Inhibition of neuromuscular transmission in the guinea-pig saphenous artery by atriopeptin II

In the guinea-pig saphenous artery, stimulation of perivascular nerves elicited contraction and two types of synaptic potentials: the excitatory junction potential and the slow depolarization. The synaptic potentials were inhibited by atriopeptin II but not by sodium nitroprusside. Exogenous noradrenaline induced membrane depolarization and contraction, and both sodium nitroprusside and atriopeptin II inhibited the contraction but not the depolarization. These results suggest that atriopeptin II has an inhibitory effect both presynaptically at the nerve terminals and postsynaptically on the vascular smooth muscle cells.     

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.     

Electro- and pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery

A contraction of the rabbit ear artery can be induced by depolarizing the cells with a K-rich solution if Ca is present. 10(-9)-10(-6) M noradrenaline and 10(-8)-10(-7) M histamine cause a contraction of this tissue without modifying the membrane potential. If the histamine concentration exceeds 10(-7) M some depolarization of the membrane also occurs. Both noradrenaline and histamine also induce a contraction in Ca-free medium, even if La is present. None of these stimuli produces action potentials or fluctuations of the membrane potential. Besides these tonic contractions, the ear artery can also produce phasic contractions when 10 mM TEA is added to the medium. Such contractions are caused by the appearance of action potentials which are Ca dependent and which are similar to those appearing in visceral smooth muscle. A study of 45Ca fluxes has revealed that K depolarization and noradrenaline cause only a small increase in 45Ca uptake by the cells, while noradrenaline also releases cellular Ca, even in Ca-free medium. A comparison of tension development and 45Ca release induced by noradrenaline in Ca-free medium suggests that Ca extrusion could be very efficient in the rabbit ear artery and that it could play a direct role in its relaxation.     

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.     

Changes in cytosolic calcium monitored by inward currents during action potentials in guinea-pig ventricular cells

Action potentials were recorded from single cells isolated from guinea-pig ventricular muscle. Contraction was measured with an optical technique. Tail currents thought to be activated by cytosolic calcium were recorded when action potentials were interrupted by application of a voltage-clamp. A family of tail currents was recorded by interrupting the action potential at various times after the upstroke. The envelope of tail current amplitudes was taken as an index of changes in cytosolic calcium. Consistent with this interpretation, tail currents were negligible following intracellular loading with the calcium chelator BAPTA to suppress calcium transients. The cytosolic calcium transient estimated from the envelope of tails reached a peak approximately 50 ms after the upstroke of the action potential, and fell close to diastolic levels before repolarization was complete; 10 mM caffeine delayed the time to peak contraction, and caused a prolongation of the cytosolic calcium transient estimated from the envelope of tail currents. Caffeine also induced the appearance of a distinct late plateau phase of the action potential. Intracellular BAPTA suppressed the late plateau, contraction and tail currents in cells exposed to caffeine. Exposure to caffeine increased the time constant for decay of tail currents (from approximately 25 to 70 ms). When action potentials were greatly abbreviated by interruption with a voltage-clamp, a progressive decline occurred in the subsequent three contractions and tail currents. There was a progressive reversal of these effects over four responses when the full action potential duration was restored. None of these effects was observed in cells exposed to caffeine. Calcium-activated tail currents appear to be a useful qualitative index of changes in cytosolic calcium. The observations are consistent with the suggestion that cytosolic calcium is reduced during the plateau by a combination of calcium extrusion through Na-Ca exchange and calcium uptake into caffeine-sensitive stores. It also appears that reduction of stores loading during abbreviated action potentials reduces subsequent contraction in cells not exposed to caffeine.     

The origin of two components in contraction of guinea pig papillary muscle in the presence of noradrenaline.

In experiments on isolated guinea pig papillary muscles the effects of verapamil (5 mg/L) and caffeine (1 g/L) on the two-component contraction were investigated. The muscles were continuously superfused with normal Tyrode's solution containing 2 mg of noradrenaline/L at 20--22 degrees C. The first derivative of contractile response and transmembrane action potential were simultaneously recorded. Verapamil suppressed the amplitude of the second component and had no influence on the first component of contraction. Caffeine eliminated the first component and increased the second component of contraction. It was suggested that in the activation of two-component contraction calcium ions from two different pools take part: (1) those released from sarcoplasmic reticulum, and (2) those that crossed a cell membrane during the plateau of the action potential.     

Effect of noradrenaline on the electrical and contractile properties of smooth muscle cells in the pulmonary artery]

Experiments were performed on the smooth muscle cells of rabbit a. pulmonalis using the microelectrode technique. No spontaneous electrical or mechanical activity was recorded in normal Krebs solution. The current-voltage relation in these smooth muscle cells showed marked rectification. No changes in the isometric tension were observed due to the anodal or cathodal stimulating currents. Strong depolarization of the muscle cells produced only local potentials on the cathelectrotone which never developed into a spike. Noradrenaline (10(-8) g/ml) caused depolarization of the 5-7 mV in the muscle cell membrane and a considerable contraction of the muscle strip as well. Under such conditions the contractile apparatus of the muscle cells became sensible to the resting potential level. Anodal stimulation was accompanied by relaxation of the muscle strip, whereas cathodal stimulation--by its contraction. The alpha-adrenoblocking agent (phentolamine) blocked the effect of noradrenaline evidencing the fact that noradrenaline exerted its excitatory action on the smooth muscle cells of the a. pulmonalis through the alpha-adrenoreceptors.     

K+-induced twitch potentiation is not due to longer action potential

The objective of this study was to determine whether an increased duration of the action potential contributes to the K+-induced twitch potentiation at 37 degrees C. Twitch contractions were elicited by field stimulation, and action potentials were measured with conventional microelectrodes. For mouse extensor digitorum longus (EDL) muscle, twitch force was greater at 7-13 mM K+ than at 4.7 mM (control). For soleus muscle, twitch force potentiation was observed between 7 and 11 mM K+. Time to peak and half-relaxation time were not affected by the increase in extracellular K+ concentration in EDL muscle, whereas both parameters became significantly longer in soleus muscle. Decrease in overshoot and prolongation of the action potential duration observed at 9 and 11 mM K+ were mimicked when muscles were respectively exposed to 25 and 50 nM tetrodotoxin (TTX; used to partially block Na+ channels). Despite similar action potentials, twitch force was not potentiated by TTX. It is therefore suggested that the K+-induced potentiation of the twitch in EDL muscle is not due to a prolongation of the action potential and contraction time, whereas a longer contraction, especially the relaxation phase, may contribute to the potentiation in soleus muscle.     

Corticotropin-releasing hormone effects on human pregnant vs. nonpregnant myometrium explants estimated from a mathematical model of uterine contraction.

In this paper, we applied a new theoretical model of uterine contraction to a large panel of human pregnant and nonpregnant myometrial strips, treated or not by corticotrophin-releasing hormone (CRH). This model is based on a fine analysis of the contraction curves. This analysis yielded four mathematical parameters (beta, theta, tau 1, and tau 2) related to excitability, duration of plateau phase, and time constants for relaxation describing, respectively, the different portions of the contraction cycle. This leads to specific differences in spontaneous contractile activity between pregnant and nonpregnant states. The relaxing effect of CRH in the pregnant state is presumably correlated with the origin of the strips (the lower uterine segment). Besides our observation of a specific receptor-dependent relaxing effect of CRH in both pregnant and nonpregnant myometrium, we could identify highly significant effects at given CRH concentration for beta in nonpregnant myometrium and for theta, tau 1, and tau 2 in pregnant myometrium. In addition, highly significant differences were found between pregnant and nonpregnant myometrium. Also, we discovered a strong correlation between theta and tau 1, specifically in the pregnant state. Although the biochemical signification of these results remains to be elucidated, they contribute to emphasize the complex network of CRH action at the myometrial level. Furthermore, our approach could pave the way toward a better analysis of the efficacy of the uterine contractile behavior.     

Characterization of isolated ventricular myocytes from adult zebrafish (Danio rerio)

The zebrafish is widely used for human related disease studies. Surprisingly, there is no information about the electrical activity of single myocytes freshly isolated from adult zebrafish ventricle. In this study, we present an enzymatic method to isolate ventricular myocytes from zebrafish heart that yield a large number of calcium tolerant cells. Ventricular myocytes from zebrafish were imaged using light and confocal microscopy. Myocytes were mostly rod shaped and responded by vigorous contraction to field electrical stimulation. Whole cell configuration of the patch clamp technique was used to record electrophysiological characteristics of myocytes. Action potentials present a long duration and a plateau phase and action potential duration decreases when increasing stimulation frequency (as observed in larger mammals). Together these results indicate that zebrafish is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human.     

Muscle layer- and region-dependent distributions of oxytocin receptors in the porcine myometrium

The aim of the present study was to clarify smooth muscle- and region-dependent distributions of the oxytocin receptor that mediates oxytocin-induced contraction in the nonpregnant porcine myometrium by means of mechanical and radioligand ([3H]-oxytocin) binding studies. In Krebs solution, oxytocin (0.1-300 nM) caused concentration-dependent contractions of the cornual myometrium, and the longitudinal muscle was more sensitive than the circular muscle. [Arg8]-vasopressin and [deamino-Cys1, D-Arg8]-vasopressin also contracted the myometrium, and the order of the potency was oxytocin > [Arg8]-vasopressin > [deamino-Cys(1), D-Arg(8)]-vasopressin. Treatment with a high concentration of oxytocin selectively inhibited the contraction of oxytocin and [Arg8]-vasopressin without affecting the responses of acetylcholine and high-K+. Selective cross inhibition was also observed in the presence of a high concentration of [Arg(8)]-vasopressin. The oxytocin-induced contraction was resistant to tetrodotoxin and atropine, but was reduced by verapamil or by the removal of external Ca2+, indicating that oxytocin has a direct action on smooth muscle cells and that extracellular Ca2+ plays an important role for the contraction. In Kumagai solution, oxytocin caused contraction of the cornual longitudinal muscle (-logEC50 = 8.5) but not the circular muscle. Longitudinal muscles of other regions (corpus and cervix) were also responsive to oxytocin, but the -logEC50 value differed from region to region (cornua > corpus = cervix). On the other hand, oxytocin failed to cause contraction of the corpus and cervical circular muscles. 3H-Oxytocin bound to crude membrane preparations of the myometrium in a concentration-dependent (0.084-2.7 nM) saturable manner. Scatchard analysis of equilibrium binding data revealed the presence of a single class of binding site with an apparent dissociation constant (Kd, 1.1-1.5 nM), but receptor density (Bmax) differed in the two muscle layer types (longitudinal muscle: circular muscle = 5:1) and tended to decrease from the cornua to the cervix. In conclusion, the receptor specific for oxytocin is present in the porcine myometrium and mediates the contractile responses of both oxytocin and [Arg8]-vasopressin. The distribution of the oxytocin receptors differs according to the type of muscle layer (longitudinal muscle > circular muscle) and the region of the uterus.     

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.     

Detection of motor unit action potentials with surface electrodes: influence of electrode size and spacing

A model of the motor unit action potential was developed to investigate the amplitude and frequency spectrum contributions of motor units, located at various depths within muscle, to the surface detected electromyographic (EMG) signal. A dipole representation of the transmembrane current in a three-dimensional muscle volume was used to estimate detected individual muscle fiber action potentials. The effects of anisotropic muscle conductance, innervation zone location, propagation velocity, fiber length, electrode area, and electrode configuration were included in the fiber action potential model. A motor unit action potential was assumed to be the sum of the individual muscle fiber action potentials. A computational procedure, based on the notion of isopotential layers, was developed which substantially reduced the calculation time required to estimate motor unit action potentials. The simulations indicated that: 1) only those motor units with muscle fibers located within 10–12 mm of the electrodes would contribute significant signal energy to the surface EMG, 2) variation in surface area of electrodes has little effect on the detection depth of motor unit action potentials, 3) increased interelectrode spacing moderately increases detection depth, and 4) the frequency content of action potentials decreases steeply with increased electrode-motor unit territory distance.     

Actions of noradrenaline and some other biogenic amines on the body-wall muscles of an echiuroid, Urechis unicinctus

1. Noradrenaline inhibited contractions of the body-wall strips of U. unicinctus in response to electrical pulse of stimulation and L-proline, while serotonin enhanced them. In addition, serotonin increased the rate of relaxation of twitch contraction. Octopamine enhanced the contractions but was less potent than serotonin. 2. Acetylcholine-contraction of the isolated inner circular body-wall muscle was also inhibited by noradrenaline and enhanced by serotonin. 3. Phentolamine, an alpha-adrenoreceptor blocker, potentiated tetanic contraction of the strip, though it did not alter twitch contraction. The inhibitory action of noradrenaline was blocked by phentolamine. 4. Noradrenaline hyperpolarized the fibre membrane of the inner circular muscle, while serotonin did not alter the membrane potential. The action of noradrenaline was blocked by phentolamine. 5. Bioassay of the body-wall extract and the ventral nerve-cord extract suggests that these extracts contain serotonin-like and noradrenaline-like substances, respectively. 6. These results suggest that noradrenaline and serotonin may be neurotransmitters or neurohormones modulating muscle contraction in the body wall of U. unicinctus.     

Overexpression of human beta2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

This study investigated cardiac excitation-contraction coupling at 37 degrees C in transgenic mice with cardiac-specific overexpression of human beta2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (I(Ca-L)) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I(Ca-L), Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37 degrees C with fluo 3. These observations suggest that the gain of Ca(2+)-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I(Ca-L) in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.     

Development of a new coulter counter system: Measurement of the volume,internal conductivity,and dielectric breakdown voltage of a single guard cell protoplast ofVicia faba

Summary Two intracellular microelectrodes were used to study electrotonic interaction between cultured embryonic (16- to 20-day-old) chick myocardial cells reaggregated into small spheresin vitro. Under different culture conditions, reaggregates with two types of functional membrane properties were produced: (i) highly differentiated reaggregates, and (ii) reverted reaggregates. In the highly differentiated state, the cells had high stable resting potentials and produced rapidly-rising tetrodotoxin (TTX)-sensitive action potentials in response to electric field stimulation. In the reverted state, the cells exhibited slowrising spontaneous action potentials having prominent pacemaker potentials and TTX-insensitive upstrokes. These states resemble electrophysiological properties of the highly differentiated (18 daysin ovo) and less fully differentiated (3 daysin ovo) intact embryonic chick heart, respectively. Both types of reaggregates had similar ultrastructural appearance, with many elongated cells and intercalated disc-like structures; gap-like junctions were not seen. The highly differentiated cells had input resistances of about 5 M, and exhibited only little electrotonic interaction in response to intracellular current injection either when the cells were at rest or during the action potential plateau. Intracellular stimulation produced propagating action potentials which triggered contraction of the entire reaggregate. Large hyperpolarizing current pulses applied during the action potential plateau caused premature repolarization which also propagated to the other impaled cell. In the reverted reaggregates, electrotonic interaction was weak or absent in about 52% of the impaled cell pairs, moderate in 30%, and strong in 18% (encountered only at interelectrode distances of less than 100 m). The difference in degree of electrotonic interaction may be due to the state of differentiation with respect to the membrane electrical properties.     

Simple techniques suitable for student use to record action potentials from the frog heart

Demonstrating action potentials during class experiments is very educational for science students. It is not easy, however, to obtain a stable intracellular recording of action potentials from the conventionally used skeletal muscle cells, because the tip of a glass microelectrode often comes out or breaks due to muscle contraction. Here, I present a much simpler recording method using a flexible polyethylene electrode with a wide orifice (approximately 1 mm) for a bullfrog heart beating on automaticity. Extracellular recordings of action potentials (electrocardiogram) can be obtained by placing an electrode on the cardiac surface, and transmembrane potentials can be obtained by rupturing the membrane with negative pressure, i.e., whole cell configuration. Once attached to the heart by suction, the polyethylene electrode does not easily come off during contraction of the heart. Perfusion of the heart via the postcaval vein offers us opportunities for observing the effects of either changing ionic compositions of solutions or applying drugs. The techniques shown here provide a simple and convenient way to perform a variety of class experiments.     

Loss of the plateau of the cardiac action potential in hypertonic solutions

The effect of hypertonicity on the electrical properties of vertebrate myocardial cells was studied in ventricular muscle fibers of guinea pig, cat, frog, and chicken. The latter two species do not have a T-tubule system, whereas the former two do. In hypertonic solutions (2 x isotonic) produced by addition of sucrose or excess of NaCl, cell diameter decreased and there was a slight hyperpolarization and decrease in action potential overshoot. In guinea pig and cat, the hypertonic solution caused a decrease in input resistance and the plateau of the action potential to disappear in some of the cells; contractions of the entire ventricle also became depressed. These effects were reversed by returning the muscle fibers to isotonic solution. Addition of 5 mM SrCl₂ to the hypertonic solution also caused the plateau component and contraction to reappear. In frog and chick cells, loss of the plateau component and contraction never occurred in hypertonic solution, and input resistance increased. Urea and glycerol hyperosmolarity (2 x) caused no loss of the plateau component or contraction. If the frog and chicken ventricular, and guinea pig atrial myocardial cells (all of which lack T tubules) were to serve as an adequate control for possible effects of hypertonicity on the surface membrane and on contractile proteins, then the results suggest that swelling of the T tubules of mammalian myocardial cells leads to loss of the plateau component.     

Activation of the fast calcium input in the denervated smooth muscle of the cat nictitating membrane

The excitation and contraction features of innervated and sympathetically denervated smooth muscle strips from cat's nictitating membrane have been studied by single sucrose gap arrangement. Increasing of smooth muscle cells sensitivity to drugs were accompanied by elevation of membrane response and the ability to generation of action potentials. Action potentials have been induced by agonists or high potassium concentration in external solution and spontaneously. In innervated muscle action potentials have been evoked as a result of depolarization by high potassium concentration of TEA blockade of potassium conductance. Induced and spontaneously generated action potentials were blocked by organic and inorganic antagonists of potential dependent Ca++ channels. In Ca-free solution action potentials were absent but might be supported by Ba++. Decrease of Na+ had no effect on smooth muscle excitability. It is supposed that activation of potential depended Ca++ channels in smooth muscle cells with pharmaco-mechanical coupling are under influence of sympathetic nerves.     

Simultaneous monitoring of cellular depolarization and contraction: a new method to investigate excitability and contractility in isolated smooth muscle cells

The present experiments were performed to establish a method for simultaneous monitoring of excitation and contraction in isolated smooth muscle cells. The smooth muscle cells were dissociated from the colons of Wistar rats by enzymatic digestion. All the experiments were performed on mixtures of circular and longitudinal cells. In a first set of experiments, focal extracellular potentials (FEPs) and transmembrane action potentials (APs) were simultaneously recorded from the cells by use of extracellular and intracellular pipettes, respectively. In a second set of experiments, cellular contraction induced by chemical stimulation was monitored simultaneous with the FEP recordings. The FEPs had spike and plateau amplitudes of 44.5 +/- 2.3 and 8.9 +/- 0.7 mV, respectively, and reproduced the general morphology of gastrointestinal APs. The parallel mechanical measurements from the rat colonic cells showed that they shortened with an average peak contraction of 8.8 +/- 1.4 microm and an average contraction velocity of 8.2 +/- 0.9 microm/s, to develop an average peak force of 1.2 +/- 0.2 microN, and generated an average peak power of 36 +/- 15 pW. Simultaneous monitoring of FEPs and cellular contraction demonstrates correlations between the electrical and mechanical events taking place in the investigated cells.