin E, we have shown an enhanced response to NO and an attenuated response to HMR 1766. In contrast, the cGMP response to HMR 1766 was enhanced by ODQ, as well as by pro-oxidant signaling via Angiotensin 11. Accordingly, under oxidative conditions, NO responsiveness was attenuated, confirming that the relative abundance of the ferrous and ferric forms of GC was modulated by the redox status of the cell. This work validates that the redox isoform of GC is specifically activated by HMR 1766. This also provides evidence that GC is a redox-regulated enzyme and that its oxidation may contribute to the loss of smooth muscle relaxation associated with inflammatory vascular diseases. The specific activation of the oxidized GC demonstrates the potential of this compound as a treatment for atherosclerotic and other vasoconstrictive diseases.