An electrophysiological investigation of the effects of cisplatin and the protective actions of dexamethasone on cultured dorsal root ganglion neurones from neonatal rats

It this study we have investigated the acute and chronic effects of cisplatin on whole cell currents in cultured dorsal root ganglion neurones. Consistent with effects on action potentials measured under current clamp, acute (5 min) application of cisplatin (5 M) attenuated voltage-activated potassium, and mixed cation currents by approximately 50% in both cases. Chronic treatment (5–7 days) of cultured neurones with 5 M cisplatin also resulted in greatly reduced voltage-activated potassium currents (by 50%) and calcium currents (by 60%) compared to events recorded from neurones not treated with cisplatin. In contrast, the amplitude of inward cation current activated by hyperpolarization was doubled by 5–12 days treatment with cisplatin. Studies on action potential after-depolarizations and calcium-activated chloride currents suggest that cisplatin disturbs calcium homeostatic mechanisms. These observations may account for anode break spike excitation and the low efficiency with which cells buffer intracellular calcium following cisplatin treatment.Dexamethasone has been found to enhance the anti-emetic effects of 5-HT₃ receptor antagonists in patients treated with cisplatin. For this reason the actions of dexamethasone were studied in combination with cisplatin treatment. Although acute application of dexamethasone (1–10 M) produced transient depolarizations and bursts of action potentials, after 5 minutes application it had no effect on membrane potential, input resistance, or the properties of action potentials evoked by depolarizing current commands. Compared to cells exposed to cisplatin alone, combined treatment of cisplatin and dexamethasone significantly improved survival of dorsal root ganglion neurones in culture by 20%. Dexamethasone also showed signs of protecting neurones from cisplatin by improving membrane potentials and action potential thresholds. In conclusion, cisplatin reduces the viability of dorsal root ganglion neurones in culture and alters their electrophysiological properties. Evidence suggests that dexamethasone has some protective properties against the neurotoxic actions of cisplatin.     

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca²⁺-induced Ca²⁺ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 μM), and by ryanodine (10 μM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca²⁺ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 μM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-d-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 μM) and ryanodine (10 μM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of –90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential. Received: 6 July 1998 / Accepted: 30 November 1998     

A study comparing the actions of gabapentin and pregabalin on the electrophysiological properties of cultured DRG neurones from neonatal rats

Background

Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca²⁺ channels. In this study we have used whole cell patch clamp recording and fura-2 Ca²⁺ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca²⁺ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents.

Results

Pregabalin (25 nM – 2.5 μM) inhibited 20–30% of the high voltage-activated Ca²⁺ current in cultured DRG neurones. The residual Ca²⁺ current recorded in the presence of pregabalin was sensitive to the L-type Ca²⁺ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca²⁺ channels but the majority of Ca²⁺ current was resistant to both drugs. The continual application of GABA, the GABA_B receptor antagonist CGP52432, or intracellular photorelease of GTP-γ-S had no effect on pregabalin-induced inhibition of Ca²⁺ currents. Although clear inhibition of Ca²⁺ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca²⁺ influx in response to pregabalin. The enhanced Ca²⁺ influx evoked by pregabalin was mimicked by partial block of K⁺ conductances with tetraethylammonium. Pregabalin produced biphasic effects on voltage-activated K⁺ currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K⁺ currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP.

Conclusions

Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca²⁺ and K⁺ channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A.     

Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones.

1. The gamma-aminobutyric acid (GABA)-mimetic actions of some anaesthetics and the antehelminthic avermectin B1a were examined on freshly isolated mammalian dorsal root ganglion (DRG) neurones by use of suction electrodes and a single electrode voltage clamp. 2. Pentobarbitone (60 microM-3 mM), chloralose (600 microM-1 mM), etomidate (10-100 microM), alphaxalone (10-60 microM) and avermectin (10-60 microM) directly activated chloride channels in GABA-sensitive DRG neurones. The agonist action was sensitive to block by bicuculline and picrotoxinin. 3. Steady-state current-voltage (I-V) curves for the anaesthetics were either linear, or rectified in the opposite direction to steady-state I-V curves obtained with GABA. Current relaxations in response to voltage jumps were also of the opposite direction. An extra surge of current ('bounce') was commonly observed on washout of some of these agonists. 4. Pentobarbitone was ineffective as an agonist at alkali pH (10.4 and 9.4), but was approximately twice as effective at acid (5.4) than at normal (7.4) pH values. 5. These results suggest that some anaesthetics and avermectin are capable of 'blocking' GABA channels in addition to activating them.     

Paclitaxel and vinorelbine, evoked the release of substance P from cultured rat dorsal root ganglion cells through different PKC isoform-sensitive ion channels

Many patients suffer from serious adverse effects including respiratory distress and pulmonary edema during and after chemotherapy with paclitaxel or vinorelbine. These effects appear to be due to the activation of neurokinin-1 receptors. The present study investigated the influences of paclitaxel and vinorelbine on the substance P (sP) release from cultured dorsal root ganglion (DRG) cells using a radioimmunoassay. Both paclitaxel and vinorelbine evoked sP release in a dose- and time-dependent manner within 60 min at a concentration range of 0.1-10 μM. The sP release levels induced by the two drugs were attenuated by pretreatment with the protein kinase Cs (PKCs) inhibitors (bisindolylmaleimide I and Gö6976). Moreover, the paclitaxel- or vinorelbine-induced sP release was diminished in the absence of extracellular Ca²⁺ or the presence of LaCl₃ (an extracellular Ca²⁺ influx blocker). A Ca²⁺ imaging assay further indicated that both paclitaxel and vinorelbine gradually increased the intracellular Ca²⁺ concentration, and these increases lasted for at least 15 min and were suppressed by Gö6976. Paclitaxel caused the membrane translocation of only PKCβ within 10 min after stimulation, whereas vinorelbine induced the translocation of both PKCα and β. The paclitaxel- and vinorelbine-induced sP release levels were separately inhibited by ruthenium red (a transient receptor potential (TRP) channel blocker) and gabapentin (an inhibitor of voltage-gated Ca²⁺ channels (VGCCs)). These findings suggest that paclitaxel and vinorelbine evoke the sP release from cultured DRG cells by the extracellular Ca²⁺ influx through TRP channels activated by PKCβ and VGCCs activated by both PKCα and β, respectively.     

Effect of lafutidine on CGRP release from cultured rat dorsal root ganglion cells

Lafutidine increased capsaicin-stimulated CGRP release from isolated rat stomach without changing basal CGRP levels. In order to clarify the mechanism of this effect, we used cultured rat DRG cells and measured CGRP release. (1) DRG cells were treated with each drug, and the CGRP content of the supernatant was determined by EIA. (2) RGM-1 cells were co-cultured with DRG cells through a cell culture insert, and capsaicin-evoked CGRP release from the DRG cells was determined when lafutidine or PGE₂ was added to the RGM-1 cells. (3) The supernatant of isolated rat stomach incubated with lafutidine was added to cultured DRG cells, and capsaicin-evoked CGRP release was determined. PGE2, but not lafutidine, augmented capsaicin-evoked CGRP release from DRG cells. Lafutidine did not modulate CGRP release from DRG cells, even though it sensitized CGRP-containing afferent nerves in the rat stomach. Lafutidne and PGE₂ may have different mechanisms of sensitization.     

Pharmacological properties of P2X3-receptors present in neurones of the rat dorsal root ganglia

1. The electrophysiological actions of several agonists which may differentiate between P2X₁- and P2X₃-receptors were studied under concentration and voltage-clamp conditions in dissociated neurones of 1–4 day old rat dorsal root ganglia.
2. β,γ-Methylene-D-ATP (β,γ-me-D-ATP) (1–300 μM), diadenosine 5′,5′′′-P¹,P⁵-pentaphosphate (AP5A) (100 nM–300 μM), diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP4A) (300 nM–300 μM) and uridine 5′-triphosphate (UTP) (1 μM–1 mM) all activated concentration-dependent inward currents with a latency to onset of a few ms.
3. The concentration-response curves for β,γ-me-D-ATP and AP5A and ATP had similar maximum values, while that for AP4A had a lower maximum. The concentration-response curve to UTP was shallow and did not reach a maximum. β,γ-Methylene-L-ATP was virtually inactive. The rank order of agonist potency was ATP>AP5A≈amp;AP4A>β,γ-me-D-ATP>UTP>>β,γ-methylene-L-ATP.
4. The inward currents were inhibited by the P2-receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μM). PPADS also inhibited responses to ATP (800 nM) and α,β-methylene ATP (2 μM) in a concentration-dependent manner.
5. This study shows that β,γ-me-D-ATP, AP5A, AP4A and UTP all act via a suramin- and PPADS-sensitive P2X-receptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The very low activity of β,γ-methylene-L-ATP suggests that the agonists were acting at the P2X₃-subtype to produce these effects.

     

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 μM each) caused a marked release of previously incorporated [³H]noradrenaline. The P2-receptor antagonists suramin (300 μM) and cibacron blue 3GA (3 μM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 μM; by 47%), by the N-type calcium channel blocker ω-conotoxin GVIA (100 nM; by 35%), and by the α₂-adrenoceptor agonist UK-14,304 (1 μM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by ω-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 μM) caused a marked increase in intraaxonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and ω-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 μM) induced a large depolarisation and action potential firing. UDP (100 μM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and – probably – the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by α₂-adrenocep-tors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to α₂-adrenoceptor modulation. Received: 22 September 1998 / Accepted: 25 January 1999     

Effects of (+/-)-kavain on voltage-activated inward currents of dorsal root ganglion cells from neonatal rats.

Kava pyrones extracted from pepper Piper methysticum are pharmacologically active compounds. Since kava pyrones exhibit anticonvulsive, analgesic and centrally muscle relaxing properties, the influence of a synthetic kava pyrone, (+/-)-kavain, on voltage-dependent ion channel currents was studied. Effects of (+/-)-kavain on voltage-activated inward currents were analysed in cultured dorsal root ganglion cells derived from neonatal rats. Voltage-activated Ca2+ and Na+ currents were elicited in the whole-cell configuration of the patch clamp technique. Extracellularly applied (+/-)-kavain dissolved in hydrous salt solutions reduced voltage-activated Ca2+ and Na+ channel currents within 3-5 min. As the solubility of (+/-)-kavain in hydrous solutions is low, dimethyl sulfoxide (DMSO) was added to the saline as a solvent for the drug in most experiments. When (+/-)-kavain was dissolved in DMSO, the drug induced a fast and pronounced reduction of both Ca2+ and Na+ currents, which partly recovered within 2-5 min even in the presence of the drug. The present study indicates that (+/-)-kavain reduces currents through voltage-activated Na+ and Ca2+ channels.     

Use-dependent block of Ih in mouse dorsal root ganglion neurons by sinus node inhibitors

1. The sinus node inhibitors UL–FS 49 and DK–AH 269 reduce heart rate by slowing diastolic depolarization rate in the sino-atrial (SA) node, which might originate from the use-dependent blockade of a hyperpolarization-activated current I_f. A hyperpolarization-activated current I_h, which is present in many types of neurons, is similar to I_f. We studied the effects of these drugs on I_h in cultured mouse dorsal root ganglion (DRG) neurons.
2. With the whole-cell patch-clamp technique use-dependent block of I_h was observed. The steady-state block following a voltage-clamp pulse train (1-s steps from −38 to −108 mV applied at 0.5 Hz) was dependent on drug concentration and showed an apparent K_d of 0.1 and 0.79 μM with DK–AH 269 and UL–FS 49 respectively.
3. The rate of block increased linearly with drug concentration. The rate of recovery from block was, however, much slower compared to cardiac tissue.
4. There was no significant effect of UL–FS 49 on the activation curve.
5. At high concentrations of UL–FS 49 a clear association of the drug with the open channel was observed.
6. When the cell was stimulated at a frequency of 3 Hz, a distinct hyperpolarization was observed in the presence of extracellular Cs⁺ or when I_h was blocked with UL–FS 49, but not in the absence of Cs⁺ and UL–FS 49.
7. These results indicate that I_h protects the cell against hyperpolarizations and subsequent inexcitability. The action of the drugs on the hyperpolarization-activated current in cardiac and neuronal tissue show some similarities; however, some pronounced differences indicate that different subtypes of the channel might exist.

     

The action of 5-hydroxytryptamine on single neurones of the rabbit superior cervical ganglion

Intracellular recordings were made from neurones in the rabbit superior cervical ganglion maintained in vitro. Perfusion of the whole ganglion with 5-hydroxytryptamine (5-HT) produced inconsistent and small effects, whereas iontophoretic application of 5-HT induced a depolarization in the majority of neurones. The depolarization was accompanied by a fall in input resistance. The amplitude of the 5-HT potentials was related to the magnitude or duration of the ejection current. The amplitude of the 5-HT potential was augmented by hyperpolarization and decreased by depolarization of the soma membrane of the ganglion cell; it appeared to be linearly related to membrane potential. Iontophoretic responses were reduced in amplitude by superfusion of the ganglion with 5-HT (1 or 50 μM) and by cyproheptadine (50 μM), but unaffected by hexamethonium (500 μM). Tachyphylaxis was apparent when iontophoretic applications were repeated at frequencies of 0.1 Hz or greater.     

Chronotropic effect of histamine on cultured neonatal rat heart cells.

The positive chronotropic effect (PCE) of histamine in cultured neonatal rat heart cells was monitored using a microscopic method as well as an electro-optically recording device. The action potential frequency was also measured (by means of microelectrodes). An increase in PCE was noted when histamine (from 1 X 10(-6) M to 1 X 10(-5) M) was added to the cells. However, higher concentrations (from 1 X 10(-5) M to 1 X 10(-4) M) were less effective. The PCE of histamine was reduced by pretreating the cells with antihistaminic drugs. H1-blocking agents (promethazine and mepyramine) were more potent than H2-blocking drugs (metiamide and cimetidine). In addition, the PCE of histamine was abolished when the cells were in presence of high K+ medium (26 mEq) but contraction and action potential amplitudes were increased. Our results demonstrate that these cultures respond to histamine and that this response is abolished by antihistaminic drugs thus suggesting the H1 and/or H2 receptors may be present in the neonatal rat heart cell cultures.     

脑必舒注射液对鸡胚背根节神经突起生长促进作用的实验观察

以进口脑活素注射液为阳性对照,观察了脑必舒注射液对鸡胚背根节神经突起的体外促生长作用.结果表明:分别含40%脑必舒注射液与进口脑活素的培养液对鸡胚背根节神经突起的生长有相当的促进作用.在脑必舒与进口脑活素的作用下,神经突起的生长状况与阴性对照组相比有显著差异.     

脑必舒注射液对鸡胚背根节神经突起生长促进作用的实验观察

以进口脑活素注射液为阳性对照,观察了脑必舒注射液对鸡胚背根节神经突起的体外促生长作用.结果表明:分别含40%脑必舒注射液与进口脑活素的培养液对鸡胚背根节神经突起的生长有相当的促进作用.在脑必舒与进口脑活素的作用下,神经突起的生长状况与阴性对照组相比有显著差异.     

Phosphorylation of TRPV1 by neurokinin-1 receptor agonist exaggerates the capsaicin-mediated substance P release from cultured rat dorsal root ganglion neurons

The present study was conducted to determine whether the activation of neurokinin-1 receptor (NK-1R) by its agonist (GR73632) enhances the capsaicin-evoked substance P (SP) release using a radioimmunoassay. A pre-exposure to GR73632 enhanced the capsaicin-evoked SP release in a time- and dose-dependent manner. The augmentation of capsaicin-evoked SP release by GR73632 was completely inhibited by pharmacological blockade of NK-1R or transient receptor potential vanilloid receptor subtype 1 (TRPV1), and was partially attenuated by the inhibition of either protein kinase C (PKC), cyclooxygenase (COX) or phospholipase C (PLC), p38 or p42/44 mitogen-activated protein (MAP) kinase, but not protein kinase A. This augmentation of SP release was further increased by inhibition of c-Jun NH2-terminal kinase. A short-term (10min) exposure to GR73632 resulted in an increase in the TRPV1 phosphorylation. The increase in the TRPV1 phosphorylated forms induced by a 60-min exposure to GR73632 was completely abolished by the inhibition of either PKC, COX or PLC, p38 or p42/44 MAP kinases. Immunocytochemistry study demonstrated that the NK-1R and TRPV1 were mainly co-expressed in the small-sized neurons. These findings suggest that the activation of NK-1R by its agonist, by sensitizing the TRPV1 through the PKC phosphorylation of TRPV1, may play a role in the enhancement of the capsaicin-evoked SP release from cultured rat DRG neurons.     

Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor.

1. The identity of the neuropeptide Y (NPY) receptor associated with the observed inhibition of neuronal Ca2+ currents (ICa) in rat dorsal root ganglion (DRG) cells has been established on the basis of agonist responses to analogues and carboxy terminal (C-terminal) fragments of the NPY molecule. 2. Whole cell barium currents (IBa) in DRG cells were reversibly inhibited by 100 nM NPY, 100 nM PYY and C-terminal fragments of NPY in a manner that correlated with the length of the NPY fragments (for inhibition of the IBa NPY = PYY greater than NPY2-36 greater than NPY13-36 greater than NPY16-36 greater than NPY18-36 much greater than NPY25-36). 3. C-terminal fragments of NPY were also effective in reversibly reducing the ICa, the associated increase in the intracellular Ca2+ concentration [( Ca2+]i) and the increased [Ca2+]i produced by evoked action potentials in the DRG cells. In addition, a Ca(2+)-activated Cl- conductance was also reversibly reduced by NPY fragments only when accompanied by a reduction in Ca2+ entry. 4. We conclude that the Y2 receptor for neuropeptide Y is coupled to inhibition of Ca2+ influx via voltage-sensitive calcium channels in DRG cells.     

Acute effects of diesel exhaust particles and cisplatin on oxidative stress in cultured human kidney (HEK 293) cells,and the influence of curcumin thereon

Particulate air pollution with particle diameters less than 2.5 μm contribute to respiratory and extra-respiratory morbidity and mortality. We have recently reported the first in vivo experimental evidence that Diesel exhaust particles (DEP) in the lung aggravated the renal, pulmonary, and systemic effects of cisplatin (CP)-induced acute renal failure in rats. This in vitro study sought to determine whether and to what extent does DEP exposure exacerbate the effects of CP-induced oxidative stress in human embryonic kidney (HEK-293) cells, and to examine if these effects could be mitigated/prevented with curcumin (the yellow pigment isolated from turmeric). Cells viability, cysteine uptake and oxidative stress indices [glutathione (GSH), total antioxidant capacity (TAC), and the activities of antioxidant enzymes (catalase; glutathione peroxidase; superoxide dismutase)] were evaluated in all study groups. DEP aggravated the CP- induced HEK-293 cells toxicity, as evidenced by decreasing cell viability and by inducing oxidative stress (GSH depletion, TAC impairment, and antioxidant enzymes inhibition). DEP, but not CP, significantly reduced cysteine uptake. Curcumin prevented the observed DEP and CP-induced cellular insults. These findings suggest that DEP augmented the CP-induced toxicity in HEK-293 cells. Curcumin exhibited a strong potential for protection against DEP and CP-induced cytotoxicity.     

Olopatadine attenuates the enhancement of capsaicin-evoked substance P release by bradykinin from cultured dorsal root ganglion neurons

Olopatadine, a second-generation antihistamine, has recently been suggested to have an inhibitory effect on the tachykinin release from guinea-pig tracheobronchial smooth muscle preparation. In the present study, using a highly sensitive radioimmunoassay for substance P, we observed that olopatadine attenuated the enhancement of capsaicin-induced substance P release by bradykinin with an IC(50) value of 12.5 microM, without any inhibitory effect on the substance P release induced by capsaicin, potassium or bradykinin from cultured dorsal root ganglion neurons. These data suggest that olopatadine may therefore be involved in the bradykinin-induced sensitization of the transient receptor potential vanilloid 1 in cultured dorsal root ganglion neurons.     

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum

1. The effect of cannabinoid receptor agonists was studied in guinea-pig myenteric neurones in vitro by use of conventional intracellular recording techniques.
2. Exposure of myenteric neurones of the S-cell type to the cannabinoid receptor agonists WIN 55,212-2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively.
3. The depressant effect of WIN 55,212-2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude-time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect.
4. The inhibitory effect of WIN 55,212-2 appeared to be concentration-dependent over the range 1–100 nM. WIN 55,212-3, its (−)-enantiomer (100 nM), was inactive.
5. The cannabinoid CB₁ receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212-2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)-induced depolarizations in 42% of neurones tested (5/12).
6. When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9).
7. WIN 55,212-2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM).
8. It is concluded that cannabinoid-induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB₁ receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S-neurones and their synapsing cholinergic and non-cholinergic, non-adrenergic terminals are not endowed with cannabinoid receptors.

     

美西律对大鼠背根神经节细胞钠电流的抑制作用

应用膜片钳技术,全细胞钳位方式可见Mex对大鼠背根神经节细胞Na~ 电流有明显的抑制作用。其临界作用浓度为5 μmol·L~(-1),50％抑制浓度为31 μmol·L~(-1),完全抑制浓度为150 μmol·L~(-1)。其抑制作用具有电压和频率依赖性。抑制剂量与小鼠抗脑缺血缺氧作用量相近。提示Mex抗脑缺血缺氧作用的机制之一可能是通过对Na~ 电流的抑制。