Topical application of disodium isostearyl 2-O-L-ascorbyl phosphate, an amphiphilic ascorbic acid derivative, reduces neuropathic hyperalgesia in rats

BACKGROUND AND PURPOSE

Ca_v3.2 T-type calcium channels, targeted by H₂S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca_v3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H₂S donor, and on neuropathic hyperalgesia.

EXPERIMENTAL APPROACH

In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically.

KEY RESULTS

The NaHS-evoked Ca_v3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55–0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP.

CONCLUSIONS AND IMPLICATIONS

Ascorbic acid, known to inhibit Ca_v3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.     

AKAP-dependent sensitization of Cav3.2 channels via the EP4 receptor/cAMP pathway mediates PGE2-induced mechanical hyperalgesia

Background and Purpose

The Ca_v3.2 isoform of T-type Ca²⁺ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE₂-induced hyperalgesia. Here we examined and analysed Ca_v3.2 sensitization via the PGE₂/cAMP pathway in NG108-15 cells that express Ca_v3.2 and produce cAMP in response to PGE₂, and its impact on mechanical nociceptive processing in rats.

Experimental Approach

In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Ca_v3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats.

Key Results

In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE₂ increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE₂ was abolished by RQ-00015986-00, an EP₄ receptor antagonist. AKAP150 was co-immunoprecipitated with Ca_v3.2, regardless of stimulation with db-cAMP, and Ca_v3.2 was phosphorylated by db-cAMP or PGE₂. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE₂ caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl₂, known to inhibit Ca_v3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE₂-induced mechanical hyperalgesia.

Conclusion and Implications

Our findings suggest that PGE₂ causes AKAP-dependent phosphorylation and sensitization of Ca_v3.2 through the EP₄ receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.     

Colonic hydrogen sulfide-induced visceral pain and referred hyperalgesia involve activation of both Ca(v)3.2 and TRPA1 channels in mice

Luminal hydrogen sulfide (H(2)S), a gasotransmitter, causes colonic pain / referred hyperalgesia in mice, most probably via activation of T-type Ca(2+) channels. Here we analyzed the mechanisms for H(2)S-induced facilitation of colonic pain signals. Intracolonic administration of NaHS, an H(2)S donor, evoked visceral pain-like nociceptive behavior and referred hyperalgesia in mice, an effect abolished by NNC 55-0396, a selective T-type Ca(2+)-channel blocker, or by knockdown of Ca(v)3.2. AP18, a TRPA1 blocker, also prevented the NaHS-induced colonic pain and referred hyperalgesia. These findings demonstrate that H(2)S-induced colonic pain and referred hyperalgesia require activation of both Ca(v)3.2 and TRPA1 channels in mice.     

Inhibition of human recombinant T-type calcium channels by the endocannabinoid N-arachidonoyl dopamine

Background and purpose:

N-arachidonoyl dopamine (NADA) has complex effects on nociception mediated via cannabinoid CB₁ receptors and the transient receptor potential vanilloid receptor 1 (TRPV1). Anandamide, the prototypic CB₁/TRPV1 agonist, also inhibits T-type voltage-gated calcium channel currents (I_Ca). These channels are expressed by many excitable cells, including neurons involved in pain detection and processing. We sought to determine whether NADA and the prototypic arachidonoyl amino acid, N-arachidonoyl glycine (NAGly) modulate T-type I_Ca

Experimental approach:

Human recombinant T-type I_Ca (Ca_V3 channels) expressed in HEK 293 cells and native mouse T-type I_Ca were examined using standard whole-cell voltage clamp electrophysiology techniques.

Key results:

N-arachidonoyl dopamine completely inhibited Ca_V3 channels with a rank order of potency (pEC₅₀) of Ca_V3.3 (6.45) ≥ Ca_V3.1 (6.29) > Ca_V3.2 (5.95). NAGly (10 µmol·L⁻¹) inhibited Ca_V3 I_Ca by approximately 50% or less. The effects of NADA and NAGly were voltage- but not use-dependent, and both compounds produced significant hyperpolarizing shifts in Ca_V3 channel steady-state inactivation relationships. By contrast with anandamide, NADA and NAGly had modest effects on Ca_V3 channel kinetics. Both NAGly and NADA inhibited native T-type I_Ca in mouse sensory neurons.

Conclusions and implications:

N-arachidonoyl dopamine and NAGly increase the steady-state inactivation of Ca_V3 channels, reducing the number of channels available to open during depolarization. These effects occur at NADA concentrations at or below to those affecting CB₁ and TRPV1 receptors. Together with anandamide, the arachidonoyl neurotransmitter amides, NADA and NAGly, represent a new family of endogenous T-type I_Ca modulators.     

H2S modulates duodenal motility in male rats via activating TRPV1 and KATP channels

Background and Purpose

H₂S induces vasodilatation by opening K_ATP channels but it may also affect other ion channels. The aim of this study was to investigate the effect of H₂S on intestinal motility in rats and its underlying mechanism.

Experimental Approach

The tension of intestinal muscle strips, afferent firing of intestinal mesenteric nerves, length of duodenal smooth muscle cells and whole-cell membrane potential of dorsal root ganglion (DRG) neurons were monitored. H₂S-producing enzymes were located by immunofluorescence staining.

Key results

NaHS exerted early transient excitation and late long-lasting inhibition on the intestinal contraction. The excitation was attenuated by TRPV1 antagonists capsazepine, A784168, SB-366791 and NK₁ receptor antagonist L703606, while the inhibition was attenuated by glibenclamide. NaHS increased duodenal afferent nerve firing and depolarized DRG neurons. These effects were reduced by capsazepine and A784168. NaHS relaxed isolated duodenal smooth muscle cells. The K_ATP channels were expressed in smooth muscle cells. Cystathionine β-synthase and cystathionine γ-lyase were expressed in rat duodenal myenteric neurons. L-cysteine and S-adenosyl-L-methionine increased the contraction of duodenal muscle strips, an effect attenuated by capsazepine and L703606.

Conclusions and Implications

NaHS induces biphasic effects on intestinal motility in rats while endogenous H₂S only exerts an excitatory effect. This transient excitatory effect might be mediated by activation of TRPV1 channels in sensory nerve terminals with the consequent release of substance P. The long-lasting inhibitory effect might be mediated by activation of K_ATP channels in the smooth muscle cells. These findings reveal a novel mechanism for the excitatory effect of H₂S on gastrointestinal motility.     

Activation of transient receptor potential A1 by a non-pungent capsaicin-like compound, capsiate

BACKGROUND AND PURPOSE

Capsiate is produced by ‘CH-19 Sweet’ (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels.

EXPERIMENTAL APPROACH

Using Ca²⁺ imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons.

KEY RESULTS

We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC₅₀ for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid.

CONCLUSIONS AND IMPLICATIONS

Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.     

Actions of hydrogen sulphide on ion transport across rat distal colon

Background and purpose:

The aim of this study was to identify the actions of H₂S on ion transport across rat distal colon.

Experimental approach:

Changes in short-circuit current (Isc) induced by the H₂S-donor, NaHS, were measured in Ussing chambers. Cytosolic Ca²⁺ concentration was evaluated using fura-2.

Key results:

NaHS concentration-dependently induced a change in Isc, that was only partially inhibited by the neurotoxin, tetrodotoxin. Lower concentrations (≤10⁻³ mol·L⁻¹) of NaHS induced a monophasic increase in Isc, whereas higher concentrations induced an additional, secondary fall of Isc, before a third phase when Isc rose again. Blockers of H₂S-producing enzymes (expression demonstrated immunohistochemically) decreased basal Isc, suggesting that endogenous production of H₂S contributes to spontaneous anion secretion. The positive Isc phases induced by NaHS were due to Cl⁻ secretion as shown by anion substitution and transport inhibitor experiments, whereas the transient negative Isc induced by higher concentrations of the H₂S-donor was inhibited by mucosal tetrapentylammonium suggesting a transient K⁺ secretion. When applied from the serosal side, glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, and tetrapentylammonium, a blocker of Ca²⁺-dependent K⁺ channels, suppressed NaHS-induced Cl⁻ secretion suggesting different types of K⁺ channels are stimulated by the H₂S-donor. NaHS-induced increase in cytosolic Ca²⁺ concentration was confirmed in isolated, fura-2-loaded colonic crypts. This response was not dependent on extracellular Ca²⁺, but was inhibited by blockers of intracellular Ca²⁺ channels present on Ca²⁺ storage organelles.

Conclusions and implications:

H₂S induces colonic ion secretion by stimulation of apical as well as basolateral epithelial K⁺ channels.     

Inhibition of T-type Ca²⁺ channels by endostatin attenuates human glioblastoma cell proliferation and migration

BACKGROUND AND PURPOSE

Endostatin (ES) is a c-terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions.

EXPERIMENTAL APPROACH

Using cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T-type Ca²⁺ channels.

KEY RESULTS

Extracellular application of ES reversibly inhibited T-type Ca²⁺ channel currents (T-currents) in U87 cells, whereas L-type Ca²⁺ currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation but was independent of G-protein and protein tyrosine kinase-mediated pathways. All three α₁ subunits of T-type Ca²⁺ channels (Ca_V3), α_1G (Ca_V3.1), α_1H (Ca_V3.2) and α_1I (Ca_V3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only Ca_V3.1 and Ca_V3.2, but not Ca_V3.3 or Ca_V1.2 (L-type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose-dependent manner. Pretreatment of the cells with the specific T-type Ca²⁺ channel blocker mibefradil occluded these inhibitory effects of ES.

CONCLUSION AND IMPLICATIONS

This study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T-type Ca²⁺ channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells.

LINKED ARTICLE

This article is commented on by Santoni et al., pp. 1244–1246 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01908.x     

The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels

BACKGROUND AND PURPOSE

Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca_v1.2 channel current (I_Ca1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca_v1.2 channels.

EXPERIMENTAL APPROACH

Twenty-four flavonoids were analysed for their effects on I_Ca1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method.

KEY RESULTS

Most of the flavonoids stimulated or inhibited I_Ca1.2 in a concentration- and voltage-dependent manner with EC₅₀ values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC₅₀ values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I_Ca1.2 stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I_Ca1.2 inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2′-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca_v1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I_Ca1.2.

CONCLUSIONS AND IMPLICATIONS

Quercetin-like plant flavonoids were active on vascular Ca_v1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca_v1.2 channels, valuable for the treatment of hypertension and stroke.     

The TRPA1 channel mediates the analgesic action of dipyrone and pyrazolone derivatives

Background and Purpose

Although still used by hundreds of millions of people worldwide, the mechanism of the analgesic action of the pyrazolone derivatives (PDs), dipyrone, propyphenazone and antipyrine remains unknown. The transient receptor potential ankyrin 1 (TRPA1) channel, expressed by nociceptors, is emerging as a major pain transduction pathway. We hypothesized that PDs target the TRPA1 channel and by this mechanism produce their analgesic effect.

Experimental Approach

Calcium responses and currents were studied in cultured TRPA1-expressing rodent dorsal root ganglion neurons and human cells. Acute nociception and mechanical hypersensitivity were investigated in naïve and genetically manipulated mice.

Key Results

Pyrazolone and PDs selectively inhibited calcium responses and currents in TRPA1-expressing cells and acute nocifensor responses in mice evoked by reactive channel agonists (allyl isothiocyanate, acrolein and H₂O₂). In line with recent results obtained with TRPA1 antagonists and TRPA1 gene deletion, the two most largely used PDs, dipyrone and propyphenazone, attenuated TRPA1-mediated nociception and mechanical allodynia in models of inflammatory and neuropathic pain (formalin, carrageenan, partial sciatic nerve ligation and the chemotherapeutic drug, bortezomib). Notably, dipyrone and propyphenazone attenuated carrageenan-evoked mechanical allodynia, without affecting PGE₂ levels. The main metabolites of PDs did not target TRPA1 and did not affect TRPA1-dependent nociception and allodynia.

Conclusions and Implications

Evidence that in rodents the nociceptive/hyperalgesic effect produced by TRPA1 activation is blocked by PDs suggests that a similar pathway is attenuated by PDs in humans and that TRPA1 antagonists could be novel analgesics, devoid of the adverse haematological effects of PDs.     

Modulation of mouse gastrointestinal motility by allyl isothiocyanate, a constituent of cruciferous vegetables (Brassicaceae): evidence for TRPA1-independent effects

BACKGROUND AND PURPOSE

Allyl isothiocyanate (AITC, mustard oil), a constituent of many common cruciferous vegetables (Brassicaceae), activates transient receptor potential of ankyrin type-1 (TRPA1) channels, claimed to regulate gastrointestinal contractility. In this study, we have investigated the effect of AITC on intestinal motility.

EXPERIMENTAL APPROACH

Effects of AITC were investigated in vivo on upper gastrointestinal transit in mice and in mouse isolated ileum [contractions induced by electrical field stimulation (EFS), acetylcholine and spontaneous contractility]. The contractor activity of AITC was studied in mouse isolated colon. The ability of TRPA1 channel antagonists to block AITC-induced elevation of intracellular Ca²⁺[Ca²⁺]_i was assessed in HEK293 cells transfected with rat TRPA1 channels.

KEY RESULTS

AITC increased [Ca²⁺]_i in HEK293 cells, reduced ileal contractility (acetylcholine-, EFS-induced contractions and spontaneous contractility), but contracted the isolated colon. Gentamicin and camphor (non-selective TRPA1 channel antagonists), HC-030031 and AP18 (selective TRPA1 channel agonists) inhibited AITC-induced effects in HEK293 cells but not in the ileum or colon. AITC-induced contractions were reduced by tetrodotoxin and strongly reduced by nifedipine, cyclopiazonic acid and ryanodine. In vivo, AITC reduced (following i.p. administration) or increased (following intragastric administration) upper gastrointestinal transit in mice These effects were not affected by HC-030031.

CONCLUSION AND IMPLICATIONS

AITC, depending, in vitro, on the regions of gut examined and, in vivo, on the route of administration, exerted both stimulatory and inhibitory effects on intestinal motility, which were not sensitive to TRPA1 channel antagonists. The proposition that TRPA1 channels are the primary targets for AITC to induce contraction should be revised.     

Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain

Background and purpose:

The activation of CB₂ receptors induces analgesia in experimental models of chronic pain. The present experiments were designed to study whether the activation of peripheral or spinal CB₂ receptors relieves thermal hyperalgesia and mechanical allodynia in two models of bone cancer pain.

Experimental approach:

NCTC 2472 osteosarcoma or B16-F10 melanoma cells were intratibially inoculated to C3H/He and C57BL/6 mice. Thermal hyperalgesia was assessed by the unilateral hot plate test and mechanical allodynia by the von Frey test. AM1241 (CB₂ receptor agonist), AM251 (CB₁ receptor antagonist), SR144528 (CB₂ receptor antagonist) and naloxone were used. CB₂ receptor expression was measured by Western blot.

Key results:

AM1241 (0.3–10 mg·kg⁻¹) abolished thermal hyperalgesia and mechanical allodynia in both tumour models. The antihyperalgesic effect was antagonized by subcutaneous, intrathecal or peri-tumour administration of SR144528. In contrast, the antiallodynic effect was inhibited by systemic or intrathecal, but not peri-tumour, injection of SR144528. The effects of AM1241 were unchanged by AM251 but were prevented by naloxone. No change in CB₂ receptor expression was found in spinal cord or dorsal root ganglia.

Conclusions and implications:

Spinal CB₂ receptors are involved in the antiallodynic effect induced by AM1241 in two neoplastic models while peripheral and spinal receptors participate in the antihyperalgesic effects. Both effects were mediated by endogenous opiates. The use of drugs that activate CB₂ receptors could be a useful strategy to counteract bone cancer-induced pain symptoms.     

Inhibition of human recombinant T-type calcium channels by N-arachidonoyl 5-HT

BACKGROUND AND PURPOSE

N-arachidonoyl 5-HT (NA-5HT) has anti-nociceptive effects reported to be mediated by inhibitory actions at the transient receptor potential vanilloid receptor 1 (TRPV1) and fatty acid amide hydrolase (FAAH). Anandamide and N-arachidonoyl dopamine (NA-DA), endocannabinoids that activate TRPV1 or are metabolized by FAAH, also inhibit T-type calcium channels (I_Ca). T-type I_Ca are expressed by many excitable cells, including neurons involved in pain detection and processing. We sought to determine whether NA-5HT also modulates T-type I_Ca.

EXPERIMENTAL APPROACH

Human recombinant T-type I_Ca (Ca_V3 channels) expressed in HEK 293 cells were examined using standard whole-cell voltage-clamp electrophysiology techniques.

KEY RESULTS

NA-5HT completely inhibited Ca_V3 channels with a rank order of potency (pEC₅₀) of Ca_V3.1 (7.4) > Ca_V3.3 (6.8) ≥ Ca_V3.2 (6.6). The effects of NA-5HT were voltage-dependent, and it produced significant hyperpolarizing shifts in Ca_V3 steady-state inactivation relationships. NA-5HT selectively affected Ca_V3.3 channel kinetics.

CONCLUSIONS AND IMPLICATIONS

NA-5HT increases the steady-state inactivation of Ca_V3 channels, reducing the number of channels available to open during depolarization. These effects occur at NA-5HT concentrations at or below those at which NA-5HT affects TRPV1 receptors and FAAH. NA-5HT is one of the most potent inhibitors of T-type I_Ca described to date, and it is likely to exert some of its biological effects, including anti-nociception, via inhibition of these channels.     

Compounds from Sichuan and Melegueta peppers activate,covalently and non-covalently,TRPA1 and TRPV1 channels

Background and purpose:

Oily extracts of Sichuan and Melegueta peppers evoke pungent sensations mediated by different alkylamides [mainly hydroxy-α-sanshool (α-SOH)] and hydroxyarylalkanones (6-shogaol and 6-paradol). We assessed how transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1), two chemosensory ion channels, participate in these pungent sensations.

Experimental approach:

The structure–activity relationships of these molecules on TRPA1 and TRPV1 was measured by testing natural and synthetic analogues using calcium and voltage imaging on dissociated dorsal root ganglia neurons and human embryonic kidney 293 cells expressing the wild-type channels or specific cysteine mutants using glutathione trapping as a model to probe TRPA1 activation. In addition, using Trpv1 knockout mice, the compounds'' aversive responses were measured in a taste brief-access test.

Key results:

For TRPA1 activation, the cis C6 double bond in the polyenic chain of α-SOH was critical, whereas no structural specificity was required for activation of TRPV1. Both 6-shogaol and 6-paradol were found to activate TRPV1 and TRPA1 channels, whereas linalool, an abundant terpene in Sichuan pepper, activated TRPA1 but not TRPV1 channels. Alkylamides and 6-shogaol act on TRPA1 by covalent bonding whereas none of these compounds activated TRPV1 through such interactions. Finally, TRPV1 mutant mice retained sensitivity to 6-shogaol but were not responsive to α-SOH.

Conclusions and implications:

The pungent nature of components of Sichuan and Melegueta peppers was mediated via interactions with TRPA1 and TRPV1 channels and may explain the aversive properties of these compounds.     

Treatment with the Kv7 potassium channel activator flupirtine is beneficial in two independent mouse models of pulmonary hypertension

Background and purpose:

Voltage-gated potassium (K_v) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K_v7 channels has been recently proposed. We investigated the effect of the K_v7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT⁺ mice).

Experimental approach:

Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg·kg⁻¹·day⁻¹). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K_v channel blocking drugs, including the K_v7 channel blocker linopirdine, were measured.

Key results:

In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT⁺ mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT⁺ mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K_v channel blockers did not.

Conclusions and implications:

Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT⁺ mice, apparently via K_v7 channel activation. These results provide the first direct evidence that drugs activating K_v7 channels may be of benefit in the treatment of PAH with different aetiologies.     

Inhibition of voltage-gated Na(+) currents in sensory neurones by the sea anemone toxin APETx2

BACKGROUND AND PURPOSE

APETx2, a toxin from the sea anemone Anthropleura elegantissima, inhibits acid-sensing ion channel 3 (ASIC3)-containing homo- and heterotrimeric channels with IC₅₀ values < 100 nM and 0.1–2 µM respectively. ASIC3 channels mediate acute acid-induced and inflammatory pain response and APETx2 has been used as a selective pharmacological tool in animal studies. Toxins from sea anemones also modulate voltage-gated Na⁺ channel (Na_v) function. Here we tested the effects of APETx2 on Na_v function in sensory neurones.

EXPERIMENTAL APPROACH

Effects of APETx2 on Na_v function were studied in rat dorsal root ganglion (DRG) neurones by whole-cell patch clamp.

KEY RESULTS

APETx2 inhibited the tetrodotoxin (TTX)-resistant Na_v 1.8 currents of DRG neurones (IC₅₀, 2.6 µM). TTX-sensitive currents were less inhibited. The inhibition of Na_v 1.8 currents was due to a rightward shift in the voltage dependence of activation and a reduction of the maximal macroscopic conductance. The inhibition of Na_v 1.8 currents by APETx2 was confirmed with cloned channels expressed in Xenopus oocytes. In current-clamp experiments in DRG neurones, the number of action potentials induced by injection of a current ramp was reduced by APETx2.

CONCLUSIONS AND IMPLICATIONS

APETx2 inhibited Na_v 1.8 channels, in addition to ASIC3 channels, at concentrations used in in vivo studies. The limited specificity of this toxin should be taken into account when using APETx2 as a pharmacological tool. Its dual action will be an advantage for the use of APETx2 or its derivatives as analgesic drugs.     

Alternative splicing modulates diltiazem sensitivity of cardiac and vascular smooth muscle Cav1.2 calcium channels

Background and purpose:

As a calcium channel blocker, diltiazem acts mainly on the voltage-gated calcium channels, Ca_v1.2, for its beneficial effects in cardiovascular diseases such as hypertension, angina and/or supraventricular arrhythmias. However, the effects of diltiazem on different isoforms of Ca_v1.2 channels expressed in heart and vascular smooth muscles remain to be investigated. Here, we characterized the effects of diltiazem on the splice variants of Ca_v1.2 channels, predominant in cardiac and vascular smooth muscles.

Experimental approach:

Cardiac and smooth muscle isoforms of Ca_v1.2 channels were expressed in human embryonic kidney cells and their electrophysiological properties were characterized using whole-cell patch-clamp techniques.

Key results:

Under closed-channel and use-dependent block (0.03 Hz), cardiac splice variant Ca_v1.2CM was less sensitive to diltiazem than two major smooth muscle splice variants, Ca_v1.2SM and Ca_v1.2b. Ca_v1.2CM has a more positive half-inactivation potential than the smooth muscle channels, and diltiazem shifted it less to negative potential. Additionally, the current decay was slower in Ca_v1.2CM channels. When we modified alternatively spliced exons of cardiac Ca_v1.2CM channels into smooth muscle exons, we found that all three loci contribute to the different diltiazem sensitivity between cardiac and smooth muscle splice isoforms.

Conclusions and implications:

Alternative splicing of Ca_v1.2 channels modifies diltiazem sensitivity in the heart and blood vessels. Gating properties altered by diltiazem are different in the three channels.     

Inhibition of transmitter release from rat sympathetic neurons via presynaptic M1 muscarinic acetylcholine receptors

Background and purpose:

M₂, M₃ and/or M₄ muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M₁ receptors mediate an inhibition of K_v7, Ca_V1 and Ca_V2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M₁ receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M₁ receptors.

Experimental approach:

In primary cultures of rat superior cervical ganglion neurons, Ca²⁺ currents were recorded via the perforated patch-clamp technique, and the release of [³H]-noradrenaline was determined.

Key results:

The muscarinic agonist oxotremorine M (OxoM) transiently enhanced ³H outflow and reduced electrically evoked release, once the stimulant effect had faded. The stimulant effect was enhanced by pertussis toxin (PTX) and was abolished by blocking M₁ receptors, by opening K_v7 channels and by preventing action potential propagation. The inhibitory effect was not altered by preventing action potentials or by opening K_v7 channels, but was reduced by PTX and ω-conotoxin GVIA. The inhibition remaining after PTX treatment was abolished by blockage of M₁ receptors or inhibition of phospholipase C. When [³H]-noradrenaline release was triggered independently of voltage-activated Ca²⁺ channels (VACCs), OxoM failed to cause any inhibition. The inhibition of Ca²⁺ currents by OxoM was also reduced by ω-conotoxin and PTX and was abolished by M₁ antagonism in PTX-treated neurons.

Conclusions and implications:

These results demonstrate that M₁, in addition to M₂, M₃ and M₄, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close VACCs.     

KV7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries

BACKGROUND AND PURPOSE

Hypoxia causes vasodilatation of coronary arteries, but the underlying mechanisms are poorly understood. We hypothesized that hypoxia reduces intracellular Ca²⁺ concentration ([Ca²⁺]_i) by opening of K channels and release of H₂S.

EXPERIMENTAL APPROACH

Porcine coronary arteries without endothelium were mounted for measurement of isometric tension and [Ca²⁺]_i, and the expression of voltage-gated K channels K_V7 channels (encoded by KCNQ genes) and large-conductance calcium-activated K channels (K_Ca1.1) was examined. Voltage clamp assessed the role of K_V7 channels in hypoxia.

KEY RESULTS

Gradual reduction of oxygen concentration from 95 to 1% dilated the precontracted coronary arteries and this was associated with reduced [Ca²⁺]_i in PGF_2α (10 μM)-contracted arteries whereas no fall in [Ca²⁺]_i was observed in 30 mM K-contracted arteries. Blockers of ATP-sensitive voltage-gated potassium channels and K_Ca1.1 inhibited hypoxia-induced dilatation in PGF_2α-contracted arteries; this inhibition was more marked in the presence of the K_v7 channel blockers, XE991 and linopirdine, while a K_V7.1 blocker, failed to change hypoxic vasodilatation. XE991 also inhibited H₂S- and adenosine-induced vasodilatation. PCR revealed the expression of K_V7.1, K_V7.4, K_V7.5 and K_Ca1.1 channels, and K_Ca1.1, K_V7.4 and K_V7.5 were also identified by immunoblotting. Voltage clamp studies showed the XE991-sensitive current was more marked in hypoxic conditions.

CONCLUSION

The K_V7.4 and K_V7.5 channels, which we identified in the coronary arteries, appear to have a major role in hypoxia-induced vasodilatation. The voltage clamp results further support the involvement of K_V7 channels in this vasodilatation. Activation of these K_V7 channels may be induced by H₂S and adenosine.