Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides

Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4 degrees C, and internalized the peptide after 10 min at 37 degrees C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4 degrees C had specific surface labelling. After 30 s at 37 degrees C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4 degrees C. After 5 min at 37 degrees C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.