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Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein
Author(s) -
Carter T. D.,
Zupancic G.,
Smith S. M.,
WheelerJones C.,
Ogden D.
Publication year - 1998
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1998.845ba.x
Subject(s) - umbilical vein , thrombin , chemistry , biophysics , intracellular , membrane potential , secretion , perfusion , endocrinology , medicine , biology , biochemistry , in vitro , platelet
1 Vesicular secretion from single human umbilical vein endothelial cells (HUVECs) was monitored by changes in membrane capacitance ( C m ). Secretion was evoked by dialysis with strongly buffered intracellular free Ca 2+ concentrations ([Ca 2+ ] i ), flash photolysis of Ca 2+ ‐loaded DM‐nitrophen or caged Ins P 3 , or by thrombin. [Ca 2+ ] i was monitored spectrofluorimetrically with furaptra. The results show that a large, slowly rising component of vesicular secretion requires prolonged exposure to high [Ca 2+ ] i . 2 Cmincreased during intracellular perfusion with [Ca 2+ ] buffered in the range 1.0–20 μM. Changes in C m comprised an initial slowly rising small component of 0.1–0.5 pF followed by a faster rising larger component of up to ∼7 pF, seen when [Ca 2+ ] i > 2 μM and which was maximal at 10–20 μM Ca 2+ . 3 Thrombin evoked rapid initial elevations of [Ca 2+ ] i to a peak of 7.1 ± 1.5 μM (mean ± s.e.m., n = 5 ) that declined within ∼20–30 s with thrombin present either to resting levels or to a maintained elevated level of 2.0 ± 0.7 μM (mean ± s.e.m., range 1.0–3.6 μM, n = 3 ). Transient [Ca 2+ ] i rises were associated with small, slowly rising increases in C m of 0.1–0.2 pF, that recovered to pre‐application levels over 2–3 min. Maintained elevations of [Ca 2+ ] i caused larger, faster‐rising sustained increases in C m to 1.14 ± 0.12 pF (mean ± s.e.m., n = 3 ). Separate specific enzyme‐linked immunosorbent assay (ELISA) showed that 1.0 U ml −1 thrombin produced secretion of von Willebrand factor in HUVEC cultures. 4 Short‐lived [Ca 2+ ] i elevations with a peak of 3–25 μM and a duration of approximately 20 s generated by flash photolysis of caged Ins P 3 or DM‐nitrophen produced either no net change in C m , or small slow increases of ∼0.1–0.6 pF at up to 5 fF s −1 that recovered to pre‐flash levels over 2–3 min. 5 Maintained elevations of [Ca 2+ ] i in the range 1–28 μM produced by flash photolysis of DM‐nitrophen caused large increases in C m , up to ∼4 pF, corresponding to ∼25–30 % of the initial cell C m . The maximum rate of change of C m was up to 50 fF s −1 at steady [Ca 2+ ] up to 20 μM; C m recovered towards pre‐flash levels only when [Ca 2+ ] had declined.