Lack of interaction between prostaglandin E2 receptor subtypes in regulating adenylyl cyclase activity in cultured rat dorsal root ganglion cells

The hyperalgesic response to prostaglandin E2 (PGE2) is thought to be mediated by activation of the cAMP/protein kinase A pathway in primary sensory neurones. The aim of this study was to investigate the relative contribution of different PGE2 (EP) receptor subtypes to the overall activity of adenylyl cyclase in adult rat isolated dorsal root ganglion (DRG) cells, in vitro. PGE2 and the prostanoid EP4 receptor agonist ONO-AE1-329 increased [3H]cAMP production with EC50 values of 500 nM and 70 nM, respectively, and showed similar efficacies. No combination of prostanoid EP1, EP2, EP3 or EP4 receptor selective agonists produced synergistic increases in [3H]cAMP. The prostacyclin mimetic cicaprost increased [3H]cAMP production with an EC50 value of 42 nM and produced a significantly greater maximal response compared with PGE2. No evidence for prostanoid EP3 receptor-dependent inhibition of adenylyl cyclase activity could be obtained to account for the relatively weak effect of PGE2 compared with prostacyclin receptor agonists. Interestingly, sulprostone (prostanoid EP3/EP1 receptor agonist) caused a Rho-kinase-dependent retraction of neurites, suggesting an alternative role for prostanoid EP3 receptors in DRG cells. In conclusion, PGE2 mediated increases in adenylyl cyclase activity in primary sensory neurones is likely to be mediated by activation of prostanoid EP4 receptors, and is not under inhibitory control by prostanoid EP3 receptors.     

Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons.     

Interactions between neuronal and non-neuronal cells in adult rat isolated dorsal root ganglion cells

Objective The glial cells of the central nervous system are involved in tripartite signaling,therefore we have been investigating the relationship between sensory neurons and non-neuronal cells in isolated preparations of dorsal root ganglia(DRG).Methods The mixed cell cultures of dissociated DRG cells were separated to yield enriched fractions of IB4-positive cells(small diameter,non-peptidergic cells),IB4-negative cells(small diameter,peptidergic cells,and large diameter cells),and non-neuronal cells(principally satellite glial cells,Schwann cells and fibroblasts).Adenylyl cyclase activity was assayed by measuring production of [3H]cAMP from cells preloaded with [3H]adenine.Results PGE2 and the PGI2 mimetic cicaprost stimulated adenylyl cyclase activity which was inhibited by ONO-AE3-208(EP4 antagonist)or CAY10441(IP antagonist)with estimated pA2 values of 8.9 and 8.2,respectively.Surprisingly,both PGE2 and cicaprost-stimulated [3H] cAMP production was greatest in the non-neuronal cell preparation.Furthermore,when the number of non-neuronal cells was kept constant and the number of neuronal cells was increased,we observed a progressive decrease in prostanoid-stimulated activity.Conclusions Sensory neurons appear to regulate prostanoid receptor-mediated cell signaling in non-neuronal cells within the DRG.     

川芎嗪对大鼠背根神经节细胞P2X嘌呤受体介导反应的作用

目的探讨川芎嗪(tetram ethy1pyrazine,TMP)对嘌呤2X(P2X)受体介导反应的作用。方法在大鼠新鲜分离的背根神经节(dorsal root ganglion,DRG)神经元标本上应用全细胞膜片钳技术记录川芎嗪对P2X受体激动剂激活电流的影响。结果外加ATP(1~1 000μmol.L-1)可引起DRG神经元产生激活电流(n=102),ATP-激活电流(IATP)显示快失敏和慢失敏两种形式的内向电流。预加川芎嗪(0.1~10mmol.L-1)后,大部分(89.2%,91/102)受检细胞可观察到ATP(100μmol.L-1)-激活电流出现明显的抑制作用。川芎嗪(1 mmol.L-1)使α,-βm eATP(10μmol.L-1)-激活电流减小。预加川芎嗪(1 mmol.L-1)后ATP(1~1 000μmol.L-1)激活电流的剂量-效应曲线明显下移。预加川芎嗪(1 mmol.L-1)前后ATP(100μmol.L-1)的I-V曲线反转电位值不变,均接近0 mV。川芎嗪(1 mmol.L-1)可明显抑制被前列腺素E2(100μmol.L-1)或P物质(0.1μmol.L-1)增大的ATP激活电流。通过微电极胞内透析注入PKA抑制剂H89(10μmol.L-1)至胞内,使川芎嗪(1 mmol.L-1)抑制ATP(100μmol.L-1)激活电流的作用减小。结论川芎嗪可能是通过PKA系统以及P2X受体离子通道复合体细胞外环的变构调制点影响P2X受体激动剂在大鼠DRG神经元的激活电流。     

Cell type-specific ATP-activated responses in rat dorsal root ganglion neurons

1. The aim of our study is to clarify the relationship between expression pattern of P2X receptors and the cell type of male adult rat (Wistar) dorsal root ganglion (DRG) neurons. We identified the nociceptive cells of acutely dissociated DRG neurons from adult rats type using capsaicin sensitivity. 2. Two types of ATP-activated currents, one with fast, the other with slow desensitization, were found under voltage-clamp conditions. In addition, cells with fast but not slow desensitization responded to capsaicin, indicating that there was a relationship between current kinetics and capsaicin-sensitivity. 3. Both types of neurons were responsive to ATP and alpha, beta methylene-ATP (alpha,betameATP). The concentration of alpha,(beta)meATP producing half-maximal activation (EC50) of neurons with fast desensitization was less (11 microM) than that of neurons with slow desensitization (63 microM), while the Hill coefficients were similar. Suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS) antagonized alpha,betameATP-induced currents in both types of neurons. 4. In situ hybridization revealed that small cells of the DRG predominantly expressed mRNAs of P2X3 and medium-sized cells expressed mRNAs of P2X2 and P2X3. In contrast, both of mRNAs were not detected in large cells of the DRG. 5. These results suggest that capsaicin-sensitive, small-sized DRG neurons expressed mainly the homomeric P2X3 subunit and that capsaicin-insensitive, medium-sized DRG neurons expressed the heteromultimeric receptor with P2X2 and P2X3.     

5-HT2受体介导大鼠DRG神经元膜GABA-激活电流的增强作用

目的　探讨 5 HT对大鼠DRG神经元膜GABA 激活电流的调节作用及其机制。方法　在新鲜分离的大鼠DRG神经元标本上,以全细胞膜片钳技术记录膜电流,用排管快速换液装置行胞外给药,以胞内透析技术分析信号转导途径。结果　给予GABA可使多数受检细胞产生浓度依赖性内向电流 (IGABA)。预加 5 HT,可使IGABA增加。此效应可被 5 HT2受体特异性激动剂α methyl 5 HT( 1×10-6mol·L-1 )所模拟,被 5 HT2受体选择性拮抗剂cyproheptadine所阻断。在部分细胞, 5 HT本身可引起由 5 HT3受体介导的快速内向电流,但并未发现该电流与 5 HT对IGABA的增强作用有必然的联系。从GABA激活电流的量效曲线可见,预加 5 HT后和对照曲线相比,阈浓度不变、EC50值相近,IGABA最大值增加 33. 6%。胞内透析GDP β S或H 7可取消 5 HT增强IGABA的效应,而透析H 9无效。结论　5 HT可增强GABA 激活电流,其机制为 5 HT2受体激活后通过PKC引起GABAA受体胞内磷酸化所致。     

Neurotensin-induced Cl(-) current in guinea-pig dorsal root ganglion cells

In guinea-pig dorsal root ganglion cells held under voltage-clamp at -80 mV, neurotensin elicited an inward current (I(NT)) whose amplitude increased with increasing neurotensin concentration (40-4000 nM). The effect was blocked by a nonpeptide neurotensin antagonist. I(NT) occurred in the absence of the extracellular Na(+), but not in the absence of the intracellular Cl(-), and it was outward directed by reversing the driving force for Cl(-). I(NT), like the gamma-amino-butyric acid (GABA)-induced Cl(-) current (I(GABA)), remained little changed after virtual elimination of cytosolic free-ionized Ca(2+) or after treatment with a Ca(2+)-activated Cl(-) channel blocker, but, in contrast to I(GABA) it was resistant to the I(GABA) blocker picrotoxin, slower in time course and more easily desensitized when repeatedly elicited. I(NT) and I(GABA) were additive to each other. AG-protein inhibitor markedly reduced I(NT), and a G-protein activator produced an inward current during which no current could be elicited by neurotensin. These results show that neurotensin exerts an effect to activate Ca(2+)-insensitive Cl(-) channels distinct from those activated by GABA in guinea-pig dorsal root ganglion cells, and the effect may arise through a G-protein-dependent mechanism.     

Bradykinin-induced modulation of calcium signals in rat dorsal root ganglion neurons in vitro

We used combined patch-clamp-microfluorimetric recordings to examine the effects of bradykinin on [Ca2+]i transients and the Ca2+ current (ICa) in rat dorsal root ganglion neurons in vitro. Bradykinin increased [Ca2+]i in approximately 20% of dorsal root ganglion cells examined and inhibited the ICa in approximately 65% of dorsal root ganglion cells. Bradykinin also inhibited the ICa when [Ca2+]i was buffered with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or when Ba2+ was the charge carrier. When ICa's of increasing duration were elicited in these neurons, [Ca2+]i transients were produced that increased in amplitude but eventually approached an asymptote at longer voltage steps. Similarly, the amplitude of the [Ca2+]i transient also approached an asymptote in current-clamp recordings when cells were induced to fire a large number of action potentials. The bradykinin-induced inhibition of the amplitude of the [Ca2+]i transient was more pronounced at shorter voltage steps. At pulse durations that produced asymptotic [Ca2+]i signals, bradykinin no longer decreased the amplitude of the rise in [Ca2+]i, although it still reduced the ICa. In current-clamp recordings, bradykinin also reduced the [Ca2+]i signal that accompanied the generation of action potentials, but again bradykinin was more effective for shorter spike trains. Bradykinin also depolarized the majority of neurons (65%). The reduction in [Ca2+]i produced by bradykinin in sensory neurons may be an important factor contributing to bradykinin-induced excitation of primary sensory afferents.     

Methoctramine analogues inhibit responses to capsaicin and protons in rat dorsal root ganglion neurons

We have investigated the possibility that vanilloid receptors have a binding site for polyamines and determined the consequences of binding to such a site. Whole-cell and single-channel patch-clamp recordings were used to investigate the effect of the tetraamine, methoctramine, and 16 of its analogues on capsaicin and proton induced responses of foetal rat dorsal root ganglion neurons. All but two methoctramine analogues inhibited responses to 10 microM capsaicin with IC50 values in the range of 2-70 microM at a holding potential of -100 mV. Inhibition was generally non-competitive and voltage-dependent. Methoctramine at 10 microM reduced the single channel mean open time (>3-fold), but also increased the mean closed time (1.7-fold). Sustained responses to pH 5.4 were antagonized by methoctramine with similar potency to capsaicin responses. Similar data were obtained with adult rat dorsal root ganglion neurons. These data indicate that methoctramine analogues bind to vanilloid receptors to inhibit their function.     

神经营养因子4在成年大鼠背根节神经元的分布

目的 探讨神经营养因子4（NT－4）与成年大鼠背根节神经元的关系。方法 本文用特异的NT－4抗血清以免疫组织化学方法观察了NT－4在成年大鼠背根L6节段神经元中的颁由。结果 NT－4的免疫阳性反应物分布背根节的各型神经元,胞浆染色。结论 NT－4可能与成年大鼠背根节神经元的生理功能有关。     

大麻酚类受体-1在脊髓、背角、背根神经节和周围神经的分布(英文)

AIM: The localization of CB1 receptors in the spinalcord, spinal roots, dorsal root ganglion (DRG), andperipheral nerve of the rat was determined.METHODS: We studied the distribution of CB1cannabinoid receptors by immunohistochemistry usingan antibody raised against the N-termina1 of thereceptor. RESULTS: The spinal cord showednumerous transverse fibers labelled for CB1 receptorsthroughout and concentrated in the dorsal horn.Lightly-stained cells were observed throughout thespinal cord gray matter. The DRG also showed cellsand fibers labelled for CB1 receptors. Labelled fiberswere observed in both dorsal and ventral roots as well as     

Capsazepine block of voltage-activated calcium channels in adult rat dorsal root ganglion neurones in culture

1. We have found that capsazepine, a competitive antagonist at the vanilloid (capsaicin) receptor, blocks voltage-activated calcium currents in sensory neurones.
2. The block of calcium current was slow to develop with a half time of about one minute at 100 μM and lasted for the duration of the experiment. The rate of block of calcium current was strongly concentration-dependent.
3. The EC₅₀ for the blocking effect at 0 mV was 7.7±1.4 μM after 6 min exposure to capsazepine. The EC₅₀ at equilibrium was estimated to be 1.4±0.2 μM.
4. The block of calcium current showed some voltage-dependence but there was no indication of any selectivity of action for a calcium channel subtype. The characteristics of the blocking action of capsazepine on the residual current of cells which were pretreated with either □Omega;-conotoxin or nimodipine were similar to control.
5. The data suggest that capsazepine, in addition to its competitive antagonism of vanilloid receptors, has a non-specific blocking action on voltage-activated calcium channels which should be taken into account when interpreting the effects of this substance on intact preparations in vitro or in vivo.

     

5-HT2受体介导大鼠DRG神经元膜GABA-激活电流的增强作用

目的探讨5-HT对大鼠DRG神经元膜GABA-激活电流的调节作用及其机制。方法在新鲜分离的大鼠DRG神经元标本上，以全细胞膜片钳技术记录膜电流，用排管快速换液装置行胞外给药，以胞内透析技术分析信号转导途径。结果给予GABA可使多数受检细胞产生浓度依赖性内向电流(I_GABA)。预加5-HT，可使I_GABA增加。此效应可被5-HT₂受体特异性激动剂α-methyl-5-HT(1×10^-6 mol·L^-1)所模拟，被5-HT₂受体选择性拮抗剂cyproheptadine所阻断。在部分细胞，5-HT本身可引起由5-HT₃受体介导的快速内向电流，但并未发现该电流与5-HT对I_GABA的增强作用有必然的联系。从GABA激活电流的量效曲线可见，预加5-HT后和对照曲线相比，阈浓度不变、EC₅₀值相近，I_GABA最大值增加33.6%。胞内透析GDP-β-S或H-7可取消5-HT增强I_GABA的效应，而透析H-9无效。结论5-HT可增强GABA-激活电流，其机制为5-HT₂受体激活后通过PKC引起GABA_A受体胞内磷酸化所致。     

Strychnine-induced potassium current in isolated dorsal root ganglion cells of the rat.

1. Effects of strychnine (Str) on the dissociated dorsal root ganglion (DRG) cells of the rat have been investigated in whole-cells configuration by a conventional patch-clamp technique. 2. gamma-Aminobutyric acid (GABA)-induced Cl- current (ICl) increased sigmoidally with increasing concentration. The half-maximal response (Ka) was 3 x 10(-5) M and the Hill coefficient was 1.5. Both Str and bicuculline inhibited the GABA-induced ICl in a concentration-dependent manner. 3. Str itself could elicit the current at concentrations over 10(-5) M, at which concentrations the GABA response was completely suppressed. The concentration-response curve for the Str-induced current was bell-shaped, and a nearly maximum response occurred at 3 x 10(-4) M. A transient 'hump' current appeared immediately after the wash-out of external solution containing high concentrations of Str over 3 x 10(-4) M. 4. The Str-induced outward current and a transient 'hump' current were augmented by the removal of extracellular K+ and were suppressed by the substitution of intracellular K+ for Cs+. But the current was not sensitive to extracellular Na+, Ca2+ and Cl-. 5. The reversal potential of Str-induced current (EStr) was -75 mV, which was close to the K+ equilibrium potential (EK = -76.3 mV). The change of EStr for a ten fold change in extracellular K+ concentration was 58 mV, indicating that the membrane behaves like a K+ electrode in the presence of Str. The reversal potential of the 'hump' current was also close to EK.(ABSTRACT TRUNCATED AT 250 WORDS)     

CB(1) cannabinoid receptor stimulation modulates transient receptor potential vanilloid receptor 1 activities in calcium influx and substance P Release in cultured rat dorsal root ganglion cells

Cannabinoids have been reported to have analgesic properties in animals of acute nociception or of inflammatory and neuropathic pain models, but the mechanisms by which they exert such alleviative effects are not yet fully understood. We investigated whether the CB(1)-cannabinoid-receptor agonist HU210 modulates the capsaicin-induced (45)Ca(2+) influx and substance P like-immunoreactivity (SPLI) release in cultured rat dorsal root ganglion (DRG) cells. HU210 attenuated the capsaicin-induced (45)Ca(2+) influx and this effect was reversed by the CB(1) antagonist AM251. Treatment of DRG cells with 100 nM bradykinin for 3 h potentiated capsaicin-induced SPLI release accompanied with the induction of cyclooxygenase-2 mRNA expression. The potentiation of SPLI release by bradykinin was reversed by HU210 or the protein kinase A (PKA) inhibitor H-89. HU210 also reduced forskolin-induced cyclic AMP production and forskolin-induced potentiation of SPLI release. These results suggest that CB(1) could inhibit either the capsaicin-induced Ca(2+) influx or the potentiation of capsaicin-induced SPLI release by a long-term treatment with bradykinin through involvement of a cyclic-AMP-dependent PKA pathway. In conclusion, CB(1)-receptor stimulation modulates the activities of transient receptor potential vanilloid receptor 1 in cultured rat DRG cells.     

Properties of 5-hydroxytryptamine3 receptor-gated currents in adult rat dorsal root ganglion neurones.

1. Responses to 5-hydroxytryptamine (5-HT) were examined on rat dorsal root ganglion (DRG) neurones maintained in tissue cultures, by use of whole cell recording techniques. 2. 5-HT (usually 10 microM) evoked a depolarization associated with an increase in membrane conductance in 40% of DRG neurones. There was a considerable variation in the size and persistence of this response between different batches of cells. 3. The 5-HT response was mimicked by applying the agonists 2-methyl-5-HT (10 microM) and phenylbiguanide (10 microM). Responses were blocked by ICS 205-930 (100 nM), but not by methysergide (0.1-1.0 microM). 4. 5-HT currents could be carried by sodium and caesium ions, but not by choline ions. The amplitude and duration of the 5-HT responses were dependent on the concentration of divalent cations in the extracellular solution: both became greater when calcium and magnesium concentrations were decreased. 5. Staurosporine, a putative antagonist of protein kinases, inhibited responses to 5-HT.     

Methyllycaconitine, alpha-bungarotoxin and (+)-tubocurarine block fast ATP-gated currents in rat dorsal root ganglion cells

The effects of nicotinic acetylcholine receptor antagonists were studied on currents evoked by application of ATP to rat isolated dorsal root ganglion cells, and human embryonic kidney 293 cells expressing rat P2X(3) and P2X(2/3) receptors. The rapidly desensitising (within 100 ms) current in dorsal root ganglion cells was inhibited by methyllycaconitine, alpha-bungarotoxin and (+)-tubocurarine (concentrations giving half-maximal inhibition were approximately 40, 60 and 800 nm, respectively), but not by hexamethonium (100 microm) or mecamylamine (100 microm). The sustained (>250 ms) current in dorsal root ganglion cells was inhibited by (+)-tubocurarine (80% by 10 microm), but not by methyllycaconitine (200 nm), alpha-bungarotoxin (200 nm), mecamylamine (100 microm) or hexamethonium (100 microm). Rapidly desensitising currents evoked by alpha,betamethylene-ATP in human embryonic kidney cells expressing P2X(3) receptors were inhibited by methyllycaconitine and alpha-bungarotoxin, at concentrations similar to those effective in dorsal root ganglion cells. The results indicate that some nicotinic acetylcholine receptor antagonists are potent blockers of P2X receptors on neurons, particularly the homo-oligomeric P2X(3) receptor. This finding suggests that these drugs should be used with care to discriminate between P2X and neuronal acetylcholine receptor types.     

Zaltoprofen inhibits bradykinin-induced responses by blocking the activation of second messenger signaling cascades in rat dorsal root ganglion cells

Bradykinin interacts with the bradykinin B2 receptor on dorsal root ganglion (DRG) neurons, setting off a series of reactions inside the cells that ultimately make the vanilloid receptor 1 more sensitive to a normal stimulus by activating various enzymes coupled with second messenger signaling cascades. Zaltoprofen, a propionic acid derivative non-steroidal anti-inflammatory drug (NSAID), was proved to inhibit bradykinin-induced pain responses in vivo experimental systems more potently than indomethacin or other NSAIDs, but the molecular mechanisms underlying its action are not yet fully understood. Currently it appears unlikely that zaltoprofen binds to specific sites on the protein of the bradykinin B2 receptor, hence we have examined the effect of zaltoprofen on bradykinin-induced responses of adult DRG neurons to investigate possible interaction sites. Compared with several other NSAIDs, such as indomethacin, loxoprofen and diclofenac, zaltoprofen most potently inhibits bradykinin-enhancement of capsaicin-induced 45Ca2+ uptake into DRG neurons. Zaltoprofen also significantly inhibits bradykinin-induced 12-lipoxygenase (12-LOX) activity and the slow bradykinin-induced onset of substance P release from DRG neurons. These data indicate zaltoprofen may produce its analgesic effects through the inhibition of bradykinin B2 receptor-mediated bradykinin responses of not only cyclooxygenases (COXs) but also bradykinin induced 12-LOX inhibitors.     

大麻酚类受体—1在脊髓,背角,背根神经节和周围神经的分布

AIM: The localization of CB1 receptors in the spinal cord, spinal roots, dorsal root ganglion (DRG), and peripheral nerve of the rat was determined. METHODS: We studied the distribution of CB1 cannabinoid receptors by immunohistochemistry using an antibody raised against the N-terminal of the receptor. RESULTS: The spinal cord showed numerous transverse fibers labelled for CB1 receptors throughout and concentrated in the dorsal horn. Lightly-stained cells were observed throughout the spinal cord gray matter. The DRG also showed cells and fibers labelled for CB1 receptors. Labelled fibers were observed in both dorsal and ventral roots as well as in peripheral nerves. CONCLUSION: The presence of CB1 receptors in the DRG, the dorsal root, and the dorsal horn is in accordance with the analgesic effects of cannabinoids. The presence of labelled cells and fibers in the ventral horn and ventral root provides a substrate for cannabinoid-induced muscle relaxant and antispastic effects.     

体外柴胡皂苷元d对C_6大鼠神经胶质瘤细胞前列腺素E_2生成的影响

目的　观察柴胡皂苷元d(saikogenind ,SGD)对C6大鼠神经胶质瘤细胞体外前列腺素E2 (PGE2 )生成的影响。方法　用放射性免疫法测定细胞产生的PGE2 ,液体闪烁测量法测定14 C花生四烯酸 (AA)标记细胞释放14 C AA。结果　SGD在 1～ 2 0 μmol·L-1范围内 ,抑制由钙离子载体A2 3187诱发C6大鼠神经胶质瘤细胞前列腺素E2 (prostaglandinE2 ,PGE2 )释放 ,其IC50 为 3μmol·L-1,但对花生四烯酸 (arachi donicacid ,AA)释放无影响。SGD不影响细胞微粒体组分将AA转化为PGE2 。结论　SGD抑制由钙离子载体A2 3187诱发体外C6大鼠神经胶质瘤细胞PGE2 产生 ,但不抑制AA释放和直接抑制环氧脂酶 (cyclooxygenase ,COX)活性。