Inhibition of ATP‐induced calcium influx by homocysteine in human umbilical vein endothelial cells

Abstract

Mechanisms involved in the association between hyperhomocysteinemia and vascular occlusive diseases remain unclear. Homocysteine (Hcy) may disturb calcium (Ca(2+) ) cytosolic regulation in endothelial cells, a process that can directly affect the synthesis of vasoactive substances, such as nitric oxide (NO). We have investigated the effect of acute and chronic incubation with high concentrations of Hcy (100 and 500 μmol/L) on the changes in the intracellular Ca(2+) concentration ([Ca(2+) ]i ) induced by ATP, using primary cultures of human umbilical vein endothelial cells (HUVEC). The changes in [Ca(2+) ]i , expressed as ΔFt /Fb , were measured using the microspectrofluorimetric technique with Fluo-3 as Ca(2+) indicator. HUVEC acutely exposed to Hcy did not produce significant effects on any of the parameters studied. However, chronic exposition (24 h) caused a significant decrease in the speed of store-mediated Ca(2+) entry, expressed as (ΔFt /Fb )/t (s(-1) ). Exposure of HUVEC to 100 and 500 µmol/L Hcy gave significantly lower values (0.019 ± 0.002 s(-1) , n = 5 and 0.021 ± 0.004 s(-1) , n = 6, respectively) compared to the controls (0.046 ± 0.004 s(-1) , n = 8, P < 0.003). This was detected only when the sustained phase of the ATP-induced [Ca(+2) ]i increase was isolated. These results demonstrate that high concentrations of Hcy can affect the mechanisms involved in [Ca(2+) ]i regulation of HUVEC, and that alteration occurs specifically in the sustained phase, which has been directly associated with NO synthesis.

LAV-BPIFB4 isoform modulates eNOS signalling through Ca2+/PKC-alpha-dependent mechanism

“…Most cells were infected and displayed clear GFP fluorescence that did not interfere with the fluorescent signal of the Ca 2+ dye. Ca 2+ transients were elicited by applying 100 μM ATP for 2 min 14 . The time courses of Ca 2+ transients were quantified by measuring at each time point the fluorescence emission in the region of interest surrounding each cell, and then transforming the obtained values as follows: Δ F / F = [ F ( t ) − F (0)]/ F (0).…”

Ca2+ signals in human umbilical endothelial cells derived from pregnancy with fetal growth restriction associated with hypertensive disorder

“…To understand how Ca 2+ dynamics is altered by FGR-HDP, components of the ATP-induced Ca 2+ signals were separated ( 18 ). There were no significant changes in the Ca 2+ signal induced by ATP in the absence of extracellular Ca 2+ , suggesting that the P2Y2 receptor mediated response was not affected in HUVECs from FGR-HDP group.…”

“…[Ca 2+ ] i was measured using the fluorescent indicator Fluo-3 AM, as previously reported ( 17 , 18 ). Briefly, confluent HUVECs grown in coverslips and incubated in Locke's solution (NaCl, 135.0; KCl, 5.6; CaCl 2 ·2H 2 O, 2.5; HEPES-acid, 10.0; MgCl 2 ·6H 2 O, 1.2 and D-glucose, 5.5 mM) were mounted in a perfusion chamber on the stage of an epifluorescence microscope (Nikon Eclipse E600FN) implemented with 490 nm excitation and 530 nm emission filters.…”

“…Cytosolic Ca 2+ response to ATP was evaluated in HUVECs, as described in a previous study ( 18 ). In the present study, the parameters analyzed were time to initial peak (t P ), amplitude of initial peak, amplitude of sustained phase, and return to the baseline [Ca 2+ ] i .…”

Effects of individualized administration of folic acid on prothrombotic state and vascular endothelial function with H-type hypertension

“…High Hcy levels are associated with the pathophysiology of atherosclerosis, which is known to be associated with a prothrombotic state in individuals with hypertension [15,16] . In addition, high Hcy levels can also enhance the sensitivity to vascular endothelial cell damage and amplify damage in cells by mobilizing intracellular calcium release [17,18] . As FA supplementation can prevent Hcy and ET-1/NO increase, [19] FA supplementation seems to be a reasonable treatment for individuals with H-type hypertension, particularly given that it can be achieved by using a relatively low-priced treatment that is easy to obtain in the community and hospital.…”