Antagonistic actions of S(−)-Bay K 8644 on cyclic nucleotide-induced inhibition of voltage-dependent Ba2+ currents in guinea pig gastric antrum

(±)-Bay K 8644, a conventional racemic mixture of Bay K 8644, is widely used as an L-type Ca²⁺ channel agonist. Although interactions between Bay K 8644 and cyclic nucleotide have been described, they have not been properly characterized. We have investigated whether two optical isomers of Bay K 8644 (i.e., R(+)- and S(−)-Bay K 8644) modify cyclic nucleotide (cAMP and cGMP)-induced inhibitory effects on nifedipine-sensitive voltage-dependent Ba²⁺ currents (I _Ba) recorded from guinea pig gastric myocytes. Conventional whole-cell recordings were used to compare the effects of R(+)-Bay K 8644 and S(−)-Bay K 8644 on I _Ba. S(−)-Bay K 8644 enhanced the peak amplitude of I _Ba evoked by depolarizing pulses to +10 mV from a holding potential of −70 mV in a concentration-dependent manner (EC₅₀ = 32 nM), while R(+)-Bay K 8644 inhibited I _Ba (IC₅₀ = 975 nM). When R(+)-Bay K 8644 (0.5 μM) was applied, I _Ba was suppressed to 71 ± 10% of control. In the presence of R(+)-Bay K 8644 (0.5 μM), additional application of forskolin and sodium nitroprusside (SNP) further inhibited I _Ba. Conversely, in the presence of S(−)-Bay K 8644 (0.5 μM), subsequent application of forskolin and SNP did not affect I _Ba. Similarly, in the presence of 0.5 μM S(−)-Bay K 8644, db-cAMP and 8-Br-cGMP had no effect on I _Ba. These results indicate that S(−)-Bay K 8644, but not R(+)-Bay K 8644, can prevent the inhibitory actions of two distinct cyclic nucleotide pathways on I _Ba in gastric myocytes of the guinea pig antrum.     

Effects of trimebutine maleate on colonic motility through Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels and L-type Ca<Superscript>2+</Superscript> channels

The effects of trimebutine maleate (TM) on spontaneous contractions of colonic longitudinal muscle were investigated in guinea pigs. The contractile responses of smooth muscle strips were recorded by an isometric force transducer. Membrane and action potentials were detected by an intracellular microelectrode technique. The whole-cell patch clamp recording technique was used to record the changes in large conductance Ca²⁺-activated K⁺ (BK_ca) and L-type Ca²⁺ currents in colonic smooth muscle cells. At high concentrations (30, 100, and 300 μM), TM inhibited the amplitude of spontaneous contractions. At low concentrations (1 and 10 μM), TM attenuated the frequency and tone of smooth muscle strips, whereas TM had no influence on the amplitude of spontaneous contractions. TM depolarized the membrane potentials, but decreased the amplitude and frequency of action potentials at high concentrations. TM inhibited BK_ca and L-type Ca²⁺ currents in a dose-dependent manner. In the presence of the BK_ca channel opener, NS1619, TM also inhibited BK_ca currents. Bayk8644, a L-type Ca²⁺ channel opener, increased L-type Ca²⁺ currents. This augmentation was also attenuated by TM. These results suggest that TM attenuates intestinal motility through inhibition of L-type Ca²⁺ currents, and depolarizes membrane potentials by reducing BK_ca currents. Thus, TM may be a multiple-ion channel regulator in the gastrointestinal tract.     

Magnesium isoglycyrrhizinate inhibits L-type Ca<Superscript>2+</Superscript> channels,Ca<Superscript>2+</Superscript> transients,and contractility but not hERG K<Superscript>+</Superscript> channels

To explore the cardiovascular protective effects of Magnesium isoglycyrrhizinate (MI), especially the underlying cellular mechanisms related to L-type calcium channels and myocardial contractility, and to examine the effects of MI on hERG K⁺ current expressed in HEK293 cells. We used the whole-cell patch clamp technique, video-based edge detection and dual excitation fluorescence photomultiplier systems to explore the effect of MI on L-type Ca²⁺ currents (I_Ca-L) and cell contraction in rat cardiomyocytes. We also examined the rapidly activating delayed rectifier potassium current (I_Kr) expressed in HEK293 cells using a perforated patch clamp. MI inhibited I_Ca-L in a dose-dependent manner, with a half-maximal inhibitory concentration (IC₅₀) of 0.22 mg/ml, and the maximal inhibitory effect was 61.10 ± 0.59%. MI at a concentration of 0.3 mg/ml reduced cell shortening by 24.12 ± 3.97% and the peak value of the Ca²⁺ transient by 36.54 ± 4.96%. MI had no significant influence on hERG K⁺ channels expressed in HEK293 cells at all test potentials. MI exerts protective effects on the heart via the inhibition of I_Ca-L and cell shortening in rat cardiomyocytes. However, MI had no significant influence on I_Kr; thus, MI may exert cardioprotective effects without causing drug-induced long QT syndrome.     

Effect of Ca2+ Agonist Bay K 8644 in human placental arteries

• 1.1. Bay K 8644 (0.1 μM) induced weak contractions in human placental artery segments that were increased in the presence of 7.5 mM K⁺. K⁺ and serotonin (5-HT) induced contractions that were enhanced by preincubation of segments with Bay K 8644. These enhancements were reduced by nifedipine (0.1 μM) and diltiazem (1 μM).
• 2.2. Bay K 8644 induced a ⁴⁵Ca²⁺ uptake increase which was potentiated by depolarization with K⁺ (less than 30 mM) and antagonized by nifedipine. K⁺ (15 and 30 mM) and 5-HT (1 μM) induced ⁴⁵Ca²⁺ uptake that was enhanced by Bay K 8644.
• 3.3. These results suggest that Bay K 8644: (1) is unable to activate the quiescent potential-operated Ca²⁺ channels (POCs) of these arteries, and (2) activates receptor (5-HT)-operated Ca²⁺ channels or facilitates Ca²⁺ influx through POCs activated by 5-HT.

     

Multiple voltage-sensitive calcium channels are probably involved in endogenous GABA release from striatal neurones differentiated in primary culture

Summary Calcium-dependent release of neurotransmitters is thought to be due to Ca²⁺ entry into nerve terminals, but the identities of the various voltage-sensitive Ca²⁺ channels (VSCC) involved in this process remain obscure. To elucidate the types of VSCCs involved in the release process, we studied the effects of various organic Ca²⁺ channel antagonists and agonists on the release of endogenous -aminobutyric acid (GABA) from mouse striatal neurones differentiated in primary culture. Diltiazem, verapamil and methoxyverapamil (D 600) inhibited K⁺-evoked (30 mM) GABA release at very high concentrations (> 1 M). The dihydropyridine (DHP) nifedipine, at low concentrations (0.01–1.00 M), was able to inhibit part of the K⁺-evoked GABA release (25.6±7.3% inhibition at 1 M). This is in agreement with the high affinity of nifedipine for DHP binding sites. The DHPs, BAY K 8644 (EC₅₀ = 41±15 nM) and CGP 28.392, which possess agonist properties at VSCCs, increased the 15 mM K⁺-evoked GABA release. The release evoked by the combination of K⁺ (15 M) and BAY K 8644 (up to 10 M) remained smaller than the release elicited by 30 mM K⁺. The effect of BAY K 8644 (1 M) was inhibited by nifedipine (IC₅₀ 0.55±0.05 M). When Na⁺ ions were replaced by choline, basal and K⁺-evoked GABA release was significantly increased. Even in the absence of external Na+, nifedipine (1 M) was not able to totally block the K⁺ effect. Moreover amiloride, a drug known to inhibit Na⁺/Ca⁺ exchange, and tetrodotoxin (TTX), did not modify the 30 mM K⁺ response. Therefore, nifedipine-insensitive K⁺-evoked GABA release is not due to Na⁺-dependent Ca²⁺ entry. These results can be explained by the presence of DHP-sensitive and insensitive Ca²⁺ channels on nerve terminals, each involved in the release process.Abbreviations CGP 28.392 4-[2-(difluoromethoxy)phenyl]-1,4,5,7-tetrahydro-2-methyl-5-oxo-furo[3,4-b]pyridine-3-carboxylic acid ethylester - BAY K 8644 (±)-methyl 1,4-dihydro-2,6-dimethyl-3nitro-4-(2 trifluoromethyl) pyridine-5-carboxylate - DHP 1,4-dihydropyridine - DIV days in vitro - D 600 methoxyverapamil - GABA -aminobutyric acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - HPLC high performance liquid chromatography - TTX tetrodotoxin - VSCC voltage-sensitive calcium channel Send offprint requests to J.-P. Pin at the above address     

The response of single cardiac sodium channels in neonatal rats to the dihydropyridines CGP 28392 and (−)-Bay K 8644

Summary Cell-attached patch clamp recording of elementary Na⁺ currents were performed at 19°C in neonatal cultured rat heart cells to study Na⁺ channel properties in the presence of dihydropyridines.Bath application of racemic CGP 28392, at 5 gmol/l, activated Na⁺ channels. By increasing the open probability, P _O, and/or the number of functioning Na⁺ channels, peak I _Na in reconstructed macroscopic Na+ currents rose without changes in the decay kinetics. This was accompanied by a prolongation of open time. (–)-Bay K 8644 (1–10 mol/l) had the same effect. In the presence of either agonist, Na⁺ channels retained an uniform open state and, as estimated from the mean number of openings per sequence, their initial tendency to reopen. Rarely appearing ultralong opening sequences are unlikely to be drug-induced as Na⁺ channels can likewise switch into this particular activity mode under drug-free conditions. Racemic CGP 28392, at 50 mol/l, blocked Na⁺ channels in an all-or-none fashion suggesting that one enantiomer acts as agonist and the other enantiomer as blocker. A quite different response consisting of the occurrence of a second open state with a several-fold increased life time and a significantly increased reopening was observed with (–)-Bay K 8644 in damaged cardiocytes with hyperpermeable membranes and after patch excision into drug-containing solution. Evidence was obtained from control inside-out patches that this increased reopening is most probably caused by the solvent, ethanol.It is concluded that cardiac Na⁺- channels affected by agonistic dihydropyridines not only respond with an increased likelihood to open during membrane depolarization but can also preserve their open state. Compared with the response of l-type Ca²⁺ channels (Hess et al. 1984), Na⁺ channel life time is only slightly prolonged by agonistic dihydropyridines. Send offprint requests to M. Kohlhardt at the above address     

Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells

Background and purpose:

Reduction of intracellular calcium ([Ca²⁺]_i) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca²⁺ mobilization during SMC contraction influenced NO efficacy.

Experimental approach:

Isometric contractions by depolarization (high potassium, K⁺) or α-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca²⁺]_i (Fura-2) were measured in aortic segments from C57Bl6 mice.

Key results:

Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K⁺ solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca²⁺]_i, which was suppressed by sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K⁺-stimulated segments did not parallel a decline of [Ca²⁺]_i. However, stimulation (BAY K8644) of L-type Ca²⁺ influx diminished, while inhibition (nifedipine, 1–100 nM) augmented the relaxing capacity of NO.

Conclusions and implications:

In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca²⁺ re-uptake into the SR and was prominent when intracellular Ca²⁺ was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca²⁺]_i, presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca²⁺ influx, and L-type Ca²⁺ channel blockers increase the vasodilator efficacy of NO.     

Effects of cibenzoline,a new class Ia antiarrhythmic drug,on various membrane ionic currents and action potentials of guinea-pig ventricular cells

We examined the effects of cibenzoline, a new class Ia antiarrhythmic drug, on various membrane ionic currents and action potentials of guinea-pig single ventricular cells, using patch clamp techniques in whole-cell configuration. Action potentials and the membrane currents were evoked at a clamping rate of 0.2 Hz, and all experiments were performed at 32–33°C.

Inhibitory effect of YM-244769, a novel Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger inhibitor on Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange current in guinea pig cardiac ventricular myocytes

Recently, YM-244769 (N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]phenoxy} nicotinamide) has been reported as a new potent and selective Na⁺/Ca²⁺ exchange (NCX) inhibitor by using various cells transfected with NCX using the ⁴⁵Ca²⁺ fluorescent technique. However, the electrophysiological study of YM-244769 on NCX had not been performed in the mammalian heart. We examined the effects of YM-244769 on NCX current (I_NCX) in single cardiac ventricular myocytes of guinea pigs by using the whole-cell voltage clamp technique. YM-244769 suppressed the bidirectional I_NCX in a concentration-dependent manner. The IC₅₀ values of YM-244769 for the bidirectional outward and inward I_NCX were both about 0.1 μM. YM-244769 suppressed the unidirectional outward I_NCX (Ca²⁺ entry mode) with an IC₅₀ value of 0.05 μM. The effect on the unidirectional inward I_NCX (Ca²⁺ exit mode) was less potent, with 10 μM of YM-244769 resulting in the inhibition of only about 50 %. At 5 mM intracellular Na⁺ concentration, YM-244769 suppressed I_NCX more potently than it did at 0 mM [Na⁺]_i. Intracellular application of trypsin via the pipette solution did not change the blocking effect of YM-244769. In conclusion, YM-244769 inhibits the Ca²⁺ entry mode of NCX more potently than the Ca²⁺ exit mode, and inhibition by YM-244769 is [Na⁺]_i-dependent and trypsin-insensitive. These characteristics are similar to those of other benzyloxyphenyl derivative NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The potency of YM-244769 as an NCX1 inhibitor is higher than those of KB-R7943 and SN-6 and is similar to that of SEA0400.     

Cytotoxicity studies of 2‐hydroxymethyl‐1‐naphthol diacetate on K+ currents in neoplastic plasma cells

The present study investigated the effects of 2‐hydroxymethyl‐1‐naphthol diacetate (TAC) on cell proliferation and K⁺ currents in RPMI‐8226 human myeloma cells. In cells with intracellular Ca²⁺ concentration ([Ca²⁺]_i) = 10 nM, depolarizing square pulses from a holding potential of –80 mV elicited instantaneous outward current with slow inactivation, corresponding to voltage‐activated K⁺ current. TAC (1–100 μM) inhibited I_K(V) in a concentration‐dependent manner. A23187 (1 μM), a Ca²⁺ ionophore, can potentiate Ca²⁺‐activated K⁺ current (I_K(Ca)). Tetraethylammonium chloride (10 mM) caused a small decrease in the amplitude of I_K(Ca) elicited by A23187, whereas TAC (30 μM) and quinidine (10 μM) decreased I_K(Ca) more effectively. The present results show that TAC directly blocks voltage‐ and Ca²⁺‐activated K⁺ currents in human myeloma cells. TAC inhibited both cell proliferation and voltage‐activated K⁺ current with an effective dose inducing half‐maximum effects at 3.8 ± 0.8 μM and 10 ± 1.5 μM, respectively. The present study suggests that the cytotoxic effect of TAC in cancer cells may be partially explained by blockade of K⁺ channels. The delocalization energy of TAC and other analogs was employed to compare their ability to block the voltage‐activated K⁺ channel in myeloma cells. It was found that naphthol derivatives‐mediated blockade of voltage‐activated K⁺ channel might relate to the level of delocalization energy and molecular volume. Drug Dev. Res. 47:1–8, 1999. © 1999 Wiley‐Liss, Inc.     

BAY K 8644 stimulates glucose-dependent rise of cytoplasmic Ca2+ in hyperpolarized pancreatic β-cells

Summary The effect of BAY K 8644 on the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in pancreatic -cells hyperpolarized by the K⁺ channel-activating agent diazoxide. After 50–60 min preexposure to 0–20 mM glucose in the presence of 400 M diazoxide [Ca²⁺]_i was close to the level in unstimulated -cells. The addition of 5 M BAY K 8644 then triggereed a rise of [Ca²⁺]_i dependent on Ca²⁺ influx. The magnitude of the BAY K 8644 effect increased with the glucose concentration and was almost 10-fold higher in 20 mM than in the absence of the sugar. It is concluded that glucose can modulate Ca²⁺ entry through the voltage-dependent channels by a mechanism additional to depolarization. This action may help to explain why previous exposure to the sugar results in an augmented insulin response to a second challenge.     

Vascular actions of calcimimetics: role of Ca²(+) -sensing receptors versus Ca²(+) influx through L-type Ca²(+) channels

BACKGROUND AND PURPOSE

The calcimimetic, (R)-N-(3-(3-(trifluoromethyl)phenyl)propyl)-1-(1-napthyl)ethylamine hydrochloride (cinacalcet), which activates Ca²⁺-sensing receptors (CaR) in parathyroid glands, is used to treat hyperparathyroidism. Interestingly, CaR in perivascular nerves or endothelial cells is also thought to modulate vascular tone. This study aims to characterize the vascular actions of calcimimetics.

EXPERIMENTAL APPROACH

In rat isolated small mesenteric arteries, the relaxant responses to the calcimimetics, cinacalcet and (R)-2-[[[1-(1-naphthyl)ethyl]amino]methyl]-1H-indole hydrochloride (calindol) were characterized, with particular emphasis on the role of CaR, endothelium, perivascular nerves, K⁺ channels and Ca²⁺ channels. Effects of L-ornithine, which activates a Ca²⁺-sensitive receptor related to CaR (GPRC6A), were also tested.

KEY RESULTS

Cinacalcet induced endothelium-independent relaxation (pEC₅₀ 5.58 ± 0.07, E_max 97 ± 6%) that was insensitive to sensory nerve desensitization by capsaicin or blockade of large-conductance Ca²⁺-activated K⁺ channels by iberiotoxin. Calindol, another calcimimetic, caused more potent relaxation (pEC₅₀ 6.10 ± 0.10, E_max 101 ± 6%), which was attenuated by endothelial removal or capsaicin, but not iberiotoxin. The negative modulator of CaR, calhex 231 or changes in [Ca²⁺]_o had negligible effect on relaxation to both calcimimetics. The calcimimetics relaxed vessels precontracted with high [K⁺]_o and inhibited Ca²⁺ influx in endothelium-denuded vessels stimulated by methoxamine, but not ionomycin. They also inhibited contractions to the L-type Ca²⁺ channel activator, BayK8644. L-ornithine induced small relaxation alone and had no effect on the responses to calcimimetics.

CONCLUSION AND IMPLICATIONS

Cinacalcet and calindol are potent arterial relaxants. Under the experimental conditions used, they predominantly act by inhibiting Ca²⁺ influx through L-type Ca²⁺ channels into vascular smooth muscle, whereas Ca²⁺-sensitive receptors (CaR or GPRC6A) play a minor role.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

Electrophysiological effects of anandamide on rat myocardium

Background and purpose:

The endocannabinoid, anandamide, has anti-arrhythmic effects. The aim of the present study was to explore the electrophysiological effects of anandamide on rat myocardium.

Experimental approach:

Evoked action potentials (APs) were recorded using intracellular recording technique in rat cardiac papillary muscles. In addition, L-type Ca²⁺ current was measured and analysed using whole-cell patch-clamp recording technique in isolated rat cardiac ventricular myocytes.

Key results:

In cardiac papillary muscles, anandamide (1, 10, 100 nM) decreased AP duration in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased AP amplitude, overshoot and V_max in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB₁ receptors, but not AM630 (100 nM), a CB₂ receptor antagonist. Furthermore, an agonist of L-type Ca²⁺ channels, Bay K 8644 (0.5 µM), a K⁺ channel blocker tetraethylammonium chloride (20 mM) and the nitric oxide synthase inhibitor l-NAME (1 mM) had no effect on anandamide-induced decrease in AP duration. In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca²⁺ current concentration-dependently, and shifted the current–voltage relationship curve of the Ca²⁺ current. Anandamide (100 nM) shifted the steady-state inactivation curve to the left and the recovery curve to the right. Blockade of CB₁ receptors with AM251 (100 nM), but not CB₂ receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca²⁺ currents.

Conclusions and implications:

These data suggest that anandamide suppressed AP and L-type Ca²⁺ current in cardiac myocytes through CB₁ receptors.     

Influence of (−)-S-Bay K 8644, (±)-Bay W 5035 and (±)-Bay T 5006 on hemodynamics and FITC-Dextran 3 elution kinetics in isolated rat hearts

• 1.1. We investigated the effects of the new calcium-agonists (±)-Bay W 5035 and (±)-Bay T 5006 in comparison to (-)-S-Bay K 8644 on hemodynamics and epimyocardial perfusion in Langendorff rat hearts.
• 2.2. At equieffective inotropic concentration, vasoconstriction of coronary resistance vessels was significantly less after (±)-Bay W 5035 or (±)-Bay T 5006 than after (-)-S-Bay K 8644 application.
• 3.3. FITC-Dextran 3 elution kinetics indicated that the epimyocardial vascular volume was significantly reduced only by (-)-S-Bay K 8644.
• 4.4. Moreover, (-)-S-Bay K 8644 enhanced transcoronary exchange more markedly than (+-)-Bay W 5035 or (±)-Bay T 5006, reflecting the differences in coronary constrictor activity.
• 5.5. We conclude that in comparison to (-)-S-Bay K 8644 the relation between inotropy and vasoconstriction is more favorable for (±)-Bay W 5035 or (±)-Bay T 5006.

     

Pharmacological properties of Ca2+-activated K+ currents of ramified murine brain macrophages

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I_K,Ca) were investigated in ramified murine brain macrophages. In order to induce I_K,Ca the intracellular concentration of nominal free Ca²⁺ was adjusted to 1μM. The Ca²⁺-activated K⁺ current of brain macrophages did not show any voltage dependence at test potentials between –120 and +30mV. A tenfold change in extracellular K⁺ concentration shifted the reversal potential of I_K,Ca by 51mV. The bee venom toxin apamin applied at concentrations of up to 1μM did not affect I_K,Ca. Ca²⁺-activated K⁺ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I_K,Ca at concentrations between 1 and 50nM. Tetraethylammonium (TEA) blocked 8.0% of I_K,Ca at a concentration of 1mM, whereas 31.4% of current was blocked by 10mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2mM for their ability to block I_K,Ca. La³⁺ reduced I_K,Ca by 72.8%, whereas Cd²⁺ decreased I_K,Ca by 17.4%; in contrast, Ni²⁺ did not have any effect on I_K,Ca. Ba²⁺ applied at a concentration of 1mM reduced I_K,Ca voltage-dependently at hyperpolarizing potentials. Received: 17 January / Accepted: 5 May 1997     

Effect of capsaicin on membrane currents in cultured vascular smooth muscle cells of rat aorta

The application of capsaicin (1 μM) produced a minor relaxant effect in endothelium-denuded rat aortae. However, capsaicin caused a greater relaxation of blood vessels precontracted with high K⁺ or phenylephrine. The effects of capsaicin on the ionic currents were also examined in A7r5 vascular smooth muscle cells. The tight-seal whole-cell voltage clamp technique was used. Capsaicin inhibited the Ba²⁺ inward current (I_Ba) through the voltage-dependent L-type Ca²⁺ channel in a concentration-dependent fashion, whereas calcitonin gene-related peptide and phenylephrine produced a minor increase in I_Ba. Capsaicin did not alter the overall shape of current-voltage relationship of I_Ba. However, capsaicin (3 μM) shifted the quasi-steady-state inactivation curve of I_Ba to more negative membrane potential by about 5 mV. These effects of capsaicin on I_Ba were reversible. In addition, capsaicin had inhibitory effects on voltage dependent K⁺ currents. These results suggest that inhibition of the voltage-dependent L-type Ca²⁺ channel is involved in the capsaicin-induced relaxation of the vascular smooth muscle, whereas capsaicin-induced inhibition of voltage-dependent K⁺ channels might produced an increase in cell excitability.     

Opposite cardiac actions of the enantiomers of Bay K 8644 at different membrane potentials in guinea-pig papillary muscles

Summary The influence of membrane potential on the effects of the enantiomers and the racemate of Bay K 8644 [1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluor-methylphenyl)-pyridine-5-carboxylate] on force of contraction and on action potentials were studied in guinea-pig papillary muscles in order to detect possible changes in the direction of drug action or in potency. Membrane potential was varied by changing the potassium concentration ([K⁺]_o) in the bathing solution.At normal resting potential, (–)-Bay K 8644 enhanced force of contraction and prolonged the action potential duration measured at 50% of repolarization (APD) to the same extent as the racemate and with similar pD₂ values. After membrane depolarization by raising ([K⁺]_o) from 5.4 to 17.4 mmol/l, the (–)-enantiomer and the racemate prolonged the APD to a similar degree but enhanced force to a lesser extent. The maximum rate of depolarization of slow action potentials, V _max, was increased at the highest concentrations (10^–5 mol/l). The effects of (+)-Bay K 8644 were more complicated. At high concentrations (10^–5 mol/l) it decreased force of contraction and APD, the pD₂ values were one order of magnitude lower than for the (–)enantiomer and the racemate. A high concentration (+)-Bay K 8644 (10^–5 mol/l) virtually abolished contractile activity at all membrane potentials, the extent of shortening in APD increased with membrane depolarization in elevated ([K⁺]_o) V _max, of slow action potentials was decreased. In normal bathing solution and with low concentrations of (+)-Bay K 8644(< 10^–7 mol/l), the APD showed a small but significant prolongation which disappeared with membrane depolarization. No significant opposite effects in force of contraction were observed. There was no evidence for a potential-dependent shift in potency of drug action of either enantiomers or racemate.In papillary muscles potential-dependent opposite actions could be demonstrated for the effects of the inhibitory enantiomer on action potential duration. Corresponding changes in inotropic effects of DHPs are obscured by the intrinsic influence of depolarization on contractile activation. Send offprint requests to U. Ravens at the above address     

A study comparing the actions of gabapentin and pregabalin on the electrophysiological properties of cultured DRG neurones from neonatal rats

Background

Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca²⁺ channels. In this study we have used whole cell patch clamp recording and fura-2 Ca²⁺ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca²⁺ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents.

Results

Pregabalin (25 nM – 2.5 μM) inhibited 20–30% of the high voltage-activated Ca²⁺ current in cultured DRG neurones. The residual Ca²⁺ current recorded in the presence of pregabalin was sensitive to the L-type Ca²⁺ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca²⁺ channels but the majority of Ca²⁺ current was resistant to both drugs. The continual application of GABA, the GABA_B receptor antagonist CGP52432, or intracellular photorelease of GTP-γ-S had no effect on pregabalin-induced inhibition of Ca²⁺ currents. Although clear inhibition of Ca²⁺ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca²⁺ influx in response to pregabalin. The enhanced Ca²⁺ influx evoked by pregabalin was mimicked by partial block of K⁺ conductances with tetraethylammonium. Pregabalin produced biphasic effects on voltage-activated K⁺ currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K⁺ currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP.

Conclusions

Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca²⁺ and K⁺ channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A.