Acute and 3-month effects of calcium carbonate on the calcification propensity of serum and regulators of vascular calcification: secondary analysis of a randomized controlled trial.

SUMMARY: Calcium supplements have been associated with increased cardiovascular risk, but the mechanism is unknown. We investigated the effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50). Changes in serum calcium were related to changes in T50. INTRODUCTION: Calcium supplements have been associated with increased cardiovascular risk; however, it is unknown whether this is related to an increase in vascular calcification. METHODS: We investigated the acute and 3-month effects of calcium supplements on the propensity of serum to calcify, based on the transition time of primary to secondary calciprotein particles (T50), and on three possible regulators of calcification: fetuin-A, pyrophosphate and fibroblast growth factor-23 (FGF23). We randomized 41 postmenopausal women to 1 g/day of calcium as carbonate, or to a placebo containing no calcium. Measurements were performed at baseline and then 4 and 8 h after their first dose, and after 3 months of supplementation. Fetuin-A, pyrophosphate and FGF23 were measured in the first 10 participants allocated to calcium carbonate and placebo who completed the study. RESULTS: T50 declined in both groups, the changes tending to be greater in the calcium group. Pyrophosphate declined from baseline in the placebo group at 4 h and was different from the calcium group at this time point (p = 0.04). There were no other significant between-groups differences. The changes in serum total calcium from baseline were significantly related to changes in T50 at 4 h (r = -0.32, p = 0.05) and 8 h (r = -0.39, p = 0.01), to fetuin-A at 3 months (r = 0.57, p = 0.01) and to pyrophosphate at 4 h (r = 0.61, p = 0.02). CONCLUSIONS: These correlative findings suggest that serum calcium concentrations modulate the propensity of serum to calcify (T50), and possibly produce counter-regulatory changes in pyrophosphate and fetuin-A. This provides a possible mechanism by which calcium supplements might influence vascular calcification.

Upregulation of alkaline phosphatase and pyrophosphate hydrolysis: potential mechanism for uremic vascular calcification.

Pyrophosphate is a potent inhibitor of medial vascular calcification where its level is controlled by hydrolysis via a tissue-nonspecific alkaline phosphatase (TNAP). We sought to determine if increased TNAP activity could explain the pyrophosphate deficiency and vascular calcification seen in renal failure. TNAP activity increased twofold in intact aortas and in aortic homogenates from rats made uremic by feeding adenine or by 5/6 nephrectomy. Immunoblotting showed an increase in protein abundance but there was no increase in TNAP mRNA assessed by quantitative polymerase chain reaction. Hydrolysis of pyrophosphate by rat aortic rings was inhibited about half by the nonspecific alkaline phosphatase inhibitor levamisole and was reduced about half in aortas from mice lacking TNAP. Hydrolysis was increased in aortic rings from uremic rats and all of this increase was inhibited by levamisole. An increase in TNAP activity and pyrophosphate hydrolysis also occurred when aortic rings from normal rats were incubated with uremic rat plasma. These results suggest that a circulating factor causes pyrophosphate deficiency by regulating TNAP activity and that vascular calcification in renal failure may result from the action of this factor. If proven by future studies, this mechanism will identify alkaline phosphatase as a potential therapeutic target.

Furosemide-sensitive Na+ and K+ transport and human erythrocyte volume.

The relationship between cation transport and cell volume in human erythrocytes was investigated by measuring ouabain-sensitive K+ influx, ouabain-resistant, furosemide-sensitive K+ influx, and ouabain + furosemide-resistant K+ influx, and maximal ouabain binding in microcytic, normocytic and macrocytic red cells. A significant correlation was found between the mean corpuscular volume and furosemide-sensitive K+ influx normalized either to cell number (r = 0.636, P less than 0.001) or to cell volume (r = 0.488, P less than 0.001). No relationship was seen between mean corpuscular volume and ouabain-sensitive K+ influx, and the number of ouabain-binding sites per cell was only weakly correlated with mean corpuscular volume (r = 0.337, P less than 0.05). A slight, negative relationship existed between mean corpuscular volume and ouabain + furosemide-resistant K+ influx expressed per volume of cells (r = -0.359, P less than 0.01), and an apparent relationship between furosemide-sensitive K+ influx and mean corpuscular hemoglobin concentration (r = 0.446, P less than 0.01) disappeared when microcytic samples were excluded from analysis. Furosemide-sensitive transport, including Na+ influx and K+ and Na+ efflux, was completely absent in microcytic cells from one patient with alpha-thalassemia minor. In addition, these cells exhibited a furosemide-resistant, Cl(-)-dependent K+ influx. Exposure of normal erythrocytes to hypotonic conditions (196 mosM) increased furosemide-sensitive K+ influx by a mean of 45% (P less than 0.05), while exposure to hypertonic conditions (386 mosM) had no significant effect. The results indicate that furosemide-sensitive transport and cell volume are interrelated in human erythrocytes. However, the inability to fully recreate this relationship with in vitro manipulation of cell volume suggest that this relationship is established prior to red cell maturation.

Evidence for the role of phospholipids in follicle-stimulating hormone binding to membrane-bound and soluble receptors from calf testis.

The role of phospholipids in the interaction of FSH with receptors in the calf testis was explored by studying the effects of phospholipase-C (PL-C) on the binding of radioiodinated human FSH ([125I]iodo-hFSH) to three previously characterized but different preparations of FSH receptor: a membrane fraction derived from calf testis homogenate, a buffer-soluble receptor present in the cytosol of the calf testis homogenate, and a detergent-soluble receptor prepared from the membrane fraction by extraction with Triton X-100. Prior incubation with PL-C markedly reduced specific [125I]iodo-hFSH binding to the membrane-bound and buffer-soluble receptors. This loss in binding was associated with hydrolysis of phospholipids, was prevented by the addition of o-phenanthroline, an inhibitor of PL-C, but not by the addition of a protease inhibitor, and could not be reproduced by the addition of the products of PL-C hydrolysis. Enzyme treatment did not dissociate [125I]iodo-hFSH already bound to the buffer-soluble receptor and dissociated only 20% of [125I]iodo-hFSH bound to membrane receptor. PL-C treatment did not reduce [125I]iodo-hFSH binding to detergent-solubilized receptor, nor did it hydrolyze constituent phospholipids. The apparent resistance of the detergent-solubilized receptor to PL-C treatment was studied by incubating membranes with or without PL-C before detergent solubilization. Triton X-100 itself (2%) inhibited phospholipid hydrolysis by PL-C, but this could be overcome by reducing the detergent concentration 10-fold by dilution. Despite a marked decrease in specific [125I]iodo-hFSH binding to PL-C-treated membranes compared to that of untreated controls, the total amount of [125I]iodo-hFSH binding to Triton X-100 extracts of each membrane preparations was not significantly different. Addition of Triton X-100 to the buffer-soluble receptor restored 40% of the hormone binding that had initially been lost concomitant with enzymic hydrolysis of membrane phospholipids. It appears that constituent phospholipids play an important role in the interaction of FSH with membrane-bound or solubilized receptor and that Triton X-100 is able to substitute, although imperfectly, for phospholipids in that regard, probably because of its related amphipathic properties.

Sonographic evaluation of renal failure.

Sonography is a critical component of the evaluation of both acute and chronic renal failure; however, most nephrologists have a limited knowledge of this procedure. The acoustic properties, limited spectrum of pathological changes, and ease of visualization of the kidneys, coupled with the safety, simplicity, and low cost of sonography, make it the modality of choice for renal imaging. This review discusses the basics of sonography as they apply to the kidney and describes the findings encountered in the more common causes of renal failure. Although many sonographic findings are nonspecific, their diagnostic use is greatly enhanced by a familiarity with the clinical presentation and a thorough understanding of renal pathophysiological characteristics. Therefore, nephrologists should be knowledgeable about renal sonography and participate in its interpretation.

How echogenic is echogenic? Quantitative acoustics of the renal cortex.

The echogenicity of the cortex is an important parameter in interpreting renal sonograms that suggest changes in cortical structure. Echogenicity is currently measured qualitatively, and no attempts have been made at quantification. We developed a method to quantify renal cortical echogenicity in reference to the liver and evaluated its reproducibility, dependence on scanning variables, and potential utility. Sonograms of the right kidney were digitized, and the mean pixel density of regions of the renal cortex and liver was measured and normalized to the gray scale. Echogenicity was expressed as the ratio of the brightness (inverse of mean pixel density) of the cortex to that of the liver. The mean coefficient of variation among measurements performed on multiple sonograms from the same study was 2.8%, and the coefficient of variation among multiple measurements performed on the same kidney over 1 year was 1.8%. The correlation between measurements obtained by two different individuals on identical images was 0.92, with a mean variation of 3.0%. Echogenicity was not significantly affected by type of scanner or probe frequency, but varied inversely with gain. However, the effect of gain was very small within the useful range. Water loading after an overnight fast increased echogenicity in all cases, with a mean increase of 6.4%. Echogenicity of normal kidneys was significantly less than that of the liver (range, 0.810 to 0.987), and in clinical sonograms analyzed retrospectively but blindly, echogenicity correlated with the qualitative gradations of echogenicity originally assigned. The most echogenic kidneys were 62% brighter than normal kidneys, many times greater than the variability of the measurement. We conclude that quantification of renal cortical echogenicity is feasible and reproducible and may be useful in detecting and following renal disease. Echogenicity of the renal cortex is less than that of the liver in healthy subjects and is influenced by the state of diuresis.

Biochemical properties of the testicular follitropin-receptor system.

The interaction of FSH with membrane receptors from rat and calf testis has been studied in some detail. The FSH receptor has been solubilized through use of the nonionic detergent Triton X-100 and highly purified by affinity chromatography on Affigel-10 coupled to ovine FSH. Hormone binding activity of the solubilized receptor has been preserved for extended periods through use of the structure-stabilizing agent glycerol. Other components of the FSH testes receptor system including the guanyl nucleotide binding protein and adenylate cyclase have been solubilized by nonionic detergents and also found to be stabilized by glycerol. FSH binding activity has been observed in testes cytosol and represents a putative class of receptors prepared from testes in the absence of detergent. The concentration of this buffer-soluble component decreased with age and increased concomitantly with loss of membrane receptors consequent to their down-regulation after administration of exogenous FSH. Phospholipids seem involved in the interaction of FSH with membrane-bound, detergent-solubilized, and buffer-soluble FSH binding activity. Phospholipids may maintain or stabilize a particular receptor conformation necessary for interaction with the hormone. A specific role for GTP seems indicated in regulation of FSH-stimulated adenylate cyclase activity in immature rat testis. Follitropin binding to testes receptor appears modulated by a variety of factors present in serum, testes extracts, follicular fluid, and seminal plasma, which are poorly understood at present. Inhibition of FSH binding by seminal plasma best-fit by a model proposing two hormone binding sites per receptor molecule, where binding to one site decreases the affinity of the other site for FSH. As a result of studies in this and other laboratories, the molecular endocrinology of FSH interaction with testis receptors is becoming increasingly understood.

The role of pump number and intracellular sodium and potassium in determining Na,K pump activity in human erythrocytes.

The factors that determine the activity of the Na,K pump in vivo were investigated by measuring Na,K pump activity under in vivo conditions in human red cells and relating it to the intracellular content of sodium ([Na]i) and potassium ([K]i) and the number of pump units per cell (pump number). Na,K pump activity was measured as ouabain-sensitive K+ influx, pump number was determined from the maximal binding of 3H-ouabain to intact cells, and [Na]i and [K]i were measured by atomic absorption spectrophotometry in washed, packed cells. In the 81 samples studied, pump activity per cell was significantly correlated with pump number (r = .64, P less than 0.001), but was negatively correlated with [Na]i (r = -.28, P less than 0.02) and was not correlated with [K]i. An inverse relationship was found between pump number and [Na]i. When pump activity was expressed as activity per pump unit, rather than per cell, a significant relationship was seen between pump activity and [Na]i (r = .50, P less than 0.001), and a negative correlation existed between the activity per pump unit and [K]i (r = -.29, P less than 0.01). The effect of intracellular Na+ at physiologic levels on pump activity was not strong, with the activity per pump unit increasing only 25% with a doubling of [Na]i. These results indicate that pump number is the major determinant of pump activity in human red cells in vivo, while [Na]i and [K]i are of secondary importance.(ABSTRACT TRUNCATED AT 250 WORDS)

The fallacy of the calcium-phosphorus product.

Scattered through the practice of medicine are dogmas with little or no scientific basis. One of these is the product of the serum calcium and phosphorus concentrations, the so-called calcium-phosphorus product or Ca x P. The assumption that ectopic calcification will occur when the product of the serum calcium and phosphorus concentrations exceeds a particular threshold has become standard practice in nephrology even though there is little scientific basis. Experimental support is lacking, the chemistry underlying the use of the product is oversimplified and the concept that ectopic calcification is simply the result of supersaturation is biologically flawed. The evidence that the Ca x P is an independent risk factor for mortality and morbidity is also questionable. Although ectopic calcification can occur in many sites, this review will focus on vascular calcification, as it is the most common site and the site most likely to affect patient outcomes.

Vascular calcification: not so crystal clear.

Patients with renal failure are predisposed to calcification of the medial layer of arteries. This calcification is far more complex than simple precipitation of calcium and phosphate and involves multiple forms of calcium phosphate. Like bone, calcification in the vessels also involves biologic events. The two are necessarily linked and unraveling the pathophysiology will require an understanding of both.

Chemical and hormonal determinants of vascular calcification in vitro.

Vascular calcification is a complex process that is dependent not only on the physicochemical effects of Ca, PO(4), and pH, but also on smooth muscle factors that may be regulated by these ions as well as by 1,25-dihydroxyvitamin D(3) (calcitriol) and parathyroid hormone (PTH). These minerals and hormones were tested in a model of medial calcification in rat aorta maintained in culture for 9 days. Calcification was quantitated as incorporation of (45)Ca, alkaline phosphatase activity was measured in aortic homogenates, and osteopontin production was measured from immunoblots of culture medium. At 1.8 mM Ca (1.46 mM free), calcification occurred at or above 2.8 mM PO(4). At 3.8 mM PO(4), calcification occurred at or above 1.10 mM free [Ca]. At a constant [Ca] x [PO(4)], calcification varied directly with [Ca] and inversely with [PO(4)]. Calcification was directly related to pH between 7.19 and 7.50 but not altered by PTH or calcitriol. Alkaline phosphatase activity and osteopontin production were increased by Ca, PO(4), calcitriol, and PTH. We conclude that calcification of rat aorta in vitro requires elevation of both [Ca] and [PO(4)], and that [Ca] rather than [PO(4)] or the product of the two is the dominant determinant. The induction of alkaline phosphatase and osteopontin indicates that Ca and PO(4) have effects in addition to simple physicochemical actions. Although PTH and calcitriol did not increase calcification in vivo, they have effects on smooth muscle that could influence calcification in vivo. Calcification is enhanced by alkalinity within the range produced during hemodialysis.

Volume-sensitive Cl-dependent K transport in human erythrocytes.

Passive K fluxes, measured with 86Rb, were investigated in osmotically swollen human erythrocytes. K influx and efflux increased progressively with increased hypotonicity up to 167 mosmol/kg. No increase in K flux was seen when NO3 or methylSO4 were substituted for Cl. Substitution of choline or N-methylglucamine for external Na reduced the K flux in swollen cells by only 22%, compared with a 60% reduction in euvolumic cells. However, the magnitude of this Na-dependent component was slightly, but significantly, higher in swollen cells. The presence of Na-dependent K influx in swollen cells was confirmed by measurements of Na influx demonstrating a K-dependent Na influx of similar magnitude in isovolumic and swollen cells. The volume-sensitive K flux was inhibited by bumetanide, but significantly less so than was Cl-dependent flux in isovolumic cells (half-maximal inhibition at 1.0 X 10(-4) vs. 5.8 X 10(-7) M). Kinetic analysis revealed that Cl-dependent K influx had a lower affinity for external K in swollen cells than in euvolumic cells (Km was 29.8 vs. 6.1 mM). The increased K flux in swollen cells was found to be transient, decreasing substantially and reverting back to a predominantly Na-dependent and more bumetanide-sensitive form after 2 h. The results indicate that swelling of human erythrocytes activates a transient Cl-dependent K flux that differs significantly from that in isovolumic cells in that it is less Na dependent, less sensitive to bumetanide, and has a lower affinity for K. Na-K cotransport is either unaffected or slightly increased in swollen cells. The altered flux in swollen cells would thermodynamically favor a volume-regulatory KCl efflux.

Flow-mediated NO release from endothelial cells is independent of K+ channel activation or intracellular Ca2+.

The role of K+ channels and intracellular [Ca2+] in flow-induced nitric oxide (NO) production was investigated in bovine aortic endothelial cells in culture. NO release (measured as nitrite production) and K+ channel activity (measured as 86Rb+ efflux) were measured in cells grown on collagen-coated microcarrier beads and perfused in a column. An eightfold increase in flow produced a rapid (within 1 min), sustained, and reversible sixfold increase in NO release. Efflux of 86Rb+ also increased but rapidly returned to baseline and then transiently decreased when flow was decreased. This was probably due to boundary layer washout rather than to K+ channel activation, because an identical pattern was seen for release of [3H]ouabain. Neither tetraethylammonium nor increasing medium [K+] to block K+ currents prevented flow-induced NO release. Removal of medium Ca2+ or chelation of intracellular Ca2+ also did not block flow-mediated NO release. The results demonstrate that flow rapidly increases NO release from endothelial cells but that this increase in NO release is not dependent on activation of K+ channels or changes in intracellular [Ca2+].

Swelling-activated K-Cl cotransport: metabolic dependence and inhibition by vanadate and fluoride.

Activation of K-Cl cotransport by cell swelling was studied by measuring K influx in isotonic and hypotonic media in human red blood cells after depletion of cellular ATP and after exposure to vanadate or fluoride. Preincubation of red blood cells with 2-deoxyglucose resulted in an inhibition of swelling-activated K-Cl cotransport that paralleled the decline in cellular ATP. Subsequent repletion of ATP by incubation in glucose, phosphate, and guanosine partially restored swelling-activated K-Cl cotransport. Swelling-activated K-Cl cotransport was also inhibited by 200 microM vanadate. This inhibition was partially blocked by DIDS, indicating an intracellular action, and required a 40-min preincubation, suggesting that inhibition was due to vanadyl rather than vanadate. Activation of K-Cl cotransport in swollen cells was also blocked by 16 mM fluoride, an effect that was immediate and independent of Cl concentration. Incubation of cells with 1 mM adenosine 3',5'-cyclic monophosphate (cAMP) to raise intracellular cAMP levels did not inhibit swelling-activated K-Cl cotransport, indicating that fluoride was not acting through adenyl cyclase. No inhibition of Cl-dependent or bumetanide-sensitive K influx in isotonic medium (Na-K-2Cl cotransport) was observed with ATP depletion, vanadate, fluoride, or cAMP. Activation of K-Cl cotransport by N-ethylmaleimide (NEM) was inhibited by ATP depletion but only partially inhibited by fluoride and not inhibited by vanadate. Fluoride inhibited K-Cl cotransport only when added before NEM treatment. These results suggest that activation of K-Cl cotransport by cell swelling requires ATP and involves a phosphohydrolase or phosphotransferase reaction that is inhibited by vanadyl and fluoride.(ABSTRACT TRUNCATED AT 250 WORDS)

Cl-dependent K transport in a pure population of volume-regulating human erythrocytes.

Swelling of human red cells activates a putative K-Cl cotransport that is not present at normal cell volume and that disappears after several hours. To determine whether regulatory volume decrease (RVD) is occurring in human erythrocytes and is responsible for the inactivation of K-Cl cotransport, the relationship between cell volume and the inactivation and reactivation of volume-sensitive (VS) K-Cl cotransport was studied. VS K influx into high K cells was transient, whereas influx into low K cells (prepared with nystatin), which are unable to shrink via K efflux, remained fully activated. Likewise, VS K efflux into hypotonic medium disappeared after 100 min in a low K medium but remained activated in a high K medium that prevented cell shrinkage. Cells that had been preincubated in hypotonic medium to inactivate VS K-Cl cotransport showed no significant recovery of VS cotransport after a 6-h incubation in isotonic medium but showed full restoration of VS cotransport after treatment with nystatin in isotonic medium to reequilibrate cell water. A pure fraction of volume-regulating (VR) cells was subsequently isolated by preincubating red cells in hypotonic medium and then subjecting them to further hypotonicity to lyse all non-VR cells. The 2.5% of cells that remained consisted of 16% reticulocytes and exhibited a Cl-dependent RVD in hypotonic medium. VS K-Cl cotransport was enriched 10-fold and Na-K-Cl cotransport was enriched 12-fold in these cells, whereas the enrichment of N-ethylmaleimide (NEM)-activated K-Cl cotransport was only threefold.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological significance of volume-regulatory transporters.

Research over the past 25 years has identified specific ion transporters and channels that are activated by acute changes in cell volume and that serve to restore steady-state volume. The mechanism by which cells sense changes in cell volume and activate the appropriate transporters remains a mystery, but recent studies are providing important clues. A curious aspect of volume regulation in mammalian cells is that it is often absent or incomplete in anisosmotic media, whereas complete volume regulation is observed with isosmotic shrinkage and swelling. The basis for this may lie in an important role of intracellular Cl- in controlling volume-regulatory transporters. This is physiologically relevant, since the principal threat to cell volume in vivo is not changes in extracellular osmolarity but rather changes in the cellular content of osmotically active molecules. Volume-regulatory transporters are also closely linked to cell growth and metabolism, producing requisite changes in cell volume that may also signal subsequent growth and metabolic events. Thus, despite the relatively constant osmolarity in mammals, volume-regulatory transporters have important roles in mammalian physiology.

Volume-sensitive, Cl-dependent K transport in resealed human erythrocyte ghosts.

Potassium influx and efflux in Cl and NO3 media were measured in resealed ghosts prepared from human red cells. Cl-dependent K influx was three times that in intact cells and, as in intact cells, was partially supported by Br but not by thiocyanate (SCN). In other properties, this flux differed from that in intact cells: substitution of N-methylglucamine for Na did not decrease but rather increased Cl-dependent K influx, the affinity for external K was reduced, with a Km of 21.3 +/- 12.5 mM, and inhibition by furosemide and bumetanide was incomplete. Furosemide at 1 mM inhibited Cl-dependent influx by 26 and 51% at 4 and 20 mM K, respectively. Bumetanide inhibited Cl-dependent K influx by 0 and 55% at concentrations of 10 microM and 1 mM, respectively, in 4 mM K, with no further inhibition at 20 mM K. Neither the magnitude nor the properties of the flux were altered by preparing ghosts in the presence of 1,4-dithiothreitol, indicating that sulfhydryl oxidation was not responsible for the altered flux in ghosts. Treatment with N-ethylmaleimide (NEM) either before or after ghost preparation did not increase Cl-dependent K influx. However, Cl-dependent influx in ghosts could be augmented by increasing ghost volume or ATP content. Resealed human erythrocyte ghosts thus exhibit a volume- and ATP-sensitive, Cl-dependent K flux that differs substantially from the putative Na-K-Cl cotransport in intact cells in that it is independent of Na, is relatively resistant to furosemide and bumetanide, and has a low affinity for K.(ABSTRACT TRUNCATED AT 250 WORDS)

Bedside renal biopsy: ultrasound guidance by the nephrologist.

The safety and efficacy of percutaneous biopsy of native kidneys performed entirely by nephrologists at the patient's bedside was evaluated in 101 consecutive patients. The location and depth of the kidney were determined with a portable ultrasound machine, and biopsy was performed with a 15G, automatic, spring-loaded biopsy device without direct ultrasonographic guidance. Renal tissue was obtained in 99 patients, and all samples were adequate for diagnosis, with an average of 33 glomeruli and more than 10 glomeruli in 97%. The number of biopsy attempts was four or fewer in 80% of patients. Three patients developed symptomatic bleeding, all of whom had a risk factor for bleeding, but none required procedures to control the bleeding. Asymptomatic hematuria occurred in two other patients. Overall, the mean decrease in hematocrit was 1.5, with a decrease of 5.0 or greater in six patients. The results are similar to those of previous studies using automatic devices but under direct ultrasound guidance. A subset of 20 patients with abnormal platelet counts, coagulation times, or bleeding times accounted for four of the five patients with complications. We conclude that percutaneous biopsy of native kidneys can be adequately and safely performed in its entirety by nephrologists at the patient's bedside. Furthermore, excellent results can be obtained without direct sonographic guidance. Hemorrhage occurs almost exclusively in those patients with abnormal platelet counts, coagulation times, or bleeding times.

Isolation and partial characterization of an acetylcholine receptor-enriched membrane fraction from skeletal muscle.

A procedure for purification of the bungarotoxin-binding fraction of sarcolemma from rabbit skeletal muscle is described. Muscle is homogenized in 0.25M sucrose without high salt extraction and membrane fractions separated initially by differential centrifugation procedures. An ultracentrifugation pellet enriched in cell surface and sarcoplasmic reticulum markers is further fractionated on a dextran gradient (density = 1.0 to 1.09). Two fractions are identified as sarcolemma according to high specific activities for lactoperoxidase-iodination, Na+, K+-ATPase and alpha-bungarotoxin-binding. No Ca++, Mg++-ATPase activity is found in these fractions. A third fraction, the dextran gradient pellet, is enriched in Ca++, Mg++-ATPase activity and lactoperoxidase iodinatable material and characterized by low bungarotoxin binding. This fraction represents a mixture of sarcoplasmic reticulum and transverse tubules with some sarcolemma contamination.