Abstract
Whole-cell patch-clamp electrophysiological investigation of endothelial cells cultured from human saphenous vein (HSVECs) has identified a voltage-gated Na+ current with a mean peak magnitude of -595 +/- 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a concentration-dependent manner, with an IC50 value of 4.7 microM, suggesting that it was of the TTX-resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of known Na+ channel alpha-subunits was able to retard current inactivation when applied intracellularly. This antibody identified a 245-kDa protein from membrane lysates on Western blotting and positively immunolabeled both cultured HSVECs and intact venous endothelium. HSVECs were also shown by reverse transcription-polymerase chain reaction to contain transcripts of the hH1 sodium channel gene. The expression of Na+ channels by HSVECs was shown using electrophysiology and cell-based enzyme-linked immunosorbent assay to be dependent on the concentration and source of human serum. Together, these results suggest that TTX-resistant Na+ channels of the hH1 isoform are expressed in human saphenous vein endothelium and that the presence of these channels is controlled by a serum factor.
Highlights
Vascular endothelial cells form the primary interface between the blood and the underlying tissue
Endothelial cells are known to possess a broad spectrum of ion channels that open in response to a variety of stimuli, including membrane potential, receptor occupation, elevation of [Ca2ϩ]i, and mechanical deformation induced by flow [3]
MRNA isolated from human saphenous vein endothelial cells (HSVECs) was reverse-transcribed, and the resulting cDNA was amplified (RT-PCR) with specific primers targeted against the 3Јuntranslated region of the hH1 cDNA [16]
Summary
Human Saphenous Vein Endothelial Cell Isolation and Culture— HSVECs were obtained by enzymatic release from saphenous vein harvested during high ligation of varicose veins or bypass surgery. Electrophysiological Recording—Experiments were performed at room temperature (20 –22 °C) using the whole-cell configuration of the patch-clamp technique [13] on subconfluent HSVECs grown in 35-mm diameter Petri dishes These were placed on the stage of an inverted microscope (Diaphot 200, Nikon, Tokyo, Japan), visualized with phasecontrast optics, and continuously superfused at 2 ml/min with extracellular solution. A protocol similar to that described above was employed for cultured HSVECs, which were, fixed by incubation in 100% methanol at 4 °C for 2 min Controls for both intact vein sections and HSVECs were prepared by omitting either the primary or secondary antibodies. Membranes were incubated at room temperature for 30 min with a biotinylated secondary antibody (goat anti-rabbit, 1:1000) and a tertiary streptavidin-horseradish peroxidase conjugate (1:1000; Dako). All other chemicals were obtained from Sigma (Poole, UK)
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