The physostigmine depolarization potentiating effect of salicylate in frog skeletal muscle.

1) The frog's sartorius muscle was depolarized depending on the degree of concentration 2--4 times more intensely by physostigmine salicylate than by physostigmine sulphate. 2) In normal Ringer's solution, 1 mM physostigmine salicylate decreased the sensitivity of the membrane to potassium depolarization by about 90%. Under similar experimental conditions, physostigmine sulphate and Na salicylate, respectively, decrease the sensitivity of the membrane to potassium depolarization by about 30%. 3) The difference manifested in the depolarizing effect of salicylate and other physostigmine salts (chloride, sulphate, phosphate, formiate, acetate, monochloracetate, benzoate and para-oxy-benzoate) is expressed already at 1 mM concentration (about 10-fold), if the muscle had been equilibrated in chloride-free glucuronate or sulphate milieu. 4) The depolarization develops slowly. It takes 30--60 minutes for the new steady state to develop even in the superficial sartorius fibres. If depolarization has reached its maximum on an average 100 mV, the membrane potential remains unchanged for hours. 5) Depolarization ensues at an unchanged degree in the presence of Na-free (choline) Ringer as well as in the presence of 2X10(-8) g/ml tetrodotoxin; therefore, it is not a Na-dependent process. 6) Under the influence of 1 mM physostigmine salicylate the membrane's resistance to the inward potassium current increased about twofold, while the increase was only 15% to the outward potassium current. It is assumed that the salicylate anion is characteristically capable of potentiating the decreasing effect of physostigmine on potassium permeability, though the role of the metabolic effect of salicylate cannot be excluded.     

Unusual potassium channels mediate nonadrenergic noncholinergic nerve-mediated inhibition in opossum esophagus

Field stimulation of the circular muscle of the opossum esophagus produces a transient hyperpolarization (inhibitory junction potential, IJP) followed by an "off" depolarization. A similar nonadrenergic, noncholinergic (NANC) response in guinea pig taenia caecum has been shown to be due to an increase in the potassium ion permeability of the smooth muscle cell membrane. Double sucrose gap studies showed a decrease in resistance during the IJP, and a reversal at an estimated membrane potential of about -90 mV (4 mM K+). The reversal potential was dependent on the extracellular potassium concentration, shifting to -75 mV when the potassium in the superfusion medium was increased to 10 mM. The IJP in the opossum esophageal circular smooth muscle is therefore like the IJP of the guinea pig taenia caecum in that it is probably due to a selective increase in potassium ion permeability. Potassium conductance blocking agents, tetraethylammonium chloride (TEA, 20 mM) and 4-aminopyridine (4-AP, 5 mM) both caused a depolarization of the smooth muscle cell membrane, but TEA increased the membrane resistance, whereas 4-AP did not affect the membrane conductance in a consistent way. A decrease in IJP amplitude owing to these agents was not apparent. Apamin (10 microM) did not affect the membrane potential, the membrane resistance, or the IJP. Quinine (0.1 mM) produced effects quantitatively similar to those of TEA. Quinine (1 mM) did abolish the IJP, however, this was likely due to a blockade of impulse transmission of the intramural nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of Ca2+, temperature and sucrose upon potassium contracture of the isolated rat right ventricle.

Potassium (100 mM KC1) contracture of the isolated rat right ventricle was lower in Tyrode solution (37 mM Na) than on substituting sucrose (270 mM) for NaC1 and was biphasic in 70% of the experiments. As in slow (tonic) skeletal muscle, the maximum contracture value persisted as long as a raised KC1 concentration was maintained. Even after complete potassium depolarization it changed when Ca was altered. At 37 degrees C, the second phase of potassium contracture was higher than at 34 degrees C (p less than 0.01). The effect of K+ and Ca2+ was inhibited if the ions were added after adding sucrose to the Tyrode solution. Contracture of the rat ventricle resembled contracture of slow (tonic) skeletal muscle.     

Delayed rectification in the transverse tubules: origin of the late after-potential in frog skeletal muscle

Tetanic stimulation of skeletal muscle fibers elicits a train of spikes followed by a long-lasting depolarization called the late after- potential (LAP). We have conducted experiments to determine the origin of the LAP. Isolated single muscle fibers were treated with a high potassium solution (5 mM or 10 mM K) followed by a sudden reduction of potassium concentration to 2.5 mM. This procedure produced a slow repolarization (K repolarization), which reflects a diffusional outflow of potassium from inside the lumen of the transverse tubular system (T system). Tetanic stimulation was then applied to the same fiber and the LAP was recorded. The time courses of K repolarization and LAP decay were compared and found to be roughly the same. This approximate equality held under various conditions that changed the time courses of both events over a wide range. Both K repolarization and the LAP became slower as fiber radius increased. These results suggest that LAP decay and K repolarization represent the same process. Thus, we conclude that the LAP is caused by potassium accumulation in the T system. A consequence of this conclusion is that delayed rectification channels exist in the T system. A rough estimation suggests that the density of delayed rectification channels is less in the T system than in the surface membrane.     

Nitric oxide as a nonadrenergic inhibitory transmitter in smooth muscle cells of the guinea pig gastro-intestinal tract: Mechanisms of action

The role of nitric oxide (NO) as a possible transmitter for nonadrenergic inhibitory transmission was studied on isolated muscle strips of the guinea pig gastro-intestinal tract (GIT) using sucrose-gap technique. In addition, the voltage clamp and intracellular dialysis techniques were employed to study the effects of sodium nitroprusside (NP) on isolated smooth muscle (SM) cells of thetaenia coli. N-nitro-L-arginine methyl ester (L-NAME), a blocker of NO synthesis from L-arginine (0.1 mM), was shown to selectively suppress the apamin-resistant component of nonadrenergic inhibitory junctional (synaptic) potentials (IJP) in the guinea pig GIT SM cells. At the same time, L-NAME did not affect the vasoactive intestinal polypeptide (VIP)- and NP-evoked hyperpolarization in SM cells of the colon. The NP-induced hyperpolarization (0.1 mM) was accompained by a decrease in the SM cell membrane resistance. Application of NP to isolated SM cells activated a small outward current and increased the frequency of spontaneous transient calcium-dependent outward currents. NP increased the Ca-dependent potassium current evoked in SM cells by step depolarization, but did not affect the potassium currents of delayed rectification. Our results suggest that NO is involved in generation of nonadrenergic IJP in SM cells of the guinea pig GIT. The action of NP on SM cells is complex and results in hyperpolarization and relaxation (partially through the activation of Ca-dependent potassium channels in SM cell membrane).     

Amiloride inhibits contraction and serotonin modulation of Aplysia muscle

1. Serotonin (5-HT) potentiates acetylcholine (ACh)-elicited contractions of Aplysia buccal muscles. Serotonin potentiation was significantly reduced by 0.03 mM, 0.1 mM, and 0.3 mM amiloride. 2. Unpotentiated ACh-elicited contractions were significantly reduced by 0.1 mM and 0.3 mM amiloride. 3. Amiloride reduced ACh-elicited depolarization. The reduction in contraction caused by 0.3 mM amiloride (to 16% of control) was larger than could be explained by the reduction in depolarization (86% of control). 4. Amiloride had no effect on tension in skinned muscle fibers, indicating that amiloride probably did not have a direct effect on contractile mechanisms. 5. Potentiation of contraction produced by zero sodium (Tris substituted, 0 Na-Tris) medium could be abolished by 0.3 mM amiloride. 6. Zero Na-Tris increased 45Ca influx 2.7-fold. In the presence of 0.3 mM amiloride, 0 Na-Tris increased 45Ca influx only 1.4-fold. 7. Amiloride (0.3 mM) reduced the elevation of muscle cAMP caused by 10(-6) M 5-HT by 60%. Zero Na-Tris did not cause a change in muscle cAMP.     

Relative roles of Ca2+, Sr2+, Ba2+ and Mg2+ in controlling lens permeability characteristics

The effects of barium, strontium and magnesium upon lens permeability characteristics were studied in the presence and absence of 2 mM calcium in the bathing medium. Permeability characteristics were determined by measuring lens potential, resistance and ⁴²K efflux rates. Barium and strontium at equimolar concentrations to calcium were able to substitute for calcium in controlling lens sodium permeability. Magnesium was ineffective in this respect.Small changes in resistance and ⁴²K efflux rates occurred when calcium was eliminated from bathing solution containing either 2 mM barium or strontium. These changes were interpreted to be the result of an increase in lens permeability to potassium. When 2 mM strontium was added to calcium-containing solution, there was no significant change in the electrical or flux parameters of the lens. However, the addition of 2 mM barium to calcium-containing solution resulted in a 54% increase in lens resistance and a 13 mV depolarization. These observations indicated a barium-induced decrease in lens permeability to potassium, and this was confirmed by an observed decrease in ⁴²K efflux rate constant under similar experimental conditions.The rapid time course of all the observed changes implies that they are the result of changes in the permeability characteristics of membranes lying close to the surface of the lens.     

Mechanomyographic and electromyographic responses of the vastus medialis muscle during isometric and concentric muscle actions

The purpose of this study was to examine the patterns for the mechanomyographic (MMG) and electromyographic (EMG) amplitude and mean power frequency (MPF) vs. torque relationships during submaximal to maximal isometric and isokinetic muscle actions. Seven men (mean +/- SD age, 22.4 +/- 1.3 years) volunteered to perform isometric and concentric isokinetic leg extension muscle actions at 20, 40, 60, 80, and 100% of maximal voluntary contraction (MVC) and peak torque (PT) on a Cybex II dynamometer. A piezoelectric MMG recording sensor was placed between bipolar surface EMG electrodes on the vastus medialis. Polynomial regression and separate 1-way repeated-measures analysis of variance were used to analyze the EMG amplitude, MMG amplitude, EMG MPF, and MMG MPF data for the isometric and isokinetic muscle actions. For the isometric muscle actions, EMG amplitude (R(2) = 0.999) and MMG MPF (R(2) = 0.946) increased to MVC, mean MMG amplitude increased to 60% MVC and then plateaued, and mean EMG MPF did not change (p > 0.05) across torque levels. For the isokinetic muscle actions, EMG amplitude (R(2) = 0.988) and MMG amplitude (R(2) = 0.933) increased to PT, but there were no significant mean changes with torque for EMG MPF or MMG MPF. The different torque-related responses for EMG and MMG amplitude and MPF may reflect differences in the motor control strategies that modulate torque production for isometric vs. dynamic muscle actions. These results support the findings of others and suggest that isometric torque production was modulated by a combination of recruitment and firing rate, whereas dynamic torque production was modulated primarily through recruitment.     

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.     

Studies on the mechanisms of the effects of protein kinase C activation on electrical and contractile activity of smooth muscle

Experiments have been carried out with guinea-pig smooth muscles taenia coli by the use of the double sucrose-gap technique. Phorbol esters (PE), activating protein kinase C (PcC) suppressed the spontaneous and induced (by turning on of depolarizing pulses, or turning off a long lasting hyperpolarizing step) electrical and contractile activity of a muscle. The inactive analog of PE did not affect the muscle activity. Na/H-ionophore monensin imitated the effects of active PE. Blockade of K+ channels by 10 mM TEA greatly decreased or in some cases even removed the inhibitory effects of PE. A treatment of the muscle by Na+/H+ exchange inhibitor ethyl isopropyl amiloride (EIPA) increased the amplitude of action potentials during membrane depolarization and markedly weakened the PE-induced suppression of muscle electrical activity. The data obtained suggested that inhibitory effects of PE on smooth muscle electrical and contractile activity resulted from an increase in potassium permeability of the membrane. Na+/H+ exchange seems to be involved in PE-induced K+ channels activation.     

High K(+)-induced membrane depolarization attenuates endothelium-dependent pulmonary vasodilation

Impairment of endothelium-dependent pulmonary vasodilation has been implicated in the development of pulmonary hypertension. Pulmonary vascular smooth muscle cells and endothelial cells communicate electrically through gap junctions; thus, membrane depolarization in smooth muscle cells would depolarize endothelial cells. In this study, we examined the effect of prolonged membrane depolarization induced by high K(+) on the endothelium-dependent pulmonary vasodilation. Isometric tension was measured in isolated pulmonary arteries (PA) from Sprague-Dawley rats, and membrane potential was measured in single PA smooth muscle cells. Increase in extracellular K(+) concentration from 4.7 to 25 mM significantly depolarized PA smooth muscle cells. The 25 mM K(+)-mediated depolarization was characterized by an initial transient depolarization (5-15 s) followed by a sustained depolarization that could last for up to 3 h. In endothelium-intact PA rings, ACh (2 microM), levcromakalim (10 microM), and nitroprusside (10 microM) reversibly inhibited the 25 mM K(+)-mediated contraction. Functional removal of endothelium abolished the ACh-mediated relaxation but had no effect on the levcromakalim- or the nitroprusside-mediated pulmonary vasodilation. Prolonged ( approximately 3 h) membrane depolarization by 25 mM K(+) significantly inhibited the ACh-mediated PA relaxation (-55 +/- 4 vs. -29 +/- 2%, P < 0.001), negligibly affected the levcromakalim-mediated pulmonary vasodilation (-92 +/- 4 vs. -95 +/- 5%), and slightly but significantly increased the nitroprusside-mediated PA relaxation (-80 +/- 2 vs. 90 +/- 3%, P < 0. 05). These data indicate that membrane depolarization by prolonged exposure to high K(+) concentration selectively inhibited endothelium-dependent pulmonary vasodilation, suggesting that membrane depolarization plays a role in the impairment of pulmonary endothelial function in pulmonary hypertension.     

Barium distinguishes separate calcium targets for synthesis and secretion of peptides in neuroendocrine cells

The effect of barium and potassium on the secretion and biosynthesis of enkephalin in bovine chromaffin cells, and prolactin and beta-endorphin in rat anterior pituitary cells, was examined to determine whether calcium-dependent secretion and biosynthesis are mediated by the same or by different calcium targets within the neuroendocrine cell. In the presence of 1.8 mM calcium, barium and potassium stimulated the secretion of all three peptides over 30 min, and increased the levels of proenkephalin and prolactin mRNA in 24 hr. These effects were inhibited by the calcium channel blocker D600. When the extracellular calcium concentration was lowered to 0.1 mM or less, secretion elicited by potassium was blocked, whereas secretion elicited by barium was enhanced, indicating that barium wholly substitutes for extracellular calcium in mediating peptide secretion. On the other hand, stimulation of proenkephalin and prolactin mRNA by both potassium and barium was inhibited when the extracellular calcium concentration was reduced. We conclude that calcium acts at two different intracellular targets to activate secretion versus biosynthesis of both enkephalin and prolactin. This appears to be the first report in which two different calcium-dependent processes in the intact cell are distinguished by a calcium ion agonist. Calcium-dependent processes such as protein phosphorylation, protein translocation, and enzyme activation may thus be related to events in the intact cell such as peptide synthesis and secretion on the basis of selective stimulation by barium.     

Effect of carbachol on miniature end-plate potentials and currents of rat skeletal muscle

Experiments on rats showed that carbachol (10 µM) appreciably quickens miniature end-plate potentials in the soleus muscle kept in medium with potassium chloride concentration increased to 13 mM. Potentiation of spontaneous mediator release also took place when the membrane potential of the muscle fibers was clamped at the level of the potassium equilibrium potential. It is concluded that the presynaptic action of carbachol on synaptic liberation of acetylcholine from motor nerve endings is unconnected with depolarization of the postsynaptic membrane but takes place as a result of the direct effect of the mimetic on motor nerve endings.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 185–189, March–April, 1982.     

Membrane Potential Measured During Potassium-Evoked Release of Noradrenaline from Rat Brain Neurons: Effects of Normorphine

A single slice of rat pons that contained the locus ceruleus (LC) or two slices of cerebellum were loaded with [3H]noradrenaline; superfusion with high (35 or 60 mM) potassium solutions evoked a release of 3H. In the presence of normorphine, the release of 3H evoked by 35 mM potassium and 60 mM potassium was reduced. In some of those experiments in which the release of 3H from the LC slice was measured, an intracellular microelectrode was used to measure membrane potential. This showed that solutions of increased potassium concentration depolarized the neurons to a potential at which inward calcium currents flowed (calcium action potentials occurred). Normorphine hyperpolarized the neurons; during this hyperpolarization the depolarization caused by 35 mM potassium did not reach the threshold for significant calcium entry. The results suggest that the inhibition by normorphine of transmitter release evoked by solutions of raised potassium concentration could result in part from the membrane hyperpolarization caused by the normorphine.     

Depolarization affects neuromuscular junction of streptozotocin-diabetic mice.

The effect of increasing extracellular calcium concentration on spontaneous transmitter release was studied at both soleus (slow) and fast extensor digitorum longus (EDL) nerve terminals of control and streptozotocin-induced diabetic (STZ-D) young C57 BL mice (7 months old) depolarized by high (20 mM) extracellular potassium [K]o. Diabetes was induced by i.p. injection with a single dose of streptozotocin (200 mg/kg) at the age 5 months and the electrophysiological studies were carried out after 8 more weeks. By using intracellular recording, miniature endplate potentials (MEPPs) were first recorded in a normal [K]o Krebs solution. Subsequently, MEPPs were recorded in high [K]o Krebs solution with 4 different Ca concentrations: Ca-free/ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), 0.5, 1.5 and 2 mM Ca. MEPP frequency was lower at STZ-D than control nerve terminals in EDL but not soleus. However, MEPP frequency was progressively higher at both EDL and soleus of STZ-D than control with increasing Ca concentration in Krebs that contained 20 mM [K]o. In STZ-D slow soleus muscle, depolarization produced 0.7, 4.3, 41.6 and 62.7 vs 1.4, 2.8, 20.7 and 31.6 Hz for control in the 4 different Ca concentrations. In STZ-D fast EDL muscle, depolarization produced 0.5, 4.9, 48.2 and 66.8 vs 1.2, 2.5, 27 and 35.4 Hz for control in the 4 different Ca concentrations. Bimodal and unimodal MEPP amplitude were present at both slow and fast nerve terminals. However, depolarization increased the percentage of bimodal MEPP amplitude in STZ-D compared to control (p<0.01) mice in EDL but not soleus. The results revealed that these changes in muscle firing pattern may provide a protective effect against diabetes-induced neuropathy at the neuromuscular junction.     

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.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization.

Homeostatic mechanisms regulate synaptic function to maintain nerve and muscle excitation within reasonable physiological limits. The mechanisms that initiate homeostasic changes to synaptic function are not known. We specifically impaired cellular depolarization by expressing the Kir2.1 potassium channel in Drosophila muscle. In Kir2.1-expressing muscle there is a persistent outward potassium current ( approximately 10 nA), decreased muscle input resistance (50-fold), and a hyperpolarized resting potential. Despite impaired muscle excitability, synaptic depolarization of muscle achieves wild-type levels. A quantal analysis demonstrates that increased presynaptic release (quantal content), without a change in quantal size (mEPSC amplitude), compensates for altered muscle excitation. Because morphological synaptic growth is normal, we conclude that a homeostatic increase in presynaptic release compensates for impaired muscle excitability. These data demonstrate that a monitor of muscle membrane depolarization is sufficient to initiate synaptic homeostatic compensation.     

Two types of K+ channels in the apical membrane of rabbit proximal tubule in primary culture

The patch-clamp technique was used to investigate ionic channels in the apical membrane of rabbit proximal tubule cells in primary culture. Cell-attached recordings revealed the presence of a highly selective K+ channel with a conductance of 130 pS. The channel activity was increased with membrane depolarization. Experiments performed on excised patches showed that the channel activity depended on the free Ca2+ concentration on the cytoplasmic face of the membrane and that decreasing the cytoplasmic pH from 7.2 to 6.0 also decreased the channel activity. In symmetrical 140 mM KCl solutions the channel conductance was 200 pS. The channel was blocked by barium, tetraethylammonium and Leiurus quinquestriatus scorpion venom (from which charybdotoxin is extracted) when applied to the extracellular face of the channel. Barium and quinidine also blocked the channel when applied to the cytoplasmic face of the membrane. Another K+ channel with a conductance of 42 pS in symmetrical KCl solutions was also observed in excised patches. The channel was blocked by barium and apamin, but not by tetraethylammonium applied to the extracellular face of the membrane. Using the whole-cell recording configuration we determined a K+ conductance of 4.96 nS per cell that was blocked by 65% when 10 mM tetraethylammonium was applied to the bathing medium.     

Possible role of Ca ions in the resting metabolism of frog sartorius muscle during potassium depolarization

Oxygen consumption and Ca exchangeability at different levels of potassium depolarization were studied in frog sartorius muscle. It was found that the changes in oxygen consumption parallel the changes in Ca exchangeability. Procaine (10^?3 M) and CaCl₂ (2.10?2 M) suppressed both extra oxygen consumption and Ca exchangeability at low values of depolarization. At higher values of depolarization procaine and CaCl₂ differed in their action. Procaine favored inhibition of these processes, CaCl₂ caused their activation. The effects of these compounds was not a result of a change in the membrane potential, since their effect on potassium depolarization was found to be small. Relations between oxygen consumption and Ca exchangeability similar to those observed at potassium depolarization seem to exist under conditions where caffeine was applied. It is proposed that the extra oxygen consumption caused by potassium depolarization or on application of caffeine and unaccompanied by mechanical changes is related to the release of Ca from its bound form. Oxygen consumption in isotonic sucrose solution was also studied, but some different data from the above were obtained.