Therapeutically relevant concentrations of neomycin selectively inhibit P-type Ca2+ channels in rat striatum

The effects of neomycin on voltage-activated Ca(2+) channels (VACCs) were studied by Ca(2+)-dependent K(+)- and veratridine-evoked [3H]dopamine release from rat striatal slices. Neomycin (0.01-1 mM) concentration dependently reduced K(+)-evoked [3H]dopamine release (IC(50) approximately 25 microM), producing approximately 98% inhibition at 1 mM. Contribution of N-, P- and Q-type Ca(2+) channels to this neomycin-sensitive [3H]dopamine release was tested by the combined application of 100 microM neomycin and selective Ca(2+) channel blockers. The effects of neomycin combined with 1 microM of omega-conotoxin GVIA (N-type Ca(2+) channels) or with 100 nM of omega-conotoxin MVIIC (Q-type Ca(2+) channels) were additive, excluding involvement of N- and Q-type Ca(2+) channels. However, the combined effects of neomycin with 30 nM of omega-agatoxin-IVA (P-type Ca(2+) channels) were not additive, suggesting involvement of P-type Ca(2+) channels in neomycin-induced inhibition of [3H]dopamine release. On the other hand, veratridine-evoked [3H]dopamine release was shown to be mediated by Q-type Ca(2+) channels only. In addition, neither the inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase thapsigargin (500 nM) nor the blocker of sarcoplasmic reticulum ryanodine Ca(2+) channels ryanodine (30 microM) modulate veratridine-evoked [3H]dopamine release, suggesting no contribution of intracellular Ca(2+) stores. Neomycin (up to 100 microM) did not affect veratridine-evoked [3H]dopamine release, suggesting that intracellular Ca(2+) stores are not a prerequisite for the action of neomycin. Lack of inhibitory effect of neomycin is taken as additional indirect evidence for the involvement of P-type Ca(2+) channels. In conclusion, therapeutically relevant concentrations of neomycin preferentially block P-type Ca(2+) channels which regulate dopamine release in rat striatum. This block could be responsible for aminoglycoside-induced toxicity.     

Role of voltage-gated cation channels and axon reflexes in the release of sensory neuropeptides by capsaicin from isolated rat trachea

In order to reveal the role of axon reflexes and sensory receptors in sensory neuropeptide release in response to capsaicin, liberation of substance P, calcitonin gene-related peptide and somatostatin from isolated rat tracheae was investigated in the presence of voltage-sensitive Na(+) and Ca(2+) channel blocking agents. Neuropeptide release induced by capsaicin (10 nM) remained unchanged in the presence of 25 mM lidocaine, 1 microM tetrodotoxin or the N-type Ca(2+) channel inhibitor, omega-conotoxin GVIA (100-300 nM). Peptide release by 100 pulses of 2 Hz field stimulation was prevented by lidocaine or tetrodotoxin. Omega-agatoxin TK (250 nM) significantly inhibited and Cd(2+) (200 microM) prevented capsaicin-induced neuropeptide release. These results suggest that chemical stimulation-induced neuropeptide release does not involve activation of fast Na(+) channels or N- and P-type voltage-dependent Ca(2+) channels, but contribution of Q-type Ca(2+) channels is possible. Sensory neuropeptides are released by capsaicin from sensory receptors without axon reflexes.     

Evidence for multiple effects of ProTxII on activation gating in Na(V)1.5

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na(+) and Ca(2+) channels. ProTxII is distinct from other known Na(+) channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na(V)1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G(max) appeared at relatively low concentrations (40-100nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (>/=500nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba(2+), known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba(2+) indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.     

Ethanol withdrawal seizure susceptibility is associated with upregulation of L- and P-type Ca2+ channel currents in rat inferior colliculus neurons

Calcium currents in the inferior colliculus (IC) are thought to play an important role in ethanol withdrawal hyperexcitability. Here, we report on the modulation of Ca(2+) channel currents in acutely dissociated IC neurons of rats, exhibiting higher incidence of audiogenic seizures when subjected to ethanol withdrawal. Whole cell Ca(2+) channel currents were activated by depolarizing pulses from a holding potential of -90 mV, in 10 mV increments, using barium (Ba(2+)) as the charge carrier. The high threshold voltage-activated (HVA) Ca(2+) channel current density increased significantly in IC neurons following ethanol withdrawal. The gating parameters of HVA Ca(2+) channel currents were only slightly altered, while the fraction of current that did not fully inactivate at positive potentials increased significantly following ethanol withdrawal. Pharmacological dissection of HVA Ca(2+) channel currents suggested that the enhanced current, associated with increased incidence of audiogenic seizures following ethanol withdrawal, was carried by L- and P-type Ca(2+) channels. The upregulation of L- and P-type currents may be responsible for IC neuronal hyperexcitability associated with increased susceptibility to ethanol withdrawal seizures.     

Inhibitory gating modulation of small conductance Ca2+-activated K+ channels by the synthetic compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reduces afterhyperpolarizing current in hippocampal CA1 neurons

SK channels are small conductance Ca(2+)-activated K(+) channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca(2+) sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In whole-cell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit (125)I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca(2+) sensitivity by shifting the activation curve for Ca(2+) to the right, only slightly affecting the maximal Ca(2+)-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca(2+)-dependently (K(d) = 0.42, 0.60, and 0.73 microM, respectively, at 0.5 microM Ca(2+)), whereas the compound did not affect the Ca(2+)-activated K(+) channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 microM.     

JZTX-IV, a unique acidic sodium channel toxin isolated from the spider Chilobrachys jingzhao

Neurotoxins are important tools to explore the structure and function relationship of different ion channels. From the venom of Chinese spider Chilobrachys jingzhao, a novel toxin, Jingzhaotoxin-IV (JZTX-IV), is isolated and characterized. It consists of 34 amino acid residues including six acidic residues clustered with negative charge (pI=4.29). The full-length cDNA of JZTX-IV encodes an 86-amino acid precursor containing a signal peptide of 21 residues, a mature peptide of 34 residues and an intervening sequence of 29 residues with terminal Lys-Gly as the signal of amidation. Under whole-cell patch clamp conditions, JZTX-IV inhibits current and slows the inactivation of sodium channels by shifting the voltage dependence of activation to more depolarized potentials on DRG neurons, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. In addition, JZTX-IV shows a slowing inactivation of sodium channel with a hyperpolarizing shift of the steady-state inactivation on acutely isolated rat cardiac cell and DRG neurons, differs from the classic site 3 toxins that do not affect the steady-state of inactivation. At high concentration, JZTX-IV has no significant effect on tetrodotoxin-resistant (TTX-R) sodium channels on rat DRG neurons and tetrodotoxin-sensitive (TTX-S) sodium channels on hippocampal neurons. Our data establish that, contrary to known toxins, JZTX-IV neither binds to the previously characterized classic site 4, nor site 3 by modifying channel gating, thus making it a novel probe of channel gating in sodium channels with potential to shed new light on this process.     

Purification and characterization of Hainantoxin-V, a tetrodotoxin-sensitive sodium channel inhibitor from the venom of the spider Selenocosmia hainana.

A neurotoxic peptide, named Hainantoxin-V (HNTX-V), was isolated from the venom of the Chinese bird spider Selenocosmia hainana. The complete amino acid sequence of HNTX-V has been determined by Edman degradation and found to contain 35 amino acid residues with three disulfide bonds. Under whole-cell patch-clamp mode, HNTX-V was proved to inhibit the tetrodotoxin-sensitive (TTX-S) sodium currents while it had no any effects on tetrodotoxin-resistant (TTX-R) sodium currents on adult rat dorsal root ganglion neurons. The inhibition of TTX-S sodium currents by HNTX-V was tested to be concentrate-dependent with the IC(50) value of 42.3nM. It did not affect the activation and inactivation kinetics of currents and did not have the effect on the active threshold of sodium channels and the voltage of peak inward currents. However, 100nM HNTX-V caused a 7.7mV hyperpolarizing shift in the voltage midpoint of steady-state sodium channel inactivation. The results indicated that HNTX-V inhibited mammalian voltage-gated sodium channels through a novel mechanism distinct from other spider toxins such as delta-ACTXs, micro -agatoxins I-VI which bind to receptor site three to slow the inactivation kinetics of sodium currents.     

Aconitine facilitates spontaneous transmitter release at rat ventromedial hypothalamic neurons

The effects of aconitine, an Aconitum alkaloid, on spontaneous inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs respectively) were investigated in the mechanically dissociated rat ventromedial hypothalamic (VMH) neurons in which native presynaptic nerve terminals remained intact. Under current-clamp conditions, aconitine (3 x 10(-6) M) depolarized the neuron with generating the action potentials. The aconitine-induced depolarization was markedly suppressed in the presence of CNQX but it was facilitated in the presence of bicuculline, suggesting that release of excitatory and inhibitory neurotransmitters may be involved in the aconitine action in addition to its direct action on postsynaptic membrane. Under the voltage-clamp conditions, aconitine reversibly increased the frequency of spontaneous IPSC and EPSC frequency, but it did not alter their amplitude distribution. Tetrodotoxin (TTX, 3 x 10(-7) M) completely abolished the aconitine action on spontaneous IPSC frequency. Likewise removal of extracellular Na(+) completely suppressed the aconitine action. Both Ca(2+)-free external solution or addition of 10(-4) M Cd(2+) to normal solutions eliminated the facilitatory effect of aconitine on the IPSC frequency. Overall these results suggest that aconitine depolarizes the presynaptic membrane by activating voltage-dependent Na(+) channels. Increase of intraterminal Ca(2+) concentration via an activation of voltage-dependent Ca(2+) channels in turn enhances the spontaneous transmitter release from presynaptic nerve terminals. The presynaptic action of aconitine may play a crucial role for membrane excitability of rat VMH neurons.     

A selective T-type Ca2+ channel blocker R(−) efonidipine

Recently, novel compound R(-) efonidipine was reported to selectively block low-voltage-activated (LVA or T-type) Ca(2+) channels in peripheral organs. We examined how R(-) efonidipine acts on T-type and high-voltage-activated (HVA) Ca(2+) channels in mammalian central nervous system (CNS) neurons. Furthermore, we compared the effects of R(-) efonidipine with those of flunarizine and mibefradil on both T-type and HVA Ca(2+) channels in rat hippocampal CA1 neurons by using the nystatin perforated-patch clamp technique. Flunarizine and mibefradil nonselectively inhibited both T-type and HVA Ca(2+) channels, though the dose-dependent blocking potency of flunarizine on T-type Ca(2+) channels was slightly stronger than that of mibefradil. In contrast, R(-) efonidipine inhibited only T-type Ca(2+) channels and did not show any effect on HVA Ca(2+) channels. The inhibitory actions of R(-) efonidipine or flunarizine were similar on both Ba(2+) and Ca(2+) current components passing through T-type Ca(2+) channels. In addition, flunarizine but not R(-) efonidipine inhibited voltage-dependent Na(+) channels and Ca(2+)-activated K(+) channels. Thus, it appears that R(-) efonidipine is a selective blocker for T-type Ca(2+) channels. It could be used as a pharmacological tool in future studies on T-type Ca(2+) channels.     

Novel spider toxin slows down the activation kinetics of P-type Ca2+ channels in Purkinje neurons of rat

We have identified a novel polypeptide toxin (Lsp-1) from the venom of the spider Lycosa (LS). Its effect has been examined on the P-type calcium channels in Purkinje neurons, using whole-cell patch-clamp. This toxin (at saturating concentration 7 nM) produces prominent (four-fold) deceleration of the activation kinetics and partial (71+/-6%) decrease of the amplitude of P-current without affecting either deactivation or inactivation kinetics. These effects are not use-dependent. They are partially reversible within a minute upon the wash-out of the toxin. Intracellular perfusion of Purkinje neurons with 100 microM of GDP or 2 microM of GTPgammaS, as well as strong depolarising pre-pulses (+100 mV), do not eliminate the action of Lsp-1 on P-channels indicating that down-modulation via guanine nucleotide-binding proteins (G-proteins) is not involved in the observed phenomenon. In view of extremely high functional significance of P-channels, the toxin can be suggested as a useful pharmacological tool.     

Enhancement of acetylcholine release by homoanatoxin-a from Oscillatoria formosa

The strain NIVA-CYA 92 of Oscillatoria formosa Bory ex Gormont produces phycotoxins with neurotoxic properties. Chemical analysis by gas chromatography/mass spectrometry of a water extract of lyophilized material of the organism showed the presence of only homoanatoxin-a. The mechanism of action of homoanatoxin-a on peripheral cholinergic nerves is so far not known. The neurotoxicity of O. formosa containing homoanatoxin-a was investigated in rat bronchi, rat brain synaptosomes and in GH(4)C(1) cells. The water extract of lyophilized material of the organism produced a concentration-dependent reversible increase in the release of [(3)H]acetylcholine from both K(+) (51 mM) depolarised and non-depolarised cholinergic nerves of the rat bronchial smooth muscle. The K(+)-evoked release of [(3)H]acetylcholine was enhanced by about 75% by a water extract from 15-20 mg/ml of lyophilized algal material. The enhanced release of [(3)H]acetylcholine was substantially reduced by the L-type Ca(2+)-channel blocker verapamil (100 μM) and not by the N-type Ca(2+)-channel blocker ω-conotoxin GVIA (1.0 μM) or the P-type Ca(2+)-channel blocker ω-agatoxin IV-A (0.2 μM). Chelation of intra-cellular Ca(2+) by 1,2-bis-(aminofenoxi)etan-N,N,N',N'-tetraacidic acid/acetoxymethyl (BAPTA/AM) (30 μM) had no effect on the phycotoxin-induced release of [(3)H]acetylcholine, indicating that an extracellular pool of Ca(2+) was important for the action of the phycotoxin on the release of [(3)H]acetylcholine from peripheral cholinergic nerves. In rat brain synaptosomes the algal extract enhanced the influx of (45)Ca(2+) in a tetrodotoxin (1.0 μM) and ω-conotoxin MVIIC (blocker of N-, P- and Q-type Ca(2+) channels) (1.0 μM) insensitive manner. Patch-clamp studies showed that the phycotoxin opened endogenous voltage dependent L-type Ca(2+) channels in neuronal GH(4)C(1) cells. These Ca(2+) channels and the effect of the toxin on the channels were blocked by the L-type Ca(2+)-channel antagonist gallopamil (200 μM). The present results suggest, therefore, that the investigated strain of O. formosa contains homoanatoxin-a, which enhances the release of acetylcholine from peripheral cholinergic nerves through opening of endogenous voltage dependent neuronal L-type Ca(2-) channels.     

Ca(v)3.2 channel is a molecular substrate for inhibition of T-type calcium currents in rat sensory neurons by nitrous oxide

Although nitrous oxide (N(2)O; laughing gas) remains widely used as an anesthetic and analgesic in clinical practice, its cellular mechanisms of action remain inadequately understood. In this report, we examined the effects of N(2)O on voltage-gated Ca(2+) channels in acutely dissociated small sensory neurons of adult rat. At subanesthetic concentrations, N(2)O blocks low-voltage-activated, T-type Ca(2+) currents (T currents), but not high-voltage-activated (HVA) currents. This blockade of T currents was concentration dependent, with an IC(50) value of 45 +/- 13%, maximal block of 38 +/- 12%, and Hill coefficient of 2.6 +/- 1.0. No desensitization of the response or change in current kinetics was observed during N(2)O application. The magnitude of T current blockade by N(2)O does not seem to reflect any use- or voltage-dependent properties. In addition, T current blockade was not altered when intracellular GTP was replaced with guanosine 5'-(gamma-thio)triphosphate or guanosine 5'-0-(2-thiodiphosphate) suggesting a lack of involvement of G-proteins in the inhibition. N(2)O selectively blocked currents arising from the Ca(v)3.2 but not Ca(v)3.1 recombinant channels stably expressed in human embryonic kidney (HEK) cells in a concentration-dependent manner with an apparent affinity and potency similar to native dorsal root ganglion currents. Analogously, the block of Ca(v)3.2 T currents exhibited little voltage- or use-dependence. These data indicate that N(2)O selectively blocks T-type but not HVA Ca(2+) currents in small sensory neurons and Ca(v)3.2 currents in HEK cells at subanesthetic concentrations. Blockade of T currents may contribute to the anesthetic and/or analgesic effects of N(2)O.     

Quercetin as a novel activator of L-type Ca(2+) channels in rat tail artery smooth muscle cells

1. The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage-dependent Ca(2+) channels of smooth muscle cells freshly isolated from the rat tail artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2. Quercetin increased L-type Ca(2+) current [I(Ca(L))] in a concentration- (pEC(50)=5.09+/-0.05) and voltage-dependent manner and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction, without, however, modifying the threshold and the equilibrium potential for Ca(2+). 3. Quercetin-induced I(Ca(L)) stimulation was reversible upon wash-out. T-type Ca(2+) current was not affected by quercetin. Quercetin shifted the voltage dependence of the steady-state inactivation and activation curves to more negative potentials by about 5.5 and 7.5 mV respectively, in the mid-potential of the curves as well as increasing the slope of activation. Quercetin slowed both the activation and the deactivation kinetics of the I(Ca(L)). The inactivation time course was also slowed but only at voltages higher than 10 mV. Moreover quercetin slowed the rate of recovery from inactivation. 4. These results prove quercetin to be a naturally-occurring L-type Ca(2+) channel activator.     

Tyramine reduces glycinergic transmission by inhibiting presynaptic Ca(2+) channels in the rat trigeminal subnucleus caudalis

We have recently reported that tyramine acts on putative presynaptic trace amine receptors to inhibit glycinergic transmission in substantia gelatinosa (SG) neurons of the rat trigeminal subnucleus caudalis. However, it is still unknown how tyramine elicits presynaptic inhibition of glycine release. In the present study, therefore, we investigated cellular mechanisms underlying the tyramine-induced inhibition of glycinergic transmission in SG neurons using a conventional whole-cell patch clamp technique. Tyramine (100 μM) reversibly and repetitively decreased the amplitude of action potential-dependent glycinergic inhibitory postsynaptic currents (IPSCs), and increased the paired-pulse ratio. Pharmacological data suggest that the tyramine-induced decrease in glycinergic IPSCs was not mediated by the modulation of adenylyl cyclase, protein kinase A and C, or G-protein coupled inwardly rectifying K(+) channels. On the other hand, glycinergic IPSCs were mainly mediated by the Ca(2+) influx passing through presynaptic N-type and P/Q-type Ca(2+) channels. The tyramine-induced decrease in glycinergic IPSCs was completely blocked by ω-conotoxin GVIA, an N-type Ca(2+) channel blocker, but not ω-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. The results suggest that tyramine acts presynaptically to decrease action potential-dependent glycine release onto SG neurons via the selective inhibition of presynaptic N-type Ca(2+) channels. This tyramine-induced inhibition of glycinergic transmission in SG neurons might affect the process of orofacial nociceptive signals.     

Vasopressin V(1) receptor-mediated activation of central sympatho-adrenomedullary outflow in rats

High-threshold Ca(2+) channels and tetrodotoxin-resistant Na(+) channels are highly expressed in small dorsal root ganglion neurons. In acutely isolated rat dorsal root ganglion neurons, the effects of neomycin, one of the aminoglycoside antibiotics, on high-threshold Ca(2+) currents and tetrodotoxin-resistant Na(+) currents were examined using whole-cell patch recording. We showed for the first time that neomycin dose-dependently inhibited peak high-threshold Ca(2+) currents and peak tetrodotoxin-resistant Na(+) currents with half-maximal inhibitory concentrations at 3.69 microM (n=20) and 1213.44 microM (n=25), respectively. Inactivation properties of high-threshold Ca(2+) currents and activation properties of tetrodotoxin-resistant Na(+) currents were also affected by neomycin with reduction of excitability of small dorsal root ganglion neurons. Half-maximal inactivation voltage of high-threshold Ca(2+) currents was -45.56 mV before and -50.46 mV after application of neomycin (n=10). Half-maximal activation voltage of tetrodotoxin-resistant Na(+) currents was -19.93 mV before and -11.19 mV after administration of neomycin (n=15). These results suggest that neomycin can inhibit high-threshold Ca(2+) currents and tetrodotoxin-resistant Na(+) currents in small dorsal root ganglion neurons, which may contribute to neomycin-induced peripheral and central analgesia.     

Opposing action of conantokin-G on synaptically and extrasynaptically-activated NMDA receptors

Synaptic and extrasynaptic activation of the N-methyl-D-aspartate receptor (NMDAR) has distinct consequences on cell signaling and neuronal survival. Since conantokin (con)-G antagonism is NR2B-selective, which is the key subunit involved in extrasynaptic activation of the receptor, its ability to specifically elicit distinct signaling outcomes in neurons with synaptically or extrasynaptically-activated NMDARs was evaluated. Inhibition of Ca(2+) influx through extrasynaptic NMDAR ion channels was neuroprotective, as it effectively enhanced levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), activated cAMP response element binding protein (CREB), enhanced mitochondrial viability, and attenuated the actin disorganization observed by extrasynaptic activation of NMDARs. Conversely, the pro-signaling pathways stimulated by synaptically-induced Ca(2+) influx were abolished by con-G. Furthermore, subunit non-selective con-T was unable to successfully redress the impairments in neurons caused by extrasynaptically-activated NMDARs, thus indicating that NR2B-specific antagonists are beneficial for neuron survival. Neurons ablated for the NR2B subunit showed weak synaptic Ca(2+) influx, reduced sensitivity to MK-801 blockage, and diminished extrasynaptic current compared to WT and NR2A(-/-) neurons. This indicates that the NR2B subunit is an integral component of both synaptic and extrasynaptic NMDAR channels. Altogether, these data suggest that con-G specifically targets the NR2B subunit in the synaptic and extrasynaptic locations, resulting in the opposing action of con-G on differentially activated pools of NMDARs.     

Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of transient receptor potential vanilloid subtype 1 in analgesic action of methylsalicylate

Methylsalicylate (MS) is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite the common use of MS as a pain reliever, the underlying molecular mechanism is not fully understood. Here we characterize the action of MS on transient receptor potential V1 (TRPV1). In human embryonic kidney 293 cells expressing human TRPV1 (hTRPV1), MS evoked increases of [Ca(2+)](i), which declined regardless of its continuous presence, indicative of marked desensitization. TRPV1 antagonists dose-dependently suppressed the MS-induced [Ca(2+)](i) increase. MS simultaneously elicited an inward current and increase of [Ca(2+)](i) in the voltage-clamped cells, suggesting that MS promoted Ca(2+) influx through the activation of TRPV1 channels. MS reversibly inhibited hTRPV1 activation by polymodal stimuli such as capsaicin, protons, heat, anandamide, and 2-aminoethoxydiphenyl borate. Because both the stimulatory and inhibitory actions of MS were exhibited in capsaicin- and allicin-insensitive mutant channels, MS-induced hTRPV1 activation was mediated by distinct channel regions from capsaicin and allicin. In cultured rat sensory neurons, MS elicited a [Ca(2+)](i) increase in cells responding to capsaicin. MS significantly suppressed nocifensive behavior induced by intraplantar capsaicin in rats. The present data indicate that MS has both stimulatory and inhibitory actions on TRPV1 channels and suggest that the latter action may partly underlie the analgesic effects of MS independent of inhibition of cyclooxygenases in vivo.     

Modification of NMDA responses by tri-n-butyltin in rat brain neurons

1. The effects of the organotin, tri-n-butyltin (TBT), on N-methyl-D-aspartate (NMDA) induced membrane currents were investigated in order to evaluate possible neuronal actions of this toxic environmental pollutant. Experiments were conducted on neurons acutely dissociated from the rat dorsal motor nucleus of vagus (DMV) using the nystatin-perforated patch clamp recording technique. 2. In Mg(2+)-free physiological recording solutions, the application of NMDA to single DMV neurons held at a holding potential (V(H)) of -40 mV evoked an inward current which rapidly reached a peak before declining to a steady-state inward current. This was followed, immediately after NMDA washout, by a transient outward current. TBT (100 nM) reversibly caused a slight reduction in the inward currents and greatly increased the amplitude of the outward currents. 3. The reversal potential of the NMDA-induced outward current in the presence of TBT was -86.7 mV, close to the theoretical K(+) equilibrium potential of -85.7 mV. 4. The NMDA-induced outward current was completely blocked when the K(+) in the internal solution was replaced with equimolar Cs(+). Under these conditions, the NMDA induced current was more sustained and was unaffected by TBT. 5. The NMDA-induced outward current was markedly inhibited by 5 mM tetraethylammonium chloride and 300 nM charybdotoxin, and it was abolished by removal of extracellular Ca(2+), suggesting that the outward current was due to the activation of Ca(2+)-activated K(+) channels by Ca(2+) influx through NMDA receptors. 6. In conclusion, in rat DMV neurons, TBT potentiates the Ca(2+)-activated K(+) current induced by NMDA application without having any direct effects on the NMDA-induced inward current. Given the significant role of NMDA receptor mediated excitation in various physiological and pathological processes, the modulation of this response by TBT may have an important influence on neuronal function.