CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways

Calcitonin gene-related peptide (CGRP) is a multifunctional neuropeptide that plays an important role in the pathology of and other debilitating orofacial diseases such as temporomandibular joint disorders (TMD) (Appelgren et al. 1995). CGRP is a 37 amino acid product of alternative splicing of the calcitonin-CGRP gene (Amara et al. 1982, Rosenfeld et al. 1983) and is widely distributed in the central and peripheral nervous systems (Van Rossum et al. 1997, Wimalawansa 1996). Excitation of trigeminal nerves, which provide sensory innervation to most of the head and face, is thought to lead to peripheral release of CGRP that promotes an inflammatory response and central release that can cause activation of second order neurons resulting in pain (Kopp 2001, Hargreaves 2007, Pietrobon 2005). In addition, results from recent studies support an autocrine and paracrine function for CGRP within the trigeminal ganglion (Thalakoti et al. 2007, Zhang et al. 2007). These cellular effects of CGRP are mediated via activation of the CGRP receptor. Although historically CGRP receptors have been divided into two classes referred to as CGRP1 and CGRP2, recent data have clarified that the CGRP1 receptor is the only CGRP receptor (Hay et al. 2008). Functional CGRP receptors are composed of a G protein-coupled receptor known as the calcitonin-like receptor (CLR), a single transmembrane domain protein called receptor activity modifying protein type 1 (RAMP1), and a receptor component protein that defines the G-protein to which the receptor couples (Poyner et al. 2002). RAMP1 functions to traffic mature CRL proteins to the surface of the cell membrane and plays a critical role for receptor function since it defines the relative potency of ligands for the receptor (Mallee et al. 2002). CGRP receptors are expressed by vascular smooth muscle cells (Moreno et al. 1999, Oliver et al. 2002), as well as neurons and glia in the peripheral and central nervous systems, such as second order neurons and astrocytes (Morara et al. 2008, Levy et al. 2004), trigeminal ganglion neurons (Lennerz et al. 2008, Zhang et al. 2007) and satellite glial cells (Thalakoti et al. 2007). It is well established that activation of CGRP receptors couples to increases in cAMP and cGMP levels in a number of different cell types (Cheng et al. 1995, Fiscus et al. 1991, Poyner et al. 2002, Wimalawansa 1996). However, CGRP receptors have also been reported to couple to activation of mitogen-activated protein kinases (MAP kinases) (Parameswaran et al. 2000, Schaeffer et al. 2003). MAP kinases are important signal transducing enzymes that connect activation of cell surface receptors to key regulatory events within the cell via a series of reversible phosphorylation events (Chang & Karin 2001, Seger & Krebs 1995). It is now known that at least four distinctly regulated groups of MAP kinases are present in mammalian cells, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinases (JNK1/2/3), p38 proteins (p38α/β/γ/δ), and ERK5, that are activated by specific MAP kinases (MAPK) (Chang & Karin 2001, Schaeffer & Weber 1999, Widmann et al. 1999). Importantly, many of the agents implicated in the initiation or maintenance of inflammation and pain have been shown to directly activate MAP kinase cellular signaling cascades in neurons and glial cells (Ji 2004a, Ji 2004b, Obata & Noguchi 2004). The inducible form of nitric oxide synthase (iNOS), which is responsible for the production of large quantities of nitric oxide (NO), is known to be regulated by MAP kinases. While iNOS is not expressed at high levels in normal human temporomandibular joints (TMJs), iNOS expression in the synovial lining of diseased TMJs is greatly increased (Homma et al. 2001, Nagai et al. 2003, Takahashi et al. 2003). Furthermore, NO levels in synovial fluid obtained from patients with internal derangement and osteoarthritis of their TMJ were significantly increased when compared to control levels and correlated with disease stage and pain in the patients' joint area (Suenaga et al. 2001, Takahashi et al. 1999). In addition, elevated levels of NO are also implicated in the underlying pathology of migraine and infusion of NO can cause migraine attacks (Iversen & Olesen 1996, Olesen & Jansen-Olesen 2000). NO is produced by the nitric oxide synthase family of enzymes that includes neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) (Liu et al. 2002). iNOS is expressed by a diverse array of cell types including both neuronal and glial cells found in the central and peripheral nervous systems. Despite the importance of iNOS and elevated NO levels in the development and maintenance of inflammation and pain, the cellular events involved in the expression of iNOS in glial cells is poorly understood. In previous studies, we have shown that CGRP activation of CGRP receptors can increase the expression of iNOS and stimulate NO release from trigeminal ganglion glial cells (Li et al. 2008). In this study, we used glia-enriched cultures to determine the role of MAP kinase signaling pathways in CGRP regulation of iNOS expression and NO release from cultured trigeminal ganglion glial cells. Data from this study provide the first evidence, to our knowledge, that CGRP can stimulate iNOS gene expression via activation of MAP kinase pathways in trigeminal ganglion glial cells. Based on our results, we propose that CGRP released from cell bodies of trigeminal neurons could function to promote and maintain an inflammatory cycle within the ganglion that mediates peripheral sensitization, and thus, play an important role in TMD pathology.