Cysteinyl Leukotriene Receptor Assays

Roseanna M. Muccitelli1, Mark A. Luttmann1, Douglas W.P. Hay1

1 SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.16
DOI:  10.1002/0471141755.ph0416s09
Online Posting Date:  May, 2001
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The cysteinyl leukotrienes (CysLTs), LTC4, LTD4 and LTE4, lipid products derived from arachidonic acid metabolism, have been implicated in the pathophysiology of several inflammatory diseases, in particular, asthma. This unit describes techniques and applications for the measurement of contractile responses to the CysLTs in isolated smooth muscle preparations. The contractions are assessed by standard methods for the isometric measurement of responses (contractile or relaxant) of isolated tissues to exogenous agonists, and a detailed description of the methods employed to assess CysLT‐induced contractions in guinea‐pig trachea is outlined. However, the same general methodology (other than parameters such as dissection for non‐airway tissues) are appropriate for measuring CysLT‐induced contractions in airway preparations from other animals, and in non‐airways tissues (e.g., the gastrointestinal tract) from different species, and also in exploring the relaxant responses to the CysLTs that have been demonstrated in some tissues (e.g., pulmonary vein or artery).

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Table of Contents

  • Basic Protocol 1: Measurement of CysLT‐Induced Contractions in Guinea PIG Trachea
  • Support Protocol 1: Storage and Spectroscopic Quantification of CysLT Solutions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Measurement of CysLT‐Induced Contractions in Guinea PIG Trachea

  • 1× Krebs‐Henseleit solution (see recipe), bubbled with 95% O 2/5% CO 2
  • 10 mM meclofenamic acid (Sigma), prepared fresh
  • Guinea pigs: adult male albino Hartley, 400 to 600 g body weight (Charles River)
  • 10 mM carbachol (Sigma), prepared fresh
  • Antagonists
  • Cysteinyl leukotrienes (CysLT; Cayman Chemical): LTC 4, LTD 4, and LTE 4 (see protocol 2 and Table 4.16.1)
  • Laboratory cleaner (e.g., Extran 200)
  • 3 mM L‐cysteine (see recipe)
  • 45 mM serine borate (see recipe)
  • Tissue bath apparatus (Fig. ):
    • Frame (Fig. ): ½‐in.‐diameter × 18‐in. (1.3 × 46–cm) metal or plastic rods mounted 8 in. (20 cm) apart on an 8 × 40–in. (20 × 101–cm) plastic base with 2‐in. (5‐cm) frame foot
    • Medium nylon jaw clamps with 6‐in. (15.2‐cm) extension (for tissue baths; Fig. C)
    • Glass rod clamps: block clamp with V‐shaped slot (Fig. C)
    • Water‐jacketed isolated tissue baths (Fig. A) with 10‐ml borosilicate glass inner chamber (Metro Scientific)
    • Tension adjusters (transducer positioners; Fig. C): rack and pinion adjustable clamps (Harvard Apparatus) or threaded drive transducer positioners (Radnoti Glass)
    • Force transducers (e.g., Grass Instrument, FT03)
    • Two‐hole connecting rack clamps
    • Circulating water bath (e.g., Lauda; Fisher), 37°C, with thermometer
    • Vinyl tubing (Nalge): 50‐ft. (15‐m) PVC tubing with 5/16‐in. i.d. (7.9‐mm) and 1/16‐in. (1.6‐mm) wall
    • Latex rubber tubing
    • Liquid waste tray (Fig. ): 6‐in. W × 4‐in. H × 40‐in. L (15.2 × 10.1 × 101.6–cm) Plexiglas with drain attached to 5/16‐in.‐i.d. × 3‐ft. (7.9 m × 91.4 cm) vinyl tubing to bucket on floor
    • 2‐in. (5‐cm) Mohr pinchcock clamps (for waste tube; Fig. ) (Fisher)
  • Gas apparatus:
    • 95% O 2/5% CO 2 cylinder
    • Medical two‐stage gas regulator: gas pressure regulator (first stage, 0 to 4000 psi) and flow meter regulator (second stage, capacity 0 to 15 liters/min)
    • Gas distribution manifold consisting of one inlet and enough needle valves for one outlet (with serrated tubing connection) per tissue bath (Industrial Automation Components)
  • Data acquisition system:
    • MP100 A/D data acquisition hardware (Biopac Systems) or multichannel polygraph recorder (Grass Instrument)
    • DA100B amplifiers and universal interface module (to connect amplifiers and MP100)
    • Computer
    • Acknowledge data acquisition and analysis software (Biopac)
  • Graphpad Prism version 3 (GraphPad Software)
  • Transducer cables
  • Transducer connection interface (if connecting non‐Biopac transducers and cables to the DA100 amplifier)
  • Standard 5‐g brass weight
  • Repeating pipettor: standard Repipet II (VWR Scientific) with wide‐mouth 1.25‐liter glass bottle and 10‐ml pump
  • Dissecting dishes (see recipe)
  • Stainless steel dissecting tools (Roboz Surgical Instruments):
    • No. 3 scalpel handles
    • No. 11 scalpel blades
    • 6‐in. (15‐cm; large) and 4‐in. (10‐cm; small) toothed forceps
    • 4¾‐in. (12‐cm) no. 2 straight micro forceps
    • 4½‐in. (11.4‐cm) no. 7 curved micro forceps
    • 3½‐in. (8.9‐cm; small) and 5½‐in. (14‐cm; large) surgical cissors
  • 26‐ or 27‐G, ½‐in. syringe needles
  • No. 4 silk suture
  • Glass rods (Fig. B): 3‐mm‐diameter, L‐shaped, 9 × 13–mm with “S” hook to attach suture (DelVal Glass)
    Table 4.6.1   Materials   Sample Cumulative Additions of CysLTs for a 10‐ml Tissue Bath a   Sample Cumulative Additions of CysLTs for a 10‐ml Tissue Bath

    Stock added (µl) [Stock] Final bath concentration
    Whole‐log increments:
    10 0.1 µM 0.1 nM
    9 1 µM 1 nM
    9 10 µM 10 nM
    9 100 µM 100 nM
    9 1 mM 1 µM
    90 10 mM 10 µM
    Half‐log increments b
    10 0.1 µM 0.1 nM
    20 0.1 µM 0.3 nM
    7 1 µM 1 nM
    20 1 µM 3 nM
    7 10 µM 10 nM
    20 10 µM 30 nM
    7 100 µM 100 nM
    20 100 µM 300 nM
    7 1 mM 1 µM
    20 1 mM 3 µM
    70 1 mM 10 µM

     aVolumes should be adjusted for bath capacities other than 10 ml.
     bPharmacological data is usually plotted using log or semilog axes; such a data transformation will yield the characteristic sigmoidal (S‐shaped) curve. Because of this, it is convenient to use concentrations shown in the half‐log increments section of the table, since half the distance between factors of 10 on a log scale is actually 3 (log 3 = 0.5). The points would not be evenly spaced if concentration multiples of 5 were used (log 5 = 0.7).

Support Protocol 1: Storage and Spectroscopic Quantification of CysLT Solutions

  • Cysteinyl leukotriene (CysLT) stock solutions (Cayman Chemical):
  •  100 µg/ml (0.16 mM) ethanolic LTC 4
  •  100 µg/ml (0.2 mM) ethanolic LTD 4
  •  100 µg/ml (0.23 mM) ethanolic LTE 4
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Literature Cited

Literature Cited
   Arunlakshana, O. and Schild, H.O. 1959. Some quantitative uses of antagonists. Br. J. Pharmacol. 14:48‐58.
   Brocklehurst, W.E. 1960. The release of histamine and slow reacting substance (SRS‐A) during anaphylactic shock. J. Physiol. 151:416‐435.
   Coleman, R.A., Eglen, R.M., Jones, R.L., Narumiya, S., Shimizu, T., Smith, W.L., Dahln, S.E., Drazen, J.M., Gardiner, P.J., Jackson, W.T., Jones, T.R., Krell, Karell, R.D. and Nicosia, S., Karell, R.D. and 1995. Prostanoid and leukotriene receptors: A progress report from the IUPHAR working parties on classification and nomeclature. Adv. Prostaglandin Thromboxane Leukot. Res. 23:283‐285.
   Drazen, J.M. and Austen, K.F. 1987. Leukotrienes and airway responses. Am. Rev. Respir. Dis. 136:985‐998.
   Hay, D.W.P. 1997. Pharmacology of leukotriene receptor antagonists. Chest 111:35S‐45S.
   Horwitz, R.J., McGill, K.A., and Busse, W.W. 1998. The role of leukotriene modifiers in the treatment of asthma. Am. J. Respir. Crit. Care Med 157:1363‐1371.
   Kellaway, C.H. and Trethewie, R.E. 1940. The liberation of a slow reacting smooth muscle stimulating substance of anaphylaxis. Q. J. Exp. Physiol. 30:121‐145.
   Lew, M.J. and Angus, J.A. 1995. Analysis of competitive agonist‐antagonist interactions by nonlinear regression Trends Pharmacol. Sci. 16:328‐337.
   Metters, K.M. 1995. Leukotriene receptors. J. Lipid Mediators Cell Signaling 12:413‐427.
   Motulsky, H. 1999. Analyzing Data with GraphPad Prism 1999. GraphPad Software, San Diego, Calif.
   Orehek, J., Douglas, J.S., Lewis, J.A., and Gouhuys, A. 1973. Prostaglandin regulation of airway smooth muscle tone Nature New Biol. 245:84‐85.
   Samuelsson, B. 1983. Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation Science 220:568‐575.
   Snyder, D.W., Aharony, D. Dobson, P. Tsai, B.S., and Krell, R.D. 1984. Pharmacological and biochemical evidence for metabolism of peptide leukotrienes by guinea‐pig airway smooth muscle in vitro J. Pharmacol. Exp Ther. 231:224‐229.
   Van Rossum, J.M. 1963. Cumulative dose response curves. II. Technique for the making of dose response curves in isolated organs and the evaluation of drug parameters Arch. Int. Pharmacodyn. Ther. 143:299‐330.
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