New index for categorising cardiac reentrant wave: in silico evaluation.

Based on the similarity between a reentrant wave in cardiac tissue and a vortex in fluid dynamics, the authors hypothesised that a new non-dimensional index, like the Reynolds number in fluid dynamics, may play a critical role in categorising reentrant wave dynamics. Therefore the goal of the present study is to devise a new index to characterise electric wave conduction in cardiac tissue and examined whether this index can be used as a biomarker for categorising the reentrant wave pattern in cardiac tissue. Similar to the procedure used to derive the Reynolds number in fluid dynamics, the authors used a non-dimensionalisation technique to obtain the new index. Its usefulness was verified using a two-dimensional simulation model of electrical wave propagation in cardiac tissue. The simulation results showed that electrical waves in cardiac tissue move into an unstable region when the index exceeds a threshold value.

Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Role of arachidonic acid-derived metabolites in the control of pulmonary arterial pressure and hypoxic pulmonary vasoconstriction in rats.

BACKGROUND: The roles of arachidonic acid (AA) metabolites in hypoxia-induced pulmonary vasoconstriction (HPV), a critical physiological mechanism that prevents ventilation/perfusion mismatch, are still incompletely understood. METHODS: Pulmonary arterial pressure was measured in ventilated/perfused rat lungs. Isometric tones of rat intralobar pulmonary arteries were also measured, using a myograph. RESULTS: Hypoxia (Po2, 3%)-induced pulmonary arterial pressure increases (ΔPAP(hypox)) were stable with blood-mixed perfusate, but decayed spontaneously. ΔPAP(hypox) was inhibited by 29%, 16%, and 28% by the thromboxane A2 (TXA2) antagonist SQ-29548, the 5-lipoxygenase inhibitor, MK886, and the leukotriene D4 antagonist, LY-171883, respectively. The prostacyclin synthase inhibitor tranylcypromine augmented ΔPAP(hypox) by 5%, whereas inhibition of cytochrome P450 did not affect ΔPAP(hypox). Consistently, the TXA2 analogue U46619 increased ΔPAP(hypox) whereas prostacyclin abolished ΔPAP(hypox). However, leukotriene D4 had no direct effect on ΔPAP(hypox). In the isolated pulmonary arteries, pretreatment with U46619 was essential to demonstrate hypoxia-induced contraction. CONCLUSIONS: The above results suggest that TXA2 and cysteinyl leukotrienes, other than leukotriene D4, are endogenous factors that facilitate HPV in rats. The indispensable role of TXA2-induced pretone in the HPV of isolated pulmonary arteries indicates that the signal from thromboxane receptors might be a critical component of oxygen sensation mechanisms.

Ca2+-dependent membrane currents in vascular smooth muscle cells of the rabbit.

The membrane potential in vascular smooth muscle cells contributes to the regulation of cytosolic [Ca2+], which in turn regulates membrane potential by means of Ca2+i-dependent ionic currents. We investigated the characteristics of Ca2+i-dependent currents in rabbit coronary and pulmonary arterial smooth muscle cells. Ca2+i-dependent currents were recorded using the whole-cell patch-clamp technique while cytosolic [Ca2+] was increased by caffeine. The reversal potentials of caffeine-induced currents were between -80 and -10 mV under normal ionic conditions, whereas they were about 0 mV when K+-free NaCl solutions were used both in pipette and bath. The total substitution of extracellular Na+ with membrane-impermeable cation N-Methyl-D-glucamine did not affect caffeine-induced currents, implying no significant contribution of Na+ as a permeant ion to the currents. The substitution of extracellular NaCl with sucrose reduced outward component of the currents and shifted the reversal potentials according to the change in Cl- equilibrium potential. Upon application of the niflumic acid under K+-free conditions, most of the current induced by caffeine was inhibited. Taken together, the results of the present study indicate that K+ and Cl- currents are major components of Ca2+i-dependent currents in vascular smooth muscles isolated from coronary and pulmonary arteries of the rabbit, and the relative contribution of each type of current to total currents are not different between the two arteries.

Inhibition of acetylcholine-activated K(+) currents by U73122 is mediated by the inhibition of PIP(2)-channel interaction.

1. We have investigated the effect of U73122, a specific inhibitor of phospholipase C (PLC), on acetylcholine-activated K(+) currents (I(KACh)) in mouse atrial myocytes. 2. In perforated patch clamp mode, I(KACh) was activated by 10 microM acetylcholine. When atrial myocytes were pretreated with U73122 or U73343, I(KACh) was inhibited dose-dependently (half-maximal inhibition at 0.12+/-0.0085 and 0.16+/-0.0176 microM, respectively). The current-voltage relationships for I(KACh) in the absence and in the presence of U73122 showed that the inhibition occurred uniformly from -120 to +40 mV, indicating a voltage-independent inhibition. 3. When U73122 was applied after I(KACh) reached steady-state, a gradual decrease in I(KACh) was observed. The time course of the current decrease was well fitted to a single exponential, and the rate constant was proportional to the concentration of U73122. 4. When K(ACh) channels were directly activated by adding 1 mM GTP gamma S to the bath solution in inside-out patches, U73122 (1 microM) decreased the open probability significantly without change in mean open time. When K(ACh) channels were activated independently of G-protein activation by 20 mM Na(+), open probability was also inhibited by U73122. 5. Voltage-activated K(+) currents and inward rectifying K(+) currents were not affected by U73122. 6. These findings show that inhibition by U73122 and U73343 of K(ACh) channels occurs at a level downstream of the action of G beta gamma or Na(+) on channel activation. The interference with phosphatidylinositol 4,5-bisphosphate (PIP(2))-channel interaction can be suggested as a most plausible mechanism.

Inhibition of acetylcholine-activated K(+) current by chelerythrine and bisindolylmaleimide I in atrial myocytes from mice.

The effects of the protein kinase C inhibitors chelerythrine and bisindolylmaleimide I on acetylcholine-activated K+ currents (I(KACh)) were examined in atrial myocytes of mice, using the patch clamp technique. Chelerythrine and bisindolylmaleimide I inhibited I(KACh) in a reversible and dose-dependent manner. Half-maximal effective concentrations were 0.49+/-0.01 microM for chelerythrine and 98.69+/-12.68 nM for bisindolylmaleimide I. However, I(KACh) was not affected either by calphostin C, which is also known as a protein kinase C inhibitor, or by a protein kinase C activator, phorbol 12,13-dibutyrate. When K(ACh) channels were activated directly by adding 1 mM GTPgammaS to the bath solution in inside-out patches, chelerythrine (10 microM) decreased the open probability from 0.043+/-0.01 to 0.014+/-0.007 (n=5), but bisindolylmaleimide I did not affect the channel activity. From these results, it is concluded that both chelerythrine and bisindolylmaleimide I inhibit K(ACh) channels independently of protein kinase C inhibition, but the level of inhibition is different.

Modulation of ATP-sensitive potassium channels by cGMP-dependent protein kinase in rabbit ventricular myocytes.

This investigation used a patch clamp technique to test the hypothesis that protein kinase G (PKG) contributes to the phosphorylation and activation of ATP-sensitive K(+) (K(ATP)) channels in rabbit ventricular myocytes. Nitric oxide donors and PKG activators facilitated pinacidil-induced K(ATP) channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. In contrast, neither a selective protein kinase A (PKA) activator nor inhibitor had any effect on K(ATP) channels at concentrations up to 100 and 10 microm, respectively. Exogenous PKG, in the presence of both cGMP and ATP, increased channel activity, while the catalytic subunit of PKA had no effect. PKG activity was prevented by heat inactivation, replacing ATP with adenosine 5'-O-(thiotriphosphate) (a nonhydrolyzable analog of ATP), removing Mg(2+) from the internal solution, applying a PKG inhibitor, or by adding exogenous protein phosphatase 2A. The effects of cGMP analogs and PKG were observed under conditions in which PKA was repressed by a selective PKA inhibitor. The results suggest that K(ATP) channels are regulated by a PKG-signaling pathway that acts via PKG-dependent phosphorylation. This mechanism may, at least in part, contribute to a signaling pathway that induces ischemic preconditioning in rabbit ventricular myocytes.

Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in alpha(1)-adrenergic pathway via the modulation of acetylcholine-activated K(+) channels in mouse atrial myocytes.

We have investigated the effect of alpha(1)-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 microm 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(KACh) was 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 (PIP(2)) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization of I(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 K(ACh) channel is modulated by PE through PLC stimulation and depletion of PIP(2).

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

1. 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 (I(Kr)) in rat atrial myocytes. 2. 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. IC(50) for amitriptyline block of HERG current was progressively decreased according to depolarization: IC(50) values at -30, -10, +10 and +30 mV were 23.0, 8.71, 5.96 and 4.66 microM, respectively. 3. 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. 4. Steady-state block by amitriptyline was obtained while depolarization to +20 mV for 0.5 s was applied at 0.5 Hz: IC(50) was 3.26 microM in 2 mM [K(+)](o). It was increased to 4. 78 microM in 4 mM [K(+)](o), suggesting that the affinity of amitriptyline on HERG was decreased by external K(+). 5. In rat atrial myocytes bathed in 35 degrees C, 5 microM amitriptyline blocked I(Kr) by 55%. However, transient outward K(+) current (I(to)) was not significantly affected. 6. In summary, the data suggest that the block of HERG currents may contribute to arrhythmogenic side effects of amitriptyline.

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 microM (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 microM) strongly inhibited channel opening both from the inside and outside. In the presence of Gd3+ (100 microM) 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 stret 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.

Evidence of enhancement of malate-aspartate shuttle activity in beta cells of streptozotocin-induced non-insulin-dependent diabetic rats.

Glucose-induced insulin secretion is selectively impaired in beta cells from animals with non-insulin-dependent diabetes mellitus (NIDDM). This study was performed to clarify whether the malate-aspartate shuttle among the glucose metabolic pathways is intact in beta cells of NIDDM rats. The insulin secretory capacity of the islets and the K(ATP) channel activity in single beta cells were measured in control and NIDDM rats injected with streptozotocin (STZ) during the neonatal period, using a radioimmunoassay and patch-clamp technique. The increase of insulin secretion induced by 11.1 mmol/L glucose or 10 mmol/L dihydroxyacetone (DHA) was significantly reduced in NIDDM islets, suggesting an impaired glycerol-phosphate shuttle. The application of glyceraldehyde (10 mmol/L) in NIDDM or control islets elicited an increase in insulin secretion, but the difference between the 2 groups was indistinguishable. On the contrary, the increase of insulin secretion and the inhibition of K(ATP) channel activity induced by aspartate, which preferentially participates in the malate-aspartate shuttle, were significantly greater in NIDDM versus the control. However, intracellularly applied aspartate in the inside-out mode did not inhibit K(ATP) channel activity. These findings show that malate-aspartate shuttle activity is potentiated in pancreatic beta cells of NIDDM rats, suggesting the development of a compensatory mechanism for the reduced activity of the glycerol-phosphate shuttle in NIDDM.

Permeability characteristics of monovalent cations in atrial myocytes of the rat heart.

We investigated the permeability of Cs+ and Na+ through various ion channels in rat atrial myocytes using the whole-cell voltage-clamp technique. With isotonic CsCl (140 mM) on both sides of the membrane and nominally [Ca2+]o-free conditions, depolarising clamp pulses induced an increase of outward currents which showed a biphasic time course. Repolarisation to the holding potential induced inward tail currents. With isotonic NaCl, depolarisation also induced outward currents which showed a monotonic decay, but inward tail currents were not observed. Both in NaCl and CsCl, currents were hardly affected by TEA (10 mM), 4-AP (5 mM) and DIDS (100 microM). Nicardipine (1 M) almost completely blocked time-dependent outward currents in isotonic NaCl solution, leaving only time-independent currents which showed linear I-V relationship. In isotonic CsCl conditions, nicardipine blocked outward current considerably, but there still remained time-dependent outward currents and inward tail currents. Addition of E-4031 (2-20 M) which is known as a specific blocker of the rapidly activating delayed rectifier K+ current (IKr) completely blocked these time-dependent outward and inward currents, leaving only a time-independent current. Time-independent currents recorded in the presence of nicardipine and E-4031 were inhibited by GdCl3, which is known to block non-selective cation (NSC) currents. From these results, it was suggested that NSC current in atrial myocytes can be investigated in isotonic Cs+ or Na+ solution in the presence of Ca2+ channel and IKr blockers.

Different contractile properties between intralobar and extralobar pulmonary arteries of the rabbit.

In pulmonary circulation, small muscular resistance arteries are known to have different receptor properties and sensitivity to neurotransmitters from those of large elastic conduit arteries. It is, however, not yet certain whether the different properties are primarily due to the diameter or the location of arteries. In the present study, we compared the contractile responses to various agonists among large extralobar (ELPA, diameter: 2-3 mm), large intralobar (ILPA, diameter: 2-3 mm), and small intralobar pulmonary arteries (SPA, diameter: 300-500 microm) of the rabbit. There were no differences in normalized dose-response curves to KCl among three groups. Half maximum doses (EC50 in mM) were 38.0+/-2.0 (n=8, mean+/-SEM) in ELPA, 36.9+/-2.4 (n=10) in ILPA, and 39.0+/-0.9 (n=12) in SPA. Responses to phenylephrine, epinephrine, histamine, serotonin, and PGF2alpha were normalized and expressed as a relative contraction against maximum tension to KCl. In ELPA, the contractile responses to various agents showed the following sequence: KCl>epinephrine>phenylephrine>serotonin>PGF2alpha>histamine. In ILPA and SPA, the sequence was: KCl>histamine>PGF2alpha>serotonin. There was little response to phenylephrine and epinephrine in ILPA and SPA. These results demonstrate that the difference of contractile responses between ELPA and ILPA was more prominent than that between ILPA and SPA, suggesting that the location is more important than the diameter itself in determining the characteristic contractile responses of pulmonary arteries.

Blockade of HERG channels expressed in Xenopus oocytes by external H+.

We have investigated the effect of external H+ concentration ([H+]o) on the human-ether-a-go-go-related gene (HERG) current (IHERG), the molecular equivalent of the cardiac delayed rectifier potassium current (IKr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+]o was increased, the amplitude of the IHERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pHo) values (the potential required for half-maximum activation, V1/2, was: -41.8 mV, -38.0 mV, -33.7 mV, -26.7 mV in pHo 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (gmax) was also affected by [H+]o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pHo from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V1/2 caused by increasing [H+]o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in gmax caused by increasing [H+]o was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+]o by two different mechanisms: voltage-dependent blockade (shift of V1/2) and the decrease in gmax. With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing gmax, their interaction is non-competitive.

Blockade of the delayed rectifier K+ currents, IKr, in rabbit sinoatrial node cells by external divalent cations.

We investigated the actions of various divalent cations on the delayed rectifier K+ currents (IKr) in rabbit sinoatrial node cells using the whole-cell voltage-clamp technique in isotonic K+ solutions. External divalent cations decreased the amplitude of currents, accelerated the time course of deactivation and shifted the activation to positive potentials in a dose-dependent manner. The concentrations for half-maximum inhibition of the steady-state currents (KM) obtained at 0 mV were 0.63, 1.36, 1.65 and 2.16 mM for Ni2+, Co2+, Mn2+ and Ba2+, respectively. The effect was voltage dependent (KM decreased e-fold for 12.2-16.8 mV hyperpolarization), but the dependence did not vary significantly among different cations. Acceleration of the time course of current deactivation by the increase of divalent cation concentration was well fitted by the voltage-dependent block model, and the binding rate constant (k1) was obtained. The binding rates for the ions took the following order: Ni2+ >Co2+ >Mn2+ >Ba2+. The degree of the shift of activation occurred in the same order: Ni2+ >Co2+ >Mn2+ >Ba2+. From these results, we conclude that IKr channels are non-selectively blocked by most divalent cations from the external side and that the binding site is located deep inside the channel, resulting in a steep voltage dependence of the blockade.

Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations.

We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance.

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.

Effects of intracellular N+, M2+ and metabolites on C2+-activated K+ channels in pulmonary and ear arterial smooth muscle cells of the rabbit.

Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism for matching the ventilation/perfusion ratio in the lung, but the signal transduction pathway through which hypoxia induces vasoconstriction remains unclear. We hypothesized that the decrease in K+ current induced by hypoxia is a key mechanism for HPV, and examined the effects of the substances which are expected to accumulate during hypoxia on the activity of large conductance Ca2+-activated K+ (BKCa) channels. Pulmonary and ear arterial smooth muscle cells were isolated from the rabbit using enzymatic digestion, and large conductance Ca2+-activated K+ current (IBK,Ca) was recorded in symmetrical K+ concentrations using the inside-out mode of the patch-clamp technique. Increasing the Na+ concentration on the intracellular side suppressed IBK,Ca dose dependently: 4.6, 20.9, 35.5 and 44.6 % reduction with 4, 8, 12 and 16 mM Na+, respectively. Mg2+ also reduced IBK,Ca, and the maximum reduction was obtained at 0.5 mM. Lactate, adenosine, ADP and ATP did not significantly affect IBK,Ca. There was no difference between pulmonary and ear arterial smooth muscle cells in their response to the above substances; this finding rules out modulation of BKCa channels by the various factors thought to accumulate during hypoxia as a major mechanism involved in the decrease in the K+ conductance of pulmonary arteries in hypoxia.

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.

Voltage-dependent blockade of HERG channels expressed in Xenopus oocytes by external Ca2+ and Mg2+.

1. We expressed the human eag-related gene (HERG), which is known to encode the delayed rectifier K+ current (IKr) in cardiac muscle, in Xenopus oocytes. Using a two-microelectrode voltage clamp technique, the effect of external Ca2+ and Mg2+ on the HERG current (IHERG) was investigated. 2. When [Ca2+]o was increased, the amplitude of outward IHERG elicited by depolarization decreased, and the rate of current onset slowed. The rate of current decay observed on repolarization was greatly accelerated. The threshold and fully activated potential of IHERG shifted to a more positive potential. On the other hand, the inactivation property represented by the negative slope of the I-V curve and the instantaneous conductance of IHERG were little affected by [Ca2+]o. 3. The effect of [Ca2+]o on IHERG can be interpreted using the channel blockade model. The blockade is voltage dependent; smaller dissociation constants (KM) at more negative potentials indicate that block is facilitated by hyperpolarization. KM changes e-fold for 14.5 mV and the fractional electrical distance of the binding site calculated from this value is 0.86. 4. Blockade by a low concentration of Ca2+ (0.5 mM) was inhibited by increasing [K+]o (from 2 to 20 mM), whereas blockade by a high concentration of Ca2+ (5 mM) was not affected by varying [K+]o, indicating that there is competition between permeating ions and blocking ions. 5. The effect of [Mg2+]o on IHERG was qualitatively similar to that of [Ca2+]o, but the potency was lower. 6. These results suggest that external Ca2+ and Mg2+ block the HERG channel in a voltage- and time-dependent manner, resulting in a voltage dependence which has been regarded as a property of the activation gate.