N-methyl-d -aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C

Neuroscience 128 (2004) 347–357

N-METHYL-D-ASPARTATE RECEPTORS ENHANCE MECHANICAL RESPONSES AND VOLTAGE-DEPENDENT Ca2ⴙ CHANNELS IN RAT DORSAL ROOT GANGLIA NEURONS THROUGH PROTEIN KINASE C V. V. CHABAN,1,2 J. LI,2 H. S. ENNES, J. NIE, E. A. MAYER AND J. A. MCROBERTS

to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2ⴙ channels which contribute to mechanically induced [Ca2ⴙ]i transients through a PKC-mediated process. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

CNS/WH: Center for Neurovisceral Sciences and Women’s Health, and Division of Digestive Diseases, Department of Medicine, University of California, Warren Hall, Room 14-103, 900 Veterans Avenue, Los Angeles, CA 90095, USA

Key words: phorbol ester, GF109203X, voltage-dependent calcium channels, patch clamp, signal transduction.

Abstract—N-methyl-D-aspartate (NMDA) receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2ⴙ]i). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1–3 days. [Ca2ⴙ]i responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2ⴙ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 ␮M NMDA in the presence, but not the absence, of co-agonist (10 ␮M D-serine) caused transient [Ca2ⴙ]i responses (101ⴞ9 nM) and potentiated [Ca2ⴙ]i peak responses to subsequent mechanical stimulation more than two-fold (P<0.001). NMDA-mediated potentiation of mechanically induced [Ca2ⴙ]i responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 ␮M), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 ␮M PDBu for 1–2 min) also potentiated mechanically induced [Ca2ⴙ]i responses nearly two-fold (P<0.001), while longer exposure (>10 min) inhibited the [Ca2ⴙ]i transients by 44% (P<0.001). Both effects of PDBu were prevented by prior treatment with GFX. Inhibition of voltage-dependent Ca2ⴙ channels with 25 ␮M La3ⴙ had no effect on mechanically induced [Ca2ⴙ]i transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2ⴙ currents by a process that was sensitive

In neurons, changes in intracellular calcium concentration ([Ca2⫹]i) regulate electrical activity, neuropeptide secretion, synaptic transmission, and gene expression. Sensory neurons located in the dorsal root ganglia (DRG) express a number of voltage- and ligand-gated Ca2⫹ channels that are important in the transmission of nociceptive signals from the periphery to the spinal cord (reviewed in: Scholz and Woolf, 2002; Julius and Basbaum, 2001). Many of these channels are also important in efferent functions of sensory nerves in the periphery in that they regulate the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP; Fisher and Bourque, 2001). Sensory nerve terminals also express other cation channels such as the vanilloid receptor 1 (VR1) that are stimulated by heat and acid, as well as capsaicin, the active ingredient in hot peppers (Di Marzo et al., 2002). The mechanisms involved in mechanical sensitivity are incompletely understood, but presumably involve different types of stretch-activated channels (SACs) on the peripheral nerve endings. The SACs are permeable to both Na⫹ and Ca2⫹, and include channels of the transient receptor potential family that also include VR1 (Gunthorpe et al., 2002). SACs are inhibited by the trivalent cation Gd3⫹ (but not La3⫹) and by the diuretic amiloride and its analogues. We previously reported that mechanoreceptors are linked to [Ca2⫹]i changes in cultured DRG neurons (Chaban et al., 2001; Gschossmann et al., 2000; Raybould et al., 1999). This Ca2⫹ influx pathway is most likely the same as the SAC recently described in the DRG neurons (McCarter et al., 1999; Cho et al., 2002; Takahashi and Gotoh, 2000; Drew et al., 2002) Our studies using both rat and mouse DRG neurons indicated that mechanically induced [Ca2⫹]i transients depend primarily on Ca2⫹ influx through plasma membrane channels other than voltage-sensitive Ca2⫹ channels, since they are abolished in Ca2⫹-free extracellular medium, inhibited by Gd3⫹, but insensitive to La3⫹. We have recently shown that extrinsic primary afferent nerves innervating visceral tissues express N-methyl-Daspartate (NMDA) receptors (NMDARs) on their peripheral

1

Present address: Department of Neurobiology, University of California, Los Angeles, CA, USA. 2 These authors contributed equally to this publication. *Corresponding author. Tel: ⫹1-310-825-4786; fax: ⫹1-310-794-2864. E-mail address: [email protected] (J. A. McRoberts). Abbreviations: [Ca2⫹]i, intracellular calcium concentration; CGRP, calcitonin gene-related peptide; CNS, central nervous system; DMEM, Dulbecco’s Modified Eagle’s medium; DRG, dorsal root ganglia; GFX, GF109203X; HBSS, Hanks’ balanced salt solution; NMDA, N-methylD-aspartate; NMDARs, N-methyl-D-aspartate receptors; NO, nitric oxide; NOS, nitric oxide synthase; PDBu, phorbol dibutyrate; PKC, protein kinase C; SACs, stretch-activated channels; TEA, tetraethylammonium; VDCC, voltage-dependent Ca2⫹ channel; VR1, vanilloid receptor 1.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.06.051

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Fig. 1. NMDAR activation enhances mechanically stimulated [Ca2⫹]i transients in DRG neurons. (A) A representative [Ca2⫹]i response to repeated mechanical stimulation (double stimulation protocol) under control conditions with a time interval of 10 min between stimuli. Graphs show features of a typical mechanoresponse, with both a rapid rise after stimulation (marked by arrow) followed by a slower recovery. Addition of NMDA (100 ␮M) by microejection in the presence of D-serine (indicated by bar) produced a transient Ca2⫹ increase in DRG neurons and increased amplitude of mechanically induced [Ca2⫹]i response (B). Stimulation with NMDA in the absence of co-agonist did not change the mechanical [Ca2⫹]i response (C). The specific PKC inhibitor GFX (5 ␮M) had no effect on mechanical responses (D) but decreased amplitude of mechanically induced Ca2⫹ transients after addition of NMDA (E). Prior addition of 25 ␮M La3⫹ to inhibit VDCC also eliminated the enhanced mechanical response following NMDA addition (F), but had no effect on mechanically mediated [Ca2⫹]i responses in the absence of NMDA (not shown). The arrows in each trace indicate times of mechanical stimulation. Experimental time and [Ca2⫹]i concentration indicated by bars.

terminals where they are involved in the transduction of noxious mechanical stimulation (McRoberts et al., 2001). NMDARs, one of the three major subtypes of ionotropic glutamate receptors, are highly expressed in the central nervous system (CNS) and in peripheral nerves, and are distinguished from most other glutamate receptors by gating of Ca2⫹ in addition to Na⫹ (Michaelis, 1998). NMDAR are composed of an essential NR1 subunit, which can be produced from eight splice variants, and one or more of four NR2 subunits denoted NR2A-D (Cull-Candy et al., 2001). Voltage-dependent Ca2⫹ channels (VDCC), VR1 and NMDAR are regulated by protein kinases in different ways. For example, phosphorylation can modulate channel kinetics or alter the number of functional receptors on the cell surface by trafficking of channels in and out of the plasma membrane. In particular Ca2⫹-dependent and Ca2⫹-independent isoforms of protein kinase C (PKC) have been shown to increase or decrease VR1 and VDCC activity by direct phosphorylation (Catterall, 2000; Numazaki et al., 2002; Olah et al., 2002). PKC-mediated channel phosphorylation can either potentiate or suppress the activity of NMDARs depending on the NR2 subunits and the NR1 splice variants involved (Grant et al., 1998; Lan et al., 2001; MacDonald et al., 2001). Conversely, NMDARs can also couple to a number of signal

transduction pathways including PKC, calmodulin kinase II, nitric oxide synthase, and MAP kinases (Leonard et al., 2002; Perkinton et al., 2002; Chandler et al., 2001; Gardoni et al., 1998, 2001a; Llansola et al., 2001). For some of these effects, NMDAR-mediated Ca2⫹ influx is essential suggesting that the coupling occurs through a rise in [Ca2⫹]i. However there is also evidence that the intracellular domain of NMDARs can interact with scaffolding proteins, some of which also bind protein kinases (Sutton and Chandler, 2002; Gardoni et al., 2001b; Zheng et al., 1997). Exactly how NMDARs located on peripheral terminals of DRG neurons are involved in the transduction of noxious mechanical stimulation is not clear. Using an in vitro colon– splanchnic nerve preparation, direct stimulation of NMDAR altered the sensitivity of single afferent fibers to distension of the colonic segment (Wei et al., 2001). The responses of fibers with high threshold sensitivity were enhanced by pretreatment with NMDA, while those with low threshold sensitivity were inhibited. These observations suggest that NMDARs may regulate mechanical sensitivity of peripheral DRG nerve terminals. Here, by studying mechanically induced [Ca2⫹]i changes in rat DRG neurons in primary culture, we aimed to confirm the interaction between NMDAR activation and mechanosensitive Ca2⫹ influx, and evaluate the possible role of PKC in this process.

V. V. Chaban et al. / Neuroscience 128 (2004) 347–357

EXPERIMENTAL PROCEDURES Primary culture Primary cultures of rat DRG were prepared as previously described (McRoberts et al., 2001). The procedure was modified by reducing trypsin concentration to 0.5 mg/ml, and adding 100 ␮M ketamine to the digestion buffer and culture media. Ketamine is a reversible NMDAR channel blocker which can prevent the cytotoxic effects of glutamate in the culture media on neurons expressing the receptor. Briefly, lumbosacral DRG were collected from the rat (levels T12–S2) under sterile technique, and placed in ice cold Dulbecco’s Modified Eagle’s medium (DMEM; Sigma Chemical Company, St. Louis, MO, USA). Adhering fat and connective tissue were removed and each DRG was minced with scissors and placed immediately in a medium consisting of 5 ml of DMEM containing 0.5 mg/ml of trypsin (Sigma Type III), 1 mg/ml of collagenase (Sigma Type IA) and 0.1 mg/ml of DNAase (Sigma Type III) and kept at 37 °C for 15 min with occasional agitation. After dissociation of the cell ganglia, soybean trypsin inhibitor (Sigma Type III) sufficient to neutralize twice the amount of trypsin in the medium was added. This cell suspension was centrifuged for 1 min at 1500 r.p.m. and the cell pellet was resuspended in DMEM supplemented with 10% fetal bovine serum, 10% horse serum, 2 mM glutamine, penicillin, streptomycin, 1 ␮g/ml DNAase and 5 ng/ml nerve growth factor (Sigma-RBI, St. Louis, MO, USA). Neurons were plated on Matrigel-coated coverslips (Collaborative Research Company Co., Bredford, PA, USA). The cells were left undisturbed for 24 h at 37 °C in a 10% CO2 incubator, given fresh media, and studied after a further 24 –72 h in culture. All efforts were made to minimize the number of animals used and their suffering. All procedures involving the use of animals in this study conformed to local and international guidelines on the ethical use of animals and were approved by the Chancellor’s Animal Research Committee at UCLA.

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Calcium fluorescence imaging The methods for Ca2⫹ imaging have been previously described in detail (Chaban et al., 2001). Briefly, cells cultured on coverslips were incubated in 5 ␮M Fura-2 AM for 1 h at 37 °C in Hanks’ balanced salt solution (HBSS) containing 20 mM HEPES, pH 7.4. Then, cells were washed twice in HBSS–HEPES and incubated at 37 °C for an additional 10 min to allow dye deesterification before use. The coverslips were mounted in a chamber and placed on the stage of a Zeiss Axiovert 100 TV inverted microscope (Carl Zeiss, Göttingen, Germany) equipped with a 40⫻ Achrostigmat objective lens. Measurements were carried out at room temperature (20 –22 °C) with constant perfusion (1 ml/min) via a peristaltic pump (Rainin Instrument Company, Woburn, MA, USA) with HBSS either alone or together with factors. Fluorescence intensity at 505 nm with excitation at 334 nm and 380 nm was followed by videomicroscopy and captured as digital images (sampling rates 0.1 s during transients). DRG neurons with diameters ⬍40 ␮m were identified by micrometry and regions of interest were identified within the soma from which quantitative measurements were made by re-analysis of stored image sequences using the Attofluor RatioVision system (Atto Instruments, Rockville, MD, USA). Intracellular calcium was determined from the ratio of Fura-2 fluorescence and calibrated using a series of buffered calcium standards.

Focal mechanical stimulation Borosilicate glass capillaries (1B150-4; World Precision Instruments, Sarasota, FL, USA) were pulled with a Narishige puller (Tokyo, Japan) and heat polished to produce 1 ␮m diameter tips. Light, focal mechanical stimulation was provided by a 1 ␮m tip diameter fire-polished glass pipette that depressed the cell membrane for 0.5 s and then retracted as previously described (Chaban et al., 2001; Gschossmann et al., 2000). The pipette was connected to an electronically controlled micropositioner (Model 5171, Eppendorf AG, Hamburg, Germany) and programmed to travel downward 1–2 ␮m and the prodding velocity was 300 ␮m/s. The same deflection and force was used for a second subsequent

Fig. 2. Summarized data on effect by NMDA on enhanced mechanosensitivity in DRG neurons. Data from a number of experiments similar to those shown in Fig. 1 are summarized as the ratio of the peak amplitude of [Ca2⫹]i response during the second mechanical stimulation relative to that of the first (control) stimulus. Values are the mean⫾S.E.M. with the number of observations for each condition indicated within the bar.

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Fig. 3. Activation of PKC by phorbol ester modulates mechanically induced Ca2⫹ responses. Short-term pretreatment with 1 ␮M PDBu (1 min) increased the amplitude of mechanically induced Ca2⫹ transients (A). Long-term pretreatment with 1 ␮M PDBu (10 min) inhibited this response (C). Both short- and long-term effects of PDBu could be prevented by addition of 5 ␮M GFX (B and D). Prior addition of 25 ␮M La3⫹ prevented the short-term enhancing effect of PDBu treatment (E), while prior addition of 100 ␮M Gd3⫹ nearly eliminated the response (F).

stimulation to obtain stable and reproducible results (Chaban et al., 2001; Gschossmann et al., 2000). At the end of the series, the tip deflection was observed under transmitted light in order to verify that the membrane depression was slight and had not visibly damaged the cell.

Whole cell patch clamp Voltage-gated calcium channel currents were recorded with the whole cell patch clamp technique, using an Axopatch 1D amplifier (Axon Instruments, Inc.) controlled by the pClamp 8.0 software. Data were digitized at 10 kHz, filtered at 5 kHz, and stored in a personal computer for analysis. Pipettes were pulled (model P-97 Micropipette Puller; Sutter Instruments Corporation) from borosilicate capillaries (Warner Instruments, Hamden, CT, USA). The pipette tips were heat-polished and had resistances of 2.0 –3.5 M⍀ when filled with intracellular buffer which contained (in mM): 110 CsF, 10 CsCl, 10 EGTA, 10 HEPES, 1 MgCl2, 0.2 CaCl2, 15 tetraethylammonium (TEA)-Cl, 5 Na2ATP, 0.5 Na3GTP, pH 7.2 with CsOH, osmolarity 300 –310. Bath solution contained (in mM): 155 TEA-Cl, 2 CaCl2, 10 HEPES, 10 glucose, 200 nM tetrodotoxin, pH 7.4 with TEA–OH, osmolarity 300. Cells were voltageclamped at ⫺80 mV, unless indicated. Series resistances were monitored throughout the experiments. Drugs were delivered by perfusion onto neurons using computer-controlled gravity-fed multibarrel perfusion system.

5-phosphopeonic acid and HEPES were obtained from SigmaRBI. Fura-2 AM was purchased from Molecular Probes, Eugene, OR, USA.

Statistical analysis All data represent mean⫾standard error throughout the text with n equal to the number of experiments. The average amplitude of [Ca2⫹]i change represents the difference between baseline concentration and the transient peak in response to mechanical or chemical stimulation. Significant differences in the increase in [Ca2⫹]i in response to mechanical stimulation with or without drug treatment were obtained by comparison of the percentage increase in [Ca2⫹]i during the first and second mechanical stimulation. For the multiple comparison tests (Figs. 2 and 4), the data were logarithmically transformed in order to equalize the variances between groups. In the patch clamp experiments, the effect of drug treatment on peak inward Ca2⫹ currents was expressed as the ratio of the response after treatment relative to before treatment. Statistical significance was determined using the Student’s t-test, or with groups of observations by ANOVA followed by Tukey’s post-test comparisons (GraphPad Prism software, San Diego, CA, USA). A P level of ⬍0.05 was considered significant.

RESULTS

Drugs

NMDA enhances mechanically induced [Ca2ⴙ]i responses in rat sensory neurons

Phorbol-12,13 dibutyrate (PDBu) and NMDA were purchased from Calbiochem, Irvine, CA, USA. GF109203X (GFX), 2-amino-

Short-term primary cultures of isolated DRG neurons express functional NMDARs that have properties character-

V. V. Chaban et al. / Neuroscience 128 (2004) 347–357

Fig. 4. Effect of modulation of PKC activity on mechanosensitivity of sensory neurons. Data from a number of experiments (n indicated within the bar) similar to those shown in Fig. 3 are summarized as the ratio of the response to second and first mechanical stimulus. For purposes of presentation, data are presented in two panels. Significance was determined by ANOVA of all 10 conditions shown in both panels, and the P values are the result of the individual Tukey post-test comparisons. Panel A shows the effect of 1 min and 10 min stimulation with 1 ␮M PDBu on mechanical responses and the inhibition of both effects by 5 ␮M GFX. It also shows the effect of 25 ␮M La3⫹ in blocking potentiation by PDBu. Panel B compares the effect of 30 ␮M nifedipine, 100 ␮M Gd3⫹ and the combination of nifedipine and Gd3⫹. In addition to the comparisons shown, the effect of nifedipine⫹1 min PDBu was not significantly different from La3⫹⫹1 min PDBu.

istic of NMDARs in the CNS including voltage-dependent blockage by extracellular Mg2⫹ (McRoberts et al., 2001). In order to activate NMDARs in our experimental setting, we used Mg2⫹-free HBSS for all experiments. Since glutamate activates other ion channels and metabotropic receptors, glutamate was replaced by NMDA to selectively activate NMDARs. D-Serine, a physiologically significant co-agonist for NMDAR, was used instead of glycine in order to avoid possible glycine-inhibitory currents (Mothet

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et al., 2000). Under these conditions, superfusion with 100 ␮M NMDA resulted in [Ca2⫹]i transients of 101⫾9 nM in 38% of all small (⬍40 ␮m) DRG neurons tested (24 of 63), similar to our previous results (McRoberts et al., 2001). The [Ca2⫹]i response of DRG neurons to mechanical stimulation was preserved in Mg2⫹-free HBSS. Mechanical stimulation of single cultured DRG neurons by brief (0.5 s) depression of cells with a glass microprobe produced transient [Ca2⫹]i increases of 270⫾12 nM in approximately 59% of all tested neurons (59 of 100 neurons, ⬍40 ␮m diameter). Mechanical stimulation did not cause membrane injury, as indicated by the lack of dye leakage monitored by the fluorescence intensity of the Fura-2 signal. In approximately 64% of neurons, [Ca2⫹]i returned to within 10% of baseline levels. Only these neurons were selected for further study. In these neurons, a second mechanical stimulation with the same force gave a [Ca2⫹]i response similar in amplitude to the first one. A response of a single neuron to two mechanical stimulations given 10 min apart is illustrated in Fig. 1a. A refractory period between mechanically induced [Ca2⫹]i responses is needed to allow [Ca2⫹]i levels to return to basal levels, thereby permitting a second [Ca2⫹]i response of similar magnitude. Although reproducible [Ca2⫹]i responses can be elicited at shorter intervals (Chaban et al., 2001; Gschossmann et al., 2000), a time interval of approximately 10 min was chosen to standardize the method and allow for drug additions. With this double-stimulation experimental protocol, the relative effect of various treatments was calculated as the ratio between the amplitude of the experimental (second) and control (first) responses. For the control situation where no drug or inhibitor was added before the second mechanical stimulation, the ratio of the response was 0.92⫾0.06 (n⫽5; summarized in Fig. 2), a value not significantly different from 1.0. Superfusion with NMDA (100 ␮M) after the first mechanical response resulted in [Ca2⫹]i transients in a portion of the cells (42%). In those cells responding to NMDA with a detectable increase in [Ca2⫹]i (peak amplitude 102⫾20 nM, n⫽10), the amplitude of second mechanically induced [Ca2⫹]i response elicited 2 min later was increased more than two-fold (Fig. 1b, summarized results in Fig. 2, n⫽14). The enhancing effect of NMDA was transient and by 10 min after addition was no longer apparent (ratio of response⫽0.97⫾0.10, n⫽5). NMDA added without co-agonist, D-serine, had no effect on mechanically induced [Ca2⫹]i response (Fig. 1c, and Fig. 2), nor did addition of D-serine without NMDA (0.92⫾0.06, n⫽3). In neurons not responding to NMDA in the presence of Dserine with a measurable [Ca2⫹]i transient, the subsequent response to mechanical stimulation was generally not enhanced (n⫽66). However, in two instances mechanically mediated Ca2⫹ influx were potentiated by the combination of NMDA and D-serine in the absence of a detectable [Ca2⫹]i response. Furthermore, in neurons responding to NMDA, the amplitude of the [Ca2⫹]i response to NMDA did not correlate with the degree of enhancement (Pearson r⫽0.45, n⫽10).

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PKC inhibition reduces potentiation of mechanically induced [Ca2ⴙ]i changes by NMDA As shown in Fig. 1d, and Fig. 2, the PKC inhibitor GFX (1 ␮M) had no significant effect on the amplitude of the mechanically induced Ca2⫹ response. GFX is a potent and specific inhibitor of PKC that binds to the ATP site in a competitive manner (Toullec et al., 1991). Treatment with GFX prior to stimulation with NMDA had no effect on the [Ca2⫹]i response to NMDA (117⫾21 nM, n⫽5 vs. 102⫾20, n⫽10 for NMDA response in the presence or absence of GFX, respectively), but eliminated NMDA enhancement of the mechanical response. In fact, the second mechanical response after the combination of GFX and NMDA tended to be smaller than the first control mechanical stimulation (ratio⫽0.52⫾0.08, n⫽5; Fig. 1e, and Fig. 2). Effect of PKC activation on mechanosensitive Ca2ⴙ response To determine if PKC is directly involved in enhancing the mechanical [Ca2⫹]i response, we tested the effect of PDBu, a phorbol ester that directly stimulates PKC. Shortterm (60 –100 s) activation of PKC by PDBu (0.1–1 ␮M) significantly increased the amplitude of the mechanically mediated [Ca2⫹]i transients (Fig. 3A, summarized data on Fig. 4A). Conversely, longer term pretreatment (⬎10 min) with PDBu suppressed mechanically induced Ca2⫹ influx (n⫽6, Fig. 3C and Fig. 4A). Both the short- and long-term effects of PDBu were prevented by prior addition of GFX, demonstrating the effect of PDBu was specific (Fig. 3B, D). Role of VDCC in NMDA and PKC-mediated enhancement of mechanical [Ca2ⴙ]i responses To test whether influx through VDCC accounts for the enhanced Ca2⫹ response to mechanical stimulation after short-term treatment with NMDA or PDBu, we evaluated the effect of La3⫹ on these responses. Concentrations of 20 –25 ␮M La3⫹ have been shown to block VDCC (Cunningham et al., 1995), but not to have an effect on mechanosensitive Ca2⫹ influx in DRG neurons (Chaban et al., 2001). Addition of 25 ␮M La3⫹ had no effect on the response to subsequent addition of NMDA (97⫾13 nM, n⫽4; P⫽0.9 vs. control), but it eliminated NMDAR-mediated enhancement to the second mechanical stimulus (Fig. 1F, Fig. 2). Similarly, the enhancing effect of short-term pretreatment with PDBu on mechanically induced responses was also eliminated by 25 ␮M La3⫹ (Fig. 3e, Fig. 4A). Prior addition of the selective L-type VDCC antagonist, nifedipine, also prevented enhancement of mechanical responses by short term PDBu pretreatment (Fig. 4B). Mechanical responses after addition of 30 ␮M nifedipine in the presence of PDBu were smaller, but not statistically different from that after addition of 25 ␮M La3⫹, suggesting that PDBu mediates enhancement Ca2⫹ uptake mainly through activation of L-type VDCCs. Addition of 100 ␮M Gd3⫹ nearly abolished mechanically mediated [Ca2⫹]i responses, and to a lesser extent inhibited responses after short-term PDBu pretreatment (Fig. 3F, Fig. 4B). The combination of Gd3⫹ and nifedipine significantly reduced me-

chanical responses after short-term PDBu pretreatment compared with nifedipine or Gd3⫹ added individually (Fig. 4B; P⬍0.001 in both cases). These results suggest that activation of PKC by NMDA or PDBu acts to sensitize DRG neurons such that L-type VDCC become activated during mechanical stimulation. That the effects of Gd3⫹ and nifedipine are partially additive suggests that PDBu does not directly enhance Ca2⫹ influx through SACs, but rather acts to enhance Ca2⫹ influx through L-type VDCCs. Enhancement of L-type VDCC currents by NMDA In order to directly assess whether stimulation of DRG neurons with NMDA enhances VDCC activity, whole cell patch clamp experiments were performed under voltage clamp conditions. The intracellular (patch pipette) and extracellular buffers were modified such that voltage-gated K⫹ and Na⫹ channels were blocked and the predominant permeant cation was Ca2⫹. Voltage-gated influx of Ca2⫹ was elicited by brief (150 ms) depolarization of the plasma membrane to ⫹10 mV from a resting potential of ⫺80 mV. Under these conditions, inward currents of 1836⫾201 pA were elicited from the majority of DRG neurons (Fig. 5a). Nearly half (49⫾4%) of this current was inhibited by 30 ␮M nifedipine (Fig. 5b). The reverse potential extrapolated from the current-voltage relationship both before and after nifedipine was consistent with Ca2⫹ being the charge carrier (Fig. 5c). Treatment of the neurons with NMDA in the presence of glycine caused a small, transient inward current in eight of 26 neurons (66⫾13 pA). In those neurons responding to NMDA, voltage-gated Ca2⫹ currents elicited by depolarization were enhanced 56⫾19% relative to control cells not treated with NMDA. There was no shift in the voltage dependence, which peaked at ⫹10 mV (Fig. 5c). The effect of NMDA on VDCC currents was time dependent with a maximal effect seen at 10 min (Fig. 6a). Prior treatment of the neurons with the PKC inhibitor GFX abolished the effect of NMDA on VDCC (Fig. 6a, b). Inhibition of L-type VDCC with nifedipine also eliminated the effect of NMDA on VDCCs (Fig. 5c, Fig. 6b). These results confirm that NMDA acting through PKC selectively enhances Ltype VDCC currents in DRG neurons.

DISCUSSION The results of this study suggest that NMDARs are coupled to PKC activation in rat DRG neurons. Stimulation of PKC either by activation of NMDARs or by phorbol esters transiently increases mechanically induced Ca2⫹ influx by “sensitizing” the neuron such that Ca2⫹ influx is higher due to contributions from both VDCCs and SAC channels. The results also demonstrate that activation of NMDARs transiently increase L-type VDCC Ca2⫹ currents by approximately 50% through a PKC-dependent process. The simplest explanation linking these two effects would involve PKC-mediated sensitization of L-type VDCCs. However, NMDA pretreatment did not cause a leftward shift in the current-voltage relationship, but simply increased the peak current responses. This result suggests either an increase in open channel probability or an increase in the density of

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Fig. 5. NMDA enhances voltage-activated, nifedipine-sensitive Ca2⫹ currents. Prior stimulation of the neuron with 250 ␮M NMDA in combination with 10 ␮M glycine increased Ca2⫹ currents elicited 10 min later by depolarization to ⫹10 mV from holding potential of ⫺80 mV (A). Voltagegated Ca2⫹ currents were partially inhibited by 30 ␮M nifedipine (B). The current-voltage relationship was established by recording at voltages from ⫺50 mV to 60 mV with 10 mV increments from a holding potential at ⫺80 mV. Measurements were made 10 min after addition of NMDA, nifedipine, or nifedipine⫹NMDA (C). In all cases, inward calcium currents activated at potentials greater than ⫺40 mV, reached a peak at ⫹10 mV, and then decreased at more positive potentials, giving the reversal potential at approximately 60 mV. However, peak currents were increased by NMDA and decreased by nifedipine relative to control cells not treated with drugs. Nifedipine also inhibited the increase in peak current mediated by NMDA.

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Fig. 6. Enhancement of VDCC current by NMDA is transient and inhibited by GFX. Perfusion of 250 ␮M NMDA combined with 10 ␮M glycine steadily increased currents of voltage-gated calcium channels elicited by depolarization to 10 mV from holding potential of ⫺80 mV over 10 min, and slowly declined thereafter (A). Pre-treatment with 1 ␮M GFX before addition of NMDA prevented the increase in current. In both control and GFX-treated cells there was a slow rundown in the activity of VDCC. The peak currents measured at 10 min from the start of all the experiments depicted in Figs. 5 and 6 are summarized in B and expressed relative to the initial Ca2⫹ current. Under control conditions, VDCC activity declined to 92⫾7% of the original value, whereas it increased to 144⫾14% in the presence of NMDA.

the channels in the plasma membrane, without a change in voltage-dependent activation. We conclude that increased Ca2⫹ flux through VDCCs probably accounts for at least part of the enhancement of mechanical [Ca2⫹]i responses by NMDA in the FURA-2 imaging experiments. However two observations suggest that NMDA is having effects on other ion channels as well. First, there was no apparent contribution of Ca2⫹ influx through VDCC to mechanically evoked responses before NMDA treatment, suggesting that NMDAR stimulation may be removing a tonic block on VDCC. Second, since NMDA pretreatment did not change

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Ca2⫹ influx through the SAC, influx of Na⫹ and the magnitude of SAC-mediated depolarization is not likely to be increased after NMDA. Two possibilities that could account for both observations include an effect of NMDAR acting though PKC on either the resting membrane potential or on voltage-dependent Na⫹ channels that underlie action potentials. For example, PKC has been shown to inhibit K⫹ channels in capsaicin-sensitive DRG neurons isolated from embryonic rat pups (Zhang et al., 2001). Alternately, NMDA might cause a shift in the voltage activation of voltage-gated Na⫹ channels such that they open at lower potentials. Indeed PKC has been shown to increase tetrodotoxin-resistant voltage-gated Na⫹ currents in rat DRG neurons (Gold et al., 1998). Finally, we also cannot rule out a contribution of Ca2⫹-induced Ca2⫹ release to the enhanced mechanical response after NMDA treatment. NMDA-mediated sensitization of neurons and its effect on VDCC exhibit slightly different time courses. Enhancement of mechanically induced Ca2⫹ transients by NMDA occurs within minutes and by 10 min is no longer apparent, while the increase in Ca2⫹ current through L-type VDCCs peaks 10 min after NMDA addition. This discrepancy could be due to differences in the experimental settings altering NMDAR coupling to PKC or PKC-mediated activation of VDCCs. However, as discussed above, a more likely explanation is that NMDARs are stimulating other processes in the intact cell. One process that could explain rapid termination of the VDCC-mediated enhancement of the mechanical Ca2⫹ response in intact cells could be NMDAR-mediated stimulation of nitric oxide (NO) synthase (NOS). NMDARs have been shown to activate neural NOS through interactions with the scaffolding protein PSD 95 (Christopherson et al., 1999). Thus, stimulation of the neurons with NMDA could cause an increase in NO production. We have previously demonstrated that inhibition of neural NOS enhances mechanically mediated [Ca2⫹]i transients more than two-fold (Chaban et al., 2001). This enhancement, like the phenomenon described here, is sensitive to 25 ␮M La3⫹, implying that NO inhibits Ca2⫹ influx through VDCC which can be prevented by NOS inhibitors. However, NMDAR-mediated production of NO would not occur in neurons in the whole cell patch configuration since the intracellular concentration of arginine, the substrate for NOS, would be vastly diluted. Coupling of NMDARs to PKC Regardless of the uncertainties correlating NMDARmediated increases in VDCC currents with enhancement of mechanically stimulated Ca2⫹ responses, the results strongly support a role for PKC in mediating both processes. A number of studies in cultured neurons and in recombinant cells expressing NMDARs have shown that activation of NMDARs results in stimulation of PKC activity (Hasham et al., 1997; Wagey et al., 2001; Jiang et al., 2002; Raval et al., 2003). NMDAR-mediated influx of Ca2⫹ would be expected to increase the activity of Ca2⫹dependent isoforms of PKC. Roberts and McLean (1997) have shown that adult rat DRG express Ca2⫹ dependent PKC isoforms (PKC-␣, -␤1, -␤2 and -␥). DRG are also

known to express Ca2⫹-independent and atypical isoforms, PKC-␦, -⑀, and -␨ (Bareggi et al., 1995; Raval et al., 2003). Since enhanced responses to mechanical stimulation were reproducibly observed after a measurable [Ca2⫹]i response to NMDA, our results support a role of a Ca2⫹-dependent PKC isoform in the process. However, we also observed two instances in which mechanically mediated Ca2⫹ influx was potentiated by NMDA in the absence of a detectable [Ca2⫹]i increase. Furthermore the amplitude of the bulk [Ca2⫹]i response to NMDA did not correlate with the degree of enhancement. These observations could be taken as evidence for a role of a Ca2⫹independent PKC isoform; however, in both instances the subplasmalemmal concentrations of [Ca2⫹]i may be very different from the measurements made with Fura-2 in the bulk cytoplasm. Indeed in the whole cell patch experiments the pipette buffer contained EGTA, which would buffer large changes in [Ca2⫹]i within the cell, although not necessarily near the plasma membrane (Fox et al., 1987). Thus, from the current observations, we cannot conclude which PKC isoform is involved in mediating this effect. The results do suggest that there must be a close association of NMDAR with one or more isoforms of PKC. Exactly how this interaction occurs is not known. NMDARs have been shown to directly bind and regulate several enzymes that initiate signal transduction cascades. Most of these interactions depend on the presence of specific subunits of the NMDAR. We have found that DRG neurons express predominately NR2B and NR2D (Marvizon et al., 2002). NR2B subunits bind specifically to calmodulin kinase II (Bayer et al., 2001) and indirectly though the scaffolding protein PSD95 to neural NOS (Christopherson et al., 1999). Although PKC has not been found to be associated with NMDAR complexes in neurons, PKC is known to phosphorylate NMDARs containing NR2A and NR2B subunits (Grosshans and Browning, 2001) implying a close interaction. Alternatively, the tyrosine phosphorylated SH2 cytoplasmic domain of NR2B can directly bind phospholipase C-␥ (Gurd and Bissoon, 1997), and NMDAR activation was found to activate phospholipase C (Fragoso and Lopez-Colome, 1999; Dixon et al., 1994). In a recent report Raval et al. (2003) found in hippocampal neurons that NMDAR can activate the Ca2⫹-independent isoform, PKC-⑀, through Ca2⫹-influx dependent activation of phospholipase C. In this manner NMDAR could couple to both Ca2⫹-dependent and independent isoforms of PKC. Regulation of VDCC by PKC DRG neurons express L-, N-, T- and P/Q-type VDCCs (Acosta and Lopez, 1999; Scroggs and Fox, 1992) that can be distinguished by selective inhibitors. In our preparation, we found that nifedipine-sensitive L-type VDCCs contribute about 50% of the voltage-activated Ca2⫹ current in small to medium size DRG neurons. The nifedipineinsensitive Ca2⫹ current represents a mixture of the N- and P/Q-type of VDCCs (Li, unpublished observations). Since nifedipine prevented NMDA-stimulated enhancement of voltage-activated Ca2⫹ currents, the most likely explana-

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tion is that NMDARs acting through PKC selectively stimulates L-type VDCC. PKC has been shown in a number of studies to transiently increase activity of L-type VDCCs in cardiac and smooth muscle followed by a prolonged decrease in activity (Catterall, 2000). The mechanisms involved in the transient increase in channel activity have not been defined. However the decrease in Ca2⫹ channel activity is due to phosphorylation of two threonine residues near the Nterminal of the Ca2⫹ pore-forming ␣1 subunit of the L-type channel (designated CaV1.2 ␣1), which are missing in the splice variants of CaV1.2 ␣1 expressed in brain (McHugh et al., 2000). Thus PKC does not inhibit L-type VDCCs in brain (Stea et al., 1995) and there are only a few instances where PKC has been shown to increase the L-type channel activity in neural or neural-like tissues: the pheochromocytoma derived PC12 cells (Taylor et al., 2000; Hsu et al., 2000) and peripheral neurons from either sympathetic (Sculptoreanu et al., 2001) or DRG (Hall et al., 1995). In the latter case, direct intracellular application of a constitutively active form of PKC into isolated DRG neurons was shown to increase both L- and N-type VDCC currents (Hall et al., 1995). Since in our experiments NMDAR activation did not increase nifedipine resistant Ca2⫹ currents, one could speculate that NMDARs may couple to a specific PKC isoform, which selectively activates L-type, but not other VDCC currents. The molecular mechanism involved in L-type VDCC activation could involve direct phosphorylation of one of the channel subunits, analogous to the well-described effect of cAMP-dependent protein kinase on L-type channels (Sculptoreanu et al., 1995) or could involve increased insertion of channels into the membrane (Altier et al., 2002). Indeed NMDAR activation is known to regulate rapid insertion and removal of AMPA receptors into synapses of the CNS leading to the phenomenon of long-term potentiation and depression. Furthermore, PKC was shown many years ago to “recruit” a new type of VDCCs in Aplasia bag cell neurons (Strong et al., 1987) by a process probably involving insertion of channels into the plasma membrane. Elucidating which of these pathways is involved in activation of L-type channels in DRG neurons by NMDARs will require more knowledge about the nature and trafficking of the L-type channels expressed in DRG neurons. Summary and conclusions NMDAR regulation of PKC and its effect on VDCC has important implications for the physiological action of NMDAR on DRG nerve terminals. Although our measurements were made on the cell bodies of primary sensory neurons, the same ion channels and signal transduction mechanisms most likely occur in the terminals (Gold et al., 1996). Therefore, while L-type VDCC are not thought to have an important role in neurotransmitter release in central synapses (Fisher and Bourque, 2001; Gruner and Silva, 1994; Malmberg and Yaksh, 1994), these channels may have a role in neurotransmitter release from nonsynaptic DRG terminals in the periphery (Harding et al., 1999; Morimoto et al., 1996; Frew and Lundy, 1995; Fisher

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and Bourque, 2001). Furthermore, while nearly all primary afferents are glutamatergic, a subset of C-fibers is peptidergic and contains substance P and/or CGRP. Release of these neuropeptides in the periphery contributes to neurogenic inflammation, while central release is important in nociceptive transmission to spinal dorsal horn neurons. Activation of NMDAR would be expected to enhance neuropeptide release both at termini in the spinal cord and the periphery. Indeed in spinal cord slices, activation of NMDARs plays an import role in substance P release and nociceptive transmission to spinal neurons (Marvizon et al., 1997). In the periphery, NMDA stimulates release of both substance P and CGRP (McRoberts et al., 2001). Our results also suggest an explanation for the role of NMDAR in nociceptive painful distensions of the lower gastrointestinal tract. In afferent fibers with high threshold, which probably correspond to nociceptors, NMDA has been shown to transiently enhance distension-induced firing (Wei et al., 2001). Whether this effect is due to PKCmediated enhancement of VDCC activity or other ion channels on the peripheral terminals remains to be investigated. Acknowledgments—This study was supported by NIH grants RO1 DK58173 and P50 DK64539-01 (E.A.M.). The authors thank Drs. Enrique Rozengurt and Richard Waldron for their helpful discussions.

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(Accepted 24 June 2004) (Available online 21 August 2004)