Anal. Chem. 2003, 75, 145-151
Detection of ATP-Induced Nitric Oxide in a Biomimetic Circulatory Vessel Containing an Immobilized Endothelium Damian H. Kotsis and Dana M. Spence*
Department of Chemistry, Saint Louis University, St. Louis, Missouri 63103
Conditions for the adhesion of bovine pulmonary artery endothelial cells (bPAECs) in microbore tubing of 250µm i.d. are described. When immobilized to the lumen of microbore tubing, these cells represent a mimic of a circulatory vessel’s endothelium. The microbore tubing is coated with 100 µg mL-1 fibronectin in order to promote bPAEC adhesion to the lumen of the tubing. A series of micrographs of the cells inside of the tubing indicates that ∼3.5 h is necessary for cell adhesion. In this study, adenosine triphosphate (ATP) is used to induce the release of nitric oxide from the endothelium mimic. The endothelium-derived NO is detected amperometrically at a parallel flow cell containing a glassy carbon working electrode modified with Nafion. Results indicate that detectable amounts of NO are only produced by the endothelium mimic when ATP is present in the buffer. The typical concentration of NO produced by the endothelium mimic upon the introduction of 100 µM ATP is ∼0.80 µM. Based on the injection volume of ATP and the estimated number of cells on the tubing lumen, this value corresponds to ∼1 amol of NO/cell. Moreover, shear stress alone does not provide the agonistic effect required for NO production in the submicromolar range. Resistance vessels in the pulmonary circulation, namely, arterioles and metarterioles that have inside diameters between 100 and 25 µm and 25 and 10 µm,1 respectively, are surrounded by a layer of smooth muscle. Importantly, the lumen of these vessels also contains a layer of endothelial cells that synthesizes and releases factors that relax the surrounding smooth muscle and, thereby, leads to an increase in vascular caliber and a decrease in vascular resistance.2,3 The inability to control vascular tone may lead to an increase in vascular resistance and the development of pulmonary hypertension. It is generally accepted that nitric oxide, the endotheliumderived relaxing factor (EDRF), is capable of relaxing vascular smooth muscle.2-5 Importantly, it is the relaxation of this vascular * Corresponding author. E-mail:
[email protected]. (1) Enderle, J. D.; Blanchard, S. M.; Bronzino, J. D. Introduction to Biomedical Engineering; Academic Press: San Diego, 2000; pp 521-522. (2) Ignaro, L. J.; Harbison, R. G.; Wood, K. S.; Kadowitz, P. J. J. Pharmacol. Exp. Ther. 1986, 237, 893-900. (3) Ignarro, L. J.; Buga, G. M.; Chaudhuri, G. Eur. J. Pharmacol. 1988, 149, 79-88. (4) Furchgott, R. F.; Zawadzki, J. V. Nature 1980, 288, 373-376. 10.1021/ac0258249 CCC: $25.00 Published on Web 11/21/2002
© 2003 American Chemical Society
smooth muscle that allows for control of vascular tone and a regulation of resistance in circulatory vessels. Although the mechanism by which NO is produced inside an endothelial cell is well understood,6-8 there exists conflicting theories as to the mechanism by which NO is released into the circulatory system and, most importantly, the signals that cause this release. For example, some previous reports have suggested that a flowinduced shear stress on the endothelium would result in NO production in the endothelial cells and release of that NO into the smooth muscle surrounding the vessel.9,10 However, in other studies involving isolated rabbit lungs, Sprague et al reported that a shear stress alone did not result in a change in measured pulmonary pressure; i.e., simply passing over the endothelium did not induce any measurable NO production.11 Further studies suggested that adenosine triphosphate (ATP) was required in the solution flowing over the endothelium to induce NO production and that erythrocytes were the source of the ATP.12,13 Other reports suggest that release of NO into the peripheral circulation involves the RBC directly.14,15 That is, NO is bound to a cysteine residue found in hemoglobin within erythrocytes, and when subjected to low oxygen levels (hypoxia), the hemoglobin undergoes a conformational change that results in the release of the NO from the cysteine residue into the peripheral circulation. There exists a need for a model that will help define the exact mechanism by which NO is delivered to the vascular smooth muscle, where it is known to control vascular tone. (5) Palmer, R.; Ferrige, M. J.; Moncada, S. Nature 1987, 327, 524-526. (6) Mo, M.; Eskin, S. G.; Schilling, W. P. Am. J. Physiol. 1991, 260, H1698H1707 (7) Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Biochem. Pharmacol. 1989, 38, 1709-1715 (8) Wohlfart, P.; Malinski, T.; Ruetten, H.; Schindler, U.; Linz, W.; Schoenafinger, K.; Strobel, H.; Wiemer, G. Br. J. Pharm. 1999, 128, 1316-1322 (9) Buga, G. M.; Gold, M. E.; Fukuto, M. M.; Ignarro, L. J. Hypertension 1991, 17, 187-193. (10) Rubanyi, G. M.; Romero, J. C.; Vanhoutte, P. M. Am. J. Physiol. 1986, 250, 1145-1149. (11) Sprague, R. S.; Stephenson, A. H.; Dimmit, R. A.; Weintraub, N. A.; Branch, C. A.; McMurdo, L.; Lonigro A. J. Am. J. Physiol. 1995, 269, H1941-H1948. (12) Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J. Am. J. Phsyiol. 1996, 271, H2717-H2722. (13) Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J. Exp. Clin. Cardiol. 1998, 3, 73-76. (14) Gow, A. J.; Stamler, J. S. Nature 1998, 391, 169-173. (15) Stamler, J. S.; Jia, L.; Eu, J. P.; Mcmahon, T. J.; Demchenko, I. T.; Bonaventura, J.; Gernert, K.; Piantadosi, C. A. Science 1997, 276, 20342037.
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Recently, we characterized the amount of ATP released from erythrocytes in response to mechanical deformation.16,17 It was reported that, although no cell lysis was occurring in the continuous-flow system, fused-silica microbore tubing could be employed to behave as a mimic of resistance vessels in vivo. As the erythrocytes were passed through microbore tubing having inside diameters ranging from 25 to 75 µm, the amount of ATP released from these cells decreased (due to less mechanical deformation placed upon the cells). It was also reported that an increase in the duration of the deformation stimulus resulted in an increase in the amount of ATP released from the erythrocytes subjected to the stress. The study involving the mechanically induced ATP release from erythrocytes is important since it suggests that erythrocytes, when subjected to mechanical deformation, do indeed release ATP.16 Moreover, our previous studies also suggested that fused-silica microbore tubing could be used as a model of resistance vessels in vivo.17 However, these studies did not attempt to examine the role of ATP in the production of NO in the endothelium. Of course, simply measuring the release of ATP from the erythrocytes in microbore tubing does not address the mechanism by which endothelial cells produce and release NO. Thus, the objective of our work is to develop a system that will help define the relationship between ATP and NO production in endothelial cells under conditions that closely mimic those of a circulatory vessel. Specifically, we report here the development of biomimetic reactor containing immobilized endothelial cells to the lumen of a small section of fused-silica microbore tubing. Although the endothelial cells immobilized in the microbore tubing are initially cultured in a conventional culture flask, there does exist a nearconfluent layer of the endothelial cells inside of the tubing when the studies attempting to describe the role of ATP in the production of NO are performed. Since endothelial cells do not adhere well to glass, immobilizing the cells to the inside wall of the fused-silica glass tubing requires that the wall be coated to promote cell attachment and to allow the cells to become confluent.18 This is accomplished by precoating the microbore tubing with a solution of fibronectin, which has proven to be a viable attachment substrate for the endothelial cells.19-21 Once the cells are immobilized to the lumen of the microbore tubing, a series of experiments are performed involving the amperometric detection of NO (produced by the endothelium) in a parallel flow cell containing a glassy carbon working electrode coated with Nafion. Nafion is a perfluoronated cation exchange polymer that only allows cations and small neutral molecules to pass through to the electrode surface. Therefore, interferences from nitrite and nitrate ions are minimized. (16) Sprung, R. W.; Sprague, R. S.; Spence, D. M. Anal. Chem.. 2002, 74, 22742278. (17) Edwards, J. L.; Sprung, R. W.; Sprague, R. S.; Spence, D. M. Analyst 2001, 126, 1257-1260. (18) Kam, L.; Boxer, S. G. J. Biomed. Mater. Res. 2001, 55, 487-495. (19) Davies, P. F.; Robotewskyj, A.; Griem, M. L. J. Clin. Invest. 1994, 93, 20312038. (20) Burmeister, J. S.; Vrany, J. D.; Reichert, W. M.; Truskey, G. A. J. Biomed. Mater. Res. 1996, 30, 13-22. (21) Sagnella, S.; Kwok, J.; Marchant, R. E.; Kottke-Marchant, K. J. Biomed. Mater. Res. 2001, 57, 419-431.
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EXPERIMENTAL SECTION Endothelial Cell Culture. Bovine pulmonary artery endothelial cells (bPAECs) and fetal bovine serum (FBS) were purchased from BioWhittaker (Walkersville, MD). Attachment factor was from Cascade Biologics (Portland, OR), and all other solutions were from Sigma (St. Louis, MO). The cells were cultured in T-75 culture flasks (Fisher Scientific) treated with attachment factor in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin solution (10 000 units penicillin and 10 mg mL-1 streptomycin). The cultures were kept in a humidified incubator at 37 °C and 5% CO2. Cells were removed from the flasks with 0.025% trypsin/0.01% EDTA when ∼90% confluent. Endothelial Cell Immobilization. Fused-silica microbore tubing (Polymicro, Inc., Phoenix, AZ) having an inside diameter of 250 µm and an outside diameter of 365 µm was used as the reactor tubing in all studies reported here. This diameter of tubing was chosen for ease of endothelial cell immobilization and also because the tubing did not have to be
