Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.     

L-Fucose Is a Potent Inhibitor of myo-Inositol Transport and Metabolism in Cultured Neuroblastoma Cells

It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.     

Synthesis of Sulfatide by Cultured Rat Schwann Cells

Abstract: The ³⁵S sulfolipids synthesized by purified cultures of rat Schwann cells, fibroblasts, and a rat cell line (RN2) were studied. Schwann cell ³⁵S sulfolipids were almost entirely [³⁵S]sulfatide, as shown by TLC in two different solvent systems with unlabeled authentic sulfatide run in the same track. RN2 and fibroblasts did not synthesize significant amounts of sulfatide, by the same criteria. Previous studies failed to detect any characteristic myelin components, including sulfatide, on Schwann cells after several days in culture (Brockes et al., 1980a; Mirsky et at., 1980). My results show that Schwann cells continue to synthesize some sulfatide in the absence of neurons.     

Effect of Dibutyryl Cyclic AMP on the Kinetics of myo-Inositol Transport in Cultured Astrocytes

Dibutyryl cyclic AMP (dBcAMP) is known to induce maturation and differentiation in astrocytes. As myo-inositol is an important osmoregulator in astrocytes, we examined the effects of maturation and biochemical differentiation on the kinetic properties of myo-inositol transport. Treatment of astrocytes with dBcAMP significantly decreased the Vmax of myo-inositol uptake, but the effect on Km was not significant. The myo-inositol content of astrocytes was significantly decreased in cells treated for 5 days with dBcAMP as compared with untreated controls. Maximum suppression of myo-inositol uptake occurred 7 days after exposure of astrocytes to dBcAMP; this was gradually reversible when dBcAMP was removed from the medium. After exposure to hypertonic medium for 6 h, mRNA expression of the myo-inositol co-transporter was diminished by approximately 36% in astrocytes treated with dBcAMP as compared with untreated cells. It appears that myo-inositol transporters in astrocytes treated with dBcAMP are either decreased in number or inactivated during maturation and differentiation, suggesting that the stage of differentiation and biochemical maturation of astrocytes is an important factor in osmoregulation.     

Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.     

许旺细胞复合改性PLA\PGA的体外生物学研究

目的：评价小鼠许旺细胞体外复合改性聚乳酸\聚羟基乙酸（PLA\PGA）的细胞活性及生物相容性。方法：转绿色荧光蛋白基 因（GFP）小鼠的许旺细胞传代培养至第2 代,然后通过MTT 检测在不同改性技术（H2O2、NaOH、NaClO4、K2CrO4及超声波）处理 的PLA\PGA 浸提液中许旺细胞的增殖情况,检测许旺细胞在PLA\PGA表面的黏附及其细胞形态。结果：于培养1 d,3 d测得在 不同改性技术处理的PLA\PGA浸提液OD值,1 天时,各浸提液组和对照组相比无显著性差异,许旺细胞的活力及增殖无影响。3 天时,经NaClO4及K2CrO4处理的PLA\PGA 与对照组相比具有统计学差异,影响许旺细胞的增殖,对许旺细胞有毒性;荧光显微 镜下观察到许旺细胞在改性PLA\PGA 表面逐渐伸展,形成伪足,最终粘附在材料表面。结论：经H2O2、NaOH 及超声波改性 PLA\PGA无细胞毒性,具有良好的生物相容性和黏附性,可以用于组织工程化神经的构筑。     

Effect of Increased Glucose Levels on Na+/K+-Pump Activity in Cultured Neuroblastoma Cells

Neuroblastoma cells were used to analyze the effect of elevated glucose levels on myo-inositol metabolism and Na+/K+-pump activity. The activity of the Na+/K+ pump in neuroblastoma cells is almost totally sensitive to ouabain inhibition. Culturing neuroblastoma cells in 30 mM glucose caused a significant decrease in Na+/K+-pump activity, myo-inositol metabolism, and myo-inositol content, compared to cells grown in the presence of 30 mM fructose. Glucose supplementation also caused a large intracellular accumulation of sorbitol. The aldose reductase inhibitor sorbinil prevented the abnormalities in myo-inositol metabolism and partially restored Na+/K+-pump activity in neuroblastoma cells cultured in the presence of elevated glucose levels. These results suggest that the accumulation of sorbitol by neuroblastoma cells exposed to elevated concentrations of extracellular glucose causes a decrease in myo-inositol metabolism and these abnormalities are associated with a reduction in Na+/K+-pump activity.     

Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.     

Ascorbic Acid Uptake by a High-Affinity Sodium-Dependent Mechanism in Cultured Rat Astrocytes

Ascorbic acid (vitamin C) is synthesized in rodent liver, circulates in the blood, and is concentrated in the brain. Experiments were performed to characterize the mechanism of ascorbate uptake by rat cerebral astrocytes in primary culture. Astroglial uptake of L-[14C]ascorbate was observed to be both saturable and stereoselective. In addition, uptake was dependent on both the incubation temperature and the concentration of Na+ because it was largely inhibited by cooling to 4 degrees C, by treatment with ouabain to increase intracellular Na+, and by the substitution of K+, Li+, or N-methyl-D-glucamine for extracellular Na+. The affinity for ascorbate was relatively high in cells incubated with a physiological concentration of extracellular Na+, because the apparent Km was 32 microM in 138 mM Na+. However, the affinity for ascorbate was significantly decreased when the extracellular Na+ concentration was lowered. Treatment of astrocytes with dibutyryl cyclic AMP induced stellation and increased the maximum rate of ascorbate uptake by 53%. We conclude that astrocytes possess a stereoselective, high-affinity, and Na+-dependent uptake system for ascorbate. This system may regulate the cerebral ascorbate concentration and consequently modulate neuronal function.     

Oxidative,Metabolic, and Apoptotic Responses of Schwann Cells to High Glucose Levels

The specific response of murine Schwann cells IMS32 to acute and chronic hyperglycemia conditions was evaluated. The pathophysiological alterations were studied to deepening the role of Schwann cells in diabetes‐related neurotoxicity and to assess a model to screen new protective molecules. IMS32 were incubated with 30 and 56 mM glucose for 48 h and 7 and 14 days, and markers of oxidative stress, apoptosis, and polyol pathway were evaluated. High glucose induced O²‐production and lipid peroxidation at all time point whereas Caspase 3 activity was induced only after 14 days. Aldose reductase activity and expression were significantly increased after 48 h and 14 days, respectively. Our results describe the response of Schwann cells to high glucose conditions and suggest the use of IMS32 for the screening of protective molecules in diabetes‐induced neuropathy.     

Stimulatory Effect of Ascorbic Acid on Norepinephrine Biosynthesis in Digitonin-Permeabilized Adrenal Medullary Chromaffin Cells

The regulatory role of ascorbic acid in norepinephrine biosynthesis was studied using digitonin-permeabilized chromaffin cells. When permeabilized chromaffin cells were incubated with [3H]3,4-dihydroxyphenylethylamine ([3H]dopamine) in calcium-free medium, the amounts of radioactive dopamine and norepinephrine measured in the cell fraction were increased as a function of incubation time and dopamine concentration. Both the accumulation of dopamine and the formation of norepinephrine were shown to require the presence of Mg-ATP in the medium. These results indicate that the permeabilization of chromaffin cells by digitonin treatment does not disrupt the functions of chromaffin granules, including dopamine uptake, norepinephrine formation, and storage of these amines. Using this permeabilized cell system, the effect of ascorbic acid on the rates of dopamine uptake and hydroxylation was investigated. The formation of norepinephrine was stimulated by ascorbic acid at concentrations of 0.5-2 mM in the presence of Mg-ATP. By contrast, dopamine uptake was not affected by the presence or absence of ascorbic acid in the medium. These findings provide evidence that ascorbic acid may stimulate the conversion of dopamine to norepinephrine by increasing dopamine beta monooxygenase activity rather than by increasing the substrate supply of dopamine. These observations also suggest that the rate of norepinephrine biosynthesis in adrenal medullary cells may be regulated by the concentration of ascorbic acid within the cell cytoplasm.     

葡萄糖浓度波动对原代培养的大鼠血管细胞和肾细胞的影响

探讨葡萄糖浓度波动对体外培养的原代大鼠血管细胞和肾细胞的影响。取SD大鼠的主动脉和肾脏进行血管细胞和肾细胞的体外原代培养,每种细胞均分为6组:正常对照组、持续高糖组、持续低糖组、波动组I、波动组II、波动组III。实验24h后,测定细胞乳酸脱氢酶(LDH)的泄漏率,细胞液中的β-N-乙酰氨基葡萄糖苷酶(NAG)的活力,细胞内还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的活力。结果表明葡萄糖浓度波动各组均能对大鼠血管细胞和肾细胞造成损伤,使细胞外液LDH、NAG的泄漏量明显增加,细胞内GSH、SOD活力明显减少,与持续高糖组和持续低糖组比较差异显著(P<0.001)。且葡萄糖浓度波动对肾细胞的损伤比血管细胞更为明显。说明葡萄糖浓度波动能够导致大鼠血管细胞和肾细胞的损伤,并且其损伤远远大于持续高糖或持续低糖的单独作用效果,损伤的结果与低糖作用细胞的时间呈正相关,在相同的损伤条件下肾细胞比血管细胞对葡萄糖浓度波动更为敏感,损伤更为严重。     

高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响

目的:探讨高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响及作用机制。方法:SD大鼠胰岛细胞原代培养,不同浓度的葡萄糖处理后,采用MTT比色法检测细胞存活率,Hoechst-PI染色观察细胞凋亡,电镜观察细胞超微结构改变,RT-PCR方法检测Bax及Bcl-2相关基因mRNA表达水平。结果:5.5 mmol·L~(-1),11.1 mmol·L~(-1)和22.2 mmol·L~(-1)葡萄糖处理后,胰岛细胞活性分别下降到78.08%±2.29%、58.39%±3.13%和36.05%±2.63%,与阴性对照组比较差异具有统计学意义(P0.05);Hoechst-PI染色结果显示随着葡萄糖作用浓度的增加,凋亡细胞数量也增加;RT-PCR显示胰岛细胞经不同浓度葡萄糖处理后,Bax mRNA的表达明显上调,Bcl-2 mRNA表达下调(P0.05);电镜观察结果显示随着葡萄糖作用浓度的增加,胰岛细胞超微结构损害程度依次加重。结论:高浓度葡萄糖能明显引起胰岛细胞活性的下降,诱导凋亡反应的发生,凋亡机制与Bcl-2家族蛋白相关。     

Depletion of Phospholipid Arachidonoyl-Containing Molecular Species in a Human Schwann Cell Line Grown in Elevated Glucose and Their Restoration by an Aldose Reductase Inhibitor

Abstract: In experimental diabetic neuropathy, defective arachidonic acid metabolism characterized by a decrease in the proportion of glycerophospholipid arachidonoyl-containing molecular species (ACMS) occurs and has been implicated in the pathogenesis of the disorder. In this study, we evaluated the suitability of a tumor-derived human Schwann cell line (NF1T) as a model to investigate the mechanism underlying the loss of ACMS. NF1T cells grown in 30 versus 5.5 m M glucose undergo a marked reduction in ACMS in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, in a manner resembling that of diabetic nerve. The depletion of ACMS can be reversed on transferring the cells from 30 m M glucose to medium containing physiological levels of glucose. Cells maintained in 5.5 m M glucose plus 25 m M mannitol or sorbitol did not exhibit decreased ACMS levels, indicating that osmotic effects were not responsible for ACMS depletion. However, growth in 25 m M fructose elicited a reduction of ACMS similar to that produced by 30 m M glucose. Excessive glucose flux through the polyol pathway has been implicated in the neural and vascular abnormalities associated with diabetes. Therefore, we examined the effects of polyol pathway inhibitors, including two aldose reductase inhibitors, zopolrestat and sorbinil, and a sorbitol dehydrogenase inhibitor (SDI), CP166,572, on ACMS levels in NF1T cells cultured in elevated glucose concentrations. At 200 µ M , zopolrestat fully and sorbinil partially corrected ACMS depletion. The SDI at concentrations up to 100 µ M failed to affect diminished ACMS levels. Neither zopolrestat nor the SDI restored ACMS levels reduced in the presence of elevated fructose concentrations. These findings suggest that enhanced flux through the polyol pathway and, in particular, elevated aldose reductase activity may play a significant role in the reduction of ACMS levels in the cells brought about by elevated glucose levels.     

Oleic Acid Inhibits Gap Junction Permeability and Increases Glucose Uptake in Cultured Rat Astrocytes

Abstract: The role of oleic acid in the modulation of gap junction permeability was studied in cultured rat astrocytes by the scrape-loading/Lucifer yellow transfer technique. Incubation with oleic acid caused a dose-dependent inhibition of gap junction permeability by 79.5% at 50 µ M , and no further inhibition was observed by increasing the oleic acid concentration to 100 µ M . The oleic acid-mediated inhibition of gap junction permeability was reversible and was prevented by bovine serum albumin. The potency of oleic acid-related compounds in inhibiting gap junction permeability was arachidonic acid > oleic acid > oleyl alcohol > palmitoleic acid > stearic acid > octanol > caprylic acid > palmitic acid > methyloleyl ester. Oleic acid and arachidonic acid, but not methyloleyl ester, increased glucose uptake by astrocytes. Neither oleic acid nor arachidonic acid increased glucose uptake in the poorly coupled glioma C6 cells. These results support that the inhibition of gap junction permeability is associated with the increase in glucose uptake. We suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication.     

Developmental regulation of insulin receptor gene in sciatic nerves and role of insulin on glycoprotein P0 in the Schwann cells

In view of the observations that Schwann cells contain insulin receptors, in the present study, we have investigated the developmental regulation of insulin receptor gene in the sciatic nerves of different postnatal age group rats. We have also investigated the role of insulin in the expression of the major PNS myelin glycoprotein P zero (P0) in normal as well as high glucose conditions in primary rat Schwann cells. The expression of insulin receptor gene in sciatic nerves appeared to be differentially regulated. The steady-state levels of insulin receptor mRNA increased remarkably during development and after postnatal day 10, when the peak of myelin structural gene (P0) expression occur and slowly increased further until at least postnatal day 90 in parallel with the growth of the myelin sheath. By employing immunofluorescence and RT-PCR, we observed significant increase in the P0 protein and mRNA levels in Schwann cells in response to the insulin than in insulin deprived counterparts. The presence of insulin in the high glucose medium ameliorated the altered protein and mRNA of P0 in Schwann cells compared to the insulin deprived counterparts. These studies demonstrate the importance of insulin and its receptor as possible regulatory factors in the PNS and also emphasizes their novel therapeutic applications in demyelinating diseases, especially in diabetic poly-neuropathy.     

Regulation by Thyroid Hormones of Glucose Transport in Cultured Rat Myotubes

Primary cultures of skeletal muscle obtained from neonatal rats possess a saturable process for active glucose uptake, the myotubes having a relatively high affinity for the substrate with a Km of 1 mM. The expression of the glucose transport system was most apparent after fusion of single myoblasts to multinucleated myotubes [3-4 days in vitro (DIV)], at which time glucose uptake increased sharply to reach plateau values at about 6-8 DIV. Treatment of the cells at age 6 DIV with triiodothyronine or thyroxine caused a marked increase in glucose uptake beginning 4 h after treatment and reaching a maximum at 24 h. Thyroid hormone-induced increase in glucose uptake was not reduced by either tetrodotoxin or verapamil, thus indicating that the effect was not secondary to the ability of the hormone to increase contractile activity. The effect of thyroid hormones was eliminated completely by inhibition of protein synthesis. The results indicate that thyroid hormones play an important role in regulation of glucose transport in skeletal muscle.     

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.