Effects of carbamylcholine on membrane potential and Na-K pump activity of cultured rat skeletal myotubes

1. We measured changes in resting membrane potential (Em) and Na-K pump activity, assayed by ouabain-sensitive 86Rb uptake, in response to carbamylcholine (CCh) and its continued presence in single rat skeletal myotubes in culture. 2. CCh caused immediate depolarization from control Em (-80 to -85 mV) to near 0 followed by repolarization of varying degrees depending on the age of the culture and temperature of the recording medium; repolarization of Em was most apparent by culture age 8-9 days in vitro (DIV), Em reaching values as high as -60 mV by 5-10 min after peak depolarization at 37 degrees C. 3. Input resistance, which decreased during CCh depolarization, increased only slightly during the initial phase of repolarization and then remained essentially unchanged during the major component of membrane repolarization in the presence of CCh. 4. Ouabain, given before CCh, prevented repolarization of Em and, when given after repolarization had begun, reversed it and caused Em to return to about -7 mV. 5. Na-K pump activity was decreased in myotubes in which Em did not repolarize or did so only slightly, and was increased by over 40-50% in myotubes whose Em repolarized by 40-60 mV, even though CCh was still present in the medium. Inhibition of pump activity in non repolarizing myotubes was related to Na influx, inhibition being reversed to stimulation when CCh was administered to myotubes in Na-free medium. 6. Repeated (three or four times) or prolonged (up to 60-min) administration of CCh to myotubes in which repolarization was hardly expressed (age 6-7 DIV) caused increases both in the amount of repolarization and in 86Rb uptake, both being related to the number or duration of CCh exposures. 7. We conclude that repolarization of Em following CCh-induced depolarization of cultured rat skeletal myotubes depends to a large extent on an increase in activity of the electrogenic Na-K pump.     

Veratridine-induced oscillations in membrane potential of cultured rat skeletal muscle: Role of the Na-K pump

1. The acute effects of veratridine on membrane potential (Em) and Na-K pump activity in cultured skeletal muscle were examined. 2. At a concentration of 10(-4) M, veratridine caused depolarization of Em and a decrease in Na-K pump activity. At concentrations of 10(-5) and 10(-6) M, veratridine caused oscillations of Em and an increase in Na-K pump activity compared to untreated, control cells. The oscillations consisted of depolarization to about -40 mV followed by hyperpolarization to about -90 mV; the level of hyperpolarization was higher at 37 than at 23 degrees C. 3. Veratridine-induced oscillations could be prevented by pretreatment with tetrodotoxin (10(-6) M) and blocked or prevented by ouabain, which depolarizes Em of cultured myotubes. In contrast, depolarization of Em to -60 mV by excess K+ did not alter the amplitude or frequency of the oscillations. 4. The results demonstrate that veratridine-induced increase in Na influx both depolarizes cultured myotubes and increases the activity of the Na-K pump, which repolarizes Em to levels higher than control. This sequence accounts for veratridine-induced oscillations in Em. High concentrations of veratridine cause only depolarization of Em and inhibition of Na-K pump activity.     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Regulation of the sodium-potassium pump in cultured rat skeletal myotubes by intracellular sodium ions

The properties of the Na-K pump and some of the factors controlling its amount and function were studied in rat myotubes in culture. The number of Na-K pump sites was quantified by measuring the amount of [3H]ouabain bound to whole-cell preparations. Activity of the pump was determined by measurement of ouabain-sensitive 86Rb-uptake and component of membrane potential. Chronic treatment of myotubes with tetrodotoxin (TTX), which lowers [Na]i, decreased the number of Na-K pumps, the ouabain-sensitive 86Rb uptake, and the size of the electrogenic pump component of Em. In contrast, chronic treatment with either ouabain or veratridine, which increases [Na+]i, resulted in an elevated level of Na-K pump sites. This effect was blocked by inhibitors of protein synthesis. Neither rates of degradation nor affinity of pump sites in cells treated with TTX, veratridine, or ouabain differred from those in control cells. The number and activity of Na-K pump sites were unaffected by chronic elevation in [Ca]i or chronic depolarization. We conclude that alterations in the level in intracellular Na ions play the major role in regulation of Na-K pump synthesis in cultured mammalian skeletal muscle.     

Generation of resting membrane potential

This brief review is intended to serve as a refresher on the ideas associated with teaching students the physiological basis of the resting membrane potential. The presentation is targeted toward first-year medical students, first-year graduate students, or senior undergraduates. The emphasis is on general concepts associated with generation of the electrical potential difference that exists across the plasma membrane of every animal cell. The intention is to provide students a general view of the quantitative relationship that exists between 1) transmembrane gradients for K(+) and Na(+) and 2) the relative channel-mediated permeability of the membrane to these ions.     

Hemin enhances differentiation and maturation of cultured regenerated skeletal myotubes

Satellite cells, isolated from marcaine-damaged rat skeletal muscle, differentiate in culture to form contracting, cross-striated myotubes. Addition of 20 microM hemin (ferriprotoporphyrin IX chloride) to the culture medium resulted in increases in the number, size, and alignment of myotubes; in the number of myotubes that exhibited cross-striations; and in the strength and frequency of myotube contractions. Hemin increased satellite cell fusion by 27%, but decreased cell proliferative rate by 30%. Hemin increased the specific activity of creatine kinase (CK), a sensitive indicator of muscle differentiation, by 157%. Separation of CK isoenzymes by agarose gel electrophoresis showed that hemin increased only the muscle-specific CK isoenzymes (MM-CK and MB-CK). Thus, hemin seems to duplicate some of the effects of innervation on cultured myotubes by increasing contraction frequency and strength, appearance of cross-striations, and muscle-specific isoenzymes. In contrast, 3-amino-1,2,4-triazole, an inhibitor of heme biosynthesis, decreased the number of cross-striated myotubes, the strength and frequency of myotube contractions, and CK activity. These inhibitory effects were reversed by hemin. Collectively, these results demonstrate a physiologically significant role for heme in myotube maturation.     

Basolateral membrane lipid dynamics alter Na-K ATPase activity in rabbit small intestine.

The role of basolateral membrane fluidity in regulating Na-K ATPase activity along the crypt-villus axis in rabbit distal small intestine was assessed. Basolateral membranes were prepared from isolated villus and crypt enterocytes at 24- to 28-fold enhancement. Villus basolateral membranes were significantly (p < 0.001) more fluid than crypt basolateral membranes as measured by 1,6-diphenyl-1,3,5-hexatriene. No difference was seen between the two groups as measured by either 2-(9-anthroyloxy)-stearic fatty acid or 16-(9-anthroyloxy)-palmitic acid. Fluidity alterations were accompanied by an increased phospholipid content in villus membranes, which resulted in a decreased cholesterol:phospholipid ratio and an increased lipid:protein molar ratio. Na-K ATPase activity was significantly (p < 0.01) greater in villus basolateral membranes than in crypt membranes, and demonstrated a greater sensitivity to ouabain inhibition. Ouabain inhibition curves calculated from villus data fit well (p < 0.001) with a two binding site model, with a high affinity (Ki 16 nM) and a low affinity (Ki 4.2 microM) ouabain binding site. In crypt basolateral membranes, only a low affinity site was apparent (Ki 3.0 microM). Fluidizing crypt basolateral membranes in vitro with benzyl alcohol to levels seen in villus basolateral membranes resulted in the appearance of a high affinity ouabain binding site (Ki 110 nM) and an increased sensitivity of Na-K ATPase to ouabain inhibition. The fluidization of villus basolateral membranes eliminated the binding associated with the high affinity site. Treatment with methanol, as a control, did not alter Na-K ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

细胞静息膜电位的测量

实验表明,大细胞表面积与体积的比率是相当大的,以至于离子梯度的下降是极为缓慢的。即使代谢类毒物阻断了依赖ATP的能量代谢,静息膜电位也能维持相当长的时间,这表明依赖ATP的泵不是维持膜电位的直接能源。例如,应用地高辛(cardiac glycol— side)来抑制枪鸟贼巨轴突膜上的Na—K泵,细胞膜电     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

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.