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

T. D. Carter, G. Zupancic, Stephen Smith, C. Wheeler-Jones, D. Ogden

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)845-855
Number of pages11
JournalJournal of Physiology
Volume513
Issue number3
DOIs
StatePublished - Dec 15 1998
Externally publishedYes

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Human Umbilical Vein Endothelial Cells
Thrombin
Photolysis
Calcium
Membranes
von Willebrand Factor
Dialysis
Cell Culture Techniques
Perfusion
Enzyme-Linked Immunosorbent Assay
1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis((oxycarbonyl)methyl)-1,2-ethanediamine

ASJC Scopus subject areas

  • Physiology

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Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein. / Carter, T. D.; Zupancic, G.; Smith, Stephen; Wheeler-Jones, C.; Ogden, D.

In: Journal of Physiology, Vol. 513, No. 3, 15.12.1998, p. 845-855.

Research output: Contribution to journalArticle

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T1 - Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein

AU - Carter, T. D.

AU - Zupancic, G.

AU - Smith, Stephen

AU - Wheeler-Jones, C.

AU - Ogden, D.

PY - 1998/12/15

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N2 - 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.

AB - 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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