Effects of neuropeptide SF and related peptides on acid sensing ion channel 3 and sensory neuron excitability
Introduction
The Phe-Met-Arg-Phe-NH2 (FMRFamide) and structurally related peptides are abundant in invertebrates where they might function as neurotransmitters and neuromodulators (Price and Greenberg, 1977, Roumy and Zajac, 1998). Although FMRFamide has not been isolated in mammals, endogenous mammalian FMRF-amide related peptides have been identified. Neuro-peptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and neuropeptide AF (NPAF, Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were first isolated from bovine brain (Yang et al., 1985), and neuropeptide SF (NPSF, Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) was isolated from the rat central nervous system (CNS) (Yang and Martin, 1995). Numerous data have accumulated about NPFF. It is released from the spinal cord following stimulation with high potassium or substance P (Zhu et al., 1992), or following opioid receptor stimulation (Devillers et al., 1995b), and it has been implicated in a variety of physiological functions, including pain modulation, opiate function and cardiovascular regulation (for reviews, see Panula et al., 1996, Roumy and Zajac, 1998).
NPFF, NPAF and NPSF derive from a common precursor, the gene of which has been cloned in human, rat, bovine and mouse (Perry et al., 1997, Vilim et al., 1999). The gene product is expressed mainly in the CNS, the dorsal spinal cord being one of the regions containing the highest concentrations (Panula et al., 1999, Vilim et al., 1999). High affinity NPFF receptors are thought to be coupled to various G-proteins (Roumy and Zajac, 2001). NPFF displays a nanomolar affinity for binding sites in the rat spinal cord whereas NPSF has a lower affinity for NPFF binding sites (28 times less than NPFF). Moreover, and contrary to NPFF, NPSF does not reduce the effect of nociceptin on dorsal raphe neurons (Roumy and Zajac, 1999, Roumy et al., 2000). An enzymatic degradation product of NPFF (QRFamide, Gln-Arg-Phe-amide) has been identified, but its affinity for the NPFF receptor in rat spinal cord membranes is 1430-fold lower than that of NPFF indicating that it cannot efficiently stimulate NPFF receptors to produce a biological effect (Sol et al., 1999).
Functional ASICs (acid sensing ionic channels) are homo- or hetero-tetrameric proton-gated Na+-permeable channels formed by the association of different subunits: ASIC1a, ASIC1b, ASIC2a, ASIC2b and ASIC3 (Chen et al., 1998, Waldmann and Lazdunski, 1998, Bassler et al., 2001). These channels are members of a super-family comprising, among others, the molluscan peptide-gated channel FaNaCh (Lingueglia et al., 1995). Proton-gated ASIC-like currents were described in sensory neurons (Krishtal and Pidoplichko, 1981b, Krishtal and Pidoplichko, 1981a, Krishtal and Pidoplichko, 1981c) and more recently, the expression of different ASIC subunits in these neurons has been reported (Waldmann and Lazdunski, 1998). ASIC3 (previously named DRASIC) is principally found in small and medium nociceptive sensory neurons (Voilley et al., 2001) and its expression in heterologous systems has been associated with a biphasic current comprising a fast transient component followed by a sustained phase (Waldmann et al., 1997). ASIC3 is thus a good candidate for mediating the non-adapting pain associated with tissue acidosis, and it has been implicated in acid-evoked nociception in the ischemic myocardium (Sutherland et al., 2001).
In the mollusc Helix aspersa, FMRFamide has been reported to directly activate the amiloride-sensitive channel FaNaCh with a micromolar concentration range (Cottrell et al., 1990, Lingueglia et al., 1995, Cottrell, 1997). The significant structural homology between FaNaCh and ASICs suggested that neuropeptides could directly activate or modulate ASIC-mediated currents. This is particularly interesting as ASIC3 expression, like that of NPFF precursor, is increased during inflammation (Vilim et al., 1999, Voilley et al., 2001). Moreover, both ASIC3 and NPFF are found in the dorsal root ganglia (Waldmann et al., 1997, Chen et al., 1998, Allard et al., 1999, Voilley et al., 2001). Recent studies have revealed that FMRFamide and NPFF modulate the homomeric ASIC1 and ASIC3 current as well as H+-gated currents from sensory neurons (Askwith et al., 2000), and the heterologously expressed heteromeric ASIC2a + 3 current (Catarsi et al., 2001). NPFF and FMRFamide are not able to generate currents on their own, but they slow down the inactivation of ASIC1a and ASIC3 currents, inducing and/or increasing the sustained phase during acidification (Askwith et al., 2000). The sustained component of those currents is thought to be important in pain coding, but its physiological relevance is largely unknown.
This study is the first report of the effects of NPSF on ASIC3 currents expressed in a heterologous system, and on ASIC3-like currents recorded from rat cultured DRG neurons. The effects of QRFamide, and of the common C-terminal motif RFamide, are also studied, and compared to the known effects of FMRFamide and NPFF. This paper is also the first to show that the effects of these neuropeptides on the sustained phase of the ASIC3 current can be correlated with an increase in neuronal excitability associated with the increase of the ASIC current-induced sustained depolarisation.
Section snippets
Expression of ASIC channels in COS cells
COS cells at a density of 20 000 cells per 35 mm diameter petri dish were transfected with a mix of the vectors pCI-CD8 and pCI-rASIC3 (1:2 ratio) using the DEAE-Dextran method. Cells were used for electrophysiological measurements 1 to 3 days after transfection. Successfully transfected cells were recognised by their ability to fix CD8-antibody-coated beads (Dynal).
Primary culture of rat DRG neurons
Dorsal root ganglion neurons were dissected from Wistar rats (5–7 weeks) and enzymatically dissociated with 0.1% collagenase.
Effects of NPSF and related peptides on ASIC3 currents expressed in COS cells
Fig. 1 shows the effects of mammalian NPSF and NPFF and of the molluscan FMRFamide on whole-cell ASIC3 currents recorded from transfected COS cells at −50 mV. RFamide, which represents the common C-terminal motif of those neuropeptides, and QRFamide, which has been found to be an inactive metabolite of NPFF in mouse brain (Sol et al., 1999), have also been tested. None of these peptides induced a significant change of the fast transient component of ASIC3 current (Fig. 1A). However, the
Discussion
This work describes the effects of NPSF and other mammalian FMRFamide-related neuropeptides on ASIC3, a channel which is expressed in nociceptive DRG neurons (Voilley et al., 2001) and which is thought to be involved in nociception (Lingueglia et al., 1997, Kress and Zeilhofer, 1999, Price et al., 2001, Reeh and Kress, 2001). We show that NPSF, like other RFamide-motif containing peptides, is able to drastically increase the inactivation rate and/or the sustained phase of homomeric ASIC3
Acknowledgements
We are grateful to N. Voilley, J. Mamet, and M. Salinas for helpful discussion, to M. Jodar and N. Leroudier for excellent technical assistance and to V. Lopez for secretarial assistance. L. Rash is gratefully acknowledged for proofreading the manuscript. The rat VR1 construct was kindly provided by Dr N. Voilley. This work was supported by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Association Française contre les
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