Dual action of FRC8653, a novel dihydropyridine derivative, on the Ba2+ current recorded from the rabbit basilar artery

Actions of FRC8653 on the macroscopic and unitary Ba2+ currents were studied using the rabbit basilar artery. Application of (+/-)-FRC8653 (less than 1 microM) increased the amplitude of the inward current when depolarization pulses more negative than -10 mV were applied but inhibited it when depolarization was more positive than 0 mV (in each case from a holding potential of -80 mV). At a holding potential of -40 mV, (+/-)-FRC8653 (greater than 0.1 nM) consistently inhibited the inward current. (-)-FRC8653 (greater than 1 nM) inhibited the amplitude of the inward current evoked by a depolarizing pulse more positive than -10 mV (the holding potential being -80 mV). At the holding potential of -80 mV, but not at -40 mV, (+)-FRC8653 (1 microM) enhanced the current amplitude evoked by a depolarizing pulse more negative than -10 mV but inhibited the current evoked by a pulse more positive than 0 mV. (+/-)-FRC8653 shifted the voltage-dependent inhibition curves to the left, and the slope of the curve became steeper (test pulse of +10 mV). Two types of single Ca2+ channel currents (12 and 23 pS) were recorded from the basilar artery by the cell-attached patch-clamp method. Opening of the 12-pS channel occurred with a depolarizing pulse (-20 mV) from a holding potential of -80 mV, but not from one of -60 mV. (+)-FRC8653 activated, and (-)-FRC8653 inhibited, the 23-pS channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.     

Current fluctuations and oscillations in smooth muscle cells from hog carotid artery. Role of the sarcoplasmic reticulum

Electrical activity of enzymatically isolated, smooth muscle cells from hog carotid arteries was recorded under current clamp and voltage clamp. Under the experimental conditions, membrane potential usually was not stable, and spontaneous hyperpolarizing transients of approximately 100-msec duration were recorded. The amplitude of the transients was markedly voltage dependent and ranged from about 20 mV at a membrane potential of 0 mV to undetectable at membrane potentials negative to -60 mV. Under voltage clamp, transient outward currents displayed a similar voltage dependency. These fluctuations reflect a K+ current; they were abolished by 10 mM tetraethylammonium chloride, a K+ channel blocker, and the current fluctuations reversed direction in high extracellular K+ concentration. Modulators of intracellular Ca2+ concentration also affected electrical activity. Lowering intracellular Ca2+ concentration by addition of 10 mM EGTA to the pipette solution or suppressing sarcoplasmic reticulum function by superfusion with caffeine (10 mM), ryanodine (1 microM), or histamine (3-10 microM) blocked the rapid voltage and current spikes. However, caffeine and histamine induced a much slower hump of outward current before blocking the rapid spikes. This slower transient outward current could be elicited only once after external Ca2+ was removed and is consistent with an activation of K+ channels by Ca2+ released from internal stores. In contrast, removal of external Ca2+ alone failed to abolish the rapid spikes. These results suggest that 1) a Ca2+-dependent K+ conductance can markedly affect the electrical behavior of arterial smooth muscle cells and 2) internal Ca2+ stores, probably the sarcoplasmic reticulum, can support rapid and frequent releases of Ca2+. Exposure to a low concentration of histamine (3 microM) caused synchronization of the irregular, rapid fluctuations giving rise to slow, periodic oscillations of Ca2+-activated K+ conductance with a frequency of 0.1-0.3 Hz. These regular oscillations are reminiscent of periodic Ca2+-induced Ca2+ release, were inhibited by 10 mM caffeine, and point to a modulation of sarcoplasmic reticulum Ca2+ release by histamine.     

Separate Cl- conductances activated by cAMP and Ca2+ in Cl(-)-secreting epithelial cells.

We studied the cAMP- and Ca2(+)-activated secretory Cl- conductances in the Cl(-)-secreting colonic epithelial cell line T84 using the whole-cell patch-clamp technique. Cl- and K+ currents were measured under voltage clamp. Forskolin or cAMP increased Cl- current 2-15 times with no change in K+ current. The current-voltage relation for cAMP-activated Cl- current was linear from -100 to +100 mV and showed no time-dependent changes in current during voltage pulses. Ca2+ ionophores or increased pipette Ca2+ increased both Cl- and K+ currents 2-30 times. The Ca2(+)-activated Cl- current was outwardly rectified, activated during depolarizing voltage pulses, and inactivated during hyperpolarizing voltage pulses. Addition of ionophore after forskolin further increased Cl- conductance 1.5-5 times, and the current took on the time-dependent characteristics of that stimulated by Ca2+. Thus, cAMP and Ca2+ activate Cl- conductances with different properties, implying that these second messengers activate different Cl- channels or that they induce different conductive and kinetic states in the same Cl- channel.     

Dopamine inhibits two characterized voltage-dependent calcium currents in identified rat lactotroph cells

The effects of dopamine (DA) on voltage-dependent Ca2+ currents were investigated in cultured rat lactotroph cells using the patch clamp recording technique. Each recorded cell was identified by the reverse hemolytic plaque assay. In the whole-cell configuration, two types of Ca2+ currents, L and T, were characterized on the basis of their kinetics, voltage sensitivity, and pharmacology. The L component had a threshold of -25 mV, showed little inactivation during a 150-msec voltage step, and was maximal at +10 mV. Cadmium ions (100 microM) significantly reduced its amplitude (75%). The T component was activated at a membrane potential close to -50 mV, was maximal at -10 mV, and showed a voltage-dependent inactivation between -90 and -30 mV. It was quickly inactivated during a maintained depolarization (time constant, 27 ms at -30 mV) and was strongly reduced (80%) by nickel ions (100 microM). Bath application of DA (10 nM) caused a markedly general depression of inward Ca2+ currents, acting differently on the T- and L-type currents. DA application shifted the voltage-dependence of the L-type current activation toward depolarization values (8 mV) without modifying its time- and voltage-dependent inactivation. In contrast, DA enhanced the inactivation of the T-type current by accelerating its time-dependent inactivation (25% decrease in the time constant of inactivation) and by shifting the voltage-dependence of the T-type current inactivation toward hyperpolarizing values (-63 mV in control vs. -77 mV in the presence of DA). These effects of DA were dose-dependent and involved the activation of a D2 receptor type. They were mimicked by bromocriptine application (10 nM), whereas sulpiride (100 nM) blocked the DA-evoked response. The D1 antagonist SCH 23390 was ineffective up to 100 microM. All of these DA-induced modifications in Ca2+ currents were abolished using a GTP-free pipette solution or after pretreatment of cells with pertussis toxin, suggesting that DA can regulate the function of Ca2+ channels through GTP-binding proteins (G-proteins). Our results show that DA acts simultaneously by reducing both voltage-dependent Ca2+ currents on lactotroph cells. Thus, DA reduces the entry of Ca2+ ions across the surface membrane and thereby influences electrical activity and the cytosolic free Ca2+ concentration involved in both basal and evoked PRL release.     

Calcium homeostasis in rabbit ventricular myocytes. Disruption by hypochlorous acid and restoration by dithiothreitol.

Hypochlorous acid (HOCl) is a toxic oxidant produced by neutrophils at sites of cardiac inflammation. To examine the effect of this oxidant on Ca2+ homeostasis in the heart, isolated rabbit ventricular myocytes were iontophoretically loaded with the Ca2+ indicator fura 2 and superfused with 100 microM HOCl under voltage-clamp conditions. Ca2+ transients and the corresponding Ca2+ currents were elicited by 300-msec depolarizing pulses from -40 to 0 mV. Within 200 seconds after HOCl addition, the amplitude of the Ca2+ transients was reduced from 402 +/- 89 to 82 +/- 29 nM (p less than 0.01) while intracellular free ([Ca2+]i increased from 78 +/- 16 to 265 +/- 48 nM (p less than 0.01). During this time, the amplitude of the slow inward currents increased by 10%, while steady-state holding current remained stable. This sustained steady-state rise in [Ca2+]i occurred even in the absence of extracellular Ca2+ but was virtually abolished by a 20-second preexposure to 10 mM caffeine, suggesting that the major source of this Ca2+ was the sarcoplasmic reticulum. Although washout of HOCl failed to induce recovery, subsequent exposure to the dithiol reducing agent dithiothreitol caused a rapid restoration of both the steady-state [Ca2+]i and Ca2+ transient amplitude. We conclude that 1) HOCl caused a rise of [Ca2+]i by inducing the release of Ca2+ from internal stores and impairing cellular extrusion mechanisms and 2) these effects occur through alteration of protein thiol redox status.     

Presynaptic inhibition in Aplysia involves a decrease in the Ca2+ current of the presynaptic neuron.

By voltage clamping presynaptic cell L10 and using pharmacologic separation techniques, we have analyzed the specific ionic currents in the presynaptic cell that correlate with presynaptic inhibition while assaying transmitter release with intracellular recordings from postsynaptic cells. We have found that presynaptic inhibition can be elicited in conditions in which the Na+ and the various K+ channels are pharmacologically blocked and depolarizing current pulses produce only an inward Ca2+ current. Both inward currents and tail currents at and above the K+ reversal potential were always less inward during presynaptic inhibition. The changes in conductance associated with presynaptic inhibition were voltage sensitive and paralleled the voltage sensitivity of the Ca2+ channel. We therefore conclude that presynaptic inhibition is caused by a direct transmitter-mediated decreased of presynaptic Ca2+-channel conductance.     

Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein

Effects of concentrations of intracellular calcium, [Ca2+]i, on the voltage-dependent Ca2+ current (ICa) recorded from dispersed single smooth muscle cells of the rabbit portal vein were studied, using a whole cell voltage clamp method combined with an intracellular perfusion technique. Outward currents were minimized by replacement of Cs+ -rich solution in the pipette and 20 mM tetraethylammonium in the bath. The ICa was evoked by command pulses of above -30 mV, and the maximum amplitude was obtained at about 0 mV. This ICa was dose dependently inhibited by increases in the [Ca2+]i above 30 nM. The Kd value of the [Ca2+]i required to inhibit the ICa was about 100 nM. The Ba2+ current was also inhibited by increases in the [Ca2+]i. Conversely, perfusion of Ba2+ into the cell up to 100 microM did not suppress the ICa. Changes in the [Ca2+]i did not modify the steady-state inactivation curve. The inhibition of the ICa evoked by the test pulse is most prominent when the preceding influx of Ca2+ during the conditioning pulse was large, as estimated using a double pulse protocol. This inhibition was proportionally reduced by increases in the concentration of the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Therefore, the Ca2+ -dependent inactivation of the Ca2+ channel may contribute toward regulating [Ca2+]i in smooth muscle cells of the rabbit portal vein.     

硝苯地平阻断豚鼠心室肌细胞L型钙流特性的研究

选择性L型钙通道阻断剂硝苯地平 (Nif)为常用的工具药 ,因此必须了解它对L型钙流 (ICa(L) )浓度、状态依赖性阻断 ,使用和非使用依赖性阻断等特性。以豚鼠分离的单个心室肌细胞为对象 ,采用膜片钳全细胞记录技术 ,给予 35℃的各种含药物细胞外液快速灌流 ,记录ICa(L) 。结果 :①保持电位 - 80mV ,使用含铯离子的细胞内、外液 ,在 +10mV的钳制电压 ,Nif抑制ICa(L) 的IC50 为 0 .3μmol·L-1;当保持电位为 - 40mV时 ,IC50 为 0 .0 5 μmol·L-1,显示Nif优先选择与失活态钙通道结合。②使用富含钾离子的细胞内、外液 ,对ICa(L) 的非使用依赖性阻断 ,随Nif使用浓度 (30～ 10 0 μmol·L-1)的增加和药物作用时间的延长而加强 ,同时对ICa(L) 的使用依赖性阻断则减小。③在 10s的静息间隔药物作用时间后的第一个实验刺激 ,Nif 3μmol·L-1或 30 μmol·L-1加速ICa(L) 的失活 ,提示Nif对ICa(L) 可能存在激活态阻断。结论 :在生理条件下 ,Nif对ICa(L) 的阻断呈浓度、状态依赖性 ,对ICa(L) 的非使用依赖性阻断随使用浓度的增加和作用时间的延长而加强 ,对ICa(L) 的使用依赖性阻断则随之减弱。     

Early Afterdepolarization Formation in Cardiac Myocyte:

Early Afterdepolarization Formation, introduction: Early afterdepolarizations (EADs) are among the mechanisms proposed to underlie ventricular arrhythmias. Sea anemone toxin, ATXII, known to delay Na inactivation and to induce plateau level voltage oscillations, was used to study the formation of EADs. Methods and Results: Action potential and membrane currents were studied in rat ventricular myocytes using whole cell current and voltage clamp techniques. Phase plane trajectories were generated by plotting membrane potential (V) versus the first time derivative of membrane potential (dV/dt). Under current clamp conditions, ATXII (40 nM) consistently prolonged the action potential and induced EADs. The EADs developed at a plateau voltage between -10 and -40 mV. Calcium channel blockers, verapamil 10 μM and cobalt 4 μM, and the sarcoplasmic reticulum modulator, ryanodine (1 μM) did not antagonize ATXII effects on the action potential and EADs. However, Na channel blockers, tetrodotoxin 0.3μM and lidocaine 40μM. and rapid stimulation consistently shortened the prolonged action potential and suppressed EADs. Under voltage clamp conditions in the presence of ATXII, a slowly decaying inward current followed the fast inward current during depolarizing pulses. Membrane currents flowing at or later than 100 msec after the test pulse were analyzed. The control isochronal current-voltage (I-V) curves showed no late inward currents. In the presence of ATXII, all the isochronal I-V curves showed an inward current that was more prominent between -40 and 0 mV. The ATXII-induced current at the 100-msec isochronc activated at a potential of approximately -60 mV, peaked at about -20 mV, and reversed at +40 mV consistent with the Na current I-V curve. The isochronal I-V curves obtained after lidocaine superfusion resembled those of the control. The phase plane trajectory of the action potential obtained with ATXII showed an oscillatory behavior corresponding to the t AD range of potential; within this voltage range, the isochronal I-V curves were shown to cross the abscissa three times instead of once. Conclusion: These results suggest that, in this experimental model, neither sarcolemmal L-type Ca current nor sarcoplasmic reticulum Ca release plays a significant role in the genesis of ATXII-induced EADs. EADs are generated by a voltage-dependent balance between a markedly prolonged Na inward current and K outward currents within the voltage plateau range of the action potential hut not by Ca current reactivation and inactivation.     

Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca2+ and K+ currents

We have examined the relationships between the modulation of transmitter release and of specific ionic currents by membrane potential in the cholinergic interneuron L10 of the abdominal ganglion of Aplysia californica. The presynaptic cell body was voltage-clamped under various pharmacological conditions and transmitter release from the terminals was assayed simultaneously by recording the synaptic potentials in the postsynaptic cell. When cell L10 was voltage-clamped from a holding potential of -60 mV in the presence of tetrodotoxin, graded transmitter release was evoked by depolarizing command pulses in the membrane voltage range (-35 mV to + 10 mV) in which the Ca(2+) current was also increasing. Depolarizing the holding potential of L10 results in increased transmitter output. Two ionic mechanisms contribute to this form of plasticity. First, depolarization inactivates some K(+) channels so that depolarizing command pulses recruit a smaller K(+) current. In unclamped cells the decreased K(+) conductance causes spike-broadening and increased influx of Ca(2+) during each spike. Second, small depolarizations around resting potential (-55 mV to -35 mV) activate a steady-state Ca(2+) current that also contributes to the modulation of transmitter release, because, even with most presynaptic K(+) currents blocked pharmacologically, depolarizing the holding potential still increases transmitter release. In contrast to the steady-state Ca(2+) current, the transient inward Ca(2+) current evoked by depolarizing clamp steps is relatively unchanged from various holding potentials.     

Action potential broadening and frequency-dependent facilitation of calcium signals in pituitary nerve terminals.

Hormone release from nerve terminals in the neurohypophysis is a sensitive function of action potential frequency. We have investigated the cellular mechanisms responsible for this frequency-dependent facilitation by combining patch clamp and fluorimetric Ca2+ measurements in single neurosecretory terminals in thin slices of the rat posterior pituitary. In these terminals both action potential-induced changes in the intracellular Ca2+ concentration ([Ca2+]i) and action potential duration were enhanced by high-frequency stimuli, all with a frequency dependence similar to that of hormone release. Furthermore, brief voltage clamp pulses inactivated a K+ current with a very similar frequency dependence. These results support a model for frequency-dependent facilitation in which the inactivation of a K+ current broadens action potentials, leading to an enhancement of [Ca2+]i signals. Further experiments tested for a causal relationship between action potential broadening and facilitation of [Ca2+]i changes. First, increasing the duration of depolarization, either by broadening action potentials with the K(+)-channel blocker tetraethylammonium or by applying longer depolarizing voltage clamp steps, increased [Ca2+]i changes. Second, eliminating frequency-dependent changes in duration, by voltage clamping the terminal with constant duration pulses, substantially reduced the frequency-dependent enhancement of [Ca2+]i changes. These results indicate that action potential broadening contributes to frequency-dependent facilitation of [Ca2+]i changes. However, the small residual frequency dependence of [Ca2+]i changes seen with constant duration stimulation suggests that a second process, distinct from action potential broadening, also contributes to facilitation. These two frequency-dependent mechanisms may also contribute to activity-dependent plasticity in synaptic terminals.     

Voltage-gated Ca2+ channel in mouse myeloma cells.

Electrical properties of the cell membrane were studied in the neoplastic lymphocyte, mouse myeloma cell line S194, by using the whole-cell patch clamp technique. Inward Ca2+ currents due to voltage-gated Ca2+ channels were found. The current, which decayed exponentially after reaching a peak, was first activated at about -50 mV and attained its maximum peak amplitude at about -20 mV in a 10 mM Ca2+ solution. Outward current was negligible for the potential range more negative than +30 mV. The channel was permeable to Sr2+ and Ba2+ in addition to Ca2+. Among these species, Sr2+ carried the greatest current. The time constants of the decay of the current depended neither on the species nor on the concentration of charge carrier. The steady-state inactivation was observed at potentials more negative than those at which the inward Ca2+ current was activated. Thus, we concluded that the inactivation of the channel was mainly voltage dependent. For reasons that are not yet understood, the amplitude of the Ca2+ current varied greatly among cells.     

Characteristics of a transient inward current that causes delayed afterdepolarizations in atrial cells of the canine coronary sinus

Delayed afterdepolarizations and triggered activity occur in atrial cells of the canine coronary sinus in response to catecholamines. We studied the properties of the membrane current that causes the afterdepolarizations with the two-microelectrode voltage clamp technique in small preparations (about 0.5 x 1 mm). At a holding potential of -50 mV a transient inward current (TI) occurred after repolarization from a depolarizing step to between -40 and -20 mV in the absence of catecholamines. When the depolarizing pulse was made more positive or its duration increased the amplitude of the TI current increased and it reached peak amplitude faster. The current-voltage relationship of the TI current was studied by changing the voltage to which the membrane was repolarized after a depolarizing clamp pulse of fixed amplitude and duration. At repolarization levels positive to -30 mV there were current fluctuations without a distinct TI current. As the repolarization voltage was made more negative, a TI current occurred and its time to peak increased monotonically. The TI current amplitude increased and reached a maximum amplitude at around -60 to -70 mV, and then declined at more negative repolarization voltages. Norepinephrine increased the TI current while simultaneously augmenting the slow inward current. Elevating [Ca]0 increased the TI current amplitude. Caffeine (2 mM) increased the TI current amplitude, while caffeine (4 mM) increased and then decreased the current amplitude. The dependence of the TI current on the voltage and duration of the activating depolarizing step in these atrial cells are qualitatively similar to those of the TI current associated with digitalis toxicity in Purkinje fibers and ventricular muscle, although there are some quantitative differences. There is no distinct TI reversal in these atrial cells, similar to TI in ventricular muscle but dissimilar to TI in Purkinje fibers.     

Voltage-activated currents through calcium channels in rat pituitary melanotrophic cells.

Voltage-gated currents through Ca channels in rat melanotrophs in short-term primary culture were investigated in whole cell voltage clamp recordings, using Ba2+ ions as charge carrier. At potentials higher than -50 mV a transient current (It) was activated, which inactivated entirely during a 150-ms command pulse and reached a maximal amplitude at -10 mV. At potentials higher than -20 mV a second component (Is) appeared, which inactivated slowly and had its maximal amplitude between 0 and +10 mV. The relative amplitudes of It and Is differed between cells. Application of 40 microM NiCl2 reversibly blocked It while leaving Is intact, whereas 20 microM CdCl2 reversibly blocked Is, but not It. Nifedipine blocked Is concentration dependently, but did not affect It. The steady state inactivation of It occurred between -80 and -40 mV. The steady-state inactivation curve of Is was located between -60 and 0 mV. The latter inactivation curve showed two phases, which were fitted best by the sum of two Boltzmann equations, suggesting the existence of two populations of Is channels.     

Dihydropyridine-sensitive Ca2+ channels in mammalian skeletal muscle cells in culture: electrophysiological properties and interactions with Ca2+ channel activator (Bay K8644) and inhibitor (PN 200-110).

The whole-cell patch-clamp technique has been used to analyze the properties of the dihydropyridine-sensitive Ca2+ channel in rat skeletal muscle cells (myoballs) in culture. The potential dependence of Ca2+-channel activation is similar to that observed in cardiac cells. However, the skeletal muscle Ca2+ channel is activated more slowly (by a factor of about 10). The voltage dependence of Ca2+-channel inactivation indicates a half-maximal inactivation (Vh0.5) at -72 mV as compared to Vh0.5 = -35 mV for cardiac cells. Blockade of the skeletal muscle Ca2+ channel by the dihydropyridine (+)-PN 200-110 is voltage dependent, with a half-maximal effect (K0.5) of 13 nM for an application of the drug to the myoball membrane held at -90 mV and of 0.15 nM for an application at a potential of -65 mV. The 100-fold difference in apparent affinity is interpreted as a preferential association of PN 200-110 with the inactivated form of the Ca2+ channel. The K0.5 value found from electrophysiological experiments for the binding to the inactivated state (K0.5 = 0.15 nM) is nearly identical to the equilibrium dissociation constant found from binding experiments with (+)-[3H]PN 200-110 using transverse-tubular membranes (Kd = 0.22 nM). The dihydropyridine activator Bay K8644 acts by increasing Ca2+ current amplitude and by slowing down deactivation.     

Frequency-dependent increase in cardiac Ca2+ current is due to reduced Ca2+ release by the sarcoplasmic reticulum.

"Ca(2+)-current facilitation" describes several features of increase in current amplitude often associated with a reduction in inactivation rate. The aim of this study was to investigate the mechanism of frequency-dependent increase in L-type Ca2+ current, I(Ca) taking advantage of recent knowledge on the control of Ca2+ current inactivation in cardiac cells. The frequency-dependent increase in I(Ca) was studied in adult rat ventricular myocytes using the whole-cell patch-clamp technique. I(Ca) was elicited by a train of 200-ms depolarizing pulses to +20 mV applied at various frequencies (0.2 up to 1.3 Hz). The increase in frequency induced a rate-dependent enhancement of I(Ca), or facilitation phenomena. In most cells, that showed two inactivation phases of I(Ca), facilitation was mainly related to slowing of the fast I(Ca) inactivation phase that occurred besides increase in peak I(Ca) amplitude. Both the decrease and slowing of the fast component of inactivation phase were attenuated on beta -adrenergic-stimulated current. Frequency-dependent I(Ca) facilitation paralleled a reduction in Ca2+ transient measured with fluo-3. After blocking sarcoplasmic reticulum-Ca2+ release by thapsigargin, the fast I(Ca) inactivation phase was reduced and facilitation was eliminated. Facilitation could not then be restored by 1 microM isoprenaline. Thus in rat ventricular myocytes, frequency-dependent facilitation of I(Ca)reflects a reduced Ca(2+)-dependent inactivation consecutive, in most part, to reduced Ca2+ load and Ca2+ release by the sarcoplasmic reticulum rather than being an intrinsic characteristic of the L-type Ca2+ channel.     

Ca2+ entry in squid axons during voltage-clamp pulses is mainly Na+/Ca2+ exchange.

Intact squid axons were injected with aequorin and bathed in 3 mM Ca seawater (a concentration close to that of squid blood). Sodium and potassium currents were pharmacologically blocked and repetitive voltage-clamp pulses of a duration of 1.5 ms were applied (to simulate the duration of an action potential) at amplitudes of +30 to +90 mV and at frequencies of 100/s. In a very fresh axon (low internal Na concentration) no detectable change in aequorin glow resulted from this treatment, whether the axons were in Na-containing or in Na-free seawater. In axons subjected to modest Na loading, repetitive voltage-clamp pulsing did not result in an increased aequorin glow when the pulses were delivered in Na seawater, whereas in Na-free seawater there was an easily measurable increase in aequorin light emission during repetitive pulsing. The increase in aequorin photons emitted per voltage-clamp pulse was e-fold for 22 mV of depolarization, and the process showed no signs of saturating at pulse amplitudes of +180 mV (i.e., at a membrane potential close to ECa). The aequorin light emission per voltage-clamp pulse increased linearly with pulse duration (at constant amplitude).     

正常肠系膜动脉平滑肌细胞钙激活钾通道活性的观察

为了解人体正常肠系膜动脉平滑肌细胞钙激活钾通道的特性 ,取 2 4例人体正常肠系膜动脉小枝节段 ,用酶消化法获取标本细胞 ,以膜片钳制技术检测钙激活钾通道的活性 ,通过Pclamp专用软件实时采样记录其平均开放时间、平均关闭时间及平均开放概率等。结果发现 ,人体肠系膜动脉平滑肌细胞钙激活钾通道开放具有明显电压依赖性 ,在对称性高钾液中 ,电流 -电压关系曲线在 10～ 6 0mV范围内可被直线拟合。在细胞吸附式膜片和内面向外膜片中 ,通道电导分别为 192 .3± 2 9.2Ps和 2 0 2 .5± 5 8.3Ps。开放概率和开放数目随Ca2 + 浓度的增加而增加 ,膜内面应用四乙胺可减少通道开放概率及电流幅值。提示人体肠系膜动脉平滑肌细胞钙激活钾通道与人体其他血管相似 ,主要为大电导钙激活钾通道 ,具有电压和钙浓度双重依赖性。     

Effects of external pH on ionic currents in smooth muscle cells from the basilar artery of the guinea pig.

pHo is an important determinant of vascular tone in cerebral blood vessels. We investigated the effects of changes in pHo on isolated smooth muscle cells from the basilar artery of the guinea pig. Single cells contracted rapidly in response to an elevation in pHo (constant CO2), and contraction was blocked by nifedipine, suggesting a role for dihydropyridine-sensitive Ca2+ channels. In whole-cell patch-clamp experiments, changes in pHo (pHo 5.7-8.1, pHi 7.2 with 10 mM HEPES) strongly affected the amplitude of the peak Ca2+ channel current (10 mM Ba2+, +15 mV, holding potential of -55 mV), with an apparent pK of 6.9. The current-voltage curves were minimally shifted, indicating no important effect of surface charge. To separate the slowly inactivating L-type Ca2+ channel current from the more rapidly inactivating B-type current, the decaying portions of inward currents from cells studied with repetitive 1-second pulses (+15 mV, holding potential of -55 mV) were fit to a two-component model. Titration curves for the L-type and B-type currents indicated maximum increases by factors of 3.65 and 1.28 at alkaline pHo and gave apparent pK values of 7.71 and 6.47 (Hill coefficient unity). The time constant of inactivation for the B-type current at +15 mV was little affected by pHo, whereas that for the L-type current increased somewhat with increasing pHo. Additional experiments showed no significant effect of pHo on holding current or on voltage-activated outward currents (pCai 7 with 11 mM EGTA). Our results provide additional evidence for participation of Ca2+ channels in regulating basal tone in cerebral smooth muscle and indicate that pHo regulates current through slowly inactivating, dihydropyridine-sensitive L-type Ca2+ channels.