[Frontiers in Bioscience 10, 1302-1312 May 1, 2005]

CULTURE CONDITIONS INFLUENCE UPTAKE AND INTRACELLULAR LOCALIZATION OF THE MEMBRANE PERMEABLE cGMP-DEPENDENT PROTEIN KINASE INHIBITOR DT-2

Kevin F. Foley 1,2, Sergio De Frutos 1, Kristina E. Laskovski 1, Werner Tegge 3, and Wolfgang R. Dostmann 1

1 Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT, USA, 3 German Research Centre for Biotechnology (GBF), AG Molecular Recognition, Mascheroder Weg 1, Braunschweig, Germany, 2 Present Address: Department of Biomedical Technologies, College of Nursing and Health Sciences, University of Vermont, Burlington, VT, USA.

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods
3.1. Peptide Synthesis
3.2. Animal and Tissue Preparation
3.3. Cell culture of dissociated vascular smooth muscle cells
3.4. Smooth muscle cell culture in fibrillar collagen
3.5. Cell viability experiments
3.6. Uptake and imaging of fDT-2
3.7. Actin Staining and Endocytosis Inhibition
4. Results
4.1. DT-2 cytotoxicity
4.2. Effects of fixation on DT-2 uptake
4.3. Uptake and cellular localization of fDT-2 in two-dimensional cultured VSMCs
4.4. Effects of endocytosis inhibitors on fDT-2 uptake
4.5. Peptide distribution in VSMCs from intact arteries
4.6. VSMCs reconstituted in three-dimensional matrices and fDT-2 uptake
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

The membrane-permeable peptide DT-2 which utilizes the HIV-Tat membrane translocation sequence is known to inhibit cGMP-dependent protein kinase (PKG) effectively in vitro and in various cell lines and tissue preparations. However, the uptake characteristics of DT-2 have not been studied in detail. We investigated the intracellular uptake and localization of fluorescein-labeled DT-2 (fDT-2) in cultured C6-glial cells and vascular smooth muscle cells (VSMCs) as well as VSMCs in intact arteries. To avoid fixation-induced fluorescence, live unfixed cells and arteries were incubated with fDT-2 and examined using conventional and confocal fluorescence microscopy. In non-differentiated cultured VSMCs, uptake appeared vesicular with nuclear exclusion, consistent with an endocytotic internalization mechanism. Inhibition of endocytosis by phenylarsine oxide (PAO), low temperature or disruption of actin polymerization by cytochalasin-D or lantrunculin-A showed a residual non-endocytotic fDT-2 translocation with diffuse cytosolic and nuclear uptake. Similarly, differentiated contractile VSMCs within the medial layer of intact cerebral arteries also showed a distinctively different, more diffuse cytosolic uptake and time dependent nuclear localization. To verify the morphology dependency of fDT-2 uptake, VSMCs were reconstituted in fibrillar collagen matrices. The cells adopted a differentiated morphology and fDT-2 translocation was similar to cells in intact arteries. These results demonstrate that VSMCs cells utilize distinct cellular uptake mechanisms depending on their phenotype.