Electrophysiological characteristic of ATP-activated currents of trigeminal ganglion neurons with different diameter in rat / 中国应用生理学杂志

<p><b>AIM</b>To explore the characteristic of ATP-activated current in trigeminal ganglion (TG) neurons of rat.</p><p><b>METHODS</b>Whole-cell patch-clamp was performed.</p><p><b>RESULTS</b>(1) The majority (92.1%) of TG neurons responded to ATP applied externally with inward currents. We recorded three distinct ATP-activated currents: fast, slow and intermediate, which were concentration-dependent. (2) In general, the fast ATP-activated currents were distributed mainly in small-diameter TG neurons, the slow ATP-activated currents were distributed mainly in large-diameter TG neurons, and the intermediate ATP-activated currents were distributed mainly in intermediate-diameter TG neurons. (3) The time course of rising phase from 10% to 90% of the three distinct ATP-activated currents were as follows: fast: (33.6 +/- 4.5) ms; intermediate: (62.2 +/- 9.9) ms; slow: (302.1 +/- 62.0) ms, and that of desensitizing phase were (399.4 +/- 58.2) ms (fast), and > 500 ms (slow) respectively. (4) From the current-voltage relationship curves, it can be seen that the reversal potential values of the three distinct ATP-activated currents were the same, all being 0-5mV. And they all were characterized by inward rectification. (5) The dose-response curve for fast ATP-activated current shifted downwards as compared with the intermediate ATP-activated current, and that for the slow ATP-activated current shifted upwards.</p><p><b>CONCLUSION</b>The EC50s of the three curves tended to be identical. The results suggested that three kinds of distinct ATP-activated currents could be mediated by various subtypes of P2X receptors assembled by different subunits, and the subtypes existed in TG neurons of different diameters and transmit different information.</p>

Cation ions modulate the ACh-sensitive current in type II vestibular hair cells of guinea pigs / 生理学报

Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-nAChR) in cochlear hair cells and frog saccular hair cells. In this study, the properties of the ACh-sensitive current were investigated by whole-cell patch clamp technique in isolated type II vestibular hair cells of guinea pigs. The direct effect of extracellular ACh was to induce a hyperpolarization effect in type II vestibular hair cells. Type II vestibular hair cells displayed a sustained outward current in response to the perfusion of ACh. It took about 60 s for the ACh-sensitive current to get a complete re-activation. The reversal potential of the ACh-sensitive current was (-66 +/- 8) mV, which indicated that potassium ion was the main carrier of this current. The blocking effect by the submillimolar concentration of tetraethylammonium (TEA) further indicated that extracellular ACh stimulated the calcium-dependent potassium current. Following replacement of the compartment of NaCl in the normal external solution with TrisCl, LiCl or saccharose respectively, the amplitude of the ACh-sensitive current was not affected. Blocking of the release of intracellular Ca(2+) stores by intracellular application of heparin failed to inhibit the ACh-sensitive current. Therefore, extracellular Na(+)and the inositol 1,4,5-trisphosphate (IP(3))-dependent intracellular Ca(2+)release were not involved in the activation of the ACh-sensitive current. However, the ACh-sensitive current was strongly affected by the concentration of the extracellular K(+), extracellular Ca(2+) and intracellular Mg(2+). The amplitude of the ACh- sensitive current was strongly inhibited by high concentration of extracellular K(+). In the Ca(2+)-free external solution, ACh only activated a very small current; however, the ACh-sensitive current demonstrated a Ca(2+)-dependent inhibition effect in high concentration of Ca(2+)solution. In addition, the ACh-sensitive current was inhibited by increasing of the concentration of intracellular Mg(2+). In conclusion, the present results demonstrate that ACh plays an important role in the vestibular efferent system. The fact that Na(+) is not involved in the ACh-sensitive current will not favor the well-known profile of alpha9-nAChR, which is reported to display a small but important permeability to Na(+). It is also suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may strongly depend on the concentration of extracellular Ca(2+)and intracellular Mg(2+).

Characteristics of P2X purinoceptors in the membrane of rat trigeminal ganglion neurons / 生理学报

The characteristics of purinoceptors in the membrane of rat trigeminal ganglion (TG) neurons were studied by using whole- cell patch clamp technique. The results showed that most of neurons examined (78.9%, 142/180) were responsive to ATP in a concentration-dependent manner; the others (21.1%, 38/180) were ATP insensitive. Of the ATP-sensitive cells, the majority (95.1%, 135/142) responded to ATP with an inward current, a few (2.1%, 3/142) with an outward current, and the rest (2.8%, 4/142) with biphasic current. Small sized cells (<30 mum) responded to ATP with a rapid desensitizing inward current and were highly sensitive to vanilloid; the medium sized cells (30~40 mum) responded to ATP with slow desensitizing inward current and were not sensitive to vanilloid; while the majority of large sized cells (>40 mum) did not respond to ATP and vanilloid. The waveform of ATP-activated inward currents was related to the cell diameter. The I-V curves for both small and medium sized cells manifested obvious inward rectification. Furthermore, we studied the kinetic features of ATP-activated currents and the effects of P2 purinoceptor agonists and antagonists on I(ATP). The findings suggest that ATP receptor-ion channels are expressed differently among different types of rat TG neurons.

Interaction of 5-HT2 and 5-HT3 receptor subtype in 5-HT-induced nociceptive responses in peripheral primary sensory nerve ending / 中国应用生理学杂志

<p><b>AIM</b>To study the correlation between 5-HT-induced pain response and the contribution by individual 5-HTR subtypes including 5-HT1R, 5-HT2R and 5-HT3R at the level of peripheral primary afferent.</p><p><b>METHODS</b>The experiments were done on acutely isolated trigeminal ganglion (TG) neurons using whole-cell patch clamp technique and the nociceptive effect was observed on behavior experiments by intraplantar injection of test drugs.</p><p><b>RESULTS</b>The majority of cells examined responded to 5-HT in a manner of concentration dependence (10(-6) - 10(-3) mol/) (61.4%, 54/88) and with a fast activating and rapid desensitizing inward current (I(5-HT)), which was thought to be mediated by the activation of 5-HT3R, since it could be blocked by 5-HT3R antagonist ICS 205930 and mimicked by 5-HT3R agonist 2-methyl-5-HT. It was found that I(5-HT) was potentiated by 5-HT2R agonist alpha-methyl-5-HT markedly, while 5-HT1R agonist R-(+)-UH 301 did not. In behavioral experiment performed on conscious rats, intraplantar injection of 5-HT(10(-5), 10(-4) and 10(-3) mol/L) induced an increment of cumulative lifting time first 20 min in a manner of concentration dependence. By dissociating 5-HTR subtypes using their corresponding antagonists (ICS and CYP) the potency order of hindpaw lifting time was identified as follows: 5-HT > 5-HT + ICS > 5-HT + CYP.</p><p><b>CONCLUSION</b>The results suggest that in 5-HT-induced nociceptive response at the primary sensory level 5-HT3R may play a role of initiation, but 5-HT2R mediates maintaining and modulatory effect in the processes of nociceptive information convey.</p>

Inhibitory effect of rhynchophylline on human ether-a-go-go related gene channel / 生理学报

We studied the effects of Chinese traditional medicine rhynchophylline (Rhy) on human ether-a-go-go related gene (HERG) channel and characterized the electrophysiological properties of Rhy's pharmacological effect on HERG channel using Xenopus oocytes. Xenopus oocytes were injected with either 23 nl (5.75 ng) HERG cRNA or 23 nl distilled water. Xenopus oocytes were randomly assigned to receive one of the following different concentrations of Rhy: (1) control, (2)10 mumol/L Rhy, (3)100 mumol/L Rhy, (4) 500 mumol/L Rhy, (5) 1 000 mumol/L Rhy, (6) 10 000 mumol/L Rhy. Cell currents were recorded in oocytes. The peak tail currents of HERG channel were inhibited by Rhy. The inhibition was in a dose-dependent manner [IC(50)=(773.4 +/- 42.5) mumol/L]. Experiment with 100 mumol/L Rhy indicated that the degree of HERG blockade showed some voltage dependence (within -40 mV to -20 mV ). Kinetic analyses revealed that Rhy decreased the rate of channel activation. The findings indicate that Rhy inhibits HERG encoded potassium channels. It may underline the molecular mechanism of myocardial electrophysiological characteristics associated with this drug.

5-HT2 receptor mediated the potentiation of GABA-activated current in the membrane of the dorsal root ganglion neurons of rat / 药学学报

<p><b>AIM</b>To explore the modulation of 5-HT on GABA-activated current (I(GABA)) in the membrane of rat dorsal root ganglion (DRG) neurons and its mechanism.</p><p><b>METHODS</b>Rat DRG neurons were isolated mechanically and enzymatically, on which whole-cell patch clamp recording and repatch technique for intracellular dialysis were performed.</p><p><b>RESULTS</b>In the majority of neurons examined (92.0%, 69/75) GABA induced a concentration-dependent inward current. In neurons sensitive to GABA preapplication of 5-HT produced potentiation effect (82.6% , 57/69) on I(GABA). Preapplication of 5-HT at concentrations of 1 x 10(-6), 1 x 10(-5), 1 x 10(-4) and 1 x 10(-3) mol x L(-1) potentiated I(GABA) by (35 +/- 8)% (n=8), (47 +/- 11)% (n=10), (65 +/- 17)% (n=9) and (75 +/- 18)% (n=11), respectively. This effect was mimicked by alpha-methyl-5-HT (1 x 10(-6) mol x L(-1)), a specific 5-HT2 receptor agonist, and reversed by cyproheptadine, a selective 5-HT2 receptor antagonist. The potentiation of I(GABA) by 5-HT was irrespective to whether the I(5-HT) presents or not in a subset of neurons. The concentration-response curves for GABA before and after pretreatment with 5-HT manifested the same threshold value and similar EC50 (2.0 x 10(-5) and 1.9 x 10(-5) mol x L(-1), respectively) , while the maximal value of I(GABA) for the latter was 33.6% higher than that for the former. Intracellular dialysis with GDP-beta-S or H-7 abolished the potentiation of I(GABA) by 5-HT, while H-9 did not.</p><p><b>CONCLUSION</b>5-HT can potentiate GABA-activated current via PKC-dependent phosphorylation of GABA(A) receptor following the activation of 5-HT2 receptor.</p>

Inhibitory effect of caffeine on GABA-activated current in acutely isolated rat dorsal root ganglion neurons / 生理学报

By means of whole-cell patch clamp technique, the modulatory effect of caffeine on GABA-activated currents (I(GABA)) was investigated in acutely isolated rat dorsal root ganglion (DRG) neurons. The majority of the neurons examined (113/116) were sensitive to GABA (1~1000 micromol/L). GABA activated a concentration-dependent inward current, which manifested obvious desensitization. In 58 out of 108 neurons, caffeine induced a small inward current, while in others no detectable current was observed. After the neurons were treated with caffeine (0.1~100 micromol/L) prior to the application of GABA (100 micromol/L) for 30 s, GABA-activated inward currents were obviously inhibited. Caffeine shifted the GABA dose-response curve downward and decreased the maximum response to 57% without changing K(d) value. These results indicate that the inhibitory effect is non-competitive. The pretreatment with caffeine (10 micromol/L) inhibited I(GABA) which was potentiated by diazepam (1 micromol/L). Intracellular application of H-8 almost completely abolished the inhibitory effect of caffeine on I(GABA). Because GABA can induce primary afferent depolarization (PAD), our results suggest that caffeine may be able to antagonize the effect of presynaptic inhibition of GABA in primary afferent.

Opposite modulatory effects of substance P on GABA-and 5-HT-activated currents in the same sensory neurons / 生理学报

The modulation by substance P of gamma-aminobutyric acid (GABA)- and 5-hydroxytryptamine (5-HT)-activated currents (I(GABA) and I(5-HT)) was studied by using patch-clamp technique in rat trigeminal ganglion (TG) neurons. The majority of neurons examined responded to GABA and 5-HT with inward currents in the same cells (63.8%, 30/47). In 22 out of 30 neurons sensitive to both GABA and 5-HT, pretreatment with substance P (SP, 0.01 micromol/L) suppressed I(GABA) by (35.7 +/-6.1)% and enhanced I(5-HT) by (65.2 +/- 8.7)%. GR 82334, a potent and specific antagonist of NK1 tachykinin receptor, reversibly blocked the modulatory effects of SP. The SP modulation on I(GABA) and I(5-HT) was also abolished by intracellular dialysis of GDP-beta-S, a non-hydrolyzable GDP analog, or GF 109203X, a selective protein kinase C inhibitor. These results suggest that SP exerts opposite modulatory actions on GABA(A) receptor and 5-HT3 receptor activity of the same primary sensory neuron via the same intracellular signal transduction pathway.

Intracellular dialysis with a microcatheter inserted into the patch-clamp pipette / 生理学报

In this paper we present an easily available method of intracellular dialysis via a microcatheter inserted into glass pipette during patch clamp experiment. An oblique hole through the glass pipette holder (above the lateral hole for cell-seal suction) is drilled, through which a microcatheter (O.D.=0.1 mm) made from the universal pipetter tip by hand-drawing passes and sticks out of the holder mouth in parallel with the Ag-AgCl electrode. With a syringe connected to the microcatheter, substitution of intracellular solution and intracellular dialysis of drugs can be achieved easily. Compared with repatch technique and intracellular solution substitution techniques used abroad, this method operates more easily and can produce more reliable results.