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Endothelins release 51Cr from cultured human cerebromicrovascular endothelium
D B Stanimirovic1, R McCarron, N Bertrand
1Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Insights
Endothelins increase brain microvascular permeability by activating protein kinase C and calcium signaling. This study details the molecular mechanisms behind endothelin-induced changes in endothelial cells.
Area of Science:
- Endocrinology
- Cell Biology
- Neuroscience
Background:
- Vasoactive peptides, specifically endothelins (ETs), play crucial roles in regulating vascular function.
- Human brain microvascular endothelial cells (HBECs) form the blood-brain barrier, a critical interface for neurological health.
Purpose of the Study:
- To investigate the effects of endothelins on HBEC permeability.
- To elucidate the signaling pathways involved in endothelin-mediated responses in HBECs.
Main Methods:
- Primary human brain microvascular endothelial cells (HBECs) were cultured.
- Effects of endothelins on 51Cr release (permeability marker), inositol triphosphate (IP3) production, and arachidonic acid (AA) release were measured.
- Involvement of protein kinase C (PKC), calcium channels, and IP3 receptors was assessed using specific inhibitors and antagonists.
Main Results:
- Endothelin-1 (ET-1) induced a dose-dependent increase in 51Cr release, IP3 production, and AA release from HBECs.
- Cell viability remained high (>97%) under experimental conditions.
- Dexamethasone inhibited ET-1-induced AA release but not 51Cr release.
- PKC inhibitor H7, calcium channel blocker verapamil, and IP3 receptor antagonist ryonidine attenuated ET-1-induced 51Cr release.
Conclusions:
- Endothelins significantly increase HBEC permeability.
- Receptor-specific activation of PKC and intracellular calcium mobilization are key mechanisms mediating endothelin-induced increases in HBEC permeability.
Abstract:
The effects of vasoactive peptides endothelins (ET-1, ET-2, ET-3, S6b, S6c) on release of 51Cr, production of inositol triphosphate (IP3), and release of arachidonic acid (AA) were examined in cultured microvascular endothelium derived from human brain (HBEC). ET-1 induced dose-dependent release of 51CR (EC50 = 7 +/- 2 nM), transient increase of IP3 (EC50 = 0.67 +/- 0.09 nM), and sustained release of AA (EC50= 59 +/- 7 nM) from HBEC. Under the same experimental conditions, viability of the cells was preserved (> 97%) as assessed by exclusion of vital dye trypan blue and release of lactate dehydrogenase (LDH). Dexamethasone (1 microM) inhibited ET-1-induced AA release, whereas it was ineffective on 51Cr release. Protein kinase C (PKC) inhibitor H7 (200 nM), calcium channel blocker verapamil (10 microM), or IP3 receptor antagonist ryonidine (5 microM) reduced ET-1 (100 nM)-induced release of 51Cr. These findings indicate that endothelins can induce an increase of HBEC permeability by a receptor-specific activation of PKC and intracellular calcium mobilization.