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

1. Vesicular secretion from single human umbilical vein endothelial cells ( HUVECs) was monitored by changes in membrane capacitance (C-m). Secretion w as evoked by dialysis with strongly buffered intracellular free Ca2+ concen trations ([Ca2+](i)), flash photolysis of Ca2+-loaded DM-nitrophen or caged InsP(3), or by thrombin. [Ca2+](i) was monitored spectrofluorimetrically w ith furaptra. The results show that a large, slowly rising component of ves icular secretion requires prolonged exposure to high [Ca2+](i). 2. C-m increased during intracellular perfusion with [Ca2+] buffered in the range 1.0-20 mu M. 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 similar to 7 pF, seen when [Ca2+](i) > 2 mu M and which was maximal at 10-20 mu M Ca2+. 3. Thrombin evoked rapid initial elevations of [Ca2+](i) to a peak of 7.1 /- 1.5 mu M (mean +/- S.E.M., n = 5) that declined within similar to 20-30 s with thrombin present either to resting levels or to a maintained elevate d level of 2.0 +/- 0.7 mu M (mean +/- S.E.M., range 1.0-3.6 mu M, n = 3). T ransient [Ca2+](i) rises were associated with small, slowly rising increase s in C-m of 0.1-0.2 pF, that recovered to pre-application levels over 2-3 m in. Maintained elevations of [Ca2+](i) caused larger, faster-rising sustain ed 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 [Ca2+](i) elevations with a peak of 3-25 mu M and a duration of approximately 20 s generated by flash photolysis of caged InsP(3) or DM -nitrophen produced either no net change in C-m, or small slow increases of similar to 0.1-0.6 pF at up to 5 fF s(-1) that recovered to pre-flash leve ls over 2-3 min. 5. Maintained elevations of [Ca2+](i) in the range 1-28 mu M produced by fl ash photolysis of DM-nitrophen caused large increases in C-m, up to similar to 4 pF, corresponding to similar to 25-30% of the initial cell C-m. The m aximum rate of change of C-m was up to 50 fF s(-1) at steady [Ca2+] up to 2 0 mu M; C-m recovered towards pre-flash levels only when [Ca2+] had decline d.