Measurement of Second Messengers in Signal Transduction: cAMP and Inositol Phosphates

Fabian Gusovsky1

1 Eisai Research Institute, Andover, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.12
DOI:  10.1002/0471142301.ns0712s05
Online Posting Date:  May, 2001
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Abstract

cAMP acts as an intracellular mediator of hormone action and the importance of accurate quantitative determination of cAMP levels in cells and tissues is widely recognized. The most utilized procedures for the determination of adenylate cyclase activity in membranes are described here for measuring the conversion of [alpha‐32P]ATP into [32P]cAMP after a two‐step chromatographic separation. Also critical in signal tranduction is phosphoinositide turnover, which is linked to receptor activation resulting from changes in cytosolic calcium concentrations. Phosphoinositide turnover can be measured as described in this unit by labeling phospholipid pools with [3H]‐inositol and then analyzing for tritiated inositol phosphates.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Cyclic AMP Concentration in Cultured Cells
  • Basic Protocol 2: Measurement of [3H]Cyclic AMP Accumulation in Synaptoneurosomes After Prelabeling with [3H]Adenine
  • Basic Protocol 3: Measurement of Phosphoinositide Turnover in Synaptoneurosomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Determination of Cyclic AMP Concentration in Cultured Cells

  Materials
  • Adherent or suspension cells
  • Incubation buffer (see recipe), 37°C
  • Incubation buffer containing 1 mM isobutylmethylxanthine (IBMX; see recipe for 100 mM stock), 37°C
  • Test agents
  • 0.2 N HCl
  • 10 and 5 N NaOH
  • 10 mM cAMP in H 2O (store in small aliquots at −20°C)
  • TE cAMP buffer (see recipe)
  • ∼60 µg/ml protein kinase A (PKA; a cAMP‐dependent protein kinase; see recipe)
  • 0.5 µCi/ml [3H]cAMP (50 Ci/mmol) in TE cAMP buffer (prepare 10 ml immediately before use; keep at 4°C; total [3H]cAMP 5 µCi)
  • Charcoal suspension (see recipe)
  • 24‐ or 48‐well microtiter plates
  • 12 × 75–mm glass tubes
  • Scintillation fluid and vials
  • Additional reagents and equipment for determining protein concentration (see CPMB UNIT and appendix 1A in this manual)

Basic Protocol 2: Measurement of [3H]Cyclic AMP Accumulation in Synaptoneurosomes After Prelabeling with [3H]Adenine

  Materials
  • 2 Hartley guinea pigs (male, ∼200 g) or rats (male, ∼150 g)
  • Krebs‐Henseleit (KH) buffer (see recipe), bubbled with 95% O 2/5% CO 2 immediately before use and kept on ice
  • KH buffer containing 10 µg/ml adenosine deaminase and 1 µM [3H]adenine (10 to 15 Ci/mmol)
  • KH buffer containing 10 µg/ml adenosine deaminase (no label)
  • 0.15 mM cAMP in 5.6% (v/v) trichloroacetic acid
  • Cation‐exchange resin (AG 50W‐4X 200‐400 mesh, H+ form; Bio‐Rad)
  • 0.2 M NaOH
  • 0.2 M HCl
  • Dry alumina
  • 0.1 M imidazole⋅HCl, pH 7.3
  • Glass/glass homogenizer
  • Test tubes prewarmed to 37°C
  • Poly‐Prep columns (Bio‐Rad; 0.8 × 4 cm, 2‐ml bed volume)
  • Scintillation fluid and vials

Basic Protocol 3: Measurement of Phosphoinositide Turnover in Synaptoneurosomes

  • [3H]Inositol (10 to 20 Ci/mmol)
  • Nonfiltered synaptoneurosomes (see protocol 2, step )
  • Krebs‐Henseleit (KH) buffer (see recipe), bubbled with 95% O 2/5% CO 2 immediately before use and kept on ice
  • 200 mM LiCl
  • 6% (v/v) trichloroacetic acid (TCA)
  • Anion‐exchange resin (AG 1‐X8, formate form; Bio‐Rad)
  • 60 mM ammonium formate/5 mM Na 2B 4O 7⋅10H 2O (column washing solution; store at room temperature)
  • 200 mM ammonium formate/100 mM formic acid (IP 1 elution buffer; store at room temperature)
  • 400 mM ammonium formate/100 mM formic acid (IP 2 elution buffer; store at room temperature)
  • 1 M ammonium formate/100 mM formic acid (IP 3 elution buffer; store at room temperature)
  • Lipid cleaning solution (LCS; see recipe)
  • Chloroform
  • 5‐ml propylene tubes
  • 1.5‐ml microcentrifuge tubes
  • Poly‐Prep columns (Bio‐Rad; 0.8 × 4 cm, 2‐ml bed volume)
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Literature Cited

Literature Cited
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