title: Cytosolic calcium changes in endothelial cells induced by a protein product of human gliomas containing vascular permeability factor activity.
abstract: A vascular permeability factor (VPF) derived from serum-free conditioned medium of cultured human malignant gliomas (HG-VPF) has been described previously. The rapid kinetics of HG-VPF activity in an in vivo assay of vascular permeability suggests a direct action upon the vascular endothelial cell. To determine whether HG-VPF was capable of inducing a physiologically significant alteration in isolated endothelial cells, cytosolic calcium [Ca++]i was measured in vitro in these cells before and after their exposure to media containing this substance. This was accomplished by preloading cultured endothelial cells with a fluorescent intracellular Ca++ probe fura-2/AM. It was found that HG-VPF induced a rapid and transient elevation of [Ca++]i in normal endothelial cells derived from human umbilical vein, bovine adrenal medulla, bovine pulmonary artery, and rat brain. This effect was inhibited by chelating extracellular calcium [Ca++]e with ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetra-acetic acid (EGTA), indicating that the HG-VPF-induced response resulted from the influx of extracellular calcium. The addition of cations that act as nonspecific calcium channel blockers (Li+, Co++, Mn++, La ) completely inhibited VPF activity, further supporting the role of [Ca++]e influx. The HG-VPF activity was not, however, blocked by verapamil, a calcium antagonist that appears to be specific for voltage-gated calcium channels. Furthermore, exposure of endothelial cells to 120 mM [K+]e did not result in a calcium transient. Coincubation of endothelial cells with dexamethasone inhibited HG-VPF-induced rises in [Ca++]i, while having no effect upon cyclic nucleotide-induced changes in calcium. The present studies indicate that vascular extravasation induced by human glioma-derived VPF may be mediated by a direct action upon vascular endothelial cells. Furthermore, the observed dexamethasone-induced inhibition of this process suggests a specific cellular action for corticosteroids. This, together with previous observations that dexamethasone suppresses both the production of VPF by tumor cells in vitro and its permeability-inducing activity in vivo, may explain the efficacy of glucocorticoids in the treatment of neoplastic vasogenic brain edema. Finally, studies with a polycationic peptide (protamine) known to induce blood-brain barrier disruption in vivo revealed similar effects upon endothelial cytosolic calcium levels. As HG-VPF is a positively charged macromolecule, a common interaction between these substances and the negatively charged endothelial cell surface in the induction of permeability is suggested. Nonspecific cross-linking of charged groups of the endothelial glycocalyx and specific HG-VPF receptor binding are both valid mechanisms of HG-VPF-mediated calcium changes. Their potential relevance in the setting of microvascular permeability is discussed.
authors.affiliation: National Institute of Neurological Disorders and Stroke, Surgical Neurology Branch, Bethesda, Maryland.
authors.forename: G R
authors.initials: GR
authors.lastname: Criscuolo
authors.forename: P I
authors.initials: PI
authors.lastname: Lelkes
authors.forename: D
authors.initials: D
authors.lastname: Rotrosen
authors.forename: E H
authors.initials: EH
authors.lastname: Oldfield
mesh_terms: D001696:Biomechanical Phenomena; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology*; D009363:Neoplasm Proteins / Q000494:pharmacology; D009994:Osmolar Concentration; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D002412:Cations; D008222:Lymphokines; D009363:Neoplasm Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D002118:Calcium