Biexponential Time Course Research Articles

Enhancement of retention and cytotoxicity of 2-chlorodeoxyadenosine in cultured human leukemic lymphoblasts by nitrobenzylthioinosine, an inhibitor of equilibrative nucleoside transport.

In leukemic cells exposed to 2-chlorodeoxyadenosine (2-CdA), levels of the nucleoside drug and its phosphate metabolites decay with time in the absence of external 2-CdA; an intrinsic part of this process is the efflux of 2-CdA. The effects of nitrobenzylthioinosine (NBMPR) and of dipyridamole (DPM), both potent inhibitors of es (e, equilibrative; s, sensitive to NBMPR) nucleoside transport processes, were studied in four lines of cultured leukemic lymphoblasts. Suspensions of 2-CdA-loaded cells were diluted 10-fold with 2-CdA-free medium to initiate the cellular 2-CdA decay processes, which followed a biexponential time course. When diluting media contained NBMPR or DPM, intracellular levels of 2-CdA and its metabolites were substantially increased (P < 0.001) compared with cells in media lacking the transport inhibitors, and 2-CdA loss followed a monoexponential time course. As a consequence, the AUCs (area under time-course plots of intracellular 2-CdA and its metabolites) were significantly (P < 0.001) lower in untreated control cells compared to inhibitor-treated cells. These results suggest that nucleoside transport processes contribute to the efflux of 2-CdA from the cultured lymphoblasts. The cytotoxicity of 1-h exposure to 2-CdA of Reh-A2 and CCRF-CEM cells was enhanced three-fold by subsequent exposure to 0.5 microM NBMPR relative to that of control cells subjected to the same manipulations without NBMPR exposure. However, before such a strategy may be considered to have a therapeutic application, careful examination of effects in normal lymphocytes and ex vivo leukemic lymphoblasts must first be undertaken. Leukemia (2000) 14, 52-60.

Characteristics of a transient outward current (sensitive to 4-aminopyridine) in Ca2+-tolerant myocytes isolated from the rabbit atrioventricular node.

A transient outward current (Ito) has been observed in the atrioventricular node (AVN), but its characteristics in Ca-tolerant AVN myocytes have not been investigated previously. In this study, Ito was measured from Ca-tolerant rabbit AVN myocytes at 37 degrees C, using the whole-cell patch-clamp technique. With interfering currents inhibited, 500-ms voltage-clamp pulses applied from -80 mV elicited Ito at potentials positive to -30 mV, which increased in magnitude with test potential amplitude. This current was completely blocked by external application of 5 mM 4-aminopyridine (4-AP). During a command pulse, Ito activated rapidly then inactivated with a bi-exponential time-course. Fast and slow time constants of current inactivation (tauf and taus, respectively) showed voltage dependence. At 0 mV, tauf was 14.5+/-2.7 ms and taus was 112.8+/-21. 2 ms, whilst at +60 mV tauf was 6.7+/-1.1 ms and taus was 63.7+/-9.2 ms (n=25). The steady-state inactivation relationship showed half-maximal inactivation at -33.8 mV (n=8). Re-activation of Ito after an inactivating pre-pulse showed a bi-exponential time-course of recovery: tau1 was 196+/-70 ms, and tau2 was 2707+/-1010 ms (n=6, at -80 mV). Repetitive application of voltage-clamp test pulses showed that Ito inactivation accumulated on repetitive stimulation, but reached a steady state rapidly for a given pulse frequency (0. 2-1.0 Hz). AVN Ito was sensitive to the class 1 anti-arrhythmic flecainide (EC50 for peak current of 24 microM), which showed selectivity for the rapidly inactivating current component. Quinidine also inhibited Ito in a dose-dependent fashion, but did not affect the current time-course. Under voltage-clamp conditions, a simulated diastolic depolarisation from -70 to -45 mV did not significantly reduce Ito amplitude, and under current-clamp conditions 4-AP inhibited spontaneous action potentials. Although this is consistent with a significant role for Ito in shaping AVN activity, under the conditions of this study 4-AP also partially blocked the "rapid" delayed rectifier current, IKr, and so the effects of 4-AP on action potentials could not be attributed exclusively to its effects on Ito.

Voltage-activated K+ currents of hypoglossal motoneurons in a brain stem slice preparation from the neonatal rat.

Whole cell, patch-clamp recordings were performed on motoneurons of the hypoglossus nucleus in a brain stem slice preparation from the neonatal rat brain. The aim was to investigate transient outward currents activated by membrane depolarization under voltage clamp conditions. In a Ca2+-free medium containing tetrodotoxin and Cs+, depolarizing voltage commands from a holding potential of -50 mV induced slow outward currents (Islow) with 34 +/- 6 ms (SE) onset time constant at 0 mV and minimal decline during a 1 s pulse depolarization. When the depolarizing command was preceded by a prepulse to -110 mV, the outward current became biphasic as it comprised a faster component (Ifast), which could be investigated in isolation by subtracting the two sets of records. Ifast showed rapid kinetics (9 +/- 4 ms 10-90% rise time and 70 +/- 20 ms decay time constant at 0 mV) and strong voltage-dependent inactivation (half inactivation was at -92.9 +/- 0.2 mV) from which it readily recovered with a biexponential timecourse (4.4 +/- 0.6 and 17 +/- 2 ms time constants at -110 mV membrane potential). Islow was selectively blocked by TEA (10-30 mM) while Ifast was preferentially depressed by 2-3 mM 4-aminopyridine. Analysis of tail current reversal indicated that both Islow and Ifast were predominantly due to K+ with minor permeability to Na+ (92/1 and 50/1, respectively). These results suggest that membrane depolarization activated distinct K+ conductances that, in view of their largely dissimilar kinetics, are likely to play a differential role in regulating the firing behavior of hypoglossal motoneurons.

Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells.

1. Kainate receptors with pharmacological properties similar to those of the GluR5 subunit have been shown to modulate inhibitory synaptic transmission in the CA1 region of the hippocampus. The kinetic properties of currents gated by GluR5 receptors have not been examined in detail. Here we describe several biophysical features of recombinant GluR5 receptors expressed in HEK293 cells. 2. We found that homomeric GluR5 receptors can exhibit striking inter-cell variability in channel kinetics in response to the agonists kainate and glutamate. Desensitization rates in response to kainate varied between individual cells by nearly 1000-fold (range, 1.5 ms to 1.5 s), while glutamate desensitization rates differed by 9-fold (range, 1.0 to 9.0 ms). 3. The time course of recovery from desensitization in response to glutamate also showed inter-cell variation. The majority of glutamate currents in GluR5-expressing cells recovered from desensitization with two widely separated exponential components: 50 +/- 10 ms and 5.1 +/- 1.0 s (contributing 37.6 % and 62.4 % of the sum of the exponential fits, respectively). In contrast, currents with the fastest desensitization kinetics had a recovery time course of 4.8 +/- 0.3 s. 4. Kainate receptors in murine dorsal root ganglion neurons are likely to be composed of homomeric GluR5 subunits. These receptor currents recovered from glutamate desensitization with a biexponential time course of 36 +/- 4 ms and 4.7 +/- 0.7 s. 5. These results suggest that aspects of GluR5 kainate receptor function are modulated by intracellular mechanism(s). At synapses such mechanisms could regulate the frequency- response relationship of synaptic kainate receptors by altering their rate of entry into and recovery from desensitization.

Ultrarapid delayed rectifier current inactivation in human atrial myocytes: properties and consequences.

The ultrarapid delayed rectifier current (IK,ur) plays a significant role in human atrial repolarization and is generally believed to show little rate dependence because of slow and partial inactivation. This study was designed to evaluate in detail the properties and consequences of IK,ur inactivation in isolated human atrial myocytes. IK,ur inactivated with a biexponential time course and a half-inactivation voltage of -7.5 +/- 0.6 mV (mean +/- SE), with complete inactivation during 50-s pulses to voltages positive to +10 mV (37 degreesC). Recovery from inactivation proceeded slowly, with time constants of 0.42 +/- 0.06 and 7.9 +/- 0.9 s at -80 mV (37 degreesC). Substantial frequency dependence was observed at 37 degreesC over a clinically relevant range of frequencies. Inactivation was faster and occurred at more positive voltages at 37 degreesC compared with room temperature. The voltage and time dependencies of Kv1.5 inactivation were studied in Xenopus oocytes to avoid overlapping currents and strongly resembled those of IK,ur in native myocytes. We conclude that, while IK,ur inactivation is slow, it is extensive, and slow recovery from inactivation confers important frequency dependence with significant consequences for understanding the role of IK,ur in human atrial repolarization.

Evidence for Multiple Open and Inactivated States of the hKv1.5 Delayed Rectifier

The kinetic properties of hKv1.5, a Shaker-related cardiac delayed rectifier, expressed in Ltk − cells were studied. hKv1.5 currents elicited by membrane depolarizations exhibited a delay followed by biphasic activation. The biphasic activation remained after 5-s prepulses to membrane potentials between −80 and −30 mV; however, the relative amplitude of the slow component increased as the prepulse potential approached the threshold of channel activation, suggesting that the second component did not reflect activation from a hesitant state. The decay of tail currents at potentials between −80 and −30 mV was adequately described with a biexponential. The time course of deactivation slowed as the duration of the depolarizing pulse increased. This was due to a relative increase in the slowly decaying component, despite similar initial amplitudes reflecting a similar open probability after 50- and 500-ms prepulses. To further investigate transitions after the initial activated state, we examined the temperature dependence of inactivation. The time constants of slow inactivation displayed little temperature and voltage dependence, but the degree of the inactivation increased substantially with increased temperature. Recovery from inactivation proceeded with a biexponential time course, but long prepulses at depolarized potentials slowed the apparent rate of recovery from inactivation. These data strongly indicate that hKv1.5 has both multiple open states and multiple inactivated states.

L-type calcium current in human ventricular myocytes at a physiological temperature from children with tetralogy of Fallot.

The aim was to investigate the electrophysiological properties of the L-type calcium current (ICa,L) in ventricular myocytes at a physiological temperature (36-37 degrees C) isolated from children undergoing surgical repair of tetralogy of Fallot. ICa,L was recorded with the patch-clamp technique in the single electrode whose-cell mode at a physiological calcium concentration (1.8 mmol/l) at 36-37 degrees C. Under these conditions, maximum current density averaged -5.80 +/- 0.45 pA/pF. ICa,L showed a bell-shaped current-voltage relationship: the current activated at -37.7 +/- 1.36 mV, peaked at +9.41 +/- 1.60 mV and reversed at +57.7 +/- 2.12 mV (n = 17). At +10 mV, time to peak of ICa,L was 5.23 +/- 0.46 ms. Membrane potentials for half-maximal steady-state activation and inactivation of ICa,L were -6.02 and -20.4 mV, respectively, the slope factors were 7.16 mV for steady-state activation and 6.49 mV for steady-state inactivation. ICa,L did not completely inactivate and showed a big window current between -45 and +40 mV. The inactivation of ICa,L showed a biexponential time course with a fast time constant ranging from 9.11 to 12.9 ms and a slow time constant ranging from 60.9 to 220 ms between -30 and +30 mV. Only the slow time constant showed a pronounced voltage dependency. The recovery from inactivation of ICa,L was biphasic with a fast time constant of 60.7 ms and a slow time constant of 619 ms. beta-Adrenergic stimulation with isoprenaline (1 mumol/l) increased the ICa,L density from -5.71 +/- 1.55 to -13.8 +/- 1.96 pA/pF (142%; P < 0.05) at +10 mV. The present study demonstrates that most of the electrophysiological properties of ICa,L in ventricular myocytes isolated from children with tetralogy of Fallot resemble those of adult ventricular cells. The existence of a big calcium window current could be involved in the occurrence of early afterdepolarizations which could lead to the high incidence of arrhythmias after surgical repair of tetralogy of Fallot.

Static and dynamic membrane properties of large-terminal bipolar cells from goldfish retina: experimental test of a compartment model.

Capacitance measurements allow direct studies of exocytosis and endocytosis in single synaptic terminals isolated from bipolar neurons of goldfish retina. Extending the technique to intact bipolar cells, with their more complex morphology, requires information about the cells' electrotonic architecture. To this end, we developed a compartment model of bipolar neurons isolated from goldfish retina and tested the model experimentally. The isolated cells retained morphology similar to that of bipolar neurons in intact goldfish retina. In whole cell recordings, current relaxations in response to 10-mV hyperpolarizing voltage pulses decayed with a biexponential time course. This suggests that the cells may be described by a two-compartment equivalent circuit with compartments corresponding to the soma/dendrites (6-10 pF) and synaptic terminal (2-4 pF), linked by the axial resistance (30-60 M omega) of the axon. Four lines of evidence validate the equivalent circuit. 1) Similar estimates of somatic/dendritic and terminal capacitance were obtained whether the patch pipette was attached to the soma or to the synaptic terminal. 2) Estimates of the capacitance of the two compartments in intact cells were similar to estimates from somata and terminals that were isolated by cleavage of the connecting axon. 3) When current transients were generated from a more complete computer simulation of a bipolar neuron, analysis of the simulated transients with the use of the simple two-compartment model yielded capacitance estimates similar to those used to set up the simulation. 4) In isolated cells, the model gave estimates of depolarization-evoked increases in capacitance of the synaptic terminal that were quantitatively similar to those measured in terminals that were detached from the rest of the cell. Although in previous studies researchers have attempted to apply a similar equivalent circuit to more geometrically complex cells, morphological correlates of the equivalent-circuit compartments have been elusive. Our results demonstrate that in dissociated bipolar cells, precise morphological correlates can be assigned to the equivalent-circuit compartments. Additionally, the work shows that time-resolved capacitance measurements of synaptic transmitter release are possible in intact, isolated bipolar neurons and may also be feasible in intact tissue.

Inhibition of pacemaker current by the bradycardic agent ZD 7288 is lost use-dependently in sheep cardiac Purkinje fibres.

The inhibition of the pacemaker current (if) in sheep cardiac Purkinje fibers by ZD 7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium++ + chloride] is lost use-dependently. This disinhibition of if was investigated by using the two-microelectrode voltage-clamp technique. The pulse protocol consisted of a rest period (holding potential of about -50 mV, 1-10 micromol/l ZD 7288) followed by a train of test pulses (potential negative to -100 mV, stimulation frequency 0.05 Hz). At the beginning of the first test pulse there was an immediate reduction of if but inhibition was lost during continued stimulation. Activation of if is sigmoidal and the early delay in current activation was prolonged from 33 ms (no ZD 7288) to 424 ms (10 micromol/l ZD 7288). Therefore hardly any disinhibition occurred during short test pulses (0.5s). During longer test pulses (5 s, -120 mV, 10 micromol/l) disinhibition developed with a time constant of about 2 s. The inhibition of if by ZD 7288 was lost voltage-dependently. With 10 micro mol/l ZD 7288 the half-maximal disinhibition occurred at -92 mV and the slope factor of the disinhibition/voltage curve (Boltzmann relation) was 4.8 mV. The voltage-dependent disinhibition could be abolished largely by extracellular application of protease (0.5 mg/ml, 7 min). After prior disinhibition, reinhibition at the holding potential (about -50 mV) followed a bi-exponential time course indicating that inhibition may be produced by a fast (tau=0.7 min) and a slow component (tau=20-30 min). Increasing ZD 7288 concentration from 1 to 10 mu mol/l accelerated reinhibition, mainly by an increase of the amplitude (A) of the fast component. The ratio Afast/Aslow was 0.399 at 1 micromol/l and 2.65 at 10 micromol/l ZD 7288. The reinhibition of if was unchanged by shifting the holding potential from -50 mV to -20 mV. Trials to wash out the effects of 10 micromol/l ZD 7288 gave two results. The inhibition of if was slightly reversed after a wash-out of 1.5 h with drug-free solution. A second effect of the drug, the fast reinhibition, could be completely removed by wash-out. In summary if is inhibited by ZD 7288 at membrane potentials at which the virtual if gate is closed. Disinhibition occurs during long-lasting hyperpolarization but will hardly be operative in unclamped fibres under physiological conditions.

A rapidly activating type of outward rectifier K+ current and A-current in rat suprachiasmatic nucleus neurones.

1. The properties of calcium-independent (i.e. persisting in the absence of external calcium) depolarization-activated potassium currents in suprachiasmatic nucleus (SCN) neurones (n = 75) were studied under voltage-clamp conditions with whole-cell patch-clamp recordings in rat hypothalamic slices (150-175 microns). 2. Two distinct types of potassium currents were found. One was a rapidly activating and slowly inactivating type of outward rectifier (named IK(FR) for fast rectifier potassium current), similar to a potassium current described in cardiac muscle, and the other was a transient A-current (IA). 3. The rates of activation and deactivation of IK(FR) were voltage dependent. Time constants of activation fitted to n4 kinetics and declined from 3.5 ms (at -20 mV) to 1.1 ms (at 60 mV). Inactivation had a biexponential time course with voltage-independent time constants of 0.3 s (minor component) and 3.0 s (major component) between 10 and 50 mV. IK(FR) was activated above -40 mV with a V1/2 (membrane potential at half-maximal activation) of 14 +/- 2 mV and slope factor of -17 +/- 1 mV reaching a conductance (not maximal) of 10.8 +/- 1.7 nS at 60 mV. Steady-state inactivation had a slope factor of 11 +/- 1 mV. 4. IK(FR) was highly selective for K+ (PNa/PK = 0.002). Tetraethylammonium (TEA) reduced IK(FR) reversibly (IC50 = 20 mM), while 4-aminopyridine (4-AP) at 10 mM had little effect. The remaining current in 30 mM TEA was similar to that in control conditions, indicating that TEA reduced IK(FR) rather than revealed an additional TEA-resistant current. 5. The rate of IA activation was voltage dependent with time to peak declining from 8.5 ms (at -40 mV) to 3.6 ms (at 60 mV). Inactivation had a biexponential time course with voltage-dependent and voltage-independent time constants. The two components were similar in amplitude. IA was activated above -60 mV, reaching a maximal conductance of 3.6 +/- 0.4 nS at above 20 mV. Steady-state inactivation was complete above -10 mV. Rates of onset of inactivation (at -40 mV) and recovery from inactivation (at -100 mV) were similar with time constants of 39 +/- 5 and 41 +/- 9 ms, respectively. 6. IK(FR) and IA were found in every neurone tested in the SCN and in all locations throughout the nucleus. The possible function of these currents is discussed, particularly in relation to the circadian rhythm of firing rate in the SCN.

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

Inactivation of outward Na(+)-Ca2+ exchange current in guinea-pig ventricular myocytes.

1. Outward Na(+)-Ca2+ exchange currents were measured in freshly dissociated guinea-pig myocytes to probe in intact cells the functional status of exchanger inactivation reactions, described previously in giant excised cardiac membranes patches. 2. When the cytoplasmic (pipette) solution contained 40 mM Na+ and 0.1 microM free Ca2+ (50 mM EGTA), the outward exchange current activated by extracellular Ca2+ decayed with time (time constant, 13.1 +/- 2.6 s; n = 6), and an inward current transient was observed upon removal of extracellular Ca2+. Both the current decay and the subsequent inward current transient were remarkably diminished with a saturating (100 mM) pipette Na+ concentration. 3. With 100 mM cytoplasmic Na+ and 140 mM extracellular Na+, a significant fraction of the exchanger population is predicted to be in an inactive state. Intracellular application of 2 mg ml-1 chymotrypsin and 5 microM sodium tetradecylsulphate, both of which decrease Na(+)-dependent inactivation in giant membrane patches, increased the outward exchange current by about 160-170%, suggesting that about 60-70% of exchangers might be inactivated. 4. With 100 mM cytoplasmic Na+ and no extracellular Na+ (replaced with 140 mM Li+), application of extracellular Ca+ was predicted to reorient exchanger binding sites from the extracellular side to the cytoplasmic side and thereby favour inactivation. During such protocols, the outward exchange current decayed by 60-80% when activated by extracellular Ca2+. The current decayed similarly when extracellular Ca2+ and Na+ were applied together, whereby current magnitudes were about 3-fold smaller. 5. The decay of outward exchange current usually followed a biexponential time course (5.8 +/- 3.5 and 27.3 +/- 16.3 s, means +/- S.D., n = 11). Intracellular application of 0.5-2 mg ml-1 trypsin attenuated the fast component more than the slow component, suggesting that the fast component reflects an inactivation process. 6. Current-voltage (I-V) relations of the outward exchange current became less steep during the inactivation protocols, but this flattening could not be correlated with inactivation. 7. Replacement of extracellular Li+ with N-methyl-D-glucamine (NMG), tetraethylammonium (TEA), sucrose or Cs+ resulted in a flattening of I-V relations and a decrease of the outward exchange current amplitude by approximately 3-fold, but the kinetics and extent of inactivation were not remarkably changed. Thus, the mechanism of inactivation appears to be independent of the mechanism(s) of activation by extracellular monovalent cations.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of the PET ligand [11C]SCH 23390. Identification of two radiolabelled metabolites with HPLC

Abstract[11C]SCH 23390 is a radioligand used for PET determination of the D1‐dopamine receptor in the living human brain. An HPLC procedure was developed for analysis of [11C]SCH 23390 and its radiolabelled metabolites in plasma after injection of the radioligand into human subjects in realtion to PET studies. After i.v. injection [11C]SCH 23390 rapidly declined from plasma, about 50 per cent of the radioligand remaining unchanged after 4 min and about 10 per cent after 40 min. The decline followed a biexponential time course. Two of the probable metabolites, the O‐sulphate and the O‐glucuronide of SCH 23390 were synthesized and characterized. Two radioactive substances in plasma co‐migrated with these reference compounds.

Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure.

A variety of outward currents exists in ventricular myocardium of different species influencing action potential duration and electrical activity. Transient outward currents have been reported in ventricular tissue of some animals but are small or absent in others. This study was conducted to investigate whether a transient outward current exists in human ventricular myocardium and to characterize its basic electrophysiological properties. Currents were recorded from enzymatically isolated human ventricular myocytes obtained from explanted hearts of 22 patients with terminal heart failure. In almost all cells studied, a transient outward current could be recorded on depolarization to between -20 and +80 mV. The size of the transient outward current was usually large enough to mask the Ca2+ current. It could be recorded under conditions in which Ca2+ influx and intracellular Ca2+ transients were suppressed. Basic current characteristics were similar to transient outward currents observed in other species. Inactivation of the transient outward current was monoexponential, with a time constant of 54.8 +/- 3.7 milliseconds at +40 mV. Half-maximal activation occurred at 16.7 +/- 1.6 mV; half-maximal steady-state inactivation occurred at -34.5 +/- 2.3 mV. Frequency-dependent reduction of peak transient outward current was 29.8 +/- 1.4% at 2 Hz compared with resting conditions. Recovery from inactivation was voltage dependent and had a biexponential time course; the faster time constant (41.0 +/- 6.5 milliseconds at -80 mV) accounted for 86.0 +/- 5.2% of total current. The transient outward current was sensitive to 4-aminopyridine (IC50, 1.15 mM). These results indicate that a large Ca(2+)-independent transient outward K+ current is present in human ventricular myocytes that might be regulated by physiological or pathological events and is a potential site for pharmacological intervention.

Tonic GABA secretion of cultured rat hippocampal neurons rapidly transformed by Zn 2+ into quantal release

Different mechanisms of neurotransmitter secretion at synapses have been ascribed to quantal, transient signals [4–9] and to continuous, tonic activity [11, 21, 25]. Quantal transmission is imputed to be the release of transmitter packets, or quanta [6], assembled and stored in vesicles present in presynaptic terminals [2, 8]. We report that 300 μM Zn 2+ rapidly transformed tonic Cl − conductance mediated by the transmitter γ-aminobutyric acid (GABA) into transient synaptic-like signals. After addition of Zn 2+, the size of the elementary fluctuations in the Cl − current progressively increased while randomly activated Cl − channel kinetics remained unchanged and accounted for the bi-exponential time course of average transient decay. The results suggest that Zn 2+ rapidly transforms tonic GABA secretion into quantal-like transient release.

A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression.

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4-aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Interconversion between distinct gating pathways of the high threshold calcium channel in rat ventricular myocytes.

1. High-voltage-activated Ca2+ current (ICa) waveforms in adult rat ventricular myocytes comprise two components, referred to here as ICa(fc) and ICa(sc) to denote the fast and slow components, respectively, of ICa decay. At all test potentials, the two time constants of ICa decay, tau fc and tau sc, differ by approximately an order of magnitude. Neither tau fc nor tau sc varies appreciably with test potential, however, suggesting that current inactivation is not markedly voltage dependent. 2. Current activation at all test potentials follows a sigmoidal time course and is best described by a power function with n = 4. Deactivation of the currents, examined following variable length depolarizations to various test potentials, however, follows a single exponential time course. In addition, the kinetics of activation and deactivation of ICa(fc) and ICa(sc) are indistinguishable. 3. Although both begin to activate at approximately -30 mV, the voltage dependences of ICa(fc) and ICa(sc) are distinct: ICa(fc) peaks at -10 mV and ICa(sc) peaks at +10 mV. 4. The relative amplitudes of ICa(fc) and ICa(sc) vary with the holding potential from which the currents are evoked and with the frequency of current activation: hyperpolarized holding potentials and low stimulation frequencies reveal preferential activation of ICa(fc), whereas depolarized holding potentials and high stimulation frequencies potentiate ICa(sc). In addition, the observed voltage- and frequency-dependent changes in ICa(fc) and ICa(sc) amplitudes are reciprocal. 5. The apparent voltage dependences of steady-state inactivation of ICa(fc) and ICa(sc) are also distinct. ICa(fc) is reduced to approximately 50% of its maximal amplitude at -45 mV, whereas ICa(sc) is approximately 50% inactivated at -30 mV. 6. Recovery of ICa(peak) from steady-state inactivation follows a complex time course. Following inactivation at -10 mV, ICa(peak) recovers at -90 mV to its maximal value over a biexponential time course; ICa(peak) then decreases over the next several seconds to a steady-state level. 7. The time course of recovery from steady-state inactivation of ICa(fc) at -90 mV is best described by the sum of two exponentials; the two time constants of recovery differ by approximately a factor of 25. ICa(sc), in contrast, recovers rapidly and over a single exponential time course to its maximal value. When the recovery time at -90 mV is increased, however, ICa(sc) amplitude decreases slowly and over a single exponential time course to a steady-state level.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple kinetic components of sodium channel inactivation in rabbit Schwann cells.

1. We have studied the kinetics of inactivation of sodium currents evoked in Schwann cells from neonatal and adult rabbits with patch-clamp recording techniques. The decay both of whole-cell currents and of ensemble currents obtained from outside-out patches was reasonably well-described by single exponential fits which gave values for the time constant, tau h, similar to those found for such currents in nerve (about 0.5 ms at 0 mV). Although inclusion of an additional exponential component usually improved the fits to the decay of these currents, the relative amplitude of the slower component (time constant 3-6 ms) was always small. 2. The time course of recovery from steady-state inactivation clearly consisted of two exponential components. At -120 mV, recovery from steady-state inactivation at -50 mV consisted of a fast component with a time constant of 2.2 +/- 0.2 ms and a much slower component with a time constant of 1.2 +/- 0.2 s (n = 9). The relative amplitude of the slow component (expressed as a fraction of the sodium current when inactivation was removed completely) was 0.56 +/- 0.03. The corresponding amplitudes of the slow component when similar experiments were done from holding potentials of 0 and -70 mV were 0.80 +/- 0.04 and 0.36 +/- 0.03, respectively (n = 5 and 8). 3. The onset of steady-state inactivation also followed a bi-exponential time course. The time constant of the slower component was similar at each potential examined (0, -50 and -70 mV), being 6-7 s. The relative amplitude of the slow component of onset depended on membrane potential, and it was similar (at each potential examined) to the corresponding amplitude of the slow component of recovery. 4. The inclusion of 40 mM-iodate ions in the pipette solution slowed the decay of whole-cell sodium currents, as did the extracellular application of venom (10 micrograms ml-1) from the scorpion Leiurus quinquestriatus. Prolonged exposure of the cells to Leiurus venom appeared to increase the steady-state amount of slow inactivation. 5. Records of single-channel sodium currents tended to cluster into records which either did, or did not, contain openings. This apparently non-random behaviour depended on membrane potential, and on the frequency at which the test steps were repeated, in the way expected if it resulted from slow inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

A thrombin receptor in resident rat peritoneal macrophages

Resident rat peritoneal macrophages possess 6 × 102 high-affinity binding sites per cell for bovine thrombin with a Kd of 11 pM, and 7.5 × 104 low-affinity sites with a Kd of 5.8 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labeled bovine thrombin is achieved after 1 min at 37 °C, and after 12 min at 4 °C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 0.27 and 0.06 min−1 at 4 °C. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3 treatment, and the receptor does not mediate a quantitatively important degradation of the ligand. The binding is not dependent on the catalytic site of thrombin, since irreversibly inactivated thrombin also binds to the receptor. 125I-labeled thrombin covalently cross-linked to its receptor migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr 160,000, corresponding to an approximate receptor Size of Mr 120,000.

High-performance liquid chromatographic (HPLC) determination of lobenzarit in plasma and its application to a bioavailability study in beagle dogs.

A method for the quantitative determination of lobenzarit (2-[(2-carboxyphenyl)amino]-4-chlorobenzoic acid) in dog plasma by high-performance liquid chromatography with UV detection (308 nm) is described. Plasma samples (200 microliters) were treated with acetonitrile and centrifuged, and the clear supernatant injected onto a reversed-phase phenyl column. The method achieved a limit of quantitation of 0.5 micrograms/ml in plasma, and the response was linear to 100 micrograms/ml. Comparing a solution and a tablet formulation given to beagle dogs, the assay demonstrated that the solution formulation was slightly more bioavailable and yielded a more variable absorption rate. The elimination of lobenzarit from plasma followed a biexponential time course, with an apparent terminal disposition half-life of between 5.8 and 10.7 hr.