β-Adrenergic receptors in innervated and denervated skeletal muscle

In order to determine if the development of β-adrenergic receptors may explain the catecholamine evoked contracture of denervated mammalian skeletal muscle, the binding capacities and dissociation constants of β-adrenergic receptors of innervated and denervated rat skeletal muscle membrane preparations were determined by using [³H] dihydroalprenolol. The dissociation constants of [³H] dihydroalprenolol binding to innervated and denervated muscle microsomal suspensions were similar. The maximal number of binding sites increased from 27 pmol/g protein to 85 pmol/g protein following 25 days denervation. These results suggest that motor nerve may be involved in part, in the regulation of β-adrenergic receptors in skeletal muscle membrane preparations.     

Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.

In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin.     

Effect of exercise and obesity on skeletal muscle amino acid uptake

The genetically obese Zucker rat has a reduced capacity to deposit dietary protein in skeletal muscle. To determine whether amino acid uptake by muscle of obese Zucker rats is impaired, soleus strip (SOL) and epitrochlearis (EPI) muscles from 10-wk-old lean and obese Zucker rats were studied in vitro by use of [14C]alpha-aminoisobutyric acid (AIB). Muscles from fasted rats were incubated under basal conditions at rest or after a 1-h treadmill run at 8% grade. To equate total work completed, lean and obese rats ran at 27 and 20 m/min, respectively. Muscles were pinned at resting length, preincubated for 30 min at 37 degrees C in Krebs-Ringer bicarbonate buffer containing 5 mM glucose under 95% O2-5% CO2, and then incubated up to 3 h in Krebs-Ringer bicarbonate with 0.5 mM AIB, [14C]AIB, and [3H]inulin as a marker of extracellular fluid. Basal AIB uptake in EPI and SOL from obese rats was significantly reduced by 40 and 30% (P less than 0.01), respectively, compared with lean rats. For both lean and obese rats, exercise increased (P less than 0.05) basal AIB uptake in EPI and SOL, but the relative increases were greater in the obese rats (EPI 54% and SOL 71% vs. EPI 32% and SOL 37%). These results demonstrate that genetically obese Zucker rats have reduced basal skeletal muscle amino acid uptake and suggest that physical inactivity may partially contribute to this defect.     

鸡前、后背阔肌去神经后对辣根过氧化物酶(HRP)的胞纳作用

本文报道了用辣根过氧化物酶(HRP)作为大分子标记物,以组织化学和生物化学方法观察了鸡前后背阔肌在去神经后的胞纳现象。结果表明,在去神经后发生肥大的前背阔肌和去神经后发生萎缩的后背阔肌同样都出现胞纳的明显增加。组织化学所观察的结果表明,浸泡在含 HRP 的任氏液中的有完整神经支配的前、后背阔肌只有极少数的肌纤维摄取 HRP,而在去神经的前、后背阔肌中则有不少的肌纤维内部出现 HRP 染色反应。这种反应在有的纤维表现为弥散性染色,有的表现为浓的 HRP 反应颗粒。生物化学的结果显示,去神经后的前、后背阔肌中 HRP 的相对含量分别比有神经支配的对照肌肉明显地增多54%和87%,我们在鸡前背阔肌用组织化学和生物化学所得的实验结果与 Thesleff 等人提出的关于肌肉萎缩机制的假设显然不符。本工作证实了肌肉的胞纳作用的增加并不一定最终导致肌纤维的变性和萎缩。     

Denervation enhances the physiological effects of the K(ATP) channel during fatigue in EDL and soleus muscle

The objective was to determine whether denervation reduces or enhances the physiological effects of the K(ATP) channel during fatigue in mouse extensor digitorum longus (EDL) and soleus muscle. For this, we measured the effects of 100 microM of pinacidil, a channel opener, and of 10 microM of glibenclamide, a channel blocker, in denervated muscles and compared the data to those observed in innervated muscles from the study of Matar et al. (Matar W, Nosek TM, Wong D, and Renaud JM. Pinacidil suppresses contractility and preserves energy but glibenclamide has no effect during fatigue in skeletal muscle. Am J Physiol Cell Physiol 278: C404-C416, 2000). Pinacidil increased the (86)Rb(+) fractional loss during fatigue, and this effect was 2.6- to 3.4-fold greater in denervated than innervated muscle. Pinacidil also increased the rate of fatigue; for EDL the effect was 2.5-fold greater in denervated than innervated muscle, whereas for soleus the difference was 8.6-fold. A major effect of glibenclamide was an increase in resting tension during fatigue, which was for the EDL and soleus muscle 2.7- and 1.9-fold greater, respectively, in denervated than innervated muscle. A second major effect of glibenclamide was a reduced capacity to recover force after fatigue, an effect observed only in denervated muscle. We therefore suggest that the physiological effects of the K(ATP) channel are enhanced after denervation.     

Sustained cell proliferation in denervated skeletal muscle of mice

Summary Cellular proliferation in skeletal muscle was measured throughout the first 4 weeks after denervation. Twenty four mice had one leg denervated, and 4 groups of 6 of these mice were injected with tritiated thymidine once daily for 7 days, either during the first, second, third or fourth week after denervation. Autoradiographic labelling of muscle and connective tissue nuclei in denervated muscles was compared with innervated muscles from the opposite innervated legs of the same mice. Labelling of connective tissue and muscle (myonuclear and satellite cell) nuclei was significantly higher in denervated muscles, compared with innervated muscles on the unoperated side. There were no significant differences among labelling of nuclei in muscles denervated for 1, 2, 3 or 4 weeks. However, connective tissue labelling after 1 week of denervation was significantly higher than at later times. This study shows that nuclei of muscle and connective tissue cells proliferate and turnover at high levels for at least one month after denervation.     

Ciliary neurotrophic factor (CNTF) affects the excitable and contractile properties of innervated skeletal muscles

The well-established trophic role of CNTF upon neurons led to performing clinical trials in patients of neurodegenerative diseases. However, trials were suspended due to side effects such as severe weight loss, hyperalgesia, coughing, muscle cramps and pain. So far it is not known how CNTF triggers the problems related to skeletal muscle cramps and pain. CNTF has also been described as a myotrophic factor for denervated skeletal muscles, but the possibility that it affects innervated muscles has also been considered. Since a myotrophic factor could be a valuable tool for treatment of several muscle diseases, we studied the effects of low doses of CNTF delivered systemically by an osmotic pump, over the electrical and mechanical properties of innervated and denervated fast and slow muscles. CNTF induced spontaneous electrical discharges and slowed twitches in innervated muscles, but did not prevent the changes induced by denervation. We postulate that the spontaneous discharges induced by CNTF in innervated muscles may be the cause of the cramps, coughing, and muscle ache reported by patients. At low doses, CNTF does not exert its myotrophic role over denervated muscles but clearly affects the excitable and contractile properties of innervated muscles.     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.     

Glucose transport by English sparrow (Passer domesticus) skeletal muscle: have we been chirping up the wrong tree?

Glucose uptake by mammalian skeletal muscle has been extensively covered in the literature, whereas the uptake of glucose by avian skeletal muscle has yet to be examined. As skeletal muscle provides the majority of postprandial glucose uptake in mammals, this study was designed to characterize the glucose transport mechanisms and glycogen content of avian skeletal muscle. In addition, plasma glucose levels were measured. English sparrow extensor digitorum communis (EDC) skeletal muscles were used for this study to quantify in vitro radiolabeled-glucose uptake. Uptake of labeled glucose was shown to decrease in the presence of increasing unlabeled glucose and was maximal by 60 minutes of incubation. Various agents known to increase glucose transport in mammalian tissues, via the insulin and contraction-responsive pathways, were used to manipulate and characterize in vitro transport in birds. The typical effectors of the mammalian insulin pathway, insulin (2 ng/ml) and insulin-like growth factor-1 (48 ng/ml), did not increase skeletal muscle glucose transport. Likewise, inducers of the mammalian contraction-responsive pathway had no effect on glucose transport by in vitro avian skeletal muscle (5 mM caffeine, 2 mM AICAR (5'-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). Interestingly, 200 microM phloretin, an agent used to block glucose transport proteins, significantly inhibited its uptake (P<0.001). These results suggest that a glucose transporter is responsible for glucose uptake by avian skeletal muscle, albeit at unexpectedly low levels, considering the high plasma glucose concentrations (265.9+/-53.5 mg/dl) and low skeletal muscle glycogen content (9.1+/-4.11 nM glucose/mg) of English sparrows.     

Surgical Sympathetic Denervation Increases α1Adrenoceptor-Mediated Accumulation of myo-Inositol Trisphosphate and Muscle Contraction in Rabbit Iris Dilator Smooth Muscle

Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo-[3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.     

Aging is associated with elevated muscle triglyceride content and increased insulin-stimulated fatty acid uptake

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under hyperglycemic and hyperinsulinemic conditions. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose uptake and palmitate delivery were similar among age groups. Palmitate uptake and oxidation as well as muscle protein concentration of fatty acid translocase (FAT/CD36) and plasma membrane fatty acid-binding protein (FABPPM) were significantly increased (P < or = 0.05) in 24- vs. 5- and 15-mo-old animals. Compared with 5- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 72-145% in red and 112-129% in white muscles of 24-mo-old animals. Palmitate uptake was associated with total preperfusion TG concentration (r2 = 0.27, P < 0.05) and total TG synthesis rate (r2 = 0.68, P < 0.05). These results indicate that, under insulin-stimulated conditions, FA uptake is significantly increased in old animals, which is associated with increased rates of TG synthesis and may contribute to the accumulation of TG in muscle of old animals.     

In vivo monitoring of norepinephrine and its metabolites in skeletal muscle

Although skeletal muscle sympathetic nerve activity plays an important role in the regulation of vascular tone and glucose metabolism, relatively little is known about regional norepinephrine (NE) kinetics in the skeletal muscle. With use of the dialysis technique, we implanted dialysis probes in the adductor muscle of anesthetized rabbits and examined whether dialysate NE and its metabolites were influenced by local administration of pharmacological agents through the dialysis probes. Dialysate dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured as two major metabolites of NE. The skeletal muscle dialysate NE, DHPG and MHPG were 11.7+/-1.2, 38.1+/-3.2, and 266.1+/-28.7 pg/ml, respectively. Basal dialysate NE levels were suppressed by tetrodotoxin (Na(+) channel blocker, 10 microM) (5.1+/-0.6 pg/ml), and augmented by desipramine (NE uptake blocker, 100 microM) (25.8+/-3.2 pg/ml). Basal dialysate DHPG levels were suppressed by pargyline (monoamine oxidase blocker, 1mM) (24.3+/-4.6 pg/ml) and augmented by reserpine (vesicle NE transport blocker, 10 microM) (75.8+/-2.7 pg/ml). Basal dialysate MHPG levels were not affected by pargyline, reserpine, or desipramine. Addition of tyramine (sympathomimetic amine, 600 microM), KCl (100 mM), and ouabain (Na(+)-K(+) ATPase blocker, 100 microM) caused brisk increases in dialysate NE levels (200.9+/-14.2, 90.6+/-25.7, 285.3+/-46.8 pg/ml, respectively). Furthermore, increases in basal dialysate NE levels were correlated with locally administered desipramine (10, 100 microM). Thus, dialysate NE and its metabolite were affected by local administration of pharmacological agents that modified sympathetic nerve endings function in the skeletal muscle. Skeletal muscle microdialysis with local administration of a pharmacological agent provides information about NE release, uptake, vesicle uptake and degradation at skeletal muscle sympathetic nerve endings.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

Comparison of dopamine uptake and release in vitro in sheep and rat striatum.

Cocaine inhibits tritium-labeled dopamine ([3H]DA) uptake in rat (IC50 approximately 400 nM) and sheep (IC50 approximately 1 microM) striatum. GBR 12909, a selective DA uptake inhibitor, potently inhibits [3H]DA uptake in rat (IC50 less than 10 nM), but is less effective (only 60% of the uptake is inhibited at a concentration of 10 microM) and less potent (IC50 approximately 300 nM) in sheep. [3H]DA release from slices of rat or sheep striatum is stimulated by potassium (15-50 mM). In the presence of nomifensine (10 microM), cocaine (10 microM) had no effect on potassium-stimulated [3H]DA release in either species. [3H]DA release is increased by N-methyl-D-aspartate (NMDA) (10-1000 microM) in rat striatum but NMDA did not stimulate [3H]DA release in sheep striatum. These findings suggest that NMDA receptors either are absent from or do not regulate release of preloaded [3H]DA in sheep striatum.     

p38 mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 (MK2) signaling in atrophic and hypertrophic denervated mouse skeletal muscle

Background

p38 mitogen-activated protein kinase has been implicated in both skeletal muscle atrophy and hypertrophy. T317 phosphorylation of the p38 substrate mitogen-activated protein kinase-activated protein kinase 2 (MK2) correlates with muscle weight in atrophic and hypertrophic denervated muscle and may influence the nuclear and cytoplasmic distribution of p38 and/or MK2. The present study investigates expression and phosphorylation of p38, MK2 and related proteins in cytosolic and nuclear fractions from atrophic and hypertrophic 6-days denervated skeletal muscles compared to innervated controls.

Methods

Expression and phosphorylation of p38, MK2, Hsp25 (heat shock protein25_rodent/27_human, Hsp25/27) and Hsp70 protein expression were studied semi-quantitatively using Western blots with separated nuclear and cytosolic fractions from innervated and denervated hypertrophic hemidiaphragm and atrophic anterior tibial muscles. Unfractionated innervated and denervated atrophic pooled gastrocnemius and soleus muscles were also studied.

Results

No support was obtained for a differential nuclear/cytosolic localization of p38 or MK2 in denervated hypertrophic and atrophic muscle. The differential effect of denervation on T317 phosphorylation of MK2 in denervated hypertrophic and atrophic muscle was not reflected in p38 phosphorylation nor in the phosphorylation of the MK2 substrate Hsp25. Hsp25 phosphorylation increased 3-30-fold in all denervated muscles studied. The expression of Hsp70 increased 3-5-fold only in denervated hypertrophic muscles.

Conclusions

The study confirms a differential response of MK2 T317 phosphorylation in denervated hypertrophic and atrophic muscles and suggests that Hsp70 may be important for this. Increased Hsp25 phosphorylation in all denervated muscles studied indicates a role for factors other than MK2 in the regulation of this phosphorylation.

     

Serum half-life, distribution, hepatic uptake and biliary excretion of alpha-tocopherol in rats

The serum clearance of alpha-[3H]tocopherol has been studied after intravenous injection of intestinal lymph labeled in vivo with radioactive alpha-tocopherol. The half-life of the injected alpha-[3H]tocopherol was approx. 12 min. Fractionation of plasma by ultracentrifugation 10 min after injection of lymph showed that 91% of the radioactive alpha-tocopherol remaining in plasma was located in chylomicrons (d less than 1.006 g/ml) and 7.8% in high-density lipoproteins (HDL, 1.05 less than d less than 1.21 g/ml). 2 h after administration of alpha-tocopherol, about 35% of the radioactivity recovered in plasma was associated with chylomicrons and approx. 51% with HDLs. alpha-[3H]Tocopherol was initially taken up by the liver, which contained more than 50% of the injected radioactivity after 45-60 min. Separation of parenchymal and nonparenchymal cells demonstrated a preferential uptake of alpha-[3H]tocopherol by the parenchymal liver cells. After 24 h about 11% of the injected dose was recovered in the liver. Considering whole organs the liver, adipose tissue and skeletal muscle had the highest content of radioactivity after 24 h. Furthermore, about 14% of the administered dose was recovered in bile during 24 h draining.     

The glucose transport system of muscle plasma membranes: characterization by means of [3H]cytochalasin B binding

A membrane-rich preparation was isolated from adult rat skeletal muscle in low salt media and further fractionated in sucrose gradients. Fraction F2, with a relative density of 1.092-1.119, consisted of sealed membrane vesicles which were enriched in plasma membrane markers. These vesicles were capable of stereospecific D-glucose uptake which was sensitive to cytochalasin B (CB). The membranes were also enriched in high affinity [3H]CB binding activity (Kd of 0.28 microM). [3H]CB binding to the glucose carrier of these plasma membranes, estimated as the fraction of binding protectable by D-glucose, ranged between 2.5 and 7.4 pmol/mg protein in several membrane preparations. The amount of [3H]CB binding to muscle membranes from newborn and adult rats was not markedly different. Trypsin, at low concentrations, altered the molecular weight of several membrane components, without affecting [3H]CB binding. Higher concentrations of trypsin abolished [3H]CB binding. Both 2,4-dinitrofluorobenzene (0.1 mM) and N-ethylmaleimide (15 mM) inhibited [3H]CB binding; inhibition by these reagents was prevented by inclusion of micromolar concentrations of CB in the reaction mixture. Several procedures that extracted specific proteins enriched the D-glucose-sensitive [3H]CB binding to the protein-depleted membranes. Antibody raised against the glucose carrier of human red cell membranes cross-reacted with a polypeptide of Mr about 45K of muscle membranes which might represent the glucose carrier.