[Frontiers in Bioscience 10, 1290-1301 May 1, 2005]

MEMBRANE-PERMEABLE CYGNETS: RAPID CELLULAR INTERNALIZATION OF FLUORESCENT cGMP-INDICATORS

Akira Honda, Markus A. Moosmeier and Wolfgang R. Dostmann

Department of Pharmacology, University of Vermont, College of Medicine, Burlington, Vermont

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Material and Methods
3.1. MPP-cygnet construction
3.2. Expression and purification
3.3. Solubility assays
3.4. In vitro fluorescence resonance energy transfer (FRET)
3.5. Cultured human mesenteric smooth muscle cells (HuMSM)
3.6. Cultured rat aortic smooth muscle cells (RaASM)
3.7. Rat cerebral arteries
3.8. FRET imaging
3.9. Confocal imaging
3.10. Confocal 3D imaging
3.11. Materials
4. Results
4.1. Purification and in vitro fluorescence resonance energy transfer
4.2. Translocation and in vivo FRET in cultured smooth muscle cells
4.3. Functional MPP-Cygnet delivery in smooth muscle cells of intact cerebral arteries
5. Discussion
6. Acknowledgement
7. References

1. ABSTRACT

We have recently developed genetically encoded cGMP-indicators (cygnets) which have enabled us to study the spatial and temporal dynamics of intracellular cGMP in single cultured cells (1). However, primary mammalian cell types (dissociated cells or acute tissue samples) are often difficult to maintain undifferentiated in culture and the current established methods of introducing molecular reporters in single cells are laborious (micro-injection) and/or require cell culture techniques to accommodate the 1-2 day lag time of genetically mediated reporter expression. Here, we present an alternative, non-genetic method to rapidly introduce cGMP-indicators into cells and intact tissues using membrane permeable peptides (MPP). Five different 125 kDa MPP-cygnets were expressed and purified from insect SF9 cells. Three constructs showed high level cGMP-dependent FRET in vitro. One of the probes, Ant7-Cygnet, demonstrated emission ratio changes identical to the unmodified indicator. Ant7-Cygnet was rapidly (3 hours) and efficiently internalized in cultured smooth muscle cells and intact cerebral arteries. Furthermore, the internalized Ant7-Cygnet detected nitric oxide mediated elevations of intracellular cGMP in cultured smooth muscle cells and sensed increased levels of intracellular cGMP derived from C-type natriuretic peptide (CNP) induced guanylyl cyclase stimulation in intact arteries. These results demonstrate that MPP-cygnets provide a novel and potentially powerful technique to study intracellular cGMP in intact tissue.