Prostanoid Receptor Assays

June Chen1, David F. Woodward1, Robert A. Coleman2, Robert L. Jones3, Simon J. Lydford4

1 Allergan, Irvine, California, 2 Pharmagene Laboratories Limited, Royston, 3 The Chinese University of Hong Kong, Shatin, 4 Molecular Devices Limited, Winnersh
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.18
DOI:  10.1002/0471141755.ph0418s14
Online Posting Date:  November, 2001
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Abstract

Prostanoids, which include the prostaglandins (PGs) and thromboxanes (TXs), interact with a specific family of G‐protein coupled receptors, of which there are known to be five distinct types, DP, EP, FP, IP and TP, each particularly sensitive to one of the five natural prostanoids, PGD2, PGE2, PGF2(, PGI2 and TXA2, respectively. Of these, it is known that the EP receptor comprises four well‐characterized subtypes: EP1, EP2, EP3 and EP4. These receptor subtypes are widely distributed throughout mammals and other species, and show particularly high levels of expression in smooth muscle and blood platelets. Despite the fact that few of these preparations express a single receptor type/subtype in isolation, a range of useful smooth muscle and platelet assays for the various prostanoid receptors are available and are presented in this unit.

     
 
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Table of Contents

  • Basic Protocol 1: Evaluation of Prostanoid DP Receptor Activity in Human Washed Platelets
  • Basic Protocol 2: Evaluation of Prostanoid EP1 Receptors in Guinea‐Pig Isolated Trachea (GPT)
  • Basic Protocol 3: Evaluation of Prostanoid EP2 Receptor Activity in Isolated Ring Preparations from the Rabbit Ear Artery
  • Basic Protocol 4: Evaluation of Prostanoid EP3 Receptors in Guinea‐Pig Isolated Vas Deferens (GPVD)
  • Alternate Protocol 1: Evaluation of Prostanoid EP3 Receptors in Guinea‐Pig Isolated Aorta (GPA)
  • Basic Protocol 5: Evaluation of Prostanoid EP4 Receptor Activity in Isolated Ring Preparations from the Rabbit Saphenous Vein
  • Basic Protocol 6: Evaluation of Prostanoid FP Receptors in Mouse Uterus
  • Alternate Protocol 2: Evaluation of Prostanoid FP Receptors in Cat and Dog Isolated Iris Sphincter (CIS/DIS)
  • Basic Protocol 7: Evaluation of Prostanoid IP Receptors in Human Isolated Pulmonary Artery (HPA)
  • Alternate Protocol 3: Evaluation of Prostanoid IP Receptors in Rat Isolated Vagus Nerve(RVN)
  • Basic Protocol 8: Evaluation of TP Receptor Activity in Human Washed Platelets
  • Alternate Protocol 4: Evaluation of TP Receptor Activity in Isolated Ring Preparations from the Rabbit Saphenous Vein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Evaluation of Prostanoid DP Receptor Activity in Human Washed Platelets

  Materials
  • Human volunteer to donate blood
  • 3.2% (w/v) sodium citrate
  • 0.1 mg/ml PGI 2 in saline
  • Calcium‐free Tyrode's buffer, pH 7.4 (see recipe)
  • 50 µM indomethacin in 10% (w/v) Na 2CO 3
  • GR32191B (a gift from GlaxoSmithKline Compound Control, Gunnels Wood Road, Stevenege, Herts, SG1 2NY, United Kingdom)
  • 50 mM CaCl 2
  • 10 mg/ml fibrinogen in saline
  • 10 µM PGD 2 in ethanol
  • 1 mM ADP
  • 20 mg/ml BW A868C in ethanol (a gift from GlaxoSmithKline Compound Control, Gunnels Wood Road, Stevenege, Herts, SG1 2NY, United Kingdom)
  • 50‐ml centrifuge tubes
  • Centrifuge
  • Siliconized glass cuvettes (Fisher)
  • Dual‐channel aggregometer (PAP4; Biodata, or equivalent)
  • Teflon‐coated magnetic stir bars
  • Advance Bryans flat‐bed chart recorders (Diverse Devices; or similar)

Basic Protocol 2: Evaluation of Prostanoid EP1 Receptors in Guinea‐Pig Isolated Trachea (GPT)

  Materials
  • Guinea‐pigs (Dunkin‐Hartley, male, 350 to 500 g)
  • 1 µM indomethacin in Krebs‐Henseleit buffer (see recipe; prepared fresh each day), 37°C
  • 3 M KCl
  • 1 mM acetylcholine chloride or histamine in saline
  • 100 µM SQ 29548 or GR 32191 in saline
  • 100 µM, 10 µM, 1 µM, and 0.1 µM 17‐phenyl PGE 2 in saline (agonist solutions; BIOMOL)
  • 1 mM AH 6809 in saline (antagonist stock solution; BIOMOL)
  • Surgical instruments
  • Petri dishes
  • Organ bath equipment (e.g., Martino Lucianco), consisting of 10‐ml jacketed organ chamber(s) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile or Grass Instruments; see Fig. for equipment setup)
  • Z‐hooks made from 0.25‐ or 0.5‐mm diameter stainless steel wire using long‐nose pliers
  • Cotton thread

Basic Protocol 3: Evaluation of Prostanoid EP2 Receptor Activity in Isolated Ring Preparations from the Rabbit Ear Artery

  Materials
  • White rabbits (New Zealand, male, 2.5 to 3.8 kg)
  • Pentobarbitone sodium solution (Sagatal)
  • 2.8 µM indomethacin in Krebs‐Henseleit buffer maintained at 37° ± 0.5°C and aerated with 5% CO 2 in oxygen (see recipe). Add indomethacin from a 280 µM stock made in 10% (w/v) Na 2CO 3 on the day of use.
  • 1 mM phenylephrine
  • 20 mg/ml PGE 2 in ethanol or 'test' agonist
  • 1 mM acetylcholine chloride
  • 1 mM AH6809 (BIOMOL)
  • 1 mM AH23848B in 1% bicarbonate in 0.9% saline
  • Assorted surgical instruments
  • Nylon cannula (0.75‐mm o.d.)
  • 10‐ml water‐jacketed organ baths, circulating water heater, Ormed beam isometric transducers (or similar), fine wire hooks (0.25‐mm o.d.) and Advance Bryans flat bed chart recorders (or similar).

Basic Protocol 4: Evaluation of Prostanoid EP3 Receptors in Guinea‐Pig Isolated Vas Deferens (GPVD)

  Materials
  • Guinea‐pigs (Dunkin‐Hartley, male, 350 to 500 g)
  • 1 µM indomethacin in Krebs‐Henseleit buffer (see recipe; prepared fresh each day), 37°C, continuously gassed with carbogen (95% O 2/5%O 2). Add indomethacin from a 20 mM ethanolic stock on the day of use.
  • Agonist solutions: 0.1 µM, 1 µM, and 10 µM sulprostone in saline
  • Test compounds
  • Surgical instruments
  • Cotton threads
  • Petri dishes
  • Organ bath equipment (e.g., Martino Lucianco), consisting of jacketed organ chamber(s) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile or Grass Instruments; see Fig. and Fig. B for equipment setup) with facility for applying EFS to the preparation

Alternate Protocol 1: Evaluation of Prostanoid EP3 Receptors in Guinea‐Pig Isolated Aorta (GPA)

  Materials
  • Guinea‐pigs (Dunkin‐Hartley, male, 350 to 500 g)
  • 1 µM indomethacin in Krebs‐Henseleit buffer (see recipe; prepared fresh each day), 37°C, continuously gassed with carbogen (95% O 2/5%O 2). Add indomethacin from a 20 mM ethanolic stock on the day of use.
  • 3 M KCl
  • 100 µM GR 32191 or SQ 29548
  • Agonist solutions: 0.1 µM, 1 µM, 10 µM, and 100 µM sulprostone in saline
  • Antagonists
  • Surgical instruments
  • Petri dishes
  • Z‐hooks made from 0.25‐ or 0.5‐mm diameter stainless steel wire
  • Cotton thread
  • Organ bath equipment (e.g., Martino Lucianco), consisting of jacketed organ chamber(s) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile or Grass Instruments; see Fig. for equipment setup)

Basic Protocol 5: Evaluation of Prostanoid EP4 Receptor Activity in Isolated Ring Preparations from the Rabbit Saphenous Vein

  Materials
  • White rabbits (New Zealand, male, 2.5 to 3.8 kg) Pentobarbitone sodium solution (Sagatal)
  • 2.8 µM indomethacin in Krebs‐Henseleit buffer maintained at 37° ± 0.5°C and aerated with 5% CO 2 in oxygen (see recipe). Add indomethacin from a 280 µM stock made in 10% w/v Na 2CO 3 on the day of use.
  • 1 mM GR32191B
  • 4 M KCl
  • 1 mM acetylcholine chloride (optional)
  • 20 mg/ml PGE 2 stock in ethanol, prepare 100 nM to 1 mM dilutions in 2.8 µM indomethacin in Krebs‐Henseleit buffer just before use
  • 1 mM AH23848B in 1% bicarbonate/0.9% saline
  • Assorted surgical instruments
  • Polypropylene cannula (1.02‐mm o.d.), lightly scored (Fisher Scientific)
  • Fine wire Z‐hooks (0.25‐mm o.d.)
  • Organ bath equipment (e.g., Martino Lucianco), consisting of jacketed organ chamber(s) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile or Grass Instruments; see Fig. for equipment setup)

Basic Protocol 6: Evaluation of Prostanoid FP Receptors in Mouse Uterus

  Materials
  • Virgin, female, albino mice (e.g., BKW, Bantin & Kingman) that have reached maturity (breeding age is 42 to 56 days)
  • CO 2 and O 2 gases (Euthanex dual tank system or similar)
  • Optional: 0.9% normal saline in beaker, curved dropper pipet with rubber bulb, glass microscope slide, light microscope (for obtaining vaginal smears)
  • Krebs‐Henseleit buffer, pH 7.4 (see recipe)
  • 2.79 µM indomethacin in recipeKrebs‐Henseleit buffer, pH 7.4, maintained at 37°C and aerated with 5% CO 2 in oxygen. Add 27.9 µl of 0.1 M indomethacin (prepared in 2% Na 2CO 3) per liter of buffer
  • 10 µM PGF in 2% Na 2CO 3/0.9% saline
  • 10 µM PGF tromethamine salt in 0.9% saline
  • Plastic bag
  • Assorted surgical instruments
  • Surgical suture (silk, 4/0)
  • Organ bath equipment: reservoir bottles (4‐ to 6‐liter), water‐jacketed glass fluid warming coils, 10‐ml water‐jacketed glass organ baths, constant temperature circulating water heater, force‐displacement transducers and polygraph with low level DC amplifier (Grass Instruments or similar, calibrated with weights 1 to 4 g). See Figure for equipment setup
  • Software for analyzing area‐under‐the‐curve: e.g., Digitize (RockWare) or ArtpadZII + puck (WACOM)

Alternate Protocol 2: Evaluation of Prostanoid FP Receptors in Cat and Dog Isolated Iris Sphincter (CIS/DIS)

  Materials
  • Adult cats or dogs of either sex
  • 60 mg/ml pentobarbitone sodium
  • 1 µM indomethacin in Krebs‐Henseleit buffer, pH 7.4 (see recipe; prepared fresh each day), 37°C, continuously gassed with carbogen (95% O 2/5%O 2). Add indomethacin from a 20 mM ethanolic stock on the day of use.
  • 10 µM acetylcholine
  • Agonist solutions: 0.1 µM, 1 µM, 10 µM, and 100 µM PGF in saline
  • 5‐ or 10‐ml Hypodermic syringe and 21‐G needles
  • Surgical instruments
  • Petri dishes with saline‐soaked cotton wool
  • Cotton thread
  • Organ bath equipment (e.g., Martino Lucianco), consisting of jacketed organ chamber(s) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile or Grass Instruments; see Fig. for equipment setup)

Basic Protocol 7: Evaluation of Prostanoid IP Receptors in Human Isolated Pulmonary Artery (HPA)

  Materials
  • Human lung specimen
  • 1 µM indomethacin in Krebs‐Henseleit buffer (see recipe; prepared fresh each day), 37°C, continuously gassed with carbogen (95% O 2/5%O 2). Add indomethacin from a 20 mM ethanolic stock on the day of use.
  • 3 M KCl
  • 100 µM GR32191
  • 1 mM phenylephrine
  • Agonist solutions: 0.1 µM, 1 µM, 10 µM, 100 µM cicaprost in saline
  • Antagonist
  • Surgical instruments
  • Petri dishes
  • Z‐hooks made from 0.25 or 0.5 mm diameter stainless steel wire (see Fig. )
  • Cotton threads
  • Organ both equipment (e.g., Martino Lucianco), consisting of jacketed organ chamber(s) and circulating water heater, with isometric force transducer(s) and potentiometric chart recorder(s) (e.g., Ugo Basile or Grass Instruments; see Fig. for equipment setup)

Alternate Protocol 3: Evaluation of Prostanoid IP Receptors in Rat Isolated Vagus Nerve(RVN)

  Materials
  • Rats (Sprague‐Dawley, male or female, 250 to 300 g)
  • 1 µM indomethacin in Krebs‐Henseleit solution (see recipe; prepared fresh each day), ice‐cold and 27°C, continuously gassed with carbogen (95% O 2/5%O 2). Add indomethacin from a 20 mM ethanolic stock on the day of use.
  • Agonist solutions: 0.1 µM, 1 µM, 10 µM, and 100 µM cicaprost in saline, from which dilutions in recipeKrebs‐Henseleit buffer are made immediately before the cumulative dose sequence
  • Surgical instruments
  • Petri dishes
  • Dissecting microscope
  • Filter paper strips
  • Grease‐gap apparatus with recording electrodes linked to a suitable pre‐amplifier and data acquisition system (e.g., MacLab GP amplifier/4s interface/PowerMac 7200/120 computer (sampling rate 40 Hz))
  • Peristaltic pumps capable of delivering fluid at 1 to 10 ml/min

Basic Protocol 8: Evaluation of TP Receptor Activity in Human Washed Platelets

  Materials
  • Human volunteers to donate blood
  • 3.2% (w/v) sodium citrate
  • 0.1 mg/ml PGI 2 in saline
  • Calcium‐free Tyrode's buffer, pH 7.4 (see recipe), 37°C
  • 50 µM indomethacin in 10% (w/v) Na 2CO 3
  • 20 mg/ml BW A868C in ethanol
  • 50 mM CaCl 2
  • 10 mg/ml fibrinogen in saline
  • 20 mg/ml U46619 in ethanol (or test compound)
  • 50 µM GR32191B
  • 50‐ml centrifuge tubes
  • Centrifuge
  • 1‐ml siliconized glass cuvettes (Fisher)
  • Dual‐channel aggregometer (e.g., Biodata PAP4 or equivalent)
  • Teflon‐coated magnetic bar
  • Advance Bryans flat bed chart recorders (or equivalent)

Alternate Protocol 4: Evaluation of TP Receptor Activity in Isolated Ring Preparations from the Rabbit Saphenous Vein

  Materials
  • White rabbits (New Zealand, male, 2.5 to 3.8 kg)
  • Pentobarbitone sodium solution (Sagatal)
  • 2.8 µM indomethacin in Krebs‐Henseleit buffer maintained at 37° ± 0.5°C and aerated with 5% CO 2 in oxygen (see recipe). Add indomethacin from a 280 µM stock made in 10% w/v Na 2CO 3 on the day of use.
  • 4 M KCl
  • 1 µM to 1 mM U46619 (or test agonist), prepare in 2.8 µM indomethacin in recipeKrebs‐Henseleit buffer just before use.
  • 1 mM GR32191B
  • Assorted surgical instruments
  • Polypropylene cannula (1.02‐mm o.d.), lightly scored (Fisher Scientific)
  • 10‐ml water‐jacketed organ baths, circulating water heater, Ormed beam isometric transducers (or similar), fine wire hooks (0.25‐mm o.d.) and Advance Bryans flat bed chart recorders (or similar)
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Figures

Videos

Literature Cited

Literature Cited
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