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 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). 2. 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+. 3. 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. 4. 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. 5. 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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