Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles.

Abstract

TO obtain small membrane markers easily accessible to the charged groups of the cell surface, we prepared, from hemeundecapeptide (HUP), three derivatives that maintain the peroxidatic activity: (a) the anionized hemeundecapeptide, Mr 1,963, estimated diameter 1.68 nm, pl 3.5, for the detection of basic groups; and both (b) a cationized hemeundecapeptide containing predominantly tertiary amino groups, Mr 2,215, estimated diameter 1.75 nm, pl 9.0, and (c) a cationized hemeundecapeptide containing only primary amino groups, Mr 2,271, estimated diameter 1.75 nm, pl 10.6, for labeling acidic residues. The markers were perfused in situ in mice to label the luminal surface of fenestrated endothelium of pancreatic capillaries. Specimens were processed through the cytochemical reaction for peroxidatic activity and examined by electron microscopy. The anionized HUP and HUP (pl 4.85) marked the plasmalemma proper, the coated pits, and the membrane and diaphragms of plasmalemmal vesicles and transendothelial channels. The cationized HUP containing predominantly tertiary amino groups (pl 9.0) decorated all cell surface components with the exception of plasmalemmal vesicles and channels; the latter were, however, labeled by the cationized HUP containing only primary groups (pl 10.6), which suggests that these structures contain on their luminal surface very weak acidic residues of high pKa values. The fact that the membrane of plasmalemmal vesicles can discriminate against permeant cationic macromolecules only up to a pl of ~9.0 indicates that in the electrostatic restriction there is a charge limit. In the case of fenestrated capillary endothelium, the upper charge limit seems to be a pl of ~9.0. In these vessels, the charge discrimination is effective for molecules as small as 2 nm.Several markers have been used to gain information about the cell surface charge. They have the limitation of either requiring prefixation (e.g., by colloidal iron), or concomitant fixation (e.g., by ruthenium red), or of being large enough (e.g., cationized ferritin) to raise the problem of their accessibility to the cell surface moieties. In order to circumvent these limitations, we prepared from hemeundecapeptide (HUP) ~ (microperoxidase M-11) two groups of derivatives that maintain their peroxidatic activity: anionized hemeundecapeptide J Abbreviations used in this paper: aHUP, anionized hemeundecapeptide; cHUP, cationized hemeundecapeptide; cHUPp, cationized hemeundecapeptide (pl 10.6) containing primary amino groups only; cHUPt, cationized hemeundecapeptide (pl 9.0) containing predominantly tertiary amino groups; HUP, hemeundecapeptide (microperoxidase-11 ).(aHUP) for the detection of basic residues, and two cationized hemeundecapeptides (cHUP) for the detection of acidic groups. These compounds have the following advantages: (a) they can be applied to fresh tissues in vivo, in situ, and in vitro; (b) owing to their small size (about 2 nm in diameter), their access to the groups of cell surface is assumed to be easier than that of...

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“…This implies that, although both cell types are negatively charged, the endothelial cells have a relatively higher negative charge than the prostate cells, while the latter have a greater capacity for endocytosis. This higher binding of the cationic conjugates to endothelial cells is in agreement with literature reports that endothelial cells express anionic regions on the glycocalyx (Haldenby et al, 1994), fenestral membranes (Ghinea and Simionescu, 1985) and vesicular openings (Baldwin and Chien, 1984), all of which have a pronounced tendency to bind polycations. These anionic regions are thought to play a role in modifying permeability and transcytosis of macromolecules (Simionescu and Simionescu, 1986).…”

“…The mechanism operating may have implications on the charge selectivity of transendothelial transport. It has been shown that membrane of the vesicular-vacuolar neck has no strongly anionic residues (Simionescu et al, 1981), thus allowing negatively charged macromolecules to penetrate into the vesicle, while cationic macromolecules remain bound to other regions of the luminal endothelial surface (Ghinea and Simionescu, 1985). This would explain the faster extravasation of the anionic conjugates compared to the cationic ones.…”

Cellular uptake and toxicity of gold nanoparticles in prostate cancer cells: a comparative study of rods and spheres

“…It is known that plasma membranes possess large negatively charged domains. Comparatively, cationic sites are fewer but are present as evidenced by adsorption of anionic ferritin (Farquhar et al, 1978; Mutsaers et al, 1988) and hemeundecapeptide (Ghinea and Simionescu, 1983). As for PEGylated GNPs, the steric hindrance of PEG strands prevents the particles from binding to the cells.…”

Lipid-Based Drug Delivery Systems in Cancer Therapy: What Is Available and What Is Yet to Come

“…Interestingly, it is believed that there are some vascular domains lacking negatively charged surface proteoglycans and glycoprotein potentially involved in endocytosis and transcytosis (Vincent et al, 1988;Campbell et al, 2009). These areas of low negative charge can be observed on the luminal surface of capillary endothelium, particularly on the surface of plasmalemmal vesicles, which facilitate the internalization of circulating molecules, including liposomes (Simionescu et al, 1981;Ghinea and Simionescu, 1985;Simionescu et al, 1985;Campbell et al, 2009). …”