Yung E. Earm

Stretch-activated and background non-selective cation channels in rat atrial myocytes

1 Stretch‐activated channels (SACs) were studied in isolated rat atrial myocytes using the whole‐cell and single‐channel patch clamp techniques. Longitudinal stretch was applied by using two patch electrodes. 2 In current clamp configuration, mechanical stretch of 20 % of resting cell length depolarised the resting membrane potential (RMP) from ‐63·6 ± 0·58 mV (n= 19) to ‐54·6 ± 2·4 mV (n= 13) and prolonged the action potential duration (APD) by 32·2 ± 8·8 ms (n= 7). Depolarisation, if strong enough, triggered spontaneous APs. In the voltage clamp configuration, stretch increased membrane conductance in a progressive manner. The current‐voltage (I–V) relationship of the stretch‐activated current (ISAC) was linear and reversed at ‐6·1 ± 3·7 mV (n= 7). 3 The inward component of ISAC was abolished by the replacement of Na+ with NMDG+, but ISAC was hardly altered by the Cl− channel blocker DIDS or removal of external Cl−. The permeability ratio for various cations (PCs:PNa:PLi= 1·05:1:0·98) indicated that the SAC current was a non‐selective cation current (ISAC,NC). The background current was also found to be non‐selective to cations (INSC,b); the permeability ratio (PCs:PNa:PLi= 1·49:1:0·70) was different from that of ISAC,NC. 4 Gadolinium (Gd3+) acted on INSC,b and ISAC,NC differently. Gd3+ inhibited INSC,b in a concentration‐dependent manner with an IC50 value of 46·2 ± 0·8 μM (n= 5). Consistent with this effect, Gd3+ hyperpolarised the resting membrane potential (‐71·1 ± 0·26 mV, n= 9). In the presence of Gd3+ (0·1 mM), stretch still induced ISAC,NC and diastolic depolarisation. 5 Single‐channel activities were recorded in isotonic Na+ and Cs+ solutions using the inside‐out configuration. In NMDG+ solution, outward currents were abolished. Gd3+ (100 μM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 μM) in the pipette solution, an increase in pipette pressure induced an increase in channel opening (21·27 ± 0·24 pS; n= 7), which was distinct from background activity. 6 We concluded from the above results that longitudinal stretch in rat atrial myocytes induces the activation of non‐selective cation channels that can be distinguished from background channels by their different electrophysiology and pharmacology.

A Model of the Single Atrial Cell: Relation between Calcium Current and Calcium Release

The hypothesis that calcium release from the sarcoplasmic reticulum in cardiac muscle is induced by rises in free cytosolic calcium (Fabiato 1983, Am. J. Physiol 245) allows the possibility that the release could be at least partly regenerative. There would then be a non-linear relation between calcium current and calcium release. We have investigated this possibility in a single-cell version of the rabbit-atrial model developed by Hilgemann & Noble (1987, Proc. R. Soc. Lond. B 230). The model predicts different voltage ranges of activation for calcium-dependent processes (like the sodium-calcium exchange current, contraction or Fura-2 signals) and the calcium current, in agreement with the experimental results obtained by Earm et al. (1990, Proc. R. Soc. Lond. B 240) on exchange current tails, Cannell et al. (1987, Science, Wash. 238) by using Fura-2 signals, and Fedida et al. (1987, J. Physiol., Lond. 385) and Talo et al. (1988, Biology of isolated adult cardiac myocytes) by using contraction. However, when the Fura-2 concentration is sufficiently high (greater than 200 μm) the activation ranges become very similar as the buffering properties of Fura-2 are sufficient to remove the regenerative effect. It is therefore important to allow for the buffering properties of calcium indicators when investigating the correlation between calcium current and calcium release.

Contribution of Ca2+‐activated K+ channels and non‐selective cation channels to membrane potential of pulmonary arterial smooth muscle cells of the rabbit

1 Using the perforated patch‐clamp or whole‐cell clamp technique, we investigated the contribution of Ca2+‐activated K+ current (IK(Ca)) and non‐selective cation currents (INSC) to the membrane potential in small pulmonary arterial smooth muscle cells of the rabbit. 2 The resting membrane potential (Vm) was ‐39·2 ± 0·9 mV (n= 72). It did not stay at a constant level, but hyperpolarized irregularly, showing spontaneous transient hyperpolarizations (STHPs). The mean frequency and amplitude of the STHPs was 5·6 ± 1·1 Hz and ‐7·7 ± 0·7 mV (n= 12), respectively. In the voltage‐clamp mode, spontaneous transient outward currents (STOCs) were recorded with similar frequency and irregularity. 3 Intracellular application of BAPTA or extracellular application of TEA or charybdotoxin suppressed both the STHPs and STOCs. The depletion of intracellular Ca2+ stores by caffeine or ryanodine, and the removal of extracellular Ca2+ also abolished STHPs and STOCs. 4 Replacement of extracellular Na+ with NMDG+ caused hyperpolarization Vm of without affecting STHPs. Removal of extracellular Ca2+ induced a marked depolarization of Vm along with the disappearance of STHPs. 5 The ionic nature of the background inward current was identified. The permeability ratio of K+ : Cs+ : Na+ : Li+ was 1·7 : 1·3 : 1 : 0·9, indicating that it is a non‐selective cation current (INSC). The reversal potential of this current in control conditions was calculated to be ‐13·9 mV. The current was blocked by millimolar concentrations of extracellular Ca2+ and Mg2+. 6 From these results, it was concluded that (i) hyperpolarizing currents are mainly contributed by Ca2+‐activated K+ (KCa) channels, and thus STOCs result in transient membrane hyperpolarization, and (ii) depolarizing currents are carried through NSC channels.

Different modulation of Ca-activated K channels by the intracellular redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit.

AstractWe investigated the electrical responses of Ca-activated K (KCa) currents induced by hypoxia and reduction or oxidation of the channel protein in pulmonary (PASMC) and ear (EASMC) arterial smooth muscle cells using the patch-clamp technique. In cell-attached patches, in the presence of a high K solution (containing 0.316 (μM Ca2+), the activity of KCa channels from PASMC was decreased (by 49±7% compared to control, pipette potential = −70 mV) by changing to a hypoxic solution (1 mM Na2S2O4, aeration with 100% N2 gas). EASMC channels did not respond to hypoxia. In order to investigate the possible mechanisms involved, using inside-out patches bathed symmetrically in 150 mM KCl, we applied redox couples to the intracellular side. Reducing agents, such as dithiothreitol (DDT, 5 mM), reduced glutathione, (GSH, 5 mM), and nicotinamide adenine dinucleotide reduced (NADH, 2 mM) decreased PASMC, but not EASMC, KCa channel activity. However, oxidizing agents such as 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, 1 mM), oxidized glutathione (GSSG, 5 mM) and NAD (2 mM) increased KCa channel activity in both PASMC and EASMC. The increased activity due to oxidizing agents was restored by applying reducing agents. From these results, we could suggest that the basal redox state of the EASMC KCa channel is more reduced than that of the PASMC channel, since the response of KCa channels of the EASMC to intracellular reducing agents differs from that of the PASMC. This difference may be related to the different responses of PASMC and EASMC KCa channels to hypoxia.

Blockade of the HERG human cardiac K+ channel by the antidepressant drug amitriptyline

Amitriptyline has been known to induce QT prolongation and torsades de pointes which causes sudden death. We studied the effects of amitriptyline on the human ether‐a‐go‐go‐related gene (HERG) channel expressed in Xenopus oocytes and on the rapidly activating delayed rectifier K+ current (IKr) in rat atrial myocytes. The amplitudes of steady‐state currents and tail currents of HERG were decreased by amitriptyline dose‐dependently. The decrease became more pronounced at more positive potential, suggesting that the block of HERG by amitriptyline is voltage dependent. IC50 for amitriptyline block of HERG current was progressively decreased according to depolarization: IC50 values at −30, −10, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 μM, respectively. Block of HERG by amitriptyline was use dependent: exhibiting a much faster block at higher activation frequency. Subsequent decrease in frequency after high activation frequency resulted in a partial relief of HERG blockade. Steady‐state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC50 was 3.26 μM in 2 mM [K+]o. It was increased to 4.78 μM in 4 mM [K+]o, suggesting that the affinity of amitriptyline on HERG was decreased by external K+. In rat atrial myocytes bathed in 35°C, 5 μM amitriptyline blocked IKr by 55%. However, transient outward K+ current (Ito) was not significantly affected. In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side effects of amitriptyline.

Phosphatidylinositol 4,5-Bisphosphate Is Acting as a Signal Molecule in α1-Adrenergic Pathway via the Modulation of Acetylcholine-activated K+ Channels in Mouse Atrial Myocytes

We have investigated the effect of α1-adrenergic agonist phenylephrine (PE) on acetylcholine-activated K+ currents (I KACh). I KACh was recorded in mouse atrial myocytes using the patch clamp technique.I KACh was activated by 10 μm ACh and the current decreased by 44.27 ± 2.38% (n = 12) during 4 min due to ACh-induced desensitization. When PE was applied with ACh, the extent of desensitization was markedly increased to 69.34 ± 2.22% (n = 9), indicating the presence of PE-induced desensitization. I KAChwas fully recovered from desensitization after a 6-min washout. PE-induced desensitization of I KACh was not affected by protein kinase C inhibitor, calphostin C, but abolished by phospholipase C (PLC) inhibitor, neomycin. When phophatidylinositol 4,5-bisphosphate (PIP2) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization ofI KACh in the presence of PE was further increased (97.25 ± 7.63%, n = 6). Furthermore, the recovery from PE-induced desensitization was inhibited, and the amplitude of I KACh at the second exposure after washout was reduced to 19.65 ± 2.61% (n = 6) of the preceding level. These data suggest that the KAChchannel is modulated by PE through PLC stimulation and depletion of PIP2.

NADH and NAD modulates Ca2+-activated K+ channels in small pulmonary arterial smooth muscle cells of the rabbit

We have investigated the effect of NADH and NAD on the gating of large conductance Ca2+-activated K(KCa) channels in arterial smooth muscle cells isolated from small pulmonary artery(outer diameter <300μm) and ear artery, using the patch clamp technique. In the inside-out configuration, intracellularly applied 2 mM NADH inhibited the activity of KCa, channels in pulmonary arterial smooth muscle cells, while it had no significant effect on ear arterial smooth muscle cells. On the other hand, 2 mM NAD increased the opening of KCa, channels in pulmonary arterial smooth muscle cells. The effects of another intracellular redox couple, glutathione(GSH) and glutathione disulfide(GSSG) were also dependent on their redox potentials. GSH(5 mM) inhibited KCa. channels activity, while GSSG(5 mM) increased the activity of pulmonary arterial smooth muscle cells. It could be concluded that the modulation of KCa channels by intracellular redox state contributes, at least in part, to the hypoxic suppression of outward current in pulmonary arterial smooth muscle cells.

Brain-derived neurotrophic factor rapidly potentiates synaptic transmission through NMDA, but suppresses it through non-NMDA receptors in rat hippocampal neuron.

Brain-derived neurotrophic factor (BDNF) rapidly enhances synaptic transmission among the hippocampal neurons. In order to examine which component of glutamate receptors participates in synaptic potentiation by BDNF, we have studied the effect of glutamate antagonists on excitatory postsynaptic currents (EPSCs) enhanced by BDNF, using cultured embryonic hippocampal neurons. In the presence of AP5, a N-methyl-D-aspartate (NMDA) antagonist, BDNF depressed the EPSCs. In contrast, BDNF enhanced the EPSCs in the presence of a non-NMDA antagonist CNQX. Our results suggest that BDNF acutely activates synaptic transmission via NMDA, but suppresses it via non-NMDA receptors in the hippocampus.

Modulation of voltage-dependent K+ channel by redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit

Abstract It has been suggested that hypoxic pulmonary vasoconstriction (HPV) results from the depolarization that is induced by the suppression of K+ current in pulmonary arterial smooth muscle cells (PASMC). We tested the hypothesis that the effect of the cellular redox potential on voltage-sensitive K+ (Kv) current is involved in HPV as a primary sensing mechanism. Kv current in PASMC and ear arterial smooth muscle cells (EASMC) of the rabbit was recorded using the whole-cell patch-clamp technique, and the effect of redox agents [dithiothreitol, DTT and 2,2’-dithio-bis(5-nitropyridine), DTBNP] was tested. Kv current was decreased by DTT, but increased by DTBNP. DTT accelerated the inactivation kinetics, but did not affect steady-state activation and inactivation, whereas DTBNP accelerated activation kinetics. Kv current has a non-inactivating window in the range of from –40 mV to +10 mV. The resting membrane potential measured using the nystatin-perforated-patch method, however, lay between –50 mV and –30 mV and was not depolarized by 5 mM 4-aminopyridine. The membrane-impermeable oxidizing agent DTNB has no effect on Kv current, suggesting that redox modulation sites are intracellular sulphydryl groups. In EASMC, Kv current was decreased by DTT, but increased by DTBNP, indicating that the redox-potential-induced modulation of Kv current in EASMC and in PASMC is the same. It is therefore concluded that Kv current is modulated by the cellular redox potential, but that this modulation is not involved in HPV as a primary sensing mechanism.

ATP-sensitive potassium channels are modulated by intracellular lactate in rabbit ventricular myocytes

During myocardial ischemia, increased anaerobic glycolysis results in the accumulation of large amount of intracellular lactate. Effects of lactate on the ATP-sensitive potassium (KATP) channels were examined in rabbit ventricular myocytes, using the inside-out patch-clamp technique. Millimolar concentrations of lactate, applied to the cytosolic side of the patch membrane, induced openings of the kATP channel. This effect was inhibited by 0.1 mM glybenclamide. Lactate-induced openings of the channel were increased in a dose-dependent fashion. In dose-response relation for lactate, Kd (the lactate concentration producing half-maximal activation) and n (Hill coefficient) were 20 mM and 1.3, respectively (n=5). Activation of KATP channels by lactate occurred even in the presence of 2 mM ATP. Lactate also caused a significant increase in Ki, the ATP concentration causing half-maximal inhibition, from 70 μM in control (n=7) to 232 μM (n=5). From the above results it could be concluded that intracellular lactate modulate kATP channels directly and such modulation may resolve the discrepancy between the low KI in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.