The endothelium plays an important role in the modulation of vascular tone and blood cell activation. Extensive work has demonstrated that the release of endothelium-derived relaxing factor (EDRF) from the endothelium is evoked by a number of physical and chemical stimuli requiring Ca2+. Because endothelial cells do not express voltage-dependent Ca2+ channels, Ca2+ influxes following receptor activation may be facilitated by cell hyperpolarizations mediated by the activation of K+ conductances. There has been recent interest in the role of ATP-sensitive K+ channels (KATP) suggesting that KATP may play a role in the regulation of blood flow. We have investigated the electrophysiological properties of an ATP-sensitive K+ conductance in whole cell and membrane patches from rat aorta and brain microvascular endothelial cells. Whole cell as well as single-channel currents were increased by either intracellular dialysis of ATP or application of glucose-free/NaCN (2 mM) solutions. Both currents were reversibly blocked by glibenclamide (1-100 microM). The KATP channel opener pinacidil (30 microM) caused activation of an outward current in the presence of physiological intracellular ATP concentrations. In inside-out patches, 10 microM-1 mM ATP invariably caused a dramatic decrease in channel activity. We conclude that both rat aorta and brain microvascular endothelial cells express KATP channels. KATP may play a role in the regulation of endothelial cell resting potential during impaired energy supply and therefore modulate EDRF release and thus cerebral blood flow.
A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or "weeding." The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin approximately laminin much greater than gelatin greater than poly-D-lysine approximately Matrigel greater than hyaluronic acid approximately plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-D-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7 +/- 2.6% (n = 6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of gamma-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.