On the Inhibition of Muscle Contraction Caused by Exposure to Hypertonic Solutions

The evidence supporting a site of inhibition of excitation contraction (E-C) coupling near the plasma membrane (the "glycerol effect," the K⁺-potentiating effect) for muscle in hypertonic solution was reinvestigated. It was found, using whole frog sartorii, that there was a rehydration of muscle soaked in glycerol Ringer after 30 min and a large swelling (to 140% after 1 hr soaking) upon return of the muscle to normal Ringer, suggesting that significant amounts of glycerol enter the fibers during this time. While contrary to the original report of the glycerol effect, this finding was consistent with other studies involving the use of single fibers. Also reexamined was the potentiating effect of K⁺ on the hypertonic inhibition of muscle contraction. It was found that muscles exposed to this KCl pretreatment swell so that they are less dehydrated in hypertonic solutions, thus accounting for the observed potentiation. After being treated instead with a K₂-tartrate Ringer solution, muscles did not swell and, as determined with twitch recordings, did not display any potentiation in hypertonic solutions—even though the [K₊] was higher than an osmotically equivalent KCl solution. The evidence was thus consistent with alternative hypotheses in which inhibition of contraction occurs at a later stage in E-C coupling or involves the contractile process itself.     

Mechanisms of spontaneous relaxation of smooth muscle (guinea pig taenia coli) depolarized in a hyperkalemic medium]

Experiments were performed on isolated strips of guinea pig taenia coli by the double sucrose-gap method. The artificial node was depolarized with potassium solution (from 120 to 167.7 mM KCl). When the bathing solution contained 0.4 mM Ca and the temperature was equal to 25 degrees C then potassium contracture was followed by fast relaxation. The muscular tone changed slightly during rectangular pulse of hyperpolarizing current, after switching off the current muscle generated a transient contractile response. The amplitude of such off-responses increased in some range with increasing in strength and duration of conditioning current. Treatment of muscle with compound D-600 resulted in a reduction of muscular tone and elimination of off-responses. The addition of Na ions to potassium solution (substitution of 47.7 mM KCl with the same quantity of NaCl) reduced muscular tone and enhanced the relaxation after off-responses. In sodium-free potassium solution each off-response was followed by increasing muscular tone but when the bathing solution contained Na ions this increase of the tone was not observed. The data obtained strongly suggest that the spontaneous relaxation of smooth muscle which was contracted in K-solution resulted from: 1) inactivation of calcium channels of surface membrane, 2) sequastration of Ca ions by intracellular storange sites, 3) extrusion of Ca in extracellular space (in part by means of Na-Ca exchange diffusion).     

Extracellular calcium and microwave enhancement of membrane conductance in snail neurons

Summary Microwave irradiation has been shown to decrease the input resistance of snail neurons. In this study, we examined the role of extracellular calcium in triggering the microwave-induced enhancement of membrane conductance. Two sets of experiments were conducted. In the first set, nerve cells were superfused using Ringer solution with added Cd²⁺ (0.9 mM) which is a known blocker of calcium channels. In the second set, cells were superfused with low Ca²⁺ (0.7 mM) Ringer solution. Microwave irradiation was conducted at 2,450 MHz for 30 min with a specific absorption rate of 13 mW/g. It was found that 7 mM to 0.7 mM lowering of Ca²⁺ in bathing solution as well as blocking of calcium channels in neuronal membrane by means of Cd²⁺ did not influence the fall in membrane resistance induced by microwave radiation. In fact, the observed changed in membrane resistance in these experiments were nearly equal to those observed for neurons superfused by normal Ringer's. Thus, these results rule out the possible contribution of external Ca²⁺ in the observed microwave effect. Experiments with high Ca²⁺ solution also support this conclusion.     

Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes

Summary Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from –23 to – 39 mV and 29 to 13 M, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at –80 mV and subsequently stepped to a series of depolarizing voltages (from–70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above –20 mV. This current was sensitive to holding potential and was not activated when the cells were held at –40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl₂ (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC₅₀ for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above–40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba²⁺ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.     

The interaction between caffeine and calcium in the desensitization of muscle postjunctional membrane receptors

The interaction between caffeine and calcium on the rate of desensitization of muscle postjunctional membrane (PJM) receptors during the sustained application of 0.27 mM carbamylcholine (CARB) has been studied in vitro on the sartorius muscle of the frog. The rate of PJM repolarization with CARB added to the solution bathing the muscle or the recovery of the effective transmembrane resistance (EMR) during the microperfusion of CARB directly onto the end-plate region of individual fibers was used as an index of the rate of desensitization. Caffeine (1.5 mM) increased the rate of PJM repolarization with bulk application of CARB in a 1.8 or 10 mM calcium Ringer solution but had no effect on PJM repolarization in a calcium-deficient, 4 mM magnesium Ringer solution. For EMR measurements the preparation was rendered mechanically quiescent by repeated challenges with isotonic KCl during an exposure of several hours to a calcium-free, 4 mM magnesium-1 mM EGTA Ringer solution. In these fibers, the microperfusion of 0.27 mM CARB together with 1.8 mM calcium plus 1.5 mM caffeine significantly increased the rate of EMR recovery above that observed in the absence of caffeine. Raising the calcium concentration to 10 mM had a similar effect; however, no additional increase was noted by the inclusion of 1.5 mM caffeine. It is suggested that the major role of caffeine in PJM desensitization is to increase the calcium permeability of the surface membrane. The transmembrane movement of calcium and the consequent intracellular accumulation of calcium is seen as a critical factor in controlling the rate of PJM desensitization.     

The effects of modified ringer's solutions on the membrane potentials of innervated and denervated frog sartorius muscle fibers

Comparison has been made between innervated and chronically denervated frog sartorius muscle fibers for resting potentials and a number of features of the action potential. Muscles were obtained from force-fed frogs maintained at room temperature for periods up to one year, and were studied with intracellular microelectrodes. Denervated muscles increased in sensitivity to acetylcholine by 100–400-fold. Studies were made in normal Ringer's solution, and in media in which concentrations of K⁺, Na⁺, Ca⁺⁺, and Cl? were altered. The only significant differences noted between the denervated and the innervated fibers were a reduction in the maximum rate of fall of the action potential (ca. 20%) and an increase in the fall time of the active membrane potential (ca. 25%). These differences were present in normal Ringer's solution and remained when the bathing medium was modified. The resting membrane potential of denervated and innervated muscles varied with log [K⁺]_o in exactly the same manner, and followed the theoretical relation proposed by Hodgkin (Proc. Roy. Soc., B, 148: 1–37, ′58), with the term representing the ratio of the sodium to potassium permeabilities assigned a value of 0.01. The results suggest that (a) the resting sodium and potassium permeabilities are reduced proportionately after denervation, since it is known that denervated frog muscle has a smaller potassium permeability, and (b) the mechanism controlling the increase in potassium conductance during the action potential is less available after denervation. Data indicate that the system controlling the sodium permeability is capable of activation to the same extent as in innervated muscles. Muslces which had been allowed to reinnervate did not show the differences presented by the denervated muscles. Innervated and denervated muscles did not show any significant changes in maximum rates of rise or fall of the action potential, nor of the active membrane potential amplitude over a 30 mV range of resting membrane potentials, indicating that the sodium and potassium permeability systems are fully available in frog muscle at membrane potentials larger than ?80 mV.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. I. A study of the "cell-attached" configuration

A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials.     

The independence of membrane potential and potassium activation of the sodium pump in muscle

The membrane potential (E_m) of sartorius muscle fibers was made insensitive to [K⁺] by equilibration in a 95 mM K⁺, 120 mM Na⁺ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with E_m sensitive to [K⁺]. In addition, in the presence of 10 mM K⁺, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10^?4 M ouabain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

Effects of Cl- deficiency on the membrane potential in mouse pancreatic beta-cells

The membrane potential of mouse pancreatic beta-cells was measured with microelectrodes. In the resting cell (3 mM D-glucose), the membrane potential was -63 +/- 3 mV (mean +/- S.E. for four experiments). In the presence of 3 mM D-glucose, total Cl- substitution by isethionate induced a depolarization by 3-4 mV, and readmission of Cl- induced a hyperpolarization by 3-5 mV. At 10 mM glucose, reduction of Cl- to 12 mM by substituting isethionate for Cl- reversibly shifted the repolarization potential by 6-9 mV in the positive direction and stimulated the burst activity during the initial 2-3 min by increasing the fraction of plateau phase. This was followed by a gradual inhibition of electrical activity, including decrease in fraction of plateau phase and slow wave amplitude. Total substitution of Cl- by isethionate or methyl sulphate reversibly shifted the repolarization potential by 3-4 mV in the positive direction and rapidly inhibited the electrical burst pattern without any initial stimulation. Glucose-induced (10 mM) insulin release (15 min) and 45Ca2+ uptake (3 min) were strongly inhibited by reducing the Cl- concentration to 10 mM (isethionate as substitute) and were further inhibited by further reduction of the Cl- concentration. It is suggested that beta-cells are equipped with on electrogenic Cl- flux, which can affect the burst pattern of electrical activity. The inhibitory effects of Cl- substitution may be explained by an influence of Cl- on the voltage-controlled Ca2+ channels.     

The independence of membrane potential and potassium activation of the sodium pump in muscle.

The membrane potential (Em) of sartorius muscle fibers was made insensitive to [K+] by equilibration in a 95 mM K+, 120 mM Na+ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with Em sensitive to [K+]. In addition, in the presence of 10 mM K+, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10- minus 4 M oubain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.     

The properties of the extraocular muscles of the frog. I. Mechanical properties of the isolated superior oblique and superior rectus muscles.

The mechanical properties of two extraocular muscles (superior oblique and superior rectus muscles) of the frog were studied and compared with those of a frog's skeletal muscle (iliofibularis muscle) which contains the same types of muscle fibres as the oculorotatory muscles. The extraocular muscles are very fast twitching muscles. They exhibit a smaller contraction time, a smaller half-relaxation time, a higher fusion frequency, and a lower twitch-tetanus ratio than the skeletal muscles. The maximum isometric tetanic tension produced per unit cross-sectional area is lower in the extraocular muscles than in skeletal muscles. However, the extraocular muscles show a higher fatigue resistance than the skeletal muscles. With respect to the dynamic properties there are some differences between the various oculorotatory muscles of the frog. The superior rectus muscle exhibits a faster time-course of the contraction, a higher fusion frequency, and a higher fatigability than the superior oblique muscle. An increase of the extracellular K+-concentration evokes sustained contractures not only in the extraocular muscles but also in the iliofibularis muscle; between these muscles there are no striking differences in the mechanical threshold of the whole muscle preparation. The mechanical threshold depends on the Ca++-concentration of the bathing solution and it is found in a range between 12.5 and 17.5 mM K+ in a normal Ringer solution containing 1.8 mM Ca++. The static-mechanical properties of the extraocular muscles of the frog and the dependence of the active developed tension on the muscle extension are very similar to those which are known to exist in the extraocular muscles of other vertebrates. In tetanic activated frog's oculorotatory muscles a linear relationship exists between length and tension. A variation of the stimulation frequency does not change the slope of this curve but causes parallel shifts of the curve. The peculiar properties of the extraocular muscles of the frog are discussed with respect to the muscle fibre types in these muscles and to the diameter of the muscle fibres.     

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.     

Effect of External Calcium and of Temperature on Contraction in Snake Muscle Fibers

The effect of external calcium and of temperature on the contractile responses has been studied in voltage clamped snake twitch muscle fibers. Increasing [Ca⁺⁺]_o from 0.2 to 7.0 mM raised contractile threshold by 15–20 mV, the latter coinciding with the appearance of delayed rectification. The duration of contracture, the rates of rise and decay of tension depended on the level of depolarization and [Ca⁺⁺]_o. The minimum duration of repolarization necessary to restore the contractile response was much shorter in high [Ca⁺⁺]_o. When the bathing solution was cooled to 10 from 20°C the time-course of contracture was markedly prolonged and the outward current was reduced without significant change in maximum tension. The threshold for contraction tended to be somewhat lower at the lower temperature. The contractile repriming was much slower at low temperature. However, reduction in temperature slowed the rate of recovery much less at low [Ca⁺⁺]_o than at normal [Ca⁺⁺]_o.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

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.     

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca²⁺ in the cytoplasm ([Ca²⁺]_c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca²⁺]_c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca²⁺]_c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca²⁺-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.     

Effects of membrane potential on the capacitance of skeletal muscle fibers

A method for measuring muscle fiber capacitance using small test pulses applied with the three-microelectrode voltage clamp is presented. Using this method, three membrane potential-dependent changes in capacitance were observed: (a) Capacitance of polarized fibers increased by 5--15% with depolarization from V less then -100 mV to voltages slightly below the contraction threshold. (b) Capacitance of fibers depolarized to -30 mV by 100 mM Rb solution decreased by roughly 8% with further depolarization to about +50 mV and increased with repolarization, exhibiting a maximum increase of about 10% at -80 to -90 mV. (c) Capacitance of fibers depolarized to -15 mV by 100 mM K solution increased by about 19% with further depolarization to +43 mV and decreased by about 23% with repolarization to -62 mV. Effects a and b are attributed to changes in specific membrane capacitance due to voltage-dependent redistribution of mobile charged groups within surface of T-tubule membranes. Effect c is caused by changes in the T-system space constant lambdaT due to the voltage dependence of K conductance (inward rectification). Analysis of c showed that in 100 mM K solution lambdaT congruent to 30 mum when inward rectification was fully activated by hyperpolarization and that the density of inward rectifier channels is about the same in surface and tubular membranes. Fiber internal resistance was found to be independent of voltage, a necessary condition for the interpretation of the capacitance measurements.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current

Disappearance of Ca²⁺-induced phase separation in phosphatidylserine-phosphatidylcholine membranes has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the preparations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca²⁺-treated membrane was transferred to acidic salt solutions ($\text{?}\text{pH 2.5}$) or to low ionic strength media ($\text{? 10}\text{mM}$) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca²⁺ by proton, proton-induced separation, followed by disappearance of the phase separation inthe buffered salt solution. Biological significance of the competition between Ca²⁺ and proton for the phase separation or domain formation in the membranes was emphasized.     

The combined effect of Cd2+ and ACh on action potentials of Nitellopsis obtusa cells

Interrelations between the action of acetylcholine (ACh) and cadmium ions (Cd²⁺) on bioelectrogenesis of Nitellopsis obtusa cells were investigated. We analyzed repetitively triggered action potentials (AP), their reproducibility, shape and dynamics of membrane potential after AP induction. ACh significantly increased membrane permeability only at high concentrations (1 mM and 5 mM). Repolarisation level of action potential after the first stimulus was much more positive in all cells treated with ACh as compared to the control. Differences of membrane potentials between points just before the first and the second stimuli were 23.4±.0 mV (control); 40.4±5.9 mV (1 mM ACh solution) and 57.7 ± 8.5 mV (5 mM ACh solution). Cd²⁺ at 20 μM concentration was examined as a possible inhibitor of acetylcholinesterase (AChE) in vivo. We found that cadmium strengthens depolarizing effect of acetylcholine after the first stimulus. The highest velocity of AP repolarization was reduced after ACh application and Cd²⁺strengthened this effect. There were no differences in dynamics of membrane potential after repetitively triggered action potentials in ACh or ACh and Cd²⁺ solutions. This shows that cadmium in small concentration acts as inhibitor of acetylcholinesterase.     

The effects of acidosis and bicarbonate on action potential repolarization in canine cardiac Purkinje fibers

Studies were performed on canine cardiac Purkinje fibers to evaluate the effects of acidosis and bicarbonate (HCO3) on action potential repolarization. Extracellular pH (pHe) was reduced from 7.4 to 6.8 by increasing carbon dioxide (CO2) concentration from 4 to 15% in a HCO3-buffered solution or by NaOH titration in a Hepes-buffered solution. Both types of acidosis produced a slowing of the rate of terminal repolarization (i.e., period of repolarization starting at about -60 mV and ending at the maximum diastolic potential) with an attendant increase in action potential duration of 10--20 ms. This was accompanied by a reduction in the maximum diastolic potential of 2--8 mV. In contrast, if the same pH change was made by keeping CO2 concentration constant and lowering extracellular HCO3 from 23.7 to 6.0 mM, in addition to the slowing of terminal repolarization, the plateau was markedly prolonged resulting in an additional 50- to 80-ms increase in action potential duration. If pHe was held constant at 7.4 and HCO3 reduced from 23.7 mM to 0 (Hepes-buffered solution), the changes in repolarization were nearly identical to those seen in 6.0 mM HCO3 except that terminal repolarization was unchanged. This response was unaltered by doubling the concentration of Hepes. Reducing HCO3 to 12.0 mM produced changes in repolarization of about one-half the magnitude of those in 6.0 mM HCO3. These findings suggest that in Purkinje fibers, HCO3 either acts as a current that slows repolarization or modulates the ionic currents responsible for repolarization.