Effect of manganese and verapamil on electrical and contractile responses of pulmonary artery smooth muscle induced by noradrenaline and histamine

Action of noradrenaline and histamine on the resting potential, membrane resistance and contractility of rabbit pulmonary artery muscle cells was investigated in normal and Ca-blockers (manganese and verapamil)-containing Ringer-Lock solutions. It was shown that catecholamine and histamine induced depolarization by different mechanisms. Thus, noradrenaline action is accounted for by the decreased membrane permeability to potassium ions, while the histamine-induced depolarization is a consequence of sodium and, probably, chlorine permeability. The contraction induced by the transmitters is activated primarily by the extracellular calcium ions entering the cells by two ways: via chemosensitive Ca-channels activated by adrenergic and histaminergic receptors or via potential-dependent slow Ca-channels activated by the transmitter-induced membrane depolarization. It is not excluded that during activation of muscle cells by the transmitters part of calcium is release from both intramembrane and intracellular stores.     

兔肠系膜下神经节细胞的两种非胆碱能性慢突触后电位

以常规细胞内记录技术对兔肠系膜下神经节细胞的跨膜电位进行了观察。对节前神经的短串脉冲刺激,可诱发出一串快兴奋性突触后电位(f-EPSP)或顺向动作电位;在此之后,大多数细胞还出现一个持续约2min 的缓慢去极化电位。该电位具有抗箭毒和阿托品性质,受低钙高镁溶液的可逆性阻抑,因而可称为非胆碱能性兴奋性突触后电位,或者也可归入迟慢兴奋性突触后电位(ls-EPSP)。多数细胞的 ls-EPSP 伴有膜电阻增大,电位的幅度随细胞静息电位的超极化而变小;提示在这些细胞上,钾电导的失活很可能参与了电位的发生。以P物质溶液灌流神经节未见该电位有显著改变。另外,在箭毒化加阿托品化的神经节中,还发现少数细胞对节前神经的串刺激发生一个持续约一分钟的超极化电位。它也具有抗胆碱能受体阻断剂的性质,受低钙高镁溶液可逆性阻抑,为此我们命之为“极慢抑制性突触后电位”(vs-IPSP),以区别于“慢抑制性突触后电位”(s-IPSP),后者是通常用以表示一种胆碱能性的慢电位。本文所述的这两种非胆碱能性的突触电位有关递质,尚待探索。     

Cellular regulation of basal tone in internal anal sphincter smooth muscle by RhoA/ROCK

Sustained contractions of smooth muscle cells (SMC) maintain basal tone in the internal anal sphincter (IAS). To examine the molecular bases for the myogenic tone in the IAS, the present studies focused on the role of RhoA/ROCK in the SMC isolated from the IAS vs. the adjoining phasic tissues of the rectal smooth muscle (RSM) and anococcygeus smooth muscle (ASM) of rat. We also compared cellular distribution of RhoA/ROCK, levels of RhoA-GTP, RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex formation, levels of p(Thr696)-MYPT1, and SMC relaxation caused by RhoA inhibition. Levels of RhoA/ROCK were higher at the cell membrane in the IAS SMC compared with those from the RSM and ASM. C3 exoenzyme (RhoA inhibitor) and Y 27632 (ROCK inhibitor) caused a concentration-dependent relaxation of the IAS SMC. In addition, active ROCK-II (primary isoform of ROCK in SMC) caused further shortening in the IAS SMC. C3 exoenzyme increased RhoA-RhoGDI binding and reduced the levels of RhoA-GTP and p(Thr696)-MYPT1. ROCK inhibitor attenuated PKC-induced contractions in IAS SMC. Conversely, a PKC inhibitor (G? 6850, which causes partial relaxation of the SMC) had no significant effect on ROCK-II-induced contractions. Further experiments showed the highest levels of RhoA, active form of RhoA (RhoA-GTP), ROCK-II, 20-kDa myosin regulatory light chain (MLC(20)), phospho-MYPT1, and phospho-MLC(20) in the IAS vs. RSM and ASM SMC. However, the trend was the reverse with the levels of inactive RhoA (GDP-RhoA-RhoGDI complex) and MYPT1. We conclude that RhoA/ROCK play a critical role in maintenance of spontaneous tone in the IAS SMC via inhibition of myosin light chain phosphatase.     

A comparative study of endothelin and sarafotoxin action in vascular and non-vascular smooth muscle

The effects of endothelin and sarafotoxin on smooth muscle tone have been examined in the rat aorta and anococcygeus muscle and their actions compared to those of norepinephrine. The contractions elicited by endothelin and sarafotoxin (10 nM), or norepinephrine (1 μM) were approximately equieffective in terms of tension development and correspond to EC₅₀ values and these concentrations were thus used throughout the study. In calcium-free Krebs the three agonists generated approximately similar levels of tone in the aorta and the anococcygeus corresponding to 20 and 5% of the maximum response, respectively. Nifedipine, 10 μM, significantly inhibited responses to endothelin and norepinephrine in the aorta but only norepinephrine in anococcygeus; the responses to sarafotoxin were however not significantly affected in either tissue. A combination of 10 μM ryanodine and nifedipine caused near complete inhibition of the response to endothelin in the aorta and also significantly reduced the response to both endothelin and norepinephrine in the anococcygeus. The lipoxgenase inhibitor, nordihydroguaiaretic acid, inhibited the response to endothelin in the aorta and endothelin and norepinephrine in the anococcygeus muscle. The cyclooxygenase inhibitor, indomethacin, however, had no effect on the responses to any of the three agonists in either the aorta or anococcygeus. At concentrations greater than 30 nM both endothelin and sarafotoxin induced myogenic activity in normally quiescent anococcygeus muscle. As determined by the loss of myogenic activity the tissues recovered more rapidly from sarafotoxin than endothelin with complete recovery apparent after 2.62 ± 0.85 and 5.22 ± 0.06 h respectively. Omitting Ca²⁺ from the Krebs solution reduced recovery times to 1.62 ± 0.2 and 2.4 ± 0.51 h respectively.

Overall the results suggest that endothelin and sarafotoxin activate different cell signaling systems to differing extents in rat aorta versus anococcygeus suggesting that the membrane receptors mediating the responses to endothelin and sarafotoxin are not necessarily identical.     

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.     

Effect of primycin on some electric properties of the frog skeletal muscle

The effect of primycin, a guanidine-type antibiotic was studied on the electric properties and 42K+ uptake of the frog sartorius and semitendinosus muscle. Both in normal and choline chloride Ringer solution, primycin evoked a concentration and time dependent depolarization of the surface membrane of the muscle. This depolarization was significantly increased by Na ions. Primycin treatment was shown to evoke a dose-dependent decrease of the depolarization induced by 20 mM K+-Ringer. When the muscles were incubated in a Ringer solution containing choline chloride, during an incubation period of 30 min the uptake of 42K+ was decreased to 12% upon the exposure to 5 x 10(-6) mol primycin as compared to the control value. As the primycin-induced depolarization increased, the shape and amplitude of the action potentials elicited by square-wave electric impulses were altered and decreased, respectively. In sodium isaethionate Ringer 1--2 x 10(-6) M primycin induced a slow depolarization resulting in firing potentials. The results suggest that primycin depolarizes the surface membrane exclusively through the blockade of the resting K+ channels, the other phenomena being the results of this depolarizing effect.     

Lobster muscle and synapse respond to cortisol, a vertebrate hormone

Cortisol (0.28 mumol X L-1) applied to lobster (Homarus americanus) neuromuscular preparations produces a hyperpolarization in muscle fibers and an increase in amplitude of excitatory postsynaptic potentials. The effect appears to be surface-mediated, because of its rapid onset (within seconds). It is also Na+-K+ ATPase dependent, because ouabain blocks the effects. The effects are relatively short-lasting, and gradually subside within 15 min. The increase in excitatory postsynaptic potentials is attributed in part to increased quantal output of transmitter, and not to changes in muscle fiber membrane resistance. The effects of cortisol on neuromuscular transmission and membrane potential indicate that cortisol may have a physiological role in crustaceans.     

Ionic mechanism for the generation of horizontal cell potentials in isolated axolotl retina

The ionic mechanism of horizontal cell potentials was investigated in the isolated retina of the axolotl Ambystoma mexicanum. The membrane potentials of both receptors and horizontal cells were recorded intracellularly while the ionic composition of the medium flowing over the receptor side of the retina was changed. The membrane potential of the horizontal cell is highly depender side of the retina was changed. The membrane potential of the horizontal cell is highly dependent on the extracellular concentration of sodium. When the external ion concentration of either chloride or potassium was changed independently of the other, there were shifts in the membrane potential of the horizontal cell which could not be explained by changes in the equilibrium potential of these ions. If the external concentrations of both potassium and chloride ions were varied so that the product of their external concentrations did not change, the shift in the membrane potential of the horizontal cell was in the direction predicted by the Nernst equation. The results are consistent with the suggestion that in the dark the receptors release a synaptic transmitter which increases primarily the sodium conductance of the horizontal cell postsynaptic membrane.     

Effects of K+ Channel Blockers and Nitric Oxide on the Electrical and Contractile Activities of Smooth Muscles of the Rabbit Main Pulmonary Artery

Using a sucrose-bridge technique, we studied electrical and mechanical responses of smooth muscle ring strips of the rabbit main pulmonary artery to applications of blockers of voltage-operated (including Ca²⁺-dependent) K⁺ channels, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), as well to application of nitric oxide (NO); nitroglycerin (NG) was used as a donor of the latter. All experiments were carried out under conditions of blockade of the adreno- and cholinoreceptors in the preparation. Both TEA and 4-AP evoked dose-dependent effects: depolarization of smooth muscle cells (SMC) and their contraction. Simultaneous addition of TEA and 4-AP to the normal superfusate (Krebs solution) resulted in intensification of depolarization and initiated generation of action potentials (AP); contractions became rather intensive and possessed a tetanic pattern. Addition of NG to TEA- and 4-AP-containing Krebs solution effectively suppressed AP generation and contractions, whereas the depolarization level underwent only mild modifications. These findings show that Ca²⁺-dependent high-conductance K⁺ channels (K_Ca channels) and 4-AP-sensitive voltage-operated K⁺ channels (K_V channels) are involved in the formation of the resting membrane potential (RMP) in SMC of the rabbit main pulmonary artery. The impact of the K_Ca channels is greater than that of the K_V channels. We suppose that the effects of NO on SMC are related to inhibition of the activity of high-threshold voltage-operated L-type Ca²⁺ channels and, probably, to lowering of the sensitivity of the contractile SMC apparatus to Ca²⁺.     

秋水仙素对大鼠神经肌肉接头传递的作用

在不均匀牵拉法固定的离体大鼠膈神经膈肌标本上,用秋水仙素探讨了突触后膜的微管在神经肌肉接头乙酰胆碱受体兴奋中的作用。秋水仙素使小终板电位的幅度下降;使串刺激(10Hz,50Hz)诱发的平均终板电位幅度和平均量子含量减少;并使有神经支配的膈肌终板区乙酰胆碱电位幅度降低;但不影响膜电位、小终板电位的频率及终板电位和乙酰胆碱电位的时程。结果表明该药对神经肌肉接头乙酰胆碱受体的兴奋有抑制作用。而其同分异构体光化秋水仙素则无此作用。据此本文提出突触后膜下的微管可能参与神经肌肉接头乙酰胆碱受体的反应过程。     

The ionic requirements for the production of action potential in Achatina fulica Ferussac neuron

The ionic requirement for the production of directly elicited action potentials of a tonically auto-active neuron (TAN) in the subesophageal ganglia of the giant African snail, Achatina fulica Ferussac, was studied electrophysiologically. Calcium free Ringer solution containing 1 mM EDTA reversibly abolished the directly elicited action potential. Verapamil (10 micrograms/ml) or cocaine (4 mg/ml) decreased both amplitude and Vmax of the action potentials. The amplitude of the action potential was also slightly decreased in sodium free choline Ringer. However, tetrodotoxin did not significantly affect either the amplitude or Vmax of the directly elicited action potentials. The results suggest that the ionic requirement for generating action potential in snail neuron is not an ordinary sodium spike. Both calcium and sodium ions may participate in carrying charges across the membrane of the action potential.     

Voltage-clamp of cut-end skeletal muscle fibre: a diffusion experiment

Membrane potential and current were studied in cut end fibres of frog skeletal muscle under current and voltage clamp conditions, by the double sucrose gap technique. Similar action potentials were recorded under current clamp conditions with either the microelectrode or the double sucrose gap techniques. Under voltage clamp conditions, the control of the membrane potential was maintained adequately. The early current was sensitive to both TTX and external Na concentration suggesting that the current was carried by Na ions. Sodium current (INa) was subsequently analysed using the Hodgkin-Huxley formulae. INa half-activation and inactivation occurred at -34 mV and -60 mV, respectively. Na-rich solution applied internally by diffusion through cut ends produced a reduction of INa associated with a shift of the sodium current reversal potential (VNa) towards more negative membrane potentials. This suggested that the sodium electromotive force was reduced by the increase in internal Na content of the fibre. Iodate applied externally changed neither the activation nor the inactivation time courses of INa, but reduced the peak current. Conversely, internally applied by diffusion from the cut end of skeletal muscle fibre, iodate slowed down the time course of INa inactivation and decreased the current peak. In conclusion, the double sucrose gap technique adapted to cut end frog skeletal muscle fibre allows a satisfactory analysis of INa.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Peroxynitrite produces relaxations on the rat anococcygeus muscle

Effects of peroxynitrite (ONOO-), its stable product 3-nitrotyrosine (NT) and sodium nitroprusside (SNP) on isolated rat anococcygeus muscle were investigated. Administration of 0.1-1.0 mM ONOO- or 0.01-100.0 microM SNP produced concentration-dependent relaxations on the phenylephrine (2 microM)-precontracted muscle. Time courses of these relaxations to ONOO- and SNP were not similar and decomposed ONOO- caused a biphasic response composed of an initial contraction followed by a relaxation. NT (0.01-0.1 mM) either incubated for 20 min prior to precontraction or given during precontraction plateau did not attenuate precontraction or ONOO(-)-induced relaxations. Results of the present study demonstrate that ONOO- relaxes rat anococcygeus muscle specifically while its stable metabolite NT has no effect.     

TTX-sensitive Na(+) and nifedipine-sensitive Ca(2+) channels in rat vas deferens smooth muscle cells.

The inward currents in single smooth muscle cells (SMC) isolated from epididymal part of rat vas deferens have been studied using whole-cell patch-clamp method. Depolarising steps from holding potential -90 mV evoked inward current with fast and slow components. The component with slow activation possessed voltage-dependent and pharmacological properties characteristic for Ca(2+) current carried through L-type calcium channels (I(Ca)). The fast component of inward current was activated at around -40 mV, reached its peak at 0 mV, and disappeared upon removal of Na ions from bath solution. This current was blocked in dose-dependent manner by tetrodotoxin (TTX) with an apparent dissociation constant of 6.7 nM. On the basis of voltage-dependent characteristics, TTX sensitivity of fast component of inward current and its disappearance in Na-free solution it is suggested that this current is TTX-sensitive depolarisation activated sodium current (I(Na)). Cell dialysis with a pipette solution containing no macroergic compounds resulted in significant inhibition of I(Ca) (depression of peak I(Ca) by about 81% was observed by 13 min of dialysis), while I(Na) remained unaffected during 50 min of dialysis. These data draw first evidence for the existence of TTX-sensitive Na(+) current in single SMC isolated from rat vas deferens. These Na(+) channels do not appear to be regulated by a phosphorylation process under resting conditions.     

A network model for biofilm development in Escherichia coli K-12

Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl^--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts. In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl^-. Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl^- ions needs to be >20 times higher outside the cell. When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.     

Excitation-contraction coupling in the smooth muscle of the femoral artery under the effects of noradrenaline

Noradrenaline (5 x 10(-8) - 10(-5) M) induced a dose-dependent contraction of muscle strips from rabbit femoral artery. At concentrations higher than 10(-7) M noradrenaline evoked also a depolarization of smooth muscle cells due to an increase in sodium and/or chloride permeability of the membrane. Repolarization of the membrane to original level by inwardly applied current resulted in restoration of membrane resistance and partial relaxation of noradrenaline-evoked contraction. The same part of contraction was also blocked by verapamil. In calcium-free EGTA-containing solution noradrenaline induced only a small transient contraction. These findings indicate that noradrenaline-activated sodium (or chloride) permeability is voltage dependent. Noradrenaline evoked contraction is activated by calcium ions entered the cell through receptor-operated and partly through voltage-operated calcium channels.     

Effect of Black Widow Spider Venom on the Lobster Neuromuscular Junctions

The effect of black widow spider venom (BWSV) on the junctions of the lobster nerve-muscle preparation was studied by intracellular recordings. After application of BWSV both excitatory and inhibitory postsynaptic potentials (epsp and ipsp) were augmented then suppressed. The frequency of miniature potentials was markedly increased by BWSV. Summated postsynaptic conductance changes appeared to be responsible for the membrane depolarization and the decrease in effective membrane resistance seen in the early stages of the venom action. In the later stages both excitatory and inhibitory "giant miniature potentials" were evoked. No discernible changes were found in the reversal potential of the epsp and ipsp and in the sensitivity of the postsynaptic membrane. The results indicate that BWSV has a presynaptic action at crustacean neuromuscular junctions.     

CNV-like potentials on the cortical surface associated with conditioning in head-restrained rats

Head-restrained rats were conditioned to perform a CNV task: to press a lever in response to an imperative auditory stimulus (S2) given 1.5 sec after a warning stimulus (S1) for a drop of jelly food. With an electrode on the surface of the forelimb cortex, (1) sharp wave complexes immediately after S1 and S2, and (2) a negative slow potential (SP) between S1 and S2, on which early and late components were discernible, were recorded in association with performance of this task. With the electrode at a depth of 2 mm in the same cortical area, the corresponding field potential showed a long-lasting positive shift in addition to the components of the surface potential. These monopolar recordings were obtained with respect to a common reference at the frontal sinus. The surface-minus-depth potential (the transcortical potential), consequently, showed a surface-negative tonic wave, confirming Pirch's report (1980). During extinction of this conditioning, the SP between S1 and S2 disappeared, while the sharp waves following S1 and S2 remained with little modification, suggesting that the sharp waves are a kind of evoked potential (EP) elicited by the stimuli.Recording from 5 surface electrodes set in an array over the left hemisphere contralateral to the used forelimb during development of the conditioning revealed not only a spatial distribution of the SP but also a transition of the potentials. As the conditioning progressed, the negativity of the early SP component tended to increase, while that of the late component tended to decrease and was confined to the sensorimotor cortex. The similarities of the rat cortical surface potentials to the human and monkey CNV in their wave form and function suggests that the rat brain can produce electrical activity analogous to the human CNV.