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PubMed1995

Pharmacological analysis of heartbeat in Drosophila.

Analysis of the mechanisms underlying cardiac excitability can be facilitated greatly by mutations that disrupt ion channels and receptors involved in this excitability. With an extensive repertoire of such mutations, Drosophila provides the best available genetic model for these studies. However, the use of Drosophila for this purpose has been severely handicapped by lack of a suitable preparation of heart and a complete lack of knowledge about the ionic currents that underlie its excitability. We describe a simple preparation to measure heartbeat in Drosophila. This preparation was used to ask if heartbeat in Drosophila is myogenic in origin, and to determine the types of ion channels involved in influencing the heart rate. Tetrodotoxin, even at a high concentration of 40 microM, did not affect heart rate, indicating that heartbeat may be myogenic in origin and that it may not be determined by Na+ channels. Heart rate was affected by PN200-110, verapamil, and diltiazem, which block vertebrate L-type Ca2+ channels. Thus, L-type channels, which contribute to the prolonged plateau of action potentials in vertebrate heart, may play a role in Drosophila cardiac excitability. It also suggests that Drosophila heart is subject to a similar intervention by organic Ca2+ channel blockers as the vertebrate heart. A role for K+ currents in the function of Drosophila heart was suggested by an effect of tetraethylammonium, which blocks all the four identified K+ currents in the larval body wall muscles, and quinidine, which blocks the delayed rectifier K+ current in these muscles. The preparation described here also provides an extremely simple method for identifying mutations that affect heart rate. Such mutations and pharmacological agents will be very useful for analyzing molecular components of cardiac excitability in Drosophila.

Action Potentials↗
PubMed2006

Modelling and imaging cardiac repolarization abnormalities.

Repolarization abnormalities, including those induced by the congenital or acquired long QT (LQT) syndrome, provide a substrate for life-threatening cardiac arrhythmias. In this article, we use computational biology to link HERG mutations mechanistically to the resulting abnormalities of the whole-cell action potential. We study how the kinetic properties of I(Ks) (the slow delayed rectifier) that are conferred by molecular subunit interactions, facilitate its role in repolarization and 'repolarization reserve'. A new noninvasive imaging modality (electrocardiographic imaging) is shown to image cardiac repolarization on the epicardial surface, suggesting its possible role in risk stratification, diagnosis and treatment of LQT syndrome.

Action Potentials↗
PubMed2002

Modulation of K(+) currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation.

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K(+) currents by 10 +/- 3 %. UTP and the analogues alpha,beta-methylene-ATP and 2-methylthio-ATP also inhibited K(+) currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K(+) currents could be inhibited by ADP (9 +/- 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that alpha,beta-methylene-ADP, often used to block the ecto-5'-nucleotidase, also inhibited voltage-gated K(+) currents (7 +/- 2.3 %). This inhibition was occluded by ADP, suggesting that alpha,beta-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5'-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, alpha,beta-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of alpha,beta-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K(+) currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation.

Adenosine Diphosphate↗
PubMed1991

Dihydropyridines interact with calcium-independent potassium currents in embryonic mammalian sensory neurons.

Early embryonic sensory neurons have two K currents resembling delayed rectifier and transient K currents of mature neurons. However, in contrast to those of adult neurons, the embryonic currents can hardly be separated either by electrophysiological or pharmacological methods, limiting their characterisation at these developmental stages. Using the whole-cell recording technique, we found that dihydropyridines (DHPs) inhibit the noninactivating component of the Ca-independent K currents of 13-day mouse embryo dorsal-root ganglion (DRG) cells. The inhibitory effect of nicardipine began around 0.5 microM and was nearly complete at 5 microM while Na currents were not altered. This effect was reversible and voltage-dependent. The same results were obtained using another DHP Ca antagonist, nimodipine, whereas Bay K 8644, a DHP Ca agonist, had no effect. Kinetic properties of the DHP-insensitive K current have been described and compared with those of transient K currents found in differentiated neurons. These results suggest that both Ca and K channels have DHP sites, possibly homologous, at this developmental stage. The DHP inhibition of Ca-independent K channels provides a new tool with which to study K channels both at a molecular level and during DRG development.

Animals↗
PubMed2002

Ethylbromide tamoxifen, a membrane-impermeant antiestrogen, activates smooth muscle calcium-activated large-conductance potassium channels from the extracellular side.

Smooth-muscle calcium-activated large-conductance potassium channels (BK channels) are activated by tamoxifen and 17-beta-estradiol. This increase in NP(o), the number of channels, N, multiplied by open probability, depends on the presence of the regulatory beta1-subunit. Furthermore, a previous study indicated that 17-beta-estradiol might bind an extracellular site on the beta1-subunit. Because tamoxifen and 17-beta-estradiol may share a common binding site, we hypothesized that tamoxifen activates BK channels through a site on the extracellular surface of the membrane. A membrane-impermeant analog of tamoxifen, ethylbromide tamoxifen, was synthesized and used to test this hypothesis in whole-cell, outside-out, cell-attached, and inside-out patches from canine colonic smooth muscle cells. Ethylbromide tamoxifen is positively charged and is therefore membrane-impermeant. In whole-cell experiments, ethylbromide tamoxifen increased K(+) current at potentials positive to +40 mV, which has previously been attributed to BK channels. Unlike tamoxifen, ethylbromide tamoxifen did not inhibit delayed rectifier current. In outside-out patches, ethylbromide tamoxifen increased BK channel NP(o) with an EC(50) value of 1 microM. Ethylbromide tamoxifen did not increase BK channel NP(o) in cell-attached or inside-out patches; however, subsequent addition of equimolar tamoxifen did. Both drugs diminished BK channel unitary conductance to a degree that paralleled the effect on NP(o), suggesting an additional interaction with the pore-forming alpha-subunit. An interaction of tamoxifen with the pore was supported by a right shift in the concentration-response curve for tetraethylammonium; similar results were evident with iberiotoxin and charybdotoxin block. Our data suggest that ethylbromide tamoxifen does not easily traverse the plasma membrane and that tamoxifen binding responsible for activation of BK channels is at an extracellular site. The tamoxifen binding site may be within the extracellular loop of the BK channel beta1-subunit or, alternatively, on an as-yet-unidentified mediator that has an extracellular binding site.

Analysis of Variance↗
PubMed1991

Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel.

Inactivation of ion channels is important in the control of membrane excitability. For example, delayed-rectifier K+ channels, which regulate action potential repolarization, are inactivated only slowly, whereas A-type K+ channels, which affect action potential duration and firing frequency, have both fast and slow inactivation. Fast inactivation of Na+ and K+ channels may result from the blocking of the permeation pathway by a positively charged cytoplasmic gate such as the one encoded by the first 20 amino acids of the Shaker B (ShB) K+ channel. We report here that mutation of five highly conserved residues between the proposed membrane-spanning segments S4 and S5 (also termed H4) of ShB affects the stability of the inactivated state and alters channel conductance. One such mutation stabilizes the inactivated state of ShB as well as the inactivated state induced in the delayed-rectifier type K+ channel drk1 by the cytoplasmic application of the ShB N-terminal peptide. The S4-S5 loop, therefore, probably forms part of a receptor for the inactivation gate and lies near the channel's permeation pathway.

Action Potentials↗
PubMed1997

Fast inactivation of delayed rectifier K conductance in squid giant axon and its cell bodies.

Inactivation of delayed rectifier K conductance (gk) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (approximately -10 mV in 50-70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12-18 degrees C, 4 mM internal MgATP) a large fraction of gK inactivates within 250 ms at -10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12 degrees C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kvl channels studied in heterologous expression systems.

Adenosine Triphosphate↗
PubMed1994

Ion channel modulators as potential positive inotropic compound for treatment of heart failure.

1. Current positive inotropy therapy of heart failure is associated with major problems: digoxin and the phosphodiesterase inhibitors can cause life-threatening toxicity while beta-adrenoceptor agonists become less effective inotropic compounds as heart failure progresses. A new approach to positive inotropy is ion channel modulation. 2. An increased influx of Na+ during the cardiac action potential, as measured with DPI 201-106 and BDF 9148 which increase the probability of the open state of the Na+ channel, will increase force of contraction. 3. Activation of L-type Ca2+ channels with Bay K 8644 will increase influx of Ca2+ and increase the force of contraction. However the Ca2+ channel activators developed to date have little potential for the treatment of heart failure as they are vasoconstrictors. 4. Blocking cardiac K+ channels is a possible mechanism of positive inotropy. Terikalant inhibits the inward rectifying K+ channel, tedisamil inhibits the transient outward K+ channel and dofetilide is one of the newly developed inhibitors of the slow delayed outward rectifying K+ channel. All these drugs prolong the cardiac action potential to increase Ca2+ entry and force of contraction. 5. Thus drugs which increase Na+ influx or block K+ channels represent exciting possibilities for positive inotropy and the potential of these compounds for the treatment of heart failure needs to be fully evaluated.

Animals↗
PubMed1995

Recent advances in understanding the molecular mechanisms of the long QT syndrome.

Competing theories to explain the congenital long QT syndrome have included an imbalance in sympathetic innervation of the heart or a defect in repolarizing ion currents. Recent studies have identified at least four chromosomal loci at which mutations cause the congenital long QT syndrome in different families. The specific genes mutated in affected individuals have been identified at two of these loci, and both encode cardiac ion channels. The affected genes are SCN5A, the cardiac sodium channel gene, and HERG, whose protein product likely underlies IKr, the rapidly activating delayed rectifier. Thus, currently available evidence indicates that the congenital long QT syndrome is a primary disease of cardiac ion channels. Abnormalities in either inward or outward currents can cause the disease. Ongoing studies are evaluating the function of the mutant ion channels and the relationship between individual mutations and the clinical manifestations of the syndrome.

Animals↗
PubMed2005

Acute hypoxia differentially regulates K(+) channels. Implications with respect to cardiac arrhythmia.

The first ion channels demonstrated to be sensitive to changes in oxygen tension were K(+) channels in glomus cells of the carotid body. Since then a number of hypoxia-sensitive ion channels have been identified. However, not all K(+) channels respond to hypoxia alike. This has raised some debate about how cells detect changes in oxygen tension. Because ion channels respond rapidly to hypoxia it has been proposed that the channel is itself an oxygen sensor. However, channel function can also be modified by thiol reducing and oxidizing agents, implicating reactive oxygen species as signals in hypoxic events. Cardiac ion channels can also be modified by hypoxia and redox agents. The rapid and slow components of the delayed rectifier K(+) channel are differentially regulated by hypoxia and beta-adrenergic receptor stimulation. Mutations in the genes that encode the subunits for the channel are associated with Long QT syndrome and sudden cardiac death. The implications with respect to effects of hypoxia on the channel and triggering of cardiac arrhythmia will be discussed.

Animals↗
PubMed2003

Effects of kinase inhibitors on TGF-beta induced upregulation of Kv1.3 K+ channels in brain macrophages.

Deactivation of brain macrophages (microglia) by transforming growth factor-beta (TGF-beta) is characterized by enhanced Kv1.3 K+ channel expression. The intracellular mechanisms by which TGF-beta causes K+ channel upregulation in microglia have remained unclear. We show here that the protein kinase inhibitor H7 abolishes TGF-beta-induced increases in delayed rectifier K+ current density. However, this effect cannot be related to inhibition of protein kinase C (PKC) or protein kinase A (PKA) activity, because specific PKC and PKA inhibitors did not exhibit effects identical to H7. TGF-beta-induced Kv1.3 channel expression was also unaffected by inhibitors of tyrosine kinase, Ca2+/calmodulin kinase II and mitogen-activated protein (MAP) kinase ERK. In contrast, delayed rectifier K+ current density was larger in TGF-beta-stimulated cells pretreated with the p38 MAP kinase inhibitor SB203580 or the phosphatidylinositol 3-OH (PI3) kinase inhibitor wortmannin, suggesting that both p38 MAP kinase and PI3 kinase regulate negatively the upregulation of Kv1.3 K+ channels in TGF-beta-treated microglial cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Voltage-gated sodium and potassium currents and their variation in calcitonin-secreting cells of the chick.

1. The electrical properties of dissociated ultimobranchial cells from chick embryos (18-20 days after fertilization) were studied using whole-cell patch electrodes. Antibodies for immunohistological identification of calcitonin-secreting cells in the preparation were obtained by immunizing rabbits with a conjugated analogue of eel calcitonin. 2. In a proportion of cells, spike-like action potentials were generated in response to depolarization when cells were immersed in standard saline containing 140 mM-Na+ but no Ca2+. When the membrane potential was shifted from a holding potential (-83 - -103 mV) to a test depolarization (-50 mV or more positive) under voltage-clamp conditions, a transient inward current was produced which was followed by a slowly developing outward current. 3. The inward current was identified as a Na+-carried current, since (1) the kinetics of the current seemed fast and the amplitude consistently depended on the holding potential, (2) replacement of external Na+ with choline ions reversibly abolished the current, and (3) external application of tetrodotoxin (1 microM) abolished the current completely. The cells in which inward currents were detected showed intense to intermediate degrees of staining with anti-calcitonin antibodies. 4. In some other cells, no regenerative potentials were evoked even with intense depolarization, but a delayed decrease in membrane depolarization during the current pulse was observed. Voltage-clamp experiments in these cells revealed the existence of slowly developing outward currents, and the cells showed an intermediate degree of antibody staining. 5. The outward currents in both types of cells were selectively diminished in the presence of K+ channel blockers such as tetraethylammonium (1-10 mM) or 4-aminopyridine (1 mM). When the pipette contained 120 mM-CsCl, none of the dissociated cells exhibited any appreciable outward currents. Thus, the outward currents were most likely to be membrane potential-dependent K+ currents. The potential dependency of activation and inactivation of the currents were consistent with those of delayed K+ rectifier. 6. In the remaining cells, only passive responses of membrane potentials were observed with current injection. No discernible voltage-dependent inward or outward currents were detected under voltage-clamp conditions. Although these cells had a similar appearance to the two types of cells previously mentioned under phase-contrast microscopy, none of them showed significant antibody staining. These cells were presumed to represent non-secretory or supporting cells within the gland.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways.

Ghrelin is an endogenous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca(2+) concentration ([Ca(2+)]i), which is determined by Ca(2+) influx and release from intracellular Ca(2+) storage sites. Ca(2+) influx is via voltage-gated Ca(2+) channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K(+) channels. The present study investigates the in vitroeffect of ghrelin on membrane voltage-gated K(+) channels in the GH3 rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K(+) currents under voltage-clamp conditions. In the presence of Co(2+) (1 mM, Ca(2+) channel blocker) and tetrodotoxin (1 microM, Na(+) channel blocker) in the bath solution, two types of voltage-gated K(+) currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K(+) current (IA) represented a significant proportion of total K(+) currents in some cells, whereas delayed rectifier K(+) current (IK) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both IA and IK currents to 48% and 64% of control, respectively. Application of apamin (1 microM, SK channel blocker) or charybdotoxin (1 microM, BK channel blocker) did not alter the K(+) current or the response to ghrelin. The ghrelin-induced reduction in K(+) currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K+ current response to ghrelin. These results suggest that ghrelin-induced reduction of voltage-gated K(+) currents in GH3 cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K(+) currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.

Animals↗
PubMed1997

Ion channel and exchange currents in single myocytes isolated from the rabbit atrioventricular node.

OBJECTIVE: To review findings from the authors' laboratory in studies of electrophysiological properties of single rod- and spindle-shaped myocytes from rabbit atrioventricular node (AVN). DESIGN: Single cells were isolated from the AVN of the rabbit heart with the use of enzymatic and mechanical dispersion. For recording, cells were superfused with a Tyrode's solution at 33 to 37 degrees C, and recordings were made with microelectrodes or patch pipettes under 'current' or voltage' clamp conditions. Results are expressed as mean +/- SEM. RESULTS: AVN cells had a mean membrane capacitance of 40 +/- 3.9 pF and membrane resistance of 565 +/- 167 M omega (n = 9). Spontaneously active cells exhibited pacemaker activity showing a clear diastolic depolarization and overshooting action potential (AP) with a relatively slow upstroke velocity (7.4 +/- 0.9 V/s, n = 6) and a maximum diastolic potential of -70.5 +/- 2.9 mV. Under voltage clamp conditions, depolarizing pulses from 40 mV elicited L-type calcium currents sensitive to inhibition by nifedipine and managanese or cadmium ions, which could also block spontaneous APs. Depolarizing pulses also activated delayed rectifier potassium current (IK). IK showed rapid activation, and IK 'tails' in AVN cells were blocked by 5 microM E4031, consistent with the rapidly activating subtype of IK (IKr). IK was similar in AVN and ventricular myocytes, except for the time-course of deactivation, which was faster in AVN cells. In 80% to 90% of cells, hyperpolarizing voltage steps activated a small time-independent current. Ten per cent to 20% of cells showed the hyperpolarization-activated current (I(f)), but I(f) amplitude was only significant at potentials more negative than the pacemaker potential. AVN cells showed an apparent absence of inwardly rectifying potassium current. CONCLUSIONS: The high membrane resistance of AVN cells suggests that only small changes in ionic currents could significantly affect membrane potential. L-type calcium current is important in generating the AP upstroke, and IKr may play a role in both AP repolarization and diastolic depolarization. The ionic basis underlying spontaneous activity is not yet clear, but in some cells I(f) is not required because cells without I(f) can generate spontaneous APs.

Animals↗
PubMed1993

Effects of ATP-sensitive K+ channel openers on pacemaker activity in isolated single rabbit sino-atrial node cells.

Electrophysiologic effects of ATP-sensitive K+ channel openers (cromakalim, pinacidil, and nicorandil) in single rabbit sino-atrial (SA) node cells were examined by a whole-cell voltage- and current-clamp technique. Cromakalim (> 30 microM), pinacidil (> 1 microM), and nicorandil (> 500 microM) caused a negative chronotropic effect. At low concentrations, the maximum diastolic potential was hyperpolarized and the maximum rate of depolarization was enhanced, but at high concentrations, both were reversed and action potential amplitude (APA) was decreased significantly. Pinacidil (> 30 microM) and nicorandil (> 3 microM) prolonged AP duration (APD), but cromakalim did not affect it. In whole-cell voltage-clamp experiments, cromakalim (100 microM), pinacidil (> 30 microM), and nicorandil (> 300 microM) inhibited the Ca2+ current significantly but had little or no effect on the delayed rectifying K+ current and the hyperpolarization-activated inward current. Glibenclamide (1 microM) did not antagonize the effects of K+ channel openers on Ca2+ current and APs. These results indicate that the K+ channel openers have direct inhibitory actions on spontaneous APs and Ca2+ current in rabbit SA node cells (but K+ current was unaffected), resulting in decreased pacemaker activity.

Action Potentials↗
PubMed1999

Modelling action potentials and membrane currents of mammalian skeletal muscle fibres in coherence with potassium concentration changes in the T-tubular system.

During prolonged activity the action potentials of skeletal muscle fibres change their shape. A model study was made as to whether potassium accumulation and removal in the tubular space is important with respect to those variations. Classical Hodgkin-Huxley type sodium and (potassium) delayed rectifier currents were used to determine the sarcolemmal and tubular action potentials. The resting membrane potential was described with a chloride conductance, a potassium conductance (inward rather than outward rectifier) and a sodium conductance (minor influence) in both sarcolemmal and tubular membranes. The two potassium conductances, the Na-K pump and the potassium diffusion between tubular compartments and to the external medium contributed to the settlement of the potassium concentration in the tubular space. This space was divided into 20 coupled concentric compartments. In the longitudinal direction the fibre was a cable series of 56 short segments. All the results are concerned with one of the middle segments. During action potentials, potassium accumulates in the tubular space by outward current through both the delayed and inward rectifier potassium conductances. In between the action potentials the potassium concentration decreases in all compartments owing to potassium removal processes. In the outer tubular compartment the diffusion-driven potassium export to the bathing solution is the main process. In the inner tubular compartment, potassium removal is mainly effected by re-uptake into the sarcoplasm by means of the inward rectifier and the Na-K pump. This inward transport of potassium strongly reduces the positive shift of the tubular resting membrane potential and the consequent decrease of the action potential amplitude caused by inactivation of the sodium channels. Therefore, both potassium removal processes maintain excitability of the tubular membrane in the centre of the fibre, promote excitation-contraction coupling and contribute to the prevention of fatigue.

Action Potentials↗
PubMed1999

Effects of anion channel antagonists in canine colonic myocytes: comparative pharmacology of Cl-, Ca2+ and K+ currents.

1. Volume-Sensitive, Outwardly Rectifying (VSOR) Cl- currents were measured in canine colonic myocytes by whole-cell patch clamp. Decreasing extracellular osmolarity 50 milliosmoles l-1 activated current that was carried by Cl- and 5 - 7 times greater in the outward direction. 2. Niflumic acid, an inhibitor of Ca2+-activated Cl- channels, did not inhibit VSOR Cl- current. Glibenclamide, an antagonist of CFTR, and anthracene-9-carboxylate (9-AC) inhibited current less than 25% at 100 microM. 3. DIDS (4, 4-diisothiocyanato-stilbene-2,2'disulphonate) inhibited VSOR Cl- current more potently than SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonate). IC50s were 0.84 and 226 microM, respectively. 4. VSOR Cl- current was strongly inhibited by tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene), an anti-oestrogen compound (IC50=0.57 microM). 5. Gd3+ antagonized VSOR Cl- current more potently than La3+. The IC50 for Gd3+ was 23 microM. In contrast, 100 microM La3+ inhibited current only 35+/-7%. 6. Antagonists of VSOR Cl- current had non-specific effects. These compounds blocked voltage-dependent K+ and Ca2+ currents in colonic myocytes. Tamoxifen (10 microM) and DIDS (10 microM) inhibited L-type Ca2+ current 87+/-7 and 31+/-5%, respectively. Additionally, in the presence of 300 nM charybdotoxin, tamoxifen (1 microM) and DIDS (10 microM) inhibited delayed rectifier K+ current 38+/-8 and 10+/-2%, respectively. 7. The pharmacology of VSOR Cl- channels overlaps with voltage-dependent cation channels. DIDS and tamoxifen inhibited VSOR Cl- equally. However, because DIDS had much less effect on L-type Ca2+ and delayed rectifier K+ channels than did tamoxifen, it might be useful in experiments to investigate the physiological and pathophysiological role of this conductance in whole tissues.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Electrophysiologic alterations in the rabbit nodal cells induced by membrane lipid peroxidation.

To investigate cellular electrophysiologic alterations due to lipid peroxidation of the cell membrane by free radicals as a possible cause of coronary reperfusion arrhythmias, we studied the effects of t-butyl hydroperoxide on the spontaneous action potential and membrane currents of the rabbit sinoatrial and atrioventricular node preparations (0.2 x 0.2 x 0.1 mm). 1-5 min of superfusion with t-butyl hydroperoxide (100-500 microM) caused a transient increase in the spontaneous firing frequency by 9%, accompanied by a 4% increase in the action potential amplitude and a 33% increase in the maximal rate of depolarization (P < 0.05, n = 6). t-Butyl hydroperoxide then gradually suppressed physiological automaticity, but induced abnormal repetitive firing due to early and delayed after-depolarizations. 15 min of superfusion with t-butyl hydroperoxide caused a complete standstill of nodal cells at a resting potential of -46 +/- 3 mV (n = 12). Such effects of t-butyl hydroperoxide on the spontaneous action potential were attenuated by pretreating the cells with butylated hydroxytoluene, a lipid peroxidation inhibitor. Voltage clamp experiments using double microelectrode methods revealed that t-butyl hydroperoxide transiently increased the Ca2+ current by 22% after 5 min of superfusion but subsequently reduced it to 46% of the control value after 15 min (P < 0.05, n = 6). Similar biphasic changes were observed in the delayed rectifying K+ current and hyperpolarization-activated inward current (n = 6). Background current was progressively increased without any change in its reversal potential (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗