Characterization of A-425619 at native TRPV1 receptors: A comparison between dorsal root ganglia and trigeminal ganglia
Introduction
The TRPV1 (transient receptor potential vanilloid-1) receptor (also known as vanilloid receptor-1 and VR1) is widely distributed throughout the peripheral nervous system, with high expression in small and medium diameter primary afferent neurons of the dorsal root ganglion and trigeminal ganglion (Guo et al., 1999), where it has been purposed to act as a molecular integrator of painful stimuli from the peripheral to the central nervous system (Caterina and Julius, 2001). The central projections of the TRPV1-positive neurons enter the CNS, where they synapse on second-order neurons of the spinal cord (for dorsal root ganglia) or the spinal nucleus of the trigeminal tract (for trigeminal ganglia). The peripheral terminals of these neurons are the sites of release for a number of proinflammatory neuropeptides (for review, Szallasi and Blumberg, 1999), including calcitonin gene-related peptide (CGRP). Quantitative analysis of L5 rat dorsal root ganglion indicates that 79% of TRPV1-positive dorsal root ganglia cells contain CGRP (Guo et al., 1999), whereas, about 10% of all TRPV1 immunoreactive cells in the human trigeminal ganglion express CGRP immunoreactivity (Hou et al., 2002). Activation of TRPV1 induces release of CGRP, as well as other neuropeptides, at both the central and peripheral terminals of sensory afferents. This release can be blocked by the TRPV1 antagonist capsazepine and the CGRP blocker αCGRP8–37 (Akerman et al., 2004). It is widely accepted that sensory secretion of CGRP plays an important role in nociceptive processing (Bennett et al., 2000, Sun et al., 2003), development and maintenance of neurogenic inflammation (Gamse and Saria, 1986), and vasodialation (Akerman et al., 2004). Furthermore, CGRP is thought to play a role in the sensation of joint pain and primary headaches (He et al., 1990, Goadsby et al., 2002).
The unique sensitivity to a diverse number of agents, including N-arachidonoyl-dopamine (NADA), an endovanilloid (Huang et al., 2002), noxious heat (> 43 °C) and high proton concentrations (< pH 6) (Caterina et al., 1997, Tominaga et al., 1998), suggests that TRPV1 may function as a key integrator of pain pathways (Caterina and Julius, 2001, Gunthorpe et al., 2002 Tominaga et al., 1998). In addition, noxious exogenous compounds, such as, capsaicin, resiniferatoxin, and tinyatoxin (an analogue of resiniferatoxin) activate TRPV1. Furthermore, increase in TRPV1 expression correlates with pathophysiologies that result in pain. In animal models of acute and chronic inflammation, TRPV1 immunoreactivity increased in rat dorsal root ganglia neurons (Amaya et al., 2003), and was evident in nerve fiber endings in rats receiving intraplanar carrageenan (Carlton and Coggeshall, 2001). Finally, previous studies report that TRPV1 mRNA levels increase in rat nociceptors after carrageenan injection (Tohda et al., 2001). For these reasons, TRPV1 is the subject of intense research focused on developing novel agents for the treatment of pain. As evidence of this interest, several new competitive TRPV1 antagonists have been discovered (Gunthope et al., 2004, Gavva et al., 2005, Doherty et al., 2005; El-Kouhen et al., 2005) within the past five years. Recently, in vivo studies have demonstrated that A-425619 dose dependently reduced capsaicin-induced mechanical hyperalgesia, and was also effective in models of acute and chronic inflammatory pain, postoperative pain and osteoarthritic pain (Gomtsyan et al., 2005, Honore et al., 2005). In vitro characterization of A-425619 indicates that it is a potent competitive antagonist against capsaicin activation (pA2 = 2.5 nM), and is also capable of inhibiting activation by anandamide, NADA, heat and acid in a recombinant cell line expressing hTRPV1 (El Kouhen et al., 2005).
The goals of the present study were to first extend the in vitro characterization of A-425619 to include its actions at native TRPV1 receptors by correlating calcium influx with electrophysiological methods, and CGRP release. The inhibitory actions of A-425619 were compared to the first generation TRPV1 antagonist capsazepine. The second aim, was to compare the pharmacological properties of TRPV1 receptors in the dorsal root ganglia with trigeminal ganglia, by examining the ability of various TRPV1 agonists and antagonists to affect TRPV1 function. To our knowledge, this is the first study to directly compare TRPV1 pharmacology between the two regions using the same technique (FLIPR). Although a few studies have reported using FLIPR-based technology to study activation of TRPV1 in the dorsal root ganglia (Jerman et al., 2000, Jerman et al., 2002), there are no comparable studies in the trigeminal ganglia.
Section snippets
Materials
Capsaicin and capsazepine were purchased from Sigma-Aldrich Co. (St. Louis, MO). NADA was purchased from Tocris Cookson, Inc. (Ellisville, MO), and resiniferatoxin and tinyatoxin were purchased from LKT Laboratories, Inc. (St. Paul, MN). A-425619 was synthesized at Abbott Laboratories (Abbott Park, IL). Dulbecco's phosphate-buffered saline (with Ca2+, Mg2+ and 1 mg/ml d-glucose)(pH 7.4), Hank's balanced salt solution (without Ca2+, Mg 2+), 0.25% trypsin–EDTA and penicillin/ streptomycin, were
Activation of rat TRPV1 receptors by both endogenous and exogenous TRPV1 agonists in dorsal root and trigeminal ganglia cultures
TRPV1 receptor agonists were tested for their ability to activate the native TRPV1 channel and elevate intracellular calcium in cultures of dorsal root ganglia and trigeminal ganglia. Capsaicin, 500 nM, induced an increase in intracellular calcium in both cultures with similar kinetics (Fig. 1A and B). After addition, there was a rapid increase in [Ca2+]i, followed by a slowly decreasing secondary response at approximately 20 s (Fig. 1A and B). The kinetics of the capsaicin-induced calcium
Discussion
The present study sought to extend the in vitro characterization of A-425619, the structurally novel competitive TRPV1 antagonist, by examining its actions at the native TRPV1 receptor expressed in the Dorsal root ganglia and trigeminal ganglia. To this end, we examined the ability of A-425619 to block capsaicin- and the endovanilloid, NADA-evoked increases in intracellular calcium concentrations and CGRP release from both cultures. Finally, we extended our initial electrophysiological findings
Acknowledgments
The authors wish to thank Drs. Mark Chiu and Alla Korepanova for their helpful discussions and support.
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