Characteristics of L-type calcium channel blockade by lacidipine in guinea-pig ventricular myocytes

1. The Ca²⁺-antagonistic properties of lacidipine were investigated in patch-clamp guinea-pig ventricular myocytes.
2. In basal conditions, 0.1 μM lacidipine reduced the action potential duration, associated with a decrease in the L-type calcium current (I_Ca,L) to 66±4% of the control value, without a change in the current-voltage relationship. Sodium current and background potassium currents were not affected. All the effects reached a steady state within 2 min.
3. The Ca²⁺-antagonistic effect of lacidipine was voltage-dependent: a marked negative shift (about 20 mV) of the steady-state inactivation curve was observed with long (10 s) conditioning prepulses, but not with short (350 ms) prepulses.
4. The onset of and recovery from the voltage-dependent effect caused by 0.1 μM lacidipine were significantly slower when compared to those of equiactive concentrations of nimodipine (0.5 μM) and nisoldipine (0.1 μM). I_Ca,L measured after prepulses at −40 mV lasting 500 ms or less was unchanged (95±5% of maximum current value) while it was reduced to 49±10% by nimodipine and 43±9% by nisoldipine (P<0.05 vs lacidipine for both).
5. Similarly, the recovery from block in the presence of lacidipine was slower than with nimodipine and nisoldipine. After a prepulse of 1 s at −80 mV, I_Ca,L recovered up to 54±2% of the maximum current value in the presence of lacidipine, and up to 91±3% and 93±5% in the presence of nimodipine and nisoldipine, respectively (P<0.05 vs lacidipine).
6. Blockade of I_Ca,L by lacidipine was use-dependent. After ten 200 ms long pulses (1 Hz) from −80 mV, I_Ca,L was reduced to 55±7% of the current measured at the first pulse. In the presence of nimodipine and nisoldipine, I_Ca,L elicited by the tenth pulse amounted to 93±3% and 80±6% of the first pulse value, respectively (P<0.05 vs lacidipine). Lacidipine did not cause use-dependent blockade of I_Ca,L in cells stimulated with 10 ms long pulses.
7. These results demonstrate that lacidipine selectively inhibits I_Ca,L in isolated cardiomyocytes and suggest that this effect occurs mainly through binding to the inactivated Ca²⁺ channels.

     

Effects of dopamine on L-type Ca2+ current in single atrial and ventricular myocytes of the rat

1. The effects of dopamine on the L-type Ca²⁺ current (I_Ca,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated.
2. Dopamine increased atrial I_Ca,L at concentrations higher than 1 μM, but had little or no effect on I_Ca,L at lower concentrations. The increase in I_Ca,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of I_Ca,L were not altered by dopamine.
3. In rat ventricular myocytes in which the D₄ receptor mRNA does not express, dopamine (20–100 μM) also increased the I_Ca,L amplitude and propranolol reversed this effect.
4. Clozapine, a potent D₄ receptor antagonist, blocked the augmenting effect of dopamine on I_Ca,L. However, this effect could be explained by β-antagonism, since clozapine also inhibited the isoprenaline effect.
5. In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 μM) was reversed by 1 μM propranolol, but not by 2 μM phentolamine. Low doses of dopamine (0.01 to 0.3 μM) did not affect the contraction in the controls or during a modest stimulation of the β-adrenoceptor with 0.01 μM isoprenaline.
6. These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the β-adrenoceptor in both atrial and ventricular myocytes. Involvement of D₄ receptor appears unlikely in the regulation of the atrial contraction.

     

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on γ-aminobutyric acid (GABA)-induced currents (I_GABA) was also examined.
2. DM inhibited 30 μM glycine-induced current (I_Gly), without affecting the current caused by 30 μM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 μM, and reversible. The half-maximum inhibitory concentration (IC₅₀) of DM was 3.3 μM, about 85 times higher than that of strychnine.
3. DM 3 μM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 μM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 μM shifted the curve for glycine in a nearly parallel fashion.
4. The effect of 10 μM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 μM, inhibited I_Gly equally at −50 mV and +50 mV. The effect of 3 μM DM on I_Gly showed no use-dependence. Blockade by strychnine 0.1 μM showed no voltage- or use-dependence.
5. The results indicate that DM inhibits I_Gly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl⁻ channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl⁻ channel of the complex.

     

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus

1. At a holding potential of −40 mV, carbachol (50 μM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (I_Cl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I_DOC). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I_oscil) superimposed on I_DOC.
2. Carbachol-induced I_oscil (amplitude 97±11 pA; frequency 0.26±0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution.
3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I_oscil could be restored by re-admission of calcium. The frequency, but not the amplitude, of I_oscil increased with increasing concentrations of extracellular calcium (0.5–10 mM).
4. Inclusion of the inositol triphosphate (IP₃) receptor antagonist heparin (5 mg ml⁻¹) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 μM), prevented the activation of I_oscil by carbachol. Caffeine (10 mM) activated both I_Cl(Ca) and I_DOC and prevented the induction of I_oscil by carbachol. Caffeine and CPA also abolished I_oscil in the presence of carbachol, as did both a low (3 μM) and a high (30 μM) concentration of ryanodine.
5. Carbachol-induced I_oscil was abolished by the general calcium entry blocker SKF 96365 (10 μM) and by Cd²⁺ (100 μM), but was unaffected by La³⁺ (400 μM). As found previously, I_DOC was also blocked by SKF 96365 and Cd²⁺, but not La³⁺; the inhibition of I_DOC preceded the abolition of I_oscil by 27 s with SKF 96365 and by 30 s with Cd²⁺. Nifedipine (1 μM) produced a partial inhibition of the carbachol-induced I_oscil frequency at holding potentials of −20 mV and −60 mV and, in addition, reduced I_DOC at −60 mV by 18%.
6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I_DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.

     

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (I_Ca(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on I_Ca(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison.
2. In human atrial cells, 100 μM pimobendan increased I_Ca(L) (evoked by depolarization to +10 mV from a holding potential of −40 mV) by 250.4±45.0% (n=15), with the concentration for half-maximal stimulation (EC₅₀) being 1.13 μM. I_Ca(L) was increased by 100 μM UD-CG 212 by 174.5±30.2% (n=10) with an EC₅₀ value of 1.78 μM in human atrial cells. These two agents inhibit PDE III selectively.
3. A selective PDE IV inhibitor, rolipram (1–100 μM), did not itself affect I_Ca(L) in human atrial cells. However, 100 μM rolipram significantly enhanced the effect of 100 μM UD-CG 212 on I_Ca(L) (increase with UD-CG 212 alone, 167.9±33.9, n=5; increase with the two agents together, 270.0±52.2%; n=5, P<0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated I_Ca(L) by 52.9±9.3% (n=5) in human atrial cells.
4. In rabbit atrial cells, I_Ca(L) at +10 mV was increased by 22.1±9.0% by UD-CG 212 (n=10) and by 67.4±12.0% (n=10) by pimobendan (each at 100 μM). These values were significantly lower than those obtained in human atrial cells (P<0.0001). Rolipram (1–100 μM) did not itself affect I_Ca(L) in rabbit atrial cells. However, I_Ca(L) was increased by 215.7±65.2% (n=10) by the combination of 100 μM UD-CG 212 and 100 μM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 μM UD-CG 212 alone.
5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating I_Ca(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under β-adrenoceptor-mediated stimulation.

     

Negative chronotropic actions of endothelin-1 on rabbit sinoatrial node pacemaker cells

1. The effects of endothelin-1 (ET-1) on sinoatrial (SA) node preparations of the rabbit heart were studied by means of whole-cell clamp techniques.
2. ET-1 at 1 nM slowed the spontaneous beating activity and rendered half of the cells quiescent. At a higher concentration of 10 nM, the slowing and cessation of spontaneous activity were accompanied by hyperpolarization.
3. In voltage-clamp experiments, ET-1 decreased the basal L-type Ca²⁺ current (I_Ca(L)) dose-dependently with a half-maximal inhibitory concentration (EC₅₀) of 0.42 nM and maximal inhibitory response (E_max) of 49.5%. The delayed rectifying K⁺ current (I_K) was also reduced by 33.2±11.1% at 1 nM. In addition, an inwardly rectifying K⁺ current was activated by ET-1 at higher concentrations (EC₅₀=4.8 nM). These ET-1-induced changes in membrane currents were abolished by BQ485 (0.3 μM), a highly selective ET_A receptor antagonist.
4. When I_Ca(L) was inhibited by ET-1 (1 nM), subsequent application of 10 μM ACh showed no additional decrease in I_Ca(L), suggesting the involvement of cyclic AMP in the effects of ET-1 on I_Ca(L). In contrast, 1 nM ET-1 further decreased I_Ca(L) in the presence of 10 μM ACh, suggesting that ET-1 activates some additional mechanism(s) which inhibit I_Ca(L). The ET-1-induced I_Ca(L) inhibition was abolished by protein kinase A inhibitory peptide (PKI, 20 μM) or H-89 (5 μM). However, the I_Ca(L) inhibition was not affected by methylene blue (10 μM), suggesting a minor role for cyclic GMP in the effect of ET-1 under basal conditions.
5. ET-1 failed to inhibit I_Ca(L) when the pipette contained GDPβS (200 μM). However, incubation of the cells with pertussis toxin (PTX, 5 μg ml⁻¹, >6 h) only reduced the ET-1-induced inhibition to 21.5±9.5%, whereas it abolished the inhibitory effect of ACh on I_Ca(L).
6. Intracellular perfusion of 8-bromo cyclicAMP (8-Br cyclicAMP, 500 μM) attenuated, but did not abolish the inhibitory effect of ET-1 on I_Ca(L). This 8-Br cyclicAMP-resistant component (17.5±14.4%, n=20) was not affected by combined application of 8-Br cyclicAMP with 8-bromo cyclicGMP (500 μM), ryanodine (1 μM) or phorbol-12-myristate-13-acetate (TPA; 50 nM).
7. In summary, ET-1 exerts negative chronotropic effects on the SA node via ET_A-receptors. ET-1 inhibits both I_Ca(L) and I_K, and increases background K⁺ current. The inhibition of I_Ca(L) by ET-1 is mainly due to reduction of the cyclicAMP levels via PTX-sensitive G protein, but some other mechanism(s) also seems to be operative.

     

Epoxyeicosatrienoic acids,potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

1. Using intracellular microelectrodes, we investigated the effects of 17-octadecynoic acid (17-ODYA) on the endothelium-dependent hyperpolarization induced by acetylcholine in the guinea-pig isolated internal carotid artery with endothelium.
2. In the presence of N^ω-nitro-L-arginine (L-NOARG, 100 μM) and indomethacin (5 μM) to inhibit nitric oxide synthase and cyclo-oxygenase, acetylcholine (1 μM) evoked an endothelium-dependent hyperpolarization which averaged −16.4 mV starting from a resting membrane potential of −56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential.
3. The acetylcholine-induced endothelium-dependent hyperpolarization was not altered by charybdotoxin (0.1 μM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 μM) to −12.8±1.2 mV (n=10) or the combination of apamin plus iberiotoxin (−14.3±3.4 mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+7.1±3.7 mV, n=8).
4. 17-ODYA (10 μM) produced a significant hyperpolarization of the resting membrane potential which averaged −59.6 mV and a partial but significant inhibition of the acetylcholine-induced endothelium-dependent hyperpolarization (−10.9 mV).
5. Apamin did not modify the effects of 17-ODYA but in the presence of charybdotoxin or iberiotoxin, 17-ODYA no longer influenced the resting membrane potential or the acetylcholine-induced hyperpolarization.
6. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6-, 8,9-, 11,12- and 14,15-EpETrE, 3 μM) did not affect the cell membrane potential and did not relax the guinea-pig isolated internal carotid artery.
7. These results indicate that, in the guinea-pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P₄₅₀ pathway in endothelium-dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium-derived hyperpolarizing factor (EDHF) is probably not due to the opening of BK_Ca channels.

     

Differential effects of the neuroprotectant lubeluzole on bovine and mouse chromaffin cell calcium channel subtypes

1. The effects of lubeluzole (a neuroprotective benzothiazole derivative) and its (−) enantiomer {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 on whole-cell currents through Ca²⁺ channels, with 10 mM Ba²⁺ as charge carrier (I_Ba), have been studied in bovine and mouse voltage-clamped adrenal chromaffin cells. Currents generated by applying 50 ms depolarizing test pulses to 0 mV, from a holding potential of −80 mV, at 10 s intervals had an average magnitude of 1 nA.
2. Lubeluzole and {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154 blocked the peak I_Ba of bovine chromaffin cells in a time and concentration-dependent manner; their IC₅₀s were 1.94 μM for lubeluzole and 2.54 μM for {"type":"entrez-nucleotide","attrs":{"text":"R91154","term_id":"958694","term_text":"R91154"}}R91154. In a current-voltage protocol, lubeluzole (3 μM) inhibited peak I_Ba at all test potentials. However, no obvious shifts of the I-V curve were detected.
3. After 10 min exposure to 3 μM lubeluzole, the late current (measured at the end of the pulse) was inhibited more than the peak current. Upon wash out of the drug, the inactivation reversed first and then the peak current recovered.
4. Blockade of peak current was greater at more depolarizing holding potentials (i.e. 35% at −110 mV and 87% at −50 mV, after 10 min superfusion with lubeluzole). Inactivation of the current was pronounced at −110 mV, decreased at −80 mV and did not occur at −50 mV.
5. Intracellular dialysis of bovine voltage-clamped chromaffin cells with 3 μM lubeluzole caused neither blockade nor inactivation of I_Ba. The external application of 3 μM lubeluzole to those dialysed cells produced inhibition as well as inactivation of I_Ba.
6. The effects of lubeluzole (3 μM) on I_Ba in mouse chromaffin cells were similar to those in bovine chromaffin cells. At −80 mV holding potential, a pronounced inactivation of the current led to greater blockade of the late I_Ba (66%) as compared with peak I_Ba (46% after 10 min superfusion with lubeluzole).
7. In mouse chromaffin cells approximately half of the whole-cell I_Ba was sensitive to 3 μM nifedipine (L-type Ca²⁺ channels) and the other half to 3 μM ω-conotoxin MVIIC (non-L-type Ca²⁺ channels). In ω-conotoxin MVIIC-treated cells, 3 μM lubeluzole caused little blockade and inactivation of I_Ba. However in nifedipine-treated cells, lubeluzole caused a pronounced blockade and inactivation of I_Ba that reversed upon wash out of the compound.
8. The results are compatible with the idea that lubeluzole preferentially blocks non-L-types of voltage-dependent Ca²⁺ channels expressed by bovine and mouse chromaffin cells. The higher concentrations of the compound also block L-type Ca²⁺ channels. The mechanism of inhibition involves the access of lubeluzole to the open channel from the outside of the cell and promotion of its inactivation. The differential blockade of Ca²⁺ channel subtypes might contribute to the neuroprotective actions of lubeluzole (which exhibit stereoselectivity). However, in view of the lack of stereoselectivity in blocking Ca²⁺ channels, this effect cannot be the only explanation for the protective activity of lubeluzole in stroke.

     

Effects of nitric oxide donors,S-nitroso-L-cysteine and sodium nitroprusside,on the whole-cell and single channel currents in single myocytes of the guinea-pig proximal colon

1. The nature of the membrane channels underlying the membrane conductance changes induced by the nitric oxide (NO) donors, S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) were investigated in single myocytes isolated from the circular muscle layer of the guinea-pig proximal colon, by use of standard whole-cell and single channel recording techniques.
2. Under voltage clamp, depolarizing steps from −60 mV elicited a rapidly-developing, little-inactivating outward K⁺ current (I_K) at potentials positive to −40 mV (at 20–25°C). The steady-state level (I_SS) of this K⁺ current increased in amplitude as the step potential was made to more positive potentials. If the depolarizing steps were made from a holding potential of −80 mV an additional rapidly activating and inactivating outward K⁺ current was also elicited, superimposed on I_K.
3. At 20–25°C, NOCys (2.5 μM), SNP (100 μM) and 8-bromo-cyclic GMP (500 μM) increased the amplitude of I_SS of I_K elicited from a holding potential of −60 mV. In contrast, NOCys (2–5 μM) had little effect on I_SS at 35°C. Higher concentrations (⩾5 μM at 20–25°C and ⩾10 μM at 35°C) of NOCys decreased the peak amplitude (I_Peak) and I_SS of I_K in a concentration-dependent manner. This blockade of I_K with NOCys was always associated with an increase of the holding current (I_Hold), due to the activation of a membrane conductance with a reversal potential between 0 and +30 mV and which was reduced approximately 50% upon the addition of Cd²⁺ (1 mM).
4. NOCys (2.5 to 10 μM) or SNP (100 μM) increased the activity of large conductance Ca²⁺-activated (BK) K⁺ channels in both cell-attached and excised inside-out patches, bathed in either a symmetrical high K⁺ (130 mM) or an asymmetrically K⁺ (6 mM_out: 130 mM_in) physiological saline. Increases in BK channel activity in NOCys (10 μM) or SNP (100 μM) were associated with an increase in the probability of BK channel opening (N.P_o), and with a negative shift of the plots of ln(N.P_o) against the patch potential, with little change in the slopes of these plots. In cell-attached patches, the increase in N.P_o with NOCys was often associated with a decrease in the BK single channel conductance.
5. In both cell-attached and excised patches, NOCys (2.5 to 10 μM) also activated an additional population of channels which allowed inward current flow at potentials positive to E_K. In excised inside-out patches bathed in asymmetrical K⁺ physiological saline, these single channel currents were 2–3 pA in amplitude at −30 mV and reversed in direction near +10 mV, even if the NaCl (126 mM) concentration in the pipette solution had been replaced with an equimolar concentration of Na gluconate.
6. Under current clamp, NOCys (2.5 μM) and SNP (100 μM) had variable effects on the membrane potential of colonic myocytes, inducing either a small membrane hyperpolarization of <5 mV, or a slowly-developing membrane depolarization of about 5 mV. In contrast, NOCys (5 μM) produced a transient membrane hyperpolarization which was followed by a large depolarization of the membrane potential to positive potentials. The electrotonic potentials elicited in response to an injection of constant hyperpolarizing current (10 pA for 400 ms) were little changed during the NOCys (5 μM)-induced membrane hyperpolarization, but significantly reduced (to 61% of control) during the periods of membrane depolarization.
7. It was concluded that NOCys and SNP, directly increased the number of active BK channels in the membrane of colonic myocytes which leads to a small rapidly oscillating membrane hyperpolarization. The following rebound depolarization in NOCys arises from both the direct opening of a population of cationic channels and the blockade of voltage- and Ca-activated K⁺ conductances. Finally, the apamin-sensitive K⁺ channels underlying the initial transient hyperpolarization recorded in the intact proximal colon, in response to nerve-released or directly-applied NO, have yet to be identified at the single channel or whole-cell current level.

     

Selective inhibition of M-type potassium channels in rat sympathetic neurons by uridine nucleotide preferring receptors

1. UTP and UDP depolarize rat superior cervical ganglion neurons and trigger noradrenaline release from these cells. The present study investigated the mechanisms underlying this excitatory action of uridine nucleotides by measuring whole-cell voltage-dependent K⁺ and Ca²⁺ currents.
2. Steady-state outward (holding) currents measured in the amphotericin B perforated-patch configuration at a potential of −30 mV were reduced by 10 μM UTP in a reversible manner, but steady-state inward (holding) currents at −70 mV were not affected. This action of UTP was shared by the muscarinic agonist oxotremorine-M. In current-voltage curves between −20 and −100 mV, UTP diminished primarily the outwardly rectifying current components arising at potentials positive to −60 mV.
3. Slow relaxations of muscarinic K⁺ currents (I_M) evoked by hyperpolarizations from −30 to −55 mV were also reduced by 10 μM UTP (37% inhibition) and oxotremorine-M (81% inhibition). In contrast, transient K⁺-currents, delayed rectifier currents, fast and slow Ca²⁺-dependent K⁺ currents, as well as voltage-dependent Ca²⁺ currents were not altered by UTP.
4. In conventional (open-tip) whole-cell recordings, replacement of GTP in the pipette by GDPβS abolished the UTP-induced inhibition of I_M, whereas replacement by GTPγS rendered it irreversible.
5. The UTP-induced reduction of I_M was half maximal at 1.5 μM with a maximum of 37% inhibition; UDP was equipotent and equieffective, while ADP was less potent (half maximal inhibition at 29 μM). ATP had no effect at ⩽30 μM.
6. The inhibition of I_M induced by 10 μM UTP was antagonized by pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) at ⩾30 μM and by reactive blue 2 at ⩾10 μM, but not by suramin at concentrations up to 30 μM.
7. These results show that rat superior cervical ganglion neurons possess uridine nucleotide preferring P2Y receptors which inhibit K_M channels. This effect presumably forms the basis of the excitatory action of uridine nucleotides in rat sympathetic neurons.

     

Serine-O-sulphate transport by the human glutamate transporter,EAAT2

1. Expression of the recombinant human excitatory amino aid transporters, EAAT1 and EAAT2, in Xenopus laevis oocytes allows electrogenic transport to be studied under voltage clamp conditions.
2. We have investigated the transport of the pharmacological substrate, L-serine-O-sulphate transport by EAAT1 and EAAT2. The EC₅₀ values for L-serine-O-sulphate transport by EAAT2 showed a steep voltage-dependence, increasing from 152±11 μM at −100 mV to 1930±160 μM at 0 mV. In contrast to EAAT2, EC₅₀ values for L-serine-O-sulphate transport by EAAT1 were relatively constant over the membrane potential range of −100 mV to 0 mV. The EC₅₀ values for L-glutamate and D-aspartate transport, by EAAT2, were also relatively constant over this membrane potential range.
3. Chloride ions modulated the voltage-dependent changes in EC₅₀ values for transport by EAAT2. This effect was most apparent for L-serine-O-sulphate transport, and to a lesser extent for L-glutamate and not at all for D-aspartate transport by EAAT2.
4. Extracellular sodium and proton concentrations also modulated the voltage-dependence of L-serine-O-sulphate EC₅₀ values for EAAT2.
5. We speculate that these different properties of L-serine-O-sulphate transport by EAAT2 compared to other substrates may be due to the much stronger acidity of the sulphate group of L-serine-O-sulphate compared to carboxyl groups of L-glutamate or D-aspartate.
6. These results highlight some of the differences in the way different glutamate transporter subtypes transport substrates. This may be used to understand further the transport process and develop subtype selective inhibitors of glutamate transport.

     

The antiarrhythmic agent bertosamil induces inactivation of the sustained outward K+ current in human atrial myocytes

1. In whole-cell patch-clamped human atrial myocytes, the antiarrhythmic agent bertosamil (10 μM) inhibited the sustained component, I_sus (38.6±3.1%), and enhanced the inactivating component, I_t (9.1±6.1%), of the outward K⁺ current elicited by 750 ms test pulses from −60 mV to +50 mV. Higher concentrations of bertosamil (>10 μM) inhibited both I_t and I_sus.
2. Suppression of I_sus and stimulation of I_t by 10 μM bertosamil was observed on renewed stimulation following a 2 min rest period during which the drug was applied and persisted after washout, indicating a rest-dependent effect of bertosamil on the outward K⁺ current.
3. Cell dialysis with an internal solution containing 10 μM bertosamil increased both I_t (78.0±14.7%) and I_total (26.7±8.4%) and inhibited I_sus (28.9±6.3%, n=6). In the presence of intracellular bertosamil, external application of the drug inhibited I_t and I_sus in a concentration-dependent and use-dependent manner.
4. Following the suppression of I_sus by 200 μM 4-aminopyridine (4-AP), bertosamil (10 μM) inhibited I_t. Washout of 4-AP was associated with a larger I_t amplitude than that observed in control conditions. In myocytes characterized by a prominent I_sus and lack of I_t, bertosamil (10 μM) induced a rapid and partial inactivation of the current, together with inward rectification of the current measured at the end of the test pulse.
5. In the presence of bertosamil the activation/voltage relationships, steady-state inactivation and recovery from inactivation of I_t were markedly modified, pointing to changes in the conductance underlying I_t.
6. We conclude that bertosamil induces rapid inactivation of sustained outward current which leads to an apparent increase in I_t and decrease in I_sus. This effect, which was distinct from the use-dependent inhibition of the outward K⁺ current, could represent a new antiarrhythmic mechanism.

     

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches).
2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba²⁺ currents at −50 mV (K_i=30.6 nM).
3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (⩾30 μM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately −82 mV by application of 100 μM levcromakalim.
4. The 100 μM levcromakalim-induced inward current (symmetrical 140 mM K⁺ conditions, −50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 μM). In contrast, Bay K 8644 (1 μM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current.
5. When 100 μM nifedipine was included in the pipette solution during conventional whole-cell recording at −50 mV, application of levcromakalim (100 μM) caused a significant inward membrane current which was suppressed by 5 μM glibenclamide. On the other hand, inclusion of 5 μM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current.
6. The levcromakalim-induced K⁺ channel openings in cell-attached configuration were suppressed by subsequent application of 5 μM glibenclamide but not of 100 μM nifedipine.
7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K⁺ channel activity mainly through extracellular blocking actions on the K⁺ channel itself.

     

A comparative study of the effects of three guanylyl cyclase inhibitors on the L-type Ca2+ and muscarinic K+ currents in frog cardiac myocytes

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (I_Ca), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the β-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of I_Ca and on the muscarinic activated potassium current I_K,ACh, in frog atrial and ventricular myocytes.
2. ODQ (10 μM) and LY 83583 (30 μM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 μM) on the isoprenaline (Iso)-stimulated I_Ca which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 μM) had no effect under similar conditions.
3. In the absence of SNAP, LY 83583 (30 μM) potentiated the stimulations of I_Ca by either Iso (20 nM), forskolin (0.2 μM) or intracellular cyclic AMP (5–10 μM). ODQ (10 μM) had no effect under these conditions, while MBlue (30 μM) inhibited the Iso-stimulated I_Ca.
4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 μM acetylcholine (ACh) on I_Ca.
5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I_K,ACh by ACh in the presence of intracellular GTP, and this inhibition was weakened when I_K,ACh was activated by intracellular GTPγS.
6. The potentiating effect of LY 83583 on Iso-stimulated I_Ca was absent in the presence of either DL-dithiothreitol (DTT, 100 μM) or a combination of superoxide dismutase (150 u ml⁻¹) and catalase (100 u ml⁻¹).
7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of β-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of MBlue.

     

Role of endothelium in regulation of smooth muscle membrane potential and tone in the rabbit middle cerebral artery

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA).
2. In endothelium-intact strips, histamine (His, 3–10 μM) and high K⁺ (20–80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 μM), 5-hydroxytryptamine (10 μM) and 9,11-epithio-11, 12-methano-thromboxane A₂ (10 nM) each produced only a small contraction (less than 5% of the maximum K⁺-induced contraction).
3. N^G-nitro-L-arginine (L-NOARG, 100 μM), but not indomethacin (10 μM), greatly enhanced the phasic and the tonic contractions induced by His (1–10 μM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3–3 μM) caused concentration-dependent relaxation (maximum relaxation by 89.7±7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 μM), but not glibenclamide (3 μM), abolished the relaxation induced by ACh (0.3–3 μM) in L-NOARG-treated strips (n=4, P<0.05).
4. In endothelium-intact tissues, His (3 μM) depolarized the smooth muscle membrane potential (by 4.4±1.8 mV, n=12, P<0.05) whereas ACh (3 μM) caused membrane hyperpolarization (−20.9±3.0 mV, n=25, P<0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (−23.5±5.9 mV, n=8, P<0.05) or glibenclamide (−20.6±5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to −5.8±3.2 mV, n=3, P<0.05).
5. Sodium nitroprusside (0.1–10 μM) did not hyperpolarize the smooth muscle cell membrane potential (0.2±0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n=4, P<0.05).
6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.

     

The pacemaker current If in single human atrial myocytes and the effect of β-adrenoceptor and A1-adenosine receptor stimulation

1. We used single human atrial myocytes to study I_f occurrence, properties and pharmacological modulation. Cells were obtained by chunk enzymatic digestion from samples of right atrial appendages of patients undergoing corrective cardiac surgery.
2. Patch-clamped cells in the whole-cell configuration were superfused with a modified Tyrode solution to reduce contamination by interfering currents and to amplify I_f. The average cell membrane capacitance was 85.06±2.41 pF (n=531). Data were consistent with the geometrical dimensions of the cells (length 94.2±1.89 μm, width 17.9±0.42 μm, n=126).
3. When hyperpolarizing to −120 mV from a holding potential of −40 mV, 252 of 306 tested cells (82%) expressed a hyperpolarization-activated inward current (I_f density =3.77±0.25 pA pF⁻¹); the current was considered to be present in a given cell if its density at −120 mV was larger than 0.5 pA pF⁻¹.
4. Current activation was sigmoidal and fitted a Boltzmann model; the average activation curve (n=25) showed a maximum current amplitude of 205.97±19.94 pA, corresponding to 3.87±0.63 pA pF⁻¹, voltage of half-maximal activation (V_1/2) at −86.68±2.19 mV and a slope of −11.39±0.69 mV. The reversal potential of I_f measured by tail-current analysis was −13.07±1.92 mV (n=6). The addition of CsCl (5 mM) fully and reversibly blocked the current.
5. In the presence of the β-adrenoceptor agonist isoprenaline (Iso, 1 μM), V_1/2 was significantly shifted toward less negative potentials by 6.06±1.96 mV (n=16, P=0.0039). The selective A₁-adenosine receptor agonist cyclopentyladenosine (CPA, 1 μM) caused a statistically significant shift of V_1/2 toward more negative potentials with respect to the control curve, both in the absence (−7.37±1.83 mV, P=0.0005, n=11) and in the presence of 1 μM Iso (−4.97±1.78, P=0.031, n=6).
6. These results demonstrate that a current with the properties of I_f described in cardiac primary and secondary pacemakers occurs in the majority of human atrial cells. While the pathophysiological relevance of I_f in human atrial tissue remains to be defined, our data clearly show that it is modulated through stimulation of β-adrenoceptors and A₁-adenosine receptors.

     

Inhibitory effects of ω-3 polyunsaturated fatty acids on receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells

1. The effects of ω-3 polyunsaturated fatty acids on receptor-mediated non-selective cation current (I_cat) and K⁺ current were investigated in aortic smooth muscle cells from foetal rat aorta (A7r5 cells). The whole-cell voltage clamp technique was employed.
2. With a K⁺-containing solution, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 30 μM) produced an outward current at a holding potential of −40 mV. This response was inhibited by tetraethylammonium (20 mM) or Cs⁺ in the patch pipette solution, and the reversal potential of the EPA-induced current followed the K⁺ equilibrium potential in a near Nernstian manner.
3. Under conditions with a Cs⁺-containing pipette solution, both vasopressin and endothelin-1 (100 nM) induced a long-lasting inward current at a holding potential of −60 mV. The reversal potential of these agonist-induced currents was about +0 mV, and was not significantly altered by the replacement of the extracellular or intracellular Cl⁻ concentration, suggesting that the induced current was a cation-selective current (I_cat).
4. La³⁺ and Cd²⁺ (1 mM) completely abolished these agonist-induced I_cat, but nifedipine (10 μM) failed to inhibit it significantly.
5. ω-3 polyunsaturated fatty acids (3100 μM), EPA, DHA and docosapentaenoic acids (DPA), inhibited the agonist-induced I_cat in a concentration-dependent manner. The potency of the inhibitory effect was EPA>DHA>DPA, and the half maximal inhibitory concentration (IC₅₀) of EPA was about 7 μM.
6. Arachidonic and linoleic acids (10, 30 μM) showed a smaller inhibitory effect compared to ω-3 fatty acids. Also, oleic and stearic acids (30 μM) did not show a significant inhibitory effect on I_cat.
7. A similar inhibitory action of EPA was observed when I_cat was activated by intracellularly applied GTPγS in the absence of agonists, suggesting that the site of action of ω-3 fatty acids is not located on the receptor.
8. These results demonstrate that ω-3 polyunsaturated fatty acids can activate a K⁺ current and also effectively inhibit receptor-mediated non-selective cation currents in rat A7r5 vascular smooth muscle cells. Thus, the data suggest that ω-3 fatty acids may play an important role in the regulation of vascular tone.

     

Effects of propafenone and 5-hydroxy-propafenone on hKv1.5 channels

1. The goal of this study was to analyse the effects of propafenone and its major metabolite, 5-hydroxy-propafenone, on a human cardiac K⁺ channel (hKv1.5) stably expressed in Ltk⁻ cells and using the whole-cell configuration of the patch-clamp technique.
2. Propafenone and 5-hydroxy-propafenone inhibited in a concentration-dependent manner the hKv1.5 current with K_D values of 4.4±0.3 μM and 9.2±1.6 μM, respectively.
3. Block induced by both drugs was voltage-dependent consistent with a value of electrical distance (referenced to the cytoplasmic side) of 0.17±0.55 (n=10) and 0.16±0.81 (n=16).
4. The apparent association (k) and dissociation (l) rate constants for propafenone were (8.9±0.9)×10⁶ M⁻¹ s⁻¹ and 39.5±4.2 s⁻¹, respectively. For 5-hydroxy-propafenone these values averaged (2.3±0.3)×10⁶ M⁻¹ s⁻¹ and 21.4±3.1 s⁻¹, respectively.
5. Both drugs reduced the tail current amplitude recorded at −40 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a `crossover'' phenomenon when the tail currents recorded under control conditions and in the presence of each drug were superimposed.
6. Both compounds induced a small but statistically significant use-dependent block when trains of depolarizations at frequencies between 0.5 and 3 Hz were applied.
7. These results indicate that propafenone and its metabolite block hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutical range.

     

Voltage- and use-dependent inhibition of Na+ channels in rat sensory neurones by 4030W92, a new antihyperalgesic agent

1. Whole cell patch clamp techniques were used to study the effects of 4030W92 (2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine), a new antihyperalgesic agent, on rat dorsal root ganglion (DRG) neurones.
2. In small diameter, presumably nociceptive DRG neurones under voltage-clamp, 4030W92 (1–100 μM) produced a concentration-related inhibition of slow tetrodotoxin-resistant Na⁺ currents (TTX_R). From a holding potential (V_h) of −90 mV, currents evoked by test pulses to 0 mV were inhibited by 4030W92 with a mean IC₅₀ value of approximately 103 μM.
3. The inhibitory effect of 4030W92 on TTX_R was both voltage- and use-dependent. Currents evoked from a V_h of −60 mV were inhibited by 4030W92 with a mean IC₅₀ value of 22 μM, which was 5 fold less than the value obtained at −90 mV. Repeated activation of TTX_R by a train of depolarizing pulses (5 Hz, 20 ms duration) enhanced the inhibitory effects of 4030W92. These data could be explained by a preferential interaction of the drug with inactivation states of the channel. In support of this hypothesis 4030W92 (30 μM) produced a significant hyperpolarizing shift of 10 mV in the slow inactivation curve for TTX_R and markedly slowed the recovery from channel inactivation.
4. Fast TTX-sensitive Na⁺ currents (TTX_S) were also inhibited by 4030W92 in a voltage-dependent manner. The IC₅₀ values obtained from V_hs of −90 mV and −70 mV were 37 μM and 5 μM, respectively. 4030W92 (30 μM) produced a 13 mV hyperpolarizing shift in the steady-state inactivation curve of TTX_S.
5. High threshold voltage-gated Ca²⁺ currents were only weakly inhibited by 4030W92. The reduction in peak Ca²⁺ current amplitude produced by 100 μM 4030W92 was 20±6% (n=6). Low threshold T-type Ca²⁺ currents were inhibited by 17±8% and 43±3% by concentrations of 4030W92 of 30 μM and 100 μM, respectively (n=6).
6. Under current clamp, some cells exhibited broad TTX-resistant action potentials whilst others showed fast TTX-sensitive action potentials in response to a depolarizing current injection. In most cells a long duration (800 ms) supramaximal current injection evoked a train of action potentials. 4030W92 (10–30 μM) had little effect on the first spike in the train but produced a concentration-related inhibition of the later spikes. The number of spikes per train was significantly reduced from 9.7±1.5 to 4.2±1.0 and 2.6±1.1 in the presence of 10 μM and 30 μM 4030W92, respectively (n=5).
7. Thus, 4030W92 is a potent voltage- and use-dependent inhibitor of Na⁺ channels in sensory neurones. This profile can be explained by a preferential action of the drug on a slow inactivation state of the channel that results in a delayed recovery to the resting state. This state-dependent modulation by 4030W92 of Na⁺ channels that are important in sensory neurone function may underlie or contribute to the antihyperalgesic profile of this compound observed in vivo.

     

The effects of propofol on macroscopic and single channel sodium currents in rat ventricular myocytes

1. The effects of the injectable anaesthetic agent propofol (di-isopropyl phenol) were examined on sodium currents and single sodium channels by use of patch-clamp techniques in ventricular myocytes isolated from rat hearts.
2. Propofol dose-dependently blocked the whole cell sodium currents evoked by a voltage step to −30 mV from a holding potential of −90 mV with an EC₅₀ of 14.8±2.3 μM (mean±s.e.mean).
3. Propofol caused a substantial hyperpolarizing shift in the voltage-dependence of inactivation of sodium currents (168 μM (30 μg ml⁻¹) propofol caused a −14 mV shift (P<0.01); 56 μM caused a −8 mV shift (P<0.05)). A smaller shift in the voltage-dependence of activation was produced (4 mV by 168 μM (not statistically significant)), but this was to more depolarized potentials. The maximal sodium conductance, as judged from the activation and inactivation curves, was reduced by 13% by 168 μM propofol (not statistically significant), but propofol did not affect the reversal potential of the current - voltage relationship.
4. The macroscopic rate of inactivation, as measured by the time constant of the exponential fall of current amplitude from the peak current, was also slowed by propofol, from a control time constant of 1.78±0.31 ms to 2.93±0.47 ms (mean±s.e.mean, n=8, P<0.05) by 168 μM propofol. Despite the increase in the time constant, the macroscopic inactivation remained well fitted by a single exponential. The macroscopic rate of activation was also slowed, but to a lesser degree (<10%, not statistically significant) by 168 μM propofol.
5. Propofol slowed the rate of recovery from inactivation of the sodium current, as measured by a two pulse protocol. Propofol (168 μM) increased the time constant of recovery, measured at −100 mV and room temperature, from a control value of 55±5.9 ms to 141±24.2 ms (mean±s.e.mean, n=8, P<0.01). Although the time constant was increased at all voltages measured, the intrinsic voltage-dependence of the rate of recovery was not changed.
6. Single channel recordings showed that the mean open time of single sodium channels was dramatically reduced by propofol (from 0.50±0.02 ms in control to 0.28±0.01 ms by 56 μM propofol and to 0.24±0.01 ms by 168 μM, both significantly different from control, P<0.01). Single channel conductance was not changed by either concentration of propofol.