Pharmacological characterisation of calcitonin gene-related peptide receptors in primary cultured rat trigeminal ganglia derived neurons

Christopher S Walker, Debbie L Hay. University of Auckland, Auckland, New Zealand.

Migraine is painful and debilitating disorder hypothesised to involve dysfunction of the trigeminovascular system. The neuropeptide calcitonin gene-related peptide (CGRP) and its receptors are expressed at key locations in the trigeminovascular system, including in the trigeminal ganglia (TG), the trigeminal nucleus of the brain stem and in the meningeal vasculature. The CGRP receptor antagonists olcegepant and telcagepant are proposed to block CGRP activity within the trigeminovascular system and have shown promise for the treatment of migraine pain (Fischer, 2010). However, the precise therapeutic site of action for these antagonists is unclear and their ability to antagonise CGRP activity in TG neurons is poorly defined. The aim of this study was to pharmacologically characterise CGRP receptors and investigate the ability of olcegepant to block αCGRP activity in TG derived neurons.

Wistar rat pups (3-5 days old) were anaesthetised with isoflurane and euthanised by decapitation. The TG were excised and collected. TG were then dissociated by incubation at 37°C for 30 minutes in Hank’s balanced salt solution (HBSS) containing dispase (10 mg ml-1). Cells were triturated in HBSS and cultures enriched for TG neurons by differential centrifugation through a bovine serum albumin gradient in L15 medium. TG neuron enriched cells were then re-suspended in culture media (L15 medium containing penicillin/streptomycin, glutamate (2 µM), glucose (50 mM), ascorbic acid (250 µM), glutathione (8 µM), mouse 2.5S nerve growth factor (NGF) (10 ng ml-1) and 8% foetal bovine serum) and further enriched by pre-plating for one hour at 37oC. Cells were then plated into 384 well poly-D-lysine/laminin-coated plates. Cultures were maintained at 37oC in a humidified incubator for 24 hours. TG neurons were incubated in serum and NGF free culture media for 10 minutes, followed by stimulation by agonist with or without antagonists for 30 minutes. For agonist potency TG neurons were incubated with rat αCGRP (rαCGRP), rat adrenomedullin (rAM) or rat amylin (rAmy). For antagonist potency TG neurons were co-incubated with rαCGRP and 100nM rαCGRP8-37 or 100nM olcegepant. cAMP content was then determined using the LANCE ultra cAMP detection kit (Perkin-Elmer). Data were expressed as a percentage of the maximum response. Curve-fitting for EC50 determination and statistical analysis was performed using Graphpad Prism 5.0. pA2 values were determined. Statistical significance was defined as p<0.05 and determined using Student’s t-tests or one-way ANOVA with post hoc Tukey’s tests. All data points represent the mean ± S.E.M. combined from n individual experiments. All procedures involving the use of animals were conducted in accordance with the New Zealand animal welfare act (1999) and approved by the University of Auckland Animal Ethics Committee.

In cultured rat TG neurons, rαCGRP (pEC50; 8.0±0.1, n = 9) and rAmy (pEC50; 7.9±0.2, n = 3) potently stimulated cAMP accumulation, displaying significantly higher (p<0.05) potency than rAM (pEC50; 6.6±0.3, n = 3). rαCGRP8-37 and olcegepant antagonised rαCGRP stimulated cAMP accumulation with pA2’s of 8.1±0.1 (n = 4) and 7.9±0.2 (n = 5), respectively.

The relative potencies of rαCGRP and rAM, coupled with the antagonist profiles of rαCGRP8-37 and olcegepant suggest that these cAMP responses are mediated through a rat CGRP receptor. However the relatively high potency of rAmy suggests that a more complex receptor phenotype is present, potentially involving rat calcitonin or amylin receptors. In summary, these data confirm that rαCGRP can stimulate cAMP accumulation in cultured TG neurons through an olcegepant sensitive receptor. This suggests that TG neurons are a potential therapeutic site of action for CGRP receptor antagonists.

Fischer, M.J. (2010). Expert. Opin. Investig. Drugs, 19(7): 815-823.