Characterization of receptors using cyanine 3-labeled neuropeptides

Peptides. 1995;16(4):733-40. doi: 10.1016/0196-9781(95)00042-i.

Abstract

We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carbocyanines*
  • Cell Line, Transformed
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Computer Systems
  • Endocytosis / physiology
  • Flow Cytometry
  • Fluorescent Dyes*
  • Gastrin-Releasing Peptide
  • Guinea Pigs
  • Neurokinin A / metabolism*
  • Peptides / metabolism*
  • Rats
  • Receptors, Neuropeptide / metabolism*
  • Substance P / metabolism*
  • Transfection

Substances

  • Carbocyanines
  • Fluorescent Dyes
  • Peptides
  • Receptors, Neuropeptide
  • cyanine dye 3
  • Substance P
  • Gastrin-Releasing Peptide
  • Neurokinin A