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     

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.     

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.     

Activation of KCa3.1 by SKA-31 induces arteriolar dilatation and lowers blood pressure in normo- and hypertensive connexin40-deficient mice

BACKGROUND AND PURPOSE

The calcium-activated potassium channel K_Ca3.1 is expressed in the vascular endothelium where its activation causes endothelial hyperpolarization and initiates endothelium-derived hyperpolarization (EDH)-dependent dilatation. Here, we investigated whether pharmacological activation of K_Ca3.1 dilates skeletal muscle arterioles and whether myoendothelial gap junctions formed by connexin40 (Cx40) are required for EDH-type dilatations and pressure depressor responses in vivo.

EXPERIMENTAL APPROACH

We performed intravital microscopy in the cremaster muscle microcirculation and blood pressure telemetry in Cx40-deficient mice.

KEY RESULTS

In wild-type mice, the K_Ca3.1-activator SKA-31 induced pronounced concentration-dependent arteriolar EDH-type dilatations, amounting to ∼40% of maximal dilatation, and enhanced the effects of ACh. These responses were absent in mice devoid of K_Ca3.1 channels. In contrast, SKA-31-induced dilatations were not attenuated in mice with endothelial cells deficient in Cx40 (Cx40^fl/fl:Tie2-Cre). In isolated endothelial cell clusters, SKA-31 induced hyperpolarizations of similar magnitudes (by ∼38 mV) in Cx40^fl/fl:Tie2-Cre, ubiquitous Cx40-deficient mice (Cx40^-/-) and controls (Cx40^fl/fl), which were reversed by the specific K_Ca3.1-blocker TRAM-34. In normotensive wild-type and Cx40^fl/fl:Tie2-Cre as well as in hypertensive Cx40^-/- animals, i.p. injections of SKA-31 (30 and 100 mg·kg⁻¹) decreased arterial pressure by ∼32 mmHg in all genotypes. The depressor response to 100 mg·kg⁻¹ SKA-31 was associated with a decrease in heart rate.

CONCLUSIONS AND IMPLICATIONS

We conclude that endothelial hyperpolarization evoked by pharmacological activation of K_Ca3.1 channels induces EDH-type arteriolar dilatations that are independent of endothelial Cx40 and Cx40-containing myoendothelial gap junctions. As SKA-31 reduced blood pressure in hypertensive Cx40-deficient mice, K_Ca3.1 activators may be useful drugs for severe treatment-resistant hypertension.     

TRPM4 inhibitor 9-phenanthrol activates endothelial cell intermediate conductance calcium-activated potassium channels in rat isolated mesenteric artery

Background and Purpose

Smooth muscle transient receptor potential melastatin 4 (TRPM4) channels play a fundamental role in the development of the myogenic arterial constriction that is necessary for blood flow autoregulation. As TRPM4 channels are present throughout the vasculature, we investigated their potential role in non-myogenic resistance arteries using the TRPM4 inhibitor 9-phenanthrol.

Experimental Approach

Pressure and wire myography were used to assess the reactivity of rat arteries, the latter in combination with measurements of smooth muscle membrane potential. Immunohistochemistry (IHC) and endothelial cell (EC) calcium changes were assessed in pressurized vessels and patch clamp measurements made in isolated ECs.

Key Results

The TRPM4 inhibitor 9-phenanthrol reversibly hyperpolarized mesenteric arteries to circa E_K and blocked α₁-adrenoceptor-mediated vasoconstriction. Hyperpolarization was abolished and vasoconstriction re-established by damaging the endothelium. In mesenteric and cerebral artery smooth muscle, 9-phenanthrol hyperpolarization was effectively blocked by the K_Ca3.1 inhibitor TRAM-34. 9-Phenanthrol did not increase mesenteric EC [Ca²⁺]_i, and Na⁺ substitution with N-methyl-D-glucamine only increased the muscle resting potential by 10 mV. Immunolabelling for TRPM4 was restricted to the endothelium and perivascular tissue.

Conclusions and Implications

These data reveal a previously unrecognized action of the TRPM4 inhibitor 9-phenanthrol – the ability to act as an activator of EC K_Ca3.1 channels. They do not indicate a functionally important role for TRPM4 channels in the reactivity of non-myogenic mesenteric arteries.     

K(Ca) 3.1 channels maintain endothelium-dependent vasodilatation in isolated perfused kidneys of spontaneously hypertensive rats after chronic inhibition of NOS

BACKGROUND AND PURPOSE

The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury.

EXPERIMENTAL APPROACH

Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar–Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N^ω-nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg⁻¹·day⁻¹).

KEY RESULTS

Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N^w-nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca²⁺-activated K⁺ channels of intermediate conductance (K_Ca3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca²⁺-activated K⁺ channels of small conductance (K_Ca2.3), was ineffective. Dilatation induced by an opener of K_Ca3.1/K_Ca2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K_Ca3.1, but not K_Ca2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR).

CONCLUSIONS AND IMPLICATIONS

In SHR kidneys, an EDHF-mediated response, involving activation of K_Ca3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.     

β1-Adrenoceptor stimulation suppresses endothelial IKCa-channel hyperpolarization and associated dilatation in resistance arteries

Background and Purpose

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK_Ca) and intermediate conductance Ca²⁺-activated K⁺ channels (IK_Ca) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK_Ca channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation.

Experimental Approach

Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]_i, mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording.

Key Results

Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM–10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]_i. The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK_Ca {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK_Ca (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh.

Conclusions and Implications

In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK_Ca channels and may be one consequence of raised circulating catecholamines.     

Genetic deficit of KCa3.1 channels protects against pulmonary circulatory collapse induced by TRPV4 channel activation

Background and Purpose

The intermediate conductance calcium/calmodulin-regulated K⁺ channel K_Ca3.1 produces hyperpolarizing K⁺ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl⁻ and fluid movements. We investigated whether a deficiency in K_Ca3.1 (K_Ca3.1^−/−) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels.

Experimental Approach

An opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and K_Ca3.1^−/− mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC).

Key Results

In wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, K_Ca3.1^−/− mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of K_Ca3.1^−/− mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and K_Ca3.1^−/− mice, which co-activated K_Ca3.1 and disrupted membrane resistance in wt PAEC, but not in K_Ca3.1^−/− PAEC.

Conclusions and Implications

Our findings show that a genetic deficiency of K_Ca3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of K_Ca3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.     

Effects of docosahexaenoic acid on large-conductance Ca2+-activated K+ channels and voltage-dependent K+ channels in rat coronary artery smooth muscle cells

Aim:

To investigate the effects of docosahexaenoic acid (DHA) on large-conductance Ca²⁺-activated K⁺(BK_Ca) channels and voltage-dependent K⁺ (K_V) channels in rat coronary artery smooth muscle cells (CASMCs).

Methods:

Rat CASMCs were isolated by an enzyme digestion method. BK_Ca and K_V currents in individual CASMCs were recorded by the patch-clamp technique in a whole-cell configuration at room temperature. Effects of DHA on BK_Ca and K_V channels were observed when it was applied at 10, 20, 30, 40, 50, 60, 70, and 80 μmol/L.

Results:

When DHA concentrations were greater than 10 μmol/L, BK_Ca currents increased in a dose-dependent manner. At a testing potential of +80 mV, 6.1%±0.3%, 76.5%±3.8%, 120.6%±5.5%, 248.0%±12.3%, 348.7%±17.3%, 374.2%±18.7%, 432.2%±21.6%, and 443.1%±22.1% of BK_Ca currents were increased at the above concentrations, respectively. The half-effective concentration (EC₅₀) of DHA on BK_Ca currents was 37.53±1.65 μmol/L. When DHA concentrations were greater than 20 μmol/L, K_V currents were gradually blocked by increasing concentrations of DHA. At a testing potential of +50 mV, 0.40%±0.02%, 1.37%±0.06%, 11.80%±0.59%, 26.50%±1.75%, 56.50%±2.89%, 73.30%±3.66%, 79.70%±3.94%, and 78.1%±3.91% of K_V currents were blocked at the different concentrations listed above, respectively. The EC₅₀ of DHA on K_V currents was 44.20±0.63 μmol/L.

Conclusion:

DHA can activate BK_Ca channels and block K_V channels in rat CASMCs, and the EC₅₀ of DHA for BK_Ca channels is lower than that for K_V channels; these findings indicate that the vasorelaxation effects of DHA on vascular smooth muscle cells are mainly due to its activation of BK_Ca channels.     

Ca2 +-activated K+ channel-3.1 blocker TRAM-34 alleviates murine allergic rhinitis

The precise pathogenesis of allergic rhinitis (AR) remains unclear and AR is less easily cured. Recent evidence has suggested that calcium-activated K⁺ channel-3.1(KCa3.1) is implicated in the immune response of allergic and inflammatory diseases and TRAM-34 is a selective KCa3.1 blocker. However, little is known about its role in AR. We aimed to investigate the effect of TRAM-34 in a mouse model of AR induced by ovalbumin (OVA). The BALB/c mice were divided into six groups: untreated AR group, 200 μg TRAM-34 treated AR group, 400 μg TRAM-34 treated AR group, 200 μg TRAM-34 treated normal group, 400 μg TRAM-34 treated normal group and untreated normal control group. Histopathological characteristics were assessed by HE staining. KCa3.1 protein expression was investigated by immunohistochemistry and western blotting method, and mRNA expression of KCa3.1, stromal interaction molecule1 (STIM1) and Orai1 in nasal tissues were assessed by real-time PCR. Furthermore, concentrations of OVA-specific IgE, ECP, IL-4, IL-5, IL-17 and IL-1β in nasal lavage fluid (NLF) were analyzed by enzyme-linked immunosorbent assay (ELISA). Results showed that TRAM-34 administration into the nostril attenuated sneezing, nasal rubbing, epithelial cell proliferation, eosinophil infiltration and inhibited nasal mucosa KCa3.1, STIM1 and Orai1 expression in TRAM-34 treated mice compared with untreated AR mice and suppressed inflammatory cytokines in the NLF of TRAM-34 treated groups compared with untreated AR mice. In conclusion, TRAM-34 could effectively alleviate murine allergic rhinitis by suppressing KCa3.1 and leads to reduction of K⁺ efflux and Ca^2 + influx, leading to inflammation reduction and allergic responses attenuation.     

TRPV4 and KCa ion channels functionally couple as osmosensors in the paraventricular nucleus

Background and Purpose

Transient receptor potential vanilloid type 4 (TRPV4) and calcium-activated potassium channels (K_Ca) mediate osmosensing in many tissues. Both TRPV4 and K_Ca channels are found in the paraventricular nucleus (PVN) of the hypothalamus, an area critical for sympathetic control of cardiovascular and renal function. Here, we have investigated whether TRPV4 channels functionally couple to K_Ca channels to mediate osmosensing in PVN parvocellular neurones and have characterized, pharmacologically, the subtype of K_Ca channel involved.

Experimental Approach

We investigated osmosensing roles for TRPV4 and K_Ca channels in parvocellular PVN neurones using cell-attached and whole-cell electrophysiology in mouse brain slices and rat isolated PVN neurons. Intracellular Ca²⁺ was recorded using Fura-2AM. The system was modelled in the NEURON simulation environment.

Key Results

Hypotonic saline reduced action current frequency in hypothalamic slices; a response mimicked by TRPV4 channel agonists 4αPDD (1 μM) and GSK1016790A (100 nM), and blocked by inhibitors of either TRPV4 channels (RN1734 (5 μM) and HC067047 (300 nM) or the low-conductance calcium-activated potassium (SK) channel (UCL-1684 30 nM); iberiotoxin and TRAM-34 had no effect. Our model was compatible with coupling between TRPV4 and K_Ca channels, predicting the presence of positive and negative feedback loops. These predictions were verified using isolated PVN neurons. Both hypotonic challenge and 4αPDD increased intracellular Ca²⁺ and UCL-1684 reduced the action of hypotonic challenge.

Conclusions and Implications

There was functional coupling between TRPV4 and SK channels in parvocellular neurones. This mechanism contributes to osmosensing in the PVN and may provide a novel pharmacological target for the cardiovascular or renal systems.     

Activation of K+ channel by 1-EBIO rescues the head and neck squamous cell carcinoma cells from Ca2+ ionophore-induced cell death

Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular Ca²⁺ ([Ca²⁺]_i)-dependent signaling affects the fate of cancer cells. Here we investigate the role of Ca²⁺-activated K⁺ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of different cell lines; SNU-1076, OSC-19 and HN5. Treatment with 1 µM ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of Ca²⁺-activated Cl^- channels (Ano-1) and Ca²⁺-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward K⁺ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Consistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the Ca²⁺ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.     

ORM-10103, a novel specific inhibitor of the Na+/Ca2+ exchanger,decreases early and delayed afterdepolarizations in the canine heart

BACKGROUND AND PURPOSE

At present there are no small molecule inhibitors that show strong selectivity for the Na⁺/Ca²⁺ exchanger (NCX). Hence, we studied the electrophysiological effects of acute administration of ORM-10103, a new NCX inhibitor, on the NCX and L-type Ca²⁺ currents and on the formation of early and delayed afterdepolarizations.

EXPERIMENTAL APPROACH

Ion currents were recorded by using a voltage clamp technique in canine single ventricular cells, and action potentials were obtained from canine and guinea pig ventricular preparations with the use of microelectrodes.

KEY RESULTS

ORM-10103 significantly reduced both the inward and outward NCX currents. Even at a high concentration (10 μM), ORM-10103 did not significantly change the L-type Ca²⁺ current or the maximum rate of depolarization (dV/dt_max), indicative of the fast inward Na⁺ current. At 10 μM ORM-10103 did not affect the amplitude or the dV/dt_max of the slow response action potentials recorded from guinea pig papillary muscles, which suggests it had no effect on the L-type Ca²⁺ current. ORM-10103 did not influence the Na⁺/K⁺ pump or the main K⁺ currents of canine ventricular myocytes, except the rapid delayed rectifier K⁺ current, which was slightly diminished by the drug at 3 μM. The amplitudes of pharmacologically- induced early and delayed afterdepolarizations were significantly decreased by ORM-10103 (3 and 10 μM) in a concentration-dependent manner.

CONCLUSIONS AND IMPLICATIONS

ORM-10103 is a selective inhibitor of the NCX current and can abolish triggered arrhythmias. Hence, it has the potential to be used to prevent arrhythmogenic events.

LINKED ARTICLE

This article is commented on by Terracciano and Hancox, pp. 765–767 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12299     

Kaempferol enhances endothelium-dependent relaxation in the porcine coronary artery through activation of large-conductance a2+-activated K+ channels

Background and Purpose

Kaempferol, a plant flavonoid present in normal human diet, can modulate vasomotor tone. The present study aimed to elucidate the signalling pathway through which this flavonoid enhanced relaxation of vascular smooth muscle.

Experimental Approach

The effect of kaempferol on the relaxation of porcine coronary arteries to endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) relaxing agents was studied in an in vitro organ chamber setup. The whole-cell patch-clamp technique was used to determine the effect of kaempferol on potassium channels in porcine coronary artery smooth muscle cells (PCASMCs).

Key Results

At a concentration without direct effect on vascular tone, kaempferol (3 × 10⁻⁶ M) enhanced relaxations produced by bradykinin and sodium nitroprusside. The potentiation by kaempferol of the bradykinin-induced relaxation was not affected by N^ω-nitro-L-arginine methyl ester, an inhibitor of NO synthase (10⁻⁴ M) or TRAM-34 plus UCL 1684, inhibitors of intermediate- and small-conductance calcium-activated potassium channels, respectively (10⁻⁶ M each), but was abolished by tetraethylammonium chloride, a non-selective inhibitor of calcium-activated potassium channels (10⁻³ M), and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channel (K_Ca1.1; 10⁻⁷ M). Iberiotoxin also inhibited the potentiation by kaempferol of sodium nitroprusside-induced relaxations. Kaempferol stimulated an outward-rectifying current in PCASMCs, which was abolished by iberiotoxin.

Conclusions and Implications

The present results suggest that, in smooth muscle cells of the porcine coronary artery, kaempferol enhanced relaxations caused by endothelium-derived and exogenous NO as well as those due to endothelium-dependent hyperpolarization. This vascular effect of kaempferol involved the activation of K_Ca1.1 channels.     

Activation of endothelial and epithelial K(Ca) 2.3 calcium-activated potassium channels by NS309 relaxes human small pulmonary arteries and bronchioles

BACKGROUND AND PURPOSE

Small (K_Ca2) and intermediate (K_Ca3.1) conductance calcium-activated potassium channels (K_Ca) may contribute to both epithelium- and endothelium-dependent relaxations, but this has not been established in human pulmonary arteries and bronchioles. Therefore, we investigated the expression of K_Ca2.3 and K_Ca3.1 channels, and hypothesized that activation of these channels would produce relaxation of human bronchioles and pulmonary arteries.

EXPERIMENTAL APPROACH

Channel expression and functional studies were conducted in human isolated small pulmonary arteries and bronchioles. K_Ca2 and K_Ca3.1 currents were examined in human small airways epithelial (HSAEpi) cells by whole-cell patch clamp techniques.

RESULTS

While K_Ca2.3 expression was similar, K_Ca3.1 protein was more highly expressed in pulmonary arteries than bronchioles. Immunoreactive K_Ca2.3 and K_Ca3.1 proteins were found in both endothelium and epithelium. K_Ca currents were present in HSAEpi cells and sensitive to the K_Ca2.3 blocker UCL1684 and the K_Ca3.1 blocker TRAM-34. In pulmonary arteries contracted by U46619 and in bronchioles contracted by histamine, the K_Ca2.3/ K_Ca3.1 activator, NS309, induced concentration-dependent relaxations. NS309 was equally potent in relaxing pulmonary arteries, but less potent in bronchioles, than salbutamol. NS309 relaxations were blocked by the K_Ca2 channel blocker apamin, while the K_Ca3.1 channel blocker, charybdotoxin failed to reduce relaxation to NS309 (0.01–1 µM).

CONCLUSIONS AND IMPLICATIONS

K_Ca2.3 and K_Ca3.1 channels are expressed in the endothelium of human pulmonary arteries and epithelium of bronchioles. K_Ca2.3 channels contributed to endo- and epithelium-dependent relaxations suggesting that these channels are potential targets for treatment of pulmonary hypertension and chronic obstructive pulmonary disease.     

Non-genomic vasorelaxant effects of 17β-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition

Aim:

The sex hormones 17β-estradiol (βES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations.

Methods:

Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K⁺ current and L-type Ca²⁺ current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells.

Results:

Both βES and PRG (1–100 μmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (–)-Bay K8644 (0.1 μmol/L) or by KCl (60 mmol/L). The IC₅₀ values of the two hormones were not statistically different. The K_V channel blocker 4-aminopyridine (2 mmol/L), BK_Ca channel blocker tetraethylammonium (1mmol/L) and K_ATP channel blocker glibenclamide (10 μmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular βES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 μmol/L) and PRG receptor antagonist mifepristone (30 μmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1–100 μmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K⁺ current.

Conclusion:

The vasorelaxant effects of βES and PRG are due to the inhibition of LTCC. The K⁺ channels are not involved in the effects.     

N-arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and Purpose

N-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly''s action on unstimulated and agonist-stimulated endothelial cells.

Experimental Approach

The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.

Key Results

In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²⁺ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca²⁺]_i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK_Ca channel activity.

Conclusion and Implications

Our data showed that NCX is a Ca²⁺ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²⁺ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK_Ca channels.     

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.