Plasma Membrane Of Muscle Cells Research Articles

SUCCESSFUL MYOBLAST ALLOTRANSPLANTATION IN MDX MICE USING RAPAMYCIN

Duchenne muscular dystrophy (DMD)* is due to a mutation occurring on a gene located on the X chromosome that normally encodes for the expression of a large, 420-kDa protein called dystrophin (Dys) (1). This protein, which is normally located along the inner part of the plasma membrane of skeletal muscle cells, is absent from DMD patient muscles (2). The lack of Dys is responsible for the absence of Dys-associated proteins and glycoproteins. The absence of this complex would be responsible for the progressive degeneration of skeletal muscles in DMD patients (3). A point mutation in the Dys gene leads to the expression of a truncated protein in the mdx mouse, considered as an animal model of human DMD (4). Normal myoblast transplantation restores Dys expression in the grafted muscles of mdx mice. Indeed, normal myoblasts contain a normal Dys gene, and when they fuse together or with the host myoblasts, nuclei from these normal myoblasts restore Dys expression (5, 6). Normal myoblast transplantation had only limited success in human. Some groups demonstrated, a short time after grafting, the presence of Dys+ fibers in some patients (7–10). Moreover, transient strength increases have been measured in a few patients (10). In some cases, hosts produced antibodies (Ab) against the myoblasts and/or myotubes of the donor, even when donor and recipient were fully compatible for class I and class II DR MHC (7, 10). Some Abs were able to induce a complement-dependent lysis of myotubes, and some were also directed against the donor Dys (10). Such immune reactions have also been demonstrated in mdx mice transplanted either with human or rat (xenografts) or mouse (allografts) myoblasts (11, 12). High doses of CsA apparently diminish or suppress in some animals this humoral reaction (12, 13). Thus, controlling the immune system seems to be a crucial step to performing successful myoblast transplantations. CsA is not very efficient for myoblast transplantation in mice. It is toxic and may not be used for long-term treatments. Therefore, the new immunosuppressant, rapamycin (RAPA), has been tested to evaluate its potential to reduce immune reactions and allow myoblast graft success. RAPA is a fungal, FK506 structurally related macrolid (14). Rapa inhibits cytokine and immunoglobulin synthesis, blocks lymphocyte proliferation, and leads to T lymphocyte unresponsiveness upon cytokine stimulation (15,16). RAPA allows, at low doses, long-term graft survival in many models and many animal species (17–19). Short-term graft success under RAPA treatment has been evaluated in this study in 4 different muscle-grafting models. Graft success has been assessed mainly by the characterization of donor fibers in the host muscles, by the examination of Dys expression, and by the analysis of production of anti-donor cell Ab. Finally, the infiltration of cells involved in inflammation and immune reactions has been studied in grafted muscles.

ATP-sensitive potassium channel and hormone/neuropeptide

ATP-sensitive potassium channels (KATP) are the ion channels which are closely associated with cellular metabolism. A number of chemical compounds which block KATP facilitate the release of hormones or neuropeptides. For example, KATP-blocking agents such as antidiabetic sulfonylureas and imidazolines stimulate insulin secretion from pancreatic beta-cells by decreasing KATP activity. On the other hand, so-called potassium channel openers, KATP-activating drugs which constitute a chemically diverse group of compounds, inhibit growth hormone secretion from anterior pituitary cells and release of gamma-aminobutylic acid from substantia nigra. Several endogenous substances also modulate release of hormone or neuropeptide by affecting KATP activity. Acetylcholine and histamine stimulate the release of endothelium-derived hyperpolarizing factor, which activates KATP in the plasma membrane of vascular smooth muscle cells. Both galanin and somatostatin inhibit insulin release from pancreatic beta-cells by opening KATP through the activation of G-protein. Glucagon-like peptide-1[7-36], which stimulates insulin secretion by indirectly blocking KATP in beta-cells, shows antidiabetic effects in patients with non-insulin-dependent diabetes mellitus. Endosulphine, an endogenous inhibitor of KATP, stimulates insulin secretion from pancreatic beta-cells. Accumulating knowledge of the modulation and function of KATP would help our understanding of the regulation and physiological role of hormones and neuropeptides.

An ultrastructural, cytochemical and freeze-fracture study of the surface structures of Brugia malayi microfilariae

Ultrastructural analysis of the cuticle of Brugia malayi micronlariae indicated that it is composed of 2 regions: the inner one 15–20 nm thick with a homogeneous aspect and the outer one, designated as epicuticle, which is 15–20 nm thick. Three laminae separated by electron-lucent regions were seen in the epicuticle. Labeling of the cuticle and epicuticle of B. malayi and Wuchereria bancrofti micronlariae was observed when thin sections of Lowicryl-embedded parasites were incubated in the presence of gold-labeled phospholipase-C. Replicas offreeze-fractured micronlariae showed the presence of 2 fracture planes in the epicuticle and no fracture plane in the inner region of the cuticle. The P face of the epicuticle outer fracture plane presented few particles similar to intramembranous particles (IMPs). The epicuticle inner fracture plane P and E faces presented large numbers of densely-packed small particles and many protuberances. Also, fracture faces of hypodermal and muscle cell plasma membranes were analyzed. Faces P and E of fractured membranes showed the presence of typical IMPs. P faces of both membranes showed larger amounts of particles than E faces. Fracture of muscle plasma membrane revealed a linear array of particles disposed in parallel rows on its P face.

Cholecystokinin-Stimulated Intracellular Signal Transduction Pathways

Cholecystokinin stimulates a variety of physiological effects throughout the gastrointestinal tract, including exocrine pancreatic secretion, contraction of gallbladder and smooth muscle throughout the gastrointestinal tract, relaxation of the sphincter of Oddi and inhibition of gastric emptying. To initiate these responses cholecystokinin must first interact with receptors on the plasma membrane of either pancreatic acinar or smooth muscle cells. Following receptor occupation the receptor is coupled to generation of intracellular messengers, such as ions, cyclic nucleotides or derivatives of phospholipid hydrolysis. These intracellular messengers activate effectors, the systems that cause a biological response. This paper uses the exocrine pancreas as a model for cholecystokinin stimulated signal transduction and examines cholecystokinin stimulated mobilization of calcium and the activation of protein kinase C. Calcium and protein kinase C act differently to mediate either the initial or sustained phases of amylase secretion from the pancreas. The activation of protein kinase C and the rise of intracellular free calcium is necessary for the initial phase of secretion, but unimportant for the sustained phase of secretion. Calcium from extracellular sources is necessary for the sustained phase of secretion. The cholecystokinin-stimulated intracellular signaling outlined for the exocrine pancreas also occurs in other tissues for transmitting the signal from the cholecystokinin receptor to the inside of the cell.

Occurrence of unusual tubular invaginations of the plasma membrane in smooth muscle cells of the lamprey, Lampetra japonica

Numerous tubular structures were observed in the surface region of smooth muscle cells making up the vascular walls in the lamprey, Lampetra japonica; they were designated as surface tubules. The limiting membrane of the surface tubules was connected to the plasma membrane, allowing communication of the lumen of the tubule with the extracellular space. Tannic acid reacted with osmium, serving as an extracellular marker, penetrated into the tubules but not into the intracellular organelles, such as the endoplasmic reticulum and the Golgi complex. The surface tubules were grouped in longitudinal parallel rows, separated from each other by tubule-free areas where dense plaques were present. Each tubule was fairly cylindrical (approximately 60 nm in diameter) and often ramified into two or three branches with a blind end. Occasionally, these tubules were encircled by the sarcoplasmic reticulum which was located immediately beneath the plasma membrane. Similar tubules were also observed in the surface region of vascular endothelial cells and fibroblasts in the adventitial connective tissue. The possibility that the surface tubules in the present observations are analogous to the smooth muscle caveolae or the striated muscle T-tubule is discussed.

Problems and paradigms: Dystrophin as a mechanochemical transducer in skeletal muscle

This review is primarily concerned with two key issues in research on dystrophin: (1) how the protein interacts with the plasma membrane of skeletal muscle fibres and (2) how an absence of dystrophin gives rise to Duchenne muscular dystrophy. In relation to the first point, we suggest that the post-translational acylation of dystrophin may contribute to its interaction with the plasma membrane. Regarding the second point, it is generally considered that an absence of dystrophin makes the plasma membrane susceptible to damage by contraction/relaxation cycles. In this connection, we propose that the progressive nature of Duchenne dystrophy, and the phenotypic characteristics of mdx mice, are more consistent with dystrophin functioning as a mechanical transducer that transmits growth stimuli from the enlarging skeleton to the muscle. On the basis of this hypothesis, dystrophin-deficient muscles would be unable to grow at the same rate as the skeleton.

Characterization of large-conductance, calcium-activated potassium channels from human myometrium

The purpose of our study was to detect and characterize potassium channels in the plasma membrane of smooth muscle cells from human myometrium. Plasma membrane vesicles were incorporated into lipid bilayers to record single potassium channel activity. We predominantly found a "maxi" calcium-activated potassium channel (261 picosiemens). This channel was calcium (micromoles per liter range) and voltage sensitive, highly selective for K+ over Na+ and Cs+, and was sensitive to external tetraethylammonium (dissociation constant approximately 220 mumol/L) and charybdotoxin (dissociation constant approximately 23 nmol/L). External apamin and 4-aminopyridine had no effect on this channel. Another type of potassium channel that was less frequently observed was also identified. It had a smaller conductance (142 picosiemens) and it seemed to be calcium independent (up to 50 nmol/L). Human myometrium possesses abundant "maxi" calcium-activated potassium channels. This channel shares common characteristics with other "maxi" calcium-activated potassium channels, including calcium and voltage gating, high conductance and selectivity, and channel pharmacologic profile.

Immunocytochemical localization of 67 KD Ca2+ binding protein (p67) in ventricular, skeletal, and smooth muscle cells.

By using immunocytochemical techniques, we examined the localization of a 67 kDa Ca2+ binding protein (p67) and calpactin I heavy chain (p36) in ventricular myocytes, skeletal myocytes, and intestinal smooth muscle cells. Immunofluorescence microscopy revealed that the p67 was expressed in all these muscle cells, whereas anti-p36 antibody stained cells in connective tissues but failed to stain these muscle cells. Immunogold electron microscopy was carried out to examine the subcellular localization of the p67 in muscle cells. The results showed that the p67 was exclusively confined to the plasma membrane of muscle cells and the presumptive transverse tubules of the striated myocytes. Immunoblot analysis with anti-p67 antibody showed that the p67 was indeed a constitutive protein of the sarcolemma isolated from rat hearts. These results indicate that the p67 is a sarcolemma-associated Ca2+ binding protein expressed in both striated myocytes and intestinal smooth muscle cells.

Effect of diabetes on glucoregulation. From glucose transporters to glucose metabolism in vivo.

Peripheral resistance to insulin is a prominent feature of both insulin-dependent and non-insulin-dependent diabetes. Skeletal muscle is the primary site responsible for decreased insulin-induced glucose utilization in diabetic subjects. Glucose transport is the rate-limiting step for glucose utilization in muscle, and that cellular process is defective in human and animal diabetes. The transport of glucose across the muscle cell plasma membrane is mediated by glucose transporter proteins, and two isoforms (GLUT1 and GLUT4) are expressed in muscle. Insulin acutely increases glucose transport in muscle by selectively stimulating the recruitment of the GLUT4 transporter (but not GLUT1) from an intracellular pool to the plasma membrane. In skeletal muscles of streptozocin-induced diabetic rats, there is a decreased GLUT4 protein content in intracellular and plasma membranes. In these rats, insulin induced the mobilization of GLUT4 from the internal pool, but the incorporation of the transporter protein into the plasma membrane is diminished. Conversely, the content of the GLUT1 transporter increases in the plasma membrane of these diabetic rats. Normalization of glycemia with phlorizin fully restores the amount of GLUT1 and GLUT4 proteins to normal levels in the plasma membrane without altering insulin levels. This suggests that glycemia regulates the number of glucose transporters at the cell surface, GLUT1 varying directly and GLUT4 inversely, to glycemia. The regulatory role of glycemia also can be seen in diabetic dogs in vivo, where correction of hyperglycemia with phlorizin restores, at least in part, the defective metabolic clearance rate of glucose seen in these animals. In addition to acutely stimulating glucose transport in muscle, insulin controls exercise- and possibly stress-mediated glucose uptake in vivo, by preventing hyperglycemia and by restraining the effects of catecholamines on lipolysis and/or muscle glycogenolysis. Finally, we postulated a neural pathway that requires the permissive effect of insulin to increase glucose uptake by the muscle. Thus, insulin, glucose, and neural pathways regulate muscle glucose utilization in vivo and are, therefore, important determinants of glucoregulation in diabetes.

Biochemical and immunocytochemical localization of the 'GLUT5 glucose transporter' in human skeletal muscle.

Using biochemical and immunocytochemical techniques, we have assessed both the protein expression and the cellular localization of the GLUT5 transporter in human skeletal muscle. Human muscle membranes, prepared by subcellular fractionation, were subjected to SDS/PAGE and Western-blot analyses using antiserum raised against a specific C-terminal amino acid sequence of the human GLUT5 transporter. GLUT5 was detected as a discrete 49 kDa protein band in a plasma-membrane-enriched fraction prepared from either soleus or gracilis muscle. In contrast, GLUT5 protein was not detectable to any significant extent in fractions which were devoid of muscle plasma membranes (mean GLUT5 abundance in intracellular fractions from three muscle preparations amounted to approximately 10% of that in the plasma-membrane-enriched fraction). Immunofluorescence studies using cryostat sections of human triceps muscle supported the biochemical observations and revealed that GLUT5 antibody selectivity labelled the plasma membrane of muscle cells. This immuno-labelling was significantly suppressed after tissue incubation with antiserum in the presence of a 14-amino-acid synthetic peptide corresponding to a specific C-terminus sequence of human GLUT5. These results indicate that human skeletal muscle expresses the GLUT5 transporter and that it is specifically localized to the plasma membrane, where it may participate in regulating hexose transfer across the sarcolemma.

Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells

Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells

ATP-sensitive K+ conductance in pancreatic zymogen granules: block by glyburide and activation by diazoxide.

The properties of transporters (or channels) for monovalent cations in the membrane of isolated pancreatic zymogen granules were characterized with an assay measuring bulk cation influx driven by a proton diffusion potential. The proton diffusion potential was generated by suspending granules in an isotonic monovalent cation/acetate solution and increasing the proton conductance of the membrane with a protonophore. Monovalent cation conductance had the sequence Rb+ > K+ > NA+ > Cs+ > LI+ > N-methyl glucamine+. The conductance could be inhibited by Ca2+, Mg2+, Ba2+, and pharmacological agents such as quinine, quinidine, glyburide and tolbutamide, but not by 5 mM tetra-ethyl ammonium or 5 mM 4-aminopyridine, when applied to the cytosolic surface of the granule membrane. Over 50% of K+ conductance could be inhibited by millimolar concentrations of ATP or MgATP. The inhibition by MgATP, but not by ATP itself, was reversed by the K+ channel opener diazoxide. The inhibitory effect is probably by a noncovalent interaction since it could be mimicked by nonhydrolyzable analogs of ATP and by ADP. The reversal of MgATP inhibition by diazoxide may be mediated by phosphorylation since it was not affected by dilution, and was blocked by the protein kinase inhibitor H7. The properties of the K+ conductance of pancreatic zymogen granule membranes are similar to those of ATP-sensitive K+ channels found in the plasma membrane of insulin-secreting islet cells, neurons, muscle, and renal cells.

Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells.

The effects of the oral hypoglycemic drug metformin on glucose and amino acid transporter activity and subcellular localization of GLUT1 and GLUT4 glucose transporters were tested in cultured L6 myotubes. In muscle cells preexposed to maximal doses of metformin (2 mM, for 16 h), 2-deoxyglucose uptake was stimulated by over 2-fold from 5.9 +/- 0.3 to 13.3 +/- 0.5 pmol/min.mg protein. Uptake of the nonmetabolizable amino acid analog methylaminoisobutyrate was unaffected by treatment with the drug under identical conditions. Extracellular calcium was required to preserve the full response to the biguanide. Exposure of muscle cells to insulin in the presence of metformin resulted in further activation of 2-deoxyglucose transport. The latter effect was additive to the maximum effect of metformin, suggesting that the biguanide stimulates hexose uptake into muscle cells by an insulin-independent mechanism. Glucose transporter number quantified by performing studies of D-glucose-protectable binding of cytochalasin-B in plasma membranes (PM) and internal membranes (IM) prepared from L6 myotubes revealed that a 16-h treatment with 800 microM metformin significantly elevated glucose transporter number in the PM (by 47%), with an equivalent decrement in glucose transporter number (47%) in the IM. Western blot analysis using antisera reactive with the GLUT1 and GLUT4 isoforms of glucose transporters showed that metformin caused a reduction in GLUT1 content in the IM fraction and a concomitant increase in the PM. Unlike insulin, metformin treatment had no effect on the subcellular distribution of GLUT4. We propose that the molecular basis of metformin action in skeletal muscle involves the subcellular redistribution of GLUT1 proteins from an intracellular compartment to the plasma membrane. Such a recruitment process may form an integral part of the mechanism by which the drug stimulates glucose uptake (and utilization) in skeletal muscle and facilitates lowering of blood glucose in the management of type II diabetes.

Ultrastructural detection in vitro of WGA-, RCA I-, and Con A-binding sites involved in the invasion of heart muscle cells by Trypanosoma cruzi.

The presence of carbohydrate residues in the plasma membrane of normal and Trypanosoma cruzi-infected heart muscle cells was investigated cytochemically using ruthenium red, lanthanum nitrate, periodic acid-Schiff/thiocarbohydrazide/silver, and gold- and ferritin-lectin complexes. The study combined conventional electron microscopy with the new analytical technique of electron spectroscopic imaging (ESI). Galactosyl, mannosyl, and sialyl residues were detected in regions of host-cell plasma membrane that undergo interiorization together with the parasite. Lectin-binding sites were sometimes found to show a punctate or patchy distribution in the endocytic vacuole membrane. These findings suggest the that glycoconjugates cytochemically detected in the host-cell plasma membrane participate in the invasion of heart muscle cells by T. cruzi.

Ordered distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells revealed by freeze-fracture following treatment with cholesterol probes.

The surface distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells was investigated by freeze-fracture. Replicas of fractured smooth muscle cell plasma membrane showed caveola-free regions with few intramembrane particles, interspersed with caveola-populated areas with a higher intramembrane particle density. Electron microscopy of thin sections of quail gizzard smooth muscle revealed the regions free of caveolae to be occupied by membrane-associated dense plaques; anchoring sites for the contractile filaments of the cell. Demarcation between the caveola-populated and caveola-free regions on the relicated intramembrane surface was not clear and thus provided little information concerning the distribution of dense plaque sites. However, treatment of the smooth muscle tissue with the cholesterol-binding agents filipin or tomatin prior to freeze-fracture allowed the dense plaque sites to be easily observed as the sites remained free of the membrane deformations characteristic of these agents. The dense plaque sites consist of caveola-free oval areas juxtaposed in regular bands that traverse the long axis of the cell. The dense plaque sites on the freeze-fracture replica were confirmed by electron microscopy of thin sections of filipin-treated quail gizzard smooth muscle cells, which showed the plasma membrane associated with the dense plaques to be unaffected by the actions of filipin, whereas that of the caveola-populated region was severely deformed. The observations presented in this study provide evidence for a highly ordered distribution of dense plaques at the cell surface of intact quail gizzard smooth muscle cells and thus corroborate existing evidence for an organized substructure of smooth muscle cells.

Dystrophin at the plasma membrane of human muscle fibers shows a costameric localization

We studied the distribution of dystrophin at the sarcolemma of normal human muscle fibers using high resolution immunofluorescence and confocal laser scanning optical microscopy (CLSOM). We found that the dystrophin lattice is organized at the muscle plasma membrane in an array of thick bands interconnected by a finer network. The bands encircle the muscle fiber perpendicular to the long axis of the fiber and they matched the sites of attachment of the sarcomeres to the plasma membrane. Dystrophin co-localized with vinculin, and dystrophin and vinculin co-localized with alpha-actinin at the region of the I-band. Dystrophin may be one of the proteins involved in the linkage of the sarcomeres to the extracellular matrix.

Cytochemical investigation of p-NPPase activity in rat cardiac muscle.

The cytochemical demonstration of the atrial cardiac myocyte pumping activity has been made by detecting p-nitrophenylphosphatase (NPPase), which is used by investigating of Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase activities. The fine ultrastructural localization of these enzymes was studied using cytochemical methods with cerium as a capturing agent. Na(+)-K(+)-ATPase was localized on the atrial muscle cell plasma membrane, T-tubule membrane, endothelial cell nuclear membrane, and cardiac myocyte nuclear membrane. H(+)-K(+)-ATPase was localized on the atrial muscle cells plasma membrane, T-tubule membrane, and sarcoplasmatic reticulum. The present findings indicate that the transporting metabolic activity of the heart as an endocrine organ is realized by the interaction between p-NPPases.

Effect of benzylamine and its metabolites on the responses of the isolated perfused mesenteric arterial bed of the rat

1. Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme activity which can be found in the plasma membrane of rat vascular smooth muscle cells. We have investigated the possibility that the products of deamination by this enzyme, namely ammonia, hydrogen peroxide and the aldehyde, may be important in the modulation of the responses of vascular smooth muscle to extracellular stimuli. 2. The isolated perfused mesenteric arterial bed of the rat was used and dose-pressure response curves (DRC) to bolus injections of adrenaline (Ad) or ATP were plotted by non-linear curve fitting. The relaxant effects of carbachol (CCh), which releases endothelium dependent relaxing factor (ERDF), were studied by co-administering CCh with Ad. The effects of including the preferred SSAO substrate, benzylamine (BZ; 25 microM), in the perfusion fluid throughout the experiment and of inhibition of SSAO by treatment of rats with (E)-2-(3',4'-dimethoxyphenyl)-3-fluoroallylamine (MDL 72145; 1 mg kg-1) 1 h before dissection, have been studied. 3. Neither BZ nor SSAO inhibition affected the DRC to ATP. BZ shifted Ad responses to the left, inhibition of SSAO increased this shift indicating that the amine, but not its metabolites, were responsible for the potentiation of the responses to Ad. DRC to CCh showed a shift to the left and a significant decrease in the Hill slope with BZ, indicative of a potentiation of low doses of CCh more than high doses. Inhibition of SSAO prevented this change and so the metabolites of BZ deamination appeared to be involved in the potentiation. 4. Ammonia generated by SSAO may contribute to the production of EDRF or hydrogen peroxide may sensitize guanylate cyclase to stimulation by EDRF and so explain these findings.

Blockade of the ATP-sensitive potassium channel modulates reactive hyperemia in the canine coronary circulation.

The mechanism of reactive hyperemia remains unknown. We hypothesized that reactive hyperemia was related to the opening of ATP-sensitive potassium channels during coronary occlusion. The resulting hyperpolarization of the smooth muscle cell plasma membrane might reduce calcium influx through voltage-dependent calcium channels and result in relaxation of smooth muscle tone and vasodilation. In eight open-chest, anesthetized dogs, 30-second coronary occlusions resulted in an average flow debt repayment of 200 +/- 41%. After low-dose (0.8 mumol/min) and high-dose (3.7 mumol/min) infusion of intracoronary glibenclamide, flow debt repayment fell to 76 +/- 14% and 50 +/- 8%, respectively (p less than 0.05 compared with control for both). The decline in flow debt repayment was due to a significant reduction both in maximum coronary conductance during reactive hyperemia and in its duration. In addition, there was a significant decline in the sensitivity of the coronary circulation to adenosine-induced vasodilation after glibenclamide. While more variable, there was no overall change in the sensitivity of the coronary vasculature to acetylcholine-induced vasodilation after glibenclamide. We conclude that reactive hyperemia is determined in a large part by the ATP-sensitive potassium channel, probably through its effect on membrane potential and voltage-sensitive calcium channels. Because reactive hyperemia was never fully abolished at the highest doses of glibenclamide tested, it is possible that additional mechanisms are involved in the genesis of this complex phenomenon.

Characterisation of Ca 2+ or Mg 2+-dependent nucleoside triphosphatase from rat mesenteric small arteries

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent K m in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF 4 −, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N′-dicyclohexylcarbodiimide caused significant (30%) inhibition. In the presence of micromolecular concentrations of Ca 2+ and Mg 2+, small (<20 mM) concentrations of Na +, K +, Rb +, Cs + and choline +, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.