Endothelial Cell Function: Role of Intracellular Signalling

Buchan, Kevin William (1991) Endothelial Cell Function: Role of Intracellular Signalling. PhD thesis, University of Glasgow.

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Abstract

A. Regulation of calcium mobilisation in bovine aortic endothelial cells 1. In the presence of 1.8mM extracellular calcium, bradykinin (0.3nM-100nM), adenosine triphosphate (ATP; 0.3muM-100muM) and thrombin (0.03U ml-1-3U ml-1) each induced biphasic elevations of intracellular free calcium ([Ca2+]i) in bovine aortic endothelial cells (BAEC), consisting of a large, initial transient component, which peaked within 30 seconds, followed by a lower, sustained plateau phase. 2. Platelet-activating factor (up to 120nM), histamine (up to 10muM) and lipopolysaccharide (up to 10mug ml -1) each had no effect on [Ca2+]i in BAEC. 3. In the presence of 1.8mM extracellular calcium, treatment of endothelial cells with the calcium influx blocker, nickel chloride (4mM), had no effect on basal 2+ [Ca 2+]i or on the magnitude of the bradykinin-induced transient elevation of [Ca2+]i, but abolished the plateau phase. 4. When BAEC were bathed in nominally calcium-free solution, containing 0.5mM EGTA, bradykinin and ATP each induced only a transient elevation of [Ca2+]i: the magnitude of this component was significantly smaller than that obtained in the presence of extracellular calcium, and the plateau phase was abolished. In the continued presence of bradykinin or ATP, re-addition of extracellular calcium, to achieve a level of around 1. 8mM, resulted in the induction of a large, initial transient component, followed by a lower, sustained component. Procaine (1mM) had no effect on the large, transient component, suggesting that calcium- induced calcium release is not involved in the generation of this component. In the presence of 1mM extracellular calcium, treatment with the calcium chelator, EGTA (2mM; 1min), slightly reduced basal [Ca2+]i and significantly reduced the magnitude of the bradykinin-induced transient elevation of [Ca2+]i. Increasing the exposure time to 10min or the concentration of EGTA to 5mM resulted in no further reduction in the magnitude of the bradykinin-induced transient component. 6. An attempt was made to selectively inhibit release of calcium from intracellular stores. In the presence of 1. 8mM extracellular calcium, treatment of BAEC with the putative inhibitor of intracellular calcium release, 3,4,5-trimethoxybenzoic acid-8-(diethylamino) octyl ester (TMB-8; 0.1mM) increased basal [Ca2+]i slightly, but had no effect on either component of the bradykinin-induced biphasic elevation of [Ca2+]i. To determine the effects of TMB-8 on only the intracellular release component, BAEC were bathed in nominally calcium-free solution, in the presence of EGTA (0.5mM). TMB-8 (0.1mM) had no effect on basal [Ca2+]i or the bradykinm-induced transient elevation of [Ca2+]i. These findings suggest that TMB-8 does not inhibit release of calcium from intracellular stores. 7. An attempt was made to determine whether removal of extracellular calcium results in the depletion of intracellular calcium stores. The elevation of [Ca2+]i induced by caffeine (5mM) was almost completely abolished when BAEC were bathed in nominally calcium-free solution containing 0. 5mM EGTA, suggesting that depletion of these stores does occur. 8. In the presence of 1. 8mM extracellular calcium, addition of potassium chloride (KCl; 30mM and 60mM) had no effect on basal [Ca2+]i. However, 60mM KCl, but not 30mM KCl, reduced the magnitude of the initial transient elevation of [Ca2+]i induced by bradykinin (10nM). Addition of KCl(30mM) during the plateau phase of the increase in [Ca2+]i induced by bradykinin (10nM), thrombin (1U ml and ATP (30muM), resulted in a fall in [Ca2+]i which was not well maintained. Hence, calcium entry in BAEC does not occur through voltage-operated channels, although the effects of membrane depolarisation on agonist-induced calcium mobilisation appear to be complex. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Cellular biology, Pharmacology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78254
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 30 Jan 2020 15:35
Last Modified: 30 Jan 2020 15:35
URI: http://theses.gla.ac.uk/id/eprint/78254

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