Anti-inflammatory synergy of MEN16132, a kinin B2 receptor antagonist, and dexamethasone in carrageenan-induced knee joint arthritis in rats

BACKGROUND AND PURPOSE

Bradykinin, through its B₂ receptor, is involved in inflammatory processes related to arthropathies. In carrageenan and lipopolysaccharide (LPS)-induced arthritis in rat, the anti-inflammatory activity of MEN16132, a potent and selective kinin B₂ receptor antagonist, was compared with that of steroidal and nonsteroidal anti-inflammatory drugs. The interaction between MEN16132 and dexamethasone was also investigated.

EXPERIMENTAL APPROACH

Drugs, alone or in combination, were injected into the knee joint 30 min before intra-articular administration of carrageenan or LPS, in pentobarbital anaesthetized rats. Effects on incapacitation, oedema, neutrophil recruitment and kallikrein system activation, in the knee joint, were assessed.

KEY RESULTS

MEN16132 and dexamethasone (10–300 µg per knee) dose-dependently reduced carrageenan-induced joint pain, oedema and neutrophil infiltration, reaching a maximal inhibition of about 50%. Dexketoprofen exerted a similar analgesic activity, whereas it did not affect the other inflammatory responses. MEN16132 showed a partial inhibition of LPS-induced joint pain, whereas dexamethasone produced a full analgesic effect. Combination of MEN16132 and dexamethasone showed a strong synergistic interaction in inhibiting both carrageenan and LPS-induced knee joint inflammation. Dexamethasone did not prevent the contact activation of prekallikrein by carrageenan and the subsequent release of kallikreins and bradykinin in the synovium.

CONCLUSIONS AND IMPLICATIONS

Steroids and kinin B₂ receptor antagonists appear to relieve arthritic symptoms induced by carrageenan or LPS and act synergistically to inhibit joint inflammation. This could have interesting therapeutic implications, possibly opening the way for combination therapies in the control of inflammatory arthropathies.     

Slow receptor dissociation is not a key factor in the duration of action of inhaled long-acting β2-adrenoceptor agonists

BACKGROUND AND PURPOSE

β₂-Adrenoceptor agonists are important bronchodilators used for the treatment of chronic obstructive pulmonary disease and asthma. Clinical data on β₂-adrenoceptor agonists show a range of onset and duration of action. We have investigated whether the receptor binding kinetics of β₂-adrenoceptor agonists can explain their observed onset of action and duration of effect in the clinic.

EXPERIMENTAL APPROACH

[³H]-DHA was used to label β₂-adrenoceptors expressed in CHO-cell membranes (K_d of 0.084 nM). Competition kinetic experiments were performed in the presence of unlabelled β₂ agonists at 37°C in HBSS containing GTP. To determine the kinetic parameters, three concentrations (10, 3 and 1 ×K_i) of the unlabelled compound were employed against a fixed concentration of [³H]-DHA (0.6 nM).

KEY RESULTS

The clinically used β₂-adrenoceptor agonists exhibited a range of association and dissociation rates. The kinetic K_d and the competition K_i values of the eight β₂-adrenoceptor agonists examined were strongly correlated, suggesting that the method had produced accurate k_off and k_on rates. The kinetic on-rate was highly correlated with equilibrium binding affinity.

CONCLUSIONS AND IMPLICATIONS

Although the β₂-adrenoceptor agonists displayed a range of kinetic rate parameters, simulations at relevant drug concentrations suggest that receptor kinetics do not play an important role in determining onset of action in the clinic. In addition, it is unlikely that receptor kinetics exert an important influence on the duration of action of these agonists, as indacaterol (once daily dosing) had a shorter residency time at the receptor than salmeterol (twice daily dosing).     

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE

KCNQ-encoded voltage-gated potassium channels (K_v7) have recently been identified as important anti-constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

EXPERIMENTAL APPROACH

Fifty arteries (41 from visceral adipose tissue, 9 mesenteric arteries) were obtained from subjects undergoing elective surgery. Quantitative RT-PCR experiments using primers specific for all known KCNQ genes and immunohistochemsitry were used to show K_v7 channel expression. Wire myography and single cell electrophysiology assessed the function of these channels.

KEY RESULTS

KCNQ4 was expressed in all arteries assessed, with variable contributions from KCNQ1, 3 and 5. KCNQ2 was not detected. K_v7 channel isoform-dependent staining was revealed in the smooth muscle layer. In functional studies, the K_v7 channel blockers, XE991 and linopirdine increased isometric tension and inhibited K⁺ currents. In contrast, the K_v7.1-specific blocker chromanol 293B did not affect vascular tone. Two K_v7 channel activators, retigabine and acrylamide S-1, relaxed preconstricted arteries, actions reversed by XE991. K_v7 channel activators also suppressed spontaneous contractile activity in seven arteries, reversible by XE991.

CONCLUSIONS AND IMPLICATIONS

This is the first study to demonstrate not only the presence of KCNQ gene products in human arteries but also their contribution to vascular tone ex vivo.

LINKED ARTICLE

This article is commented on by Mani and Byron, pp. 38–41 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.01065.x     

Fasitibant chloride, a kinin B₂ receptor antagonist, and dexamethasone interact to inhibit carrageenan-induced inflammatory arthritis in rats

BACKGROUND AND PURPOSE

Bradykinin, through the kinin B₂ receptor, is involved in inflammatory processes related to arthropathies. B₂ receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated.

EXPERIMENTAL APPROACH

Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1β, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h.

KEY RESULTS

The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg⁹-[Leu⁸]-bradykinin (B₂ receptor, leukotriene, catecholamine and B₁ receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg⁹-[Leu⁸]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg⁹-[Leu⁸]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment.

CONCLUSIONS AND IMPLICATIONS

Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B₂ receptor, or through the indirect effects mediated by release of eicosanoids and cytokines.     

In vitro and in vivo characterization of PF-04418948, a novel, potent and selective prostaglandin EP₂ receptor antagonist

BACKGROUND AND PURPOSE

Studies of the role of the prostaglandin EP₂ receptor) have been limited by the availability of potent and selective antagonist tools. Here we describe the in vitro/in vivo pharmacological characterization of a novel EP₂ receptor antagonist, PF-04418948 (1-(4-fluorobenzoyl)-3-{[(6-methoxy-2-naphthyl)oxy]methyl} azetidine-3-carboxylic acid).

EXPERIMENTAL APPROACH

Functional antagonist potency was assessed in cell-based systems expressing human EP₂ receptors and native tissue preparations from human, dog and mouse. The selectivity of PF-04418948 was assessed against related receptors and a panel of GPCRs, ion channels and enzymes. The ability of PF-04418948 to pharmacologically block EP₂ receptor function in vivo was tested in rats.

KEY RESULTS

PF-04418948 inhibited prostaglandin E₂ (PGE₂)-induced increase in cAMP in cells expressing EP₂ receptors with a functional K_B value of 1.8 nM. In human myometrium, PF-04418948 produced a parallel, rightward shift of the butaprost-induced inhibition of the contractions induced by electrical field stimulation with an apparent K_B of 5.4 nM. In dog bronchiole and mouse trachea, PF-04418948 produced parallel rightward shifts of the PGE₂-induced relaxation curve with a K_B of 2.5 nM and an apparent K_B of 1.3 nM respectively. Reversal of the PGE₂-induced relaxation in the mouse trachea by PF-04418948 produced an IC₅₀ value of 2.7 nM. Given orally, PF-04418948 attenuated the butaprost-induced cutaneous blood flow response in rats. PF-04418948 was selective for EP₂ receptors over homologous and unrelated receptors, enzymes and channels.

CONCLUSIONS AND IMPLICATIONS

PF-04418948 is an orally active, potent and selective surmountable EP₂ receptor antagonist that should aid further elaboration of EP₂ receptor function.

LINKED ARTICLE

This article is commented on by Birrell and Nials, pp. 1845–1846 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01494.x     

Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

BACKGROUND AND PURPOSE

Controlling vascular tone involves K⁺ efflux through endothelial cell small- and intermediate-conductance calcium-activated potassium channels (K_Ca2.3 and K_Ca3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.

EXPERIMENTAL APPROACH

Transgenic mice expressing enhanced green fluorescent protein (eGFP) in astrocytes were used to assess K_Ca2.3 and K_Ca3.1 expression by immunohistochemistry and RT-PCR. K_Ca currents in eGFP-positive astrocytes were determined in situ using whole-cell patch clamp electrophysiology. The contribution of K_Ca3.1 to neurovascular coupling was investigated in pharmacological experiments using electrical field stimulation (EFS) to evoke parenchymal arteriole dilatation in FVB/NJ mouse brain slices and whisker stimulation to evoke changes in cerebral blood flow in vivo, measured by laser Doppler flowmetry.

KEY RESULTS

K_Ca3.1 immunoreactivity was restricted to astrocyte processes and endfeet and RT-PCR confirmed astrocytic K_Ca2.3 and K_Ca3.1 mRNA expression. With 200 nM [Ca²⁺]_i, the K_Ca2.1-2.3/K_Ca3.1 opener NS309 increased whole-cell currents. CyPPA, a K_Ca2.2/K_Ca2.3 opener, was without effect. With 1 µM [Ca²⁺]_i, the K_Ca3.1 inhibitor TRAM-34 reduced currents whereas apamin (K_Ca2.1-2.3 blocker) had no effect. CyPPA also inhibited currents evoked by NS309 in HEK293 cells expressing K_Ca3.1. EFS-evoked Fluo-4 fluorescence confirmed astrocyte endfoot recruitment into neurovascular coupling. TRAM-34 inhibited EFS-evoked arteriolar dilatation by 50% whereas charybdotoxin, a blocker of K_Ca3.1 and the large-conductance K_Ca channel, K_Ca1.1, inhibited dilatation by 82%. TRAM-34 reduced the cortical hyperaemic response to whisker stimulation by 40%.

CONCLUSION AND IMPLICATIONS

Astrocytes express functional K_Ca3.1 channels, and these contribute to neurovascular coupling.

LINKED ARTICLES

This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3     

Activation of ��-opioid receptors and block of KIR3 potassium channels and NMDA receptor conductance by l- and d-methadone in rat locus coeruleus

BACKGROUND AND PURPOSE

Methadone activates opioid receptors to increase a potassium conductance mediated by G-protein-coupled, inwardly rectifying, potassium (K_IR3) channels. Methadone also blocks K_IR3 channels and N-methyl-D-aspartic acid (NMDA) receptors. However, the concentration dependence and stereospecificity of receptor activation and channel blockade by methadone on single neurons has not been characterized.

EXPERIMENTAL APPROACH

Intracellular and whole-cell recording were made from locus coeruleus neurons in brain slices and the activation of µ-opioid receptors and blockade of K_IR3 and NMDA channels with l- and d-methadone was examined.

KEY RESULTS

The potency of l-methadone, measured by the amplitude of hyperpolarization was 16.5-fold higher than with d-methadone. A maximum hyperpolarization was caused by both enantiomers (∼30 mV); however, the maximum outward current measured with whole-cell voltage-clamp recording was smaller than the current induced by [Met]⁵enkephalin. The K_IR3 conductance induced by activation of α₂-adrenoceptors was decreased with high concentrations of l- and d-methadone (10–30 µM). In addition, methadone blocked the resting inward rectifying conductance (K_IR). Both l- and d-methadone blocked the NMDA receptor-dependent current. The block of NMDA receptor-dependent current was voltage-dependent suggesting that methadone acted as a channel blocker.

CONCLUSIONS AND IMPLICATIONS

Methadone activated µ-opioid receptors at low concentrations in a stereospecific manner. K_IR3 and NMDA receptor channel block was not stereospecific and required substantially higher concentrations. The separation in the concentration range suggests that the activation of µ-opioid receptors rather than the channel blocking properties mediate both the therapeutic and toxic actions of methadone.     

PKC-dependent activation of human K(2P) 18.1 K(+) channels

BACKGROUND AND PURPOSE

Two-pore-domain K⁺ channels (K_2P) mediate K⁺ background currents that modulate the membrane potential of excitable cells. K_2P18.1 (TWIK-related spinal cord K⁺ channel) provides hyperpolarizing background currents in neurons. Recently, a dominant-negative loss-of-function mutation in K_2P18.1 has been implicated in migraine, and activation of K_2P18.1 channels was proposed as a therapeutic strategy. Here we elucidated the molecular mechanisms underlying PKC-dependent activation of K_2P18.1 currents.

EXPERIMENTAL APPROACH

Human K_2P18.1 channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded with the two-electrode voltage clamp technique.

KEY RESULTS

Stimulation of PKC using phorbol 12-myristate-13-acetate (PMA) activated the hK_2P18.1 current by 3.1-fold in a concentration-dependent fashion. The inactive analogue 4α-PMA had no effect on channel activity. The specific PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and chelerythrine reduced PMA-induced channel activation indicating that PKC is involved in this effect of PMA. Selective activation of conventional PKC isoforms with thymeleatoxin (100 nM) did not reproduce K_2P18.1 channel activation. Current activation by PMA was not affected by pretreatment with CsA (calcineurin inhibitor) or KT 5720 (PKA inhibitor), ruling out a significant contribution of calcineurin or cross-talk with PKA to the PKC-dependent hK_2P18.1 activation. Finally, mutation of putative PKC phosphorylation sites did not prevent PMA-induced K_2P18.1 channel activation.

CONCLUSIONS AND IMPLICATIONS

We demonstrated that activation of hK_2P18.1 (TRESK) by PMA is mediated by PKC stimulation. Hence, PKC-mediated activation of K_2P18.1 background currents may serve as a novel molecular target for migraine treatment.     

A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents

BACKGROUND AND PURPOSE

Cannabinoid CB₁ receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects.

EXPERIMENTAL APPROACH

Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated.

KEY RESULTS

AM6545 binds to CB₁ receptors with a K_i of 1.7 nM and CB₂ receptors with a K_i of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB₁ receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB₁ receptor gene-deficient mice, but not in CB₁/CB₂ receptor double knockout mice.

CONCLUSIONS AND IMPLICATIONS

Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications.     

Electrophysiological effects of anandamide on rat myocardium

Background and purpose:

The endocannabinoid, anandamide, has anti-arrhythmic effects. The aim of the present study was to explore the electrophysiological effects of anandamide on rat myocardium.

Experimental approach:

Evoked action potentials (APs) were recorded using intracellular recording technique in rat cardiac papillary muscles. In addition, L-type Ca²⁺ current was measured and analysed using whole-cell patch-clamp recording technique in isolated rat cardiac ventricular myocytes.

Key results:

In cardiac papillary muscles, anandamide (1, 10, 100 nM) decreased AP duration in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased AP amplitude, overshoot and V_max in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB₁ receptors, but not AM630 (100 nM), a CB₂ receptor antagonist. Furthermore, an agonist of L-type Ca²⁺ channels, Bay K 8644 (0.5 µM), a K⁺ channel blocker tetraethylammonium chloride (20 mM) and the nitric oxide synthase inhibitor l-NAME (1 mM) had no effect on anandamide-induced decrease in AP duration. In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca²⁺ current concentration-dependently, and shifted the current–voltage relationship curve of the Ca²⁺ current. Anandamide (100 nM) shifted the steady-state inactivation curve to the left and the recovery curve to the right. Blockade of CB₁ receptors with AM251 (100 nM), but not CB₂ receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca²⁺ currents.

Conclusions and implications:

These data suggest that anandamide suppressed AP and L-type Ca²⁺ current in cardiac myocytes through CB₁ receptors.     

NS6180, a new KCa3.1 channel inhibitor prevents T-cell activation and inflammation in a rat model of inflammatory bowel disease

Background and Purpose

The K_Ca3.1 channel is a potential target for therapy of immune disease. We identified a compound from a new chemical class of K_Ca3.1 inhibitors and assessed in vitro and in vivo inhibition of immune responses.

Experimental Approach

We characterized the benzothiazinone NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) with respect to potency and molecular site of action on K_Ca3.1 channels, selectivity towards other targets, effects on T-cell activation as well as pharmacokinetics and inflammation control in colitis induced by 2,4-dinitrobenzene sulfonic acid, a rat model of inflammatory bowel disease (IBD).

Key Results

NS6180 inhibited cloned human K_Ca3.1 channels (IC₅₀ = 9 nM) via T250 and V275, the same amino acid residues conferring sensitivity to triarylmethanes such as like TRAM-34. NS6180 inhibited endogenously expressed K_Ca3.1 channels in human, mouse and rat erythrocytes, with similar potencies (15–20 nM). NS6180 suppressed rat and mouse splenocyte proliferation at submicrolar concentrations and potently inhibited IL-2 and IFN-γ production, while exerting smaller effects on IL-4 and TNF-α and no effect on IL-17 production. Antibody staining showed K_Ca3.1 channels in healthy colon and strong up-regulation in association with infiltrating immune cells after induction of colitis. Despite poor plasma exposure, NS6180 (3 and 10 mg·kg⁻¹ b.i.d.) dampened colon inflammation and improved body weight gain as effectively as the standard IBD drug sulfasalazine (300 mg·kg⁻¹ q.d.).

Conclusions and Implications

NS6180 represents a novel class of K_Ca3.1 channel inhibitors which inhibited experimental colitis, suggesting K_Ca3.1 channels as targets for pharmacological control of intestinal inflammation.     

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat

Background and purpose:

Recent findings suggest that the noxious gas H₂S is produced endogenously, and that physiological concentrations of H₂S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H₂S to modulate carrageenan-induced synovitis in the rat knee.

Experimental approach:

Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H₂S formation (dl-propargylglycine) or an H₂S donor [Lawesson''s reagent (LR)].

Key results:

Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters.

Conclusions and implications:

Whereas exogenous H₂S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H₂S exerts little immunomodulatory effect. These data further support the development and use of H₂S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.     

Physiologically based pharmacokinetic modelling and in vivo [I]/Ki accurately predict P-glycoproteinmediated drug-drug interactions with dabigatran etexilate

Background and purpose

In vitro inhibitory potency (K_i)-based predictions of P-glycoprotein (P-gp)-mediated drug-drug interactions (DDIs) are hampered by the substantial variability in inhibitory potency. In this study, in vivo-based [I]/K_i values were used to predict the DDI risks of a P-gp substrate dabigatran etexilate (DABE) using physiologically based pharmacokinetic (PBPK) modelling.

Experimental approach

A baseline PBPK model was established with digoxin, a known P-gp substrate. The K_m (P-gp transport) of digoxin in the baseline PBPK model was adjusted to K_mⁱ to fit the change of digoxin pharmacokinetics in the presence of a P-gp inhibitor. Then ‘in vivo’ [I]/K_i of this P-gp inhibitor was calculated using K_mⁱ/K_m. Baseline PBPK model was developed for DABE, and the ‘in vivo’ [I]/K_i was incorporated into this model to simulate the static effect of P-gp inhibitor on DABE pharmacokinetics. This approach was verified by comparing the observed and the simulated DABE pharmacokinetics in the presence of five different P-gp inhibitors.

Key results

This approach accurately predicted the effects of five P-gp inhibitors on DABE pharmacokinetics (98–133% and 89–104% for the ratios of AUC and C_max respectively). The effects of 16 other P-gp inhibitors on the pharmacokinetics of DABE were also confidently simulated.

Conclusions and implications

‘In vivo’ [I]/K_i and PBPK modelling, used in combination, can accurately predict P-gp-mediated DDIs. The described framework provides a mechanistic basis for the proper design of clinical DDI studies, as well as avoiding unnecessary clinical DDI studies.     

Hypericin prolongs action potential duration in hippocampal neurons by acting on K+ channels

Background and purpose:

Synaptic deficiency is generally accepted to be involved in major depression, and accordingly classic antidepressants exert their effects through enhancing synaptic efficiency. Hypericin is one of the major active constituents of extracts of St. John''s Wort (Hypericum perforatum L.) with antidepressive actions, but little is known about its therapeutic mechanisms. Our aim was to explore whether hypericin has a modulatory effect on neuronal action potential (AP) duration by acting on voltage-gated ion channels.

Experimental approach:

We used voltage-clamp and current-clamp techniques in a whole-cell configuration to study primary cultures of neonatal rat hippocampal neurones. We measured the effects of extracellularly applied hypericin on AP duration as well as on voltage-gated Na⁺, I_A and I_K currents.

Key results:

Extracellularly applied hypericin dose-dependently increased AP duration but barely affected its amplitude. Further analysis revealed that hypericin inhibited both transient I_A and delayed rectifier I_K potassium currents. In contrast, hypericin exerted no significant effect on both Na⁺ peak current and its decay kinetics.

Conclusions and implications:

Extracellularly applied hypericin increased AP duration, which might be ascribed to its effect on I_A and I_K currents. As a small increase in AP duration could lead to a dramatic increase in synaptic efficiency, our results imply that hypericin might exert its antidepressant effects by enhancing presynaptic efficiency.     

Inhibition of vascular calcium-gated chloride currents by blockers of KCa1.1, but not by modulators of KCa2.1 or KCa2.3 channels

Background and purpose:

Recent pharmacological studies have proposed there is a high degree of similarity between calcium-activated Cl⁻ channels (CaCCs) and large conductance, calcium-gated K⁺ channels (K_Ca1.1). The goal of the present study was to ascertain whether blockers of K_Ca1.1 inhibited calcium-activated Cl⁻ currents (I_ClCa) and if the pharmacological overlap between K_Ca1.1 and CaCCs extends to intermediate and small conductance, calcium-activated K⁺ channels.

Experimental approaches:

Whole-cell Cl⁻ and K⁺ currents were recorded from murine portal vein myocytes using the whole-cell variant of the patch clamp technique. CaCC currents were evoked by pipette solutions containing 500 nM free [Ca²⁺].

Key results:

The selective K_Ca1.1 blocker paxilline (1 µM) inhibited I_ClCa by ∼90%, whereas penitrem A (1 µM) and iberiotoxin (100 and 300 nM) reduced the amplitude of I_ClCa by ∼20%, as well as slowing channel deactivation. Paxilline also abolished the stimulatory effect of niflumic acid on the CaCC. In contrast, an antibody against the Ca²⁺-binding domain of murine K_Ca1.1 had no effect on I_ClCa while inhibiting spontaneous K_Ca1.1 currents. Structurally different modulators of small and intermediate conductance calcium-activated K⁺ channels (K_Ca2.1 and K_Ca2.3), namely 1-EBIO, (100 µM); NS309, (1 µM); TRAM-34, (10 µM); UCL 1684, (1 µM) had no effect on I_ClCa.

Conclusions and implications:

These data show that the selective K_Ca1.1 blockers also reduce I_ClCa considerably. However, the pharmacological overlap that exists between CaCCs and K_Ca1.1 does not extend to the calcium-binding domain or to other calcium-gated K⁺ channels.     

TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

BACKGROUND AND PURPOSE

TASK1 (K_2P3.1) two-pore-domain K⁺ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes.

KEY RESULTS

ET-1 inhibited TASK1-mediated I_KN currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET_A (IC₅₀= 0.08 nM) and ET_B (IC₅₀= 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser³³⁶ and Ser³⁹³. Mutation of Ser³⁹³ rendered TASK1 channels insensitive to ET_A- or ET_B-mediated current inhibition. In contrast, removal of Ser³³⁶ selectively attenuated ET_A-dependent TASK1 regulation without affecting the ET_B pathway.

CONCLUSIONS AND IMPLICATIONS

ET-1 regulated vascular TASK1 currents through ET_A and ET_B receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.     

The new antihypertensive drug iptakalim activates ATP-sensitive potassium channels in the endothelium of resistance blood vessels

Aim:

To investigate the mechanisms underlying the activation of ATP-sensitive potassium channels (K_ATP) by iptakalim in cultured rat mesenteric microvascular endothelial cells (MVECs).

Methods:

Whole-cell K_ATP currents were recorded in MVECs using automated patch clamp devices. Nucleotides (ATP, ADP and UDP) were added to the internal perfusion system, whereas other drugs were added to the cell suspension on NPC-1 borosilicate glass chips.

Results:

Application of iptakalim (10 and 100 μmol/L) significantly increased the whole-cell K_ATP currents, which were prevented by the specific K_ATP blocker glibenclamide (1.0 μmol/L). The opening of K_ATP channels by iptakalim depended upon the intracellular concentrations of ATP or NDPs: iptakalim activated K_ATP channels when the intracellular ATP or NDPs were at 100 or 1000 μmol/L, and was ineffective when the non-hydrolysable ATP analogue ATPγS (1000 μmol/L) was infused into the cells. In contrast, the K_ATP opener pinacidil activated K_ATP channels when the intracellular concentrations of ATP or NDPs ranged from 10 to 5000 μmol/L, and even ATPγS (1000 μmol/L) was infused into the cells.

Conclusion:

Iptakalim activates K_ATP channels in the endothelial cells of resistance blood vessels with a low metabolic status, and this activation is dependent on both ATP hydrolysis and ATP ligands.     

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α1A-adrenoceptor

Background and purpose:

Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G-protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs.

Experimental approach:

We studied the interactions of mamba venom fractions with α₁-adrenoceptors in binding experiments with ³H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle.

Key results:

AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K_i= 0.35 nM) and high specificity for the human α_1A-adrenoceptor subtype. We showed high selectivity and affinity (K_d= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k_on= 6 × 10⁶·M⁻¹·min⁻¹) with an unusually stable α_1A-adrenoceptor/AdTx1 complex (t_1/2diss= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine''s actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM.

Conclusions and implications:

AdTx1 is the most specific and selective peptide inhibitor for the α_1A-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.     

N-arachidonoyl glycine, an endogenous lipid that acts as a vasorelaxant via nitric oxide and large conductance calcium-activated potassium channels

Background and purpose:

N-arachidonoyl glycine (NAGly) is an endogenous lipid that is structurally similar to the endocannabinoid, N-arachidonoyl ethanolamide (anandamide). While NAGly does not activate cannabinoid receptors, it exerts cannabimimetic effects in pain regulation. Here, we have determined if NAGly, like anandamide, modulates vascular tone.

Experimental approach:

In rat isolated small mesenteric arteries, the relaxant responses to NAGly were characterized. Effects of N-arachidonoyl serine and N-arachidonoyl γ-aminobutyric acid were also examined.

Key results:

In endothelium-intact arteries, NAGly-induced relaxation (pEC_50%= 5.7 ± 0.2; relaxation at 30 µM = 98 ± 1%) was attenuated by l-NAME (a nitric oxide synthase inhibitor) or iberiotoxin [selective blocker of large conductance Ca²⁺-activated K⁺ channels (BK_Ca)], and abolished by high extracellular K⁺ concentration. Endothelial removal reduced the potency of NAGly, and the resultant relaxation was inhibited by iberiotoxin, but not l-NAME. NAGly responses were sensitive to the novel cannabinoid receptor antagonist O-1918 independently of endothelial integrity, whereas pertussis toxin, which uncouples G_i/o proteins, attenuated NAGly relaxation only in endothelium-intact arteries. Treatments with antagonists for CB₁, CB₂ and TRPV1 receptors, or inhibitors of fatty acid amide hydrolase and COX had no effect. The two other arachidonoyl amino acids also induced iberiotoxin- and L-NAME-sensitive relaxations.

Conclusion and implications:

NAGly acts as a vasorelaxant predominantly via activation of BK_Ca in rat small mesenteric arteries. We suggest that NAGly activates an unknown G_i/o-coupled receptor, stimulating endothelial release of nitric oxide which in turn activates BK_Ca in the smooth muscle. In addition, NAGly might also activate BK_Ca through G_i/o- and nitric oxide-independent mechanisms.     

Evidence that the plant cannabinoid cannabigerol is a highly potent α2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist

Background and purpose:

Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Δ⁹-tetrahydrocannabinol, cannabidiol and Δ⁹-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor.

Experimental approach:

The [³⁵S]GTPγS binding assay, performed with mouse brain membranes, was used to test the ability of cannabigerol to produce G protein-coupled receptor activation or blockade. Its ability to displace [³H]CP55940 from mouse CB₁ and human CB₂ cannabinoid receptors and to inhibit electrically evoked contractions of the mouse isolated vas deferens was also investigated.

Key results:

In the brain membrane experiments, cannabigerol behaved as a potent α₂-adrenoceptor agonist (EC₅₀= 0.2 nM) and antagonized the 5-HT_1A receptor agonist, R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (apparent K_B= 51.9 nM). At 10 µM, it also behaved as a CB₁ receptor competitive antagonist. Additionally, cannabigerol inhibited evoked contractions of the vas deferens in a manner that appeared to be α₂-adrenoceptor-mediated (EC₅₀= 72.8 nM) and displayed significant affinity for mouse CB₁ and human CB₂ receptors.

Conclusions and implications:

This investigation has provided the first evidence that cannabigerol can activate α₂-adrenoceptors, bind to cannabinoid CB₁ and CB₂ receptors and block CB₁ and 5-HT_1A receptors. It will now be important to investigate why cannabigerol produced signs of agonism more potently in the [³⁵S]GTPγS binding assay than in the vas deferens and also whether it can inhibit noradrenaline uptake in this isolated tissue and in the brain.