PubMed · 3551945
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C A Bassett. 1987. Low energy pulsing electromagnetic fields modify biomedical processes.. https://doi.org/10.1002/bies.950060110
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Hydrogen peroxide (H2O2) released by neutrophils is an important mediator of endothelial cell (EC) injury and vascular inflammation via its effect on EC-free Ca2+, [Ca2+]i. Although the underlying mechanisms are not well understood, platelet endothelial cell adhesion molecule (PECAM)-1/CD-31 is a critical modulator of neutrophil-EC transmigration. PECAM-1 is also known to regulate EC calcium signals and to undergo selective tyrosine phosphorylation. Here, we report that PECAM-1 molecules transduce EC responses to hydrogen peroxide. In human umbilical vein EC and REN cells (a PECAM-1-negative EC-like cell line) stably transfected with PECAM-1 (RHP), noncytolytic H2O2 exposure (100-200 microM H2O2) activated a calcium-permeant, nonselective cation current, and a transient rise in [Ca2+]i of similar time course. Neither response was observed in untransfected REN cells, and H2O2-evoked cation current was ablated in REN cells transfected with PECAM-1 constructs mutated in the cytoplasmic tyrosine-containing domain. The PECAM-dependent H2O2 current was inhibited by dialysis of anti-PECAM-1 cytoplasmic domain antibodies, required Src family tyrosine kinase activity, was independent of inositol trisphosphate receptor activation, and required only an intact PECAM-1 cytoplasmic domain. PECAM-1-dependent H2O2 currents and associated [Ca2+]i transients may play a significant role in regulating neutrophil-endothelial interaction, as well as in oxidant-mediated endothelial response and injury.
This study was designed to elucidate the role of the subplasmalemmal endoplasmic reticulum (sER) in autacoid-induced stimulation of Ca(2+)-dependent K(+) channels in the umbilical vein endothelial cell-derived cell line EA.hy926. Cells were transfected with the Ca(2+) probe cameleon targeted to the ER for visualization of the ER network. A patch pipette was then placed close to or far (> 5 microm away) from the sER, single channel recordings (patch clamp technique) were monitored simultaneously with measurements of either ER Ca(2+) concentration (using the Ca(2+) probe Cam4-ER) or cytosolic free Ca(2+) concentration ([Ca(2+)](i); using fura-2) using a deconvolution imaging device. A voltage-dependent, large conductance Ca(2+)-dependent K(+) channel (BK(Ca); single channel conductance (gamma), 250 pS) was found. At membrane potentials of +40 and -40 mV, the EC(50) for Ca(2+) was 2.7 and 49.7 microM, respectively. In the vicinity of the sER, the BK(Ca) channel activity induced by 10 microM histamine was 32 times higher (open probability (P(o)) = 0.083 +/- 0.026) than in areas away from the sER (P(o) = 0.0026 +/- 0.002). However, at supramaximal histamine stimulation (100 microM), BK(Ca) channel activation was similar in patches in the vicinity of or away from the sER (P(o) = 0.18 +/- 0.09 and 0.25 +/- 0.07, respectively). In contrast to BK(Ca) channel activity, ER Ca(2+) depletion (Cam4-ER) and elevation of [Ca(2+)](i) in response to 10 and 100 microM histamine were not influenced by the pipette position. We conclude that in endothelial cells, the activation of BK(Ca) channels in response to moderate histamine concentration essentially depends on the proximity of the sER domains to the mouth of this K(+) channel. These findings further support our concept of the subplasmalemmal Ca(2+) control unit (SCCU) and add the local activation of Ca(2+)-activated K(+)-channels to the function of the SCCU.
Preeclampsia (PE) is a leading cause of maternal hypertension in pregnancy, fetal growth restriction, premature birth, and fetal and maternal mortality (1). Activation and dysfunction of the maternal and fetal endothelium in PE may be the consequence of increased oxidative stress associated with circulating lipid peroxides (2-4), and in cases of severe maternal hypertension, uterine and umbilical artery waveforms are abnormal (5). We have investigated PE-associated abnormalities in the regulation of intracellular Ca2+ ([Ca2+]i) and cyclic guanosine monophosphate (cGMP) production (index of nitric oxide [NO]) in human fetal umbilical vein endothelial cells. Basal [Ca2+]i was slightly elevated in PE cells, whereas agonist-stimulated Ca2+ entry was reduced in cells from PE compared with normal term or age-matched preterm pregnancies. Furthermore, PE cells exhibited a decreased permeability to Ba2+ but an increased permeability to Mn2+ and Gd3+, suggesting that PE is associated with phenotypic alterations in fetal endothelial cation channel(s). Basal and histamine-stimulated cGMP levels were elevated in PE compared with preterm or normal cells, implying an increased NO production in PE. However, immunoblots for endothelial NO synthase (eNOS) and soluble guanylyl cyclase (sGC) revealed reduced eNOS expression in PE and preterm cells, with negligible changes in sGC levels. This study provides important and novel insights into abnormalities of fetal endothelial cells isolated from women with PE, reveal ing an altered cation membrane permeability and activity of eNOS-sGC pathway. As these changes are sustained in culture in vitro, this may reflect long-term "programming" of the fetal cardiovascular system.