The electrophysiological effects of cannabidiol on action potentials and transmembrane potassium currents in rabbit and dog cardiac ventricular preparations.

Cannabis use is associated with known cardiovascular side effects such as cardiac arrhythmias or even sudden cardiac death. The mechanisms behind these adverse effects are unknown. The aim of the present work was to study the cellular cardiac electrophysiological effects of cannabidiol (CBD) on action potentials and several transmembrane potassium currents, such as the rapid (IKr) and slow (IKs) delayed rectifier, the transient outward (Ito) and inward rectifier (IK1) potassium currents in rabbit and dog cardiac preparations. CBD increased action potential duration (APD) significantly in both rabbit (from 211.7 ± 11.2. to 224.6 ± 11.4 ms, n = 8) and dog (from 215.2 ± 9.0 to 231.7 ± 4.7 ms, n = 6) ventricular papillary muscle at 5 µM concentration. CBD decreased IKr, IKs and Ito (only in dog) significantly with corresponding estimated EC50 values of 4.9, 3.1 and 5 µM, respectively, without changing IK1. Although the EC50 value of CBD was found to be higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that potassium channel inhibition by lengthening cardiac repolarization might have a role in the possible proarrhythmic side effects of cannabinoids in situations where CBD metabolism and/or the repolarization reserve is impaired.

The electrophysiological effect of cannabidiol on hERG current and in guinea-pig and rabbit cardiac preparations.

Cannabis use is associated with cardiovascular adverse effects ranging from arrhythmias to sudden cardiac death. The exact mechanism of action behind these activities is unknown. The aim of our work was to study the effect of cannabidiol (CBD), tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol on cellular cardiac electrophysiological properties including ECG parameters, action potentials, hERG and IKr ion channels in HEK cell line and in rabbit and guinea pig cardiac preparations. CBD increased action potential duration in rabbit and guinea pig right ventricular papillary muscle at lower concentrations (1 µM, 2.5 µM and 5 µM) but did not significantly change it at 10 µM. CBD at high concentration (10 µM) decreased inward late sodium and L-type calcium currents as well. CBD inhibited hERG potassium channels with an IC50 value of 2.07 µM at room temperature and delayed rectifier potassium current with 6.5 µM at 37 °C, respectively. The frequency corrected QT interval (QTc) was significantly lengthened in anaesthetized guinea pig without significantly changing other ECG parameters. Although the IC50 value of CBD was higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that hERG and potassium channel inhibition might have a role in the possible proarrhythmic adverse effects of cannabinoids in situations where metabolism of CBD impaired and/or the repolarization reserve is weakened.

GIRK channel activity of Hungarian mushrooms: From screening to biologically active metabolites.

In the current study effects of fungal extracts on the G-protein-activated inwardly rectifying potassium channel (GIRK1/4) were screened using the automated patch-clamp method. 40 organic (n-hexane, chloroform, and 50% methanol) and aqueous extracts were prepared from 10 mushroom species native to Hungary. Among the examined fungal fractions of different polarities some n-hexane and chloroform extracts exerted considerable ion channel activity. One of the most active fungal species, Hypholoma lateritium was selected for further detailed examination to determine the compounds responsible for the observed pharmacological property. Evaluation of the ion channel activity of mushroom metabolites 1-10 revealed that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) demonstrates remarkable blocking activity on GIRK current (IC50 395.1 ±â€¯31.8 nM). Investigation of the selectivity of the GIRK inhibitory effect proved that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) has only weak inhibitory activity on hERG channel (7.9 ±â€¯2.8% at 100 µM), exerting more than three orders of magnitude lower blocking activity on hERG channel than on GIRK channel.

Evaluation of Possible Proarrhythmic Potency: Comparison of the Effect of Dofetilide, Cisapride, Sotalol, Terfenadine, and Verapamil on hERG and Native IKr Currents and on Cardiac Action Potential.

The proarrhythmic potency of drugs is usually attributed to the IKr current block. During safety pharmacology testing analysis of IKr in cardiomyocytes was replaced by human ether-a-go-go-related gene (hERG) test using automated patch-clamp systems in stable transfected cell lines. Aim of this study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch-clamp methods on HEK293 cells. The native ion currents (IKr, INaL, ICaL) were recorded from rabbit ventricular myocytes by manual patch-clamp technique. Action potentials in rabbit ventricular muscle and undiseased human donor hearts were studied by conventional microelectrode technique. Dofetilide, cisapride, sotalol, terfenadine, and verapamil blocked hERG channels at 37°C with an IC50 of 7 nM, 18 nM, 343 µM, 165 nM, and 214 nM, respectively. Using manual patch-clamp, the IC50 values of sotalol and terfenadine were 78 µM and 31 nM, respectively. The IC50 values calculated from IKr measurements at 37°C were 13 nM, 26 nM, 52 µM, 54 nM, and 268 nM, respectively. Cisapride, dofetilide, and sotalol excessively lengthened, terfenadine, and verapamil did not influence the action potential duration. Terfenadine significantly inhibited INaL and moderately ICaL, verapamil blocked only ICaL. Automated hERG assays may over/underestimate proarrhythmic risk. Manual patch-clamp has substantially higher sensitivity to certain drugs. Action potential studies are also required to analyze complex multichannel effects. Therefore, manual patch-clamp and action potential experiments should be a part of preclinical safety tests.

Diterpenoids from Euphorbia dulcis with Potassium Ion Channel Inhibitory Activity with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effect.

Nine new (1-9) and two known (10, 11) jatrophane diterpenoids were isolated from the methanol extract of Euphorbia dulcis. The structure elucidation of the compounds was performed by means of extensive spectroscopic analysis, including HRESIMS, 1D (1H, JMOD), and 2D (HSQC, HMBC, 1H-1H-COSY, NOESY) NMR experiments. The absolute configuration of compound 1 was determined by single-crystal X-ray diffraction. The electrophysiological effects of compounds 1-11 and the five diterpenoids (12-16) previously isolated from Euphorbia taurinensis were investigated on stable transfected HEK-GIRK1/4 (Kir3.1/3.4) and HEK-hERG (Kv11.1) cell lines using automated patch-clamp equipment. The majority of the diterpenoids showed significant blocking activity on GIRK channels (60.8-88.7% at 10 µM), while compounds 1, 2, 9-11, 13, and 14 exerted notable inhibitory effects even at 1 µM concentration. None of the jatrophane diterpenoids interfered with the function of hERG proteins; however, compound 14 remarkably hampered K+ flow through hERG channels. These selective activities suggest that jatrophane diterpenoids may represent a group of potential lead compounds for the development of novel therapeutic agents against atrial fibrillation.

Diterpene Lipo-Alkaloids with Selective Activities on Cardiac K+ Channels.

Aconitum diterpene alkaloids are known for their remarkable toxicity, which is due to their effect on ion channels. Activation of voltage-gated Na+ channels is the major cause of their cardiotoxicity, however, influence on K+ channels may also play a role in the overall effect.Here we report the synthesis of a series of lipo-alkaloids, including four new compounds, based on the 14-benzoylaconine structure, which is the core of a vast number of diterpene alkaloids naturally occurring in Aconitum species. The activities of these compounds were measured in vitro on K+ ion channels using the whole-cell patch-clamp technique. Structure-activity analysis was carried out based on the data of 51 compounds (32 genuine diterpene alkaloids, 5 fatty acids, and 14 lipo-alkaloids). Depending on their substitution, these compounds exert different activities on GIRK (G protein-coupled inwardly-rectifying potassium channel) and hERG (human ether-à-go-go-related gene) channels. Fatty acids and diterpene alkaloids show lower activity on the GIRK channel than lipo-alkaloids. Lipo-alkaloids also have less pronounced hERG inhibitory activity compared to the cardiotoxic aconitine. Considering the GIRK/hERG selectivity as an indicator of perspective therapeutic applicability, lipo-alkaloids are significantly more selective than the genuine diterpene alkaloids. 14-Benzoyl-8-O-eicosa-8Z,11Z,14Z-trienoate and 14-benzoyl-8-O-eicosa-11Z,14Z,17Z-trienoate are strong and selective inhibitors of GIRK channels, thus, they are promising subjects for further studies to develop diterpene alkaloid-based antiarrhythmic pharmacons.

Analgesic effect of dimethyl trisulfide in mice is mediated by TRPA1 and sst4 receptors.

TRPA1 receptors are calcium-permeable ligand-gated channels expressed in primary sensory neurons and involved in inflammation and pain. Activation of these neurons might have analgesic effect. Suggested mechanism of analgesic effect mediated by TRPA1 activation is the release of somatostatin (SOM) and its action on sst4 receptors. In the present study analgesic effect of TRPA1 activation on primary sensory neurons by organic trisulfide compound dimethyl trisulfide (DMTS) presumably leading to SOM release was investigated. Opening of TRPA1 by DMTS in CHO cells was examined by patch-clamp and fluorescent Ca2+ detection. Ca2+ influx upon DMTS administration in trigeminal ganglion (TRG) neurons of TRPA1 receptor wild-type (WT) and knockout (KO) mice was detected by ratiometric Ca2+ imaging. SOM release from sensory nerves of murine skin was assessed by radioimmunoassay. Analgesic effect of DMTS in mild heat injury-induced mechanical hyperalgesia was examined by dynamic plantar aesthesiometry. Regulatory role of DMTS on deep body temperature (Tb) was measured by thermocouple thermometry with respirometry and by telemetric thermometry. DMTS produced TRPA1-mediated currents and elevated [Ca2+]i in CHO cells. Similar data were obtained in TRG neurons. DMTS released SOM from murine sensory neurons TRPA1-dependently. DMTS exerted analgesic effect mediated by TRPA1 and sst4 receptors. DMTS-evoked hypothermia and hypokinesis were attenuated in freely-moving TRPA1 KO animals. Our study has presented original evidence regarding analgesic action of DMTS which might be due to TRPA1-mediated SOM release from sensory neurons and activation of sst4 receptors. DMTS could be a novel analgesic drug candidate.

Characterization of Na+-K+-2Cl- Cotransporter Activity in Rabbit Lacrimal Gland Duct Cells.

PURPOSE: We recently reported that isolated duct segments from rabbit lacrimal gland (LG) were able to secrete fluid in response to secretagogues, which were blocked completely by bumetanide. This suggests the functional involvement of Na+-K+-2Cl- cotransporter (NKCC1) in ductal fluid secretion. Therefore, the aim of this study was to investigate the activity profile of NKCC1 in isolated rabbit LG duct segments. METHODS: Interlobular ducts were isolated from fresh rabbit LG tissue. Microfluorometry with the ammonium (NH4+)-pulse technique was used to elicit pH changes in duct cells, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1. RESULTS: While basal activity of NKCC1 was undetectable, low cytosolic chloride (Cl-) level and hyperosmotic challenge (390 mOsm) were able to increase the activity of NKCC1. Carbachol (100 µM) had no significant effect on NKCC1 activity. Elevation of cytosolic calcium (Ca2+) level with Ca2+-ionophore (A 23187, 1 µM) did not cause any alteration in the activity of the cotransporter while direct activation of protein kinase C (phorbol myristate acetate, 100 nM) increased its activity slightly but in a significant manner. Addition of either forskolin (10 µM), cell-permeable cAMP analogue (8-bromo cAMP, 100 µM) or vasoactive intestinal peptide (200 nM) resulted in a significant increase in the activity of NKCC1. CONCLUSIONS: These results highlight the functional involvement of NKCC1 in LG duct secretion. These findings may facilitate our understanding of LG function and may contribute to the development of targeted pharmacologic interventions in case of dry eye disease.

Myrsinane, Premyrsinane, and Cyclomyrsinane Diterpenes from Euphorbia falcata as Potassium Ion Channel Inhibitors with Selective G Protein-Activated Inwardly Rectifying Ion Channel (GIRK) Blocking Effects.

GIRK channels are activated by a large number of G protein-coupled receptors and regulate the electrical activity of neurons, cardiac atrial myocytes, and ß-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the pathophysiology of neuropathic pain, drug addiction, and cardiac arrhythmias. In the heart, GIRK channels are selectively expressed in the atrium, and their activation inhibits pacemaker activity, thereby slowing the heart rate. In the present study, 19 new diterpenes, falcatins A-S (1-19), and the known euphorprolitherin D (20) were isolated from Euphorbia falcata. The compounds were assayed on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cells. Blocking activity on GIRK channels was exerted by 13 compounds (61-83% at 10 µM), and, among them, five possessed low potency on the hERG channel (4-20% at 10 µM). These selective activities suggest that myrsinane-related diterpenes are potential lead compounds for the treatment of atrial fibrillation.

Effects of Chelidonium majus extracts and major alkaloids on hERG potassium channels and on dog cardiac action potential - a safety approach.

Chelidonium majus or greater celandine is spread throughout the world, and it is a very common and frequent component of modern phytotherapy. Although C. majus contains alkaloids with remarkable physiological effect, moreover, safety pharmacology properties of this plant are not widely clarified, medications prepared from this plant are often used internally. In our study the inhibitory effects of C. majus herb extracts and alkaloids on hERG potassium current as well as on cardiac action potential were investigated. Our data show that hydroalcoholic extracts of greater celandine and its alkaloids, especially berberine, chelidonine and sanguinarine have a significant hERG potassium channel blocking effect. These extracts and alkaloids also prolong the cardiac action potential in dog ventricular muscle. Therefore these compounds may consequently delay cardiac repolarization, which may result in the prolongation of the QT interval and increase the risk of potentially fatal ventricular arrhythmias.

Inhibition of G protein-activated inwardly rectifying K+ channels by extracts of Polygonum persicaria and isolation of new flavonoids from the chloroform extract of the herb.

The G protein-activated inwardly rectifying K+ channel-modulatory activities of Polygonum persicaria extracts were investigated by using an automated patch-clamp method, with the aim of identifying natural sources of promising ion channel-blocking compounds. The chloroform extract of the whole plant at 0.1 mg/mL exhibited high G protein-activated inwardly rectifying K+ channel-inhibitory activity. Fractionation of this extract by vacuum liquid chromatography on RP-silica gel resulted in 6 fractions, which were evaluated for G protein-activated inwardly rectifying K+ channel-modulatory activity. RP-HPLC of the most active fractions afforded the main compounds 1-4 in pure form and a mixture containing the minor constituents. The structures were identified by means of UV, HRMS, and advanced NMR methods as 3-O-senecioyl-isorhamnetin (1), 3-O-angeloyl-isorhamnetin (2), 5,3',4',5'-tetramethoxy-6,7-methylenedioxyflavone (3), and 3,5,3',4',5'-pentamethoxy-6,7-methylenedioxyflavone (4). Compounds 1-4 are new natural products, though 4 was reported earlier as a synthetic compound. Neither the individual, nor the combined application of compounds 1-4 modified the G protein-activated inwardly rectifying K+ channel activity. However, a marked G protein-activated inwardly rectifying K+ current-inhibitory effect was detected on use of the HPLC eluates containing the minor compounds. These results indicate the presence of electrophysiologically active agents among the minor compounds.

Identification of diterpene alkaloids from Aconitum napellus subsp. firmum and GIRK channel activities of some Aconitum alkaloids.

Diterpene alkaloids neoline (1), napelline (2), isotalatizidine (3), karakoline (4), senbusine A (5), senbusine C (6), aconitine (7) and taurenine (8) were identified from Aconitum napellus L. subsp. firmum, four (2-4, 6) of which are reported for the first time from this plant. The structures were determined by means of LC-MS, 1D and 2D NMR spectroscopy, including (1)H-(1)H COSY, NOESY, HSQC and HMBC experiments. Electrophysiological effects of the isolated compounds, together with nine diterpene alkaloids previously obtained from Aconitum toxicum and Consolida orientalis were investigated on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cell lines using automated patch clamp equipment. Significant blocking activity on GIRK channel was exerted by aconitine (7) (45% at 10 µM), but no blocking activities of the other investigated compounds were detected. The tested compounds were inactive on hERG channel in the tested concentration. The comparison of the previously reported metabolites of A. napellus subsp. firmum and compounds identified in our experiment reveals substantial variability of the alkaloid profile of this taxon.

Electrophysiological effects of ivabradine in dog and human cardiac preparations: potential antiarrhythmic actions.

Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (V(max)), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1-10 µM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent V(max) block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400 ms cycle length, V(max) block developed with an onset kinetic rate constant of 13.9 ± 3.2 beat(-1) in dog ventricular muscle. In addition to a fast recovery of V(max) from inactivation (τ=41-46 ms) observed in control, a second slow component for recovery of V(max) was expressed (offset kinetics of V(max) block) having a time constant of 8.76 ± 1.34 s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10 µM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm.