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.     

Intracellular chloride activity in rabbit papillary muscle: effect of serum

The intracellular chloride activity (aiCl), measured with Cl-selective microelectrodes on stimulated rabbit papillary muscles (1 Hz) incubated in serum, was 7.2 +/- 2.2 mM (48 measurements). Under the same condition, on the quiescent muscle, aiCl was 7.5 +/- 2.8 mM (45 measurements). The membrane potential of quiescent papillary muscles and diastolic potential of stimulated papillary muscles were -79.0 +/- 0.7 (50 measurements) and -83.5 +/- 0.5 mV (50 measurements), respectively. The experimental conditions were chosen to reproduce the in vivo conditions where the Cl equilibrium potential is close to the membrane potential or to the diastolic potential. After correcting for cytoplasmic interference (4 mM) on the aiCl measurements, the Cl equilibrium potential (ECl) was -84 mV. In conclusion, the Cl distribution in cardiac cells bathed in serum is passive as for in vivo cardiac cells.     

The "pacemaker" function of the transient outward current in the rabbit myocardium

The single sucrose gap technique was employed to study the electrically induced automaticity in rabbit papillary muscles. When the potential was clamped at the level of the "maximum diastolic potential" following the first spike of automaticity an initial decline of the outward ionic current with subsequent activation of the delayed potassium current was observed. The initial decline was potential-sensitive with a maximum at approximately -2 mV; it diminished when the rate of stimulation increased and was abolished with 4-aminopyridine plus Sr2+. It is suggested that the transient outward current determines the development of the "pacemaker potential" after the first spike of electrically induced automaticity in rabbit papillary muscles.     

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.     

POLARIZED INTERCELLULAR BRIDGES IN OVARIAN FOLLICLES OF THE CECROPIA MOTH

Fluorescein-labeled rabbit serum globulin was injected into vitellogenic oocytes of the cecropia moth. Though the label spread throughout the ooplasm in less than 30 min, it was unable even after 2 h to cross the complex of intercellular bridges connecting the oocyte to its seven nurse cells. After injection into a single nurse cell, fluorescence was detected in the oocyte adjacent to the bridge complex within 3 min and had spread throughout the ooplasm in 30 min. Here also, the cell bodies of the six uninjected nurse cells remained nonfluorescent. Four of the nurse cells are not bridged directly to the oocyte but only through the apical ends of their siblings. Unidirectional movement must therefore occur in the apical cytoplasm of the nurse cells, as well as in the intercellular bridges. The nurse cells of healthy follicles had an intracellular electrical potential -40 mV relative to blood or dissecting solution, while oocytes measured -30 mV. A mV difference was also detected by direct comparison between a ground electrode in one cell and a recording electrode in the other. Three conditions were found in which the 10 mV difference was reduced or reversed in polarity. In all three cases fluorescent globulin was able in some degree to cross the bridges from the oocyte to the nurse cells.     

Use of micromachined probes for the recording of cardiac electrograms in isolated heart tissues

Micromachined probes, with iridium (Ir) microelectrodes on silicon shanks, were evaluated to assess their suitability for cardiac electrogram recording. The electrochemical activation (anodic oxidation) procedure for the circular Ir microelectrode was investigated using the square wave potential according to the electrode size, number of cycles, and cathodic-anodic potential level of the square wave. Increase in the charge storage capacity was pronounced either in smaller electrodes or with higher potential level of the square wave. The electrode impedance reduced in a similar manner with increasing number of cycle irrespective of the electrode size. With either lower potential level (-0.70/+0.60 V) or smaller number of cycle (200 cycles) than those for the activation of stimulating electrode, the likelihood of overactivation of the recording microelectrode can be minimized. These anodic IrOx film (AIROF) microelectrodes were used for the recording of extracellular electrograms in two different ex vivo cardiac tissue preparations. A single-shank microprobe was applied to the left ventricle of a mouse heart. Both the spontaneous and paced transmural responses propagating between epicardium and endocardium were obtained. Longitudinal cardiac wavefronts propagating along the rabbit papillary muscle were also recorded with a unique multiple-shank design. The measured mean amplitude and the propagation velocity of the extracellular voltage were 12.2 +/- 1.8 mV and 58.9 +/- 2.2 cm/s, respectively (n = 27). These microprobes with precisely defined electrode spacing make a useful tool for the spatial and temporal mapping of electrical properties in isolated heart tissues ex vivo.     

Functional evidence for Na+-activated K+ channels in circular smooth muscle of the opossum lower esophageal sphincter

Na(+) reduction induces contraction of opossum lower esophageal sphincter (LES) circular smooth muscle strips in vitro; however, the mechanism(s) by which this occurs is unknown. The purpose of the present study was to investigate the electrophysiological effects of low Na(+) on opossum LES circular smooth muscle. In the presence of atropine, quanethidine, nifedipine, and substance P, conventional intracellular electrodes recorded a resting membrane potential (RMP) of -37.5 +/- 0.9 mV (n = 4). Decreasing [Na(+)] from 144.1 to 26.1 mM by substitution of equimolar NaCl with choline Cl depolarized the RMP by 7.1 +/- 1.1 mV. Whole cell patch-clamp recordings revealed outward K(+) currents that began to activate at -60 mV using 400-ms stepped test pulses (-120 to +100 mV) with increments of 20 mV from holding potential of -80 mV. Reduction of [Na(+)] in the bath solution inhibited K(+) currents in a concentration-dependent manner. Single channels with conductance of 49-60 pS were recorded using cell-attached patch-clamp configurations. The channel open probability was significantly decreased by substitution of bath Na(+) with equimolar choline. A 10-fold increase of [K(+)] in the pipette shifted the reversal potential of the single channels to the positive by -50 mV. These data suggest that Na(+)-activated K(+) channels exist in the circular smooth muscle of the opossum LES.     

Substance P-immunoreactive nerve fibres in the anterior segment of the rabbit eye

Summary Substance P-immunoreactive nerve terminals were found in several locations in the anterior segment of the rabbit eye. In the iris they occurred in the sphincter muscle and were randomly distributed in the iris stroma with some fibres running close to the dilator muscle. In the ciliary body these immunoreactive elements were few and occurred within bundles of nerve fibres, while in the ciliary processes they were more numerous with a predominantly subepithelial location. Blood vessels in the anterior uvea were often surrounded by substance P-immunoreactive fibres. No substance P-fibres were found in the cornea, while the sclera contained very few such elements.Using conventional in vitro techniques it was found that the sphincter pupillae muscle of the iris responded to electrical stimulation with a contraction that was resistant to cholinergic and adrenergic blockade, but was inhibited by the neuronal blocker tetrodotoxin. This indicates the existence of a non-cholinergic, non-adrenergic neuronal mediator of the contractile response. Exogenously applied substance P produced a long-lasting contraction of the spincter muscle, an observation compatible with the view that substance P is the noncholinergic, non-adrenergic neurotransmitter involved.     

cAMP suppresses CA2+-dependent electrical activity of airway smooth muscle induced by TEA

Using intracellular microelectrodes, we investigated whether exogenous dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) or forskolin influenced the electrical effects of tetraethylammonium (TEA) on canine tracheal smooth muscle. We found that 20 mM TEA depolarized airway smooth muscle cells from a resting membrane potential (Em) of -59 +/- 4 mV (mean +/- SD) to -45 +/- 2 mV and caused spontaneous action potentials (AP's) to develop, which were 33 +/- 2 mV in amplitude. These were totally abolished in 0 Ca2+ solution. DBcAMP (1 mM) suppressed the development of this TEA-induced electrical activity and the phasic contractions electrically coupled to it. DBcAMP had no significant effect on Em in the absence of TEA however. Forskolin (1 microM) produced similar effects. Our findings suggest that Ca2+ is the principal ion responsible for the inward current associated with the TEA-induced AP's in airway smooth muscle, and that adenosine 3',5'-cyclic monophosphate may suppress the electrogenesis of this current.     

Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials

The plasma membrane of the rabbit egg allows only one sperm to enter the egg during fertilization, but the mechanism of this block to polyspermy is unknown. Electrophysiology and in vitro fertilization techniques were employed in this study to investigate the possibility that a voltage block to polyspermy exists in rabbit eggs. Ovulated zona-intact eggs had a mean membrane potential of -71 +/- 2.1 mV (interior negative). A stereotypic response occurred 12-135 min following in vitro insemination in 19 of 40 eggs. Association of this stereotypic response with the appearance of pronuclei suggested that the electrical response was related to some interaction of gametes. This response consisted of a slow transient 8 +/- 1.5 mV depolarization upon which were superimposed up to 36 repetitive diphasic insemination potentials. Each potential consisted of a brief 2.0 +/- 0.44 mV hyperpolarization followed by a slow 2.5 +/- 0.45 mV depolarization. The small amplitude of the stereotypic response when compared with the large variation of resting potentials suggested that the response was insufficient to block polyspermy by a mechanism dependent upon the magnitude of the rabbit egg membrane potential.     

Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells

Permeabilization of the plasma membrane by electrical forces (electroporation) can be either transient or stable. Although the exact molecular mechanics have not yet been described, electroporation is believed to initiate primarily in the lipid bilayer. To better understand the kinetics of membrane permeabilization, we sought to determine the time constants for spontaneous transient pore sealing. By using isolated rat flexor digitorum brevis skeletal muscle cells and a two-compartment diffusion model, we found that pore sealing times (tau p) after transient electroporation were approximately 9 min. tau p was not significantly dependent on the imposed transmembrane potential. We also determined the transmembrane potential (delta Vm) thresholds necessary for transient and stable electroporation in the skeletal muscle cells. delta VmS ranging between 340 mV and 480 mV caused a transient influx of magnesium, indicating the existence of spontaneously sealing pores. An imposed delta Vm of 540 mV or greater led to complete equilibration of the intracellular and extracellular magnesium concentrations. This finding suggests that stable pores are created by the larger imposed transmembrane potentials. These results may be useful for understanding nerve and skeletal muscle injury after an electrical shock and for developing optimal strategies for accomplishing transient electroporation, particularly for gene transfection and cell transformation.     

Sodium-pump potentials and currents in guinea-pig ventricular muscles and myocytes.

When guinea-pig papillary muscles were depolarized to ca. -30 mV by superfusion with K+-free Tyrode's solution supplemented with Ba2+, Ni2+, and D600, addition of Cs+ transiently hyperpolarized the membrane in a reproducible manner. The size of the hyperpolarization (pump potential) depended on the duration of the preceding K+-free exposure; peak amplitudes (Epmax) elicited by 10 mM Cs+ after 5-, 10-, and 15-min K+-free exposures were 12.9, 17.7, and 23.2 mV, respectively. Pump potentials were unaffected by external Cl- but suppressed by cardiac glycosides, hyperosmotic conditions, and low-Na+ solution. Using Epmax as an indicator of Na+ pump activation, the half-maximal concentration for activation by Cs+ was 12-16.3 mM. At 6 mM, Cs+ was three times less potent than Rb+ or K+ and five times more potent than Li+. From these findings, and correlative voltage-clamp data from myocytes, we calculate that (i) a pump current of 7.8 nA/cm2 generates an Epmax of 1 mV and (ii) resting pump current in normally polarized muscle (approximately 0.16 microA/cm2) is five times smaller than previously estimated.     

Failure of tachykinins including substance P and its fragments to influence proteoglycan and protein synthesis in bovine chondrocytes in vitro.

Adult bovine articular chondrocytes were exposed to substance P, neurokinins A and B or substance P fragments, SP1-4, SP1-6 and SP7-11 in vitro. Proteoglycan synthesis was assessed by measuring proteoglycans which were released into the culture medium or incorporated into the cell layer. The intact tachykinins or substance P fragments had no direct effect on proteoglycan synthesis. Nor was total protein production affected. Gel chromatography, under dissociative conditions, revealed that sulphated proteoglycans detected in the medium or cell layer following treatment of chondrocytes with substance P, contained proteoglycans of similar molecular weight to those produced by cells exposed only to diluent controls. Therefore, we conclude that the acceleration of arthritis by substance P does not appear to be mediated through an effect on chondrocyte synthetic function.     

Calcium channels in crayfish muscle fibre fragments studied by means of the Vaseline gap technique

Calcium currents in crayfish muscle fibres were studied by means of the vaseline gap voltage clamp technique. Overlapping potassium currents were fully suppressed using fibre fragments equilibrated in K+-free intracellular solution. The design of the recording chamber tailored to crayfish muscle fibres is described in detail. Ca currents recorded has a two-component time course. The transient (ICa, T) component (peaking in about 10 ms) attained, on average, maximal overall density of 26.4 microA/cm2 at depolarization to -4.6 mV from a holding potential of -80 mV. The steady (ICa, S) component attained 16.7 microA/cm2 (evaluated at the end of a 70 ms pulse) at +13.8 mV. The average overall surface area of the clamped membrane surface (including invaginated parts) was about 0.07 cm2. The ICa, S component could be separated from ICa, T using short inactivating prepulses. Voltage and time dependence of the transient component inactivation, as well as its recovery from inactivation, were in agreement with a Ca-dependent mechanism. Independent behaviour of the two Ca current components and differences in their properties support the hypothesis concerning the existence of two populations of Ca channels in the surface membrane of the crayfish muscle.     

Effect of hemodynamic disorders on the transepithelial potential of the rat liver in vivo

The presence of transepithelial electrical potential (TEP) formed on the border between the interstitium and the interior of the rat liver bile capillary has been demonstrated. The initial TEP value (II+ 3 mV) reduced rapidly to zero after the ligation of different portal fissure vessels, while ischemia arrest led to TEP restoration. True periodical oscillations of TEP value with the period and amplitude of about 30 s and 1-2 mV, respectively, were registered. These oscillations appeared in all cases of liver ischemia 5-7 min after ischemia removal and lingered till the end of the experiment. It was assumed that variations in TEP and membrane potential of hepatocytes have the common nature.     

A STRAND OF CARDIAC MUSCLE : Its Ultrastructure and the Electrophysiological Implications of Its Geometry

The structure of a small strand of rabbit heart muscle fibers (trabecula carnea), 30–80 µ in diameter, has been examined with light and electron microscopy. By establishing a correlation between the appearance of regions of close fiber contact in light and electron microscopy, the extent and distribution of regions of close apposition of fibers has been evaluated in approximately 200 µ length of a strand. The distribution of possible regions of resistive coupling between fibers has been approximated by a model system of cables. The theoretical linear electrical properties of such a system have been analyzed and the implications of the results of this analysis are discussed. Since this preparation is to be used for correlated studies of the electrical, mechanical, and cytochemical properties of cardiac muscle, a comprehensive study of the morphology of this preparation has been made. The muscle fibers in it are distinguished from those of the rabbit papillary muscle, in that they have no triads and have a kind of mitochondrion not found in papillary muscle. No evidence of a transverse tubular system was found, but junctions of cisternae of the sarcoplasmic reticulum and the sarcolemma, peripheral couplings, were present. The electrophysiological implications of the absence of transverse tubules are discussed. The cisternae of the couplings showed periodic tubular extensions toward the sarcolemma. A regularly spaced array of Z line-like material was observed, suggesting a possible mechanism for sarcomere growth.     

Tonic activity of parasympathetic efferent nerve fibers hyperpolarizes the resting membrane potential of frog taste cells

We investigated the relationship between the membrane potential of frog taste cells in the fungiform papillae and the tonic discharge of parasympathetic efferent fibers in the glossopharyngeal (GP) nerve. When the parasympathetic preganglionic fibers in the GP nerve were kept intact, the mean membrane potential of Ringer-adapted taste cells was -40 mV but decreased to -31 mV after transecting the preganglionic fibers in the GP nerve and crushing the postganglionic fibers in the papillary nerve. The same result occurred after blocking the nicotinic acetylcholine receptors on parasympathetic ganglion cells in the tongue and blocking the substance P neurokinin-1 (NK-1) receptors in the gustatory efferent synapses. This indicates that the parasympathetic nerve (PSN) hyperpolarizes the membrane potential of frog taste cells by -9 mV. Repetitive stimulation of a transected GP nerve revealed that a -9-mV hyperpolarization of taste cells maintained under the intact GP nerve derives from an approximately 10-Hz discharge of the PSN efferent fibers. The mean frequency of tonic discharges extracellularly recorded from PSN efferent fibers of the taste disks was 9.1 impulses/s. We conclude that the resting membrane potential of frog taste cells is continuously hyperpolarized by on average -9 mV by an approximately 10-Hz tonic discharge from the parasympathetic preganglionic neurons in the medulla oblongata.     

Effects of cyclopiazonic acid on triggered activities in ventricular muscle and cardiomyocytes isolated from hamster hearts

The present experiments were performed to study the actions of cyclopiazonic acid on triggered activities generated in vitro in ventricular papillary muscle and cardiomyocytes isolated from the hearts of healthy male Syrian hamsters (Biobreeders F1B). Action potentials (APs) of ventricular muscle with a diameter around 1.5 mm were recorded using a microelectrode technique and force was recorded using a transducer. Ventricular preparations were driven at 2 Hz in high [Ca]o (9 mM)-low [K]o (1 mM) solution to induce delayed after depolarizations (DADs). Triggered activities were induced on resumption of electrical stimulation after a rest period of 20 sec. Effects of cyclopiazonic acid (3-10 microM) on steady-state rhythms and post-rest triggered activities were determined. Results revealed that cyclopiazonic acid initially enhanced the amplitude of DADs and induced post-rest triggered rhythms. However, after several minutes of cyclopiazonic acid exposure, AP duration (APD) was prolonged and DADs were significantly depressed. The effects on APD and DADs were reversible after washout of cyclopiazonic acid, but the diastolic potential during rest period oscillated and was able to generate high-frequency spontaneous APs at a reduced potential level. In ventricular myocytes isolated enzymatically, ionic currents were measured using of whole-cell patch-clamp techniques. In a high [Ca]o-low [K]o solution, a series of oscillatory transient inward currents (I(ti)) were obtained on repolarization to the holding potential (-45 mV) after a depolarizing pulse to the test potential of +20 mV for 1.2 sec. Cyclopiazonic acid (10 microM) reduced significantly the magnitude of I(ti). The present results in hamster ventricular cells suggested that cyclopiazonic acid by inhibiting the sarcoplasmic reticulum (SR)-Ca2+ pump would gradually deplete the amount of Ca2+ within the SR. The consequent reduction in the amount of Ca2+ released into the cytoplasm by cyclopiazonic acid might inhibit triggered arrhythmia through a reduction of DADs and I(ti).     

Reactive oxygen in skeletal muscle. II. Extracellular release of free radicals.

We have tested the hypothesis that diaphragm muscle fibers release superoxide anion radicals (O2-.) into the extracellular space. Fiber bundles were isolated from rat diaphragm and incubated in Krebs-Ringer solution containing cytochrome c (10(-5) M), a standard assay for O2-.. Bundles were either passive or active, i.e., directly stimulated to contract rhythmically. After 1 h, absorbance of reduced cytochrome c in the incubation medium was measured at 550 nm. Absorbance was greater in medium exposed to passive muscle than in medium without muscle (P < 0.01), indicating O2-. release by passive muscle. Absorbance was greater in medium exposed to active muscle than in that exposed to passive muscle (P < 0.01), an increase inhibited by superoxide dismutase (10(3) U/ml). Active bundles fatigued; bundles developing the lowest final stresses produced the greatest absorbance increases (P < 0.001), suggesting that the magnitude of fatigue was inversely related to O2-. release. We conclude that O2-. is released by diaphragm myocytes into the interstitium and surrounding medium, a process accelerated by fatiguing muscular contractions.