Enhancement of Potency and Efficacy of NADA by PKC-Mediated Phosphorylation of Vanilloid Receptor

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1 J Neurophysiol 91: , 2004; /jn Enhancement of Potency and Efficacy of NADA by PKC-Mediated Phosphorylation of Vanilloid Receptor Louis S. Premkumar, Zhan-Heng Qi, Jeremy Van Buren, and Manish Raisinghani Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois Submitted 4 August 2003; accepted in final form 30 November 2003 Premkumar, Louis S., Zhan-Heng Qi, Jeremy Van Buren, and Manish Raisinghani. Enhancement of potency and efficacy of NADA by PKC-mediated phosphorylation of vanilloid receptor. J Neurophysiol 91: , 2004; /jn The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)- mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 M NADA is 5 10% of the current induced by 1 M capsaicin or protons at ph 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nm), a mutant TRPV1 (S800A/ S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at ph 5 ( 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nm) induced a 15 mv depolarization and initiated a train of action potentials compared with 1 M NADA that produced a 5 mv response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKCmediated phosphorylation state. INTRODUCTION Capsaicin or Vanilloid receptor 1 (TRPV1) mediates thermal sensation 43 C and is involved in inflammatory thermal hyperalgesia. A variety of stimuli activate this receptor to varying degrees of potency (Caterina and Julius 2001; Caterina et al. 1997; Di Marzo et al. 2002; Szallasi and Blumberg 1999; Szallasi and Di Marzo 2000; Tominaga et al. 1998). Vanillyl moiety-containing agents such as capsaicin (a pungent ingredient in hot chili peppers) and resiniferatoxin are potent agonists (Szallasi and Blumberg 1999). On the other hand, lipid mediators such as arachidonic acid metabolites and anandamide are weak agonists with respect to their ability to activate VR-mediated membrane currents (Hwang et al. 2000; Premkumar and Ahern 2000) but potently increase VR- mediated intracellular Ca 2 levels (Di Marzo et al. 2002; Marshall et al. 2003; Olah et al. 2001; Smart et al. 2000; Szallasi and Di Marzo 2000). In a continuing effort to identify endogenous Address reprint requests and other correspondence to: L. S. Premkumar ( lpremkumar@siumed.edu,). ligands for TRPV1, Huang et al. (2002) postulated the possible existence of an endogenous molecule that contains a vanillylamine and an acyl chain. As a result of this inquiry, N- arachidonyl-dopamine (NADA), which has a catechol moiety, very similar to the vanillyl moiety and an acyl chain, was identified in several brain regions, particularly higher concentrations are found in the striatum. Functionally, it was shown that NADA potently increased intracellular Ca 2 levels (EC nm) and at higher concentrations ( 1 M), released neuropeptides from spinal cord slices, induced hyperalgesia, and caused paired-pulse depression (Huang et al. 2002). Other VR mediated effects in the CNS include facilitated excitatory neurotransmitter/neuropeptide release from specific regions of the CNS (Jennings et al. 2003; Marinelli et al. 2003; Tognetto et al. 2001). In this study, we have investigated the ability of NADA to induce VR-mediated membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in DRG neurons, and also we tested its ability to depolarize and initiate action potentials in DRG neurons. We have found that micromolar concentrations of NADA are required to induce currents in oocytes and DRG neurons and to cause a membrane depolarization in DRG neurons. However, after stimulation of PKC, both the sensitivity and the maximal response of the receptor are significantly increased in response to NADA. More interestingly, it was observed that repeated application of NADA potentiated its own response as well as the responses of other agonists in a PKC-dependent manner. METHODS Oocyte electrophysiology TRPV1 was expressed in Xenopus laevis oocytes as described before (Premkumar et al. 2002). Animals were cared for according to the standards of the National Institutes of Health. All animal use protocols were approved by Southern Illinois University School of Medicine Animal Care Committee. Oocytes were obtained by an abdominal incision after anesthetizing the animal by immersion in a 0.05% solution of MS222. Animals were killed by subcutaneous injection of a 2% solution of MS222. A day after separating the oocytes from the follicular layer, nl of TRPV1 or a mutant TRPV1 RNA (0.5 1 mg/ml) was injected using a Drummond Nanoject (Drummond Scientific, Broomall, PA). Oocytes were used for recording from 3 days after the injection. Double-electrode voltage clamp was performed using a Warner amplifier (OC725C, Warner Instruments, Hamden, CT) with 100% DC gain. All the experiments were performed at C. Oocytes were placed in a perspex chamber and continuously superfused (5 10 The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /04 $5.00 Copyright 2004 The American Physiological Society

2 NADA-INDUCED CURRENT 1443 ml/min) with a Ca 2 -free Ringer solution containing (in mm) 100 NaCl, 2.5 KCl, 5 HEPES, and 1.5 EGTA, ph Ca 2 -free conditions were used to minimize desensitization, tachyphylaxis, and contamination from Ca 2 -activated Cl currents. Electrodes were filled with 3 M KCl and had resistances of M. To activate PKC, oocytes were exposed to PDBu (1 M) for 5 min after recording control responses. To inhibit PKC, oocytes were incubated with BIM ( nm) for 60 min before recording currents. Data were digitized and stored in videotapes. For analysis, the data were digitized at 100 Hz and analyzed using Channel 2 (software kindly provided by Michael Smith, Australian National University, Canberra, Australia). DRG culture and electrophysiology Dorsal root ganglia were isolated from embryonic day 18 (E18) rats, triturated and cultured for 5 7 days in Neurobasal medium supplemented with B-27, glutamine and 10% fetal bovine serum (FBS) (Life Technologies; Grand Island, NY) on poly-d-lysine-coated glass coverslips. For perforated-patch recording, the bath solution contained (in mm) 140 NaGlu, 2.5 KCl, 10 HEPES, 2 MgCl 2, and 1 EGTA, ph 7.35, and the pipette solution contained (in mm) 130 NaGlu, 10 NaCl, 2.5 KCl, 10 HEPES, 1 MgCl 2, and 0.2 EGTA and amphotericin B (240 g/ml). When the agonists were included in the pipette solution, the aforementioned solutions were used without amphotericin B (i.e., recordings were performed under traditional whole cell conditions). Currents were recorded using a WPC 100 patch-clamp amplifier (E.S.F. Electronic, Goettingan, Germany) or Axopatch 200B (Axon Instruments, Union City, CA). Data were digitized at 94 khz (VR-10B, Instrutech; Great Neck, NY) and stored in videotapes. For analysis, data were filtered at 2.5 khz ( 3 db frequency with an 8-pole low-pass Bessel filter, Warner Instruments, LPF-8) and digitized at 5 khz. For current-clamp recordings, the bath solution contained (in mm) 140 NaGlu, 4 KCl, 1 MgCl 2, 2 CaCl 2, and 10 HEPES, ph 7.35 and the pipette solution contained (in mm) 60 K 2 SO 4, 35 KCl, 10 NaCl, 1 MgCl 2, 40 sucrose, 10 HEPES, and 1 EGTA ph 7.3. All the chemicals used in this study were obtained from Sigma (St. Louis, MO). NADA was purchased as a 50 mg/ml ethanol stock. The working concentrations of capsaicin and PDBu were prepared fresh before the experiments from their respective ethanol stocks. Bisindolylmaleimide (BIM) was dissolved in DMSO. The final solution contained 0.001% ethanol or DMSO. Data are given as means SE and statistical significance was evaluated using the Student s t-test. Significance levels greater than P 0.01 are not shown. RESULTS NADA induces VR1 current and potentiates its own response in a PKC-dependent mechanism Application of NADA induced currents in a dose-dependent manner in Xenopus oocytes heterologously expressing TRPV1. As shown in Fig. 1A, micromolar concentrations of NADA are required to induce currents, and the response does not appear to saturate even at a concentration as high as 30 M (EC M). Fitting the data to the Hill equation gave an EC 50 of nm for capsaicin (Fig. 1B). To compare the efficacy of NADA, the responses were compared with near saturating concentrations of capsaicin and protons. The amplitude of the current induced by 10 M NADA is 5% of the current induced by 1 M capsaicin and protons at ph 4.6. Figure 1C shows the summary, comparing responses to near maximal response induced by capsaicin ( fold, n 5) or protons ( fold, n 4) normalized to 10 M NADA (n 7). Furthermore, current activation time course is slower, suggesting that either NADA is a weak agonist or unable to sufficiently access the putative binding site that is in the intracellular surface of the membrane. On the other hand, current inactivation time course is also slow suggesting either a slow dissociation rate from the binding site or partitioning of NADA in the membrane compartment. During the course of these experiments, we consistently observed that repeated application of NADA (3 or 10 M) at short intervals (60 80 s apart) potentiated its own response. Figure 2A shows one such experiment, where the initial current amplitude was 2.8 A, and at the end of eight applications, the FIG. 1. Comparison of N -arachidonyl-dopamine (NADA)- induced currents with those of capsaicin and proton in oocytes heterologously expressing vanilloid receptor 1 (TRPV1). A: 1,3,and10 M NADA produced negligible responses compared with 1 M capsaicin and protons at ph 5. B: dose response curves of capsaicin (EC nm) and NADA (EC M). C: normalized responses showing the relative potency compared with 10 M NADA (n 7), capsaicin ( fold, n 5), and ph ( fold, n 4).

3 1444 L. S. PREMKUMAR, Z.-H. QI, J. VAN BUREN, AND M. RAISINGHANI current amplitude increased to 8.6 A. A time course of repeated application (normalized to the 8th application) is shown in Fig. 2B. The current amplitude increased fold (n 4). This self-potentiating property is specific to TRPV1 expressing oocytes because repeated application of NMDA plus glycine to oocytes expressing NR1/NR2A subunits did not potentiate the current (data not shown). We have shown previously (Premkumar and Ahern 2000) that anandamide exhibited a similar potentiation with repeated application and suggested a possible involvement of PKC. Therefore we designed experiments to determine the role of PKC in this phenomenon. The potentiation was not seen after pretreatment with PDBu ( fold n 2), which maximized the NADA response (Fig. 2, B and D) or PKC antagonist BIM ( fold, n 3; Fig. 2, D and E), implicating the involvement of PKC. To determine the specificity of TRPV1 involvement, we used a double mutant (S502A; S800A), in which the consensus sites have been mutated rendering the receptor insensitive to PKC-mediated phosphorylation (Numazaki et al. 2002). In oocytes expressing the double mutant, repeated application of NADA (10 M) did not potentiate the current ( fold, n 4, Fig. 2, C E). These experiments indicate that NADA has the ability to self-potentiate the TRPV1-mediated currents in a PKC-dependent manner. Potentiation of NADA-induced TRPV1 current by pretreatment with PDBu VR-mediated current is strongly potentiated by PKC-mediated phosphorylation (Numazaki et al. 2002; Premkumar and Ahern 2000; Tominaga et al. 2001; Vellani et al. 2001). Here we show that pretreatment with a PKC activator PDBu strongly potentiated ( fold, n 4) NADA-induced responses (Figs. 3, A and D). We and others (Crandall et al. 2002; Premkumar and Ahern 2000) have shown that PDBu could induce a small current by itself. In this study, we repeatedly FIG. 2. Repeated application of NADA potentiates its own response in a protein kinase C (PKC)-dependent manner. A: current traces showing repeated application of NADA potentiates its own response (current amplitude increased from 2.8 to 8.6 A. B: after maximally activating PKC with phorbol 12,13-dibutyrate (PDBu), repeated application of NADA does not potentiate its own response. C: the self-potentiation is abolished in the double mutant (S502A; S800A) in which, PKC phosphorylation sites have been mutated. D: graph showing normalized (to the 8th application) responses in wild-type, mutant, and after treatment with bisindolylmaleimide (BIM). E: summary graph showing lack of potentiation after PDBu in wild type ( fold, n 2), in the double mutant ( fold, n 4), and after BIM ( fold, n 3). observed a small and sustained current induced by PKC ( fold, n 4 responses are normalized to 10 M NADA, Fig. 3, inset and D), which was not seen in the phosphorylation site mutant TRPV1. The potentiation is due to an increase in the sensitivity as well as an increase in the maximal response of the receptor. Increased sensitivity was indicated by a leftward shift in the dose response curve (EC 50 value shifted from 10 to M). Interestingly, the maximal response was also increased almost 15-fold (Fig. 3B). It could be argued that the concentrations of NADA were not high enough to induce a maximal response prior to PDBu application. However, in a separate study, using other agonists, we are addressing the possibility that PKC- mediated phosphorylation not only increases the receptor sensitivity but also activates silent channels or recruits newer channels into the plasma membrane. To determine the involvement of phosphorylation of TRPV1 in this phenomenon, we made use of the double PKC phosphorylation site mutant. The potentiation of current induced by pretreatment with phorbol esters was reduced significantly in the double mutant ( fold, n 4; Fig. 3, C and D). However, interestingly after PDBu treatment, there was a twofold potentiation in the double mutant, indicating other potential phosphorylation sites. Potentiation of proton responses by pretreatment with NADA Because NADA is able to activate PKC, we hypothesized that it should be able to potentiate the responses of other agonists. We used a ph range (6 5) that showed the greatest potentiation after treatment with PDBu. ph 5.5 induced a small current; however, after application of 10 M NADA (5 8 times), the ph response was significantly potentiated ( fold n 4; Fig. 4, A and C). The potentiation is dependent on the number of times the cell was exposed to NADA, the maximum potentiation is seen after five to eight applications. Given the lipophilic nature of NADA, there is a possibility that accumulation of NADA in the membrane might be responsible

4 NADA-INDUCED CURRENT 1445 for the potentiating effect. However, if NADA is accumulating in the membrane, then the response should be additive not synergistic. Moreover, the potentiation was also significantly reduced ( fold, n 3) in cells expressing the phosphorylation site mutant TRPV1 (S502A; S800A; Fig. 4, B FIG. 4. Potentiation of proton-induced current after pretreatment with NADA. A: ph response is potentiated after 8 applications of 10 M NADA. B: in the double phosphorylation site mutant, the potentiation is dramatically reduced. C: summary graph showing potentiation of proton current after 8 applications of 10 M NADA: wild type ( fold, n 3) and double mutant ( fold, n 3). FIG. 3. Potentiation of NADA-induced current by PKC activator PDBu. A: responses to 1, 3, and 30 M NADA are potentiated dramatically after pretreatment with PDBu. Inset: PDBu itself induces a small-sustained current ( fold, n 4, normalized to 10 M NADA). B: dose-response curve showing that both the sensitivity (EC 50 shifted from 10 to 2.73 M) and the maximal responses are enhanced ( fold, n 4). C: PDBu response is dramatically reduced in oocytes expressing the mutant TRPV1 ( fold, n 4). D: summary graph showing the fold increase normalized to 10 M NADA: 1 M PDBu ( fold, n 4); 10 M NADA after PDBu ( fold, n 4); 1 M capsaicin ( fold, n 4); capsaicin after PDBu ( fold, n 4). and C). In essence, pretreatment with NADA potentiates the response to other agonists. Ability of NADA to generate action potentials and membrane currents in DRG neurons To study the effect of NADA on native VRs, we recorded membrane currents and depolarizations in voltage- and currentclamp conditions, respectively. Using perforated-patch recording technique, we found that micromolar concentrations of NADA were required to induce measurable currents. NADA induced currents in a dose-dependent manner. The mean current amplitude of 10 M NADA was 22% of the current induced by 1 M capsaicin. After application of PDBu, current amplitude for 10 M NADA was significantly increased ( fold, n 3, P 0.01; Fig. 5, A and B). In DRG neurons compared with oocytes, NADA induced responses were larger and the potentiation by PDBu was smaller. This could be due to basal phosphorylation state of the VR (Vellani et al. 2001). As observed in oocytes, NADA potentiated its own response with repeated applications ( fold n 5, P 0.01; Fig. 5, C and D). It is also possible that in the phosphorylated state, in addition to sensitization of the receptor, the effectiveness of a transporter may be increased (Ortar et al. 2003). We considered the possibility that the smaller currents induced by NADA could be due to its inability to sufficiently access the putative intracellular binding site on VR. To address this issue, we included capsaicin or NADA in the pipette solution, and the current amplitude was measured by blocking the response with capsazepine (10 M). Figure 5, E and F, shows that the relative potency of 10 M NADA was increased to 41% when applied intracellularly as compared with 1 M capsaicin. In this condition, PDBu further significantly potentiated NADA current ( fold, n 2, P 0.01). These

5 1446 L. S. PREMKUMAR, Z.-H. QI, J. VAN BUREN, AND M. RAISINGHANI results suggest that an inability of NADA to sufficiently access its binding site on VR contributes partly to its weaker action. Next, we recorded changes in membrane potential under current-clamp conditions. Application of 1 M NADA to cells that had a membrane potential of mv (n 6), induced a depolarization of mv (n 5). In the same cells, 100 nm capsaicin produced a robust depolarization of mv (n 5) followed by a burst of action potentials, which lasted almost the entire duration of capsaicin application (Fig. 6). To confirm the role of PKC-mediated phosphorylation, DRG neurons were pretreated with PDBu (1 M). The depolarization induced by NADA after PDBu was significantly larger ( mv) as compared with before ( mv, n 2). DISCUSSION FIG. 5. NADA-induced currents in capsaicin-sensitive dorsal root ganglion (DRG) neurons. A: dosedependent response of NADA (3, 10 M) and the potentiation by pretreatment with PDBu. Note application of PDBu alone induced a small current. B: summary graph showing the current responses normalized to 10 M NADA (1 M capsaicin: fold, n 5; 10 M NADA after PDBu: fold, n 5). C: NADA potentiates its own response with repeated application. D: normalized response of repeated application of NADA ( fold, n 5). E: when applied intracellularly (included in the pipette), NADA exhibits increased potency relative to capsaicin and in the presence of PDBu. F: summary graph showing the responses of NADA [normalized to 10 M NADA, (n 3); Cap 1 M ( fold, n 3); NADA 1 M ( fold, n 2) and NADA 1 M PDBu ( fold, n 2)]. Although VRs can be maximally activated by multiple integrating stimuli, the quest for identifying endogenous ligands remains unabated. The primary function of TRPV1 located at the peripheral nerve endings is to sense painful thermal stimuli 43 C (Caterina et al. 1997; Cesare and McNaughton 1996; Szallasi and Blumberg 1999; Tominaga et al. 1998). Furthermore, studies on TRPV1 knock-out mouse have indicated that it is more closely associated with inflammatory thermal hyperalgesia rather than acute thermal pain (Caterina et al. 2000; Davis et al. 2000). Significantly, distribution of these receptors in regions that do not encounter noxious temperature ranges, especially in the CNS (Jennings et al. 2003; Marinelli et al. 2003; Mezey et al. 2000), further strengthens our resolve to identify and characterize potent endogenous ligands. Compromised urinary bladder function in the TRPV1 knock-out mouse and the presence of TRPV1 in urothelium are significant findings further corroborating nonsensory functions of TRPV1 (Birder et al. 2001, 2002). Identification of NADA as a TRPV1 agonist broadens the horizon of the role of VR beyond that as a thermal sensor. Role of phosphorylation in TRPV1 function Direct phosphorylation of VR is critical for its functions. Several studies have pointed out the importance of PKC- and PKA-mediated phosphorylation in VR function. Bradykinin (BK)- and extracellular ATP-induced sensitization of VR is mediated by PKC (Cesare and McNaughton 1996; Olah et al. 2002; Premkumar and Ahern 2000; Tominaga et al. 2001; FIG. 6. NADA-induced depolarizations in DRG neurons. Capsaicin 100 nm induced a robust depolarization ( mv, n 6) and generated action potentials compared with 1 M NADA produced a small response ( mv, n 6).

6 NADA-INDUCED CURRENT 1447 Vellani et al. 2001). PKC also sensitizes heat responses in a manner that the threshold for VR activation is reduced from 42 to 35 C, which means VRs could be active at normal body temperature in certain conditions (Tominaga et al. 2001). PKC has been shown to directly phosphorylate two amino acid residues (S502 and S800) in TRPV1 protein (Numazaki et al. 2002). On the other hand, potentiation of VR responses by prostaglandins is mediated via PKA (Lopshire and Nicol 1997, 1998). PKA-mediated phosphorylation has been shown to enhance TRPV1 activity by anandamide (De Petrocellis et al. 2001b). PKA causes a robust effect in sensory neurons, but it lacks that response in oocytes expressing TRPV1 (Lee et al. 2000); this raises the possibility that TRPV1 protein may not be phosphorylated directly, instead an associated protein may be required (Distler et al. 2003). However, abolition of PKAmediated desensitization by repeated application of capsaicin after substituting serine 116 suggests that TRPV1 is a primary target of this enzyme (Bhave et al. 2002; Mohapatra and Nau 2003). PKC-mediated phosphorylation could act as a weak agonist by phosphorylating VR in the case of PDBu and PMA (Crandall et al. 2002; Premkumar and Ahern 2000). In contrast to PDBu, PMA has been shown to interact with the capsaicinbinding site (Chuang et al. 2001; Vellani et al. 2001). We have consistently observed a PDBu-induced current in oocytes expressing wild-type TRPV1 but not in PKC phosphorylation site mutant TRPV1 and in DRG neurons, suggesting that phosphorylation of VRs either acts directly as a ligand or alters temperature sensitivity to enable channel gating at room temperature (Cesare and McNaughton; 1996; Tominaga et al. 2001). Our observations suggest that phosphorylation acts as a weak ligand and is able to promote the transition from a closed to an open state of the channel. When the second ligand binds, the barrier height is dramatically lowered, favoring the open conformation of the channel. The VR has multiple ligand binding sites both at intra- and extracellular sites. Substitution of amino acid residues can affect binding and gating of one agonist but not the others (Jordt et al. 2000; Prescott and Julius 2003; Welsh et al. 2000). Other mechanisms priming TRPV1 include relief of suppressive action of PIP 2, which mediates the actions of nerve growth factor (NGF) and BK, rendering the channel active even at the 25 C (Chuang et al. 2001; Prescott and Julius 2003). Elevation of intracellular Ca 2 and ATP binding to the Walker-domain region of the TRPV1 influence the channel function positively (Kress and Guenther 1999; Kwak et al. 2000). The implication of having multiple binding sites is that it increases the probability of the channel being in the open state by weak agonists binding to their respective sites. Interestingly, these interactions give rise to a synergistic rather than an additive response. This supports the idea that in the phosphorylated state the channel is partially liganded and binding of a second weak agonist can bring about a synergistic response. In the phosphorylated state, the sensitivity of the TRPV1 is increased as indicated by a shift in the dose-response curve. Interestingly, the maximal response also increased several folds, suggesting either activation of silent channels or incorporation of newer channels into the plasma membrane. Only a slight potentiation was seen in the PKC phosphorylation site mutant after PDBu confirming the direct involvement of TRPV1 in PKC-mediated phosphorylation. Lipid mediators as TRPV1 ligands Lipid mediators such as lipoxygenase metabolities of arachidonic acid, anandamide, and NADA are weak agonists with respect to their ability to activate VR-mediated membrane currents (Hwang et al. 2000; Premkumar and Ahern 2000). However, anandamide and NADA have been shown to potently increase intracellular Ca 2 levels (Di Marzo et al. 2002; Huang et al. 2002; Smart et al. 2000). It is possible that while measuring intracellular Ca 2 levels, Ca 2 -induced Ca 2 release caused by small increases in VR- mediated Ca 2 flux could account for this apparent potency. Another possibility is that TRPV1 is also located in the membranes of intracellular organelles, which on activation cause release of Ca 2 from the stores by these membrane-permeable analogues (Liu et al. 2003; Marshall et al. 2003). A third possibility is that these lipid molecules may get accumulated in the plasma membrane and reach sufficient concentrations to activate the receptor. This study shows that in the phosphorylated state the receptor sensitivity is increased (EC 50 shifted from 10 to 2.73 M) and the maximal response increased several fold ( 15). We also show that repeated application of NADA increases its own response in a PKC-dependent manner, suggesting that NADA could activate PKC by similar mechanisms as that of anandamide (De Petrocellis et al. 1995). To test whether NADA could activate PKC, pretreatment with NADA potentiated low ph responses several-fold ( 20). To confirm the involvement of PKC, a specific PKC blocker BIM and a PKC phosphorylation site mutant TRPV1 were used. The self-potentiation of NADA response, potentiation of responses to other agonists, and potentiation induced by PDBu were all significantly reduced. Is NADA an endogenous ligand of the VR? Huang et al. (2002) have identified NADA to be a potent activator of VR-mediated functions. Even though NADA lacked the vanillyl moiety, a catechol moiety appears to be a good substitute for activation of VR. NADA increased intracellular Ca 2 levels with an EC 50 of 33 nm, which is similar to that of capsaicin (26 nm). NADA increased neuropeptide release in a capsaicin-sensitive manner and suggested the involvement of VRs. Intraplantar injection of NADA caused hyperalgesia, which could be due to a combination of activation and sensitization of the receptor by the release of inflammatory mediators. NADA could also significantly modify paired-pulse inhibition. These responses could be blocked by capsazepine and iodortx, suggesting the involvement of TRPV1 in the CNS. The role of TRPV1 in the CNS is not yet clearly understood but is suggestive of a direct or indirect role of NADA in modifying neurotransmitter release (Huang et al. 2002). Recent studies have also shown selective anandamide action modulated VR-mediated excitatory neurotransmitter release (Jennings et al. 2003; Marinelli et al. 2003; Tognetto et al. 2001). The levels of NADA in physiological conditions are not clearly measured because it is produced depending on demand. The levels could be analogous to those of anandamide, which is thought to be 100 nm (Szolcsanyi et al. 2000). Although micromolar concentrations of NADA are required to induce membrane currents, the receptor sensitivity and the maximal response are increased several-fold after PKC-medi-

7 1448 L. S. PREMKUMAR, Z.-H. QI, J. VAN BUREN, AND M. RAISINGHANI ated phosphorylation. Therefore multiple integrating factors synergistically activating the receptor remains a viable option to explain the results. Because of high-input resistance of the nerve terminals, opening of a few VR channels would enable generation of action potentials and modulate transmitter release. Weaker potency of NADA may also relate to its insufficient accessibility to its putative intracellular binding site. Activating phosphorylation cascades could potentiate NADA responses by enhancing the activity of TRPV1 and fatty acid transporters (Ortar et al. 2003). NADA homologue anandamide has been shown to activate TRPV1 from an intracellular site (De Petrocellis et al. 2001a). To address these possibilities, we included NADA in the pipette solution and assessed the current amplitude by blocking the response with capsazepine (10 M). At equimolar concentrations (1 M), NADA induced an 50% response of capsaicin. This could be due to increased accessibility to the binding site. However, stimulation of PKC potentiated NADA-induced current suggesting phosphorylation could further augment the response. Although NADA appears to be a weak agonist of TRPV1, multiple integrating physical, chemical, and biochemical stimuli acting synergistically could bring about a significant physiological response. ACKNOWLEDGMENTS The TRPV1 clone was kindly provided by D. Julius. The phosphorylation site mutant TRPV1 was kindly provided by M. Tominaga. GRANTS This work was supported by National Institute of Neurological Disorder and Stroke Gramt NS to L. S. Premkumar. REFERENCES Bhave G, Zhu W, Wang H, Brasier DJ, Oxford GS, and Gereau RW 4th. camp-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation. 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