TY - JOUR
T1 - Binding and internalization of heparin by vascular smooth muscle cells
AU - Castellot, John J.
AU - Wong, Kent
AU - Herman, Brian
AU - Hoover, Richard L.
AU - Albertini, David F.
AU - Wright, Thomas C.
AU - Caleb, Benjamin L.
AU - Karnovsky, Morris J.
PY - 1985/7
Y1 - 1985/7
N2 - Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H‐heparin to these cells occurs via specific, high‐affinity binding sites (Kd = 10−9 M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1–2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4°C, a diffuse surface staining pattern was observed. After warming the cells to 37°C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37°C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high‐affinity binding sites and the initial rapid uptake of 3H‐heparin, suggests that heparin enters smooth muscle cells by both receptor‐mediated and other endocytic pathways.
AB - Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H‐heparin to these cells occurs via specific, high‐affinity binding sites (Kd = 10−9 M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1–2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4°C, a diffuse surface staining pattern was observed. After warming the cells to 37°C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37°C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high‐affinity binding sites and the initial rapid uptake of 3H‐heparin, suggests that heparin enters smooth muscle cells by both receptor‐mediated and other endocytic pathways.
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U2 - 10.1002/jcp.1041240104
DO - 10.1002/jcp.1041240104
M3 - Article
C2 - 3930515
AN - SCOPUS:0021889240
SN - 0021-9541
VL - 124
SP - 13
EP - 20
JO - Journal of cellular physiology
JF - Journal of cellular physiology
IS - 1
ER -