Agonist- and cyclopiazonic acid-induced maintained elevation of
intracellular calcium in intact endothelium from rabbit cardiac
valves.
Li, Li, and Cornelis Van Breemen.
Department of Pharmacology & Therapeutics, The University of
British Columbia, Faculty of Medicine, Vancouver, BC, V6T 1Z3,
Canada
APStracts 2:0405H, 1995.
Intracellular Ca2+ signals of intact endothelium from rabbit aortic or
pulmonary valves loaded with fura-2 were studied using imaging
fluorescence microscopy. Agonists such as ATP or carbachol (CCh) and
inhibitors of endoplasmic reticulum (ER) Ca2+-ATPase such as
cyclopiazonic acid (CPA), thapsigargin (Tg) and 2',5',-di(tert
-butyl)-1,4,-benzohydroquinone (BHQ) induced an increase in [Ca2+]i
(cytoplasmic free calcium concentration) that was maintained above
the pre-stimulated levels as long as extracellular Ca2+ were present,
indicating that the maintained [Ca2+]i increase is dependent on the
entry of extracellular Ca2+. The voltage-gated channel was not found
to contribute to [Ca2+]i increase. SK&F 96365, a receptor-operated
cation channel (ROC) blocker, and 2-nitro-4-carboxyphenyl-N,N
-diphenyl-carbamate (NCDC), a postulated phospholipase C inhibitor,
were shown to effectively block ROC since they greatly reduced the
maintained [Ca2+]i increase caused by ATP, but not that by CPA, which
was blocked by Ni2+. To further investigate the Ca2+ influx involved
in this process, divalent cation entry was measured as Mn2+ quenching
of fura-2 fluorescence at 360 nm excitation wavelength. At rest
fluorescence quenching at 360 nm by Mn2+ was observed, which was
inhibited by Ni2+ but not by NCDC, indicating Mn2+ entry through the
leak pathway. The quenching was enhanced following ATP stimulation,
and this enhancement was abolished by pretreatment with NCDC. In
contrast the rate of Mn2+ quenching was unaffected by the application
of CPA. These results demonstrate that ATP stimulates divalent cation
influx which is not related to the Ca2+ content of ER. Abolition of
Ca2+ uptake into ER was postulated to increase the effectiveness of
the Ca2+ leak in raising [Ca2+]i .
Received 28 April 1995; accepted in final form 22 August 1995.
APS Manuscript Number H406-5.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 September 1995.