Currents In DRG Neurons Research Articles

Synthesis and evaluation of a novel analgesic conotoxin Lt7b that inhibits calcium currents and increases sodium currents

Conotoxins are promising neuropharmacological tools and drug candidates due to their high efficiency and specificity in targeting ion channels or neurotransmitter receptors. In this study, a novel O2‐superfamily conotoxin, Lt7b, was synthesized and its pharmacological functions were evaluated. Lt7b with three modified amino acids and three disulfide bonds was successfully synthesized. CD spectra showed that Lt7b had a typical α‐helix in the secondary structure. Patch clamp experiments on rat DRG neurons showed that Lt7b could significantly inhibit calcium currents with an IC50 value of 856 ± 95 nM. Meanwhile, 10 μM Lt7b could significantly increase the sodium currents by 77 ± 8%, but it had no obvious effects on the potassium currents in DRG neurons. In addition, patch clamp experiments on ion channel subtypes showed that 10 μM Lt7b could inhibit 7.0 ± 1.2%, 8.0 ± 1.5%, 4.6 ± 3.4%, and 9.5 ± 0.1% of the hCav1.2, hCav2.1, hCav2.2, and hCav3.2 currents, respectively, while it did not increase the rNav1.7, rNav1.8, hNav1.5, hNav1.7, and hNav1.8 currents. Lt7b had no obvious toxicity to HaCaT and ND7/23 cells up to 1 mM and significantly increased the pain threshold at the testing time of 0.5–4 h in a dose‐dependent manner in the mouse hotplate assay. This novel conotoxin Lt7b may be a useful tool for ion channel studies and analgesic drug development.

Stereospecific Effects of Benzimidazolonepiperidine Compounds on T-Type Ca2+ Channels and Pain.

T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca2+ influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited CaV3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca2+ influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca2+ influx. Whole-cell patch clamp recordings from small- to medium-sized DRG neurons revealed that both compounds decreased total Ca2+. Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed CaV3.1-3.3 channels with a similar IC50 value. 3-14-3-S decreased T-type, but not N-type, Ca2+ currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.

Increased Resurgent Sodium Currents in Nav1.8 Contribute to Nociceptive Sensory Neuron Hyperexcitability Associated with Peripheral Neuropathies.

Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the Possum transgenic mouse strain. We show that, in male DRG neurons, these mutations, which impair inactivation, significantly increase TTX-resistant resurgent sodium currents mediated by Nav1.8. The G1662S mutation doubled resurgent currents, and the T790A mutation increased them fourfold. These unusual currents are typically evoked during the repolarization phase of action potentials. We show that the T790A mutation greatly enhances DRG neuron excitability by reducing current threshold and increasing firing frequency. Interestingly, the mutation endows DRG neurons with multiple early afterdepolarizations and leads to substantial prolongation of action potential duration. In DRG neurons, siRNA knockdown of sodium channel β4 subunits fails to significantly alter T790A current density but reduces TTX-resistant resurgent currents by 56%. Furthermore, DRG neurons expressing T790A channels exhibited reduced excitability with fewer early afterdepolarizations and narrower action potentials after β4 knockdown. Together, our data demonstrate that open-channel block of TTX-resistant currents, enhanced by gain-of-function mutations in Nav1.8, can make major contributions to the hyperexcitability of nociceptive neurons, likely leading to altered sensory phenotypes including neuropathic pain in SFN.SIGNIFICANCE STATEMENT This work demonstrates that two disease mutations in the voltage-gated sodium channel Nav1.8 that induce nociceptor hyperexcitability increase resurgent currents. Nav1.8 is crucial for pain sensations. Because resurgent currents are evoked during action potential repolarization, they can be crucial regulators of action potential activity. Our data indicate that increased Nav1.8 resurgent currents in DRG neurons greatly prolong action potential duration and enhance repetitive firing. We propose that Nav1.8 open-channel block is a major factor in Nav1.8-associated pain mechanisms and that targeting the molecular mechanism underlying these unique resurgent currents represents a novel therapeutic target for the treatment of aberrant pain sensations.

α-Dendrotoxin inhibits the ASIC current in dorsal root ganglion neurons from rat

Dendrotoxins are a group of peptide toxins purified from the venom of several mamba snakes. α-Dendrotoxin (α-DTx, from the Eastern green mamba Dendroaspis angusticeps) is a well-known blocker of voltage-gated K+ channels and specifically of Kv1.1, Kv1.2 and Kv1.6. In this work we show that α-DTx inhibited the ASIC currents in DRG neurons (IC50=0.8μM) when continuously perfused during 25s (including a 5s pulse to pH 6.1), but not when co-applied with the pH drop. Additionally, we show that α-DTx abolished a transient component of the outward current that, in some experiments, appeared immediately after the end of the acid pulse. Our data indicate that α-DTx inhibits ASICs in the high nM range while some Kv are inhibited in the low nM range. The α-DTx selectivity and its potential interaction with ASICs should be taken in consideration when DTx is used in the high nM range.

887 Analgesic Action of Resolvin D1 to Combat Visceral Pain in a Rat Model of Visceral Hypersensitivity

G A A b st ra ct s root ganglia (DRG) neurons. The aim of this study was to examine the downstream signaling pathways mediating these effects, with the focus on Smad 3 and TRPV1. Methods: In vitro patch clamp studies were performed on dissociated DRG neurons. For in vivo studies, CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in adult male rats, with controls receiving the same volume of vehicle, followed by intrapancreatic injection of DiI, a retrograde neuronal tracer, to label the DRG neurons that project to pancreas. Three weeks later, SIS3 (2.5mg/kg i.p. daily) was administered for 1 week. Subsequently, thoracic DRGs were harvested and stained for pSmad3 using immunohistochemistry. Phosphorylated Smad3 (pSmad3), Smad3 and TRPV1 protein levels in the thoracic DRGs (T8T12) were measured using immunoblot assay. Results: DRG cells were cultured with vehicle (1μg/ml DMSO-solvent for SIS3), TGF β1(10ng/ml), SIS3-TGF β1(10ng/ml-0.3μM), respectively for 48 hours and then TRPV1 currents were determined by the response to 1μM of capsaicin. TGFβ1 significantly enhanced TRPV1 currents in DRG neurons in culture (99.06±24 pA/pF; n=6) compared to controls (44.77±14 pA/pF; n=8), an effect that was blocked by SIS3 (26.23±4.4 pA/pF; n=6) (p<0.01 by ANOVA). However, sis3 was unable to block TRPA1 currents induced by mustard oil, attesting to the relative specificity of this effect. In vivo, as shown previously, SIS3 significantly attenuates both somatic referred hypersensitivity (measured by VFF) and pancreatic hyperalgesia (measured by behavioral response to pancreatic electrical stimulation) in rats with CP. pSmad3 positive neurons in the DiI-labeled thoracic neurons were significantly increased in CP rats (79.06±4.72% compare with control 67.15±1.53%, p<0.05), which can be reduced by treatment with SIS3 (63.64±2.21%, P<0.05). Immunoblot confirmed that pSmad3 levels, but not total Smad3, were significantly reduced in the DRGs (T8-T12) of rats treated with SIS3 relative to vehicle treated rats (P<0.05). Finally, compared with vehicle treatment we found that SIS3 significantly reduced TRPV1 protein levels in rats with CP (106.11±9.44 vs 70.32±6.17, P<0.05). Conclusions: The present results demonstrate a novel link between TGFβ1 signaling and TRPV1 function and expression, an effect that appears to be mediated by Smad3. These results provide the fundamental basis for developing novel therapeutic approaches for CPinduced pain.

Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons

The effects of dragon’s blood and its components cochinchinenin A, cochinchinenin B, loureirin B as well as various combinations of the three components on capsaicin-induced TRPV1 receptor currents were studied in acutely dissociated DRG neurons using both voltage and current whole-cell patch clamp technique. The results indicated that dragon’s blood and its three components concentration-dependently reduce the peak amplitudes of capsaicin-induced TRPV1 receptor currents. There was no significant difference between the effects of dragon’s blood and the combination wherein the three components were present in respective mass fractions in dragon’s blood. The respective concentrations of the three components used alone were all higher than the total concentration of three components used in combination when the percentage inhibition of the peak amplitude was 50%. The proportion of three components was adjusted and the total concentration reduced, the resulting combination still inhibit the currents with a lower IC50 value, and inhibit capsaicin-induced membrane depolarization on current clamp. The combination of three components not only increase the capsaicin IC50 value, but also reduce the capsaicin maximal response. These result suggested that analgesic effect of dragon’s blood may be partly explained on the basis of silencing pain signaling pathways caused by the inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons and could be due to the synergistic effect of the three components. Antagonism of the capsaicin response by the combination of three components is not competitive. The analgesic effect of dragon’s blood was also confirmed using animal models.

Is ZD7288 a selective blocker of hyperpolarization-activated cyclic nucleotide-gated channel currents?

ZD7288 has been widely used as a tool in the study of hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), and to test the relationships between HCN channels and heart and brain function. ZD7288 is widely considered a selective blocker of HCN currents. Here we show that ZD7288 inhibits not only HCN channel currents, but also Na+ currents in DRG neurons and ZD7288 was confirmed to inhibit Na+ current in HEK293 cells transfected with Nav1.4 plasmids. Thus our findings challenge the view that ZD7288 is a selective blocker of HCN channels. Conclusions about the role of NCN channels in neuronal function should be re-evaluated if based exclusively on the effect of ZD7288.

Myricetin reduces voltage activated potassium channel currents in DRG neurons by a p38 dependent mechanism

Myricetin is a naturally occurring flavonoid known for its anti-neoplastic, anti-oxidant and anti-inflammatory effects. Currently, potential analgesic effects are proposed for several animal models of acute and chronic pain. Pilot studies show a flavonoid-induced modulation of intracellular mitogen-activated protein kinases (MAPK) as p38 and interactions with voltage activated potassium channel currents ( I K( V) ). The aim of this study was to investigate the underlying modulation of I K( V) and the influence of MAPK phosphorylation in an in vitro cell model. Whole cell patch-clamp recordings of rat dorsal root ganglion neurons were performed and I K( V) isolated. I K( V) were concentration-dependently reduced by myricetin (1–75 μM myricetin; reduction range 18–78%) with no voltage dependency (−80 to +60 mV). The reduction of I K( V) was enhanced by blocking p38 with the p38 inhibitor SB203580 (40 ± 20% without SB203580 vs. 62 ± 5% with 5 μM SB203580 or 83 ± 7% with 10 μM SB203580), but abolished by activation of p38 using anisomycin (40 ± 20% without anisomycin vs. 0.73 ± 17% with 5 μM anisomycin). We conclude that myricetin reduces I K( V) by p38 dependent mechanisms in sensory neurons. Since a reduction of I K( V) rather increases neuronal excitability, it is unlikely that this effect of myricetin contributes to its analgesic effects.

Synthetic Ciguatoxins Selectively Activate Nav1.8-derived Chimeric Sodium Channels Expressed in HEK293 Cells

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

Impact Of NaV1.7-PEPD Missense Mutations That Slow The Rate Of Inactivation On Sensory Neuronal Resurgent Sodium Currents

Voltage-gated sodium (Nav1.1-9) channels are dynamic transmembrane proteins that, in response to changes in the potential across the lipophilic cell membrane, undergo specific conformational (gating) modifications, between ion-conducting (open) and non-conducting (closed and inactivated) states, to selectively conduct sodium ions through their aqueous pore. Importantly, changes in these voltage-dependent gating properties can impact action potential (AP) characteristics. TTX-sensitive sodium channels in cerebellar neurons can produce resurgent currents (Raman & Bean, 1997), intriguing currents that are re-activated during intermediate repolarizations following strong, but short, depolarizations. We observe resurgent currents in some DRG neurons and found that wild-type Nav1.6 but not wild-type Nav1.7 channels can generate resurgent currents in DRG neurons (Cummins et al., 2005). It has been demonstrated that, in cerebellar neurons from Nav1.6-null mice, slowing inactivation of the remaining Nav current can induce resurgent currents (Grieco & Raman, 2004). Interestingly, single-point missense mutations in the SCN9A gene that encode for Nav1.7, implicated in paroxysmal extreme pain disorder (PEPD), slow the rate of Nav1.7 inactivation (Jarecki et al., 2008). Therefore, we hypothesized that slowing of Nav1.7 by PEPD mutations might induce abnormal resurgent currents, thus altering AP properties. To explore this hypothesis, we transiently transfected adult rat DRG neurons with a TTX-resistant form of human Nav1.7-wild-type or PEPD mutant cDNA and rat Nav1.8-targeted shRNA. Voltage-dependent properties were observed using whole-cell voltage-clamp electrophysiology and AP generation was tested using current-clamp electrophysiology. Recordings were made in the presence and absence of extracellular TTX. These experiments should yield insight into (1) the mechanism of resurgent sodium current generation in DRG neurons, (2) a potential additive effect in channel dysfunction observed in PEPD, and (3) how these mutant channels contribute to alterations in AP characteristics.

Analgesic α-Conotoxins Vc1.1 and Rg1A Inhibit N-Type Calcium Channels in Rat Sensory Neurons via GABABReceptor Activation

alpha-Conotoxins Vc1.1 and Rg1A are peptides from the venom of marine Conus snails that are currently in development as a treatment for neuropathic pain. Here we report that the alpha9alpha10 nicotinic acetylcholine receptor-selective conotoxins Vc1.1 and Rg1A potently and selectively inhibit high-voltage-activated (HVA) calcium channel currents in dissociated DRG neurons in a concentration-dependent manner. The post-translationally modified peptides vc1a and [P6O]Vc1.1 were inactive, as were all other alpha-conotoxins tested. Vc1.1 inhibited the omega-conotoxin-sensitive HVA currents in DRG neurons but not those recorded from Xenopus oocytes expressing Ca(V)2.2, Ca(V)2.1, Ca(V)2.3, or Ca(V)1.2 channels. Inhibition of HVA currents by Vc1.1 was not reversed by depolarizing prepulses but was abolished by pertussis toxin (PTX), intracellular GDPbetaS, or a selective inhibitor of pp60c-src tyrosine kinase. These data indicate that Vc1.1 does not interact with N-type calcium channels directly but inhibits them via a voltage-independent mechanism involving a PTX-sensitive, G-protein-coupled receptor. Preincubation with a variety of selective receptor antagonists demonstrated that only the GABA(B) receptor antagonists, [S-(R*,R*)][-3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxy propyl]([3,4]-cyclohexylmethyl) phosphinic acid hydrochloride (2S)-3[[(1S)-1-(3,4-dichlorophenyl)-ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid and phaclofen, blocked the effect of Vc1.1 and Rg1A on Ca2+ channel currents. Together, the results identify Ca(V)2.2 as a target of Vc1.1 and Rg1A, potentially mediating their analgesic actions. We propose a novel mechanism by which alpha-conotoxins Vc1.1 and Rg1A modulate native N-type (Ca(V)2.2) Ca2+ channel currents, namely acting as agonists via G-protein-coupled GABA(B) receptors.

Modulation of Acid-Sensing Ion Channel Activity by Nitric Oxide

Acid-sensing ion channels (ASICs) are a class of ion channels activated by extracellular protons and are believed to mediate the pain caused by tissue acidosis. Although ASICs have been widely studied, little is known about their regulation by inflammatory mediators. Here, we provide evidence that nitric oxide (NO) potentiates the activity of ASICs. Whole-cell patch-clamp recordings were performed on neonatal rat cultured dorsal root ganglion neurons and on ASIC isoforms expressed in CHO cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) potentiates proton-gated currents in DRG neurons and proton-gated currents in CHO cells expressing each of the acid-sensitive ASIC subunits. Modulators of the cGMP/PKG pathway had no effect on the potentiation, but in excised patches from CHO cells expressing ASIC2a, the potentiation could be reversed by externally applied reducing agents. NO therefore has a direct external effect on the ASIC ion channel, probably through oxidization of cysteine residues. Complementary psychophysiological studies were performed using iontophoresis of acidic solutions through the skin of human volunteers. Topical application of the NO donor glyceryl trinitrate significantly increased acid-evoked pain but did not affect heat or mechanical pain thresholds. ASICs may therefore play an important role in the pain associated with metabolic stress and inflammation, where both tissue acidosis and a high level of NO are present.

Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons.

Low voltage–activated (LVA) T-type Ca2+ (ICaT) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and ICaT currents. NaN/Nav1.9 was inhibited by inorganic ICa blockers as follows (IC50, μM): La3+ (46) > Cd2+ (233) > Ni2+ (892) and by mibefradil, a non-dihydropyridine ICaT antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni2+-resistant ICaT (AR-ICaT) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Aδ–like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aα/β cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-ICaT was restricted to CII-nociceptors, putative D-hair cells, and Aα/β-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aα/β-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.

Block of native and cloned vanilloid receptor 1 (TRPV1) by aminoglycoside antibiotics

Vanilloid receptor 1 (TRPV1) is a Ca 2+ permeable non-specific cation channel located at the peripheral nerve terminals and functions as a polymodal nociceptor. Neomycin, an aminoglycoside antibiotic induces analgesia in various animal models. However, the mechanism of action of neomycin has not been fully understood. In this study, we have determined the effect of neomycin on native TRPV1 in cultured embryonic DRG neurons and cloned TRPV1 heterologously expressed in Xenopus oocytes using patch clamp, double electrode voltage clamp, and Ca 2+ fluorescence imaging techniques. Here, we show that neomycin potently (IC 50 ∼400 nM) blocks TRPV1-mediated membrane currents in DRG neurons and the block is unrelated to capsaicin concentrations used to evoke currents, suggesting a non-competitive block. Similarly, capsaicin- and proton-induced currents are blocked in oocytes, but to a lesser extent. Increases in capsaicin-induced intracellular Ca 2+ levels are also reduced by neomycin. Single-channel current analyses reveal that single-channel conductance is unaffected by neomycin and there is no indication of open channel block. The predominant effect is to lower, the open probability ( P o) at both, negative and positive potentials. Kinetic analyses reveal that the number of exponential components required to fit the open time distributions remains the same or reduced, however, the longest open time constant and the area of distribution are shortened at negative and positive potentials, respectively. The area of distribution of longest closed-time constants were significantly prolonged at negative and positive potentials. We conclude that neomycin inhibits TRPV1 channel activity by allosteric binding and altering channel gating.

Direct interaction of adenosine with the TRPV1 channel protein.

Vanilloid receptor 1 (TRPV1), a nonspecific cation channel expressed primarily in small sensory neurons, mediates inflammatory thermal pain sensation. The function and expression of TRPV1 are enhanced during inflammation and certain neuropathies, leading to sustained hyperalgesia. Activation of TRPV1 in the spinal cord and periphery promotes release of adenosine, which produces analgesia by activating A(1) and A(2A) adenosine receptor (AR) on central and peripheral neurons. This study provides evidence of a direct interaction of AR analogs with TRPV1. Adenosine analogs inhibit TRPV1-mediated Ca(2+) entry in human embryonic kidney (HEK293) cells stably expressing TRPV1 (HEK/TRPV1) and DRG neurons. This inhibition was independent of A(2A)AR activation. Specific binding of [(3)H]resiniferatoxin (RTX) in plasma membrane preparations was inhibited by CGS21680, an A(2A)AR agonist. Similar degrees of inhibition were observed with both agonists and antagonists of ARs. Adenosine analogs inhibited [(3)H]RTX binding to affinity-purified TRPV1, indicative of a direct interaction of these ligands with the receptor. Furthermore, specific capsaicin-sensitive binding of [(3)H]CGS21680 was observed in Xenopus oocyte membranes expressing TRPV1. Capsaicin-induced inward currents in DRG neurons were inhibited by adenosine and agonist and antagonist of A(2A)AR at nanomolar concentrations. Increasing the concentrations of capsaicin reversed the inhibitory response to capsaicin, suggesting a competitive inhibition at TRPV1. Finally, exposure of HEK/TRPV1 cells to capsaicin induced an approximately 2.4-fold increase in proapoptotic cells that was abolished by adenosine analogs. Together, these data suggest that adenosine could serve as an endogenous inhibitor of TRPV1 activity by directly interacting with the receptor protein.

GABAB receptors and G proteins modulate voltage-dependent calcium channels in cultured rat dorsal root ganglion neurons: Relevance to transmitter release and its modulation

Acutely dissociated and cultured chick and rat dorsal root ganglion (DGR) neurons were studied by means of whole-cell patch-clamp technique. The high voltage-activated calcium current in DRG neurons is a result of mixture of N-type (ω-conotoxin-sensitive), P-type (ω-aga-IVA-sensitive), and L-type (dihydropyridine-sensitive) calcium currents. The modulation of these calcium channel currents in DRG neurons involves the GTP binding proteins and GABAB receptors. This kind of modulation occurs at presynaptic terminalsin vivo due to synaptically released GABA resulting in a primary afferent inhibition.