17beta-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts.

The skeletal muscle is one of the important target tissues for the actions of estrogen via both nuclear and extranuclear (non-genomic) pathways. However, there is a paucity of information about the receptor (ER) involved. The aim of this study was thus to explore the ER expression in skeletal muscle, and the influence of estrogen on it, by using C2C12 myoblasts derived from mouse skeletal muscle. Significant expression of a approximately 66-kD protein immunoreactive to ER type alpha (ERalpha) monoclonal antibody, which was comparable to that in ovary, was detected in the whole-cell (total) and nucleus-free (nonnuclear) fractions of C2C12 myoblasts. The expression level of these ER proteins increased in several hours with treatment with 17beta-estradiol (E2), which was preceded by the elevation of the ER mRNA level. This increase appeared to reflect the acceleration of de novo synthesis of ER protein, as proved by the (35)S-methionine immunoprecipitation method. A similar extent of fast increase in ER expression was also induced by a membrane-impermeable, BSA-conjugated estradiol (E2-BSA). Unexpectedly, the E2-induced increases in total and nonnuclear ER were further enhanced by the classic ER antagonists tamoxifen and ICI182,780 in a wide concentration range, implying some structural difference of the involved ER from the classical one. Treatment with the ERK1/2 inhibitor, PD98059 (10 microM), or the p38 MAPK-specific inhibitor, SB203580 (10 microM), greatly inhibited the E2-induced ER increase, while the protein kinase C (PKC) activator TPA (1 microM) enhanced it. These results collectively suggest that C2C12 skeletal myoblasts express a high level of ER, a considerable part of which is extranuclear. Further, the expression of ER in these cells may be significantly upregulated by estrogen itself via increased biosynthesis linked to membrane-bound ER and downstream MAPK-mediated signaling pathways.

Effects of 17beta-estradiol on tension responses and fatigue in the skeletal twitch muscle fibers of frog.

The effects of 17beta-estradiol (10(-5) M), an active estrogen, on the tension and fatigue responses of single fiber or fiber bundle prepared from frog skeletal muscle were investigated. The administration of 17beta-estradiol caused a transient potentiation of tetanus tension by field stimulation at every minute. This potentiation was not affected by the presence of nicardipine, suggesting that the action of 17beta-estradiol would place the excitation-contraction (E-C) coupling beyond T-tubule depolarization. Fatigue was produced by repeated tetanic stimulation every second until tension declined to approximately 40% of the initial level. Fibers were then allowed to recover by having tetani given to them every minute. In the normal Ringer solution, the time to 50% of the initial tetanus tension was 41.7 s. With the presence of 17beta-estradiol, the time to 50% tension was faster than that of control. The presence of 17alpha-estradiol, a stereoisomer, caused no potentiation of tetanic tension to be stimulated every minute, and the rate of decline of fatigued response was almost the same as that of control, suggesting the existence of specific estrogen receptors in the frog muscle. In fatigued muscle with or without estrogen, the tension to field stimulation was transient and not sustained. When the fatigued muscle was again treated with field stimulation at every minute after the more-frequent stimulation, the recovery rate was increased in 17beta-estradiol. A prompt reduction in temperature to 5 degree C, from 20 degree C, in the presence of caffeine elicited the tension response, a rapid cooling contracture (RCC). The presence of 17beta-estradiol inhibited peak tension and maximum rate of rise of the RCC only after the repetitive electrical stimuli. These results suggest that the potentiation of contraction upon the electrical stimulation by 17beta-estradiol was induced by the increase of myoplasmic-free Ca(2+) concentration via an activation of some E-C coupling process. The 17beta-estradiol-induced facilitation of fatigue response to repetitive tetanus stimuli with high frequency may be due to an increase in the imbalance of Ca(2+) turnover in the cytoplasm.

Participation of PKC in modulation of the excitation-contraction process of chick embryo cardiac muscle.

Phorbol esters are activators of protein kinase C (PKC) and have been widely used to study the function of this enzyme. We investigated the effect of phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on tension responses and free cytosolic Ca in small strips of chick embryo ventricle and on the 45Ca influx in primary cultures of chick embryo ventricular cells. TPA (10(-8) M) decreased twitch tension about 40% when driven at 1 Hz. The free cytosolic Ca measured with fura-PE3 was rapidly increased in 110 mM KCl solution and this high K-induced increase was slightly inhibited by TPA (10(-8) M), by 12%. The resting 45Ca uptake in 5 mM KCl was slightly increased by a low concentration of TPA (10(-9) M), but high K-induced 45Ca uptake was decreased by TPA in a dose-dependent manner, but to 40% at the utmost. 4 alpha-Phorbol, an inactive phorbol ester, did not inhibit 45Ca uptake. These results indicate that TPA decreased the depolarization induced transsarcolemmal Ca uptake and thus the excitation-contraction process was attenuated, but to 40% at most. PKC may participate in the modulation of myocardial Ca homeostasis and contractile state.

Spectroscopic properties of fluorescence dye fura-2 with various divalent cations.

We examined the effects of divalent cations on the fluorescence excitation spectra of fura-2 from a 2 ml solution containing 1 microM fura-2 with an excitation wavelength of 300-425 nm and an emission wavelength of 500 nm. The fluorescence spectra in solutions of fura-2-Ca2+, fura-2-Ba2+, and fura-2-Sr2+ complexes show almost the same pattern. The dissociation constants (Kd) of Ba2+ and Sr2+ with fura-2 were 1.63 and 9.02 microM, respectively, and these values were higher than that of the Ca2+ complex (222 nM). The effect of Mg2+ binding on the fluorescence of fura-2 was quite small, and Mn2+ quenched the fluorescence of fura-2. The spectra of the fura-2-Ca2+ complex were changed by the co-existence of another divalent cation. Divalent cations competitively bind to the binding site of fura-2 and show the respective fluorescence spectra; consequently, the composed fluorescence spectra according to the respective Kd.

A program for calculation of the proton activity coefficient.

A program for calculation of the proton activity coefficient at any given ionic strength and temperature was written in Quick Basic. The calculation of proton activity coefficient was confirmed by titration of HEPES buffer.

Determination of the appropriate valence of 1,4-piperazinediethanesulfonic acid (PIPES) in physiological pH.

1,4-Piperazinediethanesulfonic acid (PIPES), a zwitterion buffer, was titrated with KOH to measure its pH and osmolality. Both pH and osmolality changes showed three phases, and two transition points coincided with pH 5.1 and 10.2. The second phase is important in biological reactions, and from the present results, the pK1 is 3.3 while pK2 is 6.85 at 20 degrees C. The slope of the osmolality curve was 2 mOsm per 1 mM KOH addition in the first and third phases, while it was 1 mOsm per 1 mM KOH in the second phase. The first phase indicates that +/- PIPES +/- changes to -PIPES +/- by the addition of KOH. In the second phase, additional KOH produces -PIPES-. PIPES acts as a divalent ion, at least, in the physiological pH range of around 7.0. Concentrations of -PIPES +/- and -PIPES- around the physiological pH range are calculated as follows. [formula: see text] The ionic strength of PIPES is calculated as follows. [formula: see text]

An analysis of post-contracture potentiation in frog twitch skeletal muscle.

Post-tetanic potentiation (PTP) was mimicked by post-contracture potentiation (PCP) in the twitch muscle of frog. A marked PCP was observed in small bundles of semitendinosus muscle in tetrodotoxin (TTX)-containing solution under current clamp condition. Contractures were induced by a constant depolarizing current pulse of 1 s every 5 min. Each contracture (C) was preceded and followed by a twitch-corresponding short contracture, T1 and T2, with an interval of 2 s. The ratio of twitch height, T2/T1, was measured and compared under various conditions. In TTX-containing Ringer solution, T2/T1 depended on magnitude of both T and C. When T2/T1 was plotted against C/T1 a quasi-linear relationship was found. The slope of the curve was 0.076 and was found to decrease when the duration of T was increased to 1 s. In TTX-containing solution, addition of 2 mM Ba2+ or 1 mM caffeine potentiated T as well as C but decreased T2/T1 markedly. Similar effects were observed when Na+ was replaced with TEA+ or when bath temperature was lowered from 20 to 8 degrees C. In all cases T2/T1 vs. C/T1 curve was shifted to the left and downwards. It was considered that these results could be attributable to an elevation of the resting Ca2+ concentration in the sarcoplasm. Thus the phenomenon of PCP probably reflects the time course of Ca2+ transients in the conditioning contracture C.

Changes in contractile force by barium in the frog skeletal muscle.

Effects of Ba2+ ions on the contractility were investigated in the frog skeletal muscle under the current clamp condition. The membrane potential was depolarized by 10 to 20 mV by perfusing 2 mM Ba2+ for 5 to 10 min. Membrane resistance was first increased and then decreased. The mechanical threshold examined in TTX-containing solution was shifted to more positive potential by applying Ba2+. The electrically induced contracture in TTX-solution as well as twitch in normal Ringer were enhanced by superfusing Ba2+. In both types of contraction an afterpotentiation was observed on washing out of Ba2+ ions, reflecting the existence of an inhibitory action during Ba2+ perfusion which could be masked by potentiation. Caffeine was able to induce contracture even in muscles soaked in "zero" Ca2+ solution for 24 to 48 h. In these muscles Ba2+ was more effective than Ca2+ to potentiate the contracture tension presumably by releasing Ca2+ ions from the sarcoplasmic reticulum whereas Mg2+ inhibited it.

Dependence of contractile responses by some calcium antagonists on external calcium in the skeletal muscle.

Dependence of contractile potentiation by calcium antagonists on external calcium was investigated on the frog's twitch muscle. Low concentrations (10(-6) to 10(-5) M) of nicardipine and verapamil enhanced the peak tension of both twitch and electrically induced contracture in the presence of calcium. In the calcium-free media the drugs suppressed the contractile tension. Caffeine contracture was inhibited by the calcium antagonists at 20 degrees C. This inhibition was caused by an early onset of relaxation, which was not observed at 7 degrees C. The results suggest that some interaction between calcium ions and drug molecules at the voltage sensor on the transverse tubular membrane, which regulates E-C coupling but is not directly related to the functional calcium channel, may play an important role for the phenomena. The inhibitory action of calcium antagonists on the caffeine contracture in the presence of calcium is probably independent of the potentiating effect seen in the depolarization-induced contractions.

Development of the response to adenosine during organ culture of young embryonic chick hearts.

Young (3 days-old) embryonic chick hearts (i.e., prior to innervation) show little electrophysiological response to adenosine. However, during embryonic development the sensitivity to adenosine greatly increases. In the present experiments, in which the chick hearts were placed into organ culture, the sensitivity to adenosine was found to increase with time in culture. Thus, the ability of adenosine to slow the spontaneous heart rate became greater during the course of organ culture. These results suggest that physiological responses to adenosine continue to develop in organ culture, and that the increased sensitivity to adenosine seen in ovo may be independent from the development of sympathetic or parasympathetic innervation.

Effects of extracellular calcium and other divalent cations on mechanical response of frog skeletal muscle.

In order to investigate the mechanism by which elevated extracellular Ca ions decrease twitch and tetanus tension in frog skeletal muscle we made mechanical and electrophysiological measurements on single fibers or small bundles from twitch muscles. High concentration of Ca caused a hyperpolarization and an increase in the duration of action potential. The mechanical threshold, estimated by using the strength-duration curve, was shifted upward by adding Ca ions. These effects were fully reversible. Steady state twitch tension was slightly increased by replacing Mg and Ni with Ca and decreased by elevating their concentrations, although Ba resulted in a marked twitch augmentation and a positive correlation with the ion concentration. By contrast, the strength-duration curve was shifted upward by Ni while Mg and Ba showed no shift. These evidences point to a failure of the early step of excitation-contraction coupling, including the T-membrane depolarization, as the primary mechanism of action of high concentration of Ca, Mg, and Ni ions, whereas Ba ion has an additional intracellular potentiating effect.

Reduction in transmembrane Ca influx induced by the negative inotropic action of nicardipine in frog ventricular muscle.

The effects of nicardipine, a new 1,4-dihydropyridine derivative, on electrical and mechanical properties of frog ventricular muscle were examined. Nicardipine (3 X 10(-7) M) reduced the twitch tension, and this reduction was frequency dependent, and considerable, in case of high frequencies. The resting potential was not affected by nicardipine (3 X 10(-7) M), but the plateau height of the action potential was decreased and the duration of the action potential was shortened. The suppression of this plateau height was frequency dependent. The nicardipine-induced suppression of tension and action potential could be almost completely antagonized by raising the concentration of [Ca]o or by applying isoprenaline (8 X 10(-7) M). These results suggest that the negative inotropic action of nicardipine is induced mainly by a reduction in the transmembrane Ca influx.

Effects of nicardipine on frog skeletal muscle in normal and calcium-free media.

The effects of the new "Ca-antagonist," nicardipine, on frog skeletal muscle were studied. Nicardipine (3 X 10(-10)-3 X 10(-7) M) had no effect on the twitch tension yet a high concentration (3 X 10(-6) M) had a slight potentiating effect, with no effect on the action potential. Nicardipine suppressed the repetitive action potentials induced by perfusion of Ca-free solution and thereby suppressed augmentation of the tension responses, in a dose dependent manner. A high concentration of nicardipine (3 X 10(-6) M) abolished the twitch tension due to abolition of the action potential, in Ca-free solution. These abolitions of the action potential and twitches induced by nicardipine in Ca-free solution were antagonized in the case of NaCl hypertonic solution. Therefore, nicardipine probably inhibits Na permeability in frog skeletal muscle placed in Ca-free solution.

Effects of low Na on the tetanic contractility of frog skeletal muscle.

The effects of low Na on the tetanic contractility after trains of twitch were investigated in frog skeletal muscle. Tetanic tension was induced by a stimulation of 100 Hz for 400 msec every 5 min and a train of twitches (150 or 250) with 1 Hz was interposed between two successive tetani. When the Na concentration was reduced to 50% by replacement with choline, each twitch constituting the positive staircase during stimulus train was augmented as compared with those in normal Ringer. The amplitude of action potential during the staircase was smaller than that in normal Ringer and was gradually decreased, suggesting that low Na solution made the release of twitch-Ca from the sarcoplasmic reticulum in twitch more effective. Although the tetanic contractility in low Na was almost the same as that in normal Ringer before applying twitch train, it was more markedly inhibited after the train. The relative size of the inhibited tetanus had a correlation with the relative size of the potentiated twitch in both normal Ringer and low Na. This tetanus inhibition after the twitch train might be due to the suppression of action potential and/or due to some inhibitory factor in the excitation-contraction (E-C) coupling process.

A study on the mechanism of twitch potentiation by hypertonic solution in the frog atrial muscle.

Hypertonic solutions were found to exhibit both positive and negative inotropic effects on the contraction of the isolated atrial myocardium of bullfrog. The optimum tonicity for twitch potentiation was about 1.5 T. The mechanism for the positive inotropic action was investigated. The possibility of involvement of an increase in calcium influx during each action potential was excluded, since both the overshoot and the plateau of action potential were strongly depressed by perfusion of hypertonic solution. The effect on the time course of twitch potentiation was similar to that of muscle shrinkage, regardless of the type solute (sucrose, NaCl or LiCl) used for elevating the tonicity, except that excess sodium showed an initial rapid inhibitory phase of contraction. A marked post-rest potentiation was observed even after "zero" calcium perfusion, provided that the tonicity of the bathing medium was elevated previously. Potassium contracture occurred during the prolonged hypertonic perfusion in "zero" calcium condition. In addition, caffeine contracture was strongly augmented in hypertonic solution. The results suggest that an elevation in both the [Ca2+]i and amount of calcium bound intracellularly may play an important role in the positive inotropic action.

Paradoxical after-potentiation of the myocardial contractility by lanthanum.

Lanthanum of 0.1 to 0.2 mM caused a twitch inhibition and further showed a marked, long-lasting twitch potentiation during washout in both ventricular strips of bullfrog and atrial or papillary muscles of guinea-pig. It was found that the effect was more conspicuous in the frog heart. A similar potentiation was observed by applying gadolinium instead of lanthanum. The participation of endogeneous catecholamine release is unlikely since the positive inotropic effect was never affected by the presence of beta-adrenergic blocker. The potentiation was not accompanied by any increase in the action potential plateau, suggesting independence of the phenomenon on the calcium current. A marked elevation of the resting tension was produced during a long-term lanthanum perfusion, reflecting the increase in the intracellular calcium level. On the other hand, transient increase in the resting tension was frequently observed on returning to normal Ringer solution. The latter change seemed to result from a transient increase in the resting permeability to calcium ions. The negative or positive inotropic effect by increasing or lowering the external sodium concentration observed under the control condition was strongly depressed during the twitch potentiation. In addition, sodium-free contracture induced by solutions isotonically replaced with sucrose or potassium chloride was augmented both during lanthanum perfusion and during its washout. These results suggest the possibility that the elevation of the intracellular calcium level due to inhibition of Na-Ca exchange mechanism and/or increase in the calcium permeability is the principal cause for the twitch potentiation caused by lanthanum washout.

Effects of lanthanum on the electrical and mechanical activities of frog ventricular muscle.

The effects of lanthanum on the resting membrane potential, action potential, membrane resistance, twitch tension, and potassium contracture were investigated and the localization of the drug was studied electron microscopically in isolated frog ventricular muscle. Lanthanum in concentration of 0.2 to 5 mM decreased the resting potential by about 5-8 mV, which was accompanied by an increase in the membrane resistance of about 43% for the depolarizing and 40% for the hyperpolarizing direction. Lanthanum caused a decrease in height and a prominent shortening of the action potential, and also, a depression of the plateau level. In addition, it increased the threshold for action potential generation depending on its concentration. The slow response action potential was inhibited by lanthanum in parallel with twitch inhibition. This finding suggests that the twitch inhibition resulted from the suppression of the slow inward calcium current. In contrast, potassium contracture was not inhibited by lanthanum. When the muscle preparation was treated with neuraminidase, the twitch inhibition caused by lanthanum was strongly depressed. Electron microscopic observation revealed that the precipitates of lanthanum were localized on the external lamina of myocytes as well as in the extracellular spaces but could never be found within the cytoplasm. No such precipitates could be detected in the neuraminidase-treated muscle. From these results it is suggested that lanthanum takes the place of calcium at the membrane surface: it modifies permeabilities to sodium, potassium and calcium ions and the excitation-contraction coupling of the ventricular muscle by replacing calcium bound to the membrane-surface.

Effects of procaine on the ventricular muscle of bullfrog.

The effects of procaine on the contractility of the bullfrog's ventricular muscle were investigated. The addition of 10(-5) g/ml of procaine potentiated the twitch tension which was accompanied by an elevation as well as a prolongation of the action potential plateau. This positive inotropism of procaine was not induced by endogenous catecholamine because a beta-blocking agent did not influence this twitch potentiation. The twitch potentiation was increased in proportion to the external Ca concentration, suggesting the possibility of augmentation of Ca influx during the action potential. In normal Ringer solution, procaine suppressed potassium contracture which was composed of two components: an initial phasic component and a late tonic one. Potassium contracture after perfusion with Ca-free solution was also suppressed by procaine. However, potassium contracture which had been treated previously with La was composed of only a tonic component and was potentiated by procaine in spite of perfusion with Ca-free solution. The tonic component of potassium contracture may be considered to occur with intracellular Ca. Procaine may increase the Ca inward current, acting on the intracellular Ca storage site and consequently accelerate the excitation-contraction coupling in frog ventricular muscle.

Effects of hypertonic perfusion on the ultrastructure of frog cardiac muscle.

The ultrastructural changes induced by hypertonic perfusion were investigated using the ventricular muscle of the bullfrog. It was demonstrated that the fixative tonicity critically affects the ultrastructure. Effects of sucrose-, NaCl- and urea-hypertonicities were investigated and compared. In both sucrose- and NaCl-hypertonic media, although the cardiac muscle strongly shrank and the extracellular spaces markedly increased according to increasing tonicity the width of the intercalated disc cleft remained unchanged and the cleft was never separated even in 3 times hypertonicity. The sucrose-hypertonicity made the feature of the fine structure extremely obscure and the electron densities in both the Z-line and the intercalated disc region markedly decreased. When both the perfusate and fixative were made hypertonic by urea, which is known to easily penetrate the cell membrane, a shrinkage of the myocardial cells was observed but to a lesser extent as compared with sucrose or NaCl. The striation pattern was disordered in this condition though the intercalated discs were never affected.