The calcium channel agonist, Bay K-8644, antagonizes effects of diacetyl monoxime on cardiac tissues

Effects of a novel slow channel activator, Bay K-8644 (Bay K), were studied on slow action potential (APs) in young and old embryonic chick hearts, and on its antagonism of the effects of diacetyl monoxime (DAM). The slow APs of young hearts are mediated by slow Na+ channels, whereas those of old hearts are mediated by slow Ca2+ channels. In slow APs of old (13-18 days old) embryonic chick hearts superfused with a high (22 mM) K+ solution, Bay K (10-6 M) gradually increased the amplitude, maximum rate of rise (Vmax), and duration of the slow APs. The actions of Bay K persisted for a long time (greater than 30 min) after washout of the drug. DAM (10 mM) depressed the Vmax, duration and amplitude of the slow APs. Some of the changes in slow AP parameters produced by DAM, e.g., Vmax decrease, were antagonized by the addition of Bay K (10(-6) M). In 3-day-old embryonic chick hearts. Bay K potentiated the slow APs and DAM depressed them; Bay K antagonized these effects of DAM. Thus, the actions of Bay K and DAM are likely to be produced, respectively, via the activation and depression of slow Ca2+ channels in old embryonic chick hearts. In addition, the drugs seem to influence slow Na+ channels found in young embryonic chick hearts.     

Reactivation processes of three inward current systems involved in the rising phase of the action potentials in embryonic chick hearts

In embryonic chick hearts during development, there are three inward current systems which are involved in the rising phases of the action potentials (APs): fast INa, slow ICa, and tetrodotoxin-insensitive slow INa. To assess reactivation processes for these three types of inward current channels (fast Na+, slow Ca2+, and slow Na+ channels), diastolic recovery of Vmax was examined in embryonic chick hearts using a paired-pulse protocol. In all cases, the diastolic recoveries were approximated by single exponential functions. The time constants of recovery (tau(V)) and T90% (the diastolic interval which allows 90% recovery of Vmax of the premature AP) were, respectively, 53.1 +/- 5.2 and 61.5 +/- 8.6 ms for Na+-dependent fast AP (n = 10), 376.9 +/- 49.3 and 659.2 +/- 113.1 ms for the Ca2+-dependent slow AP (n = 10), and 40.7 +/- 5.3 and 45.6 +/- 12.0 ms for the Na+-dependent slow AP (n = 10). In the presence of lidocaine, the recovery kinetics also appeared to be single exponentials for diastolic intervals up to 500 ms (fast APs) or 250 ms (slow APs). The reactivation processes for the Na+-dependent fast and slow channels were significantly slowed by 100 microM lidocaine. In addition, in the presence of 100 microM lidocaine, Vmax was depressed in a frequency-dependent manner; the higher the stimulation frequency, the greater the depression. Hence, the fast Na+ channels and the slow Na+ channels had the following similarities: rapid reactivation, reactivation slowed by lidocaine, and frequency-dependent depression in the presence of lidocaine.     

Calcium dependence of electrical and mechanical activity in rat ileum examined by the sucrose-gap technique

1. Single sucrose gap recordings showed that spontaneous action potentials of rat ileal smooth muscle consisted of slow waves and superimposed spikes which generated rhythmic contractions. As external potassium was raised, the resting potential progressively depolarized.2. Calcium-free salines inhibited spontaneous mechanical activity and inhibited the plateau phase of the action potential, but spontaneous spike depolarizations persisted.3. Verapamil, nifedipine and diltiazem all inhibited spontaneous mechanical activity and the plateau phase of the action potential, while in addition diltiazem augmented spike amplitude.4. Mn ions also inhibited mechanical activity and the action potential plateau, without affecting spike activity while the calcium ionophore A23187 enhanced both mechanical and electrical activity with a pronounced effect on spike amplitude.5. These results are consistent with the view that the plateau phase of the ileal smooth muscle action potential is dependent upon an influx of extracellular calcium possibly through voltage dependent slow calcium channels.     

Depressant action of Ca-antagonists on slow action potentials in guinea pig ventricular muscles

The depressant action of four Ca antagonists, including a novel drug, tiapamil, on Ca channels was investigated using a conventional microelectrode technique. "All or none" slow action potentials were recorded in K+-depolarized guinea-pig papillary muscles. Verapamil and diltiazem decreased the amplitude and maximum rate of rise (Vmax) of the slow action potentials at concentrations up to 2 microM. The depressant effect of a novel Ca-antagonist, tiapamil, on the slow action potentials was as marked as that of verapamil and diltiazem. However, prenylamine was less potent than the other 3 drugs. In addition, the action of all drugs on the slow action potentials was enhanced as the frequency of stimulation was increased between 0.0083 and 1 Hz. It was concluded that tiapamil, as verapamil and diltiazem, produced a frequency-dependent blockade of the slow Ca channel.     

The effect of Lambert-Eaton myasthenic syndrome antibody on slow action potentials in mouse cardiac ventricle

Immunoglobulin G (IgG) from Lambert-Eaton myasthenic syndrome (LEMS) patients acts at motor nerve terminal Ca2+ channels. It was injected into mice to investigate effects on cardiac Ca2+ channels. Intracellular recordings were made of slow action potentials in right ventricular muscle cells in the presence of high K+ concentrations and isoprenaline (1 microM). Reduction in Ca2+ concentration reduced the rate of rise and amplitude, but not the duration, of slow action potentials whereas verapamil (1 microM) blocked them. They were not blocked by tetrodotoxin (10 microM), and 4-aminopyridine (1 mM) prolonged the decay phase without affecting the rate of rise and amplitude. The rate of rise, amplitude and duration of slow action potentials were not affected by LEMS IgG. These results show that LEMS IgG does not act on Ca2+ channel currents that underlie slow action potentials in mouse ventricles, suggesting antigenic differences between Ca2+ channels at motor nerve terminals and heart.     

Differentiation of receptor sites for [3H]nitrendipine in chick hearts and physiological relation to the slow Ca2+ channel and to excitation-contraction coupling

The properties of interaction of the Ca2+ channel antagonist [3H]nitrendipine have been investigated in chick hearts at various stages of in ovo and post-natal development and in cultured cells. The dissociation constant of the [3H]nitrendipine-receptor complex is between 0.4 nM and 0.5 nM for intact ventricle and cultured cells. [3H]Nitrendipine binding is antagonized by nitrendipine analogs. The order of efficacy of the different dihydropyridine molecules is nitrendipine greater than nimodipine greater than nifedipine greater than nisoldipine with Kd values ranging from 0.5 to 4 nM. Inhibition of [3H]nitrendipine binding by other antiarrhythmic molecules like amiodarone, F13004 and bepridil was observed. Half-maximum inhibitions (K0.5) were found for verapamil and D600 at concentrations between 0.23 and 0.26 microM. The potency of organic Ca2+ blockers to depress by 50% the maximum amplitude of spontaneous beating of heart cells is closely related to K0.5 values obtained from [3H]nitrendipine binding experiments. Electrophysiological results indicate that the slow channel is insensitive to nitrendipine at the younger stage of development (3-day-old) whereas, in adult like cells, nitrendipine (50 nM) abolished both slow action potential due to the slow Ca2+ channel and contraction. The maximum binding capacity for [3H]nitrendipine is found to increase during development of the embryonic heart from 40 fmol/mg protein at day 3 to 100 fmol/mg protein at day 14, to stay relatively stable until day 18. Then the number of sites increases rapidly to reach a second plateau at 210 fmol/mg protein on day 4 after hatching. Treatment with 6-hydroxydopamine results in 35% increase in [3H]nitrendipine binding, whereas reserpine treatment is without effect. Developmental properties of nitrendipine-sensitive Ca2+ channels have been compared with those of tetrodotoxin-sensitive Na+ channels and muscarinic receptors. These results indicate that nitrendipine receptors exist at the early stage of development (3-day-old-hearts) but that they do not correspond to functional slow Ca2+ channels, that in ovo development corresponds both to an increase of the number of [3H]nitrendipine receptors and to the transformation of silent Ca2+ channels into functional Ca2+ channels, and that there is a regulation of the level nitrendipine-sensitive Ca2+ channels by innervation.     

Calcium-sensitive action potential of long duration in the fertilized egg of the ctenophore Mnemiopsis leidyi

The membrane properties of fertilized eggs of the ctenophore Mnemiopsis leidyi were studied using standard microelectrode techniques. The resting potential was approximately -80 mV, and was dependent on the extracellular K concentration. Depolarizing current injections elicited an action potential with an initial peak amplitude of +20 to +40 mV (duration about 5 sec) and a long lasting (duration 3 to 10 min) plateau phase. The depolarizing phase and the plateau phase appeared to have different ionic mechanisms. The entire action potential could be prevented by removal of extracellular Ca, but only the amplitude of the depolarizing phase, not the plateau phase, was dependent on the extracellular Ca concentration. The plateau phase was not observed in the absence of Ca, but in the presence of Ca its duration was dependent on the external Ca concentration. The data suggest that the plateau phase is activated as a consequence of Ca influx during the initial depolarizing phase. Removal of external Na resulted in only minor changes in the waveform of repolarization. The action potential was resistant to low concentrations of Mn and Cd in the presence of Ca. The role of this action potential in ctenophore development is not known, but in its waveform and duration it resembles the sperm-gated potentials that have been seen in eggs of other phyla. These experiments show ctenophore embryos to be excitable at very early stages, and suggest their utility in the study of the differentiation of cellular electrical properties.     

Calcineurin trapped in liposomes inhibits the action potentials of isolated 3-days-old embryonic chick ventricle.

Calcineurin, an intracellular protein phosphatase (type 2B), is reported to inhibit L-type (slow) calcium channels and thereby play a key role in channel inactivation. The present study was undertaken to examine effects of calcineurin on slow channel dependent action potentials of 3-days-old embryonic chick ventricle and to assess the role of this enzyme in regulation of developing slow channels. Calcineurin trapped in phosphatidylcholine-liposomes to facilitate its intracellular uptake was found to inhibit maximal upstroke velocity (+Vmax), overshoot and duration of action potentials. At higher doses of calcineurin containing liposomes the preparations ceased to exhibit spontaneous activity but elicited electrically driven action potentials with lower +Vmax and overshoot. These observations show that calcineurin down-modulates the embryonic cardiac slow channels under basal conditions.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.     

Action potentials dependent on monovalent cations in developing mouse embryos

Action potentials were examined using intracellular recording techniques to study the ionic mechanisms of excitability in oocytes and embryos of the mouse from the 1-cell through to the 16-cell stages of development. At all stages examined, action potentials dependent on monovalent cations (Na+ or Li+) were observed under Ca2+-free conditions, and the maximum rate of rise (MRR) of the Na action potential was larger than that of the Li action potential at a given concentration of monovalent cations. Both the Na and Li action potentials were insensitive to tetrodotoxin, and they were blocked by inorganic (Co2+, Cd2+, Mn2+, La3+) and organic (diltiazem) Ca antagonists. These properties were exactly the same as those of the Ca channels present in the membranes of the mouse embryos. In addition, competition was observed between permeant monovalent and divalent cations: the overshoot and MRR of the Na or Li action potentials were reduced in the presence of Ca2+. These results suggest that Na+ or Li+ go through the Ca channels when the external Ca2+ concentration was very low, and that the Ca channels are more permeable to Na+ than to Li+. Separate Na channels could not be detected or induced at any stages of development.     

Voltage-dependent modulation of ion binding and translocation in the cardiac Na(+)-Ca2+ exchange system

The transport of Na+ and Ca2+ ions in the cardiac Na(+)-Ca2+ exchanger can be described as separate events (Khananshvili, D. (1990) Biochemistry 29, 2437-2442). Thus, the Na(+)-Na+ and Ca(2+)-Ca2+ exchange reactions reflect reversible partial reactions of the transport cycle. The effect of diffusion potentials (K(+)-valinomycin) on different modes of the Na(+)-Ca2+ exchanger (Na(+)-Ca2+, Ca(2+)-Ca2+, and Na(+)-Na+ exchanges) were tested in reconstituted proteoliposomes, obtained from the Triton X-100 extracts of the cardiac sarcolemmal membranes. The initial rates of the Nai-dependent 45Ca-uptake (t = 1 s) were measured in EGTA-entrapped proteoliposomes at different voltages. At the fixed values of voltage [45 Ca]o was varied from 4 to 122 microM, and [Na]i was saturating (150 mM). Upon varying delta psi from -94 to +91 mV, the Vmax values were increased from 9.5 +/- 0.5 to 26.5 +/- 1.5 nmol.mg-1.s-1 and the Km from 17.8 +/- 2.5 to 39.1 +/- 5.2 microM, while the Vmax/Km values ranged from only 0.53 +/- 0.08 to 0.73 +/- 0.17 nmol.mg-1.s-1.microM-1. The equilibrium Ca(2+)-Ca2+ exchange was voltage sensitive at very low [Ca]o = [Ca]i = 2 microM, while at saturating [Ca]o = [Ca]i = 200 microM the Ca(2+)-Ca2+ exchange became voltage-insensitive. The rates of the equilibrium Na(+)-Na+ exchange appears to be voltage insensitive at saturating [Na]o = [Na]i = 160 mM. Under the saturating ionic conditions, the rates of the Na(+)-Na+ exchange were at least 2-3-fold slower than the Ca(2+)-Ca2+ exchange. The following conclusions can be drawn. (a) The near constancy of the Vmax/Km for Na(+)-Ca2+ exchange at different voltages is compatible with the ping-pong model proposed previously. (b) The effects of voltage on Vmax of Na(+)-Ca2+ exchange are consistent with the existence of a single charge carrying transport step. (c) It is not yet possible to clearly assign this step to the Na+ or Ca2+ transport half of the cycle although it is more likely that 3Na(+)-transport is a charge carrying step. Thus, the unloaded ion-binding domain contains either -2 or -3 charges (presumably carboxyl groups). (d) The binding of Na+ and Ca2+ appears to be weakly voltage-sensitive. The Ca(2+)-binding site may form a small ion-well (less than 2-3 A).     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Current recording from sensory cilia of olfactory receptor cells in situ. II. Role of mucosal Na+, K+, and Ca2+ ions

Action potential-driven current transients were recorded from sensory cilia and used to monitor the spike frequency generated by olfactory receptor neurons, which were maintained in their natural position in the sensory epithelium. Both basal and messenger-induced activities, as elicited with forskolin or cyclic nucleotides, were dependent on the presence of mucosal Na+. The spike rate decreased to approximately 20% when mucosal Na+ was lowered from 120 to 60 mM (replaced by N-methyl-D-glucamine+), without clear changes in amplitude and duration of the recorded action potential-driven transients. Mucosal Ca2+ and Mg2+ blocked spike discharge completely when increased from 1 to 10 mM in Ringer solution. Lowering mucosal Ca2+ below 1 mM increased the spike rate. These results can be explained by the presence of a cyclic nucleotide-dependent, Ca(2+)-sensitive cation conductance, which allows a depolarizing Na+ inward current to flow through the apical membrane of in situ receptor cells. A conductance with these properties, thought to provide the receptor current, was first described for isolated olfactory cells by Nakamura and Gold (1987. Nature (Lond.). 325:442-444). The forskolin-stimulated spike rate decreased when l-cis-diltiazem, a known blocker of the cyclic nucleotide-dependent receptor current, was added to the mucosal solution. Spike rate also decreased when the mucosal K+ concentration was lowered. Mucosal Ba2+ and 4-aminopyridine, presumably by means of cell depolarization, rapidly increased the spike rate. This suggests the presence of apical K+ channels that render the receptor cells sensitive to the K+ concentration of the olfactory mucus. With a slower time course, mucosal Ba2+ and 4-aminopyridine decreased the amplitude and caused rectification of the fast current transients (prolongation of action potentials). Abolishment of the apical Na+ current (by removal of mucosal Na+), as indicated by a strong decrease in spike rate, could be counteracted by adding 10 mM Ba2+ or 1 mM 4-aminopyridine to the mucosal solution, which re-established spiking. Similarly, blockage of the apical cation conductance with 10 mM Ca could be counteracted by adding 10 mM Ba2+ or by raising the mucosal K+ concentration. Thus mucosal concentrations of Na+, K+, and Ca2+ will jointly affect the sensitivity of odor detection.     

Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine

In clonal rat pituitary cells (GH cells), thyrotropin-releasing hormone (TRH) induced a pattern of changes in cytosolic free calcium concentrations [( Ca2+]i) composed of two phases: an acute spike phase to micromolar levels which decayed (t1/2 = 8 s) to a near-basal concentration and then rose to a prolonged plateau phase of elevated [Ca2+]i (as measured using Quin 2). Closely following these changes in [Ca2+]i, TRH stimulated a rapid "spike phase" of pronounced, but brief, enhancement of the rate of prolactin and growth-hormone secretion and then a "plateau phase" of prolonged enhancement. These two phases were dissociated using two classes of pharmacologic agents: the ionophore ionomycin, and a calcium channel antagonist nifedipine. Ionomycin (100 nM) specifically blocked (less than 90%) the spike phase of TRH action by rapidly emptying the TRH-regulated reservoir of cellular Ca2+ to generate a TRH-like spike in [Ca2+]i; nifedipine inhibited (less than 50%) the plateau phase of TRH-induced changes in [Ca2+]i and hormone secretion by preventing Ca2+ influx through voltage-dependent Ca2+ channels. These agents demonstrated that the TRH-induced spike in [Ca2+]i in GH cells is caused by release of an ionomycin-sensitive pool of cellular Ca2+ with a small component (10%) due to influx of extracellular Ca2+. The TRH-induced plateau in [Ca2+]i is due to influx of extracellular Ca2+, about half of which enters through voltage-dependent calcium channels and half of which enters via nifedipine/verapamil-insensitive influx. The TRH-induced spike in [Ca2+]i led to a burst in hormone secretion, and the plateau in [Ca2+]i produced a prolonged enhancement of secretion; the spike and plateau phases were generated independently by TRH. A spike in [Ca2+]i is necessary, but not sufficient, to induce burst release of hormone, while the prolonged rate of hormone secretion is intimately related to the steady-state [Ca2+]i.     

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels

An analysis of the relationship between electrical membrane activity and Ca2+ influx in differentiated GnRH-secreting (GT1) neurons revealed that most cells exhibited spontaneous, extracellular Ca(2+)-dependent action potentials (APs). Spiking was initiated by a slow pacemaker depolarization from a baseline potential between -75 and -50 mV, and AP frequency increased with membrane depolarization. More hyperpolarized cells fired sharp APs with limited capacity to promote Ca2+ influx, whereas more depolarized cells fired broad APs with enhanced capacity for Ca2+ influx. Characterization of the inward currents in GT1 cells revealed the presence of tetrodotoxin-sensitive Na+, Ni(2+)-sensitive T-type Ca2+, and dihydropyridine-sensitive L-type Ca2+ components. The availability of Na+ and T-type Ca2+ channels was dependent on the baseline potential, which determined the activation/inactivation status of these channels. Whereas all three channels were involved in the generation of sharp APs, L-type channels were solely responsible for the spike depolarization in cells exhibiting broad APs. Activation of GnRH receptors led to biphasic changes in cytosolic Ca2+ concentration ([Ca2+]i), with an early, extracellular Ca(2+)-independent peak and a sustained, extracellular Ca(2+)-dependent phase. During the peak [Ca2+]i response, electrical activity was abolished due to transient hyperpolarization. This was followed by sustained depolarization of cells and resumption of firing of increased frequency with a shift from sharp to broad APs. The GnRH-induced change in firing pattern accounted for about 50% of the elevated Ca2+ influx, the remainder being independent of spiking. Basal [Ca2+]i was also dependent on Ca2+ influx through AP-driven and voltage-insensitive pathways. Thus, in both resting and agonist-stimulated GT1 cells, membrane depolarization limits the participation of Na+ and T-type channels in firing, but facilitates AP-driven Ca2+ influx.     

Sodium-conducting channels in cardiac membranes in low calcium.

With no Ca in the patch electrode, two kinds of channels conduct Na in spontaneously beating embryonic chick heart cells. One channel conducts Na primarily during the upstroke of the action potential and is blocked by tetrodotoxin (TTX). The other channel conducts Na primarily during the late plateau and early repolarization phase of the action potential, but only in Ca concentrations below 10(-6) M. This second channel is TTX-insensitive and has a conductance of 50 to 90 pS, depending upon the interpretation of open-channel flickering. These two Na-conducting channels correspond to the channels that normally carry the fast Na current (INa) and the slow Ca current (Isi).     

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).     

Thyrotropin-releasing hormone-induced spike and plateau in cytosolic free Ca2+ concentrations in pituitary cells. Relation to prolactin release

Using the acetoxymethyl ester of "Quin 2," a fluorescent Ca2+-indicator, we have loaded prolactin (PRL)-producing rat pituitary cells with non-toxic concentrations of Quin 2 and quantitated changes in cytosolic free calcium concentration ( [Ca2+]i) during stimulation of PRL release by thyrotropin-releasing hormone (TRH) and 40 mM K+. TRH induced a biphasic response, with an immediate (less than 1 s) spike in [Ca2+]i from basal levels (350 +/- 80 nM) to a peak of 1-3 microM, which decayed rapidly (t 1/2 = 8 s) to a near basal nadir, then rising to a plateau in [Ca2+]i of 500-800 nM. The TRH-induced spike phase was attenuated but not abolished by prior addition of EGTA, while the plateau phase was eliminated by EGTA. Addition of 40 mM K+ caused an immediate spike in [Ca2+]i to 1-3 microM which equilibrated slowly (t 1/2 = 1 min) directly to a plateau of 600-800 nM. The K+-induced spike and plateau phases were both abolished by prior addition of EGTA. The biphasic nature of TRH action on [Ca2+]i parallels the biphasic actions of TRH on 45Ca2+ fluxes and the biphasic release of PRL by GH cells in suspension. These findings provide evidence that Ca2+-dependent agonist-mediated increases in [Ca2+]i and hormone release are linked, and may generally have two modes: an acute "spike" mode, dependent primarily on redistribution of intracellular Ca2+ stores; and a sustained "plateau" mode, dependent on influx of extracellular Ca2+.     

Bethanidine increased Na+ and Ca2+ currents and caused a positive inotropic effect in heart cells

The effects of bethanidine sulphate, a pharmacological analog of the cardiac antibrillatory drug, bretylium tosylate, were studied on action potentials (APs) and K+, Na+, and Ca2+ currents of single cultured embryonic chick heart cells using the whole-cell current clamp and voltage clamp technique. Extracellular application of bethanidine (3 X 10(-4) M) increased the overshoot and the duration of the APs and greatly decreased the outward K+ current (IK) and potentiated the inward fast Na+ currents (INa) and the inward slow calcium current (ICa). However, intracellular introduction of bethanidine (10(-4) M) blocked INa. In isolated atria of rat, bethanidine increased the force of contraction in a dose-dependent manner. These findings suggest that when applied extracellularly, bethanidine exerts a potentiating effect on the myocardial fast Na+ current and slow Ca2+ current and an inhibitory effect of IK. The positive inotropic effect of bethanidine could be due, at least in part, to an increase of Ca2+ influx via the slow Ca2+ channel and the Na-Ca exchange. It is suggested that the decrease of IK by bethanidine may account for its antifibrillatory action.     

Verapamil and zero Ca2+ alter responses of cat muscle to halothane and caffeine

Strips of soleus (slow twitch, oxidative) and gracilis (fast-twitch, glycolytic) muscle were obtained from 27 anesthetized cats and mounted in organ baths filled with oxygenated Krebs-Ringer solution (37 degrees C). The responses to caffeine, halothane (1%), caffeine in the presence of halothane, and electrical stimulation in the presence of halothane were examined in the two fiber types. These responses were compared with those observed in paired strips of muscle that had been treated with verapamil (10 or 28 microM), a slow calcium (Ca2+) channel blocker, with zero Ca2+, or with zero Ca2+ where magnesium (3.7 mM Ca2+) was added to replace the Ca2+. Halothane-induced contractures in the soleus were blocked by verapamil and zero Ca2+. Caffeine-induced contractures and tetanic contractions were attenuated in zero Ca2+ and by verapamil in both fiber types. Halothane overcame verapamil-induced reductions of caffeine contractures and tetanic contractions in both fiber types. In contrast, halothane did not overcome zero Ca2+-induced reductions in caffeine contractures or tetanic contractions in either fiber type. Furthermore, the addition of Mg2+ to the zero Ca2+ did not restore the responses. The findings with verapamil indicate that in cat muscle, both halothane- and caffeine-induced contractures and tetanic contractions are dependent on the influx of extracellular Ca2+. This extracellular Ca2+ may enter through the slow Ca2+ channels. However, because halothane in combination with caffeine or electrical stimulation overcame the effects of verapamil, there may be other sites involved.