Characterization of calcitonin gene-related peptide (CGRP) receptors and their receptor-activity-modifying proteins (RAMPs) in human brain microvascular and astroglial cells in culture.
1. In the present study, we examined the expression of the CGRP receptor-activity-modifying proteins (RAMP1, RAMP2 and RAMP3) and receptor component protein (RCP) in human brain astrocytes (AST), cerebromicrovascular endothelial (EC) and smooth muscle (SMC) cells in culture. Further, we pharmacologically characterized CGRP receptors in these cells by assessing the potency of the CGRP receptor antagonists h-alpha CGRP(8-37) and the new non-peptide compound BIBN4096BS to block the production of cAMP elicited by CGRP(1) and CGRP(2) receptor agonists. 2. AST, EC and SMC all expressed mRNAs for RAMP1, RAMP2 and RCP. In contrast, message for RAMP3 was detected in AST, but not in SMC and in only one out of four preparations of EC. 3. h-alpha CGRP, h-beta CGRP and [Cys (Et)(2,7)]-h-alpha CGRP exerted concentration-dependent production of cAMP in all cultures, with a maximal effect at 25-50 nM (20-60-fold increase from basal levels). In contrast, 50 nM [Cys (Acm)(2,7)]-h-alpha CGRP only induced a weak stimulatory effect on cAMP formation, especially in SMC and AST (1.5- and 5-fold increase above baseline, respectively). 4. h-alpha CGRP(8-37) and BIBN4096BS concentration-dependently inhibited cAMP formation evoked by CGRP receptor agonists. Depending on the agonists used, h-alpha CGRP(8-37) distinguished two different CGRP receptors for which it exhibited low (pIC(50)< or =6.4) and high (pIC(50) approximately 7.3) affinity, respectively. BIBN4096BS was much more potent (>2.5 orders of magnitude) than h-alpha CGRP(8-37). Further, BIBN4096BS was able to discriminate three different CGRP receptor sites for which it exhibited low (pIC(50) approximately 9.3-9.9), intermediate (pIC(50) approximately 10.9), and a very high (pIC(50) approximately 13.7) affinity, respectively. Together, these results suggest the presence of CGRP(1) and/or CGRP(2) receptors in human brain AST, EC and SMC, and of an additional population of CGRP receptors in AST, possibly associated to the combined expression of RAMP3 and RCP in these cells, for which BIBN4096BS exhibits an exquisitely high affinity.
Moreno
Mariá Jesús
MJ
Laboratory of Cerebrovascular Research, Department of Neurology and Neurosurgery, Montreal Neurological Institute, 3801 University Street, Montréal, QC, Canada H3A 2B4.
Terrón
José Antonio
JA
Stanimirovic
Danica B
DB
Doods
Henry
H
Hamel
Edith
E
D001253:Astrocytes / Q000378:metabolism*; D001923:Brain Chemistry / Q000502:physiology*; D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D002196:Capillaries / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000819:agonists / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013268:Stimulation, Chemical
D000077221:Calcitonin Gene-Related Peptide Receptor Antagonists; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D018015:Receptors, Calcitonin Gene-Related Peptide; D000242:Cyclic AMP