Carvedilol targets human K2P 3.1 (TASK1) K+ leak channels

BACKGROUND AND PURPOSE

Human K_2P3.1 (TASK1) channels represent potential targets for pharmacological management of atrial fibrillation. K_2P channels control excitability by stabilizing membrane potential and by expediting repolarization. In the heart, inhibition of K_2P currents by class III antiarrhythmic drugs results in action potential prolongation and suppression of electrical automaticity. Carvedilol exerts antiarrhythmic activity and suppresses atrial fibrillation following cardiac surgery or cardioversion. The objective of this study was to investigate acute effects of carvedilol on human K_2P3.1 (hK_2P3.1) channels.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record hK_2P3.1 currents from Xenopus oocytes, Chinese hamster ovary (CHO) cells and human pulmonary artery smooth muscle cells (hPASMC).

KEY RESULTS

Carvedilol concentration-dependently inhibited hK_2P3.1 currents in Xenopus oocytes (IC₅₀= 3.8 µM) and in mammalian CHO cells (IC₅₀= 0.83 µM). In addition, carvedilol sensitivity of native I_K2P3.1 was demonstrated in hPASMC. Channels were blocked in open and closed states in frequency-dependent fashion, resulting in resting membrane potential depolarization by 7.7 mV. Carvedilol shifted the current–voltage (I–V) relationship by −6.9 mV towards hyperpolarized potentials. Open rectification, characteristic of K_2P currents, was not affected.

CONCLUSIONS AND IMPLICATIONS

The antiarrhythmic drug carvedilol targets hK_2P3.1 background channels. We propose that cardiac hK_2P3.1 current blockade may suppress electrical automaticity, prolong atrial refractoriness and contribute to the class III antiarrhythmic action in patients treated with the drug.     

Increased endothelin-1 reactivity and endothelial dysfunction in carotid arteries from rats with hyperhomocysteinemia

Background and purpose:

There are interactions between endothelin-1 (ET-1) and endothelial vascular injury in hyperhomocysteinemia (HHcy), but the underlying mechanisms are poorly understood. Here we evaluated the effects of HHcy on the endothelin system in rat carotid arteries.

Experimental approach:

Vascular reactivity to ET-1 and ET_A and ET_B receptor antagonists was assessed in rings of carotid arteries from normal rats and those with HHcy. ET_A and ET_B receptor expression was assessed by mRNA (RT-PCR), immunohistochemistry and binding of [¹²⁵I]-ET-1.

Key results:

HHcy enhanced ET-1-induced contractions of carotid rings with intact endothelium. Selective antagonism of ET_A or ET_B receptors produced concentration-dependent rightward displacements of ET-1 concentration response curves. Antagonism of ET_A but not of ET_B receptors abolished enhancement in HHcy tissues. ET_A and ET_B receptor gene expressions were not up-regulated. ET_A receptor expression in the arterial media was higher in HHcy arteries. Contractions to big ET-1 served as indicators of endothelin-converting enzyme activity, which was decreased by HHcy, without reduction of ET-1 levels. ET-1-induced Rho-kinase activity, calcium release and influx were increased by HHcy. Pre-treatment with indomethacin reversed enhanced responses to ET-1 in HHcy tissues, which were reduced also by a thromboxane A₂ receptor antagonist. Induced relaxation was reduced by BQ788, absent in endothelium-denuded arteries and was decreased in HHcy due to reduced bioavailability of NO.

Conclusions and implications:

Increased ET_A receptor density plays a fundamental role in endothelial injury induced by HHcy. ET-1 activation of ET_A receptors in HHcy changed the balance between endothelium-derived relaxing and contracting factors, favouring enhanced contractility.British Journal of Pharmacology (2009) 157, 568–580; doi:10.1111/j.1476-5381.2009.00165.x; published online 9 April 2009This article is part of a themed section on Endothelium in Pharmacology. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion

BACKGROUND AND PURPOSE

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1.

EXPERIMENTAL APPROACH

SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O₂^•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively.

KEY RESULTS

I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ET_B mRNA expression, and down-regulated ET_A mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ET_B receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ET_B receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ET_B receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, N^ω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O₂^•− production by increasing NADPH oxidase 1 (NOX-1) and p^47phox mRNA.

CONCLUSIONS AND IMPLICATIONS

Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ET_B receptors and decrease in NO bioavailability.     

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE

Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET_B) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction.

EXPERIMENTAL APPROACH

ET_B receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca²⁺, F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A₂ and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively.

KEY RESULTS

Cigarette smoke extract dose-dependently increased ET_B receptor expression in HPAECs after 24 h incubation. CSE-induced ET_B expression was attenuated by bosentan, the ET_B receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET_B receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET_B receptor expression, mimicking the effect of CSE. CSE-induced ET_B receptor overexpression caused greater cell contraction; increased intracellular Ca²⁺; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA₂ and ROS production; and decreased NO after acute ET-1 (10 nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine.

CONCLUSIONS AND IMPLICATION

Cigarette smoke extract induced ET_B receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET_B receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.     

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.     

Prolonged pharmacodynamic effects of S-0139, an intravenously administered endothelin A (ETA) antagonist, in the human forearm blood flow model

AIMS

To estimate the pharmacologically active dose range of a new investigational compound S-0139, a selective endothelin A (ET_A) receptor antagonist, in man, and to examine the duration of its pharmacodynamic effect.

METHODS

Venous occlusion plethysmography was performed to assess changes in forearm blood flow following intra-brachial administration of endothelin-1 (ET-1). ET_A antagonists have been shown to block ET-1-induced vasoconstriction in this model. The study was conducted in three parts: (1) a pilot study to explore dose–response (dose range 0.08–13.33 µg kg⁻¹ min⁻¹), (2) a randomized study to confirm dose–response (placebo, 2.5, 6.67 and 15 µg kg⁻¹ min⁻¹), and (3) a delayed administration study (15.7 µg kg⁻¹ min⁻¹) to explore the duration of the pharmacodynamic effect. In all studies a 3-h infusion of S-0139 was given and during the last 90 min of the infusion, ET-1 was infused concurrently for 90 min. In study (3) a second ET-1 infusion was given starting 3 h after completion of the first.

RESULTS

Intravenously administered S-0139 resulted in significant inhibition of ET-1-induced vasoconstriction in the forearm (plasma concentration 800–2000 ng ml⁻¹). In the delayed administration study, the same extent of inhibition was still present when ET-1 was administered 3 h after the end of infusion of S-0139, even though the S-0139 plasma concentrations (mean 17 ng ml⁻¹) were well below pharmacologically active concentrations as determined in studies 1 and 2.

CONCLUSIONS

S-0139 dose-dependently blocks ET-1-mediated vasoconstriction in the forearm and has a prolonged duration of effect beyond that expected from its pharmacokinetic profile.     

Enhanced endothelin receptor type B-mediated vasodilation and underlying [Ca2+]i in mesenteric microvessels of pregnant rats

Background and Purpose

Normal pregnancy is associated with decreased vascular resistance and increased release of vasodilators. Endothelin-1 (ET-1) causes vasoconstriction via endothelin receptor type A (ET_AR), but could activate ET_BR in the endothelium and release vasodilator substances. However, the roles of ET_BR in the regulation of vascular function during pregnancy and the vascular mediators involved are unclear.

Experimental Approach

Pressurized mesenteric microvessels from pregnant and virgin Sprague–Dawley rats were loaded with fura-2/AM for simultaneous measurement of diameter and [Ca²⁺]_i.

Key Results

High KCl (51 mM) and phenylephrine (PHE) caused increases in vasoconstriction and [Ca²⁺]_i that were similar in pregnant and virgin rats. ET-1 caused vasoconstriction that was less in pregnant than virgin rats, with small increases in [Ca²⁺]_i. Pretreatment with the ET_BR antagonist BQ-788 caused greater enhancement of ET-1-induced vasoconstriction in pregnant rats. ACh caused endothelium-dependent relaxation and decreased [Ca²⁺]_i, and was more potent in pregnant than in virgin rats. ET-1 + ET_AR antagonist BQ-123, and the ET_BR agonists sarafotoxin 6c (S6c) and IRL-1620 caused greater vasodilation in pregnant than in virgin rats with no changes in [Ca²⁺]_i, suggesting up-regulated ET_BR-mediated relaxation pathways. ACh-, S6c- and IRL-1620-induced relaxation was reduced by the NO synthase inhibitor N_ω-nitro-l-arginine methyl ester, and abolished by tetraethylammonium or endothelium removal. Western blots revealed greater amount of ET_BR in intact microvessels of pregnant than virgin rats, but reduced levels in endothelium-denuded microvessels, supporting a role of endothelial ET_BR.

Conclusions and Implications

The enhanced ET_BR-mediated microvascular relaxation may contribute to the decreased vasoconstriction and vascular resistance during pregnancy.     

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.     

Trypanosoma cruzi invades host cells through the activation of endothelin and bradykinin receptors: a converging pathway leading to chagasic vasculopathy

BACKGROUND AND PURPOSE

Independent studies in experimental models of Trypanosoma cruzi appointed different roles for endothelin-1 (ET-1) and bradykinin (BK) in the immunopathogenesis of Chagas disease. Here, we addressed the hypothesis that pathogenic outcome is influenced by functional interplay between endothelin receptors (ET_AR and ET_BR) and bradykinin B₂ receptors (B₂R).

EXPERIMENTAL APPROACH

Intravital microscopy was used to determine whether ETR/B₂R drives the accumulation of rhodamine-labelled leucocytes in the hamster cheek pouch (HCP). Inflammatory oedema was measured in the infected BALB/c paw of mice. Parasite invasion was assessed in CHO over-expressing ETRs, mouse cardiomyocytes, endothelium (human umbilical vein endothelial cells) or smooth muscle cells (HSMCs), in the presence/absence of antagonists of B₂R (HOE-140), ET_AR (BQ-123) and ET_BR (BQ-788), specific IgG antibodies to each GPCRs; cholesterol or calcium-depleting drugs. RNA interference (ET_AR or ET_BR genes) in parasite infectivity was investigated in HSMCs.

KEY RESULTS

BQ-123, BQ-788 and HOE-140 reduced leucocyte accumulation in HCP topically exposed to trypomastigotes and blocked inflammatory oedema in infected mice. Acting synergistically, ET_AR and ET_BR antagonists reduced parasite invasion of HSMCs to the same extent as HOE-140. Exogenous ET-1 potentiated T. cruzi uptake by HSMCs via ETRs/B₂R, whereas RNA interference of ET_AR and ET_BR genes conversely reduced parasite internalization. ETRs/B₂R-driven infection in HSMCs was reduced in HSMC pretreated with methyl-β-cyclodextrin, a cholesterol-depleting drug, or in thapsigargin- or verapamil-treated target cells.

CONCLUSIONS AND IMPLICATIONS

Our findings suggest that plasma leakage, a neutrophil-driven inflammatory response evoked by trypomastigotes via the kinin/endothelin pathways, may offer a window of opportunity for enhanced parasite invasion of cardiovascular cells.

LINKED ARTICLE

This paper is commented on by D''Orléans-Juste et al., pp. 1330–1332 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01636.x     

Endothelin-1 and endothelin-2 initiate and maintain contractile responses by different mechanisms in rat mesenteric and cerebral arteries

BACKGROUND AND PURPOSE

Endothelin (ET)-1 and ET-2 cause potent long-lasting vasoconstrictions by tight binding to smooth muscle ET_A receptors. We tested the hypotheses that different mechanisms mediate initiation and maintenance of arterial contractile responses to ET-1 and ET-2 and that this differs among vascular beds.

EXPERIMENTAL APPROACH

Segments of rat mesenteric resistance artery (MRA) and basilar artery (BA) were studied in wire myographs with and without functional antagonists.

KEY RESULTS

Sensitivity and maximum of MRA contractile responses to ET-1 were not, or only moderately, reduced by stimulation of soluble GC, AC or K⁺-channels and by an inhibitor of receptor-operated ion channels. However, each of these reduced maintenance of ET-1 effects and relaxed ET-1-induced contractions in MRA. A calcium channel antagonist did not alter sensitivity, maximum and maintenance of ET-1 effects, but relaxed ET-1-induced contractions in MRA. A PLC inhibitor prevented contractile responses to ET-1 and ET-2 in MRA and BA, and relaxed ET-1- and ET-2-induced responses in MRA and ET-1 effects in BA. A Rho-kinase inhibitor did not modify sensitivity, maximum and maintenance of responses to both peptides in both arteries but relaxed ET-2, but not ET-1, effects in MRA and ET-1 effects in BA.

CONCLUSIONS AND IMPLICATIONS

PLC played a key role in arterial contractile responses to ETs, but ET-1 and ET-2 initiated and maintained vasoconstriction through different mechanisms, and these differed between MRA and BA. Selective functional antagonism may be considered for agonist- and vascular bed selective pharmacotherapy of ET-related diseases.     

Isoproterenol-induced FKBP12.6/12 downregulation is modulated by ETA and ETB receptors and reversed by argirhein, a derivative of rhein

Aim:

To investigate which endothelin receptors mediated isoproterenol (ISO)-induced downregulation of FKBP12.6/12 in cardiomyocytes and study whether argirhein, a novel compound containing rhein and L-arginine that has anti-inflammatory activity, could reverse the downregulation of FKBP12.6/12 induced by ISO.

Methods:

Neonatal rat cardiomyocytes were incubated with ISO to downregulate FKBP12.6/12. Then the cells were treated with a selective ET_A blocker (PD156707) and a ET_B blocker (IRL1038), a dual ET_A/ET_B antagonist (CPU0213), and argirhein, respectively. FKBP12.6/12 expression was assayed by RT-PCR, Western blot, and immunocytochemistry.

Results:

The expression of FKBP12.6 mRNA was reduced by 37.7% (P<0.01) and 28.9% (P<0.05) relative to the control by ISO 1 and 0.1 μmol/L, respectively, but no response to ISO 0.01 μmol/L was observed in vitro. FKBP12.6/12 protein expression was reduced by 47.2% (P<0.01) and 37.8% (P<0.05) by ISO 1 and 0.1 μmol/L, respectively. This decrease was reversed significantly by PD156707, or IRL1038, and CPU0213. CPU0213 was more potent than either PD156707 or IRL-1038. Argirhein 10 μmol/L blunted the downregulation of FKBP12.6/12 by ISO, as demonstrated by the rising mRNA and protein levels and by the fluorescent density of the ISO-incubated cardiomyocytes.

Conclusion:

In cardiomyocytes, the ISO induced downregulation of FKBP12.6/12 is modulated by both ET_A and ET_B. A new compound, argirein, reversed the down-regulation of FKBP12.6/12 expression in myocardial cells stimulated with ISO.     

Role of endothelin-1 in the hyper-responsiveness to nitrovasodilators following acute NOS inhibition

BACKGROUND AND PURPOSE

Acute NOS inhibition in humans and animals is associated with hypersensitivity to NO donors. The mechanisms underlying this phenomenon have not been fully elucidated. The purpose of the present study was to assess whether hypersensitivity to NOS-blockade is linked to endothelin-1 (ET-1) signalling.

EXPERIMENTAL APPROACH

Sprague Dawley rats were instrumented with indwelling arterial and venous catheters for continuous assessments of haemodynamic parameters and drug delivery, respectively. Mesenteric arteries were isolated and tested for reactivity by wire myography.

KEY RESULTS

NOS blockade with L-N^G-nitroarginine methyl ester (L-NAME) caused a pronounced increase in arterial blood pressure (BP) (∼40 mmHg). In L-NAME-treated animals, the dose of sodium nitroprusside (SNP) required to cause a significant reduction in arterial BP was lower than in vehicle-treated rats (P < 0.001), and the magnitude of the reduction in BP was greater. Similar results were obtained with other NO mimetics, but not isoprenaline; moreover, decreasing the BP back to baseline levels with prazosin after L-NAME treatment did not attenuate the hyper-responsiveness to NO donors. The increased responsiveness to NO donors was abolished by pretreatment with the ET_A/B receptor antagonist, PD145065, or the ET_A receptor-specific antagonist ABT627. Ex vivo, L-NAME treatment potentiated the constriction induced by big endothelin-1 (bET-1), the precursor to active ET-1, but had no effect on the ET-1-mediated constriction.

CONCLUSIONS AND IMPLICATIONS

These data suggest that the increased sensitivity to NO donors is mediated, at least in part, by ET-1 in vivo, and the mechanism may involve the conversion of bET-1 to ET-1.     

Adenosine negatively regulates duodenal motility in mice: role of A(1) and A(2A) receptors

BACKGROUND AND PURPOSE

Adenosine is considered to be an important modulator of intestinal motility. This study was undertaken to investigate the role of adenosine in the modulation of contractility in the mouse duodenum and to characterize the adenosine receptor subtypes involved.

EXPERIMENTAL APPROACH

RT-PCR was used to investigate the expression of mRNA encoding for A₁, A_2A, A_2B and A₃ receptors. Contractile activity was examined in vitro as changes in isometric tension.

KEY RESULTS

In mouse duodenum, all four classes of adenosine receptors were expressed, with the A_2B receptor subtype being confined to the mucosal layer. Adenosine caused relaxation of mouse longitudinal duodenal muscle; this was antagonized by the A₁ receptor antagonist and mimicked by N⁶-cyclopentyladenosine (CPA), selective A₁ agonist. The relaxation induced by A₁ receptor activation was insensitive to tetrodotoxin (TTX) or N^ω-nitro-l-arginine methyl ester (l-NAME). Adenosine also inhibited cholinergic contractions evoked by neural stimulation, effect reversed by the A₁ receptor antagonist, but not myogenic contractions induced by carbachol. CPA and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680), A_2A receptor agonist, both inhibited the nerve-evoked cholinergic contractions. l-NAME prevented only the CGS-21680-induced effects. S-(4-Nitrobenzyl)-6-thioinosine, a nucleoside uptake inhibitor, reduced the amplitude of nerve-evoked cholinergic contractions, an effect reversed by an A_2A receptor antagonist or l-NAME.

CONCLUSIONS AND IMPLICATIONS

Adenosine can negatively regulate mouse duodenal motility either by activating A₁ inhibitory receptors located post-junctionally or controlling neurotransmitter release via A₁ or A_2A receptors. Both receptors are available for pharmacological recruitment, even if only A_2A receptors appear to be preferentially stimulated by endogenous adenosine.

LINKED ARTICLE

This article is commented on by Antonioli et al., pp. 1577–1579 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01529.x     

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.     

A2B adenosine receptors inhibit superoxide production from mitochondrial complex I in rabbit cardiomyocytes via a mechanism sensitive to Pertussis toxin

BACKGROUND AND PURPOSE

A_2B adenosine receptors protect against ischaemia/reperfusion injury by activating survival kinases including extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). However, the underlying mechanism(s) and signalling pathway(s) remain undefined.

EXPERIMENTAL APPROACH

HEK 293 cells stably transfected with human A_2B adenosine receptors (HEK-A_2B) and isolated adult rabbit cardiomyocytes were used to assay phosphorylation of ERK by Western blot and cation flux through cAMP-gated channels by patch clamp methods. Generation of reactive oxygen species (ROS) by mitochondria was measured with a fluorescent dye.

KEY RESULTS

In HEK-A_2B cells, the selective A_2B receptor agonist Bay 60-6583 (Bay 60) increased ERK phosphorylation and cAMP levels, detected by current through cAMP-gated ion channels. However, increased cAMP or its downstream target protein kinase A was not involved in ERK phosphorylation. Pertussis toxin (PTX) blocked ERK phosphorylation, suggesting receptor coupling to G_i or G_o proteins. Phosphorylation was also blocked by inhibition of PI3K (with wortmannin) or of ERK kinase (MEK1/2, with PD 98059) but not by inhibition of NO synthase (NOS). In cardiomyocytes, Bay 60 did not affect cAMP levels but did block the increased superoxide generation induced by rotenone, a mitochondrial complex I inhibitor. This effect of Bay 60 was inhibited by PD 98059, wortmannin or PTX. Inhibition of NOS blocked superoxide production because NOS is downstream of ERK.

CONCLUSION AND IMPLICATIONS

Activation of A_2B adenosine receptors reduced superoxide generation from mitochondrial complex I through G_i/o, ERK, PI3K, and NOS, all of which have been implicated in ischaemic preconditioning.     

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling

Background and purpose:

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A_2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine.

Experimental approach:

The trafficking of the wild type A_2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy.

Key results:

The wild type A_2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln³²⁵-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu³³⁰-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln³²⁵-stop, Ser³²⁶-stop and Phe³²⁸-stop receptors. Following internalization, the wild type A_2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln³²⁵-stop receptor did not recycle.

Conclusions and implications:

Deletion of the COOH-terminus of the A_2B adenosine receptor beyond Leu³³⁰ switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A_2B adenosine receptor following prolonged agonist addition.     

Protein kinase C-independent inhibition of arterial smooth muscle K(+) channels by a diacylglycerol analogue

BACKGROUND AND PURPOSE

Analogues of the endogenous diacylglycerols have been used extensively as pharmacological activators of protein kinase C (PKC). Several reports show that some of these compounds have additional effects that are independent of PKC activation, including direct block of K⁺ and Ca²⁺ channels. We investigated whether dioctanoyl-sn-glycerol (DiC8), a commonly used diacylglycerol analogue, blocks K⁺ currents of rat mesenteric arterial smooth muscle in a PKC-independent manner.

EXPERIMENTAL APPROACH

Conventional whole-cell and inside-out patch clamp was used to measure the inhibition of K⁺ currents of rat isolated mesenteric smooth muscle cells by DiC8 in the absence and presence of PKC inhibitor peptide.

KEY RESULTS

Mesenteric artery smooth muscle K_v currents inactivated very slowly with a time constant of about 2 s following pulses from −65 to +40 mV. Application of 1 µM DiC8 produced an approximate 40-fold increase in the apparent rate of inactivation. Pretreatment of the cells with PKC inhibitor peptide had a minimal effect on the action of DiC8, and substantial inactivation still occurred, indicating that this effect was mainly independent of PKC. We also found that DiC8 blocked BK and K_ATP currents, and again a significant proportion of these blocks occurred independently of PKC activation.

CONCLUSIONS AND IMPLICATIONS

These results show that DiC8 has a direct effect on arterial smooth muscle K⁺ channels, and this precludes its use as a PKC activator when investigating PKC-mediated effects on vascular K⁺ channels.     

High salt-induced hypertension in B2 knockout mice is corrected by the ETA antagonist,A127722

BACKGROUND AND PURPOSE

The contribution of endothelin-1 (ET-1) in a B₂KO mouse model of a high salt-induced arterial hypertension was investigated.

EXPERIMENTAL APPROACH

Wild-type (WT) or B₂KO mice receiving a normal diet (ND) or a high-salt diet (HSD) were monitored by radiotelemetry up to a maximum of 18 weeks. At the 12th week of diet, subgroups under ND or HSD received by gavage the ET_A antagonist A127722 during 5 days. In addition, blood samples were collected and, following euthanasia, the lungs, heart and kidneys were extracted, homogenized and assayed for ET-1 by RIA. In a separate series of experiments, the ET_A antagonist, BQ123 was tested against the pressor responses to a NOS inhibitor L-N^G-nitroarginine methyl ester (L-NAME) in anaesthetized WT and B₂KO mice.

KEY RESULTS

In B₂KO, but not WT mice, 12 weeks of HSD triggered a maximal increase of the mean arterial pressure (MAP) of 19.1 ± 2.8 mmHg, which was corrected by A127722 to MAP levels found in B₂KO mice under ND. Significant increases in immunoreactive ET-1 were detected only in the lungs of B₂KO mice under HSD. On the other hand, metabolic studies showed that sodium urinary excretion was markedly reduced in B₂KO compared with WT mice under ND. Finally, BQ123 (2 mg·kg⁻¹) reduced by 50% the pressor response to L-NAME (2 mg·kg⁻¹) in B₂KO, but not WT mice under anaesthesia.

CONCLUSIONS AND IMPLICATIONS

Our results support the concept that functional B₂ receptors oppose high salt-induced increments in MAP, which are corrected by an ET_A receptor antagonist in this mouse model of experimental hypertension.     

Pharmacological profile of phosphatidylinositol 3-kinases and related phosphatidylinositols mediating endothelin(A) receptor-operated native TRPC channels in rabbit coronary artery myocytes

BACKGROUND AND PURPOSE

Endothelin_A (ET_A) receptor-operated canonical transient receptor potential (TRPC) channels mediate Ca²⁺ influx pathways, which are important in coronary artery function. Biochemical pathways linking ET_A receptor stimulation to TRPC channel opening are unknown. We investigated the involvement of phosphatidylinositol 3-kinases (PI3K) in ET_A receptor activation of native heteromeric TRPC1/C5/C6 and TRPC3/C7 channels in rabbit coronary artery vascular smooth muscle cells (VSMCs).

EXPERIMENTAL APPROACH

A pharmacological profile of PI3K was created by studying the effect of pan-PI3K, pan-Class I PI3K and Class I PI3K isoform-selective inhibitors on ET_A receptor-evoked single TRPC1/C5/C6 and TRPC3/C7 channel activities in cell-attached patches from rabbit freshly isolated coronary artery VSMCs. The action of phosphatidylinositol 3-phosphate- [PI(3)P], 4-phosphate- [PI(4)P] and 5-phosphate- [PI(5)P] containing molecules involved in PI3K-mediated reactions were studied in inside-out patches. Expression of PI3K family members in coronary artery tissue lysates were analysed using quantitative PCR.

KEY RESULTS

ET_A receptor-operated TRPC1/C5/C6 and TRPC3/C7 channel activities were inhibited by wortmannin. However, ZSTK474 and AS252424 reduced ET_A receptor-evoked TRPC1/C5/C6 channel activity but potentiated TRPC3/C7 channel activity. All the PI(3)P-, PI(4)P- and PI(5)P-containing molecules tested induced TRPC1/C5/C6 channel activation, whereas only PI(3)P stimulated TRPC3/C7 channels.

CONCLUSIONS AND IMPLICATIONS

ET_A receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channel activities are likely to be mediated by Class I PI3Kγ and Class II/III PI3K isoforms, respectively. ET_A receptor-evoked and constitutively active PI3Kγ-mediated pathways inhibit TRPC3/C7 channel activation. PI3K-mediated pathways are novel regulators of native TRPC channels in VSMCs, and these signalling cascades are potential pharmacological targets for coronary artery disease.