Abstract
The galanin neuropeptide system is widely distributed throughout the brain and periphery and is thought to play a role in feeding, pain and reproduction. To evaluate the human galanin receptor 1 as a potential therapeutic target, we fully characterized its interaction with several galanin-like peptides. The human galanin receptor 1 receptor was stably expressed using an episomal system in human embryonic kidney 293E cells. Saturation isotherms using 125I-human galanin revealed two distinct populations of receptor affinity states in membranes and whole cells with picomolar and nanomolar affinities at the high- and low affinity states, respectively. A scintillation proximity assay revealed that 125I-human galanin binding in membranes reached steady-state within 2 to 2.5 hr; however, only 50% of galanin radiolabel dissociated from the receptors by excess galanin or guanosine 5′-O-3-thiotriphosphate even after 20 hr. In contrast, galanin binding in whole cells was completely reversible within 1 hr. Competition binding assays showed that galanin-like peptides bound with picomolar affinities in membranes and whole cells. These peptides behaved as full agonists as determined by the inhibition of forskolin-stimulated cyclic 3′5′-adenosine monophosphate production and the stimulation of guanosine 5′-O-(3-[35S]thiotriphosphate binding. The agonist profile of M40, a representative chimeric peptide, was found not to be the result of receptor reserve because receptor inactivation by partial alkylation experiments confirmed its full intrinsic efficacy under conditions of a “zero” reserve state. These observations suggest that the antagonist effects in vivo of M40, and perhaps other chimeric peptides, are not mediated via direct interactions with the galanin receptor 1 receptor.
Footnotes
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Send reprint requests to: Dr. Lawrence W. Fitzgerald, CNS Diseases Research, E400/4442, The DuPont Pharmaceuticals Co., Experimental Station, P.O. Box 80400, Wilmington, DE 19880.
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1 Current address: Pharmacopeia, Inc., Cranbery, NJ 08512.
- Abbreviations:
- cAMP
- cyclic 3′5′ adenosine monophosphate
- GALR
- galanin receptor
- HEK
- human embryonic kidney
- [35S]GTPγS
- guanosine 5′-O-3-[35S]thiotriphosphate
- Gpp(NH)p
- 5′guanylimidodiphosphate
- SPA
- scintillation proximity assay
- PBS
- phosphate-buffered saline
- EBNA1
- Epstein Barr nuclear antigen 1
- CMV
- cytomegalovirus
- NPY
- neuropeptide Y
- PEI
- polyethyleneimine
- DMEM
- Dulbecco’s modified Eagle media, galantide or M15, galanin-(1-13)-substance P-(5-11)
- M35
- galanin-1-13)-bradykinin-(2-9)
- M40
- galanin-(1-13)-Pro-Pro-(Ala-Leu)2-Ala-NH2
- C7
- galanin-(1-13)spantide
- 7TM
- seven transmembrane
- PBZ
- phenoxybenzamine
- GTP
- guanosine triphosphate
- Received February 2, 1998.
- Accepted June 4, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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