The activation of transient receptor potential vanilloid ... - Springer Link

7 downloads 0 Views 281KB Size Report
Nov 23, 2007 - receptor subtype 1 by capsaicin without extracellular Ca. 2+ is involved in the mechanism of distinct substance P release in cultured rat dorsal ...
Naunyn-Schmiedeberg’s Arch Pharmacol (2008) 377:325–332 DOI 10.1007/s00210-007-0211-5

ORIGINAL ARTICLE

The activation of transient receptor potential vanilloid receptor subtype 1 by capsaicin without extracellular Ca2+ is involved in the mechanism of distinct substance P release in cultured rat dorsal root ganglion neurons He-Bin Tang & Yoshihiro Nakata

Received: 20 July 2007 / Accepted: 22 October 2007 / Published online: 23 November 2007 # Springer-Verlag 2007

Abstract Using a highly sensitive substance P (SP) radioimmunoassay, we attempted to elucidate the molecular mechanism of SP release from cultured adult rat dorsal root ganglion (DRG) neurons triggered by capsaicin. As a result, we found that capsaicin can induce SP release in the absence of extracellular Ca2+ by activating transient receptor potential vanilloid receptor subtype 1 (TRPV1). Therefore, we compared the pharmacological profile of SP release involved in several intracellular effectors (phosphoinositide 3-kinase (PI3K), Ca2+ release from intracellular stores, and mitogenactivated protein (MAP) kinases) in the presence/absence of extracellular Ca2+ by stimulating DRG neurons with various concentrations (10 to 1,000 nM) of capsaicin. In the presence of extracellular Ca2+, a capsaicin-induced maximal release of SP obtained at 100 nM capsaicin was attenuated by either the phosphoinositide 3-kinase (PI3K) inhibitors (wortmannin and LY294002), the inositol 1,4,5-triphosphate (IP3)-induced Ca2+ release blocker (2-aminoethyl diphenylborinate, 2-APB) or a specific antagonist of TRPV1 (capsazepine). In contrast, in the absence of extracellular Ca2+, only a high concentration (1 μM) of capsaicin induced a significant increase in the SP release, which was then completely abolished by either a mitogen-activated protein kinase kinase (MEK) inhibitor U0126 or capsazepine, and significantly inhibited by either thapsigargin (a Ca2+-ATPase inhibitor) or BAPTA-AM (a rapid Ca2+ chelator). In summary, the activation of TRPV1 by capsaicin modulates the SP release

H.-B. Tang : Y. Nakata (*) Department of Pharmacology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734–8553, Japan e-mail: [email protected]

from DRG neurons via two different mechanisms, one requiring extracellular Ca2+, the activation of PI3K and the IP3-dependent intracellular Ca2+ release, and the other which is independent of extracellular Ca2+ but involves the activation of MEK. These data suggest that a distinct SP release mechanism exists on DRG through the activation of TRPV1 without extracellular Ca2+ by capsaicin to facilitate neuropeptide release. Keywords Capsaicin . Dorsal root ganglion neuron . Substance P release . Transient receptor potential vanilloid receptor subtype 1

Introduction Substance P (SP) is a neuropeptide that functions as a neurotransmitter and/or neuromodulator (Severini et al. 2002; Grady et al. 2000). It is released from primary afferent neurons to convey information about various noxious stimuli through a very complex process. The process of SP release induced by the application of bradykinin, capsaicin, and potassium involves several intracellular effectors, such as mitogen-activated protein kinase kinase (MEK) activation, phosphoinositide 3-kinase (PI3K), and Ca2+ release from the inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+ stores (Suzuki et al. 1998; Tang et al. 2005 and 2006; Tang and Nakata 2006). Interestingly, Purkiss et al. (2000) reported that SP release can be evoked by capsaicin from cultured dorsal root ganglion (DRG) neurons in both the presence and absence of extracellular Ca2+, thus, suggesting that extracellular Ca2+ could therefore function as an important modulator in the process of transient receptor potential vanilloid receptor subtype 1 (TRPV1, a cloned capsaicin receptor)-triggered SP release.

326

TRPV1 is one member of the transient receptor potential family of nonselective Ca2+ ion channels. It is highly expressed in small-to-medium-sized DRG neurons involved in the nociception and neurogenic inflammation (Helliwell et al. 1998; Caterina and Julius 2001). A previous study by Oláh et al. (2001) indicated the expression of TRPV1 to be localized in the plasma membrane and mostly located in the internal membrane of the cells. Eun et al. (2001), Marshall et al. (2003), and Kárai et al. (2004) further supported this pattern of TRPV1 localization because TRPV1 can function to release Ca2+ from endoplasmic stores and enable the Ca2+ entry from outside the cell. We have shown that the activation of TRPV1 by a short-term treatment (10 min) with capsaicin (a powerful stimulus for a specific subset of primary sensory neurons) triggers the SP release from cultured DRG neurons (Tang et al. 2006; Tang and Nakata 2006). Recently, Dinh et al. (2004) and Price and Flores (2007) reported the colocalization of TRPV1 and SP in the rat DRG neurons. It is therefore of interest to investigate whether the concerned intracellular effectors (PI3K, MAP kinases, and Ca2+ release from intracellular stores) are involved in the regulation of the capsaicin-induced SP release from cultured DRG neurons in the presence and absence of extracellular Ca2+. Therefore, the objective of the present study is designed to investigate the possible involvement of the above-described intracellular effectors in the process of the TRPV1-triggered SP release from cultured DRG neurons in the presence and absence of extracellular Ca2+, by using a highly sensitive radioimmunoassay to measure the amount of SP release from cultured rat DRG neurons.

Naunyn-Schmiedeberg’s Arch Pharmacol (2008) 377:325–332

Isolation and culture of rat DRG cells According to a previously described method (Tang et al. 2004, 2005, 2006), DRGs of young adult Wistar rats (6– 9 weeks of age) were dissociated into single isolated neurons and non-neuronal cells after being treated with enzymes (collagenase and trypsin). The cells (3 DRGs/dish) were maintained at 37°C in a water-saturated atmosphere with 5% CO2 for 5 days before the initiation of the experiments. All procedures for animal experiments were performed according to the Guide for Animal Experimentation, Hiroshima University and the Committee of Research Facilities for Laboratory Animal Sciences, Graduate School of Biomedical Sciences, Hiroshima University, Japan. Measurement of the SP release Except for some cells treated by peptidase inhibitors alone (as a control), the cells were pre-exposed to various inhibitors in either a Ca2+-free or Ca2+ (2 mM)-containing Krebs–HEPES buffer [NaCl 110, KCl 4.5, CaCl2 2, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, D-glucose 11.7, HEPES 5 (mM)] for 10 min at 37°C. After washing two times with a Ca2+-free or Ca2+ (2 mM)-containing Krebs-HEPES buffer, those cells were then continuously stimulated by the appropriate concentrations of capsaicin plus peptidase inhibitors (1 μM phosphoramidon, 4 μg/ml bacitracin, and 1 μM captopril) or by peptidase inhibitors alone in either a Ca2+-free or Ca2+-containing Krebs–HEPES buffer for 10 min at 37°C. Thereafter, the SP content collected from the Krebs–HEPES buffer was measured by a highly sensitive radioimmunoassay (Tang et al. 2005, 2006; Tang and Nakata 2006), respectively.

Materials and methods

Western blotting analysis

Materials

At the end of the SP release experiments, the cells were processed for Western blotting according to a previously described method (Tang et al. 2006). Primary antibodies were raised against total or phospho-specific ERK1/2 (1:1,000 dilution; phospho-p44/42MAPK and p44/42MAPK polyclonal antibodies; Cell Signaling Technology, Beverly, MA). The horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:2,000 dilution; Cell Signaling Technology, Beverly, MA) was used for the detection of chemiluminescence according to the manufacturer’s instructions.

The following drugs were used: Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan); collagenase, phosphoramidon and bacitracin, captopril, capsaicin, and 2-APB (2-aminoethyl diphenylborinate), SP600125 (1,9-Pyrazoloanthrone), dantrolene and wortamnnin, LY294002 (2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran4-one hydrochloride) (Sigma Chemical, St Louis, MO, USA); trypsin (2.5%) (Invitrogen, Canada); [125I]Tyr8-SP (81.4 TBq/mmol) (New England Nuclear, Boston, MA, USA); H89 (N-[2-(p-bromocinnamylamino)ethyl]-5isoquinolinesulfonamide) (Seikagaku, Tokyo, Japan); U0126 (1,4-diamino-2,3-dicyno-1,4-bis[2-amino-phenylthio]butadiene) and SB203580 (4-(4-fluorophenyl)-2-(4methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole) (Promega, Madison, WI, USA).

Statistical analysis The data are presented as the mean±SEM. Statistical analyses were performed by a one-way ANOVA followed by Bonferroni’s test. Significance was set at a value of p