title: Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein.
abstract: 1. Vesicular secretion from single human umbilical vein endothelial cells (HUVECs) was monitored by changes in membrane capacitance (Cm). 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. Cm increased during intracellular perfusion with [Ca2+] buffered in the range 1.0-20 microM. Changes in Cm comprised an initial slowly rising small component of 0.1-0.5 pF followed by a faster rising larger component of up to approximately 7 pF, seen when [Ca2+]i > 2 microM and which was maximal at 10-20 microM Ca2+. 3. Thrombin evoked rapid initial elevations of [Ca2+]i to a peak of 7.1 +/- 1.5 microM (mean +/- s.e. m., n = 5) that declined within approximately 20-30 s with thrombin present either to resting levels or to a maintained elevated level of 2.0 +/- 0.7 microM (mean +/- s.e.m., range 1.0-3.6 microM, n = 3). Transient [Ca2+]i rises were associated with small, slowly rising increases in Cm 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 Cm 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 microM and a duration of approximately 20 s generated by flash photolysis of caged InsP3 or DM-nitrophen produced either no net change in Cm, or small slow increases of approximately 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 microM produced by flash photolysis of DM-nitrophen caused large increases in Cm, up to approximately 4 pF, corresponding to approximately 25-30 % of the initial cell Cm. The maximum rate of change of Cm was up to 50 fF s-1 at steady [Ca2+] up to 20 microM; Cm recovered towards pre-flash levels only when [Ca2+] had declined.
authors.affiliation: National Institute for Medical Research, Mill Hill, London NW7 1AA,, UK.tcarter@nimr.mrc.ac.uk
authors.forename: T D
authors.initials: TD
authors.lastname: Carter
authors.forename: G
authors.initials: G
authors.lastname: Zupancic
authors.forename: S M
authors.initials: SM
authors.lastname: Smith
authors.forename: C
authors.initials: C
authors.lastname: Wheeler-Jones
authors.forename: D
authors.initials: D
authors.lastname: Ogden
mesh_terms: D000085:Acetates / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology; D004553:Electric Conductivity; D004558:Electric Stimulation; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005029:Ethylenediamines / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques; D010782:Photolysis; D011247:Pregnancy; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000085:Acetates; D002614:Chelating Agents; D005029:Ethylenediamines; D007473:Ion Channels; C057014:DM-nitrophen; D013917:Thrombin; D002118:Calcium