Development of slow Ca2+-Na+ channels during organ culture of young embryonic chick hearts

Young (3-days-old) embryonic chick hearts have slowly-rising spontaneous action potentials, dependent on tetrodotoxin-insensitive slow Na+ channels. When the hearts were placed into organ culture for 5-11 days, action potential duration was markedly increased by 260-370%, and a notch appeared between the initial spike phase and the plateau phase in some hearts. The spike amplitude was mainly dependent on [Na]0, whereas the plateau amplitude was dependent on [Ca]0. Thus, the young embryonic hearts develop slow Ca2+-Na+ channels (while retaining the slow Na+ channels) during organ culture, and the spike phase and the plateau phase of the slow action potentials are mainly dependent on currents through slow Na+ channels and through slow Ca2+-Na+ channels, respectively. The effects of Mn2+ (a specific blocker of slow Ca2+-Na+ channels) and verapamil (a blocker of slow Na+ channels as well as of slow Ca2+-Na+ channels) on the spike phase and the plateau phase were examined. Mn2+ (0.5 mM) and verapamil (5 microM) depressed the plateau duration and overshoot. Verapamil did not decrease the maximum rate of rise (Vmax), but Mn++ produced a small, but significant, decrease. High concentrations (10/30 microM) of verapamil depressed the action potential amplitude and Vmax, and abolished the spontaneous action potentials. These results indicate that slow Ca2+-Na+ channels appear de novo during organ culture of young embryonic hearts.     

Electrophysiology of the flounder heart (Platichthys flesus)—the effect of agents which modify transmembrane ion transport

1. A sucrose gap system was used to record action potentials and mechanical responses of flounder heart.2. Diltiazem eliminated mechanical responses and strongly inhibited the action potential plateau while nifedipine only slightly reduced cardiac contractions without significantly changing the action potential.3. Verapamil slightly hyperpolarized flounder heart but was without effect on either the action potential or mechanical activity except at very high concentrations.4. Lanthanum was ineffective at 2 mM on flounder heart, but manganese at 3 mM substantially inhibited electrical and mechanical responses accompanied by a small hyperpolarization. Substitution of manganese for calcium abolished all flounder cardiac activity.5. BAY K 8644 enhanced cardiac force and enhanced the action potential plateau while depolarizing the preparations. Calcium-free salines abolished heart contractions and the action potential plateau while the spike phase persisted.6. Low sodium salines enhanced while sodium-free salines abolished all heart activity as did tetrodotoxin above I μM. Tetrodotoxin abolished the action potential spike leaving only a small plateau phase.7. Substituting lithium for sodium hyperpolarized the heart, enhanced contractions and prolonged the action potential plateau. Ouabain enhanced cardiac activity and depolarized the heart but ferosemide was without effect on either electrical or mechanical activity.8. TEA at 6 mM had a modest positive inotropic effect and negative chronotropic effect on the heart while the action potential plateau phase was enhanced.9. These results indicate that extracellular sodium and calcium are crucial in flounder heart electrogenesis but such a major role for potassium could not be established.     

Purinergic modulation of spontaneous activity and of responses to high potassium and acetylcholine in rat ileal smooth muscle

1. In rat ileal smooth muscle both adenosine and ATP at 10⁻⁴ M significantly enhanced spontaneous mechanical activity. The excitatory actions of adenosine were blocked by the P₁ receptor antagonist 8-phenyltheophylline and the excitatory effects of ATP were significantly reduced by the P₂ receptor antagonist quinidine.2. The P₂ receptor desensitizer α,β-methylene-ATP was without effect on ACh responses nor did the stable analogue β,gg-methylene-ATP exert any effect on spontaneous mechanical activity.3. Pretreatment with adenosine caused a dose-dependent enhancement of K-induced contractures in the ileum. Low adenosine concentrations slightly inhibited and high concentrations slightly enhanced ACh-induced contractures in the ileum.4. ATP potentiated the phasic component of the ileal K-induced contracture but strongly inhibited tonic force at high concentrations. This agent slightly inhibited the phasic component of the ACh-induced contracture while strongly inhibiting ACh-induced tonic force.5. α,β-methylene-ATP inhibited ileal muscle ACh induced contractures while it potentiated both phasic and tonic K-induced contractures. β, γ-methylene ATP inhibited ACh-induced contractures but it enhanced K-induced phasic contractures while inhibiting K-induced tonic force.6. The results of this study suggest that rat ileum may contain the A₁ subtype of the P₁ receptor but the evidence for a P₂ receptor subtype is conflicting despite the inhibition of ATP actions by quinidine.7. The inhibition of K- and ACh-induced tonic force suggests that adenosine and ATP interactions with ileal smooth muscle may inactivate slow voltage-dependent calcium channels leading to EC uncoupling.     

Differences in Na and Ca Spikes As Examined by Application of Tetrodotoxin, Procaine, and Manganese Ions

The effects of tetrodotoxin, procaine, and manganese ions were examined on the Ca spike of the barnacle muscle fiber injected with Ca-binding agent as well as on the action potential of the ventricular muscle fiber of the frog heart. Although tetrodotoxin and procaine very effectively suppress the "Na spike" of other tissues, no suppressing effects are found on "Ca spike" of the barnacle fiber, while the initiation of the Ca spike is competitively inhibited by manganese ions. The initial rate of rise of the ventricular action potential is suppressed by tetrodotoxin and procaine but the plateau phase of the action potential is little affected. In contrast the suppressing effect of manganese ions is mainly on the plateau phase. The results suggest that the plateau phase of the ventricular action potential is related to the conductance increase in the membrane to Ca ions even though Na conductance change may also contribute to the plateau.     

Bombesin-induced changes in membrane potential-dependent phasic contractions of cat gastric muscle

Electrical and contractile activities of smooth muscle strips isolated from the circular muscle layer of cat gastric antrum were studied using the sucrose gap technique. Bombesin (10(-8) mol/l) depolarized the gastric muscle; this was accompanied by an increase in the strip tone, in the plateau action potential frequency and in both the frequency and the amplitude of the spike potentials as well as by a shortening of the plateau action potential duration. Both the frequency and the amplitude of the phasic contractions increased thereafter. The changes in the frequency of the plateau action potentials and contractions were not influenced either by antagonists of cholinergic and adrenergic receptors or by TTX. In the presence of the Ca antagonists D600 (10(-6) mol/l) and nifedipine (10(-7) mol/l) or in Ca-free medium containing EGTA the effect of bombesin on the frequency of the plateau action potentials and phasic contractions remained unchanged; however, spike potentials were not observed and no increase in the amplitude of phasic contractions occurred. UV-light inactivation of nifedipine restored the typical bombesin effect on the electrical and contractile activities of the gastric smooth muscle. The present data suggest that the effect of bombesin on the frequency of both plateau action potentials and phasic contractions is not linked with Ca2+ influx.     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

Effects of cortisone on mechanical activity and membrane ionic currents in frog auricular fibres.

The influence of cortisone on the mechanical and electrical activity of frog auricular fibres was investigated under voltage clamp conditions. 1. Cortisone exerted in vitro an inotropic action depending on concentration; a maximal positive inotropic effect was observed with 2 x 10(-4) g/ml of cortisone. 2. The positive inotropic effect of cortisone might be either an indirect sympathomimetic effect or an adrenaline-like effect. 3. The positive inotropic action of cortisone was correlated with modifications of the cardiac action potential: the amplitude of the action potential was enhanced while the resting membrane potential was unchanged; the amplitude and duration of the plateau were increased and the duration of the action potential was lengthened. 4. The electrical changes were related to an increase in the slow calcium current intensity resulting from an increase in the slow calcium conductance.     

Effects of tetraethylammonium and 4-aminopyridine on the plateau potential of circular myometrium from the pregnant rat

In the pregnant rat, spontaneous electrical activity of circular muscle (CM) changes from single, plateau-type action potentials at early and mid-term to repetitive spike trains at term. To examine mechanisms underlying the plateau, we studied the effects of potassium channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on membrane potentials in CM from rats on gestation Days 14, 15, 16, 21 (term). Apparent membrane conductance was measured at rest and during the plateau in Day 14 muscles with and without TEA. 4-AP depolarized the resting membrane on all gestation days. Therefore, a direct action of 4-AP on plateau configuration could not be separated from an indirect effect of depolarization. TEA did not affect the resting potential but increased action potential size and depolarization rate on all gestation days. On Day 16, TEA reduced plateau amplitude, unmasking small, repetitive depolarizations. D-600 decreased plateau amplitude and duration and attenuated these effects of TEA. Plateau conductance increased initially then decreased before membrane repolarization. Membrane conductance and outward rectification during the plateau were reduced by TEA. The plateau potential may result from an outwardly rectifying TEA-sensitive current combined with a slow inward current, the plateau magnitude being determined by the relative intensity of each current.     

Augmentation and suppression of action potentials by estradiol in the myometrium of pregnant rat.

The purpose of this study was to investigate the actions of estradiol on spontaneous and evoked action potentials in the isolated longitudinal smooth muscle cells of the pregnant rat. Single cells were obtained by enzymatic digestion from pregnant rat longitudinal myometrium. Action potentials and currents were recorded by whole-cell current-clamp and voltage-clamp methods, respectively. The acute effects of 17beta-estradiol on action potentials and inward and outward currents were investigated. The following results were obtained. The average resting membrane potential of single myometrial cells was -54 mV (n = 40). In many cells, an electrical stimulation evoked a membrane depolarization, and action potentials were superimposed on the depolarization. In some cells, spontaneous action potentials were observed. Estradiol (30 microM) slightly depolarized the membrane (ca. 5 mV) and attenuated the generation of action potentials by reducing the frequency and amplitude of the spikes. Afterhyperpolarization was also attenuated by estradiol (30 microM). On the other hand, in 5 of 35 cells, estradiol increased the first spike amplitude and action potential duration, while frequency of the spike generation and afterhyperpolarization were inhibited. In voltage-clamped muscle cells, estradiol inhibited both inward and outward currents. Acute inhibition or augmentation of spike generation by estradiol is due to the balance of inhibition of inward and outward currents. Inhibition of both currents also prevented afterhyperpolarization, causing potential-dependent block of Ca spikes.     

The effects of H2O2 on electromechanical activity of the ileum longitudinal muscle

The effects of H2O2 on electrical and mechanical activity of the longitudinal layer from the guinea-pig ileum were studied using sucrose-gap technique and the influence of H2O2 on ionic current was investigated in single smooth muscle cells by the patch-clamp method. In most of the preparations tested, the spontaneous activity observed was composed of slow waves with superimposed action potentials (APs). Both were resistant to tetrodotoxin and atropine. H2O2 (1 mmol/l) evoked sustained 3-5 mV membrane depolarisation, doubled the amplitude of the slow waves and increased their frequency, augmented the APs and reduced their splitting. These changes were accompanied with significant contraction, which had an amplitude comparable to that of the tonic component of 50 mmol/l K+-induced contraction. Calcium-free solution caused membrane depolarisation, reduction of the slow wave amplitude and frequency, disappearance of APs and decreased the mechanical tension of the preparations. Application of H2O2 (1 mmol/l) into the zero-calcium bath solution recovered the APs, which was accompanied by a low amplitude contraction. H2O2 (up to 1 mmol/l) increased the L-type calcium current (I(Ca)) both under conventional whole-cell patch-clamp configuration and under amphotericin-perforated patches by 16 +/- 3%. These data demonstrated that contractile response of the ileum longitudinal smooth muscle preparation evoked by H2O2 was mainly due to the enhanced electrical activity.     

In vivo recording of electrical activity of canine tracheal smooth muscle.

Electrical activity of the tracheal smooth muscle was studied using extracellular bipolar electrodes in 37 decerebrate, paralyzed, and mechanically ventilated dogs. A spontaneous oscillatory potential that consisted of a slow sinusoidal wave of 0.57 +/- 0.13 (SD) Hz mean frequency but lacked a fast spike component was recorded from 15 dogs. Lung collapse accomplished by bilateral pneumothoraxes evoked or augmented the slow potentials that were associated with an increase in tracheal muscle contraction in 26 dogs. This suggests that the inputs from the airway mechanoreceptors reflexly activate the tracheal smooth muscle cells. Bilateral vagal transection abolished both the spontaneous and the reflexly evoked slow waves and provided relaxation of the tracheal smooth muscle. Electrical stimulation of the distal nerve with a train pulse (0.5 ms, 1-30 Hz) evoked slow-wave oscillatory potentials accompanied by a contraction of the tracheal smooth muscle in all the experimental animals. Our observations in this in vivo study confirm that the electrical activity of tracheal smooth muscle consists of slow oscillatory potentials and that tracheal contraction is at least partly coupled to the slow-wave activity of the smooth muscle.     

Morphological and electrophysiological aspects of dorsal median paired neurons generating plateau action potentials in the terminal abdominal ganglion of the insect central nervous system

Among the three clusters of dorsal unpaired median neurons of the Periplaneta americana terminal abdominal ganglion, another type of neuron has been characterized by anterograde cobalt stainings and microelectrode technique. These neurons are bilaterally distributed in the ganglion. Their axons ipsilaterally exit the ganglion via the anterior proctodeal nerves, to innervate the proctodeum. They are characterized by a long-duration overshooting action potentials and a low firing frequency. Most often the depolarizing phase is composed of two peaks: a fast spike followed by a slow phase. Tetrodotoxin suppressed the fast peak and blocked the spontaneous activity suggesting that sodium channels are involved in the depolarizing phase as well as in the initiation of the action potential. Calcium channel blockers induced a disappearing of the slow depolarizing phase indicating the participation of calcium ions and a reduction of the afterhyperpolarization reflecting the participation of calcium-activated potassium channels. Furthermore, cadmium, as lanthanum or barium, induced a long-lasting plateau potential, which would be due to a persistent sodium conductance. Tetraethylammonium increased the duration of the action potential indicating that potassium channels are implicated in the falling phase. The results demonstrate that these neurons are different from other cells, especially dorsal unpaired median neurons, of the central nervous system of the cockroach.Abbreviations DUM dorsal unpaired median - SDP slow depolarizing phase - AP action potential - PAP plateau action potential - TAG terminal abdominal ganglion - CNS central nervous system     

Diltiazem potentiates mechanical activity in mammalian skeletal muscle

The calcium antagonist drug diltiazem produces a concentration dependent increase in twitch amplitude and a decrease in the mechanical threshold in mouse and rat skeletal muscle fibers in vitro. The greater potency of the d-cis isomer of diltiazem over the 1-cis isomer suggests that the effects of diltiazem are specific and may result from its binding to a specific receptor.     

Calcium-dependent slow potassium conductance in rat skeletal myotubes

A prolonged hyperpolarizing afterpotential (amplitude 5–20 mV, half decay time about 400 msec at 25°C) follows the action potential in myotubes and myosacs cultured from rat skeletal muscle. This slow hyperpolarizing afterpotential (hap) is mediated by an increase in membrane K conductance, because its reversal potential follows the Nernst potential for K and is not affected by other ions. The conductance increase measured during the hap (up to four times the resting input conductance) correctly predicts the time course of the slow hap. The slow hap is Ca dependent. Its amplitude decreases when bath [Ca] is lowered, and both amplitude and duration increase when bath [Ca] is raised. The slow hap is blocked by intracellular injection of the calcium chelator, EGTA. It is inhibited by solutions containing 2–4 mM manganese or 1–5 mM barium, but is not blocked by 5–20 mM tetraethylammonium. Myotubes bathed in zero [Na], high [Ca] solutions show calcium action potentials, which are inhibited by 2–10 mM manganese, nickel or cobalt. Myotubes bathed in isotonic Ca salts (or in 2 mM Ca plus 5 mM caffeine) show long-lasting (up to 10 sec) spontaneous hyperpolarizations accompanied by prolonged contractions. These hyperpolarizations are associated with a large increase in input conductance, and they reverse in sign near the K equilibrium potential. They appear to reflect activation of the Ca-sensitive K conductance by Ca released from intracellular stores. The observation that spontaneous hyperpolarizations usually occur with no prior depolarization argues that at least a portion of the slow, Ca-sensitive K conductance system can be activated by internal Ca alone, with no requirement for plasma membrane depolarization. Cultured myotubes also have a faster K conductance system, which is inhibited by 5–20 mM tetraethylammonium or 1–5 mM barium, and is not dependent on Ca for its activation.     

Spontaneous electromechanical activity in the rat duodenum in vitro.

Isolated rat duodenum shows spontaneous mechanical and electrical activities. Mechanical activity consists in changes both in endoluminal pressure and in isometric tension. Electrical activity is characterized by slow waves with superimposed bursts. This spontaneous activity is tetrodotoxin (TTX) resistant and therefore it is myogenic in origin. Indeed, TTX pretreatment, even in the presence of atropine and guanethidine, caused an increase in amplitude and in frequency of the electrical and mechanical activities. This finding indicates the presence of tonically active inhibitory intramural non adrenergic, non cholinergic (NANC) nerves. Duodenal longitudinal strips showed a spontaneous mechanical activity resembling that one recorded from isolated segment. Instead, circular strips are quiescent under resting condition and a contractile activity can be detected only after TTX pretreatment suggesting that: i) the circular smooth muscle layer is tonically inhibited by intramural NANC nerves and, ii) the contractions observed in the rat duodenum are due to the activity of the longitudinal one.     

Effects of high and low doses of vitamins A and E on the excitability of frog cardiomyocytes

The effects of complex vitamins A and E on electrophysiological characteristics was investigated on frog cardiomyocytes. Large doses of vitamins A and E decreased the steepness of the action potential (AP) front, decreased the spike amplitude, shortened the plateau, decreased the steepness in the last phase of AP-repolarization, and decreased the rest potential level but considerably increased overshoot. The Ap-duration decreased noticeably. Small doses of vitamins decreased the spike amplitude, shortened the plateau, decreased the Ap-duration. We assume that complex of vitamins A and E can affect the cardiomyocyte membrane transport function, mainly due to the suppression of the slow membrane channels. The AP-front increase is less steep under the influence of vitamins. It is shown that the 0-phase depolarization speed decreases due to the inhibition of sodium channels.     

Effect of methionine-enkephalin on the spontaneous electrical and mechanical activity of the smooth muscle of the rat portal vein

In the longitudinal smooth muscle of the isolated rat portal vein, methionine-enkephalin (Met-enkephalin) increased the spontaneous contraction with a concentration as low as 10(-8)M. When the membrane activity was recorded using a microelectrode, Met-enkephalin enhanced the spike burst activity but without any effect on the resting membrane potential. Naloxone, phentolamine, atropine and reserpine pre-treatment did not inhibit the excitatory effect of Met-enkephalin on the spontaneous contraction. These results suggest that the excitatory effect of Met-enkephalin on the mechanisms involved in the automaticity may be a direct action on smooth muscle or relate to presynaptic action on a non-adrenergic non-cholinergic system.     

The effects of cholecystokinin-octapeptide and pentagastrin on electrical and motor activities of canine colonic circular muscle

The effects of cholecystokinin-octapeptide (CCK-OP) and pentagastrin on electrical and motor activities of circular muscle of the canine colon were studied with the sucrose gap technique. Additional organ bath experiments were performed to further characterize the motor response to the peptides and to elucidate their site of action. The electrical activity consisted of slow waves having an initial potential followed by a plateau potential, at a regular frequency of 4.5 cycles/min. Both peptides prolonged the duration and increased the amplitude of the plateau phase of the slow waves. Concomitantly, the slow wave frequency was reduced. In addition, CCK-OP increased spiking activity. Both spiking activity and the prolonged plateau potential generated contractile activity, prolonged phasic contraction occurring with slow waves with a prolonged plateau. In organ bath experiments, both CCK-OP and pentagastrin increased the basal tone of the muscle strips and prolonged the duration of the phasic contractions. The prolongation of the duration of the contractions was not antagonized by tetrodotoxin (TTX) and atropine. CCK-OP but not pentagastrin increased the force of contractions, this action was not affected by atropine but was reduced in the presence of TTX, suggesting that the increase in force may be partially mediated by noncholinergic excitatory nerves. The increase in basal tension by the peptides was enhanced in the presence of TTX indicating that myenteric inhibitory neurones were tonically active under our experimental conditions. The results provide the electrophysiological basis for CCK-OP and pentagastrin induced changes in colonic motility.     

Neurotoxic action of some antiarrhythmic agents: comparative effects of propafenone,lidocaine and amiodarone on leech Retzius nerve cell

A series of antiarrhythmic drugs was studied on spontaneous spike activity and depolarizing outward potassium current in leech Retzius nerve cells. Propafenone (0.7 μM/ml) produced a cardiac-like action potential with a rapid depolarization followed by a sustained depolarization or plateau, which is terminated after 250 msec by a rapid repolarization. The effect of lidocaine (0.7 μM/ml) on spontaneous spike activity was less pronounced, and early afterdepolarization has been recorded. Amiodarone at the same and much higher concentrations (3 μM/ml) did not generate either a cardiac-like action potential or an early afterdepolarization. In the voltage clamp experiments, fast and slow calcium-activated outward potassium currents were suppressed with propafenone and lidocaine but not with amiodarone. These results suggest that the antiarrhythmic drugs, propafenone and lidocaine modulate calcium-activated potassium channels in leech Retzius nerve cells.     

Effect of Bay K 8644 on tetraethylammonium-induced excitability of the rabbit pulmonary artery

The effect of Bay K 8644 on the electrical activity of the smooth muscle cells in the main pulmonary artery of the rabbit was examined. In normal physiological solution, the resting membrane potential was -56 +/- 0.6 mV, and the cells were electrically quiescent. Tetraethylammonium (5 mM) depolarized the membrane to about -45 mV, and electrical stimulation elicited action potentials. To suppress contractile responses and thereby facilitate sustained impalements, the muscle strips were bathed with a hypertonic solution containing sucrose. The mean amplitude of the tetraethylammonium-induced action potentials in the hypertonic solution was 35 +/- 0.9 mV. The action potentials were dependent upon the extracellular Ca2+ concentration and were abolished by diltiazem (10(-6) M). Spontaneous action potentials were occasionally generated in the presence of tetraethylammonium alone and could be induced by the further addition of Ba2+ (0.5 mM). The Ca2+ agonist Bay K 8644 (10(-8) to 10(-6) M) had no effect on the resting membrane potential or excitability in normal solution. However, in the hypertonic solution containing tetraethylammonium, Bay K 8644 caused a further depolarization and oscillatory potential changes, which were not prevented by tetrodotoxin. The oscillations were suppressed or abolished by diltiazem or nilvadipine. Thus, active responses can occur in the normally quiescent smooth muscle cells of the rabbit pulmonary artery when the outward K+ current(s) are suppressed.