Culture conditions influence uptake and intracellular localization of the membrane permeable cGMP-dependent protein kinase inhibitor DT-2

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.

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

“…6D). The former effect is similar to the previously reported characteristics of FITC-labelled DT-2 [48] that also relies upon highly positively charged CPP for transport into the cells.…”

Insulin degrading enzyme is localized predominantly at the cell surface of polarized and unpolarized human cerebrovascular endothelial cell cultures

“…Cells were grown to confluence. On the day of treatment, media was replaced with fresh media containing either no inhibitor or one or more of the following inhibitors: insulin (100 μM), a competitive inhibitor of IDE (Edland, 2004); phenylmethylsulphonyl fluoride (PMSF, 0.5 mM), an inhibitor of serine proteases (Barr and Warner, 2003); 1,10 phenanthroline (2 mM), a non‐competitive inhibitor of IDE (Harada et al, 1993); ethylenediamine tetraacetic acid (EDTA, 5mM), a chelator of divalent ions (Kim et al, 2005); 5,5′‐dithiobis (2‐nitrobenzoic acid) (DTNB, 2 mM), a membrane impermeable modifier of thiol groups (Zoccarato et al, 1999); thiorphan (100 nM), a specific neprilysin inhibitor (Roques et al, 1980); phosphoramidon (1 μM), a specific inhibitor of endothelin converting enzyme (Ikegawa et al, 1990); phenylarsine oxide (PAO, 20 μM), an endocytosis inhibitor (Foley et al, 2005); sucrose (0.5 mM), an endocytosis inhibitor (Nieland et al, 2005); or filipin complex IV (Fil, 10 μg/ml), an inhibitor of caveolae mediated endocytosis (Schnitzer et al, 1994). At the time of treatment cells were brought to 10–20 pM with [ 125 I] Aβ 1–40 and samples were taken at the times specified.…”

cGMP-Dependent Protein Kinase Inhibitors in Health and Disease

“…DT-2 and DT-3 are used for in vitro studies; the in vivo use is controversial [31,32]. Uptake of DT-2 into cells occurs via endocytic or non-endocytic mechanisms depending on their cellular phenotype [33]. (D)-DT-2 is the D-amino acid analogue of DT-2 and can be used as a potent PKG-Iα inhibitor [34].…”