Measurement of Nitric Oxide in Single Cells and Tissue Using a Porphyrinic Microsensor

Tadeusz Malinski1, Igor Huk2

1 Center of Biomedical Research, Oakland University, Rochester, Michigan, 2 University of Vienna, Vienna, Austria
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.14
DOI:  10.1002/0471142301.ns0714s06
Online Posting Date:  May, 2001
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Abstract

This unit describes the preparation and applications of porphyrinic sensors for quantitative measurement of nitric oxide (NO) in single cells and in tissues. The determination of NO is based on the electrochemical oxidation of NO on a carbon fiber electrode covered with a thin layer of a conducting polymeric metalloporphyrin catalyst, overlaid with another thin film of Nafion, a cation exchange material. The electric current generated during NO oxidation on the surface of the polymeric porphyrin is linearly proportional to the concentration of NO, so this current is used as an analytical signal which can be measured in either the amperometric or the voltammetric mode. Both methods provide a quantitative signal. This unit describes the electrochemical setup for measurement of NO in single cells and tissue. Support protocols describe porphyrin synthesis, sensor preparation, and sensor calibration.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Nitric Oxide in Single Cells and Tissue with a Porphyrinic Microsensor
  • Support Protocol 1: Preparation and Calibration of Porphyrinic Sensors
  • Support Protocol 2: Preparation of Bare Carbon‐Fiber Electrodes
  • Support Protocol 3: Preparation of TMHPPNi Electrochemical Coating Solution
  • Support Protocol 4: Preparation of Saturated NO Solution
  • Reagents and Solutions
  • Commenatry
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Nitric Oxide in Single Cells and Tissue with a Porphyrinic Microsensor

  Materials
  • Modified HBSS (see recipe)
  • Biological matrix: cells (grown on 25‐mm tissue culture dish) or tissue of interest
  • Chemical agonist for constitutive nitric oxide synthase (cNOS; optional)
  • Selective inhibitor for inducible nitric oxide synthase (iNOS; optional)
  • 0.1 mM L‐arginine in recipemodified HBSS
  • Saturated nitric oxide (NO) solution (see protocol 5)
  • Temperature‐controlled 3‐ml microscope stage (Olympus)
  • Porphyrinic sensor (see protocol 2)
  • Stereotactic micromanipulators, resolution 1 µm or better
  • Platinum wire (platinum auxiliary or counter electrode; 0.5‐ to 1‐mm diameter, 3‐cm length)
  • Silver/silver chloride electrode (SSCE; 1‐ to 2‐mm‐diameter silver wire covered with thin layer of silver chloride; Princeton Applied Research) or saturated calomel electrode (SCE; 2‐mm diameter; Princeton Applied Research)
  • Fast‐response (≤1 µsec) voltammetric analyzer (Princeton Applied Research, PAR 273)
  • Low‐diameter (e.g., 1‐mm) copper wires shielded with copper mesh
  • Faraday cage (optional; Princeton Applied Research)
  • Chart recorder, xy recorder, or computer with appropriate software (Electrochemistry Software, Princeton Applied Research)
  • Nanopipet, picopipet, or femtopipet (World Precision Instruments; optional)
  • Microscopes: stereoscopic (for measurements in tissue) and inverted (for measurements in single cells)

Support Protocol 1: Preparation and Calibration of Porphyrinic Sensors

  Materials
  • 0.1 M NaOH
  • TMHPPNi electrochemical coating solution (see protocol 4)
  • 5% (w/v) Nafion solution in alcohols (Aldrich)
  • Absolute ethanol
  • Saturated NO solution (1.74 mM at 0°C; see protocol 5)
  • Physiological buffer solution, pH 7.4 (optional)
  • Electrolytic cells: 19 × 48–mm, 2‐dram (∼10‐ml) clear glass vials (Kimble) with a Teflon cap with four holes
  • Multifiber or single‐fiber carbon electrode (see protocol 3)
  • Platinum wire (platinum auxiliary or counter electrode; 0.5‐ to 1‐mm diameter, 3‐cm length)
  • Silver/silver chloride electrode (SSCE; 1‐ to 2‐mm‐diameter silver wire covered with thin layer of silver chloride; Princeton Applied Research) or saturated calomel electrode (SCE; 2‐mm diameter; Princeton Applied Research)
  • Fast‐response (≤1 µsec) voltammetric analyzer (Princeton Applied Research, PAR 273)
  • Nitrogen or argon gas tank
  • 1‐ to 2‐mm‐i.d. glass pipet
  • Flatbed xy recorder or computer with appropriate software
  • Vacuum oven at 40°C

Support Protocol 2: Preparation of Bare Carbon‐Fiber Electrodes

  Materials
  • Conductive silver epoxy (P/N EG 8020; AIT), parts A and B
  • Beeswax
  • Nonconductive epoxy (2‐TON, Devcon Plexus; for single‐fiber electrodes only)
  • Violin rosin
  • 0.5‐ to 1‐mm‐i.d. open‐ended glass capillaries
  • Carbon fibers (6‐ to 7‐µm diameter; specific resistivity 12 ohm⋅cm; Amoco Performance Products)
  • Bare copper or silver wire (1‐mm diameter)
  • Emery paper
  • Vacuum dryer at 40°C
  • Propane microburner

Support Protocol 3: Preparation of TMHPPNi Electrochemical Coating Solution

  Materials
  • Propionic acid (Aldrich)
  • Silicon grease (Fisher)
  • Reagent‐grade vanillin (Aldrich)
  • Pyrrole (Aldrich), distilled immediately before use
  • Methylene chloride (Aldrich)
  • Methanol (Aldrich), ice cold
  • 1:1 (v/v) methylene chloride/methanol
  • Gradient of 200:1 to 40:1 (v/v) methylene chloride/methanol
  • Dimethylhydrofuran (Aldrich), room temperature and ice cold
  • Nickel(II) acetate tetrahydrate (Aldrich)
  • 0.1 M NaOH
  • Boiling stones
  • 250‐ and 500‐ml single‐neck round‐bottom flasks with female 24/40 joint (Pyrex)
  • Heating mantles for 250‐ and 500‐ml round‐bottom flasks
  • Claisen adapter with three 24/40 joints, one male leading to two female (Pyrex)
  • Tap water–cooled reflux condenser with 24/40 male joint (Pyrex)
  • 24/40 male ground‐glass stopper (Pyrex)
  • Rubber hoses
  • Small glass funnels
  • 60‐ml medium‐coarseness frits (Pyrex) and rubber adapter to fit a 1‐liter Erlenmeyer flask
  • 1‐liter side‐arm Erlenmeyer flasks (Pyrex)
  • Florisil chromatography column
  • Silica‐gel chromatography column
  • Rotary evaporator
  • Ground‐glass hose adapter with 24/40 male joint (Pyrex)
  • Tygon tubing
  • Nitrogen gas tank
  • Electrolytic cell: 19 × 48–mm, 2‐dram (∼10‐ml) clear glass vial (Kimble) with a Teflon cap with four holes
  • 0.5‐ to 1.0‐mm‐i.d. glass pipet

Support Protocol 4: Preparation of Saturated NO Solution

  Materials
  • 4 M NaOH
  • Laboratory‐generated or commercial NO (Fisher)
  • Methyl red
  • 0.1 M sodium phosphate buffer, pH 7.40 ( appendix 2A)
  • 1 M KMnO 4 (optional)
  • Two 500‐ml purge cylinders (Fisher)
  • 5‐ml conical Pyrex reaction vial with 14/10 threaded, screw‐cap rubber septum
  • Teflon‐coated magnetic spin vane to fit conical vial
  • 22‐G syringe needles
  • Hot plate or heating mantle equipped with a stirrer
  • Nitrogen or argon tank (99% v/v purity) equipped with hose and 22‐G input syringe needle
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Figures

Videos

Literature Cited

Literature Cited
   Adler, A.D., Longo, F.R., Finarelli, J.D., Goldmacher, J., Assour, J., and Korsakoff, L.J. 1967. A Simplified Synthesis for meso‐Tetraphenylporphyrin. Org. Chem. 32: 476.
   Archer, S. 1993. Measurement of nitric oxide in biological models. FASEB J. 7: 349‐360.
   Baek, K.J., Thiel, B.A., Lucas, S., and Stuehr, D. 1993. Macrophage nitric oxide synthase subunits. J. Biol. Chem. 268: 21120‐21129.
   Feelisch, M. and Stamler, J.S. 1996. Measurement of NO‐related Activities—Which Assay for Which Purpose? In Methods of Nitric Oxide Research (M. Feelisch and J.S. Stamler, eds.) pp. 303‐307. John Wiley & Sons Ltd., Chichester, UK.
   Fleming, I., Hecker, M., and Busse, R. 1994. Intracellular alkalination induced by bradykinin sustains activation of constitutive nitric oxide synthase in endothelial cells. Circ. Res. 74: 1220.
   Friedemann, M.N., Robinson, S.W., and Gerhardt, G.A. 1996. O‐phenylenediamine‐modified carbon fiber electrodes for the detection of nitric oxide. Anal. Chem. 68: 2621‐2628.
   Hecker, M., Mülsch, A., and Busse, R. 1994. Sub‐cellular localization and characterization of neuronal nitric oxide synthase. J. Neurochem. 62: 1524‐1529.
   Kiechle, F. and Malinski, T. 1993. Nitric oxide: Biochemistry, pathophysiology and detection. Am. J. Clin. Pathol. 100: 567‐575.
   Malinski, T. and Taha, Z. 1992. Nitric oxide release from a single cell measured in situ by a porphyrinic microsensor. Nature 358: 676‐678.
   Malinski, T.L. and Czuchajowski, L. 1996. Nitric oxide measurement by electrochemical methods. In Methods of Nitric Oxide Research (M. Feelisch and J.S. Stamler, eds.) pp. 319‐339. John Wiley & Sons Ltd., Chichester, UK.
   Malinski, T.Z., Taha, Z., Grunfeld, S., Burewicz, A., Tomboulian, P., and Kiechle, F. 1993. Measurements of nitric oxide in biological materials using a porphyrinic microsensor. Anal. Chim. Acta 279: 135‐140.
   Malinski, T., Patton, S., Pierchala, B., Kubaszewski, E., Grunfeld, S., Rao, K.V.S., and Tomboulian, P. 1994. Kinetics of nitric oxide release in the presence of superoxide in the endocardium as measured by a porphyrinic sensor. In Frontiers of Reactive Oxygen Species in Biology and Medicine (K. Asada and T. Yoshikawa, eds.) pp. 207‐210. Elsevier Science Publishing, Amsterdam.
   Marletta, M.A. 1994. Nitric oxide synthase—Aspects concerning structure and catalysis. Cell 78: 927‐930.
   Mesaros, S., Grunfeld, S., Mesarosova, A., Bustin, D., and Malinski, T. 1997. Determination of nitric oxide saturated (stock) solution by chronoamperometry on a porphyrin microelectrode. Anal. Chim. Acta 339: 265‐270.
   Nathan, C. and Xie, Q.W. 1994. Nitric oxide synthases: Roles, tolls, and controls. Cell 78: 915‐918.
   Schmidt, H.H.H.W. 1994. NO at work. Cell 78: 919‐925.
   Stamler, J.S. 1994. Redox signaling‐nitrosylation and related target interactions of nitric oxide. Cell 78: 931‐936.
   Vallance, P., Patton, S., Bhagat, K., MacAllister, R., Radomski, M., Moncada, S., and Malinski, T. 1995. Direct measurement of nitric oxide in human beings. Lancet 345: 153‐154.
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