Organ culture of the trigeminal ganglion induces enhanced expression of calcitonin gene-related peptide via activation of extracellular signal-regulated protein kinase 1/2.
OBJECTIVE
Clinical and experimental studies have revealed a central role of calcitonin gene-related peptide (CGRP) in primary headaches. The role of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in neuronal and glial cell expression of CGRP- immunoreactivity (-ir) in rat trigeminal ganglia was studied with an organ culture method.


METHODS
Sections of adult rat trigeminal ganglia were cultured for up to 48 hours, examined with immunohistochemistry and quantitative real-time polymerase chain reaction (PCR) assay. Specific antibodies against CGRP, phosphorylated ERK1/2 (pERK1/2), total ERK1/2 (tERK1/2), phosphorylated p38 (pp38), phosphorylated C-Jun-N-terminal protein kinase (pJNK), pro-calcitonin (pro-CT), CGRP receptor activity modifying protein 1 (RAMP1), glutamine synthetase (GS) and pro-CT were used. To explore molecular mechanisms involved in the organ culture-induced CGRP-ir in neurons and glial cells, the effects of the MEK/ERK1/2 inhibitor U0126, its inactive analogue U0124, the p38 inhibitor SB203580 and the JNK inhibitor SP600125 were studied.


RESULTS
In fresh ganglia, small- and medium-sized neurons were CGRP-ir while some larger neurons displayed RAMP1-ir. Glial cells were negative to both. After organ culture, neurons showed enhanced CGRP- and RAMP1-ir. In addition, some glial cells were RAMP1- and CGRP-ir. Isolated glial cells and neurons were found to contain CGRP mRNA, and showed pro-CT-ir, suggestive of local formation of CGRP. Neurons and glial cells showed enhanced pERK1/2-ir already after two hours of organ culture and this remained elevated for 48 hours. There was transient pJNK-ir in neurons at two hours, while pp38-ir was not altered. U0126 reduced the enhanced pERK1/2-ir, while U0124 had no such effect; the CGRP-ir in neurons and glial cells was reduced at 48 hours and in parallel the CGRP mRNA expression was lower at 24 hours.


CONCLUSIONS
We suggest that in conditions of elevated CGRP expression, inhibition of ERK1/2 might be an option for novel treatment.
Tajti
János
J
Department of Medicine, Institute of Clinical Science, University Hospital, Lund University, Lund, Sweden.
Kuris
Anikó
A
Vécsei
László
L
Xu
Cang-Bao
CB
Edvinsson
Lars
L
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000096:biosynthesis*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis; D048052:Mitogen-Activated Protein Kinase 3 / Q000096:biosynthesis*; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D012668:Trigeminal Ganglion / Q000166:cytology / Q000378:metabolism*
D004791:Enzyme Inhibitors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015740:Calcitonin Gene-Related Peptide