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Transgenic mice for cGMP imaging.

ABSTRACT: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown.To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues.Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22? promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken ?-actin/?-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ? 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ? 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation.These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.

SUBMITTER: Thunemann M

PROVIDER: S-EPMC3896241 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Transgenic mice for cGMP imaging.

Thunemann Martin M Wen Lai L Hillenbrand Matthias M Vachaviolos Angelos A Feil Susanne S Ott Thomas T Han Xiaoxing X Fukumura Dai D Jain Rakesh K RK Russwurm Michael M de Wit Cor C Feil Robert R

Circulation research 20130625 4

<h4>Rationale</h4>Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown.<h4>Objective</h4>To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues.<h4>Methods and results</h4>Mouse lines with smooth muscle-specific or ubiquitous expression of cGi5 ...[more]

PMID: 23801067

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Project description:Transgenic (Tg) mice overexpressing human amyloid precursor protein (APP) mutants reproduce features of early Alzheimer's disease (AD) including memory deficit, presence of ?-amyloid (A?) oligomers, and age-associated formation of amyloid deposits. In this study we used hippocampal microdialysis to characterize the signaling of N-methyl-d-aspartic acid receptors (NMDA-Rs) in awake and behaving AD Tg mice. The NMDA-R signaling is central to hippocampal synaptic plasticity underlying memory formation and several lines of evidence implicate the role of A? oligomers in effecting NMDA-R dysfunction. CA1 NMDA-Rs were stimulated by NMDA infused through reverse microdialysis while changes in the cyclic guanosine monophosphate (cGMP) concentration in the brain interstitial fluid (ISF) were used to determine NMDA-Rs responsiveness. While 4 months old wild type C57BL/6 mice mounted robust cGMP response to the NMDA challenge, the same stimulus failed to significantly change the cGMP level in 4 and 15 months old APP(SW) and 4 months old APP(SW)/PS1(L166P) Tg mice, which were all on C57BL/6 background. Lack of response to NMDA in AD Tg mice occurred in the absence of changes in expression levels of several synaptic proteins including synaptophysin, NR1 NMDA-R subunit and postsynaptic density protein 95, which indicates lack of profound synaptic degeneration. A? oligomers were detected in all three AD Tg mice groups and their concentration in the hippocampus ranged from 40.5±3.6ng/g in 4 months old APP(SW) mice to 60.8±15.9ng/g in 4 months old APP(SW)/PS1(L166P) mice. Four months old APP(SW) mice had no A? amyloid plaques, while the other two AD Tg mice groups showed evidence of incipient A? amyloid plaque formation. Our studies describes a novel approach useful to study the function of NMDA-Rs in awake and behaving AD Tg mice and demonstrate impairment of NMDA-R response in the presence of endogenously formed A? oligomers but predating onset of A? amyloidosis.

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Transgenic mice for cGMP imaging.

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Transgenic mice for cGMP imaging.

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