Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides |
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Authors: | Nigel W Bunnett Donald G Payan and Eileen F Grady |
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Institution: | (1) Department of Surgery, University of California, 94143-0660 San Francisco, CA, USA;(2) Department of Physiology, University of California, 94143-0660 San Francisco, CA, USA;(3) Department of Medicine, University of California, 94143-0660 San Francisco, CA, USA;(4) Department of Surgery, 521 Parnassus Avenue, 94143-0660 San Francisco, CA, USA |
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Abstract: | Summary 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°C, and internalized the peptide after 10 min at 37°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°C
had specific surface labelling. After 30 s at 37°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°C. After 5 min at 37°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. |
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Keywords: | |
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