Inhibition of the histamine-induced Ca2+ influx in primary human endothelial cells (HUVEC) by volatile anaesthetics.
OBJECTIVE
Vasoactive substances such as histamine, acetylcholine or ATP increase the [Ca2+]i of endothelial cells, which leads to the activation of nitric oxide synthase (eNOS). The NO produced by this enzyme relaxes the underlying smooth muscle. Evidence suggests that eNOS activation is dependent on agonist-induced Ca2+ entry. Recently we have shown that in human endothelial cells (HUVEC), this Ca2+ entry is sensitive to isoflurane. The objective here was to study the mechanism by which volatile anaesthetics can depress the histamine-induced Ca2+ entry into HUVEC cells.


METHODS
HUVECs on coverslips were loaded with the Ca2+ indicator Fluo-3 and inserted in a gastight, temperature-controlled perfusion chamber. Excitation was at 488 nm and fluorescence signals were monitored with a confocal laser scanning microscope (MRC1024, Biorad). Direct measurement of the Ca2+ influx was with Mn2+ as surrogate for calcium at 360 nm in cells loaded with Fura-2.


RESULTS
Addition of histamine induces a biphasic [Ca2+]i increase consisting of Ca2+ release from internal stores and a Ca2+ influx from the external medium (plateau phase). The plateau phase was dose-dependently inhibited by enflurane and sevoflurane (13.7 resp. 21.9% inhibition by 1 MAC anaesthetic). Direct measurement of the Ca2+ influx using the Mn2+ quench of the Fura-2 fluorescence gave similar results. The inhibition of the anaesthetics was not reduced by inhibition of the cGMP pathway, inactivation of protein kinase C, depolarization of the cells or the presence of specific Ca2+-dependent K+ channel inhibitors. Interestingly, unsaturated fatty acids inhibit the histamine-induced Ca2+ influx in a similar way as the volatile anaesthetics.


CONCLUSIONS
Volatile anaesthetics dose-dependently inhibit the histamine-induced Ca2+ influx in HUVECs by a mechanism that may involve unspecific perturbation of the lipid bilayer.
Tas
P W L
PW
University of Würzburg, Center of Operative Medicine, Department of Anesthesiology, Oberdürrbacher Strasse 6, Würzburg, Germany. tas_p@klinik.uni-wuerzburg.de
Stössel
C
C
Roewer
N
N
D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000814:Aniline Compounds; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005224:Fats, Unsaturated / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D006221:Halothane / Q000494:pharmacology*; D006632:Histamine / Q000502:physiology*; D006801:Humans; D047569:Laser Scanning Cytometry; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008738:Methyl Ethers / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000187:drug effects; D000077149:Sevoflurane; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology; D014966:Xanthenes
D018685:Anesthetics, Inhalation; D000814:Aniline Compounds; D005224:Fats, Unsaturated; D005456:Fluorescent Dyes; D008738:Methyl Ethers; D024681:Potassium Channels, Calcium-Activated; D014966:Xanthenes; C049925:fura-2-am; C059715:Fluo-3; D009569:Nitric Oxide; D000077149:Sevoflurane; D008345:Manganese; D006632:Histamine; D017869:Cyclic GMP-Dependent Protein Kinases; D011493:Protein Kinase C; D006152:Cyclic GMP; D002118:Calcium; D016257:Fura-2; D006221:Halothane; D019331:NG-Nitroarginine Methyl Ester