Signaling pathways targeting mitochondrial potassium channels.

In this review, we describe key signaling pathways regulating potassium channels present in the inner mitochondrial membrane. The signaling cascades covered here include phosphorylation, redox reactions, modulation by calcium ions and nucleotides. The following types of potassium channels have been identified in the inner mitochondrial membrane of various tissues: ATP-sensitive, Ca2+-activated, voltage-gated and two-pore domain potassium channels. The direct roles of these channels involve regulation of mitochondrial respiration, membrane potential and synthesis of reactive oxygen species (ROS). Changes in channel activity lead to diverse pro-life and pro-death responses in different cell types. Hence, characterizing the signaling pathways regulating mitochondrial potassium channels will facilitate understanding the physiological role of these proteins. Additionally, we describe in this paper certain regulatory mechanisms, which are unique to mitochondrial potassium channels.

Aldosterone impairs coronary adenosine-mediated vasodilation via reduced functional expression of Ca2+-activated K+ channels.

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A2 mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A2A, not A2B, receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A2A gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (KCa) channels that blunted and abrogated adenosine and A2A-dependent dilation, respectively. Gene expression of several coronary KCa channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A2A-KCa-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function.NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A2A, not A2B, receptor-dependent vasodilation associated with downregulation of coronary KCa channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.

Inhaled nitric oxide protects cerebral autoregulation through prevention of impairment of ATP and calcium sensitive K channel mediated cerebrovasodilation after traumatic brain injury.

Hypotension and low cerebral perfusion pressure are associated with low cerebral blood flow, cerebral ischemia, and poor outcomes after traumatic brain injury (TBI). Cerebral autoregulation is impaired after TBI, contributing to poor outcome. In prior studies, ERK mitogen activated protein kinase (MAPK) and ET-1 had been observed to be upregulated and contribute to impairment of cerebral autoregulation and histopathology after fluid percussion brain injury (FPI). Activation of ATP and Calcium sensitive (Katp and Kca) channels produce cerebrovasodilation and contribute to autoregulation, both impaired after TBI. Upregulation of ERK MAPK and endothelin-1 (ET-1) produces K channel function impairment after CNS injury. Inhaled nitric oxide (iNO) has recently been observed to prevent impairment of cerebral autoregulation and hippocampal CA1 and CA3 neuronal cell necrosis after FPI via block of upregulation of ERK MAPK and ET-1. We presently investigated whether iNO prevented impairment of Katp and Kca-mediated cerebrovasodilation after FPI in pigs equipped with a closed cranial window. Results show that pial artery dilation in response to the Katp agonist cromakalim, the Kca agonist NS1619, PGE2 and the NO releaser sodium nitroprusside (SNP) were blocked by FPI, but such impairment was prevented by iNO administered at 2 h post injury. Protection lasted for at least 1 h after iNO administration was stopped. Using vasodilaton as an index of function, these data indicate that iNO prevents impairment of cerebral autoregulation and limits histopathology after TBI through protection of K channel function via blockade of ERK MAPK and ET-1.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes.

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

Relaxant effect of Lippia origanoides essential oil in guinea-pig trachea smooth muscle involves potassium channels and soluble guanylyl cyclase.

ETHNOPHARMACOLOGICAL RELEVANCE: Lippia origanoides H.B.K. is an aromatic species used in folk medicine to treat respiratory diseases, including asthma. AIM OF THE STUDY: The aim of this work was to evaluate the relaxing potential and mechanism of action of the L. origanoides (LOO) essential oil in isolated guinea-pig trachea. MATERIALS AND METHODS: Leaves from L. origanoides were collected at experimental fields under organic cultivation, at the Forest Garden of Universidade Estadual de Feira de Santana. Essential oil was extracted by hydrodistillation, analyzed by GC/FID and GC/MS and the volatile constituents were identified. Spasmolytic activity and relaxant mechanism of LOO were assayed in isolated guinea-pig trachea contracted with histamine, carbachol or hyperpolarizing KCl. RESULTS: Chemical analysis revealed the presence of carvacrol (53.89%) as major constituent. LOO relaxed isolated guinea-pig trachea pre-contracted with KCl 60 mM [EC50 = 30.02 µg/mL], histamine 1 µM [EC50 = 9.28 µg/mL] or carbachol 1 µM [EC50 = 51.80 µg/mL]. The pre-incubation of glibenclamide, CsCl, propranolol, indomethacin, hexamethonium, aminophylline or L-NAME in histamine-induced contractions did not alter significantly the relaxant effect of LOO. However, the presence of 4-aminopyridine, tetraethylammonium or methylene blue reduced LOO effect, while the presence of dexamethasone or atropine potentialized the LOO relaxant effect. LOO pre-incubation inhibited carbachol-evoked contractions, with this effect potentialized in the presence of sodium nitroprusside and blocked in the presence of ODQ. CONCLUSIONS: The relaxant mechanism of LOO on the tracheal smooth muscle possibly involves stimulating of soluble guanylyl cyclase with consequent activation of the voltage-gated and Ca2+-activated K+ channels.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Labetalol, nebivolol, and propranolol relax human radial artery used as coronary bypass graft.

OBJECTIVE: Beta-blockers are a heterogeneous class of agents that are used in the treatment of many cardiovascular diseases, especially hypertension and atherosclerosis, and that are commonly prescribed after cardiac surgery. In the present study, the aim is to investigate the vasorelaxant effects of some common beta-adrenoceptor blockers on the human radial artery in vitro, as well as their relaxation mechanisms. METHODS: Radial artery rings sourced from human patients were mounted in an organ bath and tested for changes in isometric tension in relaxation response to labetalol, nebivolol, and propranolol in the presence and absence of NG-nitro-L-arginine methyl ester (3 × 10(-5) mol/L) and tetraethyl ammonium (3 × 10(-4) mol/L). RESULTS: The labetalol (10(-8) to 10(-4) mol/L), nebivolol (10(-8) to 10(-4) mol/L), and propranolol (10(-8) to 10(-4) mol/L) induced concentration-dependent relaxations on the radial artery rings, which had been precontracted with phenylephrine (10(-6) mol/L). The relaxation response induced by labetalol in the isolated radial artery rings was significantly higher when compared with the nebivolol and propranolol samples (P < .05). NG-nitro-L-arginine methyl ester significantly reduced the relaxation of nebivolol (P < .05), and tetraethyl ammonium significantly reduced the relaxation of labetalol, nebivolol, and propranolol (P < .05). CONCLUSIONS: We speculated that the relaxant effect of labetalol, nebivolol, and propranolol was due partly to the Ca(2+)-activated K(+) channels. In addition, the relaxation induced by nebivolol was largely related with nitric oxide release. Nebivolol, and partly propranolol, may provide significant therapeutic benefit, but labetalol can be a good alternative for coronary artery bypass grafting with radial artery use.

[Relation of intermediate-conductance Ca(2+)-activated K(+) channels with ability of proliferation, migration, invasion and IgE secretion of multiple myeloma cells].

This study was aimed to investigate the effects of the intermediate-conductance Ca(2+)-activated K(+) (IKCa1) channels on the proliferation, migration, invasion ability and monoclonal immunoglobulin (IgE) secretion of multiple myeloma (MM) cells. Trypan blue exclusion was used to evaluate the impact of clotrimazole (CLO, an inhibitor of the KCa1) on the survival ability of MM cell line U266; transwell chamber and matrigel experiments were used to evaluate the impact of CLO on the ability of the migration and invasion of U266 cells; the influence of CLO on IgE secretion in U266 cells was detected by ELISA. The results showed that small dose of CLO ( ≤ 1.0 µmol/L) could not inhibit the viability of U266 cells. The Transwell and Matrigel invading tests displayed that the cell number moving into lower chamber of transwell decreased after U266 cells treated with small dose of CLO ( ≤ 1.0 µmol/L). After treating the cells with 1.00 µmol/L CLO for 24 h and 48 h, the concentration of IgE in cell supernatant was (4.98 ± 0.39) and (4.38 ± 0.32) ng/ml, while those in control group were (15.41 ± 1.88) and (19.73 ± 2.01) ng/ml, respectively, suggesting significant difference between them(P < 0.05). It is concluded that CLO can decrease the ability of migration and monoclonal immunoglobulin secretion of multiple myeloma cells by blocking the IKCa1, thus this study provides a new think for the targeted therapy of MM.

Vasodilatory effect of 14,15-epoxyeicosatrienoic acid on mesenteric arteries in hypertensive and aged rats.

The objective of this study was to investigate the 14,15-epoxyeicosatrienoic acid (14,15-EET)-induced vasodilatations as well as the underlying signaling pathways in rat mesenteric arteries from young, adult and old normotensive (WKY) and hypertensive rats. Protein expressions for prostaglandin EP(1-4) receptors, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, and adenylate cyclase (AC) were determined together with 14,15-EET-induced vasodilatations in primary- versus secondary-branches of the mesenteric artery. Responses to 14,15-EET were greater in the smaller secondary- versus primary-branches (and also more sensitive with lower EC50) and were reduced in vessels from old (80 weeks) rats as well as from vessels from the spontaneous hypertensive rats (SHR). Regardless of age or hypertension responses to 14,15-EET were inhibited by the EP2 antagonist AH6809, BK(Ca) channel inhibitor iberiotoxin, or 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway antagonists. These data indicate 14,15-EET-induced vasodilatation is mediated via the activation of EP2 receptors and opening of BK(Ca) channels. The expressions of the EP2 receptor and AC were markedly reduced in vessels from SHR as well as old rats, whereas BK(Ca) expression was reduced in old WKY and SHR, but not adult SHR. Furthermore, expression of the p53 protein, an indicator of cell senescence and apoptosis, was elevated in adult and old SHR as well as in old WKY. In summary, attenuated 14,15-EET-induced vasodilatation in mesenteric arteries from old normotensive WKY as well as adult and old SHR is associated with reduced expression of EP2 receptors and AC.

Effects of parathyroid hormone-related peptide on the large conductance calcium-activated potassium channel and calcium homeostasis in vascular smooth muscle cells.

AIM: To demonstrate the impact of the parathyroid hormone-related peptide (PTHrP) on the large conductance calcium-activated potassium (BKCa) channels in vascular smooth muscle cells (VSMC) and hyperpolarization of the cell membrane and its dependence on calcium. MATERIALS AND METHODS: VSMC were isolated from rat aorta and further subcultured. Four experiments were conducted in calcium-release measurements and each of them consisted of a control group, PTHrP, chemical substance, and PTHrP + chemical substance. Chemical substances used were: iberiotoxin, xestospongin C, xestospongin D, and thapsigargin, respectively. Fura-2 imaging was used to determine changes in calcium release of VSMC. In membrane-potential experiments, groups were designed similarly to the Fura-2 imaging experiments: iberiotoxin, BAPTA, and xestospongin D were added, in respective order. Changes in the membrane potential were examined using the fluorescence dye (DiBAC). RESULTS: Given in a dose between 0.01 and 1.0 µmol/L, PTHrP caused a concentration-dependent decrease in fluorescence intensity, with a maximum effect at 0.5 µmol/L. The decrease, therefore, demonstrated a PTHrP-induced hyperpolarization of the VSMC. The effect was blocked by use of iberiotoxin (100 nmol/L), a highly selective inhibitor of BKCa. Furthermore, when the calcium chelator BAPTA (10 µmol/L) was added, there was a significant reduction in PTHrP-induced hyperpolarization. Use of PTHrP (0.5 µmol/L) also decreased the fluorescence intensity of the indicator for intracellular calcium, Fura-2AM (a membrane-permeable derivative of Fura 2). This effect was re-blocked by use of iberiotoxin. Xestospongin C (3 µmol/L) and xestospongin D (6 µmol/L), both inhibitors of the inositol 1,4,5 trisphosphate-triggered calcium release, inhibited the effects of PTHrP. Additionally, thapsigargin (1 µmol/L), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor, inhibited the effect of PTHrP. CONCLUSION: The results of our study show that PTHrP induces hyperpolarization and activates BKCa in VSMC. The activation of BKCa channels is calcium dependent; activation is linked to the inositol 1,4,5 trisphosphate-triggered calcium release and is also dependent on the endo/sarcoplasmic reticulum calcium pump.

Age-related impairment of conducted dilation in human coronary arterioles.

Conducted vasodilation is essential to coordinate vascular resistance along distances to ensure adequate tissue perfusion. We hypothesized that conducted vasodilation of coronary resistance arteries declines with age. Coronary arterioles were dissected from right atrial appendage of patients (n = 27) undergoing cardiac surgery. Arterioles (~100 µm) were cannulated and pressurized (80 mmHg), and developed spontaneous myogenic tone. Conducted vasodilation was initiated by locally administering the endothelium-dependent agonist bradykinin (BK; 100 µM) ejected from a glass micropipette (~3 µm tip opening, positioned in close proximity to the vessel wall). Diameter changes were measured at local and upstream sites (500 and 1,000 µm from the stimulus) with videomicroscopy. Local administration of BK elicited vasodilation, the magnitude of which increased with the duration of stimulus (69 ± 6, 81 ± 6, 90 ± 2%, after 1, 3, and 5 × 100 ms, respectively). BK-induced dilation remained substantial at upstream sites (500 µm: 53 ± 7%; 1,000 µm: 46 ± 9%). The gap junction uncoupler carbenoxolone or 18-α-glycyrrhetinic acid did not affect local responses, but diminished conducted vasodilation. Inhibitors of small/intermediate conductance calcium-activated potassium channels (SKCa/IKCa), apamin and TRAM34, reduced dilations both at local and remote sites. We found that conducted dilation, but not the local response, was significantly reduced in older (≥64 yr) patients. The nitric oxide (NO) synthesis inhibitor N(ω)-nitro-l-arginine methyl ester did not affect local responses, but markedly reduced conducted dilation in younger (<64 yr) individuals. Collectively, we show that human coronary arterioles exhibit SKCa/IKCa-mediated hyperpolarization spread through gap junctions, which contributes to conducted vasodilation initiated by focal application of BK. We demonstrate that conducted dilation declines with age, likely due to reduced NO availability, which plays a permissive role in propagating longitudinal vasomotor signaling.

Design of a truncated cardiotoxin-I analogue with potent insulinotropic activity.

Insulin secretion by pancreatic ß-cells in response to glucose or other secretagogues is tightly coupled to membrane potential. Various studies have highlighted the prospect of enhancing insulin secretion in a glucose-dependent manner by blocking voltage-gated potassium channels (K(v)) and calcium-activated potassium channels (K(Ca)). Such strategy is expected to present a lower risk for hypoglycemic events compared to KATP channel blockers. Our group recently reported the discovery of a new insulinotropic agent, cardiotoxin-I (CTX-I), from the Naja kaouthia snake venom. In the present study, we report the design and synthesis of [Lys(52)]CTX-I(41-60) via structure-guided modification, a truncated, equipotent analogue of CTX-I, and demonstrate, using various pharmacological inhibitors, that this derivative probably exerts its action through Kv channels. This new analogue could represent a useful pharmacological tool to study ß-cell physiology or even open a new therapeutic avenue for the treatment of type 2 diabetes.

SKA-31, a novel activator of SK(Ca) and IK(Ca) channels, increases coronary flow in male and female rat hearts.

AIMS: Endothelial SK(Ca) and IK(Ca) channels play an important role in the regulation of vascular function and systemic blood pressure. Based on our previous findings that small molecule activators of SK(Ca) and IK(Ca) channels (i.e. NS309 and SKA-31) can inhibit myogenic tone in isolated resistance arteries, we hypothesized that this class of compounds may induce effective vasodilation in an intact vascular bed, such as the coronary circulation. METHODS AND RESULTS: In a Langendorff-perfused, beating rat heart preparation, acute bolus administrations of SKA-31 (0.01-5 µg) dose-dependently increased total coronary flow (25-30%) in both male and female hearts; these responses were associated with modest, secondary increases in left ventricular (LV) systolic pressure and heart rate. SKA-31 evoked responses in coronary flow, LV pressure, and heart rate were qualitatively comparable to acute responses evoked by bradykinin (1 µg) and adenosine (10 µg). In the presence of apamin and TRAM-34, selective blockers of SK(Ca) and IK(Ca) channels, respectively, SKA-31 and bradykinin-induced responses were largely inhibited, whereas the adenosine-induced changes were blocked by ∼40%; TRAM-34 alone produced less inhibition. Sodium nitroprusside (SNP, 0.2 µg bolus dose) evoked changes in coronary flow, LV pressure, and heart rate were similar to those induced by SKA-31, but were unaffected by apamin + TRAM-34. The NOS inhibitor L-NNA reduced bradykinin- and adenosine-evoked changes, but did not affect responses to either SKA-31 or SNP. CONCLUSION: Our study demonstrates that SKA-31 can rapidly and reversibly induce dilation of the coronary circulation in intact functioning hearts under basal flow and contractility conditions.

H2S relaxes vas deferens smooth muscle by modulating the large conductance Ca2+ -activated K+ (BKCa) channels via a redox mechanism.

INTRODUCTION: Hydrogen sulfide (H(2) S) is generated in mammalian cells mainly by one of the two pyridoxal-5'-phosphate-dependent enzymes, cystathione-γ-lyase (CSE), and cystathione-ß-synthase (CBS) using L-cysteine as the main substrate. In previous studies, we found that CBS and CSE were functionally expressed in vas deferens (VD) and H(2) S-mediated VD smooth muscle relaxation. However, the detail mechanisms that H(2) S-relaxed VD smooth muscle were unknown so far. AIM: The aim of this study is to explore the molecular target sites of H(2) S in VD smooth muscle. METHODS: Isolated rat VD smooth muscle strips were used for tension recording in vitro. Double immunofluorescence staining was used to identify the localization of large conductance Ca(2+) -activated K(+) (BK(Ca)) channels. MAIN OUTCOME MEASURES: Changes in tonic contraction of isolated rat VD smooth muscle strip were measured after the treatment of drugs. The expression of BKca channels in rat VD smooth muscle cells was also assessed. RESULTS: The results showed that L-NG-nitroarginine methyl ester (a nitric oxide synthase inhibitor) did not affect the response of VD to sodium hydrosulphide (NaHS), suggesting that nitric oxide pathway was not involved. Further studies revealed that transient receptor potential (TRP) channels did not contribute to the NaHS-induced relaxant effect. Glibenclamide, an ATP-sensitive K channel blocker, did the same thing, whereas BK(Ca) channel blockers iberiotoxin or tetraethylammonium largely reversed the relaxant effect, suggesting that H(2) S may target BK(Ca) channels. We also confirmed that BK(Ca) channels were localized in VD smooth muscle cells. Then, studies revealed that NaHS-induced VD smooth muscle relaxation was abolished by N-ethylmaleimide, which was widely used as a sulfhydryl alkylation compound protecting thiols from oxidation, whereas DL-Dithiothreitol, a strong reducing agent, did not affect the response of VD to NaHS. CONCLUSIONS: We concluded that H(2) S relaxed the VD smooth muscle by targeting BK(Ca) channels via redox-mediated mechanism.

Upregulation of endothelium-derived hyperpolarizing factor compensates for the loss of nitric oxide in mesenteric arteries of Dahl salt-sensitive hypertensive rats.

This study was designed to determine whether a high-salt diet would alter endothelial function and, if so, the relative contributions of endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide (NO) to any changes in vasomotor responses. Male Dahl salt-sensitive (DS) rats were given either a high-salt diet (8% NaCl, DS-H) or a low-salt diet (0.4% NaCl, DS-L) for 6 weeks. Membrane potentials and contractile responses were recorded from the isolated superior mesenteric arteries. After salt loading, DS-H developed hypertension, while DS-L remained normotensive. No difference was found in acetylcholine (ACh)-induced, endothelium-dependent relaxation between the groups. However, after treatment with indomethacin and NO synthase inhibitor, EDHF-like relaxation was significantly greater in DS-H than in DS-L. In contrast, NO-mediated relaxation was significantly smaller in DS-Hthan in DS-L. Iberiotoxin (IbTx), a specific blocker of large-conductance calcium-dependent potassium (BKCa) channels, attenuated EDHF-like relaxation in DS-H but not in DS-L. IbTx marginally inhibited EDHF-mediated hyperpolarization only in DS-H. Endothelium-independent relaxation in response to sodium nitroprusside or levcromakalim was similar in both groups. In conclusion, EDHF-like relaxation is upregulated through the activation of BKCa channels in the mesenteric arteries of DS-H. As the overall relaxation in response to ACh did not differ between the groups, the upregulation of EDHF appears to compensate for the loss of NO in the mesenteric arteries of DS-H.

Peroxisome proliferator-activated receptor-ß/δ, the acute signaling factor in prostacyclin-induced pulmonary vasodilation.

As powerful vasodilators, prostacyclin analogues are presently the mainstay in the treatment of severe pulmonary arterial hypertension. Although the hemodynamic effects of prostacyclin analogues are well known, the molecular mechanism of their acute effects on pulmonary vascular tone and systemic vascular tone remains poorly understood. Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) was previously identified as a putative receptor responsible for the modulation of target gene expression in response to prostacyclin analogues. The present study investigated the signaling pathway of prostacyclin in human pulmonary arterial smooth muscle cells (PASMCs), and sought to define the role of PPARß/δ in the acute vasodilating effect. In human PASMCs, prostacyclin rapidly activated TWIK-related acid-sensitive K channel 1 (TASK-1) and calcium-dependent potassium channels (K(Ca)). This pathway was mediated via the prostanoid I receptor-protein kinase A pathway. The silencing of PPARß/δ demonstrated that the downstream K(Ca) activation was exclusively dependent on PPARß/δ signaling, whereas the activation of TASK-1 was not. In addition, the PPARß/δ-induced activation of K(Ca) was independent of NO. The acute prostacyclin-induced K(Ca) activation is critically dependent on PPARß/δ as a rapid signaling factor. This accounts in part for the vasodilating effect of prostacyclin in pulmonary arteries, and provides insights into a new molecular explanation for the effects of prostanoids.

A novel family of insect-selective peptide neurotoxins targeting insect large-conductance calcium-activated K+ channels isolated from the venom of the theraphosid spider Eucratoscelus constrictus.

Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (κ-TRTX-Ec2a, κ-TRTX-Ec2b, and κ-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that κ-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K(+) (BK(Ca)) channel currents with IC(50) values of 3 to 25 nM. In addition, κ-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K(+) currents, but only at significantly higher concentrations. κ-TRTX-Ec2a and κ-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous κ-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, κ-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, κ-TRTX-Ec2a and κ-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BK(Ca) channels. This study therefore establishes these toxins as tools for studying the role of BK(Ca) channels in insects and lead compounds for the development of novel insecticides.

Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.

BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

P2Y1 receptors mediate an activation of neuronal calcium-dependent K+ channels.

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K(V)7 K(+) and Ca(V)2 Ca(2+) channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y(1) receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca(2+) were investigated. At a membrane potential of 30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 µm. These currents had reversal potentials close to the K(+) equilibrium potential and changed direction when extracellular Na(+) was largely replaced by K(+), but remained unaltered when extracellular Cl() was changed. Currents were abolished by P2Y(1) antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca(2+), and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca(2+) stores were depleted. Blockers of K(Ca)2, but not those of K(Ca)1.1 or K(Ca)3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at 30 mV which were attenuated by P2Y(1) antagonists, depletion of Ca(2+) stores, or a blocker of K(Ca)2 channels. These results demonstrate that activation of neuronal P2Y(1) receptors may gate Ca(2+)-dependent K(+) (K(Ca)2) channels via phospholipase C-dependent increases in intracellular Ca(2+) and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y(1) receptors.