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Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein

T. D. Carter, G. Zupancic, S. M. Smith, C. Wheeler-Jones * and D. Ogden

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 Ca²⁺ concentrations ([Ca²⁺]_i), flash photolysis of Ca²⁺-loaded DM-nitrophen or caged InsP₃, or by thrombin. [Ca²⁺]_i was monitored spectrofluorimetrically with furaptra. The results show that a large, slowly rising component of vesicular secretion requires prolonged exposure to high [Ca²⁺]_i.

C_m increased during intracellular perfusion with [Ca²⁺] 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 [Ca²⁺]_i > 2 µM and which was maximal at 10-20 µM Ca²⁺.

Thrombin evoked rapid initial elevations of [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_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.

Short-lived [Ca²⁺]_i elevations with a peak of 3-25 µM and a duration of approximately 20 s generated by flash photolysis of caged InsP₃ 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.

Maintained elevations of [Ca²⁺]_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 [Ca²⁺] up to 20 µM; C_m recovered towards pre-flash levels only when [Ca²⁺] had declined.

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