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.

Effects of Schisandra chinensis extract on the contractility of corpus cavernosal smooth muscle (CSM) and Ca2+ homeostasis in CSM cells.

UNLABELLED: What's known on the subject? And what does the study add? Schisandra chinensis extract (SCE) has been known to have relaxative effects on penile smooth muscle. A recent study showed that SCE could enhance slidenafil citrate-induced relaxation of penile corpus cavernosum. The current study investigated the mechanism of action of SCE and its constituents on corporal smooth muscle cells. And this study shows that SCE induced relaxation of CSM primarily through an endothelium independent pathway and the relaxation effects of SCE on corporal smooth muscle are, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)]. OBJECTIVE: • To evaluate the relaxant effects of Schisandra chinensis extract (SCE) on corporal tissue in the penis and to investigate the mechanism of action of SCE and its constituents on corporal smooth muscle (CSM) cells. MATERIALS AND METHODS: • The fruit of SC was collected and extracted with ethanol. Six SC lignans (schisandrol A, schisandrol B, schisandrin A, schisandrin B, gomisin N, and schisandrin C) were isolated and purified, and the chemical structures were confirmed by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR data. • Isolated rabbit CSM strips were mounted in an organ-bath system, and the effects of SCE were evaluated. • To estimate the intracellular Ca(2+) level ([Ca(2+)](i)), we used a Fura-2 fluorescent technique, and a conventional whole-cell patch-clamp technique was used to measure the calcium-sensitive K(+) channels (K(Ca)), inward rectifier K(+) channels (K(IR)), and canonical transient receptor potential cation channel 6 (TRPC6) currents. RESULTS: • SCE induced concentration-dependent relaxation in contracted CSM tissue, and the removal of the endothelium did not significantly affect their relaxation potencies. • In CSM cells, extracellular application of SCE significantly increased whole-cell K(Ca) currents (117.4%) and K(IR) currents (110.0%). These effects were completely abolished by charybdotoxin or BaCl(2). • In contrast, carbachol-induced TRPC6 channel activity was significantly inhibited (87.3%) by SCE in green fluorescent protein-TRPC6 pcDNA transfected HEK 293 cells. [Ca(2+)](i) measurements showed that SCE effectively reduced basal [Ca(2+)](i) in both cell lines (CSM cells and A7r5 cells) and the [Arg8]-vasopressin (AVP)-induced [Ca(2+)](i) increase in A7r5 cells. • Among the six SC lignans, schisandrin A and schisandrin B most effectively attenuated the AVP-induced [Ca(2+)](i) increase. CONCLUSIONS: • SCE induced relaxation of CSM that occurred primarily via an endothelium-independent pathway. • The relaxation effects of SCE on CSM were, in part, due to the activation of K(+) channels and inhibition of TRPC6 channels, resulting in decreased [Ca(2+)](i).

Functional involvement of Ca(2+) and Ca(2+)-activated K(+) channels in anethol-induced changes in Ca(2+) dependent excitability of F1 neurons in Helix aspersa.

The effects of anethol, the major component of anise oil, on the Ca(2+)-dependent excitability and afterhyperpolarization (AHP) in snail neurons were examined using intracellular recording. Anethol (0.5%) significantly broadened the spike, reduced the firing frequency and enhanced the AHP amplitude. In contrast, anethol (2%) significantly increased the firing frequency and decreased the AHP. Blockade of Ca(2+) channels after anethol application depolarized the membrane potential and significantly reduced the firing rate. Furthermore, in the presence of anethol (0.5%) a significant decrease in the AHP was observed by Ca(2+) channels blockage. Here, anethol-induced functional modification of Ca(2+) and Ca(2+)-activated K(+) channels is suggested.

The effect of Korean red ginseng extract on the relaxation response in isolated rabbit vaginal tissue and its mechanism.

INTRODUCTION: Ginseng is an herbal medicine with a variety of biological activities. AIM: The purpose of this study was to investigate the effect of Korean red ginseng (KRG) extract on the relaxation response in isolated rabbit vaginal tissue and its mechanism as a potential therapeutic agent for female sexual dysfunction. METHOD: Strips of rabbit vagina were mounted in organ chambers to measure isometric tension. After the strips were precontracted with phenylephrine, the contractile responses to KRG extract (1-20 mg/mL), nitric oxide inhibitor (N[omega]-nitro-L-arginine methyl ester [L-NAME]), an inhibitor of soluble guanylate cyclase (methylene blue), an inhibitor of Ca(2+)-activated K(+) channels (tetraethylammonium [TEA]), and an adenosine triphosphate (ATP)-sensitive K(+) channel blocker (glybenclamide) were examined. MAIN OUTCOME MEASURES: The relaxation of the vaginal tissue strip was assessed after treating KRG extract or other chemicals. RESULTS: KRG (1-20 mg/mL) extract relaxed the vaginal tissue strip in a dose-dependent manner up to 85%. The relaxation effect was significantly inhibited by L-NAME (30 microM) and methylene blue (30 microM) (P < 0.05). In addition, KRG inhibited the contraction induced by depolarization with 10, 20, and 40 mM KCl. The KRG-induced relaxation effect was significantly inhibited by TEA (300 microM) (P < 0.05), and not by glybenclamide (30 microM). CONCLUSIONS: These data show that KRG extract has a relaxing effect on rabbit vaginal smooth muscle tissue. These effects might be mediated partly through the NO pathway and hyperpolarization via Ca(2+)-activated K(+) channels.

The phytoestrogen ginsensoside Re activates potassium channels of vascular smooth muscle cells through PI3K/Akt and nitric oxide pathways.

In vascular smooth muscle cells, large-conductance Ca(2+)-activated K(+) channels (K(Ca) channels) play a pivotal role in determining membrane potential, and thereby the vascular tone. Ginsenoside Re, a phytochemical from ginseng, is reported to activate this channel, but its precise mechanism is unsolved. Patch clamp studies showed that ginsenoside Re activates K(Ca) channels in the arterial smooth muscle cell line A10 in a dose-dependent manner. The channel-opening effect of ginsenoside Re was inhibited by 1 microM L-NIO, an inhibitor of eNOS, but not by 3 microM SMTC, an inhibitor of nNOS, indicating that ginsenoside Re activated K(Ca) channels through activation of eNOS. SH-6 (10 microM), an Akt inhibitor, and wortmannin, a PI3-kinase inhibitor, completely blocked activation of K(Ca) channels by ginsenoside Re, indicating that it activates eNOS via a c-Src/PI3-kinase/Akt-dependent mechanism. In addition, the ginsenoside Re-induced activation of eNOS and K(Ca) channel was blocked by 10 microM ICI 182, 780, an inhibitor of membrane estrogen receptor-alpha, suggesting that eNOS activation occurs via a non-genomic pathway of this receptor. In conclusion, ginsenoside Re releases NO via a membrane sex steroid receptors, resulting in K(Ca) channel activation in vascular smooth muscle cells, promoting vasodilation and preventing severe arterial contraction.

Inhibitory effects of peony root extract on the large conductance calcium-activated potassium current essential in production of bursting activity.

To elucidate the mechanism of inhibitory action of peony root extract on pentylenetetrazol-induced bursting activity, effects of peony root extract on the iberiotoxin-sensitive large conductance calcium-activated potassium (BKCa) current that plays an essential role in the production of bursting activity were investigated. Peony root extract showed a clear inhibitory effect on the iberiotoxin-sensitive calcium-activated potassium current. Peony root extract also showed clear inhibitory effects on spontaneous bursting activity and BKCa current in the cerebral cortical neurons of the EL mouse, a hereditary epilepsy animal model. These results together with our previous studies, including the protective effect against neuron damage, indicate that peony root extract is a promising herbal drug for inhibition of convulsions.

Activation of the iberiotoxin-sensitive BKCa channels by salvianolic acid B of the porcine coronary artery smooth muscle cells.

In this study, we examined the effects of Salvia miltiorrhiza (Danshen) crude extract, some of its lipid-soluble components (tanshinone I, tanshinone II(A), cryptotanshinone, dihydroisotanshinone I) and the water-soluble compounds (danshensu and salvianolic acid B) on the K(+) channels such as the iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and the glibenclamide-sensitive ATP-dependent K(+) (IK(ATP)) channels of the porcine left anterior descending coronary artery smooth muscle cells. Cumulative application of salvianolic acid B (30-300 microM) caused a l-NNA (100 microM)-insensitive, potentiation of the outward BK(Ca) current amplitude with no apparent effect on the IK(ATP) channels opening. Salvianolic acid B (300 microM) caused an ODQ (10 microM, a guanylate cyclase inhibitor)-sensitive enhancement of the outward BK(Ca) current amplitude. In contrast, none of the other isolated chemical constituents of S. miltiorrhiza modified the openings of the two types of K(+) channels studied. In conclusion, our results suggest that salvianolic acid B, a major hydrophilic constituent found in Radix S. miltiorrhiza, activated the opening of the BK(Ca) channels of the porcine coronary artery smooth muscle cells through the activation of guanylate cyclase without the involvement of the nitric oxide synthase activation.

Quercetin-induced induction of the NO/cGMP pathway depends on Ca2+-activated K+ channel-induced hyperpolarization-mediated Ca2+-entry into cultured human endothelial cells.

Quercetin is one of the dietary-derived flavonoids that are held responsible for the beneficial effects of red wine drinking in coronary artery disease known as the "French paradox". We examined whether quercetin modulates endothelial function by influencing Ca2+-activated K+ channels with large conductance (BK(Ca)) in cultured human endothelial cells. Membrane potential and intracellular Ca2+ concentrations of cultured human endothelial cells derived from umbilical cord veins (HUVEC) were measured using the fluorescence dyes DiBAC, and FURA-2, respectively. NO production was examined using a cGMP radioimmunoassay. HUVEC proliferation was analyzed by cell counts and thymidine incorporation. A dose-dependent hyperpolarization of HUVEC was recorded when quercetin was added (5-100 micromol/L). The maximum effect (50 micromol/L) was significantly reduced by the addition of the highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/L), but not by blockers of other Ca2+-activated K+ channels (n = 30; p < 0.05). This BK(Ca)-induced hyperpolarization caused a transmembrane Ca2+ influx, because the quercetin-induced increase of intracellular Ca2+ was blocked by iberiotoxin, or by applying 2-aminoethoxydiphenylborate (100 micromol/L)--an inhibitor of capacitative Ca2+ entry (n = 30; p < 0.05). Quercetin-induced cGMP levels were significantly reduced by the eNOS-inhibitor l-NMMA (300 micromol/L), and by iberiotoxin (n = 10; p < 0.05). Endothelial proliferation was significantly reduced by 56 % when cells were incubated with quercetin (n = 12; p < 0.05). This effect was due to the increased NO production, because it was reversed when the cells were treated with a combination of quercetin and l-NMMA. In conclusion quercetin improves endothelial dysfunction by increasing NO synthesis involving BK(Ca)-dependent membrane hyperpolarization-induced capacitative Ca 2+ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.

Contribution of Ca2+-activated K+ channels to hyperpolarizing after-potentials and discharge pattern in rat supraoptic neurones.

The contribution of Ca(2+)-activated K(+) channels to hyperpolarizing after-potentials (HAP) of action potentials, to spike-frequency adaptation and thus to the shaping of discharge pattern, was examined in rat supraoptic magnocellular neurosecretory cells. In addition, the expression of BK channels and SK3 subunits of SK channels was studied using double immunofluorescence detection. The presence of BK channels and SK3 subunits was detected in many supraoptic neurones containing either vasopressin or oxytocin. Current-clamp recordings of current-induced spike trains revealed that HAPs comprise a fast and a slow HAP (fHAP and sHAP). Correlation analyses revealed that the increase of the fHAP in amplitude and spike broadening were correlated to a moderate gradual increase of the interspike interval and thus to weak spike-frequency adaptation. By contrast, marked prolongation of the interspike interval and strong spike-frequency adaptation depended on the appearance and on the amplitude of the sHAP. The sHAP and spike-frequency adaptation were blocked by cadmium, as well as by the SK channel antagonist apamin. The fHAP was attenuated by the BK channel antagonist iberiotoxin (IbTX), by the BK/IK channel antagonist charybdotoxin (ChTX) and by apamin. ChTX attenuated fHAPs throughout the entire spike train. By contrast, the IbTX-induced attenuation of the fHAP was restricted to the initial part of the spike train, while the apamin-induced attenuation slowly increased with the progression of the spike train. These results suggest that strong spike-frequency adaptation in supraoptic neurones essentially depends on the generation of the sHAP by activation of SK channels. Comparison of effects of IbTX, ChTX and apamin suggests a complementary contribution of SK-, BK- and IK-channels to fHAPs.

Isosteviol as a potassium channel opener to lower intracellular calcium concentrations in cultured aortic smooth muscle cells.

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, and is commonly used as a non-caloric sugar substitute in Japan and Brazil. The present study attempted to elucidate the role of potassium (K (+)) channels in the action of isosteviol on intracellular calcium concentrations ([Ca (2+)]i) in cultured vascular smooth muscle (A7r5) cells using the Ca (2+)-sensitive dye Fura-2 as an indicator. The increase of [Ca (2+)]i in A7r5 cells produced by vasopressin (1 micromol/L) or phenylephrine (1 micromol/L) was attenuated by isosteviol from 0.01 micromol/L to 10 micromol/L. The attenuation by isosteviol of the vasopressin- and phenylephrine-induced increase in [Ca (2+)]i was inhibited by glibenclamide, apamin and 4-aminopyridine but not by charybdotoxin. Furthermore, the inhibitory action of isosteviol on [Ca (2+)]i was blocked when A7r5 cells co-treated with glibenclamide and apamin in conjunction with 4-aminopyridine were present. Therefore, not only did the ATP-sensitive potassium (K (ATP)) channel affect the action of isosteviol on [Ca (2+)]i modulation in A7r5 cells, but also those on the small conductance calcium-activated potassium (SK (Ca)) channels and voltage-gated (Kv) channels. However, the blockers of large-conductance Ca (2+)-activated potassium channels failed to modify the inhibitory action of isosteviol on [Ca (2+)]i. The obtained results indicated that a decrease of [Ca (2+)]i in A7r5 cells by isosteviol is mainly mediated by the selective opening of K (ATP) channel or/and SK (Ca) channel. Alteration in the Kv channel also plays a critical role in the inhibitory action of isosteviol.

[Effect of dehydroepiandrosterone on Ca(2+)-activated K+ channel of pulmonary arterial smooth muscle cells in pulmonary hypertensive rats].

OBJECTIVE: To investigate the effects of dehydroepiandrosterone (DHEA) on Ca(2+)-activated K(+) (K(Ca)) channel and mean pulmonary arterial pressure (mPAP) in rats with chronic pulmonary hypertension. METHODS: Fifty Wistar rats were divided randomly into a normal group (group A, n = 10) and a chronic hypoxia group (group B, n = 40). The rats in group B were subdivided into group B(1), B(2), B(3), and B(4) (each n = 10) at random. The rats in group B were exposed to hypoxia (FiO(2) = 0.10 +/- 0.05) for 3 weeks, whereas the rats in group A maintained in air. Under normoxic conditions, the smooth muscle cells (SMCs) were isolated from the pulmonary artery by the acute enzymatic dissection methods. In the symmetrical high K(+) solution, the K(Ca) currents were separated with inside-out configuration using the patch clamp technique. The activity of K(Ca) currents in SMCs between group B(1) and group A was compared under normoxic conditions, and the effect of DHEA on K(Ca) channel from group B(1) was observed. The mPAP and mean systemic arterial pressure (mSAP) were determined by right cardiac catheterization in rats from group B(2), B(3), B(4) before and after DHEA was administrated to rats by intravenous injection. RESULTS: The activity of K(Ca) channel in group B rats was much lower than that in group A (P < 0.01). DHEA could reverse the reduced K(Ca) channel in group B(1) rats. The mPAP were decreased significantly (P < 0.01) after DHEA was administrated to the rats in group B(2), B(3), B(4) with little change on mSAP (P < 0.05). CONCLUSIONS: Persistent decrease of K(Ca) channel activity may take part in the development of chronic hypoxic pulmonary hypertension in rats. DHEA can decrease the increased mPAP induced by chronic hypoxia via activating K(Ca) channel of SMCs from pulmonary arteries.

Natural modulators of large-conductance calcium-activated potassium channels.

Large-conductance calcium-activated potassium channels, also known as BK or Maxi-K channels, occur in many types of cell, including neurons and myocytes, where they play an essential role in the regulation of cell excitability and function. These properties open a possible role for BK-activators (also called BK-openers) and/or BK-blockers as effective therapeutic agents for different neurological, urological, respiratory and cardiovascular diseases. The synthetic benzimidazolone derivatives NS004 and NS1619 are the pioneer BK-activators and have represented the reference models which led to the design of several novel and heterogeneous synthetic BK-openers, while very few synthetic BK-blockers have been reported. Even today, the research towards identifying new BK-modulating agents is proceeding with great impetus and is giving an ever-increasing number of new molecules. Among these, also a handsome number of natural BK-modulator compounds, belonging to different structural classes, has appeared in the literature. The goal of this paper is to provide a possible simple classification of the broad structural heterogeneity of the natural BK-activating agents (terpenes, phenols, flavonoids) and blockers (alkaloids and peptides), and a concise overview of their chemical and pharmacological properties as well as potential therapeutic applications.

Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli, and effects of paxilline intermediates on mammalian maxi-K ion channels.

The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (Ki=730 nM) compared to paxilline (Ki=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 microM.

Martentoxin, a novel K+-channel-blocking peptide: purification, cDNA and genomic cloning, and electrophysiological and pharmacological characterization.

Martentoxin, a novel K+-channel-specific peptide has been purified and characterized from the venom of the East-Asian scorpion (Buthus martensi Karsch). The whole cDNA precursor sequence suggested that martentoxin was composed of 37 residues with a unique sequence compared with other scorpion neurotoxins. The genomic DNA of martentoxin showed an additional intron situated unexpectedly in the 5' UTR region, besides one located close to the C-terminal of the signal peptide. The patch-clamp recording found that martentoxin at the applied dose of 100 nm could strongly block large-conductance Ca2+-activated K+ (BKCa) currents in adrenal medulla chromaffin cells, and BKCa currents blocked by martentoxin could be fully recovered within 30 seconds after washing, which is at least 10 times faster than recovery after charybdotoxin. Meanwhile, a biosensor binding assay showed a fast association rate and a slow dissociation rate of martentoxin binding on rat brain synaptosomes. The binding of martentoxin on rat brain synaptosomes could be inhibited regularly by charybdotoxin, and gradually by toosendanin in a concentration-dependent manner, but not by either apamin or P03 from Buthus martensi. The results thus indicate that martentoxin is a new member in the family of K+-channel-blocking ligands.

Prevention of red cell dehydration: a possible new treatment for sickle cell disease.

One of the pathogenetic mechanisms responsible for sickling of erythrocytes in patients with sickle cell disease is the decreased hydration status of the cells. In this brief review, we discuss the pathophysiologic background and explore some new treatment options to prevent vaso-occlusive crises or other problems in this patient population.

[The cellular mechanism of angiotensin II inhibition on BKCa of ECV304 cell and the protective effect of gingko leaf extract].

OBJECTIVE: To observe the cellular mechanism of A II inhibition on Maxi-conductance calcium activated potassium channel (BKCa) in ECV304 cell membrane. METHOD: Using the cell-attached configuration of patch clamp technique. RESULTS: A II receptor antagonist saralasin (10(-7) mol/L) may block the inhibitory effect of A II (10(-7) mol/L). Phorbol ester (5 x 10(-8) mol/L) potentiated the effect of A II, while NO (10(-10) mol/L SNP) decreased the effect of A II and gingko leaf extract (800 micrograms/ml) activated BKCa and opposed the effects of A II. CONCLUSION: A II receptor mediates the inhibitory effect of A II on BKCa in ECV304, and PKC is involved in this inhibition. NO and ginkgo leaf extract protect BKCa from the inhibition of A II.

[Interfering effects of radix Salviae miltiorrhizae and lingustrazine on mm-LDL activating BKCa in ECV304 cell].

OBJECTIVE: To observe the action of minimally modified low density lipoprotein (mm-LDL) on BKCa in ECV304 cell and the interfering effects of radix salviae miltiorrhizae extract powder 764-3 (30 micrograms/ml) and lingustrazine (200 micrograms/ml) on this action. METHOD: The cell-attached configuration of patch clamp technique was applied. RESULTS: mm-LDL (100 micrograms/ml) potentiated the activity of BKCa in ECV304. While 764-3 and lingustrazine abolished it. CONCLUSIONS: mm-LDL acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKCa and might result in the increase of electro-chemical gradient for the resting Ca2+ influx, thus resting cytoplasmic concentration of calcium could be elevated and endothelial dysfunction would be induced. 764-3 and lingustrazine might have the protective action through decreasing the activity of BKCa.