Inhibitor Of Calcium-activated Potassium Channels Research Articles

Riluzole and spinocerebellar ataxia type 2: the ATRIL trial

The search for neuroprotective therapies for the spinocerebellar ataxias remains challenging because of the huge clinical and genetic heterogeneity and low prevalence of these disorders, as well as the absence of objective biomarkers for disease progression. However, efforts persist in the clinical development of gene therapies, in the search for novel drugs, and in the repurposing of neuroprotective agents already approved for neurodegenerative diseases with overlapping pathophysiology.1 This is the case for riluzole, a glutamate release inhibitor and activator of calcium-activated potassium channels that has shown evidence of efficacy in targeting glutamatergic excitotoxicity and modulating potassium channel activity in spinocerebellar ataxias.

Mechanisms of Flow-Mediated Dilation of Pial Collaterals and the Effect of Hypertension.

Leptomeningeal anastomoses are small distal anastomotic vessels also known as pial collaterals in the brain. These vessels redirect blood flow during an occlusion and are important for stroke treatment and outcome. Pial collaterals have unique hemodynamic forces and experience significantly increased luminal flow and shear stress after the onset of ischemic stroke. However, there is limited knowledge of how pial collaterals respond to flow and shear stress, and whether this response is altered in chronic hypertension. Using an in vitro system, pial collaterals from normotensive and hypertensive rats (n=6-8/group) were isolated and luminal flow was induced with intravascular pressure maintained at 40 mm Hg. Collateral lumen diameter was measured following each flow rate in the absence or presence of pharmacological inhibitors and activators. Collaterals from male and female Wistar rats dilated similarly to increased flow (2 µL/minute: 58.4±18.7% versus 67.9±7.4%; P=0.275), and this response was prevented by inhibition of the transient receptor potential vanilloid type 4 channel, as well as inhibitors of nitric oxide and intermediate-conductance calcium-activated potassium channels, suggesting shear stress-induced activation of this pathway was involved. However, the vasodilation was significantly impaired in hypertensive rats (2 µL/minute: 17.7±7.7%), which was restored by inhibitors of reactive oxygen species and mimicked by angiotensin II. Thus, flow- and shear stress-induced vasodilation of pial collaterals appears to be an important stimulus for increasing collateral flow during large vessel occlusion. Impairment of this response during chronic hypertension may be related to poorly engaged pial collaterals during ischemic stroke in hypertensive subjects.

Inhibition of Potassium Channels Affects the Ability of Pig Spermatozoa to Elicit Capacitation and Trigger the Acrosome Exocytosis Induced by Progesterone.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.

Mechanisms underlying suppression of noradrenaline-induced contraction by prolonged treatment with advanced glycation end-products in organ-cultured rat carotid artery.

We investigated the direct effects of prolonged exposure to advanced glycation end-products (AGEs) on noradrenaline-induced contraction of rat carotid artery smooth muscle. Noradrenaline-induced contraction of endothelium-denuded carotid artery rings was suppressed by AGE-bovine serum albumin (AGE-BSA) pretreatment (0.01 and 0.1mg/mL for 23 ± 1h) compared with vehicle pretreatment (control), whereas isotonic-K+-induced contraction was not significantly altered by AGE-BSA pretreatment. This reduction in noradrenaline-induced contraction by AGE-BSA (0.1mg/mL) was reversed by iberiotoxin, an inhibitor of large-conductance calcium-activated potassium (BKCa) channels, but not by inhibitors of other K channels [4-AP (Kv inhibitor), TRAM-34 (IKCa inhibitor), or glibenclamide (KATP inhibitor)]. Acute incubation of carotid arterial rings with H2O2 had also reduced noradrenaline-induced contraction in control arteries, but it had no effect on noradrenaline-induced contraction in AGE-BSA-pretreated arteries. Alternatively, acute incubation with the H2O2 scavenger catalase increased noradrenaline-induced contraction of AGE-BSA-pretreated arteries but had no effect on noradrenaline-induced contraction of control arteries. Noradrenaline-induced contraction in the presence of H2O2 was increased by co-treatment with iberiotoxin. The AGE-BSA-mediated suppression of noradrenaline-induced contraction was prevented by the organic cation transporter 3 (OCT3) inhibitor corticosterone, whereas the expression of OCT3 protein was similar between control and AGE-BSA-treated endothelium-denuded carotid arteries. These findings suggest that noradrenaline-induced arterial contraction is reduced by prolonged AGE-BSA exposure due to activation of BKCa channels via H2O2 generation and increased OCT3-mediated noradrenaline transport activity.

Tocomin Restores Endothelium-Dependent Relaxation in the Diabetic Rat Aorta by Increasing NO Bioavailability and Improving the Expression of eNOS.

We aimed to determine whether tocomin, an extract from palm oil that has a high tocotrienol content, was able to prevent diabetes-induced endothelial dysfunction. To induce type 1 diabetes streptozotocin (50 mg/kg) was injected into the tail vein of Wistar rats. Six weeks later the diabetic rats, and normal rats injected with citrate buffer, commenced treatment with tocomin (40 mg/kg/day sc) or its vehicle (peanut oil) for a further 4 weeks. Aortae isolated from diabetic rats had impaired acetylcholine (ACh)-induced endothelium-dependent relaxation compared to normal rat aortae but there was no change in endothelium-independent relaxation in response to sodium nitroprusside. By contrast, responses to ACh in aortae from diabetic rats treated with tocomin were not different to normal rats. In addition to impaired endothelium-dependent relaxation the diabetic aortae had increased expression of the NADPH oxidase Nox2 subunit, increased generation of superoxide and decreased expression of eNOS and all of these effects were prevented by tocomin treatment. Tocomin did not affect plasma glucose levels. The impaired response to ACh in vitro was maintained in the presence of TRAM-34 and apamin, selective inhibitors of calcium-activated potassium (KCa) channels, indicating diabetes impaired the contribution of NO to endothelium-dependent relaxation. By contrast, neither diabetes nor tocomin treatment influenced EDH-type relaxation as, in the presence of L-NNA, an inhibitor of eNOS, and ODQ, to inhibit soluble guanylate cyclase, responses to ACh were similar in all treatment groups. Thus tocomin treatment improves NO mediated endothelium dependent relaxation in aortae from diabetic rats associated with a decrease in vascular oxidant stress but without affecting hyperglycaemia.

The role of potassium channels and calcium in the relaxation mechanism of magnesium sulfate on the isolated rat uterus

MgSO4 is used as a tocolytic agent. It is considered to be a calcium channel antagonist, but a different mechanism of its action might be involved. The aim of this study was to examine the contribution of calcium concentrations and potassium channels in the mechanism of MgSO4-mediated uterine relaxation. Isolated uteri from female Wister rats were treated with increasing MgSO4 concentrations (0.1-30 mM). MgSO4 induced dose-dependent inhibition of spontaneous activity. Addition of Ca2+ (6 mM and 12 mM) stimulated uterine contractile activity and attenuated the inhibitory activity of MgSO4. In order to analyze the role of different subtypes of potassium channels, Ca2+-stimulated uteri were pretreated with glibenclamide (Glib), a selective ATP-sensitive potassium channel inhibitor (KATP), tetraethylammonium (TEA), a non-specific inhibitor of large conductance calcium-activated potassium channels (BKCa), and 4-aminopyridine (4-AP), a voltage-sensitive potassium channel inhibitor (Kv), at concentrations that had no effect per se. Pretreatment with 4-AP had no effect on MgSO4-mediated relaxation of Ca2+-stimulated uteri. The relaxing effect of MgSO4 was potentiated by pretreatment with glibenclamide. Pretreatment with TEA attenuated the MgSO4-mediated decrease in frequency. Our results suggest that MgSO4 acts as a general calcium antagonist that influences Ca2+-mediated potassium channels. Furthermore, it seems that MgSO4 uterine relaxation activity is partially mediated by selective ATP-sensitive potassium channels, suggesting an ATP-dependent role.

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of KATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H2S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of KATP-channels.

Mechanism of resveratrol-induced relaxation of the guinea pig fundus

BackgroundResveratrol is a polyphenolic compound that can be isolated from plants and also is a constituent of red wine. Resveratrol induces relaxation of vascular smooth muscle and may prevent cardiovascular diseases. PurposeImpaired gastric accommodation plays an important role in functional dyspepsia and fundic relaxation and is a therapeutic target of functional dyspepsia. Although drugs for fundic relaxation have been developed, these types of drugs are still rare. The purpose of this study was to investigate the relaxant effects of resveratrol in the guinea pig fundus. Study DesignWe studied the relaxant effects of resveratrol in the guinea pig fundus. In addition, we investigated the mechanism of resveratrol-induced relaxation on the guinea pig fundus by using tetraethylammonium (a non-selective potassium channel blocker), apamine (a selective inhibitor of the small conductance calcium-activated potassium channel), iberiotoxin (an inhibitor of large conductance calcium-activated potassium channels), glibenclamide (an ATP-sensitive potassium channel blocker), KT 5720 (a cAMP-dependent protein kinase A inhibitor), KT 5823 (a cGMP-dependent protein kinase G inhibitor), NG-nitro-L-arginine (a competitive inhibitor of nitric oxide synthase), tetrodotoxin (a selective neuronal Na+ channel blocker), ω-conotoxin GVIA (a selective neuronal Ca2+ channel blocker) and G-15 (a G-protein coupled estrogen receptor antagonist). ResultsThe results of this study showed that resveratrol has potent and dose-dependent relaxant effects on the guinea pig fundic muscle. In addition, the results showed that resveratrol-induced relaxation of the guinea pig fundus occurs through nitric oxide and ATP-sensitive potassium channels. ConclusionThis study provides the first evidence concerning the relaxant effects of resveratrol in the guinea pig fundic muscle strips. Furthermore, resveratrol may be a potential drug to relieve gastrointestinal dyspepsia.

Potassium channel inhibitors induce oxidative stress in breast cancer cells

Abstract Background Antioxidant levels increase to protect cell homeostasis when oxidant generation is increased by drug or inhibitor treatment. If the oxidant–antioxidant equilibrium is disrupted, oxidative stress will occur. Objectives To determine the effects of various potassium channel inhibitors in the disruption of oxidant–antioxidant equilibrium in breast cancer cell lines with various phenotypes. Methods MCF-7 or MDA-MB-231 breast cancer cells were treated with tetraethylammonium chloride (5 mM; TEA), 4-aminopyridine (5 mM; 4-AP), margatoxin (25 nM; MgTX), or astemizole (200 nM; AST). After treatment, total antioxidant, oxidant, and oxidative stress levels were determined. Results Incubation with TEA, 4-AP, MgTX, and AST increased oxidative stress in both MCF-7 and MDA-MB-231 cells (P < 0.001). Specific inhibitors of calcium-activated potassium channels and ether á go-go 1-related potassium channels produce greater oxidative stress than other inhibitors in MCF-7 breast cancer cells, whereas in MDA-MB-231 cells, the nonselective channel inhibitor 4-AP produces the greatest oxidative stress. Conclusions Potassium channel inhibitors used in our study disrupted the antioxidant–oxidant equilibrium and increased oxidative stress in the cancer cell lines. Although all of the channel inhibitors increased oxidative stress in cells, TEA and AST were the most effective inhibitors in MCF-7 cells. 4-AP was the most effective inhibitor in MDA-MB-231 cells. Voltage-gated potassium channels are attractive targets for anticancer therapy, and their inhibitors may enhance the effects of anticancer drugs.

Study on the electrophsiological properties in the stria vascularis pericytes in cochlear of guinea pig

The purpose of this paper was to study the electrophysiological properties and the type of potassium channels on cell membrane in the stria vascularis pericytes in cochlear of guinea pig. Firstly examined the expression of the stria vascularis pericytes by desmin, a marker of pericytes, in cochlear of guinea pig with immunofluorescent method. Using whole-cell patch clamp recording techniques to observe electrophysiological properties in the cochlear pericytes in stria vascularis of guinea pig. Pericytes were predominately distributed in the capillaries of cochlea.The average membrane capacitance, resistance, and potential of a single pericyte in stria vascularis were(5.9±0.3)pF, (2.2±0.3)GΩ and (-30.9±1.2)mV, respectively by using patch clamp technique. In addition, the average current density of cochlear pericyte was voltage-sensitive (Vh from 0 to + 60 mV, in 20 mV steps). The pericytes exhibited outward current and this property could be blocked by TEA (tetraethylammonium) 1 mmol/L, a large-conductance calcium-activated potassium channel(BKCa)inhibitor and 4-AP (4-aminopyridine) 1 mmol/L, a voltage-dependent K(+) channels(KV) channel blocker. TEA blocked the outward current from (296.2±35.9)pA to (163.7±16.8)pA and 4-AP blocked the outward current from (248.7±39.8)pA to (158.0±38.0)pA. These results suggest that pericytes in stria vascularis have BKCa and KV channels.

Endothelial-derived hyperpolarization contributes to acetylcholine-mediated vasodilation in human skin in a dose-dependent manner

Cutaneous acetylcholine (ACh)-mediated dilation is commonly used to assess microvascular function, but the mechanisms of dilation are poorly understood. Depending on dose and method of administration, nitric oxide (NO) and prostanoids are involved to varying extents and the roles of endothelial-derived hyperpolarizing factors (EDHFs) are unclear. In the present study, five incremental doses of ACh (0.01-100 mM) were delivered either as a 1-min bolus (protocol 1, n = 12) or as a ≥20-min continuous infusion (protocol 2, n = 10) via microdialysis fibers infused with 1) lactated Ringer, 2) tetraethylammonium (TEA) [a calcium-activated potassium channel (KCa) and EDHF inhibitor], 3) L-NNA+ketorolac [NO synthase (NOS) and cyclooxygenase (COX) inhibitors], and 4) TEA+L-NNA+Ketorolac. The hyperemic response was characterized as peak and area under the curve (AUC) cutaneous vascular conductance (CVC) for bolus infusions or plateau CVC for continuous infusions, and reported as %maximal CVC. In protocol 1, TEA, alone and combined with NOS+COX inhibition, attenuated peak CVC (100 mM Ringer 59 ± 6% vs. TEA 43 ± 5%, P < 0.05; L-NNA+ketorolac 35 ± 4% vs. TEA+L-NNA+ketorolac 25 ± 4%, P < 0.05) and AUC (Ringer 25,414 ± 3,528 vs. TEA 21,403 ± 3,416%·s, P < 0.05; L-NNA+ketorolac 25,628 ± 3,828%(.)s vs. TEA+L-NNA+ketorolac 20,772 ± 3,711%·s, P < 0.05), although these effects were only significant at the highest dose of ACh. At lower doses, TEA lengthened the total time of the hyperemic response (10 mM Ringer 609 ± 78 s vs. TEA 860 ± 67 s, P < 0.05). In protocol 2, TEA alone did not affect plateau CVC, but attenuated plateau in combination with NOS+COX inhibition (100 mM 50.4 ± 6.6% vs. 30.9 ± 6.3%, P < 0.05). Therefore, EDHFs contribute to cutaneous ACh-mediated dilation, but their relative contribution is altered by the dose and infusion procedure.

Kaempferol enhances endothelium‐dependent relaxation in the porcine coronary artery through activation of large‐conductance Ca2+‐activated K+ channels

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. 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). 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 (KCa 1.1; 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. 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 KCa 1.1 channels.

Angiotensin II modulates mouse skeletal muscle resting conductance to chloride and potassium ions and calcium homeostasis via the AT1 receptor and NADPH oxidase.

Angiotensin II (ANG II) plays a role in muscle wasting and remodeling; however, little evidence shows its direct effects on specific muscle functions. We presently investigated the acute in vitro effects of ANG II on resting ionic conductance and calcium homeostasis of mouse extensor digitorum longus (EDL) muscle fibers, based on previous findings that in vivo inhibition of ANG II counteracts the impairment of macroscopic ClC-1 chloride channel conductance (gCl) in the mdx mouse model of muscular dystrophy. By means of intracellular microelectrode recordings we found that ANG II reduced gCl in the nanomolar range and in a concentration-dependent manner (EC50 = 0.06 μM) meanwhile increasing potassium conductance (gK). Both effects were inhibited by the ANG II receptors type 1 (AT1)-receptor antagonist losartan and the protein kinase C inhibitor chelerythrine; no antagonism was observed with the AT2 antagonist PD123,319. The scavenger of reactive oxygen species (ROS) N-acetyl cysteine and the NADPH-oxidase (NOX) inhibitor apocynin also antagonized ANG II effects on resting ionic conductances; the ANG II-dependent gK increase was blocked by iberiotoxin, an inhibitor of calcium-activated potassium channels. ANG II also lowered the threshold for myofiber and muscle contraction. Both ANG II and the AT1 agonist L162,313 increased the intracellular calcium transients, measured by fura-2, with a two-step pattern. These latter effects were not observed in the presence of losartan and of the phospholipase C inhibitor U73122 and the in absence of extracellular calcium, disclosing a Gq-mediated calcium entry mechanism. The data show for the first time that the AT1-mediated ANG II pathway, also involving NOX and ROS, directly modulates ion channels and calcium homeostasis in adult myofibers.

Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

Characterization of the Relaxant Response to Taurine in Rat Corpus Cavernosum

The aim of this study was to evaluate the relaxant effect of taurine, one of the most commonly employed dietary supplements, in rat corpus cavernosum (CC), and to further investigate the contribution of possible underlying mechanisms. Strips of CC were suspended in an organ bath system for isometric tension studies. Taurine (10-80 mmol/l) produced a concentration-dependent relaxation response in rat CC. Endothelial removal did not cause a significant inhibition of the relaxation response to taurine. Incubation of endothelium-denuded CC strips with the nonselective potassium channel blocker tetraethylammonium (10 mmol/l), the ATP-sensitive potassium (K_ATP) channel blocker glibenclamide (10 μmol/l), the inward rectifier potassium (K_ir) channel inhibitor barium chloride (30 μmol/l), and the large conductance calcium-activated potassium channel inhibitor iberiotoxin (0.1 μmol/l) significantly inhibited the relaxant responses to taurine. However, taurine-induced relaxation was not inhibited by the voltage-dependent potassium channel inhibitor 4-aminopyridine (1 mmol/l). On the other hand, taurine (20 mmol/l, 30 min) inhibited both intracellular and extracellular calcium-dependent contraction in CC strips. These findings suggest that taurine induced relaxation of CC via an endothelium-independent pathway. The activation of K_ATP channels, K_ir channels and calcium channels is thought to play an important role in endothelium-independent relaxation of CC, but other direct effects on calcium dynamics may also contribute to its relaxant effect.

Ropivacaine-Induced Contraction Is Attenuated by Both Endothelial Nitric Oxide and Voltage-Dependent Potassium Channels in Isolated Rat Aortae

This study investigated endothelium-derived vasodilators and potassium channels involved in the modulation of ropivacaine-induced contraction. In endothelium-intact rat aortae, ropivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: the nonspecific nitric oxide synthase (NOS) inhibitor N ω-nitro-L-arginine methyl ester (L-NAME), the neuronal NOS inhibitor N ω-propyl-L-arginine hydrochloride, the inducible NOS inhibitor 1400W dihydrochloride, the nitric oxide-sensitive guanylyl cyclase (GC) inhibitor ODQ, the NOS and GC inhibitor methylene blue, the phosphoinositide-3 kinase inhibitor wortmannin, the cytochrome p450 epoxygenase inhibitor fluconazole, the voltage-dependent potassium channel inhibitor 4-aminopyridine (4-AP), the calcium-activated potassium channel inhibitor tetraethylammonium (TEA), the inward-rectifying potassium channel inhibitor barium chloride, and the ATP-sensitive potassium channel inhibitor glibenclamide. The effect of ropivacaine on endothelial nitric oxide synthase (eNOS) phosphorylation in human umbilical vein endothelial cells was examined by western blotting. Ropivacaine-induced contraction was weaker in endothelium-intact aortae than in endothelium-denuded aortae. L-NAME, ODQ, and methylene blue enhanced ropivacaine-induced contraction, whereas wortmannin, N ω-propyl-L-arginine hydrochloride, 1400W dihydrochloride, and fluconazole had no effect. 4-AP and TEA enhanced ropivacaine-induced contraction; however, barium chloride and glibenclamide had no effect. eNOS phosphorylation was induced by ropivacaine. These results suggest that ropivacaine-induced contraction is attenuated primarily by both endothelial nitric oxide and voltage-dependent potassium channels.

Relaxant effect of all-trans-retinoic acid via NO-sGC-cGMP pathway and calcium-activated potassium channels in rat mesenteric artery

Intraperitoneal injection of all-trans-retinoic acid (ATRA) results in a reduction of blood pressure in spontaneously hypertensive rats. However, the mechanisms involved in this effect are not clear. We hypothesized that ATRA may relax resistance arteries. In this study, we found that ATRA relaxed phenylephrine-preconstricted mesenteric arterial rings, which were abrogated by the removal of the endothelium. Pretreatment of endothelium-intact arterial rings with an inhibitor of endothelial nitric oxide (NO) synthase, N(G)-nitro-l-arginine methyl ester (l-NAME), or soluble guanylyl cyclase, 1H-[1,2,4]-oxadiazole-[4,3-α]-quinoxaline-1-one, reduced the vasorelaxant effect of ATRA. Incubation of mesenteric arterial rings with ATRA increased the production of NO and cGMP, which were blocked by N(G)-nitro-l-arginine methyl ester. The vasorelaxant effect of ATRA was markedly attenuated in the presence of an inhibitor of big conductance calcium-activated potassium channels (charybdotoxin), but not with an inhibitor of voltage-dependent potassium channel (4-aminopyridine) or ATP-sensitive potassium channel (glibenclamide). Activation of retinoic acid receptors (RARs) with CH55 or retinoic X receptors (RXRs) with LGD1069 induced the vasorelaxation of phenylephrine-preconstricted mesenteric arterial rings. The RAR (BMS493) and RXR (UVI3003) antagonists blocked the ATRA-induced vasorelaxation. The vasorelaxant effect ATRA is physiologically relevant because the intravenous infusion of ATRA decreased blood pressure in normotensive rats. We conclude that ATRA relaxes resistance vessels via both RARs and RXRs receptors that are mediated by the endothelium-dependent NO-cGMP pathway, which may participate in the control of blood pressure.

Glutamate-induced cell death in HT22 mouse hippocampal cells is attenuated by paxilline, a BK channel inhibitor

In the present study, we show that the large conductance calcium-activated potassium channel (BKCa channel) inhibitor paxilline protects neuronal cells against glutamate-induced cell death. In our studies, we used HT22 mouse hippocampal cells as an experimental model and observed that the effect of paxilline was dose-dependent. We also found that other inhibitors of BKCa channels, iberiotoxin and charybdotoxin, were not cytoprotective. Paxillinol, which is a structural analog of paxilline but does not inhibit BKCa channel, also protected HT22 cells against glutamate-induced toxicity. These data suggest that the observed cytoprotection was not related to BKCa channel inhibition by paxilline. In addition, paxilline neither restored glutathione levels nor reduced the amount of reactive oxygen species upon glutamate treatment.Our results suggest that paxilline protects neuronal HT22 cells against glutamate-induced cell death independently of BKCa channel activity and oxidative stress induced by glutamate treatment.

Decreases in splanchnic vascular resistance contribute to hypotensive effects ofl-serine in hypertensive rats

l-Serine administration reduces mean arterial pressure (MAP) in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats rendered hypertensive by chronic oral treatment with the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME). To determine if the fall in MAP was due to decreases in vascular resistance or cardiac output (CO), and to record regional hemodynamic effects, we measured the distribution of fluorescent microspheres to single bolus intravenous injections of l-serine (1 mmol/kg) in 14-wk-old male WKY, SHR, and l-NAME-treated WKY rats. MAP and total peripheral resistance (TPR) were significantly higher (P < 0.01), whereas CO was lower in l-NAME-treated WKY (P < 0.01) and SHR (P < 0.05). l-Serine administration led to a rapid fall in MAP (WKY 22%, l-NAME-WKY 46%, SHR 34%,) and TPR (WKY 24%, l-NAME-WKY 68%, SHR 53%), whereas CO was elevated. In WKY rats, l-serine induced an increase in blood flow only in the small intestine (53%) while it was more profound in several vascular beds of hypertensive rats [l-NAME-WKY: small intestine (238%), spleen (184%), diaphragm (85%), and liver (65%); SHR: small intestine (217%), spleen (202%), diaphragm (116%), large intestine (105%), pancreas (96%), and liver (93%)]. Pretreatment with a combination of apamin (a small calcium-activated potassium channel inhibitor) and charybdotoxin (an intermediate calcium-activated potassium channel inhibitor) abolished the l-serine-induced changes in blood flow and TPR. l-Serine acts predominantly on apamin- and charybdotoxin-sensitive potassium channels in the splanchnic circulation to increase blood flow, thereby contributing to the fall in TPR and the pronounced blood pressure-lowering effect of l-serine in hypertensive rats.

Endothelium-independent vasorelaxation by the selective alpha estrogen receptor agonist propyl pyrazole triol in rat aortic smooth muscle

Abstract Objectives This study investigated the signalling mechanism of the relaxant responses to the estrogen receptor alpha (ERα) agonist PPT (propyl pyrazole triol) in endothelium-denuded rat aortic rings. Methods Several compounds, including protein kinase G (PKG) inhibitors and potassium channel inhibitors, were tested against PPT-dependent rat aortic relaxation. Cyclic GMP and cytosolic calcium responses to PPT in isolated aortic smooth muscle were investigated in parallel. Key findings PPT vasorelaxation was largely reduced by the selective ERα antagonist methyl-piperidinopyrazole (MPP; −91.6 ± 2.5%), by the selective PKG inhibitor Rp-8-Br-cGMP (−78.6 ± 4.9%), by the specific soluble guanylyl cyclase inhibitor ODQ (1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one; −85.3 ± 5.2%) and to a lesser extent by the selective BKCa (large-conductance calcium- and voltage-activated potassium channel) inhibitor iberiotoxin (−59.3%), the selective IKCa (intermediate-conductance calcium-activated potassium channel) inhibitor TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole; −50.7%) and the voltage-gated potassium channel inhibitor 4-aminopyridine (−40.8%). In isolated aortic smooth muscle, PPT strongly enhanced the cyclic GMP content (+144%) and Rp-8-Br-cGMP largely reduced the PPT-dependent calcium signal (−80.8%). Conclusions ERα receptor stimulation in rat aortic smooth muscle evokes a PKG-signalling pathway, likely triggering relaxation by BKCa and IKCa channel opening.