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AF-DX 384 binding in rabbit cingulate cortex: two site kinetics and section autoradiography

K L Dopke1, K E Vrana, L J Vogt

  • 1Department of Physiology and Pharmacology Bowman Gray School of Medicine Wake Forest University Winston-Salem, North Carolina, USA.

The Journal of Pharmacology and Experimental Therapeutics
|July 1, 1995
PubMed
Summary

Researchers used AF-DX 384 and pirenzepine to label m2 muscarinic receptors in rabbit brain tissue. This method successfully identified and characterized m2 receptor binding sites in specific brain regions.

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Area of Science:

  • Neuroscience
  • Pharmacology

Background:

  • Autoradiographic studies of muscarinic receptors are hindered by a lack of selective ligands.
  • AF-DX 384 exhibits higher affinity for m2 than m4 receptors, while pirenzepine (PZ) has an inverse affinity profile.

Purpose of the Study:

  • To develop a method for labeling m2 muscarinic receptors using competition between AF-DX 384 and PZ.
  • To characterize m2 receptor binding sites in homogenized and sectioned rabbit cingulate cortex.

Main Methods:

  • Utilized competition assays with [3H]AF-DX 384 and varying concentrations of PZ.
  • Employed saturation isotherms and Scatchard analysis to determine binding parameters (KD and Bmax).
  • Investigated receptor binding in both anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC), with PCC tissue deafferented via undercut lesions.

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Main Results:

  • Saturation analysis of [3H]AF-DX 384 revealed a single binding site with KD = 9 +/- 2.3 nM and Bmax = 1405 +/- 146 fmol/mg protein.
  • Competition studies indicated a two-site model (low and high affinity) for PZ binding in both ACC and PCC.
  • PZ (150 nM) binding with [3H]AF-DX 384 fit a single-site model, suggesting PZ blocks the lower affinity site.

Conclusions:

  • The competition between AF-DX 384 and PZ provides a viable method for labeling m2 muscarinic receptors.
  • Differential affinity of PZ for low-affinity sites was observed between ACC and PCC.
  • This approach facilitates the study of m2 receptor distribution and presynaptic binding in specific brain regions.