Intrathecal P/Q- and R-type calcium channel blockade of spinal substance P release and c-Fos expression☆,☆☆
Introduction
Voltage-sensitive calcium channels (VSCCs) facilitate calcium influx and play an important role in the regulation of neurotransmitter release, synaptic transmission and neuronal excitability (Catterall, 2000, Catterall and Few, 2008). The α1 is the pore-forming subunit and dictates the VSCC's major characteristics of pharmacology, electrophysiology and kinetic activity, representing the basis for the calcium channel subtype. Ten different α1 have been identified, namely Cav1.1–1.4, Cav2.1–2.3 and Cav3.1–3.3. These are distributed into five subgroups, L-(Cav1.1–1.4), P/Q-(Cav2.1), N-(Cav2.2), R-(Cav2.3) and T-type (Cav3.1–3.3). Based on their voltage activation properties, VSCCs are divided into two classes, high voltage-activated channels and low voltage-activated channels (Ertel et al., 2000). High voltage-activated channels include L-, P/Q-, N- and R-types. These are heteromeric complexes consisting of an α1 subunit along with auxiliary subunits such as α2δ, β and γ subunit. Low voltage-activated channels or T-type are activated by much more negative membrane potentials (Carbone and Lux, 1984, Nowycky et al., 1985) and are not known to interact with auxiliary subunits.
VSCCs are expressed in the dorsal root ganglion (DRG) and spinal cord dorsal horn (Murakami et al., 2001, Westenbroek et al., 1998, Yusaf et al., 2001), suggestive of their critical role in nociception. Intrathecal (IT) ziconotide (Prialt®), a selective N-type blocker is approved for treatment of severe chronic pain. Consistent with location of N-type VSCCs on peptidergic primary afferents, N-type VSCC blockade attenuates neurotransmitter release from primary afferents, as defined in in vitro and in vivo models (Evans et al., 1996, Maggi et al., 1990, Santicioli et al., 1992, Takasusuki and Yaksh, 2011). However, the role of other VSCCs subtypes in afferent neurotransmitter release remains unclear. We found previously that intrathecal L- and T-type blockers minimally affected intraplantar formalin-evoked release of substance P (SP) in vivo as measured by neurokinin 1 receptor (NK1r) internalization (Takasusuki and Yaksh, 2011). Those results suggested L- and T-type VSCCs were not involved in stimulus-evoked SP release. In the present study we focus on the role of P/Q- and R-types.
Cav2.1 (P/Q-type) channels contain the α1A subunit and are distributed in the nervous system including DRG and spinal dorsal horn (Catterall and Few, 2008, Kulik et al., 2004, Urban et al., 2005). P/Q-type VSCCs are inhibited by ω-agatoxin IVA, a 48- amino acid peptide isolated from the venom of funnel web spider, Agelenopsis aperta (Mintz et al., 1992). ω-agatoxin IVA blocks P-type with high affinity and Q-type with lower affinity (Adams, 2004). IT ω-agatoxin IVA reduces the number of flinches in formalin phase 2, but did not alter thermal escape latencies (Malmberg and Yaksh, 1994). Cav2.3 (R-type) channels contain the α1E subunit, and are detected in spinal cord and DRG (Murakami et al., 2001, Saegusa et al., 2000, Westenbroek et al., 1998, Yusaf et al., 2001). Cav2.3 knockout mice exhibited reduced pain behaviors in the formalin test (Saegusa et al., 2000). In another study, Cav2.3 was shown to participate in nerve injury-induced hypersensitivity (Matthews et al., 2007). Dense labeling of Cav2.3 was observed in the superficial layers of the dorsal horn (Saegusa et al., 2000). However, whether Cav2.3 is involved in primary afferent neurotransmitter release has not been established.
Here, we investigated intrathecal R- and P/Q-type channel blockers and spinal release of SP evoked by intraplantar formalin. Increase in local extracellular SP due to its release (induced by intraplantar formalin) from small peptidergic, transient receptor potential protein vanilloid 1 (TRPV1) (+) C-fibers evokes NK1r internalization (Kondo et al., 2005, Mantyh, 2002). Thus NK1r internalization provides a powerful tool to evaluate in vivo the effects of VSCCs blockade on afferent terminal release of SP. Furthermore, intraplantar formalin induces c-Fos protein expression in dorsal horn neurons, which receive input from small diameter Aδ and C primary afferents (Bullitt, 1990, Hunt et al., 1987, Long et al., 2012), causing c-Fos expression to be a useful index of spinal activation. This in vivo methodology allows us to assess the effects of R- and P/Q-type VSCCs antagonists upon SP release, dorsal horn neuron activation and assess the covariance of these effects with pain behaviors (flinching) at the corresponding drug doses.
Section snippets
Animals
Male Holtzman Sprague-Dawley rats (250–300 g; Harlan Indianapolis, IN) were individually housed in standard cages and maintained on a 12-h light/dark cycle (lights on at 07:00 h). Testing occurred during the light cycle. Food and water were available ad libitum to all rats in the study. Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85–23, Bethesda, MD) and as approved by the institutional Animal Care and Use
Behavior and motor effects of intrathecal SNX-482
IT administration of R-type VSCCs blocker SNX-482 (0.5 and 4.6 μg) in rats induced various adverse effects such as agitation, spontaneous and touch-evoked vocalization and motor deficits (Table 1). At the low dose (0.5 μg) these effects were minor and the incidence was low. The following behaviors were noticed within 2 h after the IT drug injection: vocalization evoked by gently touching the flank and back with a plastic tubing (2/14), facilitated startle response (2/14), absence of righting
Discussion
Voltage sensitive calcium channels have received much attention in pain research. A well-defined role for VSCCs, specifically N-type VSCCs, is to mediate neurotransmitter release from primary sensory neurons in response to noxious stimuli (Evans et al., 1996, Maggi et al., 1990, Santicioli et al., 1992). Spinal blockade of N-type VSCCs results in potent analgesia in preclinical models (Chaplan et al., 1994, Lewis et al., 2000), which led to the development of ziconotide (Prialt®), an FDA
Funding support
This research was supported by funding from the National Institutes of Health: NIH-DA02110, Bethesda, MD (TLY).
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank Arbi Nazarian, Ph.D. for his assistance in setting up the internalization protocol. The work was supported by DA02110.
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Preliminary data were presented in a poster at Neuroscience 2012 (Society for Neuroscience, New Orleans, Louisiana, USA, October 15, 2012).
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This work is attributed to ‘Department of Anesthesiology, University of California, San Diego, California, USA’.