The cannabinoid CB1 receptor antagonists rimonabant (SR141716) and AM251 directly potentiate GABA(A) receptors

BACKGROUND AND PURPOSE

Rimonabant (SR141716) and the structurally related AM251 are widely used in pharmacological experiments as selective cannabinoid receptor CB₁ antagonists / inverse agonists. Concentrations of 0.5–10 µM are usually applied in in vitro experiments. We intended to show that these drugs did not act at GABA_A receptors but found a significant positive allosteric modulation instead.

EXPERIMENTAL APPROACH

Recombinant GABA_A receptors were expressed in Xenopus oocytes. Receptors were exposed to AM251 or rimonabant in the absence and presence of GABA. Standard electrophysiological techniques were used to monitor the elicited ionic currents.

KEY RESULTS

AM251 dose-dependently potentiated responses to 0.5 µM GABA at the recombinant α₁β₂γ₂ GABA_A receptor with an EC₅₀ below 1 µM and a maximal potentiation of about eightfold. The Hill coefficient indicated that more than one binding site for AM251 was located in this receptor. Rimonabant had a lower affinity, but a fourfold higher efficacy. AM251 potentiated also currents mediated by α₁β₂, α_xβ₂γ₂ (x = 2,3,5,6), α₁β₃γ₂ and α₄β₂δ GABA_A receptors, but not those mediated by α₁β₁γ₂. Interestingly, the CB₁ receptor antagonists {"type":"entrez-nucleotide","attrs":{"text":"LY320135","term_id":"1257555575","term_text":"LY320135"}}LY320135 and O-2050 did not significantly affect α₁β₂γ₂ GABA_A receptor-mediated currents at concentrations of 1 µM.

CONCLUSIONS AND IMPLICATIONS

This study identified rimonabant and AM251 as positive allosteric modulators of GABA_A receptors. Thus, potential GABAergic effects of commonly used concentrations of these compounds should be considered in in vitro experiments, especially at extrasynaptic sites where GABA concentrations are low.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Inhibition of fatty acid amide hydrolase unmasks CB1 receptor and TRPV1 channel-mediated modulation of glutamatergic synaptic transmission in midbrain periaqueductal grey

BACKGROUND AND PURPOSE

While arachidonyl ethanolamine (anandamide) produces pharmacological effects mediated by cannabinoid CB1 receptors, it is also an agonist at the transient receptor potential vanilloid type 1 (TRPV1) ion channel. This study examined the cellular actions of anandamide in the midbrain periaqueductal grey (PAG), a region implicated in the analgesic actions of cannabinoids, and which expresses both CB1 receptors and TRPV1.

EXPERIMENTAL APPROACH

In vitro whole cell patch clamp recordings of glutamatergic excitatory postsynaptic currents (EPSCs) were made from rat and mouse PAG slices.

KEY RESULTS

Capsaicin (1 µM) increased the rate, but not the amplitude of miniature EPSCs in subpopulations of neurons throughout the rat and mouse PAG. Capsaicin had no effect on miniature EPSCs in PAG neurons from TRPV1 knock-out mice. In mouse PAG neurons, anandamide (30 µM) had no effect on the rate of miniature EPSCs alone, or in the presence of either the CB1 antagonist AM251 (3 µM) or the TRPV1 antagonist iodoresiniferatoxin (300 nM). Anandamide produced a decrease in miniature EPSC rate in the presence of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 µM). By contrast, anandamide produced an increase in miniature EPSC rate in the presence of both URB597 and AM251, which was absent in TRPV1 knock-out mice.

CONCLUSIONS AND IMPLICATIONS

These results suggest that the actions of anandamide within PAG are limited by enzymatic degradation by FAAH. FAAH blockade unmasks both presynaptic inhibition and excitation of glutamatergic synaptic transmission which are mediated via CB1 receptors and TRPV1 respectively.     

F 15845, a new blocker of the persistent sodium current prevents consequences of hypoxia in rat femoral artery

Background and purpose:

Selective cyclooxygenase-2 (COX-2) inhibitors such as rofecoxib (Vioxx) and celecoxib (Celebrex) were developed as NSAIDs with reduced gastric side effects. Celecoxib has now been shown to affect cellular physiology via an unexpected, COX-independent, pathway – by inhibiting K_v2.1 and other ion channels. In this study, we investigated the mechanism of the action of celecoxib on K_v2.1 channels.

Experimental approach:

The mode of action of celecoxib on rat K_v2.1 channels was studied by whole-cell patch-clamping to record currents from channels expressed in HEK-293 cells.

Key results:

Celecoxib reduced current through K_v2.1 channels when applied from the extracellular side. At low concentrations (≤3 µM), celecoxib accelerated kinetics of activation, deactivation and inactivation. Recovery of rat K_v2.1 channels from inactivation could be characterized by two components, with celecoxib selectively accelerating the slow component of recovery at ≤10 µM. At >3 µM, celecoxib led to closed-channel block with relative slowing of activation. At 30 µM, it additionally induced open-channel block that manifested in use-dependent inhibition and slower recovery from inactivation.

Conclusions and implications:

Celecoxib reduced current through K_v2.1 channels by modifying gating and inducing closed- and open-channel block, with the three effects manifesting at different concentrations. These data will help to elucidate the mechanisms of action of this widely prescribed drug on ion channels and those underlying its neurological, cardiovascular and other effects.     

Pharmacological characterization of native α7 nicotinic ACh receptors and their contribution to depolarization-elicited exocytosis in human chromaffin cells

BACKGROUND AND PURPOSE

Expression of α7 nicotinic acetylcholine receptors (nAChRs) and their role in exocytosis have not yet been examined in human chromaffin cells.

EXPERIMENTAL APPROACH

To characterize these receptors and investigate their function, patch-clamp experiments were performed in human chromaffin cells from organ donors.

KEY RESULTS

The nicotinic current provoked by 300 µM ACh in voltage-clamped cells was blocked by the nicotinic receptor antagonists α-bungarotoxin (α-Bgtx; 1 µM; 6 ± 1.7%) or methyllycaconitine (MLA; 10 nM; 7 ± 1.6%), respectively, in an irreversible and reversible manner, without affecting exocytosis. Choline (10 mM) pulses induced a biphasic current with an initial quickly activated (5.5 ± 0.4 ms rise time) and inactivated component (8.5 ± 0.4 ms time constant) (termed α7), which was blocked by α-Bgtx or MLA, followed by a slower component (non-α7). α7 nAChR currents were dissected by blocking the non-α7 nAChR current component of the ACh and choline response with the α6* nAChR blocker α-conotoxin (α-Ctx) MII[S4A, E11A, L15A]. PNU-282987, an α7 nAChR-specific agonist, elicited rapidly activated and rapidly inactivated currents. α7 nAChR-positive allosteric modulators, such as 5-hydroxyindole (1 mM) and PNU-120596 (10 µM), potentiated responses that were blocked by α-Bgtx or MLA. Exocytosis was evoked by depolarization-elicited α7 nAChR currents, using choline in the presence of α-Ctx MII[MS4A, E11A, L15A] or PNU-282987 as agonists.

CONCLUSIONS AND IMPLICATIONS

Our electrophysiological recordings of pure α7 nAChR currents elicited by rapid application of agonists demonstrated that functional α7 nAChRs are expressed and contribute to depolarization-elicited exocytosis in human chromaffin cells.     

Cannabinoid CB(2) receptor attenuates morphine-induced inflammatory responses in activated microglial cells

BACKGROUND AND PURPOSE

Among several pharmacological properties, analgesia is the most common feature shared by either opioid or cannabinoid systems. Cannabinoids and opioids are distinct drug classes that have been historically used separately or in combination to treat different pain states. In the present study, we characterized the signal transduction pathways mediated by cannabinoid CB₂ and µ-opioid receptors in quiescent and LPS-stimulated murine microglial cells.

EXPERIMENTAL APPROACH

We examined the effects of µ-opioid and CB₂ receptor stimulation on phosphorylation of MAPKs and Akt and on IL-1β, TNF-α, IL-6 and NO production in primary mouse microglial cells.

KEY RESULTS

Morphine enhanced release of the proinflammatory cytokines, IL-1β, TNF-α, IL-6, and of NO via µ-opioid receptor in activated microglial cells. In contrast, CB₂ receptor stimulation attenuated morphine-induced microglial proinflammatory mediator increases, interfering with morphine action by acting on the Akt-ERK1/2 signalling pathway.

CONCLUSIONS AND IMPLICATIONS

Because glial activation opposes opioid analgesia and enhances opioid tolerance and dependence, we suggest that CB₂ receptors, by inhibiting microglial activity, may be potential targets to increase clinical efficacy of opioids.     

Rosiglitazone inhibits Kv4.3 potassium channels by open-channel block and acceleration of closed-state inactivation

BACKGROUND AND PURPOSE

Rosiglitazone is a widely used oral hypoglycaemic agent, which improves insulin resistance in type 2 diabetes. Chronic rosiglitazone treatment is associated with a number of adverse cardiac events. The present study was designed to characterize the effects of rosiglitazone on cloned K_v4.3 potassium channels.

EXPERIMENTAL APPROACH

The interaction of rosiglitazone with cloned K_v4.3 channels stably expressed in Chinese hamster ovary cells was investigated using whole-cell patch-clamp techniques.

KEY RESULTS

Rosiglitazone decreased the currents carried by K_v4.3 channels and accelerated the current inactivation, concentration-dependently, with an IC₅₀ of 24.5 µM. The association and dissociation rate constants for rosiglitazone were 1.22 µM⁻¹·s⁻¹ and 31.30 s⁻¹ respectively. Block by rosiglitazone was voltage-dependent, increasing in the voltage range for channel activation; however, no voltage dependence was found in the voltage range required for full activation. Rosiglitazone had no effect on either the deactivation kinetics or the steady-state activation of K_v4.3 channels. Rosiglitazone shifted the steady-state inactivation curves in the hyperpolarizing direction, concentration-dependently. The K_i for the interaction between rosiglitazone and the inactivated state of K_v4.3 channels was 1.49 µM, from the concentration-dependent shift in the steady-state inactivation curves. Rosiglitazone also accelerated the kinetics of the closed-state inactivation of K_v4.3 channels. Rosiglitazone did not affect either use dependence or recovery from inactivation of K_v4.3 currents.

CONCLUSION AND IMPLICATIONS

Our results indicate that rosiglitazone potently inhibits currents carried by K_v4.3 channels by interacting with these channels in the open state and by accelerating the closed-state inactivation of K_v4.3 channels.

LINKED ARTICLE

This article is commented on by Hancox, pp. 496–498 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01281.x     

Effects of P2Y(1) receptor antagonism on the reactivity of platelets from patients with stable coronary artery disease using aspirin and clopidogrel

BACKGROUND AND PURPOSE

P2Y₁ is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy.

EXPERIMENTAL APPROACH

Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day⁻¹) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y₁ inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed.

KEY RESULTS

Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α_IIbβ₃ activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α_IIbb₃ activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s⁻¹), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y₁₂ inhibitor 2-MeSAMP.

CONCLUSIONS AND IMPLICATIONS

Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y₁ in the blood of patients with CAD appears to play a more important role than previously anticipated.     

Inhibition of vascular ectonucleotidase activities by the pro-drugs ticlopidine and clopidogrel favours platelet aggregation

BACKGROUND AND PURPOSE

After conversion to their active forms by the liver, ticlopidine and clopidogrel exert antiplatelet effects through irreversible inhibition of the P2Y₁₂ receptor. Concentrations of nucleotides such as ADP, the physiological agonist at platelet P2Y₁ and P2Y₁₂ receptors, are regulated by vascular ectonucleotidases, mainly nucleoside triphosphate diphosphohydrolase (NTPDase)1 and ecto-5′-nucleotidase. Here we evaluate the effect of these pro-drugs on vascular ectonucleotidase activity and on the natural function of these enzymes in regulating platelet aggregation.

EXPERIMENTAL APPROACH

Nucleotidase assays were performed by HPLC and by P_i determination, using human umbilical vein endothelial cells (HUVEC) and protein extracts from transfected COS-7 cells as sources of enzymes. Platelet aggregation was assayed using human platelet-rich plasma.

KEY RESULTS

Each pro-drug inhibited endothelial ectonucleotidase activities and decreased their ability to block platelet aggregation in vitro. At their therapeutic concentrations, ticlopidine (60 µM) and clopidogrel (20 µM) inhibited ADP hydrolysis by HUVEC by about 80%, and AMP hydrolysis by one-third. Accordingly, these compounds showed a mixed-type inhibition of recombinant human NTPDase1 with an apparent K_i (K_i,app) of 10 µM (clopidogrel) and 14 µM (ticlopidine). Recombinant rat ecto-5′-nucleotidase, but not its human orthologue, was inhibited by ticlopidine with a K_i,app of 4.5 mM.

CONCLUSIONS AND IMPLICATIONS

These pro-drugs facilitated platelet aggregation via the inhibition of vascular NTPDase1 in vitro. Further studies should be performed to assess whether this effect also occurs in vivo, especially at the beginning of treatment, before sufficient levels of active metabolites are produced by the liver.     

Involvement of cholinergic system in suppression of formalin-induced inflammatory pain by cobratoxin

Aim:

To investigate the analgesic effect of cobratoxin (CTX), a long-chain α-neurotoxin from Thailand cobra venom, in a rat model of formalin-induced inflammatory pain.

Methods:

Inflammatory pain was induced in SD rats via injecting 5% formalin (50 μL) into the plantar surface of their right hind paw. CTX and other agents were ip administered before formalin injection. The time that the animals spent for licking the injected paw was counted every 5 min for 1 h.

Results:

CTX (25, 34, and 45 μg/kg) exhibited a dose-dependent analgesic effect during the phase 1 (0–15 min) and phase 2 (20–60 min) response induced by formalin. Pretreatment with naloxone (0.5 or 2.5 mg/kg) did not block the analgesic effect of CTX. Pretreatment with atropine at 5 mg/kg, but not at 2.5 mg/kg, antagonized the analgesic effect of CTX. Treatment with the nonselective nAChR antagonist mecamylamine (3 mg/kg) inhibited the analgesic effects of CTX in Phase 1 and Phase 2 responses, while with the selective α7-nAChR antagonist methyllycaconitine (3 mg/kg) antagonized the effect of CTX only in the Phase 1 response. Treatment with the α7-nAChR agonist PNU282987 (3 mg/kg) significantly reduced the formalin-induced phase 2 pain response, but only slightly reduced the Phase 1 pain response.

Conclusion:

The results suggest that CTX exerts an antinociceptive effect in formalin-induced inflammatory pain, which appears to be mediated by mAChR and α7-nAChR.     

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.     

Modulation of drug block of the cardiac potassium channel KCNA5 by the drug transporters OCTN1 and MDR1

BACKGROUND AND PURPOSE

A common site for drug binding on voltage-gated ion channels is at the interior face of the channel pore. In this study, we tested the hypothesis that the extent of drug block of the human cardiac KCNA5 (K_v1.5) channel underlying the atrial-specific, ultra-rapidly activating, delayed K⁺ current (I_Kur) is modulated by the drug uptake and efflux transporters encoded by organic cation transporter 1 (OCTN1) and multiple drug-resistant gene 1 (MDR1) and expressed in human heart.

EXPERIMENTAL APPROACH

Drug block of KCNA5 was assessed in Chinese hamster ovary cells transiently transfected with KCNA5 alone or in combination with the OCTN1 or MDR1 transporter construct, as well as in an MDR1 stably expressed cell line.

KEY RESUTLS

Co-expression of OCTN1 significantly facilitated block by quinidine (10 µM), verapamil (20 µM), propafenone (5 µM) and clofilium (30 µM). Further evidence of drug transport modulating drug block was the finding that with OCTN1, block developed faster and only partially washed-out, and that block potentiation was prevented by cimetidine, an inhibitor of OCTN1. MDR1 expression attenuated KCNA5 block by erythromycin (an MDR1 substrate). Block was restored by reversin-205 (10 µM, an MDR1 inhibitor). MDR1 did not affect KCNA5 inhibition by KN-93 (1 µM), a blocker acting on the outer mouth of the channel pore.

CONCLUSIONS AND IMPLICATIONS

The extent of drug block of KCNA5 can be modulated by drug uptake and efflux transporters. These data provide further support for the idea that modifying intracellular drug concentrations could modulate the effects of blocking ion channels in patients.     

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.     

Self-augmentation of the lengthening of repolarization is related to the shape of the cardiac action potential: implications for reverse rate dependency

Background and purpose:

The aims of the present work were to study the mechanism of the reverse rate dependency of different interventions prolonging cardiac action potential duration (APD).

Experimental approach:

The reverse rate-dependent lengthening effect of APD-prolonging interventions and the possible involvement of I_Kr (rapid component of the delayed rectifier potassium current) and I_K1 (inward rectifier potassium current) were studied by using the standard microelectrode and the whole-cell patch-clamp techniques in dog multicellular ventricular preparations and in myocytes isolated from undiseased human and dog hearts.

Key results:

All applied drugs – dofetilide (1 µmol·L⁻¹), BaCl₂ (10 µmol·L⁻¹), BAY-K-8644 (1 µmol·L⁻¹), veratrine (1 µg·mL⁻¹) – lengthened APD in a reverse rate-dependent manner regardless of their mode of action, suggesting that reverse rate dependency may not represent a specific mechanism of APD prolongation. The E-4031-sensitive current (I_Kr) and the Ba²⁺-sensitive current (I_K1) were recorded during repolarizing voltage ramps having various steepness and also during action potential waveforms with progressively prolonged APD. Gradually delaying repolarization results in smaller magnitude of I_Kr and I_K1 currents at an isochronal phase of the pulses. This represents a positive feedback mechanism, which appears to contribute to the reverse rate-dependent prolongation of action potentials.

Conclusions and implications:

Action potential configuration may influence the reverse rate-dependent APD prolongation due to the intrinsic properties of I_Kr and I_K1 currents. Drugs lengthening repolarization by decreasing repolarizing outward, or increasing depolarizing inward, currents are expected to cause reverse rate-dependent APD lengthening with high probability, regardless of which current they modify.     

2'-Methoxy-6-methylflavone: a novel anxiolytic and sedative with subtype selective activating and modulating actions at GABA(A) receptors

BACKGROUND AND PURPOSE

Flavonoids are known to have anxiolytic and sedative effects mediated via actions on ionotropic GABA receptors. We sought to investigate this further.

EXPERIMENTAL APPROACH

We evaluated the effects of 2′-methoxy-6-methylflavone (2′MeO6MF) on native GABA_A receptors in new-born rat hippocampal neurons and determined specificity from 18 human recombinant GABA_A receptor subtypes expressed in Xenopus oocytes. We used ligand binding, two-electrode voltage clamp and patch clamp studies together with behavioural studies.

KEY RESULTS

2′MeO6MF potentiated GABA at α2β1γ2L and all α1-containing GABA_A receptor subtypes. At α2β2/3γ2L GABA_A receptors, however, 2′MeO6MF directly activated the receptors without potentiating GABA. This activation was attenuated by bicuculline and gabazine but not flumazenil indicating a novel site. Mutation studies showed position 265 in the β1/2 subunit was key to whether 2′MeO6MF was an activator or a potentiator. In hippocampal neurons, 2′MeO6MF directly activated single-channel currents that showed the hallmarks of GABA_A Cl^- currents. In the continued presence of 2′MeO6MF the single-channel conductance increased and these high conductance channels were disrupted by the γ2(381–403) MA peptide, indicating that such currents are mediated by α2/γ2-containing GABA_A receptors. In mice, 2′MeO6MF (1–100 mg·kg⁻¹; i.p.) displayed anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark tests. 2′MeO6MF induced sedative effects at higher doses in the holeboard, actimeter and barbiturate-induced sleep time tests. No myorelaxant effects were observed in the horizontal wire test.

CONCLUSIONS AND IMPLICATIONS

2′MeO6MF will serve as a tool to study the complex nature of the activation and modulation of GABA_A receptor subtypes.     

Natural and synthetic modulators of SK (K(ca)2) potassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7

BACKGROUND AND PURPOSE

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells.

EXPERIMENTAL APPROACH

We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments.

KEY RESULTS

TRPM7 currents were inhibited by modulators of small conductance Ca²⁺-activated K⁺ channels (K_Ca2.1–2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC₅₀ 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²⁺-dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K_Ca2.1–2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.     

Modulation of presynaptic GABA(A) receptors by endogenous neurosteroids

BACKGROUND AND PURPOSE

Although 3α-hydroxy, 5α-reduced pregnane steroids, such as allopregnanolone (AlloP) and tetrahydrodeoxycorticosterone, are endogenous positive modulators of postsynaptic GABA_A receptors, the functional roles of endogenous neurosteroids in synaptic transmission are still largely unknown.

EXPERIMENTAL APPROACH

In this study, the effect of AlloP on spontaneous glutamate release was examined in mechanically isolated dentate gyrus hilar neurons by use of the conventional whole-cell patch-clamp technique.

KEY RESULTS

AlloP increased the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in a dose-dependent manner. The AlloP-induced increase in sEPSC frequency was completely blocked by a non-competitive GABA_A receptor blocker, tetrodotoxin or Cd²⁺, suggesting that AlloP acts on presynaptic GABA_A receptors to depolarize presynaptic nerve terminals to increase the probability of spontaneous glutamate release. On the other hand, γ-cyclodextrin (γ-CD) significantly decreased the basal frequency of sEPSCs. However, γ-CD failed to decrease the basal frequency of sEPSCs in the presence of a non-competitive GABA_A receptor antagonist or tetrodotoxin. In addition, γ-CD failed to decrease the basal frequency of sEPSCs after blocking the synthesis of endogenous 5α-reduced pregnane steroids. Furthermore, γ-CD decreased the extent of muscimol-induced increase in sEPSC frequency, suggesting that endogenous neurosteroids can directly activate and/or potentiate presynaptic GABA_A receptors to affect spontaneous glutamate release onto hilar neurons.

CONCLUSIONS AND IMPLICATIONS

The modulation of presynaptic GABA_A receptors by endogenous neurosteroids might affect the excitability of the dentate gyrus-hilus-CA3 network, and thus contribute, at least in part, to some pathological conditions, such as catamenial epilepsy and premenstrual dysphoric disorder.     

Effects of aldosterone and related steroids on LPS-induced increased expression of inducible NOS in rat aortic smooth muscle cells

BACKGROUND AND PURPOSE

Expression of inducible NOS (iNOS) is important in certain inflammatory diseases. We determined if the hormone aldosterone, a mineralocorticoid receptor (MR) agonist, affects LPS activation of iNOS expression in rat aortic smooth muscle cells (RASMC).

EXPERIMENTAL APPROACH

Cultured RASMC were treated with LPS, with or without agonists/antagonists of steroid receptors. iNOS expression was determined by nitrite assays on culture medium removed from treated cells and by immunoblotting of cell protein extracts.

KEY RESULTS

LPS (1 µg·mL⁻¹) increased nitrite and iNOS protein above that in control (untreated) cells. These effects of LPS were reduced by aldosterone (0.1–10 µM). The MR antagonists, eplerenone (10 µM) and spironolactone (10 or 50 µM), did not inhibit these actions of 1 µM aldosterone, but the latter were prevented by 10 µM mifepristone, a glucocorticoid (GR) and progestogen receptor (PR) antagonist. Mifepristone also prevented the reduction of LPS-induced nitrite increase produced by 1 µM dexamethasone (GR agonist) and 10 µM progesterone (PR agonist). Spironolactone (10–50 µM) by itself decreased LPS-induced increases in nitrite and iNOS protein. Mifepristone (10 µM) partially reversed these effects of 10 µM spironolactone, but not those of 50 µM; the effects of 50 µM spironolactone were also unchanged when mifepristone was increased to 50 µM.

CONCLUSIONS AND IMPLICATIONS

This pharmacological profile suggests that aldosterone, and possibly 10 µM spironolactone, use mechanisms that are dependent on PR and/or GR, but not MR, to inhibit iNOS induction in RASMC. With 50 µM spironolactone, other inhibitory mechanisms requiring further investigation may become predominant.     

Characteristics and molecular basis of celecoxib modulation on K(v)7 potassium channels

BACKGROUND AND PURPOSE

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used for the treatment of pain and inflammation. Emerging and accumulating evidence suggests that celecoxib can affect cellular targets other than COX, such as ion channels. In this study, we characterized the effects of celecoxib on K_v7 K⁺ channels and compared its effects with the well-established K_v7 channel opener retigabine.

EXPERIMENTAL APPROACH

A perforated whole-cell patch technique was used to record K_v7currents expressed in HEK 293 cells and M-type currents from rat superior cervical ganglion neurons.

KEY RESULTS

Celecoxib enhanced K_v7.2–7.4, K_v7.2/7.3 and K_v7.3/7.5 currents but inhibited K_v7.1 and K_v7.1/KCNE1 currents and these effects were concentration dependent. The IC₅₀ value for inhibition of K_v7.1 channels was approximately 4 µM and the EC₅₀ values for activation of K_v7.2–7.4, K_v7.2/K_v7.3 and K_v7.3/K_v7.5 channels were approximately 2–5 µM. The effects of celecoxib were manifested by increasing current amplitudes, shifting the voltage-dependent activation curve in a more negative direction and slowing the deactivation of K_v7 currents. 2,5-Dimethyl-celecoxib, a celecoxib analogue devoid of COX inhibition activity, has similar but greater effects on K_v7currents. K_v7.2(A235T) and K_v7.2(W236L) mutant channels, which have greatly attenuated responses to retigabine, showed a reversed response to celecoxib, from activation to inhibition.

CONCLUSIONS AND IMPLICATIONS

These results suggest that K_v7 channels are targets of celecoxib action and provide new mechanistic evidence for understanding the effects of celecoxib. They also provide a new approach to developing K_v7 modulators and for studying the structure–function relationship of K_v7 channels.     

Differential contributions of adenosine to hypoxia-evoked depressions of three neuronal pathways in isolated spinal cord of neonatal rats

BACKGROUND AND PURPOSE

Hypoxic effects on neuronal functions vary significantly with experimental conditions, but the mechanism for this is unclear. Adenosine has been reported to play a key role in depression of neuronal activities in the CNS during acute hypoxia. Hence, we examined the effect of acute hypoxia on different spinal reflex potentials and the contribution of adenosine to them.

EXPERIMENTAL APPROACH

Spinal reflex potentials, monosynaptic reflex potential (MSR), slow ventral root potential (sVRP) and dorsal root potential (DRP), were measured in the isolated spinal cord of the neonatal rat. Adenosine release was measured by using enzymatic biosensors.

KEY RESULTS

In the spinal cord preparation isolated from postnatal day 5–8 rats at 27°C, acute hypoxia induced adenosine release and depressed three reflex potentials. However, in postnatal day 0–3 rats at 27°C, the hypoxic-induced adenosine release and depression of MSR were negligible, while the depression of sVRP and DRP were perceptible responses. In postnatal day 0–3 rats at 33°C, hypoxia evoked adenosine release and depression of MSR. An adenosine A₁ receptor selective antagonist and a high [Ca²⁺]_o, which suppressed adenosine release, abolished the hypoxic-induced depression of MSR but not those of sVRP and DRP.

CONCLUSIONS AND IMPLICATIONS

Hypoxic-induced depression of MSR depends on adenosine release, which is highly susceptible to age, temperature and [Ca²⁺]_o. However, a large part of the depressions of DRP and sVRP are mediated via adenosine-independent mechanisms. This differential contribution of adenosine to depression is suggested to be an important factor for the variable effects of hypoxia on neuronal functions.     

Valerenic acid derivatives as novel subunit-selective GABAA receptor ligands �Cin vitro and in vivo characterization

BACKGROUND AND PURPOSE

Subunit-specific modulators of γ-aminobutyric acid (GABA) type A (GABA_A) receptors can help to assess the physiological function of receptors with different subunit composition and also provide the basis for the development of new drugs. Valerenic acid (VA) was recently identified as a β_2/3 subunit-specific modulator of GABA_A receptors with anxiolytic potential. The aim of the present study was to generate VA derivatives as novel GABA_A receptor modulators and to gain insight into the structure–activity relation of this molecule.

EXPERIMENTAL APPROACH

The carboxyl group of VA was substituted by an uncharged amide or amides with different chain length. Modulation of GABA_A receptors composed of different subunit compositions by the VA derivatives was studied in Xenopus oocytes by means of the two-microelectrode voltage-clamp technique. Half-maximal stimulation of GABA-induced chloride currents (I_GABA) through GABA_A receptors (EC₅₀) and efficacies (maximal stimulation of I_GABA) were estimated. Anxiolytic activity of the VA derivatives was studied in mice, applying the elevated plus maze test.

KEY RESULTS

Valerenic acid amide (VA-A) displayed the highest efficacy (more than twofold greater I_GABA enhancement than VA) and highest potency (EC₅₀= 13.7 ± 2.3 µM) on α₁β₃ receptors. Higher efficacy and potency of VA-A were also observed on α₁β₂γ_2s and α₃β₃γ_2s receptors. Anxiolytic effects were most pronounced for VA-A.

CONCLUSIONS AND IMPLICATIONS

Valerenic acid derivatives with higher efficacy and affinity can be generated. Greater in vitro action of the amide derivative correlated with a more pronounced anxiolytic effect in vivo. The data give further confidence in targeting β₃ subunit containing GABA_A receptors for development of anxiolytics.