8 37 cgrp

Forskolin, wtCGRP, and [8–37]CGRP were purchased from Sigma-Aldrich and used in bioassays as described above. To determine whether CGRP responses were specifically mediated by RAMP1/CLR, two HEK293FT cell lines were used, one expressing the Glo22F cAMP-responsive luciferase only and the other also expressing the RAMP1/CLR CGRP receptor heterodimer. All luminescence values were normalized from zero to one using baseline values obtained from buffer only or addition of 100 μM Forskolin on the same respective cell line. In a separate experiment, CGRP dose–response curves were obtained in the presence of [8–37]CGRP on reporter cells expressing RAMP1 and CLR. [8–37]CGRP is an N-terminally truncated CGRP variant that acts as a competitive inhibitor of CGRP agonist activity and is expected to shift CGRP dose–response curves to the right. Here luminescence data were normalized between values obtained for buffer only and 100 nM CGRP only.

L -NA hydrochloride, ACh chloride, diethylamine NONOate diethyilammonium salt, 8-37 CGRP, rat CGRP, TTX, L -NAME hydrochloride, phentolamine, and suramin were from Sigma-Aldrich, Spain. Stock solutions (10 mmol/L) of drugs were made in distilled water, except for NA, which was dissolved in a NaCl (0.9%)-ascorbic acid (0.01% w/v) solution. These solutions were kept at –20 °C and appropriate dilutions were made in KHS on the day of the experiment.